Comparison of single,homologous prime-boost and heterologous prime-boost immunization strategies against H5N1 influenza virus in a mouse challenge model

Preparation for an H5N1 influenza pandemic in humans may involve priming the population with a vaccine produced from an existing, available H5N1 strain. We have used a mouse challenge model to compare the immunogenicity and efficacy of inactivated, Vero cell-derived, whole virus H5N1 vaccines in single immunization and homologous or heterologous prime-boost regimes. A single immunization was sufficient to protect against a lethal challenge with strains from matched and unmatched H5N1 clades. Homologous and heterologous prime-boost regimes induced cross-neutralizing antibodies and cross-protection against representative viruses of H5N1 clade 1, clade 2.1, clade 2.2 and clade 2.3. Moreover, the results indicate that heterologous prime-boost immunization regimes might broaden the specificity of the anti-H5N1 antibody response.     

Prior infection of chickens with H1N1 avian influenza virus elicits heterologous protection against highly pathogenic H5N2

Current vaccines for influenza are primarily killed whole virus vaccines that elicit antibody responses to the homologous virus but lack protection against heterologous viruses. Using chickens as a model we have explored the possibility of using a live low pathogenic avian influenza (LPAI) A/goose/AB/223/2005 H1N1 virus as a vaccine to generate protective immunity against heterologous highly pathogenic avian influenza (HPAI) A/chicken/Pensylvania/1370/1983 H5N2 virus challenge. Virus replicated in chickens infected with LPAI H1N1 but did not cause clinical disease. In addition, these chickens developed neutralizing antibodies to LPAI H1N1 virus, but not HPAI H5N2, 21 days post infection (DPI). Furthermore, peripheral blood mononuclear cells from H1N1-infected chickens at 20 DPI had antigen specific proliferation and IFN-γ secretion following antigen stimulation to H5N2 indicating a heterologous HPAI H5N2 specific cell mediated immunity (CMI) following LPAI H1N1 infection. Following challenge with HPAI H5N2 virus, all control chickens developed clinical disease, while chickens previously infected with H1N1 did not develop clinical disease and shed significantly less virus by oral and cloacal routes. These results indicated that previous infection with LPAI virus can generate heterologous CMI capable of protecting against HPAI H5N2.     

Homologous and heterologous antigenic matched vaccines containing different H5 hemagglutinins provide variable protection of chickens from the 2014 U.S. H5N8 and H5N2 clade 2.3.4.4 highly pathogenic avian influenza viruses

From December 2014 to June 2015, a novel H5 Eurasian A/goose/Guangdong (Gs/GD) lineage clade 2.3.4.4 high pathogenicity avian influenza (HPAI) virus caused the largest animal health emergency in US history resulting in mortality or culling of greater than 48 million poultry. The outbreak renewed interest in developing intervention strategies, including vaccines, for these newly emergent HPAI viruses. In these studies, several existing H5 vaccines or vaccine seed strains with varying genetic relatedness (85–100%) to the 2.3.4.4 HPAI viruses were evaluated for protection in poultry. Chickens received a single dose of either an inactivated whole H5 AI vaccine, or a recombinant fowl poxvirus or turkey herpesvirus-vectored vaccines with H5 AI hemagglutinin gene inserts followed by challenge with either a U.S. wild bird H5N8 (A/gyrfalcon/Washington/40188-6/2014) or H5N2 (A/northern pintail/Washington/40964/2014) clade 2.3.4.4 isolate. Results indicate that most inactivated H5 vaccines provided 100% protection from lethal effects of H5N8 or H5N2 challenge. In contrast, the recombinant live vectored vaccines only provided partial protection which ranged from 40 to 70%. Inactivated vaccine groups, in general, had lower number of birds shedding virus and at lower virus titers then the recombinant vaccine groups. Interestingly, prechallenge antibody titers using the HPAI challenge viruses as antigen in heterologous vaccine groups were typically low (≤2 log₂), yet the majority of these birds survived challenge. Taken together, these studies suggest that existing vaccines when used in a single immunization strategy may not provide adequate protection in poultry against the 2.3.4.4 HPAI viruses. Updating the H5 hemagglutinin to be genetically closer to the outbreak virus and/or using a prime-boost strategy may be necessary for optimal protection.     

