HA2-FimA DNA Vaccine Treats Experimental Periodontitis.

PURPOSE: To study the therapeutic effect of hemagglutinin-2 and fimbrial (HA2-FimA) vaccine on experimental periodontitis in rats. MATERIALS AND METHODS: The first batch of rats was divided into two groups and immunised with pure water or pVAX1-HA2-FimA at the age of 6, 7, and 9 weeks. After sacrificing the animals, total RNA was extracted from the spleens for RNA high-throughput sequencing (RNA-Seq) analysis. The second batch of rats was divided into four groups (A, B, C, D), and an experimental periodontitis rat model was established by suturing silk thread around the maxillary second molars of rats in groups B, C, and D for 4 weeks. The rats were immunised with pure water, pVAX1-HA2-FimA vaccine, empty pVAX1 vector, and pure water at 10, 11, and 13 weeks of age, respectively. Secretory immunoglobulin A (SIgA) antibodies and cathelicidin antimicrobial peptide (CAMP) levels in saliva were measured by enzyme-linked immunosorbent assay (ELISA). All rats were euthanised at 17 weeks of age, and alveolar bone loss was examined using micro-computed tomography (Micro-CT). RESULTS: Through sequencing analysis, six key genes, including Camp, were identified. Compared with the other three groups, the rats in the periodontitis+pVAX1-HA2-FimA vaccine group showed higher levels of SIgA and CAMP (p < 0.05). Micro-CT results showed significantly less alveolar bone loss in the periodontitis+pVAX1-HA2-FimA vaccine group compared to the periodontitis+pVAX1 group and periodontitis+pure water group (p < 0.05). CONCLUSION: HA2-FimA DNA vaccine can increase the levels of SIgA and CAMP in the saliva of experimental periodontitis model rats and reduce alveolar bone loss.

Contribution of Neuraminidase to the Efficacy of Seasonal Split Influenza Vaccines in the Ferret Model.

Seasonal influenza vaccination takes into account primarily hemagglutinin (HA)-specific neutralizing antibody responses. However, the accumulation of substitutions in the antigenic regions of HA (i.e., antigenic drift) occasionally results in a mismatch between the vaccine and circulating strains. To prevent poor vaccine performance, we investigated whether an antigenically matched neuraminidase (NA) may compensate for reduced vaccine efficacy due to a mismatched HA. Ferrets were vaccinated twice with adjuvanted split inactivated influenza vaccines containing homologous HA and NA (vacH3N2), only homologous HA (vacH3N1), only homologous NA (vacH1N2), heterologous HA and NA (vacH1N1), or phosphate-buffered saline (vacPBS), followed by challenge with H3N2 virus (A/Netherlands/16190/1968). Ferrets vaccinated with homologous HA (vacH3N2 and vacH3N1) displayed minimum fever and weight loss compared to vacH1N1 and vacPBS ferrets, while ferrets vaccinated with NA-matched vacH1N2 displayed intermediate fever and weight loss. Vaccination with vacH1N2 further led to a reduction in virus shedding from the nose and undetectable virus titers in the lower respiratory tract, similarly to when the homologous vacH3N2 was used. Some protection was observed upon vacH1N1 vaccination, but this was not comparable to that observed for vacH1N2, again highlighting the important role of NA in vaccine-induced protection. These results illustrate that NA antibodies can prevent severe disease caused by influenza virus infection and that an antigenically matched NA in seasonal vaccines might prevent lower respiratory tract complications. This underlines the importance of considering NA during the yearly vaccine strain selection process, which may be particularly beneficial in seasons when the HA component of the vaccine is mismatched. IMPORTANCE Despite the availability of vaccines, influenza virus infections continue to cause substantial morbidity and mortality in humans. Currently available influenza vaccines take primarily the hemagglutinin (HA) into account, but the highly variable nature of this protein as a result of antigenic drift has led to a recurrent decline in vaccine effectiveness. While the protective effect of neuraminidase (NA) antibodies has been highlighted by several studies, there are no requirements with regard to quantity or quality of NA in licensed vaccines, and NA immunity remains largely unexploited. Since antigenic changes in HA and NA are thought to occur asynchronously, NA immunity could compensate for reduced vaccine efficacy when drift in HA occurs. By matching and mismatching the HA and NA components of monovalent split inactivated vaccines, we demonstrated the potential of NA immunity to protect against disease, virus replication in the lower respiratory tract, and virus shedding in the ferret model.

