Broadly Protective Adenovirus-Based Multivalent Vaccines against Highly Pathogenic Avian Influenza Viruses for Pandemic Preparedness

Recurrent outbreaks of H5, H7 and H9 avian influenza viruses in domestic poultry accompanied by their occasional transmission to humans have highlighted the public health threat posed by these viruses. Newer vaccine approaches for pandemic preparedness against these viruses are needed, given the limitations of vaccines currently approved for H5N1 viruses in terms of their production timelines and the ability to induce protective immune responses in the absence of adjuvants. In this study, we evaluated the feasibility of an adenovirus (AdV)-based multivalent vaccine approach for pandemic preparedness against H5, H7 and H9 avian influenza viruses in a mouse model. Replication-defective AdV vectors expressing hemagglutinin (HA) from different subtypes and nucleoprotein (NP) from one subtype induced high levels of humoral and cellular immune responses and conferred protection against virus replication following challenge with H5, H7 and H9 avian influenza virus subtypes. Inclusion of HA from the 2009 H1N1 pandemic virus in the vaccine formulation further broadened the vaccine coverage. Significantly high levels of HA stalk-specific antibodies were observed following immunization with the multivalent vaccine. Inclusion of NP into the multivalent HA vaccine formulation resulted in the induction of CD8 T cell responses. These results suggest that a multivalent vaccine strategy may provide reasonable protection in the event of a pandemic caused by H5, H7, or H9 avian influenza virus before a strain-matched vaccine can be produced.     

Avian influenza h6 viruses productively infect and cause illness in mice and ferrets

Influenza pandemic preparedness has focused on influenza virus H5 and H7 subtypes. However, it is not possible to predict with certainty which subtype of avian influenza virus will cause the next pandemic, and it is prudent to include other avian influenza virus subtypes in pandemic preparedness efforts. An H6 influenza virus was identified as a potential progenitor of the H5N1 viruses that emerged in Hong Kong in 1997. This virus continues to circulate in the bird population in Asia, and other H6 viruses are prevalent in birds in North America and Asia. The high rate of reassortment observed in influenza viruses and the prevalence of H6 viruses in birds suggest that this subtype may pose a pandemic risk. Very little is known about the replicative capacity, immunogenicity, and correlates of protective immunity for low-pathogenicity H6 influenza viruses in mammals. We evaluated the antigenic and genetic relatedness of 14 H6 influenza viruses and their abilities to replicate and induce a cross-reactive immune response in two animal models: mice and ferrets. The different H6 viruses replicated to different levels in the respiratory tracts of mice and ferrets, causing varied degrees of morbidity and mortality in these two models. H6 virus infection induced similar patterns of neutralizing antibody responses in mice and ferrets; however, species-specific differences in the cross-reactivity of the antibody responses were observed. Overall, cross-reactivity of neutralizing antibodies in H6 virus-infected mice did not correlate well with protection against heterologous wild-type H6 viruses. However, we have identified an H6 virus that induces protective immunity against viruses in the North American and Eurasian lineages.     

Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus

Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.     

Vaccination against seasonal influenza A/H3N2 virus reduces the induction of heterosubtypic immunity against influenza A/H5N1 virus infection in ferrets

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.     

Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM+ Memory B Cells

Background

The hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection.

Methods and Findings

Here we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM⁺ memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge.

Conclusions

The human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM⁺ memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens.     

Hemagglutinin Stalk Immunity Reduces Influenza Virus Replication and Transmission in Ferrets

We assessed whether influenza virus hemagglutinin stalk-based immunity protects ferrets against aerosol-transmitted H1N1 influenza virus infection. Immunization of ferrets by a universal influenza virus vaccine strategy based on viral vectors expressing chimeric hemagglutinin constructs induced stalk-specific antibody responses. Stalk-immunized ferrets were cohoused with H1N1-infected ferrets under conditions that permitted virus transmission. Hemagglutinin stalk-immunized ferrets had lower viral titers and delayed or no virus replication at all following natural exposure to influenza virus.     