Vaccine protection of chickens against antigenically diverse H5 highly pathogenic avian influenza isolates with a live HVT vector vaccine expressing the influenza hemagglutinin gene derived from a clade 2.2 avian influenza virus

Vaccination is an important tool in the protection of poultry against avian influenza (AI). For field use, the overwhelming majority of AI vaccines produced are inactivated whole virus formulated into an oil emulsion. However, recombinant vectored vaccines are gaining use for their ability to induce protection against heterologous isolates and ability to overcome maternal antibody interference. In these studies, we compared protection of chickens provided by a turkey herpesvirus (HVT) vector vaccine expressing the hemagglutinin (HA) gene from a clade 2.2 H5N1 strain (A/swan/Hungary/4999/2006) against homologous H5N1 as well as heterologous H5N1 and H5N2 highly pathogenic (HP) AI challenge. The results demonstrated all vaccinated birds were protected from clinical signs of disease and mortality following homologous challenge. In addition, oral and cloacal swabs taken from challenged birds demonstrated that vaccinated birds had lower incidence and titers of viral shedding compared to sham-vaccinated birds. Following heterologous H5N1 or H5N2 HPAI challenge, 80–95% of birds receiving the HVT vector AI vaccine at day of age survived challenge with fewer birds shedding virus after challenge than sham vaccinated birds. In vitro cytotoxicity analysis demonstrated that splenic T lymphocytes from HVT-vector-AI vaccinated chickens recognized MHC-matched target cells infected with H5, as well as H6, H7, or H9 AI virus. Taken together, these studies provide support for the use of HVT vector vaccines expressing HA to protect poultry against multiple lineages of HPAI, and that both humoral and cellular immunity induced by live vaccines likely contributes to protection.     

Efficacy of recombinant Marek’s disease virus vectored vaccines with computationally optimized broadly reactive antigen (COBRA) hemagglutinin insert against genetically diverse H5 high pathogenicity avian influenza viruses

The genetic and antigenic drift associated with the high pathogenicity avian influenza (HPAI) viruses of Goose/Guangdong (Gs/GD) lineage and the emergence of vaccine-resistant field viruses underscores the need for a broadly protective H5 influenza A vaccine. Here, we tested experimental vector herpesvirus of turkey (vHVT)-H5 vaccines containing either wild-type clade 2.3.4.4A-derived H5 inserts or computationally optimized broadly reactive antigen (COBRA) inserts with challenge by homologous and genetically divergent H5 HPAI Gs/GD lineage viruses in chickens. Direct assessment of protection was confirmed for all the tested constructs, which provided clinical protection against the homologous and heterologous H5 HPAI Gs/GD challenge viruses and significantly decreased oropharyngeal shedding titers compared to the sham vaccine. The cross reactivity was assessed by hemagglutinin inhibition (HI) and focus reduction assay against a panel of phylogenetically and antigenically diverse H5 strains. The COBRA-derived H5 inserts elicited antibody responses against antigenically diverse strains, while the wild-type-derived H5 vaccines elicited protection mostly against close antigenically related clades 2.3.4.4A and 2.3.4.4D viruses. In conclusion, the HVT vector, a widely used replicating vaccine platform in poultry, with H5 insert provides clinical protection and significant reduction of viral shedding against homologous and heterologous challenge. In addition, the COBRA-derived inserts have the potential to be used against antigenically distinct co-circulating viruses and future drift variants.     

Suboptimal protection against H5N1 highly pathogenic avian influenza viruses from Vietnam in ducks vaccinated with commercial poultry vaccines

Domestic ducks are the second most abundant poultry species in many Asian countries including Vietnam, and play a critical role in the epizootiology of H5N1 highly pathogenic avian influenza (HPAI) [FAO]. In this study, we examined the protective efficacy in ducks of two commercial H5N1 vaccines widely used in Vietnam; Re-1 containing A/goose/Guangdong/1/1996 hemagglutinin (HA) clade 0 antigens, and Re-5 containing A/duck/Anhui/1/2006 HA clade 2.3.4 antigens. Ducks received two doses of either vaccine at 7 and at 14 or 21 days of age followed by challenge at 30 days of age with viruses belonging to the HA clades 1.1, 2.3.4.3, 2.3.2.1.A and 2.3.2.1.B isolated between 2008 and 2011 in Vietnam. Ducks vaccinated with the Re-1 vaccine were protected after infection with the two H5N1 HPAI viruses isolated in 2008 (HA clades 1.1 and 2.3.4.3) showing no mortality and limited virus shedding. The Re-1 and Re-5 vaccines conferred 90–100% protection against mortality after challenge with the 2010 H5N1 HPAI viruses (HA clade 2.3.2.1.A); but vaccinated ducks shed virus for more than 7 days after challenge. Similarly, the Re-1 and Re-5 vaccines only showed partial protection against the 2011 H5N1 HPAI viruses (HA clade 2.3.2.1.A and 2.3.2.1.B), with a high proportion of vaccinated ducks shedding virus for more than 10 days. Furthermore, 50% mortality was observed in ducks vaccinated with Re-1 and challenged with the 2.3.2.1.B virus. The HA proteins of the 2011 challenge viruses had the greatest number of amino acid differences from the two vaccines as compared to the viruses from 2008 and 2009, which correlates with the lesser protection observed with these viruses. These studies demonstrate the suboptimal protection conferred by the Re-1 and Re-5 commercial vaccines in ducks against H5N1 HPAI clade 2.3.2.1 viruses, and underscore the importance of monitoring vaccine efficacy in the control of H5N1 HPAI in ducks.     

Inclusion of membrane-anchored LTB or flagellin protein in H5N1 virus-like particles enhances protective responses following intramuscular and oral immunization of mice

We previously demonstrated that intramuscular immunization with virus-like particles (VLPs) composed of the haemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins of A/meerkat/Shanghai/SH-1/2012 (clade 2.3.2.1) protected mice from lethal challenge with viruses from other H5 HPAI clades. The inclusion of additional proteins that can serve as immunological adjuvants in VLPs may enhance adaptive immune responses following vaccination, and oral vaccines may represent the safest choice. Here, we report the generation of H5N1 VLPs composed of the viral HA, NA, and M1 proteins and membrane-anchored forms of the Escherichia coli heat-labile enterotoxin B subunit protein (LTB) or the Toll-like receptor 5 ligand flagellin (Flic). Mice intramuscularly or orally immunized with VLPs containing LTB or Flic generated greater humoural and cellular immune responses than those administered H5N1 VLPs without LTB or Flic. Intramuscular immunization with VLPs protected mice from lethal challenge with homologous or heterologous H5N1 viruses irrespective of whether the VLPs additionally included LTB or Flic. In contrast, oral immunization of mice with LTB- or Flic-VLPs conferred substantial protection against lethal challenge with both homologous and heterologous H5N1 influenza viruses, whereas mice immunized orally with VLPs lacking LTB and Flic universally succumbed to infection. Mice immunized orally with LTB- or Flic-VLPs showed 10-fold higher virus-specific IgG titres than mice immunized with H5N1-VLPs lacking LTB or Flic. Collectively, these results indicate that the inclusion of immunostimulatory proteins, such as LTB and Flic, in VLP-based vaccines may represent a promising new approach for the control of current H5N1 HPAI outbreaks by eliciting higher humoural and cellular immune responses and conferring improved cross-clade protection.     

