Abstracts from AIDS Vaccine 2011 Bangkok,Thailand 12–15 September,2011

1 Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland 2 Centre Hospitalier Universitaire Vaudois (CHUV)/Gastroenterology, Lausanne, Switzerland 3 Institut de Génétique Moléculaire de Montpellier (IGMM), Montpellier, France 4 Sanofi Pasteur, Swiftwater, USA 5 Institute of Medical Microbiology, University of Regensburg, Regensburg, Germany 6 Department of Molecular and Cellular Biology (CNB-CSIC), Madrid, Spain 7 Institut National de la Santé et de la Recherche Médicale, Unité U955, Créteil, France OA01.01 DNA/NYVAC Vaccine Regimen Induces HIV-Specific CD4 and CD8 T-Cell Responses in Intestinal Mucosa

Background: HIV vaccine-candidates based on rare adenovirus serotypes such as Ad26 and Ad35 vectors, and poxvirus vectors are important components of future promising vaccine regimens that in the near future hopefully will move into a number of efficacy clinical trials in combination with protein vaccines. For these reasons, it is important to comprehensively characterize the vaccine-induced immune responses in different anatomical compartments and particularly at mucosal sites which represent the primary port of entry for HIV.

Methods: In the present study, we have investigated the anatomic distribution in blood and gut mucosal tissues (rectum and ileum) of memory poxvirus-specific CD4 and CD8 T cells in subjects vaccinated with smallpox and compared with vector (NYVAC)-specific and HIV insert-specific T-cell responses induced by an experimental DNA-C/NYVAC-C vaccine regimen.

Results: Smallpox-specific CD4 T-cell responses were present in the blood of 52% of subject studied, while Smallpox-specific CD8 T cells were rarely detected (12%). With one exception, Smallpox-specific T cells were not measurable in gut tissues. Interestingly, NYVAC vector-specific and HIV-specific CD4 and CD8 T-cell responses were detected in almost 100% of the subjects immunized with DNA-C/NYVAC-C in blood and gut tissues. The large majority (83%) of NYVAC-specific CD4 T cells expressed a4b7 integrins and the HIV co-receptor CCR5.

Conclusion: These results demonstrate that the experimental DNA-C/NYVAC-C HIV vaccine regimen induces the homing of potentially protective HIV-specific CD4 and CD8 T cells in the gut, the port of entry of HIV and one of the major sites for HIV spreading and depletion of CD4 T cells.

1 Military HIV Research Program, Rockville, USA 2 Armed Forces Research Institute for Medical Sciences, Bangkok, Thailand 3 University of Pennsylvania School of Medicine, Philadelphia, USA 4 Inovio Pharmaceuticals, Blue Bell, USA 5 Divisions of AIDS, NIAID, Bethesda, USA 6 Laboratory of Viral Diseases, NIAID, Bethesda, USA OA01.02 Cell-Mediated Immune Responses After DNA Delivered by Either Biojector or Electroporation and Boosted with a Heterologous Insert Recombinant Poxvirus

Background: We conducted a phase I, randomized, open label trial of PENNVAX^TM-G DNA and MVA-CMDR to evaluate safety and immunogenicity of a heterologous DNA prime/recombinant poxvirus boost in human volunteers in the US prior to expansion of the trial into east Africa.

Methods: Volunteers were given a priming vaccination with PENNVAX^TM-G DNA at months 0 and 1, followed by boosting with MVA-CMDR at months 3 and 6. Immunomonitoring was performed at pre-immunization, and at 2 weeks post-DNA and MVA vaccinations respectively. The phenotype and function of HIV-specific T cells was examined by flow cytometry measuring IFNγ, IL-2, TNFα, MIP-1β and CD107a. Simultaneous phenotyping for effector cell, effector-memory cell, and central memory cell populations was performed using the surface markers CD45RA, CD45RO, CCR7 and CD28.

Results: Early analysis of the ICS assay data has revealed that a strong cell-mediated immune response was generated in subjects receiving either the BioJector or Electroporation delivered DNA and a rMVA-CMDR boost. IFNγ producing CD4+ and CD8+ T cells specific for both Gag and Env peptide sets matching the rMVA-CMDR inserts were detected at frequencies as high as 0.57% (Env-specific CD8+ T cells) and 0.37% (Env-specific CD4+ T cells). The phenotype of the responding cells has been identified as effector-memory cells (CD45RO+/CCR7-/CD28-) for both the CD4+ and CD8+ T cell populations. The highest magnitude responses were seen after the MVA-CMDR boost.

Conclusion: PENNVAX^TM-G DNA prime/MVA-CMDR boost generates a balanced CD4+/CD8+HIV-specific T cell-mediated immune response. Elispot and further ICS assays using DNA insert matched peptide sets are planned to determine the frequency of antigen-specific T cells after the DNA prime.

1 NCI/NIH, Bethesda, USA 2 AIDS and Cancer Virus Program NCI-Frederick, USA 3 Human Monoclonal Antibody Core Laboratory, University of Maryland, USA 4 Duke University Medical Center, Durham, USA 5 Biostatistics and Data Management Section, NCI/NIH, USA 6 Human Retrovirus Section, NCI/NIH, USA 7 Walter Reed Army Institute of Research, USA 8 Sanofi Pasteur, Inc, USA OA01.03 Titered Mucosal Challenge of Rhesus Macaques with SIVmac251 Recapitulates HIV Vaccine Efficacy in Humans

Background: A combination vaccine based on the recombinant canarypox vector ALVAC-HIV and HIV gp120 envelope glycoprotein protected nearly one third of vaccinees from HIV acquisition but afforded no protection from CD4⁺T-cell loss or high virus levels in vaccines that became infected in the RV144 trial in Thailand. This result was surprising, given the limited ability of either vaccine component to induce CD8⁺T-cell responses or elicit broadly neutralizing antibodies. An animal model able to predict HIV vaccine efficacy for humans could hasten progress in our understanding of the immune responses that contribute to protection.

Methods: We vaccinated Indian rhesus macaques with an ALVAC-SIV and SIVgp120 immunization regimen that mimics the RV144 trial. At the end of the immunization regimen, the vaccinated animals received a titered mucosal challenge with a dose of SIV_mac251 shown to transmit a limited number of variants to naïve controls, recapitulating human mucosal HIV transmission.

Results: Vaccine immunogenicity and efficacy in macaques were similar to observations in humans; as in RV144, one third of vaccinated macaques were protected from acquisition SIV_mac251. Viral load and CD4⁺ T cell numbers in animals that did become infected were not different from controls. Animals protected from infection had equivalent T-cell responses but higher avidity binding antibodies to gp120 compared to unprotected animals.

Conclusion: Titered intrarectal challenge of macaques with SIV_mac251 shows potential to accurately model clinically observed vaccine efficacy, suggesting a role for this model in understanding protective responses elicited by HIV preventive vaccines. Consistent with the results from RV144, our findings suggest non-neutralizing antibody responses may play a role in protection from SIV/HIV acquisition.

1 Centre for the AIDS Programme of Research in South Africa (CAPRISA), U, Durban, South Africa 2 HIV Pathogenesis Programme, Doris Duke Medical Research Institute and, South Africa 3 University of Kwazulu-Natal, Durban, South Africa OA01.04 Preservation of HIV-1-Specific CD4+ T-IFNγ-Cell Responses in Intercurrent Infections Following Exposure to Tenofovir Gel

Background: The CAPRISA004 microbicide trial demonstrated reduction of sexual HIV-1 acquisition in women using a vaginal microbicide gel containing tenofovir. Future vaccine development efforts need to factor in the consequences of prophylactic use of anti-retrovirals in the evolution of immune responses during intercurrent HIV-1 infection. From the microbicide trial, we compared innate and adaptive immune responses in women with primary/intercurrent HIV-1 infection following exposure to 1% tenofovir gel or placebo gel.

Methods: Natural killer (NK) cells and myeloid dendritic cells (mDCs) frequencies and activation status, and HIV-1-specific T cell responses were cross-sectionally assessed using multi-parametric flow cytometry in 36 randomly selected HIV-1 clade C infected female adults exposed either to tenofovir microbicide gel (n = 17) or placebo (n = 19) at an estimated 3 months post HIV acquisition.

Both the 17 tenofovir-arm and 19 placebo-arm women had similar CD4+ T cell counts and HIV-1 viral loads at time of change in serostatus as well as at the time of this study.

Results: Overall, NK cell and mDCs frequencies and activation, and HIV-1-specific CD8+ T cell responses did not differ significantly between the two groups. In contrast, HIV-1-specific Gag IFNγ CD4+ T-cell responses were significantly higher (p = 0.01) in infected women randomized to the tenofovir arm.

Conclusion: These data suggest that the use of tenofovir gel by women around the time of breakthrough HIV infection may lead to preservation of HIV-1-specific IFNγ CD4+ T cells. Overall, the use of tenofovir containing gel around the time of infection can modulate HIV-1 immunity, and these immunological changes need to be considered in future trials combining HIV-1 vaccines and microbicides.

1 GeoVax Inc., Smyrna, USA 2 Yerkes National Primate Research Center, Atlanta, USA 3 Louisiana State University Health Sciences Center, New Orleans, USA 4 National Institute of Allergy and Infectious Diseases, NIH, Bethesda, USA 5 National Cancer Institute at Frederick, Frederick, USA 6 Vaccine Research Center, Emory University, Atlanta, USA OA01.05 Homologous Priming and Boosting with Recombinant MVA, Prevention of Infection as Good as With the Heterologous Regimen of DNA Priming and MVA Boosting

Background: Heterologous priming and boosting has become popular for HIV/AIDS vaccine development. Here we report that a simpler homologous regimen of MVA priming and boosting elicits rates of prevention of rectal infection as good as those elicited by the more complicated heterologous regimen of DNA priming and MVA boosting.

Methods: SIV239 vaccines produced non-infectious virus-like-particles (VLP) displaying trimeric membrane-bound Env. A DDMM regimen delivered DNA (3mg/i.m.) at 0 and 8 weeks and MVA (1x108 plaque forming units/i.m.) at 16 and 24 weeks. An MMM regimen delivered MVA at weeks 0, 8 and 24. Twelve weekly intrarectal challenges with 5000 50% tissue-culture infectious doses of SIVsmE660 were administered at 6 months following the final immunization.

Results: The DDMM regimen elicited CD4+ T cells with higher breadth, magnitude, and degree of polyfunctionality than the MMM regimen. The MMM regimen elicited higher titer and higher avidity Env-specific IgG, higher ADCC activity, and higher Env-specific IgA responses in rectal secretions. Magnitudes, breadths and functionality of elicited CD8+ T cells and titers of neutralizing antibody were overall similar between groups. The best protective responses were in the MMM group. Following 6 challenges, 5 out of 8 MMM vaccinated animals (60%), 3 out of 8 DDMM vaccinated (40%) and 1 out of 23 unvaccinated controls (4%) remained uninfected. By 12 challenges, all unvaccinated animals were infected, whereas 2 out of 8 rhesus in both DDMM and MMM groups remained protected. The challenge infected 30% of the unvaccinated rhesus at each exposure, a much higher transmission rate than observed for typical human infections.

Conclusion: Our results reveal the simpler MMM regimen eliciting more antibody and comparable, if not better, protection than the more complex DDMM regimen. Our results suggest that homologous priming and boosting with VLP expressing MVA immunogens may merit testing in Phase 2b efficacy trials.

1 Fred Hutchinson Cancer Research Center, Seattle, USA 2 Institute for Systems Biology, Seattle, USA 3 Institute for systems Biology, Seattle, USA 4 University of Alabama at Birmingham, Birmingham, USA 5 GeoVax Labs Inc., Smyrna, USA OA02.01 Innate Immune Response Signatures to Ad5 (HVTN 071) and MVA (HVTN 205/908) Vectored Candidate HIV Vaccines Predict Induction of Adaptive Responses

Background: Identifying human innate immune signatures that predict adaptive immune responses may enable development of more efficacious HIV vaccines.

Methods: We compared the innate immune responses after vaccination with either Ad5/HIV (MRKAd5 HIV-1 gag/pol/nef, n = 35), or a DNA/HIV prime, MVA/HIV boost regimen (pGA2/JS7 DNA, MVA/HIV62 gag/pol/env, n = 22) and identified signatures predictive of T-cell responses to HIV inserts.

Results: No changes in serum cytokines, circulating leukocyte populations, or PBMC gene transcription were detected at 24 hours after DNA/HIV vaccination. In contrast, vaccination with Ad5/HIV or MVA/HIV caused a decrease in circulating lymphocytes at 24 hours and an increase in CD14+ CD16+ transitional monocytes at 72 hours post-vaccination. Profiling of 74 soluble factors in the serum revealed that Ad5/HIV induced changes in 21 while MVA/HIV altered only 8 (Hochberg p < 0.05). Commonly induced factors included chemokines likely responsible for the alterations in circulating leukocytes such as IP-10 and MCP-1, as well as TNF-?, IFN-?, and TRAIL. Ad5/HIV showed higher magnitude induction of TRAIL, I-TAC, and MCP-2 than MVA/HIV and significantly induced the immunoregulatory cytokine IL-10. As seen for serum cytokines, PBMC transcriptional responses induced by MVA/HIV, including many interferon-inducible networks, were a subset of Ad5/HIV-induced responses. Ad5/HIV vaccination uniquely up-regulated expression of 244 genes in PBMC, including C2, CD40, and IL-10. Linear discriminant analysis examining signatures common to the two vaccines revealed that serum changes in IP-10 and TRAIL at 24 hours post-vaccination predicted the induction 2–4 weeks later of Gag-specific CD8+ T-cells expressing IL-2 and/or IFN-γ with 75–90% accuracy. Analysis of transcriptional response signatures that predict T-cell and nAb responses is on-going.

Conclusion: These data may help to explain differential induction and potency of adaptive immune responses by these two vaccine vectors, and such an approach can be applied to optimize future vaccine regimens.

1 University of KwaZulu-Natal, Durban, South Africa 2 Ragon Institute of MGH, MIT and Harvard, Charlestown, USA OA02.02 Frequent and Strong NK Responses to HIV-1 Env Peptides in Individuals with Acute and Chronic HIV-1 Infection

Background: NK cells play a critical role in the control of HIV-1 infection and NK cells that respond to HIV peptides have been recently described (Tiemessen et al, JI, 2009). However, the mechanisms by which NK cells recognize HIV-1 antigen are not understood. We investigated the frequency of NK responses to HIV peptide pools during acute and chronic HIV clade B infection.

Methods: Individuals with acute HIV-1 infection (n = 15), progressive chronic (n = 10) and controlled chronic HIV-1 infection (n = 10) were studied. Whole blood was stimulated with HIV peptide pools spanning the entire clade B consensus sequence and NK responses were quantified using multiparameter flow cytometry.

Results: No NK cell responses to HIV-1 peptides were detected in HIV uninfected individuals. In contrast, 60% of chronically infected individuals and 58% of individuals with acute HIV-1 infection had detectable IFN-γ+ NK cell responses to HIV-1 antigen (p = 0.002 compared to negative individuals). IFN-γ and TNF-α producing NK cell responses most frequently targeted Env (ranging from 2%-30% of all NK cells). Other HIV-1 proteins such as Gag, Pol and Nef were rarely targeted. Env-specific NK cell responses were fine-mapped to individual 18mer peptides and did not depend on HIV-1 specific T cell responses. In contrast, Env-specific T cell responses were most commonly absent in individuals with NK cell responses to Env.

Conclusion: NK cells from HIV-1-infected individuals can respond frequently and strongly to HIV-1 peptides in vitro. The receptor/ligand interactions responsible for the recognition of HIV-1 require further investigation to rationally modulate NK cell responses to HIV-1.

1 AP-HP, Groupe Henri-Mondor Albert-Chenevier, Immunologie clinique, Créteil, France 2 Institut Pasteur, Paris, France OA02.03 NKp46 and NKG2D Receptors Are Implicated in the Priming and Suppression of HIV Replication by Natural Killer Cells Stimulated by MVA/HIV ANRS Vaccine

Background: Innate mechanisms are critical for the normal development of host immune responses to antigen. The interaction between Natural Killer (NK) and Dendritic cells (DC) is expected to greatly impact the establishment of both innate and adaptive immune responses to vaccine.

Methods: We describe in vitro responses of NK cells to recombinant Modified Vaccinia Virus Ankara expressing HIV-1 peptides (rMVA) compared to wild type vector (wtMVA) using an autologous in vitro co-culture system to analyze the NK response to DC exposed to the viral vector.

Results: Monocyte-Derived DC were efficiently infected with the rMVA virus and produced the encoded HIV-1 proteins. This being a cytopathic virus, we subsequently used a fluorescent dye system, to demonstrate that rMVA-infected DC were phagocytosed by uninfected DC after 48h in co-culture, allowing for cross-presentation in our in vitro system. MDDC infected with rMVA or the wtMVA control did not differ in their production of cytokines; and when these DC were cultured with autologous NK cells, they stimulated similar levels of NK proliferation. Both MVA strains were capable of inducing NK repertoire modifications, but comparative analysis indicated that NK stimulated with rMVA expressed higher levels of NKp30 and NKG2D. NK cells also showed increased degranulation when stimulated with rMVA-infected DC compared to wtMVA. This stimulation was contact-dependent and augmented when NKp46 was blocked during the NK:DCrMVA interaction. Functionally, rMVA-infected DC stimulated NK cells were better at subsequently suppressing HIV-1 in vitro as compared to wtMVA-stimulated cells. Blocking experiments showed NKG2D as major factor in this NK control of HIV-1 infection. NK cell stimulation by rMVA was specific since no differences were observed in CMV replication suppression between rMVA and wtMVA-stimulated NK cells.

Conclusion: These data demonstrate that recombinant MVA vaccine against HIV induced activated NK cells capable of specifically controlling HIV infection in vitro.

1 McGill University, Montreal, Canada 2 University of Melbourne, Melbourne, Australia OA02.04 HIV Infection Eliminates the Antibody-Dependent Cellular Cytotoxicity (ADCC) Functional Advantage of KIR3DL1-Expressing NK Cells from HLA-Bw4 Carriers

Background: Mechanisms for harvesting ADCC responses to develop better HIV vaccines are unclear. KIR3DL1 and HLA-Bw4 allelic combinations are associated with protection from HIV/AIDS. The KIR3DL1/HLA-Bw4 combination facilitates licensing of NK cells, potentially enhancing killing of HIV-infected cells. Licensed KIR3DL1⁺ NK cells are, however, non-responsive to autologous HIV-infected cells, suggesting mechanisms other than direct killing are critical. We hypothesized that KIR3DL1⁺ NK cells from individuals co-carrying the HLA-Bw4 ligand overcome inhibitory signals and mediate enhanced anti-HIV ADCC.

Methods: Blood from 21 healthy controls and 22 HIV-infected individuals was tested using an intracellular cytokine staining assay to detect NK cell activation in response to HIV-specific ADCC antibodies. Whole-blood was incubated for 5hr with HIV⁺ plasma and HIV envelope peptides and lymphocytes were stained to identify CD107a and IFN-γ expression from KIR3DL1^+/- NK cells.

Results: NK cells mediated robust ADCC responses. In HLA-Bw4⁺ individuals, KIR3DL1⁺ NK cells mediated anti-HIV ADCC, despite the inhibitory potential of the interaction between KIR3DL1 and HLA-Bw4. NK cells from healthy control HLA-Bw4⁺ subjects exhibited higher ADCC bi-functionality in KIR3DL1⁺ than KIR3DL1^- NK cells (16.5% vs. 11.6%; p < 0.01). However, HIV-infected HLA-Bw4⁺ subjects demonstrated equal bi-functionity between KIR3DL1⁺ and KIR3DL1^- NK cells (10.3% vs. 9.8%; p = 0.7).

Conclusion: KIR3DL1⁺ NK cells, from HLA-Bw4⁺ healthy controls exhibit robust anti-HIV ADCC, and represent a more functional subset than KIR3DL1^- NK cells. This advantage is lost in HIV-infected individuals. ADCC mediated by KIR3DL1⁺ NK cells may represent a correlate of protection from HIV.

1 USMHRP, Walter Reed Army Institute of Research, Rockville, USA 2 USMHRP, HJF, Rockville, USA 3 Walter Reed Army Institute of Research, Silver Spring, USA OA02.05 Inhibition of Human Immunodeficiency Virus Type 1 Infection of Human Monocyte-Derived Macrophages by Anti-Lipid and Anti-MPER Monoclonal Antibodies

Background: HIV-1 entry into cells requires the interaction of both HIV-1 envelope proteins and membrane lipids. We have generated several murine monoclonal antibodies (mAbs) that recognize the MPER region of HIV-1 envelope protein and/or lipids such as phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate. These lipids are present not only on the inner surface of plasma membranes of cells but also on the virions, which the virus acquires during the budding process. We investigated the ability of these mAbs to neutralize HIV-1 infection of primary monocyte-derived macrophages (MDM).

Methods: Monocytes were isolated from peripheral blood mononuclear cells of HIV-1 seronegative donors and differentiated into MDM following in vitro culture. Binding of the mAbs to MDM was determined by flow cytometry and confocal microcopy. Detection of intracellular HIV-1 as well as murine mAb binding to US-1 virus was evaluated by electron microscopy. The neutralization ability of murine and human mAbs was assessed by flow cytometry. HIV-1 p24 and chemokines in the supernatants harvested from HIV-1 infected MDM cultures were assayed by ELISA.

Results: Following preincubation with MDM, only WR301 (anti-PIP IgM mAb) bound to the cells and effectively neutralized HIV-1. In contrast, the murine IgG mAbs WR324 (anti-MPER IgG mAb) and WR321 (anti-MPER, anti-PIP IgG mAb) effectively neutralized HIV-1 following preincubation with US-1. Electron microscopy showed that WR324 bound to virions. Infection of MDM enhanced the secretion of the chemokines several fold compared to MDM preincubated with mAbs alone. The chemokine levels were further increased in the presence of US-1 and the murine mAbs, with WR301 being a stronger inducer compared to WR324.

Conclusion: The mAbs neutralize HIV-1 infection of MDM by different mechanisms. Antibodies generated against the lipid or against the HIV-1 envelope protein neutralize HIV-1 infection of primary human MDM either by production of chemokines or by binding to the virus.

1 Baylor Institute for Immunology Research, INSERM U899, dallas, USA 2 North Texas Infectious Diseases, dallas, USA 3 UMR-S 955, HIV vaccine network/Vaccine Research Institute, Creteil, France OA03.01 Targeting Antigen to CD40 on Dendritic Cells Mediates Expansion of Multi-Epitope HIV-Specific Polyfunctional CD4+ and CD8+ T Cells

Background: Targeting Dendritic Cells (DCs) with anti-DC receptor antibody-antigen fusion proteins is a novel approach to vaccine development. In mouse models, these innovative vaccines induce potent cellular immune responses.

Methods: We engineered an agonistic anti-human CD40 recombinant antibody fused via the heavy chain C-terminus to a string of five 19- to 32-amino-acid long sequences from HIV-1 Gag, Nef and Pol proteins (anti-CD40.HIV5pep). These peptides bear multiple highly conserved CD4+ and CD8+ T cell epitopes. HIV patient PBMCs or DC-T cell co-cultures were incubated with anti-CD40 and control hIgG4.HIV5pep fusion proteins. After 10 days, the total T cells were challenged with each individual HIV peptide, and then antigen-specific cytokine production was detected using intracellular staining. The cytotoxic function of the in-vitro expanded CD8+ T cell was also assayed.

Results: Low doses of anti-CD40.HIV5pep prototype vaccine, but not the control hIgG4.HIV5pep, expand of peptide-specific CD4+ and CD8+ T cells. The range of antigen-specific T cells recall responses over the 5 HIV peptide regions of our anti-CD40.HIV5pep varied between patients (n = 9, range 1–4), but in sum epitopes from all 5 regions could be effectively processed and presented across the entire HIV-infected population we surveyed, which was heterogeneous for MHC alleles. These in vitro-expanded antigen-specific CD4+ and CD8+ T cells were polyfunctional, simultaneously producing multiple cytokines (IFNa, TNFa and MIP-1b), and the CD8+ T cells also had cytotoxic characteristics (granzyme B, surface CD107a, perforin). Finally the anti-CD40.HIV5pep-expanded CD8+ T cells were able to kill peptide-loaded autologous target cells and inhibit HIV-1 replication in vitro in autologous CD4+ T cells.

Conclusion: Taken together, our results demonstrated that all desirable T cell effector qualities were elicited by our anti-CD40.HIV5pep prototype vaccine in the HIV-patient PBMCs and DC/T cell co-cultures. These in vitro data provide a pre-clinical rationale for further testing this DC-targeting HIV prototype vaccine for therapeutic applications in humans.

1 Ragon Institute of MGH, MIT and Harvard, Charlestown, USA OA03.02 Identification of Escape-Refractory Subdominant CD8 Epitopes for Common HLA Alleles

Background: The mechanism of control of HIV by protective HLA class I alleles remains incompletely defined and thus so too does the immune environment to be induced by an effective HIV T cell vaccine. Here, we quantified the replicative cost of CTL escape in epitopes across the HIV proteome to explore the relationship between the differential cost of escape from immunodominant CD8+ T cell responses and the differential control of HIV by HLA alleles.

Methods: We constructed recombinant HIV NL4-3 variants expressing each of 44 described HLA-associated polymorphisms in Gag and Nef located within 30 epitopes restricted by 16 HLA alleles. Replication capacity was quantified by a combination of FACS analysis in GFP-reporter cells and qRT-PCR in PBMC.

Results: The majority of mutations did not significantly impact replication capacity, including escape mutations in the immunodominant epitopes of the non-protective HLA-A02 and A03 alleles. In contrast, 19 mutations significantly impacted viral replication, including the Gag mutations A163G, R264K, and K302R which reduced replication capacity by upwards of 60%. Notably, each of these mutations confers escape from immunodominant CD8 responses of the protective HLA-B57, B27, and B14 alleles. More importantly, Gag mutations K331R and E260D, located within epitopes targeted by sub-dominant HLA-B08 and B35 responses, also significantly impaired replication, revealing potentially durable responses lower in the targeting hierarchy of these common but non-protective HLA alleles.

Conclusion: These data suggest that protective HLA alleles dominantly target epitopes in which escape occurs at high cost to the virus whereas non-protective alleles dominantly target epitopes in which escape occurs at minimal cost. The significant escape-associated costs observed in sub-dominant epitopes restricted by non-protective alleles suggest that vaccine antigens that induce responses against such escape refractory sub-dominant epitopes and avoid inducing responses against easily escaping immunodominant “decoy” epitopes may be central to the rational design of an effective HIV vaccine.

1 Vaccine Research Center, National Institutes of Health, Bethesda, USA 2 Department of Biochemistry, University of Washington, Seattle, USA 3 Duke Human Vaccine Institute and the Duke Center for AIDS Research, Durham, USA 4 International AIDS Vaccine Initiative, Brooklyn, USA 5 NIDDK, National Institute of Health, Bethesda, USA OA03.03 HIV-1 gp120 V1V2-Scaffolds for Structural Analysis and Immunogen Design

Background: To date, the only piece of gp120 that has not been determined at the atomic level is the V1V2-loop. Interestingly, portions of the V1, V2, and V3 loops are the target of broadly and potently neutralizing quaternary structure-preferring antibodies, such as the PG9, PG16 and CH01-05 antibodies. Although these antibodies prefer a quaternary epitope formed only in the context of the trimeric viral spike, they can bind to certain strains of gp120 monomers, albeit with reduced affinity.

Methods: To obtain structural information on the V1V2-loop and understand how quaternary structure-preferring antibodies broadly neutralize diverse HIV-1 isolates whilst targeting loops that vary in sequence length and composition, we initiated structural and biophysical studies. The V1V2-loop from the primary isolate YU2 was transplanted into 17 acceptor scaffolds, and antigenic analysis was performed using monoclonal antibodies directed against the V1V2-loop of YU2.

Results: Here we show that of the 17 YU2-V1V2 scaffolds expressed, two retained antigenic properties similar to V1V2 in full-length gp120. These two scaffolds are being used for structural studies by NMR and X-ray crystallography. A panel of gp120 was tested for binding to the quaternary structure-preferring antibodies, and the V1V2-loops from strains that showed binding were transplanted into the two previously characterized scaffolds.

