Influenza Virus Challenge In Mice Research Articles

A CpG 1018 adjuvanted neuraminidase vaccine provides robust protection from influenza virus challenge in mice

Influenza virus infections pose a significant threat to global health. Vaccination is the main countermeasure against influenza virus spread, however, the effectiveness of vaccines is variable. Current seasonal influenza virus vaccines mostly rely on the immunodominant hemagglutinin (HA) glycoprotein on the viral surface, which usually leads to a narrow and strain-specific immune response. The HA undergoes constant antigenic drift, which can lead to a dramatic loss in vaccine effectiveness, requiring the annual reformulation and readministration of influenza virus vaccines. Recently, it has been demonstrated that the subdominant glycoprotein, neuraminidase (NA), is an attractive target for vaccine development. Here, we tested a newly developed recombinant influenza virus N1 neuraminidase vaccine candidate, named N1-MPP, adjuvanted with CpG 1018, a TLR9 agonist. Additionally, N2-MPP and B-NA-MPP vaccine constructs have been generated to cover the range of influenza viruses that are seasonally circulating in humans. These constructs have been characterized in vitro and in vivo regarding their functionality and protective potential. Furthermore, a trivalent NA-MPP mix was tested. No antigenic competition between the individual NA constructs was detected. By adjuvating the recombinant protein constructs with CpG 1018 it was possible to induce a strong and robust immune response against the NA, which provided full protection against morbidity and mortality after high lethal challenges in vivo. This study provides important insights for the development of a broadly protective NA-based influenza virus vaccine candidate.

Vaccination with viral vectors expressing NP, M1 and chimeric hemagglutinin induces broad protection against influenza virus challenge in mice

Seasonal influenza virus infections cause significant morbidity and mortality every year. Annual influenza virus vaccines are effective but only when well matched with circulating strains. Therefore, there is an urgent need for better vaccines that induce broad protection against drifted seasonal and emerging pandemic influenza viruses. One approach to design such vaccines is based on targeting conserved regions of the influenza virus hemagglutinin. Sequential vaccination with chimeric hemagglutinin constructs can refocus antibody responses towards the conserved immunosubdominant stalk domain of the hemagglutinin, rather than the variable immunodominant head. A complementary approach for a universal influenza A virus vaccine is to induce T-cell responses to conserved internal influenza virus antigens. For this purpose, replication deficient recombinant viral vectors based on Chimpanzee Adenovirus Oxford 1 and Modified Vaccinia Ankara virus are used to express the viral nucleoprotein and the matrix protein 1. In this study, we combined these two strategies and evaluated the efficacy of viral vectors expressing both chimeric hemagglutinin and nucleoprotein plus matrix protein 1 in a mouse model against challenge with group 2 influenza viruses including H3N2, H7N9 and H10N8. We found that vectored vaccines expressing both sets of antigens provided enhanced protection against H3N2 virus challenge when compared to vaccination with viral vectors expressing only one set of antigens. Vaccine induced antibody responses against divergent group 2 hemagglutinins, nucleoprotein and matrix protein 1 as well as robust T-cell responses to the nucleoprotein and matrix protein 1 were detected. Of note, it was observed that while antibodies to the H3 stalk were already boosted to high levels after two vaccinations with chimeric hemagglutinins (cHAs), three exposures were required to induce strong reactivity across subtypes. Overall, these results show that a combinations of different universal influenza virus vaccine strategies can induce broad antibody and T-cell responses and can provide increased protection against influenza.

In Vitro Neutralization Is Not Predictive of Prophylactic Efficacy of Broadly Neutralizing Monoclonal Antibodies CR6261 and CR9114 against Lethal H2 Influenza Virus Challenge in Mice.

