HA Stem Research Articles

The complement protein C1q modulates the activities of anti-influenza HA-stem and anti-SARS-Cov2-nonRBD antibodies and alters viral evolution

Abstract The broadly neutralizing hemagglutinin (BN-HA) stem antibodies represent an universal vaccine prospect. However, our understanding of how these antibodies interact with the influenza virus is limited by excluding other immune elements, such as complement, in our experimental systems. Here, we show that the Fc binding protein of the complement system, C1q, grants BN-HA stem Abs a novel attachment inhibition (AI) and boosts virus neutralization (VN) activity. C1q promotes AI even if added to preformed BN-HA Ab-virion-RBC complexes, pointing at the steric nature of the phenomenon. Most probably by the same mechanism of steric hindrance, C1q also contributes to fusion inhibition. By employing a panel of engineered avidity-reverted anti-BNHA Abs we show that the C1q-mediated VN enhancement is independent of Ab avidity. We also show that C1q’s presence expands the escape viral repertoire beyond the HA stem domain. Among selected viral variants, near-RBS mutations (E156K-Sb, R224I-Ca, S145N-Ca) modulate HA avidity while causing antigenic drift. Expanding our discoveries to SARS-CoV-2, we identified non-RBD anti-SARS-CoV-2 spike Abs which exhibit C1q-VN enhancement. With an increased SARS-CoV-2 spike per viral particle amount, C1q-VN increased concomitantly. Altogether, our results emphasize the general role of C1q in Ab-mediated antiviral activity and viral evolution. NIH/NIAID intramural funding

Heterosubtypic immune pressure accelerates emergence of influenza A virus escape phenotypes in mice

Rapid antigenic evolution of the influenza A virus surface antigen hemagglutinin undermines protection conferred by seasonal vaccines. Protective correlates targeted by universal vaccines such as cytotoxic T cells or HA stem directed broadly neutralizing antibodies have been shown to select for immune escape mutants during infection. We developed an in vivo serial passage mouse model for viral adaptation and used next generation sequencing to evaluate full genome viral evolution in the context of broadly protective immunity. Heterosubtypic immune pressure increased the incidence of genome-wide single nucleotide variants, though mutations found in early adapted populations were predominantly stochastic in nature. Prolonged adaptation under heterosubtypic immune selection resulted in the manifestation of highly virulent phenotypes that ablated vaccine mediated protection from mortality. High frequency mutations unique to escape phenotypes were identified within the polymerase encoding segments. These findings suggest that a suboptimial usage of population-wide universal influenza vaccine may drive formation of escape variants attributed to polygenic changes.

Genetic characterization and evolution of H6N6 subtype avian influenza viruses.

H6-subtype avian influenza virus (AIV) was prevalent in the world and could sporadically infect humans. Here, a new chicken-derived H6N6-subtype AIV strain A/chicken/Zhejiang/49/2021 (ZJ49) was isolated in Zhejiang Province, China in 2021. Phylogenetic analysis by Maximum likelihood methods showed that H6-subtype AIVs were classed into 13 groups according to HA gene. The ZJ49 strain belonged to the G12 group, which mainly consisted of strains from Asian and dominated in recent years. Based on NA gene, H6-subtype AIVs were divided into N6.1 and N6.2 clades according to the NA gene. The ZJ49 isolate was located in the N6.2e clade, which mainly consisted of the H5N6-subtype AIVs. Phylogenetic analysis by Bayesian methods showed that the effective quantity size of H6-subtype AIVs increased around 1990, reached a peak around 2015, declined after 2015, then kept in a stable level after 2018. The reassortment analysis predicted that the PB2, PA, and NA genes of ZJ49 may recombine with H5-subtype AIVs. The amino acid at 222 position of HA gene of ZJ49 strain mutated from A to V, suggesting that ZJ49 has a potential ability to cross species barriers. The four glycosylation sites were highly conserved, implying less impact on the fold and conception of HA stem structure. Our results revealed the complicated evolution, reassortment, and mutations of receptor binding sites of H6-subtype AIVs, which emphasize the importance to continuously monitor the epidemiology and evolution of H6-subtype AIVs.

