HPV18 E1 Protein Plus α -Galactosylceramide Elicit in Mice CD8 + T Cell Cross-Reactivity Against Cells Expressing E1 from Diverse Human Papillomavirus Types

Abstract

CD8⁺ T cell immune response plays a critical role in the clearance of human papillomavirus (HPV)-infected cells. During the natural history of HPV infection, the E1 protein, an early-expressed helicase highly conserved among papillomaviruses, is involved in the replication of HPV genomes. We have previously shown, in a murine model, that immunization with HPV18 E1 protein combined with α-galactosylceramide elicits a specific CD8⁺ T cell response. We further proved those findings by analyzing whether CD8⁺ T cells from mice immunized with α-galactosylceramide plus HPV18 E1 protein could have a cytotoxic effect on cells expressing the carboxyl-terminal domain from the E1 proteins of other HPV types. Interestingly, CD8⁺ T cells raised against HPV18 E1 antigen presented cross-reactivity against the E1 protein from HPV53, 33, 16, and 31. Poor cross-reactivity was observed for HPV11, and none for HPV6. This outcome may be relevant for the design of broad-spectrum immune-protective agents against HPV infections.

Introduction

Persistent infection with human papillomavirus (HPV) is the main risk factor for the development of cervical cancer (16). HPV types are classified as high risk (HR-HPV) or low risk (LR-HPV), according to their oncogenic potential. HPV16 is the type most frequently found in cervical cancer, followed by HPV18, while HPV6 and HPV11 are the LR-HPV types most frequently found in genital warts and premalignant lesions (6,7). During viral infection, the E1 protein participates in HPV DNA replication through its helicase function (1,8). In some cases, for unclear reasons, the HPV genome integrates randomly into the host DNA, commonly disrupting E1/E2 open reading frame (12,18). Nevertheless, it has been reported that the expression of E1 is found to be increased in patients with cervical intraepithelial neoplasia-3 and squamous cell carcinoma, in relation to low-grade cervical lesions (4).

E1 belongs to the helicase superfamily III (15). This protein has an N-terminal domain that contains nuclear localization signals, a DNA-binding domain, and a carboxyl-terminal (CT) domain that harbors the adenosin triphosphate (ATP)-dependent DNA helicase (1). The CT is the most conserved domain of E1 and displays a set of three motifs conserved among large groups of DNA-dependent ATPases, such as helicase superfamily III and the DnaA family (15).

CD8⁺ T cell immune response plays a significant role in the clearance of HPV-infected cells (20). However, HPV can evade the immune system and promote a persistent infection (19,20). In fact, an important proportion of HPV-infected women do not develop a successful immune response against HR-HPV (19).

Although licensed HPV vaccines are effective to prevent infections with the viral types of the L1 proteins included in their formulation, they are not useful to treat HPV infections already in progress (11). Thus, experimental HPV vaccines for therapeutic use have been designed, mostly for the treatment of established HPV16 and HPV18 infections, which elicit an antigen-specific CD8⁺ T cell immune response to the E6 and/or E7 oncoproteins (21). Some of these therapeutic vaccines use novel adjuvants to achieve preferentially a cytotoxic T lymphocytes (CTL) response. The adjuvant KRN7000, a chemically synthesized α-galactosylceramide (α-GalCer), has shown superiority in promoting a CTL immune response 3 weeks after subcutaneous inoculation, compared with other adjuvants such as the incomplete Freund adjuvant, interferon-α, poly(I:C), or Toll-like receptor-9 (TLR-9) agonists, used in combination with internalization molecules such as the B subunit of Shiga toxin (2). Besides, α-GalCer alone has demonstrated to increase cross-presentation by dendritic cells, thus enhancing CD8⁺ T cell response against tumors generated in mice (14). α-GalCer can stimulate Natural killer T (NKT) cells, and when co-administered with proteins, it was shown to increase the population of antigen-specific CD8⁺ T cells (2,3,9). α-GalCer was shown to inhibit CTL responses up to 1 week after subcutaneous administration (2); nevertheless, it has been demonstrated that this effect changes drastically over time, promoting a strong CTL immune response after several weeks of α-GalCer subcutaneous administration (2,10).

