Biology subjects

Koutsakos, M.

Publications and source records attributed to Koutsakos, M..

12 recordsLinked to original sources

bioRxiv2026

Immunogen selection and prior immunity shape antibody breadth following immunisation with avian H5 hemagglutinin

Avian influenza A viruses pose a persistent zoonotic threat to humans owing to their expanding host range and high case fatality rates. In particular, viruses from the 2.3.4.4b clade of the H5 subtype have now been detected in over 60 mammalian species, raising serious pandemic concerns. Understanding immune recognition of the H5 hemagglutinin (HA) is therefore critical for effective vaccine design and pandemic preparedness. To understand the breadth of cross-recognition induced by different H5 strains, we selected genetically diverse H5 human isolates from 2003-2023 and assessed neutralising antibody responses elicited by adjuvanted recombinant HA protein-based vaccines in C57BL/6 mice. Neutralisation activity of sera was determined against seven H5 HA variants using pseudotyped viruses and a PR8-reassortant virus in micro-neutralisation assays. Our results showed a wide variety of cross-strain neutralisation across H5 HA antigen variants. The conventional vaccine strain A/Indonesia/05/2005 displayed narrow activity against emerging clade 2.3.4.4b viruses, whereas ancestral variants exhibited cross-neutralisation profiles showing a diversity of breath but with limited potency. Polyvalent H5 HA formulations and nanoparticle-displayed H5 HA platforms substantially broadened cross-neutralisation against diverse H5 strains. To examine the impact of pre-existing immunity on H5 vaccine immunogenicity in mouse models, mice were primed with either seasonal influenza infection or quadrivalent influenza vaccine (QIV) prior to H5 HA immunisation. QIV pre-vaccination, but not prior influenza infection, enhanced subsequent neutralizing responses towards A/Fujian-Sanyuan/21099/2017 (clade 2.3.4.4b) H5. Collectively, our results demonstrate that immunogen selection and prior immunity shape antibody breadth following immunisation with avian A(H5) hemagglutinin.

immunology↗

bioRxiv2026

Epistasis facilitates the long-term antigenic evolution of the influenza B virus hemagglutinin

The antigenic drift of viral glycoproteins must be balanced by purifying selection pressure to maintain functionality. Understanding these evolutionary processes is key to predicting and combating viral evolution but is primarily based on influenza A(H3N2), which may limit generalisability. By characterising the influenza B virus haemagglutinin (HA) over 8 decades of circulation in humans, we found continuous genetic diversification, punctuated with antigenic changes that did not follow a linear path in antigenic space. Antigenic change is primarily underpinned by re-occurring mutations and deletions at positions 136, 150, 162-165, 197 and 203. These residues form complex epistatic networks that modulate the antigenic impact of mutation recycling. They also generate permissive backbones on which immune escape can emerge with limited replicative fitness cost. Our study identifies critical similarities and differences with A(H3N2) evolution and demonstrates the role of epistasis in balancing antigenic novelty with viral fitness. Our findings and genetic, antigenic and phenotypic datasets support the development of genotype-to-phenotype prediction tools, but such predictions need to capture the complex outcomes of epistasis.

microbiology↗

bioRxiv2026

Pre-existing systemic and nasal antibodies against avian H5 influenza A viruses vary according to childhood imprinting

Avian influenza A viruses (IAV) pose a constant pandemic threat, with the recent 2.3.4.4b clade of the H5 subtype causing high pathogenicity and spreading across animal species and geographic locations. Understanding human pre-existing immunity to avian H5 IAV can inform on population susceptibility, a critical aspect of pandemic preparedness. To that end, we analysed the IAV HA-specific antibodies across individuals born between 1928-1999 with different early life exposures to IAV subtypes. Individuals born prior to 1957 had the highest pre-existing serum antibodies to group 1 HA antigens, including the 2.3.4.4b H5 and a group 1 HA stem antigen. These birth-year-specific patterns were not reflected in the limited pre-existing serum neutralising antibodies detectable against a 2.3.4.4b H5 IAV or in H5-specific memory B cell populations. They were however evident in pre-existing nasal IgG and IgA titres to H5, which were greater in individuals born prior to 1957. Our findings demonstrate that the immunological biases afforded by early life exposure extend to antibodies detected in the nasal mucosa, the site of IAV replication. ImportanceUnderstating pre-existing immunity to influenza A viruses of pandemic potential is an important aspect of pandemic preparedness. This includes an understanding the heterogeneity of pre-existing immunity across the population. Here, we demonstrate that pre-existing antibodies to H5 IAV vary according to year of birth and childhood imprinting. We demonstrate that this is the case for both systemic and nasal antibodies, highlighting the importance of understanding pre-existing mucosal immunity at the sites of influenza virus replication.

