Heterologous Influenza Virus Research Articles

Conserved HA-peptide NG34 formulated in pCMV-CTLA4-Ig reduces viral shedding in pigs after a heterosubtypic influenza virus SwH3N2 challenge.

Swine influenza viruses (SIVs), the causal agents of swine influenza, are not only important to control due to the economic losses in the swine industry, but also can be pandemic pathogens. Vaccination is one of the most relevant strategies to control and prevent influenza infection. Current human vaccines against influenza induce strain-specific immunity and annual update is required due to the virus antigenic shift phenomena. Previously, our group has reported the use of conserved hemagglutinin peptides (HA-peptides) derived from H1-influenza virus as a potential multivalent vaccine candidate. Immunization of swine with these HA-peptides elicited antibodies that recognized and neutralized heterologous influenza viruses in vitro and demonstrated strong hemagglutination-inhibiting activity. In the present work, we cloned one HA-peptide (named NG34) into a plasmid fused with cytotoxic T lymphocyte-associated antigen (CTLA4) which is a molecule that modifies T cell activation and with an adjuvant activity interfering with the adaptive immune response. The resulting plasmid, named pCMV-CTLA4-Ig-NG34, was administered twice to animals employing a needle-free delivery approach. Two studies were carried out to test the efficacy of pCMV-CTLA4-Ig-NG34 as a potential swine influenza vaccine, one in seronegative and another in seropositive pigs against SIV. The second one was aimed to evaluate whether pCMV-CTLA4-Ig-NG34 vaccination would overcome maternally derived antibodies (MDA). After immunization, all animals were intranasally challenged with an H3N2 influenza strain. A complete elimination or significant reduction in the viral shedding was observed within the first week after the challenge in the vaccinated animals from both studies. In addition, no challenged heterologous virus load was detected in the airways of vaccinated pigs. Overall, it is suggested that the pCMV-CTLA4-Ig-NG34 vaccine formulation could potentially be used as a multivalent vaccine against influenza viruses.

Enhanced cross-lineage protection induced by recombinant H9N2 avian influenza virus inactivated vaccine

BackgroundAntigenic drift of H9N2 low pathogenic avian influenza viruses (AIV) may result in vaccination failure in the poultry industry and thus a cross-protective vaccine against H9N2 AIV is highly desirable. MethodsA series of H9N2 recombinant viruses with the internal genes of A/Puerto Rico/8/34 (H1N1, PR8) were generated, based on the compatibility between HA and NA, the effect of HA deglycosylation, and protective antigenic epitopes in HA. After evaluation of their biological and immunological characteristics, three recombinant AIVs with the internal genes of the Y280-like strain SN were selected for protective efficacy studies. ResultsThe recombinant viruses rHASNNA3, rHASN-△200, rHASN-△287, and rHASN-R92G-E93K displayed good cross reactivity and induced higher neutralization antibody titers against both SN and the F98-like strain YZ4. Furthermore, those recombinant viruses had a higher EID50 in chicken embryos after the replacement of internal-gene backbone from PR8 to SN. The rSNHA-△200 induced better protection in immunized chickens against challenge of homologous and heterologous H9N2 avian influenza viruses when compared with the wild type strain. ConclusionThe recombinant virus rSNHA-△200 can be used as a potential broad-spectrum vaccine against H9N2 avian influenza.

Heterosubtypic protection against avian influenza virus by live attenuated and chimeric norovirus P-particle-M2e vaccines in chickens