H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination

Background

During the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection.

Methods and Findings

To study hemagglutinin (HA) antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV) and compared them to the plasma cell repertoires of subjects experimentally infected (EI) with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject.

Conclusion

The presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.     

Virus-like particle vaccine induces protective immunity against homologous and heterologous strains of influenza virus

Recurrent outbreaks of highly pathogenic avian influenza virus pose the threat of pandemic spread of lethal disease and make it a priority to develop safe and effective vaccines. Influenza virus-like particles (VLPs) have been suggested to be a promising vaccine approach. However, VLP-induced immune responses, and their roles in inducing memory immune responses and cross-protective immunity have not been investigated. In this study, we developed VLPs containing influenza virus A/PR8/34 (H1N1) hemagglutinin (HA) and matrix (M1) proteins and investigated their immunogenicity, long-term cross-protective efficacy, and effects on lung proinflammatory cytokines in mice. Intranasal immunization with VLPs containing HA induced high serum and mucosal antibody titers and neutralizing activity against PR8 and A/WSN/33 (H1N1) viruses. Mice immunized with VLPs containing HA showed little or no proinflammatory lung cytokines and were protected from a lethal challenge with mouse-adapted PR8 or WSN viruses even 5 months postimmunization. Influenza VLPs induced mucosal immunoglobulin G and cellular immune responses, which were reactivated rapidly upon virus challenge. Long-lived antibody-secreting cells were detected in the bone marrow of immunized mice. Immune sera administered intranasally were able to confer 100% protection from a lethal challenge with PR8 or WSN, which provides further evidence that anti-HA antibodies are primarily responsible for preventing infection. Taken together, these results indicate that nonreplicating influenza VLPs represent a promising strategy for the development of a safe and effective vaccine to control the spread of lethal influenza viruses.     

Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets

Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV) were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP), polymerase basic protein 1 (PB1) and matrix protein 1 (M1). C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks.     

H5N1 virus-like particle vaccine elicits cross-reactive neutralizing antibodies that preferentially bind to the oligomeric form of influenza virus hemagglutinin in humans

Transmission of pathogenic avian influenza viruses (AIV) from wild birds to domestic poultry and humans is continuing in multiple countries around the world. In preparation for a potential AIV pandemic, multiple vaccine candidates are under development. In the case of H5N1 AIV, a clear shift in transmission from clade 1 to clade 2 viruses occurred in recent years. The virus-like particle (VLP) represents an economical approach to pandemic vaccine development. In the current study, we evaluated the humoral immune response in humans vaccinated with H5N1 A/Indonesia/05/2005 (clade 2.1) VLP vaccine manufactured in Sf9 insect cells. The VLPs were comprised of the influenza virus hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1) proteins. In an FDA-approved phase I/II human clinical study, two doses of H5N1 VLPs at 15, 45, or 90 μg HA/dose resulted in seroconversion and production of functional antibodies. Moreover, cross-reactivity against other clade 2 subtypes was demonstrated using virus neutralization assays. H5N1 whole-genome fragment phage display libraries (GFPDL) were used to elucidate the antibody epitope repertoire in postvaccination human sera. Diverse epitopes in HA1/HA2 and NA were recognized by postvaccination sera from the two high-dose groups, including large segments spanning the HA1 receptor binding domain. Importantly, the vaccine elicited sera that preferentially bound to an oligomeric form of recombinant HA1 compared with monomeric HA1. The oligomeric/monomeric HA1 binding ratios of the sera correlated with the virus neutralizing titers. Additionally, the two high-dose VLP vaccine groups generated NA-inhibiting antibodies that were associated with binding to a C-terminal epitope close to the sialic acid binding site. These findings represent the first report describing the quality of the antibody responses in humans following AIV VLP immunization and support further development of such vaccines against emerging influenza virus strains.     

