Recombinant influenza viruses are promising vectors that can bolster antibody and resident lymphocyte responses within mucosal sites. This study evaluates recombinant influenza viruses with SARS-CoV-2 RBD genes in eliciting mucosal and systemic responses. Using reverse genetics, we generated replication-competent recombinant influenza viruses carrying heterologous RBD genes in monomeric, trimeric, or ferritin-based nanoparticle forms. Following intranasal immunisation, mice developed potent serological anti-RBD responses, with ferritin nanoparticles superseding monomeric or trimeric RBD responses. While parenteral and mucosal immunisation elicited robust anti-RBD IgG in serum, mucosal immunisation seeded respiratory IgA, RBD-specific lung-resident memory and germinal centre (GC) B cells. In animals with prior intramuscular vaccination, intranasal boosting with recombinant influenza vectors augmented mucosal IgG, IgA, GC and memory B cells, and SARS-CoV-2 lung neutralising titres. Recall of RBD-specific memory B cells via antigen re-exposure in the lung increased antibody-secreting cells in the lung-draining lymph nodes, with maintenance of lung GC B cells. Recombinant influenza-based vaccines effectively deliver highly immunogenic self-assembling nanoparticles, generating antibodies and B cells in the respiratory mucosa. This strategy provides a tractable pathway to augment lung-localised responses against recurrent respiratory viral infections.

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.

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.

The influenza virus neuraminidase (NA) is a promising target for next-generation influenza vaccines but standardised 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 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 suggest discrepancies between NA-specific antibody measurements in serum and plasma. Therefore, the consistent use of serum should be considered in standardising NA-based serological assays.

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.

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.

Objectives: Immunisation remains the most cost-effective mechanism to combat 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. Therefore, there is a need to better understand pre-existing cross-reactive human immunity against avian influenza strains, as highlighted by the recent global spread of avian H5Nx clade 2.3.4.4b variants.

Methods: Here, we quantified the frequencies and specificities of B and T cells recognising avian hemagglutinin (HA) within unexposed adults and characterised the ability of seasonal immunisation to boost cross-reactive immune responses to H5Nx strains, including from clade 2.3.4.4b.

Results: 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 below 0.13% and two- to five-fold lower than populations of seasonal HA-specific B cells. Boosting of B-cell responses against clade 2.3.4.4b H5Nx HA following seasonal immunisation was sporadic with only three 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 HA stem recognition. CD4+ T-cell responses towards H5 HA were weakly boosted with little increase in circulating T follicular helper cell populations.

Conclusion: These findings highlight the need for avian influenza-specific vaccine products to bolster immunity in human populations. Such vaccines could aid pre-pandemic preparedness by expanding baseline frequencies of avian influenza-specific memory lymphocytes.

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.

BACKGROUND: There is uncertainty about the timing of booster vaccination against COVID-19 in highly vaccinated populations during the present endemic phase of COVID-19. Studies focused on primary vaccination have previously suggested improved immunity with a longer interval between the first and second vaccine doses.
METHODS: We conducted a randomized, controlled trial (November 2022-August 2023) and assigned 52 fully vaccinated adults to an immediate or a 3-month delayed bivalent Spikevax mRNA booster vaccine. Follow-up visits were completed for 48 participants (n = 24 per arm), with collection of saliva and plasma samples following each visit.
RESULTS: The rise in neutralizing antibody responses to ancestral and Omicron strains were almost identical between the immediate and delayed vaccination arms. Analyses of plasma and salivary antibody responses (IgG, IgA), plasma antibody-dependent phagocytic activity, and the decay kinetics of antibody responses were similar between the 2 arms. Symptomatic and asymptomatic SARS-CoV-2 infections occurred in 49% (21 of 49) participants over the median 11.5 months of follow-up and were also similar between the 2 arms.
CONCLUSIONS: Our data suggest that there was no benefit in delaying COVID-19 mRNA booster vaccination in preimmune populations during the present endemic phase of COVID-19.
TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry number 12622000411741

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) breakthrough infection of vaccinated individuals is increasingly common with the circulation of highly immune evasive and transmissible Omicron variants. Here, we report the dynamics and durability of recalled spike-specific humoral immunity following Omicron BA.1 or BA.2 breakthrough infection, with longitudinal sampling up to 8 months after infection. Both BA.1 and BA.2 infections robustly boosted neutralization activity against the infecting strain while expanding breadth against BA.4, although neutralization activity was substantially reduced for the more recent XBB and BQ.1.1 strains. Cross-reactive memory B cells against both ancestral and Omicron spike were predominantly expanded by infection, with limited recruitment of de novo Omicron-specific B cells or antibodies. Modeling of neutralization titers predicts that protection from symptomatic reinfection against antigenically similar strains will be durable but is undermined by new emerging strains with further neutralization escape.

Vγ9Vδ2 T cells can recognize various molecules associated with cellular stress or transformation, providing a unique avenue for the treatment of cancers or infectious diseases. Nonetheless, Vγ9Vδ2 T-cell-based immunotherapies frequently achieve suboptimal efficacies in vivo. Enhancing the cytotoxic effector function of Vγ9Vδ2 T cells is one potential avenue through which the immunotherapeutic potential of this subset may be improved. We compared the use of four pro-inflammatory cytokines on the effector phenotype and functions of in vitro expanded Vγ9Vδ2 T cells, and demonstrated TCR-independent cytotoxicity mediated through CD26, CD16, and NKG2D, which could be further enhanced by IL-23, IL-18, and IL-15 stimulation throughout expansion. This work defines promising culture conditions that could improve Vγ9Vδ2 T-cell-based immunotherapies and furthers our understanding of how this subset might recognize and target transformed or infected cells.

Although the protective role of neutralizing 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 in previously vaccinated individuals. 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 degrees of expansion. The frequency of activated SARS-CoV-2-specific CD8+ T cells at baseline and peak inversely correlated with peak SARS-CoV-2 RNA levels in nasal swabs and accelerated viral clearance. Our study demonstrates that a 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.