Influenza A viruses (IAV-S) belonging to the H1 subtype are endemic in swine worldwide. Antigenic drift and antigenic shift lead to a substantial antigenic diversity in circulating IAV-S strains. As a result, the most commonly used vaccines based on whole inactivated viruses (WIVs) provide low protection against divergent H1 strains due to the mismatch between the vaccine virus strain and the circulating one. Here, a consensus coding sequence of the full-length of HA from H1 subtype was generated in silico after alignment of the sequences from IAV-S isolates obtained from public databases and was delivered to pigs using the Orf virus (ORFV) vector platform. The immunogenicity and protective efficacy of the resulting ORFVΔ121conH1 recombinant virus were evaluated against divergent IAV-S strains in piglets. Virus shedding after intranasal/intratracheal challenge with two IAV-S strains was assessed by real-time RT-PCR and virus titration. Viral genome copies and infectious virus load were reduced in nasal secretions of immunized animals. Flow cytometry analysis showed that the frequency of T helper/memory cells, as well as cytotoxic T lymphocytes (CTLs), were significantly higher in the peripheral blood mononuclear cells (PBMCs) of the vaccinated groups compared to unvaccinated animals when they were challenged with a pandemic strain of IAV H1N1 (CA/09). Interestingly, the percentage of T cells was higher in the bronchoalveolar lavage of vaccinated animals in relation to unvaccinated animals in the groups challenged with a H1N1 from the gamma clade (OH/07). In summary, delivery of the consensus HA from the H1 IAV-S subtype by the parapoxvirus ORFV vector decreased shedding of infectious virus and viral load of IAV-S in nasal secretions and induced cellular protective immunity against divergent influenza viruses in swine.

Seasonal influenza, causes hundreds of thousands of deaths annually, posing a severe threat to human health. Currently available influenza vaccines are targeted only at specific strains or conserved epitopes; however, these vaccines are not completely efficacious because influenza viruses can undergo mutation during circulation, leading to antigenic mismatch between recommended strains and circulating strains and elusion from the immune system. Therefore, developing an influenza vaccine that is quick, effective, and broadly protective has become crucial, and the integral part of hemagglutinin (HA) remains an ideal target for vaccine development. This study developed a lipid nanoparticle-encapsulated nucleoside-modified mRNA vaccine (mRNA-LNPs) encoding a consensus full-length HA sequence (H1c) and evaluated its protective efficacy and immunogenicity through in vitro and in vivo assays. Following two intramuscular immunizations (2, 10 µg, or 20 µg) at a 3-week interval in BALB/c mice, H1c-mRNA-LNP vaccine induced strong antibodies as shown in the hemagglutination-inhibition test and protective neutralizing antibodies against numerous heterologous H1N1 influenza viruses as shown in the microneutralization assay. Additionally, both Th1- and Th2-biased cellular immune responses were elicited, with the Th1-biased response being stronger. Two doses of the H1c-mRNA-LNP vaccine could neutralize a panel of heterologous H1N1 influenza viruses and could confer protection in mice. Taken together, these findings suggest that the H1c-mRNA-LNP vaccine encoding a consensus full-length HA is a feasible strategy for developing a cross-protective vaccine against a panel of heterologous H1N1 influenza viruses.

BACKGROUND: Local transmission of seasonal influenza viruses (IVs) can be difficult to resolve. Here, we study if coupling high-throughput sequencing (HTS) of hemagglutinin (HA) and neuraminidase (NA) genes with variant analysis can resolve strains from local transmission that have identical consensus genome. We analyzed 24 samples collected over four days in January 2020 at a large university in the US. We amplified complete hemagglutinin (HA) and neuraminidase (NA) genomic segments followed by Illumina sequencing. We identified consensus complete HA and NA segments using BLASTn and performed variant analysis on strains whose HA and NA segments were 100% similar.
RESULTS: Twelve of the 24 samples were PCR positive, and we detected complete HA and/or NA segments by de novo assembly in 83.33% (10/12) of them. Similarity and phylogenetic analysis showed that 70% (7/10) of the strains were distinct while the remaining 30% had identical consensus sequences. These three samples also had IAV and IBV co-infection. However, subsequent variant analysis showed that they had distinct variant profiles. While the IAV HA of one sample had no variant, another had a T663C mutation and another had both C1379T and C1589A.
CONCLUSIONS: In this study, we showed that HTS coupled with variant analysis of only HA and NA genes can help resolve variants that are closely related. We also provide evidence that during a short time period in the 2019-2020 season, co-infection of IAV and IBV occurred on the university campus and both 2020/2021 and 2021/2022 WHO recommended H1N1 vaccine strains were co-circulating.

