Neospora caninum antigens displaying virus-like particles as a bivalent vaccine candidate against neosporosis

Neospora caninum is a causative and transmissible agent of dog and bovine neosporosis. The resulting reproductive failures in infected cattle lead to significant economic losses worldwide. However, there is no satisfactory treatment or vaccine currently available to combat this pathogen. Thus, the development of appropriate vaccines to manage its infection and transmission is urgently needed. In this study, we expressed Rous sarcoma virus-like particles (RSV-LP) that displayed dual N. caninum antigens in silkworms. The antigen candidates are modified by adding a transmembrane domain of GP64 protein from Bombyx mori nucleopolyhedrovirus (BmNPV) to the C-terminus of surface antigen 1 (NcSAG1) and SAG1-related sequence 2 (NcSRS2). The NcSRS2 alone or the NcSAG1/NcSRS2 bivalent form displaying RSV-LPs were purified using sucrose density gradient centrifugation. These purified VLPs were then used for immunizations in gerbils, Meriones unguiculatus, to evaluate the anti-N. caninum effects in vivo. The results demonstrated that antigens displaying RSV-LPs in immunized gerbils produced the antigen-specific antibody, leading to a relatively lower parasite load after infections of N. caninum. To the best of our knowledge, this is the first study to present an RSV-LP vaccine displaying bivalent antigens from neosporosis. Taken together, our strategy suggests that silkworm-expressed virus-like particles (VLPs) are promising bivalent vaccine candidates against N. caninum infections.     

Immunogenic virus-like particles continuously expressed in mammalian cells as a veterinary rabies vaccine candidate

Rabies is one of the most lethal infectious diseases in the world, with a mortality approaching 100%. There are between 60,000 and 70,000 reported annual deaths, but this is probably an underestimation. Despite the fact that there are vaccines available for rabies, there is a real need of developing more efficacious and cheaper vaccines. This is particularly true for veterinary vaccines because dogs are still the main vector for rabies transmission to human beings. In a previous work, we described the development and characterization of rabies virus-like particles (RV-VLPs) expressed in HEK293 cells. We showed that RV-VLPs are able to induce a specific antibodies response. In this work, we show that VLPs are able to protect mice against virus challenge. Furthermore, we developed a VLPs expressing HEK-293 clone (sP2E5) that grows in serum free medium (SFM) reaching high cell densities. sP2E5 was cultured in perfusion mode in a 5 L bioreactor for 20 days, and the RV-VLPs produced were capable of triggering a protective immune response without the need of concentration or adjuvant addition. Further, these VLPs are able to induce the production of rabies virus neutralizing antibodies. These results demonstrate that RV-VLPs are a promising rabies vaccine candidate.     

Golden-star nanoparticles as adjuvant effectively promotes immune response to foot-and-mouth disease virus-like particles vaccine

Virus-like particles (VLPs) have become a hot topic in modern vaccine research because of its safety, facile production, and immune properties. To further enhance the immune effect of VLPs, we synthesized and used gold-star nanoparticles (AuSNs) as adjuvant for vaccine. Foot-and-mouth disease (FMD) VLPs as target antigen were combined with AuSNs. The FMD VLPs-AuSNs complex was characterized through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, ultraviolet light absorption, and morphological measurement analyses. Result indicated that the FMD VLPs-AuSNs complex is non-toxic in different cell lines. AuSNs can effectively promote the entry of FMD VLPs into cells and improve macrophages activation when combined with FMD VLPs compared with FMD VLPs alone. Further animal vaccination and challenge tests revealed that the specific immune response and protection rate of AuSNs adjuvant group is higher than that of conventional mineral oil (ISA206) adjuvant group. AuSNs can effectively improve the immune protection effects of FMD VLPs vaccines, and exhibit potential as a new adjuvant for other vaccines.     

Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective influenza vaccine candidate

Virus-like particles (VLPs) constitute a promising alternative as influenza vaccine. They are non-replicative particles that mimic the morphology of native viruses which make them more immunogenic than classical subunit vaccines. In this study, we propose HEK-293 cells in suspension culture in serum-free medium as an efficient platform to produce large quantities of VLPs. For this purpose, a stable cell line expressing the main influenza viral antigens hemagglutinin (HA) and neuraminidase (NA) (subtype H1N1) under the regulation of a cumate inducible promoter was developed (293HA-NA cells). The production of VLPs was evaluated by transient transfection of plasmids encoding human immunodeficiency virus (HIV) Gag or M1 influenza matrix protein. To facilitate the monitoring of VLPs production, Gag was fused to the green fluorescence protein (GFP). The transient transfection of the gag containing plasmid in 293HA-NA cells increased the release of HA and NA seven times more than its counterpart transfected with the M1 encoding plasmid. Consequently, the production of HA-NA containing VLPs using Gag as scaffold was evaluated in a 3-L controlled stirred tank bioreactor. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion and ultrafiltered by tangential flow filtration. Transmission electron micrographs of final sample revealed the presence of particles with the average typical size (150–200 nm) and morphology of HIV-1 immature particles. The concentration of the influenza glycoproteins on the Gag-VLPs was estimated by single radial immunodiffusion and hemagglutination assay for HA and by Dot-Blot for HA and NA. More significantly, intranasal immunization of mice with influenza Gag-VLPs induced strong antigen-specific mucosal and systemic antibody responses and provided full protection against a lethal intranasal challenge with the homologous virus strain. These data suggest that, with further optimization and characterization the process could support mass production of safer and better-controlled VLPs-based influenza vaccine candidate.     

A virus-like particle vaccine candidate for influenza A virus based on multiple conserved antigens presented on hepatitis B tandem core particles

Existing Influenza A virus (IAV) vaccines target variable parts of the virus that may change between seasons. Vaccine design relies on predicting the predominant circulating influenza strains but when there is a mismatch between vaccine and circulating strains, efficacy is sub-optimal. Furthermore, current approaches provide limited protection against emerging influenza strains that may cause pandemics. One solution is to design vaccines that target conserved protein domains of influenza, which remain largely unchanged over time and are likely to be found in emergent variants. We present a virus-like particle (VLP), built using the hepatitis B virus tandem core platform, as an IAV vaccine candidate containing multiple conserved antigens. Hepatitis B core protein spontaneously assembles into a VLP that is immunogenic and confers immunogenicity to proteins incorporated into the major insertion region (MIR) of core monomers. However, insertion of antigen sequences may disrupt particle assembly preventing VLP formation or result in unstable particles. We have overcome these problems by genetically manipulating the hepatitis B core to express core monomers in tandem, ligated with a flexible linker, incorporating different antigens at each of the MIRs. Immunisation with this VLP, named Tandiflu1, containing 4 conserved antigens from matrix protein 2 ectodomain and hemagglutinin stalk, leads to production of cross-reactive and protective antibodies. The polyclonal antibodies induced by Tandiflu1 can bind IAV Group 1 hemagglutinin types H1, H5, H11, H9, H16 and a conserved epitope on matrix protein 2 expressed by most strains of IAV. Vaccination with Tandiflu1 results in 100% protection from a lethal influenza challenge with H1N1 IAV. Serum transfer from vaccinated animals is sufficient to confer protection from influenza-associated illness in naïve mice. These data suggest that a Tandem Core based IAV vaccine might provide broad protection against common and emergent H1 IAV strains responsible for seasonal and pandemic influenza in man.     

Non-chromatographic preparation of a bacterially produced single-shot modular virus-like particle capsomere vaccine for avian influenza

Highly pathogenic avian influenza (HPAI) causes significant economic loss, reduced food security and poses an ongoing pandemic threat. Poultry vaccination significantly decreases these problems and recognizes that the health of humans, animals and ecosystems are connected. Low-cost manufacture of poultry vaccine matched quickly to the ever-changing circulating strain is needed for effective vaccination. Here, we re-engineered the process to manufacture bacterially synthesized modular capsomere comprising influenza M2e, previously shown to confer complete protection in challenged mice, for application in poultry. Modular capsomere was prepared using a simplified non-chromatographic salting-out precipitation method and its immunogenicity tested in vivo in poultry. Modular capsomere crudely purified by precipitation (pCapM2e) contained more contaminants than equivalent product purified by chromatography (cCapM2e). Unadjuvanted pCapM2e containing 80 EU of endotoxin per dose was inferior to highly purified and adjuvanted cCapM2e (2 EU per dose). However, addition of adjuvant to pCapM2e resulting in high immunogenicity after only a single dose of vaccination, yet without any local adverse reaction. This finding suggests a strong synergy between adjuvant, antigen and contaminants, and the possible existence of a “Goldilocks” level of contaminants, where high immunogenicity and low reactogenicity can be obtained in a single-shot vaccination. The simplified process offers potential cost and speed advantages to address the needs in influenza poultry vaccination in low-cost veterinary markets.     

Plant-expressed HA as a seasonal influenza vaccine candidate

Influenza is a globally important respiratory pathogen that causes a high degree of morbidity and mortality annually. Although current vaccines are effective against virus infection, new strategies need to be developed to satisfy the global demand for an influenza vaccine. To address this point, we have engineered and produced the full-length hemagglutinin (HA) protein from the A/Wyoming/03/03 (H3N2) strain of influenza in plants. The antigenicity of this plant-produced HA was confirmed by ELISA and single-radial immunodiffusion (SRID) assays. Immunization of mice with plant-produced HA resulted in HA-specific humoral (IgG1, IgG2a and IgG2b) and cellular (IFNgamma and IL-5) immune responses. In addition, significant serum hemagglutination inhibition (HI) and virus neutralizing (VN) antibody titers were obtained with an antigen dose as low as 5mug. These results demonstrate that plant-produced HA protein is antigenic and can induce immune responses in mice that correlate with protection.     

Evaluation of avian influenza virus isolated from ducks as a potential live vaccine candidate against novel H7N9 viruses

Recent outbreaks of a novel H7N9 avian influenza virus in humans in China raise pandemic concerns and underscore an urgent need to develop effective vaccines. Theoretically, live influenza vaccines are of multiple advantages over traditional inactivated influenza vaccines to be used in a pandemic, because they can be produced rapidly, safely, and inexpensively. However, studies on live vaccines against the novel H7N9 virus are limited. In this study, we evaluated a potential live influenza vaccine candidate using an H7N3 avian influenza virus isolated from ducks with controls of two recombinant viruses generated through reverse genetics. The potential candidate could be produced efficiently using chicken embryonated eggs, and is homogenous to the novel H7N9 virus in their viral hemagglutinin genes. The potential candidate is likely low pathogenic to birds and mammals, and likely sensitive to oseltamivir and amantadine, as suggested by its genomic sequences. Its low pathogenicity was further supported through inoculation in mice, chicken embryonated eggs and chickens. Specific antibodies elicited in mice were detectable at least during the period between day 14 and day 56 after intranasal administration of the candidate for one time. Titers of the specific antibodies increased significantly with a boost intranasal administration or a higher inoculation dose. The induced specific antibodies were of substantial cross-reactivity with the novel H7N9 virus. These primary but promising evaluation data suggest that the duck influenza virus could be used as a potential live vaccine candidate, favorably through a prime-boost route, to mitigate the severity of the possible pandemic caused by the newly emerging H7N9 virus, and is valuable to be further evaluated.     

Immunogenicity and efficacy of two types of West Nile virus-like particles different in size and maturation as a second-generation vaccine candidate

Virus-like particles (VLPs) of flaviviruses generated from the prM and E genes are a promising vaccine candidate. We have established cell clones continuously releasing VLPs of West Nile virus (WNV) in serum-free conditions. Two types of VLPs were distinguished by sedimenting analyses in sucrose density gradients. Fast sedimenting VLPs (F-VLPs) were large (40–50 nm) and composed of the E and processed mature M proteins, whereas slowly sedimenting VLPs (S-VLPs) were small (20–30 nm) particles consisting of the E and immature prM proteins. F-VLPs induced higher neutralizing antibody and anti-WNV IgG titers than S-VLPs. Furthermore, IgG2a was dominant over IgG1 by immunization with F-VLPs as with whole virion-derived antigens. Mice vaccinated with a low dose (3 ng) of F-VLPs showed higher protective efficacy (83% survivals) against WNV infection than S-VLP-immune mice (17% survivals). These results indicate that F-VLPs more closely resemble the virions and take a better immunogenic form than S-VLPs as WNV vaccine candidates.     

Hemozoin as a novel adjuvant for inactivated whole virion influenza vaccine

Because vaccination is an effective means to protect humans from influenza viruses, extensive efforts have been made to develop not only new vaccines, but also for new adjuvants to enhance the efficacy of existing inactivated vaccines. Here, we examined the adjuvanticity of synthetic hemozoin, a synthetic version of the malarial by-product hemozoin, on the vaccine efficacy of inactivated whole influenza viruses in a mouse model. We found that mice immunized twice with hemozoin-adjuvanted inactivated A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1) virus elicited higher virus-specific antibody responses than did mice immunized with non-adjuvanted counterparts. Furthermore, mice immunized with hemozoin-adjuvanted inactivated viruses were better protected from lethal challenge with influenza viruses than were mice immunized with non-adjuvanted inactivated vaccines. Our results show that hemozoin improves the immunogenicity of inactivated influenza viruses, and is thus a promising adjuvant for inactivated whole virion influenza vaccines.     

