Novel G3/DT adjuvant promotes the induction of protective T cells responses after vaccination with a seasonal trivalent inactivated split-virion influenza vaccine

Vaccines used against seasonal influenza are poorly effective against influenza A viruses of novel subtypes that may have pandemic potential. Furthermore, pre(pandemic) influenza vaccines are poorly immunogenic, which can be overcome by the use of adjuvants. A limited number of adjuvants has been approved for use in humans, however there is a need for alternative safe and effective adjuvants that can enhance the immunogenicity of influenza vaccines and that promote the induction of broad-protective T cell responses. Here we evaluated a novel nanoparticle, G3, as an adjuvant for a seasonal trivalent inactivated influenza vaccine in a mouse model. The G3 adjuvant was formulated with or without steviol glycosides (DT, for diterpenoid). The use of both formulations enhanced the virus-specific antibody response to all three vaccine strains considerably. The adjuvants were well tolerated without any signs of discomfort. To assess the protective potential of the vaccine-induced immune responses, an antigenically distinct influenza virus strain, A/Puerto Rico/8/34 (A/PR/8/34), was used for challenge infection. The vaccine-induced antibodies did not cross-react with strain A/PR/8/34 in HI and VN assays. However, mice immunized with the G3/DT-adjuvanted vaccine were partially protected against A/PR/8/34 infection, which correlated with the induction of anamnestic virus-specific CD8⁺ T cell responses that were not observed with the use of G3 without DT. Both formulations induced maturation of human dendritic cells and promoted antigen presentation to a similar extent. In conclusion, G3/DT is a promising adjuvant formulation that not only potentiates the antibody response induced by influenza vaccines, but also induces T cell immunity which could afford broader protection against antigenically distinct influenza viruses.     

Protection by universal influenza vaccine is mediated by memory CD4 T cells

There is a diverse array of influenza viruses which circulate between different species, reassort and drift over time. Current seasonal influenza vaccines are ineffective in controlling these viruses. We have developed a novel universal vaccine which elicits robust T cell responses and protection against diverse influenza viruses in mouse and human models. Vaccine mediated protection was dependent on influenza-specific CD4⁺ T cells, whereby depletion of CD4⁺ T cells at either vaccination or challenge time points significantly reduced survival in mice. Vaccine memory CD4⁺ T cells were needed for early antibody production and CD8⁺ T cell recall responses. Furthermore, influenza-specific CD4⁺ T cells from vaccination manifested primarily Tfh and Th1 profiles with anti-viral cytokine production. The vaccine boosted H5-specific T cells from human PBMCs, specifically CD4⁺ and CD8⁺ T effector memory type, ensuring the vaccine was truly universal for its future application. These findings have implications for the development and optimization of T cell activating vaccines for universal immunity against influenza.     

The effects of preexisting immunity to influenza on responses to influenza vectors in mice

The use of viral vectors as vaccine candidates has shown promise against a number of pathogens. However, preexisting immunity to these vectors is a concern that must be addressed when deciding which viruses are suitable for use. A number of properties, including the existence of antigenically distinct subtypes, make influenza viruses attractive candidates for use as viral vectors. Here, we evaluate the ability of influenza viral vectors containing inserts of foreign pathogens to elicit antibody and CD8⁺ T cell responses against these foreign antigens in the presence of preexisting immunity to influenza virus in mice. Specifically, responses to an H3N1-based vector expressing a 90 amino acid polypeptide derived from the protective antigen (PA) of Bacillus anthracis or an H1N1-based vector containing a CD8⁺ T cell epitope from the glycoprotein (GP) of lymphocytic choriomeningitis virus were evaluated following infections with either homosubtypic or heterosubtypic influenza viruses. We found that mice previously infected with influenza viruses, even those expressing HA and NA proteins of completely different subtypes, were severely compromised in their ability to mount an immune response against the inserted epitopes. This inhibition was demonstrated to be mediated by CD8⁺ T cells, which recognize multiple strains of influenza viruses. These CD8⁺ T cells were further shown to protect mice from a lethal challenge by a heterologous influenza subtype. The implication of these data for the use of influenza virus vectors and influenza vaccination in general are discussed.     