Highly pathogenic avian influenza virus H5N1 from Egypt escapes vaccine-induced immunity but confers clinical protection against a heterologous clade 2.2.1 Egyptian isolate

The poultry populations of Egypt are endemically infected by highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1. Vaccination was chosen as an auxiliary tool to control HPAIV in poultry. Potency of commercial vaccines regarding emerging variants is under discussion. In the current study efficacy of four different inactivated whole H5 virus vaccines representing different sublineages of HPAIV H5N1 were tested in chickens against challenge viruses currently co-circulating in Egypt and representing two antigenically widely distinct HPAIV H5N1 lineages, i.e., “variant” (clade 2.2.1var) and “proper” (clade 2.2.1pro) viruses. All vaccines induced clinical protection against challenge with 2.2.1pro Egyptian strains. In contrast, when challenged with a variant strain, only chickens vaccinated with the homologous Egyptian clade 2.2.1var virus or an inactivated re-assorted H5N1 strain (Re-5, clade 2.3) were protected. However, only the homologous virus induced sterile immunity whereas chickens clinically protected after Re-5 vaccination shed virus at day two after infection indistinguishable to H5N2 vaccines. In conclusion, monitoring vaccine-driven evolution of HPAIV H5N1 by surveillance, antigenic characterization, and challenge studies is essential to assess efficacy of AIV vaccination campaigns.     

Single- and multiple-clade influenza A H5N1 vaccines induce cross protection in ferrets

The rapid evolution, genetic diversity, broad host range, and increasing human infection with avian influenza A (H5N1) viruses highlight the need for an efficacious cross-clade vaccine. Using the ferret model, we compared induction of cross-reactive immunity and protective efficacy of three single-clade H5N1 vaccines and a novel multiple-clade H5N1 vaccine, with and without MF59 adjuvant. Reverse genetics (rg) was used to generate vaccine viruses containing the hemagglutinin (HA) and neuraminidase genes of wild-type H5N1 viruses. Ferrets received two doses of inactivated whole-virus vaccine separated by 3 weeks. Single-clade vaccines (7.5 μg HA per dose) included rg-A/Vietnam/1203/04 (clade 1), rg-A/Hong Kong/213/03 (clade 1), and rg-A/Japanese White Eye/Hong Kong/1038/06 (clade 2.3). The multiple-clade vaccine contained 3.75 μg HA per dose of each single-clade vaccine and of rg-A/Whooper Swan/Mongolia/244/05 (clade 2.2). Two doses of vaccine were required to substantially increase anti-HA and virus neutralizing antibody titers to H5N1 viruses. MF59 adjuvant enhanced induction of clade-specific and cross-clade serum antibody responses, reduced frequency of infection (as determined by upper respiratory tract virus shedding and seroconversion data), and eliminated disease signs. The rg-A/Hong Kong/213/03 vaccine induced the highest antibody titers to homologous and heterologous H5N1 viruses, while rg-A/Japanese White Eye/Hong Kong/1038/06 vaccine induced the lowest. The multiple-clade vaccine was broadly immunogenic against clade 1 and 2 viruses. The rg-A/Vietnam/1203/04 vaccine (the currently stockpiled H5N1 vaccine) most effectively reduced upper respiratory tract virus shedding after challenge with clade 1 and 2 viruses. Importantly, all vaccines protected against lethal challenge with A/Vietnam/1203/04 virus and provided cross-clade protection.     

Inactivated and adjuvanted whole-virion clade 2.3.4 H5N1 pre-pandemic influenza vaccine possesses broad protective efficacy against infection by heterologous clades of highly pathogenic H5N1 avian influenza virus in mice

In this study, we evaluated the immunogenicity and protective efficacy of a candidate attenuated H5N1 pre-pandemic influenza vaccine of clade 2.3.4, rgAnhui, which was reverse genetically generated from highly virulent A/Anhui/01/2005 (H5N1) wild-type virus. When a low-dose antigen (0.3 μg HA) vaccine was combined with aluminum hydroxide adjuvant, virus neutralization and anti-HA IgG antibodies induced in the sera of vaccinated mice showed similar levels as those in mice vaccinated with non-adjuvanted high-dose antigen (3 μg HA) vaccine. Serum antibodies had broad reactivity against highly pathogenic H5N1 viruses of both homologous and heterologous clades. All mice vaccinated with adjuvanted and non-adjuvanted rgAnhui vaccines at low and high antigen doses survived, without any significant weight loss, lethal challenge infection with homologous clade 2.3.4 viruses, including antigenic variant virus and heterologous clade 2.1.3. Mice vaccinated with low-dose antigen without adjuvant, however, exhibited 20% and 60% survival rates against clade 1 and clade 2.2 viruses, respectively; but, addition of adjuvant improved these rates to 80% and 100%, respectively. The data strongly suggest that aluminum hydroxide-adjuvanted rgAnhui vaccine can elicit broad cross-reactive and protective immunities against homologous and heterologous clades, and that the rgAnhui vaccine is a useful pre-pandemic H5N1 vaccine.     

Experimental challenge of chicken vaccinated with commercially available H5 vaccines reveals loss of protection to some highly pathogenic avian influenza H5N1 strains circulating in Hong Kong/China

Highly pathogenic avian influenza (HPAI) H5N1 virus continues to circulate in poultry in Asia and Africa posing a threat to both public and animal health. Vaccination, used as an adjunct to improved bio-security and stamping-out policies, contributed to protecting poultry in Hong Kong from HPAI H5N1 infection in 2004–2008 although the virus was repeatedly detected in dead wild birds. The detection of clade 2.3.4 H5N1 viruses in poultry markets and a farm in Hong Kong in 2008 raised the question whether this virus has changed to evade protection from the H5 vaccines in use. We tested the efficacy of three commercial vaccines (Nobilis, Poulvac and Harbin Re-5 vaccine) in specific pathogen free white leghorn chickens against a challenge with A/chicken/Hong Kong/8825-2/2008 (clade 2.3.4) isolated from vaccinated poultry in Hong Kong and A/chicken/Hong Kong/782/2009 (clade 2.3.2). Harbin Re5 vaccine provided the best, albeit not complete protection against challenge with the clade 2.3.4 virus. All three vaccines provided good protection from death and significantly reduced virus shedding following challenge with the clade 2.3.2 virus. Only Harbin Re-5 was able to completely protect chickens from virus shedding as well as mortality. Sera from vaccinated chickens had lower geometric hemagglutination inhibition titers against A/chicken/Hong Kong/8825-2/08, as compared to two other clade 2.3.4 and one clade 0 virus. Alignment of amino-acid sequences of the haemagglutinin of A/chicken/Hong Kong/8825-2/08 and the other H5 viruses revealed several mutations in positions including 69, 71, 83, 95, 133,140, 162, 183, 189, 194 and 270 (H5 numbering) which may correlate with loss of vaccine protection. Our results indicated that the tested HPAI H5N1 (2.3.4) virus has undergone antigenic changes that allow it to evade immunity from poultry vaccines. This highlights the need for continued surveillance and monitoring of vaccine induced immunity, with experimental vaccine challenge studies being done where indicated.     

Co-administration of certain DNA vaccine combinations expressing different H5N1 influenza virus antigens can be beneficial or detrimental to immune protection

Achieving broad-spectrum immunity against emerging zoonotic viruses such as avian influenza H5N1 and other possible pandemic viruses will require generation of cross-protective immune responses. Strong antibody responses generated against the H5HA protein are protective, however, antigenic variation between diverging isolates can interfere with virus neutralization. The current study investigates co-administration of an H5 HA DNA vaccine with other variable and conserved influenza antigens (NA, NP, and M2). All antigens were derived from the A/Hanoi/30408/2005 (H5N1) virus and the contribution towards overall protection and immune activation was assessed against lethal homologous and heterologous challenges. An (HA + NA) combination afforded the best protection against homologous challenge and (HA + NP) was comparable to HA alone against heterologous A/Hong Kong/483/1997 challenge. Interestingly, combining all four H5 antigens at a single site did not improve protection against matched challenge and unexpectedly reduced survival by 30% against a heterologous challenge. Survival was also significantly decreased against heterologous challenge following combination of (HA + NP) with an unrelated antigen. Although there were no significant changes in antibody titres, significantly lower T-cell responses were detected against all antigens except HA in each combination. Co-administration of the vaccines at different injection sites restored T-cell responses but did not improve overall protection. Similar observations were also recorded following combination of HA and NP antigens using two different adenovirus-based backbones. Overall, the data suggest that co-administering certain H5N1 antigens offer better or comparable protection to HA alone, however, combining extra antigens may be unnecessary and lead to unfavourable immune responses.     

