Intranasal M2SR (M2-Deficient Single Replication) H3N2 Influenza Vaccine Provides Enhanced Mucosal and Serum Antibodies in Adults.

BACKGROUND: We previously demonstrated that an intranasal dose of 108 50% tissue culture infectious dose (TCID50) M2-deficient single replication (M2SR) influenza vaccine protected against highly drifted H3N2 influenza challenge in a subset of subjects who demonstrated ≥2-fold increase in microneutralization (MN) antibodies to Belgium2015 (the challenge strain) after vaccination. Here, we describe a phase 1b, observer-blinded, dose-escalation study demonstrating an increased proportion of responders with this signal of immune protection. METHODS: Serosusceptible subjects aged 18-49 years were randomized to receive 2 doses (108-109 TCID50) of M2SR or placebo administered 28 days apart. Clinical specimens were collected before and after each dose. The primary objective was to demonstrate safety of M2SR vaccines. RESULTS: The vaccine was well tolerated at all dose levels. Against Belgium2015, ≥ 2-fold increases in MN antibodies were noted among 40% (95% confidence interval [CI], 24.9%-56.7%) of subjects following a single 108 TCID50 M2SR dose and among 80.6% (95% CI, 61.4%-92.3%) after 109 dose (P < .001). A single 109 TCID50 dose of M2SR generated ≥4-fold hemagglutination inhibition antibody seroconversion against the vaccine strain in 71% (95% CI, 52.0%-85.8%) of recipients. Mucosal and cellular immune responses were also induced. CONCLUSIONS: These results indicate that M2SR may provide substantial protection against infection with highly drifted strains of H3N2 influenza. CLINICAL TRIALS REGISTRATION: NCT03999554.

In recent years, influenza A H3N2 viruses have evolved into multiple cocirculating clades, resulting in low vaccine efficacy and highlighting the need for more effective influenza vaccines. In a previous challenge study, a single intranasal dose of the investigational vaccine M2SR demonstrated protection against a highly drifted H3N2 influenza challenge virus in a subset of vaccine recipients with a signature immune response. Increasing the dose of the M2SR vaccine in this phase1b study demonstrated a statistically significant increase in the proportion of subjects with the signature immune responses seen previously. The vaccine-induced antibodies were cross-reactive with a panel of drifted H3N2 viruses from 2007 to 2019. Additionally, M2SR generated a rise in serum hemagglutination inhibition antibody titer in 71% of subjects. In contrast, the H3N2 seroresponse rate for the licensed intranasal vaccine FluMist is 10% in seronegative adults. Moreover, M2SR elicited mucosal and cell-mediated immune responses. This study demonstrates that the intranasal M2SR generates a multifaceted immune response and has the potential to provide better efficacy against vaccine-matched strains and influenza drift variants reducing the need to update the vaccine on an annual basis. This is a noteworthy step in the development of a broadly protective influenza vaccine.

Antigen signal strength during priming determines effector CD4 T cell function and antigen sensitivity during influenza virus challenge.

TCR signal strength during priming is a key determinant of CD4 T cell activation, but its impact on effector CD4 T functions in vivo remains unclear. In this study, we compare the functionality of CD4 T cell responses induced by peptides displaying varying binding half-lives with MHC class II before and after influenza virus infection. Although significant quantitative and qualitative differences in CD4 T cell responses were observed before infection between mice vaccinated with low- or high-stability peptides, both mice mounted robust early Th1 effector cytokine responses upon influenza challenge. However, only effector CD4 T cells induced by low-stability peptides proliferated and produced IL-17A after influenza challenge. In contrast, effector T cells elicited by higher-stability peptides displayed a terminally differentiated phenotype and divided poorly. This defective proliferation was T cell intrinsic but could not be attributed to a reduced expression of lymph node homing receptors. Instead, we found that CD4 T cells stimulated with higher-stability peptides exhibited decreased responsiveness to low levels of Ag presentation. Our study reveals the critical role of TCR signal strength during priming for the function and Ag sensitivity of effector CD4 T cells during viral challenge.

In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses.

Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S-OIV pandemic.

Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus.

