Superior efficacy of a skin-applied microprojection device for delivering a novel Zika DNA vaccine.

Zika virus (ZIKV) infections are spreading silently with limited global surveillance in at least 89 countries and territories. There is a pressing need to develop an effective vaccine suitable for equitable distribution globally. Consequently, we previously developed a proprietary DNA vaccine encoding secreted non-structural protein 1 of ZIKV (pVAX-tpaNS1) to elicit rapid protection in a T cell-dependent manner in mice. In the current study, we evaluated the stability, efficacy, and immunogenicity of delivering this DNA vaccine into the skin using a clinically effective and proprietary high-density microarray patch (HD-MAP). Dry-coating of pVAX-tpaNS1 on the HD-MAP device resulted in no loss of vaccine stability at 40°C storage over the course of 28 days. Vaccination of mice (BALB/c) with the HD-MAP-coated pVAX-tpaNS1 elicited a robust anti-NS1 IgG response in both the cervicovaginal mucosa and systemically and afforded protection against live ZIKV challenge. Furthermore, the vaccination elicited a significantly higher magnitude and broader NS1-specific T helper and cytotoxic T cell response in vivo compared with traditional needle and syringe intradermal vaccination. Overall, the study highlights distinctive immunological advantages coupled with an excellent thermostability profile of using the HD-MAP device to deliver a novel ZIKV DNA vaccine.

SARS-CoV-2 Omicron variant escapes neutralizing antibodies and T cell responses more efficiently than other variants in mild COVID-19 convalescents.

Coronavirus disease 2019 (COVID-19) convalescents living in regions with low vaccination rates rely on post-infection immunity for protection against re-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluate humoral and T cell immunity against five variants of concern (VOCs) in mild-COVID-19 convalescents at 12 months after infection with ancestral virus. In this cohort, ancestral, receptor-binding domain (RBD)-specific antibody and circulating memory B cell levels are conserved in most individuals, and yet serum neutralization against live B.1.1.529 (Omicron) is completely abrogated and significantly reduced for other VOCs. Likewise, ancestral SARS-CoV-2-specific memory T cell frequencies are maintained in >50% of convalescents, but the cytokine response in these cells to mutated spike epitopes corresponding to B.1.1.529 and B.1.351 (Beta) VOCs were impaired. These results indicate that increased antigen variability in VOCs impairs humoral and spike-specific T cell immunity post-infection, strongly suggesting that COVID-19 convalescents are vulnerable and at risk of re-infection with VOCs, thus stressing the importance of vaccination programs.

Enhanced T Cell Responses Induced by a Necrotic Dendritic Cell Vaccine, Expressing HCV NS3.

A vaccine that induces potent, broad and sustained cell-mediated immunity, resulting in effective memory has the potential to restrict hepatitis C (HCV) virus infection. Early, multi-functional CD4+ and CD8+ T cell responses against non-structural protein 3 (NS3) have been associated with HCV clearance. Necrotic cells generate strong immune responses and represent a major antigenic source used by dendritic cells (DC) for processing and presentation, but there is conflicting evidence as to their immunogenicity in vaccination. Immunization with DC loaded with viral antigens has been done in the past, but to date the immunogenicity of live vs. necrotic DC vaccines has not been investigated. We developed a DC2.4 cell line stably expressing HCV NS3, and compared the NS3-specific responses of live vs. necrotic NS3 DC. Vaccination of mice with necrotic NS3 DC increased the breadth of T-cell responses and enhanced the production of IL-2, TNF-α, and IFN-γ by effector memory CD4+ and CD8+T cells, compared to mice vaccinated with live NS3 DC. A single dose of necrotic NS3 DC vaccine induced a greater influx and activation of cross-presenting CD11c+ CD8α+ DC and necrosis-sensing Clec9A+ DC in the draining lymph nodes. Furthermore, using a hydrodynamic challenge model necrotic NS3 DC vaccination resulted in enhanced clearance of NS3-positive hepatocytes from the livers of vaccinated mice. Taken together, the data demonstrate that necrotic DC represent a novel and exciting vaccination strategy capable of inducing broad and multifunctional T cell memory.