Conclusion: Structures of V1V2-loop scaffolds alone and in complex with neutralizing antibodies should aid in the design of immunogens capable of eliciting quaternary structure-preferring antibodies.

1 Georg-Speyer-Haus Institute for Biomedical Research, Frankfurt, Germany 2 Global HIV Vaccine Research Cryorepository, Sulzbach, Germany 3 Technische Universität Braunschweig, Braunschweig, Germany 4 University of Ioannina, Greece 5 Instituto de Salud Carlos III, Madrid, Spain 6 University Hospital, Goethe University, Frankfurt, Germany OA03.04 Identification of Mimotopes for Neutralizing Antibodies from Elite Controllers with Env-Tailored Phage Display Libraries

Background: The aim of this study was to identify immunogenic epitope mimics for HIV-neutralizing antibodies present in HIV-1 elite controllers (EC), which are able to elicit neutralizing antibodies upon vaccination.

Methods: Plasma samples from EC were tested for neutralization on TZM-bl cells against a cross-clade HIV pseudovirus panel. An Env-tailored phage display library was constructed and screened with serum antibodies from EC with cross-clade neutralizing capacity. The selected phage clones were analyzed by phage-ELISA for the binding specificity and peptide encoding inserts were sequenced. Phages and the corresponding linear peptides were tested for depletion of plasma neutralizing activity. The antibodies recognizing the selected epitopes were affinity purified from the corresponding serum and tested for neutralization. Mice were primed with pSyngp140JR-FL DNA and boosted with the selected epitope coupled to a palmitoyl, a sequential oligo peptide carrier (SOC) or to the phage.

Results: A subgroup of EC was identified with cross-clade plasma neutralizing activity against pseudoviruses. An HIV-1 Env-tailored phage library expressing random HIV-1 Env fragments was generated and screened with antibodies from EC26 who ranked top for neutralizing capacity. After several rounds of positive and negative selections the selected epitopes were sequenced. Epitopes were identified within the immunodominant regions of gp41, the fusion peptide and the membrane-proximal external region (MPER) of gp41. The MPER specific epitope EC26-2A4 could deplete approximately 50% plasma neutralizing activity against SF162.LS. Further, the affinity purified antibodies recognizing the EC26-2A4 epitope neutralized SF162.LS with an IC50 of 5.9 μg/ml. Together these data prove the EC26-2A4 epitope as interesting vaccine candidate. The in vivo immunogenicity analysis of the EC26-2A4 epitope is ongoing.

Conclusion: We demonstrated cross-clade neutralizing antibodies in a subgroup of EC and identified their antibodies based on Env-tailored phage display libraries. Such epitopes may be suitable for the derivation of prophylactic vaccine candidates.

1 Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China 2 Institute of Biophysics, Chinese Academy of Sciences, China OA03.06 Development and Characterization of HIV-1 Virus-like Particles Produced by Stably Transfected Drosophila S2 cells

Background: HIV-1 virus-like particles (VLP), perceived to be much safer than live-attenuated viruses, are being developed as a vaccine candidate against HIV-1/AIDS. However, in one way or the other current methods used for HIV-1 VLP production have many limitations, such as low yield, poor gp160 cleavage, difficulty to purify and variation between batches.

Methods: To overcome these limitations, we developed a Drosophila S2 expression system for HIV-1 VLP production and evaluated immunogenicity of S2-produced HIV-1 VLP in heterologous DNA-VLP prime-boost strategy in mice.

Results: Here we report that stable S2 cell transfectants efficiently produce and release HIV-1 VLP into culture supernatants with the amount much higher than those produced by transiently transfected 293 T cells. HIV-1 envelope proteins are properly processed and glycosylated in a high mannose form. HIV-1 envelope and gag proteins both are effectively incorporated into VLP. Cryo electron microscopy indicates that HIV-1 VLP contains many HIV-1 spikes on VLP surface. Finally, we show that heterologous DNA-VLP prime-boost elicits both ELISA-binding and neutralizing antibody responses as well as HIV-1 envelope and gag peptide-specific CD8 and CD4 T cell responses.

Conclusion: Thus, we conclude that production of HIV-1 VLP by stable transfectants of Drosophila S2 cells overcomes several limitations of the current HIV-1 VLP producing systems. The HIV-1 VLP produced by stable S2 transfectants elicits both hormoral and cellular immune responses.

1 The Scripps Research Institute and IAVI Neutralizing Antibody Center, La Jolla, USA 2 IAVI Ragon Institute of MGH, MIT and Harvard, Boston, USA 3 NIH Vaccine Research Center, Bethesda, USA 4 Vaccine Research Center, NIH, Bethesda, USA OA04.01 Potent and Broad Neutralization by a CD4 Binding Site Monoclonal Antibody from an HIV-1 Infected Donor

Background: Recently, several novel neutralizing antibodies (nAbs) have been isolated from HIV-1 positive donors. Of these nAbs, PG9/16, and VRC01, are unprecedented in their potency and breadth. In this study, we characterize PGV04 (also known as VRC-PG04), a new nAb that has potency and breadth that rivals that of PG9/16 and VRC01.

Methods: PGV04 was isolated by single, memory B cell sorting using the resurfaced core (RSC3) protein as bait. The nAb was screened for neutralization on a 162 virus panel and shown to be both broad and potent. The epitope was further mapped by competition with other MAbs and by evaluating binding and neutralization of single alanine substitutions in the JR-CSF gp120 protein monomer and gp120 incorporated into pseudovirus respectively. Binding to cell surface expressed trimers was measured by flow cytometry and binding to deglycosylated gp120 was measured by ELISA.

Results: PGV04 competed with CD4, b12 and VRC01 for binding to gp120, confirming it is a CD4bs mAb. However, when screened on a large panel of viruses, PGV04 varied in its neutralization profile from VRC01, VRC03 and b12. Furthermore, PGV04 binding to gp120 containing single alanine substitutions revealed differences in residue dependence between PGV04 and other CD4bs mAbs. The residues found to be important in binding to gp120 had varying effects on the ability of PGV04 to neutralize pseudovirus incorporating these same alanine substitutions and only one substitution, D279A, abrogated PGV04 neutralization completely. All of the CD4bs mAbs tested bound deglycosylated gp120 and none induced the co-receptor site on functional trimers.

Conclusion: Broad and potent CD4bs nAbs have subtle differences in the way they recognize and access the CD4bs in the context of the viral spike.

1 New York University School of Medicine, New York, USA 2 Veterans Affairs New York Harbor Healthcare System, New York, USA 3 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA OA04.02 Human Anti-V2 Monoclonal Antibodies Neutralize Tier 1 HIV-1 Pseudoviruses

Background: Antibodies (Abs) to the V2 region of gp120 are present in a minority of HIV-infected subjects, and, to date, little has been published on their immunologic or functional properties. Recent analysis of plasma from the RV144 clinical vaccine trial, however, revealed that anti-V2 Abs were present in almost all plasma from vaccine recipients, and their contribution to protection is being extensively analyzed.

Methods: A panel of seven human anti-V2 monoclonal Abs (mAbs), generated previously using cellular techniques from PBMCs of individuals infected with clade B HIV-1, was analyzed for their immunoglobulin variable gene usage, ELISA cross-reactivity to recombinant gp120 derived from viruses from clades A, B and C, and neutralizing activity against pseudoviruses using the TZM-bl cell assay.

Results: All the anti-V2 mAbs are IgG1 except for one which is IgG3. Five mAbs were sequenced: four use the same IGHV1-69 gene segment, suggesting preferential gene usage by Abs targeting this region. The remaining mAb uses the IGHV4-34 gene and has very different immunologic characteristics, including interference with sCD4 binding. All seven V2 mAbs are highly cross-reactive with comparable relative affinities. They bind to 19 recombinant gp120s representing eight from clade B, ten from clade C and, one from clade A. Four anti-V2 mAbs were tested for their neutralizing activity. Tier 1 pseudoviruses were neutralized, including six from clade B and two from clades A and C. The 50% neutralizing values ranged between <0.8 and 88.7 μg/ml, with a mean of 16.6 μg/ml.

Conclusion: The results indicate that human anti-V2 mAbs display cross-clade neutralization against Tier 1 pseudoviruses in the TZM.bl assay.

1 Vaccine Institute, Durham, USA 2 Human Vaccine Institute, Durham, USA 3 Vaccine Research Center, Bethesda, USA 4 Simon Fraser University, Burnaby, BC, Canada 5 NCI-Frederick, NIH, Frederick, USA OA04.03 2F5-like Cross-Reactive HIV-1-Neutralizing Monoclonal Antibodies with Low Number of Somatic Mutations

Background: The genes encoding broadly HIV-1-neutralizing (bn) human monoclonal (m) antibodies (Abs) are highly divergent from their germline counterparts. We have hypothesized that this could pose a challenge for their elicitation and that identification and characterization of less somatically mutated bnAbs may help in the design of effective vaccines.

Methods: IgG and IgM libraries were constructed using PBMC mRNA from a patient with bn serum containing Abs targeting the epitope of the bn mAb 2F5. The mAbs were selected from the libraries by panning against peptides containing the 2F5 epitope and against HIV-1 gp140JR-FL, and characterized by standard assays.

Results: Two mAbs (m66 and m66.6) were identified that share the same heavy chain; the variant bearing the more mutated light chain (m66.6) exhibited higher HIV-1 neutralizing activity than the less mutated variant - it was very potent neutralizer in assays based on PBMCs and macrophages but less potent than 2F5 in the TZM-bl assay. Both mAbs required the DKW664-666 core of the 2F5 epitope as well as two additional upstream residues (L660,663) for binding. The heavy chains of these mAbs have long (21 residues) third complementarity determining regions. m66.6 exhibited relatively weak polyspecific reactivity to a few self antigens and m66 was not autoreactive. Both m66 and m66.6 use different VH and VL genes from 2F5 and are significantly less divergent from their germline Ab counterparts than 2F5 – they have a total of 11 and 18 amino acid changes, respectively, from the closest VH and Vκ germline gene products, compared to 25 for 2F5.

Conclusion: m66.6 and m66 represent unique Abs whose binding mechanism and neutralization specificities overlap with those of 2F5. This pair of mAbs should be of use in exploring the complex maturation pathways of bnAbs, poly- and autoreactivity, and may inform the design of vaccine immunogens and HIV therapeutics.

1 Karolinska Institutet, Stockholm, Sweden 2 Department of Immunology and Microbial Science, The Scripps, La Jolla, USA 3 Vaccine Research Center, NIAID, NIH, Bethesda, USA OA04.04 Characterization of CD4-Binding Site Directed Monoclonal Antibodies Isolated from HIV-1 gp140 Env Immunized Rhesus Macaques

Background: An increased focus on the cells that produce vaccine-elicited antibodies (Abs) and on the process of B cell maturation can help guide efforts aimed at replicating the successful generation of broadly neutralizing Abs observed in some infected humans. Examination of neutralizing monoclonal Abs (MAbs) from chronically infected individuals suggests that extensive Ab affinity maturation is required to achieve efficient neutralization; however, little is known about the relationship between HIV-1 protein subunit immunization and Ab somatic hypermutation.

Methods: We have isolated and expressed Abs from single cell-sorted Env-specific memory B cells from non-human primates (NHPs) that were immunized with soluble YU2 gp140 trimers. The sorting was performed with Env probes designed to selectively isolate CD4 binding site (CD4bs)-specific B cells. PCR amplification of Ab heavy and light chain gene transcripts was followed by sequence analysis, expression and characterization of MAbs.

Results: Analyses of a first panel of eight MAbs demonstrates that all eight arose from different clonal lineages as determined by their VDJ gene family usage. All MAbs were capable of neutralizing Tier 1 strains of HIV-1 with reasonable potency, including some non-clade B viruses. Their neutralization pattern was consistent with the polyclonal plasma activity elicited in the animal from which the MAbs were isolated. Cross-competition analyses demonstrated that all eight NHP MAbs cross-competed with a set of well-characterized CD4bs-directed human MAbs, confirming their specificity for the CD4bs consistent with the criteria used for NHP memory B cell sorting. Several of the MAbs displayed distinct binding properties when tested against a panel of gp120-derived ligands.

Conclusion: These data show that the soluble gp140 trimers elicit neutralizing CD4bs-directed Abs in healthy non-human primates. Further examination of the fine specificity of these and additional MAbs will provide information to guide the design of improved vaccines and immunization strategies.

1 The Scripps Research Institute and Ragon Institute of MIT, La Jolla, CA and Boston, MA, USA 2 The Scripps Research Institute and Ragon Institute MIT, La Jolla, CA and Boston, MA, USA 3 The International AIDS Vaccine Initiative, New York, USA 4 The Interenational AIDS Vaccine Initiative, New York, USA 5 The Scripps Research Institute, La Jolla, USA OA04.05 Structural Insights into HIV-1 Neutralization by New Highly Potent and Broadly Neutralizing Antibodies

Background: Human monoclonal antibodies have been characterized recently that potently neutralize HIV-1 isolates across all clades. These exciting new antibodies (PGT series) were derived from direct functional screening of B cells from IAVI protocol G donors (Theraclone/Monogram) and are unusually potent with binding predicted to be to novel epitopes on Env gp120.

Methods: The crystal structures of these new PGT antibodies are being determined by x-ray crystallography with further characterization using binding and mutagenesis data.

Results: The crystal structures so far have been elucidated for one complete family of these antibodies and others are in progress, as well as Fab complexes with gp120 and fragments.

Conclusion: Structural characterization and biochemical analysis of these antibodies have uncovered novel specificities to new epitopes and reveal further mechanisms for viral neutralization. These new epitopes provide additional insights for neutralization of HIV-1 and can be used for structure-assisted vaccine design.

This study was supported by the International AIDS Vaccine Initiative (IAVI), Ragon Institute, and NIH AI84817.

1 Ragon Institute of MGH, MIT, and Harvard, Charlestown, USA 2 Praxis Jessen Jessen Stein, Berlin, Germany 3 Infectious Disease Division, Massachusetts General Hospital, Boston, USA OA05.01 HIV-Specific Cytolytic CD4+ T-Cell Responses During Acute HIV-1 Infection Predict Disease Outcome

Background: During acute HIV-1 infection, early control of viremia and establishment of viral set point have been widely attributed to HIV-specific CD8 T-cell responses. However, despite increasing evidence for direct antiviral activity by CD4 T-cells in other infections, the impact of HIV-specific CD4 T-cells on viral control has not been studied.

Methods: We studied 26 acutely infected, treatment-naïve subjects and monitored their clinical outcome for up to 3 years post-infection. Baseline HIV-specific CD4 T-cell responses were assessed for degranulation (CD107a), IFNγ secretion, expression of granzymes (GrzA,B,K) and perforin. HIV-specific cytolytic CD4 T-cell responses were investigated longitudinally in a subset of 11 subjects with similar peak viral loads for 1 year post-infection.

Results: Among the patients followed longitudinally, 6 progressed to a low viral set point, while 5 progressed to a significantly higher set point (134,020vs.11,234 copies/ml; p = 0.004). Interestingly, a significant expansion of HIV-specific cytolytic CD4 cell responses was observed in individuals who controlled viral replication compared to those who progressed to a high viral set point (IFNγ: p = 0.038, CD107a: p = 0.042). Importantly, this expansion was observed early post-infection, prior to the divergence of viral load or CD4 T cell counts between the two groups. Examination of the baseline HIV-specific CD4 responses revealed a distinct GrzA-enriched cytolytic phenotype that was highly associated with subsequent viral control. Strikingly, Kaplan-Meier analysis of only the baseline cytolytic CD4 T-cell response in a larger cohort demonstrated that individuals with HIV-specific GrzA+ responses remained off HAART significantly longer than individuals with HIV-specific GrzA- responses (p = 0.0023).

Conclusion: Here we demonstrate that the rapid induction and expansion of a distinct cytolytic CD4 T-cell response during acute infection is significantly associated with viral control and disease outcome. These data suggest a pivotal role for HIV-specific cytolytic CD4 T-cells in the early control of viremia following acute infection and for future HIV vaccine design strategies.

1 University of KwaZulu-Natal, Durban, South Africa 2 Ragon Institute of Massachusetts General Hospital, Massachusetts Insti, Boston, USA OA05.02 Control of Acute HIV-1 Subtype C Viremia Is Associated with Limited CD8+ T-Cell Immunogenicity

Background: HIV-1-specific CD8+ T-cell responses contribute to the decline in acute peak viremia following infection. However, the relative immunogenicity CD8+ T-cell epitopes during and after acute viremia has not been well defined, and data on the breadth of responses following the initial drop in acute viremia are lacking.

Methods: We characterized CD8+ T-cell responses to peptides spanning the full viral proteome in 20 HIV-1C acutely infected, antiretroviral naive subjects using the IFN-γ ELISPOT assay as early as 28 days after estimated date of infection. Viruses from plasma were also sequenced within defined CD8+ T-cell epitopes for selected subjects. Of the 20 participants in the cohort, eleven had not reached complete seroconversion at analysis.

Results: Despite documented declines in peak viremia, the initial responses associated with the decline in acute phase peak viremia were narrowly directed, and of weak magnitude. At approximately 28 days after estimated initial infection, following a 15-fold decline in viral loads, CD8+ T-cell responses were directed against an average of 3 of the 410 peptides tested (range 0–6); two individuals had no detectable CD8+ T-cell responses at this time. At 18 weeks post the estimated date of infection the average number of peptides targeted had increased to 5 (range 0–11). Of 56 optimal Gag CD8+ T-cell epitopes sequenced, 31 were wild type in the acute infecting viruses, however, only 11 of 31 elicited measurable CD8+ T-cell responses.

Conclusion: These data demonstrate that the majority of CD8+ responses that subsequently arise are not elicited during acute HIV-1 infection despite the presence of the cognate epitope in the infecting strain. Further studies are needed to address why recognition of HIV-1 peptides appears to be selective during acute infection as the lack of recognition may contribute to the failure to control viremia to a low set point.

1 Mbeya Medical Research Program, Mbeya, Tanzania, United Republic of 2 Makerere University Walter Reed Project, Kampala, Uganda 3 Walter Reed Project Kericho, Kericho, Kenya 4 Royal Thai Army, Department of Retrovirology, AFRIMS, Bangkok, Thailand 5 US Military HIV Research Program (MHRP), Rockville, USA OA05.03 Lymphocyte Dynamics in Acute HIV-1 Infection: RV217-The Early Capture HIV Cohort Study (ECHO)

Background: Acute HIV infection events between exposure and peak viremia in non-subtype B populations are poorly characterized.

Methods: Individuals in East Africa and Thailand at high risk for HIV-1 infection were prospectively followed with twice weekly HIV-1 RNA (NAT) to detect acute infection. Lymphocyte subsets (CD4⁺T, CD8⁺T, NK and B cells) were enumerated on a FACSCalibur using MultiTest reagents (Becton Dickinson) starting as early as 3–9 days after the last known NAT-negative sample and followed at multiple timepoints during early infection. Viral load (VL) was measured using Abbott Real Time Viral Load.

Results: Dynamic lymphocyte changes were observed in 16 acute HIV-1 infections. Peripheral CD4⁺T cell and B cell nadir occurred at a median of 20 days post infection. CD4⁺T cells fell from a median 929 (range, 419–1532) cells/ul to 466 (142–866) cells/ul (P < 0.001). B cells declined from a median 290 (186–592) cells/ul to 127 (15–275) cells/ul (P < 0.001). In contrast, NK and CD⁺T cells increased at different rates within the first 100 days. CD8⁺T cells increased nearly 3-fold from a median 500 (271–1150) cells/ul to 1498 (693–2367) cells/ul (P < 0.001). NK cells increased from a median 252 (49–589) cells/ul to 335 (100–864) cells/ul (P = 0.002). CD4⁺T cell and B cell counts exhibited an inverse correlation (P < 0.001) while NK cell counts showed a direct correlation (P = 0.022) with contemporaneous VL. CD8⁺T cell counts did not correlate with VL at any time consistent with expansion by indirect mechanisms. VLs preceding and following CD4⁺T cell and B cell measurements were negatively correlated suggesting that these subsets are being directly depleted. In contrast, VL preceding, but not following NK cell measurements were positively correlated, suggesting that VL may be driving NK cell expansion.

Conclusion: Understanding lymphocyte dynamics, their relationship to VL and early pathogenic events of non-B HIV-1 infection may help to target prevention or treatment interventions.

1 University of Florida, Gainesville, USA 2 University of South Florida, St. Petersburg, USA OA05.04 Long-Lived Historical Record of Evolution of Viral Diversity in Peripheral Blood Cells by Deep Pyrosequencing

Background: Assessments of HIV-1 diversity are generally inferred from limited population sampling. Deep sequencing was applied to increase quasispecies coverage for direct analysis of viral diversity, population structure, and the historical archive of HIV-1 within the host.

Methods: 454 deep sequencing was applied to HIV-1 envelope hypervariable domain 3 (V3) in peripheral blood cells from therapy-naïve children, infected for 1.5 to 6.1 years, with high plasma virus levels (4.0 to 6.7 log10 HIV-1 RNA copies/ml), but variable CD4 T-lymphocytes (2% to 30%). A V3 library was constructed for each subject from 400 viral templates and sequenced to generate 10,000 quality reads. Consensus sequences were derived by clustering reads at 3% genetic distance, and used to estimate viral biodiversity and population structure via rarefaction/CHAO1 algorithm. Coreceptor use was inferred by position-specific scoring matrix. Viral archive and most recent common ancestor were evaluated by maximum likelihood phylogenetic trees constructed from consensus sequences combined with longitudinal conventional sequences.

Results: Biodiversity of HIV-1 V3 quasispecies across individuals ranged from 12 to 99 clusters. Viral population structure was organized into a limited number of dominant genomes with a high frequency of unique sequences. Dominant viral variants at later time points evolved exclusively from low frequency variants at earlier time points. Phenotypic profiles of bioclusters revealed a breadth of coreceptor use that was unrelated to population structure or immune status. CXCR4 variants were co-archived with CCR5 variants even when CD4 T-cells exceeded 20%. Viral variants persisted in peripheral blood cells for as long as six years. In at least one individual, pyrosequencing at four years after infection identified minor viral variants that colocalized with early transmitting viruses.

Conclusion: Unprecedented coverage by deep sequencing defines HIV-1 population complexity and structure, enriches the evolutionary landscape between samplings, and reveals an evolutionary record of persistent, cell-associated viral sequences closely related to transmitting viruses.

1 Ragon Institute of MGH, MIT and Harvard, Charlestown, USA 2 University of California, San Diego, San Diego, USA 3 Practice Jessen, USA 4 University of California, San Francisco, San Franciscon, USA OA05.06 Important Contribution of Subdominant HIV-Specific CD8 T Cell Responses to Early Control of HIV Replication

Background: During acute HIV-1 infection, HIV-specific CD8 T cell (CTL) responses emerge, suppressing viral loads to a semi-stable viral setpoint; this early viral setpoint has been shown to be highly predictive of disease outcome. However, while immunodominant responses restricted by rare HLA class I alleles have been associated with better control of viral replication, immunodominant responses restricted by common HLA alleles like B*8 or B*7 do not appear to have a significant impact on early control.

Methods: Over 600 individuals with primary HIV-1 infection were screened for HLA-restricted, epitope-specific CTL responses using optimally defined epitopes. The early viral setpoint was determined in all treatment-naïve individuals by two independent ID specialists.

Results: The recognition of HIV-specific CTL responses was very predictable based on HLA class I expression and followed a clear hierarchical pattern. While the immunodominant responses in individuals with rare HLA class I alleles were significantly associated with lower viral setpoint, individuals with immunodominant responses restricted by common HLA class I alleles had, on average, higher viral loads. Interestingly, the absence of CTL responses in primary infection directed against some of the immunodominant epitopes restricted by common HLA class I alleles (e.g.HLA-B8-FL8) allowed for the development of responses against subdominant epitopes (e.g.HLA-B8-EI8) within the same individuals and was associated with significantly better subsequent viral control(p = 0.001). A distinct ranking of the relative contribution of epitope-specific CTL responses to the early viral setpoint suggested an unexpected contribution of subdominant responses to the initial viral control.

Conclusion: Our data suggest that in the context of common HLA class I alleles, CTL responses directed against subdominant epitopes but not dominant epitopes during primary infection significantly contribute to better control of viremia. Thus, engineered proteins that lack common immunodominant CTL epitopes and allow for the priming of otherwise subdominant epitopes might provide an important approach for vaccine design strategies.

1 Ragon Institute of MGH, MIT and Harvard, Charlestown, USA 2 Brigham and Women Hospital, Harvard Medical School, Boston, USA OA06.01 Deconstructing HIV-Specific CD8+ T Cell Responses in HIV Elite Controllers

Background: Evidence suggests that HIV-specific CD8+ T cells (CTL) play a critical role in a large proportion of rare individuals capable of immune control of HIV replication in the absence of therapy (elite controllers, EC). However, there is still no clear understanding of what differentiates effective CTL responses from ineffective responses in untreated chronic progressive disease (CP) and in subjects with controlled viral load on antiviral therapy (ARTC).

Methods: We combined standardized assays and advanced statistical analysis to determine whether a subset of HIV-specific CTL endowed with unique characteristics is present in EC. Inter and intra-donor analysis of HIV CTL-specific proliferation and cytokine secretion was performed at different time points after antigen stimulation by CFSE and luminex assays. 23 EC, 15 CP and 15 ARTC were studied. Independent cohorts of 15 EC and 15 ARTC were used for validation.

Results: We observed that within the same individuals, the proliferative capacity of CTL targeting epitopes restricted by protective alleles was significantly stronger than epitopes restricted by non-protective HLAs (p = 0.003). However, this marked difference was present only in EC. Kinetic analysis of cytokine secretion showed marked differences between groups. Slopes of IFN-γ, IL-2 and TNF secretion were significantly steeper in EC than in the other groups. A model incorporating proliferation and early IL-2 secretion accurately predicted EC status in an independent cohort (c-statistic = 0.908).

Conclusion: Our data illustrate that HIV-specific CTLs in EC are characterized by late and robust cytokine secretion. We also show that proliferation is driven by protective alleles only in EC, suggesting that HIV-specific responses restricted by protective HLA alleles present unique features in EC not observed in subjects with progressive disease. Our results suggest that this strategy can be used to define immune correlates of protection in natural HIV infection and may have important implications for immune monitoring of responses to HIV.

1 Duke University Medical School, Durham, USA 2 University of Alabama, Birmingham, USA 3 University of Oxford, Oxford, United Kingdom (Great Britain) OA06.02 Ontogeny of CD8+ T Cells in Acute HIV-1 that Inhibit Autologous and Heterologous Transmitted/Founder (T/F) Viruses

Background: CD8-mediated virus inhibition can be detected in HIV-1+ subjects naturally controlling virus replication. Characterizing the inhibitory function of CD8⁺ T cells in acute HIV-1 infection (AHI) is important for determining the nature of CD8⁺ responses that need to be elicited by an HIV-1 vaccine. In this study, we examine the timing, epitope specificity, and contribution of soluble factors to the antiviral CD8⁺ T cell response in AHI.

Methods: We used a CD8⁺ T cell mediated virus inhibition assay (CD8 VIA) to assess CD8⁺ T cell function during AHI in the CHAVI 001 cohort. Primary CD4⁺ enriched lymphocytes were infected with transmitted/founder (T/F) autologous and heterologous viruses in the presence of autologous CD8⁺ T cells. Peptide-specific soluble antiviral responses were determined by transwell assay, luminex cytokine analysis, and flow-cytometric intracellular cytokine staining.

Results: Potent CD8⁺ antiviral responses against autologous and heterologous (T/F) viruses appeared during AHI prior to Fiebig stage 4. Responses against autologous transmitted virus were durable to 48 weeks; however heterologous responses declined concurrent with the resolution of viremia. Viruses obtained 6 months post infection were more resistant to CD8⁺ mediated virus inhibition than cognate T/F viruses. CD8⁺ T cells specific for epitopes that have been shown to drive HIV-1 escape in AHI released soluble factors that inhibited T/F virus replication.

Conclusion: The rapid development of potent CD8⁺ antiviral activity may reflect the previously identified inhibitory capacity of early and transitional memory cells. These studies suggest that continued antigenic exposure is necessary to maintain antiviral breadth. Chronic virus resistance to early CD8⁺ cell antiviral activity supports prior evidence that CD8⁺ cells drive rapid virus escape. These data provide insight into the mechanisms of CD8-mediated viral inhibition, and suggest that comparable functional analyses will be important for determining whether similar activities can be induced by T-cell directed vaccine strategies.

