Generation of an attenuated H5N1 avian influenza virus vaccine with all eight genes from avian viruses

In the face of disease outbreaks in poultry and the potential pandemic threat to humans caused by the highly pathogenic avian influenza viruses (HPAIVs) of H5N1 subtype, improvement in biosecurity and the use of inactivated vaccines are two main options for the control of this disease. Vaccine candidates of influenza A viruses of H5N1 subtype have been generated in several laboratories by plasmid-based reverse genetics with hemagglutinin (HA) and neuraminidase (NA) genes from the epidemic strains of avian viruses in a background of internal genes from the vaccine donor strain of human strains, A/Puerto Rico/8/34 (PR8). These reassortant viruses containing genes from both avian and human viruses might impose biosafety concerns, also may be do if C4/F AIV would be a live attenuated vaccine or cold-adaptive strain vaccine. In order to generate better and safer vaccine candidate viruses, we genetically constructed attenuated reassortant H5N1 influenza A virus, designated as C4/F AIV, by plasmid-based reverse genetics with all eight genes from the avian strains. The C4/F AIV virus contained HA and NA genes from an epidemic strain A/Chicken/Huadong/04 (H5N1) (C4/H5N1) in a background of internal genes derived from a low pathogenic strain of A/Chicken/F/98(H9N2). The reassortant virus was attenuated by removal of the multibasic amino acid motif in the HA gene by mutation and deletion (from PQRERRRKKR (downward arrow) G to PQIETR (downward arrow) G). The intravenous pathogenicity index (IVPI) of C4/F AIV virus was 0, whereas that of the donor virus C4/H5N1 was 3.0. The virus HA titer of C4/H5N1 in the allantoic fluid from infected embryonated eggs was as high as 1:2048. The inactivated vaccine prepared from the reassortant virus C4/F AIV-induced high HI titer in vaccinated chickens and gave 100% protection when challenged with highly pathogenic avian influenza virus of H5N1 subtype.     

Generation and characterization of a cold-adapted influenza A H9N2 reassortant as a live pandemic influenza virus vaccine candidate

H9N2 subtype influenza A viruses have been identified in avian species worldwide and were isolated from humans in 1999, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. Reassortant H1N1 and H3N2 human influenza A viruses with the internal genes of the influenza A/Ann Arbor/6/60 (H2N2) (AA) cold-adapted (ca) virus have proven to be attenuated and safe as live virus vaccines in humans. Using classical genetic reassortment, we generated a reassortant virus (G9/AA ca) that contains the hemagglutinin and neuraminidase genes from influenza A/chicken/Hong Kong/G9/97 (H9N2) (G9) and six internal gene segments from the AA ca virus. When administered intranasally, the reassortant virus was immunogenic and protected mice from subsequent challenge with wild-type H9N2 viruses, although it was restricted in replication in the respiratory tract of mice. The G9/AA ca virus bears properties that are desirable in a vaccine for humans and is available for clinical evaluation and use, should the need arise.     

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.     

Generation and evaluation of the trivalent inactivated reassortant vaccine using human, avian, and swine influenza A viruses

Reassortant technology was used to obtain three interspecific reassortant influenza viruses using three influenza viruses of A/Puerto Rico/8/34(H1N1), A/swine/Hebei/1/2005(H3N2) and A/chicken/Guangdong/126/2002(H9N2). The high-growth reassortant strains were H9/PR8, H3/H9N2 and H1/H9N2 that contained hemagglutinin (HA) and neuraminidase (NA) genes from the inactivated parental viruses and the other 6 internal genes from the live parental viruses. The trivalent formalin-inactivated vaccine, containing H1, H3 and H9 subtype antigens from human, swine and avian influenza viruses respectively, was prepared using these reassortant viruses. Animal studies showed that the vaccine was safe and immunogenic. Two-dosing regimen of the influenza vaccine induced high titers of hemagglutination inhibiting (HI) antibodies and influenza-specific IgG antibodies without antigenic cross-interference. It protected 100% chickens from challenge of A/chicken/Guangdong/126/2002 virus and protected 100% mice against challenges with different combinations of the three infective parental viruses. These results indicated that the trivalent vaccine could offer multi-protection against multi-influenza viruses synchronously. This kind of multivalent inactivated reassortant influenza vaccine maybe enlightens the pandemic influenza preparedness as the emergency measure.     

