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Background: Arg-gingipain A (RgpA) is the primary virulence factor of Porphyromonas gingivalis and contains hemagglutinin adhesin (HA), which helps bacteria adhere to cells and proteins. Hemagglutinin's functional domains include cleaved adhesin (CA), which acts as a hemagglutination and hemoglobin-binding actor. Here, we confirmed that the HA and CA genes are immunogenic, and using adjuvant chemokine to target dendritic cells (DCs) enhanced protective autoimmunity against P. gingivalis-induced periodontal disease., Methods: C57 mice were immunized prophylactically with pVAX1-CA, pVAX1-HA, pVAX1, and phosphate-buffered saline (PBS) through intramuscular injection every 2 weeks for a total of three administrations before P. gingivalis-induced periodontitis. The DCs were analyzed using flow cytometry and ribonucleic acid sequencing (RNA-seq) transcriptomic assays following transfection with CA lentivirus. The efficacy of the co-delivered molecular adjuvant CA DNA vaccine was evaluated in vivo using flow cytometry, immunofluorescence techniques, and micro-computed tomography., Results: After the immunization, both the pVAX1-CA and pVAX1-HA groups exhibited significantly elevated P. gingivalis-specific IgG and IgG1, as well as a reduction in bone loss around periodontitis-affected teeth, compared to the pVAX1 and PBS groups (p < 0.05). The expression of CA promoted the secretion of HLA, CD86, CD83, and DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) in DCs. Furthermore, the RNA-seq analysis revealed a significant increase in the chemokine (C-C motif) ligand 19 (p < 0.05). A notable elevation in the quantities of DCs co-labeled with CD11c and major histocompatibility complex class II, along with an increase in interferon-gamma (IFN-γ) cells, was observed in the inguinal lymph nodes of mice subjected to CCL19-CA immunization. This outcome effectively illustrated the preservation of peri-implant bone mass in rats afflicted with P. gingivalis-induced peri-implantitis (p < 0.05)., Conclusions: The co-administration of a CCL19-conjugated CA DNA vaccine holds promise as an innovative and targeted immunization strategy against P. gingivalis-induced periodontitis and peri-implantitis., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

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.

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., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

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 HA 118-132 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 HA 118-132 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., (© 2021. The Author(s).)

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 10 8.0 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Cold Spring Harbor Laboratory Press; all rights reserved.)

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.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.

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., Competing Interests: JH-D, JF, EP, AaJ, SJ, RN, AlJ, PF, and DHD own shares in Nanommune Inc. Nanommune does not sell any products described in this paper, nor funded any part of the work described herein. Neither Nanommune nor its shareholders are likely to benefit from the results described in this publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hernandez-Davies, Felgner, Strohmeier, Pone, Jain, Jan, Nakajima, Jasinskas, Strahsburger, Krammer, Felgner and Davies.)

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 β-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., (© 2021 Wiley Periodicals LLC.)

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., (© 2021 International Society of Blood Transfusion.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: Some of the authors are affiliated with Atreca, Inc., but the company provided no funding for the study and no competing interests exist. The commercial affiliation of those authors does not alter our adherence to PLoS ONE policies on sharing data and materials.

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Influenza virus remains a continuous and severe threat to public health worldwide, and its prevention and treatment have always been a major international issue. Because of its ability to evade immune surveillance through rapid antigenic drift and antigenic shift, broad-spectrum vaccines seem increasingly important., Methods: A mAb named 3C12 from an immortalized hybrid cell was generated via immunizing mice with HA2 protein from A/chicken/Anhui/BRI99/2016 (AH/BRI99/16, H9N2) generated by prokaryotic expression. Then, its broad-spectrum activity was analyzed by WB and IFA. Next, the minimal linear epitope was identified via analyzing the reaction of a series of HA truncations with 3C12. Finally, the protective effects of 3C12 were evaluated in vitro and in vivo infection experiments., Results: The mAb could react with the viruses of subtypes H1, H2, H5, H8, H9, H12, H13, H16, and HA protein of H18 in group 1, but failed to react with viruses in group 2. The minimal linear epitope targeted by the mAb was 433 NAELLVL 439 in full length of HA and localized in the C-helix region of HA2 (residue 95-101, HA2 numbering). What's more, the mAb 3C12 inhibited H1, H2, H5, H8, H9, H12, H13 and H16 virus-replication in vitro and also has shown effectiveness in preventing and treating disease in mice challenged with lethal dose of AH/BRI99/16 (H9N2) virus in vivo. These results suggested that the broadly reactive anti-HA stem mAb 3C12 exhibited prophylactic and therapeutic efficacy., Conclusions: Here, we have demonstrated that the linear epitope identified in this study could be a novel target for developing broad-spectrum influenza diagnostics or vaccine design, and the HA2-based monoclonal antibody is indeed a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.

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.