1 Fred Hutchinson Cancer Research Center, Seattle, USA 2 US Military HIV Research Program, Rockville, USA 3 Thai Ministry of Public Health, Bangkok, Thailand OA06.03 The RV144 Vaccine Induced CD4+ T Cells Producing Th1 and Th2 Cytokines with Proliferative Potential

Background: The RV144 efficacy trial demonstrated moderate reduction in HIV acquisition for participants receiving recombinant canarypox encoding Env, Gag, and Protease, and recombinant gp120 proteins.

Methods: PBMC from 10 placebo and 40 vaccine recipients were examined at baseline, Week 26 (two weeks post last vaccination) and Week 52. Intracellular cytokine staining (ICS) measured T cells producing IFN-γ, IL-2, TNF-α, or expressing CD40L, following 6-hour ex vivo stimulation with insert-matched Env and Gag peptide pools. Multiplex bead array measured 12 cytokines in PBMC supernatant following 48-hour stimulation. The CFSE assay measured proliferation.

Results: Responses were largely restricted to CD4+ T cells and to Env. CD40L detected the highest response rate by ICS (60% at Week 26, 20% at Week 52), with a median magnitude of 0.1% CD4+ T cells expressing CD40L at Week 26. The Week 26 response rate for IL-2 was 43%, but only 18% for IFN-γ; both decreased at Week 52 (23% and 13%, respectively). Response rates were similar by multiplex bead array (50% for IL-2 and 14% for IFN-γ at Week 26), and additional responses of Th2 and regulatory cytokines were detected (30% for IL-4, 44% for IL-5, 53% for IL-13, and 28% for IL-10). Proliferative response rates were lower at Week 26 (16% for Env), but persisted at Week 52 (21%). For all assays, false positive responses were minimal (<5%).

Conclusion: In this pilot immunogenicity study, CD4+ T-cell responses were detected in a majority of vaccine recipients, and were polyfunctional including secretion of Th2 cytokines. This, together with expression of CD40L, indicates ability to provide B-cell help. A smaller proportion of vaccine recipients had proliferative T-cell responses, but these appeared more durable, persisting to one year. These assays will be used to examine samples from cases and matched controls and may identify a correlate of protection.

1 University of Cape Town, Cape Town, South Africa 2 National Institute for Communicable Diseases, Johannesburg, South Africa 3 Centre for AIDS Programme Research in South Africa, UKZN, Durban, South Africa OA06.04 Association of Timing of HLA B*8101 and B*3910 Mediated p24 Sequence Evolution and Pathways to CTL Escape with Disease Progression

Background: HLAs that are associated with control of vireamia, often select for mutations in Gag p24 in acute/early phases of the HIV–1 infection.

Methods: To understand the role of viral and host factors in the pathogenesis of subtype C, HIV-1 infection, we timed CTL mediated sequence evolution in TL9 (Gag 180 – 188) epitope in p24 and compared disease progression in B*8101 (n = 5) and B*3910 (n = 2) participants from the CAPRISA acute infection cohort.

Results: We report that both the B*3910 and B*8101 alleles select mutations in the TL9 epitope, in which either the 182Q or both the 182Q and 186T are replaced by S (TPqDLNtML). However, the timing to sequence evolution differed among these alleles. While both B*3910 participants selected acute/early mutations, four of the five (4/5) B*8101 individuals selected late mutations in the TL9 despite persistent CTL responses targeting this epitope. One B*8101 participant did not develop any restricted sequence evolution in the TL9 epitope by 2.5 years post-infection despite consistent CTL responses. In three of the remaining four individuals, sequence evolution in TL9 was accompanied or preceded with changes in SV9 (Gag 148 – 156) epitope or amino acids flanking this epitope. The fourth participant developed an E177D mutation in addition to the double mutations in the TL9 epitope. These three mutations were also developed by one of the B*3910 individual but in the acute phase of the infection. Irrespective of HLA, participants who selected both the Q182S and T186S mutations had a slow disease progression profile.

Conclusion: These results provide new evidence on the role of the restricting HLA type in the timing of sequence evolution and contribute new information on the constituents of effective CTL responses. In addition, these results further support inclusion of multiple conserved regions of Gag in a vaccine meant to render HIV-1 less fit.

1 National Center for AIDS/STD Control and Prevention, China CDC, Beijing, China OA06.06 Imbalance Between T Helper Type 17 and T Regulatory Cells: Implications for HIV Pathogenesis

Background: T helper 17 (Th17)and CD4⁺CD25^hiFoxp3⁺ regulatory T (Treg) cells have been described as two distinct subsets from T helper 1 (Th1) and T helper 2 (Th2) cells, their balance may play an important role in the pathogenesis of HIV-1 infection.

Methods: 115 untreated chronic HIV-infected individuals and 32 healthy donors were recruited in this study. Peripheral blood mononuclear cells were isolated and stained to characterize the frequencies of Th17 and Treg. 42 individuals including 10 elite controllers were followed up for more than one year, changes of Th17 and Treg frequencies were analyzed over time.

Results: Th17 levels with CD4 counts were depleted while Treg were increased in the cohort of 115 HIV-infected individuals, compared to healthy controls (median = 0.608% vs 0.938%, 5.150% vs 4.605%; p < 0.001, p = 0.032), leading to an obvious imbalance of Th17/Treg when compared to the healthy control counterparts (median = 0.117 vs 0.194; p < 0.001). In the 42 patients, loss of Th17 contrasted with an increased Treg frequencies from Jun 2009 to Oct 2010 were followed longitudinally (median = 0.669% vs 0.603%, 4.765% vs 5.890%; p = 0.078, 0.010). Meanwhile, the elite controller group had comparable Treg, Th17 levels and a Th17/Treg with healthy controls during the follow-up study, (in 2009, median = 4.555% vs 4.605%, 0.843% vs 0.938%, 0.176 vs 0.194; p = 0.948, 0.635, 0.585; in 2010, median = 5.205% vs 4.605%, 0.768% vs 0.938%, 0.140 vs 0.194; p = 0.159, 0.220, 0.053). Additionally, we demonstrated that loss of balance between Th17 and Treg could lead to an earlier CD4 T cell decline than Treg or Th17 during the course of HIV infection.

Conclusion: The loss of Th17 cells and a reciprocal increase in the Treg cells in HIV-1 chronic infections with no differences observed in elite controllers give prominence to the understanding of HIV-1's pathogenesis and prompt novel considerations for future thoughts on HIV vaccine development.

1 Aaron Diamond AIDS Research Center, Rockefeller University, New York, USA 2 Beth Israel Deaconess Medical Center, Boston, USA OA07.01 Bispecific Fusion Antibodies PG9-Ibalizumab (PG9-iMab) and VRC01-Ibalizumab (VRC01-iMab) Exhibit 100% Breadth and Picomolar Potency

Background: Ibalizumab, PG9 and VRC01, three of the broadest and most potent monoclonal antibodies against HIV-1 infection described to-date, neutralize 66% - 90% of HIV-1 strains. Here, we created PG9-iMab and VRC01-iMab bispecific antibodies, exploiting ibalizumab's high affinity for domain 2 of CD4 and inherent antiviral activity to create bispecific fusion antibodies that anchor PG9 or VRC01 at the site of viral entry.

Methods: Bispecific antibodies were created by fusing PG9 or VRC01 scFv to the N-terminus of the ibalizumab heavy chain. Breadth and potency were assessed to a maximum concentration of 10 μg/mL against a well characterized panel of HIV-1 Env pseudotypes (n = 118).

Results: Addition of PG9 or VRC01 scFv to the N-terminal of ibalizumab was well tolerated; expression and purification yields of bispecific antibodies were comparable to the parent antibodies and had negligible effects on CD4 binding affinity. PG9-iMab and VRC01-iMab neutralized 100% of viruses to at least 50% inhibition (median IC50, 24 pM and 135 pM, respectively) and indeed, PG9-iMab neutralized 97% of viruses and VRC01-iMab neutralized 95% of viruses, to at least 95% inhibition. Most importantly, PG9-iMab and VRC01-iMab each potently neutralized dual PG9- and ibalizumab-resistant viruses and dual VRC01- and ibalizumab-resistant viruses, respectively. PG9-iMab exhibited synergistic potency, neutralizing each virus 100 ± 375 fold and 32 ± 282 fold more potently than PG9 and ibalizumab, respectively (median ± IQR). In contrast, while potency of VRC01-iMab was enhanced 19 ± 75 fold compared to VRC01, potency was comparable to ibalizumab (3 ± 91 fold). The synergistic activity and ability to neutralize dual-resistant viruses required CD4 anchoring and could not be achieved by simply co-administering PG9 or VRC01 with ibalizumab, which yielded only additive effects.

Conclusion: Bispecific ibalizumab antibodies are broad and potent candidates for passive or gene-delivered biologics for prevention and treatment of HIV-1 infection.

1 University of Cape Town, Cape Town, South Africa 2 AIDS Virus Research Unit, National Institute for Communicable Diseases, Johannesburg, South Africa 3 Institute of Infectious Diseases and Molecular Medicine, UCT, Cape Town, South Africa 4 Los Alamos National Laboratory/ Santa Fe Institute, Los Alamos/ Sante Fe, USA 5 Department of Surgery, Duke University Medical Center, Durham, USA 6 School of Mathematics, National University of Ireland, Galway, Galway, Ireland OA07.02 Identification of Amino Acid Residues in HIV-1 Envelope Targeted by Plasma Broadly Neutralizing Antibodies using Evolutionary Models

Background: During the course of HIV-1 infection, a small proportion of individuals develop broadly cross-reactive antibodies capable of neutralizing diverse HIV-1 strains. Identification of the epitopes targeted by these antibodies will provide important clues for the design of a preventative vaccine against HIV-1 infection. We have developed an evolutionary model to identify key residues targeted by plasma neutralizing antibodies.

Methods: Five HIV-1 subtype C serum samples from the CAPRISA 002 Acute Infection cohort previously shown to have neutralization breadth were tested against a large multiclade panel of 206 pseudoviruses for which gp160 sequences were available. Amino acid residues that influence neutralization sensitivity at ID₅₀ were identified by using Bayes factors (BFs) to compare the fit of a model in which the evolution of the residue correlates with changes in neutralization sensitivity to that of a null model in which the virus evolves independently of the antibody response. Sites with significant BFs were mapped onto the three-dimensional protein structure to identify epitopes on the surface of the envelope glycoprotein. Predictions were tested experimentally by site-directed mutagenesis.

Results: We identified between 1 and 4 sites per sample that were strongly associated with neutralization sensitivity (BF >20) and confirmed a subset of the predicted residues experimentally for 4 of the 5 sera. Our model predictions included two sites in the V2 region (166 and 169) that contributed to a trimer-specific PG9/16-like epitope in one subject, a glycan at position 332 in a second individual and the 674 residue in the membrane proximal region of gp41 in a third participant.

Conclusion: Our results provide strong support for the use of evolutionary models applied to cross-sectional viral neutralization data to identify novel epitopes recognized by antibodies that confer neutralization breadth. Such epitopes are difficult to discover experimentally or with existing computational tools, thereby demonstrating the utility of our approach.

1 IAVI/TSRI, La Jolla, USA 2 Vaccine Research Center, NIAID, NIH, Bethesda, USA 3 Laboratory of Immunoregulation, NIAID, NIH, Bethesda, USA OA07.03 Isolation of a Subset of Novel HIV-1 Broadly Neutralizing Antibodies with Dual Specificity Overlapping the Env Receptor and Coreceptor Binding Sites

Background: Both CD4 binding site (CD4 bs) and coreceptor binding site (CoR bs) of HIV-1 gp120 are two of the most conserved functional elements of HIV Env. Elicitation of potent neutralizing antibodies (nAbs) targeting these structural elements with Env-based immunogen has been pursued extensively, with limited success. Our previous study showed that broadly and potent CD4 bs-specific nAbs can be elicited during natural infection at high titers and can be detected in selected patient sera. From the memory B cell repertoire of one such patient, we isolated a subset of CD4 bs-specific nAbs including VRC01, which neutralizes more than 90% of circulating primary virus isolates. From the same patient sera, we detected that there was also a fraction of potent nAbs specific for the Env CoR bs by differential protein adsorption and neutralization analysis.

Methods: In this study, we sought to isolate CoR bs-specific nAbs from this patient by sorting with a fluorescence-tagged gp120 possessing a CoR bs knockout mutation (I420R).

Results: A series of B cell clones were isolated by the FACS phenotype of WT gp120-high/I420R-low. A subset of these clones was able to neutralize ∼ 50% of Clade B tier 2 primary virus isolates, with better potency than that of the typical CoR bs antibodies isolated previously. These clones displayed gp120 binding affinity abrogated by gp120 with both CD4 bs and CoR bs mutations, suggesting its overlapping epitope, which was also supported by cross competition analysis with site-specific ligands.

Conclusion: We have isolated a subset of novel CD4/CoR nAbs whose epitopes may directly or functionally overlap with both the HIV gp120 receptor and coreceptor binding sites. Fine epitope mapping and impact on the configuration of the viral Env functional spikes upon antibody binding is under way to define the unique specificity.

1 INSERM U748, Université de Strasbourg, Strasbourg, France 2 Etablissement Français du Sang, U725 Cellules dendritiques, Strasbourg, France 3 Covance CLS SA, Meyrin, Switzerland OA07.04 Neutralizing Antibodies Inhibit HIV-1 Transfer from Dendritic Cells to Primary CD4 T Lymphocytes

Background: Sexual mucosal transmission is the main route of HIV-1 infection worldwide. Immature dendritic cells (DCs), which reside in genital mucosal surfaces, are among the first cells that encounter HIV-1. We have shown previously that in HIV-1 transfer conditions, where HIV-1 loaded immature DCs were co-cultured with primary CD4 T lymphocytes, HIV-1 replication was enhanced in DCs. Here, we aim to analyze the kinetic of HIV-1 replication and the ability of HIV-1 specific antibodies to inhibit HIV-1 infection in the co-culture of immature DCs with primary PHA-activated CD4 T lymphocytes.

Methods: DCs were generated from purified human blood CD14+ monocytes. After 2 hours of immature DCs incubation with R5 HIV-1 strains, we added autologous primary CD4 T lymphocytes in the presence or absence of HIV-1 specific antibodies. The percentage of infection was quantified by flow cytometry using intracellular p24 viral antigen staining.

Results: In this co-culture condition, the early step of HIV replication was more rapid in co-cultured DCs compared to infected DCs without lymphocytes, whereas the kinetic of HIV-1 fusion was unchanged. Monoclonal neutralizing antibodies, but not non-neutralizing or inhibitory antibodies, were able to potently inhibit HIV-1 transfer to CD4 T lymphocytes. Moreover, we found a decrease of HIV-1 replication in DCs although neutralizing antibodies or non-neutralizing inhibitory antibodies were added on HIV-1-loaded DCs after two hours. This inhibition of HIV-1 replication in DCs was correlated with DC maturation.

Conclusion: These results demonstrate that neutralizing antibodies are able to efficiently inhibit HIV-1 infection of DCs and HIV-1 transfer to primary CD4 T lymphocytes. The efficient inhibition of HIV-1 transfer strengthens the requirement of the induction of HIV-1 specific antibodies directly at mucosal portal of HIV-1 entry. Vaccines that induce such antibody response may prevent the very early dissemination of HIV-1 after its sexual transmission.

1 Ragon Institute of MGH, MIT and Harvard, Somerville, USA 2 Monogram Biosciences, San Francisco, USA 3 Monogram Bioscinces, San Francisco, USA 4 University of California, San Francisco, USA OA07.05 Induction of Neutralizing Antibody Responses in HIV Infection Is Associated with HIV-Specific CD4 T Cell Responses

Background: One of the foremost challenges in the development of an HIV-1 vaccine is that HIV-1 targets CD4-T cells, which are critically important in shaping the immune response to infection. It has been demonstrated that T follicular helper (TFH) CD4-T cells are pivotal for the induction of broadly neutralizing antibody (nAb) responses and for the generation of long-lived B-cell memory maturation. By cognate interaction and cytokine secretion, CD4-T cells expressing CD40L can induce class switching and somatic hypermutation in antigen specific B-cells, which is reflected in the expression of activation-induced cytidine deaminase (AID). Nonetheless, the role of TFH cells and their interaction with B-cells in HIV infection is currently unknown.

Methods: Ten subjects were identified during acute HIV infection and followed longitudinally over a median of three years. Neutralizing activity of plasma antibodies against a panel of present and past autologous and heterologous viruses was assessed using a single-replication cycle assay in which full-length envelope genes were incorporated into expression vectors (Monogram Biosciences). Phenotypic and functional characteristics of HIV-specific CD4-T cell responses and their impact on AID expression were assessed longitudinally by multiparameter flow cytometry.

Results: The breadth of the broadly neutralizing antibody response to heterologous HIV significantly increased over time in each patient. Interestingly, the expansion in heterologous breadth was not only significantly correlated with an increase in AID expression in B-cells, but also with CD4-T cell expression of PD-1, a marker that has been associated with TFH cells. Moreover, a positive association between AID expression and gp120-specific CD40L-responses was detected at the earliest time point.

Conclusion: Our data strongly indicate that the induction of broadly neutralizing antibody responses is linked to the function and activation of HIV-specific CD4-T cell responses. These findings will be pivotal in efforts to generate neutralizing antibody responses by vaccination strategies.

1 US Military HIV Research Program, Rockville, USA 2 Duke University, Durham, USA 3 University of Alabama at Birmingham, Birmingham, USA OA07.06 Influence of NK Cells and NK Cell Receptor Polymorphisms in the Assessment of HIV-1 Neutralization

Background: Genetic polymorphisms within the killer immunoglobulin receptor (KIR) and Fc gamma receptor genes of natural killer (NK) cells have been shown to influence the containment of HIV replication, both in vivo and in vitro. Peripheral blood mononuclear cells (PBMC) prepared from different donors for in vitro use contain varying numbers of immune cells, including NKs. This study addresses the impact of this variation and of specific genetic polymorphisms on the measurement of neutralization using PBMC from HIV-seronegative donors.

Methods: Leukopaks (LP) were obtained from 25 donors and PBMC immune cell populations were quantified using multi-color flow cytometry. Viral permissivity and HIV-1 neutralization profiles were assessed using infectious molecular clones (IMC) and intracellular p24 or Renilla luciferase (LucR) expression as endpoints. HIV-1 IMC with LucR (NL-LucR-BaL.ecto and NL-LucR-SF162.ecto) were used in neutralization assays; sCD4 and polyclonal and monoclonal antibodies were tested. CD4+, CD8+ and NK cells were depleted from PBMC using antibody-coated beads.

Results: The PBMC NK cell percentages ranged from 0.5–10.0%. When donor PBMC were stratified into quartiles using mean neutralization titer or viral titer rankings, NK cells were found to be elevated in PBMC yielding higher neutralization titers and lower viral growth (p = 0.01, Mann-Whitney). KIR3DS1 and a polymorphism for higher affinity (158-Val) in Fc gamma RIIIa, were both associated with PBMC that showed higher neutralization (p = 0.009-0.03, depending on the antibody tested). Furthermore, 100% of KIR3DS1+PBMC fell into the upper 2 neutralization quartiles (p = 0.03, Fisher's exact test). NK cell-depletion decreased neutralization up to 1000-fold, depending upon the PBMC and antibody tested.

Conclusion: NK cells, KIR polymorphisms and Fc gamma RIIIa affinity can influence HIV-1 neutralization results. These findings indicate that the LucR-IMC PBMC platform, with antibody continuously present, simultaneously assesses neutralizing and non-neutralizing antibody-dependent HIV-1 inhibition. This may provide the opportunity to delineate the dominant antibody function(s) in polyclonal responses to vaccines.

1 Emory University, Atlanta, USA 2 Laboratory of Molecular Microbiology, National Institutes of Health, Bethesda, USA 3 Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, USA 4 School of Medicine, UT Health Science Center at San Antonio, San Antonio, USA 5 Department of Biostatistics, University of Pennsylvania, Philadelphia, USA 6 The AIDS and Cancer Virus Program, The National Cancer Institute, Frederick, USA OA08.01 Evidence for CD4 T-Cell Mediated Immune Control of Macrophage Infection in Rhesus Macaques Undergoing CD4+ Lymphocyte Depletion Prior to SIV Infection

Background: The relative contribution of CD4+ T-cells to the control of virus replication during primary SIV infection rhesus macaques (RM) remains incompletely defined. To investigate the relationship between CD4+ T-cells and post-peak decline of viremia, we depleted CD4+ lymphocytes from non-inductive immunological sites of five RMs prior to SIV-infection.

Methods: Nine RMs were infected intravenously with SIVmac251. Prior to infection, five RMs were CD4+ lymphocyte-depleted using the Cdf OKT4A HulgG1 Ab. Samples were collected longitudinally from blood, bone marrow, lymph nodes (LNs), and rectal biopsies (RBs). Plasma SIV viremia and cell-associated SIV-DNA were measured by RT-PCR. Cell-associated vRNA in tissues was assessed by dual immunohistochemistry and in situ hybridization. Immune function parameters were measured by multi-color flow cytometry, neutralization assays and ex vivo stimulation assays.

Results: At the time of infection, CD4+ lymphocyte-depleted RM showed ten-fold lower CD4+ T cell levels in blood and LNs and normal CD4+ T-cell levels in RBs. After infection, CD4+ lymphocyte-depleted animals exhibited a similar peak of viremia but, in contrast to non-depleted RMs, showed no post-peak virus decline, and progressed more rapidly to AIDS. Importantly, no significant differences were found between depleted and undepleted RMs in terms of (i) availability of target cells; (ii) levels of SIV-specific CD8+ T cell responses; and (iii) SIV-specific humoral responses. Interestingly, peak viral load in depleted animals was associated with a striking increase in virus production by macrophages and dendritic cells.

Conclusion: These data indicate an essential role for CD4+ T-cells in mediating post-peak decline of viremia in SIV-infected RMs that may be associated with an ability to suppress virus production by macrophages. The complex balance between CD4+ T-cells as target cells and immune effectors may play a greater than anticipated role in the outcome of vaccine efficacy and should be considered during future vaccine design.

1 University of Wisconsin, Madison, Madison, USA 2 Oregon Health & Science University, Beaverton, USA OA08.02 CD4+ T Cell Escape in a SIVmac239-Infected Indian Rhesus Macaque Elite Controller with Breakthrough Viremia

Background: The role of CD8+ T cells in containment of retroviral infections is well documented by studies of CD8+ T cell-mediated viral escape. It contrast, the immunological role of retrovirus-specific CD4+ T cells remains unclear, particularly in the setting of elite control. Here, we describe viral escape from CD4+ T cells concomitant with viral breakthrough and loss of elite control.

Methods: A SIVmac239-infected Indian rhesus macaque maintaining elite control of viremia (<1,000 vRNA copies/mL plasma) spontaneously lost control, experienced breakthrough viremia, and eventually succumbed to AIDS. We performed whole genome sequencing on plasma virus prior to the virus becoming undetectable and immediately following breakthrough and loss of elite control. We then investigated the T cell response directed against areas in the viral proteome exhibiting sequence divergence between the pre- and post-breakthrough time points.

Results: Comparison of the pre- and post-breakthrough viral sequences revealed surprisingly few amino acid substitutions post-breakthrough. These substitutions occurred within two previously defined and three previously undescribed CD8+ T cell epitopes, confirming the critical role of CD8+ T cells in control of viral replication. Interestingly, we identified two mutations within Gag, V63A and D205E, which occurred within CD4+ T cell epitopes. Both of these mutations abrogated CD4+ T cell recognition. While the V63A mutation also abrogated recognition of an overlapping CD8+ T cell epitope, the D205E mutation was solely targeted by a CD4+ T cell response. We confirmed that no CD8+ T cell responses were targeting this region. Finally, we demonstrated that viruses bearing the GagD205E mutation escaped CD4+ T cell effector function in vitro.

Conclusion: Virus-specific CD4+ T cells play an active role in the containment of retroviruses and are likely essential for the maintenance of low-level viremia. How these CD4+ T cells persist and exert their anti-viral function during retroviral infection remains unclear and warrants further investigation.

1 Northwestern University, Chicago, USA 2 University of Massachusetts Medical School, Worcester, USA 3 New England Primate Research Center, Harvard Medical School, Southborough, USA OA08.03 Decreased Penetration of GFP-Labeled Lentiviral Particles in the Vagina and Endocervix of SIVΔ3-Vaccinated Macaques

Background: Immunization with live attenuated SIV strains, such as SIVΔ3, provide robust protection against vaginal challenge with pathogenic SIV strains. However, the precise contributions of humoral and cellular immune responses, and the anatomic compartment(s) in which virus transmission is interrupted are unknown. To gain insight, we examined that the ability of fluorescent virus particles expressing the SIV envelope to penetrate intact genital epithelium in unvaccinated and SIVΔ3-vaccinated macaques.

Methods: A total of 11 animals were studied: 8 were vaccinated with SIVΔ3 29 months previously, and 3 were uninfected controls. Three of the SIVΔ3-vaccinated animals were superinfected with SIVmac251 after vaginal challenge one year after vaccination. Subsequently, female macaques were inoculated intravaginally with PA-GFP-gag labeled virions. Genital tracts were removed 4 hours post-inoculation and immediately dissected and snap frozen in OCT media. Tissue specimens were sectioned and stained for cellular proteins, prior to imaging with fluorescent deconvolution microscopy.

Results: Within 4 hours, PA-GFP virions were able to penetrate the simple columnar and stratified squamous epithelia in both SIVΔ3-vaccinated and control animals, penetrating up to depths of 50μm. When comparing viral penetration depths between the two sample groups, a significant decrease in penetration of viral particles in the vaginal and endocervical mucosa of SIVΔ3-vaccinated animals was observed.

Conclusion: Our results indicate that in unvaccinated and SIVΔ3-vaccinated animals, PA-GFP virions can penetrate to depths where they can interact with target cells in both squamous and columnar epithelium. A decrease in median depth penetration of virus particles in vaccinated macaques suggests vaccination with live attenuated SIV is able to block early events of virus transmission, most likely via the induction of SIV-specific antibody responses. A clearer understanding of the ability of virus-specific antibody responses to interrupt early events of viral transmission at mucosal sites is essential for the development of future HIV vaccine strategies.

1 UPENN, Philadelphia, USA 2 1Department of Microbiology, University of Washington, Seattle, USA 3 Bioqual, Rockville, USA 4 Wisconsin National Primate Research Center, Madison, USA 5 Inovio Pharmaceuticals, Blue Bell, USA 6 University of Washington, Seattle, USA OA08.04 Long-Term Programming of Antigen-Specific Immunity from Gene Expression Signatures in the PBMC of Rhesus Macaques Immunized with an SIV DNA Vaccine

Background: While HIV-1-specific cellular immunity is thought to be critical for the suppression of viral replication, the correlates of protection have not yet been determined. Rhesus macaques (RM) are an important animal model for the study and development of vaccines against HIV/AIDS. Our laboratory has helped to develop and study DNA-based vaccines in which recent technological advances including genetic optimization and in vivo electroporation (EP) have helped to dramatically boost their immunogenicity.

Methods: In this study, RMs were immunized with a DNA vaccine including individual plasmids encoding SIV gag, env, and pol alone, or in combination with a molecular adjuvant, plasmid DNA expressing the chemokine ligand 5 (RANTES), followed by EP. Along with standard immunological assays, flow-based activation analysis without ex vivo restimulation and high-throughput gene expression analysis was performed.

Results: Strong cellular immunity was induced by vaccination which was supported by all assays including PBMC microarray analysis that identified the up-regulation of 563 gene sequences including those involved in interferon signaling. Furthermore, 699 gene sequences were differentially regulated in these groups at peak viremia following SIVmac251 challenge. We observed that the RANTES-adjuvanted animals were significantly better at suppressing viral replication during chronic infection and exhibited a distinct pattern of gene expression which included immune cell-trafficking and cell cycle genes. Furthermore, a greater percentage of vaccine-induced central memory CD8+ T-cells capable of an activated phenotype were detected in these animals as measured by activation analysis.

Conclusion: Thus, co-immunization with the RANTES molecular adjuvant followed by EP led to the generation of cellular immunity that was transcriptionally distinct and had a greater protective efficacy than its DNA alone counterpart. Furthermore, activation analysis and high-throughput gene expression data may provide better insight into mechanisms of viral control than may be observed using standard immunological assays.