Pre-existing immunity and vaccine history determine hemagglutinin-specific CD4 T cell and IgG response following seasonal influenza vaccination.

Effectiveness of seasonal influenza vaccination varies between individuals and might be affected by vaccination history among other factors. Here we show, by monitoring frequencies of CD4 T cells specific to the conserved hemagglutinin epitope HA118-132 and titres of IgG against the corresponding recombinant hemagglutinin protein, that antigen-specific CD4 T cell and antibody responses are closely linked to pre-existing immunity and vaccine history. Upon immunization, a strong early reaction is observed in all vaccine naïve participants and also in vaccine experienced individuals who have not received the respective seasonal vaccine in the previous year. This response is characterized by HA118-132 specific CD4 T cells with a follicular helper T cell phenotype and by ascending titers of hemagglutinin-specific antibodies from baseline to day 28 following vaccination. This trend was observed in only a proportion of those participants who received the seasonal vaccine the year preceding the study. Regardless of history, levels of pre-existing antibodies and CD127 expression on CD4 T cells at baseline were the strongest predictors of robust early response. Thus, both pre-existing immunity and vaccine history contribute to the response to seasonal influenza vaccines.

Hemagglutinin Nanoparticulate Vaccine with Controlled Photochemical Immunomodulation for Pathogenic Influenza-Specific Immunity.

Recently, viral infectious diseases, including COVID-19 and Influenza, are the subjects of major concerns worldwide. One strategy for addressing these concerns focuses on nasal vaccines, which have great potential for achieving successful immunization via safe, easy, and affordable approaches. However, conventional nasal vaccines have major limitations resulting from fast removal when pass through nasal mucosa and mucociliary clearance hindering their effectiveness. Herein a nanoparticulate vaccine (NanoVac) exhibiting photochemical immunomodulation and constituting a new self-assembled immunization system of a photoactivatable polymeric adjuvant with influenza virus hemagglutinin for efficient nasal delivery and antigen-specific immunity against pathogenic influenza viruses is described. NanoVac increases the residence period of antigens and further enhances by spatiotemporal photochemical modulation in the nasal cavity. As a consequence, photochemical immunomodulation of NanoVacs successfully induces humoral and cellular immune responses followed by stimulation of mature dendritic cells, plasma cells, memory B cells, and CD4+ and CD8+ T cells, resulting in secretion of antigen-specific immunoglobulins, cytokines, and CD8+ T cells. Notably, challenge with influenza virus after nasal immunization with NanoVacs demonstrates robust prevention of viral infection. Thus, this newly designed vaccine system can serve as a promising strategy for developing vaccines that are active against current hazardous pathogen outbreaks and pandemics.

Structure-Guided Creation of an Anti-HA Stalk Antibody F11 Derivative That Neutralizes Both F11-Sensitive and -Resistant Influenza A(H1N1)pdm09 Viruses.

The stalk domain of influenza virus envelope glycoprotein hemagglutinin (HA) constitutes the axis connecting the head and transmembrane domains, and plays pivotal roles in conformational rearrangements of HA for virus infection. Here we characterized molecular interactions between the anti-HA stalk neutralization antibody F11 and influenza A(H1N1)pdm09 HA to understand the structural basis of the actions and modifications of this antibody. In silico structural analyses using a model of the trimeric HA ectodomain indicated that the F11 Fab fragment has physicochemical properties, allowing it to crosslink two HA monomers by binding to a region near the proteolytic cleavage site of the stalk domain. Interestingly, the F11 binding allosterically caused a marked suppression of the structural dynamics of the HA cleavage loop and flanking regions. Structure-guided mutagenesis of the F11 antibody revealed a critical residue in the F11 light chain for the F11-mediated neutralization. Finally, the mutagenesis led to identification of a unique F11 derivative that can neutralize both F11-sensitive and F11-resistant A(H1N1)pdm09 viruses. These results raise the possibility that F11 sterically and physically disturbs proteolytic cleavage of HA for the ordered conformational rearrangements and suggest that in silico guiding experiments can be useful to create anti-HA stalk antibodies with new phenotypes.

