Discrimination of the H1N1 and H5N2 Variants of Influenza A Virus Using an Isomeric Sialic Acid-Conjugated Graphene Field-Effect Transistor.

There has been a continuous effort to fabricate a fast, sensitive, and inexpensive system for influenza virus detection to meet the demand for effective screening in point-of-care testing. Herein, we report a sialic acid (SA)-conjugated graphene field-effect transistor (SA-GFET) sensor designed using α2,3-linked sialic acid (3'-SA) and α2,6-linked sialic acid (6'-SA) for the detection and discrimination of the hemagglutinin (HA) protein of the H5N2 and H1N1 viruses. 3'-SA and 6'-SA specific for H5 and H1 influenza were used in the SA-GFET to capture the HA protein of the influenza virus. The net charge of the captured viral sample led to a change in the electrical current of the SA-GFET platform, which could be correlated to the concentration of the viral sample. This SA-GFET platform exhibited a highly sensitive response in the range of 101-106 pfu mL-1, with a limit of detection (LOD) of 101 pfu mL-1 in buffer solution and a response time of approximately 10 s. The selectivity of the SA-GFET platform for the H1N1 and H5N2 influenza viruses was verified by testing analogous respiratory viruses, i.e., influenza B and the spike protein of SARS-CoV-2 and MERS-CoV, on the SA-GFET. Overall, the results demonstrate that the developed dual-channel SA-GFET platform can potentially serve as a highly efficient and sensitive sensing platform for the rapid detection of infectious diseases.

An influenza hemagglutinin stem nanoparticle vaccine induces cross-group 1 neutralizing antibodies in healthy adults.

Influenza vaccines could be improved by platforms inducing cross-reactive immunity. Immunodominance of the influenza hemagglutinin (HA) head in currently licensed vaccines impedes induction of cross-reactive neutralizing stem-directed antibodies. A vaccine without the variable HA head domain has the potential to focus the immune response on the conserved HA stem. This first-in-human dose-escalation open-label phase 1 clinical trial (NCT03814720) tested an HA stabilized stem ferritin nanoparticle vaccine (H1ssF) based on the H1 HA stem of A/New Caledonia/20/1999. Fifty-two healthy adults aged 18 to 70 years old enrolled to receive either 20 µg of H1ssF once (n = 5) or 60 µg of H1ssF twice (n = 47) with a prime-boost interval of 16 weeks. Thirty-five (74%) 60-µg dose participants received the boost, whereas 11 (23%) boost vaccinations were missed because of public health restrictions in the early stages of the COVID-19 pandemic. The primary objective of this trial was to evaluate the safety and tolerability of H1ssF, and the secondary objective was to evaluate antibody responses after vaccination. H1ssF was safe and well tolerated, with mild solicited local and systemic reactogenicity. The most common symptoms included pain or tenderness at the injection site (n = 10, 19%), headache (n = 10, 19%), and malaise (n = 6, 12%). We found that H1ssF elicited cross-reactive neutralizing antibodies against the conserved HA stem of group 1 influenza viruses, despite previous H1 subtype head-specific immunity. These responses were durable, with neutralizing antibodies observed more than 1 year after vaccination. Our results support this platform as a step forward in the development of a universal influenza vaccine.

A Bayesian approach to incorporate structural data into the mapping of genotype to antigenic phenotype of influenza A(H3N2) viruses.