Alpha-C-galactosylceramide as an adjuvant for a live attenuated influenza virus vaccine

There is a substantial need to develop better influenza virus vaccines that can protect populations that are not adequately protected by the currently licensed vaccines. While live attenuated influenza virus vaccines induce superior immune responses compared to inactivated vaccines, the manufacturing process of both types of influenza virus vaccines is time consuming and may not be adequate during a pandemic. Adjuvants would be particularly useful if they could enhance the immune response to live attenuated influenza virus vaccines so that the amount of vaccine needed for a protective dose could be reduced. The glycolipid, alpha-galactosylceramide (alpha-GalCer), has recently been shown to have adjuvant activity for both inactivated and replicating recombinant vaccines. The goal of these experiments was to determine whether a derivative of alpha-GalCer, alpha-C-galactosylceramide (alpha-C-GalCer) can enhance the immune response elicited by a live attenuated influenza virus vaccine containing an NS1 protein truncation and reduce the amount of vaccine required to provide protection after challenge. Our results indicated that the adjuvant reduced both morbidity and mortality in BALB/c mice after challenge with wild type influenza virus. The adjuvant also increased the amount of influenza virus specific total IgG, IgG1, and IgG2a antibodies as well as IFN-γ secreting CD8⁺ T cells. By using knockout mice that are not able to generate NKT cells, we were able to demonstrate that the mechanism of adjuvant activity is dependent on NKT cells. Thus, our data indicate that stimulators of NKT cells represent a new avenue of adjuvants to pursue for live attenuated virus vaccines.     

Insect cell-expressed hemagglutinin with CpG oligodeoxynucleotides plus alum as an adjuvant is a potential pandemic influenza vaccine candidate

Vaccination is the most effective method used to reduce the morbidity and mortality of influenza infections. However, as exemplified in the current swine-origin influenza virus (S-OIV) pandemic, the global manufacturing capacity of influenza vaccines is severely limited. In the present proof-of-concept study, we combined cell substrate selection and antigen engineering with adjuvant development to design a potential pandemic influenza vaccine candidate, in which CpG oligodeoxynucleotides (CpG-ODN) plus alum was used as a composite adjuvant to enhance the immunogenicity of insect cell-expressed recombinant hemagglutinin (rHA). Our candidate vaccine was found to be effective in inducing protective humoral as well as cellular immunity in mice and able to protect the immunized mice from related influenza virus challenge. If this candidate vaccine is validated in humans, vaccine development can be started immediately after the release of the first HA sequence of any pandemic influenza virus. Moreover, given the potential of large-scale manufacturing capacity of the recombinant antigen, in combination with the antigen-sparing effect of the composite adjuvant, this technology could be an invaluable asset in the fight against pandemic influenza.     

Evaluation of formalin inactivated V3526 virus with adjuvant as a next generation vaccine candidate for Venezuelan equine encephalitis virus

V3526, a genetically modified strain of Venezuelan equine encephalitis virus (VEEV), was formalin inactivated for evaluation as a next generation vaccine candidate for VEEV. In this study, we tested formalin-inactivated V3526 (fV3526) with and without adjuvant for immunogenicity and efficacy in BALB/c mice and results were compared to the existing inactivated VEEV vaccine, C84. Mice were vaccinated intramuscularly (IM) or subcutaneously (SC) with fV3526 formulations and challenged with VEEV IAB Trinidad donkey (VEEV TrD) strain by SC or aerosol exposure. Efficacy following SC or aerosol challenge was not significantly different between the fV3526 formulations or compared to C84 despite C84 being administered in more doses and higher concentration of viral protein per dose. These data support further evaluation of fV3526 formulations as a next generation VEEV vaccine.     

Mutants of cholera toxin as an effective and safe adjuvant for nasal influenza vaccine.

