An additional oligosaccharide moiety in the HA of a pandemic influenza H1N1 candidate vaccine virus confers increased antigen yield in eggs

The H1N1 influenza pandemic in 2009 highlighted the need for the rapid generation of candidate vaccine viruses (CVVs) against an A/California/7/2009-like virus. The first available CVVs gave low protein yields in eggs but improved yields were achieved for second generation CVVs which contained amino acid substitutions compared to their precursor viruses. In this study, we investigated the basis for the increased virus protein yield of CVV NIBRG-121xp and whether the improved yield characteristics could be transferred between this virus and two other CVVs, NYMC X-179A and NYMC X-181. We generated variant viruses by reverse genetics to contain combinations of amino acid substitutions found in high yielding NIBRG-121xp and NYMC X-181. We found that the increase in total protein yield and functional HA yield of NIBRG-121xp in eggs is attributable to the single amino acid substitution K119N in the HA. We also found that the glycosylation of position 119 is essential for the improved virus protein yield in eggs. However, the K119N yield-enhancing effect was not transferable between viruses, nor was the N129D change found in high yielding NYMC X-181. However, position 119 may be a useful locus to monitor in future for viruses and CVVs with potentially high yield.     

Optimization of influenza A vaccine virus by reverse genetic using chimeric HA and NA genes with an extended PR8 backbone

The yield of influenza antigen production may significantly vary between vaccine strains; for example the A/California/07/09 (H1N1)-X179A vaccine virus, prepared during 2009 influenza pandemic, presented a low antigen yield in eggs compared to other seasonal H1N1 reassortants. In this study a bi-chimeric virus expressing HA and NA genes with A/Puerto Rico/8/34 (H1N1) (PR8) and X179A domains was rescued by reverse genetics using a mixture of Vero/CHOK1 cell lines (Medina et al. [7]). The bi-chimeric virus obtained demonstrated to yield much larger amounts of HA than X179A in eggs as measured by single-radial-immunodiffusion (SRID), the reference method to quantify HA protein in influenza vaccine. Such kind of optimized virus using PR8 backbone derived chimeric glycoproteins could be used as improved seed viruses for vaccine production.     

Improvement of H5N1 influenza vaccine viruses: influence of internal gene segments of avian and human origin on production and hemagglutinin content

The H5N1-clade 1 influenza vaccine strain NIBRG-14 produces exceptionally low amounts of antigen, a problem recently encountered also for initial pandemic H1N1-2009 vaccine seeds. Here, we report on a strategy that may contribute to overcome this obstacle. Influenza vaccine viruses usually consist of two segments coding for the antigenic HA and NA proteins of a wild-type strain and the six residual internal gene segments of the vaccine donor strain A/PR/8/34 (PR8). To enhance the antigen yield from H5N1 vaccine virus we generated by reverse genetics a set of PR8-based reassortant viruses expressing the HA and NA segments of the prototypic strain A/Vietnam/1203/2004 and additional replacements of the internal M or PB1 genes of PR8. The reassortants were compared to the parental PR8 and H5N1 viruses in terms of growth in embryonated chicken eggs and the amount of incorporated antigenic HA protein. Compared to NIBRG-14, three out of six viruses displayed an increased replication in embryonated chicken eggs and higher HA content that was also maintained after ether/detergent extraction of virions. Electron microscopic analysis showed that the reassortment hardly affected particle shape and size. Two selected H5N1 reassortant viruses were investigated concerning their pathogenicity in ferrets and found to behave as low pathogenic as the PR8 donor strain. In conclusion, this study shows that replication and antigen content of PR8-derived H5N1 influenza vaccine viruses can be improved by incorporation of heterologous internal gene segments without compromising their attenuated character.     

