Influenza Virus-like Particle Vaccine Research Articles

H3N2 canine influenza virus-like particle vaccine with great protection in beagle dogs.

A new antigenically and genetically distinct canine influenza virus (CIV) H3N2 clade possessing mutations associated with mammalian adaptation emerged in 2016 and substituted previously circulating strains. This clade poses a risk for zoonotic infection. In our study, intramuscular injection of the H3N2 virus-like particle (VLP) vaccine and inactivated H3N2 CIV confer completely sterilizing protection against homologous H3N2 canine influenza virus challenge. Our results provide further support for the possibility of developing VLP vaccines that can reliably induce immunity in animal species.

Dual N-linked glycosylation at residues 133 and 158 in the hemagglutinin are essential for the efficacy of H7N9 avian influenza virus like particle vaccine in chickens and mice

H7N9 subtype avian influenza virus (AIV) poses a great challenge to poultry industry. Virus-like particle (VLP) is a prospective alternative for the traditional egg-based influenza vaccines. N-linked glycosylation (NLG) regulates the efficacy of influenza vaccines, whereas the impact of NLG modifications on the efficacy of influenza VLP vaccines remains unclear. Here, H7N9 VLPs were assembled in insect cells through co-infection with the baculoviruses expressing the NLG-modified hemagglutinin (HA), neuraminidase and matrix proteins, and the VLP vaccines were assessed in chickens and mice. NLG modifications significantly enhanced hemagglutination-inhibition and virus neutralization antibody responses in mice, rather than in chickens, because different immunization strategies were used in these animal models. The presence of dual NLG at residues 133 and 158 significantly elevated HA-binding IgG titers in chickens and mice. The VLP vaccines conferred complete protection and significantly suppressed virus replication and lung pathology post challenge with H7N9 viruses in chickens and mice. VLP immunization activated T cell immunity-related cytokine response and inhibited inflammatory cytokine response in mouse lung. Of note, the presence of dual NLG at residues 133 and 158 optimized the capacity of the VLP vaccine to stimulate interleukin-4 expression, inhibit virus shedding or alleviate lung pathology in chickens or mice. Intriguingly, the VLP vaccine with NLG addition at residue 133 provided partial cross-protection against the H5Nx subtype AIVs in chickens and mice. In conclusion, dual NLG at residues 133 and 158 in HA can be potentially used to enhance the efficacy of H7N9 VLP vaccines in chickens and mammals.

A mosaic influenza virus-like particles vaccine provides broad humoral and cellular immune responses against influenza A viruses

The development of a universal influenza vaccine to elicit broad immune responses is essential in reducing disease burden and pandemic impact. In this study, the mosaic vaccine design strategy and genetic algorithms were utilized to optimize the seasonal influenza A virus (H1N1, H3N2) hemagglutinin (HA) and neuraminidase (NA) antigens, which also contain most potential T-cell epitopes. These mosaic immunogens were then expressed as virus-like particles (VLPs) using the baculovirus expression system. The immunogenicity and protection effectiveness of the mosaic VLPs were compared to the commercial quadrivalent inactivated influenza vaccine (QIV) in the mice model. Strong cross-reactive antibody responses were observed in mice following two doses of vaccination with the mosaic VLPs, with HI titers higher than 40 in 15 of 16 tested strains as opposed to limited cross HI antibody levels with QIV vaccination. After a single vaccination, mice also show a stronger level of cross-reactive antibody responses than the QIV. The QIV vaccinations only elicited NI antibodies to a small number of vaccine strains, and not even strong NI antibodies to its corresponding vaccine components. In contrast, the mosaic VLPs caused robust NI antibodies to all tested seasonal influenza virus vaccine strains. Here, we demonstrated the mosaic vaccines induces stronger cross-reactive antibodies and robust more T-cell responses compared to the QIV. The mosaic VLPs also provided protection against challenges with ancestral influenza A viruses of both H1 and H3 subtypes. These findings indicated that the mosaic VLPs were a promising strategy for developing a broad influenza vaccine in future.

Chimeric H5 influenza virus-like particle vaccine elicits broader cross-clade antibody responses in chickens than in ducks.