The 1918 influenza pandemic was unusually severe, resulting in about 50 million deaths worldwide. The 1918 virus is also highly pathogenic in mice, and studies have identified a multigenic origin of this virulent phenotype in mice. However, these initial characterizations of the 1918 virus did not address the question of its pathogenic potential in primates. Here we demonstrate that the 1918 virus caused a highly pathogenic respiratory infection in a cynomolgus macaque model that culminated in acute respiratory distress and a fatal outcome. Furthermore, infected animals mounted an immune response, characterized by dysregulation of the antiviral response, that was insufficient for protection, indicating that atypical host innate immune responses may contribute to lethality. The ability of influenza viruses to modulate host immune responses, such as that demonstrated for the avian H5N1 influenza viruses, may be a feature shared by the virulent influenza viruses.

Quadrivalent Formulation of Intranasal Influenza Vaccine M2SR (M2-Deficient Single Replication) Protects against Drifted Influenza A and B Virus Challenge.

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.

Subclinical brain injury caused by H5N1 influenza virus infection.

Although H5N1 influenza A viruses can cause systemic infection, their neurotropism and long-term effects on the central nervous system (CNS) are not fully understood. We assessed H5N1viral invasion of the CNS and its long-term effects in a ferret model. An H5N1 virus caused nonsuppurative encephalitis, which lasted for 3 months without neurologic signs. Further, another H5N1 virus caused nonsuppurative vasculitis with brain hemorrhage. Three-dimensional analysis of viral distribution in the brain identified the olfactory system as a major route for brain invasion. The efficient growth of virus in the upper respiratory tract may thus facilitate viral brain invasion.

Host regulatory network response to infection with highly pathogenic H5N1 avian influenza virus.

During the last decade, more than half of humans infected with highly pathogenic avian influenza (HPAI) H5N1 viruses have died, yet virus-induced host signaling has yet to be clearly elucidated. Airway epithelia are known to produce inflammatory mediators that contribute to HPAI H5N1-mediated pathogenicity, but a comprehensive analysis of the host response in this cell type is lacking. Here, we leveraged a system approach to identify and statistically validate signaling subnetworks that define the dynamic transcriptional response of human bronchial epithelial cells after infection with influenza A/Vietnam/1203/2004 (H5N1, VN1203). Importantly, we validated a subset of transcripts from one subnetwork in both Calu-3 cells and mice. A more detailed examination of two subnetworks involved in the immune response and keratinization processes revealed potential novel mediators of HPAI H5N1 pathogenesis and host response signaling. Finally, we show how these results compare to those for a less virulent strain of influenza virus. Using emergent network properties, we provide fresh insight into the host response to HPAI H5N1 virus infection and identify novel avenues for perturbation studies and potential therapeutic interventions for fatal HPAI H5N1 disease.

Viral replication rate regulates clinical outcome and CD8 T cell responses during highly pathogenic H5N1 influenza virus infection in mice.

Since the first recorded infection of humans with H5N1 viruses of avian origin in 1997, sporadic human infections continue to occur with a staggering mortality rate of >60%. Although sustained human-to-human transmission has not occurred yet, there is a growing concern that these H5N1 viruses might acquire this trait and raise the specter of a pandemic. Despite progress in deciphering viral determinants of pathogenicity, we still lack crucial information on virus/immune system interactions pertaining to severe disease and high mortality associated with human H5N1 influenza virus infections. Using two human isolates of H5N1 viruses that differ in their pathogenicity in mice, we have defined mechanistic links among the rate of viral replication, mortality, CD8 T cell responses, and immunopathology. The extreme pathogenicity of H5N1 viruses was directly linked to the ability of the virus to replicate rapidly, and swiftly attain high steady-state titers in the lungs within 48 hours after infection. The remarkably high replication rate of the highly pathogenic H5N1 virus did not prevent the induction of IFN-ß or activation of CD8 T cells, but the CD8 T cell response was ineffective in controlling viral replication in the lungs and CD8 T cell deficiency did not affect viral titers or mortality. Additionally, BIM deficiency ameliorated lung pathology and inhibited T cell apoptosis without affecting survival of mice. Therefore, rapidly replicating, highly lethal H5N1 viruses could simply outpace and overwhelm the adaptive immune responses, and kill the host by direct cytopathic effects. However, therapeutic suppression of early viral replication and the associated enhancement of CD8 T cell responses improved the survival of mice following a lethal H5N1 infection. These findings suggest that suppression of early H5N1 virus replication is key to the programming of an effective host response, which has implications in treatment of this infection in humans.