1 National Center for AIDS/STD Control and Prevention in China CDC, Beijing, China 2 College of Life Science, Nankai University, China 3 Institute of Laboratory Animal Sciences, CAMS, China OA08.05 Compromised NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Chronic SIV Infection

Background: Increasing evidence indicates that antibody-dependent cellular cytotoxicity (ADCC) contributes to control of HIV/SIV infection. However, virtually nothing is known about the ADCC function of Nature killer cells in non-human primate models. Efforts to get a clearer understanding of the specific role NK cells play in rhesus macaques will be helpful in evaluation of vaccines in non-human primate models.

Methods: The antibody-coated target cells were used to measure the ADCC function of macaque NK cells. The ADCC responses were analyzed by NK cell cytokine secretion and degranulation through ICS-based assay. All data were collected on BD FACS Aria.

Results: To identify the differences of NK cell-mediated ADCC function between naive and chronic SIV-infected macaques, some cytokines were measured in respond to Fc target cells. It was obvious to notice the downregulation of CD107a expression (P = 0.0203) as well as decreased secretion of IFN-γ (P = 0.0180) and TNF-α (P = 0.032) in NK cells from SIV-infected group compared with naives. The greatly reduced CD16⁺ NK subset in macaques with SIV infection (P = 0.0209) was the reason of impaired NK cell function, cause there was a direct association between CD16 expression and cytokine production in NK cells, including CD107a(P = 0.0204), IFN-γ(P = 0.0231), TNF-α(P = 0.0413). The loss of CD16 MFI following stimulation, which was used to quantify the Fc-mediated activation of NK cell, was detected much lower in SIV-infected group (P = 0.0036).

Conclusion: In chronic SIV-infected group, we could see the significant compromised ADCC function and its close relationship with CD16 expression on NK cells: the increased levels of CD16 allow NK cells to better respond to Fc target cells and this marker could be served as sensor for ADCC function of NK cells in macaques. These data offer new insights into the role of NK cells as an important bridge between innate and adaptive immunity in non-human primate models.

1 NIAID/NIH, Chevy Chase, USA 2 Vaccine Branch, NCI/NIH, Bethesda, USA 3 AIDS and Cancer Virus Program, SAIC-Frederick, Inc., Frederick, USA 4 Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, USA OA08.06 Cytotoxicity Capacity of SIV-Specific CD8+ T Cells Against Primary Autologous Targets Correlates with Immune Control in SIV-Infected Rhesus Macaques

Background: HIV-specific CD8⁺ T cells of long-term nonprogressors / elite controllers (LTNP/EC) exhibit extraordinary per-cell cytotoxic capacity against HIV-infected CD4⁺ T cells, in contrast to those of progressors. In this study, we explore whether our previous findings can be extended to the SIV-rhesus macaque (RM) model by measuring SIV-specific cytotoxic capacity of CD8⁺ T cells against SIV-infected targets.

Methods: Cytotoxicity and IFN-γ production of SIV-specific CD8⁺ T cells were examined following 6-day incubation with autologous SIVmac251-infected telomerase-transduced CD4⁺ T cell clone targets. SIV-specific CD8⁺ T cell cytotoxic responses were measured at 1 hour by flow cytometric detection of active granzyme (Gr) B delivery to live targets and infected CD4 elimination (ICE).

Results: Fifteen SIV-infected RM with different extents of control of viral replication have been evaluated. Data collection is ongoing with laboratory personnel blinded to RM disease status. LTNP/EC RM had higher median delivery of active GrB to targets (43.5% vs. 18.5%, p = 0.04), and higher median ICE than progressors (67.3% vs. 24.4%, p = 0.009). There was a significant correlation between ICE and viral load (R = −0.55, p = 0.03), and between GrB delivery and ICE (R = 0.86, p < 0.0001).

Conclusion: Lytic granule loading of CD8⁺ T cells and efficient delivery of active GrB to SIV-infected targets are associated with control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans. These findings suggest ICE is a correlate of control of viral replication in chronic SIV infection. They also suggest the use of cytotoxic capacity as a predictor of immunologic control in the vaccine setting should be tested.

1 University of Melbourne, Parkville, Australia OA08.07 Live Attenuated Influenza-SIV Vaccines: Efficacy Against Mucosal Challenge in Macaques

Background: Attenuated Influenza-HIV vaccines are attractive since they are easy to deliver, induce mucosal immunity and have established manufacturing processes.

Methods: We assessed Influenza-SIV constructs expressing 3 SIV CTL epitopes restricted by Mane-A*10 engineered into the neuraminidase stalk. Vaccines were administered primarily intranasally to 15 Mane-A*10+ pigtail macaques.

Results: Prime-boost vaccinations with H1N1 and H3N2 constructs were well tolerated via the respiratory route and all animals seroconverted to Flu, had Flu RNA recovered early after vaccination, and generated Flu-specific CTL responses. Vaginal inoculations did not result in Flu seroconversion despite nonoxynl-9 pretreatment. SIV-specific responses were low after vaccination but expressed the mucosal homing marker a4b7 and rose dramatically after intravaginal SIV exposure. SIV RNA levels were however, high after challenge and rapid serial escape at the SIV CTL epitopes was observed. Indeed, immune escape was faster in vaccinees than controls (p = 0.02) and the SIV specific CTLs rapidly became dysfunction after challenge in comparison to Flu-specific CTLs.

Conclusion: Although live attenuated influenza vaccines prime SIV-specific CTL immunity, broader immunity will be required to maintain viral control. We are currently engineering recombinants expressing a larger suite of HIV/SIV antigens in vectors that replicate more effectively in primates to induce more broadly based T and B cell immunity.

1 International AIDS Vaccine Initiative (IAVI), New York, USA 2 IAVI Human Immunology Laboratory, London, United Kingdom (Great Britain) 3 University of Rochester School of Medicine & Dentistry, Rochester, USA 4 IAVI, New York, USA 5 The EMMES Corporation, Rockville, USA 6 International AIDS Vaccine Initiative, Human Immunology Laboratory, London, United Kingdom (Great Britain) OA09.01 T-Cell Responses Induced by Immunization with Ad35-HIV-1 Vaccines Are Broad, Durable, Polyfunctional and Can Mediate Inhibition of HIV

Background: Broad, robust, polyfunctional T cells able to mediate functional inhibition and/or killing of HIV-infected CD4 targets are critical for immunological control of HIV replication. Ad35 replication-defective vectors may be able to induce these responses and circumvent pre-existing immunity to common human adenoviruses.

Methods: A Phase I trial of Ad35 expressing HIV-1 subtype A gag, RT, integrase, nef (GRIN as a fusion protein) and envelope (ENV) genes or GRIN alone was conducted in 56 healthy HIV-uninfected, Ad35 antibody negative adults in the U.S. The vaccines were administered intramuscularly at 0 and 6 months at 2x10⁹, 2x10¹⁰ or 2x 10¹¹ viral particles (vp) for groups A-C, respectively. Group D received Ad35-GRIN alone at 1x10¹⁰ vp. Immunogenicity was assessed by IFN-g ELISPOT, polychromatic flow cytometry, viral inhibition, ENV ELISA and Ad35 neutralization.

Results: IFN-g ELISPOT responses to any vaccine antigen in groups A-D were detected in 50, 56, 70 and 90% and in 75, 100, 88 and 86% of vaccine recipients post 1st (Vac.1) and 2nd (Vac.2) vaccination. The geometric mean spot forming cells/million PBMC to any antigen was 101–122 across groups A-C and 145–185 in group D after Vac.1 and Vac.2, with an increase of 0.34 log after Vac.2. Breadth increased from a median of 2 (Vac.1) to 4 (Vac.2) proteins out of 5. CD4 and CD8 T-cell responses were polyfunctional. CD8 T-cell-mediated inhibition of HIV was detected in 61% (Vac.1) and 52% (Vac.2) of vaccinees across multiple HIV-subtypes and dose dependent. Env antibodies were detected in all vaccinees in groups A-C. Ad35 neutralizing titers were low even after Vac.2.

Conclusion: HIV-specific cellular responses were seen in the majority of volunteers immunized with Ad35-GRIN/ENV or Ad35-GRIN, the response rate, breadth and magnitude of the response increased after the second dose. T-cell responses were polyfunctional and inhibition of HIV was seen.

1 SCHARP, Fred Hutchinson Cancer Research Center, Seattle, USA 2 Duke University Medical Center, Durham, USA 3 Mahidol University, Bangkok, Thailand 4 Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand 5 Ministry of Public Health, Nonthaburi, Thailand 6 Walter Reed Army Institute of Research, USMHRP, Rockville, USA 7 Harvard Medical School, Southborough, USA OA09.02 ADCC Titers to HIV-Infected Cells Are Detectable in the Majority of Vaccine Recipients in the RV144 Trial

Background: A modest reduction in the rate of HIV-1 infection was observed among vaccine recipients in the RV144 trial who received a prime-boost vaccine regimen with recombinant canarypox and soluble gp120. Whereas virus-specific CD8+ T cell responses were largely undetectable, antibodies capable of binding to gp120 were detectable in nearly all vaccinated subjects. However, these antibodies were unable to neutralize primary HIV-1 isolates, suggesting that antibodies may have contributed to protection by non-neutralizing effector mechanisms.

Methods: To determine if vaccinated subjects made antibodies capable of directing antibody-dependent cell-mediated cytotoxicity (ADCC), we used a novel assay to measure ADCC titers against HIV-infected cells in pilot samples from the RV144 trial. This assay is based on the killing of an HIV-infected CD4+ T cell line by a CD16+ NK cell line in the presence of serial dilutions of plasma, thus allowing ADCC titers to be measured against virus infected cells expressing the native, oligomeric conformation of the HIV-1 envelope glycoprotein. Using this approach, ADCC titers in blinded plasma samples from 80 vaccine recipients and 20 placebo controls were measured at baseline (week 0) and at two weeks after the last boost (week 26) against target cells infected with the HIV-1 subtype AE isolate 92TH023.

Results: Comparison of the response rate for the vaccine group versus the placebo group revealed that 76.2% (61/80; 95% CI 66%, 84%) of the vaccinated subjects versus 10.0% (2/20; 95% CI 3%, 30%) of the placebo controls had detectable ADCC titers against HIV-1 92TH023 infected cells at week 26 (Fisher's exact test, P = 6.7e-8). ADCC titers were also significantly higher at week 26 than at week 0 among the vaccine recipients (McNemar's exact p-value, P = 3.9e-12).

Conclusion: These results demonstrate that the majority of vaccinated subjects in the RV144 trial made antibodies capable of directing ADCC against HIV-infected cells.

1 NYU School of Medicine, New York, USA 2 Fred Hutchinson Cancer Research Center, Seattle, USA 3 Duke University School of Medicine, Durham, USA 4 US Military HIV Research Program/Henry M. Jackson Foundation, Rockville, USA 5 Ministry of Public Health, Nonthaburi, Thailand OA09.03 V2-Reactive Antibodies in RV144 Vaccinees' Plasma

Background: Initial studies suggested that V2 antibodies (Abs) played a protective role in RV144. Pilot experiments were needed to quantify and characterize anti-V2 Abs in plasma of RV144 participants.

Methods: One hundred coded RV144 pilot study specimens were analyzed by ELISA using plasma obtained before immunization (visit 1), and 2 and 28 weeks after the last immunizing dose (visits 8 and 9, respectively). Results were decoded after submission and analysis of data.

Results: [a] At visit 1, 0/20 placebo and 3/80 vaccinee specimens diluted 1:20 reacted with gp120_JR-FL. At visit 8, 0/20 and 80/80 were reactive. Plasma were not reactive vs. bovine serum albumin. [b] At visit 1, 0/20 placebo and 1/80 vaccinee specimens diluted 1:20 reacted with a V1V2_HxB2-gp70 fusion protein. At visit 8, 1/20 and 76/80 (95%) were reactive. [c] Reactivity was tested against four 21-mer linear V2 peptides spanning the α4β7 binding motif, chosen to represent: the most polar sequence found in V2 loops with 38 amino acids (“polar-38”); the most common sequence of V2s with 40 AAs (“common-40”); the most polar sequence in V2 loops with 40 AAs (“polar-40”); and the consensus sequence of V2 loops with 40 AAs (“consensus-40”). Activity in plasma diluted 1:100 showed differential reactivity with the peptides: “polar-38” ≈ “polar-40” > “consensus”>“common-40”. For visit 8, 1/20 placebo and 47/80 (59%) vaccine specimens were reactive with “polar-38”. Shorter linear peptides were poorly reactive. Notably, by visit 9, the reactivity to gp120_JR-FL, V1V2_HxB2-gp70, and all V2 peptides was markedly reduced in all vaccinees' plasmas.

Conclusion: Anti-V2 Abs were elicited by the RV144 immunization protocol. Reactivity was highest 2 weeks after the last boost, and was significantly diminished 26 weeks later. V2-specific reactivity was demonstrable with a fusion protein containing the entire V1V2 loop and with 21-mer linear V2 peptides spanning the α4β7 binding motif.

1 USMHRP, HJF, Rockville, USA 2 AFRIMS, Bangkok, Thailand 3 Mahidol University, Bangkok, Thailand 4 Royal Thai Army, Armed Forces Research Institute of Medical Sciences, Thailand 5 Fred Hutchinson Cancer Research Center, Seattle, USA 6 Duke University School of Medicine, Durham, USA 7 NYU, New York, USA 8 USMHRP, Walter Reed Army Institute of Research, Rockville, USA 9 Ministry of Public Health, Bangkok, Thailand OA09.04 Surface Plasmon Resonance Analysis of Anti-gp120 V2-Specific IgG Antibodies Generated in the RV144 Thai Trial

Background: The RV144 HIV-1 prime-boost vaccine trial showed a modest level of protection with a vaccine efficacy of 31.2%. The reduced rate of viral acquisition could be due to the induction of envelope-specific antibodies. The V2 loop of HIV-1 gp120 has been implicated as a binding partner of integrin α4β7. Therefore, a subset (pilot study) of 40 plasma samples was analyzed for IgG antibodies specific to cyclic V2 and cyclic V3 peptides derived from the gp120 subunit of HIV-1 92TH023 envelope protein.

Methods: Biacore analyses of 40 plasma samples (32 vaccinee, 8 placebo recipients) from the RV144 vaccine trial before vaccination (visit 1) and 2 and 28 weeks after the last immunization (visits 8, and 9, respectively) were conducted. Cyclic peptides (V2, V2 variants, and V3) were coupled to Biacore sensor chips. Diluted plasma samples (1:50) were injected over the chip surface followed by anti-human IgG antibody.

Results: V2-specific IgG antibodies were present in 0/32 from visit 1 and 31/32 (96.8%) vaccinees from visit 8. Vaccinee plasma showed binding to the 42 amino acid cyclic peptide containing the α4β7 binding motif. Binding was greatly diminished when that region was scrambled. There was significant reduction in V2-specific IgG antibodies in all of the vaccinee plasma samples by visit 9. The RV144 samples analyzed had extremely low or no antibodies reactive with cyclic V3 peptide. In contrast to vaccinees, plasma samples (1:50 dilution) from individuals infected with clade A/E from the same geographical region had >3-fold more anti-V3 antibodies compared to anti-V2 antibodies.

Conclusion: Our results support the conclusion that the V2 loop of gp120 as presented in the context of the RV144 vaccine is more amenable to induction of V2-specific antibodies when compared to a natural infection. The majority of the V2-specific antibodies were directed against the region containing the α4β7 binding motif.

1 Swedish Institute for Communicable Disease Control, Stockholm, Sweden 2 Swedish Institute for Communicable Disease Control and Karolinska Inst, Stockholm, Sweden 3 US Military HIV Research Program, Walter Reed Army Institute of Resear, Rockville, USA 4 Venhälsan, Karolinska Institutet, Södersjukhuset, Stockholm, Sweden OA09.05 Polyfunctional CD4/CD8 HIV-Specific Cytokine Responses and Presence of Cytolytic Markers in Swedish Vaccinees Immunized with HIV-1 DNA and HIV-1 MVA

Background: We have monitored polyfunctional HIV-1-vaccine-induced responses as measured by cytokine and cytolytic marker expression. Healthy volunteers were immunized at 0, 1 and 3 months with DNA plasmid expressing gp160 of HIV-1 subtypes A, B and C; revB; p17/p24 gag A and B and RTmut B. At nine months they were boosted with a heterologous MVA expressing HIV-1 env, gag, pol of CRF01_AE. Approximately 3 years after the first HIV-MVA vaccination, 24 volunteers were given a second HIV-MVA boost.

Methods: HIV-Gag specific immune responses were assessed by IFN-gamma-ELISpot and by 8-colour ICS for expression of cytokines (CD3/CD4/CD8/IFN-gamma/IL-2/TNF-α/MIP1-β/VIVID) using fresh cells, and for expression of cytolytic markers (CD3/CD8/IFN-gamma/MIP1-β/CD107/Perforin/Granzyme B/VIVID) using cryopreserved cells.

Results: Two weeks after the second HIV-MVA vaccination, 19 (83%) of 23 evaluable vaccinees were reactive in IFN-gamma-ELISpot; 18 to Gag and 11 to Env peptides. The ICS revealed CD4 and/or CD8 Gag-specific reactivity in 15/23 (65%) vaccinees. Polyfunctional CD4+ and CD8+ T cell Gag-specific responses were detected in 7/23 (30%) vaccinees, all of whom had IFN-gamma-ELISpot Gag reactivity >175 SFC/million PBMC. Within the CD8+ T-cell compartment approximately 25% of the responding cells were dual functional and 60% polyfunctional, predominantly expressing IFN-gamma/MIP1-β, IFN-gamma/TNF-α/MIP1-β and IFN-gamma/IL-2/TNF-α/MIP-1β. Among the responding CD4 T-cells approximately 25% were dual functional and 60% polyfunctional, predominantly expressing IL-2/TNF-α, IFN-gamma/IL-2/MIP1-β and IFN-gamma/IL-2/TNF-α/MIP1-β.

Conclusion: A second HIV-MVA boost given approximately 3 years after the initial HIV-DNA/HIV-MVA vaccinations gave rise to polyfunctional immune responses as measured by the production of cytokines and cytolytic markers. The data supports further exploration of this vaccine concept.

1 Medical Research Council Laboratories, The Gambia, Banjul, Gambia 2 The Jenner Institute, University of Oxford, Oxford, United Kingdom (Great Britain) 3 Karolinska Institutet, Stockholm, Sweden 4 Kenya AIDS Vaccine Initiative, University of Nairobi, Nairobi, Kenya 5 Departments of Medicine and Global Health, University of Washington, Seattle, USA OA09.06 PedVacc001:Safety and Immunogenicity of a Candidate HIV-1 Vaccine,MVA.HIVA, Administered to Healthy Gambian Infants Born to HIV-1/2-Uninfected Mothers

Background: The development of a safe and effective HIV-1 vaccine remains a global priority especially in prevention of mother to child transmission of HIV-1 during breastfeeding. Our vaccine platform consists of recombinant BCG prime-recombinant modified vaccinia virus Ankara (MVA) boost. This study is the first stage and evaluates the safety and immunogenicity of MVA.HIVA, a candidate HIV-1 vaccine, among healthy Gambian infants born to HIV 1/2 uninfected mothers.

Methods: Sixty-five mothers were sensitized on delivery of healthy babies at Sukuta Health Centre, The Gambia. Sixty-two of whom consented to HIV voluntary counseling and testing and all the mothers (100%) had negative HIV test. Forty-eight of the eligible infants were randomized into vaccine and no-treatment control group at 20 weeks of age. The vaccine group received one dose of 5 × 10^7 pfu of MVA.HIVA administered intramuscularly. The safety of MVA.HIVA was determined by comparing adverse events, reactogenicity, biochemical and haematological data of vaccine and control groups. Immunogenicity was measured by interferon (IFN)-γ ELISPOT assay on peripheral blood mononuclear cells.

Results: The mean Haemoglobin (Hb), White Blood Cell count (WBC), Alanine transaminase (ALT) and Creatinine at pre-vaccination and post–vaccination visits were within acceptable normal ranges. Adverse events observed included fever, excessive crying, cough, vomiting, bilateral eye discharge, diarrhea and poor weight gain. All study infants had negative HIV antibody tests at 28 weeks and no severe adverse events or suspected unexpected serious adverse reactions were reported. Preliminary cultured IFN-γ ELISPOT analysis showed induction of HIV-1-specific T-cell responses in about 40% of the vaccinees.

Conclusion: We have shown that MVA.HIVA is safe and immunogenic in healthy Gambian infants. A parallel study PedVacc002 of MVA.HIVA administered to infants born to HIV-1-positive mothers is ongoing in Kenya.

1 Albert Einstein College of Medicine, New York, USA 2 Duke University, Durham, USA 3 Iowa State University, Ames, USA OA10.01 Eliciting Broadly Neutralizing Antibodies Against HIV-1 Using a Novel Antigen Containing gp41 Membrane-Proximal External Region

Background: A major roadblock in developing an AIDS vaccine is the difficulty in eliciting broadly neutralizing antibodies (BR-Nabs). The membrane-proximal external region (MPER) of gp41 is highly conserved and targeted by BR-Nabs 2F5 and 4E10. Thus, it is an attractive target for vaccine development. However, many past attempts to elicit BR-Nabs using MPER-based antigens have failed. Here, we report a unique, comprehensive vaccine strategy that uses a structurally rigid mini-protein based on the MPER and an adjuvant/antigen-delivery platform that can elicit BR-Nabs in rabbits.

Methods: We generated a trimeric mini-protein that is structurally rigid, yet efficiently recognized by 2F5, 4E10 and Z13e1. It is based on M group consensus sequence and contains the C-terminal 54 a.a. of gp41 ectodomain (gp41-54Q), which includes the HR2 and the MPER. A 6xHis tag at the C-terminus was used to attach gp41-54Q to Zn-chitosan, which served as an adjuvant and antigen depot. Rabbits were immunized subcutaneously 3–4 times. Antibody responses were evaluated by ELISA and pseudovirus neutralization assays.

Results: The mini-protein was highly immunogenic, eliciting antibody titers greater than 1x10⁷. More importantly, antisera exhibited potent, broadly neutralizing activity against Tier 1 clade B and C viruses. Antisera also exhibited weak, but detectable, neutralizing activities against a multi-subtype panel of Tier 2 viruses. Results from a multitude of analyses suggested that we are eliciting a novel neutralizing antibody that is different from 2F5 or 4E10.

Conclusion: We have successfully generated a soluble, structurally rigid mini-protein containing gp41 MPER that is able to induce potent, BR-Nabs against HIV-1 in rabbits. These results represent a significant breakthrough in the HIV-1 vaccine field. Considering that the neutralizing antibody we are eliciting is different from 2F5 and 4E10, our study opens up a new avenue of AIDS vaccine research.

1 NYU School of Medicine, New York, USA 2 Molsoft, LLC, La Jolla, USA 3 Beth Israel Deaconess Medical Center, Boston, USA 4 University of Massachusetts Medical School, Worcester, USA OA10.02 Neutralizing Antibody Responses Induced with V3-Scaffold Protein Boosts Following Priming with gp120 DNA

Background: The V3 loop is one of the known targets for neutralizing antibody (NAb) responses, and V3-scaffold fusion proteins used as a boost after gp120 DNA priming were previously shown to induce NAbs in rabbits. In the current study, we evaluated whether the breadth and potency of NAb responses could be further improved when boosted with rationally designed and/or double combinations of V3-scaffold immunogens after gp120 DNA priming.

Methods: Rabbits were primed with codon-optimized clade C gp120 DNA and then boosted with one of five V3-cholera toxin B fusion proteins (V3-CTBs) or with various double combinations of these V3-CTBs. The V3 inserts in these V3-CTB protein immunogens were rationally designed to present epitopes shared by the majority of global isolates. Immune sera were assessed for (a) neutralization of Tier 1 and 2 pseudoviruses (psVs) infecting TZM.bl cells, and (b) neutralization of PBMC-grown primary isolates infecting TZM-bl cells, the latter being a far less sensitive assay than the former.

Results: The V3_C-CTB immunogen gave the most broad and potent neutralizing Ab response of the five V3-CTB immunogens tested individually. Generally, the double combinations of V3-CTB boosts gave better breadth and/or potency than did boosts with single V3-CTBs. With the double combination boosts, neutralization was achieved against 9/9 Tier 1 psVs from clades AG, B and C, and against 7/14 psVs from the clade B and C standard psV panels. Similarly, neutralization was achieved with 7/15 primary isolates from clades A, AG, and B.

Conclusion: The data demonstrate that priming with gp120 DNA followed by boosts with V3-scaffold immunogens effectively elicits cross-clade NAbs.

1 Faculty of Medicine of the University of Gent, Gent, Belgium 2 CEVAC, Ghent University & Hospital, Gent, Belgium 3 CEVAC – Ghent University & Hospital, Gent, Belgium 4 Kinesis Pharma B.V., Breda, Netherlands 5 Pevion Biotech Ldt, Ittigen, Switzerland 6 Pevion Biotech Ltd, Ittigen, Switzerland 7 Chimera Biotec, Dortmund, Germany 8 INSERM UMR U634, Faculté de Médecine Pasteur, Nice, France 9 Fondazione Centro San Raffaele del Monte Tabor Immunbiology of HIV, Milan, Italy 10 Institut Cochin, CNRS UMR8104/ INSERM U567, Paris, France 11 Institut Cochin CNRS UMR8104/ INSERM U567, Paris, France 12 Mymetics Corporation, Epalinges, Switzerland OA10.03 Phase I “Proof of Principle”: Immunization with Virosome-Gp41-Derived Antigen Induces Mucosal Antibodies with Antiviral Properties

Background: The ideal prophylactic vaccine against HIV-1 sexually transmitted should prevent the entry and early infection of HIV-1 at mucosal sites. We have previously demonstrated that mucosal IgAs/IgGs induced by vaccination with influenza virosomes harboring HXB2 rgp41 and modified-P1, a lipopeptide containing the MPER and the galactosyl ceramide mucosal receptor binding motif, protect non-human primates (NHP) against vaginal heterologous SHIV challenges, in the absence of seric neutralizing antibodies. Correlation was observed between induction of HIV-1 transcytosis-blocking and ADCC activities from cervico-vaginal antibodies and protection. We have then investigated if mucosal antibodies with similar antiviral properties could be induced in women during a Phase I trial.

Methods: This is a double-blind, placebo-controlled Phase I study conducted at CEVAC (Belgium), involving 24 healthy women randomized in 2 Panels to monitor safety, tolerability and immunogenicity of the vaccine MYM-V101 (virosomes-lipopeptides P1): Panel 1: 10 ug/dose and Panel 2: 50 ug/dose. In each Panel, 8 subjects received the vaccine and 4 subjects received the placebo (virosomes without HIV-1 antigens) through intra-muscular (weeks 0/8) and intra-nasal (weeks 16/24) administrations.

Results: The vaccine MYM-V101 is considered safe and well-tolerated for both Panels, when injected intramuscularly and intransally. All vaccinated subjects have rapidly developed within two months lipopeptide specific serum antibodies. The presence of lipopeptide-P1 specific mucosal IgG and IgA antibodies in the vaginal and rectal secretions was also confirmed for the majority of the samples tested (ImperacerTM technology). Tested vaginal antibodies from the lipopepetide-P1 vaccinated subjects exhibited strong inhibition of HIV-1 transcytosis, as compared to no or weak inhibition with the placebo, confirming previous results from NHP. However, no significant neutralizing activities could be detected in the mucosal samples. We also confirm that MYM-V101 don't induce a Th1 response.

Conclusion: This study confirms the safety profile of virosomes and the promising anti-HIV-1 mucosal responses elicited by gp41-derived virosomal vaccine.

1 National Cancer Institute at Frederick, Frederick, USA 2 Inovio Pharmaceuticals, Inc., Blue Bell, USA 3 SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, USA 4 Yerkes National Primate Research Center, Atlanta, USA 5 GeoVax, Inc., Smyrna, USA 6 National Institutes of Allergy and Infectious Diseases, Bethesda, USA 7 Institute of Human Virology, University of Maryland School of Medicine, Baltimore, USA 8 National Cancer Institute, Bethesda, USA 9 Duke University Medical Center, Durham, USA OA10.04 DNA and Protein Vaccination Confers Protection Upon Mucosal Challenge with Heterologous SIVsmE660

Background: Macaques vaccinated by intramuscular electroporation with optimized plasmid DNAs expressing SIVmac239 antigens develop potent immune responses able to reduce viremia of the high dose SIVmac251 challenge. To further improve immune responses and protection, protocols combining DNA with protein immunization (coimmunization, prime-boost) were tested.