Eurasian-lineage highly pathogenic avian influenza (HPAI) H5 viruses have spread throughout Asia, the Middle East, Europe, Africa, and most recently, North and South America. These viruses are independently evolving into genetically and antigenically divergent clades, and broad-spectrum vaccines protecting against these divergent clades are needed. In this study, we developed a chimeric virus-like particle (VLP) vaccine co-expressing hemagglutinins from two clades (clades 1 and 2.3.2.1) of HPAI H5 viruses and performed comparative cross-clade hemagglutination inhibition (HI) analysis in chickens and ducks. The chimeric VLP immunization induced a significantly broader spectrum of antibodies against various clades of HPAI H5 viruses than monovalent VLPs both in chickens and ducks. While the chimeric VLP led to broadened antibody responses in both species, significantly lower levels of HI antibodies were elicited in ducks than in chickens. Moreover, boost immunization failed to increase antibody responses in ducks regardless of the VLPs used, in contrast to chickens that showed significantly enhanced antibody responses upon boost immunization. These results suggest (1) the potential application of the chimeric VLP technology in poultry to help control HPAI H5 viruses by offering broader antibody responses against antigenically different strains and (2) possible obstacles in generating high levels of antibody responses against HPAI H5 viruses in ducks via vaccination, implying the need for advanced vaccination strategies for ducks.

Dose immunogenicity study of a plant-produced influenza virus-like particle vaccine in layer hens

Avian influenza poses one of the largest known threats to global poultry production and human health, but effective poultry vaccines can reduce infections rates, production losses and prevent mortalities, and reduce viral shed to limit further disease spread. The antigenic match between a vaccine and the circulating field influenza A viruses (IAV) is a critical determinant of vaccine efficacy. Here, an Agrobacterium tumefaciens-mediated transient tobacco plant (Nicotiana benthamiana) system was used to rapidly update an H6 influenza subtype virus-like particle (VLP) vaccine expressing the hemagglutininn (HA) protein of South African H6N2 IAVs circulating in 2020. Specific pathogen free White Leghorn layer hens vaccinated twice with ≥125 hemagglutinating unit (HAU) doses elicited protective antibody responses associated with prevention of viral shedding, i.e. hemaglutination inhibition (HI) mean geometric titres (GMTs) of ≥7 log2, for at least four months before dropping to approximately 5–6 log2 for at least another two months. A single vaccination with a 250 HAU dose induced significantly higher HI GMTs compared lower or higher doses, and was thus the optimal dose for chickens. Use of an adjuvant was essential, as the plant-produced H6 HA VLP alone did not induce protective antibody responses. Plant-produced IAV VLPs enable differentiation between vaccinated and infected animals (DIVA principle), and with sucrose density gradient-purified yields of 20,000 doses per kg of plant material, this highly efficacious, safe and economical technology holds enormous potential for improving poultry health in lower and middle-income countries.

Supplementation of H7N9 Virus-Like Particle Vaccine With Recombinant Epitope Antigen Confers Full Protection Against Antigenically Divergent H7N9 Virus in Chickens.

The continuous evolution of the H7N9 avian influenza virus suggests a potential outbreak of an H7N9 pandemic. Therefore, to prevent a potential epidemic of the H7N9 influenza virus, it is necessary to develop an effective crossprotective influenza vaccine. In this study, we developed H7N9 virus-like particles (VLPs) containing HA, NA, and M1 proteins derived from H7N9/16876 virus and a helper antigen HMN based on influenza conserved epitopes using a baculovirus expression vector system (BEVS). The results showed that the influenza VLP vaccine induced a strong HI antibody response and provided effective protection comparable with the effects of commercial inactivated H7N9 vaccines against homologous H7N9 virus challenge in chickens. Meanwhile, the H7N9 VLP vaccine induced robust crossreactive HI and neutralizing antibody titers against antigenically divergent H7N9 viruses isolated in wave 5 and conferred on chickens complete clinical protection against heterologous H7N9 virus challenge, significantly inhibiting virus shedding in chickens. Importantly, supplemented vaccination with HMN antigen can enhance Th1 immune responses; virus shedding was completely abolished in the vaccinated chickens. Our study also demonstrated that viral receptor-binding avidity should be taken into consideration in evaluating an H7N9 candidate vaccine. These studies suggested that supplementing influenza VLP vaccine with recombinant epitope antigen will be a promising strategy for the development of broad-spectrum influenza vaccines.