Virus-Like Particle Vaccine Protects against 2009 H1N1 Pandemic Influenza Virus in Mice

Background

The 2009 influenza pandemic and shortages in vaccine supplies worldwide underscore the need for new approaches to develop more effective vaccines.

Methodology/Principal Findings

We generated influenza virus-like particles (VLPs) containing proteins derived from the A/California/04/2009 virus, and tested their efficacy as a vaccine in mice. A single intramuscular vaccination with VLPs provided complete protection against lethal challenge with the A/California/04/2009 virus and partial protection against A/PR/8/1934 virus, an antigenically distant human isolate. VLP vaccination induced predominant IgG2a antibody responses, high hemagglutination inhibition (HAI) titers, and recall IgG and IgA antibody responses. HAI titers after VLP vaccination were equivalent to those observed after live virus infection. VLP immune sera also showed HAI responses against diverse geographic pandemic isolates. Notably, a low dose of VLPs could provide protection against lethal infection.

Conclusion/Significance

This study demonstrates that VLP vaccination provides highly effective protection against the 2009 pandemic influenza virus. The results indicate that VLPs can be developed into an effective vaccine, which can be rapidly produced and avoid the need to isolate high growth reassortants for egg-based production.     

Immune protection induced on day 10 following administration of the 2009 A/H1N1 pandemic influenza vaccine

Background

The 2009 swine-origin influenza virus (S-OIV) H1N1 pandemic has caused more than 18,000 deaths worldwide. Vaccines against the 2009 A/H1N1 influenza virus are useful for preventing infection and controlling the pandemic. The kinetics of the immune response following vaccination with the 2009 A/H1N1 influenza vaccine need further investigation.

Methodology/Principal Findings

58 volunteers were vaccinated with a 2009 A/H1N1 pandemic influenza monovalent split-virus vaccine (15 µg, single-dose). The sera were collected before Day 0 (pre-vaccination) and on Days 3, 5, 10, 14, 21, 30, 45 and 60 post vaccination. Specific antibody responses induced by the vaccination were analyzed using hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA). After administration of the 2009 A/H1N1 influenza vaccine, specific and protective antibody response with a major subtype of IgG was sufficiently developed as early as Day 10 (seroprotection rate: 93%). This specific antibody response could maintain for at least 60 days without significant reduction. Antibody response induced by the 2009 A/H1N1 influenza vaccine could not render protection against seasonal H1N1 influenza (seroconversion rate: 3% on Day 21). However, volunteers with higher pre-existing seasonal influenza antibody levels (pre-vaccination HI titer ≥1∶40, Group 1) more easily developed a strong antibody protection effect against the 2009 A/H1N1 influenza vaccine as compared with those showing lower pre-existing seasonal influenza antibody levels (pre-vaccination HI titer <1∶40, Group 2). The titer of the specific antibody against the 2009 A/H1N1 influenza was much higher in Group 1 (geometric mean titer: 146 on Day 21) than that in Group 2 (geometric mean titer: 70 on Day 21).

Conclusions/Significance

Recipients could gain sufficient protection as early as 10 days after vaccine administration. The protection could last at least 60 days. Individuals with a stronger pre-existing seasonal influenza antibody response may have a relatively higher potential for developing a stronger humoral immune response after vaccination with the 2009 A/H1N1 pandemic influenza vaccine.     