Methods: Indian rhesus macaques (N = 8/group) were vaccinated using IM-electroporation by (a) SIVmac239 DNAs only, (b) coimmunization of DNAs with protein (AT-2-inactivated particles) or (c) 2 DNA vaccinations followed by 2 protein boosts (weeks 0, 8, 16 and 36). The animals were challenged weekly with low dose heterologous SIVsmE660 7 months after the last vaccination.

Results: Co-delivery of SIV DNA and protein increased the magnitude, longevity, and avidity of humoral responses and the ability to cross-neutralize heterologous Envs. SIV-specific cellular responses were readily measured in blood and mucosal sites after the first immunization and remained high during the entire follow-up. Vaccinated macaques resisted infection by SIVsmE660 compared to naïve controls (p = 0.05; stratified for Trim5a genotype). Two of 8 DNA-protein coimmunized animals did not get infected after 14 exposures, while all controls were infected by 6 exposures. Vaccinees had significantly lower peak VL (1.7 log, p = 0.03) and 75% of vaccinees suppressed virus replication rapidly to undetectable levels, maintaining normal CD4 counts (40 weeks of follow-up). Virus acquisition correlated with antibody avidity to SIVsmE660; acquisition delay and control of viremia also correlated with the presence of vaccine-induced cellular immune responses (CD4+ EffectorMemory T-cells and IFN + GranzymeB + cytotoxic T-cells).

Conclusion: The vaccine combining DNA and protein in a single administration is a novel concept to achieve rapid, high, persistent, broad and effective immune responses, and captures the best properties of both procedures: high and broad range of cellular immune responses produced by DNA and strong antibody responses produced by protein immunization. This combination is able to prevent infection at the portal of entry.

1 IAVI/TSRI, La Jolla, USA 2 IAVI, La Jolla, USA 3 International AIDS Vaccine Initiative, La Jolla, USA 4 International AIDS Vaccine Initiative/The Scripps Research Institute, La Jolla, USA OA10.05 Direct Antibody Access to the HIV-1 MPER Positively Correlates with Neutralization Sensitivity

Background: The precise kinetics of neutralization by the gp41 membrane proximal external region (MPER)-directed broadly neutralizing antibodies, 2F5 and 4E10, remains unresolved. Binding data to cell-surface Env and entry data using primary isolates suggests inaccessibility of the 2F5 and 4E10 epitopes on the viral spike prior to receptor engagement, but trimer gel shift analysis and recent slow kinetic shedding data indicate otherwise. Therefore, it remains unclear if the epitopes themselves are formed (or at least sampled) prior to receptor/co-receptor engagement, or if receptor interactions both form and expose the 2F5 and 4E10 epitopes, presumably in the putative pre-fusion transitional intermediate.

Methods: Here, we performed antibody-virus “washout experiments” using both lab-adapted and a diverse panel of clade B primary isolates and one clade C isolate to analyze MPER accessibility.

Results: The neutralization activity of 2F5 and 4E10 against lab-adapted viruses and a few sensitive and moderately resistant viruses was largely unaffected by relatively rapid antibody-virus washing, suggesting direct interaction with the static spike. However, for more neutralization resistant viruses, the 2F5 and 4E10 antibodies could neutralize only in the “no antibody-virus wash” conditions, implying that the MPER epitopes were not accessible prior to receptor engagement on these isolates. In fact, accessibility in the washout conditions could be precisely predicted by the relative resistance to neutralization in a standard neutralization format.

Conclusion: These data are consistent with a model in which the local MPER antibody epitope conformations may be sampled on the native spike, but are occluded to antibody by local steric or distal quaternary constraints adopted by highly resistant HIV-1 isolates.

1 Fred Hutchinson Cancer Research Center, Seattle, USA 2 Emory University, Atlanta, USA 3 Novartis Vaccines and Diagnostics, Cambridge, USA 4 University of Rochester, Rochester, USA 5 University of Alabama at Birmingham, Birmingham, USA 6 Geovax, Atlanta, USA 7 Vaccine Research Center, Bethesda, USA 8 Duke University, Durham, USA 9 Ministry of Public Health, Nonthaburi, Thailand 10 US Military HIV Research Program, WRAIR, Rockville, USA OA10.06 A DNA Prime/Protein Boost Vaccine Leads to Higher B-Cell Responses than a Vector Prime/Protein Boost or DNA Prime/Vector Boost Regimens

Background: Reduced risk of HIV acquisition in RV144 vaccinees in the absence of strong T-cell mediated immunity suggests that protection may be linked to HIV-specific antibodies. We assessed memory B-cell responses to Envelope proteins in RV144 and compared these to responses elicited by other vaccines.

Methods: Memory B-cell responses were measured by ELISpot using Env proteins matched to the RV144 protein boosts (gp120_A244, gp120_MN), a consensus (gp140_CON-S) and an unrelated clade B protein (gp140_BaL). Samples from HVTN 049 (3xDNA + 2xprotein, n = 20), 055 (5xMVA, n = 14), 065 (3xMVA, n = 20 or 2xDNA + 2xMVA, n = 20), 068 (2xAd5, n = 13) and 204 (3xDNA + 1xAd5, n = 19) were compared to RV144 (2xALVAC + 2xALVAC/2xprotein, n = 40); samples obtained at baseline and 2–4 weeks after the last boost of each trial were analyzed. Responses are reported as %Env-specific IgG-producing B cells using total IgG-producing B cells as denominator. No false-positive responses were detected at baseline using 3xbackground (KLH) and >20 spot-forming cells/million as threshold for positivity.

Results: Gp140_CON-S responses were detected in 98% of participants post-boost, but magnitudes varied significantly between trials (median 0.18%–3.65% Env-specific IgG-producing B cells), with MVA-vectored trials showing low and HVTN049 the highest magnitude. RV144 responses were intermediate, being significantly lower than HVTN049 (p < 0.0001) but higher than HVTN055 (p < 0.0001). Ad5-vectored vaccines induced responses of similar (HVTN204) or significantly higher (HVTN068, p = 0.008) magnitude than RV144. Responses in HVTN049 were stronger than in all other trials (p < 0.0001 except HVTN068, p = 0.09). Response rates were lower for other proteins but magnitude patterns were similar to those for gp140_CON-S, with RV144 showing a significantly lower magnitude than HVTN049 even for clade AE gp120_A244 (p = 0.003).

Conclusion: Although strong antibody responses were observed in RV144, memory B-cell responses in this trial are not outstanding in frequency or magnitude compared to other Env-containing regimens, while the clade B DNA prime/protein boost vaccine in HVTN049 generated strong cross-clade memory B-cell responses.

1 University of Minnesota, Minneapolis, USA 2 University of Nebraska, Lincoln, USA OA11.01 Microarray Analysis of Cervical Tissue Reveals a Transcriptional Imprint that Correlates with Protective Immunity in SIV-ΔNef-Vaccinated Animals

Background: The long-term goal for an efficacious HIV vaccine is to provide sterilizing protection from HIV infection. Thus far, this scenario has only been achieved experimentally using live-attenuated SIV vaccines. As such, a great deal of interest lies in identifying correlates of protection from a successful host response to pathogenic SIV. To this end, we have used a global genomics approach to examine gene expression in the female reproductive tract (FRT) of animals exposed intra-vaginally to WT virus (SIVmac251) and compared this environment to animals first vaccinated with the live-attenuated virus known as SIV ΔNef and then challenged intra-vaginally with SIVmac251.

Methods: RNA from cervical tissue was purified for microarray analysis. The RMA algorithm was used to quantify signal levels for all probe sets and weighted least squares was used to test for differences between groups. Genes were selected based on associated q-values and then functionally classified/annotated while protein expression determined using single-cell analytical procedures for tissue sections.

Results: Three major themes emerged from this analysis differentiating the response of vaccinated from unvaccinated animals during virus infection: 1) dampened innate immunity through decreased expression of innate sensors (ex.: MDA5) and increased expression of innate-signaling inhibitors (ex.: PCBP2, ATF7); 2) retarded inflammatory cell infiltration into the FRT of vaccinated animals, coinciding with decreased expression of chemotactic signals (ex.: MIP-1, 3α) and increased expression of inhibitors of pro-inflammatory signaling (ex.: RORA, SPRED1, COMMD10); 3) preponderance of gene expression associated with antibody diversification, maturation, and production (ex.:MALT1, IGIP, POLH, ITPKB, RAD52), coinciding with increased plasma cell concentration and immunoglobulin levels in the FRT.

Conclusion: This analysis provides both a comprehensive picture of a “protective” environment in the FRT and tantalizing clues regarding properties of a successful host response to immunodeficiency virus infections (ex.: on-site generation/diversification of antibody responses and dampened inflammation associated with innate signaling).

1 NEPRC, Harvard Medical School, Southborough, USA OA11.02 Gut Inflammation and Indoleamine Deoxygenase Inhibit IL-17 Production and Induce Cytotoxicity in RORγt+NKp44+Mucosal NK Cells During SIV Infection

Background: Lentiviruses primarily replicate in gut mucosa and cause inflammation and loss of epithelial integrity. Recent studies have identified a novel mucosal NK cell population secreting the TH17 cytokines, IL-17 and IL-22, which are critical for gut homeostasis. However, the effects of lentivirus infection on these unique NK cells are unknown.

Methods: Mucosal NK cells, from naive and SIV-infected macaques, were enumerated by a novel bead-based flow cytometry assay, analyzed phenotypically by polychromatic flow cytometry, and evaluated functionally by ICS. Expression analyses of transcription factors and cytokine mRNA in sorted NK cells and whole biopsy pieces were performed by RT-PCR.

Results: We identified two lineages of NK cells characterized by dichotomous expression of the NK cell-related markers, NKG2A and NKp44. Both cell types expressed the NK cell-related transcription factor, NFIL3, but NKp44+ NK cells also expressed the TH17 transcription factor, RORγt. Unlike systemic classical NKG2A+ NK cells, NKp44+ NK cells were mucosae-restricted, produced IL-17 and IL-22, but were noncytotoxic. During SIV infection, NKp44+ NK cells were depleted ∼9-fold and had decreased IL-17 secretion, increased IFN-γ secretion, and surprisingly acquired cytotoxicity. NKp44+ NK cells showed no evidence of direct SIV infection in vivo, but depletion and altered function of NKp44+ NK cells were associated with SIV-induced upregulation of inflammatory mediators in the gut, including IDO-1. Furthermore, treatment of NKp44+ NK cells with IDO-1 catabolites in vitro ablated IL-17 production.

Conclusion: SIV infection results in massive depletion of NKp44+ NK cells in the gut and reshapes their functional repertoire, not by direct infection, but by indirectly altering the gut cytokine milieu. Due to the critical role of IL-17 and IL-22 in maintaining gut homeostasis, the loss of the NKp44+ NK cell subpopulation is likely to contribute to loss of gut integrity and subsequent microbial translocation and chronic immune activation in lentivirus infections.

1 National Cancer Institute, National Institute of Health, Bethesda, USA 2 Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, USA 3 California National Primate Research Center, University of California, Davis, USA 4 AIDS and Cancer Virus Program SAIC-NCI-Frederick, Frederick, USA 5 Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, USA 6 Immune Biology Retroviral Infection Section, National Cancer Institute, Bethesda, USA 7 Vaccine Research Center, National Institute of Allergy and Infectious, Bethesda, USA OA11.03 Targeting the Vaginal Mucosa with Human Papillomavirus Pseudovirion Vaccines Delivering SIV DNA

Background: Most HIV transmission occurs by the genital route, suggesting that vaccines that target the genital mucosa and induce local B- and T-cell immune responses may offer better protection from infection than vaccines that induce mainly systemic responses.

Methods: We tested a novel vaccine delivery platform that specifically targets the vaginal mucosa. Human Papillomavirus (HPV) capsid proteins, L1 and L2, can self assemble into virus like particles (VLPs), and when co-transfected with a plasmid containing a gene of interest, L1 and L2 will encapsidate the gene and form pseudovirions (PsV). We generated HPV PsVs vaccines that deliver SIV genes to the female genital tract and designed feasibility and vaccine protection studies, using the SIVmac251 macaque model.

Results: In the feasibility study, an HPV PsV Gag vaccine induced SIV-specific IgG and IgA in vaginal secretions and SIV-specific T-cell responses in the cervicovaginal tract of macaques. Importantly, HPV PsV- induced mucosal T-cell responses that rapidly expand upon vaginal exposure of a subset of animals to high doses of SIVmac251. To evaluate relative vaccine efficacy, a cohort of macaques have been immunized using HPV PsVs, delivering SIV Gag-pro, and env genes, as well as a re-assorted gene encompassing the Tat, Nef, and Rev genes (Retanef). The animals were then boosted, with recombinant gp120 protein. The efficacy of a systemic prime and mucosal boost vaccine regimen is also being evaluated by combining the canarypox vector ALVAC and HPV-PsV. At the end of the immunization regimen (June 2011), these animals will undergo repeated low-dose intravaginal challenges with SIVmac251 to mimic HIV transmission among humans.

Conclusion: The HPV PsV system is a novel approach for direct genetic vaccination of the vaginal mucosa and is immunogenic in macaques. SIV challenge data to determine whether this combination of vaccine modalities will afford significant protection from SIVmac251 infection will be presented.

1 Beth Israel Deaconess Medical Center, Boston, USA 2 New England Primate Research Center, Southborough, USA 3 Ragon Institute of MGH, MIT and Harvard, Boston, USA OA11.04 Durability and Phenotype of SIV-Specific Mucosal T Lymphocyte Responses Elicited by Recombinant Adenovirus Vectors in Rhesus Monkeys

Background: The induction of durable and potent mucosal cellular immune responses may be critical for the development of effective vaccines against HIV-1 and other pathogens. A variety of vaccine vectors have been reported to elicit HIV-1/SIV-specific cellular immune responses at mucosal sites, but the anatomic distribution, kinetics, durability and phenotype of these responses remain poorly characterized.

Methods: Rhesus monkeys were immunized with intramuscular injection of (i) single, (ii) homologous prime-boost, or (iii) heterologous prime-boost regimen with recombinant adenovirus (rAd) vectors expressing SIVmac239 Gag. Magnitude and phenotype of SIV-specific T lymphocytes were evaluated with multiparameter tetramer staining assays and multiparameter intracellular cytokine staining (ICS) assays.

Results: Intramuscular injection of rAd vectors in rhesus monkeys induced durable SIV-specific T lymphocyte responses that persisted for over 2 years in multiple mucosal tissues, including colorectal, duodenal and vaginal biopsies as well as bronchoalveolar lavage (BAL). In peripheral blood, SIV-specific T lymphocytes underwent a phenotypic evolution from effector memory (EM) and transitional memory (TM) cells to central memory (CM) cells following vaccination. In contrast, mucosal SIV-specific T lymphocytes exhibited a persistent TM phenotype as well as persistent activation characterized by CD69 expression.

Conclusion: These data demonstrate that rAd vectors induce durable and widely distributed mucosal T lymphocyte responses that are phenotypically distinct from peripheral T lymphocyte responses. These findings suggest that vaccine-elicited T lymphocytes acquire qualitative features of target tissues following trafficking to mucosal sites which may allow them to respond rapidly upon mucosal viral challenge.

1 University of Cape Town, Cape Town, South Africa 2 Department of Pediatrics, Seattle Children's Hospital, UW, Seattle, USA 3 School of Public Health and Family Medicine, UCT, Cape Town, South Africa OA11.05 Role of Genital Tract Inflammation and T Cell Activation in CD4 Depletion at the Female Genital Tract During HIV Infection

Background: The micro-environment within the female genital tract has a strong influence on both the acquisition and transmission of HIV-1 during penetrative sex. Although immune activation in response to invading pathogens is a crucial to protective immunity, such responses may ironically also contribute to HIV pathogenesis by providing the virus with a steady supply of susceptible target cells. This study investigated the role of inflammation and immune activation in CD4 depletion and HIV shedding in the female genital tract associated with HIV pathogenesis.

Methods: Cervical cytobrush and blood-derived CD8+ and CD4+ T cells from 33 HIV+ and 40 HIV- women were investigated for expression of activation markers CCR5, HLA-DR, CD38 and Ki67 by flow cytometry. Concentrations of inflammatory and regulatory cytokines (eotaxin, G-CSF, IL-1α, IP-10, MIP-1α, IL-6, IL-10, IL-8 and RANTES) in cervical supernatants and blood plasma were determined by Luminex. HIV loads were measured in cervical secretions and plasma.

Results: The frequency of activation marker expression in blood was significantly predictive of genital mucosal T cell activation in HIV-infected women. Importantly, cervical T cell activation was broadly associated with mucosal HIV shedding and the extent of CD4 depletion in the genital tract during HIV infection. HIV-infected women displayed greater levels of T cell immune activation in the blood and cervix than uninfected women, and had significantly elevated frequencies of activation in the cervix than in blood. IL-8 and G-CSF levels were consistently greater at the cervix than blood. Genital tract concentrations of the inflammatory chemokine RANTES correlated positively with local CD38+ and HLA-DR+ T cell frequencies, with local HIV shedding, and negatively with CD4 depletion.

Conclusion: We conclude that the interplay between mucosal inflammation and local immune activation is an important contributor to CD4 depletion and the degree of HIV shedding in the female genital tract during HIV infection.

1 Prime-boost HIV vaccine phase III trial, Nonthaburi, Thailand 2 US Military HIV Research Program, Rockville, MD, USA 3 Chonburi Regional hospital, Chonburi, Thailand 4 SCHARP, Seattle, WA, USA 5 Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand 6 Department of Retrovirology, USAMC-AFRIMS, Bangkok, Thailand 7 MHRP/AFRIMS, Bangkok, Thailand 8 GSID, San Francisco, CA, USA 9 NIAID, NIH, Bethesda, MD, USA 10 sanofi pasteur, Swiftwater, PA, USA 11 Royal Thai Army, AFRIMS, Bangkok, Thailand 12 Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand 13 US Military HIV Research Program, Rockville, MD, USA OA01.06 LB Extended Evaluation of Volunteers Who Become HIV-1 Infected During Participation in a Phase III Vaccine Trial of ALVAC-HIV and AIDSVAX^® B/E

Background: The Thai Phase III trial of ALVAC-HIV^® and AIDSVAX B/E^® was the first study to show modest efficacy for prevention of HIV infection in a community-based cohort. Of the 16,402 volunteers enrolled in the study, 132 became HIV-infected during the trial and 120 of these volunteers agreed to participate in RV152 to evaluate the effect of this prime-boost regimen on clinical disease progression as well as immunologic and virologic outcomes. The primary objective of the study was to evaluate differences among vaccine and placebo recipients in a composite endpoint comprised of clinical (AIDS-defining events, initiation of antiretroviral therapy) and laboratory (CD4-count <350 cells/microliter) events.

Methods: Volunteers who became HIV infected in RV 144 were followed every 3 months with clinical and CD4 and HIV-1 viral RNA assessments. Both study volunteers and investigators remained blinded over the entire study period. Volunteers received HIV care and treatment according to the Thai Ministry of Public Health National Guidelines. Both modified Intent-to-Treat (mITT) and per-protocol (PP) analyses were performed using a time-to-event model based on a Cox proportional hazards. Secondary analyses of biomarker-based endpoints were assessed using marginal mean models fit by Generalized Estimating Equations and linear mixed models.

Results: There were 48 PP and 61 mITT events among the 120 enrolled volunteers at the pre-specified, event-driven stopping point.

Conclusion: Results of the study will be presented and discussed to include estimates of vaccine efficacy for disease progression as well as longitudinal comparison of biomarkers.

1 Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand 2 Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA, USA OA02.06 LB JAK3 Inhibition In Vivo in Chronically SIV Infected Rhesus Macaques Deplete NK-17 Cells

Background: During simian immunodeficiency virus (SIV) infection, the gut-associated lymphoid tissue (GALT) is the primary target of virus replication during acute infection. While NK cells and IL-17 producing NK cells (NK-17) have been shown to play a key role in not only maintaining epithelial cell integrity of the GALT but also causing cytotoxicity for SIV infected cells. Their role during chronic infection is not clear. The purpose of this study was to determine the effect of NK cell depletion using a JAK3 inhibitor in chronically SIV infected rhesus macaques.

Methods: Six chronic SIV-infected rhesus macaques were daily orally administered a pre-determined optimal dose of 10 mg/kg of a JAK3 inhibitor for 5 weeks. Blood and colorectal biopsies were collected. Cells were stimulated with PMA and ionomycin in the presence of brefeldin-A. The cells were stained with various fluorescent conjugated monoclonal antibodies and analyzed on LSRII flow cytometer. The plasma viral loads were also monitored.

Results: Total NK cell and NK-17 in blood and biopsies were markedly depleted as early as the first week and the depletion persisted during the course of drug administration. While the total NK cells in blood returned to normal levels within 3 weeks after treatment cessation, the total NK cells in biopsies did not. The NK-17 cells in the blood and biopsies also did not return to normal levels after cessation. Drug treatment led to a transient increase in plasma viremia during the loss of total NK cells.

Conclusion: The administration of a JAK3 inhibitor to chronically SIV infected monkeys depletes total and NK-17 in both the blood and mucosal tissue and is associated with increases in viremia denoting an important role for NK cells during chronic SIV infection.

1 University of California Davis, Davis, CA, USA 2 CEA, Gif-sur-Yvette, France 3 Novartis, Inc., Cambridge, MA, USA 4 National Institutes of Health, Gaithersburg, MD, USA OA03.05 LB Role of Hypervariable Loops in Intersubunit Associations of Env: Trimeric Gp140 Immunogen as a Platform for Novel Epitope Display

Background: The HIV-1 Env complex mediates membrane fusion with host cells following interaction with both a receptor and a co-receptor. The Env subunit gp120 binds to CD4 receptors, resulting in cryptic epitope exposure and allowing coreceptor binding (see our recent structure of Env gp140 trimer, Moscoso et al., 2011, PNAS 108(15):6091–6096). Some hypervariable loops on gp120, primarily V2 and V3, seem to play roles in the conformational transition to the induced, exposed coreceptor state.

Methods: Here, we perform further comparative analysis of variants of the soluble Env construct gp140, with and without a partial V2 truncation, using cryoelectron microscopy and single particle reconstruction.

Results: The Env structure displays a similar overall architecture with crucial detailed distinctions, one of them being a tighter association between gp120 subunits that results in a smaller trimer radius. Another pertinent feature is the presence of V2 near the base of the trimer proximal to the viral membrane, as suggested previously, placing V2 in proximity to the adjacent counterclockwise gp120 subunit. The juxtaposition of V2 and the neighboring gp120 subunit's V3 loop (V3’) contribute to a quaternary epitope formed by these two loops, which is the binding site for antibodies such as PG16 and 2909. Such a quaternary location of V2 would facilitate gp41-independent gp120-gp120 interactions, and hints at a mechanism of cooperative epitope occlusion mediated by hypervariable loop interaction, a decrease in trimer diameter and increased steric hindrance near the CD4 binding sites due to the decreased distance between adjoining CD4 epitopes.

Conclusion: Comparative structural analysis of these recombinant vaccine candidates informs rational design of strategies towards an immunogen with conserved structural features based on the native form of Env trimers, previously missed in monomeric gp120 vaccine efforts, to maximize exposure of novel epitopes and thus to elicit broadly and potently neutralizing antibodies, such as VRC01.

1 AIDS Virus research Unit, NICD, South Africa, Johannesburg, South Africa 2 University of Cape Town, Cape Town, South Africa 3 CAPRISA, University Of KwaZulu Natal, Durban, South Africa OA04.07 LB Evolution of HIV-1 Transmitted/Founder Viruses Results in the Formation of Epitopes for Later Broadly Cross-Neutralizing Antibodies

Background: Much emphasis has been placed on mapping the epitopes of broadly cross-neutralizing (BCN) antibodies. However, the interplay between strain-specific antibodies, BCN antibodies and autologous viral evolution has not been well characterized.

Methods: Single genome amplification was used to obtain longitudinal autologous envelope sequences from three individuals (CAP177, CAP8 and CAP256) in the CAPRISA 002 cohort who developed BCN antibodies with known specificities (Gray et al, JV, 2011 and Moore et al, JV, 2011). The sequences were then analysed to determinewhether previously defined BCN epitopes were present on the transmitted/founder viruses, and to characterize their evolution over time.

Results: CAP177 developed BCN antibodies dependent on a glycan at position 332 in the C3 region. This N332 glycan was however not present on the CAP177 transmitted/founder virus, but arose by 6 months post-infection as a result of escape from an early strain-specific antibody targeting the alpha-2 helix of the C3 region. Similarly in CAP8, who developed BCN antibodies dependent on the N160 glycan, the transmitted founder virus did not encode the N160 glycan, but instead contained a K160 residue. The N160 glycan evolved in CAP8 by 6 months of infection and became fixed. In a third individual, CAP256, who also developed extremely potent N160-dependent BCN antibodies, the transmitted/founder virus lacked this residue. However, superinfection at 15 weeks post-infection with a second virus that contained the N160 glycan resulted in the generation of recombinant viruses that harbored the N160 glycan forming the CAP256 BCN epitope.

Conclusion: In three individuals who developed BCN antibodies, the BCN epitope was not present on the transmitted/founder virus, but evolved within 6 months of infection. In one case, the BCN epitope evolved via viral escape from an earlier strain-specific antibody targeting the same region. These studies highlight the role of viral evolution in shaping the development of BCN antibody responses.

1 AFRIMS, APO, AP, USA 2 U.S. Military HIV Research Program, Rockville, MD, USA OA07.08 LB The Thai Phase Iii Clinical Trial (RV144) Induces the Generation of Antibodies That Target a Conserved Region Within the V2 Loop of GP120

Background: The Thai phase III clinical trial (RV 144) is the only HIV-1 vaccine regimen to show efficacy against acquisition of HIV-1. Antibodies targeting HIV-1 ENV may contribute to preventing infection. We compared the HIV-1 humoral responses induced by the vaccine to those by natural HIV-1 infections.

Methods: Antibody responses to gp120 HIV-1 were evaluated using cyclic peptides spanning 42 amino acids (aa) of the V2 and 35 aa of the V3 loop of the ALVAC (vCP1521) Env gp120. Plasma from 40 HIV-1 uninfected trial participants (32 vaccinee/8 placebo recipients) that completed all injections with ALVAC-HIV/AIDSVAX B/E boost combination and 20 HIV-1 CRF01_AE infected subjects were tested by ELISA.

Results: 31/32 (97%) vaccine recipients had antibody responses against V2 at 2 weeks post final immunization (week 26) with a geometric mean end point titer (GMT) of 1100 (range: 200–3200).V2 responses were transient, declining to 19% at 28 weeks post last injection (GMT: 110, range: 100–200). The majority of antibody responses targeted the crown of the V2 loop with aa sequence KQKVHALFYKLDIVPI which includes the α4β7 binding motif LDI that could bind HIV-1 at mucosal surfaces. Unlike the V2 responses, the frequency of V3 responses was modest (18/32, 56%) with a GMT of 185(range: 100–800) at week 26. In comparison, HIV-1 infected individuals had a lower frequency and magnitude of antibody responses to V2: (10/20, 50%) with a GMT of 400 (range: 100–3200) and a higher frequency and magnitude to V3 (19/20, 95%) with a GMT of 3570 (range: 200–12800).

Conclusion: The RV 144 vaccine regimen induced antibodies targeting a conserved region of the V2 loop, which includes the α4β7 binding motif LDI. These observations suggest that V2 antibodies may be a contributing factor in the prevention of HIV-1 acquisition.

