Human mucosal-associated invariant T cells contribute to antiviral influenza immunity via IL-18–dependent activation

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes known to elicit potent immunity to a broad range of bacteria, mainly via the rapid production of inflammatory cytokines. Whether MAIT cells contribute to antiviral immunity is less clear. Here we asked whether MAIT cells produce cytokines/chemokines during severe human influenza virus infection. Our analysis in patients hospitalized with avian H7N9 influenza pneumonia showed that individuals who recovered had higher numbers of CD161+Vα7.2+ MAIT cells in peripheral blood compared with those who succumbed, suggesting a possible protective role for this lymphocyte population. To understand the mechanism underlying MAIT cell activation during influenza, we cocultured influenza A virus (IAV)-infected human lung epithelial cells (A549) and human peripheral blood mononuclear cells in vitro, then assayed them by intracellular cytokine staining. Comparison of influenza-induced MAIT cell activation with the profile for natural killer cells (CD56+CD3−) showed robust up-regulation of IFNγ for both cell populations and granzyme B in MAIT cells, although the individual responses varied among healthy donors. However, in contrast to the requirement for cell-associated factors to promote NK cell activation, the induction of MAIT cell cytokine production was dependent on IL-18 (but not IL-12) production by IAV-exposed CD14+ monocytes. Overall, this evidence for IAV activation via an indirect, IL-18–dependent mechanism indicates that MAIT cells are protective in influenza, and also possibly in any human disease process in which inflammation and IL-18 production occur

Enhanced CD8+ T-cell response in mice immunized with NS1-truncated influenza virus

Influenza viruses with truncated NS1 protein stimulate a more intensive innate immune response compared to their wild type counterparts. Here, we investigate how the shortening of the NS1 protein influence the immunogenicity of the conserved T-cellular epitopes of influenza virus. Using flow cytometry, we showed that the intraperitoneal immunization of mice with influenza virus encoding 124 N-terminal amino acid residues of the NS1 protein (A/PR8/NS124) induced higher levels of CD8+ T-cells recognizing immunodominant (NP366-374) and sub-immunodominant (NP161-175, NP196-210, HA323-337, HA474-483, NA427-433) epitopes compared to immunization with the virus expressing full-length NS1 (A/PR8/full NS). It is noteworthy that the response to the immunodominant influenza epitope NP366-374 was achieved with the lower immunization dose of A/PR8/NS124 virus compared to the reference wild type strain. Despite the fact that polyfunctional CD8+ effector memory T-lymphocytes simultaneously producing two (IFNγ and TNFα) or three (IFNγ, IL2, and TNFα) cytokines prevailed in the immune response to both viruses, the relative number of such T-cells was higher in A/PR8/NS124-immunized mice. Furthermore, we have found that polyfunctional populations of lymphocytes generated upon the immunization of mice with the mutant virus demonstrated an increased capacity to produce IFNγ compared to the corresponding populations derived from the A/PR8/full NS-immunized mice. Therefore, immunization with the attenuated influenza virus encoding truncated NS1 protein ensures a more potent CD8+ T-cell immune response.Influenza viruses with truncated NS1 protein stimulate a more intensive innate immune response compared to their wild type counterparts. Here, we investigate how the shortening of the NS1 protein influence the immunogenicity of the conserved T-cellular epitopes of influenza virus. Using flow cytometry, we showed that the intraperitoneal immunization of mice with influenza virus encoding 124 N-terminal amino acid residues of the NS1 protein (A/PR8/NS124) induced higher levels of CD8+ T-cells recognizing immunodominant (NP366-374) and sub-immunodominant (NP161-175, NP196-210, HA323-337, HA474-483, NA427-433) epitopes compared to immunization with the virus expressing full-length NS1 (A/PR8/full NS). It is noteworthy that the response to the immunodominant influenza epitope NP366-374 was achieved with the lower immunization dose of A/PR8/NS124 virus compared to the reference wild type strain. Despite the fact that polyfunctional CD8+ effector memory T-lymphocytes simultaneously producing two (IFNγ and TNFα) or three (IFNγ, IL2, and TNFα) cytokines prevailed in the immune response to both viruses, the relative number of such T-cells was higher in A/PR8/NS124-immunized mice. Furthermore, we have found that polyfunctional populations of lymphocytes generated upon the immunization of mice with the mutant virus demonstrated an increased capacity to produce IFNγ compared to the corresponding populations derived from the A/PR8/full NS-immunized mice. Therefore, immunization with the attenuated influenza virus encoding truncated NS1 protein ensures a more potent CD8+ T-cell immune response

Enhanced CD8 + T-cell response in mice immunized with NS1-truncated influenza virus

