gD Subunit Vaccine Research Articles

Comparison of effects of multiple adjuvants and immunization routes on the immunogenicity and protection of HSV-2 gD subunit vaccine

Genital herpes caused by herpes simplex virus type 2 (HSV-2) poses a global health issue. HSV-2 infection increases the risk of acquiring HIV infection. Studies have demonstrated that HSV-2 subunit vaccines have potential benefits, but require adjuvants to induce a balanced Th1/Th2 response. To develop a novel, effective vaccine, in this study, a truncated glycoprotein D (aa 1–285) of HSV-2 was formulated with an Al(OH)3 adjuvant, three squalene adjuvants, zMF59, zAS03, and zAS02, or a mucosal adjuvant, bacterium-like particles (BLPs). The immunogenicity of these subunit vaccines was evaluated in mice. After three immunizations, vaccines formulated with Al(OH)3, zMF59, zAS03, and zAS02 (intramuscularly) induced higher titers of neutralizing antibody than that formulated without adjuvant, and in particular, mice immunized with the vaccine plus zAS02 had the highest neutralizing antibody titers and tended to produce a more balanced immune reaction than others. Intranasal gD2-PA-BLPs also induced excellent IgA levels and a more balanced Th1 and Th2 responses than intranasal gD2. After challenge with a lethal dose of HSV-2, all five adjuvants exhibited a positive effect in improving the survival rate. zAS02 and gD2-PA-BLPs enhanced survival by 50% and 25%, respectively, when compared with the vaccine without adjuvant. zAS02 was the only adjuvant that resulted in complete vaginal virus clearance and genital lesion healing within eight days. These results demonstrate the potential of using zAS02 as a subunit vaccine adjuvant, and BLPs as a mucosal vaccine adjuvant.

Efficacy of a gB + gD-based subunit vaccine and the adjuvant granulocyte-macrophage colony stimulating factor for pseudorabies virus in rabbits.

Pseudorabies (PR), which is caused by the pseudorabies virus (PRV), is a severe infectious disease that causes abortions in adult sows and fatal encephalitis in piglets; the disease can occur in pigs of all ages and other mammals, which can lead to significant economic loss around the worldwide. The new PRV variant invalidated the available commercial attenuated and inactivated vaccines. Consequently, subunit vaccines have been suggested as novel strategies for PR control, while they are usually formulated with adjuvants due to their lower immunogenicity. We aimed to select a safe and efficient adjuvant for subunit vaccines for PR. In our study, glycoprotein B (gB) and glycoprotein D (gD) were expressed based on a baculovirus expression system, and granulocyte-macrophage colony-stimulating factor (GM-CSF) was expressed using an Escherichia coli (E. coli) expression system; subsequently, a gB + gD subunit vaccine adjuvanted by GM-CSF was constructed. A rabbit model infected with a PRV SD-2017 strain was established, the TCID50 and LD50 were measured, and the typical clinical symptoms were observed. After a lethal challenge of 5 LD50 with a PRV SD-2017 strain, the rabbits exhibited typical clinical symptoms, including itching and high temperature, and histopathology revealed severe inflammation in the brain, which is the dominant target organ of PRV. Rabbits immunized with the gB + gD + GM-CSF subunit vaccines produced higher levels of antibodies than those immunized with gB + gD + ISA 201, which was adjuvanted with a frequently used oil adjuvant. The survival rate of rabbits vaccinated with gB + gD + GM-CSF was 100%, which was superior to that of rabbits vaccinated with gB + gD + ISA 201 (80%), inactivated PRV + GM-CSF (60%) and commercial inactivated vaccine (60%) after challenge with PRV SD-2017. These data suggested that the gB + gD + GM-CSF-based subunit vaccine had good protective efficacy against the PRV SD-2017 strain in rabbits and that GM-CSF could be developed as a candidate adjuvant for use in a vaccine regimen to prevent and even eradicate PR.