1 Beth Israel Deaconess Medical Center and Harvard University, Boston, MA, USA 2 U.S. Military HIV Research Program (MHRP), Rockville, MD, USA 3 School of Mathematics, Statistics and Applied Mathematics,NUI, Galway, Ireland 4 Los Alamos National Laboratory, Los Alamos, NM, USA 5 The Statistical Center for HIV/AIDS Research and Prevention, Seattle, WA, USA 6 Vaccine Research Center, NIAID, Bethesda, MD, USA 7 Vaccine Research Center (NIAID), Bethesda, MD, USA 8 School of Mathematics, Statistics and Applied Mathematics, Galway, Ireland OA08.08 LB Reduction of Founder Virus in Vaccinated Monkeys After Mucosal SIV Challenge

Background: A key criteria in the NHP evaluation of candidate vaccines is determining the presence of any vaccine-induced effects on the genotype or number of founder strains acquired during repeated low-dose mucosal challenges. We have undertaken studies in a cohort of 167 monkeys to determine the presence of any vaccine-induced “sieving” effects, or differences in minimum number of SIV founders initiating infection in vaccinated or sham-control animals.

Methods: Methods: 167 rhesus monkeys received either an experimental DNA prime Ad5 boost regimen, or sham vaccination. All monkeys were challenged intra-rectally with a repeat low-dose SIVE660 or SIVmac251 challenge 40 weeks after the last vaccination. We amplified the envelopes of blood-associated SIVE660 at the earliest point after viral infection using single genome amplification. Full-length SIV envelope sequences were subject to extensive bio-informatic and statistical analysis to evaluate the minimum number of virus acquired during the infection of each monkey, and if vaccine-induced sieving effects were present.

Results: Results: Overall, evidence of vaccine-induced sieving was observed in virus sequences derived prior to the peak of virus replication. However, this sieving was most pronounce in monkeys expressing the Mamu A*01 allele. We also observed a significant reduction in the minimum number of founder virus initiating infection in vaccinated monkeys with permissible host genotypes. We also observed altered levels of mutation in virus sequences isolated from vaccine recipients.

Conclusion: Conclusions: Evidence of vaccine-induced effects can be observed at the earliest times after virus challenge and can impact viral diversity post acquisition. Importantly, vaccination impacts the number of acquired viral lineages, but significant levels of specific immunological “sieving” was observed only in a subgroup of vaccinees. These data highlight non-specific immune mechanisms of vaccine-interrupted virus acquisition. Moreover, the inclusion of a quantitative endpoint for founder/transmitted virus should be considered for future evaluations of candidate vaccine efficacy.

1 University of Minnesota, Minneapolis, MN, USA 2 University of Nebraska, Lincoln, NE, USA 3 National Cancer Institute, Frederick, MD, USA 4 ZeptoMetrix Corporation, Buffalo, NY, USA 5 Harvard Medical School, Southborough, MA, USA 6 Harvard Medical School and Ragon Institute, Southborough, MA, USA OA11.06 LB SIV Attenuated Vaccine-Induced Plasma Cells and Antibodies Limit Infection at the Portal of Entry and Systemically Following Vaginal Challenge

Background: In the SIV rhesus macaque model of HIV-1 transmission to women, vaccination with SIVmac239-delta-nef confers partial or complete protection against vaginal challenge with SIVmac251 by as yet unknown mechanisms.

Methods: To identify potential protective mechanisms against vaginal transmission, we analyzed host responses in cervical-vaginal and lymphatic tissues (LTs) of Indian rhesus macaques following intravenous SIV delta-nef infection (vaccine phase), and following vaginal challenge with SIVmac251 (Challenge phase).

Results: We found that vaccine induces a significant increase in SIV-specific antibodies and plasma cells and IgG localized beneath the cervical-vaginal epithelium and the development of ectopic lymphoid tissue follicles within cervical-vaginal tissues. Within the first week after heterologous vaginal challenge of SIVmac251, there is a rapid increase in the number of IgG producing plasma cells, concentration of anti-Env antibody and the breadth of antibody in vaginal-cervical mucosal sites in the vaccinated rhesus macaques, which correlates with the local expansion and maturation of ectopic lymphoid follicles within vaginal tissues in which there are plasmablasts and/or memory B cells with evidence of AID-mediated class-switch recombination. The high concentrations of antibodies and plasma cells correlate with as much as a 6-order of magnitude reduction of tissue viral load at the portal of entry. There is a similar rapid increase in plasma cells in the lymphoid tissues that correlates with a rapid response to disseminated infection that is also associated with major reductions in viral replication at these sites.

Conclusion: These data suggest a mechanism by which a humoral response at the “right place, right time” may contribute to the protection, a concept relevant to developing effective vaccines against HIV-1 and other mucosal pathogens.

1 University of Washington, Seattle, USA 2 University of California Los Angeles, Los Angeles, USA 3 Statistical Center for HIV/AIDS Research & Prevention (SCHARP), Seattle, USA 4 Department of Microbiology, University of Washington, Seattle, USA 5 Department of Medicine, University of Washington, Seattle, USA 6 Vaccine & Infectious Disease Div, Fred Hutch Cancer Research Center, Seattle, USA 7 University of Washington and Seattle Biomedical Research Institute, Seattle, USA P01.01 Analysis of Epitope-Specific HIV T Cell Responses During Early HIV-1 Infection and Their Association with Viral Control

Background: HIV-1-specific cytotoxic T lymphocytes (CTL) are important in the control of viremia; however, the precise qualities of effective epitope-specific T cell responses that may be responsible for control remain unclear. Here we investigated the frequency and specificities of T cell responses during early HIV-1 infection, to address the question of whether conservation score (CS) of targeted epitopes play an important role in viral control.

Methods: Using IFN-γ ELISpot assays, we mapped epitope specificities recognized by HIV-specific T cells in 12 ART-naïve individuals during early infection (within 6 months). We then identified CS of targeted epitopes, where the CS is defined as the proportion of HIV-1 group M amino acid sequences in the LANL database that include the epitope. We further evaluated the association between the CS of the targeted epitopes and the individuals' viral-load.

Results: Our epitope mapping of T cell responses showed that the median number of epitopes targeted was 9 (range, 5–24) mostly against Gag, Nef, Env and Pol. More than one third of these epitopes were novel epitopes. In addition, variable epitopes were targeted more frequently than conserved epitopes (p < 0.0001). The median CS of targeted Gag epitopes were more conserved than those of Pol, Nef, Env and Acc epitopes (p = 0.003). However, the magnitude of responses elicited by Gag epitopes was not significantly different than those elicited by Pol, Nef, Env and Acc epitopes. Furthermore, there was a significant inverse correlation between the rank of CS of targeted epitopes with the rank of viral-load (r = −0.76, p = 0.004), indicating that individuals that possess T cells targeting conserved epitopes early in infection had lower viral loads.

Conclusion: These data suggest that HIV-specific CTL targeting conserved epitopes of HIV-1 are superior at controlling viral replication in vivo, supporting the rationale for designing vaccines containing conserved immunogens.

This project is funded by NIH grant #R01AI090783.

1 University of Washington, Seattle, USA 2 U.S. Military HIV Research Program, Henry M. Jackson Foundation, Rockville, USA 3 Fred Hutchinson Cancer Research Center, Seattle, USA P01.02 Balance of CTL Escape and Viral Fitness in Acute and Early HIV Infection

Background: Investigations into host-virus interactions during acute HIV-1 infection are important for the design of a preventative HIV-1 vaccine. To understand the dynamic interplay between host CTL responses and the mechanisms by which HIV-1 evades those responses, we studied viral evolutionary patterns and host CTL responses in a linked transmitter:recipient pair.

Methods: Ten HIV-1 SGA sequences were amplified from the transmitter T3 near the time of transmission and from the recipient R3 at sequential visits from 25–247 days post-onset of symptoms (dps). A composite alignment of all sequences was generated to identify the amino acids (AA) sites that i) differed between T3 and R3 or ii) evolved over time in R3. To identify if CTL pressure promoted these AA changes, IFNγ ELISpot assays were performed on T3 and R3 using an autologous peptide set designed against the regions harboring evolving sites and including variants found in T3 and R3.

Results: We identified responses in R3 against wildtype and variant forms of peptides, sometimes at a time when the variant sequence had not yet been detected, suggesting that cross-reactive CTL failed to contain the early evolution of mutant viruses. In R3, CTL-mediated escape mutations were identified in the functionally constrained regions of the Gag-Pol frameshift stimulator loop and in the Env gp41 C-terminal Heptad Repeat. The virus appears to have exploited the limited availability of AA sites not required to maintain function in these regions in order to attain CTL escape.

Conclusion: Focusing immunological studies on viral regions under selection pressure allows for detailed investigations of host-virus interactions using limited numbers of PBMC. Comparison of viruses between the transmitter and recipient provides a better characterization of founder viruses' specificities. Identification of escape mechanisms allows further understanding of the challenges faced by the virus in achieving a balance of immunologic escape and replicative fitness costs.

1 SEARCH, The Thai Red Cross AIDS Research Centre, Bangkok, Thailand 2 Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand 3 US Military HIV Research Program, Rockville, USA 4 National Institute of Allergy and Infectious Diseases, Bethesda, USA 5 Division of Retrovirology, Armed Forces Research Institute of Medical, Bangkok, Thailand 6 Division of Retrovirology, AFRIMS, Bangkok, Thailand P01.03 Abnormal Liver Function During Acute HIV Infection Correlates with Fiebig Stage and Systemic Inflammation

Background: Abnormal liver function may present during acute HIV infection (AHI). Its pathogenesis is unclear but may be linked to systemic inflammation.

Methods: 30 Thai adults with Fiebig stages I to IV AHI were assessed at baseline for ALT, yGT, bilirubin, IP-10, MCP-1, TNFα, IL-22, IL-6 (n = 30) and markers of inflammation [CRP, d-dimer, hyaluronic acid (n = 19), sCD14 (n = 7)]. The % CD4+ CCR5+ cells in sigmoid tissue was determined by flow cytometry (n = 24).

Results: Twenty-six (87%) were male; the mean (SD) age was 29 (6.2) years; they were 8 Fiebig I; 5 Fiebig II; 14 Fiebig III and 3 Fiebig IV. Mean (SD) time from HIV exposure was 18 (9.1) days. Median (IQR) CD4 was 406 cells/mm3 (298–555) and HIV RNA was 5.5 (5.1–6.4) log10copies/ml. Twenty-six (87%) had ARS. Most common symptoms were fever (77%), myalgia (60%), fatigue (57%), and headache, pharyngitis, and oral ulcers (all 50%). Three had positive HBsAg and none had positive HCV Ab. Sixteen (53%) had abnormal ALT (>31 UL-1) and 10 (33%) had abnormal yGT (>43 UL-1); 2 of these had positive HBsAg (ALT 45 and yGT 49 respectively). Absolute ALT and yGT positively correlated with Fiebig stage [rho = 0.689 (p < 0.001) and rho = 0.518 (p = 0.003) respectively]. Abnormal ALT was more common in Fiebig III/IV compared to Fiebig I/II (OR = 13.2, p = 0.009). Both absolute and abnormal yGT correlated with TNF-α expression (both rho = 0.406, p = 0.026). Absolute ALT was positively correlated with IP-10 (rho = 0.403, p = 0.027), while abnormal ALT was correlated with IL-12 (rho = 0.375, p = 0.041). No relationship between ALT/yGT with gut CD4+ CCR5+ T cells or CRP, D-dimer, HA and sCD14 was seen.

Conclusion: ARS was common in Thais with AHI. Abnormal liver function was prominent and correlated with Fiebig stage and inflammatory cytokines levels, but not with gut T cell depletion. The clinical and prognostic implications of these findings require further study.

1 Faculty of Medicine Siriraj Hospital, Mahidol University and MHRP, Rockville, USA 2 MHRP, USA 3 AFRIMS, Bangkok, Thailand 4 WRP-Kenya, Kericho, Kenya 5 MMRP, Mbeya, Tanzania, United Republic of 6 MUWRP, Kampala, Uganda P01.05 Early Cytokine and Chemokine Patterns During Acute HIV Infection

Background: Understanding the earliest immunologic events during acute HIV infection is critical to inform prevention and treatment efforts. We focused on characterizing plasma cytokine profiles during the earliest stages of acute infections from subjects in the prospective RV217 cohort representing multiple HIV subtype infections (A, C, D and CRF-01).

Methods: Twice weekly nucleic acid testing (NAT) was used to prospectively identify subjects during very early HIV infection (NAT+, antibody-). Plasma cytokine levels were measured at baseline and at all available early time points throughout acute infection and early set point. Assays were performed in triplicate using the Quansys Q-Plex Multiplex IR ELISA Array. Images were captured using the LiCor IR imaging system and analyzed using Q-View Plus software. Data and statistical analysis were performed using GraphPad Prism.

Results: Longitudinal plasma samples acquired prior to EIA reactivity during the upslope in viral load from acutely infected individuals (n = 6) showed similar cytokine profiles. Plasma levels of the CC-chemokine MCP-1/CCL2 (6/6) and CXC-chemokine IP-10/CXCL10 (4/4) rose sharply during viral load ramp up. Conversely, a concomitant nadir in plasma IL-2 (5/6), often with a decrease in IL-17 (4/6) was observed. There were profound decreases in both peripheral absolute CD4 counts and B cells during these early days of viral expansion. Other cytokines were detected (IL-1β, IL-6, IL-10, IL-12p70, IL-15 and IFN-α) but showed more variability in expression patterns during early phases of infection.

Conclusion: Few studies have examined cytokine expression patterns prior to and during the acute phase of HIV infection. The early detection of MCP-1/CCL2 and IP-10/CXCL10 observed here, similar to a prior study in acute subtype B infections, may indicate early trafficking events and reflect involvement of specific cell types during acute infection. Falling levels of IL-2 and IL-17 could be linked to changes in frequency or redistribution of peripheral lymphocyte subsets.

1 Baylor Institute for Immunology Research, Dallas, USA 2 Ba, USA 3 Baylor Institute for Health Care Research and Improvement, Dallas, USA 4 Nationwide Children's Hospital Center for Vaccines and Immunity, Columbus, USA 5 Department of Medical Technology, Naresuan University, Phitsanulok, Thailand 6 Department of Clinical Immunology, Khon Kaen University, Khon Kaen, Thailand 7 MRC National Institute for Medical Research, London, England 8 INSERM U897, Bordeaux, France 9 University of North Carolina at Chapel Hill, Chapel Hill, USA 10 Duke University School of Medicine, Durham, USA 11 Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, England 12 Henri Mondor Hospital-ANRS, Creteil, France P01.06 Whole Blood Transcriptional Monitoring of Acute HIV-1 Infection Reveals Differential Signatures of Host Immune Activation

Background: Genome-wide transcriptional studies in non-human primates have suggested that the quality and magnitude of early immune responses to SIV infection are associated with downstream disease progression. The aim of this study is to identify, monitor, and interpret the whole blood transcriptional signature of acute HIV-1 infection (AHI).

Methods: A total of 56 AHI patients and matching healthy controls from both Africa and the United States were distributed into training, test, and validation datasets. Whole blood from these subjects was obtained for transcriptional profiling using microarrays. Both gene and modular transcriptional framework analyses were utilized to interpret the signature of AHI and for comparisons with other disease signatures including: influenza, tuberculosis, sepsis, and chronic HIV following interruption of anti-retroviral therapy (ART). Additional samples were collected at 1, 2, 4, 12, and 24 weeks post-enrollment for training and test set AHI subjects, to establish the longevity of the signature in the presence or absence therapy.

Results: We identified a robust AHI signature with increased activity in: interferon, cell cycle, cytotoxic, plasma cell, and B-cell modules amongst others. This activity was unique when compared to signatures obtained from patients with other infections. Only interferon and cell cycle activity was observed in both AHI subjects and chronically infected patients following interruption of therapy. Notably, 20% of AHI patients were observed to express a quiescent signature that clustered independent of the highly active signature described above. We found that these patients had significantly lower viral set-points (p = 0.01) when compared to patients with the active signature. Finally, longitudinal analysis demonstrated that the active AHI signature can persist independent of viral load in ART treated patients and up to 6 months in the absence of therapy.

Conclusion: Transcriptional monitoring of early HIV infection offers both quantitative and qualitative assessments patient responses that may be predictive of long-term disease progression.

1 U.S. Military HIV Research Program, Rockville, USA 2 Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand 3 AFRIMS, Bangkok, Thailand P01.07 Risk Behavior and Demographic Characteristics of Infected vs Non-Infected Participants in RV217d, a Study in At-Risk Populations in Pattaya, Thailand

Background: RV217d is a study of early capture HIV infection in most at risk populations in Pattaya, Thailand through repeat twice weekly testing.

Methods: Female sex workers (FSW), MSM, and transgenders (TG) were given a baseline risk assessment. Those at risk were enrolled and followed for two years with twice weekly HIV testing.

Results: A total of 381 participants took the baseline questionnaire. The majority reported working as bar, massage or sex workers (94%) or having received money in exchange for sex (85%). They are well educated, with average age of 27 years and 39% have never married. At baseline their recent risk for HIV is high, with unprotected sex with a known HIV positive partner, 15%; unprotected sex with greater than or equal to three partners in the last three months, 43%; recent STI symptoms, 60% and inconsistent or no condom use with male clients, 56%.

Sixteen participants have become infected to date, for an incidence rate of 8.59/ 100 py in MSM, 6.81 in TGs, and 2.10 in FSWs.

Incident cases are younger, less educated, and earn half the income of the uninfected group.

The groups were similar across the behavioral responses except 77% of infected MSM and TG participants had sex exclusively with males or transgenders versus 59% of the uninfected.

Conclusion: In this most at risk cohort, a lack of a characteristic behavioral profile distinguishing infected participants may indicate that all are at risk; however, low-income, younger MSM and TGs would be an appropriate group to target.

1 Fred Hutchinson Cancer Research Center, Seattle, USA 2 U.S. Military HIV Research Program, Rockville, USA 3 University of Washington, Seattle, USA 4 University of Minnesota, Minneapolis, USA P01.08 Integrated Analysis of Immunogenicity, Viral Sequence, and Viral Load Data in the Step Study

Background: The sieve analysis for the Step Study (Rolland et al. 2011) showed that breakthrough HIV-1 sequences were more divergent from the vaccine insert in vaccine than placebo recipients, and identified 10 “signature sites” where the rate of amino acid (AA) mismatch to the insert residue differed between treatment groups. We linked Step viral sequences with immunogenicity and acute viral load data to evaluate mechanisms for, and consequences of, the sieve effect.

Methods: Analyses included 91 male subjects diagnosed with HIV infection prior to study unblinding (37 placebo, 54 vaccine recipients). Viral sequences from the first PCR-positive visit were summarized using global measures of genetic distance and the 10 AA signature sites identified by Rolland et al. Pre-infection T-cell responses were mapped to individual 15-mers 4 weeks post second vaccination using interferon-gamma (IFNγ) ELISpot. Acute viral load, obtained at RNA-positive but antibody-negative visits, was available for 27 subjects and multiply imputed for the remainder.

Results: There was some evidence of reduced acute viremia in vaccines versus placebos (4.7 vs 5.1 log RNA copies/ml), although this difference was not significant (p = 0.27) and not predicted by pre-infection T-cell responses. The locations of the epitopes targeted pre-infection did not match the signature sites. Magnitude and breadth of the pre-infection responses were not associated with global genetic distances. Vaccine and placebo recipients did not differ in their associations between global genetic distance and acute viral load; however, mutations at five Gag AA sites were associated with acute viral load in vaccinees.

Conclusion: The Step vaccine not only impacted founder virus populations, but potentially reduced acute viral load. However, current measures of T-cell function did not predict these effects. Sequence differences between treatment groups were not associated with viral load, although we did identify several mutations in Gag associated with lower/higher viral load in vaccinees alone.

1 St George's, University of London, London, United Kingdom (Great Britain) 2 Imperial College London, London, United Kingdom (Great Britain) 3 Infectious Disease Research Institute, Seattle, USA P02.02 Vaccine Antigen Immunity Is Enhanced by Optimized TLR4 and TLR7/8 Adjuvant Combinations in Mini-Pigs Inoculated via a Systemic but Not a Mucosal Route

Background: Previous studies have demonstrated synergy between TLR4 and TLR7/8 stimulation with enhanced CD4 T cell cytokine production and cooperative upregulation. We performed a series of experiments to analyse the inter-relationship of these two innate receptors in mini-pigs, animals that are fully responsive to these TLR ligands and that are immunologically similar to man.

Methods: Groups of 4 pigs were vaccinated twice at monthly intervals with protein antigens adjuvanted with various concentrations of either aqueous formulation GLA (synthetic monophosphoryl lipid A - TLR4 ligand) or R848 (resiquimod - TLR7/8 ligand) via the intranasal (IN - 50ug antigen) or intradermal (ID - 20ug antigen) route. Pigs were sampled weekly (serum and vaginal wash) and antigen-specific IgG and IgA quantified by ELISA.

Results: R848 augmented antigen-specific responses when administered via the IN or ID route, while an adjuvant effect was only observed with GLA used ID. Combinations of optimal amounts of each TLR ligand had an additive enhancing effect when used ID but surprisingly GLA decreased R848-induced immunity after IN inoculation. Interestingly, R848 significantly enhanced mucosal antigen-specific IgA after IN inoculation but only within a narrow concentration range, as high dose R848 inhibited mucosal IgA.

Conclusion: These data begin to address important issues relating to adjuvant combinations and route of administration. TLR4 and TLR7/8 agonists combined to additively enhance antigen-driven immune responses but only after ID vaccination. The inhibitory effect of GLA on R848-driven responses suggests an active contribution by GLA rather than a simple formulation issue, as adjuvant co-formulation did not inhibit R848 responses after ID vaccination. While high dose R848 did not inhibit the serum antibody responses we found that mucosal responses, particularly IgA, were particularly dose sensitive and responses were significantly abrogated above a threshold, a crucial parameter to incorporate into any adjuvanted vaccine modality.

1 Aaron Diamond AIDS Research Center, Rockefeller University, New York, USA 2 Tulane National Primate Research Center, Covington, USA 3 Henry M. Jackson Foundation, Rockville, USA 4 U.S. Naval Medical Research Center, Silver Spring, USA P02.04 A Novel Glycolipid Adjuvant Significantly Boosts Cellular Immune Responses to an Adenoviral Vaccine in Non Human Primates

Background: The majority of adjuvants in development to date boost immune responses to protein antigens. Identification of effective adjuvants for viral vectors remains critical to improving vaccination regimens that utilize viral vectors. We have identified a novel glycolipid compound, 7DW8-5, which binds the CD1d receptor with higher affinity than its parent compound, α-galactosyl ceramide (αGalCer), enabling NKT cell activation and subsequent recruitment and activation of dendritic cells and other inflammatory mediators. We sought to determine whether 7DW8-5 would provide an adjuvant effect to an adenoviral vaccine in rhesus macaques, and to determine the optimal dose for subsequent clinical development.

Methods: Five groups of rhesus macaques (n = 5 per group) received a single intramuscular vaccination with 2x10¹⁰pu NMRC-M3V-Ad-PfCA (AdPfCA), a 1:1 mixture of Ad5-based vectors expressing the CSP and AMA-1 antigens from Plasmodium falciparum, respectively, with increasing concentrations of 7DW8-5 in each group: no adjuvant, 0.1μg, 1μg, 10μg, and 100μg. Macaques were monitored for safety, innate immune responses and immunogenicity for four weeks.

Results: 7DW8-5 elicited dose-dependent local erythema at injection sites, but no evidence of systemic reactogenicity (fever, tachycardia, respiratory distress). 7DW8-5 provided a significant increase in IFNγ ELISPOT responses to both antigens, most significant at the 10μg dose (fold increase in mean spot-forming units/million over no adjuvant: 9.8 and 5.8 for CSP and AMA-1, respectively, p = 0.04 for both). The magnitude of response did not correlate with circulating NKT cell level, indicating that this may be useful across broad populations. Initial decreases in circulating NKT cell levels on Day 1 normalized rapidly by Day 2. 7DW8-5 activated circulating plasmacytoid dendritic cells earlier than macaques who received vaccine alone, measured by CD40, CD80, and CD86 expression.

Conclusion: 7DW8-5 provides a significant adjuvant effect on the cellular immunogenicity of an adenoviral vaccine in non-human primates, and is advancing into a Phase 1 clinical trial in healthy volunteers.

1 IBCP-CNRS, Lyon, France 2 GIMAP, Saint Etienne, France 3 Invivogen, Toulouse, France 4 LMPB, Lyon, France P02.05 Co-Administration of NOD1 and NOD2 Ligands with HIV-1 p24 Increase Mucosal Immune Potency of Biodegradable Nanocarriers

Background: The use of TLR ligands as mucosal adjuvant for vaccine administration is already largely described, but the use of NOD ligands is still investigated. As NOD ligands are able to induce production of pro-inflammatory proteins and chemokines, we have evaluated if their co-delivery with biodegradable nanocarriers carrying HIV Gag antigens amplify the mucosal immune responses in mice.

Methods: We used Poly(Lactic Acid) (PLA) nanoparticles (200 nm) as vaccine vehicle for delivery of HIV-1 Gag p24. To assess their immunogenicity and the adjuvant effect of NOD agonists (NOD1 and NOD2), we have compared two routes of immunization, enteric and subcutaneous, in B6D2 mice with co-administration of NOD ligands. For each group, cellular and humoral responses have been analyzed on splenocytes and in vaginal washes, faeces and sera.

Results: We first confirmed that PLA nanoparticles are efficient protein carriers of HIV p24 without alteration of the colloidal stability of the formulation. By analyzing humoral immune responses, we noticed that co-administration of p24-PLA NPs and NOD ligands through subcutaneous route was very efficient to induce higher anti-p24 IgG responses in rectal, vaginal washes and sera, irrespective of the sub classes. However, p24-specific IgA responses at the rectal site were only found after repeated enteric vaccination with adjuvanted formulations.

Upon subcutaneous injection in mice, we could observe that p24-PLA NPs co-administered with NOD1 ligand induced a significant decrease of the IgG1/IgG2a ratio compared to non-adjuvanted formulation meaning a Th1 orientation.

Conclusion: When PLA are co-administered with NOD ligands as exemplified by the appearance of mucosal immunity, a co-adjuvant effect was clearly observed. Use of NOD1 and 2 ligands as mucosal adjuvant deserve further experiments and we are currently investigating the mechanisms involved and increasing delivery efficacy after co-encapsulations.

1 U.S. Military HIV Research Program, Henry M. Jackson Foundation, Rockville, USA 2 Walter Reed Army Institute of Research, Silver Spring, USA 3 U.S. Military HIV Research Pro, Walter Reed Army Institute of Research, Rockville, USA 4 The Catholic University of America, Washington, USA P02.06 Liposomes Containing Glucosyl Ceramide and the Adjuvant Monophosphoryl Lipid A Specifically Bind T4 Bacteriophage: A Self-Assembling Nanocarrier

Background: Our goal was to create a self-assembling nanocarrier vaccine adjuvant formulation by utilizing bacteriophage T4 displaying HIV-1 gp41 antigens and liposomes containing glucosyl ceramide (GluCer) and monophosphoryl lipid A (MPLA). Liposomes containing MPLA have been demonstrated to have potent adjuvant activities for experimental vaccines both in humans and in animals and because of this the immunogenicity of the spontaneously bound nanocarrier antigen-adjuvant formulation was evaluated in small animals.

Methods: A peptide antigen derived from either the C-trimer (Ct) or the c-terminal outer domain (COD) of HIV-1 gp41 was fused to the highly antigenic outer capsid protein (Hoc), a nonessential protein of T4 that spontaneously binds to the T4 capsid. This resulted in display of Ct-Hoc or COD-Hoc antigens on the T4 capsid. Mice and rabbits were immunized with Ct-Hoc-T4 and COD-Hoc-T4 constructs as such or bound to liposomes containing both GluCer and MPLA. The specificity of the spontaneously bound T4-liposomal formulation was determined by Biacore. Humoral responses in mice and rabbits and cellular immune responses in mice were analyzed.

Results: The display of Ct-Hoc antigen on T4 capsids was confirmed by electron microscopy. Binding of phage T4 did not occur to glycolipids, such as galactosyl ceramide, containing an aldose in which the C-2 or C-4 conformations were not identical to glucose. These results strongly support previous reports that glucose is a major receptor moiety for T4 binding to Escherichia coli. High titer antigen-specific IgG antibodies were induced in the immunized mice and rabbits. Neutralizing antibodies were analyzed in the PBMC, TZM-Bl, and a macrophage neutralization assay. Cellular responses specific to both Ct and COD antigens were also induced in mice.

Conclusion: Liposomes containing both GluCer and MPLA can spontaneously bind to T4 displaying HIV-1 gp41 antigens. This formulation could be utilized as an easily manufactured self-assembling antigen and adjuvant carrier.