Administration of Multivalent Influenza Virus Recombinant Hemagglutinin Vaccine in Combination-Adjuvant Elicits Broad Reactivity Beyond the Vaccine Components.

Combining variant antigens into a multivalent vaccine is a traditional approach used to provide broad coverage against antigenically variable pathogens, such as polio, human papilloma and influenza viruses. However, strategies for increasing the breadth of antibody coverage beyond the vaccine are not well understood, but may provide more anticipatory protection. Influenza virus hemagglutinin (HA) is a prototypic variant antigen. Vaccines that induce HA-specific neutralizing antibodies lose efficacy as amino acid substitutions accumulate in neutralizing epitopes during influenza virus evolution. Here we studied the effect of a potent combination adjuvant (CpG/MPLA/squalene-in-water emulsion) on the breadth and maturation of the antibody response to a representative variant of HA subtypes H1, H5 and H7. Using HA protein microarrays and antigen-specific B cell labelling, we show when administered individually, each HA elicits a cross-reactive antibody profile for multiple variants within the same subtype and other closely-related subtypes (homosubtypic and heterosubtypic cross-reactivity, respectively). Despite a capacity for each subtype to induce heterosubtypic cross-reactivity, broader coverage was elicited by simply combining the subtypes into a multivalent vaccine. Importantly, multiplexing did not compromise antibody avidity or affinity maturation to the individual HA constituents. The use of adjuvants to increase the breadth of antibody coverage beyond the vaccine antigens may help future-proof vaccines against newly-emerging variants.

The Potential of Neuraminidase as an Antigen for Nasal Vaccines To Increase Cross-Protection against Influenza Viruses.

Despite the availability of vaccines that efficiently reduce the severity of clinical symptoms, influenza viruses still cause substantial morbidity and mortality worldwide. In this regard, nasal influenza vaccines-because they induce virus-specific IgA-may be more effective than traditional parenteral formulations in preventing infection of the upper respiratory tract. In addition, the neuraminidase (NA) of influenza virus has shown promise as a vaccine antigen to confer broad cross-protection, in contrast to hemagglutinin (HA), the target of most current vaccines, which undergoes frequent antigenic changes, leading to vaccine ineffectiveness against mismatched heterologous strains. However, the usefulness of NA as an antigen for nasal vaccines is unclear. Here, we compared NA and HA as antigens for nasal vaccines in mice. Intranasal immunization with recombinant NA (rNA) plus adjuvant protected mice against not only homologous but also heterologous virus challenge in the upper respiratory tract, whereas intranasal immunization with rHA failed to protect against heterologous challenge. In addition, intranasal immunization with rNA, but not rHA, conferred cross-protection even in the absence of adjuvant in virus infection-experienced mice; this strong cross-protection was due to the broader capacity of NA-specific antibodies to bind to heterologous virus. Furthermore, the NA-specific IgA in the upper respiratory tract that was induced through rNA intranasal immunization recognized more epitopes than did the NA-specific IgG and IgA in plasma, again increasing cross-protection. Together, our findings suggest the potential of NA as an antigen for nasal vaccines to provide broad cross-protection against both homologous and heterologous influenza viruses. IMPORTANCE Because mismatch between vaccine strains and epidemic strains cannot always be avoided, the development of influenza vaccines that induce broad cross-protection against antigenically mismatched heterologous strains is needed. Although the importance of NA-specific antibodies to cross-protection in humans and experimental animals is becoming clear, the potential of NA as an antigen for providing cross-protection through nasal vaccines is unknown. We show here that intranasal immunization with NA confers broad cross-protection in the upper respiratory tract, where virus transmission is initiated, by inducing NA-specific IgA that recognizes a wide range of epitopes. These data shed new light on NA-based nasal vaccines as powerful anti-influenza tools that confer broad cross-protection.

