Intranasal M2SR (M2-deficient Single Replication) Influenza Vaccine Induces Broadly Reactive Mucosal Antibody Production in Adults.

Intranasal M2SR (M2-deficient Single Replication influenza virus) vaccine induces robust immune responses in animal models and human subjects. A high-throughput multiplexed platform was used to analyze hemagglutinin-specific mucosal antibody responses in adults after a single dose of H3N2 M2SR. Nasal swab specimens were analyzed for total and hemagglutinin-specific IgA. Significant, dose-dependent increases in mucosal antibody responses to vaccine-matched and drifted H3N2 hemagglutinin were observed in M2SR vaccinated subjects regardless of baseline serum and mucosal immune status. These data suggest that M2SR induces broadly cross-reactive mucosal immune responses which may provide better protection against drifted and newly emerging influenza strains.

M2-Deficient Single-Replication Influenza Vaccine-Induced Immune Responses Associated With Protection Against Human Challenge With Highly Drifted H3N2 Influenza Strain.

BACKGROUND: Current influenza vaccines are strain specific and demonstrate low vaccine efficacy against H3N2 influenza disease, especially when vaccine is mismatched to circulating virus. The novel influenza vaccine candidate, M2-deficient single replication (M2SR), induces a broad, multi-effector immune response. METHODS: A phase 2 challenge study was conducted to assess the efficacy of an M2SR vaccine expressing hemagglutinin and neuraminidase from A/Brisbane/10/2007 (Bris2007 M2SR H3N2; clade 1). Four weeks after vaccination, recipients were challenged with antigenically distinct H3N2 virus (A/Belgium/4217/2015, clade 3C.3b) and assessed for infection and clinical symptoms. RESULTS: Adverse events after vaccination were mild and similar in frequency for placebo and M2SR recipients. A single dose of Bris2007 M2SR induced neutralizing antibody to the vaccine (48% of recipients) and challenge strain (27% of recipients). Overall, 54% of M2SR recipients were infected after challenge, compared with 71% of placebo recipients. The subset of M2SR recipients with a vaccine-induced microneutralization response against the challenge virus had reduced rates of infection after challenge (38% vs 71% of placebo recipients; P = .050) and reduced illness. CONCLUSIONS: Study participants with vaccine-induced neutralizing antibodies were protected against infection and illness after challenge with an antigenically distinct virus. This is the first demonstration of vaccine-induced protection against a highly drifted H3N2 challenge virus.

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.

Mucosal immunization with dual influenza/COVID-19 single-replication virus vector protects hamsters from SARS-CoV-2 challenge.

The COVID-19 pandemic has highlighted the need for mucosal vaccines as breakthrough infections, short-lived immune responses and emergence of new variants have challenged the efficacy provided by the first generation of vaccines against SARS-CoV-2 viruses. M2SR SARS-CoV-2, an M2-deleted single-replication influenza virus vector modified to encode the SARS-CoV-2 receptor binding domain, was evaluated following intranasal delivery in a hamster challenge model for protection against Wuhan SARS-CoV-2. An adjuvanted inactivated SARS-CoV-2 whole virus vaccine administered intramuscularly was also evaluated. The intranasal M2SR SARS-CoV-2 was more effective than the intramuscular adjuvanted inactivated whole virus vaccine in providing protection against SARS-CoV-2 challenge. M2SR SARS-CoV-2 elicited neutralizing serum antibodies against Wuhan and Omicron SARS-CoV-2 viruses in addition to cross-reactive mucosal antibodies. Furthermore, M2SR SARS-CoV-2 generated serum HAI and mucosal antibody responses against influenza similar to an H3N2 M2SR influenza vaccine. The intranasal dual influenza/COVID M2SR SARS-CoV-2 vaccine has the potential to provide protection against both influenza and COVID.

Safety and immunogenicity of the intranasal H3N2 M2-deficient single-replication influenza vaccine alone or coadministered with an inactivated influenza vaccine (Fluzone High-Dose Quadrivalent) in adults aged 65-85 years in the USA: a multicentre, randomised, double-blind, double-dummy, phase 1b trial.