Avian influenza in poultry continues to be a great concern worldwide, and the currently licensed inactivated influenza vaccines are not effective against the novel strains of influenza virus that continue to emerge in the field. This warrants the development of more broadly protective influenza vaccines or vaccination regimens. Live attenuated influenza vaccines (LAIVs) and subunit vaccines derived from viral peptides, such as the highly conserved ectodomain of influenza virus matrix protein 2 (M2e), can offer a more broadly reactive immune response. In chickens, we previously showed that a chimeric norovirus P particle containing M2e (M2eP) could provide partial but broad immunity, when administered as a standalone vaccine, and also enhanced the protective efficacy of inactivated vaccine when used in a combination regimen. We also demonstrated that a naturally-selected NS1-truncated H7N3 LAIV (pc4-LAIV) was highly efficacious against antigenically distant heterologous H7N2 low pathogenicity avian influenza virus challenge, especially when used as the priming vaccine in a prime-boost vaccination regimen. In this study, we investigated the cross-subtype protective efficacy of pc4-LAIV in conjunction with M2eP using single vaccination, combined treatment, and prime-boost approaches. Chickens vaccinated with pc4-LAIV showed significant reduction of tracheal shedding of a low pathogenicity H5N2 challenge virus. This cross-subtype protective efficacy was further enhanced, during the initial stages of challenge virus replication, in chickens that received a vaccination regimen consisting of priming with pc4-LAIV at 1 day of age and boosting with M2eP. Further, H5N2-specific serum IgG and pc4-LAIV-specific hemagglutination-inhibition antibody titers were enhanced in LAIV-primed and M2eP boost-vaccinated chickens. Taken together, our data point to the need of further investigation into the benefits of combined and prime-boost vaccination schemes utilizing LAIV and epitope-based vaccines, to develop more broadly protective vaccination regimens.

Monoclonal antibodies to hemagglutinin of influenza b viruses victoria evolutionary lineage.

Co-circulation of two evolutionary distinct lineages of influenza virus in one epidemic season has led to development specifc reagents for rapid identifcation and typing of new isolates. Panel of MAbs to hemagglutinin of influenza virus B/Brisbane/46/15 belonging to Victoria evolutionary lineage was developed. All MAbs reacted in ELISA with B/Victoria-like strains only. There were no interactions with heterologous influenza viruses of B/Yamagata lineage, seasonal and potentially pandemic influenza A viruses. All MAbs reacted in hemagglutination inhibition and virus neutralization. MAbs interacted in hemagglutination inhibition only with B/Victoria-like viruses, but did not interacted B/Yamagata-like strains. Neutralization and hemagglutination inhibition studies of viruses isolated before 1983 with MAbs revealed that MAbs 6E11, 9G5, 9B5 and 6A4 had the ability to interact with the virus B/ Russia/69 which may evidence that B strains of early isolation period (before lineage separation) have common epitope with recent Victoria lineage viruses. MAbs 7C8, 7G9, 7H8 and 8D11 were directed to a conserved epitope (or epitopes) specifc for influenza hemagglutinin viruses of B/Victoria group. The presence of differences in the effectiveness of the interaction of MAbs 6A9, 7G9 and 8A8 in hemagglutination inhibition test allows the identifcation and differentiation of strains isolated in chicken embryos and MDCK cell culture. Thus, the developed MAbs can be successfully used for identifcation and antigenic analysis of B/Victoria-like strains.

A unique nanoparticulate TLR9 agonist enables a HA split vaccine to confer FcγR-mediated protection against heterologous lethal influenza virus infection.

The development of a universal influenza vaccine that can provide a robust and long-lasting protection against a broader range of influenza virus strains is a global public health priority. One approach to improve vaccine efficacy is to use an adjuvant to boost immune responses to the target antigens; nevertheless, the role of adjuvants in the context of influenza vaccines is not fully understood. We have previously developed the K3-schizophyllan (SPG) adjuvant, which is composed of nanoparticulated oligodeoxynucleotides K3, a TLR9 agonist, with SPG, a non-agonistic β-glucan ligand of Dectin-1. In this study, K3-SPG given with conventional influenza hemagglutinin (HA) split vaccine (K3-SPG HA) conferred protection against antigenically mismatched heterologous virus challenge. While K3-SPG HA elicited robust cross-reactive HA-specific IgG2c and CD8 T-cell responses, CD8 T-cell depletion had no impact on this cross-protection. In contrast, K3-SPG HA was not able to confer protection against heterologous virus challenge in FcRγ-deficient mice. Our results indicated that FcγR-mediated antibody responses induced by the HA antigen and K3-SPG adjuvant were important for potent protection against antigenically mismatched influenza virus infection. Thus, we demonstrated that the K3-SPG-adjuvanted vaccine strategy broadens protective immunity against influenza and provides a basis for the development of next-generation influenza vaccines.

