**4. Considerations for Immune Responses and Protection**

As noted above, immunization with VLPs have shown promising results in protecting from influenza infections with H7N9 [27,70,71] (Table 2). Potentially, antibody induced by VLPs can be more effective than those of subunit vaccines containing recombinant protein antigens [72]. In addition, experimental VLP vaccines generally show higher protective rates to high-risk groups such as children and the elderly [26]. However, the full spectrum of factors affecting immunogenicity of VLPs requires additional studies. For example, it has been shown that amino acid residues of HA that are related to receptor specificity can affect the protective efficacy of H5N1 and H7N9 vaccines in mice [44]. H7N9 VLP vaccine that contained L226 (mammalian specificity) and G228 (avian specificity) in HA showed better immunogenicity and protection efficacy than VLP containing HA with either L226 + S228 or Q226 + S228. This observation indicated that specific HA residues could enhance a vaccine's protection efficacy and HA glycoproteins with both avian-type and human-type receptor specificities may produce better pandemic influenza vaccines for humans [44].

While inactivated vaccines have been shown to induce predominantly systemic humoral response, VLP vaccines stimulate both humoral and cellular immune responses. H7N9 VLPs secreted from 293T cells triggered both humoral and cellular immune responses in mice. This vaccine produced higher levels and antibody and isotypes of IgG, as well as cross-reactive HAI titer against heterologous H1N1 and H1N3 subtypes [43].

The presence of NA and potentially other IAV proteins in VLP could be beneficial. The NA was demonstrated to protect host from influenza infection [73]. Mice immunized with NA VLPs (without HA) were protected against lethal challenge of homologous A/PR/8/34 virus without any weight loss [74].

Furthermore, both the exterior and interior of VLPs can be altered to enhance their stability and immunogenicity. Various adjuvants such as alum, CpG DNA, monophosphoryl lipid A (MPL), poly IC, gardiquimod, cholera toxin (CT) can be encapsulated into VLPs by exogenous and endogenous methods [75,76]. These adjuvanted VLPs show higher levels of antibodies in both sera and mucosa. In another study, adjuvants that stimulate TLR3 or NLPR3 pathways showed higher efficiency of

influenza VLP vaccine in aged group of mice [77]. The CpG-adjuvanted intranasal immunization with an egg-derived split H7N9 vaccine offered a high level of protection against H7N9 infection in mice [78].