The substantial genetic diversity exhibited by influenza A viruses of swine (IAV-S) represents the main challenge for the development of a broadly protective vaccine against this important pathogen. The consensus vaccine immunogen has proven an effective vaccinology approach to overcome the extraordinary genetic diversity of RNA viruses. In this project, we sought to determine if a consensus IAV-S hemagglutinin (HA) immunogen would elicit broadly protective immunity in pigs. To address this question, a consensus HA gene (designated H3-CON.1) was generated from a set of 1,112 H3 sequences of IAV-S recorded in GenBank from 2011 to 2015. The consensus HA gene and a HA gene of a naturally occurring H3N2 IAV-S strain (designated H3-TX98) were expressed using the baculovirus expression system and emulsified in an oil-in-water adjuvant to be used for vaccination. Pigs vaccinated with H3-CON.1 immunogen elicited broader levels of cross-reactive neutralizing antibodies and interferon gamma secreting cells than those vaccinated with H3-TX98 immunogen. After challenge infection with a fully infectious H3N2 IAV-S isolate, the H3-CON.1-vaccinated pigs shed significantly lower levels of virus in their nasal secretions than the H3-TX98-vaccinated pigs. Collectively, our data provide a proof-of-evidence that the consensus immunogen approach may be effectively employed to develop a broadly protective vaccine against IAV-S.

Continuous outbreaks of highly pathogenic avian influenza (HPAI) viruses in commercial poultry have caused devastating losses to domestic poultry with a raising public health concern. The outbreaks of HPAI viruses have increased worldwide, including the North America. Therefore, vaccination has been considered as an alternative strategy for an efficient control of HPAI viruses. In this study, we aimed to generate Newcastle disease virus (NDV) vectored H7 serotype-specific vaccines by expressing the consensus sequence of the HA protein. Conventional NDV strain LaSota vector and a chimeric NDV vector containing the avian paramyxovirus type-2 F and HN protein were able to express the consensus sequence of HA protein. The protective efficacy of vaccines was evaluated in broiler chickens and in turkeys. One-day-old poults were prime immunized with the chimeric vector expressing the HA protein followed by boost immunization with LaSota vector expressing the HA protein or co-expressing the HA and NA proteins. Our vaccine candidates provided complete protection of broiler chickens from mortality and shedding of H7N8 HPAI challenge virus. Turkeys were better protected by boosting with the LaSota vector co-expressing the HA and NA proteins than the LaSota vector expressing only the HA protein. Our study demonstrated a potential use of heterologous prime and boost vaccination strategy to protect poultry against H7 HPAI viruses.

Hemagglutinin (HA) head has long been considered to be able to elicit only a narrow, strain-specific antibody response as it undergoes rapid antigenic drift. However, we previously showed that a heterologous prime-boost strategy, in which mice were primed twice with DNA encoding HA and boosted once with virus-like particles (VLP) from an H5N1 strain A/Thailand/1(KAN)-1/2004 (noted as TH DDV), induced anti-head broad cross-H5 neutralizing antibody response. To explain why TH DDV immunization could generate such breadth, we systemically compared the neutralization breadth and potency between TH DDV sera and immune sera elicited by TH DDD (three times of DNA immunizations), TH VVV (three times of VLP immunizations), TH DV (one DNA prime plus one VLP boost) and TK DDV (plasmid DNA and VLP derived from another H5N1 strain, A/Turkey/65596/2006). Then we determined the antigenic sites (AS) on TH HA head and the key residues of the main antigenic site. Through the comparison of different regiments, we found that the combination of the immunization with the sequence close to the consensus sequence and two DNA prime plus one VLP boost caused that TH DDV immunization generate broad neutralizing antibodies. Antigenic analysis showed that TH DDV, TH DV, TH DDD and TH VVV sera recognize the common antigenic site AS1. Antibodies directed to AS1 contribute to the largest proportion of the neutralizing activity of these immune sera. Residues 188 and 193 in AS1 are the key residues which are responsible for neutralization breadth of the immune sera. Interestingly, residues 188 and 193 locate in classical antigen sites but are relatively conserved among the 16 tested strains and 1,663 HA sequences from NCBI database. Thus, our results strongly indicate that it is feasible to develop broad cross-H5 influenza vaccines against HA head.

Seasonal and emerging epidemics caused by influenza virus remain as a public health concern and an economic burden. The weak immunogenicity of conserved epitopes on hemagglutinin that induces broad protective immune responses is the main obstacle to the development of universal vaccines. In the present report, we designed the cross-subtypic sequential vaccination strategy and evaluated its neutralizing antibody (nAb) activity by pseudovirus-based neutralization assays. The results clearly indicated that compared with traditional vaccines strategy, the cross-subtypic sequential immunization could significantly induce a broad serum cross-reactive nAb response in mice as well as against homologous strains, and provide protection from heterologous virus PR8 (H1N1) challenge. Furthermore, we isolated two monoclonal antibodies from sequentially immunized mice, which had potent broadly neutralizing activity against multiple influenza strains. These data suggest the feasibility of sequential immunization in universal flu vaccine development.