DAMP-Inducing Adjuvant and PAMP Adjuvants Parallelly Enhance Protective Type-2 and Type-1 Immune Responses to Influenza Split Vaccination.

Recently, it was reported that 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD), a common pharmaceutical additive, can act as a vaccine adjuvant to enhance protective type-2 immunogenicity to co-administered seasonal influenza split vaccine by inducing host-derived damage-associated molecular patterns (DAMPs). However, like most other DAMP-inducing adjuvants such as aluminum hydroxide (Alum), HP-β-CyD may not be sufficient for the induction of protective type-1 (cellular) immune responses, thereby leaving room for improvement. Here, we demonstrate that a combination of HP-β-CyD with a humanized TLR9 agonist, K3 CpG-ODN, a potent pathogen-associated molecular pattern (PAMP), enhanced the protective efficacy of the co-administered influenza split vaccine by inducing antigen-specific type-2 and type-1 immune responses, respectively. Moreover, substantial antigen-specific IgE induction by HP-β-CyD, which can cause an allergic response to immunized antigen was completely suppressed by the addition of K3 CpG-ODN. Furthermore, HP-β-CyD- and K3 CpG-ODN-adjuvanted influenza split vaccination protected the mice against lethal challenge with high doses of heterologous influenza virus, which could not be protected against by single adjuvant vaccines. Further experiments using gene deficient mice revealed the unique immunological mechanism of action in vivo, where type-2 and type-1 immune responses enhanced by the combined adjuvants were dependent on TBK1 and TLR9, respectively, indicating their parallel signaling pathways. Finally, the analysis of immune responses in the draining lymph node suggested that HP-β-CyD promotes the uptake of K3 CpG-ODN by plasmacytoid dendritic cells and B cells, which may contributes to the activation of these cells and enhanced production of IgG2c. Taken together, the results above may offer potential clinical applications for the combination of DAMP-inducing adjuvant and PAMP adjuvant to improve vaccine immunogenicity and efficacy by enhancing both type-2 and type-1 immune responses in a parallel manner.

Inclusion of membrane-anchored LTB or flagellin protein in H5N1 virus-like particles enhances protective responses following intramuscular and oral immunization of mice

We previously demonstrated that intramuscular immunization with virus-like particles (VLPs) composed of the haemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins of A/meerkat/Shanghai/SH-1/2012 (clade 2.3.2.1) protected mice from lethal challenge with viruses from other H5 HPAI clades. The inclusion of additional proteins that can serve as immunological adjuvants in VLPs may enhance adaptive immune responses following vaccination, and oral vaccines may represent the safest choice. Here, we report the generation of H5N1 VLPs composed of the viral HA, NA, and M1 proteins and membrane-anchored forms of the Escherichia coli heat-labile enterotoxin B subunit protein (LTB) or the Toll-like receptor 5 ligand flagellin (Flic). Mice intramuscularly or orally immunized with VLPs containing LTB or Flic generated greater humoural and cellular immune responses than those administered H5N1 VLPs without LTB or Flic. Intramuscular immunization with VLPs protected mice from lethal challenge with homologous or heterologous H5N1 viruses irrespective of whether the VLPs additionally included LTB or Flic. In contrast, oral immunization of mice with LTB- or Flic-VLPs conferred substantial protection against lethal challenge with both homologous and heterologous H5N1 influenza viruses, whereas mice immunized orally with VLPs lacking LTB and Flic universally succumbed to infection. Mice immunized orally with LTB- or Flic-VLPs showed 10-fold higher virus-specific IgG titres than mice immunized with H5N1-VLPs lacking LTB or Flic. Collectively, these results indicate that the inclusion of immunostimulatory proteins, such as LTB and Flic, in VLP-based vaccines may represent a promising new approach for the control of current H5N1 HPAI outbreaks by eliciting higher humoural and cellular immune responses and conferring improved cross-clade protection.