Induction of immune response to influenza virus with anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against five monoclonal antibodies (MAbs) specific for different antigenic sites on the hemagglutinin (HA) of influenza virus Mem71H-BelN (H3N1) [A/Memphis/1/71 (H3N2) x A/Bel/42 (H1N1)]. Each of the anti-Id sera was directed predominantly towards a unique (private) idiotype of the immunizing MAb, none of the five idiotypes being detectable in pooled BALB/c antisera against Mem71H-BelN virus or on most other anti-HA MAbs tested. Partial idiotypic sharing was observed, however, between certain MAbs, from different mice, having the same or similar epitope specificity for HA. When used as immunogens in BALB/c mice, two of the five anti-Id preparations induced antibodies that reacted with Mem71H-BelN virus and displayed neutralizing activity. Mice of other inbred strains responded similarly, indicating that the response was not genetically restricted by the Igh locus. From their pattern of reactivity with mutants of Mem71H-BelN virus with known single amino acid substitutions in the HA molecule, the antiviral antibodies elicited by anti-Id antibodies were shown to be directed to the same antigenic site on A/Memphis/1/71 HA as the original immunizing MAb (site A or site E, respectively). However, several of these antisera were shown to contain additional distinct subpopulations of antibodies specific for heterologous influenza A virus strains, either of the H3 subtype or of a different HA subtype (H1 or H2). Since the induction of antibodies to HA of different subtypes is not a feature of the antibody response to influenza virus itself, their induction by anti-Id antibodies merits further investigation.     

Efficacy of vaccination with different combinations of MF59-adjuvanted and nonadjuvanted seasonal and pandemic influenza vaccines against pandemic H1N1 (2009) influenza virus infection in ferrets

Serum antibodies induced by seasonal influenza or seasonal influenza vaccination exhibit limited or no cross-reactivity against the 2009 pandemic swine-origin influenza virus of the H1N1 subtype (pH1N1). Ferrets immunized once or twice with MF59-adjuvanted seasonal influenza vaccine exhibited significantly reduced lung virus titers but no substantial clinical protection against pH1N1-associated disease. However, priming with MF59-adjuvanted seasonal influenza vaccine significantly increased the efficacy of a pandemic MF59-adjuvanted influenza vaccine against pH1N1 challenge. Elucidating the mechanism involved in this priming principle will contribute to our understanding of vaccine- and infection-induced correlates of protection. Furthermore, a practical consequence of these findings is that during an emerging pandemic, the implementation of a priming strategy with an available adjuvanted seasonal vaccine to precede the eventual pandemic vaccination campaign may be useful and life-saving.     

A VLP Vaccine Induces Broad-Spectrum Cross-Protective Antibody Immunity against H5N1 and H1N1 Subtypes of Influenza A Virus

The recent threats of influenza epidemics and pandemics have prioritized the development of a universal vaccine that offers protection against a wider variety of influenza infections. Here, we demonstrate a genetically modified virus-like particle (VLP) vaccine, referred to as H5M2eN1-VLP, that increased the antigenic content of NA and induced rapid recall of antibody against HA(2) after viral infection. As a result, H5M2eN1-VLP vaccination elicited a broad humoral immune response against multiple viral proteins and caused significant protection against homologous RG-14 (H5N1) and heterologous A/California/07/2009 H1N1 (CA/07) and A/PR/8/34 H1N1 (PR8) viral lethal challenges. Moreover, the N1-VLP (lacking HA) induced production of a strong NA antibody that also conferred significant cross protection against H5N1 and heterologous CA/07 but not PR8, suggesting the protection against N1-serotyped viruses can be extended from avian-origin to CA/07 strain isolated in humans, but not to evolutionally distant strains of human-derived. By comparative vaccine study of an HA-based VLP (H5N1-VLP) and NA-based VLPs, we found that H5N1-VLP vaccination induced specific and strong protective antibodies against the HA(1) subunit of H5, thus restricting the breadth of cross-protection. In summary, we present a feasible example of direction of VLP vaccine immunity toward NA and HA(2), which resulted in cross protection against both seasonal and pandemic influenza strains, that could form the basis for future design of a better universal vaccine.     