Humoral immune responses to influenza virus vaccines in elderly individuals are poorly adapted toward new antigenically drifted influenza virus strains. Instead, older individuals respond in an original antigenic sin fashion and produce much more cross-reactive but less potent antibodies. Here, we investigated four influenza B virus hemagglutinin (HA) head specific, hemagglutination inhibition-inactive monoclonal antibodies (MAbs) from elderly individuals. We found that they were broadly reactive within the B/Victoria/2/1987-like lineage, and two were highly cross-reactive with B/Yamagata/16/1988-like lineage viruses. The MAbs were found to be neutralizing, to utilize Fc effector functions, and to be protective against lethal viral challenge in a mouse model. In order to identify residues on the influenza B virus hemagglutinin interacting with the MAbs, we generated escape mutant viruses. Interestingly, escape from these MAbs led to numerous HA mutations within the head domain, including in the defined antigenic sites. We observed that each individual escape mutant virus was able to avoid neutralization by its respective MAb along with other MAbs in the panel, although in many cases binding activity was maintained. Point mutant viruses indicated that K90 is critical for the neutralization of two MAbs, while escape from the other two MAbs required a combination of mutations in the hemagglutinin. Three of four escape mutant viruses had increased lethality in the DBA2/J mouse model. Our work indicates that these cross-reactive antibodies have the potential to cause antigenic drift in the viral population by driving mutations that increase virus fitness. However, binding activity and cross-neutralization were maintained by a majority of antibodies in the panel, suggesting that this drift may not lead to escape from antibody-mediated protection. IMPORTANCE Understanding the immune response that older individuals mount to influenza virus vaccination and infection is critical in order to design better vaccines for this age group. Here, we show that older individuals make broadly neutralizing antibodies that have no hemagglutination-inhibiting activity and are less potent than strain-specific antibodies. These antibodies could drive viral escape from neutralization but did not result in escape from binding. Given their different mechanisms of action, they might retain protective activity even against escape variants., (Copyright © 2020 American Society for Microbiology.)

Hemagglutination-inhibitory antibodies are usually highly strain specific with little effect on infection with drifted or shifted strains. The significance of broadly cross-reactive non-HAI anti-influenza antibodies against conserved domains of virus glycoproteins, such as the hemagglutinin (HA) stalk, is of great interest. We characterize a cohort of 40 H1N1pmd09 influenza-infected patients and identify lower respiratory symptoms (LRSs) as a predictor for development of pneumonia. A binomial logistic regression of log10 pre-existing antibody values shows that the probability of LRS occurrence decreased with increased anti-HA full-length and stalk antibody ELISA titers. However, a multilevel logistic regression model adjusted by other potential serocorrelates demonstrates that only antibodies directed against the stalk of HA correlate with protection from lower respiratory infection, limiting disease progression. Our predictive model indicates that a threshold of protective immunity based on broadly cross-reactive HA stalk antibodies could be feasible., Competing Interests: A.G.-S. is an inventor of patents owned by the Icahn School of Medicine at Mount Sinai in the field of influenza virus vaccines. The A.G.-S. lab has received research funds from Avimex, GSK, and 7Hills to investigate novel influenza virus vaccines. F.K. is a named inventor of technology in the field of human influenza vaccines and a named co-inventor on patents to develop chimeric HA technology to develop a universal influenza vaccine., (© 2020 The Authors.)

Following influenza A vaccination, certain individuals exhibit adverse reactions in the nervous system, which causes a problem with the safety of the influenza A vaccine. However, to the best of our knowledge, the underlying mechanism of this is unknown. The present study revealed that a monoclonal antibody (H1‑84mAb) against the H1N1 influenza virus hemagglutinin (HA) protein cross‑reacted with an antigen from brain tissue. Total brain tissue protein was immunoprecipitated with this cross‑reactive antibody, and mass spectrometry revealed that the bound antigens were heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNPA2/B1. Subsequently, the two proteins were expressed in bacteria and it was demonstrated that H1‑84mAb bound to hnRNPA1 and hnRNPA2/B1. These two proteins were expressed in three segments and the cross‑reactivity of H1‑84mAb with the glycine (Gly)‑rich domains of hnRNPA1 (195aa‑320aa) and hnRNPA2/B1 (202aa‑349aa) was determined using ELISA blocking experiments. It was concluded that the Gly‑rich domains of these two proteins are heterophilic antigens that cross‑react with influenza virus HA. The association between the heterophilic antigen Gly‑rich domains and the safety of influenza A vaccines remains to be investigated.

Effective and reliable anti-influenza treatments are acutely needed and passive immunizations using broadly neutralizing anti-influenza monoclonal antibodies (bNAbs) are a promising emerging approach. Because influenza infections are initiated in and localized to the pulmonary tract, and newly formed viral particles egress from the apical side of the lung epithelium, we compared the effectiveness of hemagglutinin (HA) stalk-binding bNAbs administered through the airway (intranasal or via nebulization) versus the systemic route (intraperitoneal or intravenous). Airway deliveries of various bNAbs were 10- to 50-fold more effective than systemic deliveries of the same bNAbs in treating H1N1, H3N2, B/Victoria-, and B/Yamagata-lineage influenza viral infections in mouse models. The potency of airway-delivered anti-HA bNAbs was highly dependent on antiviral neutralization activity, with little dependence on the effector function of the antibody. In contrast, the effectiveness of systemically delivered anti-HA bNAbs was not dependent on antiviral neutralization, but critically dependent on antibody effector functions. Concurrent administration of a neutralizing/effector function-positive bNAb via the airway and systemic routes showed increased effectiveness. The small amount of airway-delivered bNAbs needed for effective influenza treatment creates the opportunity to combine potent bNAbs with different anti-influenza specificities to generate a cost-effective antiviral therapy that provides broad coverage against all circulating influenza strains infecting humans. A 3 mg/kg dose of the novel triple antibody combination CF-404 (i.e., 1 mg/kg of each component bNAb) delivered to the airway was shown to effectively prevent weight loss and death in mice challenged with ten 50% lethal dose (LD 50 ) inoculums of either H1N1, H3N2, B/Victoria-lineage, or B/Yamagata-lineage influenza viruses. IMPORTANCE Influenza causes widespread illness in humans and can result in morbidity and death, especially in the very young and elderly populations. Because influenza vaccination can be poorly effective some years, and the immune systems of the most susceptible populations are often compromised, passive immunization treatments using broadly neutralizing antibodies is a promising therapeutic approach. However, large amounts of a single antibody are required for effectiveness when delivered through systemic administration (typically intravenous infusion), precluding the feasible dosing of antibody combinations via this route. The significance of our research is the demonstration that effective therapeutic treatments of multiple relevant influenza types (H1N1, H3N2, and B) can be achieved by airway administration of a single combination of relatively small amounts of three anti-influenza antibodies. This advance exploits the discovery that airway delivery is a more potent way of administering anti-influenza antibodies compared to systemic delivery, making this a feasible and cost-effective therapeutic approach., (Copyright © 2020 Vigil et al.)