Prophylactic herpes simplex virus type 2 vaccine adjuvanted with a universal CD4 T cell helper peptide induces long-term protective immunity against lethal challenge in mice

Induction of robust and long-term immune responses at the portal of entry remains a big challenge for HSV-2 vaccine development. The adoption of a CD4 T cell helper peptide in the vaccine is thought to be beneficial for the enhancement of immune responses, however, its effect on HSV-2 vaccines has not yet been studied. In this study, we designed a DNA vaccine (gD-TpD) simultaneously expressing HSV-2 gD ectodomain and a universal CD4 T cell helper peptide (TpD), and tested its efficacy on a murine model. Mice were immunized 3 times with gD-TpD or control DNA formulations, and then were rested until Day 150 when they were vaginally challenged with lethal doses of HSV-2. Our data showed that gD-TpD significantly increased gD-specific IgG and IgA in both sera and vaginal washes. Furthermore, the increased antibody responses showed enhanced neutralization activity in vitro. In addition, gD-TpD induced balanced Th1/2 cellular responses and CD8+ T cell-dependent CTL activity. Although immune responses dropped over time after the final immunization, robust and rapid antibody and T cell responses were induced upon virus challenge in gD-TpD group. Moreover, gD-TpD provided full protection against lethal viral challenge in immunized mice. Together, our findings indicate that the inclusion of the CD4 T cell helper peptide TpD in HSV-2 gD subunit vaccine could induce long-term protective immunity, providing information for a rational design of vaccines against HSV-2 or even other viruses.

Expression and characterization of the gD protein of HSV-2 fused to the tetramerization domain of the transcription factor p53

The highly immunogenic glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) is a very important element for entry of this virus into host cells. These characteristics have made this protein a very interesting HSV-2 subunit vaccine candidate. Despite efforts to prevent genital herpes using gD-based subunit vaccines, to date, clinical trials using this antigen have failed. Therefore, using a small animal model, we sought to determine if a tetramerized truncated form of gD subunit vaccine, produced by recombinant baculovirus infected insect larvae, would elicit better protection against genital herpes than a monomeric gD-2 subunit vaccine. Three out of 5 mice immunized with the tetramerized antigen produced in a baculovirus expression vector system, survived a lethal challenge with a wild type HSV-2 strain (for more than 3weeks after challenge). In contrast, all the mice (5) immunized with the truncated protein, produced by the same methodology, died within 2weeks after challenge. These results suggest that multimerization (increasing the structural complexity) of the truncated gD antigen might be more likely protective than the monomer form. Also the use of an alternative cost-efficient eukaryotic expression system is described.

Herpes simplex virus 2 (HSV-2) infected cell proteins are among the most dominant antigens of a live-attenuated HSV-2 vaccine.

Virion glycoproteins such as glycoprotein D (gD) are believed to be the dominant antigens of herpes simplex virus 2 (HSV-2). We have observed that mice immunized with a live HSV-2 ICP0 - mutant virus, HSV-2 0ΔNLS, are 10 to 100 times better protected against genital herpes than mice immunized with a HSV-2 gD subunit vaccine (PLoS ONE 6:e17748). In light of these results, we sought to determine which viral proteins were the dominant antibody-generators (antigens) of the live HSV-2 0ΔNLS vaccine. Western blot analyses indicated the live HSV-2 0ΔNLS vaccine elicited an IgG antibody response against 9 or more viral proteins. Many antibodies were directed against infected-cell proteins of >100 kDa in size, and only 10 ± 5% of antibodies were directed against gD. Immunoprecipitation (IP) of total HSV-2 antigen with 0ΔNLS antiserum pulled down 19 viral proteins. Mass spectrometry suggested 44% of immunoprecipitated viral peptides were derived from two HSV-2 infected cells proteins, RR-1 and ICP8, whereas only 14% of immunoprecipitated peptides were derived from HSV-2’s thirteen glycoproteins. Collectively, the results suggest the immune response to the live HSV-2 0ΔNLS vaccine includes antibodies specific for infected cell proteins, capsid proteins, tegument proteins, and glycoproteins. This increased breadth of antibody-generating proteins may contribute to the live HSV-2 vaccine’s capacity to elicit superior protection against genital herpes relative to a gD subunit vaccine.