With the exception of the live attenuated influenza vaccine there have been no substantial changes in influenza vaccine strategies since the 1940\'s. Here we report an alternative vaccine approach that uses Adenovirus-vectored centralized hemagglutinin (HA) genes as vaccine antigens. Consensus H1-Con, H3-Con and H5-Con HA genes were computationally derived. Mice were immunized with Ad vaccines expressing the centralized genes individually. Groups of mice were vaccinated with 1 X 1010, 5 X 107 and 1 X 107 virus particles per mouse to represent high, intermediate and low doses, respectively. 100% of the mice that were vaccinated with the high dose vaccine were protected from heterologous lethal challenges within each subtype. In addition to 100% survival, there were no signs of weight loss and disease in 7 out of 8 groups of high dose vaccinated mice. Lower doses of vaccine showed a reduction of protection in a dose-dependent manner. However, even the lowest dose of vaccine provided significant levels of protection against the divergent influenza strains, especially considering the stringency of the challenge virus. In addition, we found that all doses of H5-Con vaccine were capable of providing complete protection against mortality when challenged with lethal doses of all 3 H5N1 influenza strains. This data demonstrates that centralized H1-Con, H3-Con and H5-Con genes can be effectively used to completely protect mice against many diverse strains of influenza. Therefore, we believe that these Ad-vectored centralized genes could be easily translated into new human vaccines.

BACKGROUND: Pigs have been implicated as mixing reservoir for the generation of new pandemic influenza strains, control of swine influenza has both veterinary and public health significance. Unlike human influenza vaccines, strains used for commercially available swine influenza vaccines are not regularly replaced, making the vaccines provide limited protection against antigenically diverse viruses. It is therefore necessary to develop broadly protective swine influenza vaccines that are efficacious to both homologous and heterologous virus infections. In this study, two forms of DNA vaccines were constructed, one was made by fusing M2e to consensus H3HA (MHa), which represents the majority of the HA sequences of H3N2 swine influenza viruses. Another was made by fusing M2e and a conserved CTL epitope (NP147-155) to consensus H3HA (MNHa). Their protective efficacies against homologous and heterologous challenges were tested.
RESULTS: BALB/c mice were immunized twice by particle-mediated epidermal delivery (gene gun) with the two DNA vaccines. It was shown that the two vaccines elicited substantial antibody responses, and MNHa induced more significant T cell-mediated immune response than MHa did. Then two H3N2 strains representative of different evolutional and antigenic clusters were used to challenge the vaccine-immunized mice (homosubtypic challenge). Results indicated that both of the DNA vaccines prevented homosubtypic virus infections completely. The vaccines\' heterologous protective efficacies were further tested by challenging with a H1N1 swine influenza virus and a reassortant 2009 pandemic strain. It was found that MNHa reduced the lung viral titers significantly in both challenge groups, histopathological observation showed obvious reduction of lung pathogenesis as compared to MHa and control groups.
CONCLUSIONS: The combined utility of the consensus HA and the conserved M2e and CTL epitope can confer complete and partial protection against homologous and heterologous challenges, respectively, in mouse model. This may provide a basis for the development of universal swine influenza vaccines.

H5N1 influenza viruses have spread extensively among wild birds and domestic poultry. Cross-species transmission of these viruses to humans has been documented in over 380 cases, with a mortality rate of approximately 60%. There is great concern that a H5N1 virus would acquire the ability to spread efficiently between humans, thereby becoming a pandemic threat. An H5N1 influenza vaccine must, therefore, be an integral part of any pandemic preparedness plan. However, traditional methods of making influenza vaccines have yet to produce a candidate that could induce potently neutralizing antibodies against divergent strains of H5N1 influenza viruses. To address this need, we generated a consensus H5N1 hemagglutinin (HA) sequence based on data available in early 2006. This sequence was then optimized for protein expression before being inserted into a DNA plasmid (pCHA5). Immunizing mice with pCHA5, delivered intramuscularly via electroporation, elicited antibodies that neutralized a panel of virions that have been pseudotyped with the HA from various H5N1 viruses (clades 1, 2.1, 2.2, 2.3.2, and 2.3.4). Moreover, immunization with pCHA5 in mice conferred complete (clades 1 and 2.2) or significant (clade 2.1) protection from H5N1 virus challenges. We conclude that this vaccine, based on a consensus HA, could induce broad protection against divergent H5N1 influenza viruses and thus warrants further study.