The H1N1 influenza virus is known as a serious pandemic threat across the globe. Vaccination is one of the most effective methods of protection against this virus and the way to reduce the seasonal pandemic risk. The commercial vaccine does not adequately respond to pandemic strains. This study examines the potential function of formulated H1N1 hemagglutinin with MF59 adjuvant against A/PR/8/34 (H1N1). To this end, a recombinant hemagglutinin (rHA) gene of influenza A virus was designed and expressed in SF9 cell by the Baculovirus expression system. Four groups of mice were immunized by rHA in combination with MF59, Alum adjuvant, and virus split only. The immunized mice subsequently used for the humoral immune assay and the results compared with untreated mice (negative group). Besides, both treated and control mice groups were challenged with mouse-adapted influenza virus A/PR/8/34(H1N1) through the intranasal drop. Bodyweight, survival, temperature variation, and the medical conditions of the samples were assessed. Mice immunized with the recombinant protein demonstrated a humoral response to the influenza A virus. Upon virus challenging, co-administration of rHA with MF59 adjuvant could lead to 92% survival of the vaccinated mice within 10 days. The MF59-treated group showed slight weight loss and high-temperature body two weeks after infection. This group also displayed a higher hemagglutination inhibition (HI) antibody titer as compared to the group vaccinated with virus split, and Alum adjuvant. Altogether, the results showed that the recombinant protein with the MF59 adjuvant created better safety than the Alum adjuvant, thereby can be considered as a safe and reliable vaccine against the H1N1 virus for further investigations.

H9N2, a low pathogenic avian influenza virus, causes significant economic losses in the poultry industry worldwide. Herein, we describe the construction of an attenuated Salmonella Gallinarum (SG) strain for expression and delivery of H9N2 haemagglutinin (HA) 1 (SG-HA1), HA2 (SG-HA2) and/or the conserved matrix protein 2 ectodomain (SG-M2e). We demonstrated that recombinant SG strains expressing HA1, HA2 and M2e antigens were immunogenic and safe in a chicken model. Chickens ( n  = 8) were vaccinated once orally with SG alone, SG-HA1, SG-HA2, SG-M2e, or mixture of SG-HA1, SG-HA2 and SG-M2e, or vaccinated once intramuscularly with an oil-adjuvant inactivated H9N2 vaccine. Our results demonstrated that vaccination with SG mutants encoding influenza antigens, administered individually or as a mixture, elicited significantly ( P  < 0.05) greater antigen-specific humoral and cell-mediated immune responses in chickens compared with those vaccinated with SG alone. A conventional H9N2 vaccine induced significantly ( P  < 0.05) greater HA1 and HA2 antibody responses than SG-based H9N2 vaccine strains, but significantly ( P  < 0.05) less robust M2e-specific responses. Upon challenge with the virulent H9N2 virus on day 28 post-vaccination, chickens vaccinated with either the SG-based H9N2 or conventional H9N2 vaccines exhibited comparable lung inflammation and viral loads, although both were significantly lower ( P  < 0.05) than in the group vaccinated with SG alone. In conclusion, our results showed that SG-based vaccination stimulated efficient immune responses against virulent H9N2. Further studies are needed to fully develop this approach as a preventive strategy for low pathogenic avian influenza viruses affecting poultry. RESEARCH HIGHLIGHTS S. gallinarum expressing HA1, HA2 and M2e antigens are immunogenic and safe. Salmonella has dual function of acting as a delivery system and as a natural adjuvant. Vaccine constructs elicit specific humoral and cell-mediated immune responses.

