Influenza Virus-like Particles 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.

Immunizing Mice with Influenza Virus-like Particles Expressing the Leishmania amazonensis Promastigote Surface Antigen Alleviates Inflammation in Footpad.

Cutaneous leishmaniasis (CL) is a tropical disease endemic in many parts of the world. Characteristic clinical manifestations of CL include the formation of ulcerative skin lesions that can inflict life-long disability if left untreated. Although drugs are available, they are unaffordable and out of reach for individuals who need them the most. Developing a highly cost-efficient CL vaccine could address this problem but such a vaccine remains unavailable. Here, we developed a chimeric influenza virus-like particle expressing the Leishmania amazonensis promastigote surface antigen (LaPSA-VLP). LaPSA-VLPs were self-assembled in Spodoptera frugiperda insect cell lines using the baculovirus expression system. After characterizing the vaccines and confirming successful VLP assembly, BALB/c mice were immunized with these vaccines for efficacy assessment. Sera acquired from mice upon subcutaneous immunization with the LaPSA-VLP specifically interacted with the L. amazonensis soluble total antigens. LaPSA-VLP-immunized mice elicited significantly greater quantities of parasite-specific IgG from the spleens, popliteal lymph nodes, and footpads than unimmunized mice. LaPSA-VLP immunization also enhanced the proliferation of B cell populations in the spleens of mice and significantly lessened the CL symptoms, notably the footpad swelling and IFN-γ-mediated inflammatory response. Overall, immunizing mice with the LaPSA-VLPs prevented mice from developing severe CL symptoms, signifying their developmental potential.

Synergetic light and cytokinin treatments mitigate the recombinant protein yield depression induced by high-density cultivation of hydroponically-grown Nicotiana benthamiana.

Plant molecular farming is currently operating a transition from soil-based cultures towardhydroponic systems. In this study, we designed a whole-plant NFT (nutrient film technique) platform for the transient expression of influenza virus-like particles harboring hemagglutinin H1 proteins in Nicotiana benthamiana. In particular, we examined the effects of plant density during the post-infiltration expression phase on plant growth and H1 yield in relation to the daily light integral (DLI) received by the crop and the exogenous application of 6-BAP cytokinin (CK). We expected from previous work that high DLI and CK treatments would stimulate the development of highly productive leaves on axillary (secondary) stems and thereby improve the H1 yield at the whole-plant scale. Increasing plant density from 35.7 to 61 plants m-2 during the post-infiltration phase significantly decreased the proportion of axillary leaf biomass by 30% and H1 yield per plant by 39%, resulting in no additional yield gain on a whole-crop area basis. Adding CK to the recirculated nutrient solution decreased the harvested leaf biomass by 31% and did not enhance the relative proportion of S leaves of the plants as previously reported with foliar CK application. There was a 36% increase in H1 yield when doubling the DLI from 14 to 28 mol m-2 s-1, and up to 71% yield gain when combining such an increase in DLI with the hydroponic CK treatment. Contrary to our expectations, leaves located on the main stem, particularly those from the upper half of the plant (i.e.,eighth leaf and above), contributed about 80% of total H1 yield. Our study highlights the significantly different phenotype (~30% less secondary leaf biomass) and divergent responses to light and CK treatments of NFT-grown N. benthamiana plants compared to previous studies conducted on potted plants.

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.

Crossprotection induced by virus-like particles containing influenza dual-hemagglutinin and M2 ectodomain.

Aims: To develop an effective universal vaccine against antigenically different influenza viruses. Materials & methods: We generated influenza virus-like particles (VLPs) expressing the H1 and H3 antigens with or without M2e5x. VLP-induced immune responses and crossprotection against H1N1, H3N2 or H5N1 viruses were assessed to evaluate their protective efficacy. Results: H1H3M2e5x immunization elicited higher crossreactive IgG antibodies than H1H3 VLPs. Upon challenge, both VLPs enhanced lung IgG, IgA and germinal center B-cell responses compared with control. While these VLPs conferred protection, H1H3M2e5x showed greater lung viral load reduction than H1H3 VLPs with minimal body weight loss. Conclusion: Utilizing VLPs containing dual-hemagglutinin, along with M2e5x, can be a vaccination strategy for inducing crossprotection against influenza A viruses.

Adaptive Laboratory Evolution to Improve Recombinant Protein Production Using Insect Cells.

