Ian A. York

Glycosylation of the influenza A virus hemagglutinin is crucial for viral fitness and immune evasion. The hemagglutinin of contemporary human A(H3N2) influenza A viruses is extensively glycosylated with up to 13 putative glycosylation sites. Glycan types and occupancy of these sites are not uniform: while most are nearly completely occupied by glycans, some, such as those at amino acid residues 45 and 144, are only partially occupied and are evolutionarily unstable. While the effects of highly occupied glycosylated sites of hemagglutinin on A(H3N2) influenza A virus biology are well studied, there is limited information on the functional impact of the low-occupancy glycosylation sites on the viral hemagglutinin. Here, by using reverse-genetics A(H3N2) influenza A viruses lacking glycans at either residue 45 or 144, or both in hemagglutinin, we demonstrate that, despite reduced receptor binding, thermal stability, and fusion, the presence of a low level of glycosylation at these positions does not impact virus growth in Madin-Darby canine kidney epithelial cell culture. In contrast, these low-occupancy sites do affect immune responses in mice, by reducing titers of antibodies that correlate with virus neutralization and protection against influenza disease. We suggest that the temporary introduction of sites with low glycosylation allows influenza viruses to reduce the immune pressure on antigenic and receptor binding sites of hemagglutinin without significantly compromising viral virulence. This mechanism may help support the sustained circulation of A(H3N2) influenza A virus in human populations. IMPORTANCE: Many of the glycosylation sites on the hemagglutinin of influenza A viruses are near antigenic sites, such that the glycans block antibody recognition and help evade population immunity. However, glycans may also reduce viral replication and virulence. Glycosylation sites at amino acid residues 45 and 144 of the hemagglutinin of contemporary A(H3N2) influenza A viruses are minimally occupied, with fewer than 10% of the sites containing glycans. This is still sufficient to partially evade antibody-based population immunity, while minimizing the impact on replication and virulence. Over time, A(H3N2) viruses have used both these sites only during limited periods, suggesting that even a low level of glycan occupancy may confer some negative selection pressure that is compensated by evasion of human population immunity. Data from this study provide a better understanding of the mechanisms that allow viruses to evade antibody-based immunity and maintain circulation in the human population.

Background/Objectives: Humans are primarily infected by two distinct seasonal influenza A virus (IAV) subtypes, A(H1N1)pdm09 and A(H3N2). Because IAV mutates rapidly, it continuously escapes existing protective antibodies. However, repeated exposure to these evolving strains increases the diversity of virus-specific memory B cell (MBC) population. To investigate the extent of this diversity and cross-reactivity of IAV- specific immunity in adults, and its possible impact on emerging strains, we analyzed the hemagglutinin (HA)-specific MBC repertoire of an adult frequently immunized with seasonal vaccines. Methods: High-throughput single-cell sequencing of HA probe-positive MBCs collected from an individual with repeated seasonal influenza vaccination was performed. Monoclonal antibodies (mAbs) from multicell clonotypes were tested for HA binding and influenza virus neutralization. Results: Two clonotypes were identified that bound to both A(H1) and A(H3) HAs from strains circulating before and after the season of blood collection. Both clonotypes neutralized A(H3N2) strains, while one also neutralized A(H1N1)pdm09 strains. Notably, these cross-group binders showed affinity for A(H5), A(H7) and A(H9) avian influenza HAs and neutralized bovine A(H5N1) IAV. Conclusions: Our findings suggest that seasonal vaccination can support pre-existing cross-reactive B-cell immunity in some individuals. This underscores the importance of studying influenza-specific MBC repertoires across diverse populations to better understand immune dynamics and improve vaccine design.

The unprecedented 2.3.4.4b. A(H5N1) outbreak in dairy cattle, poultry, and spillover to humans in the United States (US) poses a major public health threat. Population immunity is a critical component of influenza pandemic risk assessment. We assessed the pre-existing cross-reactive immunity to 2.3.4.4b A(H5N1) viruses and analyzed 1794 sera from 723 people (0.5–88 yrs) in multiple US geographic regions during 2021–2024. Pre-existing neutralizing and hemagglutinin (HA)-head-binding antibodies to A(H5N1) were low, but there were substantial cross-reactive binding antibodies to N1 neuraminidase (NA) of 2.3.4.4b A(H5N1). Antibodies to group 1 HA stalk were also prevalent and increased with age. A(H1N1)pdm09 infection and influenza vaccination did not induce neutralizing antibodies to A(H5N1) viruses but induced significant rise of functional NA inhibition (NAI) antibodies to N1 of 2.3.4.4b A(H5N1), and group 1 HA stalk antibodies. Moreover, pre-pandemic stockpiled 2.3.4.4c vaccine can elicit cross-reactive neutralizing antibodies to 2.3.4.4b A(H5N1) viruses. Understanding population susceptibility is essential for pandemic preparedness.