Long term immunity against Peste Des Petits Ruminants mediated by a recombinant Newcastle disease virus vaccine.

Peste des Petits Ruminants (PPR) is a highly contagious and often fatal disease of sheep and goats. Conventional live vaccines have been successfully used in endemic countries however, there are not completely safe and not allowing differentiation between vaccinated and infected animals (DIVA). In this study, a recombinant Newcastle disease virus (NDV) expressing the hemagglutinin of PPRV (NDV-PPRVH) was evaluated on small ruminants by serology response in sheep and goats, experimental infection in goats and immunity duration in sheep. The NDV-PPRVH vaccine injected twice at 28 days' interval, provided full protection against challenge with a virulent PPR strain in the most sensitive species and induced significant neutralizing antibodies. Immunological response in goats was slightly higher than sheep and the vaccine injected at 108.0 50 % egg infective dose/mL allowed anti-PPRV antibodies that lasted at least 12 months as shown by antibody response monitoring in sheep. The NDV vector presented a limited replication in the host and vaccinated animals remained negative when tested by cELISA based on PPRV nucleoprotein allowing DIVA. This recombinant vaccine appears to be a promising candidate in a free at risk countries and may be an important component of the global strategy for PPR eradication.

The construction of recombinant Lactobacillus casei expressing hemagglutinin-neuraminidase protein and its immune response in chickens.

Newcastle disease virus (NDV) is one of the most important diseases in poultry. The present study generated recombinant surface-displayed Lactobacillus casei (L. casei) expressing the hemagglutinin-neuraminidase (HN) of NDV (Lc-pPG-HN) and a live pPG vector (Lc-pPG) and evaluated their immunogenicity. A 1670 bp HN gene fragment was successfully amplified and cloned into a prokaryotic protein expression system. Protein expression in the resulting recombinant Lc-pPG-HN (surface displayed) strain was verified using Western blotting and indirect immunofluorescence. A single band was observed on the Western blots, and the molecular weight of the corresponding protein was 63 kDa. A fluorescent signal for Lc-pPG-HN was observed using fluorescence microscopy. A total of 270 healthy chicks were divided into three treatment groups. Five replicates were used for each treatment, while six chicks were used per replicate. The following three treatment groups were used: physiological saline group (Control), Lc-pPG group and recombinant vaccine group (Lc-pPG-HN). The primary immunization and booster immunization of the chicks were performed via oral administration on 1 and 10 days old. Tissue and blood samples were collected from chickens that received oral recombinant L. casei strains on 1, 7, 14 and 21 days post-immunization for immune-related index analyses. Chickens orally immunized with Lc-pPG-HN showed significantly increased body weights and immune organ indices. Oral immunization with Lc-pPG-HN also enhanced the concentrations of serum interleukin-2 (IL-2), interferon-γ (IFN-γ), intestinal lavage fluid secretory immunoglobulin A (SIgA) and histomorphological development of the small intestine. Our results also indicated that recombinant L. casei significantly increased Lactobacillus and Bifidobacterium colonization and decreased the relative abundance of Escherichia coli (E. coli) in the chicken caecum. Similar enhancement effects from hemagglutination inhibition were also observed in the antibody titers. Oral administration of Lc-pPG-HN effectively protected against NDV and alleviated the symptoms of the NDV challenge. In summary, recombinant L. casei had positive impacts on the performance, immunological function, gut development, and microbiota of growing chicks and may be a potential therapeutic candidate against NDV.

Evolutionary genomics of recent clinical Bordetella pertussis isolates from Iran: wide circulation of multiple ptxP3 lineages and report of the first ptxP3 filamentous hemagglutinin-negative B. pertussis.