immunology↗

bioRxiv2025

Impact of serum versus anticoagulant-containing plasma on influenza virusneuraminidase-based serological assays

The influenza virus neuraminidase (NA) is a promising target for next-generation influenza vaccines but standardized protocols for NA-based serological assays are lacking. Previous studies have demonstrated discordant results from haemagglutination inhibition and live virus microneutralization assays when comparing matched serum and plasma samples. It is therefore important to consider is the choice of serum or plasma samples in assays measuring influenza virus NA-specific antibodies. Here, we compared antibody titres against influenza A and B virus NAs in matched serum and different types of plasma using an enzyme-linked lectin assay (ELLA) and an enzyme-linked immunosorbent assay (ELISA). We observed good correlations between titres determined in serum and different types of plasma. However, there was variable and often poor agreement in the nominal titre values obtained from serum and different kinds of plasma in both ELLA and ELISA, with plasma samples often resulting in lower titres compared to serum samples. We also found differences in NA-specific responses to seasonal influenza vaccination assessed in serum versus plasma. Overall, our data demonstrate discrepancies between NA-specific antibody measurements in serum and plasma. We recommend the consistent use of serum as an important part of standardising NA-based serological assays.

immunology↗

bioRxiv2025

Memory T and B cells with recognition of avian influenza hemagglutinins are poorly responsive to existing seasonal influenza vaccines

Immunisation remains the most cost-effective mechanism to combat global influenza infection and is widely employed against seasonal influenza viruses. Zoonotic transmission of avian influenza A viruses represents a significant threat to human health given the lack of population level immunity, which could translate into an influenza pandemic. Therefore, there is a need to better understand pre-existing human immunity against avian influenza strains. as highlighted by the recent rapid, global spread of avian H5Nx clade 2.3.4.4b variants. Here, we sought to quantify the frequencies and specificities of B cells recognising avian hemagglutinin (HA) within unexposed adults, and to characterise the ability of seasonal immunisation to boost cross-reactive immune responses to H5Nx strains, including from clade 2.3.4.4b. Low but detectable serum antibody titres against H5 and H7 avian influenza HA were observed in donors. The frequency of memory B cells with cross-reactive recognition of H5 and H7 HA was low and 2-5 fold lower than populations of seasonal H1N1 and H3N2 HA-specific B cells. Boosting of B cell responses against H5Nx clade 2.3.4.4b HA following seasonal immunisation were sporadic with only 3 out of 19 individuals showing an increased population of probe-positive cells. Cross-reactive B cells generally expressed immunoglobulins drawn from variable heavy chain genes associated with recognition of the HA stem (VH6-1, VH1-69, VH1-18). CD4+ T cell responses towards H5 HA were also weakly boosted with little to no increase in circulating T follicular helper cell populations. These findings highlight the need for avian influenza-specific vaccine products to bolster immunity in human populations, with consideration for use in pre-pandemic preparedness to expand baseline frequencies of avian influenza-specific memory B and T lymphocytes.

immunology↗

bioRxiv2024

Triton-X 100-treated virus-based ELLA demonstrates discordant antigenic evolution of influenza B virus haemagglutinin and neuraminidase

Neuraminidase (NA)-specific antibodies have been associated with protection against influenza and thus NA is considered a promising target for next-generation vaccines against influenza A (IAV) and B viruses (IBV). NA inhibition (NI) by antibodies is typically assessed using an enzyme-linked lectin assay (ELLA). However, ELLA can be confounded by anti- hemagglutinin (anti-HA) antibodies that block NA by steric hindrance (termed HA interference). While strategies have been employed to overcome HA interference for IAV, similar approaches have not been assessed for IBV. We found HA interference is common in ELLA using IBV, rendering the technique unreliable. Anti-HA antibodies were not completely depleted from sera by HA-expressing cell lines and this approach was of limited utility. In contrast, we find that treatment of virions with Triton-X 100, but not Tween-20 or ether, efficiently separates the HA and NA components and overcomes interference caused by anti-HA antibodies. We also characterise a panel of recombinant IBV NA proteins that further validated the results from Triton-X 100-treated virus-based ELLA. Using these reagents and assays we demonstrate discordant antigenic evolution between IBV NA and HA over the last 80 years. This optimized ELLA protocol will facilitate further in-depth serological surveys of IBV immunity as well as antigenic characterisation of the IBV NA on a larger scale. ImportanceInfluenza B viruses contribute to annual epidemics and may cause severe disease, especially in children. Consequently, several approaches are being explored to improve vaccine efficacy, including the addition of neuraminidase. Antigen selection and assessment of serological responses will require a reliable serological assay to specifically quantify Neuraminidase inhibition. While such assays have been assessed for influenza A viruses, this has not been done of influenza B viruses. Our study identifies a readily applicable strategy to measure inhibitory activity of neuraminidase-specific antibodies against influenza B virus without interference from anti-hemagglutinin antibodies. This will aid broader serological assessment of influenza B virus-specific antibodies and antigenic characterisation of the influenza B virus neuraminidase.