Cross-protective immunity against multiple influenza virus subtypes by a novel modified vaccinia Ankara (MVA) vectored vaccine in mice

Development of an influenza vaccine that provides cross-protective immunity remains a challenge. Candidate vaccines based on a recombinant modified vaccinia Ankara (MVA) viral vector expressing antigens from influenza (MVA/Flu) viruses were constructed. A vaccine candidate, designated MVA/HA₁/C13L/NP, that expresses the hemagglutinin from pandemic H1N1 (A/California/04/09) and the nucleoprotein (NP) from highly pathogenic H5N1 (A/Vietnam/1203/04) fused to a secretory signal sequence from vaccinia virus was highly protective. The vaccine elicited strong antibody titers to homologous H1N1 viruses while cross-reactive antibodies to heterologous viruses were not detectable. In mice, this MVA/HA₁/C13L/NP vaccine conferred complete protection against lethal challenge with A/Vietnam/1203/04 (H5N1), A/Norway/3487-2/09 (pandemic H1N1) or A/Influenza/Puerto Rico/8/34 (seasonal H1N1) and partial protection (57.1%) against challenge with seasonal H3N2 virus (A/Aichi/68). The protective efficacy of the vaccine was not affected by pre-existing immunity to vaccinia. Our findings highlight MVA as suitable vector to express multiple influenza antigens that could afford broad cross-protective immunity against multiple subtypes of influenza virus.     

A polyvalent influenza A DNA vaccine induces heterologous immunity and protects pigs against pandemic A(H1N1)pdm09 virus infection

The composition of current influenza protein vaccines has to be reconsidered every season to match the circulating influenza viruses, continuously changing antigenicity. Thus, influenza vaccines inducing a broad cross-reactive immune response would be a great advantage for protection against both seasonal and emerging influenza viruses. We have developed an alternative influenza vaccine based on DNA expressing selected influenza proteins of pandemic and seasonal origin. In the current study, we investigated the protection of a polyvalent influenza DNA vaccine approach in pigs. We immunised pigs intradermally with a combination of influenza DNA vaccine components based on the pandemic 1918 H1N1 (M and NP genes), pandemic 2009 H1N1pdm09 (HA and NA genes) and seasonal 2005 H3N2 genes (HA and NA genes) and investigated the protection against infection with virus both homologous and heterologous to the DNA vaccine components.     

Innate immunemodulator containing adjuvant formulated HA based vaccine protects mice from lethal infection of highly pathogenic avian influenza H5N1 virus

The highly pathogenic avian influenza (HPAI) H5N1 viruses and their spillover into the human population pose substantial economic and public health threats. Although antiviral drugs have some effect in treating influenza infection, vaccination is still the most effective intervention to prevent possible pandemic outbreaks. We have developed a novel H5 influenza vaccine to improve the world’s pandemic preparedness. We produced a hemagglutinin (HA) of HPAI H5N1 virus A/Alberta/01/2014 (AB14) using both mammalian (m) and bacterial (b) expression systems. The purified recombinant proteins were formulated with a proprietary adjuvant (TriAdj) and their efficacy as vaccine candidates was evaluated in mice. Intramuscular delivery of two doses of TriAdj formulated mammalian expressed HA (m-HA/TriAdj) was shown to provide full protection against a lethal challenge of AB14 in mice. In contrast, bacterially expressed HA with TriAdj (b-HA/TriAdj), b-HA without adjuvant, and m-HA without adjuvant resulted in no protection in immunized mice. Furthermore, m-HA/TriAdj elicited significantly higher levels of balanced Th1 and Th2 responses and neutralizing antibody titres. All the mice in the m-HA/TriAdj group survived a lethal AB14 H5N1 challenge and showed no signs of disease or infection as demonstrated by no loss of body weight or detectable virus in the lungs. Our results suggest that m-HA formulated with TriAdj has potential to protect against pandemic H5N1 in the event of its cross over to the human host.     

