Vaccination is the best way to combat annual influenza epidemics, yet the breadth of vaccine-induced humoral immunity toward decades of future differentially evolving influenza A (IAV) and influenza B (IBV) viruses is unclear. Using historic 1994 influenza vaccination cohorts of young and older adults, we defined antibody responses elicited by 1994 vaccination against future influenza strains spanning three decades of differentially evolving IAV and IBV strains. Quality of antibody responses and vaccine-induced and cross-reactive B cell memory responses were investigated. Vaccination increased antibody titers against all 1994 vaccine components in younger and older adults. Antibodies to future H1N1 strains were also detected across younger and older adults, including nonneutralizing hemagglutinin (HA) stem responses. Prominent boosting against earlier B/Yamagata/16/1988 and future Yamagata lineage strains was also observed, but antibody responses toward future rapidly evolving H3N2 strains were minimal. Systems serology revealed divergent antibody signatures between younger and older adults against future antigens. However, postvaccination responses were of high quality in both age groups. Cross-reactive HA-specific memory B cells induced by 1994 vaccination bound to vaccine and future H1 and IBV strains but exhibited minimal responses against H3. Group 1 cross-reactive H1/H5 stalk-specific responses also contributed to the overall H1 future response, whereas cross-reactive H3/H7 stalk-specific B cells were not detected. Our study provides insights into the breadth of vaccine-induced humoral immunity toward future influenza viruses over 30 years of influenza virus evolution, including newly emerging pandemic strains, and highlights the unmet need to optimize future vaccines strategies, especially for H3N2.

Abstract Indigenous people are disproportionately impacted by influenza viruses and chronic multimorbidity. Yet, the impact of comorbidities on immunity towards influenza vaccination is unknown. We recruited Australian First Nations and non-Indigenous people vaccinated with seasonal inactivated influenza vaccines and assessed their humoral and cellular responses in the context of comorbidities at baseline and after immunisation. Our study highlights prevalence of multimorbidity in First Nations people, associated with elevated baseline cellular activation, pro-inflammatory cytokines and agalactosylated IgG. Following vaccination, all groups had increased antibody titres and haemagglutinin-specific IgD - B-cell frequencies as compared to baseline. However, we reveal increased prevalence of pro-inflammatory atypical B cells within influenza haemagglutinin-specific IgD - B cells and lack of significant circulating T follicular helper type-1 cell activation in individuals with comorbidities, correlating with multimorbidity-associated baseline inflammatory features. Our findings thus reveal that vaccinees with comorbidities, both Australian First Nations and non-Indigenous participants, can mount antibody responses following influenza vaccination, although their cellular immune features, haemagglutinin-specific IgD - B cells and cT FH 1 compartments, display features of perturbed humoral axis functionality linked to multimorbidity-associated inflammation and IgG glycosylation patterns at baseline. Our study supports influenza vaccination for individuals with comorbidities, especially relevant to Indigenous populations with prevalent multimorbidity.

Background Rational engineering of vaccine immunogens to focus B cell responses on potently neutralising epitopes is a promising approach to improve the potency, breadth and durability of viral vaccines. Such strategies, however, can compromise vaccine immunogenicity through the unintended exclusion of CD4+ T cell epitopes, which are critical for the development of T follicular helper (TFH) cells and to support high affinity antibody production. Methods Using a prototypic influenza haemagglutinin (HA) stem immunogen lacking effective CD4+ T cell help in C57BL/6 mice, we interrogated the minimal requirements for T cell help needed to drive serological responses to vaccination. Findings We find that priming of naïve CD4+ T cells is markedly efficient, however the immunodominance of a given CD4+ T cell epitope is not predictive of the propensity to provide high quality help to antigen-specific B cells. In the context of soluble antigens, provision of a single MHC class II epitope is sufficient to drive robust germinal centre responses and serum IgG titres. However not all CD4+ epitopes provide equivalent levels of B cell help, despite priming comparable numbers of antigen-specific CD4+ T cells. Finally, we show multimerizing and arraying antigens on nanoparticle scaffolds unlocks highly subdominant, near-undetectable CD4+ T cell helper responses to support a T-dependent antibody response. Interpretation Our findings emphasise the importance of CD4+ T cell help for programming robust and durable humoural immunity, and provide crucial insights to guide the rational incorporation of favourable T cell epitopes into vaccines. Funding The study was funded by the NHMRC.