Influenza virus constantly acquires genetic mutations/reassortment in the major surface protein, hemagglutinin (HA), resulting in the generation of strains with antigenic variations. There are, however, HA epitopes that are conserved across influenza viruses and are targeted by broadly protective antibodies. A goal for the next-generation influenza vaccines is to stimulate B-cell responses against such conserved epitopes in order to provide broad protection against divergent influenza viruses. Broadly protective B cells, however, are not easily activated by HA antigens with native structure, because the virus has multiple strategies to escape from the humoral immune responses directed to the conserved epitopes. One such strategy is to hide the conserved epitopes from the B-cell surveillance by steric hindrance. Technical advancement in the analysis of the human B-cell antigen receptor (BCR) repertoire has dissected the BCRs to HA epitopes that are hidden in the native structure but are targeted by broadly protective antibodies. We describe here the characterization and function of broadly protective antibodies and strategies that enable B cells to seek these hidden epitopes, with potential implications for the development of universal influenza vaccines., (© The Japanese Society for Immunology. 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Naturally occurring self-assembling ferritin nanoparticles have become widely appreciated for vaccine design. In this study, an apoferritin (AFt) nanocage was used as a carrier to construct a biomimetic influenza vaccine by encapsulating a conserved internal nucleoprotein (NP) antigen peptide inside the nanocage, followed by chemically conjugating the surface antigen hemagglutinin (HA) protein on the outer surface of the AFt. Benefiting from the excellent thermal stability and thermallyassociated structural flexibility of the AFt nanocages, a novel temperature shift based encapsulation process was proposed and proved efficient for encapsulation of the NP peptides. On average, about 18 NPs were encapsulated and 1.6 HA antigens were conjugated in each of the HA-AFt+NP dual-antigen influenza vaccines. Upon immunization in mice, the HA-AFt+NP vaccine elicited both HA and NP-specific antibodies, and conferred complete protection against a lethal infection of both homologous PR8 H1N1 and heterologous A/FM/1/47 (FM1, H1N1) strains, while the HA-AFt conjugate vaccine without encapsulated NP antigen only conferred 60% protection against the FM1 H1N1 viral challenge. The potential cross-protective effect of the HA-AFt+NP vaccine was further demonstrated by significant specific hemagglutination inhibition (HAI) titers in serum of the immunized mice against heterologous A/Hong Kong/4801/2014 (H3N2) viral strain, which was about 3-fold of that induced by HA antigen and 2-fold of the HA-AFt conjugate vaccine. This biomimetic HA-AFt+NP conjugate vaccine, therefore, may represent a new strategy for developing a potential universal influenza vaccine without the need of any adjuvant, and further broaden the application of AFt nanocages in the areas of vaccine development and delivery system.

Humoral immune protection against influenza virus infection is mediated largely by antibodies against hemagglutinin (HA) and neuraminidase (NA), the two major glycoproteins on the virus surface. While influenza virus vaccination efforts have focused mainly on HA, NA-based immunity has been shown to reduce disease severity and provide heterologous protection. Current seasonal vaccines do not elicit strong anti-NA responses-in part due to the immunodominance of the HA protein. Here, we demonstrate that by swapping the 5' and 3' terminal packaging signals of the HA and NA genomic segments, which contain the RNA promoters, we are able to rescue influenza viruses that express more NA and less HA. Vaccination with formalin-inactivated "rewired" viruses significantly enhances the anti-NA antibody response compared to vaccination with unmodified viruses. Passive transfer of sera from mice immunized with rewired virus vaccines shows better protection against influenza virus challenge. Our results provide evidence that the immunodominance of HA stems in part from its abundance on the viral surface, and that rewiring viral packaging signals-thereby increasing the NA content on viral particles-is a viable strategy for improving the immunogenicity of NA in an influenza virus vaccine. IMPORTANCE Influenza virus infections are a major source of morbidity and mortality worldwide. Increasing evidence highlights neuraminidase as a potential vaccination target. This report demonstrates the efficacy of rewiring influenza virus packaging signals for creating vaccines with more neuraminidase content which provide better neuraminidase (NA)-based protection., (Copyright © 2020 American Society for Microbiology.)

Of the 24 known measles genotypes, only D8 and B3 are responsible for outbreaks in the last years in Europe, Asia, and America. In this study the H gene of 92 strains circulating between 2015 and 2019 in Lombardy, Northern Italy, and 1273 H sequences available in GenBank were analyzed in order to evaluate the genetic variability and to assess the conservation of the immunodominant sites. Overall, in Lombardy we observed the presence of four different B3 and three different D8 clusters, each one of them including sequences derived from viruses found in both vaccinated and unvaccinated subjects. Worldwide, the residue 400 within the H protein, a position located within the main immune epitope, is mutated in all circulating strains that belong to the two globally endemic genotypes, B3 and D8. Our data demonstrate the usefulness of measles virus (MV) H gene sequencing. Indeed, the monitoring the H protein epitopes of circulating strains could be included in the measles laboratory surveillance activities in order to improve and optimize strategies for measles control, as countries go towards elimination phase.

Sequential intramuscular immunization with chimeric hemagglutinins (cHA) composed of the same conserved HA stalk domain and distinct HA heads is a proposed strategy to produce a supra-seasonal universal influenza vaccine. To evaluate the local tolerance and the local and systemic effects of this strategy, two studies were performed in rabbits. In the first study, two different split virion monovalent cHA vaccines, containing cH5/1N1 and cH8/1N1, with or without AS01 or AS03, were injected at a two-week interval. In the second study, animals were given these vaccines and two weeks later an additional dose of split virion monovalent cHA vaccine containing cH11/1N1, with or without AS01 or AS03. General health status, rectal temperature, local tolerance, ophthalmology, hematology, coagulation, and blood chemistry parameters were monitored. Macroscopic and microscopic evaluations were performed three days after the last dose and after a treatment-free recovery period. The treatment-related changes included body weight loss and food consumption decrease, increases in neutrophil count, C-reactive protein and fibrinogen levels. Microscopic signs of inflammation at the injection sites and immune stimulation of the draining lymph nodes and spleen were also noticed. Most post-injection findings could be linked to the transient inflammation due to the establishment of the desired vaccine-elicited immune response, and were mainly observed in the adjuvanted groups. In conclusion, the sequential administration of different cHA vaccines was locally and systemically well-tolerated in rabbits., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: BB, CPM, ED, JB and RNR are, or were at the time of the study, employees of the GSK group of companies. ED and CPM report ownership of GSK shares and/or restricted shares. CPM is listed as inventor on patents owned by GSK. AB, CT-D and EG are, or were at the time of the study employees of Charles River Laboratories Evreux, a Contract Research Organization contracted by GSK in the context of this study., (Copyright © 2020. Published by Elsevier Inc.)

