Efficacy Between Vaccines of Different Technologies with the Same Hemagglutinin and Different Neuraminidase in Avian Influenza Virus Infections

Avian influenza virus (AIV) belongs to Orthomyxoviridae family and is characterised by containing negative-sense, single-stranded RNA genome of 8 segments that encode for 10 structural and at least 9 nonstructural/regulatory proteins. Hemagglutinin (HA) and neuraminidase (NA) that are the most abundant and immunogenic surface protein, and are involved in virus entry and exit from the cell. There are 18 hemagglutinin and 11 neuraminidase subtypes that are known to exist in the nature, and all but H17N10 and H18N11 subtypes circulate in wild aquatic birds which are the largest known natural reservoir of AIVs. Out of 144 possible HA-NA combinations, over 120 were reported to exist in non-bat AIVs in nature. Although many combinations are possible, only limited numbers are prevalent so far.

The main role of hemagglutinin is to attach the virions to the terminal sialic acid (SA) residues on glycoproteins/glycolipids and initiate the infectious cycle, while NA cleaves terminal sialic acids, releasing virions to complete the infectious cycle. Attachment of single HA trimer to SA is low, however, multivalent binding of multiple HA trimers can increase the avidity up to 106 making the attachment almost irreversible if mitigating factors such as blocking antibodies (Ab) are absent. Neuraminidase is not needed to initiate infection. However, it plays a role in completing the infectious cycle by releasing the virions from the cells surface (preventing aggregation of virions) via HA desialylation and possibly also virion glycolipids, facilitating viral engagement of target cells. The NA is therefore the main target of antiviral drugs for the treatment of influenza virus infections in human (e.g., oseltamivir, zanamivir, peramivir, etc.).

Vaccines are an essential tool for the control of influenza virus infection in domestic animals, including poultry to prevent and control highly pathogenic avian influenza virus (HPAIV) infection in domestic birds. However, only limited number of countries authorised the use of vaccination against AIVs. China is the leader, followed by Egypt, Indonesia and Vietnam. In China, trivalent vaccine has been updated recently to include RE-13 (H5N6 2.3.4.4 h virus), RE-14 (H5N8 2.3.4.4b virus) and RE4 (H7N9 virus). A multivalent vaccine based on H9N2, H5N1 and H7N3, and clades 2.1.3 and 2.3.2.1 seed strains, have been developed in Pakistan and Indonesia, respectively. Recently due to ongoing H5Nx AIV outbreak, European Union approved Nobilis Influenza H5N2 and Poulvac FluFend H5N3 RG vaccines to use in certain types of poultry, Nobilis Influenza H5N2 can be used in chickens whereas Poulvac FluFend H5N3 RG in chickens and Pekin ducks. In USA, there are different H5Nx-based licensed vaccines (killed, DNA, and polyvalent) against AIVs available on the market (as of February 2023). Both, hemagglutinin, and neuraminidase are targeted during the vaccine formulation. Although a role for NA-mediated immunity has been known for some time, protection from influenza virus infection has predominantly been associated with antibody responses to the HA glycoprotein. Along with conventional inactivated/killed vaccines, a new technology for vaccine development has been applied to catch up with a worldwide demand. This includes recombinant vectored-live vaccines (such as HVT-AI), reverse engineered (eg. rg H5N3), RNA particles (eg. alphavirus RPH5), plasmid DNA vaccines, virus-like particles - VLPs (mono- or polyvalent), or mRNA-based influenza vaccines (especially in human). We have previously tested three different vaccine technologies to evaluate their efficacy against 2.3.4.4 H5N2 HPAI challenge in turkeys. This includes reverse genetic clade 2.3.4.4 H5 hemagglutinin (HA) gene (rgH5), recombinant turkey herpesvirus encoding a clade 2.2. H5 HA (rHVT-AI), and recombinant replication-deficient alphavirus RNA particle vaccine encoding a clade 2.3.4.4 H5 HA (RP-H5). All vaccines offered significant protection against lethal challenge. The most-efficacious vaccine tested in this study was rgH5 which also contained an N1 and would be considered differentiate infected from vaccinated animals (DIVA) compatible. However, not all the vaccines tested in this study, provided a full protection, which is similar to our previous findings in chickens. Details will be discussed during the presentation.

Matching the vaccine to the challenge strain provides the best clinical protection and reduction in virus shedding, which is important to control disease, but the main goal of the newly generated vaccines is to induce heterologous immunity which could protect against many influenza strains. This could be potentially achieved by generation of multivalent-matching the circulating strains vaccines or by targeting “universal”, broadly conserved epitopes (by using epitope mapping) that are less affected by antigenic drift. We have previously shown that multi-clade H5 (clades: 2.3.4.4; 2.1.3; 2.2.1) VLPs might protect from H5N8 and H5N1 challenge. Recently, the NA has reemerged as an attractive antigenic target for vaccine development, especially in relation to seasonal influenza in human since it harbours broadly conserved epitopes. Vaccines engineered to express high levels of NA in vivo are important to better understand the role of NA immunity in protection against AIV. It was demonstrated in mice that vaccination with N1 VLPs from A/California/04/2009 (N1 VLP) strain induced cross-protection against antigenically different influenza viruses of H1N1, H5N1 and H3N2 subtype (Kim et al., 2019). N1 VLP-immunized mice were 100% protected against homologous A/Cal (H1N1) and heterologous rgH5N1 virus lethal challenge suggesting that neuraminidase-presenting VLP could be potentially developed as an effective cross-protective vaccine candidate (Kim et al., 2019). Furthermore, commercial incorporation of immunomodulators (TLR, cytokines, etc) in conventional vaccines and/or incorporation of genes in recombinant vectored vaccines might also enhance specificity and duration of immunity to infection.