A critical role for the sphingosine analog AAL-R in dampening the cytokine response during influenza virus infection.

Pulmonary tissue damage resulting from influenza virus infection is caused by both the cytolytic activity of the virus and the host immune response. Immune-mediated injury results from T cell-mediated destruction of virus-infected cells and by release of cytokines and chemokines that attract polymorphonuclear leukocytes (PML) and macrophages to the infected site. The cytokines/chemokines potentiate dendritic cell (DC) activation and T cell expansion, which further enhances local damage. Here we report that immune modulation by local administration to the respiratory tract of sphingosine analog AAL-R significantly dampens the release of cytokines and chemokines while maintaining protective neutralizing antibody and cytotoxic T cell responses. As a result there was a marked reduction of infiltrating PML and macrophages into the lung and resultant pulmonary tissue injury. DC maturation was suppressed, which limited proliferation of specific antiviral T cells in the lung and draining lymph nodes. Further, AAL-R was effective in controlling CD8(+) T cell accumulation in the lungs even when given 4 days after initiation of influenza virus infection. These data indicate that sphingosine analogs display useful potential for controlling the immunopathology caused by influenza virus.

Longevity and Mechanism of Heterosubtypic Protection Induced by M2SR (M2-Deficient Single-Replication) Live Influenza Virus Vaccine in Mice.

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.

Safety and Immunogenicity of M2-Deficient, Single Replication, Live Influenza Vaccine (M2SR) in Adults.

M2SR (M2-deficient single replication) is an investigational live intranasal vaccine that protects against multiple influenza A subtypes in influenza-naïve and previously infected ferrets. We conducted a phase 1, first-in-human, randomized, dose-escalation, placebo-controlled study of M2SR safety and immunogenicity. Adult subjects received a single intranasal administration with either placebo or one of three M2SR dose levels (106, 107 or 108 tissue culture infectious dose (TCID50)) expressing hemagglutinin and neuraminidase from A/Brisbane/10/2007 (H3N2) (24 subjects per group). Subjects were evaluated for virus replication, local and systemic reactions, adverse events (AE), and immune responses post-vaccination. Infectious virus was not detected in nasal swabs from vaccinated subjects. At least one AE (most commonly mild nasal rhinorrhea/congestion) was reported among 29%, 58%, and 83% of M2SR subjects administered a low, medium or high dose, respectively, and among 46% of placebo subjects. No subject had fever or a severe reaction to the vaccine. Influenza-specific serum and mucosal antibody responses and B- and T-cell responses were significantly more frequent among vaccinated subjects vs. placebo recipients. The M2SR vaccine was safe and well tolerated and generated dose-dependent durable serum antibody responses against diverse H3N2 influenza strains. M2SR demonstrated a multi-faceted immune response in seronegative and seropositive subjects.

Region required for protein expression from the stop-start pentanucleotide in the M gene of influenza B virus.

Segment 7 of influenza B virus encodes two proteins, M1 and BM2. BM2 is expressed from a stop-start pentanucleotide, in which the BM2 initiation codon overlaps with the M1 stop codon. Here, we demonstrate that 45 nucleotides of the 3' end of the M1 coding region, but not the 5' end of the BM2 coding region, are sufficient for the efficient expression of the downstream protein. Placing these 45 nucleotides and the stop-start pentanucleotide in between the coding sequences induced the expression of at least three noninfluenza proteins, suggesting the utility of this system for expressing multiple proteins from one mRNA.

Replication-deficient ebolavirus as a vaccine candidate.