Previously, we reported that the immunization of mice with the recombinant E1_202-654 protein along with α-GalCer induces an antigen-specific CD8⁺ T cell immune response, capable of eliminating B16-F0 melanoma cells that express HPV18 E1 protein (3). In the present study, we analyzed if CD8⁺ T cell from mice immunized with HPV18 E1 protein can cross-react against E1 from diverse HPV types.

Materials and Methods

Plasmids

The E1-CT domains from HPV types 6, 11,16, 31, 33, and 53 were amplified from pBR322-HPV6 and pBR322-HPV11 (kindly donated by E.M. de Villiers, Division of Episomal-Persistent DNA in Cancer and Chronic Diseases, Deutsches Krebsforschungszentrum, Heidelberg, Germany); pBR322-HPV16 (kindly donated by M. Dürst, Department of Gynecology, Friedrich Schiller University Hospital, Jena, Germany); pT712-HPV31 and pLink-HPV33 (kindly donated by Prof. H. zur Hausen, Division of Episomal-Persistent DNA in Cancer and Chronic Diseases, Deutsches Krebsforschungszentrum, Heidelberg, Germany); and pEMBL-HPV53 (kindly donated by L. Gissmann, Deutsches Krebsforschungszentrum, Heidelberg, Germany). The CT domain from HPV18 E1 was amplified from a codon-optimized HPV18 E1 plasmid (3). Polymerase chain reaction products were cloned into pcDNA 3.3 Topo plasmid (Cat no. K830001; Invitrogen; Thermo Fisher) with a Kozak sequence and human influenza hemagglutinin (HA) tag, to obtain pE1-CT/HPV6HA, pE1-CT/HPV11HA, pE1-CT/HPV16HA, pE1-CT/HPV18HA, pE1-CT/HPV31HA, pE1-CT/HPV33HA, and pE1-CT/HPV53HA. The plasmid constructions were confirmed by sequencing.

Cell culture and transfection

B16-F0 cell line (Cat no. CRL-6322; ATCC) was grown in Dulbecco's modified Eagle's medium F12 supplemented with 10% fetal bovine serum (FBS) and transiently transfected with pE1-CT/HPV6HA, pE1-CT/HPV11HA, pE1-CT/HPV16HA, pE1-CT/HPV18HA, pE1-CT/HPV31HA, pE1-CT/HPV33HA, pE1-CT/HPV53HA, or pcDNA3.1, as previously described (3).

Western blot analysis

Protein extracts from B16-F0 cells transiently transfected with pE1-CT/HPV plasmids or pcDNA3.1 control vector were loaded onto sodium dodecyl sulfate –polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Afterward, membranes were sequentially incubated with rabbit polyclonal anti-HA antibody (Cat no. 3724; Cell Signaling Technology) and horseradish peroxidase-labelled goat anti-rabbit antibody (Cat no. sc-2030; Santa Cruz Biotechnology). The E1 proteins were revealed by enhanced chemiluminescence (Cat no. RPN2236; Amersham; GE Healthcare Life Sciences). β-Actin was used as loading control (Cat no. sc-47778; Santa Cruz Biotechnology) followed by mouse IgG kappa conjugated to horseradish peroxidase (Cat no. sc-516102; Santa Cruz Biotechnology). Western blotting was performed in triplicate in every case to ensure reproducibility.

E1_202-654 recombinant protein

The E1_202-654 protein that contains the DNA-binding and CT domains was produced by GenScript as previously described (3).