Monoclonal antibodies (mAbs) are an emerging class of therapeutics for the prevention and treatment of viral infections. Recent advances in mRNA/lipid nanoparticle (LNP) technology provide a promising new modality for the production of mAbs in vivo, potentially bypassing the need for recombinant manufacturing of mAb proteins. In this study, we compared traditional infusion of protein-based neutralizing mAbs targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or influenza viruses to mRNA/LNP-based production of mAbs in treated mice. High serum concentrations of mAbs were achieved upon delivery of a single mRNA encoding both heavy and light chains via intravenous or intramuscular routes using prototypic LNP formulations. However, the pharmacokinetics of mRNA-delivered mAbs were heavily influenced by the induction of anti-drug antibody responses directed against the encoded mAbs, resulting in reduced half-life in vivo and compromised protective capacity against SARS-CoV-2 Omicron BA.1 infection. In contrast, mRNA/LNP delivery of a neutralizing mAb conferred superior protection against lethal influenza challenge compared to equivalent recombinant protein doses. Overall, mRNA/LNP delivery comprises a feasible and attractive pathway to speed the development and deployment of antiviral antibodies. However, optimization of LNP formulation, dosing, and administration routes is required to maximize protective potential.

Antibody specificity is primarily derived from immunoglobulin (Ig) genes, which exhibit extensive genetic polymorphism both within and across species. Non-human primates (NHP) such as pigtail macaques are often used as preclinical models for immunological studies because of their genetic proximity to humans, despite a comparatively poor characterisation of their immunoglobulin loci and its impact on antibody specificity. To determine how pigtail macaque antibody responses differ from humans, we annotated the immunoglobulin loci of two pigtail macaques and compared BCR sequences and antigen binding sites of SARS-CoV-2 spike-specific antibodies derived from pigtail macaque or human B cells. Pigtail macaques showed high levels of genetic diversity across all three immunoglobulin loci. Spike-specific BCRs from a single pigtail macaque contained greater number of heavy chain variable genes compared to sequences obtained from six humans. Nevertheless, macaque antibodies targeted similar receptor binding domain (RBD) epitopes as humans. This work highlights the need for further understanding of the impacts of immunogenetics especially in light of recent advances in germline-based vaccine strategies.

Conflicting results have emerged regarding immune correlates of protection against SARS-CoV-2. In this secondary analysis of a trial of immediate versus delayed bivalent mRNA booster vaccination, we studied a range of humoral and cellular immune responses 28 days after vaccination and analyzed their association with subsequent symptomatic and asymptomatic SARS-CoV-2 infection. We found neutralizing antibodies and other plasma antibody titers correlated with prevention of SARS-CoV-2 infection. In a multivariate model, only neutralizing antibodies remained associated with protection from infection (p = 0.021). There was no significant correlation observed between B and T cell responses to Spike and protection. Our findings add to evidence of antibody correlates of prevention of SARS-CoV-2 infection.Trial registration: ANZCTR 12622000411741.

The SARS-CoV-2 spike receptor binding domain (RBD) is the major target for neutralising antibodies. However, subdomains like RBD may constrain the availability of CD4 T follicular helper (TFH) cells and impact immunogenicity. We engineered a chimeric trimeric RBD (CTR) glycoprotein, replacing the RBD of HKU-1 spike with SARS-CoV-2 RBD (ancestral WT/Omicron BA.2). This maintains trimerised RBD, while providing CD4 help via the HKU-1 scaffold. In C57BL/6 mice, CTR-BA.2 elicited high anti-BA.2-RBD IgG and neutralising titres, matching native spike responses. Germinal centre B cells were predominantly WT+/BA.2+ cross-reactive, and TFH predominantly recognised HKU-1 epitopes, demonstrating scaffold-directed help. In macaques, CTR-WT elicited comparable anti-RBD IgG, anti-spike IgG and neutralising responses to native spike, with elevated RBD-specific GC B cells in draining lymph nodes. Macaque TFH responses targeted RBD, NTD/S2 or HKU-1 peptides. This chimeric design overcomes poor RBD immunogenicity by engaging CD4 TFH, maintaining neutralising responses that is non-inferior to native spike.