Ebolavirus causes severe hemorrhagic fever, with case fatality rates as high as 90%. Currently, no licensed vaccine is available against Ebolavirus. We previously generated a replication-deficient, biologically contained Ebolavirus, EbolaDeltaVP30, which lacks the essential VP30 gene, grows only in cells stably expressing this gene product, and is genetically stable. Here, we evaluated the vaccine potential of EbolaDeltaVP30. First, we demonstrated its safety in STAT-1-knockout mice, a susceptible animal model for Ebolavirus infection. We then tested its protective efficacy in two animal models, mice and guinea pigs. Mice immunized twice with EbolaDeltaVP30 were protected from a lethal infection of mouse-adapted Ebolavirus. Virus titers in the serum of vaccinated mice were significantly lower than those in nonvaccinated mice. Protection of mice immunized with EbolaDeltaVP30 was associated with a high antibody response to the Ebolavirus glycoprotein and the generation of an Ebolavirus NP-specific CD8(+) T-cell response. Guinea pigs immunized twice with EbolaDeltaVP30 were also protected from a lethal infection of guinea pig-adapted Ebolavirus. Our study demonstrates the potential of the EbolaDeltaVP30 virus as a new vaccine platform.

Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice.

Highly pathogenic avian H5N1 influenza A viruses have spread throughout Asia, Europe, and Africa, raising serious worldwide concern about their pandemic potential. Although more than 250 people have been infected with these viruses, with a consequent high rate of mortality, the molecular mechanisms responsible for the efficient transmission of H5N1 viruses among humans remain elusive. We used a mouse model to examine the role of the amino acid at position 627 of the PB2 viral protein in efficient replication of H5N1 viruses in the mammalian respiratory tract. Viruses possessing Lys at position 627 of PB2 replicated efficiently in lungs and nasal turbinates, as well as in cells, even at the lower temperature of 33 degrees C. Those viruses possessing Glu at this position replicated less well in nasal turbinates than in lungs, and less well in cells at the lower temperature. These results suggest that Lys at PB2-627 confers to avian H5N1 viruses the advantage of efficient growth in the upper and lower respiratory tracts of mammals. Therefore, efficient viral growth in the upper respiratory tract may provide a platform for the adaptation of avian H5N1 influenza viruses to humans and for efficient person-to-person virus transmission, in the context of changes in other viral properties including specificity for human (sialic acid alpha-2,6-galactose containing) receptors.

Single-replication BM2SR vaccine provides sterilizing immunity and cross-lineage influenza B virus protection in mice.

Both influenza A and B viruses cause outbreaks of seasonal influenza resulting in significant morbidity and mortality. There are two antigenically distinct lineages of influenza B virus, Yamagata lineage (YL) and Victoria lineage (VL). Since both B lineages have been co-circulating for years, more than 70% of influenza vaccines currently manufactured are quadrivalent consisting of influenza A (H1N1), influenza A (H3N2), influenza B (YL) and influenza B (VL) antigens. Although quadrivalent influenza vaccines tend to elevate immunity to both influenza B lineages, estimated overall vaccine efficacy against influenza B is still only around 42%. Thus, a more effective influenza B vaccine is needed. To meet this need, we generated BM2-deficient, single-replication (BM2SR) influenza B vaccine viruses that encode surface antigens from influenza B/Wisconsin/01/2010 (B/WI01, YL) and B/Brisbane/60/2008 (B/Bris60, VL) viruses. The BM2SR-WI01 and BM2SR-Bris60 vaccine viruses are replication-deficient in vitro and in vivo, and can only replicate in a cell line that expresses the complementing BM2 protein. Both BM2SR viruses were non-pathogenic to mice, and vaccinated animals showed elevated mucosal and serum antibody responses to both Yamagata and Victoria lineages in addition to cellular responses. Serum antibody responses included lineage-specific hemagglutinin inhibition antibody (HAI) responses as well as responses to the stem region of the hemagglutinin (HA). BM2SR vaccine viruses provided apparent sterilizing immunity to mice against intra- and inter-lineage drifted B virus challenge. The data presented here support the feasibility of BM2SR as a platform for next-generation trivalent influenza vaccine development.

Local not systemic modulation of dendritic cell S1P receptors in lung blunts virus-specific immune responses to influenza.