Major Histocompatibility complex class I (MHC-I) peptide-binding prediction and multiple sequence alignment

The RANKPEP server (http://imed.med.ucm.es/Tools/rankpep.html) was used to predict peptide-binding affinities to mouse H2-D^b and H2-K^b, with 2% threshold. We considered all possible immunogenic peptides with a percentile score (% OPT) ≥1%. Peptides from 8 to 11 amino acid residues were analyzed in E1-aligned sequences using Clustal Omega multiple sequence alignment (https://www.ebi.ac.uk/Tools/msa/clustalo/), to find conserved immunogenic peptides among the E1-CT domains of HPV6, 11, 16, 18, 31, 33, and 53. Clustal Omega multiple sequence alignment analysis was also used to determine the homology of E6, E7, E2, and E1-CT genes among different HPV types.

Mice and immunizations

Female C57BL/6 mice were purchased from Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), Mexico City, Mexico. The Ethics Committees of the Universidad Nacional Autónoma de México (UNAM, Mexico City) and of the Instituto Nacional de Cancerología, Mexico City, approved the procedures for care and use of animals. All applicable institutional regulations concerning the ethical use of animals were followed accordingly. Seven- to 8-week-old female mice, distributed in groups of four mice each, were immunized as previously described (3): (i) E1_202-654 group: immunized with recombinant E1_202-654; (ii) E1_202-654 plus α-GalCer group: immunized with recombinant E1_202-654 plus α-GalCer; (iii) α-GalCer group: inoculated with α-GalCer; and (iv) phosphate-buffered saline (PBS) group: inoculated with PBS. This animal experiment was replicated two additional times with identical design.

Preparation of murine splenocytes and CD8⁺ T cell isolation

Seven days after the last immunization, the mice were euthanized, and murine spleens were excised to proceed with the preparation of splenocytes, followed by CD8⁺ T cell isolation using the CD8α ⁺ T Cell Isolation Kit II (Cat no. 130104075; Miltenyi Biotec) as previously described (3). After magnetic isolation, the purity of CD8⁺ T cells was evaluated by staining with CD8⁺/Brilliant Violet 421™ and CD3⁺/APC, obtaining a purity of 94%. Cells of each mice group were pooled.

Direct ex vivo cytotoxic activity assay for CD8⁺ T cells

B16-F0 cells transiently transfected with pE1-CT/HPV6HA, pE1-CT/HPV11HA, pE1-CT/HPV16HA, pE1-CT/HPV31HA, pE1-CT/HPV33HA, pE1-CT/HPV53HA, or pE1-CT/HPV18HA were co-cultured with CD8⁺ T cells (with a viability ≥90%) previously isolated from mice immunized with recombinant HPV18 E1_202-654 protein plus α-GalCer, or HPV18 E1_202-654 protein, or α-GalCer or PBS, to perform the cytotoxic activity assay as previously described (3). The appearance of CD107a/b on cell surface was assessed only in cells stained with Brilliant Violet 421 -anti-CD8a. With this analysis, we assure that cytotoxic activity was by CD8⁺ T cells.

Flow cytometry

Samples from all assays were acquired in a FACSAria II flow cytometer (Becton Dickinson) using FACS Diva software 8.0.1 and analyzed with FlowJo software (version 10.2). Typically, 40,000 events were acquired for cytotoxic activity.

For the flow cytometry panel and lineage-specific panels, the following monoclonal antibodies were used: Brilliant Violet 421 anti-CD8a (Cat no. 100753; Clone 53-6.7), Allophycocyanin (APC)-anti-CD3 (Cat no. 100210; Clone 17A2), Alexa Fluor 488^® (AF488)-anti-CD107a (Cat no. 121608; Clone 1D4B), and Alexa Fluor 488 (AF488)-anti-CD107b (Cat no. 108510; Clone M3/84) (all from Biolegend, San Diego, CA).

Statistical analysis

Statistical analysis was performed by one-way analysis of variance and Dunnett's post-test (JMP^® 13.0.0 copyright 2016; SAS Institute, Inc.). Error bars represent standard error of the mean, and p values <0.05 were considered statistically significant.