Surface antigens of pathogens are commonly targeted by vaccine-elicited antibodies but antigenic variability, notably in RNA viruses such as influenza, HIV and SARS-CoV-2, pose challenges for control by vaccination. For example, influenza A(H3N2) entered the human population in 1968 causing a pandemic and has since been monitored, along with other seasonal influenza viruses, for the emergence of antigenic drift variants through intensive global surveillance and laboratory characterisation. Statistical models of the relationship between genetic differences among viruses and their antigenic similarity provide useful information to inform vaccine development, though accurate identification of causative mutations is complicated by highly correlated genetic signals that arise due to the evolutionary process. Here, using a sparse hierarchical Bayesian analogue of an experimentally validated model for integrating genetic and antigenic data, we identify the genetic changes in influenza A(H3N2) virus that underpin antigenic drift. We show that incorporating protein structural data into variable selection helps resolve ambiguities arising due to correlated signals, with the proportion of variables representing haemagglutinin positions decisively included, or excluded, increased from 59.8% to 72.4%. The accuracy of variable selection judged by proximity to experimentally determined antigenic sites was improved simultaneously. Structure-guided variable selection thus improves confidence in the identification of genetic explanations of antigenic variation and we also show that prioritising the identification of causative mutations is not detrimental to the predictive capability of the analysis. Indeed, incorporating structural information into variable selection resulted in a model that could more accurately predict antigenic assay titres for phenotypically-uncharacterised virus from genetic sequence. Combined, these analyses have the potential to inform choices of reference viruses, the targeting of laboratory assays, and predictions of the evolutionary success of different genotypes, and can therefore be used to inform vaccine selection processes.

Low prevalence of influenza viruses and predominance of A(H3N2) virus with respect to SARS-CoV-2 during the 2021-2022 season in Bulgaria.

Social distancing, mask-wearing, and travel restrictions during the COVID-19 pandemic have significantly impacted the spread of influenza viruses. The objectives of this study were to analyze the pattern of influenza virus circulation with respect to that of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Bulgaria during the 2021-2022 season and to perform a phylogenetic/molecular analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains. Influenza infection was confirmed using real-time reverse transcription polymerase chain reaction in 93 (4.2%) of the 2193 patients with acute respiratory illness tested wherein all detected viruses were subtyped as A(H3N2). SARS-CoV-2 was identified in 377 (24.3%) of the 1552 patients tested. Significant differences in the incidence of influenza viruses and SARS-CoV-2 were found between individual age groups, outpatients/inpatients, and in the seasonal distribution of cases. Two cases of coinfections were identified. In hospitalized patients, the Ct values of influenza viruses at admission were lower in adults aged ≥65 years (indicating higher viral load) than in children aged 0-14 years (p < 0.05). In SARS-CoV-2-positive inpatients, this association was not statistically significant. HA genes of all A(H3N2) viruses analyzed belonged to subclade 3C.2a1b.2a. The sequenced viruses carried 11 substitutions in HA and 5 in NA, in comparison to the vaccine virus A/Cambodia/e0826360/2020, including several substitutions in the HA antigenic sites B and C. This study revealed extensive changes in the typical epidemiology of influenza infection, including a dramatic reduction in the number of cases, diminished genetic diversity of circulating viruses, changes in age, and seasonal distribution of cases.

Use of the particle agglutination/particle agglutination inhibition test for antigenic analysis of SARS-CoV-2.

Background: The antigenicity of SARS-CoV-2 is a critical issue for the effectiveness of the vaccine, and thus, it should be phenotypically evaluated by serological assays as new field isolates emerge. The hemagglutination/hemagglutination inhibition (HA/HI) tests are well known as a representative method for antigenic analysis of influenza viruses, but SARS-CoV-2 does not agglutinate human or guinea pig red blood cells. Therefore, the antigenic analysis requires complicated cell-based assays using special equipment such as plate reader or ELISPOT analyzer. Methods: Based on the HA/HI tests for influenza viruses, we developed the particle agglutination/particle agglutination inhibition (PA/PAI) test to easily and rapidly quantify the virus and antibody using human angiotensin-converting enzyme 2 (hACE2)-bound latex beads. The virus titers were determined by mixing the beads and the virus from culture supernatant, settling it overnight, and then observing the sedimentation/agglutination pattern (PA test). The neutralization antibody titers were determined by mixing virus-infected hamster antisera in addition to the beads and virus (PAI test). Results: The PA titer was positively correlated with the plaque-forming units. The PAI titer using the hamster antisera clearly revealed the antigenic difference between the omicron and previous variants. The antigenic differences were supported by the results shown in other methods. Conclusions: The PAI test is an easy and rapid method to analyze the antigenicity of SARS-CoV-2.

Antigenic characterization of influenza and SARS-CoV-2 viruses.