GM-CSF Increases Mucosal and Systemic Immunogenicity of an H1N1 Influenza DNA Vaccine Administered into the Epidermis of Non-Human Primates

Background

The recent H5N1 avian and H1N1 swine-origin influenza virus outbreaks reaffirm that the threat of a world-wide influenza pandemic is both real and ever-present. Vaccination is still considered the best strategy for protection against influenza virus infection but a significant challenge is to identify new vaccine approaches that offer accelerated production, broader protection against drifted and shifted strains, and the capacity to elicit anti-viral immune responses in the respiratory tract at the site of viral entry. As a safe alternative to live attenuated vaccines, the mucosal and systemic immunogenicity of an H1N1 influenza (A/New Caledonia/20/99) HA DNA vaccine administered by particle-mediated epidermal delivery (PMED or gene gun) was analyzed in rhesus macaques.

Methodology/Principal Findings

Macaques were immunized at weeks 0, 8, and 16 using a disposable single-shot particle-mediated delivery device designed for clinical use that delivers plasmid DNA directly into cells of the epidermis. Significant levels of hemagglutination inhibiting (HI) antibodies and cytokine-secreting HA-specific T cells were observed in the periphery of macaques following 1–3 doses of the PMED HA DNA vaccine. In addition, HA DNA vaccination induced detectable levels of HA-specific mucosal antibodies and T cells in the lung and gut-associated lymphoid tissues of vaccinated macaques. Importantly, co-delivery of a DNA encoding the rhesus macaque GM-CSF gene was found to significantly enhance both the systemic and mucosal immunogenicity of the HA DNA vaccine.

Conclusions/Significance

These results provide strong support for the development of a particle-mediated epidermal DNA vaccine for protection against respiratory pathogens such as influenza and demonstrate, for the first time, the ability of skin-delivered GM-CSF to serve as an effective mucosal adjuvant for vaccine induction of immune responses in the gut and respiratory tract.     

Memory immune responses against pandemic (H1N1) 2009 influenza virus induced by a whole particle vaccine in cynomolgus monkeys carrying Mafa-A1*052:02

We made an H1N1 vaccine candidate from a virus library consisting of 144 (?=?16 HA×9 NA) non-pathogenic influenza A viruses and examined its protective effects against a pandemic (2009) H1N1 strain using immunologically na?ve cynomolgus macaques to exclude preexisting immunity and to employ a preclinical study since preexisting immunity in humans previously vaccinated or infected with influenza virus might make comparison of vaccine efficacy difficult. Furthermore, macaques carrying a major histocompatibility complex class I molecule, Mafa-A1*052:02, were used to analyze peptide-specific CD8(+) T cell responses. Sera of macaques immunized with an inactivated whole particle formulation without addition of an adjuvant showed higher neutralization titers against the vaccine strain A/Hokkaido/2/1981 (H1N1) than did sera of macaques immunized with a split formulation. Neutralization activities against the pandemic strain A/Narita/1/2009 (H1N1) in sera of macaques immunized twice with the split vaccine reached levels similar to those in sera of macaques immunized once with the whole particle vaccine. After inoculation with the pandemic virus, the virus was detected in nasal samples of unvaccinated macaques for 6 days after infection and for 2.67 days and 5.33 days on average in macaques vaccinated with the whole particle vaccine and the split vaccine, respectively. After the challenge infection, recall neutralizing antibody responses against the pandemic virus and CD8(+) T cell responses specific for nucleoprotein peptide NP262-270 bound to Mafa-A1*052:02 in macaques vaccinated with the whole particle vaccine were observed more promptly or more vigorously than those in macaques vaccinated with the split vaccine. These findings demonstrated that the vaccine derived from our virus library was effective for pandemic virus infection in macaques and that the whole particle vaccine conferred more effective memory and broader cross-reactive immune responses to macaques against pandemic influenza virus infection than did the split vaccine.     

