Use of Adjuvants to Enhance the Immune Response Induced by a DNA Vaccine Against Bovine Herpesvirus-1

Introduction

Bovine herpesvirus-1 (BoHV-1) is the causative agent of infectious bovine rhinotracheitis (IBR), a cattle disease with important economic impact. The clinical symptoms include rhinotracheitis, pustular vulvo-vaginitis/balanoposthitis, neurological disease, abortion, and high predisposition to secondary bacterial infections leading to high morbidity and mortality (5,22).

It is reported that antibodies could contribute to prevention of viral spread to susceptible cells by antibody-dependant cytotoxicity (1). Nevertheless, BoHV-1 spreads intracellularly. Therefore, it can exist in presence of virus-specific antibodies (4,11). For this reason, cytotoxic T lymphocytes (CTL) are critical for elimination of the virus (17).

Inactivated vaccines do not provide complete protection, since they generally are poor inducers of cellular immunity. Regarding live-attenuated vaccines, they are thought to induce higher levels of protective immunity, but they are not entirely safe (20). In this context, DNA vaccines have emerged as an attractive approach.

gD is one of the BoHV-1 envelope glycoproteins. It has been highlighted as the best antigen for a DNA vaccine against BoHV-1 (18). A construct encoding the secreted form of the protein was able to induce a higher immune response than the complete gD sequence (9,10).

However, the potency of naked DNA vaccines is known to be limited (14). The report at hand was designed to test a plasmid encoding the BoHV-1 secreted gD with different commercial adjuvants (Seppic), in its ability to enhance viral-specific humoral and cellular immune response in mice and in a preliminary assay in cattle, in order to select adjuvant candidates for a challenge trial in bovines. Mice have been used regularly as an experimental model for the study of immune responses toward different types of viruses (8,16), reducing the costs and time involved in the use of large animals and as a first step to select the appropriate conditions for larger experiments involving host species. Since the difficulty of performing assays for measuring cytotoxicity mediated for T lymphocytes (CTL) in outbred animals, mice have been widely used as a model for assaying cellular immune responses and CTL epitopes have been identified for BoHV-1 in mice (2,3,6,8,12,13,21,23).

Vaccines were formulated using 15 μg/dose of a secreted gD coding plasmid (pCIgD) according to previous dose–response results (8) and different adjuvants following the manufacturer's indications (Seppic): Montanide Essai IMS 1312 VG (1312), Montanide Essai 903107 (107), Montanide Essai 903108 (108), Montanide Essai 903109 (109), Montanide Essai 903110 (110), Montanide Essai ISA 206 VG (ISA 206), Montanide ISA 201 VG (ISA 201), Montanide GEL 01 PR (GEL 01), Montanide ISA 71 VG (ISA 71), or Montanide ISA 25 VG (ISA 25). The resulting vaccines were named according to the adjuvant included in each formulation.

Montanide adjuvants are considered as safe by the Committee for Veterinary Medical Products (CVMP) for use in immunological products and are included in Annex of European Council Regulation no. 470/2009 (previously no. 2377/90/EC). Adjuvants classified as IMS are dispersions of liquid particles, varying in size between 50 and 500 nm, in an aqueous phase containing an immunostimulating compound. Adjuvants classified as ISA are used to formulate different type of emulsions: water in oil, oil in water, or water in oil in water, and they are based on mineral oil, non-mineral oil, or a mixture of both, with a surfactant based on mannitol oleate. All adjuvants used in this work were selected according to data provided by the manufacturer (Seppic) indicating that these adjuvants were designed to improve cellular immune responses and some of them to improve DNA binding and transfection.

Groups of five BALB/c mice were intradermally (i.d.) inoculated with 0.2 mL of the pCIgD plus mentioned adjuvants, pCIgD alone, or empty pCIneo plasmid (Promega; negative control). Fourteen days after the first vaccination, the mice received a booster vaccination. Analysis of sera by anti-gD enzyme-linked immunosorbent assay (ELISA; Fig. 1A) showed that vaccines GEL 01, 107, 108, 109, 110, and ISA 206 at 14 days post-vaccination (d.p.v.) and all vaccines and pCIgD at 28 d.p.v. were able to induce anti-gD antibody titers (Abs) >2. However, only vaccine GEL01 at 14 and 28 d.p.v. and vaccines 107, 109, and 110 at 28 d.p.v. induced significantly higher anti-gD Abs levels than pCIgD.