Safety Profile of a Multi-Antigenic DNA Vaccine Against Hepatitis C Virus.

Despite direct acting antivirals (DAAs) curing >95% of individuals infected with hepatitis C (HCV), in order to achieve the World Health Organization HCV Global Elimination Goals by 2030 there are still major challenges that need to be overcome. DAAs alone are unlikely to eliminate HCV in the absence of a vaccine that can limit viral transmission. Consequently, a prophylactic HCV vaccine is necessary to relieve the worldwide burden of HCV disease. DNA vaccines are a promising vaccine platform due to their commercial viability and ability to elicit robust T-cell-mediated immunity (CMI). We have developed a novel cytolytic DNA vaccine that encodes non-structural HCV proteins and a truncated mouse perforin (PRF), which is more immunogenic than the respective canonical DNA vaccine lacking PRF. Initially we assessed the ability of the HCV pNS3-PRF and pNS4/5-PRF DNA vaccines to elicit robust long-term CMI without any adverse side-effects in mice. Interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay was used to evaluate CMI against NS3, NS4 and NS5B in a dose-dependent manner. This analysis showed a dose-dependent bell-curve of HCV-specific responses in vaccinated animals. We then thoroughly examined the effects associated with reactogenicity of cytolytic DNA vaccination with the multi-antigenic HCV DNA vaccine (pNS3/4/5B). Hematological, biochemical and histological studies were performed in male Sprague Dawley rats with a relative vaccine dose 10-20-fold higher than the proposed dose in Phase I clinical studies. The vaccine was well tolerated, and no toxicity was observed. Thus, the cytolytic multi-antigenic DNA vaccine is safe and elicits broad memory CMI.

Single-Dose Vaccination with a Hepatotropic Adeno-associated Virus Efficiently Localizes T Cell Immunity in the Liver with the Potential To Confer Rapid Protection against Hepatitis C Virus.

Hepatitis C virus (HCV) is a significant contributor to the global disease burden, and development of an effective vaccine is required to eliminate HCV infections worldwide. CD4+ and CD8+ T cell immunity correlates with viral clearance in primary HCV infection, and intrahepatic CD8+ tissue-resident memory T (TRM) cells provide lifelong and rapid protection against hepatotropic pathogens. Consequently, we aimed to develop a vaccine to elicit HCV-specific CD4+ and CD8+ T cells, including CD8+ TRM cells, in the liver, given that HCV primarily infects hepatocytes. To achieve this, we vaccinated wild-type BALB/c mice with a highly immunogenic cytolytic DNA vaccine encoding a model HCV (genotype 3a) nonstructural protein (NS5B) and a mutant perforin (pVAX-NS5B-PRF), as well as a recombinant adeno-associated virus (AAV) encoding NS5B (rAAV-NS5B). A novel fluorescent target array (FTA) was used to map immunodominant CD4+ T helper (TH) cell and cytotoxic CD8+ T cell epitopes of NS5B in vivo, which were subsequently used to design a KdNS5B451-459 tetramer and analyze NS5B-specific T cell responses in vaccinated mice in vivo The data showed that intradermal prime/boost vaccination with pVAX-NS5B-PRF was effective in eliciting TH and cytotoxic CD8+ T cell responses and intrahepatic CD8+ TRM cells, but a single intravenous dose of hepatotropic rAAV-NS5B was significantly more effective. As a T-cell-based vaccine against HCV should ideally result in localized T cell responses in the liver, this study describes primary observations in the context of HCV vaccination that can be used to achieve this goal.IMPORTANCE There are currently at least 71 million individuals with chronic HCV worldwide and almost two million new infections annually. Although the advent of direct-acting antivirals (DAAs) offers highly effective therapy, considerable remaining challenges argue against reliance on DAAs for HCV elimination, including high drug cost, poorly developed health infrastructure, low screening rates, and significant reinfection rates. Accordingly, development of an effective vaccine is crucial to HCV elimination. An HCV vaccine that elicits T cell immunity in the liver will be highly protective for the following reasons: (i) T cell responses against nonstructural proteins of the virus are associated with clearance of primary infection, and (ii) long-lived liver-resident T cells alone can protect against malaria infection of hepatocytes. Thus, in this study we exploit promising vaccination platforms to highlight strategies that can be used to evoke highly functional and long-lived T cell responses in the liver for protection against HCV.