Cloned cDNA of A/swine/Iowa/15/1930 internal genes as a candidate backbone for reverse genetics vaccine against influenza A viruses

Reverse genetics viruses for influenza vaccine production usually utilize the internal genes of the egg-adapted A/Puerto Rico/8/34 (PR8) strain. This egg-adapted strain provides high production yield in embryonated eggs but does not necessarily give the best yield in mammalian cell culture. In order to generate a reverse genetics viral backbone that is well-adapted to high growth in mammalian cell culture, a swine influenza isolate A/swine/Iowa/15/30 (H1N1) (rg1930) that was shown to give high yield in Madin-Darby canine kidney (MDCK) cells was used as the internal gene donor for reverse genetics plasmids. In this report, the internal genes from rg1930 were used for construction of reverse genetics viruses carrying a cleavage site-modified hemagglutinin (HA) gene and neuraminidase (NA) gene from a highly pathogenic H5N1 virus. The resulting virus (rg1930H5N1) was low pathogenic in vivo. Inactivated rg1930H5N1 vaccine completely protected chickens from morbidity and mortality after challenge with highly pathogenic H5N1. Protective immunity was obtained when chickens were immunized with an inactivated vaccine consisting of at least 2(9) HA units of the rg1930H5N1 virus. In comparison to the PR8-based reverse genetics viruses carrying the same HA and NA genes from an H5N1 virus, rg1930 based viruses yielded higher viral titers in MDCK and Vero cells. In addition, the reverse genetics derived H3N2 and H5N2 viruses with the rg1930 backbone replicated in MDCK cells better than the cognate viruses with the rgPR8 backbone. It is concluded that this newly established reverse genetics backbone system could serve as a candidate for a master donor strain for development of inactivated influenza vaccines in cell-based systems.     

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

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

Mutations to A/Puerto Rico/8/34 PB1 gene improves seasonal reassortant influenza A virus growth kinetics

It is desirable for influenza vaccine virus strains to have phenotypes that include good growth and hemagglutinin (HA) protein yield. The quality of these characteristics varies among the vaccine viruses and is usually due to multigenic effects. Many influenza A virus vaccine viruses are made as reassortants of the high yield virus A/Puerto Rico/8/34 (PR/8) and a circulating seasonal virus. Co-infection of eggs with the two viruses, and selection of reassortants with the HA and neuraminidase (NA) segments from the seasonal virus, can result in viruses that contain a mixture of internal genes derived from both the high yield virus and the circulating virus. Segment 2 (PB1), which encodes the RNA-dependent RNA polymerase, frequently cosegregates with the seasonal HA and NA segments. We asked whether mutations based on the seasonal PB1 genes could improve vaccine virus strains. Here we report that mutations to the PR/8 PB1 gene, based on differences observed between seasonal and PR/8 PB1 genes, accelerate egg and cell culture based replication for a reassortant virus containing HA and NA segments from the low yield A/Wyoming/03/2003 (H3N2) vaccine virus.     

Genetic stability of live attenuated vaccines against potentially pandemic influenza viruses

BackgroundEnsuring genetic stability is a prerequisite for live attenuated influenza vaccine (LAIV). This study describes the results of virus shedding and clinical isolates’ testing of Phase I clinical trials of Russian LAIVs against potentially pandemic influenza viruses in healthy adults.MethodsThree live attenuated vaccines against potentially pandemic influenza viruses, H2N2 LAIV, H5N2 LAIV and H7N3 LAIV, generated by classical reassortment in eggs, were studied. For each vaccine tested, subjects were randomly distributed into two groups to receive two doses of either LAIV or placebo at a 3:1 vaccine/placebo ratio. Nasal swabs were examined for vaccine virus shedding by culturing in eggs and by PCR. Vaccine isolates were tested for temperature sensitivity and cold-adaptation (ts/ca phenotypes) and for nucleotide sequence.ResultsThe majority of nasal wash positive specimens were detected on the first day following vaccination. PCR method demonstrated higher sensitivity than routine virus isolation in eggs. None of the placebo recipients had detectable vaccine virus replication.All viruses isolated from the immunized subjects retained the ts/ca phenotypic characteristics of the master donor virus (MDV) and were shown to preserve all attenuating mutations described for the MDV. These data suggest high level of vaccine virus genetic stability after replication in humans.During manufacture process, no additional mutations occurred in the genome of H2N2 LAIV. In contrast, one amino acid change in the HA of H7N3 LAIV and two additional mutations in the HA of H5N2 LAIV manufactured vaccine lot were detected, however, they did not affect their ts/ca phenotypes.ConclusionsOur clinical trials revealed phenotypic and genetic stability of the LAIV viruses recovered from the immunized volunteers. In addition, no vaccine virus was detected in the placebo groups indicating the lack of person-to-person transmission.LAIV TRIAL REGISTRATION at ClinicalTrials.gov: H7N3-NCT01511419; H5N2-NCT01719783; H2N2-NCT01982331.     