Here we investigated nationwide clinical Bordetella pertussis isolated during 2005-2017 from different provinces of Iran, a country with more than 50 years whole cell vaccine immunisation history. Our results revealed the ongoing increase in the population of ptxP3/fim3-2 B. pertussis isolates in different provinces which were differentiated into nine clades. The largest clade (clade 8) which was previously found to be prevalent in Tehran was also prevalent across the country and clade 5 with ptxP3/prn9 genotype has also increased in frequency (14% of all ptxP3 isolates) in recent years. Furthermore, we detected the first ptxP3 B. pertussis isolates that does not express filamentous hemagglutinin (FhaB) as one of the major antigens of the pathogen and a key component of the acellular pertussis vaccine.

Balancing the influenza neuraminidase and hemagglutinin responses by exchanging the vaccine virus backbone.

Virions are a common antigen source for many viral vaccines. One limitation to using virions is that the antigen abundance is determined by the content of each protein in the virus. This caveat especially applies to viral-based influenza vaccines where the low abundance of the neuraminidase (NA) surface antigen remains a bottleneck for improving the NA antibody response. Our systematic analysis using recent H1N1 vaccine antigens demonstrates that the NA to hemagglutinin (HA) ratio in virions can be improved by exchanging the viral backbone internal genes, especially the segment encoding the polymerase PB1 subunit. The purified inactivated virions with higher NA content show a more spherical morphology, a shift in the balance between the HA receptor binding and NA receptor release functions, and induce a better NA inhibitory antibody response in mice. These results indicate that influenza viruses support a range of ratios for a given NA and HA pair which can be used to produce viral-based influenza vaccines with higher NA content that can elicit more balanced neutralizing antibody responses to NA and HA.

Anti-A and SARS-CoV-2: an intriguing association.

BACKGROUND: Blood groups and anti-A isohemagglutinin may be involved in susceptibility to SARS-CoV-2 infection. MATERIALS AND METHODS: We retrospectively studied 268 COVID-19 convalescent plasma donors and 162 COVID-19 inpatients (total 430 subjects, confirmed by RT-PCR) and 2,212 healthy volunteer first-time blood donors as a control group. These were further divided into two groups: those with anti-A (blood types O and B) and those without it (types A and AB). Titres of nucleoproteins, and neutralizing SARS-CoV-2 antibody were measured in the convalescent plasma donors and inpatients. Multivariate logistic regression and non-parametric tests were applied. RESULTS: Persons having types O or B showed less infection prevalence than those of types A or AB (OR = 0·62, 95% CI 0·50-0·78; P < 0·001), but there was no difference when COVID-19 inpatients were analysed. Immunoglobulins M, G and A were lower in COVID-19 subjects of types O or B group than those of A or AB (0·16 vs. 0·19; P = 0·03, 2·11 vs. 2·55; P = 0·02, 0·23 vs. 0·32; P = 0·03, respectively). CONCLUSION: In this retrospective cohort, COVID-19 individuals were less likely to belong to blood types O and B, and also had lower SARS-CoV-2 antibody titres than A and AB individuals. COVID-19 severity did not associate with the blood groups.

Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans.

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.

Broad cross protection by recombinant live attenuated influenza H3N2 seasonal virus expressing conserved M2 extracellular domain in a chimeric hemagglutinin.

Hemagglutinin (HA)-based current vaccines provide suboptimum cross protection. Influenza A virus contains an ion channel protein M2 conserved extracellular domain (M2e), a target for developing universal vaccines. Here we generated reassortant influenza virus rgH3N2 4xM2e virus (HA and NA from A/Switzerland/9715293/2013/(H3N2)) expressing chimeric 4xM2e-HA fusion proteins with 4xM2e epitopes inserted into the H3 HA N-terminus. Recombinant rgH3N2 4xM2e virus was found to retain equivalent growth kinetics as rgH3N2 in egg substrates. Intranasal single inoculation of mice with live rgH3N2 4xM2e virus was effective in priming the induction of M2e specific IgG antibody responses in mucosal and systemic sites as well as T cell responses. The rgH3N2 4xM2e primed mice were protected against a broad range of different influenza A virus subtypes including H1N1, H3N2, H5N1, H7N9, and H9N2. The findings support a new approach to improve the efficacy of current vaccine platforms by recombinant influenza virus inducing immunity to HA and cross protective M2e antigens.

Convergent antibody evolution and clonotype expansion following influenza virus vaccination.