Antigenic characterization of emerging and re-emerging viruses is necessary for the prevention of and response to outbreaks, evaluation of infection mechanisms, understanding of virus evolution, and selection of strains for vaccine development. Primary analytic methods, including enzyme-linked immunosorbent/lectin assays, hemagglutination inhibition, neuraminidase inhibition, micro-neutralization assays, and antigenic cartography, have been widely used in the field of influenza research. These techniques have been improved upon over time for increased analytical capacity, and some have been mobilized for the rapid characterization of the SARS-CoV-2 virus as well as its variants, facilitating the development of highly effective vaccines within 1 year of the initially reported outbreak. While great strides have been made for evaluating the antigenic properties of these viruses, multiple challenges prevent efficient vaccine strain selection and accurate assessment. For influenza, these barriers include the requirement for a large virus quantity to perform the assays, more than what can typically be provided by the clinical samples alone, cell- or egg-adapted mutations that can cause antigenic mismatch between the vaccine strain and circulating viruses, and up to a 6-month duration of vaccine development after vaccine strain selection, which allows viruses to continue evolving with potential for antigenic drift and, thus, antigenic mismatch between the vaccine strain and the emerging epidemic strain. SARS-CoV-2 characterization has faced similar challenges with the additional barrier of the need for facilities with high biosafety levels due to its infectious nature. In this study, we review the primary analytic methods used for antigenic characterization of influenza and SARS-CoV-2 and discuss the barriers of these methods and current developments for addressing these challenges.

Detection Methods for H1N1 Virus.

Influenza A virus H1N1, a respiratory virus transmitted via droplets and responsible for the global pandemic in 2009, belongs to the Orthomyxoviridae family, a single-negative-stranded RNA. It possesses glycoprotein spikes neuraminidase (NA), hemagglutinin (HA), and a matrix protein named M2. The Covid-19 pandemic affected the world population belongs to the respiratory virus category is currently mutating, this can also be observed in the case of H1N1 influenza A virus. Mutations in H1N1 can enhance the viral capacity which can lead to another pandemic. This virus affects children below 5 years, pregnant women, old age people, and immunocompromised individuals due to its high viral capacity. Its early detection is necessary for the patient's recovery time. In this book chapter, we mainly focus on the detection methods for H1N1, from traditional ones to the most advance including biosensors, RT-LAMP, multi-fluorescent PCR.

On the lookout for influenza viruses in Italy during the 2021-2022 season: Along came A(H3N2) viruses with a new phylogenetic makeup of their hemagglutinin.

AIMS: To assess influenza viruses (IVs) circulation and to evaluate A(H3N2) molecular evolution during the 2021-2022 season in Italy. MATERIALS AND METHODS: 12,393 respiratory specimens (nasopharyngeal swabs or broncho-alveolar lavages) collected from in/outpatients with influenza illness in the period spanning from January 1, 2022 (week 2022-01) to May 31, 2022 (week 2022-22) were analysed to identify IV genome and were molecularly characterized by 12 laboratories throughout Italy. A(H3N2) evolution was studied by conducting an in-depth phylogenetic analysis of the hemagglutinin (HA) gene sequences. The predicted vaccine efficacy (pVE) of vaccine strain against circulating A(H3N2) viruses was estimated using the sequence-based Pepitope model. RESULTS: The overall IV-positive rate was 7.2% (894/12,393), all were type A IVs. Almost all influenza A viruses (846/894; 94.6%) were H3N2 that circulated in Italy with a clear epidemic trend, with 10% positivity rate threshold crossed for six consecutive weeks from week 2022-11 to week 2022-16. According to the phylogenetic analysis of a subset of A(H3N2) strains (n=161), the study HA sequences were distributed into five different genetic clusters, all of them belonging to the clade 3C.2a, sub-clade 3C.2a1 and the genetic subgroup 3C.2a1b.2a.2. The selective pressure analysis of A(H3N2) sequences showed evidence of diversifying selection particularly in the amino acid position 156. The comparison between the predicted amino acid sequence of the 2021-2022 vaccine strain (A/Cambodia/e0826360/2020) and the study strains revealed 65 mutations in 59 HA amino acid positions, including the substitution H156S and Y159N in antigenic site B, within major antigenic sites adjacent to the receptor-binding site, suggesting the presence of drifted strains. According to the sequence-based Pepitope model, antigenic site B was the dominant antigenic site and the p(VE) against circulating A(H3N2) viruses was estimated to be -28.9%. DISCUSSION AND CONCLUSION: After a long period of very low IV activity since public health control measures have been introduced to face COVID-19 pandemic, along came A(H3N2) with a new phylogenetic makeup. Although the delayed 2021-2022 influenza season in Italy was characterized by a significant reduction of the width of the epidemic curve and in the intensity of the influenza activity compared to historical data, a marked genetic diversity of the HA of circulating A(H3N2) strains was observed. The identification of the H156S and Y159N substitutions within the main antigenic sites of most HA sequences also suggested the circulation of drifted variants with respect to the 2021-2022 vaccine strain. Molecular surveillance plays a critical role in the influenza surveillance architecture and it has to be strengthened also at local level to timely assess vaccine effectiveness and detect novel strains with potential impact on public health.