Annual influenza vaccination affects the development of heterosubtypic immunity

Annual vaccination of healthy children >6 months of age against seasonal influenza has been recommended by public health authorities of some countries. However, currently used seasonal vaccines provide only limited protection against (potentially) pandemic influenza viruses. Furthermore, we recently hypothesized that annual vaccination may hamper the development of cross-reactive immunity against influenza A viruses of novel subtypes, that would otherwise be induced by natural infection. Here we summarize our findings in animal models in which we demonstrated that vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity against highly pathogenic avian influenza A/H5N1 virus, otherwise induced by a prior infection with influenza A/H3N2 virus. The reduction of heterosubtypic immunity correlated with reduced virus-specific CD8+ T cell responses. An additional study was performed in humans, in which we collected peripheral blood mononuclear cells from annually vaccinated children with cystic fibrosis (CF) and age-matched unvaccinated healthy control children to study the virus-specific T cell response. An age-related increase of the virus-specific CD8+ T cell response was observed in unvaccinated children that was absent in vaccinated children with CF. These findings highlight the importance of the development of vaccines that provide protection against influenza A viruses of all subtypes.     

Differential effect of CD4Foxp3 T-regulatory cells on the B and T helper cell responses to influenza virus vaccination

The T-regulatory (T-reg) cells restrict the T-cell functions in various viral infections including influenza infection. However little is known about the effect of T-regs in influenza vaccination. Herein, we found that immunization of BALB/c mice with a prototype of UV-inactivated influenza PR8/A/34 virus vaccine expanded the CD4⁺Foxp3⁺ T-reg pool and fostered the development of virus-specific CD4⁺Foxp3⁺ T-reg cells. Increasing the size of Foxp3⁺ T-reg pool did not alter the primary PR8-specific B-cell response, but it did suppress the primary and memory PR8-specific T helper responses induced by vaccination. In contrast, the vaccination-induced T helper cell response was augmented in the absence of CD4⁺Foxp3⁺ T-reg cells. Since CD4 T helper cells contribute to anti-influenza protection, therapeutic “quenching” of T-reg function prior to vaccination may enhance the efficacy of influenza vaccination.     

Subcutaneous inoculation of a whole virus particle vaccine prepared from a non-pathogenic virus library induces protective immunity against H7N7 highly pathogenic avian influenza virus in cynomolgus macaques

Development of H7N7 highly pathogenic avian influenza virus (HPAIV) vaccines is an urgent issue since human cases of infection with this subtype virus have been reported and most humans have no immunity against H7N7 viruses. We made an H7N7 vaccine combining components from an influenza virus library of non-pathogenic type A influenza viruses. Antibody and T cell recall responses specific against the vaccine strain were elicited by subcutaneous inoculation with the whole virus particle vaccine with or without alum as an adjuvant in cynomolgus macaques. No significant difference was observed in magnitude of antibody responses between vaccination with alum and vaccination without alum, though vaccination with alum induced longer recall responses of CD8⁺ T cells than did vaccination without alum. After challenge with a subtype of H7N7 HPAIV, the virus was detected in nasal swabs of unvaccinated macaques for 8 days but only for 1 day in the animals vaccinated either with or without alum, although the macaques vaccinated with alum showed elevated body temperature more briefly after infection. These findings demonstrated that this H7N7 HPAIV strain is pathogenic to macaques and that the vaccine conferred protective immunity to macaques against H7N7 HPAIV infection.     

Polyanhydride nanovaccine against swine influenza virus in pigs

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study, we evaluated the efficacy of ∼200 nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine), and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses, antigen-specific proliferation of peripheral blood mononuclear cells, gross and microscopic lung lesions, and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4⁺CD8αα⁺ T helper and CD8⁺ cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition, pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8-fold reduction in nasal shedding of SwIAV four days post-challenge compared to control animals. Immunologically, increased IFN-γ secreting T lymphocyte populations against both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized pigs compared to the animals immunized with KAg alone. However, in the KAg nanovaccine-immunized pigs, hemagglutination inhibition, IgG and IgA antibody responses, and virus neutralization titers were comparable to that in the animals immunized with KAg alone. Overall, our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs, with promise to induce cross-protective immunity.     