Biolistic-mediated gene transfer using the bovine herpesvirus-1 glycoprotein D is an effective delivery system to induce neutralizing antibodies in its natural host

A genetic vaccine consisting of the bovine herpesvirus-1 (BHV-1) glycoprotein D (gD) gene was constructed and administered to cattle using the biolistic (gene-gun) process. Results were compared to standard intramuscular injection of an inactivated whole BHV-1 commercial vaccine. Cattle genetically immunized by the gene-gun-delivered gD subunit vaccine developed high titers of IgG antibodies specific to gD demonstrating that this immunization method is a potent humoral response inducer. Further, gene-gun vaccinated cattle produced high neutralizing antibody titers to BHV-1 similar to levels induced in the commercial vaccine immunized animals. Additionally, cellular immunity was measured by an increased level of IFN-γ mRNA detected in PBMC of cattle immunized with the gD gene or with the commercial vaccine, whereas augmented levels of IL-4 were not detected following vaccination. Because of its simplicity and effectiveness in inducing an immune response in cattle similar to a commercial vaccine, gene-gun delivery of a subunit BHV-1 gD vaccine would be a viable alternative to current immunization protocols.

Reactivation of latent bovine herpesvirus 1 in cattle seronegative to glycoproteins gB and gE

Six heifers were vaccinated intranasally with the live bovine herpesvirus 1 (BHV1) temperature-sensitive (ts) vaccine strain RBL106 within 3 weeks of birth. These calves most likely still had maternal antibodies against BHV1. Thereafter, these heifers were vaccinated several times with an experimental BHV1 glycoprotein-D (gD) subunit vaccine. At the age of 3 years these 6 heifers were seronegative in the BHV1 gB and gE blocking ELISAs, but had neutralizing antibodies against BHV1, probably induced by the vaccinations with the gD subunit vaccine. Five of these 6 heifers excreted BHV1 after treatment with dexamethasone. Restriction enzyme analysis of the genome of the excreted viruses revealed that all 5 isolates had a BHV1.1 genotype and that isolates of 3 heifers were not obviously different from the ts-vaccine strain. The restriction enzyme fragment pattern of the isolate of 1 heifer was clearly different from the pattern of the ts-vaccine strain. It is concluded that cattle can be seronegative against BHV1 gB and gE but can still carry BHV1 in a latent form. This finding strongly suggests that there are completely BHV1 seronegative cattle that are latently infected with BHV1. The impact of this finding on BHV1 eradication programmes is discussed.

An inactivated gE-negative marker vaccine and an experimental gD-subunit vaccine reduce the incidence of bovine herpesvirus 1 infections in the field

An inactivated glycoprotein E-negative vaccine and an experimental glycoprotein D-subunit vaccine against bovine herpesvirus 1 (V1) were examined for their effectiveness in a randomized, double-bline, placebo-controlled field trial comprising 130 dairy farms. The use of these marker vaccines enabled us to monitor the incidence of infections in vaccinated populations. The aims of this trial were to evaluate whether these vaccines: (1) reduce the proportion of outbreaks in dairy herds; and (2) reduced virus transmission within dairy herds and to what extent. Vaccination with either of the two vaccines significantly reduced the proportion of herds wherein an outbreak occurred as well as the virus transmission within herds, as compared to placebo-treated herds. The estimated number of secondary cases caused by one infectious animal, expressed as the reproduction ratio R , was for both vaccines significantly >1. This indicates that when BHV1 is introduced into vaccinated herds, major outbreaks may still occur.

Inactivated bovine herpesvirus 1 marker vaccines are more efficacious in reducing virus excretion after reactivation than a live marker vaccine

A comparative study was carried out to evaluate the efficacy of three bovine herpesvirus 1 (BHV1) marker vaccines to reduce the reexcretion of virus after reactivation of latent BHV1. A live gE-negative vaccine, an inactivated gE-negative vaccine and an experimental gD-subunit vaccine were tested in three identical experiments in which cattle, latently infected with BHV1, were vaccinated twice before they were treated with high doses of dexamethasone. Virus excretion after dexamethasone treatment was compared with that in BHV1-infected, unvaccinated cattle which served as controls. All cattle, controls and vaccinees, excreted virus. However, the inactivated vaccines reduced virus excretion more efficiently than did the live vaccine.