Adaptive laboratory evolution (ALE) is a powerful tool for enhancing the fitness of cell lines in specific applications, including recombinant protein production. Through adaptation to nonstandard culture conditions, cells can develop specific traits that make them high producers. Despite being widely used for microorganisms and, to lesser extent, for mammalian cells, ALE has been poorly leveraged for insect cells. Here, we describe a method for adapting insect High Five and Sf9 cells to nonstandard culture conditions via an ALE approach. Aiming to demonstrate the potential of ALE to improve productivity of insect cells, two case studies are demonstrated. In the first, we adapted insect High Five cells from their standard pH (6.2) to neutral pH (7.0); this adaptation allowed to improve production of influenza virus-like particles (VLPs) by threefold, using the transient baculovirus expression vector system. In the second, we adapted insect Sf9 cells from their standard culture temperature (27°C) to hypothermic growth (22°C); this adaptation allowed to improve production of influenza VLPs by sixfold, using stable cell lines. These examples demonstrate the potential of ALE for enhancing productivity within distinct insect cell hosts and expression systems by manipulating different culture conditions.

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.

Vaccine-induced neutralizing antibody responses to seasonal influenza virus H1N1 strains are not enhanced during subsequent pandemic H1N1 infection.

The first exposure to influenza is presumed to shape the B-cell antibody repertoire, leading to preferential enhancement of the initially formed responses during subsequent exposure to viral variants. Here, we investigated whether this principle remains applicable when there are large genetic and antigenic differences between primary and secondary influenza virus antigens. Because humans usually have a complex history of influenza virus exposure, we conducted this investigation in influenza-naive cynomolgus macaques. Two groups of six macaques were immunized four times with influenza virus-like particles (VLPs) displaying either one (monovalent) or five (pentavalent) different hemagglutinin (HA) antigens derived from seasonal H1N1 (H1N1) strains. Four weeks after the final immunization, animals were challenged with pandemic H1N1 (H1N1pdm09). Although immunization resulted in robust virus-neutralizing responses to all VLP-based vaccine strains, there were no cross-neutralization responses to H1N1pdm09, and all animals became infected. No reductions in viral load in the nose or throat were detected in either vaccine group. After infection, strong virus-neutralizing responses to H1N1pdm09 were induced. However, there were no increases in virus-neutralizing titers against four of the five H1N1 vaccine strains; and only a mild increase was observed in virus-neutralizing titer against the influenza A/Texas/36/91 vaccine strain. After H1N1pdm09 infection, both vaccine groups showed higher virus-neutralizing titers against two H1N1 strains of intermediate antigenic distance between the H1N1 vaccine strains and H1N1pdm09, compared with the naive control group. Furthermore, both vaccine groups had higher HA-stem antibodies early after infection than the control group. In conclusion, immunization with VLPs displaying HA from antigenically distinct H1N1 variants increased the breadth of the immune response during subsequent H1N1pdm09 challenge, although this phenomenon was limited to intermediate antigenic variants.

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.

A VLP-Based Vaccine Displaying HBHA and MTP Antigens of Mycobacterium tuberculosis Induces Protective Immune Responses in M. tuberculosis H37Ra Infected Mice.

Heparin-binding hemagglutinin (HBHA) and M. tuberculosis pili (MTP) are important antigens on the surface of Mycobacterium tuberculosis. To display these antigens effectively, the fusion protein HBHA-MTP with a molecular weight of 20 kD (L20) was inserted into the receptor-binding hemagglutinin (HA) fragment of influenza virus and was expressed along with matrix protein M1 in Sf9 insect cells to generate influenza virus-like particles (LV20 in short). The results showed that the insertion of L20 into the envelope of the influenza virus did not affect the self-assembly and morphology of LV20 VLPs. The expression of L20 was successfully verified by transmission electron microscopy. Importantly, it did not interfere with the immunogenicity reactivity of LV20 VLPs. We demonstrated that LV20 combined with the adjuvant composed of DDA and Poly I: C (DP) elicited significantly higher antigen-specific antibodies and CD4+/CD8+ T cell responses than PBS and BCG vaccination in mice. It suggests that the insect cell expression system is an excellent protein production system, and LV20 VLPs could be a novel tuberculosis vaccine candidate for further evaluation.

Protective mucosal and systemic immunity induced by virus-like particles expressing Toxoplasma gondii cyst wall protein.

Toxoplasma gondii host cellular invasion factors such as the rhoptry proteins, micronemal antigens, or other subcellular compartment proteins have shown limited vaccine efficacies. T. gondii cyst wall protein (CST1) as a cyst persistence factor is critical for cyst wall integrity and bradyzoite persistence. Here, we generated influenza virus-like particles (VLPs) expressing the T. gondii CST1 and evaluated the mucosal as well as systemic immunities induced by VLPs. Intranasal immunization with the VLPs induced parasite-specific IgG and IgA antibody responses in sera and intestines. VLP immunization showed higher levels of germinal center B cell response and antibody-secreting cell (ASC) response upon challenge infection, indicating memory B cell response was induced. VLP-immunized mice showed a significant reduction of cyst counts and lower levels of pro-inflammatory cytokines (IFN-γ, IL-6) production in the brain upon T. gondii ME49 challenge infection compared to unimmunized control. Thus, VLP immunization protected mice from the lethal dose challenge infection with T. gondii ME49 and did not incur bodyweight loss. These results indicated that T. gondii CST1 containing VLPs can induce mucosal and systemic immunity and also suggest its developmental potential as an effective vaccine candidate against T. gondii infection.