NL63 is an alphacoronavirus that uses the same ACE2 receptor as SARS-CoV and SARS-CoV-2, but generally causes mild respiratory illness. In a cohort of healthy adults, we characterised humoral responses against NL63 spike and isolated a panel of human monoclonal antibodies (mAbs), including five with potent viral neutralising activity. Four neutralising mAbs blocked ACE2 receptor engagement and were found to target the receptor binding motif. A single mAb targeting the S2 subunit displayed potent neutralisation activity comparable to those directly blocking receptor engagement. The S2 mAb targets a membrane proximal heptad repeat 2 (HR2) region in spike that is absent in betacoronaviruses, potentially revealing a site of vulnerability unique to alphacoronaviruses. For all neutralising mAbs, putative epitopes were highly conserved in over 200 NL63 sequences, including recent clinical isolates. A deeper understanding of the recognition of alphacoronavirus spike by human antibodies will guide vaccine and therapeutic development against alphacoronavirus threats.

BACKGROUND. The immunogenicity of current influenza vaccines need improvement. Inactivated influenza and COVID-19 mRNA vaccines can be co-administered but randomized controlled trial data is lacking on whether the two vaccines are more immunogenic if given in the same or opposite arms. Murine studies suggest mRNA vaccines can adjuvant influenza vaccines when co-formulated and delivered together.

The receptor binding domain (RBD) of the SARS-CoV-2 spike is the major target for neutralising antibodies elicited by current vaccines. Using small domains such as the RBD as vaccine immunogens, however, may constrain the availability of CD4 T follicular helper (TFH) cells and impact immunogenicity. We engineered a novel chimeric trimeric RBD (CTR) glycoprotein, replacing the RBD of human coronavirus HKU-1 spike with SARS-CoV-2 RBD of either ancestral (WT) or Omicron BA.2 strains. This strategy maintains a native trimeric conformation of the RBD, while providing additional sources of CD4 T cell help via the HKU-1 spike scaffold. In C57BL/6 mice, CTR-BA.2 prime-boost vaccination elicited high anti-BA.2-RBD IgG and neutralising titres, matching responses in animals immunised with native SARS-CoV-2 spike proteins. GC B cells elicited by CTR-BA.2 were predominantly WT+/BA.2+ cross- reactive, and TFH cells predominantly recognised HKU-1 epitopes, demonstrating scaffold-directed T cell help. Macaques prime-boost immunised with CTR-WT similarly elicited high anti-RBD IgG, anti-spike IgG and neutralising responses, comparable to native spike-vaccinated animals. In draining lymph nodes of CTR-WT vaccinated macaques, RBD-specific GC B cells were present at elevated levels. In contrast to the murine studies, lymph node-draining TFH responses in macaques were broadly elicited against RBD, NTD/S2 or HKU-1-derived peptides. Although native SARS- CoV-2 spike was also highly immunogenic in animal models, our findings establish the chimeric glycoprotein design as a strategy to overcome the poor immunogenicity of the SARS-CoV-2 RBD by engaging CD4 TFH cells, while maintaining the ability to elicit protective neutralising responses. One sentence summary A chimeric glycoprotein design preserves SARS-CoV-2 RBD antigenic conformation enabling elicitation of neutralising responses, while allowing recruitment of HKU-1 scaffold-directed CD4 helper responses to support the humoral response. ### Competing Interest Statement The authors have declared no competing interest. Australian National Health and Medical Research Council, 2004398 Australian Medical Research Future Fund, 2013870