Antigenic Characterization of Human Monoclonal Antibodies for Therapeutic Use against H7N9 Avian Influenza Virus.

Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,500 human infections and the culling of millions of poultry. Despite large-scale poultry vaccination, H7N9 AIVs continue to circulate among poultry in China and pose a threat to human health. Previously, we isolated and generated four monoclonal antibodies (mAbs) derived from humans naturally infected with H7N9 AIV. Here, we investigated the hemagglutinin (HA) epitopes of H7N9 AIV targeted by these mAbs (L3A-44, K9B-122, L4A-14, and L4B-18) using immune escape studies. Our results revealed four key antigenic epitopes at HA amino acid positions 125, 133, 149, and 217. The mutant H7N9 viruses representing escape mutations containing an alanine-to-threonine substitution at residue 125 (A125T), a glycine-to-glutamic acid substitution at residue 133 (G133E), an asparagine-to-aspartic acid substitution at residue 149 (N149D), or a leucine-to-glutamine substitution at residue 217 (L217Q) showed reduced or completely abolished cross-reactivity with the mAbs, as measured by a hemagglutination inhibition (HI) assay. We further assessed the potential risk of these mutants to humans should they emerge following mAb treatment by measuring the impact of these HA mutations on virus fitness and evasion of host adaptive immunity. Here, we showed that the L4A-14 mAb had broad neutralizing capabilities, and its escape mutant N149D had reduced viral stability and human receptor binding and could be neutralized by both postinfection and antigen-induced sera. Therefore, the L4A-14 mAb could be a therapeutic candidate for H7N9 AIV infection in humans and warrants further investigation for therapeutic applications. IMPORTANCE Avian influenza virus (AIV) H7N9 continues to circulate and evolve in birds, posing a credible threat to humans. Antiviral drugs have proven useful for the treatment of severe influenza infections in humans; however, concerns have been raised as antiviral-resistant mutants have emerged. Monoclonal antibodies (mAbs) have been studied for both prophylactic and therapeutic applications in infectious disease control and have demonstrated great potential. For example, mAb treatment has significantly reduced the risk of people developing severe disease with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In addition to the protection efficiency, we should also consider the potential risk of the escape mutants generated by mAb treatment to public health by assessing their viral fitness and potential to compromise host adaptive immunity. Considering these parameters, we assessed four human mAbs derived from humans naturally infected with H7N9 AIV and showed that the mAb L4A-14 displayed potential as a therapeutic candidate.

Matrix-Encoding Gene Diversity of 624 Influenza A/H3N2 Genomes Does Not Show Association with Impaired Viral Detection by Commercialized qPCR Assays.