Effect of Influenza Vaccines on H5N1 in Ferrets

Mice are widely used as small animal models for influenza infection and immunization studies because of their susceptibility to many strains of influenza, obvious clinical signs of infection, and ease of handling. Analgesia is rarely used in such studies even if nonstudy effects such as fight wounds, tail injuries, or severe dermatitis would otherwise justify it because of concerns that treatment might have confounding effects on primary study parameters such as the course of infection and/or the serological response to infection. However, analgesia for study-related or -unrelated effects may be desirable for animal welfare purposes. Opioids, such as extended-release buprenorphine, are well-characterized analgesics in mice and may have fewer immune-modulatory effects than other drug classes. In this study, BALB/c and DBA/2 mice were inoculated with influenza virus, and treatment groups received either no analgesics or 2 doses of extended-release buprenorphine 72 h apart. Clinical signs, mortality, and influenza-specific antibody responses were comparable in mice that did or did not receive buprenorphine. We therefore conclude that extended-release buprenorphine can be used to alleviate incidental pain during studies of influenza infection without altering the course of infection or the immune response.

The globular head domain of influenza virus surface protein hemagglutinin (HA1) is the major target of neutralizing antibodies elicited by vaccines. As little as one amino acid substitution in the HA1 can result in an antigenic drift of influenza viruses, indicating the dominance of some epitopes in the binding of HA to polyclonal serum antibodies. Therefore, identifying dominant binding epitopes of HA is critical for selecting seasonal influenza vaccine viruses. In this study, we have developed a biolayer interferometry (BLI)-based assay to determine dominant binding epitopes of the HA1 in antibody response to influenza vaccines using a panel of recombinant HA1 proteins of A(H1N1)pdm09 virus with each carrying a single amino acid substitution. Sera from individuals vaccinated with the 2010–2011 influenza trivalent vaccines were analyzed for their binding to the HA1 panel and hemagglutination inhibition (HI) activity against influenza viruses with cognate mutations. Results revealed an over 50% reduction in the BLI binding of several mutated HA1 compared to the wild type and a strong correlation between dominant residues identified by the BLI and HI assays. Our study demonstrates a method to systemically analyze antibody immunodominance in the humoral response to influenza vaccines.

Influenza A(H7N9) viruses remain as a high pandemic threat. The continued evolution of the A(H7N9) viruses poses major challenges in pandemic preparedness strategies through vaccination. We assessed the breadth of the heterologous neutralizing antibody responses against the 3rd and 5th wave A(H7N9) viruses using the 1st wave vaccine sera from 4 vaccine groups: 1. inactivated vaccine with 2.8 μg hemagglutinin (HA)/dose + AS03_A; 2. inactivated vaccine with 5.75 μg HA/dose + AS03_A; 3. inactivated vaccine with 11.5 μg HA/dose + MF59; and 4. recombinant virus like particle (VLP) vaccine with 15 μg HA/dose + ISCOMATRIX™. Vaccine group 1 had the highest antibody responses to the vaccine virus and the 3rd/5th wave drifted viruses. Notably, the relative levels of cross-reactivity to the drifted viruses as measured by the antibody GMT ratios to the 5th wave viruses were similar across all 4 vaccine groups. The 1st wave vaccines induced robust responses to the 3rd and Pearl River Delta lineage 5th wave viruses but lower cross-reactivity to the highly pathogenic 5th wave A(H7N9) virus. The population in the United States was largely immunologically naive to the A(H7N9) HA. Seasonal vaccination induced cross-reactive neuraminidase inhibition and binding antibodies to N9, but minimal cross-reactive antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies to A(H7N9).

Epidemiological studies suggest that humans who receive repeated annual immunization with influenza vaccine are less well protected against influenza than those who receive vaccine in the current season only. To better understand potential mechanisms underlying these observations, we vaccinated influenza-naive ferrets either twice, 10 months apart (repeated vaccination group; RV), or once (current season only group; CS), using a prime-boost regimen, and then challenged the ferrets with A/Hong Kong/4801/2014(H3N2). Ferrets that received either vaccine regimen were protected against influenza disease and infection relative to naive unvaccinated ferrets, but the RV group shed more virus, especially at the peak of virus shedding 2 days post infection (p < 0.001) and regained weight more slowly (p < 0.05) than those in the CS group. Qualitative, rather than quantitative, differences in the antibody response may affect protection after repeated influenza vaccination.

Influenza virus causes widespread, yearly epidemics by accumulating surface protein mutations to escape neutralizing antibodies established from prior exposure. In contrast to antibody epitopes, T cell mediated immunity targets influenza epitopes that are more highly conserved and have potential for cross-protection. The extent of T cell cross-reactivity between a diverse array of contemporary and historical influenza strains was investigated in ferrets challenged with 2009 pandemic H1N1 influenza or the seasonal H3N2 strain, A/Perth/16/2009. Post-challenge cell-mediated immune responses demonstrated extensive cross-reactivity with a wide variety of contemporary and historical influenza A strains as well as influenza B. Responses in peripheral blood were undetectable by 36d post-challenge, but cross-reactivity persisted in spleen. The strongest responses targeted peptides from the NP protein and demonstrated cross-reactivity in both the CD4+ and CD8+ T cell populations. Cross-reactive CD4+ T cells also targeted HA and NA epitopes, while cross-reactive CD8+ T cells targeted internal M1, NS2, and PA. T cell epitopes demonstrated extensive cross-reactivity between diverse influenza strains in outbred animals, with NP implicated as a significant antigenic target demonstrating extensive cross-reactivity for both CD4+ and CD8+ T cells.