Influenza viruses of the H1N1, H2N2, and H3N2 subtypes have caused previous pandemics. H2 influenza viruses represent a pandemic threat due to continued circulation in wild birds and limited immunity in the human population. In the event of a pandemic, antiviral agents are the mainstay for treatment, but broadly neutralizing antibodies (bNAbs) may be a viable alternative for short-term prophylaxis or treatment. The hemagglutinin stem binding bNAbs CR6261 and CR9114 have been shown to protect mice from severe disease following challenge with H1N1 and H5N1 and with H1N1, H3N2, and influenza B viruses, respectively. Early studies with CR6261 and CR9114 showed weak in vitro activity against human H2 influenza viruses, but the in vivo efficacy against H2 viruses is unknown. Therefore, we evaluated these antibodies against human- and animal-origin H2 viruses A/Ann Arbor/6/1960 (H2N2) (AA60) and A/swine/MO/4296424/06 (H2N3) (Sw06). In vitro, CR6261 neutralized both H2 viruses, while CR9114 only neutralized Sw06. To evaluate prophylactic efficacy, mice were given CR6261 or CR9114 and intranasally challenged 24 h later with lethal doses of AA60 or Sw06. Both antibodies reduced mortality, weight loss, airway inflammation, and pulmonary viral load. Using engineered bNAb variants, antibody-mediated cell cytotoxicity reporter assays, and Fcγ receptor-deficient (Fcer1g-/-) mice, we show that the in vivo efficacy of CR9114 against AA60 is mediated by Fcγ receptor-dependent mechanisms. Collectively, these findings demonstrate the in vivo efficacy of CR6261 and CR9114 against H2 viruses and emphasize the need for in vivo evaluation of bNAbs.IMPORTANCE bNAbs represent a strategy to prevent or treat infection by a wide range of influenza viruses. The evaluation of these antibodies against H2 viruses is important because H2 viruses caused a pandemic in 1957 and could cross into humans again. We demonstrate that CR6261 and CR9114 are effective against infection with H2 viruses of both human and animal origin in mice, despite the finding that CR9114 did not display in vitro neutralizing activity against the human H2 virus. These findings emphasize the importance of in vivo evaluation and testing of bNAbs.

Self-assembly polymerization enhances the immunogenicity of influenza M2e peptide

The extracellular domain of Influenza M2 protein (M2e) was considered as a promising target for universal influenza vaccine development. Several M2e-based influenza vaccines have been developed and many of them used a mutant M2e peptide, in which the two conserved cysteine residues were substituted by serine residues. In this paper, we compared the antigenicity and immunogenicity of wild type and cysteine-mutant M2e peptides. We found that the cysteine substitution slightly affected the antigenicity of M2e epitope, but greatly reduced the immunogenicity of M2e peptide. The cysteine substitution also disabled the M2e peptide from inducing protection against influenza virus challenge in mice. Further analysis revealed that the immunogenicity of M2e peptide was enhanced by the self-assembly of the peptide through inter-peptide disulfide bonds. These results provide new information to improve the design of M2e-based vaccines against potential influenza pandemics.

Molecular mechanisms underlying protection against H9N2 influenza virus challenge in mice by recombinant Lactobacillus plantarum with surface displayed HA2-LTB

It has been considered that the Avian influenza virus (AIV) causes severe threats to poultry industry. In this study, we constructed a series of recombinant Lactobacillus plantarum (L. plantarum) with surface displayed hemagglutinin subunit 2 (HA2) alone or together with heat-labile toxin B subunit (LTB) from enterotoxigenic Escherichia coli. Balb/c mice were used as model to evaluate the protective effects of recombinant L. plantarum strains against H9N2 subtype challenge.The results showed that the presence of LTB significantly increased the percentages of CD3+CD4+IL-4+, CD3+CD4+IFN-γ+ and CD3+CD4+IL-17+ T cells, as well as CD3+CD8+IFN-γ+ T cells in spleen and MLNs determined by Fluorescence-Activated Cell Sorting assay. Similar increased production of serum IFN-γ was also confirmed by enzyme linked immunosorbent assay (ELISA). The L. plantarum with surface displayed HA2-LTB also dramatically increased the percentages of B220+ IgA+ B cells in peyer patch, in consistent with elevated production of mucosal SIgA antibody determined by ELISA. Finally, the orally administrated HA2-LTB expressing strain efficiently protected mice against H9N2 subtype AIV challenge shown by increased survival percentages, body weight gains and decreased lung lesions in histopathologic analysis. In conclusion, this study provides more detail mechanisms underlying the adjuvant effects of LTB on heterologous antigen produced in recombinant lactic acid bacteria.