Nucleoside-modified mRNA immunization elicits influenza virus hemagglutinin stalk-specific antibodies

Currently available influenza virus vaccines have inadequate effectiveness and are reformulated annually due to viral antigenic drift. Thus, development of a vaccine that confers long-term protective immunity against antigenically distant influenza virus strains is urgently needed. The highly conserved influenza virus hemagglutinin (HA) stalk represents one of the potential targets of broadly protective/universal influenza virus vaccines. Here, we evaluate a potent broadly protective influenza virus vaccine candidate that uses nucleoside-modified and purified mRNA encoding full-length influenza virus HA formulated in lipid nanoparticles (LNPs). We demonstrate that immunization with HA mRNA-LNPs induces antibody responses against the HA stalk domain of influenza virus in mice, rabbits, and ferrets. The HA stalk-specific antibody response is associated with protection from homologous, heterologous, and heterosubtypic influenza virus infection in mice.

Influenza virus-like particles composed of conserved influenza proteins and GPI-anchored CCL28/GM-CSF fusion proteins enhance protective immunity against homologous and heterologous viruses

Influenza viruses cause significant morbidity and mortality and pose a substantial threat to public health. Vaccination represents the principle means of preventing influenza virus infection. Current vaccine approaches are hindered by the need to routinely reformulate vaccine compositions in an effort to account for the progressive antigenic changes that occur as influenza viruses circulate in the human population. In this study, we evaluated chimeric virus-like particle (cVLP) vaccines containing conserved elements of influenza proteins (HL5M2e (HA stem gene with 5M2e gene inserted) and NP), with or without glycosylphosphatidylinositol-anchored CCL28 (GPI-CCL28) and/or GM-CSF (GPI-GM-CSF) fusion proteins as molecular adjuvants. cVLPs elicited strong humoral and cellular immune responses against homologous and heterologous viruses, and improved survival following lethal challenge with both homologous and heterologous viruses. Inclusion of GPI-anchored adjuvants in cVLP vaccines augmented the generation of influenza-specific humoral and cellular immune responses in mice in comparison to the non-adjuvanted cVLP vaccines. VLPs containing GPI-anchored adjuvants reduced morbidity and improved survival to lethal challenge with homologous and heterologous influenza viruses. This work suggests that VLP vaccines incorporating conserved influenza virus proteins and GPI-anchored molecular adjuvants may serve as a platform for a broadly protective “universal” influenza vaccine.

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.

Applying Unique Molecular Identifiers in Next Generation Sequencing Reveals a Constrained Viral Quasispecies Evolution under Cross-Reactive Antibody Pressure Targeting Long Alpha Helix of Hemagglutinin.

To overcome yearly efforts and costs for the production of seasonal influenza vaccines, new approaches for the induction of broadly protective and long-lasting immune responses have been developed in the past decade. To warrant safety and efficacy of the emerging crossreactive vaccine candidates, it is critical to understand the evolution of influenza viruses in response to these new immune pressures. Here we applied unique molecular identifiers in next generation sequencing to analyze the evolution of influenza quasispecies under in vivo antibody pressure targeting the hemagglutinin (HA) long alpha helix (LAH). Our vaccine targeting LAH of hemagglutinin elicited significant seroconversion and protection against homologous and heterologous influenza virus strains in mice. The vaccine not only significantly reduced lung viral titers, but also induced a well-known bottleneck effect by decreasing virus diversity. In contrast to the classical bottleneck effect, here we showed a significant increase in the frequency of viruses with amino acid sequences identical to that of vaccine targeting LAH domain. No escape mutant emerged after vaccination. These results not only support the potential of a universal influenza vaccine targeting the conserved LAH domains, but also clearly demonstrate that the well-established bottleneck effect on viral quasispecies evolution does not necessarily generate escape mutants.

Seasonal Influenza Split Vaccines Confer Partial Cross-Protection against Heterologous Influenza Virus in Ferrets When Combined with the CAF01 Adjuvant.