Hemagglutinins (HAs) from human influenza viruses adapt to bind α2-6-linked sialosides, overcoming a receptor-defined species barrier distinct from the α2-3 specificity of avian virus progenitors. Additionally, human-adapted HAs gain glycosylation sites over time, although their biological function is poorly defined. Using quantitative glycomic analysis, we show that HAs from human pandemic viruses exhibit significant proportions of high-mannose type N-linked glycans throughout the head domain. By contrast, poorly adapted avian-origin HAs contain predominately complex-type glycans, which have greater structural diversity. Although oligomannose levels vary, they are present in all tested recombinant HAs and whole viruses and can be specifically targeted for universal detection. The positions of high-mannose glycosites on the HA of human H1N1 and H3N2 strains are conserved. Additionally, high-mannose-binding lectins possess a broad capacity to neutralize and prevent infection with contemporary H3N2 strains. These findings reveal the biological significance of HA glycosylation and therapeutic potential of targeting these structures., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The aim of this study was to determine the level of antibodies against hemagglutinin of influenza viruses in the sera of people in the seven age groups in the epidemic season 2018/2019 in Poland. The level of anti-hemagglutinin antibodies was determined by hemagglutination inhibition test (HAI). 1050 clinical samples from all over the country were tested. The level of antibodies against influenza viruses was highest in the 10-14 age group for A/Singapore/INFIMH-16-0019/2016 (H3N2) and B/Phuket/3073/2013 Yamagata lineage antigens. These results confirm the circulation of four antigenically different influenza virus strains, two subtypes of influenza A virus - A/Michigan/45/2015 (H1N1)pdm09 and A/Singapore/INFIMH-16-0019/2016 (H3N2) and two lineages of influenza B virus - B/Colorado/06/2017 - Victoria lineage and B/Phuket/3073/2013 Yamagata lineage.

The specificity of antibodies (Abs) generated against influenza A virus (IAV) infection can significantly alter protection and viral clearance. At present, the impact of age upon this process is relatively unexplored. Here, we evaluated the Ab response in newborn and adult African green monkeys following infection with IAV using a strain that enables us to determine the immunodominance (ID) hierarchy of the Ab response to hemagglutinin (HA), the principal target of protective Abs. This revealed altered ID patterns in the early IgM anti-HA response in newborns versus adults that converged over time. While the IgG ID profiles for HA in newborn and adult monkeys were similar, this was not the case for IgA. Importantly, HA stem-specific Abs were generated robustly and similarly in newborns and adults in terms of quality and quantity. Together, these results demonstrate that newborns and adults can differ in the Ab ID pattern established following infection and that the ID pattern can vary across isotypes. In addition, newborns have the ability to generate potent HA stem-specific Ab responses. Our findings further the understanding of the newborn response to IAV antigens and inform the development of improved vaccines for this at-risk population.

Background: Isohemagglutinins (anti-A and anti-B) mediate hemolytic transfusion reactions, antibody-mediated rejection of solid-organ transplants, and delayed engraftment after stem cell transplant. However, quantification of isohemagglutinins is often labor intensive and operator dependent, limiting availability and interfacility comparisons. We evaluated an automated, solid-phase and agglutination-based antibody titer platform versus manual gel testing., Study Design and Methods: Plasma samples were obtained from 54 randomly selected patients. Titers were determined by our laboratory's standard assay (manual dilution followed by manual gel testing) and were compared to results obtained on a fully automated blood bank analyzer (Galileo NEO, Immucor). The analyzer determined immunoglobulin G (IgG) antibodies using solid-phase and immunoglobulin M (IgM) antibodies by direct hemagglutination., Results: Isohemagglutinin titers obtained by manual gel versus the automated assay generally (>80%) agreed within one doubling dilution, and always (100%) agreed within two dilutions. Among O samples, the gel titer and the highest titer obtained with the automated assay (either IgG or IgM) were similar in paired, nonparametric analysis (p = 0.06 for anti-A; p = 0.13 for anti-B). Gel titers from group A and group B patients were slightly higher than the highest titer obtained using the automated assay (p = 0.04 for group A; p = 0.009 for group B), although these differences were within the accepted error of measurement., Conclusion: Manual and automated methodologies yielded similar isohemagglutinin titers. Separate quantification of IgM and IgG isohemagglutinins via automated titration may yield additional insight into hemolysis, graft survival after ABO-incompatible transplantation, and red blood cell engraftment after ABO-incompatible stem cell transplant., (© 2020 AABB.)

The discovery of potent and broadly protective influenza virus epitopes could lead to improved vaccines that are resistant to antigenic drift. Here, we describe human antibody C585, isolated from a vaccinee with remarkable serological breadth as measured by hemagglutinin inhibition (HAI). C585 binds and neutralizes multiple H3N2 strains isolated between 1968 and 2016, including strains that emerged up to 4 years after B cells were isolated from the vaccinated donor. The crystal structure of C585 Fab in complex with the HA from A/Switzerland/9715293/2013 (H3N2) shows that the antibody binds to a novel and well-conserved epitope on the globular head of H3 HA and that it differs from other antibodies not only in its epitope but in its binding geometry and hypermutated framework 3 region, thereby explaining its breadth and ability to mediate hemagglutination inhibition across decades of H3N2 strains. The existence of epitopes such as the one elucidated by C585 has implications for rational vaccine design. IMPORTANCE Influenza viruses escape immunity through continuous antigenic changes that occur predominantly on the viral hemagglutinin (HA). Induction of broadly neutralizing antibodies (bnAbs) targeting conserved epitopes following vaccination is a goal of universal influenza vaccines and advantageous in protecting hosts against virus evolution and antigenic drift. To date, most of the discovered bnAbs bind either to conserved sites in the stem region or to the sialic acid-binding pocket. Generally, antibodies targeting the stem region offer broader breadth with low potency, while antibodies targeting the sialic acid-binding pocket cover narrower breadth but usually have higher potency. In this study, we identified a novel neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against a broad range of H3N2 with high potency. This epitope may provide insights for future universal vaccine design., (Copyright © 2020 American Society for Microbiology.)