Results

Expression of E1 proteins from HPV6, 11, 16, 18, 31, 33, and 53 in B16-F0 cells

The E1-CT domain was detected in B16-F0 cells after 24 h post-transfection with plasmids expressing E1 fragments from different HPV types. The expression of the E1-CT domain from HPV6, 11, 16, 18, 31, 33, and 53 was detected by Western blot as 32-kDa hybrid polypeptides (Fig. 1A). Protein extracts from B16-F0 cells transfected with empty vector were used as negative control. The B16-F0 cells expressing different types of E1 were used as targets to determine the CD8⁺ T cell cytotoxic activity from mice immunized with HPV18 E1.

FIG. 1.

Expression of E1-CT domains and cross-reactivity of CD8⁺ T cells against B16-F0 cells expressing E1-ct from several HPVs. (A) B16-F0 cells were transfected with pE1-CT/HPV6HA, pE1-CT/HPV11HA, pE1-CT/HPV16HA, pE1-CT/HPV31HA, pE1-CT/HPV33HA, pE1-CT/HPV53HA, or pE1-CT/HPV18HA. The CT domains were detected as 32-kDa hybrid polypeptides 24 h post-transfection by Western blot, using an anti-HA antibody. Protein extracted from B16-F0 cells transfected with an empty vector was used as negative control. β-Actin expression ensures that a similar amount of lysate was loaded in each lane. The acquisition of cell surface CD107a/b was measured as a CD8⁺ T cell cytotoxic activity marker. Splenic T CD8⁺ cells from mice immunized or inoculated with (B) HPV18 E1_202-654 protein plus α-GalCer, (C) PBS, (D) α-GalCer, and (E) E1_202-654, co-cultured for up to 4 h with B16-F0/E1-CT/HPV18HA, B16-F0/E1-CT/HPV53HA, B16-F0/E1-CT/HPV33HA, B16-F0/E1-CT/HPV16HA, B16-F0/E1-CT/HPV31HA, B16-F0/E1-CT/HPV11HA, B16-F0/E1-CT/HPV6HA, or B16-F0 in the presence of Alexa Fluor 488-anti-CD107a/b, Brilliant Violet 421-anti-CD8, and anti-CD3-APC. An increase in exposed cell surface CD107a/b indicates CD8⁺ T cell cytotoxic activity. The lower panels show representative flow cytometry images of cytotoxic activity assays where CD107a/b molecules are detected on the cell surface. Statistical significance with **p < 0.01 and ***p < 0.0001 by one-way ANOVA and Dunnett's post-test comparison is represented in the graph. α-GalCer, α-galactosylceramide; ANOVA, analysis of variance; APC, allophycocyanin; CT, carboxyl-terminal; HA, hemagglutinin; HPV, human papillomavirus; PBS, phosphate-buffered saline.

In silico analysis of immunogenic peptides of the CT domain of HPV18 E1

The E1-CT domain is highly conserved among different HPV types (1). Moreover, this region contains a set of three motifs highly conserved among a large group of DNA-dependent ATPases (15). Thus, we analyzed this domain in the RANKPEP server to predict peptides with the ability to bind to Major Histocompatibility Complex Class I (MHC-I) in a murine context. After obtaining a large variety of predicted peptides, we aligned the CT domains in the Clustal Omega server to find conserved peptides among E1 proteins from HPV types 6, 11, 18, 16, 31, 33, and 53. The analysis showed that at least 15 predicted peptides are conserved among the seven HPV E1 types (columns in Table 1). In addition, the different HPV types share other additional predicted peptides with HPV18, which are identical or highly homologous. For example, E1-CT/HPV53 shares 20 additional peptides; E1-CT/HPV31 shares 19 additional peptides; E1-CT/HPV33 shares 19 additional peptides; and E1-CT/HPV16 shares 19 additional peptides. LR-HPVs are known to share fewer additional peptides with HPV18; for example, E1-CT/HPV11 shares 14 peptides, while E1-CT/HPV6 shares only 7 peptides (data not shown).

