Inactivated whole influenza virus particle vaccines induce neutralizing antibodies with an increase in immunoglobulin gene subclones of B-lymphocytes in cynomolgus macaques.

The All-Japan Influenza Vaccine Study Group has been developing a more effective vaccine than the current split vaccines for seasonal influenza virus infection. In the present study, the efficacy of formalin- and/or ß-propiolactone-inactivated whole virus particle vaccines for seasonal influenza was compared to that of the current ether-treated split vaccines in a nonhuman primate model. The monovalent whole virus particle vaccines or split vaccines of influenza A virus (H1N1) and influenza B virus (Victoria lineage) were injected subcutaneously into naïve cynomolgus macaques twice. The whole virus particle vaccines induced higher titers of neutralizing antibodies against H1N1 influenza A virus and influenza B virus in the plasma of macaques than did the split vaccines. At challenge with H1N1 influenza A virus or influenza B virus, the virus titers in nasal swabs and the increases in body temperatures were lower in the macaques immunized with the whole virus particle vaccine than in those immunized with the split vaccine. Repertoire analyses of immunoglobulin heavy chain genes demonstrated that the number of B-lymphocyte subclones was increased in macaques after the 1st vaccination with the whole virus particle vaccine, but not with the split vaccine, indicating that the whole virus particle vaccine induced the activation of vaccine antigen-specific B-lymphocytes more vigorously than did the split vaccine at priming. Thus, the present findings suggest that the superior antibody induction ability of the whole virus particle vaccine as compared to the split vaccine is attributable to its stimulatory properties on the subclonal differentiation of antigen-specific B-lymphocytes.

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 naïve mice and macaques. WPV is thus recommended as an alternative option for seasonal influenza vaccines, especially for children.

Inactivated whole virus particle vaccine with potent immunogenicity and limited IL-6 induction is ideal for influenza.

In contrast to current ether- or detergent-disrupted "split" vaccines (SVs) for influenza, inactivated whole influenza virus particle vaccines (WPVs) retain the original virus structure and components and as such may confer similar immunity to natural infection. In a collaboration between academia and industry, the potential of WPV as a new seasonal influenza vaccine was investigated. Each of the four seasonal influenza vaccine manufacturers in Japan prepared WPVs and SVs from the same batches of purified influenza virus. Both mice and monkeys vaccinated with the WPVs exhibited superior immune responses to those vaccinated with the corresponding SVs. Vaccination with A/California/07/2009 (H1N1) WPV enabled mice to survive a lethal challenge dose of homologous virus whereas those vaccinated with SV succumbed to infection within 6 days. Furthermore, mice vaccinated with WPV induced substantial numbers of multifunctional CD8+ T cells, important for control of antigenically drifted influenza virus strains. In addition, cytokines and chemokines were detected at early time points in the sera of mice vaccinated with WPV but not in those animals vaccinated with SV. These results indicate that WPVs induce enhanced innate and adaptive immune responses compared to equivalent doses of SVs. Notably, WPV at one fifth of the dose of SV was able to induce potent immunity with limited production of IL-6, one of the pyrogenic cytokines. We thus propose that WPVs with balanced immunogenicity and safety may set a new global standard for seasonal influenza vaccines.

Bovine alpha-2-HS-glycoprotein functions as a booster antigen for efficiently stimulating humoral immune responses to CCR5 and SIVmac239 envelope glycoprotein.

The presence of anti-CCR5 and anti-HIV-1 envelope glycoprotein (ENV) gp41 antibodies (Abs) at sites of HIV-1 exposure was effective in preventing its transmission to HIV-1-exposed seronegative (ESN) subjects. Here, we design an immunogen that can induce Abs against CCR5 and SIVmac239 ENV simultaneously and show that bovine alpha-2-HS-glycoprotein (bAHSG) functions as a booster antigen for efficiently stimulating humoral immune responses to CCR5 and ENV. Initially, we generated a rhesus CCR5-derived cyclopeptide (cDDR5) conjugated with a recombinant trimeric SIVmac239 Env. When inguinally administered to rhesus macaques, the immunogen simultaneously induced both anti-CCR5 and anti-ENV Abs in sera, and the purified serum IgG fraction exerted an inhibitory effect on SIVmac239 infection in vitro. When further boosted with bAHSG, the responses of both Abs were significantly enhanced. To examine the cross-reactivity of bAHSG, it was administered to naïve cynomolgus macaques. The results showed a statistically significant increase in IgG response against cynomolgus CCR5 and SIVmac239 ENV, and the induction of neutralizing activity against SIVmac239. These findings suggest that bAHSG is useful for immune strategies aimed at generating Abs against CCR5 and ENV simultaneously to confer HIV-protective immunity.