Vaccination of aged mice with adjuvanted recombinant influenza nucleoprotein enhances protective immunity

Elderly individuals are highly susceptible to influenza virus (IAV) infection and respond poorly to influenza vaccines. Although the generally accepted correlate of protection following influenza vaccination is neutralizing antibody titers, cytotoxic T cell activity has been found to be a better correlate in the elderly. This suggests that vaccines designed to protect against influenza in the elderly should induce both humoral and cellular immunity. The co-induction of T cell immunity is additionally advantageous, as virus-specific T cells are frequently cross-reactive against different strains of IAV. Here, we tested the capacity of a synthetic TLR-4 adjuvant, SLA-SE (second-generation lipid adjuvant formulated in a squalene-based oil-in-water emulsion) to elicit T cell immunity to a recombinant influenza nucleoprotein (rNP), in both young and aged mice. IAV challenge of vaccinated mice resulted in a modest increase in the numbers of NP-specific CD4 and CD8 effector T cells in the spleen, but did not increase numbers of memory phenotype CD8 T cells generated following viral clearance (compared to control vaccinated mice). Cytotoxic activity of CD8, but not CD4 T cells was increased. In addition, SLA-SE adjuvanted vaccination specifically enhanced the production of NP-specific IgG2c antibodies in both young and aged mice. Although NP-specific antibodies are not neutralizing, they can cooperate with CD8 T cells and antigen-presenting cells to enhance protective immunity. Importantly, SLA-SE adjuvanted rNP-vaccination of aged mice resulted in significantly enhanced viral clearance. In addition, vaccination of aged mice resulted in enhanced survival after lethal challenge compared to control vaccination, that approached statistical significance. These data demonstrate the potential of SLA-SE adjuvanted rNP vaccines to (i) generate both cellular and humoral immunity to relatively conserved IAV proteins and (ii) elicit protective immunity to IAV in aged mice.     

CD8 T cell immunity against human respiratory syncytial virus

Human respiratory syncytial virus (HRSV) was first discovered in the 1950s, but despite decades of research, a licensed vaccine against it is not available. Epidemiological studies indicate that antibodies directed against the fusion protein (F) partially correlate with protection. In addition, an F-specific monoclonal antibody is licensed as a prophylactic treatment in children who are at high risk of developing complications following HRSV infection. Therefore, most HRSV-oriented vaccination strategies focus on inducing a humoral immune response against F. In the quest for the development of a safe HRSV vaccine, the induction of a T cell immune response has received a lot less attention. T cell immunity directed against HRSV has not been associated unequivocally with protection against HRSV and CD4⁺ T helper cell responses may even worsen disease due to HRSV. However, many studies support a protective role for CD8⁺ T cells in clearance of HRSV from the lungs. In this review we highlight the clinical and experimental evidence in favor of a CD8⁺ T lymphocyte-based vaccination strategy to protect against HRSV. First, we describe how T cell responses and T cell memory are induced in the lungs upon respiratory viral infection. HRSV has evolved mechanisms that hamper CD8⁺ T cell priming and effector functions. We appraise the information on HRSV-specific CD8⁺ T cell immunity gained from laboratory mouse studies, taking into account the advantages and limitations of this animal model and, where possible, the accordance with clinical evidence. Finally, we focus on recent efforts to develop T cell based vaccines against HRSV.     

Efficient protective immunity against Trypanosoma cruzi infection after nasal vaccination with recombinant Sendai virus vector expressing amastigote surface protein-2

Chagas’ disease, caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi), is intractable showing a high mortality rate, and the development of effective vaccines is much desired. To examine the efficacy of a new mode of recombinant viral vaccine, we constructed two non-transmissible Sendai viruses (rSeV/dF) encoding the full-length parasite antigen amastigote surface protein-2 (ASP2) or ASP2 fused with a mono-ubiquitin on its N-terminus (UASP2). C57BL/6 mice immunized intranasally with rSeV/dF expressing either ASP2 or UASP2 showed significantly suppressed parasitemia and could be protected from lethal T. cruzi challenge. Depletion of CD8⁺ T cells around the time of infection with T. cruzi completely abolished this protection, confirming that acquired immunity against the infection of T. cruzi is dependent on CD8⁺ T cells. We also demonstrated that the protective immunity correlated with higher secretion of interferon-γ (IFN-γ) by spleen cells on in vitro-specific or non-specific stimulation. Increased CTL activity was also confirmed by degranulation or CTL assays. Interestingly, the control virus, rSeV/dF-GFP, induced even a higher IFN-γ production from spleen cells following non-specific but not specific stimulation in vitro, suggesting that SeV may also be a good adjuvant when used as a vaccine vehicle. Taking together, the current findings indicate that recombinant Sendai virus expressing the ASP2 or UASP2 antigens of T. cruzi are interesting candidates for the development of a new mode of recombinant viral vaccine against Chagas’ disease.     