The influenza HA stem domain is a promising vaccine antigen to elicit broadly protective antibody responses. In vivo, however, trimeric stem is poorly immunogenic due to a lack of MHC class II epitopes. To address this, we designed a series of stem antigens that incorporate individual CD4 T cell epitopes derived from viral proteins or model antigens. 14 days post-vaccination in WT BL/6 mice, these antigens elicited stem IgG titres ranging from undetectable (<1:100) to > 2x104. Novel MHC class II tetramers revealed that vaccine immunogenicity was unrelated to the total frequency of antigen-specific CD4 T cells. Rather, a bias toward TFH differentiation dictated whether prototypic vaccines were immunogenic or not. Our data suggest that between 60-450 ag-specific TFH are required in the draining lymph node to support a robust germinal centre (GC) response and the proliferation of stem-specific GC B cells. Incorporation of a single, TFH-competent class II epitope into the stem immunogen elicited antibody titres that were 19-fold higher than the full-length HA protein, and 8-fold higher than stem-ferritin nanoparticles, demonstrating the extent to which CD4 T cell help can augment the intrinsic immunogenicity of an antigen. Understanding the factors that underpin variability in TFH differentiation across polyclonal, epitope-specific CD4 T cell populations will further inform rational design of potently immunogenic vaccines. Supported by the NHMRC (Australia; GNT2004398 and 2009308) and Sylvia and Charles Viertel Foundation. Vaccines and Immunotherapy (VAC)

Intranasal vaccination aims to elicit mucosal immunity in the respiratory tract to better protect against respiratory infections (e.g., SARS-CoV-2 and influenza). Most vaccines, including recent COVID-19 mRNA lipid nanoparticles (LNPs), are optimized for intramuscular (i.m.) administration and typically perform poorly when delivered intranasally. Here, we prepared mRNA-LNPs using clinically approved ionizable lipids (ALC-0315, SM-102, and DLin-MC3-DMA) with or without a permanent cationic lipid (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]) to deliver a model immunogen (ovalbumin [OVA]) and CRE recombinase reporter mRNA. Using wild-type C57BL/6 and Ai14 reporter mouse models, we deconvoluted the effects of LNP formulation on mRNA cargo delivery and immunogenicity following i.m. or intranasal (i.n.) administration. After i.m. vaccination, mRNA-LNPs demonstrated transfection of muscle and immune cells in vivo, and consequently robust humoral immune responses. In contrast, mRNA-LNP delivery to the respiratory mucosa was poorly immunogenic, both in naive animals and in those with post-infection inflammation. Encouragingly, mRNA-LNPs efficiently transfected epithelial and immune cells within the lungs and expressed mRNA cargo could efficiently recall immunity in draining secondary lymphoid tissues. The addition of DOTAP led to enhanced recall responses. Decoding interplays of LNP formulations and their performance in vivo within specific tissue compartments will provide principles that can guide the rational design of mRNA-LNPs for maximal protection against respiratory diseases.

The receptor binding domain (RBD) of the SARS-CoV-2 spike is the major target for neutralising antibodies elicited by current vaccines. However, RBD subunit vaccines are poorly immunogenic, due to insufficient CD4 T follicular helper (TFH) cell epitopes, consequently driving poor germinal centre (GC) responses. We engineered a novel chimeric trimer-RBD (CTR) glycoprotein, replacing the RBD of human coronavirus HKU-1 spike with SARS-CoV-2 RBD (Wuhan-Hu-1 or BA.2). This strategy leverages HKU-1 scaffold CD4 epitopes while preserving SARS-CoV-2 RBD neutralising targets, with antigenicity confirmed by monoclonal antibodies. In C57BL/6 mice, CTR-BA.2 prime-boost vaccination elicited high anti-BA.2-RBD IgG titres (∼104) and BA.2 neutralisation (IC50: 4.0x102), matching native spike responses. GC B cells elicited by CTR-BA.2 were predominantly Wuhan-Hu-1+/BA.2+ cross-reactive, while 25.3% of TFH cells, measured by CD154+, recognised HKU-1 epitopes, demonstrating scaffold-directed T cell help. Macaques prime-boost immunised with CTR-Wuhan-Hu-1 elicited high anti-RBD IgG (∼104) and neutralising responses (IC50: 7.5x102), comparable to native spike (n = 5/group). RBD-specific GC B cells (BCL-6+CD95+) were present at elevated levels in draining lymph nodes of native (1.94%) or CTR spike-vaccinated (1.62%) animals vs non-draining sites. Our findings establish the chimeric glycoprotein design as a strategy to overcome the poor immunogenicity of the SARS-CoV-2 RBD by engaging CD4 TFH cells. Supported by the National Health and Medical Research Council (Australia) (2009711, 2026762, 2009308) and Medical Research Future Fund (Australia) (2013870). Vaccines and Immunotherapy (VAC)