Influenza epidemics occur annually, and estimated 5–10% of the adult population and 20–30% of children will become ill from influenza infection. Seasonal vaccines primarily work through the induction of neutralizing antibodies against the principal surface antigen hemagglutinin (HA). This important role of HA-specific antibodies explains why previous pandemics have emerged when new HAs have appeared in circulating human viruses. It has long been recognized that influenza virus-specific CD4(+) T cells are important in protection from infection through direct effector mechanisms or by providing help to B cells and CD8(+) T cells. However, the seasonal influenza vaccine is poor at inducing CD4(+) T-cell responses and needs to be combined with an adjuvant facilitating this response. In this study, we applied the ferret model to investigate the cross-protective efficacy of a heterologous trivalent influenza split-virion (TIV) vaccine adjuvanted with the CAF01 adjuvant, with proven ability to induce CD4(+) T-cell and antibody responses in mice, ferrets, pigs, primates, and humans. Our results indicate that CAF01-adjuvanted vaccine induces HA inhibition (HAI)-independent protection after heterologous challenge, manifested as reduced viral load and fever. On the other hand, we observe increased inflammation in the airways and more neutrophil and mononuclear cell infiltration in these ferrets when compared with optimally protected animals, i.e., ferrets receiving the same vaccine but a homologous challenge. This suggest that HAI-independent immunity induced by TIV + CAF01 can reduce viral shedding and systemic disease symptoms, but does not reduce local inflammation in the nasal cavity.

Multigenic DNA vaccine induces protective cross-reactive T cell responses against heterologous influenza virus in nonhuman primates.

Recent avian and swine-origin influenza virus outbreaks illustrate the ongoing threat of influenza pandemics. We investigated immunogenicity and protective efficacy of a multi-antigen (MA) universal influenza DNA vaccine consisting of HA, M2, and NP antigens in cynomolgus macaques. Following challenge with a heterologous pandemic H1N1 strain, vaccinated animals exhibited significantly lower viral loads and more rapid viral clearance when compared to unvaccinated controls. The MA DNA vaccine induced robust serum and mucosal antibody responses but these high antibody titers were not broadly neutralizing. In contrast, the vaccine induced broadly-reactive NP specific T cell responses that cross-reacted with the challenge virus and inversely correlated with lower viral loads and inflammation. These results demonstrate that a MA DNA vaccine that induces strong cross-reactive T cell responses can, independent of neutralizing antibody, mediate significant cross-protection in a nonhuman primate model and further supports development as an effective approach to induce broad protection against circulating and emerging influenza strains.

Vaccination with Recombinant Parainfluenza Virus 5 Expressing Neuraminidase Protects against Homologous and Heterologous Influenza Virus Challenge.

Seasonal human influenza virus continues to cause morbidity and mortality annually, and highly pathogenic avian influenza (HPAI) viruses along with other emerging influenza viruses continue to pose pandemic threats. Vaccination is considered the most effective measure for controlling influenza; however, current strategies rely on a precise vaccine match with currently circulating virus strains for efficacy, requiring constant surveillance and regular development of matched vaccines. Current vaccines focus on eliciting specific antibody responses against the hemagglutinin (HA) surface glycoprotein; however, the diversity of HAs across species and antigenic drift of circulating strains enable the evasion of virus-inhibiting antibody responses, resulting in vaccine failure. The neuraminidase (NA) surface glycoprotein, while diverse, has a conserved enzymatic site and presents an appealing target for priming broadly effective antibody responses. Here we show that vaccination with parainfluenza virus 5 (PIV5), a promising live viral vector expressing NA from avian (H5N1) or pandemic (H1N1) influenza virus, elicited NA-specific antibody and T cell responses, which conferred protection against homologous and heterologous influenza virus challenges. Vaccination with PIV5-N1 NA provided cross-protection against challenge with a heterosubtypic (H3N2) virus. Experiments using antibody transfer indicate that antibodies to NA have an important role in protection. These findings indicate that PIV5 expressing NA may be effective as a broadly protective vaccine against seasonal influenza and emerging pandemic threats.IMPORTANCE Seasonal influenza viruses cause considerable morbidity and mortality annually, while emerging viruses pose potential pandemic threats. Currently licensed influenza virus vaccines rely on the antigenic match of hemagglutinin (HA) for vaccine strain selection, and most vaccines rely on HA inhibition titers to determine efficacy, despite the growing awareness of the contribution of neuraminidase (NA) to influenza virus vaccine efficacy. Although NA is immunologically subdominant to HA, and clinical studies have shown variable NA responses to vaccination, in this study, we show that vaccination with a parainfluenza virus 5 recombinant vaccine candidate expressing NA (PIV5-NA) from a pandemic influenza (pdmH1N1) virus or highly pathogenic avian influenza (H5N1) virus elicits robust, cross-reactive protection from influenza virus infection in two animal models. New vaccination strategies incorporating NA, including PIV5-NA, could improve seasonal influenza virus vaccine efficacy and provide protection against emerging influenza viruses.