Preclinical evaluation of the efficacy of an H5N8 vaccine candidate (IDCDC-RG43A) in mouse and ferret models for pandemic preparedness

Because H5N1 influenza viruses continuously threaten the public health, the WHO has prepared various clades of H5N1 mock-up vaccines as one of the measures for pandemic preparedness. The recent worldwide outbreak of H5Nx virus which belongs to clade 2.3.4.4 and of which H5N6 subtype belongs and already caused human infection also increases the need of pandemic vaccine for such novel emerging viruses. In this study, we evaluated the protective efficacy and immunogenicity of an egg-based and inactivated whole-virus H5N8 (IDCDC-RG43A) developed by CDC containing HA and NA gene of the parent virus A/gyrfalcon/Washington/41088-6/2014. Mice vaccinated two times elicited low to moderate antibody titer in varying amount of antigen doses against the homologous H5N8 vaccine virus and heterologous intra–clade 2.3.4.4 H5N6 (A/Sichuan/26221/2014) virus. Mice immunized with at least 3.0?µg/dose of IDCDC-RG43A with aluminum hydroxide adjuvant were completely protected from lethal challenge with the mouse-adapted H5N8 (A/Environment/Korea/ma468/2015, maH5N8) as well as cleared the viral replication in tissues including lung, brain, spleen, and kidney. Vaccinated ferrets induced high antibody titers against clade 2.3.4.4 H5N8/H5N6 viruses and the antibody showed high cross-reactivity to clade 2.2 H5N1 but not to clade 1 and 2.3.4 viruses as measured by hemagglutinin inhibition and serum neutralization assays. Furthermore, administration of the vaccine in ferrets resulted in attenuation of clinical disease signs and virus spread to peripheral organs including lung, spleen, and kidney from high dose challenge with maH5N8 virus. The protective and immunogenic characteristic of the candidate vaccine are essential attributes to be considered for further clinical trials as a pre-pandemic vaccine for a potential pandemic virus.     

Quantitation of haemagglutinin in H5N1 influenza viruses reveals low haemagglutinin content of vaccine virus NIBRG-14 (H5N1)

The assessment of potential candidate influenza vaccine viruses includes a number of factors. Growth properties of the virus and yield of antigen, specifically the haemagglutinin (HA), are of key importance. The recently developed H5N1 candidate vaccine virus NIBRG-14 (with HA and NA genes derived from the clade 1 virus A/Viet Nam/1194/2004 in an A/Puerto Rico/8/34 background) has been suggested to yield low amounts of antigen. While investigating the antigen yield of H5N1 vaccine viruses, we found that accurate quantitation of the HA content of some H5N1 viruses was difficult due to the migration characteristics of the proteins on SDS-PAGE gels. The HA1 and HA2 bands co-migrated with nucleoprotein (NP) and matrix protein (M1) respectively, preventing accurate analysis. We have developed an accurate way of quantitating HA from these H5N1 viruses by introducing a deglycosylation step to the standard protocol. Using this method, we showed reproducibly that the low yield of NIBRG-14 is, at least in part, due to a lower than usual content of HA in virus preparations. This was also found to be the case for the parent wild type A/Viet Nam/1194/2004 virus.     

Characterization of the neuraminidase of the H1N1/09 pandemic influenza virus

In this study, we compared properties of the neuraminidase (NA) of the H1N1/2009 pandemic virus (H1N1pdm) and N1 NAs of other influenza viruses. The H1N1pdm NA was more active than NAs of seasonal H1N1 viruses, hydrolyzed Neu5Acα2-3Gal linkage as efficiently as did avian viruses and cleaved Neu5Acα2-6Gal linkage as efficiently as classical swine viruses. To assess the functional balance between heterologous NAs and pandemic virus HA, we generated four recombinant viruses that shared seven genes of A/Hamburg/5/09 and contained the NA gene from representative avian, swine and human viruses. The viruses harboring NA from avian, Eurasian avian-like swine and seasonal human viruses eluted more slowly from red blood cells, were more sensitive to neutralization by human airway mucins, and replicated less efficiently in differentiated human tracheo-bronchial epithelial cultures as compared with the viruses containing the NA of H1N1pdm and the NA of the North American classical swine virus lineage. Our data suggest that functional properties of the NA of H1N1pdm could be closer to those of classical swine viruses than to those of avian, avian-like swine and seasonal human viruses.     