Activation-induced marker (AIM) assays identify antigen (Ag)-specific T cells, but recent studies revealed AIM+ T helper cell 17 (TH17)-like (CCR6+) and circulating T follicular helper cells (cTfh) were not associated with peptide/HLA tetramer staining. We show that CD39+ regulatory T cell (Treg)-like and CD26hi TH22-like cells undergo T cell receptor (TCR)-independent activation by cytokines during Ag stimulation, leading to nonspecific up-regulation of AIM readouts. Transcriptional analysis enabled discrimination of bona fide Ag-specific T cells from cytokine-activated Treg and TH22 cells. CXCR4 down-regulation emerged as a hallmark of clonotypic expansion and TCR-dependent activation in memory CD4+ T cells and cTfh. By tracking tetramer-binding cells upon Ag restimulation, we demonstrated that CXCR4-CD137+ cells provided a more accurate measure of Ag-specificity than standard AIM readouts. This modified assay excluded the predominantly CCR6+ cytokine-activated T cells that contributed to an average 12-fold overestimation of the Ag-specific population. Our findings provide an accurate approach to characterize genuine Ag-specific T cells.

BACKGROUND The immunogenicity of current influenza vaccines needs improvement. Inactivated influenza and COVID-19 mRNA vaccines can be coadministered, but randomized controlled trial data are lacking on whether the 2 vaccines are more immunogenic if given in the same arm or opposite arms. Murine studies suggest mRNA vaccines can adjuvant influenza vaccines when coformulated and codelivered.METHODS We randomly assigned 56 adults to receive the Afluria quadrivalent inactivated influenza and Moderna monovalent SARS-CoV-2 XBB.1.5 mRNA vaccines, either in opposite arms or both in the same arm at the same site. The primary endpoint was the difference in median combined serum hemagglutination inhibition titer to the H1, H3, and B-Vic vaccine influenza strains after vaccination.RESULTS We found no significant difference in hemagglutination inhibition antibody levels between the groups (P = 0.30), with the same-arm group having a 1.26-fold higher titer than the opposite-arm group. There were no differences in analyses of antibodies against individual influenza strains or in nasal or saliva antibody levels. While both binding and neutralizing antibody titers against SARS-CoV-2 were not significantly different between groups postvaccination, there was a higher fold-change in BA.5 and ancestral strain neutralizing antibodies in the opposite-arm group.CONCLUSION Influenza vaccination is equivalently immunogenic if given in the same arm or opposite arms as the SARS-CoV-2 vaccine, but it may be preferable to administer the SARS-CoV-2 vaccine at a different site from influenza vaccines.TRIAL REGISTRATION Australian New Zealand Clinical Trials Registry ACTRN12624000445572.FUNDING Australian National Health and Medical Research Council, Australian Medical Research Future Fund, and National Institutes of Health (UH2AI176172).

Human clinical trials have reported immunological outcomes can differ between ipsilateral (same side) and contralateral (alternate sides) prime-boost vaccination. However, our mechanistic understanding of how keeping or shifting the anatomical sites of immunization impacts the resultant germinal centers (GCs) and antibody responses is limited. Here, we use an adjuvanted SARS-CoV-2 spike vaccine to dissect GC dynamics in draining lymph nodes and serological outcomes following ipsilateral or contralateral prime-boost vaccination in C57BL/6 mice. Contralateral vaccination elicited independent GCs at distinct lymph nodes, where robust secondary GCs only appeared upon secondary distal vaccination, while ongoing GCs from the primary site were not boosted. In contrast, ipsilateral vaccination resulted in sustained GC activity. Ipsilateral vaccination accelerated the development of antibody titers against ancestral (wild-type [WT]), Beta, and BA.1 but were later comparable between ipsilateral and contralateral groups in terms of magnitude, durability, and neutralization capacity beyond 28 d. Using a heterologous SARS-CoV-2 WT/BA.1 spike prime-boost model, cross-reactive GC responses were generated against WT and BA.1 spike, with analogous serological and GC dynamics to our homologous model. Within the cross-reactive GC B cells, differential recognition of WT and BA.1 antigens was observed and were further compartmentalized in primary or secondary GCs, depending on ipsilateral or contralateral regimes. Collectively, maintaining a common prime-boost site augments the kinetics of memory B cell recall and transiently drive higher antibody titers, but longer-term serological outcomes are unaffected by the anatomical localization of immunization.