Multiple Neuraminidase Containing Influenza Virus-like Particle Vaccines Protect Mice from Avian and Human Influenza Virus Infection.

Avian influenza virus remains a threat for humans, and vaccines preventing both avian and human influenza virus infections are needed. Since virus-like particles (VLPs) expressing single neuraminidase (NA) subtype elicited limited heterosubtypic protection, VLPs expressing multiple NA subtypes would enhance the extent of heterosubtypic immunity. Here, we generated avian influenza VLP vaccines displaying H5 hemagglutinin (HA) antigen with or without avian NA subtypes (N1, N6, N8) in different combinations. BALB/c mice were intramuscularly immunized with the VLPs to evaluate the resulting homologous and heterosubtypic immunity upon challenge infections with the avian and human influenza viruses (A/H5N1, A/H3N2, A/H1N1). VLPs expressing H5 alone conferred homologous protection but not heterosubtypic protection, whereas VLPs co-expressing H5 and NA subtypes elicited both homologous and heterosubtypic protection against human influenza viruses in mice. We observed that VLP induced neuraminidase inhibitory activities (NAI), virus-neutralizing activity, and virus-specific antibody (IgG, IgA) responses were strongly correlated with the number of different NA subtype expressions on the VLPs. VLPs expressing all 3 NA subtypes resulted in the highest protection, indicated by the lowest lung titer, negligible body weight changes, and survival in immunized mice. These results suggest that expressing multiple neuraminidases in avian HA VLPs is a promising approach for developing a universal influenza A vaccine against avian and human influenza virus infections.

Development and fit-for-purpose verification of an LC-MS method for quantitation of hemagglutinin and neuraminidase proteins in influenza virus-like particle vaccine candidates

Recombinant influenza Virus-Like Particle (VLP) vaccines are promising vaccine candidates to prevent influenza, contain two major viral antigenic glycoproteins, Hemagglutinin (HA) and Neuraminidase (NA), on the surface of recombinant VLPs. Accurate quantitation of the mass of these antigenic proteins is important to ensure the product quality and proper dosing. Currently, Single Radial Immunodiffusion (SRID) is a recognized assay for determination of the HA immuno-reactive concentration (potency) in vaccine products, based on immuno-reactivity of HA with strain-specific antisera. The SRID assay, however, requires availability of strain-specific and properly calibrated reagents, which can be time-consuming to generate and calibrate. In addition, the assay is not suitable for quantitation of low abundant proteins, such as NA. In order to accelerate the overall production cycle, we have developed and optimized a high-resolution (HR) LC-MS method for absolute quantitation of both HA and NA protein concentrations in influenza VLP vaccine candidates. In this work, we present the method development, optimization and verification of its suitability for the intended purpose, as a prerequisite for its potential application in Quality Control, by assessing specificity, precision and accuracy, detection characteristics, and dynamic linear range. The method can be also used for other HA/NA containing preparations including in-process samples, purified proteins, whole virus preparations, nano-particle and egg-based vaccine preparations, or for calibration of SRID reference antigens.

Vaccine Efficacy Induced by 2009 Pandemic H1N1 Virus-Like Particles Differs from that Induced by Split Influenza Virus