The 2009 pandemic H1N1 virus induces anti-neuraminidase (NA) antibodies that cross-react with the NA of H5N1 viruses in ferrets

A miniaturized neuraminidase inhibition (NI) assay using HA-mismatched H6 reassortant viruses was performed to examine the neuraminidase (NA)-specific antibody response in ferrets immunized with live-attenuated influenza vaccine (LAIV) strains. The strains tested possessed different NAs derived from seasonal H1N1 and H3N2, 2009 pandemic H1N1, and the highly pathogenic influenza H5N1 virus. The anti-NA antibodies from the 2009 pandemic strain (A/California/7/2009) immunized ferrets cross-reacted with the NA of H5N1 but not with the NA of seasonal H1N1 viruses. The plaque size reduction assay confirmed the cross-reactivity between the NAs of A/California/7/2009 and the H5N1 virus. Sequence and structural analyses of these N1 NA proteins showed that the NA of the 2009 pandemic H1N1 strain shared at least 22 more amino acids in the head domain with the NAs of the avian H5N1 strains than with the NAs of seasonal human H1N1 viruses. Our data demonstrated LAIV-induced NA antibody responses in ferrets and cross-reactive NA antibodies induced by 2009 pandemic H1N1 and H5N1 LAIV viruses.     

Reassortant Highly Pathogenic Influenza A(H5N6) Virus in Laos

In March 2014, avian influenza in poultry in Laos was caused by an emergent influenza A(H5N6) virus. Genetic analysis indicated that the virus had originated from reassortment of influenza A(H5N1) clade 2.3.2.1b, variant clade 2.3.4, and influenza A(H6N6) viruses that circulate broadly in duck populations in southern and eastern China.     

人感染高致病性H5N6禽流感病毒表面糖蛋白分子进化特征

目的对19例人感染高致病性H5N6禽流感病毒的血凝素(hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)蛋白进行分子进化分析。方法运用下一代测序平台对病毒分离物进行全基因组测序,从美国国家生物技术信息中心(national center for biotechnology information,NCBI)和全球流感序列数据库(global initiative on sharing avian influenza data,GISAID)下载参考序列,利用Blasts、Mega 6.1及Clustal X 2.1等软件进行序列分析。结果2014-2018年中国共发生23例人感染H5N6禽流感病毒病例。对19个病例的H5N6病毒的HA和NA基因进行进化分析。HA进化分析显示病毒都属于Clade 2.3.4.4,其中涉及17个病例的病毒属于Group C;首例H5N6病例毒株(A/Sichuan/26221/2014)属于Group D;福建一个病例(A/Fujian-Sanyuan/21099/2017)属于Group B。所有19个病例的病毒HA蛋白的裂解位点含有多个碱性氨基酸。所有病毒的HA蛋白的受体结合位点226~228位氨基酸是QS(R)G(氨基酸排序以H3-HA为准),理论上对禽类受体α2-3半乳糖苷唾液酸(SAα2-3Gal)有嗜性。18病例病毒的HA蛋白发生了T160A的突变,导致在158N位点失去糖基化。除了A/Sichuan/26221/2014外,18个病例的病毒NA蛋白在58~68位缺失了10个氨基酸。9个病例的病毒PB2蛋白发生E627K突变。结论2014-2018年间中国人感染H5N6病毒进化活跃,具有明显的基因多样性,需要加强对病毒分子进化的监测。     

Emergence of novel clade 2.3.4 influenza A (H5N1) virus subgroups in Yunnan Province,China

From December 2013 to March 2014, a major wave of highly pathogenic avian influenza outbreak occurred in poultry in Yunnan Province, China. We isolated and characterized eight highly pathogenic avian influenza A (H5N1) viruses from poultry. Full genome influenza sequences and analyses have been performed.Sequence analyses revealed that they belonged to clade 2.3.4 but did not fit within the three defined subclades. The isolated viruses were provisional subclade 2.3.4.4e. The provisional subclade 2.3.4.4e viruses with six internal genes from avian influenza A (H5N2) viruses in 2013 were the novel reassortant influenza A (H5N1) viruses which were associated with the outbreak of H5N1 occurred in egg chicken farms in Yunnan Province. The HA genes were similar to subtype H5 viruses isolated from January to March of 2014 in Asia including H5N6 and H5N8. The NA genes were most closely related to A/chicken/Vietnam/NCVD-KA423/2013 (H5N1) from the subclade 2.3.2. The HI assay demonstrated a lack of antigenic relatedness between clades 2.3.4.4e and 2.3.4.1 (RE-5 vaccine strain) or 2.3.2.2 (RE-6 vaccine strain).     