Quantification of experimental transmission of bovine herpes-virus 1 in cattle vaccinated with marker vaccines

An attenuated and an inactivated bovine herpesvirus 1 (BHV1) gE-negative vaccine and an experimental BHV1 gD-subunit vaccine were evaluated for their ability to reduce BHV1 transmission. For that purpose, experiments to quantify transmission among vaccinated and unvaccinated cattle were carried out. Three separate experiments, including 90 cattle, were performed. Each group was housed separately. Two groups were vaccinated, each with a different marker vaccine, and the third served as an unvaccinated control group. At 4 weeks after the second vaccination, from each group five animals were taken out, and the remaining five were challenged intranasally with 105 TCID50 of the Dutch Lam strain of BHV1. After 24 hours, the animals were placed back into their original stables. The transmission of the virus to and among the five non-challenged, in-contact animals was then monitored. An animal was defined as being infected when it shed virus from its nasal fluids or developed antibodies to glycoprotein gE of BHV1. Based on the findings, the R, i.e. the average number of secondary cases per infectious individual, was calculated. Only the attenuated gE- negative vaccine was able to significantly reduce the transmission of BHV1.

An attenuated bovine herpesvirus 1 marker vaccine induces a better protection than two inactivated marker vaccines

A comparative study on the efficacy of 3 bovine herpesvirus 1 (BHV1) marker vaccines was carried out. An attenuated gE-negative vaccine, an inactivated gE-negative vaccine and an experimental gD-subunit vaccine were tested twice in a vaccination-challenge experiment in cattle. The attenuated vaccine induced the best clinical protection as evidenced by the total absence of clinical signs and fever in cattle. In addition, the attenuated vaccine reduced the shedding of challenge virus significantly more than the inactivated vaccines. Of the inactivated vaccines, the gE-negative vaccine induced a better clinical protection than the gD-subunit vaccine.

Vaccination of mice with herpes simplex virus type 1 glycoprotein D DNA produces low levels of protection against lethal HSV-1 challenge

The herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene was inserted into vectors pSVL or pRc CMV under control of the SV40 late promoter or the human cytomegalovirus major immediate-early promoter, respectively. Intramuscular injection of mice with these gD-containing plasmids appeared to induce low levels of serum anti-gD antibody, as judged by the appearance of low levels of anti-HSV-1-neutralizing antibody and anti-gD ELISA responses in the serum of gD-DNA-vaccinated mice. As previously reported in other virus systems, vaccination with vector DNA also induced ELISA and neutralizing antibody titers. However, these titers were lower than those induced by the gD-containing plasmids. The ELISA and neutralization titers induced by the vectors appeared to be non-specific rather than directed at specific HSV-1 proteins, since serum from mice vaccinated with plasmid-gD immunoprecipitated significant amounts of gD from extracts of HSV-1-infected cells, while serum from mice vaccinated with vectors was unable to immunoprecipitate gD or any other obvious HSV-1 proteins. Neither pSVL-gD nor pRc CMV-gD induced detectable lymphocyte proliferative or CTL responses. Vaccination with pSVL-gD provided a significant ( P = 0.04, Fisher's exact test), but low level of protection against lethal challenge with HSV-1. Vaccination with pRc CMV-gD also appeared to provide a low level of protection against challenge, that was statistically significance at the 10% level ( P = 0.054, Fisher's exact test). Reports from numerous laboratories (including ours) have shown that vaccination with recombinantly expressed gD can provide very high levels of protection against HSV-1 lethal challenge. Thus, the results reported here suggest that vaccination with HSV-1 gD-DNA is not yet a useful alternative to a gD subunit vaccine.

Native herpes simplex virus glycoprotein D vaccine: immunogenicity and protection in animal models

Mice, guinea pigs, and rhesus monkeys were immunized with immunoaffinity-purified native glycoprotein D (gD) derived from herpes simplex virus type 1 (HSV1). The native glycoprotein has evoked significant in vivo responses even at low doses. Thus, mice immunized with doses as low as 1 μg were significantly protected from the morbidity and mortality of lethal HSV2 challenge and from establishment of latent HSV2 infection. Protection was dose-related and correlated with prechallenge serum neutralizing antibody titres to HSV. Similarly, immunized guinea-pigs demonstrated significant reductions in the frequency, severity and duration of genital lesions induced by HSV2 vaginal challenge. In long term immunogenicity studies, immunized rhesus monkeys exhibited significant serum neutralizing antibody responses to both HSV1 and HSV2. In vitro stimulation of monkey peripheral blood leucocytes with purified gD resulted in a significant cellular proliferative response. The results obtained in these animal models with a gD subunit vaccine provide an appropriate foundation for the initiation of human studies.