A Hepatitis C Virus DNA Vaccine Encoding a Secreted, Oligomerized Form of Envelope Proteins Is Highly Immunogenic and Elicits Neutralizing Antibodies in Vaccinated Mice.

Hepatitis C virus (HCV) persistently infects approximately 71 million people globally. To prevent infection a vaccine which elicits neutralizing antibodies against the virus envelope proteins (E1/E2) which are required for entry into host cells is desirable. DNA vaccines are cost-effective to manufacture globally and despite recent landmark studies highlighting the therapeutic efficacy of DNA vaccines in humans against cervical cancer, DNA vaccines encoding E1/E2 developed thus far are poorly immunogenic. We now report a novel and highly immunogenic DNA vaccination strategy that incorporates secreted E1 and E2 (sE1 and sE2) into oligomers by fusion with the oligomerization domain of the C4b-binding protein, IMX313P. The FDA approved plasmid, pVax, was used to encode sE1, sE2, or sE1E2 with or without IMX313P, and intradermal prime-boost vaccination studies in BALB/c mice showed that vaccines encoding IMX313P were the most effective in eliciting humoral and cell-mediated immunity against the envelope proteins. Further boosting with recombinant E1E2 proteins but not DNA nor virus-like particles (VLPs) expressing E1E2 increased the immunogenicity of the DNA prime-boost regimen. Nevertheless, the antibodies generated by the homologous DNA prime-boost vaccinations more effectively inhibited the binding of VLPs to target cells and neutralized transduction with HCV pseudoparticles (HCVpp) derived from different genotypes including genotypes 1, 2, 3, 4, 5, and 6. This report provides the first evidence that IMX313P can be used as an adjuvant for E1/E2-based DNA vaccines and represents a translatable approach for the development of a HCV DNA vaccine.

Cytolytic Perforin as an Adjuvant to Enhance the Immunogenicity of DNA Vaccines.

DNA vaccines present one of the most cost-effective platforms to develop global vaccines, which have been tested for nearly three decades in preclinical and clinical settings with some success in the clinic. However, one of the major challenges for the development of DNA vaccines is their poor immunogenicity in humans, which has led to refinements in DNA delivery, dosage in prime/boost regimens and the inclusion of adjuvants to enhance their immunogenicity. In this review, we focus on adjuvants that can enhance the immunogenicity of DNA encoded antigens and highlight the development of a novel cytolytic DNA platform encoding a truncated mouse perforin. The application of this innovative DNA technology has considerable potential in the development of effective vaccines.

Toward DNA-Based T-Cell Mediated Vaccines to Target HIV-1 and Hepatitis C Virus: Approaches to Elicit Localized Immunity for Protection.

Human immunodeficiency virus (HIV)-1 and hepatitis C virus (HCV) are major contributors to the global disease burden with many experts recognizing the requirement of an effective vaccine to bring a durable end to these viral epidemics. The most promising vaccine candidates that have advanced into pre-clinical models and the clinic to eliminate or provide protection against these chronic viruses are viral vectors [e.g., recombinant cytomegalovirus, Adenovirus, and modified vaccinia Ankara (MVA)]. This raises the question, is there a need to develop DNA vaccines against HIV-1 and HCV? Since the initial study from Wolff and colleagues which showed that DNA represents a vector that can be used to express transgenes durably in vivo, DNA has been regularly evaluated as a vaccine vector albeit with limited success in large animal models and humans. However, several recent studies in Phase I-IIb trials showed that vaccination of patients with recombinant DNA represents a feasible therapeutic intervention to even cure cervical cancer, highlighting the potential of using DNA for human vaccinations. In this review, we will discuss the limitations and the strategies of using DNA as a vector to develop prophylactic T cell-mediated vaccines against HIV-1 and HCV. In particular, we focus on potential strategies exploiting DNA vectors to elicit protective localized CD8+ T cell immunity in the liver for HCV and in the cervicovaginal mucosa for HIV-1 as localized immunity will be an important, if not critical component, of an efficacious vaccine against these viral infections.