Genetic and biological characterization of two novel reassortant H5N6 swine influenza viruses in mice and chickens

Novel H5N6 influenza A viruses have infected birds and human beings and caused four human clinical cases in China since 2014. The pig, as a mixing vessel, plays an important role for influenza virus reassortment and transmission. Towards this, routine surveillance for swine influenza in Guangdong province was conducted in 2014. In this study, we reported the biological characterization of two H5N6 influenza viruses isolated from healthy pigs in Guangdong province. Genetic analysis indicates that the two viruses are reassortants of 2.3.4.4 H5N1 and H6N6 avian influenza viruses with a high similarity to duck and human H5N6 influenza viruses isolated from Guangdong province. The data from chicken and mouse experiments show that the viruses are highly pathogenic in chickens and result in a systemic infection, and replicate in the mouse lung accompanying with a clinical inflammatory pathology. The results of the study demonstrate that the two H5N6 influenza viruses isolated from swine are the avian-originated viruses and have not adapted to swine population yet. However, they might keep evolving and pose a potential risk to public health and the continued surveillance of swine influenza should be strengthened.     

An epidemiological study of influenza viruses among Chinese farm families with household ducks and pigs.

To examine the possibility of interspecies transmission and genetic reassortment of influenza viruses on farms in Southern China, we surveyed 20 farm families living outside the city of Nanchang who raised pigs and ducks in their homes. Weekly interviews of family members and virus isolation studies of throat swabs and faecal samples, collected from September 1992 to September 1993, established the seasonal pattern of respiratory tract infections in these families and identified 11 influenza viruses (6 in humans and 5 in ducks). Most of the human isolates were type A of H3N2 subtype. Serologic studies of farm pigs indicated infection by the same human viruses circulating in family members, but there was no evidence that either swine or avian viruses had been transmitted to pigs. Eight of 156 human serum samples inhibited the neuraminidase activity of two of the duck isolates, raising the possibility of interspecies transmission of these avian viruses. Genotype analysis of duck and human isolates provided no evidence for reassortment. Our finding support the concept that intermingling of humans, pigs and ducks on Chinese farms is favourable to the generation of new, potentially hazardous strains of influenza virus.     

Development of Eurasian H7N7/PR8 high growth reassortant virus for clinical evaluation as an inactivated pandemic influenza vaccine

Avian-to-human transmission of the high pathogenicity (HP) H7N7 subtype avian influenza viruses in the Netherlands during 2003 caused zoonotic infections in 89 people, including a case of acute fatal respiratory distress syndrome. Public health emergency preparedness against H7N7 avian influenza viruses with pandemic potential includes the development of vaccine candidate viruses. In order to develop a high growth reassortant vaccine candidate virus, low pathogenicity (LP) A/mallard/Netherlands/12/2000 (H7N3) and A/mallard/Netherlands/2/2000 (H10N7) strains were selected as donors of the H7 haemagglutinin and N7 neuraminidase genes, respectively. The donor viruses exhibited high amino acid sequence homology with the surface glycoproteins of A/Netherlands/219/03 H7N7 virus (NL219), an isolate recovered from the fatal human case. Adhering to the seasonal influenza vaccine licensure regulations, we generated a H7N7/PR8 reassortant containing desired surface glycoprotein genes from the mallard viruses and internal genes of A/Puerto Rico/8/34 human vaccine strain (H1N1). Antigenic analysis revealed that the vaccine candidate virus confers broad antigenic cross-reactivity against contemporary Eurasian and the North American H7 subtype human isolates. Mice immunized with formalin inactivated (FI) H7N7/PR8 whole virus vaccine with or without aluminum hydroxide adjuvant conferred clinical protection from mortality and reduced pulmonary replication of the NL219 challenge virus. The FI H7N7/PR8 whole virus vaccine also afforded cross-protection in mice at the pulmonary level against antigenically distinct North American LP A/Canada/444/04 (H7N3) human isolate. The vaccine candidate virus satisfied the agricultural safety requirements for chickens, proved safe in mice, and has entered in phase-I human clinical trial in the United States.     

Novel Eurasian Highly Pathogenic Avian Influenza A H5 Viruses in Wild Birds,Washington, USA, 2014

Novel Eurasian lineage avian influenza A(H5N8) virus has spread rapidly and globally since January 2014. In December 2014, H5N8 and reassortant H5N2 viruses were detected in wild birds in Washington, USA, and subsequently in backyard birds. When they infect commercial poultry, these highly pathogenic viruses pose substantial trade issues.     