Universally Immune: How Infection Permissive Next Generation Influenza Vaccines May Affect Population Immunity and Viral Spread.

Next generation influenza vaccines that target conserved epitopes are becoming a clinical reality but still have challenges to overcome. Universal next generation vaccines are considered a vital tool to combat future pandemic viruses and have the potential to vastly improve long-term protection against seasonal influenza viruses. Key vaccine strategies include HA-stem and T cell activating vaccines; however, they could have unintended effects for virus adaptation as they recognise the virus after cell entry and do not directly block infection. This may lead to immune pressure on residual viruses. The potential for immune escape is already evident, for both the HA stem and T cell epitopes, and mosaic approaches for pre-emptive immune priming may be needed to circumvent key variants. Live attenuated influenza vaccines have not been immunogenic enough to boost T cells in adults with established prior immunity. Therefore, viral vectors or peptide approaches are key to harnessing T cell responses. A plethora of viral vector vaccines and routes of administration may be needed for next generation vaccine strategies that require repeated long-term administration to overcome vector immunity and increase our arsenal against diverse influenza viruses.

Broad Neutralization of H1 and H3 Viruses by Adjuvanted Influenza HA Stem Vaccines in Non-human Primates

Broad Neutralization of H1 and H3 Viruses by Adjuvanted Influenza HA Stem Vaccines in Non-human Primates

Broad neutralization of H1 and H3 viruses by adjuvanted influenza HA stem vaccines in nonhuman primates.

Seasonal influenza vaccines confer protection against specific viral strains but have restricted breadth that limits their protective efficacy. The H1 and H3 subtypes of influenza A virus cause most of the seasonal epidemics observed in humans and are the major drivers of influenza A virus-associated mortality. The consequences of pandemic spread of COVID-19 underscore the public health importance of prospective vaccine development. Here, we show that headless hemagglutinin (HA) stabilized-stem immunogens presented on ferritin nanoparticles elicit broadly neutralizing antibody (bnAb) responses to diverse H1 and H3 viruses in nonhuman primates (NHPs) when delivered with a squalene-based oil-in-water emulsion adjuvant, AF03. The neutralization potency and breadth of antibodies isolated from NHPs were comparable to human bnAbs and extended to mismatched heterosubtypic influenza viruses. Although NHPs lack the immunoglobulin germline VH1-69 residues associated with the most prevalent human stem-directed bnAbs, other gene families compensated to generate bnAbs. Isolation and structural analyses of vaccine-induced bnAbs revealed extensive interaction with the fusion peptide on the HA stem, which is essential for viral entry. Antibodies elicited by these headless HA stabilized-stem vaccines neutralized diverse H1 and H3 influenza viruses and shared a mode of recognition analogous to human bnAbs, suggesting that these vaccines have the potential to confer broadly protective immunity against diverse viruses responsible for seasonal and pandemic influenza infections in humans.

Aggregation by peptide conjugation rescues poor immunogenicity of the HA stem

Influenza virus infection is a global public health threat. Current seasonal influenza vaccines are efficacious only when vaccine strains are matched with circulating strains. There is a critical need for developing “universal” vaccines that protect against all influenza viruses. HA stem is a promising target for developing broad-spectrum influenza vaccines due to its relatively conserved feature. However, HA stem is weakly immunogenic when administered alone in a soluble form. Several approaches have been employed to improve the immunogenicity of HA stem, including conjugation of HA stem with a highly immunogenic carrier protein or displaying HA stem on a nanoparticle scaffold. Converting a weakly immunologic protein into a multimer through aggregation can significantly enhance its immunogenicity, with some multimeric protein aggregates previously shown to be more immunogenic than their soluble counterparts in animal models. Here, we show that a chemically coupling a peptide derived from the head domain of PR8 HA (P35) with the poorly immunogenic HA stem protein results in aggregation of the HA stem which significantly increases stem-specific B cell responses following vaccination. Importantly, vaccination with this conjugate in the absence of adjuvant still induced robust B cell responses against stem in vivo. Improving HA stem immunogenicity by aggregation provides an alternative avenue to conjugation with exotic carrier proteins or nanoparticle formulation.