The transmissibility and pandemic potential of influenza viruses depends on their ability to efficiently replicate and be released from an infected host, retain viability as they pass through the environment, and then initiate infection in the next host. There is a significant gap in knowledge about viral properties that enable survival of influenza viruses between hosts, due to a lack of experimental methods to reliably isolate viable virus from the air. Using a novel technique, we isolate and characterise infectious virus from droplets emitted by 2009 pandemic H1N1-infected ferrets. We demonstrate that infectious virus is predominantly released early after infection. A virus containing a mutation destabilising the haemagglutinin (HA) surface protein displayed reduced survival in air. Infectious virus recovered from droplets exhaled by ferrets inoculated with this virus contained mutations that conferred restabilisation of HA, indicating the importance of influenza HA stability for between-host survival. Using this unique approach can improve knowledge about the determinants and mechanisms of influenza transmissibility and ultimately could be applied to studies of airborne virus exhaled from infected people., Competing Interests: The authors have declared that no competing interests exist.

Objective: To recover broad-neutralizing monoclonal antibodies (BnAbs) from avian influenza A (H5N1) virus infection cases and investigate their genetic and functional features., Methods: We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients. The genetic basis, biological functions, and epitopes of the obtained BnAbs were assessed and modeled., Results: Two BnAbs, 2-12D5, and 3-37G7.1, were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset. Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity (ADCC) activity. Albeit derived from distinct Ab lineages, i.e. , V H 1-69-D2-15-J H 4 (2-12D5) and V H 1-2-D3-9-J H 5 (3-32G7.1), the BnAbs were directed toward CR6261-like epitopes in the HA stem, and HA 2 I45 in the hydrophobic pocket was the critical residue for their binding. Signature motifs for binding with the HA stem, namely, IFY in V H 1-69-encoded Abs and LXYFXW in D3-9-encoded Abs, were also observed in 2-12D5 and 3-32G7.1, respectively., Conclusions: Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus. The HA stem epitopes targeted by the BnAbs, and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development., (Copyright © 2020 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.

The majority of antibodies induced by influenza neuraminidase (NA), like those against hemagglutinin (HA), are relatively specific to viruses isolated within a limited time window, as seen in serological studies and the analysis of many murine monoclonal antibodies (MAbs). We report three broadly reactive human MAbs targeting N1 NA. Two were isolated from a young adult vaccinated with trivalent influenza vaccine (TIV), which inhibited N1 NA from viruses isolated from humans over a period of a hundred years. The third antibody, isolated from a child with acute mild H7N9 infection, inhibited both group 1 N1 and group 2 N9 NAs. In addition, the antibodies cross-inhibited the N1 NAs of highly pathogenic avian H5N1 influenza viruses. These antibodies are protective in prophylaxis against seasonal H1N1 viruses in mice. This study demonstrates that human antibodies to N1 NA with exceptional cross-reactivity can be recalled by vaccination and highlights the importance of standardizing the NA antigen in seasonal vaccines to offer optimal protection. IMPORTANCE Antibodies to the influenza virus NA can provide protection against influenza disease. Analysis of human antibodies to NA lags behind that of antibodies to HA. We show that human monoclonal antibodies against NA induced by vaccination and infection can be very broadly reactive, with the ability to inhibit a wide spectrum of N1 NAs on viruses isolated between 1918 and 2018. This suggests that antibodies to NA may be a useful therapy and that the efficacy of influenza vaccines could be enhanced by ensuring the appropriate content of NA antigen., (Copyright © 2020 Rijal et al.)

Hemagglutinin (HA) stability, or the pH at which HA is activated to cause membrane fusion, has been associated with the replication, pathogenicity, transmissibility, and interspecies adaptation of influenza A viruses. Here, we investigated the mechanisms by which a destabilizing HA mutation, Y17H (activation pH, 6.0), attenuates virus replication and pathogenicity in DBA/2 mice compared to wild-type (WT) virus (activation pH, 5.5). The extracellular lung pH was measured to be near neutral (pH 6.9 to 7.5). WT and Y17H viruses had similar environmental stability at pH 7.0; thus, extracellular inactivation was unlikely to attenuate the Y17H virus. The Y17H virus had accelerated replication kinetics in MDCK, A549, and RAW 264.7 cells when inoculated at a multiplicity of infection (MOI) of 3 PFU/cell. The destabilizing mutation also increased early infectivity and type I interferon (IFN) responses in mouse bone marrow-derived dendritic cells (DCs). In contrast, the HA-Y17H mutation reduced virus replication in murine airway murine nasal epithelial cell and murine tracheal epithelial cell cultures and attenuated virus replication, virus spread, the severity of infection, and cellular infiltration in the lungs of mice. Normalizing virus infection and weight loss in mice by inoculating them with Y17H virus at a dose 500-fold higher than that of WT virus revealed that the destabilized mutant virus triggered the upregulation of more host genes and increased type I IFN responses and cytokine expression in DBA/2 mouse lungs. Overall, HA destabilization decreased virulence in mice by boosting early infection in DCs, resulting in the greater activation of antiviral responses, including the type I IFN response. These studies reveal that HA stability may regulate pathogenicity by modulating IFN responses. IMPORTANCE Diverse influenza A viruses circulate in wild aquatic birds, occasionally infecting farm animals. Rarely, an avian- or swine-origin influenza virus adapts to humans and starts a pandemic. Seasonal and many universal influenza vaccines target the HA surface protein, which is a key component of pandemic influenza viruses. Understanding the HA properties needed for replication and pathogenicity in mammals may guide response efforts to control influenza. Some antiviral drugs and broadly reactive influenza vaccines that target the HA protein have suffered resistance due to destabilizing HA mutations that do not compromise replicative fitness in cell culture. Here, we show that despite not compromising fitness in standard cell cultures, a destabilizing H1N1 HA stalk mutation greatly diminishes viral replication and pathogenicity in vivo by modulating type I IFN responses. This encourages targeting the HA stalk with antiviral drugs and vaccines as well as reevaluating previous candidates that were susceptible to destabilizing resistance mutations., (Copyright © 2020 American Society for Microbiology.)