Vaccination with virus-like particles containing H5 antigens from three H5N1 clades protects chickens from H5N1 and H5N8 influenza viruses

Highly pathogenic avian influenza (HPAI) viruses, especially H5N1 strains, represent a public health threat and cause widespread morbidity and mortality in domestic poultry. Recombinant virus-like particles (VLPs) represent a promising novel vaccine approach to control avian influenza including HPAI strains. Influenza VLPs contain viral hemagglutinin (HA), which can be expressed in cell culture within highly immunogenic VLPs that morphologically and antigenically resemble influenza virions, except VLPs are non-infectious. Here we describe a recombinant VLP containing HA proteins derived from three distinct clades of H5N1 viruses as an experimental, broadly protective H5 avian influenza vaccine. A baculovirus vector was configured to co-express the H5 genes from recent H5N1 HPAI isolates A/chicken/Germany/2014 (clade 2.3.4.4), A/chicken/West Java/Subang/29/2007 (clade 2.1.3) and A/chicken/Egypt/121/2012 (clade 2.2.1). Co-expression of these genes in Sf9 cells along with influenza neuraminidase (NA) and retrovirus gag genes resulted in production of triple-clade H555 VLPs that exhibited hemagglutination activity and morphologically resembled influenza virions. Vaccination of chickens with these VLPs resulted in induction of serum antibody responses and efficient protection against experimental challenges with three different viruses including the recent U.S. H5N8 HPAI isolate. We conclude that these novel triple-clade VLPs represent a feasible strategy for simultaneously evoking protective antibodies against multiple variants of H5 influenza virus.     

H5N1 VLP vaccine induced protection in ferrets against lethal challenge with highly pathogenic H5N1 influenza viruses

In this study, recombinant virus-like particles (VLPs) were evaluated as a candidate vaccine against emerging influenza viruses with pandemic potential. The VLPs are composed of the hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1) proteins of the H5N1 A/Indonesia/05/2005 (clade 2.1; [Indo/05]) virus, which were expressed using baculovirus in Spodoptera frugiperda (Sf9) cells. Ferrets received either 2 injections of the VLP vaccine at escalating doses (based on HA content), recombinant HA, or were mock vaccinated. Vaccinated ferrets were then challenged with either H5N1 Indo/05 or H5N1 A/Viet Nam 1203/2004 (VN/04) wild-type viruses. All ferrets that received the VLP vaccine survived regardless of the VLP dose or challenge strain, whereas seven of eight mock vaccinated ferrets died. The VLP vaccine induced HAI antibodies against the homologous H5N1 clade 2.1 strain, as well as heterologous strains from H5N1 clades 1, 2.2, and 2.3. The magnitude of the HAI titers correlated with VLP dose. Neutralizing antibody responses against the Indo/05 and VN/04 strains showed a similar pattern. Affinity of the anti-HA antibodies raised by the H5N1 Indo/05 VLPs had a higher association rate to the homologous clade 2.1 HA than to the clade 1 (VN/04) HA; however, once bound, antibodies had similar slow disassociation rates. These results provide support for continued development of the H5N1 VLPs as a candidate vaccine against pandemic influenza. Exploration of immunologic correlates of protection for H5N1 vaccines beyond HAI and neutralizing antibody responses is warranted.     

Single dose of oil-adjuvanted inactivated vaccine protects chickens from lethal infections of highly pathogenic H5N1 influenza virus