ABSTRACT Influenza virus-like particles (VLPs) vaccines are highly immunogenic, showing strong protective efficacy against homologous virus infection compared to split vaccine. However, a comparative efficacy study against heterosubtypic virus infection between VLPs and split vaccine has yet to been reported. In this study, we generated VLPs vaccine containing hemagglutinin (HA) and matrix protein (M1) of the 2009 pandemic H1N1, and investigated the protective efficacies induced by VLPs vaccine and commercial monovalent H1N1 pandemic split vaccine from Sanofi-Pasteur. Mice were intramuscularly immunized with either VLPs vaccine or split vaccine and subsequently challenge-infected with homologous virus (A/California/04/2009, H1N1) or heterosubtypic virus (A/Philippines/82, H3N2) after 4.5 months. VLPs vaccination demonstrated a higher level of protective efficacy against homologous viruses compared to split vaccine, as lessened lung viral loads and minuscule levels of proinflammatory lung cytokines IFN-gamma and IL-6 were observed. Protective efficacies were close to non-existent in VLP-immunized mice challenged with heterosubtypic viruses (H3N2). In contrast, split vaccine showed lower vaccine efficacy against homologous virus than VLP vaccine, but conferred better protection against heterosubtypic viruses through lung viral loads reduction and heightened survival rate. These results indicate that influenza VLPs provide better protective efficacy against homologous virus challenge infection, whereas split vaccine shows better protective efficacy against heterosubtypic virus challenge. Findings from the current study contribute to the rational design of vaccines conferring a broad range of protection.

Process development for pandemic influenza VLP vaccine production using a baculovirus expression system

BackgroundInfluenza viruses cause hundreds of thousands of respiratory diseases worldwide each year, and vaccination is considered the most effective approach for preventing influenza annual epidemics or pandemics. Since 1950, chicken embryonated eggs have been used as the main method for producing seasonal influenza vaccines. However, this platform has the main drawback of a lack of scale-up flexibility, and thus, egg-based vaccine manufacturers cannot supply sufficient doses within a short period for use for pandemic prevention. As a result, strategies for reducing the manufacturing time and increasing production capacity are urgently needed. Non-virion vaccine methods have been considered an alternative strategy against an influenza pandemic, and the purpose of maintaining an immunogenic capsule structure with infectious properties appears to be met by the virus-like particle (VLP) platform.ResultsAn influenza H7N9-TW VLP production platform using insect cells, which included the expression of hemagglutinin (HA), NA, and M1 proteins, was established. To scale up H7N9-TW VLP production, several culture conditions were optimized to obtain a higher production yield. A high level of dissolved oxygen (DO) could be critical to H7N9-TW VLP production. If the DO was maintained at a high level, the HA titer obtained in the spinner flask system with ventilation was similar to that obtained in a shake flask. In this study, the HA titer in a 5-L bioreactor with a well-controlled DO level was substantially improved by 128-fold (from 4 HA units (HAU)/50 μL to 512 HAU/50 μL).ConclusionsIn this study, a multigene expression platform and an effective upstream process were developed. Notably, a high H7N9-TW VLP yield was achieved using a two-step production strategy while a high DO level was maintained. The upstream process, which resulted in high VLP titers, could be further used for large-scale influenza VLP vaccine production.

Highly immunogenic influenza virus-like particles containing B-cell-activating factor (BAFF) for multi-subtype vaccine development

Virus-like particle (VLP) technology is an attractive platform for the development of seasonal and pandemic influenza vaccines. Influenza VLPs can be obtained by the overexpression of HA, M1, NA, and/or M2 viral proteins in insect, mammalian, or plant cells. In this study, we reported to obtain highly immunogenic influenza VLPs by molecular incorporation with B-cell-activating factor (BAFF) or proliferation-inducing ligand (APRIL). Since BAFF and APRIL act as homotrimers to interact with their receptors, we engineered the VLPs by direct fusion of BAFF or APRIL to the transmembrane anchored domain of H5HA gene. Results showed that immunizations with the HA-transmembrane anchored BAFF- or APRIL-VLPs only formulated in alum but not MPL adjuvant elicited significantly higher IgG titers in sera. However, only the BAFF-VLPs formulated in alum adjuvant elicited more broadly neutralizing antibodies against the homologous and two heterologous H5N1 clade/subclade viruses and conferred protective immunity against live virus challenges. As the multi-subtype influenza vaccines containing a variety of HA subtypes can confer broader protective immunity, we also obtained multi-subtype H5H7 BAFF-VLPs and H1H5H7 BAFF-VLPs and demonstrated that these multi-subtype BAFF-VLPs were able to induce the production of neutralizing antibodies against multiple HA subtypes. Our findings provided useful information for the development of highly immunogenic, multi-subtype influenza VLP vaccines.