As for the case of SARS-CoV-2, genome sequencing of influenza viruses is of potential interest to raise and address virological issues. Recently, false-negativity of real-time reverse transcription-PCR (qPCR) assays that detect influenza A/H3N2 virus RNA were reported and associated with two mutations (A37T and C161T) in the Matrix-encoding (M1) gene located on viral segment 7. This triggered a national alert in France. The present study sought to assess the association between the presence of these mutations and potential false negative results of influenza A/H3N2 virus RNA detection by commercialized qPCR assays at the clinical virology laboratory of our university hospitals in southern France. This study focused on the genetic diversity in the M1 gene and segment 7 of 624 influenza A/H3N2 virus genomes obtained from respiratory samples having tested qPCR-positive with M1 gene-targeting assays in our clinical virology laboratory. A total of 585 among the 624 influenza A/H3N2 virus genomes (93.7%) were of clade 3C.2a1b.2a.2, and 39 (6.3%) were of clade 3C.2a1b.1a. M1 gene substitutions A37T and C161T were both present in 582 (93.3%) genomes, only of clade 3C.2a1b.2a.2. Substitution A37T was present in 621 (99.5%) genomes. Substitution C161T was present in 585 genomes (93.8%), all of clade 3C.2a1b.2a.2. Moreover, 21 other nucleotide positions were mutated in ≥90% of the genomes. The present study shows that A37T/C and C161T mutations, and other mutations in the M1 gene and segment 7, were widely present in influenza A/H3N2 virus genomes recovered from respiratory samples diagnosed qPCR-positive with commercialized assays.

Chemically Modified Bovine ß-Lactoglobulin as a Broad-Spectrum Influenza Virus Entry Inhibitor with the Potential to Combat Influenza Outbreaks.

Frequent outbreaks of the highly pathogenic influenza A virus (AIV) infection, together with the lack of broad-spectrum influenza vaccines, call for the development of broad-spectrum prophylactic agents. Previously, 3-hydroxyphthalic anhydride-modified bovine ß-lactoglobulin (3HP-ß-LG) was proven to be effective against human immunodeficiency virus (HIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and it has also been used in the clinical control of cervical human papillomavirus (HPV) infections. Here, we show its efficacy in potently inhibiting infection by divergent influenza A and B viruses. Mechanistic studies suggest that 3HP-ß-LG binds, possibly through its negatively charged residues, to the receptor-binding domain in the hemagglutinin 1 (HA1) subunit in the HA of the influenza virus, thus inhibiting the attachment of the HA to sialic acid on host cells. The intranasal administration of 3HP-ß-LG led to the protection of mice against challenges by influenza A(H1N1)/PR8, A(H3N2), and A(H7N9) viruses. Furthermore, 3HP-ß-LG is highly stable when stored at 50 °C for 30 days and it shows excellent safety in vitro and in vivo. Collectively, our findings suggest that 3HP-ß-LG could be successfully repurposed as an intranasal prophylactic agent to prevent influenza virus infections during influenza outbreaks.

Identification of Human Case of Avian Influenza A(H5N1) Infection, India.

A 11-year-old boy with acute myeloid leukemia was brought for treatment of severe acute respiratory infection in the National Capital Region, New Delhi, India. Avian influenza A(H5N1) infection was laboratory confirmed. Complete genome analysis indicated hemagglutinin gene clade 2.3.2.1a. We found the strain to be susceptible to amantadine and neuraminidase inhibitors.

Human-type sialic acid receptors contribute to avian influenza A virus binding and entry by hetero-multivalent interactions.

Establishment of zoonotic viruses, causing pandemics like the Spanish flu and Covid-19, requires adaptation to human receptors. Pandemic influenza A viruses (IAV) that crossed the avian-human species barrier switched from binding avian-type α2-3-linked sialic acid (2-3Sia) to human-type 2-6Sia receptors. Here, we show that this specificity switch is however less dichotomous as generally assumed. Binding and entry specificity were compared using mixed synthetic glycan gradients of 2-3Sia and 2-6Sia and by employing a genetically remodeled Sia repertoire on the surface of a Sia-free cell line and on a sialoglycoprotein secreted from these cells. Expression of a range of (mixed) 2-3Sia and 2-6Sia densities shows that non-binding human-type receptors efficiently enhanced avian IAV binding and entry provided the presence of a low density of high affinity avian-type receptors, and vice versa. Considering the heterogeneity of sialoglycan receptors encountered in vivo, hetero-multivalent binding is physiologically relevant and will impact evolutionary pathways leading to host adaptation.

