**6. Broadly Protective Influenza VLP Vaccines**

Current annual vaccines (inactivated, live attenuated, and recombinant subunit) protect from circulating, antigenically matching IAV and influenza B virus strains. Broad protection is difficult to achieve because of the frequent emergence of new strains [83]. Therefore, current approved and experimental vaccines have limited protection against antigenically mismatched variants and newly emerging viral strains. For new strains, antigenically matching new vaccines have to be developed within short periods of time. Therefore, vaccines are needed that can protect from an antigenically divergent strain, especially from potentially pandemic viruses with a new HA subtype. In addition to H7N9 virus described above, multiple other viruses with pandemic potential, including H5N1, H9N2, and H10N8 subtypes, that have caused human infections in the past, continue to circulate in birds and other animals. Therefore, vaccines capable of protecting against multiple potentially pandemic influenza strains would be advantageous for pandemic preparedness and public health.

One way to achieve protection against multiple strains and increase the breadth of immune response afforded by VLPs can be a blended formulation of monovalent vaccines. A vaccine was prepared by mixing VLPs that display H1, H3, H5 or H7 HA molecules. Mice vaccinated with these VLPs intranasally showed significant protection (94% aggregate survival following vaccination) against 1918 H1, 1957 H2, and avian H5, H6, H7, H10 and H11 HA subtypes [41]. These experiments suggest a promising and practical strategy for developing a broadly protective influenza vaccine.

Potentially, HA molecules from different subtypes can be co-localized within recombinant VLP envelope for broader immune coverage. A multi-subtype, mosaic VLP design containing three or four subtypes of the full-length rHA within the envelope has also been described [50,71]. A triple-subtype VLP contained HA proteins from the potentially pandemic H5N1, H7N2, and H9N2 subtypes [50]. A recombinant baculovirus vector was prepared to co-express the H5, H7, and H9 genes from A/Viet Nam/1203/2004 (H5N1), A/New York/107/2003 (H7N2) and A/Hong Kong/33982/2009 (H9N2) viruses, respectively, as well as NA and M1 genes from A/Puerto Rico/8/1934 (H1N1) virus. VLPs were

prepared in Sf9 cells, and the immunogenicity and efficacy of the resulting H5/H7/H9 VLPs were evaluated in a ferret animal model following intranasal (i.n.) vaccination. We showed that i.n. vaccination with the H5/H7/H9 triple-subtype VLP induced immune responses and protected ferrets from experimental challenges with three subtypes of avian influenza viruses [50]. We also prepared triple-HA mosaic VLPs that co-localized A/Vietnam/1203/2004 (H5N1), A/Hong Kong/33982/2009 (H9N2) and A/Shanghai/2/2013 (H7N9) rHA proteins, as well as quadri-HA VLPs (Figure 3).

**Figure 3.** Structural proteins in mono- and quadri-subtype VLPs. (**a**) Mono-subtype H7N9 VLP with H7, N9 and M1 genes; (**b**) Mono-subtype chimeric H7N1 VLP with H7, N1, and Gag genes; (**c**) Quadri-subtype VLPs co-localizing H5, H7, H9 and H10 subtypes along with N1 and Gag within a VLP.

By using Group antigen (Gag) derived from bovine retrovirus as a VLP core in place of M1 protein, quadri-subtype VLPs were prepared, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera [84]. Quadri-subtype vaccine elicited serum antibody in ferrets to the homologous H5, H7, H9, and H10 antigens. Antiserum was also evaluated for cross-reaction with multiple clades of H5N1 virus, and cross-reactivity has been confirmed. The level of immune response suggests protection against multiple influenza subtypes, which was experimentally confirmed by challenge with H10 IAV. Ferrets were protected from challenge with H10 virus [85]. Overall, such multi-subtype, mosaic VLPs that co-localize distinct HA subtypes in the envelope showed broader protection range against different influenza viruses [47,85,86]. Multi-subtype mosaic VLPs combine advantages of conserved HA epitope and blended VLPs, as VLPs contain both subtype-specific head epitopes and the conserved stem epitopes.

Recently, a VLP preparation consisting of retroviral Gag-VLPs pseudo-typed with the HA was expressed using the novel *Trichoplusia ni* (*T.ni*)-derived insect cell line *Tnms*42 and tested successfully to assess the sole contribution of anti-HA immunity in limiting post-influenza secondary *Staphylococcus aureus* bacterial infection, morbidity and mortality in a situation of a vaccine match and mismatch [87]. The results demonstrate that matched anti-HA immunity elicited by a VLP preparation may suffice to prevent morbidity and mortality caused by lethal secondary bacterial infection.

It should also be noted that cross-reactive antibodies to the H7N9 virus were also induced by recombinant viral vectors, such as Newcastle disease virus (NDV) [88–90] and parainfluenza virus PIV5 [91].