Japanese encephalitis virus structural and nonstructural proteins expressed in Escherichia coli induce protective immunity in mice.

Ectodomain of Japanese encephalitis virus (JEV) E protein [domains I through III (D1-3), domains I and II (D1-2) and domain III (D3)] and the nonstructural protein 1 (NS1) were expressed in Escherichia coli, and administered to BALB/c mice via the intranasal (i.n.) route. The E protein, but not the NS1, induced JEV-specific serum IgG with virus-neutralization capacity in vitro. When mice were lethally challenged with JEV, i.n. immunization with D1-3, D1-2, D3, or a mouse brain-derived formalin-inactivated JE vaccine conferred complete protection, while an 80% protection rate was observed in the NS1 immunized mice. Cytokine analysis of the cervical lymph nodes of mice i.n. immunized with D1-3 or NS1 revealed antigen-specific IL-2 and IL-17 responses, but no IFN-γ T cell response, were observed. This study demonstrates for the first time the i.n. vaccine efficacy of the E. coli-expressed recombinant JEV proteins.

Targeted delivery of immunogen to primate m cells with tetragalloyl lysine dendrimer.

Effective uptake of Ags by specialized M cells of gut-associated lymphoid tissues is an important step in inducing efficient immune responses after oral vaccination. Although stable nontoxic small molecule mimetics of lectins, such as synthetic multivalent polygalloyl derivatives, may have potential in murine M cell targeting, it remains unclear whether synthetic multivalent polygalloyl derivatives effectively target nonhuman and human M cells. In this study, we evaluated the ability of a tetragalloyl derivative, the tetragalloyl-D-lysine dendrimer (TGDK), to target M cells in both in vivo nonhuman primate and in vitro human M-like cell culture models. TGDK was efficiently transported from the lumen of the intestinal tract into rhesus Peyer's patches by M cells and then accumulated in germinal centers. Oral administration of rhesus CCR5-derived cyclopeptide conjugated with TGDK in rhesus macaque resulted in a statistically significant increase in stool IgA response against rhesus CCR5-derived cyclopeptide and induced a neutralizing activity against SIV infection. Furthermore, TGDK was specifically bound to human M-like cells and efficiently transcytosed from the apical side to the basolateral side in the M-like cell model. Thus, the TGDK-mediated vaccine delivery system represents a potential approach for enabling M cell-targeted mucosal vaccines in primates.

Development of cell-expressed and virion-incorporated CCR5-targeted vaccine.

Our previous study demonstrated that the immunization with a cycloimmunogen derived from extracellular loop-2 (ECL-2) of CCR5 (cDDR5) attenuated acute phase of CCR5-tropic simian-human immunodeficiency virus (SHIV)(SF162P3) replication in vivo. Although the study showed that the antisera raised against cDDR5 reacted with cell-expressed CCR5, we have not yet demonstrated whether the antisera can react with virion-incorporated CCR5. Here, we show that rhesus cDDR5 (rcDDR5)-specific antibodies react with not only cell-expressed but also virion-incorporated simian CCR5s (siCCR5s), but may predominantly exert their inhibitory effects on simian immunodeficiency virus (SIV) infection by the binding of cell-expressed rather than virion-incorporated CCR5s. These results suggest that the virion-incorporated CCR5 may contribute to the reactivation of the anti-rcDDR5 antibody-producing B-cells by SIV particles after rcDDR5 immunization, although the binding of anti-rcDDR5 antibody to virion-incorporated CCR5 results in a partial inhibitory effect on SIV infection.