Efficacy of an Adenoviral Vectored Multivalent Centralized Influenza Vaccine

Mice were immunized with Adenovirus expressing the H1-con, H2-con, H3-con and H5-con HA consensus genes in combination (multivalent) and compared to mice immunized with the traditional 2010–2011 FluZone and FluMist seasonal vaccines. Immunized mice were challenged with 10–100 MLD50 of H1N1, H3N1, H3N2 and H5N1 influenza viruses. The traditional vaccines induced robust levels of HA inhibition (HI) titers, but failed to protect against five different heterologous lethal influenza challenges. Conversely, the multivalent consensus vaccine (1 × 1010 virus particles (vp)/mouse) induced protective HI titers of ≥40 against 8 of 10 influenza viruses that represent a wide degree of divergence within the HA subtypes and protected 100% of mice from 8 of 9 lethal heterologous influenza virus challenges. The vaccine protection was dose dependent, in general, and a dose as low as 5 × 107 vp/mouse still provided 100% survival against 7 of 9 lethal heterologous influenza challenges. These data indicate that very low doses of Adenovirus-vectored consensus vaccines induce superior levels of immunity against a wide divergence of influenza subtypes as compared to traditional vaccines. These doses are scalable and translatable to humans and may provide the foundation for complete and long-lasting anti-influenza immunity.

Induction of lung CD8+ T cell responses by consecutive inoculations of a poly(I:C) influenza vaccine.

The cytotoxic T lymphocyte (CTL) response plays a key role in host recovery from influenza virus infection and in subsequent immunity. Compared to natural infection with influenza virus, however, intranasal vaccination with adjuvant-combined inactivated vaccine elicits only moderate CTL responses. Here we demonstrate that 5 days of consecutive, intranasal vaccination with a combination of inactivated influenza vaccine and poly(I:C) elicits a strong CTL response in the lung. Antigen-captured respiratory DCs did efficiently migrate from the lung to the mediastinal lymph node (mLN) after the 5 day series of inoculations with vaccine and poly(I:C). Importantly, formalin-inactivated whole virus vaccine and poly(I:C) adjuvant have synergic effects on consecutive vaccinations to elicit a strong CTL response in the lung. Although the CTL response was less effective against heterologous influenza virus, we show for the first time that intranasal administration of inactivated influenza virus vaccine and poly(I:C) for 5 consecutive days can elicit high levels of influenza virus-specific CD8+ T cells in the lung.

Highly conserved M2e and hemagglutinin epitope-based recombinant proteins induce protection against influenza virus infection

Highly pathogenic influenza viruses continue to cause serious threat to public health due to their pandemic potential, calling for an urgent need to develop effective, safe, convenient, and universal vaccines against influenza virus infection. In this study, we constructed two recombinant protein vaccines, 2H5M2e-2H7M2e-H5FP-H7FP (hereinafter M2e-FP-1) and 2H5M2e-H5FP-2H7M2e-H7FP (hereinafter M2e-FP-2), by respectively linking highly conserved sequences of two molecules of ectodomain of M2 (M2e) and one molecule of fusion peptide (FP) epitope of hemagglutinin (HA) of H5N1 and H7N9 influenza viruses in different orders. The Escherichia coli-expressed M2e-FP-1 and M2e-FP-2 proteins induced similarly high-titer M2e-FP-specific antibodies in the immunized mice. Importantly, both proteins were able to prevent lethal challenge of heterologous H1N1 influenza virus, with significantly reduced viral titers and alleviated pathological changes in the lungs, as well as increased body weight and complete survivals, in the challenge mice. Taken together, our study demonstrates that highly conserved M2e and FP epitope of HA of H5N1 and H7N9 influenza viruses can be used as important targets for development of safe and economical universal influenza vaccines, and that the position of H7N9 M2e and H5N1 HA epitope sequences in the vaccine components has no significant effects on the immunogenicity and efficacy of M2e-FP-based subunit vaccines.