microbiology↗

bioRxiv2023

Dissemination of influenza B virus to the lower respiratory tract of mice is restricted by the interferon response

The global burden of disease caused by Influenza B virus (IBV) is substantial, however IBVs remain overlooked. Understanding host-pathogen interactions as well as establishing physiologically relevant models of infection are important for the development and assessment of therapeutics and vaccines against IBV. Here, we assessed an upper respiratory tract (URT)-restricted model of mouse IBV infection, comparing it to the conventional administration of virus to the total respiratory tract (TRT). We found that URT infections with different strains of IBV resulted in limited dissemination of IBV to the lungs. Infection of the URT did not result in weight loss or systemic inflammation even at high inoculum doses and despite robust viral replication in the nose. Dissemination of IBV to the lung was enhanced in mice lacking functional type I IFN receptor (IFNAR2) but not IFN{gamma}. Conversely, in mice expressing the IFN-inducible gene Mx1 we found reduced IBV replication in the lung and reduced dissemination of IBV from the URT to the lung. Both URT and TRT inoculation with IBV resulted in seroconversion against IBV. However, priming at the TRT conferred superior protection from a heterologous lethal IBV challenge compared to URT priming, as determined by improved survival rates and reduced viral replication throughout the respiratory tract. Overall, our study establishes a URT-restricted IBV infection model, highlights the critical role of IFNs in limiting dissemination of IBV to the lungs but also demonstrates that the lack of viral replication in the lung may impact protection from subsequent infections. ImportanceOur study investigated how IBV spreads from the nose to the lung of mice, the impact this has on disease and protection from re-infection. We found that when applied to the nose only, IBV does not spread very efficiently to the lungs in a process controlled by the interferon response. Priming immunity at the nose only was less protective from re-infection than priming immunity at both the nose and lung. These insights can guide the development of potential therapies targeting the interferon response as well as of intranasal vaccines against IBV.

immunology↗

bioRxiv2023

Differential cross-reactivity to the influenza B virus haemagglutinin underpins lineage-specific susceptibility between birth cohorts

Influenza exposures early in life are believed to shape future susceptibility to influenza infections by imprinting immunological biases that engender differential cross-reactivity to future influenza viruses, but direct serological evidence linked to susceptibility is limited. We analysed hemagglutination-inhibition titres in 1451 cross-sectional samples collected between 1992-2020, from individuals born between 1917-2008, against influenza B virus (IBV) isolates from 1940-2021, including future isolates that circulated after sample collection. We demonstrate that immunological biases are conferred by early life IBV infection and result in lineage-specific cross-reactivity of a birth cohort towards future IBV isolates. This translates into differential estimates of susceptibility between birth cohorts towards the two IBV antigenic lineages, explaining lineage-specific age distributions of observed medically attended IBV infections. Our data bridge a critical gap between early life exposure, cross-reactivity, and influenza epidemiology and identify a plausible model to further dissect the interplay between host immunity, viral evolution and epidemiology.

immunology↗

bioRxiv2022

Rapid recall and de novo T cell responses during SARS-CoV-2 breakthrough infection

While the protective role of neutralising antibodies against COVID-19 is well-established, questions remain about the relative importance of cellular immunity. Using 6 pMHC-multimers in a cohort with early and frequent sampling we define the phenotype and kinetics of recalled and primary T cell responses following Delta or Omicron breakthrough infection. Recall of spike-specific CD4+ T cells was rapid, with cellular proliferation and extensive activation evident as early as 1 day post-symptom onset. Similarly, spike-specific CD8+ T cells were rapidly activated but showed variable levels of expansion. Strikingly, high levels of SARS-CoV-2-specific CD8+ T cell activation at baseline and peak were strongly correlated with reduced peak SARS-CoV-2 RNA levels in nasal swabs and accelerated clearance of virus. Our study demonstrates rapid and extensive recall of memory T cell populations occurs early after breakthrough infection and suggests that CD8+ T cells contribute to the control of viral replication in breakthrough SARS-CoV-2 infections.

immunology↗

bioRxiv2021

High expression of CD38 and MHC class II on CD8+ T cells during severe influenza disease reflects bystander activation and trogocytosis