The Carbomer-Lecithin Adjuvant Adjuplex Has Potent Immunoactivating Properties and Elicits Protective Adaptive Immunity against Influenza Virus Challenge in Mice.

The continued discovery and development of adjuvants for vaccine formulation are important to safely increase potency and/or reduce the antigen doses of existing vaccines and tailor the adaptive immune response to newly developed vaccines. Adjuplex is a novel adjuvant platform based on a purified lecithin and carbomer homopolymer. Here, we analyzed the adjuvant activity of Adjuplex in mice for the soluble hemagglutinin (HA) glycoprotein of influenza A virus. The titration of Adjuplex revealed an optimal dose of 1% for immunogenicity, eliciting high titers of HA-specific IgG but inducing no significant weight loss. At this dose, Adjuplex completely protected mice from an otherwise lethal influenza virus challenge and was at least as effective as the adjuvants monophosphoryl lipid A (MPL) and alum in preventing disease. Adjuplex elicited balanced Th1-/Th2-type immune responses with accompanying cytokines and triggered antigen-specific CD8+ T-cell proliferation. The use of the peritoneal inflammation model revealed that Adjuplex recruited dendritic cells (DCs), monocytes, and neutrophils in the context of innate cytokine and chemokine secretion. Adjuplex neither triggered classical maturation of DCs nor activated a pathogen recognition receptor (PRR)-expressing NF-κB reporter cell line, suggesting a mechanism of action different from that reported for classical pathogen-associated molecular pattern (PAMP)-activated innate immunity. Taken together, these data reveal Adjuplex to be a potent and well-tolerated adjuvant with application for subunit vaccines.

Long-term immunogenicity of an inactivated split-virion 2009 pandemic influenza A H1N1 virus vaccine with or without aluminum adjuvant in mice.

In 2009, a global epidemic of influenza A(H1N1) virus caused the death of tens of thousands of people. Vaccination is the most effective means of controlling an epidemic of influenza and reducing the mortality rate. In this study, the long-term immunogenicity of influenza A/California/7/2009 (H1N1) split vaccine was observed as long as 15 months (450 days) after immunization in a mouse model. Female BALB/c mice were immunized intraperitoneally with different doses of aluminum-adjuvanted vaccine. The mice were challenged with a lethal dose (10× 50% lethal dose [LD(50)]) of homologous virus 450 days after immunization. The results showed that the supplemented aluminum adjuvant not only effectively enhanced the protective effect of the vaccine but also reduced the immunizing dose of the vaccine. In addition, the aluminum adjuvant enhanced the IgG antibody level of mice immunized with the H1N1 split vaccine. The IgG level was correlated to the survival rate of the mice. Aluminum-adjuvanted inactivated split-virion 2009 pandemic influenza A H1N1 vaccine has good immunogenicity and provided long-term protection against lethal influenza virus challenge in mice.

Crucial roles of interleukin-7 in the development of T follicular helper cells and in the induction of humoral immunity.

T follicular helper (Tfh) cells are specialized providers of cognate B cell help, which is important in promoting the induction of high-affinity antibody production in germinal centers (GCs). Interleukin-6 (IL-6) and IL-21 have been known to play important roles in Tfh cell differentiation. Here, we demonstrate that IL-7 plays a pivotal role in Tfh generation and GC formation in vivo, as treatment with anti-IL-7 neutralizing antibody markedly impaired the development of Tfh cells and IgG responses. Moreover, codelivery of mouse Fc-fused IL-7 (IL-7-mFc) with a vaccine enhanced the generation of GC B cells as well as Tfh cells but not other lineages of T helper cells, including Th1, Th2, and Th17 cells. Interestingly, a 6-fold-lower dose of an influenza virus vaccine codelivered with Fc-fused IL-7 induced higher antigen-specific and cross-reactive IgG titers than the vaccine alone in both mice and monkeys and led to markedly enhanced protection against heterologous influenza virus challenge in mice. Enhanced generation of Tfh cells by IL-7-mFc treatment was not significantly affected by the neutralization of IL-6 and IL-21, indicating an independent role of IL-7 on Tfh differentiation. Thus, IL-7 holds promise as a critical cytokine for selectively inducing Tfh cell generation and enhancing protective IgG responses. Here, we demonstrate for the first time that codelivery of Fc-fused IL-7 significantly increased influenza virus vaccine-induced antibody responses, accompanied by robust expansion of Tfh cells and GC B cells as well as enhanced GC formation. Furthermore, IL-7-mFc induced earlier and cross-reactive IgG responses, leading to striking protection against heterologous influenza virus challenge. These results suggest that Fc-fused IL-7 could be used for inducing strong and cross-protective humoral immunity against highly mutable viruses, such as HIV and hepatitis C virus, as well as influenza viruses.