Intramuscular and intranasal immunization with an H7N9 influenza virus-like particle vaccine protects mice against lethal influenza virus challenge

The H7N9 influenza virus epidemic has been associated with a high mortality rate in China. Therefore, to prevent the H7N9 virus from causing further damage, developing a safe and effective vaccine is necessary. In this study, a vaccine candidate consisting of virus-like particles (VLPs) based on H7N9 A/Shanghai/2/2013 and containing hemagglutinin (HA), neuraminidase (NA), and matrix protein (M1) was successfully produced using a baculovirus (BV) expression system. Immunization experiments showed that strong humoral and cellular immune responses could be induced by the developed VLPs when administered via either the intramuscular (IM) or intranasal (IN) immunization routes. Notably, VLPs administered via both immunization routes provided 100% protection against lethal infection caused by the H7N9 virus. The IN immunization with 40μg of H7N9 VLPs induced strong lung IgA and lung tissue resident memory (TRM) cell-mediated local immune responses. These results provide evidence for the development of an effective preventive vaccine against the H7N9 virus based on VLPs administered through both the IM and IN immunization routes.

Blockage of regulatory T cells augments induction of protective immune responses by influenza virus-like particles in aged mice

Elderly humans over 65 years old are at great risk to pathogenesis by influenza virus infection. However, although influenza vaccines provide effective protection in healthy young adults, protection of elderly adults is substantially lower even with a good match between the vaccine and the circulating influenza virus. To gain insight of the underlying mechanism for the reduced immunogenicity of influenza vaccines in the aged population, we investigated immunogenicity of influenza virus-like particle vaccines in aged mice, which represent a useful model for studying aging associated impairment in immune responses. Specifically, we investigated the effect of inhibiting regulatory T cells in aged mice on induction of protective immune responses by influenza vaccines. Our results showed that injecting anti-CD25 antibodies could down-regulate CD25 on the surface of regulatory T cells and significantly increase the levels of antibody responses induced by VLP immunization in aged mice. Further, the profiles of antibody responses were also changed towards Th1 type by regulatory T cell blockage in aged mice. Moreover, aged mice that were treated by anti-CD25 antibodies prior to vaccination were more effectively protected against lethal influenza virus challenge.

Neuraminidase-based recombinant virus-like particles protect against lethal avian influenza A(H5N1) virus infection in ferrets

Avian influenza A (H5N1) viruses represent a growing threat for an influenza pandemic. The presence of widespread avian influenza virus infections further emphasizes the need for vaccine strategies for control of pre-pandemic H5N1 and other avian influenza subtypes. Influenza neuraminidase (NA) vaccines represent a potential strategy for improving vaccines against avian influenza H5N1 viruses. To evaluate a strategy for NA vaccination, we generated a recombinant influenza virus-like particle (VLP) vaccine comprised of the NA protein of A/Indonesia/05/2005 (H5N1) virus. Ferrets vaccinated with influenza N1 NA VLPs elicited high-titer serum NA-inhibition (NI) antibody titers and were protected from lethal challenge with A/Indonesia/05/2005 virus. Moreover, N1-immune ferrets shed less infectious virus than similarly challenged control animals. In contrast, ferrets administered control N2 NA VLPs were not protected against H5N1 virus challenge. These results provide support for continued development of NA-based vaccines against influenza H5N1 viruses.

Preparation and immunogenicity of influenza virus-like particles using nitrocellulose membrane filtration.

PurposeNitrocellulose membrane–based filtration system (NCFS) is widely used for protein concentration. In this study, we applied NCFS for production of virus-like particle (VLP) as a vaccine candidate and evaluated yield property and immunogenicity.Materials and MethodsInfluenza VLPs were generated by baculovirus-insect cell protein expression system. NCFS and sucrose gradient ultracentrifugation were used for purification of VLP. Immunogenicity of VLP was evaluated by animal experiment.ResultsInfluenza VLPs expressing hemagglutinin (HA) and neuraminidase proteins derived from highly pathogenic influenza virus (H5N8) were effectively produced and purified by NCFS. HA activity of VLP which correlated with antigenicity was well conserved during multiple purification steps. This NCFS based purified VLPs induced influenza virus–specific antibody responses.ConclusionOur results indicate that the influenza VLP vaccine could be prepared by NCFS without loss of immunogenicity and elicit antigen-specific immune responses.