Influenza viruses with truncated NS1 protein stimulate a more intensive innate immune response compared to their wild type counterparts. Here, we investigate how the shortening of the NS1 protein influence the immunogenicity of the conserved T-cellular epitopes of influenza virus. Using flow cytometry, we showed that the intraperitoneal immunization of mice with influenza virus encoding 124 N-terminal amino acid residues of the NS1 protein (A/PR8/NS124) induced higher levels of CD8 + T-cells recognizing immunodominant (NP 366-374 ) and sub-immunodominant (NP 161-175 , NP 196-210 , HA 323-337 , HA 474-483 , NA 427-433 ) epitopes compared to immunization with the virus expressing full-length NS1 (A/PR8/full NS). It is noteworthy that the response to the immunodominant influenza epitope NP 366-374 was achieved with the lower immunization dose of A/PR8/NS124 virus compared to the reference wild type strain. Despite the fact that polyfunctional CD8+ effector memory T-lymphocytes simultaneously producing two (IFNγ and TNFα) or three (IFNγ, IL2, and TNFα) cytokines prevailed in the immune response to both viruses, the relative number of such T-cells was higher in A/PR8/NS124-immunized mice. Furthermore, we have found that polyfunctional populations of lymphocytes generated upon the immunization of mice with the mutant virus demonstrated an increased capacity to produce IFNγ compared to the corresponding populations derived from the A/PR8/full NS-immunized mice. Therefore, immunization with the attenuated influenza virus encoding truncated NS1 protein ensures a more potent CD8 + T-cell immune response

Conformational occlusion of blockade antibody epitopes, a novel mechanism of GII.4 human norovirus immune evasion

Extensive antigenic diversity within the GII.4 genotype of human norovirus is a major driver of pandemic emergence and a significant obstacle to development of cross-protective immunity after natural infection and vaccination. However, human and mouse monoclonal antibody studies indicate that, although rare, antibodies to conserved GII.4 blockade epitopes are generated. The mechanisms by which these epitopes evade immune surveillance are uncertain. Here, we developed a new approach for identifying conserved GII.4 norovirus epitopes. Utilizing a unique set of virus-like particles (VLPs) representing the in vivo-evolved sequence diversity within an immunocompromised person, we identify key residues within epitope F, a conserved GII.4 blockade antibody epitope. The residues critical for antibody binding are proximal to evolving blockade epitope E. Like epitope F, antibody blockade of epitope E was temperature sensitive, indicating that particle conformation regulates antibody access not only to the conserved GII.4 blockade epitope F but also to the evolving epitope E. These data highlight novel GII.4 mechanisms to protect blockade antibody epitopes, map essential residues of a GII.4 conserved epitope, and expand our understanding of how viral particle dynamics may drive antigenicity and antibody-mediated protection by effectively shielding blockade epitopes. Our data support the notion that GII.4 particle breathing may well represent a major mechanism of humoral immune evasion supporting cyclic pandemic virus persistence and spread in human populations. IMPORTANCE In this study, we use norovirus virus-like particles to identify key residues of a conserved GII.4 blockade antibody epitope. Further, we identify an additional GII.4 blockade antibody epitope to be occluded, with antibody access governed by temperature and particle dynamics. These findings provide additional support for particle conformation-based presentation of binding residues mediated by a particle “breathing core.” Together, these data suggest that limiting antibody access to blockade antibody epitopes may be a frequent mechanism of immune evasion for GII.4 human noroviruses. Mapping blockade antibody epitopes, the interaction between adjacent epitopes on the particle, and the breathing core that mediates antibody access to epitopes provides greater mechanistic understanding of epitope camouflage strategies utilized by human viral pathogens to evade immunity

Generation and Comprehensive Analysis of Host Cell Interactome of the PA Protein of the Highly Pathogenic H5N1 Avian Influenza Virus in Mammalian Cells

Accumulating data have identified the important roles of PA protein in replication and pathogenicity of influenza A virus (IAV). Identification of host factors that interact with the PA protein may accelerate our understanding of IAV pathogenesis. In this study, using immunoprecipitation assay combined with liquid chromatography-tandem mass spectrometry, we identified 278 human cellular proteins that might interact with PA of H5N1 IAV. Gene Ontology annotation revealed that the identified proteins are highly associated with viral translation and replication. Further KEGG pathway analysis of the interactome profile highlighted cellular pathways associated with translation, infectious disease, and signal transduction. In addition, Diseases and Functions analysis suggested that these cellular proteins are highly related with Organismal Injury and Abnormalities and Cell Death and Survival. Moreover, two cellular proteins (nucleolin and eukaryotic translation elongation factor 1-alpha 1) identified both in this study and others were further validated to interact with PA using co-immunoprecipitation and co-localization assays. Therefore, this study presented the interactome data of H5N1 IAV PA protein in human cells which may provide novel cellular target proteins for elucidating the potential molecular functions of PA in regulating the lifecycle of IAV in human cells

Designing a chimeric subunit vaccine for influenza virus, based on HA2, M2e and CTxB: a bioinformatics study