BACKGROUND: Older adults (aged ≥65 years) show increased susceptibility to severe disease with influenza virus infection, accounting for 70-85% of annual influenza-related fatalities in the USA. Stimulating mucosal antibodies and T cells might enhance the low vaccine effectiveness seen in older adults for currently licensed inactivated influenza vaccines, which induce mainly serum antibodies. We aimed to evaluate the safety and immunogenicity of the intranasal H3N2 M2-deficient single-replication (M2SR) vaccine, alone or coadministered with a licensed inactivated influenza vaccine (Fluzone High-Dose Quadrivalent; hereafter referred to as Fluzone HD), in older adults. METHODS: In this multicentre, randomised, double-blind, double-dummy, phase 1b trial, individuals aged 65-85 years who were considered healthy or with stable chronic conditions with no recent (<6 months) influenza vaccinations were recruited from five clinical trial sites in the USA and randomly assigned (3:3:3:1) using a permuted block design to receive the H3N2 M2SR vaccine and Fluzone HD, the H3N2 M2SR vaccine and placebo, Fluzone HD and placebo, or placebo alone. All participants received a single intranasal spray and a single intramuscular injection, whether active or placebo, to maintain masking. The primary outcome was to assess the safety of H3N2 M2SR, administered alone or with Fluzone HD, in the safety analysis set, which included all participants who were randomly assigned and received treatment. Serum and mucosal antibodies were assessed as a secondary endpoint, and cell-mediated immunity as an exploratory endpoint, in participants in the per-protocol population, which included individuals in the safety analysis set without major protocol deviations. This trial is registered with ClinicalTrials.gov, NCT05163847. FINDINGS: Between June 14 and Sept 15, 2022, 305 participants were enrolled and randomly assigned to receive the H3N2 M2SR vaccine plus placebo (n=89), H3N2 M2SR vaccine plus Fluzone HD (n=94), Fluzone HD plus placebo (n=92), or placebo alone (n=30). All randomly assigned participants were included in the safety analysis set. The most frequently reported local symptoms up to day 8 in groups that received M2SR were rhinorrhoea (43% [38 of 89] in the H3N2 M2SR plus placebo group and 38% [36 of 94] in the H3N2 M2SR plus Fluzone HD group), nasal congestion (51% [45 of 89] and 35% [33 of 94]), and injection-site pain (8% [seven of 89] and 49% [46 of 94]), and the most frequently reported solicited systemic symptoms were sore throat (28% [25 of 89]) for M2SR and decreased activity (26% [24 of 94]) for the M2SR plus Fluzone HD group. In the Fluzone HD plus placebo group, the most frequently reported local symptom was injection-site pain (48% [44 of 92]) and systemic symptom was muscle aches (22% [20 of 92]). The frequency of participants with any treatment-emergent adverse event related to vaccination was low across all groups (2-5%). One serious adverse event was reported, in a participant in the Fluzone HD plus placebo group. M2SR with Fluzone HD induced seroconversion (≥four-fold increase in haemagglutination inhibition antibodies from baseline to day 29) in 44 (48%) of 91 participants, compared with 28 (31%) of 90 participants who seroconverted in the Fluzone HD plus placebo group (p=0·023). M2SR with Fluzone HD also induced mucosal and cellular immune responses. INTERPRETATION: The H3N2 M2SR vaccine coadministered with Fluzone HD in older adults was well tolerated and provided enhanced immunogenicity compared with Fluzone HD administered alone, suggesting potential for improved efficacy of influenza vaccination in this age group. Additional studies are planned to assess efficacy. FUNDING: US Department of Defense.

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.

Intranasal Single-Replication Influenza Vector Induces Cross-Reactive Serum and Mucosal Antibodies against SARS-CoV-2 Variants.

Current SARS-CoV-2 vaccines provide protection for COVID-19-associated hospitalization and death, but remain inefficient at inhibiting initial infection and transmission. Despite updated booster formulations, breakthrough infections and reinfections from emerging SARS-CoV-2 variants are common. Intranasal vaccination to elicit mucosal immunity at the site of infection can improve the performance of respiratory virus vaccines. We developed SARS-CoV-2 M2SR, a dual SARS-CoV-2 and influenza vaccine candidate, employing our live intranasal M2-deficient single replication (M2SR) influenza vector expressing the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein of the prototype strain, first reported in January 2020. The intranasal vaccination of mice with this dual vaccine elicits both high serum IgG and mucosal IgA titers to RBD. Sera from inoculated mice show that vaccinated mice develop neutralizing SARS-CoV-2 antibody titers against the prototype and Delta virus strains, which are considered to be sufficient to protect against viral infection. Moreover, SARS-CoV-2 M2SR elicited cross-reactive serum and mucosal antibodies to the Omicron BA.4/BA.5 variant. The SARS-CoV-2 M2SR vaccine also maintained strong immune responses to influenza A with high titers of anti H3 serum IgG and hemagglutination inhibition (HAI) antibody titers corresponding to those seen from the control M2SR vector alone. With a proven safety record and robust immunological profile in humans that includes mucosal immunity, the M2SR influenza viral vector expressing key SARS-CoV-2 antigens could provide more efficient protection against influenza and SARS-CoV-2 variants.

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.

Influenza A virus infection-induced macroautophagy facilitates MHC class II-restricted endogenous presentation of an immunodominant viral epitope.

CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires de novo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on de novo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed.

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.

Monocyte apoptotic bodies are vehicles for influenza A virus propagation.

The disassembly of apoptotic cells into small membrane-bound vesicles termed apoptotic bodies (ApoBDs) is a hallmark of apoptosis; however, the functional significance of this process is not well defined. We recently discovered a new membrane protrusion (termed beaded apoptopodia) generated by apoptotic monocytes which fragments to release an abundance of ApoBDs. To investigate the function of apoptotic monocyte disassembly, we used influenza A virus (IAV) infection as a proof-of-concept model, as IAV commonly infects monocytes in physiological settings. We show that ApoBDs generated from IAV-infected monocytes contained IAV mRNA, protein and virions and consequently, could facilitate viral propagation in vitro and in vivo, and induce a robust antiviral immune response. We also identified an antipsychotic, Haloperidol, as an unexpected inhibitor of monocyte cell disassembly which could impair ApoBD-mediated viral propagation under in vitro conditions. Together, this study reveals a previously unrecognised function of apoptotic monocyte disassembly in the pathogenesis of IAV infections.