M2SR,a novel live single replication influenza virus vaccine,provides effective heterosubtypic protection in mice

Despite the annual public health burden of seasonal influenza and the continuing threat of a global pandemic posed by the emergence of highly pathogenic/pandemic strains, conventional influenza vaccines do not provide universal protection, and exhibit suboptimal efficacy rates, even when they are well matched to circulating strains. To address the need for a highly effective universal influenza vaccine, we have developed a novel M2-deficient single replication vaccine virus (M2SR) that induces strong cross-protective immunity against multiple influenza strains in mice. M2SR is able to infect cells and expresses all viral proteins except M2, but is unable to generate progeny virus.M2SR generated from influenza A/Puerto Rico/8/34 (H1N1) protected mice against lethal challenge with influenza A/Puerto Rico/8/34 (H1N1, homosubtypic) and influenza A/Aichi/2/1968 (H3N2, heterosubtypic). The vaccine induced strong systemic and mucosal antibody responses of both IgA and IgG classes. Strong virus-specific T cell responses were also induced. Following heterologous challenge, significant numbers of IFN-γ-producing CD8 T cells, with effector or effector/memory phenotypes and specific for conserved viral epitopes, were observed in the lungs of vaccinated mice. A substantial proportion of the CD8 T cells expressed Granzyme B, suggesting that they were capable of killing virus-infected cells.Thus, our data suggest that M2-deficient influenza viruses represent a promising new approach for developing a universal influenza vaccine.     

Establishment of functional influenza virus-specific CD8+ T cell memory pools after intramuscular immunization

The emergence of the avian-origin influenza H7N9 virus and its pandemic potential has highlighted the ever-present need to develop vaccination approaches to induce cross-protective immunity. In this study, we examined the establishment of cross-reactive CD8⁺ T cell immunity in mice following immunization with live A/Puerto Rico/8/1934 (PR8; H1N1) influenza virus via two non-productive inoculation routes. We found that immunization via the intramuscular (IM) route established functional influenza-virus specific memory CD8⁺ T cell pools capable of cross-reactive recall responses. Epitope-specific primary, memory and recall CD8⁺ T-cell responses induced by the IM route, highly relevant to human influenza immunisations, were of comparable magnitude and quality to those elicited by the intraperitoneal (IP) priming, commonly used in mice. Furthermore, IM immunisation resulted in lower lung viral titres following heterologous challenge with A/Aichi/68 (X31; H3N2) compared to the IP route. Examining the ability of DCs from lymphoid organs to present viral antigen revealed that immune induction following IM immunization occurred in draining lymph nodes, while immunization via the IP route resulted in the priming of responses in distal lymphoid organs, indicative of a systemic distribution of antigen. No major differences in the pulmonary cytokine environment of immunized animals following X31 challenge were observed that could account for the improved heterologous protection induced by the IM route. However, while both routes induced similar levels of PR8-specific antibodies, higher levels of cross-reactive antibodies against X31 were induced following IM inoculation. Our data demonstrate how non-replicative routes of infection can induce efficient cross-reactive CD8⁺ T cell responses and strong strain-specific antibody responses, with the additional benefit from IM priming of enhanced heterosubtypic antibody production.     

Cationic lipid/DNA complexes (JVRS-100) combined with influenza vaccine (Fluzone) increases antibody response,cellular immunity,and antigenically drifted protection

Safe and effective adjuvants for influenza vaccines that could increase both the levels of neutralizing antibody, including against drifted viral subtypes, and T-cell immunity would be a major advance in vaccine design. The JVRS-100 adjuvant, consisting of DOTIM/cholesterol cationic liposome–DNA complexes, is particularly promising for vaccines that require induction of high levels of antibody and T-cell immunity, including CD8⁺ cytotoxic T lymphocytes (CTL). Inclusion of protein antigens with JVRS-100 results in the induction of enhanced humoral and cell-mediated (i.e., CD4⁺ and CD8⁺ T cells) immune responses. The JVRS-100 adjuvant combined with a split trivalent influenza vaccine (Fluzone^®—sanofi pasteur) elicited increased antibody and T-cell responses in mice and non-human primates compared to vaccination with Fluzone^® alone. Mice vaccinated with JVRS-100–Fluzone^® and challenged with antigenically drifted strains of H1N1 (PR/8/34) and influenza B (B/Lee/40) viruses had higher grade protection, as measured by attenuation of weight loss and increased survival, compared to recipients of unadjuvanted vaccine. The results indicate that the JVRS-100 adjuvant substantially increases immunogenicity and protection from drifted-strain challenge using an existing influenza vaccine.     