Although co-expression of CD38 and HLA-DR on CD8+ T cells reflects activation during influenza, SARS-CoV-2, Dengue, Ebola and HIV-1 viral infections, high and prolonged CD38+HLA-DR+ expression can be associated with severe and fatal disease outcomes. As the expression of CD38+HLA-DR+ is poorly understood, we used mouse models of influenza A/H7N9, A/H3N2 and A/H1N1 infection to investigate the mechanisms underpinning CD38+MHC-II+ phenotype on CD8+ T-cells. Our analysis of influenza-specific immunodominant DbNP366+CD8+ T-cell responses showed that CD38+MHC-II+ co-expression was detected on both virus-specific and bystander CD8+ T-cells, with increased numbers of both CD38+MHC-II+CD8+ T-cell populations observed in the respiratory tract during severe infection. To understand the mechanisms underlying CD38 and MHC-II expression, we also used adoptively-transferred transgenic OT-I CD8+ T-cells recognising the ovalbumin-derived KbSIINFEKL epitope and A/H1N1-SIINKEKL infection. Strikingly, we found that OT-I cells adoptively-transferred into MHC-II-/- mice did not display MHC-II after influenza virus infection, suggesting that MHC-II was acquired via trogocytosis in wild-type mice. Additionally, detection of CD19 on CD38+MHC II+ OT-I cells further supports that MHC-II was acquired by trogocytosis, at least partially, sourced from B-cells. Our results also revealed that co-expression of CD38+MHC II+ on CD8+ T-cells was needed for the optimal recall ability following secondary viral challenge. Overall, our study provides evidence that both virus-specific and bystander CD38+MHC-II+ CD8+ T-cells are recruited to the site of infection during severe disease, and that MHC-II expression occurs via trogocytosis from antigen-presenting cells. Our findings also highlight the importance of the CD38+MHC II+ phenotype for CD8+ T-cell memory establishment and recall. SummaryCo-expression of CD38 and MHC-II on CD8+ T cells is recognized as a classical hallmark of activation during viral infections. High and prolonged CD38+HLA-DR+ expression, however, can be associated with severe disease outcomes and the mechanisms are unclear. Using our established influenza wild-type and transgenic mouse models, we determined how disease severity affected the activation of influenza-specific CD38+MHC-II+CD8+ T cell responses in vivo and the antigenic determinants that drive their activation and expansion. Overall, our study provides evidence that both virus-specific and bystander CD38+MHC-II+ CD8+ T-cells are recruited to the site of infection during severe disease, and that MHC-II expression occurs, at least in part, via trogocytosis from antigen-presenting cells. Our findings also highlight the importance of the CD38+MHC II+ phenotype for CD8+ T-cell memory establishment and recall.

immunology↗

bioRxiv2020

Atypical B cells are a normal component of immune responses to vaccination and infection in humans

The full diversity of the circulating human B cell compartment is unknown. Flow cytometry analysis suggests that in addition to naive and memory B cells, there exists a population of CD11c+, CD27- CD21- "atypical" B cells, that are associated with chronic or recurrent infection and autoimmunity. We used single cell RNA-seq approaches to examine the diversity of both antigen-specific B cells and total B cells in healthy subjects and individuals naturally-exposed to recurrent malaria infections. This analysis revealed two B cell lineages: a classical lineage of activated and resting memory B cells, and an atypical-like lineage. Surprisingly, the atypical lineage was common in both malaria exposed individuals and non-exposed healthy controls. Using barcoded antibodies in conjunction with our transcriptomic data, we found that atypical lineage cells in healthy individuals lack many atypical B markers and thus represent an undercounted cryptic population. We further determined using antigen specific probes that atypical cells can be induced by primary vaccination in humans and can be recalled upon boosting. Collectively these data suggest that atypical cells are not necessarily pathogenic but can be a normal component of B responses to antigen.

immunology↗

bioRxiv2018

MAIT cells contribute to protection against lethal influenza infection in vivo

Mucosal associated invariant T (MAIT) cells are evolutionarily-conserved, innate-like lymphocytes which are abundant in human lungs and can contribute to protection against pulmonary bacterial infection. MAIT cells are also activated during human viral infections, yet it remains unknown whether MAIT cells play a significant protective or even detrimental role during viral infections in vivo. Using murine experimental challenge with two strains of influenza A virus, we show that MAIT cells accumulated and were activated early in infection, with upregulation of CD25, CD69 and Granzyme B, peaking at 5 days post infection. Activation was modulated via cytokines independently of MR1. MAIT cell-deficient MR1-/- mice showed enhanced weight loss and mortality to severe (H1N1) influenza. This was ameliorated by prior adoptive transfer of pulmonary MAIT cells in both immunocompetent and immunodeficient RAG2-/-{gamma}C-/- mice. Thus, MAIT cells contribute to protection during respiratory viral infections, and constitute a potential target for therapeutic manipulation.

immunology↗