The mechanism by which locally delivered sphingosine analogs regulate host response to localized viral infection has never been addressed. In this report, we show that intratracheal delivery of the chiral sphingosine analog (R)-2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol (AAL-R) or its phosphate ester inhibits the T-cell response to influenza virus infection. In contrast, neither intraperitoneal delivery of AAL-R nor intratracheal instillation of the non-phosphorylatable stereoisomer AAL-S suppressed virus-specific T-cell response, indicating that in vivo phosphorylation of AAL-R and sphingosine 1-phosphate (S1P) receptor modulation in lungs is essential for immunomodulation. Intratracheal delivery of water-soluble S1P(1) receptor agonist at doses sufficient to induce systemic lymphopenia did not inhibit virus-specific T-cell response, indicating that S1P(1) is not involved in the immunosuppressive activities of AAL-R and that immunosuppression acts independently of naive lymphocyte recirculation. Accumulation of dendritic cells (DCs) in draining lymph nodes was inhibited by intratracheal but not intraperitoneal delivery of AAL-R. Direct modulation of DCs is demonstrated by the impaired ability of virus-infected bone marrow-derived DCs treated in vitro with AAL-R to trigger in vivo T-cell response after adoptive transfer to the airways. Thus, our results suggest that locally delivered sphingosine analogs induce immunosuppression by modulating S1P receptors other than S1P(1) or S1P(2) on dendritic cells in the lungs after influenza virus infection.

Limited compatibility between the RNA polymerase components of influenza virus type A and B.

Reassortants between type A and B influenza viruses have not been detected in nature, although both viruses co-circulate in human populations. One explanation for this may be functional incompatibility of RNA transcription and replication between type A and B viruses. To test this possibility, we constructed type A/B mosaic polymerase machinery, containing PB2, PB1, PA and nucleoprotein from each of the two virus types, and assessed their polymerase activities with a type A promoter in a reporter assay. Type B polymerase machinery containing homologous components was functional with the type A promoter albeit to various extents depending on the segments from which the regions downstream of the promoter sequence were derived, indicating functional compatibility between the type A promoter and B polymerase machinery. However, all of the A/B mosaic polymerase machinery, except that containing PA from a type A and the others from a type B virus strain, did not function with the type A promoter, indicating limited compatibility among polymerase components of both types. Taken together, these data suggest that incompatibility among components of the polymerase machinery for RNA transcription and replication alone is not responsible for the lack of heterotypic reassortants.

Novel influenza vaccine M2SR protects against drifted H1N1 and H3N2 influenza virus challenge in ferrets with pre-existing immunity.

Current influenza vaccines do not provide effective protection against heterologous influenza viruses. The ability of the novel M2SR influenza vaccine to protect against drifted influenza viruses was evaluated in naïve ferrets and in ferrets with pre-existing immunity to influenza. In naïve ferrets, M2SR provided similar protection against drifted challenge viruses as the comparator vaccine, FluMist®. However, in ferrets with pre-existing immunity, M2SR provided superior protection than FluMist in two model systems. In the first model, ferrets were infected with influenza A H1N1pdm and influenza B viruses to mimic the diverse influenza exposure in humans. The pre-infected ferrets, seropositive to H1N1pdm and influenza B but seronegative to H3N2, were then vaccinated with H3N2 M2SR or monovalent H3N2 FluMist virus (A/Brisbane/10/2007, clade 1) and challenged 6 weeks later with a drifted H3N2 virus (clade 3C.2a). Antibody titers to Brisbane/10/2007 were higher in M2SR vaccinated ferrets than in FluMist vaccinated ferrets in the pre-infected ferrets whereas the opposite was observed in naïve ferrets. After challenge with drifted H3N2 virus, M2SR provided superior protection than FluMist monovalent vaccine. In the second model, the impact of homologous pre-existing immunity upon vaccine-induced protection was evaluated. Ferrets, pre-infected with H1N1pdm virus, were vaccinated 90 days later with H1N1pdm M2SR or FluMist monovalent vaccine and challenged 6 weeks later with a pre-pandemic seasonal H1N1 virus, A/Brisbane/59/2007 (Bris59). While cross-reactive serum IgG antibodies against the Bris59 HA were detected after vaccination, anti-Bris59 hemagglutination inhibition antibodies were only detected post-challenge. M2SR provided better protection against Bris59 challenge than FluMist suggesting that homologous pre-existing immunity affected FluMist virus to a greater degree than M2SR. These results suggest that the single replication intranasal M2SR vaccine provides effective protection against drifted influenza A viruses not only in naïve ferrets but also in those with pre-existing immunity in contrast to FluMist viruses.