The effectiveness and safety of mutants of cholera toxin (CT) as an adjuvant for nasal influenza vaccine was examined. Four CT mutants, called CT7 K (Arg to Lys), CT61F (Arg to Phe), CT112 K (Glu to Lys), and CT118E (Glu to Gln), were produced by the replacement of one amino acid at the A1-subunit using site-directed mutagenesis. All these mutants were confirmed to be less toxic than native CT when the toxicity was analysed by using Y1 adrenal cells in vitro. When high (1 microg) and low (0.1 microg) doses of these CT mutants, together with high (1 microg) and low (0.1 microg) doses of influenza A/PR/8/34 virus (H1N1) vaccine, respectively, were administered intranasally into BALB/c mice in a two dose regimen (twice, 4 weeks apart), they produced both anti-PR8 hemagglutinin (HA) IgA and IgG antibody (Ab) responses roughly in a dose-dependent manner. The relatively low level of anti-HA Ab responses, induced by the low dose CT mutants, were enough to provide complete protection against the homologous virus infection. Under these vaccination conditions, no anti-CTB IgE Ab responses were induced. The mutant CT112 K, which showed a relatively high adjuvant activity, the lowest toxicity and relatively high yields in a bacterial culture, seems to be the most effective and safest adjuvant for nasal influenza vaccine among those examined. The low dose of CT derivatives or vaccine used in the mouse model (0.1 microg/20 g mouse) corresponded to 100 microg/20 kg, the estimated dose per person. A tentative plan for safety standards for human use of CT (or LT) derivatives as an adjuvant of nasal influenza vaccine is discussed.     

Influenza H7N9 LAH-HBc virus-like particle vaccine with adjuvant protects mice against homologous and heterologous influenza viruses

The long alpha-helix (LAH) region located in influenza virus hemagglutinin (HA) shows conservation among different influenza A strains, which could be used as a candidate target of influenza vaccines. Moreover, the hepatitis B virus core protein (HBc) is a carrier for heterologous epitopes in eliciting effective immune responses. We inserted the LAH region of H7N9 influenza virus into the HBc and prepared the LAH-HBc protein, which were capable of self-assembly into virus-like particles (VLP), by using E. coli expression system. Intranasal immunization of the LAH-HBc VLP in combination with chitosan adjuvant or CTB¹ adjuvant in mice could induce both humoral and cellular immune responses effectively and provide complete protection against lethal challenge of homologous H7N9 virus or heterologous H3N2 virus, as well as partial protection against lethal challenge of heterologous H1N1 virus. These results provide a proof of concept for LAH-HBc VLP vaccine that would be fast and easy to be produced and might be an ideal candidate as a rapid-response tool against a future influenza pandemic.     

A candidate dual vaccine against influenza and noroviruses

The extracellular domain of the matrix protein 2 (M2e) of influenza viruses is highly conserved among all influenza A subtypes, making it a suitable target for a universal influenza vaccine. In this study, we demonstrated an enhanced immune response and protection of a chimeric M2e vaccine against influenza A viruses using our newly developed vaccine platform, the norovirus P particle, to present the M2e peptide. The 23-amino acid peptide was inserted into one of the surface loops of the P protein, resulting in 24 copies of M2e presented on each P particle. Significantly (P < 0.001) increased antibody responses to M2e were observed in mice immunized with the P particle-M2e chimera compared with those immunized with the free M2e peptides. Mice immunized with the P particle-M2e vaccine were fully protected (100% survived) against lethal challenge of a mouse adapted human influenza virus PR8 (H1N1), while only low survival rates (<12.5%) were found in mice immunized with the free M2e peptides or wild type P particle. In addition, the mouse sera collected after immunization with the P particle-M2e vaccine were able to block the binding of norovirus virus-like particle and P particle to histo-blood group antigen receptors. These results suggest that the P particle-M2e chimera can be used as dual vaccine against both noroviruses and influenza viruses.     

Biochemical and immunologic comparison of virus-like particles for a rotavirus subunit vaccine.

A parenterally administered rotavirus vaccine composed of virus-like particles (VLPs) is being evaluated for human use. VLPs composed of bovine VP6 and simian VP7 (SA11, G3) proteins (6/7-VLPs) or of bovine VP2, bovine VP6, and simian VP7 (SA11, G3) proteins (2/6/7-VLPs) were synthesized and purified from Sf9 insect cells co-infected with recombinant baculoviruses. 6/7- and 2/6/7-VLP administered parenterally (i.m.) in mice had comparable immunogenicity, but the 2/6/7-VLPs were more homogeneous and stable. The inclusion of the VP2 capsid contributed to particle formation and stability. The adjuvant QS-21 significantly enhanced the immunogenicity of 2/6/7-VLPs over A10H or saline alone. Equivalent serum neutralizing antibody responses were induced over the range of 1-15 microg/dose of 2/6/7-VLPs administered with the range of 5-20 microg/dose of QS-21. The immunogenicity of 2/6/7-VLPs and inactivated SA11 virus were comparable. 2/6/7-VLPs are a promising candidate for a parenterally delivered rotavirus subunit vaccine.     