Table 1.

Prediction of Conserved Peptides in the Carboxyl-Terminal Domains of Several HPV E1 Proteins That Bind MHC Class I Murine Proteins

	Peptide for MHC-I H2D^b	Peptide for MHC-I H2D^b and H2K^b	Peptide for MHC-I H2D^b	Peptide for MHC-I H2D^b	Peptide for MHC-I H2D^b and H2K^b	Peptide for MHC-I H2D^b	Peptide for MHC-I H2K^b	Peptide for MHC-I H2D^b
	10 aa	9 aa	8 aa	8 aa	9 aa	10 aa	9 aa	8 aa
E1-CT/HPV18	KCPP I LLTTN	NTGKSYFGM	PANTGKSY	KNGNPVYE	DSNSNAAAF	CGPANTGKSY	VQFLRYQQI	KNWKCFFE
E1-CT/HPV6	KCPP L L V T S N	D TGKSYFCM	PD D TGKSY	RNGNAVYE	DF D SNARAF	VGPP D TGKSY	VQFLR H QNI	ANWKCFFE
E1-CT/HPV11	KCPP L L V T S N	D TGKSCFCM	PD D TGKSC	RNGNAVYE	DF D SNARAF	VGPP D TGKSC	VQFLR H QNI	ANWKCFFE
E1-CT/HPV16	KCPP L L I T S N	NTGKSLFGM	AANTGKSL	KNGNPVYE	DT N SNASAF	YGAANTGKSL	VMFLRYQG V	KNWKSFFS
E1-CT/HPV31	KCPP L L I T S N	NTGKSYFGM	APNTGKSY	KNGNPVYE	DS D SNACAF	HGAPNTGKSY	VKFLRYQQI	KNWKSFFS
E1-CT/HPV33	KCPP L LLT S N	NTGKSYFGM	PANTGKSY	ENGNPVYA	DSNSNAAAF	CGPANTGKSY	VQ L LRYQNI	ENWKSFFS
E1-CT/HPV53	KCPP V L I TTN	NTGKSCFAM	PPNTGKSC	VNGNPVYQ	DV D SNAQAF	YGPPNTGKSC	VQFLRYQG V	ANWKCFFE

	Peptide for MHC-I H2D^b	Peptide for MHC-I H2K^b	Peptide for MHC-I H2K^b	Peptide for MHC-I H2K^b	Peptide for MHC-I H2K^b	Peptide for MHC-I H2D^b	Peptide for MHC-I H2K^b
	8 aa	9 aa	8 aa	8 aa	8 aa	9 aa	8 aa
E1-CT/HPV18	WKCFFERT	C K HY R RAQK	K HY R RAQK	RTWSRLDL	NSTSHFWL	GTPKKNCLV	SHFWLEPL
E1-CT/HPV6	WKCFFERL	C R HY K RAQQ	R HY KH A E M	RLSSSLDI	NSSSHFWL	GTPKKNC I A	SHFWL Q PL
E1-CT/HPV11	WKCFFERL	C R HY KK A E K	R HY KH A E M	RLSSSLDI	NSCSHFWL	GTPKKNC I A	SHFWL Q PL
E1-CT/HPV16	WKSFFSRT	C R HY K RA E K	R HY K RA E K	RTWSRLSL	NSKSHFWL	GIPKKNC I L	SHFWL Q PL
E1-CT/HPV31	WKSFFSRT	C R HY K RA E K	R HY K RA E K	RTWCRLNL	NSKSHFWL	GVPKKNC I L	SHFWL Q PL
E1-CT/HPV33	WKSFFSRT	C R HY KH A E M	R HY K RA E K	RTWCKLDL	NSKSHFWL	GIPKKSC M L	SHFWL Q PL
E1-CT/HPV53	WKCFFERT	C R HY KH A E M	R HY K RAQQ	RTWSRLDL	NSHSHFWL	GTPK H NCLV	SHFWLEPL

Bold indicates fully conserved residue with respect to the HPV18 sequence. Italics indicate residues with strongly similar properties with respect to the HPV18 sequence. Bold-italics indicate not conserved residue with respect to the HPV18 sequence.

