Potent priming by inactivated whole influenza virus particle vaccines is linked to viral RNA uptake into antigen presenting cells

Current detergent or ether-disrupted split vaccines (SVs) for influenza do not always induce adequate immune responses, especially in young children. This contrasts with the whole virus particle vaccines (WPVs) originally used against influenza that were immunogenic in both adults and children but were replaced by SV in the 1970s due to concerns with reactogenicity. In this study, we re-evaluated the immunogenicity of WPV and SV, prepared from the same batch of purified influenza virus, in cynomolgus macaques and confirmed that WPV is superior to SV in priming potency. In addition, we compared the ability of WPV and SV to induce innate immune responses, including the maturation of dendritic cells (DCs) in vitro. WPV stimulated greater production of inflammatory cytokines and type-I interferon in immune cells from mice and macaques compared to SV. Since these innate responses are likely triggered by the activation of pattern recognition receptors (PRRs) by viral RNA, the quantity and quality of viral RNA in each vaccine were assessed. Although the quantity of viral RNA was similar in the two vaccines, the amount of viral RNA of a length that can be recognized by PRRs was over 100-fold greater in WPV than in SV. More importantly, 1000-fold more viral RNA was delivered to DCs by WPV than by SV when exposed to preparations containing the same amount of HA protein. Furthermore, WPV induced up regulation of the DC maturation marker CD86 on murine DCs, while SV did not. The present results suggest that the activation of antigen-presenting DCs, by PRR-recognizable viral RNA contained in WPV is responsible for the effective priming potency of WPV observed in naive mice and macaques. WPV is thus recommended as an alternative option for seasonal influenza vaccines, especially for children. (c) 2021 Elsevier Ltd. All rights reserved

Immunity elicited by Env and CD40Lm expressing m8 Δ prime followed by recombinant SeV boost.

<p> (A) Schematic schedules of m8Δ prime/rSeV-Env boost vaccination protocol. Mice were primed with various vaccinia viruses followed by Env expressing SeV boost. Group G: primed with m8Δ-Env plus m8Δ-p7.5hCD40Lm; group H: m8Δ-Env plus m8Δ; group I: m8Δ-Env/hCD40Lm; group J: m8Δ-Env; group K: m8Δ; group L: naive. (B) Comparison of Env peptide-specific CD8⁺ T cell responses. The percentage of IFN-γ⁺ expressing cells in gated CD8⁺ T cell compartment was measured by ICS and FACS analysis. (C) The percentage of IFN-γ⁺ T cells in gated CD4⁺ T cell compartment.</p

Neutralizing antibodies elicited in the Env expressing LC16m8 Δ prime/SeV boost regimen.

<p> (A) ID₅₀ of individual mouse serum against SF162 envelope-pseudotyped virus was determined using TZM-bl cells. Groups I, G and J consisted of 21, 17 and 14 mice, respectively. (B) Antibodies from randomly selected mice sera from group G, I, J and control were purified using protein A Sepharose. ID₅₀ of individual mouse serum against SF162 envelope-pseudotyped virus vs 50% inhibitory concentration (IC₅₀) (concentration of purified IgG that caused a 50% reduction in the RLU compared to virus control) of individual purified IgG was plotted. The regression line and R square are shown.</p

Priming with the coexpression vector m8 Δ-Env/hCD40Lm induces greater amounts of Env-specific antibodies.

<p> Serum from individual immunized mice was analyzed using an HIV-1_JR-CSF gp160 ELISA assay. The plates were developed with HRP-conjugated anti-mouse IgG antibody. (A) The titer of Env-specific antibodies was determined by OD₄₅₀ values subtracted from the background values. Data are mean ± SD of the Env-specific antibody titer of all animals in each group (n = 16 for group G and I; n = 8 for group H and J; n = 5 for group B and M). (B) Endpoint binding titer of sera against HIV-1 JR-CSF gp160 from each of vaccine groups was plotted. The titer in the group primed with m8ΔEnv/hCD40Lm was significantly different from all other groups. There are no significant differences in env-specific antibody titer among the group G, H and J.</p

Structures and expression of recombinant DNA and LC16m8 Δ.

<p>(A) Structures of four different recombinant m8Δs. (B) Profiles of Western blotting for Env and human CD40Lm expressed by infection with the HIV-1 Env expression plasmid, pCAGGS-JRCSFrev/env, SeV-env, and recombinant m8Δs are presented.</p

Cellular immunity elicited by a regimen consisting of DNA priming followed by vaccinia m8

<p>Δ <b>boosts.</b> (A) Schematic schedules of DNA prime/m8Δ-Env boost vaccination protocol. Mice were immunized twice with pCAGGS-JRCSFrev/env and boosted with various vaccinia viruses as follows. Group A: m8Δ; group B: m8Δ-Env; group C: m8Δ-Env/hCD40Lm; group D: m8Δ-Env plus m8Δ; group E: m8Δ-Env plus m8Δ-p7.5hCD40Lm; and group F: m8Δ-Env plus m8Δ-pSFJ1-10hCD40Lm. (B) Comparison of Env peptide-specific CD8⁺ T cell responses. The frequencies of IFN-γ⁺ CD8⁺ T cells in gated CD8⁺ T cell compartment was determined by intracellular cytokine staining (ICS) and FACScalibur/FACScanto analysis.</p

Priming with m8 Δ-Env/hCD40Lm elicits Env-specific antibodies with higher avidity.

<p> The avidity of serum antibodies for HIV-1 Env was determined based on the resistance of antibody-gp160 binding to disruption by treatment with urea. (A) Pooled serum from immunized mice of each group was applied to a HIV-1_JR-CSF gp160-coated ELISA plate at a 1∶300 dilution, as described above. After antibody-antigen binding, the plates were treated with increasing concentrations of urea (3–9 M) for 30 min at room temperature, followed by the ELISA procedure. (B) Dissociation analysis of 300 fold-diluted serum from individual mice using 7 M urea. The percentage of antibody remaining after urea treatment was plotted, and the mean and P values were calculated.</p

Correlation between neutralization activity against HIV-1 SF162 and the amount and avidity of anti-Env antibodies.

<p>The trendline and R and P values are displayed. (A) Correlation between neutralization activity against HIV-1 SF162 and the avidity of Env antibodies. (B) Correlation between the amount of anti-Env antibodies and neutralization activity. (C) Correlation between the amount of anti-Env antibodies and their avidity.</p

Mice immunization protocol used in this study.

<p>Mice immunization protocol used in this study.</p