Novel Influenza A(H7N2) Virus in Chickens,Jilin Province,China, 2014

In February 2014, while investigating the source of a human infection with influenza A(H7N9) virus in northern China, we isolated subtypes H7N2 and H9N2 viruses from chickens on the patient’s farm. Sequence analysis revealed that the H7N2 virus is a novel reassortant of H7N9 and H9N2 viruses. Continued surveillance is needed.     

Identification of the source of A (H10N8) virus causing human infection

A novel H10N8 influenza A virus has been detected in three humans in China since December 2013. Although this virus was hypothesized to be a novel reassortant among influenza viruses from wild birds and domestic poultry, its evolutionary path leading to human infection is unknown. Sporadic surveillance at the live poultry market (LPM) suspected to be the source of infection for the first H10N8 patient has shown a gradual increase in influenza virus prevalence culminating with a predominance of H10N8 viruses. Influenza viruses detected in the LPM up to 8 months prior to human infection contributed genetic components to the zoonotic virus. These H10N8 viruses have continued to evolve within this LPM subsequent to the human infection, and continuous assessments of these H10N8 viruses will be necessary. Serological surveillance showed that the virus appears to have been present throughout the LPM system in Nanchang, China. Reduction of the influenza virus burden in LPMs is essential in preventing future emergence of novel influenza viruses with zoonotic and pandemic potential.     

Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice

Avian influenza viruses represent a growing threat for an influenza pandemic. To develop recombinant vaccine for avian influenza of the H9N2 subtype, we expressed in insect cells virus-like particles (VLPs) consisting of three structural proteins of influenza A/Hong Kong/1073/99 (H9N2) virus. Upon infection of Sf9 cells with recombinant baculoviruses, the hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins were co-expressed in the infected cells, self-assembled, and released into the culture medium as VLPs of 80–120 nm in diameter. VLPs exhibited functional characteristics of influenza virus including hemagglutination and neuraminidase activities. In BALB/c mice, VLPs elicited serum antibodies specific for influenza A/Hong Kong/1073/99 (H9N2) virus and inhibited replication of the influenza virus after challenge. Thus, VLPs represent a potential strategy for the development of human vaccines against avian influenza H9N2 viruses.     

Generation of reassortant influenza vaccines by reverse genetics that allows utilization of a DIVA (Differentiating Infected from Vaccinated Animals) strategy for the control of avian influenza

Vaccination of poultry with inactivated influenza vaccine can be an effective tool in the control of avian influenza (AI). One major concern of using inactivated vaccine is vaccine-induced antibody interference with serologic surveillance and epidemiology. In the United States, low pathogenicity H5 and H7 subtype AI viruses have caused serious economic losses in the poultry industry. Most of these viruses also have the accompanying N2 subtype and no H5N1 or H7N8 subtype AI viruses have been identified in poultry in the US. In order to allow the Differentiation of Infected from Vaccinated Animals (DIVA) while maintaining maximum efficacy of the vaccine, we generated reassortant viruses by reverse genetics that contained the same H5 and H7 hemagglutinin (HA) gene as the challenge virus, but a heterologous N1 or N8 neuraminidase (NA) gene. In vaccination-challenge experiments in 2-week-old specific pathogen free chickens, reassortant influenza vaccines (rH5N1 and rH7N8) demonstrated similar antibody profiles and comparable protection rates as vaccines prepared with parent H5N2 and H7N2 viruses. Further, we were able to differentiate the sera from infected and vaccinated birds by neuraminidase inhibition test and indirect immunofluorescent antibody assay on the basis of different antibodies elicited by their NA proteins. These results demonstrate the usefulness of a reverse genetics system for the rapid generation of reassortant AI virus that allows utilization of the DIVA strategy for the control of AI infections in poultry.     

Characterization of the M protein and nucleoprotein genes of an avian influenza A virus which are involved in host range restriction in monkeys