Biological strategies for osteoarthritis: from early diagnosis to treatment.

To provide an updated review of the literature on the use of orthobiologics as a potential treatment option to alleviate symptoms associated with osteoarthritis (OA), slow the progression of the disease, and aid in cartilage regeneration. A comprehensive review of the literature was performed to identify basic science and clinical studies examining the role of orthobiologics in the diagnosis and management of osteoarthritis. Certain molecules (such as interleukin-6 (IL-6), interleukin-8 (IL-8), matrix metalloproteinase (MMPs), cartilage oligomeric matrix protein (COMP), and tumor necrosis factor (TNF), microRNAs, growth differentiation factor 11 (GDF-11)) have been recognized as biomarkers that are implicated in the pathogenesis and progression of degenerative joint disease (DJD). These biomarkers have been used to develop newer diagnostic applications and targeted biologic therapies for DJD. Local injection therapy with biologic agents such as platelet-rich plasma or stem cell-based preparations has been associated with significant improvement in joint pain and function in patients with OA and has increased in popularity during the last decade. The combination of PRP with kartogenin or TGF-b3 may also enhance its biologic effect. The mesenchymal stem cell secretome has been recognized as a potential target for the development of OA therapies due to its role in mediating the chondroprotective effects of these cells. Recent experiments have also suggested the modification of gut microbiome as a newer method to prevent OA or alter the progression of the disease. The application of orthobiologics for the diagnosis and treatment of DJD is a rapidly evolving field that will continue to expand. The identification of OA-specific and joint-specific biomarker molecules for early diagnosis of OA would be extremely useful for the development of preventive and therapeutic protocols. Local injection therapies with HA, PRP, BMAC, and other stem cell-based preparations are currently being used to improve pain and function in patients with early OA or those with progressed disease who are not surgical candidates. Although the clinical outcomes of these therapies seem to be promising in clinical studies, future research will determine the true role of orthobiologic applications in the field of DJS.

Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A (H5N1) Virus

ObjectiveTo recover broad-neutralizing monoclonal antibodies (BnAbs) from avian influenza A (H5N1) virus infection cases and investigate their genetic and functional features. MethodsWe 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. ResultsTwo 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., VH1-69-D2-15-JH4 (2-12D5) and VH1-2-D3-9-JH5 (3-32G7.1), the BnAbs were directed toward CR6261-like epitopes in the HA stem, and HA2 I45 in the hydrophobic pocket was the critical residue for their binding. Signature motifs for binding with the HA stem, namely, IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs, were also observed in 2-12D5 and 3-32G7.1, respectively. ConclusionsCross-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.

N-benzyl 4,4-disubstituted piperidines as a potent class of influenza H1N1 virus inhibitors showing a novel mechanism of hemagglutinin fusion peptide interaction

The influenza virus hemagglutinin (HA) is an attractive target for antiviral therapy due to its essential role in mediating virus entry into the host cell. We here report the identification of a class of N-benzyl-4,4,-disubstituted piperidines as influenza A virus fusion inhibitors with specific activity against the H1N1 subtype. Using the highly efficient one-step Ugi four-component reaction, diverse library of piperidine-based analogues was synthesized and evaluated to explore the structure-activity relationships (SAR). Mechanistic studies, including resistance selection with the most active compound (2) demonstrated that it acts as an inhibitor of the low pH-induced HA-mediated membrane fusion process. Computational studies identified an as yet unrecognized fusion inhibitor binding site, which is located at the bottom of the HA2 stem in close proximity to the fusion peptide. A direct π-stacking interaction between the N-benzylpiperidine moiety of 2 and F9HA2 of the fusion peptide, reinforced with an additional π-stacking interaction with Y119HA2, and a salt bridge of the protonated piperidine nitrogen with E120HA2, were identified as important interactions to mediate ligand binding. This site rationalized the observed SAR and provided a structural explanation for the H1N1-specific activity of our inhibitors. Furthermore, the HA1-S326V mutation resulting in resistance to 2 is close to the proposed new binding pocket. Our findings point to the N-benzyl-4,4,-disubstituted piperidines as an interesting class of influenza virus inhibitors, representing the first example of fusion peptide binders with great potential for anti-influenza drug development.