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FIG. 1.

(A) Serum antibody titers specific for gD after vaccination in mice. Bars represent the mean antibody titer±standard error of the mean (SEM). Antibodies were detected using an indirect enzyme-linked immunosorbent assay (ELISA). Briefly, Immulon 2HB plates were coated with 1.4 μg/well recombinant gD diluted in carbonate–bicarbonate buffer. Plates were washed and blocked with phosphate-buffered saline (PBS) containing 0.05% Tween 80 and 1% ovoalbumin (PBST-OVA). After washing, serum samples diluted in PBST-OVA were incubated on the plates. Negative and positive control sera were included. A peroxidase-labeled antimouse rabbit antiserum diluted in PBST-OVA was added. After washing, o-phenylenediamine-H₂O₂ was used as peroxidase substrate. Absorbance was recorded at 492 nm. Titers were expressed as the log₁₀ of the reciprocal of the dilution whose optical density (OD) is higher than the cutoff. Cutoff was calculated as mean OD+2 SD of sera from negative mice. Significant differences are *p<0.05 and **p<0.01 regarding pCIgD group as established by analysis of variance (ANOVA) and Dunnet post-ANOVA tests. The dotted line represents an antibody titer of 1.8. (B) T cell proliferation assay. Briefly, spleen cells were isolated from vaccinated mice and stimulated in vitro for 3 days with gD protein. Then, ³H-thymidine was incorporated, and after 18 h, radioactivity incorporation was measured using a scintillation counter. The proliferation index was calculated as: [³H-thymidine incorporated with stimulus/³H-thymidine incorporated without stimulus]. The results are expressed as mean proliferation index±SEM. The dotted line represents a cutoff value of 2.5 selected based in data from bibliography (13,15). (C) IFN-γ secretion by spleen cells after 48 h of in vitro incubation with inactivated BoHV-1. IFN-γ was measured in supernatants using a commercial ELISA kit (BD Biosciences). OD of each sample was interpolated in a standard curve. Results are expressed as the mean value of IFN-γ secretion in pg/mL±SEM. BoHV-1, bovine herpesvirus-1; SEM, standard error of the mean.

To study the cell-mediated immune responses induced by the vaccines, spleen cells from immunized mice were removed at 30 d.p.v., stimulated in vitro with gD protein or inactivated BoHV-1 and evaluated for antigen-specific proliferation and IFN-γ secretion. As shown in Figure 1B, vaccines GEL 01 and 110 induced qualitatively higher proliferation indexes than other groups. IFN-γ secretion was evaluated in spleen cell culture supernatants after stimulation with inactivated BoHV-1 by a commercial ELISA kit (e-bioscience). As shown in Figure 1C, vaccines GEL 01 and 110 seemed to induce the highest IFN-γ secretion values, indicating that the respective adjuvants are able to enhance the cellular immune response. When IL4 secretion was assayed, no differences were found between groups (data not shown).

Taking together, these results indicate that pCIgD vaccine formulated with adjuvants GEL 01 and 110 could induce a higher humoral and cellular response against BoHV-1 in mice. Therefore, these adjuvants were further tested in a preliminary trial in bovines, natural host species.

A dose of 500 μg pCIgD was chosen based on a dose–response curve (data not shown). Bovines (1–2 years old, Angus×Hereford breed) were vaccinated i.d. with 1.5 mL of pCIgD alone (n=3), pCIgD formulated with GEL 01 (n=3) or 110 (n=3), pCIneo as negative control (n=3), or an inactivated commercial vaccine (n=3). Fifteen days after the first vaccination, bovines received a booster vaccination. The highest antibody titers against BoHV-1 were recorded at 21 d.p.v. in the commercial group. Bovines in groups pCIgD, GEL01, and 110 had similar antibody levels (Fig. 2A). No differences between groups were found when neutralizing antibodies were measured (data not shown). Nevertheless, when IFN-γ secretion was evaluated at 21 d.p.v. in peripheral blood mononuclear cells (PBMCs) after in vitro stimulation with inactivated BoHV-1, group 110 showed the highest IFN-γ secretion levels (Fig. 2B). This result indicates that adjuvant 110 is capable of improving cellular immune response elicited by pCIgD in bovines, which could improve the protection against BoHV-1 infection. Therefore, adjuvant Montanide Essai 903110 plus gD vaccine was selected for another trial with larger number of bovines and to perform a viral challenge experiment. It was previously reported that adjuvants can selectively induce T helper subtypes and trigger cellular uptake and processing mechanisms that facilitate MHC class I pathway loading of antigen and priming CD8+ cytolytic T cell responses. Examples of this kind of adjuvants are saponins and immune-stimulating complexes (15) such as the ones present in 110 formulation.

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FIG. 2.

(A) Serum antibody titers specific for BoHV-1 after vaccination in cattle. Bars represent the mean antibody titer±SEM. Antibodies were detected using an indirect ELISA. Briefly, partially purified BoHV-1 was prepared from Madin–Darby bovine kidney (MDBK) cells infected with BoHV-1. Infected cells and culture fluids were frozen/thawed and clarified. Supernatants were pelleted by ultracentrifugation at 120,000 g for 1 h at 4°C. The antigen preparations were diluted in carbonate–bicarbonate buffer (pH 9.6). Immulon I microtiter plates were coated with the antigen for 16 h at 4°C. Plates were washed and then incubated with serum samples diluted in PBST-OVA. Rabbit antibovine serum labeled with peroxidase was added. After washing, o-phenylenediamine-H₂O₂ was used as peroxidase substrate. Absorbance was recorded at 492 nm. Titers are expressed as the log₁₀ of the reciprocal of the dilution whose OD is higher than the cutoff. Cutoff was calculated as mean OD±2 SD of sera from negative bovines. A positive control was added in every plate. (B) IFN-γ secretion by bovine peripheral blood mononuclear cells (PBMCs). PBMCs were isolated by centrifugation of blood on Ficoll-Paque™ PLUS. After 48 h of in vitro incubation with inactivated BoHV-1, supernatants were collected and IFN-γ was measured using a sandwich ELISA. Briefly, Immulon II plates were coated with a monoclonal anti-IFN-γ antibody (kindly donated by Dr. Babiuk) in carbonate–bicarbonate buffer. Plates were washed and blocked with PBST-0.1% bovine serum albumin (BSA; PBST-BSA). Dilutions of samples and recombinant IFN-γ standard (Serotec) were added. After incubation, plates were washed and incubated with rabbit polyclonal anti-IFN-γ antibodies (produced in the authors' lab) after a subsequent washing cycle. Biotin-conjugated antibody antirabbit IgG was added. After incubation, peroxidase-conjugated streptavidine (KPL) was added. After washing, o-phenylenediamine-H₂O₂ was used as peroxidase substrate. OD of each sample was determined at 492 nm and was interpolated in a standard curve. Results are expressed in pg/mL. The black line in each group represents the mean value of IFN-γ secretion.

Montanide Gel 01 is composed of cross-linked polymers that form highly stable micron size aggregates of sodium polycrylate in water (19). Montanide ESSAI 903110 consists of non-cross-linked charged polymers (10–500 nm) dispersed in water containing an immunostimulating compound. It is designed to improve DNA binding capacities and transfection potency (Dupuis L., pers. comm.). On the other hand, it is reported that the immunostimulating compounds contained in both adjuvants recruit cells to the site of injection through inflammatory processes (19). Although the immune mechanisms exerted by these adjuvants remain unclear, an increase of antigen presenting cells at the inoculation site, together with an improved transfection capacity of DNA vaccine, could explain the higher immunity elicited by the combination of pCIgD and these adjuvants.