The highly pathogenic H5N1 influenza viruses are endemic in poultry in many countries, but continuously infect humans and cause human mortality. H5N1 influenza viruses have been regarded as a pandemic candidate. In a pandemic event by this virus, the protection of poultry with an effective vaccine will help to greatly reduce the spread of this virus to humans since it easily infects poultry. Here we showed that immunization with one dose of oil-adjuvanted inactivated H5N1 vaccine could protect chickens from lethal infection by highly pathogenic H5N1 influenza virus until 12 weeks post-immunization. The complete protection of chickens depended on the amount of HA antigens in the vaccine. Complete homologous protection required over 1.25 μg of HA antigens and complete heterologous protection required over 5.0 μg of HA antigens. The bivalent H5N1 inactivated vaccine composed of 1.25 μg of each antigen from clade 1 and clade 2.3.4 H5N1 influenza virus completely protected chickens from the lethal challenge of both viruses. When we determined the induction of antibody subtypes in tissues including nasal cavity, trachea, and lungs, the IgG subtype of antibody was induced more than the IgM or IgA subtype of antibody. Taken together, our results suggest that one dose of oil-adjuvanted inactivated H5N1 vaccine could provide chickens with sterile immunity against the homologous highly pathogenic H5N1 influenza virus.     

Poly I:C adjuvanted inactivated swine influenza vaccine induces heterologous protective immunity in pigs

Swine influenza is widely prevalent in swine herds in North America and Europe causing enormous economic losses and a public health threat. Pigs can be infected by both avian and mammalian influenza viruses and are sources of generation of reassortant influenza viruses capable of causing pandemics in humans. Current commercial vaccines provide satisfactory immunity against homologous viruses; however, protection against heterologous viruses is not adequate. In this study, we evaluated the protective efficacy of an intranasal Poly I:C adjuvanted UV inactivated bivalent swine influenza vaccine consisting of Swine/OH/24366/07 H1N1 and Swine/CO/99 H3N2, referred as PAV, in maternal antibody positive pigs against an antigenic variant and a heterologous swine influenza virus challenge. Groups of three-week-old commercial-grade pigs were immunized intranasally with PAV or a commercial vaccine (CV) twice at 2 weeks intervals. Three weeks after the second immunization, pigs were challenged with the antigenic variant Swine/MN/08 H1N1 (MN08) and the heterologous Swine/NC/10 H1N2 (NC10) influenza virus. Antibodies in serum and respiratory tract, lung lesions, virus shedding in nasal secretions and virus load in lungs were assessed. Intranasal administration of PAV induced challenge viruses specific-hemagglutination inhibition- and IgG antibodies in the serum and IgA and IgG antibodies in the respiratory tract. Importantly, intranasal administration of PAV provided protection against the antigenic variant MN08 and the heterologous NC10 swine influenza viruses as evidenced by significant reductions in lung virus load, gross lung lesions and significantly reduced shedding of challenge viruses in nasal secretions. These results indicate that Poly I:C or its homologues may be effective as vaccine adjuvants capable of generating cross-protective immunity against antigenic variants/heterologous swine influenza viruses in pigs.     

Evidence supporting the inclusion of strains from each of the two co-circulating lineages of H3N8 equine influenza virus in vaccines

Two lineages of antigenically distinct equine influenza A H3N8 subtype viruses, American and European, co-circulate. Experiments were conducted in ponies to investigate the protection induced by vaccines containing virus from one lineage against challenge infection with homologous or heterologous virus. Regression analysis showed that vaccinated ponies with average pre-challenge single radial haemolysis (SRH) antibody levels (i.e. 45-190mm2) had a higher probability of becoming infected if they were vaccinated with virus heterologous to the challenge strain than if they were vaccinated with homologous virus. Field studies in Thoroughbred racehorses also showed that SRH antibody levels of >/= 150mm2 induced by vaccines containing a European lineage strain are protective against infection with a virus from the same lineage, but that the same or higher antibody levels may not be protective against an American lineage virus. In conclusion, vaccines should contain virus strains representative of both H3N8 subtype lineages to maximise protection against infection.     

Protective efficacy of an inactivated Eurasian avian-like H1N1 swine influenza vaccine against homologous H1N1 and heterologous H1N1 and H1N2 viruses in mice

Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses are prevalent in pigs in Europe and Asia, but occasionally cause human infection, which raises concern about their pandemic potential. Here, we produced a whole-virus inactivated vaccine with an EA H1N1 strain (A/swine/Guangxi/18/2011, SW/GX/18/11) and evaluated its efficacy against homologous H1N1 and heterologous H1N1 and H1N2 influenza viruses in mice. A strong humoral immune response, which we measured by hemagglutination inhibition (HI) and virus neutralization (VN), was induced in the vaccine-inoculated mice upon challenge. The inactivated SW/GX/18/11 vaccine provided complete protection against challenge with homologous SW/GX/18/11 virus in mice and provided effective protection against challenge with heterologous H1N1 and H1N2 viruses with distinctive genomic combinations. Our findings suggest that this EA H1N1 vaccine can provide protection against both homologous H1N1 and heterologous H1N1 or H1N2 virus infection. As such, it is an excellent vaccine candidate to prevent H1N1 swine influenza.     