Recent advances in high-throughput single cell sequencing have opened up new avenues into the investigation of B cell receptor (BCR) repertoires. In this study, PBMCs were collected from 17 human participants vaccinated with the split-inactivated influenza virus vaccine during the 2016-2017 influenza season. A combination of Immune Repertoire Capture (IRCTM) technology and IgG sequencing was performed on ~7,800 plasmablast (PB) cells and preferential IgG heavy-light chain pairings were investigated. In some participants, a single expanded clonotype accounted for ~22% of their PB BCR repertoire. Approximately 60% (10/17) of participants experienced convergent evolution, possessing public PBs that were elicited independently in multiple participants. Binding profiles of one private and three public PBs confirmed they were all subtype-specific, cross-reactive hemagglutinin (HA) head-directed antibodies. Collectively, this high-resolution antibody repertoire analysis demonstrated the impact evolution can have on BCRs in response to influenza virus vaccination, which can guide future universal influenza prophylactic approaches.

Neutralizing antibody PR8-23 targets the footprint of the sialoglycan receptor binding site of H1N1 hemagglutinin.

Influenza virus cause seasonal influenza epidemic and seriously sporadic influenza pandemic outbreaks. Hemagglutinin (HA) is an important target in the therapeutic treatment and diagnostic detection of the influenza virus. Variation in the sialic acid receptor binding site leads to strain-specific binding and results in different binding modes to the host receptors. Here, we evaluated the neutralizing activity and hemagglutination inhibition activity of a prepared murine anti-H1N1 monoclonal antibody PR8-23. Then we identified the epitope peptide of antibody PR8-23 by phage display technique from phage display peptide libraries. The identified epitope, 63-IAPLQLGKCNIA-74, containing two α-helix and two ß-fold located at the footprint of the sialoglycan receptor on the RBS in the globular head domain of HA. It broads the growing arsenal of motifs for the amino acids on the globular head domain of HA in sialic acid receptor binding site and neutralizing antibody production.

An influenza HA stalk reactive polymeric IgA antibody exhibits anti-viral function regulated by binary interaction between HA and the antibody.

IgA antibodies, which are secreted onto the mucosal surface as secretory IgA antibodies (SIgAs), play an important role in preventing influenza virus infection. A recent study reported that anti-hemagglutinin (HA) head-targeting antibodies increase anti-viral functions such as hemagglutination inhibition (HI) and virus neutralization (NT), in addition to HA binding activity (reactivity) via IgA polymerization. However, the functional properties of anti-viral IgA antibodies with mechanisms of action distinct from those of anti-HA head-targeting antibodies remain elusive. Here, we characterized the functional properties of IgG, monomeric IgA, and polymeric IgA anti-HA stalk-binding clones F11 and FI6, and B12 (a low affinity anti-HA stalk clone), as well as Fab-deficient (ΔFab) IgA antibodies. We found that IgA polymerization impacts the functional properties of anti-HA stalk antibodies. Unlike anti-HA head antibodies, the anti-viral functions of anti-HA stalk antibodies were not simply enhanced by IgA polymerization. The data suggest that two modes of binding (Fab paratope-mediated binding to the HA stalk, and IgA Fc glycan-mediated binding to the HA receptor binding site (RBS)) occur during interaction between anti-stalk HA IgA antibodies and HA. In situations where Fab paratope-mediated binding to the HA stalk exceeded IgA Fc glycan-mediated binding to HA RBS, IgA polymerization increased anti-viral functions. By contrast, when IgA Fc glycan-mediated binding to the HA RBS was dominant, anti-viral activity will fall upon IgA polymerization. In summary, the results suggest that coordination between these two independent binding modules determines whether IgA polymerization has a negative or positive effect on the anti-viral functions of anti-HA stalk IgA antibodies.

Hemagglutinin Structure and Activities.