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.

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.

An M2 cytoplasmic tail mutant as a live attenuated influenza vaccine against pandemic (H1N1) 2009 influenza virus.

The 2009 influenza pandemic brought home the importance of vaccines in infection control. Previously, we demonstrated an M2 cytoplasmic tail mutant H5N1 influenza virus could serve as a live-attenuated vaccine. Here, we adapted that strategy, generating a mutant pandemic (H1N1) 2009 virus that grew well in cell culture, but replicated less well in mice than did wild-type virus. The mutant virus elicited sterile immunity in mice, completely protecting them from challenge with a pandemic (H1N1) 2009 virus. Our results indicate that M2 cytoplasmic tail mutants are suitable for live-attenuated vaccines against pandemic viruses.

Identification of broad binding class I HLA supertype epitopes to provide universal coverage of influenza A virus.

Influenza virus remains a significant health concern, with current circulating strains that affect millions each year plus the threat of newly emerging strains, such as swine-origin H1N1 and avian H5N1. Our hypothesis is that influenza-derived HLA-class I-restricted epitopes can be identified for use as a reagent to monitor and quantitate human CD8(+) T-cell responses and for vaccine development to induce protective cellular immunity. Protein sequences from influenza A virus strains currently in circulation, agents of past pandemics and zoonotic infections of man were evaluated for sequences predicted to bind to alleles representative of the most frequent HLA-A and -B (class I) types worldwide. Peptides that bound several different HLA molecules and were conserved among diverse influenza subtypes were tested for their capacity to recall influenza-specific immune responses using human donor PBMC. Accordingly, 28 different epitopes antigenic for human donor PBMC were identified and 25 were 100% conserved in the newly emerged swine-origin H1N1 strain. The epitope set defined herein should provide a reagent applicable to quantitate CD8(+) T cell human responses irrespective of influenza subtype and HLA composition of the responding population. In addition, these epitopes may be suitable for vaccine applications directed at the induction of cellular immunity.

Universal influenza DNA vaccine encoding conserved CD4+ T cell epitopes protects against lethal viral challenge in HLA-DR transgenic mice.

The goal of the present study was to design a vaccine that would provide universal protection against infection of humans with diverse influenza A viruses. Accordingly, protein sequences from influenza A virus strains currently in circulation (H1N1, H3N2), agents of past pandemics (H1N1, H2N2, H3N2) and zoonotic infections of man (H1N1, H5N1, H7N2, H7N3, H7N7, H9N2) were evaluated for the presence of amino acid sequences, motifs, that are predicted to mediate peptide epitope binding with high affinity to the most frequent HLA-DR allelic products. Peptides conserved among diverse influenza strains were then synthesized, evaluated for binding to purified HLA-DR molecules and for their capacity to induce influenza-specific immune recall responses using human donor peripheral blood mononuclear cells (PBMC). Accordingly, 20 epitopes were selected for further investigation based on their conservancy among diverse influenza strains, predicted population coverage in diverse ethnic groups and capacity to recall influenza-specific responses. A DNA plasmid encoding the epitopes was constructed using amino acid spacers between epitopes to promote optimum processing and presentation. Immunogenicity of the DNA vaccine was measured using HLA-DR4 transgenic mice and the TriGrid in vivo electroporation device. Vaccination resulted in peptide-specific immune responses, augmented HA-specific antibody responses and protection of HLA-DR4 transgenic mice from lethal PR8 influenza virus challenge. These studies demonstrate the utility of this vaccine format and the contribution of CD4(+) T cell responses to protection against influenza infection.

Rationally engineered therapeutic proteins with reduced immunogenicity.

Chronic administration of protein therapeutics may elicit unacceptable immune responses to the specific protein. Our hypothesis is that the immunogenicity of protein drugs can be ascribed to a few immunodominant helper T lymphocyte (HTL) epitopes, and that reducing the MHC binding affinity of these HTL epitopes contained within these proteins can generate drugs with lower immunogenicity. To test this hypothesis, we studied the protein therapeutic erythropoietin (Epo). Two regions within Epo, designated Epo 91-120 and Epo 126-155, contained HTL epitopes that were recognized by individuals with numerous HLA-DR types, a property common to immunodominant HTL epitopes. We then engineered analog epitopes with reduced HLA binding affinity. These analog epitopes were associated with reduced in vitro immunogenicity. Two modified forms of Epo containing these substitutions were shown to be bioactive and nonimmunogenic in vitro. These findings support our hypothesis and demonstrate that immunogenicity of protein drugs can be reduced in a systematic and predictable manner.