A reassortant virus possessing RNA segment 7, which codes for the M1 and M2 proteins, of the avian influenza A/Mallard/New York/6750/78 (H2N2) virus and the other seven RNA segments of the human influenza A/Udorn/307/72 (H3N2) virus had been shown previously to be markedly restricted in replication in the respiratory tract of squirrel monkeys. In contrast, a reassortant possessing segment 7 of another avian influenza virus, A/Pintail/Alberta/119/79 (H4N6), and the seven other RNA segments from the A/Udorn/72 virus was not restricted. The nucleotide and deduced amino acid sequence of the RNA segment 7 of each virus was determined to identify the structural basis for the attenuation phenotype specified by RNA segment 7 of the A/Mallard/78 virus. Analysis of the deduced amino acid sequences revealed only two amino acid differences in the M1 protein and one difference in the M2 protein, suggesting that the attenuation phenotype of a reassortant virus possessing segment 7 of the A/Mallard/78 virus may be specified by one to three amino acids. Reassortant viruses possessing RNA segment 6, which codes for the nucleoprotein, of either avian influenza virus and the other seven RNA segments of a human influenza virus were also restricted in replication in squirrel monkeys. A comparison of the deduced amino acid sequences of the two avian nucleopeoteins demonstrated only three amino acid differences indicating that these two avian viruses possess NP genes that are highly related. The high degree of relatedness of both the NP and M proteins of these two avian viruses contrasts with their divergent surface antigens. The structural basis for the attenuation phenotype of the NP gene of the A/Mallard/78 virus is being investigated.     

Prior infection with an H1N1 swine influenza virus partially protects pigs against a low pathogenic H5N1 avian influenza virus

Most humans lack virus neutralizing (VN) and haemagglutination inhibition (HI) antibodies to H5N1 avian influenza viruses (AIVs), but cross-reactive neuraminidase inhibition (NI) antibodies and cell-mediated immune (CMI) responses are common. These immune responses result largely from infections with seasonal human H1N1 influenza viruses, but the protective effect of H1N1 infection-immunity against H5N1 infection has never been examined. To this purpose, we have used the pig model of influenza and a low pathogenic (LP) H5N1 AIV. Pigs were inoculated intranasally with sw/Belgium/1/98 (H1N1) 4 weeks before challenge with duck/Minnesota/1525/81 (H5N1). While the viruses failed to cross-react in HI and VN tests, the H1N1 infection induced high levels of H5N1 cross-reactive NI antibodies. Cross-reactive CMI was demonstrated by measurements of lymphoproliferation and IFN-γ secretion after in vitro restimulation of peripheral blood mononuclear cells. All control pigs showed clinical signs and H5N1 virus isolation from the respiratory tract post-challenge. The H1N1-immune pigs, in contrast, showed a complete clinical protection and only 3 pigs out of 10 were H5N1 virus-positive. In a second and smaller experiment, H1N1 virus infection also conferred cross-protection against a LP H5N2 AIV, while cross-reactive immunity was solely detected in tests for CMI. Our data further support the notion that immunity induced by seasonal human H1N1 influenza virus infection may provide some protection against H5N1 or other H5 AIVs in the absence of neutralizing H5 antibodies. Further studies should reveal whether cross-protection holds against H5N1 viruses that are better adapted to replicate in mammals or with a more distantly related N1.     

Generation of a reassortant avian influenza virus H5N2 vaccine strain capable of protecting chickens against infection with Egyptian H5N1 and H9N2 viruses

BackgroundAvian influenza H5N1 viruses have been enzootic in Egyptian poultry since 2006. Avian influenza H9N2 viruses which have been circulating in Egyptian poultry since 2011 showed high replication rates in embryonated chicken eggs and mammalian cells.MethodsTo investigate which gene segment was responsible for increasing replication, we constructed reassortant influenza viruses using the low pathogenic H1N1 PR8 virus as backbone and included individual genes from A/chicken/Egypt/S4456B/2011(H9N2) virus. Then, we invested this finding to improve a PR8-derived H5N1 influenza vaccine strain by incorporation of the NA segment of H9N2 virus instead of the NA of H5N1. The growth properties of this virus and several other forms of reassortant H5 viruses were compared. Finally, we tested the efficacy of this reassortant vaccine strain in chickens.ResultsWe observed an increase in replication for a reassortant virus expressing the neuraminidase gene (N2) of H9N2 virus relative to that of either parental viruses or reassortant PR8 viruses expressing other genes. Then, we generated an H5N2 vaccine strain based on the H5 from an Egyptian H5N1 virus and the N2 from an Egyptian H9N2 virus on a PR8 backbone. This strain had better replication rates than an H5N2 reassortant strain on an H9N2 backbone and an H5N1 reassortant on a PR8 backbone. This virus was then used to develop a killed, oil-emulsion vaccine and tested for efficacy against H5N1 and H9N2 viruses in chickens. Results showed that this vaccine was immunogenic and reduced mortality and shedding.DiscussionOur findings suggest that an inactivated PR8-derived H5N2 influenza vaccine is efficacious in poultry against H5N1 and H9N2 viruses and the vaccine seed replicates at a high rate thus improving vaccine production.     

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.