Increased hemagglutinin content in a reassortant 2009 pandemic H1N1 influenza virus with chimeric neuraminidase containing donor A/Puerto Rico/8/34 virus transmembrane and stalk domains

The glycoproteins, heamagglutinin (HA) and neuraminidase (NA) of influenza virus confer host protective immune responses during vaccination, which is the most effective approach for preventing influenza-associated morbidity and mortality. Since the functional balance between the HA and NA proteins may affect viral receptor binding and replication, a pandemic influenza A virus (H1N1 pdm09), strain A/Texas/05/2009, was optimized to elevate its HA antigen content by modifying the NA gene. In this study, we have constructed two 2:6 reassortant viruses between pdmH1N1 (A/Texas/05/2009) and A/Puerto Rico/8/34 (PR8), in which the NA gene of A/Texas/05/2009 was modified to contain part of the NA gene from PR8. One chimeric NA virus has the PR8 transmembrane (TM) region (HNtm 2:6) and the other contains both the PR8 NA TM and stem regions (HNst 2:6). Using quantitative reverse phase-HPLC (RP-HPLC) analysis, we observed that the HNst2:6 virus contains a higher HA1 content than HN2:6 wild type. In addition, this mutant virus displays a higher HA1 to nucleoprotein (NP) ratio, based on gel electrophoresis densitometry analysis. Furthermore, the neuraminidase activity of purified HNst 2:6 virus is approximately 30% lower than that of HN2:6 virus, which is suggestive of a lower incorporation of NA into the viral envelope. Therefore, we propose that the reduction of NA packaging in the virion may lead to a compensatory increase of HA. Such an improvement in HA yield is possibly beneficial to H1N1 pdm09 vaccine production.     

Mutations affecting the stability of the haemagglutinin molecule impair the immunogenicity of live attenuated H3N2 intranasal influenza vaccine candidates lacking NS1

The isolation and cultivation of human influenza viruses in embryonated hen eggs or cell lines often leads to amino acid substitutions in the haemagglutinin (HA) molecule. We found that the propagation of influenza A H3N2 viruses on Vero cells may trigger the appearance of HA destabilising mutations, affecting viral resistance to low pH or high temperature treatment. Two ΔNS1 reassortants, containing the HA sequences identical to the original human H3N2 influenza virus isolates were constructed. Passages of these viruses on Vero cells led to the appearance of single mutations in the HA₁ L194P or HA₂ G75R subunits that impaired virus stability. The original HA sequences and the stable phenotypes of the primary isolates were preserved if reassortants were passaged by infection at pH 5.6 and cultivation in medium at pH 6.5. Corresponding ΔNS1 reassortants were compared for their immunogenicity in ferrets upon intranasal immunisation. Vaccine candidates containing HA mutations demonstrated significantly lower immunogenicity compared to those without mutations. Thus, the retaining of the original HA sequences of human viruses during vaccine production might be crucial for the efficacy of live attenuated influenza vaccines.     