Assessment of enhanced influenza vaccination finds thatFluAdconveys an advantage in mice and older adults.

ObjectivesEnhanced inactivated influenza vaccines (eIIV) aim to increase immunogenicity and protection compared with the widely used standard IIV (S‐IIV).MethodsWe tested four vaccines in parallel, FluZone high dose, FluBlok and FluAd versus S‐IIV in a randomised controlled trial of older adults and in a mouse infection model to assess immunogenicity, protection from lethal challenge and mechanisms of action.ResultsIn older adults, FluAd vaccination stimulated a superior antibody profile, including H3‐HA antibodies that were elevated for up to 1 year after vaccination, higher avidity H3HA IgG and larger HA stem IgG responses. In a mouse model, FluAd also elicited an earlier and larger induction of HA stem antibodies with increased germinal centre responses and upregulation and long‐term expression of B‐cell switch transcription factors. Long‐term cross‐reactive memory responses were sustained by FluAd following lethal heterosubtypic influenza challenge, with reduced lung damage and viral loads, coinciding with increased T‐ and B‐cell recall. Advantages were also noted for the high‐dose FluZone vaccine in both humans and mice.ConclusionThe early, broadly reactive and long‐lived antibody response of FluAd indicates a potential advantage of this vaccine, particularly in years when there is a mismatch between the vaccine strain and the circulating strain of influenza viruses.

Measuring influenza hemagglutinin (HA) stem-specific antibody-dependent cellular cytotoxicity (ADCC) in human sera using novel stabilized stem nanoparticle probes

BackgroundGenerating vaccine that confers a complete protection is a major goal in designing a universal influenza vaccine. Currently, there is a considerable interest in the broadly neutralizing antibodies (bnAb) targeting the conserved HA stem region. These antibodies have been shown to activate cellular immune responses, such as ADCC, in addition to their neutralization activity. We had previously demonstrated that immunization with H1-based stabilized stem (SS) nanoparticles (np) protects against heterosubtypic lethal H5N1 challenge, despite the absence of detectable neutralizing activity. Utilizing these novel SS probes to develop an ADCC assay would help in understanding the mechanism of action of stem-specific antibodies, as well as evaluating future influenza vaccines. ObjectivesTo develop a new protocol to assess the ADCC activity mediated by stem-directed antibodies in human sera using novel SS np probes. Study designHuman sera samples were screened for binding and ADCC activities to different influenza group 1 SS probes (H1, H2, and H5) using trimeric SS or multivalent SS-np (n = 8 trimers) formats. ResultsInitial screening revealed 63% (57/90) seroprevalence of anti-HA (H1) stem-epitope antibodies, as determined by the differential binding to HA SS and its corresponding epitope-mutant (Ile45Arg/Thr49Arg) probe. Using equimolar amounts, the multivalent presentation of HA SS on np induced significantly higher ADCC activity compared to the monovalent (trimer) SS probes (2–6 fold increase). Further, ADCC activity was similarly reported against different group 1 influenza subtypes: H1, H2, and H5. Importantly, ADCC was mediated mainly by antibodies targeting the bnAb-epitope on the HA stem. ConclusionWe report on an assay to measure stem-specific ADCC activity using SS np probes. Our results indicate high prevalence of HA-stem antibodies with cross-reactive ADCC activity. Such assay could be utilized in the assessment of next generation influenza vaccines.

Hemagglutinin head-specific responses dominate over stem-specific responses following prime boost with mismatched vaccines.

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.

1833. Measuring Influenza HA Stem-Specific ADCC in Human Sera Using Novel Stabilized Stem Nanoparticle Probes

BackgroundGenerating a vaccine that confers complete protection and overcomes the high variability among influenza viruses is the major goal in designing a universal influenza vaccine. Currently, there is considerable interest in the broadly neutralizing antibodies (bNAb) targeting the conserved HA stem region. These antibodies have been shown to activate cellular immune responses, such as antibody-dependent cellular cytotoxisity (ADCC), in addition to their neutralization activity. We had previously demonstrated that immunization with H1-based stabilized stem (SS) nanoparticles (np) protects against heterosubtypic lethal challenge with H5N1 influenza virus, despite the absence of detectable H5N1 neutralizing activity. Here, we utilized these novel SS np probes to develop a new protocol to assess the ADCC activity mediated by stem-directed antibodies in human sera.MethodsHuman sera samples were initially screened for binding reactivity to H1 SS trimer using ELISA procedure. Of these, selected samples representing high, moderate, and low binders were further characterized for binding to H1 and H5 SS their corresponding Δstem (Ile45Arg/Thr49Arg) probes, and were analyzed for ADCC activity using a reporter bioassay.ResultsThe initial screening revealed 82% (73/90) seroprevalence of anti-HA (H1) stem epitope antibodies, as determined by the differential binding to HA SS and it corresponding epitope-mutant probe. Using equimolar amounts, the multivalent presentation of HA SS on np probes induced significantly higher ADCC activity compared with the monovalent SS probes (2- to 6-fold increase). Further, ADCC activity was similarly reported against different influenza group 1 subtypes: H1, H2, and H5. Importantly, ADCC was mediated mainly by antibodies targeting the bNAb-epitope on the HA stem. In conclusion, we developed an assay to measure stem-specific ADCC activity using SS np probes.ConclusionOur results indicate a high prevalence of HA-stem antibodies with cross-reactive ADCC activity. Such assay could be utilized in the assessment of next-generation influenza vaccines. Disclosures All Authors: No reported Disclosures.

Requirement for memory B-cell activation in protection from heterologous influenza virus reinfection

While two memory compartments, memory B cells and long-lived plasma cells, are thought to contribute to the successful establishment of memory recall responses, the unique roles of each cellular compartment are still unclear. Herein, by tracing influenza anti-hemagglutinin (HA)-specific antibodies in mice, we demonstrate that pre-existing antibodies secreted by long-lived plasma cells are essential for protection from reinfection with the same influenza virus, whereas protection from secondary infection with an antigenically distinct influenza virus requires memory B-cell activation. These activated memory B cells were largely specific for the conserved HA stem region, and generated sufficient levels of antibodies for protection from heterologous reinfection. Given that the anti-stem plasmablasts derived from the memory B cells were higher affinity than those from naive B cells, our results suggest that maturation of anti-stem memory B cells during primary influenza infection and their subsequent activation are required for protection from reinfection by mutant viruses.

Novel approaches for studying cell-mediated immune responses to influenza vaccination in humans

Abstract Vaccination is the most cost-effective public health intervention strategy in the prevention of the spread of infectious diseases. Unfortunately, each year, a substantial proportion of individuals do not respond or respond poorly to vaccination due to age, nutrition, pre-existing medical conditions, vaccine mismatch, or other unknown reasons. To investigate this further, we propose studying the mechanisms and molecular signatures associated with immunogenicity and the efficacy of influenza vaccination in different populations. The priming environment, induced by the innate immune system is crucial for initiating and fine-tuning antigen-specific adaptive immune responses. Comprehensive antibody panels have been developed to identify the new paradigms in the innate priming environment, focusing on NK, innate lymphoid cells and γδ T cells by flow cytometry in PBMC from vaccine recipients. The specific functions of these cells are then analyzed using single cell sorting and high-throughput transcriptomic and proteomics analysis. To further probe the adaptive response, we have successfully developed receptor binding site mutant HA (H1, H3, B) and HA stem (group 1 and 2) probes that are conjugated to fluorochromes to enumerate and characterize HA-specific B cells. M2e antigen probes are under development. Combining cell sorting, single cell molecular analyses, and the Illumina MiSeq system, paired heavy and light chains of B cell receptors (BCR) from isolated antigen specific B cells are sequenced to dissect antibody diversity. Through these novel approaches, we aim to identify and characterize critical host factors that can be exploited to develop as novel adjuvants and strategies for more effective influenza vaccines.