Antibody Responses to a Quadrivalent Hepatitis C Viral-Like Particle Vaccine Adjuvanted with Toll-Like Receptor 2 Agonists.

The development of an effective preventative hepatitis C virus (HCV) vaccine will reside, in part, in its ability to elicit neutralizing antibodies (NAbs). We previously reported a genotype 1a HCV virus like particle (VLP) vaccine that produced HCV specific NAb and T cell responses that were substantially enhanced by Toll-like receptor 2 (TLR2) agonists. We have now produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine and tested the ability of two TLR2 agonists, R4Pam2Cys and E8Pam2Cys, to stimulate the production of NAb. We now show that our vaccine with R4Pam2Cys or E8Pam2Cys produces strong antibody and NAb responses in vaccinated mice after just two doses. Total antibody titers were higher in mice inoculated with vaccine plus E8Pam2Cys compared to HCV VLPs alone. However, the TLR2 agonists did not result in stronger NAb responses compared to vaccine without adjuvant. Such a vaccine could provide a substantial addition to the overall goal to eliminate HCV.

Induction of Genotype Cross-Reactive, Hepatitis C Virus-Specific, Cell-Mediated Immunity in DNA-Vaccinated Mice.

A universal hepatitis C virus (HCV) vaccine should elicit multiantigenic, multigenotypic responses, which are more likely to protect against challenge with the range of genotypes and subtypes circulating in the community. A vaccine cocktail and vaccines encoding consensus HCV sequences are attractive approaches to achieve this goal. Consequently, in a series of mouse vaccination studies, we compared the immunogenicity of a DNA vaccine encoding a consensus HCV nonstructural 5B (NS5B) protein to that of a cocktail of DNA plasmids encoding the genotype 1b (Gt1b) and Gt3a NS5B proteins. To complement this study, we assessed responses to a multiantigenic cocktail regimen by comparing a DNA vaccine cocktail encoding Gt1b and Gt3a NS3, NS4, and NS5B proteins to a single-genotype NS3/4/5B DNA vaccine. To thoroughly evaluate in vivo cytotoxic T lymphocyte (CTL) and T helper (Th) cell responses against Gt1b and Gt3a HCV peptide-pulsed target cells, we exploited a novel fluorescent-target array (FTA). FTA and enzyme-linked immunosorbent spot (ELISpot) analyses collectively indicated that the cocktail regimens elicited higher responses to Gt1b and Gt3a NS5B proteins than those with the consensus vaccine, while the multiantigenic DNA cocktail significantly increased the responses to NS3 and NS5B compared to those elicited by the single-genotype vaccines. Thus, a DNA cocktail vaccination regimen is more effective than a consensus vaccine or a monovalent vaccine at increasing the breadth of multigenotypic T cell responses, which has implications for the development of vaccines for communities where multiple HCV genotypes circulate.IMPORTANCE Despite the development of highly effective direct-acting antivirals (DAA), infections with hepatitis C virus (HCV) continue, particularly in countries where the supply of DAA is limited. Furthermore, patients who eliminate the virus as a result of DAA therapy can still be reinfected. Thus, a vaccine for HCV is urgently required, but the heterogeneity of HCV strains makes the development of a universal vaccine difficult. To address this, we developed a novel cytolytic DNA vaccine which elicits robust cell-mediated immunity (CMI) to the nonstructural (NS) proteins in vaccinated animals. We compared the immune responses against genotypes 1 and 3 that were elicited by a consensus DNA vaccine or a DNA vaccine cocktail and showed that the cocktail induced higher levels of CMI to the NS proteins of both genotypes. This study suggests that a universal HCV vaccine can most readily be achieved by use of a DNA vaccine cocktail.

Preclinical Development and Production of Virus-Like Particles As Vaccine Candidates for Hepatitis C.

