Sequential Immunizations with heterosubtypic virus-like particles elicit cross protection against divergent influenza A viruses in mice

Abstract

Seasonal influenza vaccines have proven to be effective against well-matched viruses in healthy adults. However, rapid accumulation of mutations in the main antigenic surface proteins of influenza can compromise the efficiency of flu vaccines. Occasionally, influenza pandemics arise and present a different type of challenge to current seasonal vaccines. Novel vaccination strategies that can educate the host immune system to generate immune responses focusing on conserved epitopes on theses antigenic surface proteins are crucial for controlling and limiting influenza epidemics and pandemics. In this study, we have sequentially vaccinated mice with heterosubtypic influenza HA virus-like particles (VLPs) harboring H1, H8, and H13 from the HA phylogenetic group 1, or H3, H4, and H10 from the HA phylogenetic group 2, or in various combinations. The immunized animals were fully protected when challenged with lethal doses of heterosubtypic viruses from either phylogenetic group. Our vaccination approach demonstrates a promising strategy for the development of a 'universal influenza vaccine'.

Figure 1

Characterization of purified VLPs. Figure represents Western blot of (A) HA and (B) M1 proteins in the different VLPs. Total HA and M1 contents in the prepared VLPs were analyzed by SDS-PAGE followed by Western blot using mouse anti-influenza HA and anti-M1 antibodies, respectively, (C) Negative staining electron micrographs of different influenza HA VLPs. Bars represent ∼100 nm.

Figure 2

rVet and rSH- specific serum IgG/IgG subtype endpoint titers and intracellular ELISA. Endpoint titers are expressed as the highest dilution of serum having a mean OD₄₅₀ greater than the mean plus 2 standard deviation of similarly diluted naïve serum samples. For intracellular ELISA, MDCK cells were exposed to Cal09 or Phi82 virions, used as antigens in the ELISA. After permeabilization, MDCK cells were incubated with diluted sera, followed by detection with HRP-conjugated anti-mouse IgG antibody. Figure represents (i) rVet-specific (A) IgG, (B) IgG1, (C) IgG2a, and (D) IgG2b; (ii) rSH-specific (E) IgG, (F) IgG1, (G) IgG2a, and (H) IgG2b; and (iii) (I) Cal09-specific and (J) Phi82-specific IgG levels in sera. Results are represented as the mean ± SD (n = 5). (*P < 0.05, **P < 0.01, ***P < 0.001, ns-non-significant).

Figure 3

Mucosal responses in nasal, tracheal, and lung washes. After the 3^rd immunization, (A) nasal wash, (B) tracheal wash, and (C) lung wash were collected and total antigen-specific IgA amount were determined by ELISA using rVet and rSH viruses as coating antigens. Results represent the mean ± SD (n = 5). (*P < 0.05, **P < 0.01, ns-non-significant).

Figure 4

Serum HAI titers. Mean HAI titers of serum IgG was measured as described in materials and methods using MDCK cells. Results are represented as the mean ± SD (n = 5). (*P < 0.05).

Figure 5

Estimation of cytokine levels. Splenocytes were isolated from immunized mice 3 week after the final immunization. Cells (1 × 10⁶) were seeded into 96-well culture plates. The inactivated rVet or rSH viruses were added into each well and secreted (A) IL-2, (B) IFN-γ, (C) IL-4, and (D) TNF-α levels were determined as described in materials and methods. Results are represented as the mean ± SD (n = 5). (*P < 0.05, **P < 0.01).

Figure 6

Post inflammatory response. The levels of inflammatory cytokines; (A,B) IL-6, and (C,D) IFN-γ were evaluated in the lung samples after the virus challenges. Lung samples were prepared on day 4 post-challenge and the cytokines were estimated by ELISA. Results are represented as the mean ± SD (n = 5). (*P < 0.05, **P < 0.01).

Figure 7

Challenges study. Vaccinated animals were challenged with 10x LD50 mouse-adapted viruses. Mice were monitored daily for 14 days for body weight changes and their survival. Figures show (i) the survival of vaccinated animals challenged with (A) Cal09 (H1N1), (B) Phi82 (H3N2), (C) rVet (H5N1), or (D) rSH (H7N9) viruses; and (ii) body weight changes in the vaccinated animals challenged with (E) Cal09, (F) Phi82, (G) rVet, or (H) rSH viruses.