Epidemiological and genetic characterization of pH1N1 and H3N2 influenza viruses circulated in MENA region during 2009\u20132017

BackgroundInfluenza surveillance is necessary for detection of emerging variants of epidemiologic and clinical significance. This study describes the epidemiology of influenza types A and B, and molecular characteristics of surface glycoproteins (hemagglutinin [HA] and neuraminidase [NA]) of influenza A subtypes: pH1N1 and H3N2 circulated in Arabian Gulf, Levant and North Africa regions during 2009–2017.MethodsAnalysis of phylogenetics and evolution of HA and NA genes was done using full HA and NA sequences (n = 1229) downloaded from Influenza Research Database (IRD).ResultsIn total, 130,354 influenza positive cases were reported to WHO during study period. Of these, 50.8% were pH1N1 positive, 15.9% were H3N2 positives and 17.2% were influenza B positive. With few exceptions, all three regions were showing the typical seasonal influenza peak similar to that reported in Northern hemisphere (December–March). However, influenza activity started earlier (October) in both Gulf and North Africa while commenced later during November in Levant countries. The molecular analysis of the HA genes (influenza A subtypes) revealed similar mutations to those reported worldwide. Generally, amino acid substitutions were most frequently found in head domain in H1N1 pandemic viruses, while localized mainly in the stem region in H3N2 viruses. Expectedly, seasons with high pH1N1 influenza activity was associated with a relatively higher number of substitutions in the head domain of the HA in pH1N1 subtype. Furthermore, nucleotide variations were lower at the antigenic sites of pH1N1 viruses compared to H3N2 viruses, which experienced higher variability at the antigenic sites, reflecting the increased immunological pressure because of longer circulation and continuous vaccine changes. Analysis of NA gene of pH1N1 viruses revealed sporadic detections of oseltamivir-resistance mutation, H275Y, in 4% of reported sequences, however, none of NAI resistance mutations were found in the NA of H3N2 viruses.ConclusionsMolecular characterization of H1N1 and H3N2 viruses over 9 years revealed significant differences with regard to position and function of characterized substitutions. While pH1N1 virus substitutions were mainly found in HA head domain, H3N2 virus substitutions were mostly found in HA stem domain. Additionally, more fixed substitutions were encountered in H3N2 virus compared to larger number of non-fixed substitutions in pH1N1.

Cross-reactive antibody-dependent cellular cytotoxicity antibodies are increased by recent infection in a household study of influenza transmission.

ObjectivesInfluenza causes a spectrum of disease from asymptomatic infection to fatal outcome, and pre‐existing immunity can alter susceptibility and disease severity. In a household transmission study, we recruited outpatients with confirmed influenza virus infection and prospectively identified secondary infections in their household contacts, therefore identifying infection cases with baseline samples for determining immune‐mediated protection from influenza infection.MethodsWe examined baseline broadly reactive immune correlates of relevance to universal vaccine development, specifically antibody‐dependent cytotoxic (ADCC) antibodies and T‐cell responses in functional assays. Antibodies were assessed in a cell‐based NK cell degranulation assay by flow cytometry, and T‐cell responses were assessed by IFN‐γ intracellular cytokine staining flow cytometry assay.ResultsThe magnitude of antibody responses and ADCC function for multiple influenza‐specific proteins was lower in participants who became infected, consolidating the role of pre‐existing antibodies in protection from seasonal influenza virus infection. Among H1N1‐infected contacts, we found that higher levels of pre‐existing H1‐haemagglutinin ADCC responses correlated with reduced symptom severity. Recent infection boosted the titre and magnitude of haemagglutinin‐, neuraminidase‐ and nucleoprotein‐specific ADCC antibodies. Limited T‐cell samples precluded conclusions on the role of pre‐existing T‐cell responses.ConclusionsOverall, ADCC responses are a protective correlate against influenza virus infection that should be considered in future vaccine development and evaluation.Influenza‐specific ADCC responses are elevated in uninfected subjects, associated with reduced symptoms and boosted by recent infection, whilst HA stem and NA IgG are also elevated in uninfected participants irrespective of ADCC function.