Mucosal immunization with integrase-defective lentiviral vectors protects against influenza virus challenge in mice.

Recent reports highlight the potential for integrase-defective lentiviral vectors (IDLV) to be developed as vaccines due to their ability to elicit cell-mediated and humoral immune responses after intramuscular administration. Differently from their integrase-competent counterpart, whose utility for vaccine development is limited by the potential for insertional mutagenesis, IDLV possess a mutation in their integrase gene that prevents genomic integration. Instead, they are maintained as episomal DNA circles that retain the ability to stably express functional proteins. Despite their favorable profile, it is unknown whether IDLV elicit immune responses after intranasal administration, a route that could be advantageous in the case of infection with a respiratory agent. Using influenza as a model, we constructed IDLV expressing the influenza virus nucleoprotein (IDLV-NP), and tested their ability to generate NP-specific immune responses and protect from challenge in vivo. We found that administration of IDLV-NP elicited NP-specific T cell and antibody responses in BALB/c mice. Importantly, IDLV-NP was protective against homologous and heterosubtypic influenza virus challenge only when given by the intranasal route. This is the first report demonstrating that IDLV can induce protective immunity after intranasal administration, and suggests that IDLV may represent a promising vaccine platform against infectious agents.

Intranasally administered Endocine™ formulated 2009 pandemic influenza H1N1 vaccine induces broad specific antibody responses and confers protection in ferrets

Influenza is a contagious respiratory disease caused by an influenza virus. Due to continuous antigenic drift of seasonal influenza viruses, influenza vaccines need to be adjusted before every influenza season. This allows annual vaccination with multivalent seasonal influenza vaccines, recommended especially for high-risk groups. There is a need for a seasonal influenza vaccine that induces broader and longer lasting protection upon easy administration. Endocine™ is a lipid-based mucosal adjuvant composed of endogenous lipids found ubiquitously in the human body. Intranasal administration of influenza antigens mixed with this adjuvant has been shown to induce local and systemic immunity as well as protective efficacy against homologous influenza virus challenge in mice. Here we used ferrets, an established animal model for human influenza virus infections, to further investigate the potential of Endocine™ as an adjuvant. Intranasal administration of inactivated pandemic H1N1/California/2009 split antigen or whole virus antigen mixed with Endocine™ induced high levels of serum hemagglutination inhibition (HI) and virus neutralization (VN) antibody titers that were also cross reactive against distant swine viruses of the same subtype. HI and VN antibody titers were already demonstrated after a single nasal immunization. Upon intratracheal challenge with a homologous challenge virus (influenza virus H1N1/The Netherlands/602/2009) immunized ferrets were fully protected from virus replication in the lungs and largely protected against body weight loss, virus replication in the upper respiratory tract and pathological changes in the respiratory tract. Endocine™ formulated vaccines containing split antigen induced higher HI and VN antibody responses and better protection from body weight loss and virus shedding in the upper respiratory tract than the Endocine™ formulated vaccine containing whole virus antigen.

The Chemotherapeutic Agent DMXAA as a Unique IRF3-Dependent Type-2 Vaccine Adjuvant

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a potent type I interferon (IFN) inducer, was evaluated as a chemotherapeutic agent in mouse cancer models and proved to be well tolerated in human cancer clinical trials. Despite its multiple biological functions, DMXAA has not been fully characterized for the potential application as a vaccine adjuvant. In this report, we show that DMXAA does act as an adjuvant due to its unique property as a soluble innate immune activator. Using OVA as a model antigen, DMXAA was demonstrated to improve on the antigen specific immune responses and induce a preferential Th2 (Type-2) response. The adjuvant effect was directly dependent on the IRF3-mediated production of type-I-interferon, but not IL-33. DMXAA could also enhance the immunogenicity of influenza split vaccine which led to significant increase in protective responses against live influenza virus challenge in mice compared to split vaccine alone. We propose that DMXAA can be used as an adjuvant that targets a specific innate immune signaling pathway via IRF3 for potential applications including vaccines against influenza which requires a high safety profile.