HPV, human papillomavirus; MHC, Major Histocompatibility Complex Class I.

Cross-reactivity against other HPV types

To determine if CD8⁺ T cells from mice immunized with HPV18 E1 could have cytotoxic activity against other E1 HPV types, splenic CD8⁺ T cells from mice immunized with HPV18 E1_202-654 plus α-GalCer were co-cultured with B16-F0 cells expressing the CT domains from HPV6, 11, 16, 31, 33, and 53. As a negative control, non-transfected B16-F0 cells were used; and as a positive control, B16-F0 cells expressing E1-CT/HPV18HA were used, as previously reported (3). Activation-induced granule exocytosis leads to the exposure of CD107a and CD107b on the surface of CD8⁺ T cells (5). Co-cultures were monitored up to 4 h, time at which they reached the highest CD107a/b cell surface expression.

A significant increase in cell surface exposure of CD107a/b was detected in the co-cultures of CD8⁺ T cells with B16-F0 cells individually expressing E1 proteins of HPV types 53, 33, 16, 31, and 11, in relation to the co-culture with B16-F0 non-transfected cells (Fig. 1B). The highest percentage of cell surface CD107a/b exposure was obtained in the co-cultures of cells expressing E1-CT/HPV53, and the lowest was obtained for E1-CT/HPV11. No cell surface exposure of CD107a/b was detected in splenic CD8⁺ T cells when co-cultured with cells expressing E1-CT/HPV6 (Fig. 1B). These data indicate that CD8⁺ T cells from mice immunized with HPV18/E1 plus α-GalCer can also display cytotoxic activity against cells expressing E1-CT/HPV53HA, E1-CT/HPV33HA, E1-CT/HPV16HA, E1-CT/HPV31HA, and E1-CT/HPV11HA.

Splenic CD8⁺ T cells from mice injected with PBS (Fig. 1C), α-GalCer (Fig. 1D), or immunized exclusively with HPV18 E1_202-654 protein (Fig. 1E) showed no cytotoxic activity against any E1-CT domain. Therefore, immunization with HPV18/E1 promoted a cytotoxic response against E1 from different HPV types, only when combined with α-GalCer.

Discussion

Cervical cancer is the fourth cause of cancer death in women worldwide (13), and HPV is the main etiological agent. The current efforts to develop therapeutic vaccines against HPV are mainly directed against E6 and/or E7 from HPV16 and/or HPV18. Since E6 and E7 HPV proteins are poorly conserved among HPV types (Supplementary Table S1), their cross-reactivity with other HPV types could be limited. A therapeutic vaccine based on the most conserved HPV proteins, such as E1, could ideally elicit a wider-spectrum protection against several HPV types.

In the current study we evaluated ex vivo the ability of HPV18 E1 antigen-specific CD8⁺ T cells induced in mice to cross-react against B16-F0 cells expressing E1 proteins from other HPV types. Through in silico analysis we found highly conserved peptides, which would predictably bind to MHC class I proteins, in the CT domain of E1 proteins from HPV6, 11, 16, 18, 31, 33, and 53 (Table 1). It is remarkable that the CT domains from HR-HPV types (16, 31, 33, and 53) share with E1-CT/HPV18 more predicted peptides that bind to MHC class I proteins (data not shown), while the CT domains from LR-HPV types (11 and 6) share less peptides. This latter data matches the results obtained from ex vivo cytotoxic activity assays, where splenic CD8⁺ T cells from mice immunized with HPV18 E1_202-654 plus α-GalCer, co-cultured with B16-F0 expressing E1-CT domains from HPV types 53, 33, 16, or 31, showed the highest cell surface CD107a/b exposure, while cells expressing E1-CT from HPV11 and HPV6 showed the lowest cell surface CD107a/b exposure (Fig. 1B).