Hemagglutinins (HAs) are the receptor-binding and membrane fusion glycoproteins of influenza viruses. They recognize sialic acid-containing, cell-surface glycoconjugates as receptors but have limited affinity for them, and, as a consequence, virus attachment to cells requires their interaction with several virus HAs. Receptor-bound virus is transferred into endosomes where membrane fusion by HAs is activated at pH between 5 and 6.5, depending on the strain of virus. Fusion activity requires extensive rearrangements in HA conformation that include extrusion of a buried "fusion peptide" to connect with the endosomal membrane, form a bridge to the virus membrane, and eventually bring both membranes close together. In this review, we give an overview of the structures of the 16 genetically and antigenically distinct subtypes of influenza A HA in relation to these two functions in virus replication and in relation to recognition of HA by antibodies that neutralize infection.

Identification of H7N9 hemagglutinin novel protein epitopes that elicit strong antibody-dependent, cell-mediated cytotoxic activities with protection from influenza infection in mouse model.

BACKGROUND: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the mechanisms connecting humoral immunity and cellular immunity and has been well-demonstrated in recent studies. Neutralizing antibodies and antibodies can mediate ADCC effects and both build a strong defense against H7N9 influenza virus infection. In our previous study, we found that H7N9 patients' plasma displayed low neutralizing activities that were not sufficient for host protection; however, the plasma of some patients can mediate strong ADCC effects. METHODS: Based on the plasma samples of H7N9 infected patients collected, we measured the ADCC activities of these samples and selected the best to locate the dominant epitopes on H7N9 hemagglutinin (HA) protein that can elicit antibodies and strong ADCC activities. We constructed a yeast surface-display H7N9 HA protein epitope library and screened this library against plasma samples with different potencies in mediating ADCC effects. RESULTS: Two dominant epitopes were selected from the screening. Plasma samples with depleted antibodies that were specific to the epitopes showed reduced ADCC activities. The serum of mice immunized with the epitopes elicited strong ADCC activities. Three monoclonal antibodies were isolated which showed high ADCC effects in vitro. Vaccination with isolated ADCC activating epitopes can provide partial protection from influenza infection in mouse model. And mice with vaccinated with combination of epitopes and extracellular domain can provide full protection from influenza infection in the same mouse model. CONCLUSIONS: In this study, the epitopes isolated on H7N9 HA were immunogenic and elicited antibodies and strong ADCC activities in mice. Although the protective effect of the epitopes is partial, the combination of epitopes and extracellular domain can provide 100% protection from influenza virus infection in the same mouse model. Our study provides information on the potential use of epitope vaccine design against H7N9 viral infection.

Role of the Hemagglutinin Residue 227 in Immunogenicity of H5 and H7 Subtype Avian Influenza Vaccines in Chickens.

Many H5 and H7 subtype avian influenza vaccines are poorly immunogenic in terms of inducing hemagglutination-inhibition (HI) antibody titers. Residue 227 (H3 numbering) in the receptor binding site in the hemagglutinin (HA) is critical for the detectability of HI antibodies induced by H5 influenza vaccines. However, whether the effect of residue 227 on immunogenicity can be generalized in different subtypes is unclear. In this study, the impact of HA residue 227 on immunogenicity of H5N1, H5N6, and H7N9 avian influenza vaccines was evaluated in chickens. Polymorphism analysis revealed that S227 is overwhelmingly dominant in HA of the H5N1 and H7N9 subtypes, whereas this amino acid is present in a small proportion of H5N6 viruses. The H5N1, H5N6, and H7N9 vaccines harboring S227 in HA induced relatively low HI titers at week 2 postimmunization (pi), and antibody titers increased at week 3 pi. S227N substitution in these vaccines consistently enhanced HI titers significantly. Another H5N6 vaccine harboring Q227 in HA elicited a robust HI antibody response, and Q227S substitution led to a significant drop of HI titers. Cross-HI testing against the wild-type and mutant viruses revealed that the amino acid at position 227 was associated with the detectability of HI titers induced by H5 and H7 avian influenza vaccines. The results indicate an important role of residue 227 in HA in immunogenicity of H5 and H7 subtype avian influenza vaccines in chickens. Our findings also provided useful information for vaccine seed virus selection and genetic engineering for immunogenicity enhancement of avian influenza vaccines.