Inducible Nitric Oxide Contributes to Viral Pathogenesis Following Highly Pathogenic Influenza Virus Infection in Mice

Highly pathogenic influenza A viruses, including avian H5N1 viruses and the 1918 pandemic virus, cause severe respiratory disease in humans and animals. Virus infection is followed by intense pulmonary congestion due to an extensive influx of macrophages and neutrophils, which can release large quantities of reactive oxygen species potentially contributing to the pathogenesis of lung disease. Here, the role of nitric oxide (NO), a potent signaling molecule in inflammation, was evaluated following highly pathogenic influenza virus challenge in mice. We observed higher levels of NO in mice infected with H5N1 and 1918 viruses as compared to a seasonal H1N1 virus. Mice deficient in inducible NO synthase (NOS2(-/-)) exhibited reduced morbidity, reduced mortality, and diminished cytokine production in lung tissue following H5N1 and 1918-virus challenge, compared with wild-type control mice. Furthermore, systemic treatment of mice with the NOS inhibitor NG-monomethyl-l-arginine delayed weight loss and death among 1918 virus infected mice compared to untreated control animals. This study demonstrates that NO contributes to the pathogenic outcome of H5N1 and 1918 viral infections in the mouse model.

Multiple-clade H5N1 influenza split vaccine elicits broad cross protection against lethal influenza virus challenge in mice by intranasal vaccination.

BackgroundThe increase in recent outbreaks and unpredictable changes of highly pathogenic avian influenza (HPAI) H5N1 in birds and humans highlights the urgent need to develop a cross-protective H5N1 vaccine. We here report our development of a multiple-clade H5N1 influenza vaccine tested for immunogenicity and efficacy to confer cross-protection in an animal model.Methodology/Principal FindingsMice received two doses of influenza split vaccine with oil-in-water emulsion adjuvant SP01 by intranasal administration separated by two weeks. Single vaccines (3 µg HA per dose) included rg-A/Vietnam/1203/2004(Clade 1), rg-A/Indonesia/05/2005(Clade 2.1), and rg-A/Anhui/1/2005(Clade 2.3.4). The trivalent vaccine contained 1 µg HA per dose of each single vaccine. Importantly, complete cross-protection was observed in mice immunized using trivalent vaccine with oil-in-water emulsion adjuvant SP01 that was subsequently challenged with the lethal A/OT/SZ/097/03 influenza strain (Clade 0), whereas only the survival rate was up to 60% in single A/Anhui/1/2005 vaccine group.Conclusion/SignificanceOur findings demonstrated that the multiple-clade H5N1 influenza vaccine was able to elicit a cross-protective immune response to heterologous HPAI H5N1 virus, thus giving rise to a broadly cross-reactive vaccine to potential prevention use ahead of the strain-specific pandemic influenza vaccine in the event of an HPAI H5N1 influenza outbreak. Also, the multiple-clade adjuvanted vaccine could be useful in allowing timely initiation of vaccination against unknown pandemic virus.

Surfactant protein C is an essential constituent for mucosal adjuvanticity of Surfacten, acting as an antigen delivery vehicle and inducing both local and systemic immunity