M2SR, a novel live influenza vaccine, protects mice and ferrets against highly pathogenic avian influenza.

The emergence of highly pathogenic avian influenza H5N1 viruses has heightened global concern about the threat posed by pandemic influenza. To address the need for a highly effective universal influenza vaccine, we developed a novel M2-deficient single replication (M2SR) influenza vaccine virus and previously reported that it provided strong heterosubtypic protection against seasonal influenza viruses in mice. In the current study, we assessed M2SR induced protection against H5N1 influenza in mice and ferrets. Mice were intranasally inoculated with M2SR viruses containing the HA and NA from A/Vietnam/1203/2004 (M2SR H5N1) or A/California/07/2009 (M2SR H1N1). All M2SR vaccinated mice survived lethal challenge with influenza A/Vietnam/1203/2004 (H5N1), whereas 40% of mice vaccinated with recombinant H5 HA and none of the naïve controls survived. M2SR H5N1 provided sterile immunity, whereas low levels of virus were detected in the lungs of some M2SR H1N1 vaccinated mice. In contrast, recombinant H5 HA vaccinated mice and naïve controls showed systemic infection. M2SR H5N1 induced strong serum and mucosal antibody responses (IgG and IgA classes) against H5 HA, with high hemagglutination inhibition (HAI) titers. In contrast, while M2SR H1N1 elicited cross-reactive antibodies recognizing the H5 HA2 stalk region or the neuraminidase, no HAI activity against H5N1 virus was detected after M2SR H1N1 immunization. Both M2SR H5N1 and H1N1 also protected ferrets against lethal challenge with A/Vietnam/1203/2004. A prime-boost regimen provided optimal protection with no virus detected in the respiratory tract or brain after challenge. As in the mouse model, only the M2SR H5N1 vaccine induced HAI antibodies against the challenge virus in ferrets, while the M2SR H1N1 was able to provide protection without the induction of HAI antibodies. In summary, effective protection against highly pathogenic H5N1 influenza virus was provided by both homologous H5N1 M2SR and heterologous H1N1 M2SR demonstrating the cross-protective attributes of the M2SR platform.

M2SR, a novel live single replication influenza virus vaccine, provides effective heterosubtypic protection in mice.

Despite the annual public health burden of seasonal influenza and the continuing threat of a global pandemic posed by the emergence of highly pathogenic/pandemic strains, conventional influenza vaccines do not provide universal protection, and exhibit suboptimal efficacy rates, even when they are well matched to circulating strains. To address the need for a highly effective universal influenza vaccine, we have developed a novel M2-deficient single replication vaccine virus (M2SR) that induces strong cross-protective immunity against multiple influenza strains in mice. M2SR is able to infect cells and expresses all viral proteins except M2, but is unable to generate progeny virus. M2SR generated from influenza A/Puerto Rico/8/34 (H1N1) protected mice against lethal challenge with influenza A/Puerto Rico/8/34 (H1N1, homosubtypic) and influenza A/Aichi/2/1968 (H3N2, heterosubtypic). The vaccine induced strong systemic and mucosal antibody responses of both IgA and IgG classes. Strong virus-specific T cell responses were also induced. Following heterologous challenge, significant numbers of IFN-γ-producing CD8 T cells, with effector or effector/memory phenotypes and specific for conserved viral epitopes, were observed in the lungs of vaccinated mice. A substantial proportion of the CD8 T cells expressed Granzyme B, suggesting that they were capable of killing virus-infected cells. Thus, our data suggest that M2-deficient influenza viruses represent a promising new approach for developing a universal influenza vaccine.