Hepatitis C Virus (HCV) infects 2% of the world's population and is the leading cause of liver disease and liver transplantation. It poses a serious and growing worldwide public health problem that will only be partially addressed with the introduction of new antiviral therapies. However, these treatments will not prevent re-infection particularly in high risk populations. The introduction of a HCV vaccine has been predicted, using simulation models in a high risk population, to have a significant effect on reducing the incidence of HCV. A vaccine with 50 to 80% efficacy targeted to high-risk intravenous drug users could dramatically reduce HCV incidence in this population. Virus like particles (VLPs) are composed of viral structural proteins which self-assemble into non-infectious particles that lack genetic material and resemble native viruses. Thus, VLPs represent a safe and highly immunogenic vaccine delivery platform able to induce potent adaptive immune responses. Currently, many VLP-based vaccines have entered clinical trials, while licensed VLP vaccines for hepatitis B virus (HBV) and human papilloma virus (HPV) have been in use for many years. The HCV core, E1 and E2 proteins can self-assemble into immunogenic VLPs while inclusion of HCV antigens into heterogenous (chimeric) VLPs is also a promising approach. These VLPs are produced using different expression systems such as bacterial, yeast, mammalian, plant, or insect cells. Here, this paper will review HCV VLP-based vaccines and their immunogenicity in animal models as well as the different expression systems used in their production.

Emerging Targets for Developing T Cell-Mediated Vaccines for Human Immunodeficiency Virus (HIV)-1.

Human immunodeficiency virus (HIV)-1 has infected >75 million individuals globally, and, according to the UN, is responsible for ~2.1 million new infections and 1.1 million deaths each year. Currently, there are ~37 million individuals with HIV infection and the epidemic has already resulted in 35 million deaths. Despite the advances of anti-retroviral therapy (ART), a cost-effective vaccine remains the best long-term solution to end the HIV-1 epidemic especially given that the vast majority of infected individuals live in poor socio-economic regions of the world such as Sub-Saharan Africa which limits their accessibility to ART. The modest efficacy of the RV144 Thai trial provides hope that a vaccine for HIV-1 is possible, but as markers for sterilizing immunity are unknown, the design of an effective vaccine is empirical, although broadly cross-reactive neutralizing antibodies (bNAb) that can neutralize various quasispecies of HIV-1 are considered crucial. Since HIV-1 transmission often occurs at the genito-rectal mucosa and is cell-associated, there is a need to develop vaccines that can elicit CD8+ T cell immunity with the capacity to kill virus infected cells at the genito-rectal mucosa and the gut. Here we discuss the recent progress made in developing T cell-mediated vaccines for HIV-1 and emphasize the need to elicit mucosal tissue-resident memory CD8+ T (CD8+ Trm) cells. CD8+ Trm cells will likely form a robust front-line defense against HIV-1 and eliminate transmitter/founder virus-infected cells which are responsible for propagating HIV-1 infections following transmission in vast majority of cases.

Cytolytic DNA vaccine encoding lytic perforin augments the maturation of- and antigen presentation by- dendritic cells in a time-dependent manner.

The use of cost-effective vaccines capable of inducing robust CD8+ T cell immunity will contribute significantly towards the elimination of persistent viral infections and cancers worldwide. We have previously reported that a cytolytic DNA vaccine encoding an immunogen and a truncated mouse perforin (PRF) protein significantly augments anti-viral T cell (including CD8+ T cell) immunity. Thus, the current study investigated whether this vaccine enhances activation of dendritic cells (DCs) resulting in greater priming of CD8+ T cell immunity. In vitro data showed that transfection of HEK293T cells with the cytolytic DNA resulted in the release of lactate dehydrogenase, indicative of necrotic/lytic cell death. In vitro exposure of this lytic cell debris to purified DCs from naïve C57BL/6 mice resulted in maturation of DCs as determined by up-regulation of CD80/CD86. Using activation/proliferation of adoptively transferred OT-I CD8+ T cells to measure antigen presentation by DCs in vivo, it was determined that cytolytic DNA immunisation resulted in a time-dependent increase in the proliferation of OT-I CD8+ T cells compared to canonical DNA immunisation. Overall, the data suggest that the cytolytic DNA vaccine increases the activity of DCs which has important implications for the design of DNA vaccines to improve their translational prospects.