Gene Expression Analysis of Adapted Insect Cells during Influenza VLP Production Using RNA-Sequencing.

Adaptive laboratory evolution has been used to improve production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs) in insect cells. However, little is known about the underlying biological mechanisms promoting higher HA-VLP expression in such adapted cell lines. In this article, we present a study of gene expression patterns associated with high-producer insect High Five cells adapted to neutral pH, in comparison to non-adapted cells, during expression of influenza HA-VLPs. RNA-seq shows a decrease in the amount of reads mapping to host cell genomes along infection, and an increase in those mapping to baculovirus and transgenes. A total of 1742 host cell genes were found differentially expressed between adapted and non-adapted cells throughout infection, 474 of those being either up- or down-regulated at both time points evaluated (12 and 24 h post-infection). Interestingly, while host cell genes were found up- and down-regulated in an approximately 1:1 ratio, all differentially expressed baculovirus genes were found to be down-regulated in infected adapted cells. Pathway analysis of differentially expressed genes revealed enrichment of ribosome biosynthesis and carbohydrate, amino acid, and lipid metabolism. In addition, oxidative phosphorylation and protein folding, sorting and degradation pathways were also found to be overrepresented. These findings contribute to our knowledge of biological mechanisms of insect cells during baculovirus-mediated transient expression and will assist the identification of potential engineering targets to increase recombinant protein production in the future.

A Single Vaccination of Chimeric Bivalent Virus-Like Particle Vaccine Confers Protection Against H9N2 and H3N2 Avian Influenza in Commercial Broilers and Allows a Strategy of Differentiating Infected from Vaccinated Animals.

H9N2 and H3N2 are the two most important subtypes of low pathogenic avian influenza viruses (LPAIV) because of their ongoing threat to the global poultry industry and public health. Although commercially available inactivated H9N2 vaccines are widely used in the affected countries, endemic H9N2 avian influenza remains uncontrolled. In addition, there is no available avian H3N2 vaccine. Influenza virus-like particles (VLPs) are one of the most promising vaccine alternatives to traditional egg-based vaccines. In this study, to increase the immunogenic content of VLPs to reduce production costs, we developed chimeric bivalent VLPs (cbVLPs) co-displaying hemagglutinin (HA) and neuraminidase (NA) of H9N2 and H3N2 viruses with the Gag protein of bovine immunodeficiency virus (BIV) as the inner core using the Bac-to-Bac baculovirus expression system. The results showed that a single immunization of chickens with 40μg/0.3mL cbVLPs elicited an effective immune response and provided complete protection against H9N2 and H3N2 viruses. More importantly, cbVLPs with accompanying serological assays can successfully accomplish the strategy of differentiating infected animals from vaccinated animals (DIVA), making virus surveillance easier. Therefore, this cbVLP vaccine candidate would be a promising alternative to conventional vaccines, showing great potential for commercial development.

Influenza Virus-like Particle-Based Hybrid Vaccine Containing RBD Induces Immunity against Influenza and SARS-CoV-2 Viruses.

Several approaches have produced an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since millions of people are exposed to influenza virus and SARS-CoV-2, it is of great interest to develop a two-in-one vaccine that will be able to protect against infection of both viruses. We have developed a hybrid vaccine for SARS-CoV-2 and influenza viruses using influenza virus-like particles (VLP) incorporated by protein transfer with glycosylphosphatidylinositol (GPI)-anchored SARS-CoV-2 RBD fused to GM-CSF as an adjuvant. GPI-RBD-GM-CSF fusion protein was expressed in CHO-S cells, purified and incorporated onto influenza VLPs to develop the hybrid vaccine. Our results show that the hybrid vaccine induced a strong antibody response and protected mice from both influenza virus and mouse-adapted SARS-CoV-2 challenges, with vaccinated mice having significantly lower lung viral titers compared to naive mice. These results suggest that a hybrid vaccine strategy is a promising approach for developing multivalent vaccines to prevent influenza A and SARS-CoV-2 infections.

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.

Influenza virus-like particle-based hybrid vaccine containing RBD induces immunity against influenza and SARS-CoV-2 viruses

Abstract Several approaches have produced an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the influence of immunity against other vaccinations on the durability and efficacy of the immune response against SARS-CoV-2 is still unknown in settings where vaccines against other viruses need to be administered simultaneously. This will be an important factor in developing multivalent vaccines against seasonal viruses. We have developed a hybrid vaccine for SARS-CoV-2 and influenza viruses using influenza virus-like particles (VLP) incorporated with glycosylphosphatidylinositol (GPI)-anchored Spike RBD of SARS-CoV-2 fused to GM-CSF as an adjuvant. GPI-anchored fusion protein of GM-CSF and the SARS-CoV-2 S1 RBD was expressed in CHO-S cells, purified by immunoaffinity chromatography and incorporated onto influenza VLPs by protein transfer to make a hybrid VLP vaccine. The efficacy of the hybrid VLP vaccine was tested against both SARS-CoV-2 and influenza A/PR8 viruses in a mouse model. Our results show that the hybrid vaccine induced a strong antibody response and protected the mice from both influenza virus and mouse-adapted SARS-CoV-2 challenges, with vaccinated mice having less body weight loss and significantly lower lung viral titers compared to control mice. These results suggest that the hybrid vaccine is a promising candidate for preventing influenza A and SARS-CoV-2 infections. This work was supported by NIH/NIAID (SBIR Contract# 75N93019C00017 Amendment to Pack/Ramachandiran), and Intel Corporation for the Intel COVID-19 Global Technology Response Initiative grant.

Characterization of Immune Response towards Generation of Universal Anti-HA-Stalk Antibodies after Immunization of Broiler Hens with Triple H5N1/NA-HA-M1 VLPs.

(1) Background: Avian influenza viruses (AIVs) promptly evade preexisting immunity by constantly altering the immunodominant neutralizing antibody epitopes (antigenic drift) or by procuring new envelope serotypes (antigenic shift). As a consequence, the majority of antibodies elicited by infection or vaccination protect only against closely related strains. The immunodominance of the globular head of the main glycoprotein has been shown to mask the immunogenicity of the conserved regions located within the hemagglutinin (HA) protein. It has been shown that the broadly neutralizing universal antibodies recognize the HA2 domain in headless hemagglutinin (HA-stalk). Therefore, the HA-stalk is a highly conserved antigen, which makes it a good candidate to be used in universal vaccine development against AIVs. (2) Methods: Sf9 insect cells were used to produce triple H5N1/NA-HA-M1 influenza virus-like particles (VLPs) via co-expression of neuraminidase, hemagglutinin and matrix proteins from a tricistronic expression cassette. Purified influenza VLPs were used to immunize broiler hens. An in-depth characterization of the immune response was performed with an emphasis on the pool of elicited universal antibodies. (3) Results: Our findings suggest, that after vaccination with triple H5N1/NA-HA-M1 VLPs, hens generate a pool of broad-spectrum universal anti-HA-stalk antibodies. Furthermore, these universal antibodies are able to recognize the mammalian-derived HA-stalk recombinant proteins from homologous H5N1 and heterologous H7N9 AIVs as well as from the heterosubtypic human H1N1 influenza strain. (4) Conclusions: Our findings may suggest that highly pathogenic avian influenza H5 HA protein contain functional epitopes that are attractive targets for the generation of broad-spectrum antibodies against AIVs in their native hosts.

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.

Bioanalytics for Influenza Virus-Like Particle Characterization and Process Monitoring.

Virus-like particles (VLPs) are excellent platforms for the development of influenza vaccine candidates. Nonetheless, their characterization is challenging due to VLPs’ unique biophysical and biochemical properties. To cope with such complexity, multiple analytical techniques have been developed to date (e.g., single-particle analysis, thermal stability, or quantification assays), most of which are rarely used or have been successfully demonstrated for being applicable for virus particle characterization. In this study, several biophysical and biochemical methods have been evaluated for thorough characterization of monovalent and pentavalent influenza VLPs from diverse groups (A and B) and subtypes (H1 and H3) produced in insect cells using the baculovirus expression vector system (IC-BEVS). Particle size distribution and purity profiles were monitored during the purification process using two complementary technologies — nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS). VLP surface charge at the selected process pH was also assessed by this last technique. The morphology of the VLP (size, shape, and presence of hemagglutinin spikes) was evaluated using transmission electron microscopy. Circular dichroism was used to assess VLPs’ thermal stability. Total protein, DNA, and baculovirus content were also assessed. All VLPs analyzed exhibited similar size ranges (90–115 nm for NTA and 129–141 nm for TRPS), surface charges (average of −20.4 mV), and morphology (pleomorphic particles resembling influenza virus) exhibiting the presence of HA molecules (spikes) uniformly displayed on M1 protein scaffold. Our data shows that HA titers and purification efficiency in terms of impurity removal and thermal stability were observed to be particle dependent. This study shows robustness and generic applicability of the tools and methods evaluated, independent of VLP valency and group/subtype. Thus, they are most valuable to assist process development and enhance product characterization.

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.