Roles of antibodies to influenza A virus hemagglutinin, neuraminidase, and M2e in conferring cross protection

Although neuraminidase (NA) is the second major viral glycoprotein of influenza virus, its immune mechanism as a vaccine target has been less considered. Here we compared the properties of antibodies and the efficacy of cross protection by N1 and N2 NA proteins, inactivated split influenza vaccines (split), and tandem repeat extracellular domain M2 on virus-like particles (M2e5x VLP). Anti-NA immune sera could confer better cross-protection against multiple heterologous influenza viruses correlating with NA inhibition activity compared to split vaccine immune sera. Whereas split vaccine was superior to NA in conferring homologous protection. NA and M2e immune sera each showed comparable survival protection. Protective efficacy by NA immune sera was lower in Fc receptor common γ-chain deficient mice but comparable in C3 complement deficient mice compared to that in wild type mice, suggesting a role of Fc receptor in NA immunity.

Efficacy of Live-Attenuated H9N2 Influenza Vaccine Candidates Containing NS1 Truncations against H9N2 Avian Influenza Viruses.

H9N2 avian influenza virus is a zoonotic agent with a broad host range that can contribute genetic information to H5 or H7N9 subtype viruses, which are significant threats to both humans and birds. Thus, there is a great need for a vaccine to control H9N2 avian influenza. Three mutant viruses of an H9N2 virus A/chicken/Taixing/10/2010 (rTX-NS1-73, rTX-NS1-100, and rTX-NS1-128) were constructed with different NS1 gene truncations and confirmed by western blot analysis. The genetic stability, pathogenicity, transmissibility, and host immune responses toward these mutants were evaluated. The mutant virus rTX-NS1-128 exhibited the most attenuated phenotype and lost transmissibility. The expression levels of interleukin 12 in the nasal and tracheal tissues from chickens immunized with rTX-NS1-128 were significantly upregulated on day 3 post-immunization and the IgA and IgG antibody levels were significantly increased on days 7, 14, and 21 post-immunization when compared to chickens that received an inactivated vaccine. rTX-NS1-128 also protected chickens from challenge by homologous and heterologous H9N2 avian influenza viruses. The results indicate that rTX-NS1-128 can be used as a potential live-attenuated vaccine against H9N2 avian influenza.

Pulmonary monocytes promote the establishment of lung tissue-resident CD8 T cell memory following influenza infection

Abstract Lung tissue resident memory CD8 T cells (TRM) are critical for protection against heterologous influenza virus challenge by promoting rapid viral clearance. Unlike other mucosal tissues, we have found that local environmental signals are insufficient for the establishment of lung TRM, and that pulmonary antigen encounter is necessary for TRM to differentiate in the lung. Therefore, we investigated the role of lung antigen presenting cell (APC) subsets in the establishment of lung CD8 TRM following influenza infection. The initial appearance of flu-specific CD8 T cells in the lung with a TRM phenotype begins around day 10 post-infection, which coincides with viral clearance. Correspondingly, while most lung APC subsets contain influenza antigens over the course of infection, monocytes are the most numerous lung APC subset containing influenza antigens at the time of TRM appearance. Furthermore, imaging shows direct interaction between monocytes and T cells in the lung, supporting the idea that monocytes can act as drivers of lung TRM differentiation. In a murine influenza model, CCR2−/− mice, which have a defect in monocyte recruitment to the lung, had significantly fewer flu-specific lung CD8 TRM than WT controls. Notably, this defect was not observed in the flu-specific CD8 memory T cells in the lung vasculature or mediastinal lymph node, indicating this effect is specific for tissue-resident memory cells. In addition, this phenotype is not driven by a lack of CCR2 on T cells, as adoptive transfer of wild-type OT-I T cells into CCR2−/− mice yields the same deficiency of lung TRM. Thus, these data show that antigen presentation by lung CCR2+ monocytes has an important role in the establishment of lung CD8 TRM.