We have reported that Surfacten® (St), a bovine pulmonary surfactant free of antigenic c-type lectins, is a useful mucosal adjuvant for nasal vaccination. To prepare ample supplies a synthetic adjuvant that mimics St, we analyzed essential constituents of St for mucosal adjuvanticity. Intranasal inoculation of influenza virus hemagglutinin (HA) vaccine combined with St free of surfactant protein (SP)-C resulted in failure of HA vaccine delivery to dendritic cells and loss of local and systemic immune responses. Naïve bovine SP-C, synthetic human or bovine SP-C peptide reconstituted with three major St lipids restored delivery activity and local and systemic immune responses to levels similar to those of St and provided almost complete protection against lethal doses of influenza virus challenge in mice. The delivery of fluoresceinated HA vaccine to cultured dendritic cells was significantly enhanced by co-administration of St or synthetic adjuvant, and moderately stimulated the expression of MHC class II and CD86. In addition, both St and synthetic adjuvant markedly sustained HA vaccine and achieved a wide antigen distribution in murine nasal cavity. These results suggest that synthetic mucosal adjuvant reconstituted with SP-C peptide and major St lipids is useful for ample supply of the potent mucosal adjuvant as an antigen delivery vehicle for intranasal vaccination.

The virus-induced signaling adaptor molecule enhances DNA-raised immune protection against H5N1 influenza virus infection in mice

As an adaptor molecule in the retinoic acid-inducible gene-I (RIG-I) signaling pathway, the virus-induced signaling adaptor (VISA) molecule activates NF-κB and IRF3 and thereby leads to the production of type I interferons (IFNs). To explore the potential of VISA as a genetic adjuvant for DNA vaccines, a eukaryotic expression plasmid, pVISA, was generated by cloning the VISA gene into the pVAX1vector. For comparison, the pTRIF plasmid was similarly constructed, encoding the known genetic adjuvant TRIF (TIR-domain-containing adapter-inducing interferon-β), an adapter in the Toll-like receptor (TLR) signaling pathway. Mice were immunized with the chimeric DNA vaccine pHA/NP 147–155, which encodes the HA (hemagglutinin) fused with NP (nucleoprotein) CTL epitope (NP 147–155) of H5N1 influenza virus, either alone or in combination with pVISA or pTRIF. Antigen-specific immune responses were examined in immunized mice. Our results demonstrate that co-immunization of the pHA/NP 147–155 plasmid with the VISA adjuvant augmented DNA-raised cellular immune responses and provided protection against H5N1 influenza virus challenge in mice. In addition, our data suggest that VISA acts as a stronger adjuvant for DNA immunization than TRIF. We conclude that co-inoculation with a vector expressing the adaptor molecule VISA enhanced the protective immunity against H5N1 infection induced by pHA/NP 147–155 and that VISA could be developed as a novel genetic adjuvant for DNA vaccines.

Original Article: Peroxisome proliferator‐activated receptor and AMP‐activated protein kinase agonists protect against lethal influenza virus challenge in mice

Please cite this paper as: Moseley et al. (2010) Peroxisome proliferator‐activated receptor and AMP‐activated protein kinase agonists protect against lethal influenza virus challenge in mice. Influenza and Other Respiratory Viruses 4(5), 307–311.Background A novel influenza A (H1N1) virus was isolated from humans in North America and has developed into the first pandemic of the 21st century. Reports of a global shortage of antiviral drugs, the evolution of drug‐resistant influenza virus variants, and a 6‐month delay in vaccine availability underline the need to develop new therapeutics that may be widely distributed during future pandemics.Methods In an effort to discover alternatives to the conventional therapeutic strategies available, we screened several classes of immunomodulatory agents possessing the potential to mitigate the effects of influenza virus‐induced immunopathology.Results Here, we provide preliminary evidence that two classes of drugs, peroxisome proliferator‐activated receptor‐γ agonists and AMP‐activated protein kinase agonists, provide protection in mice infected with highly pathogenic and pandemic strains of influenza virus.Conclusions The extensive production in the developed world, combined with the significant degree of protection described here, establishes these drugs as a potential therapeutic option that may be broadly implemented to combat serious disease caused by future influenza epidemics or pandemics.