1 Walter Reed Army Institute of Research, Rockville, USA P02.07 Antigen-Specific Enhancement of Natural Human IgG Antibodies to Lipids Induced by a Liposomal Vaccine Containing Lipid A and a Protein Antigen

Background: Monoclonal antibodies 2F5 and 4E10 not only bind to the protein epitope, but also to lipids. Multispecific antibodies with similar binding specificities to 2F5 and 4E10 and antibodies to lipids can readily be induced in mice by immunization with liposomes containing monophosphoryl lipid A (MPLA). However, it has never been demonstrated that antibodies with lipid binding specificity can be induced by vaccination of humans. This study was conducted to determine if antibodies to lipids can be induced or boosted in titer by vaccination of humans.

Methods: ELISA for IgG to lipids was conducted on stored sera obtained from volunteers who received a candidate vaccine to Plasmodium falciparum (PNAS 1992; 89:358–362). The vaccine consisted of liposomes that contained both the recombinant protein antigen and MPLA as an adjuvant.

Results: The pre-immune sera contained natural antibodies (NA) to one or more of the lipids in the vaccine: dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), cholesterol, and MPLA. Two weeks after the vaccine boost, increased levels of IgG to all of the liposomal lipids, especially DMPG and MPLA, were observed. Antibodies to phosphatidylinositol-4-phosphate (PIP) above the normal pre-immune NA to PIP were also observed. Although PIP was not present in the immunizing liposomes, the anti-PIP antibodies were thought to represent a cross-reaction with DMPG. The immune response was antigen-specific in that NA to unrelated lipids that were not present in the liposomes, galactosyl ceramide and ganglioside GM1, were not increased by immunization. Furthermore, antibodies to beta-2-glycoprotein I or albumin were also no increased in titer following immunization.

Conclusion: Antibodies to DMPC, DMPG, PIP, cholesterol, and MPLA can be induced or boosted in humans by immunization with liposomes containing MPLA. There were no adverse effects of these antibodies and no increases in the pathogenic antibodies to beta-2-glycoprotein I.

1 Emory University, Atlanta, USA 2 Louisiana State University Health Sciences Center, New Orleans, USA 3 National Institute of Allergy and Infectious Diseases, Bethesda, USA 4 Geovax Inc., Smyrna, USA P02.08 GM-CSF as an Adjuvant for MVA Vaccine: High Dose but Not Low Dose Inhibits T Cell Responses and Rectal IgA by Modulating DC Activation and Phenotype

Background: We recently demonstrated that co-delivery of Granulocyte-Macrophage-Colony-Stimulating Factor (GM-CSF) DNA with SIV DNA vaccine enhances protection from acquisition of heterologous mucosal SIVsmE660 infection from 25% to 71% by a DNA/MVA vaccine. Here, we evaluate the optimal dose of GM-CSF for enhancing the immunogenicity and efficacy of MVA-only vaccine in rhesus macaques.

Methods: A MMM regimen delivered MVA/SIV (108 plaque forming units, pfu) expressing SIV239 Gag, Pol and Env at weeks 0, 8 and 24. In addition, GM-CSF adjuvanted groups simultaneously received MVA expressing GM-CSF (GM) at indicated dose. There were 7–8 animals/group.

Results: The MMM vaccine rapidly induced expression of CD80 (activation), CCR7 (lymph node homing) and alpha4 beta7 integrin (a4b7, gut homing) on plasmacytoid dendritic cells (PDC) and monocytoid DC (MDC) in blood. Consistent with DC activation, MMM vaccine elicited strong SIV-specific CD8 and CD4 T cell responses in blood, high titer and avidity Env-specific IgG in serum, and moderate levels of Env-specific IgA in rectal secretions. High doses (5x107 and 107 pfu) of GM inhibited SIV-specific T cell responses and SIV-specific IgA in rectal secretions whereas low doses (106 and 105 pfu) either enhanced or maintained them. However, high doses of GM did not inhibit SIV-specific IgG in serum. The high dose but not low dose GM groups inhibited CD80 expression on MDC and down regulated a4b7 on PDC that was strongly associated with inhibition of SIV-specific T cell responses and rectal IgA, respectively in the high dose GM groups.

Conclusion: These results demonstrate that high doses of MVA/GM-CSF inhibit cellular immune responses in blood and IgA in rectal secretions by modulating activation status of MDC and gut homing potential of PDC. These animals will be challenged with SIVsmE660 to study the influence of high and low dose GM-CSF on protection.

1 University of Oxford, Oxford, United Kingdom (Great Britain) P02.09 Polyethyleneimine Is a Potent Mucosal Adjuvant and Candidate Adjuvant for HIV-1 Vaccination

Background: There are no mucosal adjuvants licensed for use in man although protection against many mucosally-transmitted infections probably requires immune activation at the site of pathogen entry. Polyethyleneimine (PEI) is used extensively as a transfection reagent and in-vivo gene delivery vehicle. Here we show that PEI has potent mucosal adjuvant activity with HIV-1 glycoprotein. PEI recruits leukocytes, triggers a local balanced Th1/Th2 cytokine environment and targets antigen to lysosome-like compartments in antigen presenting cells.

Methods: Balb/C mice were i.n. immunised with PEI (25 kD branched); PBS or CTB in formulation with HIV-1 glycoprotein (CN54gp140) antigen. Bleeds and vaginal lavages were obtained pre-prime, post-prime and post-boost immunisation and analysed for CN54gp140-specific IgG and IgA titres (ELISA). Spleens and cervical lymph nodes were obtained, restimulated with CN54gp140 and analysed for T cell proliferation (thymidine incorporation assay) and cytokine production (LUMINEX). Balb/C mice received i.p. injection of PEI, Alexa-647-ovalbumin, or both. Peritoneal lavages were obtained and cytokine levels determined (LUMINEX). Single cell suspensions were prepared and stained for leukocyte markers for flow cytometry. Naive peritoneal macrophages were incubated with Alexa488-CN54gp140 with or without PEI, fixed, permeabilised, stained with subcellular compartment markers for confocal microscopy.

Results: PEI triggered local and systemic antibody levels of equal or higher size than gold standard adjuvant CTB. PEI also triggered T cell responses in spleen and lymph nodes of equal size and interestingly more localised character. Peritoneal administration showed that PEI recruits leukocytes, particularly monocytes, to the site of injection and triggers a local balanced Th1/Th2 cytokine environment. Analysis with Alexa 647- ovalbumin demonstrated that PEI targets antigen to antigen presenting cells. Confocal microscopy confirmed the presence of HIV-1 CN54gp140 inside macrophages when ex-vivo co-incubated with PEI.

Conclusion: PEI has immunostimulatory properties and adjuvant potency for HIV-1 immunisation. PEI may merit development as a mucosal adjuvant for human use.

1 University of Oxford, Oxford, United Kingdom (Great Britain) P02.10 Degradable PEI Form as Safer Alternative for HIV-1 Vaccine Development and Adjuvant Potency as a Common Trait of Oligoethyleneimines

Background: There are no mucosal adjuvants licensed for use in man although protection against many mucosally-transmitted infections probably requires immune activation at the site of pathogen entry. Polyethyleneimine (PEI) is used extensively as a transfection reagent and in-vivo gene delivery vehicle. We have shown that PEI has potent mucosal adjuvant activity with HIV-1 glycoprotein. One of the potential drawbacks of PEI is its potential toxicity. In these studies, we compared various PEI forms including degradable PEI forms for their adjuvant potency and toxicity.

Methods: Various linear and branched, degradable and non-degradable PEI forms were used in formulation with HIV-1 glyprotein (CN54gp140) antigen for i.n, i.p.. and s.c. immunisation in female Balb/c mice. Bleeds and vaginal lavages were obtained at regular intervals pre-prime, post-prime and post-boost immunisation. Body weights were recorded at 0, 6, 12, 24, and 48 hours post immunisation. Lavage and serum samples were analysed for CN54gp140 – specific IgG and IgA titres using ELISA.

Results: A wide range of structural PEI forms showed enhanced local and systemic antibody responses when co-formulated with CN54gp140 for immunisation in mice via various administration routes. Interestingly, no relation was found between molecular weight (mW) or linear/branched structure and adjuvant potency, which is in contrast to PEI gene transfection studies that showed optimal transfection for high mW branched forms. Some degradable forms in our studies showed substantial adjuvant potency combined with lower observed body weight loss in mice.

Conclusion: PEI's immunostimulatory properties are a shared trait between various structural, degradable and non-degrable PEI forms. Some degradable PEI forms triggered substantially less weight loss in mice, indicative of a lower toxicity profile, while still being potent adjuvants in these models. Degradable PEI forms could therefore form a safer alternative for standard non-degradable forms when developing PEI as an adjuvant for HIV-1 vaccination.

1 New England Primate Research Center Harvard Medical School, Southborough, USA P03.01 Differential Expression of Transcription Factors in SIV-Specific CD8+ T Cells at Week 5 and Week 20 After Vaccination with SIV239Δnef

Background: Current evidence suggests that protective immunity against vaginal challenge in SIVΔnef-vaccinated macaques develops at 20 weeks after vaccination, whereas the magnitude of SIV-specific CD8+ T cells peaks at 5 weeks. Phenotypic analyses indicate a maturation of memory responses from week 5 to 20, as characterized by upregulation of CCR7 and CD127. These observations suggest that the quality of a more mature CD8+ T cell response, as opposed to the magnitude, may correlate with protection. We therefore analyzed differential expression of transcription factors involved in T cell maturation in SIV-specific CD8+ T cells at 5 and 20 weeks after SIVΔnef vaccination.

Methods: Highly parallel qRT-PCR (Fluidigm) was used to characterize the expression of 23 transcription factors in CD8+ T cells sorted into naïve, central, transitional, and effector memory subsets, as well as in SIV-specific CD8+ T cells obtained at wk5 and wk20 after SIV239Δnef vaccination.

Results: Unsupervised hierarchical clustering segregated naïve and memory cell populations by phenotypic class. While the expression of some transcription factors, e.g. T-bet and Blimp-1, progressively increased during memory cell differentiation, others, (TCF7 and LEF1), progressively decreased. Further, SIV-specific CD8+ cells segregated into wk5 and wk20 clusters, with the wk 20 population exhibiting increased levels of transcription factors associated with both quiescent memory T cells (TCF7, BAZF) and maintenance of effector function (Eomes, T-Bet).

Conclusion: Our data indicate distinct transcriptional profiles of the different memory CD8+ T cell subsets, as well as clear qualitative differences in wk5 and wk20 SIV-specific CD8+ T cell transcriptomes. We further demonstrate that the mature CD8+ T cell response induced by SIV239Δnef is characterized by the expression of transcription factors associated with both central memory and effector memory T cells. Analysis of transcription factor expression therefore provides a valuable complement to the analysis of memory cell differentiation based on classical phenotypic markers.

1 The University of Hong Kong, Pokfulam, Hong Kong 2 National Cancer Institute at Frederick, NIH, Frederick, USA 3 National Cancer Institute-Frederick, National Institutes of Health, Frederick, USA P04.01 Putative Rhesus Macaque Germline Antibodies of Broadly HIV-Neutralizing Antibodies: Similarities and Differences Compared with the Human Counterparts

Background: Broadly neutralizing antibodies (bnAbs) are likely to be a key component of protective immunity conferred by an effective HIV-1 vaccine. We and others have reported that putative human germline predecessors of bnAbs lack measurable binding to the HIV-1 envelope glycoprotein (Env) that could be a new challenge to elicit human bnAbs. Rhesus macaques have been used as a nonhuman primate model for testing HIV-1 vaccine candidates, but little is known about their germline antibodies.

Methods: We used one of the several best characterized bnAbs, b12, as a model Ab and identified a putative rhesus macaque b12 germline predecessor by sequence analysis. We compared the human and macaque germline predecessors and possible intermediate antibodies of b12 by sequence analysis and by ELISA and neutralization assays.

Results: Putative rhesus macaque b12 germline predecessor also did not show measurable binding to HIV-1 Envs. However, we found different sequence characteristics between the human and macaque germline genes and their intermediate antibodies of b12 isolated from B cell receptor repertoires of nonimmune rhesus macaques and human.

Conclusion: These results suggest that initiation of somatic maturation of germline b12 predecssor in rhesus macaque may also be a challenge. But maturation pathways leading to elicitation of b12 or b12-like antibodies in rhesus macaques may be different from those in human, suggesting that primary immunogens to initiate somatic maturation and Env-based vaccine immunogens to further elicit bnAbs in rhesus macaque may be different from those in human. This finding may have implications for HIV-1 vaccine development.

1 Key Laboratory of AIDS Immunology of Ministry of Health, Department of, Shenyang, China 2 Key Laboratory of AIDS Immunology of Ministry of Health, Shenyang, China 3 AIDS Research Center, School of Medicine, Tsinghua University, Beijing, China P04.02 Genetic and Neutralization Sensitivity of Diverse HIV-1 Env Clones from Chronically Infected Patients in China

Background: As HIV-1 continues to spread in China from traditional high-risk populations to the general public, its genetic makeup has become increasingly complex. However, the impact of these genetic changes on the biological and neutralization sensitivity of the virus is unknown. The current study aims to characterize the genetic, biological and neutralization sensitivity of HIV-1 identified in China between 2004 and 2007.

Methods: Full-length envelope genes were amplified by PCR directly from proviral DNA extracted from patients' unclutured PBMC and cloned into expression vector. Env-bearing pseudotyped viruses were generated by co-transfection of env-expressing plasmid together with backbone construct pNL43R-E-luciferase into the 293 cells. Neutralization sensitivity of these diverse HIV-1 to subtype-specific plasma pools from infected patients as well as to broadly neutralizing monoclonal antibodies (2F5, 4E10, 2G12, PG9, PG16, IgG1b12, and VRC01) were analyzed.

Results: Pseudotyped viruses built upon the viable env genes have demonstrated their substantial variability in mediating viral entry, and in sensitivity to neutralization by subtype-specific plasma pools and broadly neutralizing monoclonal antibodies (bnmAb). Many viruses are resistant to one or more bnmAb including those known to have high potency against diverse viruses from outside China. Sequence and structural analysis has revealed several mechanisms by which these resistant viruses escape recognition from bnmAb.

Conclusion: We believe that these results will help us to better understand the impact of genetic diversity on the neutralizing sensitivity of the viruses, and to facilitate design of immunogens capable of eliciting antibodies with similar potency and breadth as bnmAb. The viruses characterized here will also provide a strong foundation for establishing a broader and more representative Chinese virus panel to evaluate the antibody responses in infected and vaccinated individuals, and to contribute to the international virus panel where only a limited number of Chinese viruses are currently available.

1 University of Cape Town, Cape Town, South Africa 2 AIDS Virus Research Unit, National Institute for Communicable Diseases, Johannesburg, South Africa 3 Department of Molecular and Cell Biology, University of Cape Town, Cape Town, South Africa 4 Centre for the AIDS Programme of Research in South Africa (CAPRISA), Durban, South Africa P04.03 Superinfection Results in Potent Neutralizing Antibody Responses to the Superinfecting Virus, but Does Not Necessarily Enhance Neutralization Breadth

Background: A better understanding of how neutralizing antibody responses develop during natural infection and the factors that augment them would be invaluable in the design of improved immunogens and immunization protocols. HIV dual infection (infection by >1 distinct HIV strain) provides a unique opportunity to evaluate whether polyvalent immunogens and prime-boost strategies are likely to enhance the breadth and potency of vaccine-induced responses against HIV.

Methods: Ten dual-infected participants were identified from the CAPRISA002 cohort, 5 of whom were co-infected at or prior to seroconversion, and 5 were superinfected within the first year of infection. Neutralization breadth at 3 years post-infection was compared to 16 singly-infected participants using a panel of 44 heterologous viruses, including subtypes A, B, and C. Autologous plasma neutralizing titers in 3 superinfected, and 3 co-infected individuals were assessed longitudinally using representative clones from multiple timepoints in the pseudovirus-based TZM-bl assay.

Results: There was no association between co-infection, superinfection, or diversity in early infection and the development of greater neutralization breadth. Furthermore, titers to the primary-infecting variants were not boosted following superinfection. However, 2 of the 3 superinfected participants developed elevated titers against the superinfecting virus, with ID₅₀ values exceeding 1:20,000 and 1:40,000 respectively. In one individual these high titers were generated despite maintaining low viral loads, frequently below detection (<400 copies/ml). None of the 3 co-infected participants generated similarly elevated titers, suggesting that sequential infection was a driving factor.

Conclusion: The lack of an association between dual-infection and breadth highlights the fact that polyvalent immunogens may not necessarily improve the breadth of elicited humoral responses. However, the high titers generated against the superinfecting variants suggest that a prime-boost approach has the potential to increase the potency of neutralizing responses against the boosting immunogen. As existing specificities were not boosted, the mechanism is likely distinct from the anamnestic response.

1 Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom (Great Britain) 2 Biomedical Primate Research Centre , Rijswijk, Netherlands P04.04 In Vitro Neutralization Kinetics of the Human Monoclonal Antibody IgG1 b12 Which Is Protective In The SHIV Rhesus Macaque Challenge Model

Background: In passive transfer experiments, the human monoclonal antibody IgG1 b12 protects rhesus macaques against infection with both the neutralization sensitive SHIVSF162P4 and the more resistant SHIVSF162P3. Relatively high concentrations of antibody are required to protect macaques against a bolus while lower quantitites are protective in the repeated, low dose, challenge model.

Methods: HIV-1 SF162, SHIVSF162P4 and SHIVSF162P3 were grown in human PBMCs. Virus was mixed with antibody and incubated. Aliquots of the mixture were exposed to GHOST cells. Cultures were washed to terminate the absortion phase. Infected cells were quantified by FACS. Results are expressed either as plots of reductions in infectious virus over time or residually infectious virus against virus dose.

Results: Neutralization of both HIV-1 and SHIV is exponential during both the incubation and absorption phases. However, at relatively high doses of antibody, there is significant neutralization without prior incubation. Dose response plots of infected cells against virus dose are linear with a gradient of one passing through the origin. Incubation of low doses of virus with a low concentration of antibody yields a dose response plot which is parallel to controls: the percentage neutralization is low with 100 TCID50 virus but increases as the virus dose is reduced, reaching complete loss of infectivity at the point where the plot crosses the horizontal axis.

Conclusion: IgG1 b12 forms a complex with virus. However, this complex is still infectious. Events during the absorption phase of a SHIV neutralization assay determine the number (rather than the proportion) of viruses which subsequently lose infectivity. At low concentrations of antibody a limited number of infectious virus can be completely inactivated. It is proposed that this number is proportional to the dose of virus which a vaccinated individual can be exposed to without subsequent infection. Such assays should be used to monitor human vaccine trials.

1 The University of Melbourne, Melbourne, Australia 2 National Centre for HIV Epidemiology and Clinical Research, Sydney, Australia P04.05 Broad ADCC Responses and ADCC Directed at VPU Epitopes in Humans Is Associated with Slow Progression of HIV

Background: Robust antibody-dependant cellular cytotoxicity (ADCC) responses at the site of HIV infection may enable the elimination of virus prior to viral spread and latency. Identifying the targets of ADCC responses in long-term slow progressors (LTSP) could help direct the future design of vaccines to elicit beneficial ADCC responses.

Methods: A cohort of 61 LTSPs and a cohort of 78 unselected subjects with varying rates of HIV progression were analysed for ADCC responses against GP140 proteins using cytolysis-based and NK-cell activation-based assays. ADCC Responses against HIV-1 peptide pools were also analysed and specific epitopes mapped using an intracellular cytokine staining assay to detect NK cell activation.

Results: HIV-specific ADCC responses correlated with reduced loss of CD4 T cells across the two cohorts (p = 0.027). Broader ADCC responses against a greater number of HIV-1 peptide pools were found in subjects of the LTSP cohort than in the unselected cohort (p = 0.006). In addition, ADCC responses against epitopes within the Vpu protein were overrepresented in slow progressors and two key ADCC epitopes in VPU were observed.

Conclusion: Broad ADCC responses correlate with slower progression of HIV. We hypothesise that inducing ADCC Abs against specific epitopes within multiple HIV-1 proteins, including regulatory proteins such as VPU may provide a mechanism for protection from acquisition of HIV-1 or slower disease progression of those that do acquire HIV-1. Further work to design vaccines that elicit ADCC against key targets needs to be urgently explored.

1 Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands P04.06 Activity of Broadly Neutralizing Antibodies Against Recently Transmitted Subtype B HIV-1 Variants from Early and Late in the Endemic

Background: For the development of a neutralizing antibody-based HIV-1 vaccine, it is important to characterize which antibody specificities are most effective against currently circulating HIV-1 variants. As we recently reported that HIV-1 has become more resistant to antibody neutralization over the course of the epidemic, we here explored if this increased neutralization resistance was also observed for the newly identified broadly neutralizing antibodies (BrNAbs) PG9, PG16, and VRC01. Furthermore, we performed a comprehensive analysis of the neutralizing sensitivity of currently circulating recently transmitted subtype B viruses to the currently most known BrNAbs.

Methods: We determined the neutralizing sensitivity to PG9, PG16, and VRC01 of virus variants that were isolated less than six months after seroconversion from individuals in the Amsterdam cohort who seroconverted between 1985 and 1989 (n = 14), as well as b12, 2F5,4E10, 2G12 for individuals who seroconverted between 2003 and 2006 (n = 21). Moreover, we looked at potential mutations that would influence neutralizing sensitivity.

Results: Viruses from contemporary seroconverters were significantly more resistant to neutralization by VRC01 and tended to be more resistant to neutralization by PG16 than historic seroconverters, which coincided with the presence of more mutations at positions in the viral envelope that may potentially influence neutralization by these antibodies. Despite this increased neutralization resistance, all recently transmitted viruses from contemporary seroconverters were sensitive to at least one BrNAb at concentrations of ≤5 μg/ml, with PG9, PG16, and VRC01 showing the greatest breadth at lower concentrations.

Conclusion: Our results show that while HIV-1 has become more resistant to CD4-binding site-directed neutralization over the course of the epidemic, all virus variants were sensitive to at least one of the BrNAbs tested. These results suggest that a vaccine capable of eliciting multiple BRNAb specificities will be necessary for protection of the population against HIV-1 infection.

1 Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands 2 Torrey Pines Institute for Molecular studies, San Diego, USA 3 Department of Medicine, University of Alabama, Birmingham, USA 4 National Institute for Communicable Diseases, Johannesburg, South Africa 5 Vaccine Research Center, National Institute of Allergy and Infect, NIH, Bethesda, USA 6 Vaccine Research Centre, NIAID, NIH, Bethesda, USA P04.07 Neutralizing Activity Against Both Gp120 and Gp41 in Patients with HIV-Specific Cross-Reactive Neutralizing Humoral Immunity

Background: To date, no HIV-1 immunogen is available that can elicit a potent cross-reactive neutralizing humoral immune response. One of the current approaches is the identification, characterization and subsequent use of epitopes of very potent broadly neutralizing antibodies as immunogens to elicit HIV-1 specific neutralizing antibodies with similar potency and breadth.

Methods: In an attempt to identify novel epitopes for broadly neutralizing antibodies, we selected five individuals who at two years post-imputed-SC had developed potent serum cross-reactive neutralizing activity (CRNA).

Results: Interestingly, sera from all five patients to some extent had post-CD4-CCR5-binding HIV-1 neutralizing activity (IC50 titers range 1:100–1:350; 20–70% of neutralization activity), implicating the presence of anti-MPER antibodies. Chimeric HIV-2 variants with HIV-1 MPER or parts thereof, were used to identify the exact epitopes of the anti-MPER activity, with all five patients targeting different parts of the MPER-region. Adsorption of MPER antibodies with linear-MPER-coated beads reduced CRNA in sera from two individuals, however the contribution of linear anti-MPER activity to the CRNA in the other three patients could not be confirmed. In all five sera, detectable anti-gp120 activity was measured, although it did not reach more than 50% of the neutralization capacity. Assays so far indicate little if any evidence for neutralizing antibodies similar to b12, 2G12, PG9/PG16, and CD4i or anti-core antibodies, although there was evidence for what may be type-specific N332A-sensitive activity. Neutralizing antibodies to the CD4-binding site are currently under investigation in these patients.

Conclusion: These results show that our sera all contain significant titers of anti-gp41 MPER neutralizing antibodies. However, there is evidence for other epitopes to be targeted by the CRNA in these patients, including those directed to gp120, suggesting that cross-reactive neutralization is achieved by multiple specificities. Further characterization of the cross-reactive neutralizing epitopes may assist in the development of new HIV-1 vaccine targets.

1 Fundation Osvaldo Cruz, Rio de Janeiro, Brazil 2 Evandro Chagas Clinical Research Institute, Rio de Janeiro, Brazil P04.08 Broad ADCC Responses and ADCC Directed at VPU Epitopes in Humans Is Associated with Slow Progression of HIV

Background: Tests for the detection of humoral immune response to HIV-1 have to be established, demanding regional efforts to permit assessments of efficacy of future vaccine trials and allow comparisons on a global level. Our goal is to use the TZM-bl assay to investigate Neutralization antibody (NAb)able to inhibit HIV-1 replication in plasma from HIV-1 positive individuals with distinct profiles of Aids progression.

Methods: Eight env-pseudoviruses were evaluated against 22 plasmas from HIV-1-infected individuals: 13 typical-progressors (TP) and 9 long-term non-progressors (LTNP). We used as a control sCD4 and 2F5 Mab and 3 clade/variant plasma pooled TP (B,B/Bbr,F). The same plasmas were tested against the HIV-1 IIIB isolate in primary lymphocyte (ID90%).

Results: From the 13 TP plasma tested, 2 (15%) were potent and broad in their neutralizing capacity (100%), as well as the HIV-1 IIIB isolate (PBMC), 7 (53%) neutralized 50% of the env-pseudotyped tested, the remain varied from 25–30%. In the ID50% in TZMBL assay 104 titer points (13 TP plasmas × 8 pseudoviruses)were evaluated, from those 64 (62%) had neutralizing titers from 1:20–1:4374 and 40 (38%) haven't NAb. Of the 72 (9 plasmas x 8 pseudoviruses) points LTNP plasma tested, 10 (14%) had neutralizing titers between 1:50–1:6250 and 62 (86%) haven't NAb. The highest neutralization titer was verified in the B/Bbr1 pooled.

Conclusion: Neutralization assay in TZM-bl cells was successfully reproduced in the laboratory being a tool for the detection of NAb from TP and LTNP plasmas in distinct proportions, showing a high capacity to neutralize virus/pseudovirus. These results and these reference reagents enable the participation of Brazil in future assays of neutralizing antibodies induced by vaccine to HIV-1. Financial support: GHVE and Dpto DST/AIDS and Hepatites MS.

1 International AIDS Vaccine Initiative Neutralizing Antibody Center, La Jolla, USA 2 International AIDS Vaccine Intitiative, New York, USA 3 Rwanda Zambia HIV Research Group, Lusaka/Ndola/Kigali, Zambia 4 MRC/UVRI Uganda Research Unit on AIDS, Masaka and Entebbe, Uganda 5 Centre for Geographic Medicine-Coast, Kenya Medical Research Institute, Kilifi, Kenya 6 Monogram Biosciences, Inc, South San Francisco, USA 7 International AIDS Vaccine Initiative Human immunology laboratory, London, United Kingdom (Great Britain) 8 International AIDS Vaccine Initiative, San Francisco, USA P04.09 Development of Broadly Neutralizing Antibody Responses in a Large Sub-Sahara African HIV Primary Infection Cohort

Background: Recent studies have demonstrated that 10% to 20% of individuals infected with HIV-1 are capable of generating HIV-1 broadly neutralizing antibodies (bNAbs). Understanding the correlates of how these broad responses develop could be extremely important for the design of a protective vaccine.

Methods: The IAVI Protocol C program investigates a longitudinal cohort of primary HIV-1 infection in Eastern and South Africa. Currently over 400 donors with recent HIV-1 infection have been recruited. To date, development of neutralization has been evaluated on 200 individuals, including 174 with at least 3 years of follow-up. Neutralization activity was assessed at Monogram Biosciences using a 6 cross-clade pseudo-virus panel highly predictive of neutralization breadth on larger panels. Neutralization of an isolate was recorded positive if equal or greater than 40% at a 1/100 serum dilution.