A broad spectrum HVT-H5 avian influenza vector vaccine which induces a rapid onset of immunity

Current methods to combat highly pathogenic avian influenza (HPAI) outbreaks in poultry rely on stamping out and preventive culling, which can lead to high economic losses and invoke ethical resistance. Emergency vaccination could be an alternative as vaccination is one of the most efficient and cost-effective measures to protect poultry from HPAI infection, preventing spreading to other poultry and greatly reducing the potential transmission to humans. Current conventional inactivated AI vaccines may be useful for combating AI outbreaks, but do not fulfil all targets of an ideal AI vaccine, including mass applicability and rapid onset of immunity. We aimed to further investigate the potential of Herpesvirus of Turkeys (HVT) as a vector containing a recombinant H5 hemagglutinin of HPAI H5N1. This HVT-H5 vector was analysed in vitro, tested for onset of immunity against AI challenge, breadth of protection, reduction of virus shedding, and induction of both antibody and cellular responses in SPF layers or broiler chicks containing maternal derived antibodies (MDA+). In SPF layers HVT-H5 provided full protection to lethal challenges with 4 antigenically diverse HPAI H5N1 strains from 2 weeks post vaccination (w.p.v.), while in MDA+ birds full protection was provided from 3 w.p.v. to homologous challenge. Also shedding of challenge virus was reduced in both SPF and MDA+ birds. HVT-H5 induced a protective HI titre (≥4) to 11 HPAI H5N1 strains at 3 w.p.v. in 3-week-old SPF layers and to HPAI H5N8 A/ch/Neth/14015531/2014. Besides inducing a protective antibody response HVT-H5 also induced an influenza-specific T cell response. This data demonstrates that HVT-H5 vaccine appears to fulfil many of the criteria for an ideal AI vaccine including early onset of immunity, a broad protection, reduced virus shedding, protection in presence of AI-MDA and could be a useful tool in the combat of AI outbreaks worldwide.     

Nasal delivery of Protollin-adjuvanted H5N1 vaccine induces enhanced systemic as well as mucosal immunity in mice

Sporadic, yet frequent human infections with avian H5N1 influenza A viruses continue to pose a potential pandemic threat. Poor immunogenicity of unadjuvanted H5N1 vaccines warrants developing novel adjuvants and formulations as well as alternate delivery systems to improve their immunogenicity and efficacy. Here, we show that Protollin, a nasal adjuvant composed of Neisseria meningitides outer membrane proteins non-covalently linked to Shigella flexneri 2a lipopolysaccharide, is a potent nasal adjuvant for an inactivated split virion H5N1 clade 1 A/Viet Nam1203/2004 (A/VN/1203/04) vaccine in a mouse model. Protollin-adjuvanted vaccines elicited enhanced serum protective hemagglutination inhibition titers, mucosal IgA responses, and H5N1-specific cell-mediated immunity that resulted in complete protection against a lethal challenge with a homologous virus as well as a heterologous clade 2 virus A/Indonesia/05/2005 (A/IN/05/05). Detailed analysis of adaptive immunity revealed that Protollin increased the frequency of lymphoid- as well as local tissue-resident antibody-secreting cells, local germinal center reaction of B cells, broad-spectrum of CD4 T cell response. Our findings suggest that nasal delivery of H5N1 vaccine with Protollin adjuvant can overcome the poor immunogenicity of H5N1 vaccines, induce both cellular and humoral immune responses, enhance protection against challenge with clade 1 and clade 2 H5N1 viruses and achieve significant antigen dose-sparing.