A project aimed at establishing replacement batches for the European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) Bordetella (B.) pertussis mouse antiserum was started in 2013 under the aegis of the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). This BRP is used for the immunogenicity assay in mice to assess the potency of acellular pertussis (aP) vaccines as described in Ph. Eur. general method 2.7.16. Assay of pertussis vaccine (acellular). In a preliminary phase of the project (referred to herein as BSP129 phase 1) a hyper-immune serum pool was produced in mice using a combined aP vaccine as immunogen. This pool was used to generate 3 freeze-dried candidate (c) B. pertussis anti-mouse serum BRP batches (cBRP2, cBRP3 and cBRP4). After the pre-qualification that showed their suitability as candidate batches, an international collaborative study (BSP129 phase 2) was carried out in order to standardise these 3 batches against the current BRP1 in terms of anti-PT, -FHA, -PRN and -FIM2/3 antibody contents. For the sake of continuity with the standardisation of BRP1, the corresponding WHO standard (1RR 97/642) was introduced as a second reference for the calibration of the 3 candidate BRPs. Eleven laboratories took part in phase 2. Ten of them performed the ELISA method they use routinely for aP vaccine batch release and one laboratory performed the Multiplex Immunoassay (MIA) as an alternative test. Four participants titrated the antibodies against all 5 pertussis antigens, 5 participants determined the antibody content against 3 antigens (PT, FHA, PRN), one participant titrated the antibodies against PT and FHA antigens and one laboratory determined the antibody content for the PT antigen only. Details of all ELISA methods used were analysed to evaluate their impact on the calibration of the cBRPs. The variability of the results in relation to the nature and methodology of the tests appeared rather limited. Discrepant titres of cBRPs were measured depending on the reference used: the use of the 1RR induced an overestimation (in 8 out of 11 laboratories) and a large inter-laboratory variation in the calculated titres. Regardless of the reference used, equivalency between the calculated titres of cBRP2 and cBRP3 was observed, whilst cBRP4 had systematically lower titres for all antibodies against the 5 acellular pertussis vaccine components. Based on these observations, it was decided to establish the candidate BRP batches against BRP1 and to assign the following potencies based on the mean values determined through centrally calculated results of the calibration assays performed by ELISA in BSP129 phase 2: For cBRP2 and cBRP3 Anti-pertussis toxin: 37 ELISA Units (ELU) per vial Anti-filamentous haemagglutinin: 114 ELU per vial Anti-pertactin: 44 ELU per vial Anti-fimbrial agglutinogens (FIM2/3): 25 ELU per vial For cBRP4 Anti-pertussis toxin: 32 ELU per vial Anti-filamentous haemagglutinin: 98 ELU per vial Anti-pertactin 38 ELU per vial Anti-fimbrial agglutinogens (FIM2/3):23 ELU per vial In February 2018, BRP2, BRP3 and BRP4 were adopted by correspondence by the Ph. Eur. Commission., (© Council of Europe 2020.)

Objectives: To analyze the prevalence of systemic de novo thrombotic microangiopathy in ABO-incompatible kidney transplantation and risk factors associated with this condition., Methods: A total of 201 patients who received living-donor kidney transplantation (114 patients with ABO-identical kidney transplantation and 87 patients with ABO-incompatible kidney transplantation) were retrospectively analyzed. Systemic de novo thrombotic microangiopathy was diagnosed clinically according to the presence of thrombocytopenia with microangiopathic hemolytic anemia and pathological findings of thrombotic microangiopathy. Anti-A and anti-B antibodies were purified from human plasma, and these antibodies' bindings to human kidney were investigated in vitro., Results: ABO-incompatible kidney transplantation was a significant risk factor of systemic de novo thrombotic microangiopathy (odds ratio 55.9, 95% CI 1.8-8.9, P < 0.001) after transplantation. Multivariate logistic regression analysis showed that non-use of mycophenolate mofetil, pretreatment immunoglobulin G antibody titer ≥64-fold and pretransplant immunoglobulin M antibody titer ≥16-fold were significant risk factors for systemic de novo thrombotic microangiopathy in ABO-incompatible kidney transplantation. Microvascular inflammation of 1-h post-transplant biopsy could be observed more frequently in thrombotic microangiopathy patients than in non-thrombotic microangiopathy patients. Anti-A and anti-B antibodies purified from human plasma showed a strong in vitro reaction against human kidney when the antibody titer was ≥16-fold., Conclusions: Antibody titer should be decreased to ≤16-fold until the day of ABO-incompatible kidney transplantation by desensitization therapy including mycophenolate mofetil. The 1-h biopsy results might help to diagnose systemic de novo thrombotic microangiopathy., (© 2019 The Japanese Urological Association.)