Nevertheless, although cross-reactive cytotoxic activity against cells expressing HPV type 11 was detected, it was non-significant (2.11%). Thus, these data suggest that immunization with E1 from HPV18 could contribute to the clearance of low-grade and high-grade lesions associated with different HPV types (4), when MHC class I molecules are still presenting E1 antigens. It is worth mentioning that most of the low-grade cervical intraepithelial lesions express E1 and E2 proteins (12,17). Although it has been reported that the expression of E2 decreases while the lesions advance, it has been shown that E1 is expressed in high-grade lesions and cervical cancer (4,12).

We previously demonstrated that immunizing mice with HPV18 E1 plus α-GalCer promoted an E1-specific CD8⁺ T cell cytotoxic response, involved in growth inhibition of melanoma B16-F0 cells expressing HPV18 E1 antigens (3). Therefore, to test its potential as a broad-spectrum protector, it would be necessary to analyze whether the inhibition of tumor growth could also be achieved in a murine model of grafted B16-F0 cells expressing E1 antigens of various HPV types.

In summary, this report provides evidence that immunization with HPV18 E1_202-654 protein plus α-GalCer can elicit a cross-immune CTL response against the E1 protein from diverse HPV types.

Our data support further studies on therapeutic vaccines capable of generating effective and broad-spectrum protection against HPV, based on cross-reactive CTL immune responses against early-expressed HPV proteins.

Footnotes

Acknowledgments

Alfredo Amador Molina is a doctoral student at “Programa de Maestría y Doctorado en Ciencias Bioquímicas, Universidad Nacional Autónoma de México.” Funding: This work was supported by Instituto Nacional de Cancerología, Mexico City (017/001/IBI) (CEI/1076/17).

Ethical Statement

The Ethics Committees of the Universidad Nacional Autónoma de México (UNAM, Mexico City) and of the Instituto Nacional de Cancerología, Mexico City (017/001/IBI), approved the procedures for care and use of animals. All applicable institutional regulations concerning the ethical use of animals were followed accordingly.

Author Disclosure Statement

No competing financial interests exist.

Supplementary Material

Supplementary Table S1

References

Abbate

, Berger

, and Botchan

. The X-ray structure of the papillomavirus helicase in complex with its molecular matchmaker E2. Genes Dev, 2004; 18:1981–1996.

Adotevi

, Vingert

, Freyburger

, et al. B subunit of Shiga toxin-based vaccines synergize with alpha-galactosylceramide to break tolerance against self antigen and elicit antiviral immunity. J Immunol, 2007; 179:3371–3379.

Amador-Molina

, Trejo-Moreno

, Romero-Rodriguez

, et al. Vaccination with human papillomavirus-18 E1 protein plus alpha-galactosyl-ceramide induces CD8(+) cytotoxic response and impairs the growth of E1-expressing tumors. Vaccine, 2019; 37:1219–1228.

Baedyananda

, Chaiwongkot

, and Bhattarakosol

. Elevated HPV16 E1 expression is associated with cervical cancer progression. Intervirology, 2017; 60:171–180.

Betts

, Brenchley

, Price

, et al. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods, 2003; 281:65–78.

Clifford

, Smith

, Aguado

, et al. Comparison of HPV type distribution in high-grade cervical lesions and cervical cancer: a meta-analysis. Br J Cancer, 2003; 89:101–105.

Clifford

, Smith

, Plummer

, et al. Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis. Br J Cancer, 2003; 88:63–73.