Results: At year 2, only 4 individuals out of 171 had developed broad neutralizing activity, with 5 out of 6 viruses neutralized. In contrast, at year 3, 16 donors out of 163 (9.8%) neutralized more than 4 viruses. In addition, 69 individuals (36%) neutralized 3 or 4 viruses on the panel. Broad responses developed at year 4 in 9 additional donors out of 71 (12.7%). In most individuals, neutralization breadth developed simultaneously, appeared to be relatively stable with time, and no significant improvement of potency was noted over time. Screening of an additional 96 donors is pending.

Conclusion: In conclusion, our current data show that in our large longitudinal sub-Sahara African cohort, broad neutralizing responses have so far developed in about 15% of individuals and essentially at year 3. Analysis of correlates of breadth development with clinical parameters, such as CD4 counts and viral load, is ongoing as well as the mapping of broad neutralizing specificities in the top neutralizers.

1 University of Regensburg, Regensburg, Germany P04.10 FACS-Based Epitope Mapping Platform to Characterise New Identified Monoclonal Anti-HIV-1 Envelope Antibodies

Background: Recent systematic approaches for identification of HIV-1 infected patients with neutralizing sera and subsequent generation of new broadly neutralizing monoclonal antibodies (bNMAb) create the need for a rapid characterisation tool which allows the determination of conformational epitopes on a native envelope structure.

Methods: A sequentional permutation envelope library based on the isolate ZM96 was generated, where every amino acid is stepwise mutated to an alanine, resulting in 608 variants with a single point mutation. Every variant is used in a separate transient transfection and analysed by high throughput FACS analysis using an antibody of interest in combination with a second gp41 antibody used as an internal standard. First the screening process was validated using the v3 antibody HGN194 known to recognize a linear epitope as well as soluble CD4 which binds to an highly conformational epitope. Second, new antibodies with so far unmapped epitopes like HJ16 were screened.

Results: The screen with HGN194 showed several critical amino acids for binding in the v3 region and confirms the published data by Corti D. at al. 2010. The screen using sCD4 confirmed several known amino acid positions which are known to be critical for the binding of the receptor to the envelope. The screening of HJ16 resulted in the identification of the several critical amino acids within the gp120 envelope for the binding of the bNMAB. These positions suggest a high conformational epitope. Additional mutations were identified which showed an enhanced binding of the bNMAB. Confirmed amino acid positions were modelled in the gp120 core structure.

Conclusion: This method allows the identification of conformational epitopes of anti-HIV envelope antibodies on a native, trim-eric and membrane bound envelope.

1 Harvard University, Charlestown, USA 2 Tulane University, New Orleans, USA 3 Ragon Institute of MGH, MIT and Harvard, Charlestown, USA P04.11 Innate Immune Signals Regulate Non-Neutralizing Effector Functions of Antibodies Through Glycosylation

Background: Data from the RV144 trial have shown that the modest success of this trial was not dependent on elicitation of CD8+ T cell or neutralizing antibody responses. Interestingly, most vaccinees induced non-neutralizing antibodies, which raises the possibility that other humoral antiviral activities may have contributed to protection. In fact, innate immune recruiting antibodies are enriched in long-term non-progressors, and this humoral activity has been shown to play a critical role in promoting the sterilizing protective effect of some neutralizing antibodies. However, the mechanisms by which these effector antibodies can be induced in vivo through vaccination, are not understood. We hypothesized that innate immune signals, particularly toll-like receptor (TLR) ligands, are able to modulate effector functionality of antibodies.

Methods: Gene expression of a set of over 20 glycosylation enzymes was analyzed in healthy bulk B cells stimulated with TLR ligands to identify interesting changes in glycan structure.

Results: Specific TLR stimulation of bulk B cells resulted in significant alterations of glycosylation enzyme expression. These changes related to the subcellular localization of the TLRs: extracellular TLRs did not induce significant changes in glycosylation enzyme expression, however, intracellular nucleic acid sensing TLRs 3, 7, 8, and 9 caused significant alterations. Interestingly, stimulation of the nucleic acid sensing receptors that recognize intracellular pathogens, such as HIV, significantly altered glycosylation gene expression. These changes correlated with a decrease in galactose, fucose, and sialic acid additions, which may correspond to the induction of more inflammatory, less branched glycan structures, that mediate stronger effector functions.

Conclusion: These data are the first to show that innate immune signals modulate the antibody glycan patterns on stimulated B cells toward more inflammatory structures. This study strongly suggests that specific signals may be exploited in future vaccines to elicit antibody responses with specific glycosylation patterns and tailored effector functions.

1 AIDS institute, Hong Kong University, HongKong, Hong Kong P04.12 Single Mutation Turns a Non-Binding Germline-like Predecessor of Broadly Neutralizing Antibody into a Binding Antibody to HIV-1 Envelope Glycoproteins

Background: Broadly neutralizing antibodies (BnAbs) against human immunodeficiency virus type I (HIV-1) are rare in natural infection. We and other groups have reported that HIV-1 bnAbs are highly diversified from their germline-like predecessors. We do not know what are the minimum mutations required for converting non-binding germline-like predecessors to Env-binding antibodies.

Methods: We started with the bnAb b12 as a prototype and generated six “chimeric” scFv b12 variants by sequentially replacing the heavy chain V-segment (HV), D(J)-segment [HD(J)], and the light chain variable region (VL) in b12 germline-like predecessor with the mature counterparts, and tested the scFv variants for binding and neutralizing activities in ELISA and pseudovirus assays.

Results: A point mutation in germline heavy chain D-segment from “Y” to “D” converted nonbinding germline-like b12 to an Env-binding antibody. Replacement with either mature HV or mature VL also made the germline-like b12 bind to Env, but none of the single segment replacements conferred neutralization ability to the germline antibody. Mature VL in combination with mature HD(J), or mature HV, or both conferred increasing neutralization activity to the germline antibody. Hybrid scFv, mature VH/germline VL, did not neutralize the virus.

Conclusion: During antibody maturation, less mutation, as low as one single nucleotide change, may be needed to turn a non-binding germline-like predecessor of bnAbs into a binding antibody intermediate to HIV-1 Env, which may further mature to bnAbs upon HIV-1 infection or vaccination. Somatic maturation in HV-, HD(J)-segments and VL of germline-like b12 additively contributed to the binding of b12 to Envs. Although b12 light chain did not make contact with gp120 core in the co-crystal structure, mature b12 VL was required for “chimeric” b12 variants to neutralize the virus. These results may have implications for vaccine development.

1 Makerere University Walter Reed Project, Kampala, Uganda 2 U.S Military HIV Research Program, Rockville, USA 3 Armed Forces Research Institute of Medical Sciences , Bangkok, Thailand 4 Makerere University School of Public Health, Kampala, Uganda 5 Duke University, Durham, USA P04.13 B Cell Depletion in HIV-1 Subtype A Infected Ugandan Adults: Relationship to CD4 Count, Viral Load and Humoral Immune Responses

Background: HIV-1 infection leads to B cell dysfunction through mechanisms that are not fully characterized, particularly in subjects infected with non-B subtypes. To better understand the nature of B cell dysfunctions in East African subtype A infection, the relationship between B cell depletion, antibody function and disease progression was assessed.

Methods: Participants were recruited under an IRB approved vaccine cohort protocol. Enumeration of lymphocyte subsets was performed using FACS MultiTEST, TruCount tubes and a FACSCalibur flow cytometer. Plasma viral load was measured by the Roche HIV-1 Monitor Test version 1.5. Sera from 50 subjects with chronic subtype A infection were tested by ELISA for gp120 binding antibodies and in the TZM-bl neutralization assay against a panel of 10 pseudoviruses from newly transmitted infections of subtypes A-D and CRF02_AG.

Results: B cell numbers were significantly lower in HIV-1+ patients, compared to community matched HIV-negative controls (p < 0.0001). Neutralization and binding antibody titers showed no correlation with viral load or CD4 counts. However, B cell absolute counts were found to correlate inversely with neutralizing titers against subtype A and CRF02_AG viruses. A positive correlation was observed between subtype A gp120 binding titers and neutralizing antibody breadth (p < 0.02) and titer (p < 0.05). Additionally, subtype A sera showed preferential neutralization of subtype A and CRF02_AG pseudoviruses, compared with pseudoviruses expressing non-A envelopes (p < 0.0001).

Conclusion: In patients with chronic HIV-1 subtype A infection, significant B cell depletion can be observed which does not appear to be associated with a decrease in functional antibodies. The inverse correlation between functional antibody titers and B cell numbers may be due to B cell maturation and migration out of the periphery. These findings also further substantiate the importance of subtype in the specificity of cross-clade neutralizing antibody responses in HIV infection.

1 University College London, London, United Kingdom (Great Britain) 2 University of Utrecht, Utrecht, Netherlands 3 Vaccine Research Center, National Institutes of Health, Bethesda, USA P04.14 Llama Antibody Fragments that Potently Neutralize HIV-1 at the CD4 Binding Site of HIV-1 Gp120

Background: Llamas produce heavy-chain only antibodies, from which the small (∼15kd) binding fragment (VHH) may readily be cloned into a phagemid vector to construct phage libraries. We have isolated a number of VHH from libraries derived from llamas immunized with recombinant gp120 or trimeric gp140 which exhibit strong neutralization of diverse strains of HIV-1.

Methods: Individual VHH clones expressed in E.coli from a phagemid vector were screened for binding to recombinant HIV-1 envelope proteins and neutralisation ability against HIV-1 in the TZM-bl cell-based assay.

Results: The best VHH isolated to date, J3, neutralizes almost 100% of HIV-1 pseudoviruses tested, with a breadth encompassing clades A, B, C, A/G and B’/C. Structural studies of VHH:gp120 complexes indicate that both the angle at which VHH bind to their target and their small size compared to entire antibodies contribute to their neutralizing properties.

Conclusion: Our studies of llama VHH are useful in three aspects of protection against HIV infection: (a) they show that experimental immunization with recombinant HIV envelope can elicit broad neutralizing responses; (b) they indicate that neutralization epitopes can be exploited in immunogen design for B-cell based vaccines; (c) the VHH themselves can be developed as potential microbicides owing to their stability over a wide range of temperature and pH, low immunogenicity and ease of scale-up for inexpensive mass production.

1 Weizmann Institute of Science, Rehovot, Israel 2 College of Staten Island of the City University of New York,, Staten Island, USA 3 College of Staten Island of the City University of New York, Staten Island, USA P04.15 Optimization of the V3 Directed Antibody Response: Strategies to Expose the Epitope

Background: V3 directed antibodies with significant breadth and potency have been identified. We studied the structure of V3 peptides in complex with neutralizing antibodies, designed and tested a panel of constrained V3 peptides mimicking the 447-52D bound conformation. Our study resulted in identification of an optimally constrained V3 peptide, C4-V3T303C-E322C, which elicits an antibody response that neutralizes SS-1196 and 6535 viral strains. Nevertheless, masking of the V3 loop remains a problem limiting the breadth of V3 directed antibodies. Antibodies such as KD-247 and F425-B4e8, which can neutralize resistant primary isolates such as JR-CSF, recognize a shorter epitope centering the GPGR tip.

Methods: The V3 loop can become exposed for neutralization following CD4 binding. CD4M33 is a small and stable CD4-mimic peptide. We have tested the ability of CD4M33 to act in synergy with C4-V3T303C-E322C elicited sera. We are also testing a new methodology of immuno-focusing attempting to focus the antibody response to exposed elements in V3. Optimally constrained peptides at V3T303C-E322C are used for priming followed by additional boost with short V3 peptides representing the F425-B4e8 epitope.

Results: CD4M33 was found to act in synergy with C4-V3T303C-E322C induced sera in a panel of HIV-1 isolated including SS1196, 6535 and QH0692. Combination-index range from 0.1–0.5. We have also immunized rabbits with the aim of optimizing the number of priming boosts with the long V3 peptides and the final boosts with the short peptides. Initial binding studies suggest that a single priming step may be optimal. Neutralization studies are currently being performed and will also be presented.

Conclusion: The combination of V3 directed vaccine with a pre-exposure prophylactic administration of a CD4-mimic compounds should be considered as an alternative to other pre-exposure prophylactic drugs. Strategies to expose the V3 loop and expand the breadth of V3 directed antibodies by active immunization should also be pursued.

1 Academic Medical Center University of Amsterdam, Amsterdam, Netherlands P04.16 HIV-1 Envelope Characteristics that Coincide with the Development of Cross-Reactive Neutralizing Activity in HIV-1 Infected Patients

Background: The HIV-1 envelope glycoprotein (Env) on the viral surface is the target for anti-HIV-1 neutralizing antibodies. In some HIV-1 infected patients broadly neutralizing antibodies (BrNAb) develop that neutralise HIV-1 from different subtypes and therefore are considered to be directed against conserved regions. Understanding of factors involved in BrNAb development will help the rational design of an effective antibody-based vaccine immunogen.

Methods: From a previous screening of 299 patients for broadly neutralizing activity (BrNAc), we selected 12 patients with high BrNAc in serum and 10 patients without BrNAc at 3 years after SC. From these patients clonal viruses were obtained within the first year after SC from which gp160 was sequenced and analyzed for length of different regions and number of potential N-linked glycosylation sites (PNGS).

Results: There was a trend observed for longer variable regions 1 and 2 (V1V2) in viruses from patients who lacked BrNAc (P = 0.16), but there were no significant differences in length of gp160 or different regions of env between the two patient groups. Env from HIV-1 of patients who lacked BrNAc also tended to have a higher number of PNGS (P = 0.16), especially in the V1 region, with significantly more PNGS (P = 0.044). The number of PNGS in gp41 also tended to be higher in the non-BrNAc group (P = 0.10).

Conclusion: We hypothesize that the development of BrNAc in HIV-1 infected patients is associated with a more accessible open structure of the viral envelope glycoprotein, which in turn is associated with fewer glycans and shorter variable loops. Testing of epitope specificities in these sera as well as testing of the neutralization sensitivity of these viruses for well defined BrNAbs will help to identify the epitopes on Env that may be responsible for eliciting BrNAc and could help the rational design of an effective antibody-based vaccine immunogen.

1 AIIMS, New Delhi, India 2 Regional STD Teaching Training & Research Centre, Safdarjang Hospital, New Delhi, India 3 All India Institute of Medical Sciences, New Delhi, India P04.17 Neutralization Potential and Epitope Profiling of Plasma Antibodies from HIV-1 Infected Asian Indians

Background: Human immunodeficiency virus type 1 (HIV-1) is the primary cause of AIDS pandemic. Dissecting the specificities of the anti-HIV-1 neutralizing antibodies will assist in identifying targets for an HIV-1 vaccine.

Methods: Plasma from 65 drug naïve HIV-1 infected patients were tested against a panel of 3 subtype-B (TRO.11, JRFL and RHPA) and 5 subtype-C (Du156.12, ZM53, ZM109F AIIMS201, AIIMS212) tier 1 and tier 2 viruses. Three plasma, found to be broadly neutralizing, were mapped with a set of 211 con-C gp160 overlapping peptides (15mer each). The 50% binding titers were determined by ELISA using consensus-C V3 (35mer), ID loop (19mer) and MPER (24mer) peptides. Statistical analysis was performed using Graph Pad Prism 5.

Results: Of the 65 plasma samples, 53 (81.5%) were able to neutralize at least one virus while 12 (18.46%) did not show any neutralization. Only 15 (23%) samples were found to neutralize ≥50% viruses tested. Clustering analysis revealed that AIIMS201and Du156.12 (clade-C viruses) were the most sensitive while ZM109F, a tier 1b clade-C followed by TRO.11 (clade-B) isolates were the most resistant to antibody neutralization. Epitope specificities of three broadly neutralizing plasma (AIIMS206, AIIMS239 and AIIMS249) with consensus-C gp160 overlapping peptides mapped to V2, V3 and C5 of gp120 and ID loop, MPER and CT of gp41. We did not find any correlation between neutralization capacity and 50% binding titers of anti-V3 (p = 0.429), anti-MPER (p = 0.683) and anti-ID loop (p = 0.680) plasma antibodies. Depletion and competition with V3, MPER and ID-loop peptides showed modest effect on neutralization except for AIIMS239 which showed dependence on MPER directed antibodies.

Conclusion: Clustering of neutralization data and phylogenetic analysis suggest the recognition of similar neutralization determinants across different HIV-1 viruses. Identification of epitopes that elicit broadly neutralizing antibodies may serve as effective tool for HIV-1 vaccine design.

1 International AIDS Vaccine Initiative, La Jolla, USA P04.18 The Antibody 2F5 Third Complementarity-Determining Region of the Heavy Chain (CDRH3) Displays Length Plasticity for Binding and Neutralization

Background: The HIV-1 membrane proximal external region (MPER) broadly neutralizing monoclonal antibodies (Mab) 2F5 and 4E10 bind contiguous epitopes in gp41 and are crystallographically characterized in complex with their cognate peptides. The 2F5 structure reveals an extended loop conformation that contrasts with the helix generally adopted by the MPER. Most of the unusually long (22aa) CDRH3 of the 2F5 Mab does not contact the epitope and the CDRH3 possesses a highly hydrophobic apex. Recent data demonstrates that increasing CDRH3 hydrophobicity correlated with neutralization. Here, we aimed at elucidating the involvement of 2F5 CDRH3 length in regards to its capacity to bind or neutralize HIV-1.

Methods: We generated 2F5 mutant Mabs that possessed either longer or shorter CDRH3 loops. We next increased hydrophobicity of the length-altered CDRH3s by targeted tryptophan substitutions. We determined Mabs binding affinity constants to epitope and assessed their HIV neutralization activity in pseudovirus assays.

Results: The 2F5 CDRH3 tolerated both elongations and reductions of up to four residues, while maintaining nanomolar binding to peptide and HIV neutralization; these activities were enhanced by introducing a V100DW mutation. Substitutions of up to three tryptophans at the apex of the shortened CDRH3 fully reconstituted HIV neutralization activity. Antibody on-rate for the wt MPER peptide correlated positively with neutralization activity.

Conclusion: The 2F5 CDRH3 length displayed plasticity for binding its epitope and for neutralization with hydrophobicity of the CDRH3 appearing more critical for neutralization. The positive correlation observed between the on-rate of recognition for the wt MPER peptide and the neutralization activity of the mutant antibodies suggests a model of 2F5 MPER recognition in which the CDRH3 destabilizes the MPER helix to allow the antibody to induce the extended loop peptide-bound conformation observed in the crystal structure.

1 University of Rochester School of Medicine and Dentistry, Rochester, USA P04.19 Identification of Serum SLE-like Auto-Antibodies with Cross Reactivity to Viral Antigens in Patients with HIV-1 Infection

Background: A successful HIV-1 vaccine requires the induction of neutralizing antibodies that can protect from infection by a broad range of HIV-1 isolates(BNA). The characteristics of B cells making these antibodies are largely unknown. Several of the BNA are reactive to self-antigens, suggesting a relationship between self-reactivity, B cell tolerance, and the development of BNA. Patients with Systemic Lupus Erythematosis (SLE) have auto-reactive antibodies encoded by VH4.34, also called 9G4 (monoclonal antibody). 9G4 antibody producing cells in healthy individuals are restricted to the naive B cell compartment, yet in SLE patients these 9G4 B cells can undergo germinal center reactions (GC) and become antibody-secreting cells (ASC), leading to elevated serum levels of 9G4. The objective of this study is to examine the association between the presence of 9G4 B cells and BNA activity in HIV-infected patients.

Methods: Patients' blood samples were obtained. Sera were serial diluted in 9G4 or HIV-1 clade antigen coated 96 well Enzyme-linked Immunosorbant Assay plates and detected using enzyme/substrate reactions. BNA screening was performed using published protocols (ref).

Results: Similar to SLE patients (n = 35), 9G4 serum antibody titers are significantly elevated in HIV-infected patients (n = 112) in comparison to healthy controls (n = 28 p < 0.05). A fraction of patients (>5%) had significantly greater titers compared to SLE patients. These 9G4 positive antibodies from some HIV-1 patients bind to clade A, B and C HIV-1 Env trimers, and patients with high 9G4+ B cell numbers tend to have BNA to a wide range of HIV strains.

Conclusion: Preliminary findings suggest that these 9G4+ B cells in HIV-infected patients may offer an important avenue to study the role of tolerance in the context of induction of a broadly protective anti-HIV-1 antibody response.

1 Brigham and Women's Hospital/Harvard Medical School, Cambridge, USA 2 Boston University School of Medicine, Boston, USA P04.20 Surface Unit but Not Transmembrane Domain Directed Broadly Neutralizing Antibodies Have Differential Potency Against Cell Associated Virus

Background: Cell free virions are used as the challenge stock in the majority of HIV-1 infection models. Numerous broadly neutralizing antibodies (bNAbs) have been shown to block a diverse array of HIV-1 virions, but it remains unclear if these antibodies exhibit similar potency against cell associated infection, especially dendritic cell trans infection, which potentially plays an important role during mucosal HIV-1 transmission.

Methods: Mature DCs were derived from CD14+ monocytes cultured in the presence of IL-4, GM-CSF, and LPS. Mature DCs were exposed to various primary and laboratory adapted HIV-1 isolates (MOI = 0.2), and washed to remove unbound virus particles. Sensitivity to various bNAbs was examined between cell free and mDC laden HIV-1 in TZM-bl and primary CD4+ T cells. Cell staining was used to examine bNAbs binding mDC and mDC – T cell conjugates in the absence of HIV-1.

Results: Compared to cell free infection, mDC mediated HIV-1 trans infection was significantly less susceptible to gp120 directed bNAbs (VRC01, b12, 2G12). Cell free and mDC associated viruses were equally sensitive to gp41 targeted bNAb (4E10 and 2F5). Broadly NAb, b12, Fab fragment blocked both cell free and mDC-mediated virus infection with equal efficiency. Broadly NAb, 4E10, but not 2G12 bound both mDC alone and mDC – T cell conjugate in the absence of HIV-1.

Conclusion: Steric hindrance from the tight juxtaposition of HIV-1 bearing mDC and target T cell membranes interferes with the ability of gp120 directed bNAbs from blocking mDC-mediated trans infection. On the other hand, gp41 directed bNAb's recognition of host cell lipids allows them to bind mDC prior to the formation of an infectious synapse. Our studies suggest gp120 but not gp41 directed bNAbs may be less potent in preventing HIV-1 spread if transmission or spread from the initial site of infection occurs from a dendritic cell associated source.

1 Kumamoto University, Kumamoto, Japan 2 Kyoto University, Kyoto, Japan P04.21 Isolation of Potent Neutralizing Monoclonal Antibodies Against V3 Loop from SIV-Infected Macaques

Background: Broadly neutralizing monoclonal antibodies from HIV-1-infected patients are an essential but rare tool to understand the mechanism of neutralization against a broad spectrum of HIV-1 strains, including neutralization-resistant strains. Although the humoral immune response to SIV has been studied in the development of vaccine candidates and for exploration of antibodies that efficiently control viral infection, the lack of monoclonal antibodies that can neutralize neutralization-resistant SIV strains is the problem of studying the mechanism of efficient neutralization using the SIV model.

Methods: We used phage display method to obtain MAbs against SIV antigens from a SIVsmH635FC-infected rhesus macaque with robust envelope-specific antibody responses. Variable regions of immunoglobulin genes were amplified by rhesus macaque-specific primers and inserted into the phagemid pComb3X to construct phage libraries expressing the Fab fragment. SIV-specific Fab clones were selected by panning with SIV antigen on 96-well plate, and their specificity and neutralizing activities were analyzed.

Results: As a result of panning using SIV antigen, many Fab clones specific for SIV Env gp120, gp41 and Gag p27 were obtained. Flow cytometry analysis showed that these Fabs efficiently bound diverse strains of SIVsm/mac, and some of them were cross-reactive with HIV-2. Some of the anti-gp120 Fab clones neutralized the homologous SIVsmH635FC and the genetically divergent SIVmac316, but did not neutralize SIVmac239 and HIV-2GH123. These Fab clones with potent neutralizing activity recognized the same epitope on gp120 including V3 loop. Neutralizing Fab clones specific to the same V3 epitope were also isolated from other SIVsmH635FC-infected macaques.

Conclusion: Identification of monoclonal antibodies with potent neutralizing activity against SIV will help to elucidate the mechanisms for inducing broadly neutralizing antibodies in a SIV model.

1 Duke University Medical Center, Durham, USA 2 National Center for AIDS/STD Control and Prevention, Chinese Center, Beijing, China P04.22 Profiles of Neutralizing Antibody Responses in Chronically HIV-1 CRF07_BC Infected Intravenous Drug Users in Western China

Background: It is essential to characterize neutralizing antibody (Nab) responses in individuals infected with the diverse HIV-1 strains to reveal the potential target for HIV-1 vaccine development. We assess the prevalence, breadth and potency of Nab responses in CRF07_BC chronic infectors(n = 100) with infected time around 3 years and compare the neutralization pattern with that of clade B’ chronically infectors(n = 103) with infected time at least 10 years.

Methods: 114 plasma samples were collected from intravenous drug users infected with CRF07_BC virus in western China who were ART- naïve. The Env pseudovirus-based TZM-bl assay was performed against a panel of tier 2–3 pseudoviruses composed of CRF07_BC(10), clade C(5), B(7), A(4) and CRF01_AE(4) strains and 2 tier1 viruses(SF162.LS and MW965.26). As negative control virus, SVA-MLV positive samples were excluded from further analysis.

Results: All 100 plasma samples (excluding 14 SVA-MLV positive samples) neutralized both tier 1 viruses. 53% of samples (n = 53) neutralized half of the viruses and 17% of samples (n = 17) neutralized more than 80% strains tested. 1% (n = 1) neutralized all the viruses and 2% (n = 2) neutralized none of the viruses tested. Significant difference of geometric mean ID50 titer (GMT) between intraclade and interclade samples(p < 0.001) was observed. CRF07_BC chronically infected subjects showed higher neutralizing activities against subtype-matched viruses than subtype B’ infectors. However, higher prevalence of broadly cross-reactive Nabs response were observed in clade B’ chronically infected than that of CRF07_BC infection (29% vs 17%). Heatmap analysis against top 6% neutralizers demonstrated that each plasma exhibited unique pattern against the spectrum of viruses.

Conclusion: We detected relatively high broadly cross-reactive neutralization activities in 17% plasma of CRF07_BC infectors tested. Further epitope specificity dissection of these broadly cross-reactive Nab samples will provide useful insights for rational HIV-1 vaccine design.

1 UMR INSERM/UdS U748, Strasbourg, France, Metropolitan 2 Covance CLS SA, Meyrin, Switzerland P04.23 Plasmacytoid Dendritic Cells Infection by HIV-1 Is Inhibited by Neutralizing Antibodies Without Interfering with IFN-α Production

Background: Plasmacytoid dendritic cells (PDC) are involved in innate and adaptive immunity, and produce large amounts of antiviral type I interferons. These cells are productively infected by HIV-1 in vitro and in vivo, and several studies revealed a decrease in circulating PDC number, correlated with an increased plasma viral load.

We have previously showed an FcγRs-mediated inhibition of myeloid DC infection by neutralizing (NAbs) as well as non-neutralizing inhibitory (NNIAbs) antibodies. The aim of this study is to analyze the mechanism of inhibition of PDC infection by Ab.

Methods: Neutralization assay was performed with primary human healthy PDC or GEN2.2 PDC cell line in the presence of serial antibodies concentrations. The percentage of infection was quantified by flow cytometry using intracellular p24 viral antigen staining and IFN-α production was measured by CBA flex.

Results: We showed that NAbs strongly inhibited R5-HIV-1 replication in GEN2.2 cell lines. Although they expressed FcγRII, we have not detected an inhibitory activity of NNIAbs. We also found an efficient inhibition of primary PDC infection by NAbs, but not with NNIAbs, with similar activities that those measured with GEN2.2 PDC cell line. Interestingly, a strong level of IFN-α production was maintained in primary PDC infected by HIV, even when the HIV-1 replication was inhibited by NAbs.

Conclusion: We showed that 1) HIV-1 infection of PDC is inhibited by NAbs, 2) FcγRIIa seems not to be involved in this inhibitory process, and 3) the inhibition of HIV-1 replication by NAbs do not preclude IFN-α production by PDC. Overall these results suggest that HIV-1-specific NAbs induced by an efficient vaccine should prevent PDC infection, without interfering with the release of IFN-α and thus the anti-viral innate immune response.