Poly(γ-glutamic acid) nano-particles combined with mucosal influenza virus hemagglutinin vaccine protects against influenza virus infection in mice

Adding poly(γ-glutamic acid) nano-particles (γ-PGA-NPs), a safe, natural material, to subcutaneous immunization with influenza virus hemagglutinin (HA) vaccine increases the protective immune responses against influenza virus in mice. Here, we examined whether intranasal administration of the HA vaccine with γ-PGA-NPs would induce protection from influenza virus challenge in mice. Intranasal immunization with the mixture of γ-PGA-NPs and HA vaccine from an influenza virus strain A/PR/8/34 (H1N1) or A/New Caledonia/20/99 (H1N1) enhanced protection of mice from A/PR/8/34 infection. Intranasal immunization with A/New Caledonia/20/99 HA vaccine and γ-PGA-NPs induced cell-mediated immune responses and neutralizing antibody production for both A/New Caledonia/20/99 and A/PR/8/34. These data suggest that γ-PGA-NPs may have potential for clinical applications as a mucosal adjuvant.

A single immunization with HA DNA vaccine by electroporation induces early protection against H5N1 avian influenza virus challenge in mice

BackgroundDeveloping vaccines for the prevention of human infection by H5N1 influenza viruses is an urgent task. DNA vaccines are a novel alternative to conventional vaccines and should contribute to the prophylaxis of emerging H5N1 virus. In this study, we assessed whether a single immunization with plasmid DNA expressing H5N1 hemagglutinin (HA) could provide early protection against lethal challenge in a mouse model.MethodsMice were immunized once with HA DNA at 3, 5, 7 days before a lethal challenge. The survival rate, virus titer in the lungs and change of body weight were assayed to evaluate the protective abilities of the vaccine. To test the humoral immune response induced by HA DNA, serum samples were collected through the eye canthus of mice on various days after immunization and examined for specific antibodies by ELISA and an HI assay. Splenocytes were isolated after the immunization to determine the antigen-specific T-cell response by the ELISPOT assay.ResultsChallenge experiments revealed that a single immunization of H5N1 virus HA DNA is effective in early protection against lethal homologous virus. Immunological analysis showed that an antigen-specific antibody and T-cell response could be elicited in mice shortly after the immunization. The protective abilities were correlated with the amount of injected DNA and the length of time after vaccination.ConclusionA single immunization of 100 μg H5 HA DNA vaccine combined with electroporation was able to provide early protection in mice against homologous virus infection.

Recombinant Newcastle Disease Virus as a Vaccine Vector

A complete cDNA clone of the Newcastle disease virus (NDV) vaccine strain Hitchner B1 was constructed, and infectious recombinant virus expressing an influenza virus hemagglutinin was generated by reverse genetics. The rescued virus induces a strong humoral antibody response against influenza virus and provides complete protection against a lethal dose of influenza virus challenge in mice, demonstrating the potential of recombinant NDV as a vaccine vector.

Studies on the IgA‐independent immunological responses in mice to influenza virus challenge after oral vaccination with irradiated whole virus and an erythrocyte complex

Previous studies have described an oral influenza vaccine comprising whole irradiated virus and an erythrocyte complex (IV-EC), which gave broad-based protection against influenza virus challenge in mice. The present study examined the immune responses generated after live virus challenge of vaccinated mice, particularly to determine whether mice vaccinated with IV-EC had enhanced CTL activity to compensate for the previously reported diminution in lung IgA response. Oral vaccine groups examined were IV-EC, live virus alone (LV) or live virus-erythrocyte complex (LV-EC), compared with irradiated virus and erythrocyte alone controls. The antibody responses of IV-EC and LV-EC vaccinated mice showed significantly elevated lung and serum IgG2a levels post live virus challenge, with no comparable increases in IgG1 levels compared to controls. Spleen cells from IV-EC mice showed an enhanced post-challenge proliferative response to antigen compared with mice that had received live oral vaccines, indicating enhanced cellular activity post IV-EC immunization. However, CTL activity was not enhanced for IV-EC mice, and live virus-vaccinated mice had reduced CTL activity compared with controls, indicating that CTL were not important for post-vaccine protection. Cytokine analysis revealed a predominant IFN-gamma response in spleen cells from orally vaccinated mice, whereas IL-4 was not detected in any lung or spleen culture analysed. The results suggest, therefore, that protection from live influenza challenge after IV-EC or LV-EC vaccination was due to an IFN-mediated IgG2a response. Definitive confirmation of the role of these factors in post-vaccine protection can now be tested in IgG2a-depleted or IFN-gamma gene knockout mouse models.