Demeret

, Goyat

, Yaniv

, et al. The human papillomavirus type 18 (HPV18) replication protein E1 is a transcriptional activator when interacting with HPV18 E2. Virology, 1998; 242:378–386.

Dolen

, Kreutz

, Gileadi

, et al. Co-delivery of PLGA encapsulated invariant NKT cell agonist with antigenic protein induce strong T cell-mediated antitumor immune responses. Oncoimmunology, 2016; 5:e1068493.

10.

Guillonneau

, Mintern

, Hubert

, et al. Combined NKT cell activation and influenza virus vaccination boosts memory CTL generation and protective immunity. Proc Natl Acad Sci U S A, 2009; 106:3330–3335.

11.

Herrero

. Human papillomavirus (HPV) vaccines: limited cross-protection against additional HPV types. J Infect Dis, 2009; 199:919–922.

12.

, Zhu

, Wang

, et al. Genome-wide profiling of HPV integration in cervical cancer identifies clustered genomic hot spots and a potential microhomology-mediated integration mechanism. Nat Genet, 2015; 47:158–163.

13.

International Agency for Research on Cancer WHO. Estimated number of deaths in 2018, worldwide, all cancers, female, all ages. http://gco.iarc.fr/today/home (accessed on February 5, 2019 ).

14.

Kogo

, Shimizu

, Negishi

, et al. Suppression of murine tumour growth through CD8(+) cytotoxic T lymphocytes via activated DEC-205(+) dendritic cells by sequential administration of alpha-galactosylceramide in vivo. Immunology, 2017; 151:324–339.

15.

Koonin

. A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication. Nucleic Acids Res, 1993; 21:2541–2547.

16.

Parkin

, Bray

, Ferlay

, et al. Global cancer statistics, 2002. CA Cancer J Clin, 2005; 55:74–108.

17.

Romanczuk

, and Howley

. Disruption of either the E1 or the E2 regulatory gene of human papillomavirus type 16 increases viral immortalization capacity. Proc Natl Acad Sci U S A, 1992; 89:3159–3163.

18.

Sano

, and Oridate

. The molecular mechanism of human papillomavirus-induced carcinogenesis in head and neck squamous cell carcinoma. Int J Clin Oncol, 2016; 21:819–826.

19.

Stanley

, Pett

, and Coleman

. HPV: from infection to cancer. Biochem Soc Trans, 2007; 35:1456–1460.

20.

Tindle

. Immune evasion in human papillomavirus-associated cervical cancer. Nat Rev Cancer, 2002; 2:59–65.

21.

Yang

, Farmer

, Wu

, et al. Perspectives for therapeutic HPV vaccine development. J Biomed Sci, 2016; 23:75.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.02 MB

0.00 MB

HPV18 E1 Protein Plus α -Galactosylceramide Elicit in Mice CD8 + T Cell Cross-Reactivity Against Cells Expressing E1 from Diverse Human Papillomavirus Types

Abstract

Introduction

Materials and Methods

Plasmids

Cell culture and transfection

Western blot analysis

E1202-654 recombinant protein

Major Histocompatibility complex class I (MHC-I) peptide-binding prediction and multiple sequence alignment

Mice and immunizations

Preparation of murine splenocytes and CD8+ T cell isolation

Direct ex vivo cytotoxic activity assay for CD8+ T cells

Flow cytometry

Statistical analysis

Results

Expression of E1 proteins from HPV6, 11, 16, 18, 31, 33, and 53 in B16-F0 cells

In silico analysis of immunogenic peptides of the CT domain of HPV18 E1

Cross-reactivity against other HPV types

Discussion

Footnotes

Acknowledgments

Ethical Statement

Author Disclosure Statement

Supplementary Material

References

Supplementary Material

E1_202-654 recombinant protein

Preparation of murine splenocytes and CD8⁺ T cell isolation

Direct ex vivo cytotoxic activity assay for CD8⁺ T cells