Hemagglutinin (HA) is the major surface antigen of influenza virus and the most promising influenza vaccine immunogen. In 2013, the devastating H7N9 influenza virus was identified in China, which induced high mortality. The HA of this virus (H7) is relatively unstable, making it challenging to produce an effective vaccine. To improve the stability of HA protein from H7N9 influenza virus for better vaccine antigens without impairing immunogenicity, we recombined the HA from H7N9 (H7) with a more stable HA from H3N2 (H3) by structure-guided recombination, resulting in six chimeric HAs, FrA-FrF. Two of these chimeric HAs, FrB and FrC, exhibited proper hemagglutination activity and presented improved thermal stability compared to the original H7. Mice immunized with FrB and FrC elicited H7-specific antibodies comparable to those induced by parental H7, and the antisera collected from these immunized mice successfully inhibited H7N9 infection in a microneutralization assay. These results suggest that our structural-recombination approach can create stabilizing chimeric antigens while maintaining proper immunogenicity, which may not only benefit the construction of more stable HA vaccines to fight against H7N9 infection, but also facilitate effective vaccine improvements for other influenza viruses or infectious pathogens. In addition, this study also demonstrates the potential for better engineering of multimeric protein complexes like HA to achieve improved function, which are often immunologically or pharmaceutically important but difficult to modify.

Broadly neutralizing Abs targeting the HA stem can provide broad protection against different influenza subtypes, raising the question of how best to elicit such Abs. We have previously demonstrated that vaccination with pandemic live-attenuated influenza vaccine (pLAIV) establishes immune memory for HA head-specific Abs. Here, we determine the extent to which matched versus mismatched LAIV-inactivated subunit vaccine (IIV) prime-boost vaccination elicits stem-specific memory B cells and Abs. We vaccinated African green monkeys with H5N1 pLAIV-pIIV or H5N1 pLAIV followed by seasonal IIV (sIIV) or with H5N1 pLAIV alone and measured Abs and HA-specific B cell responses. While we observed an increase in stem-specific memory B cells, head-specific memory B cell responses were substantially higher than stem-specific responses and were dominant even following boost with mismatched IIV. Neutralizing Abs against heterologous influenza viruses were undetectable. Head-specific B cells from draining lymph nodes exhibited germinal center markers, while stem-specific B cells found in the spleen and peripheral blood did not. Thus, although mismatched prime-boost generated a pool of stem-specific memory B cells, head-specific B cells and serum Abs substantially dominated the immune response. These findings have implications for including full-length native HA in prime-boost strategies intended to induce stem-specific Abs for universal influenza vaccination.

In recent years, cell-based influenza vaccines have gained a great interest over the egg-based vaccines. Several inactivated H7N9 vaccines have been evaluated in clinical trials, including whole-virion vaccines, split vaccines and subunit vaccines. Recently, we developed a new suspension MDCK (sMDCK) cell line for influenza viruses production. However, the properties of purified antigen from sMDCK cells remain unclear. In this study, the stability of influenza H7N9 vaccine bulk derived from sMDCK cells was investigated, and the data were compared with the vaccine antigen derived from our characterized adhesion MDCK (aMDCK) cells in serum-free medium. The influenza H7N9 bulks derived from sMDCK and aMDCK cells were stored at 2-8 °C for different periods of time, and a number of parameters selected to monitor the H7N9 vaccine antigen stability were evaluated at each interval (1, 3 and 12 months). The monitored parameters included virus morphology, hemagglutinin (HA) activity, HA concentration, antigenicity, and immunogenicity. The sMDCK-derived H7N9 bulk showed similar morphology to that of the aMDCK-derived H7N9 bulk, and there were no obvious changes after the extended storage periods. Furthermore, the HA titer, HA concentration, and antigenicity of sMDCK-derived H7N9 bulk were stable after 28 months of storage. Finally, the results of hemagglutination inhibition and neutralization tests showed that sMDCK- and aMDCK-derived H7N9 vaccines had comparable immunogenicity. These results indicated that sMDCK-derived H7N9 bulk has good stability compared to that of aMDCK-derived H7N9 bulk. Thus, the newly developed suspension MDCK cell line shows a great alternative for manufacturing cell-based influenza vaccines., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The novel H7N9 avian influenza A virus has caused human infections in China since 2013; some isolates from the fifth wave of infections have emerged as highly pathogenic avian influenza viruses. Recombinant hemagglutinin proteins of H7N9 viruses can be rapidly and efficiently produced with low-level biocontainment facilities. In this study, recombinant H7 antigen was obtained from engineered stable clones of Chinese Hamster Ovary (CHO) cells for subsequent large-scale production. The stable CHO cell clones were also adapted to grow in serum-free suspension cultures. To improve the immunogenicity of the recombinant H7 antigens, we evaluated the use of a novel combination adjuvant of PELC and CpG (PELC/CpG) to augment the anti-H7N9 immune responses in mice. We compared the effects with other adjuvants such as alum, AddaVax (MF59-like), and several Toll-like receptor ligands such as R848, CpG, and poly (I:C). With the PELC/CpG combination adjuvant, CHO cell-expressed rH7 antigens containing terminally sialylated complex type N-glycans were able to induce high titers of neutralizing antibodies in sera and conferred protection following live virus challenges. These data indicate that the CHO cell-expressed recombinant H7 antigens and a PELC/CpG combination adjuvant can be used for H7N9 subunit vaccine development., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)