Different mechanisms of the protection against influenza A infection mediated by broadly reactive HA2-specific antibodies.

Influenza Aviruses (IAVs) cause yearly repeating infections in humans. The current vaccination approach is based on the production of virus-neutralizing antibodies. Virus-neutralizing antibodies, however, are closely strain-specific due to the IAV variability. Therefore, antibodies produced during the previous influenza season do not provide sufficient protection against new infection, and, hence, annual revaccination is needed. The utilization of the influenza conserved stem domain of hemagglutinin (HA), the HA2 gp, led to a new vaccine design based on cross-reactive cellular and especially humoral immune responses represented by HA2-specific antibodies. The HA2-specific antibodies exhibit cross-reactivity with HA2 gp within one subtype or even among subtypes and play a role in protective immunity against influenza infection. There are several elimination mechanisms of viral replication mediated by HA2-specific antibodies. After recognition of the epitope, they prevent the conformational rearrangement of HA or the insertion of the fusion protein into the endosomal membrane and, consequently, the fusion pore formation. In this case, no release of viral genetic information into the target cell is enabled and virus cannot replicate. The HA2-specific antibodies are involved in the elimination of pathogen via the Fc fragment by activation of the cytotoxic mechanisms of innate immunity as are the antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), or complement-dependent cytotoxicity (CDC), resulting in virus elimination and earlier recovery of the host from the infection. Though the protective effect of HA2-specific antibodies on the course of IAV infection was shown, few cases of worsening of IAV infection mediated by HA2-specific antibodies have been described. The identification of antigenic epitopes on HA2 gp that induce antibodies with such deteriorating effect on influenza infection can help to eliminate the unsuitable epitopes of HA2 gp as immunogens during the design of heteroprotective vaccine against influenza and can remove the side effects linked with the observations mentioned above. Keywords: influenza A virus; HA2 stem domain of hemagglutinin; immunization strategies; HA2-specific antibodies.

Differences in the ease with which mutant viruses escape from human monoclonal antibodies against the HA stem of influenza A virus

BackgroundBroadly protective human monoclonal antibodies that recognize the conserved epitopes in the HA of influenza A virus are being developed as therapeutic agents. Emergence of resistant viruses must always be considered when developing therapeutic agents against influenza. ObjectivesWe examined human hetero-reactive mAbs against the HA stem of influenza A virus for the ease with which escape mutant viruses emerged. Study designWe attempted to generate the mutant viruses escaped from the hetero-reactive anti-HA stem antibodies. We also evaluated their protective efficacy, binding affinity, and epitopes. ResultsWe obtained several human monoclonal antibodies (mAbs) that react with the HA of different HA subtypes of influenza A virus belonging to group 1. Upon attempting to generate escape mutant viruses, we found that the ease with which such viruses emerged differed among the mAbs; viruses barely escaped from two of the mAbs (clones S9-3-37 and F20C77), whereas escape from the third mAb (clone F5B7) occurred readily. Comparisons of the mAbs revealed that the HA stem epitopes, in vitro neutralization potency, binding affinity to H1-HA, and protective efficacy against lethal challenge with H1N1pdm09 virus were all comparable. ConclusionsThese results demonstrate the importance of determining the ease with which escape mutant viruses emerge when evaluating anti-HA stem antibodies as antiviral agents during preclinical testing.