#### **7. Expression of H7N9 Influenza Epitopes Using VLP Carriers**

The emerging strains potentially can be also targeted by "universal" vaccines consisting of conserved viral proteins or epitopes, such as stem region of HA, or extracellular domain M2e of the ion channel protein M2, which are both considered capable of inducing cross-reactive immune responses. Targeting the stem region of HA for antibody production could be a promising approach to generate a

broadly protective influenza vaccine. The immune response can be elicited against both the head and stem region of HA protein. However, because of the constantly changing nature of the head region due to the antigenic drift, new vaccine candidates need to be updated frequently. Since the stem region is evolutionally more stable and more conserved among different influenza strains, vaccine candidates targeting the stem region might not need to be updated every year and could induce a broad range of protective immunity against different influenza strains [92,93]. However, the stem region is less immunogenic in the virus. Therefore, chimeric VLP approaches have been used to elicit antibodies to the stem region of HA. The long alpha-helix (LAH) region located in influenza virus hemagglutinin (HA) shows conservation among different influenza A strains, and could be used as a candidate target for influenza vaccines. The hepatitis B virus core protein (HBc) was used as a carrier for heterologous LAH epitopes to elicit effective immune responses [94]. The spatial conformation of LAH epitope cloned in the major immunodominant region (MIR) of the HBc molecule and expressed in yeast is shown on Figure 4. The LAH region of the H7N9 influenza virus was inserted into the HBc to prepare chimeric LAH-HBc protein, which was capable of self-assembly into VLPs in *E. coli* expression system [95]. Intranasal immunization of the LAH-HBc VLP in combination with chitosan adjuvant or CTB∗ adjuvant in mice induced both humoral and cellular immune responses effectively and conferred complete protection against lethal challenge with homologous H7N9 virus or heterologous H3N2 virus, as well as partial protection against lethal challenge of heterologous H1N1 virus. These results provide a proof of concept for LAH-HBc VLP vaccine that can be rapidly produced and potentially can serve as an antigen against a future influenza pandemic [95].

**Figure 4.** An example of expression of influenza epitope using chimeric VLP based on recombinant LAH3-HBc gene. Design (**a**) and cartoon (**b**) of the LAH3-HBc dimer. Individual hepatitis B virus core protein (HBc) monomers are shown in green and orange. The first major immunodominant region (MIR) contains the LAH domain (the 55 amino acid long influenza H3N2 virus (A/Hong Kong/1/1968, Accession No. AAK51718) HA stalk domain, corresponding to HA amino acids 420–474, shown as pink spheres) while the second MIR contains lysine linker (blue spheres)). The model was created using PyMOL version 1.7rc1. The tandem core dimer is based on the structure from PDB-1QGT, with the linker shown in black.

In another study, the globular head domain (HA1-2, aa 62–284) of the protective H7 HA was fused to the potent TLR5 ligand, *Salmonella typhimurium* flagellin (FliC) [96]. The resulting fusion protein, HA1-2-fliC, which apparently did not form VLPs, was efficiently expressed in *E. coli*, retained the native HA and fliC conformations, and was highly immunogenic in mice by intraperitoneal vaccination. Furthermore, highly immunogenic influenza VLPs have been prepared via the overexpression of four viral proteins, HA, NA, M1, and M2, using M2 fusion with the FliC [97]. The chimeric H5N1 VLPs were further combined with the molecular adjuvant, granulocyte-macrophage colony-stimulating

factor (GM-CSF), FliC, or a GM-CSF/FliC [98]. All three forms of the chimeric H5N1 VLPs elicited protective immunity against live virus. Next, the GM-CSF/FliC H5N1 VLPs were obtained to include H7 or H1H7 antigens for developing multi-subtype influenza vaccines [98].

The chimeric norovirus VLPs, P particles, were used to express the trivalent HA2:90-105 epitopes derived from H1, H3 and B subtypes, with 24 copies in total, on the surface loops [99].

Finally, trimeric H7 transiently expressed in *N. benthamiana* was conjugated successfully onto the surface of nanodiamond particles [100]. After two or three immunizations in mice, the mixture of trimeric H7 protein and nanodiamond elicited statistically significant stronger H7-specific-IgG response demonstrated by higher amounts of H7N9-specific IgG.

Other universal influenza vaccine approaches applicable to H7N9 were based on ion channel protein M2. Extracellular part of M2 protein, ~23 aa residues, is highly conserved among human IAV strains, suggesting its potential utility as a broadly protective immunogen for the development of broadly protective influenza vaccines. Tandem repeats of heterologous M2e sequences (M2e5x) derived from human, swine, and avian origin influenza A viruses were expressed on influenza VLPs in a membrane-anchored form. Immunization of mice with M2e5x VLPs induced antibodies that were cross-reactive to antigenically distinct influenza A viruses and conferred cross-protection [101]. Furthermore, the M2e5x VLPs demonstrated a clear advantage in inducing IgG2a isotype antibodies, T cell responses, plasma cells and germinal center B cells as well as in conferring cross protection [102]. These studies paved the way to a novel vaccination strategy by enhancing the cross protective efficacy of live attenuated influenza virus vaccines by supplemented vaccination with M2e5x VLPs [103].

High immunogenicity in mice, even in the absence of adjuvants, was demonstrated by the M2e displayed on VLPs of *Macrobrachium rosenbergii* nodavirus [104,105]. This novel vaccine candidate was tested by the H1N1 and H3N2 challenge in mice, and potentially, it can be universal and applicable to H7N9 virus. In another example, the M2e proteins, as well as NA, were expressed on recombinant *Lactococcus lactis*, which conferred effective mucosal and systemic immune responses [106].
