Seasonal influenza and the threat of global pandemics present a continuing threat to public health. However, conventional inactivated influenza vaccines (IAVs) provide little cross-protective immunity and suboptimal efficacy, even against well-matched strains. Furthermore, the protection against matched strains has been shown to be of a short duration in both mouse models and humans. M2SR (M2-deficient single-replication influenza virus) is a single-replication vaccine that has been shown to provide effective cross-protection against heterosubtypic influenza viruses in both mouse and ferret models. In the present study, we investigated the duration and mechanism of heterosubtypic protection induced by M2SR in a mouse model. We previously showed that M2SR generated from influenza A/Puerto Rico/8/34 (H1N1) significantly protected C57BL/6 mice against lethal challenge with both influenza A/Puerto Rico/8/34 (H1N1, homosubtypic) and influenza A/Aichi/2/1968 (H3N2, heterosubtypic), whereas the inactivated influenza vaccine provided no heterosubtypic protection. The homosubtypic protection induced by M2SR was robust and lasted for greater than 1 year, whereas that provided by the inactivated vaccine lasted for less than 6 months. The heterosubtypic protection induced by M2SR was of a somewhat shorter duration than the homosubtypic protection, with protection being evident 9 months after vaccination. However, heterosubtypic protection was not observed at 14 months post vaccination. M2SR has been shown to induce strong systemic and mucosal antibody and T cell responses. We investigated the relative importance of these immune mechanisms in heterosubtypic protection, using mice that were deficient in B cells or mice that were depleted of T cells immediately before challenge. Somewhat surprisingly, the heterosubtypic protection was completely dependent on B cells in this model, whereas the depletion of T cells had no significant effect on survival after a lethal heterosubtypic challenge. While antibody-dependent cellular cytotoxicity (ADCC) has been demonstrated to be important in the response to some influenza vaccines, a lack of Fc receptors did not affect the survival of M2SR-vaccinated mice following a lethal challenge. We examined the influenza proteins targeted by the heterosubtypic antibody response. Shortly after the H1N1 M2SR vaccination, high titers of cross-reactive antibodies to heterosubtypic H3N2 nucleoprotein (NP) and lower titers to the stalk region of the hemagglutinin (HA2) and neuraminidase (NA) proteins were observed. The high antibody titers to heterosubtypic NP persisted one year after vaccination, whereas the antibody titers to the heterosubtypic HA2 and NA proteins were very low, or below the limit of detection, at this time. These results show that the intranasal M2SR vaccine elicits durable protective immune responses against homotypic and heterosubtypic influenza infection not seen with intramuscular inactivated vaccines. Both the homo- and heterosubtypic protection induced by the single-replication vaccine are dependent on B cells in this model. While the homosubtypic protection is mediated by antibodies to the head region of HA, our data suggest that the heterosubtypic protection for M2SR is due to cross-reactive antibodies elicited against the NP, HA2, and NA antigens that are not targeted by current seasonal influenza vaccines.

(S.R.S.) I was introduced to viral immunology while working in Peter Doherty's laboratory in the early stages of my research career, inspiring a lifelong interest in this area. During those early years under Peter's mentorship, we studied a mouse gammaherpesvirus model (murine gammaherpesvirus - 68 [MHV-68]) that provided a useful small animal model for investigating the immunological control of gammaherpesvirus infection. Interestingly, while CD4 T cells were not required for acute control of MHV-68 in the lung, CD8 T cell-mediated control was progressively lost in the absence of CD4 T cell help, leading to viral recrudescence. This was one of several early studies showing that CD8 T cell control of persistent viral infections was lost in the absence of CD4 T cell help, preceding the concept of CD8 T cell exhaustion. Further studies showed that MHV-68 infection of mice offered a unique model for comparing the mechanisms of acute and long-term control of a persistent viral infection and developing strategies for reversing T cell exhaustion. Here, we provide a brief review of the literature on CD8 T cell activation and exhaustion in this model, focusing on the role of CD40 and B7 family members and including some previously unpublished data.

Background/Objectives: Live influenza vaccines are considered to stimulate better overall immune responses but are associated with safety concerns regarding shedding and the potential for transmission or reassortment with wild-type influenza viruses. Intranasal M2SR and BM2SR (M2- and BM2-deficient single replication), intranasal influenza viruses, have shown promise as broadly cross-reactive next-generation influenza vaccines. The replication deficiency, shedding, and transmissibility of M2SR/BM2SR viruses were evaluated in a ferret model. Methods: Wild-type influenza A and B control viruses replicated in upper respiratory organs and transmitted to both direct and aerosol contact ferrets, whereas M2SR and BM2SR influenza vaccine viruses were not detected in any tissues or in nasal washes after inoculation and were not recovered from any direct or aerosol contact ferrets. Mice were simultaneously infected with wild-type influenza A and M2SR viruses to assess reassortment potential. Sequence and PCR analyses of the genome recovered from individual virus plaques isolated from lung homogenates identified the origin of the segments as exclusively from the replicating wild-type virus. Results: These results indicate that M2SR and BM2SR influenza vaccine viruses are attenuated, do not shed or transmit, and have a low probability for reassortment after coinfection. Absence of shedding was further demonstrated in nasal swabs taken from subjects who were inoculated with H3N2 M2SR in a previously described Phase 1 clinical study. Conclusions: These results indicate that M2SR/BM2SR viruses have the potential to be used in a broader population range than current live influenza vaccines.

Current influenza vaccines demonstrate low vaccine efficacy, especially when the predominantly circulating strain and vaccine are mismatched. The novel influenza vaccine platform M2- or BM2-deficient single replication (M2SR and BM2SR) has been shown to safely induce strong systemic and mucosal antibody responses and provide protection against significantly drifted influenza strains. In this study, we demonstrate that both monovalent and quadrivalent (Quad) formulations of M2SR are non-pathogenic in mouse and ferret models, eliciting robust neutralizing and non-neutralizing serum antibody responses to all strains within the formulation. Following challenge with wildtype influenza strains, vaccinated mice and ferrets demonstrated reduced weight loss, decreased viral replication in the upper and lower airways, and enhanced survival as compared to mock control groups. Mice vaccinated with H1N1 M2SR were completely protected from heterosubtypic H3N2 challenge, and BM2SR vaccines provided sterilizing immunity to mice challenged with a cross-lineage influenza B virus. Heterosubtypic cross-protection was also seen in the ferret model, with M2SR vaccinated animals exhibiting decreased viral titers in nasal washes and lungs following the challenge. BM2SR-vaccinated ferrets elicited robust neutralizing antibodies toward significantly drifted past and future influenza B strains. Mice and ferrets that received quadrivalent M2SR were able to mount immune responses equivalent to those seen with each of the four monovalent vaccines, demonstrating the absence of strain interference in the commercially relevant quadrivalent formulation.