Monoclonal antibodies (mAbs) are an emerging class of therapeutics for the prevention and treatment of viral infections. Recent advances in mRNA/lipid nanoparticle (LNP) technology provide a potential new modality for the expression of mAbs in vivo, potentially bypassing the need for recombinant manufacturing of mAb proteins. In this study, we compared traditional infusion of neutralising mAbs targeting SARS-CoV-2 or influenza to mRNA-based induction of de novo mAb expression in treated mice. High serum concentrations of mAbs were achieved upon delivery of a single mRNA encoding both heavy and light chains via intravenous or intramuscular routes using prototypic LNP formulations. However, pharmacokinetics were heavily influenced by the induction of anti-drug antibody responses directed against the encoded mAbs, driving reductions in in vivo half-life and compromising protective capacity against SARS-CoV-2 Omicron BA.1 infection. Overall, mRNA/LNP delivery comprises a feasible and attractive pathway to speed the development and deployment of antiviral antibodies, however optimisation of LNP formulation, dosing and administration routes is required to maximise protective potential. ### Competing Interest Statement The authors have declared no competing interest. Australian Medical Research Future Fund, 2005544, 2013870

Influenza has been responsible for multiple global pandemics and seasonal epidemics and claimed millions of lives. The imminent threat of a panzootic outbreak of avian influenza H5N1 virus underscores the urgent need for pandemic preparedness and effective countermeasures, including monoclonal antibodies (mAbs). Here, we characterize human mAbs that target the highly conserved catalytic site of viral neuraminidase (NA), termed NCS mAbs, and the molecular basis of their broad specificity. Cross-reactive NA-specific B cells were isolated by using stabilized NA probes of non-circulating subtypes. We found that NCS mAbs recognized multiple NAs of influenza A as well as influenza B NAs and conferred prophylactic protections in mice against H1N1, H5N1, and influenza B viruses. Cryo-electron microscopy structures of two NCS mAbs revealed that they rely on structural mimicry of sialic acid, the substrate of NA, by coordinating not only amino acid side chains but also water molecules, enabling inhibition of NA activity across multiple influenza A and B viruses, including avian influenza clade 2.3.4.4b H5N1 viruses. Our results provide a molecular basis for the broad reactivity and inhibitory activity of NCS mAbs targeting the catalytic site of NA through substrate mimicry.

The ongoing rollout of SARS-CoV-2 vaccines lags behind rapid viral evolution. Updated vaccine immunogens elicit neutralising antibodies against the component strain. However, protection against future SARS-CoV-2 variants is unclear. Here, we sought to understand factors underpinning serological breadth following bivalent BA.1 vaccination. Booster vaccination of 33 individuals elicited robust and durable antibody responses against component vaccine antigens and elevated frequencies of spike-specific CD4 and CD8 T cells. Immunisation predominantly drove recall of cross-reactive memory B cells which also recognised XBB.1.5 spike, with significantly enhanced neutralisation titres against XBB virus seen within 91% of participants. Multivariate regression indicated that both baseline neutralising titres and spike-specific CD4 T cell frequencies were strong predictors of ancestral, BA.1 and XBB neutralisation post-immunisation. These data highlight that updated SARS-CoV-2 vaccines recall cross-reactive memory that maintains recognition of antigenically evolved viral variants and suggests T cell help and prior antibody titres underpin robust vaccine-induced neutralising activity.