Background: Type A influenza viruses are contagious and even life-threatening if left untreated. So far, no broadly protective vaccine is available due to rapid antigenic changes and emergence of new subtypes of influenza virus. In this study, we exploited bioinformatics tools in order to design a subunit chimeric vaccine from the antigenic and highly conserved regions of HA and M2 proteins of H7N9 subtype of influenza virus. We used mucosal adjuvant candidates, including CTxB, STxB, ASP-1, and LTB to stimulate mucosal immunity and analyzed the combination of HA2, M2e, and the adjuvant. Furthermore, to improve the antigen function and to maintain their three-dimensional structure, 12 different linkers including six rigid linkers and six flexible linkers were used. The 3D structure model was generated using a combination of homology and ab initio modeling methods and the molecular dynamics of the model were analyzed, either. Results: Analysis of different adjuvants showed that using CtxB as an adjuvant, results in higher overall vaccine stability and higher half-life among four adjuvant candidates. Fusion of antigens and the CTxB in the form of M2e-linker-CTxB-linker-HA2 has the most stability and half life compared to other combination forms. Furthermore, the KPKPKP rigid linker showed the best result for this candidate vaccine among 12 analyzed linkers. The changes in the vaccine 3D structure made by linker insertion found to be negligible, however, although small, the linker insertion between the antigens causes the structure to change slightly. Eventually, using predictive tools such as Ellipro, NetMHCpan I and II, CD4episcore, CTLpred, BepiPred and other epitope analyzing tools, we analyzed the conformational and linear epitopes of the vaccine. The solubility, proteasome cleavage sites, peptidase and potential chemical cutters, codon optimization, post translational modification were also carried out on the final vaccine. Conclusions: It is concluded that M2e-Linker-CTxB-Linker-HA2 combination of chimeric vaccine retains its 3D structure and antigenicity when KPKPKP used as linker and CTxB used as adjuvant. © 2020, The Author(s)

Universal immunity to influenza must outwit immune evasion

Although an influenza vaccine has been available for 70 years, influenza virus still causes seasonal epidemics and worldwide pandemics. Currently available vaccines elicit strain-specific antibody responses to the surface haemagglutinin (HA) and neuraminidase (NA) proteins, but these can be ineffective against serologically-distinct viral variants and novel subtypes. Thus, there is a need for cross-protective or universal influenza vaccines to overcome the necessity for annual immunisation against seasonal influenza and to provide immunity to reduce the severity of infection with pandemic or outbreak viruses. It is well established that natural influenza infection can provide cross-reactive immunity that can reduce the impact of infection with distinct influenza type A strains and subtypes, including H1N1, H3N2, H2N2, H5N1 and H7N9. The key to generating universal influenza immunity via vaccination is to target functionally-conserved regions of the virus, which include epitopes on the internal proteins for cross-reactive T cell immunity or on the HA stem for broadly reactive antibody responses. In the wake of the 2009 H1N1 pandemic, broadly neutralizing antibodies have been characterized and isolated from convalescent and vaccinated individuals, inspiring development of new vaccination techniques to elicit such responses. Induction of influenza-specific T cell responses through vaccination has also been examined in clinical trials. Strong evidence is available from human and animal models of influenza to show that established influenza-specific T cell memory can reduce viral shedding and symptom severity. However, the published evidence also shows that CD8+ T cells can efficiently select immune escape mutants early after influenza virus infection. Here, we discuss universal immunity to influenza viruses mediated by both cross-reactive T cells and antibodies, the mechanisms of immune evasion in influenza, and how to counteract commonly occurring-escape variants

Influenza Virus-specific CD8+ T Cells

Influenza viruses are among the leading causes of acute respiratory tract infections worldwide. Natural influenza virus infections elicit both humoral and cellular immune responses. Although, neutralizing antibodies directed to the hemagglutinin (HA) globular head domain prevent reinfection with the same influenza virus they exert limited/no cross-reactivity with antigenically drifted variants or influenza viruses of different strains, it is therefore of interest to identify other correlates of protection. Cellular immunity, especially influenza virus-specific CD8+ cytotoxic T lymphocytes (CTLs), contribute to rapid clearance of influenza virus infections and thereby reduce viral shedding. Influenza virus-specific CTLs, elicited after seasonal influenza virus infections, are mainly directed to conserved internal proteins. In this dissertation we were able to demonstrate the cross-reactivity of seasonal influenza virus-specific CTLs with the novel and potentially pandemic A/H7N9 virus and between different lineages of influenza B viruses. Furthermore, using an unique PBMC donor cohort we were able to assess the longevity of these cells in healthy individuals. In addition, we were able to demonstrate that human influenza A viruses can impair the recognition of the HLA-A*0201 restricted and highly conserved M158-66 epitope by specific CTLs by variations in the extra-epitopic amino acids. This CTL evasion strategy may have implications for the viral replication kinetics in HLA-A*0201 individuals and thus the spread of influenza A viruses in the human population. Finally, we describe a novel adjuvant, G3/DT, that improves the immunogenicity of a standard inactivated seasonal influenza vaccine in terms of enhancing the antibody response and inducing a protective CTL response