Antigenic and virological properties of an H3N2 variant that continues to dominate the 2021-22 Northern Hemisphere influenza season.

Influenza viruses circulated at very low levels during the beginning of the COVID-19 pandemic, and population immunity against these viruses is low. An H3N2 strain (3C.2a1b.2a2) with a hemagglutinin (HA) that has several substitutions relative to the 2021-22 H3N2 vaccine strain is dominating the 2021-22 Northern Hemisphere influenza season. Here, we show that one of these substitutions eliminates a key glycosylation site on HA and alters sialic acid binding. Using glycan array profiling, we show that the 3C.2a1b.2a2 H3 maintains binding to an extended biantennary sialoside and replicates to high titers in human airway cells. We find that antibodies elicited by the 2021-22 Northern Hemisphere influenza vaccine poorly neutralize the 3C.2a1b.2a2 H3N2 strain. Together, these data indicate that 3C.2a1b.2a2 H3N2 viruses efficiently replicate in human cells and escape vaccine-elicited antibodies.

Influenza Vaccination Results in Differential Hemagglutinin Stalk-Specific Fc-Mediated Functions in Individuals Living With or Without HIV.

Influenza virus hemagglutinin (HA) stalk-specific antibodies have been shown to potently induce Fc-mediated effector functions which are important in protection from disease. In placebo-controlled maternal influenza (MatFlu) vaccination trials of pregnant women living with or without HIV, reduced risk of influenza illness was associated with high HA stalk antibody titers following trivalent inactivated vaccination (TIV). However, the mechanisms of immunity conferred by the HA stalk antibodies were not well understood. Here, we investigated HA stalk-specific Fc effector functions including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent complement deposition (ADCD), and FcγRIIa and FcγRIIIa binding in response to seasonal influenza vaccination. These were measured pre- and 1-month post-vaccination in 141 HIV-uninfected women (67 TIV and 74 placebo recipients) and 119 women living with HIV (WLWH; 66 TIV and 53 placebo recipients). In contrast to HIV-uninfected women, where HA stalk-specific ADCP and FcγRIIa binding were significantly boosted, WLWH showed no increase in response to vaccination. HA stalk-specific ADCC potential and FcγRIIIa binding were not boosted regardless of HIV status but were higher in WLWH compared with HIV-uninfected women prior to vaccination. HA stalk-specific ADCD was significantly increased by vaccination in all women, but was significantly lower in the WLWH both pre- and post- vaccination. Co-ordination between HA stalk-specific ADCP and ADCD in WLWH was improved by vaccination. Fc polyfunctionality was enhanced by vaccination in HIV-uninfected women and driven by the HA stalk antibody titers. However, in the WLWH, higher pre-vaccination Fc polyfunctionality was maintained post-vaccination but was decoupled from titer. Overall, we showed differential regulation of Fc effector HA stalk responses, suggesting that HIV infection results in unique humoral immunity in response to influenza vaccination, with relevance for future strategies that aim to target the HA stalk in this population.

Swine H1N1 Influenza Virus Variants with Enhanced Polymerase Activity and HA Stability Promote Airborne Transmission in Ferrets.