Development of a candidate influenza vaccine based on virus-like particles displaying influenza M2e peptide into the immunodominant region of hepatitis B core antigen: Broad protective efficacy of particles carrying four copies of M2e

A long-term objective when designing influenza vaccines is to create one with broad cross-reactivity that will provide effective control over influenza, no matter which strain has caused the disease. Here we summarize the results from an investigation into the immunogenic and protective capacities inherent in variations of a recombinant protein, HBc/4M2e. This protein contains four copies of the ectodomain from the influenza virus protein M2 (M2e) fused within the immunodominant loop of the hepatitis B virus core antigen (HBc). Variations of this basic design include preparations containing M2e from the consensus human influenza virus; the M2e from the highly pathogenic avian A/H5N1 virus and a combination of two copies from human and two copies from avian influenza viruses. Intramuscular delivery in mice with preparations containing four identical copies of M2e induced high IgG titers in blood sera and bronchoalveolar lavages. It also provoked the formation of memory T-cells and antibodies were retained in the blood sera for a significant period of time post immunization. Furthermore, these preparations prevented the death of 75–100% of animals, which were challenged with lethal doses of virus. This resulted in a 1.2–3.5 log 10 decrease in viral replication within the lungs. Moreover, HBc particles carrying only “human” or “avian” M2e displayed cross-reactivity in relation to human (A/H1N1, A/H2N2 and A/H3N2) or A/H5N1 and A(H1N1)pdm09 viruses, respectively; however, with the particles carrying both “human” and “avian” M2e this effect was much weaker, especially in relation to influenza virus A/H5N1. It is apparent from this work that to quickly produce vaccine for a pandemic it would be necessary to have several variations of a recombinant protein, containing four copies of M2e (each one against a group of likely influenza virus strains) with these relevant constructs housed within a comprehensive collection Escherichia coli-producers and maintained ready for use.     

Polymeric lamellar substrate particles as carrier adjuvant for recombinant hepatitis B surface antigen vaccine

Blank polymeric lamellar substrate particles (PLSP) of poly (l-lactide) were prepared and recombinant hepatitis B surface antigen (rHBsAg) was adsorbed onto these particles. The physical characteristics of blank PLSPs or PLSP-rHBsAg in vitro and its immunological responses in Balb/c mice were investigated. The average size of the particles was less than 10 μm. Antigen adsorption efficiency was found to be 62.66 ± 1.26%. Immunization with PLSP-rHBsAg resulted in upregulation of specific cellular (lymphoproliferation, IFN-γ and NO release) as well as IgG response in animals. These responses were higher than those produced by two-dose schedule of alum-adsorbed antigen (alum-rHBsAg). Thus in conclusion, in terms of convenience and efficacy PLSP-rHBsAg is superior to alum-rHBsAg.     

H9N2 avian influenza virus-like particle vaccine provides protective immunity and a strategy for the differentiation of infected from vaccinated animals

In the present study, virus-like particles (VLPs) were evaluated as a candidate poultry vaccine against avian influenza virus (AIV) subtype H9N2. Specific pathogen-free chickens received a single injection of the VLP vaccine expressing HA and M1 protein of AIV H9N2 (H9 HA VLP) at escalating doses in the presence or absence of ISA70 water-in-oil adjuvant. At 3 weeks post vaccination, we performed hemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) to determine serological immune responses, and challenge studies using SPF chickens. A single dose of H9 HA VLP vaccine induced high levels of HI antibodies and lowered frequencies of virus isolation after the wild-type virus challenge. The addition of ISA70 adjuvant significantly increased the immunogenicity of H9 HA VLP vaccines. Furthermore, it allows differentiation of AIV-infected chickens from vaccinated chickens with an ELISA using nucleocapsid antigen, which offers a promising strategy to differentiate infected from vaccinated animals (DIVA). These results provide support for continued development of the VLP as an animal vaccine against influenza virus.