Enhancement of T cell-mediated immune responses to whole inactivated influenza virus by chloroquine treatment in vivo

Current influenza vaccines induce poor cross-reactive CD8+ T cell responses. Cellular immunity is generally specific for epitopes that are remarkably conserved among different subtypes, suggesting that strategies to improve the cross-presentation of viral antigens by dendritic cells (DC) could elicit a broadly protective immune response. Previous studies have shown that limited proteolysis within the endocytic pathway can favorably influence antigen processing and thus immune responses. Herein, we demonstrate that chloroquine improves the cross-presentation of non-replicating influenza virus in vitro and T cell responses in mice following a single administration of inactivated HI-X31 virus. CD8+ T cells were also recruited to lymph nodes draining the site of infection and able to reduce viral load following pulmonary challenge with the heterologous PR8 virus. These findings may have implications for vaccination strategies aimed at improving the cross-presentation capacity of DCs and thus the size of effector and memory CD8+ T cells against influenza vaccines.     

Heat shock protein gp96 adjuvant induces T cell responses and cross-protection to a split influenza vaccine

The commonly used inactivated or split influenza vaccines induce only induce minimal T cell responses and are less effective in preventing heterologous virus infection. Thus, developing cross-protective influenza vaccines against the spread of a new influenza virus is an important strategy against pandemic emergence. Here we demonstrated that immunization with heat shock protein gp96 as adjuvant led to a dramatic increased antigen-specific T cell response to a pandemic H1N1 split vaccine. Notably, gp96 elicited a cross-protective CD8⁺ T cell response to the internal conserved viral protein NP. Although the split pH1N1vaccine alone has low cross-protective efficiency, adding gp96 as an adjuvant effectively improved the cross-protection against challenge with a heterologous virus in mice. Our study reveals the novel property of gp96 in boosting the T cell response against conserved epitopes of influenza virus and its potential use as an adjuvant for human pre-pandemic inactivated influenza vaccines against different viral subtypes.     

Highly conserved influenza T cell epitopes induce broadly protective immunity

Influenza world-wide causes significant morbidity and mortality annually, and more severe pandemics when novel strains evolve to which humans are immunologically naïve. Because of the high viral mutation rate, new vaccines must be generated based on the prevalence of circulating strains every year. New approaches to induce more broadly protective immunity are urgently needed. Previous research has demonstrated that influenza-specific T cells can provide broadly heterotypic protective immunity in both mice and humans, supporting the rationale for developing a T cell-targeted universal influenza vaccine. We used state-of-the art immunoinformatic tools to identify putative pan-HLA-DR and HLA-A2 supertype-restricted T cell epitopes highly conserved among > 50 widely diverse influenza A strains (representing hemagglutinin types 1, 2, 3, 5, 7 and 9). We found influenza peptides that are highly conserved across influenza subtypes that were also predicted to be class I epitopes restricted by HLA-A2. These peptides were found to be immunoreactive in HLA-A2 positive but not HLA-A2 negative individuals. Class II-restricted T cell epitopes that were highly conserved across influenza subtypes were identified. Human CD4⁺ T cells were reactive with these conserved CD4 epitopes, and epitope expanded T cells were responsive to both H1N1 and H3N2 viruses. Dendritic cell vaccines pulsed with conserved epitopes and DNA vaccines encoding these epitopes were developed and tested in HLA transgenic mice. These vaccines were highly immunogenic, and more importantly, vaccine-induced immunity was protective against both H1N1 and H3N2 influenza challenges. These results demonstrate proof-of-principle that conserved T cell epitopes expressed by widely diverse influenza strains can induce broadly protective, heterotypic influenza immunity, providing strong support for further development of universally relevant multi-epitope T cell-targeting influenza vaccines.     