Understanding how animal influenza A viruses (IAVs) acquire airborne transmissibility in humans and ferrets is needed to prepare for and respond to pandemics. Here, we investigated in ferrets the replication and transmission of swine H1N1 isolates P4 and G15, whose majority population had decreased polymerase activity and poor hemagglutinin (HA) stability, respectively. For both isolates, a minor variant was selected and transmitted in ferrets. Polymerase-enhancing variant PA-S321 airborne-transmitted and propagated in one ferret. HA-stabilizing variant HA1-S210 was selected in all G15-inoculated ferrets and was transmitted by contact and airborne routes. With an efficient polymerase and a stable HA, the purified minor variant G15-HA1-S210 had earlier and higher peak titers in inoculated ferrets and was recovered at a higher frequency after airborne transmission than P4 and G15. Overall, HA stabilization played a more prominent role than polymerase enhancement in the replication and transmission of these viruses in ferrets. The results suggest pandemic risk-assessment studies may benefit from deep sequencing to identify minor variants with human-adapted traits. IMPORTANCE Diverse IAVs circulate in animals, yet few acquire the viral traits needed to start a human pandemic. A stabilized HA and mammalian-adapted polymerase have been shown to promote the adaptation of IAVs to humans and ferrets (the gold-standard model for IAV replication, pathogenicity, and transmissibility). Here, we used swine IAV isolates of the gamma lineage as a model to investigate the importance of HA stability and polymerase activity in promoting replication and transmission in ferrets. These are emerging viruses that bind to both α-2,6- and α-2,3-linked receptors. Using isolates containing mixed populations, a stabilized HA was selected within days in inoculated ferrets. An enhanced polymerase was also selected and propagated after airborne transmission to a ferret. Thus, HA stabilization was a stricter requirement, yet both traits promoted transmissibility. Knowing the viral traits needed for pandemic potential, and the relative importance of each, will help identify emerging viruses of greatest concern.

Teach 'em young: Influenza vaccines induce broadly neutralizing antibodies in children.

Antibodies against the influenza virus hemagglutinin stalk afford broad protection against antigenically drifted viruses. In this issue of Cell Reports Medicine, Yegorov et al.1 identify that current vaccine formulations induce neutralizing stalk antibodies in children-a highly vulnerable population.

Conserved topology of virus glycoepitopes presents novel targets for repurposing HIV antibody 2G12.

Complex glycans decorate viral surface proteins and play a critical role in virus-host interactions. Viral surface glycans shield vulnerable protein epitopes from host immunity yet can also present distinct "glycoepitopes" that can be targeted by host antibodies such as the potent anti-HIV antibody 2G12 that binds high-mannose glycans on gp120. Two recent publications demonstrate 2G12 binding to high mannose glycans on SARS-CoV-2 and select Influenza A (Flu) H3N2 viruses. Previously, our lab observed 2G12 binding and functional inhibition of a range of Flu viruses that include H3N2 and H1N1 lineages. In this manuscript, we present these data alongside structural analyses to offer an expanded picture of 2G12-Flu interactions. Further, based on the remarkable breadth of 2G12 N-glycan recognition and the structural factors promoting glycoprotein oligomannosylation, we hypothesize that 2G12 glycoepitopes can be defined from protein structure alone according to N-glycan site topology. We develop a model describing 2G12 glycoepitopes based on N-glycan site topology, and apply the model to identify viruses within the Protein Data Bank presenting putative 2G12 glycoepitopes for 2G12 repurposing toward analytical, diagnostic, and therapeutic applications.

A self-assembling nanoparticle vaccine targeting the conserved epitope of influenza virus hemagglutinin stem elicits a cross-protective immune response.

Various vaccine strategies have been developed to provide broad protection against diverse influenza viruses. The hemagglutinin (HA) stem is the major potential target of these vaccines. Enhancing immunogenicity and eliciting cross-protective immune responses are critical for HA stem-based vaccine designs. In this study, the A helix (Ah) and CD helix (CDh) from the HA stem were fused with ferritin, individually, or in tandem, yielding Ah-f, CDh-f and (A + CD)h-f nanoparticles (NPs), respectively. These NPs were produced through a prokaryotic expression system. After three immunizations with AS03-adjuvanted NPs in BALB/c mice via the subcutaneous route, CDh-f and (A + CD)h-f induced robust humoral and cellular immune responses. Furthermore, CDh-f and (A + CD)h-f conferred complete protection against a lethal challenge of H3N2 virus, while no remarkable immune responses and protective effects were detected in the Ah-f group. These results indicate that the CDh-based nanovaccine represents a promising vaccine platform against influenza, and the epitope-conjugated ferritin NPs may be a potential vaccine platform against other infectious viruses, such as SARS-COV-2.