Inferring the antigenic epitopes for highly pathogenic avian influenza H5N1 viruses

The evasion of influenza virus from host immune surveillance is mainly mediated through its surface protein hemagglutinin (HA), the main component of influenza vaccine. Thus, identification of influenza virus antigenic epitopes on HA can not only help us understand the molecular mechanisms of viral immune escape but also facilitate vaccine strain selection. Despite previous efforts, there is a lack of systematic definition of the antigenic epitopes for the highly pathogenic avian influenza (HPAI) H5N1 viruses. In this study, we infer the HA antigenic epitopes for H5N1 viruses by integrating the antigenic sites mapped from the HA of human influenza H3N2 viruses, the sites which were reported to be associated with immune escape in H5 viruses and the mutation hotspot sites identified in the evolutionary history of HPAI H5N1 viruses. We show that these inferred antigenic epitopes play significant roles in antigenic variation of HPAI H5N1 viruses. Based on inferred antigenic epitopes, we further develop a computational method to effectively predict antigenic variants for HPAI H5N1 viruses (available at http://biocloud.hnu.edu.cn/predict/html/index.html). Therefore, our work has not only inferred the antigenic epitopes for HPAI H5N1 viruses but also provided an effective computational method to assist vaccine recommendations for protection against the deadly bird flu.     

天津地区2005年甲1亚型流感病毒HA1区基因特性分析

目的 了解天津地区夏季一起小学校流感样暴发疫情中分离出2株甲1亚型流感病毒株HA1基因变异情况。方法MDCK细胞和鸡胚双腔法分离流感病毒，收获病毒液提取病毒的RNA，进行RT—PCR，扩增产物纯化后测序，用DNASTAR软件进行序列分析。结果新分离株HA1基因长度为325个氨基酸，与国际疫苗株A／NewCaledonia／20／99相比氨基酸同源性高于98％，氨基酸替换位点有5个，其中165位位于抗原决定簇Cal区。结论天津地区新分离甲1亚型流感病毒抗原性发生进一步漂移，但变异程度不大。     

Evaluation of conserved and variable influenza antigens for immunization against different isolates of H5N1 viruses

The combination of rapid evolution and high mortality in human cases of infections has raised concerns that the H5N1 avian influenza virus may become a new, possibly severe, pandemic virus. Vaccination is likely to be the most efficient strategy to mitigate the impact of the next influenza pandemic. The present study evaluates B and T cell immune responses generated by the H5N1 viral antigens, hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), or the M2 ion channel in parallel, expressed from a DNA vaccine vehicle. Protection studies of immunized mice challenged with 100 LD50 of homologous or heterologous H5N1 viruses indicate that HA afforded better protection than the NA, NP or M2 DNA vaccines. The antibody response was also higher in HA-vaccinated mice as determined by hemagglutination inhibition (HI) and neutralizing antibodies (NAB) assays. Interestingly, the T cell response was higher against HA than against NA, NP or M2 and was detectable at low doses of the DNA–HA vaccine capable of inducing complete protection, despite the absence of a detectable B cell response. This study emphasizes the need to evaluate the relationship between both arms of the adaptive immune responses in regards to protective efficacy against influenza virus.     

The development and characterization of H5 influenza virus vaccines derived from a 2003 human isolate

The pandemic threat posed by highly pathogenic H5N1 influenza A viruses has created an urgent need for vaccines to protect against H5 virus infection. Because pathogenic viruses grow poorly in chicken eggs and their virulence poses a biohazard to vaccine producers, avirulent viruses produced by reverse genetics have become the preferred basis for vaccine production. Here, we investigated two key characteristics of potential H5 vaccine candidates: the hemaggutinin (HA) cleavage site sequence and its modification to attenuate virulence and the choice of background virus to provide a high-growth rate. We produced recombinant (6:2 reassortant) viruses that possessed a series of modified avirulent-type HA and neuraminidase genes, both of which were derived from an H5N1 human isolate. The other genes of these recombinant viruses were derived from donor virus strains known to grow well in eggs: the human strain A/Puerto Rico/8/34 (PR8) or an avian strain. All of the recombinant viruses grew well in eggs, were avirulent in chicks, and protected animals against infection with a wild-type virus. However, one of the recombinant viruses with an avian virus background acquired a mutation in the HA cleavage site sequence that conferred virulence potential to this virus. Moreover, vaccine candidates with the avian virus background were more virulent than those with the human virus background. We conclude that 6:2 recombinant viruses with a PR8 background are more suitable than those with an avian virus background for vaccine development and that the HA cleavage site sequence must be modified to minimize the potential for a vaccine virus to convert to a virulent form.     

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.