Intranasal Immunization with Influenza Virus-Like Particles Containing Membrane-Anchored Cholera Toxin B or Ricin Toxin B Enhances Adaptive Immune Responses and Protection against an Antigenically Distinct Virus.

Vaccination is the most effective means to prevent influenza virus infection, although current approaches are associated with suboptimal efficacy. Here, we generated virus-like particles (VLPs) composed of the hemagglutinin (HA), neuraminidase (NA) and matrix protein (M1) of A/Changchun/01/2009 (H1N1) with or without either membrane-anchored cholera toxin B (CTB) or ricin toxin B (RTB) as molecular adjuvants. The intranasal immunization of mice with VLPs containing membrane-anchored CTB or RTB elicited stronger humoral and cellular immune responses when compared to mice immunized with VLPs alone. Administration of VLPs containing CTB or RTB significantly enhanced virus-specific systemic and mucosal antibody responses, hemagglutination inhibiting antibody titers, virus neutralizing antibody titers, and the frequency of virus-specific IFN-γ and IL-4 secreting splenocytes. VLPs with and without CTB or RTB conferred complete protection against lethal challenge with a mouse-adapted homologous virus. When challenged with an antigenically distinct H1N1 virus, all mice immunized with VLPs containing CTB or RTB survived whereas mice immunized with VLPs alone showed only partial protection (80% survival). Our results suggest that membrane-anchored CTB and RTB possess strong adjuvant properties when incorporated into an intranasally-delivered influenza VLP vaccine. Chimeric influenza VLPs containing CTB or RTB may represent promising vaccine candidates for improved immunological protection against homologous and antigenically distinct influenza viruses.

Construction of a recombinant baculovirus expressing the hemaglutimin (ha) of an A/H5N1 clade 2.3.3.1b virus

Highly pathogenic avian influenza A (H5N1) virus is still a threat to public health, especially, the emergence of a new clade recently with more virulent and resistant to the vaccine developed from the previously circulating clade has raised the need for development of new vaccines. With the ultimate aim of producing influenza virus-like particles (VLPs) vaccine, in this study we created recombinant baculovirus expressing Hemagglutini (HA) from H5N1 virus clade 2.3.2.1b isolated in Quang Ngai province (DkQN37/11) – which is resistant to NIBRG14 (clade 1) currently used in Vietnam. The HA - encoding gene was amplified by PCR with a specific pair of primers, which has Kozak sequence, initation codon and BamHI site at the 5’ of the forward primer and the recognition sequence of HindIII at the 5' of the reverse primer. The PCR product was cloned into pCR2.1 and sequenced then inserted into the baculovirus vector (pBluBac4.5 / V5-His-TOPO, Invitrogen) at BamHI and HindIII sites to yield pBuBacHA. Vector pBluBacHA then co-transfected with baculovirus DNA into insect cells (Sf9) to generate recombinant baculovirus. The presence of ha gene and the expression of recombinant HA protein in the recombinant virus were detemined by PCR and Western blot using anti-HA antibody. PCR and Western blot results confirmed that we have successfully created a recombinant baculovirus expressing surface antigens of influenza A/H5N1 clade 2.3.2.1b. Our work provides essential material for further study to develop A/H5N1 VLP vaccine in Vietnam.

Characterization of Humoral Responses Induced by an H7N9 Influenza Virus-Like Particle Vaccine in BALB/C Mice.

In April 2013, human infections with a novel avian influenza (H7N9) virus emerged in China. It has caused serious concerns for public health throughout the world. However, there is presently no effective treatment, and an A (H7N9) H7 subtype influenza vaccine is not available. Vaccination with virus-like particles (VLPs) has showed considerable promise for many other subtype influenza viruses. To produce H7N9 VLPs, full length, unmodified hemagglutinin (HA), neuraminidase (NA), and matrix1 (M1) genes from the A/Wuxi/1/2013(H7N9) were cloned into a pCDNA5.1 FRT vector. By co-transfection, VLPs containing HA, NA, and M1 were secreted by 293T cells. VLPs were purified by ultracentrifugation and injected into mice by the intramuscular route. In animal experiments, humoral and cellular immunoresponse were all triggered by H7N9 VLPs. High levels of specific antibodies and the isotypes of IgG were detected by ELISA. Anamnestic cellular immune responses were examined by detecting specific cytotoxic T cell for IFN-γ production in ELISPOT assay. The hemagglutination-inhibition (HAI) against the homologous virus was more than 1:64, and cross-reactive HAI titers against the heterologous virus (H1N1 and H3N2) were more than 1:16. Moreover, VLPs immunized mice showed a rapid increase of neutralizing antibodies, with neutralizing antibody titers more than 1:8, which increased four-fold against PBS immunized mice in week four. By week six, the mice had high neutralization ability against the given strain and held a potent homologous virus neutralizing capacity. Thus, VLPs represent a potential strategy for the development of a safe and effective vaccine against novel avian influenza (H7N9) virus.

Generation and characterization of a trackable plant-made influenza H5 virus-like particle (VLP) containing enhanced green fluorescent protein (eGFP).

Medicago, Inc. has developed an efficient virus-like particle (VLP) vaccine production platform using the Nicotiana benthamiana expression system, and currently has influenza-based products targeting seasonal/pandemic hemagglutinin (HA) proteins in advanced clinical trials. We wished to generate a trackable HA-based VLP that would allow us to study both particle assembly in plants and VLP interactions within the mammalian immune system. To this end, a fusion protein was designed, composed of H5 (from influenza A/Indonesia/05/2005 [H5N1]) with enhanced green fluorescent protein (eGFP). Expression of H5-eGFP in N. benthamiana produced brightly fluorescent ∼160 nm particles resembling H5-VLPs. H5-eGFP-VLPs elicited anti-H5 serologic responses in mice comparable to those elicited by H5-VLPs in almost all assays tested (hemagglutination inhibition/IgG(total)/IgG1/IgG2b/IgG2a:IgG1 ratio), as well as a superior anti-GFP IgG response (mean optical density = 2.52 ± 0.16 sem) to that elicited by soluble GFP (mean optical density = 0.12 ± 0.06 sem). Confocal imaging of N. benthamiana cells expressing H5-eGFP displayed large fluorescent accumulations at the cell periphery, and draining lymph nodes from mice given H5-eGFP-VLPs via footpad injection demonstrated bright fluorescence shortly after administration (10 min), providing proof of concept that the H5-eGFP-protein/VLPs could be used to monitor both VLP assembly and immune trafficking. Given these findings, this novel fluorescent reagent will be a powerful tool to gain further fundamental insight into the biology of influenza VLP vaccines.

H5N1 influenza virus-like particle vaccine protects mice from heterologous virus challenge better than whole inactivated virus

The highly pathogenic avian influenza (HPAI) H5N1 virus has become highly enzootic since 2003 and has dynamically evolved to undergo substantial evolution. Clades 2.3.2.1 and 2.3.4 have become the most dominant lineage in recent years, and H5N8 avian influenza outbreaks have been reported Asia. The current approach to generate influenza virus vaccines uses embryonated chicken eggs for large-scale production, although such vaccines have been poorly immunogenic to heterologous virus challenge. In the current study, virus-like particles (VLP) based on A/meerkat/Shanghai/SH-1/2012 (clade 2.3.2.1) and comprising hemagglutinin (HA), neuraminidase (NA), and matrix (M1) were produced using a baculovirus expression system to develop effective protection for different H5 HPAI clade challenges. Mice immunized with VLP demonstrated stronger humoral and cellular immune responses than mice immunized with whole influenza virus (WIV), with 20-fold higher IgG antibody titers against A/meerkat/Shanghai/SH-1/2012 after boost. Notably, the WIV vaccine group showed partial protection (80% survival) to homologous challenge, little protection (40% survival) to heterologous challenge, and 20% survival to H5N8 challenge, whereas all mice in the VLP+CFA group survived. These results provide insight for the development of effective prophylactic vaccines based on VLPs with cross-clade protection for the control of current H5 HPAI outbreaks in humans.