Identification of protective and non-protective T cell epitopes in influenza

Understanding the immune response to different CD8 T cell epitopes is important for the development of vaccines designed to promote protective cellular immunity. Recently, we have shown that vaccination with the PA(224-233)/D(b) epitope of influenza virus was poorly protective in terms of viral clearance. To determine if other influenza virus epitopes behave in this manner, we analyzed the ability of three newly identified CD8 T cell epitopes and three previously defined epitopes to provide protection following vaccination and viral challenge. All six of the peptide-based vaccinations resulted in significantly increased numbers of epitope-specific CD8 T cells in the spleen. Interestingly, we found that vaccination with three peptides (HA(332-340), M1(128-135), or PA(224-233)) resulted in delayed viral clearance following infection. These findings indicate that some epitopes have a detrimental impact on viral clearance and have important implications for the development of vaccination strategies designed to provide protection against subsequent influenza virus challenge.     

Sequential pulmonary immunization with heterologous recombinant influenza A virus tuberculosis vaccines protects against murine M. tuberculosis infection

Tuberculosis (TB) infection affects a quarter of the global population resulting in a large burden of TB disease and mortality. The long-term control of TB requires vaccines with greater efficacy and durability than the current Mycobacterium bovis Bacille Calmette-Guérin (BCG). Pulmonary immunization may increase and prolong immunity at the site of Mycobacterium tuberculosis infection. We have investigated recombinant influenza A viruses (rIAVs) expressing the p25 CD4⁺ T cell epitope of M. tuberculosis Ag85B_240–254 for single and sequential immunization against M. tuberculosis infection. Intranasal immunization with single dose of rIAV X31 (H3N2 strain) expressing the p25 epitope (X31-p25), induced p25-specific CD4⁺ T cells and conferred protection against aerosol delivery of M. tuberculosis infection in the lungs. To enhance this effect, prime-boost immunization with hetero-subtypic rIAVs was examined. Sequential immunization with X31-p25 and a second rIAV, PR8 (H1N1 strain) expressing the same epitope (PR8-p25), increased the frequency of p25-specific IFN-γ T cell responses and polyfunctional CD4⁺ T cells producing IFN-γ, IL-2, and TNF, compared to immunization with each rIAV alone. This combination resulted in protection against M. tuberculosis in both the lungs and spleen. Therefore, our study revealed that rIAV is not only an efficient vector to induce protective immunity in the lungs, but also has a potential use for sequential immunization with heterologous rIAV to boost the immunogenicity and improve the protection against M. tuberculosis.     

HCV-core and NS3 antigens play disparate role in inducing regulatory or effector T cells in vivo: Implications for viral persistence or clearance

A distinguishing feature of HCV is its ability to persist in majority of the infected people. We investigated the role of HCV-core and NS3 in inducing effector T cells to mediate antiviral immunity. Our studies revealed that immunization with recombinant adenoviral vector containing HCV-core or NS3 leads to differential development of regulatory vs. effector T cells in mice, resulting in distinct outcomes of virus infection. For the first time, our studies directly demonstrate that HCV-core enhances both CD4⁺ and CD8⁺ T_regs which possibly contribute to persistent infection, whereas HCV NS3 induces both CD4⁺ and CD8⁺ effector T cells to allow viral clearance.     

Complete protection against a H5N2 avian influenza virus by a DNA vaccine expressing a fusion protein of H1N1 HA and M2e

Most influenza vaccines target hemagglutinin (HA) in order to protect the host against infection. However, theses vaccines are strain-specific due to major antigenic variations of HA. Since it is difficult to predict epidemic and pandemic strains of influenza virus, the development of effective vaccines against divergent influenza viruses is urgently needed. Although M2e-based vaccines are associated with weaker protection than HA-based vaccines that induce neutralizing antibodies against challenge virus matched-strain, the extracellular domain of Matrix 2 protein (M2e) is one of a potential broad-spectrum immunogen because it contains highly conserved sequences among influenza A viruses. In this study, M2e sequence was fused to H1N1 HA DNA (M2e-HA) and the immunogenicity and antiviral efficacy of this DNA vaccine was evaluated in response to challenge with a heterosubtypic H5N2 avian influenza virus. Compared to vaccination with HA or M2e DNA alone, vaccination with M2e-HA DNA or combination of M2e DNA and HA DNA (M2e DNA + HA DNA) induced a broad immunity without evidence of immune interference. In addition, HA-specific CD8⁺ and M2e-specific T cell responses elicited by M2e-HA DNA vaccination were significantly higher than those of HA or M2e DNA vaccine alone, respectively. Following challenge with a heterosubtypic influenza virus infection, vaccination with M2e-HA DNA conferred complete protection against mortality. In combination, these results suggest that DNA vaccines expressing a fusion protein, M2e-HA, may provide an attractive approach for the development of broad-spectrum influenza vaccines.