Protective effect of nasal immunization of influenza virus hemagglutinin with recombinant cholera toxin B subunit as a mucosal adjuvant in mice

To develop an efficient nasal influenza vaccine, influenza A and B virus HA with rCTB as a mucosal adjuvant were administered to mice intranasally. Serum anti-HA IgG and IgA antibody responses for both HA vaccines were significantly increased in the presence of rCTB. Higher HI and neutralizing antibody titers and higher mucosal IgA antibody responses in the respiratory tract were detected when rCTB was added than without rCTB. When mice were immunized with HA vaccine with or without rCTB and challenged by intranasal administration of mouse-adapted pathogenic influenza A virus, all mice immunized with HA plus rCTB survived for seven days without any inflammatory changes in the lungs, while not all the mice immunized with HA without rCTB survived, and all of them had lung consolidations. These results demonstrate that intranasal co-administration of rCTB as a mucosal adjuvant with influenza virus HA is necessary not only for the induction of systemic and mucosal HA antibodies, but also for the protection of mice from morbidity and mortality resulting from virus infection.     

A novel particulate influenza vaccine induces long-term and broad-based immunity in mice after oral immunization.

The immunogenicity of a novel particulate oral influenza vaccine was examined in terms of antibody response and protection in mice. Oral immunization with chicken erythrocytes (CRBC) adsorbed with gamma-irradiated influenza A virus induced high levels of immunoglobulin G antibodies and protection in the lung compared with gamma-irradiated virus administered alone or CRBC. Immunoglobulin A antibodies were the predominant antibodies in nasal washings, and their presence did not correlate with protection as well as immunoglobulin G antibodies. Immunity was not specific for the immunizing virus subtype, as antibodies and enhanced lung clearance of virus were demonstrated with different virus subtypes. However, mice were not protected when challenged with live influenza B virus. The antibody response and the degree of protection were dependent on both the concentration of virus adsorbed to CRBC and number of CRBC adsorbed to virus. Virus-adsorbed CRBC given subcutaneously failed to induce antibodies or protection. Oral immunization with A/Qld/6/72 (H3N2) virus gave a high level of protection over 12 weeks, which could be demonstrated with different subtypes. Protection correlated with antibody levels in the lung determined by both enzyme-linked immunosorbent and hemagglutination inhibition assays, although the levels detected by the latter declined over time.     

Cross-clade protective immune responses to influenza viruses with H5N1 HA and NA elicited by an influenza virus-like particle

Background

Vaccination is a cost-effective counter-measure to the threat of seasonal or pandemic outbreaks of influenza. To address the need for improved influenza vaccines and alternatives to egg-based manufacturing, we have engineered an influenza virus-like particle (VLP) as a new generation of non-egg or non-mammalian cell culture-based candidate vaccine.

Methodology/Principal Findings

We generated from a baculovirus expression system using insect cells, a non-infectious recombinant VLP vaccine from both influenza A H5N1 clade 1 and clade 2 isolates with pandemic potential. VLPs were administered to mice in either a one-dose or two-dose regimen and the immune responses were compared to those induced by recombinant hemagglutinin (rHA). Both humoral and cellular responses were analyzed. Mice vaccinated with VLPs were protected against challenge with lethal reassortant viruses expressing the H5N1 HA and NA, regardless if the H5N1 clade was homologous or heterologous to the vaccine. However, rHA-vaccinated mice showed considerable weight loss and death following challenge with the heterovariant clade virus. Protection against death induced by VLPs was independent of the pre-challenge HAI titer or cell-mediated responses to HA or M1 since vaccinated mice, with low to undetectable cross-clade HAI antibodies or cellular responses to influenza antigens, were still protected from a lethal viral challenge. However, an apparent association rate of antibody binding to HA correlated with protection and was enhanced using VLPs, particularly when delivered intranasally, compared to rHA vaccines.

Conclusion/Significance

This is the first report describing the use of an H5N1 VLP vaccine created from a clade 2 isolate. The results show that a non-replicating virus-like particle is effective at eliciting a broadened, cross-clade protective immune response to proteins from emerging H5N1 influenza isolates giving rise to a potential pandemic influenza vaccine candidate for humans that can be stockpiled for use in the event of an outbreak of H5N1 influenza.