Human Rhinovirus-A1 as an Expression Vector.

Expression vectors that are based on live human rhinoviruses (HRVs) are attractive, yet often overlooked in vaccine development due to their limited capacity for foreign gene inserts and poor genetic stability. This chapter describes a novel methodology to engineer a replication-competent genetically stable recombinant HRV (rHRV) without affecting viral replication capability. We have previously used these methods to generate live, genetically stable recombinant HRVs encoding HIV Gag and Tat proteins (rHRV-Gag-Tat), a potential mucosally targeted HIV vaccine.

Mucosal vaccination with a live recombinant rhinovirus followed by intradermal DNA administration elicits potent and protective HIV-specific immune responses.

Mucosal immunity is deemed crucial to control sexual transmission of human immunodeficiency virus (HIV). Herein we report the efficacy of a mucosal HIV vaccine strategy comprising intranasal (IN) vaccination with a cocktail of live recombinant human rhinoviruses (HRVs) encoding overlapping fragments of HIV Gag and full length Tat (rHRV-Gag/Tat) followed by intradermal (ID) vaccination with DNA vaccines encoding HIV Gag and Tat (pVAX-Gag-Tat). This heterologous prime-boost strategy will be referred to hereafter as rHRV-DNA. As a control, IN vaccination with wild type (wt)-HRV-A1 followed by a single ID dose of pVAX (wt-HRV-A1/pVAX vaccination) was included. rHRV-DNA vaccination elicited superior multi-functional CD8+T cell responses in lymphocytes harvested from mesenteric lymph nodes and spleens, and higher titres of Tat-specific antibodies in blood and vaginal lavages, and reduced the viral load more effectively after challenge with EcoHIV, a murine HIV challenge model, in peritoneal macrophages, splenocytes and blood compared compared with wt-HRV-A1/pVAX vaccination or administration of 3 ID doses of pVAX-Gag-Tat (3X pVAX-Gag-Tat vaccination). These data provide the first evidence that a rHRV-DNA vaccination regimen can induce HIV-specific immune responses in the gut, vaginal mucosa and systemically, and supports further testing of this regimen in the development of an effective mucosally-targeted HIV-1 vaccine.

A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice.

DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans.

Immune responses against hepatitis C virus genotype 3a virus-like particles in mice: A novel VLP prime-adenovirus boost strategy.

Chronic hepatitis C virus (HCV) infection represents a major health threat to global population. In India, approximately 15-20% of cases of chronic liver diseases are caused by HCV infection. Although, new drug treatments hold great promise for HCV eradication in infected individuals, the treatments are highly expensive. A vaccine for preventing or treating HCV infection would be of great value, particularly in developing countries. Several preclinical trials of virus-like particle (VLP) based vaccine strategies are in progress throughout the world. Previously, using baculovirus based system, we have reported the production of hepatitis C virus-like particles (HCV-LPs) encoding structural proteins for genotype 3a, which is prevalent in India. In the present study, we have generated HCV-LPs using adenovirus based system and tried different immunization strategies by using combinations of both kinds of HCV-LPs with other genotype 3a-based immunogens. HCV-LPs and peptides based ELISAs were used to evaluate antibody responses generated by these combinations. Cell-mediated immune responses were measured by using T-cell proliferation assay and intracellular cytokine staining. We observed that administration of recombinant adenoviruses expressing HCV structural proteins as final booster enhances both antibody as well as T-cell responses. Additionally, reduction of binding of VLP and JFH1 virus to human hepatocellular carcinoma cells demonstrated the presence of neutralizing antibodies in immunized sera. Taken together, our results suggest that the combined regimen of VLP followed by recombinant adenovirus could more effectively inhibit HCV infection, endorsing the novel vaccine strategy.

A Multiantigenic DNA Vaccine That Induces Broad Hepatitis C Virus-Specific T-Cell Responses in Mice.

UNLABELLED: There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising "multiantigen" vaccine that elicits robust CMI. IMPORTANCE: Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans.