SARS-CoV-2-specific CD8+ T cells from people with long COVID establish and maintain effector phenotype and key TCR signatures over 2 years.

Long COVID occurs in a small but important minority of patients following COVID-19, reducing quality of life and contributing to healthcare burden. Although research into underlying mechanisms is evolving, immunity is understudied. SARS-CoV-2-specific T cell responses are of key importance for viral clearance and COVID-19 recovery. However, in long COVID, the establishment and persistence of SARS-CoV-2-specific T cells are far from clear, especially beyond 12 mo postinfection and postvaccination. We defined ex vivo antigen-specific B cell and T cell responses and their T cell receptors (TCR) repertoires across 2 y postinfection in people with long COVID. Using 13 SARS-CoV-2 peptide-HLA tetramers, spanning 11 HLA allotypes, as well as spike and nucleocapsid probes, we tracked SARS-CoV-2-specific CD8+ and CD4+ T cells and B-cells in individuals from their first SARS-CoV-2 infection through primary vaccination over 24 mo. The frequencies of ORF1a- and nucleocapsid-specific T cells and B cells remained stable over 24 mo. Spike-specific CD8+ and CD4+ T cells and B cells were boosted by SARS-CoV-2 vaccination, indicating immunization, in fully recovered and people with long COVID, altered the immunodominance hierarchy of SARS-CoV-2 T cell epitopes. Meanwhile, influenza-specific CD8+ T cells were stable across 24 mo, suggesting no bystander-activation. Compared to total T cell populations, SARS-CoV-2-specific T cells were enriched for central memory phenotype, although the proportion of central memory T cells decreased following acute illness. Importantly, TCR repertoire composition was maintained throughout long COVID, including postvaccination, to 2 y postinfection. Overall, we defined ex vivo SARS-CoV-2-specific B cells and T cells to understand primary and recall responses, providing key insights into antigen-specific responses in people with long COVID.

Improvement of immune dysregulation in individuals with long COVID at 24-months following SARS-CoV-2 infection.

This study investigates the humoral and cellular immune responses and health-related quality of life measures in individuals with mild to moderate long COVID (LC) compared to age and gender matched recovered COVID-19 controls (MC) over 24 months. LC participants show elevated nucleocapsid IgG levels at 3 months, and higher neutralizing capacity up to 8 months post-infection. Increased spike-specific and nucleocapsid-specific CD4+ T cells, PD-1, and TIM-3 expression on CD4+ and CD8+ T cells were observed at 3 and 8 months, but these differences do not persist at 24 months. Some LC participants had detectable IFN-γ and IFN-ß, that was attributed to reinfection and antigen re-exposure. Single-cell RNA sequencing at the 24 month timepoint shows similar immune cell proportions and reconstitution of naïve T and B cell subsets in LC and MC. No significant differences in exhaustion scores or antigen-specific T cell clones are observed. These findings suggest resolution of immune activation in LC and return to comparable immune responses between LC and MC over time. Improvement in self-reported health-related quality of life at 24 months was also evident in the majority of LC (62%). PTX3, CRP levels and platelet count are associated with improvements in health-related quality of life.

Broad spectrum SARS-CoV-2-specific immunity in hospitalized First Nations peoples recovering from COVID-19.

Indigenous peoples globally are at increased risk of COVID-19-associated morbidity and mortality. However, data that describe immune responses to SARS-CoV-2 infection in Indigenous populations are lacking. We evaluated immune responses in Australian First Nations peoples hospitalized with COVID-19. Our work comprehensively mapped out inflammatory, humoral and adaptive immune responses following SARS-CoV-2 infection. Patients were recruited early following the lifting of strict public health measures in the Northern Territory, Australia, between November 2021 and May 2022. Australian First Nations peoples recovering from COVID-19 showed increased levels of MCP-1 and IL-8 cytokines, IgG-antibodies against Delta-RBD and memory SARS-CoV-2-specific T cell responses prior to hospital discharge in comparison with hospital admission, with resolution of hyperactivated HLA-DR+ CD38+ T cells. SARS-CoV-2 infection elicited coordinated ASC, Tfh and CD8+ T cell responses in concert with CD4+ T cell responses. Delta and Omicron RBD-IgG, as well as Ancestral N-IgG antibodies, strongly correlated with Ancestral RBD-IgG antibodies and Spike-specific memory B cells. We provide evidence of broad and robust immune responses following SARS-CoV-2 infection in Indigenous peoples, resembling those of non-Indigenous COVID-19 hospitalized patients.

Durable reprogramming of neutralizing antibody responses following Omicron breakthrough infection.

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.

Adenovirus vector produced Zika virus-like particles induce a long-lived neutralising antibody response in mice.

Countermeasures against Zika virus (ZIKV) epidemics are urgently needed. In this study we generated a ZIKV virus-like particle (VLP) based vaccine candidate and assessed the immunogenicity of these particles in mice. The ZIKV-VLPs were morphologically similar to ZIKV by electron microscopy and were recognized by anti-Flavivirus neutralising antibodies. We observed that a single dose of unadjuvanted ZIKV-VLPs, or inactivated ZIKV, generated an immune response that lasted over 6 months, but did not neutralize ZIKV infection of cells in vitro. However, when we co-administered the ZIKV VLPs with either Aluminium hydroxide (Alhydrogel®; Alum), AddaVax or Pam2Cys we observed that Alum was the most effective in a single dose regime, since it not only produced antibodies that neutralized the virus, but also generated a greater number of antigen-specific memory B cells. We additionally observed that the generation of the neutralising antibodies persisted for up to 6 months. Our results suggest that a single dose ZIKV VLPs could be a suitable single dose vaccine candidate for use in outbreak settings.

Mucosal vaccines for SARS-CoV-2: triumph of hope over experience.

Currently approved COVID-19 vaccines administered parenterally induce robust systemic humoral and cellular responses. While highly effective against severe disease, there is reduced effectiveness of these vaccines in preventing breakthrough infection and/or onward transmission, likely due to poor immunity elicited at the respiratory mucosa. As such, there has been considerable interest in developing novel mucosal vaccines that engenders more localised immune responses to provide better protection and recall responses at the site of virus entry, in contrast to traditional vaccine approaches that focus on systemic immunity. In this review, we explore the adaptive components of mucosal immunity, evaluate epidemiological studies to dissect if mucosal immunity conferred by parenteral vaccination or respiratory infection drives differential efficacy against virus acquisition or transmission, discuss mucosal vaccines undergoing clinical trials and assess key challenges and prospects for mucosal vaccine development.

Robust and prototypical immune responses toward COVID-19 vaccine in First Nations peoples are impacted by comorbidities.

High-risk groups, including Indigenous people, are at risk of severe COVID-19. Here we found that Australian First Nations peoples elicit effective immune responses to COVID-19 BNT162b2 vaccination, including neutralizing antibodies, receptor-binding domain (RBD) antibodies, SARS-CoV-2 spike-specific B cells, and CD4+ and CD8+ T cells. In First Nations participants, RBD IgG antibody titers were correlated with body mass index and negatively correlated with age. Reduced RBD antibodies, spike-specific B cells and follicular helper T cells were found in vaccinated participants with chronic conditions (diabetes, renal disease) and were strongly associated with altered glycosylation of IgG and increased interleukin-18 levels in the plasma. These immune perturbations were also found in non-Indigenous people with comorbidities, indicating that they were related to comorbidities rather than ethnicity. However, our study is of a great importance to First Nations peoples who have disproportionate rates of chronic comorbidities and provides evidence of robust immune responses after COVID-19 vaccination in Indigenous people.

Immune profiling of SARS-CoV-2 infection during pregnancy reveals NK cell and γδ T cell perturbations.

Pregnancy poses a greater risk for severe COVID-19; however, underlying immunological changes associated with SARS-CoV-2 during pregnancy are poorly understood. We defined immune responses to SARS-CoV-2 in unvaccinated pregnant and nonpregnant women with acute and convalescent COVID-19, quantifying 217 immunological parameters. Humoral responses to SARS-CoV-2 were similar in pregnant and nonpregnant women, although our systems serology approach revealed distinct antibody and FcγR profiles between pregnant and nonpregnant women. Cellular analyses demonstrated marked differences in NK cell and unconventional T cell activation dynamics in pregnant women. Healthy pregnant women displayed preactivated NK cells and γδ T cells when compared with healthy nonpregnant women, which remained unchanged during acute and convalescent COVID-19. Conversely, nonpregnant women had prototypical activation of NK and γδ T cells. Activation of CD4+ and CD8+ T cells and T follicular helper cells was similar in SARS-CoV-2-infected pregnant and nonpregnant women, while antibody-secreting B cells were increased in pregnant women during acute COVID-19. Elevated levels of IL-8, IL-10, and IL-18 were found in pregnant women in their healthy state, and these cytokine levels remained elevated during acute and convalescent COVID-19. Collectively, we demonstrate perturbations in NK cell and γδ T cell activation in unvaccinated pregnant women with COVID-19, which may impact disease progression and severity during pregnancy.

Robust SARS-CoV-2 T cell responses with common TCRαß motifs toward COVID-19 vaccines in patients with hematological malignancy impacting B cells.

Immunocompromised hematology patients are vulnerable to severe COVID-19 and respond poorly to vaccination. Relative deficits in immunity are, however, unclear, especially after 3 vaccine doses. We evaluated immune responses in hematology patients across three COVID-19 vaccination doses. Seropositivity was low after a first dose of BNT162b2 and ChAdOx1 (∼26%), increased to 59%-75% after a second dose, and increased to 85% after a third dose. While prototypical antibody-secreting cells (ASCs) and T follicular helper (Tfh) cell responses were elicited in healthy participants, hematology patients showed prolonged ASCs and skewed Tfh2/17 responses. Importantly, vaccine-induced expansions of spike-specific and peptide-HLA tetramer-specific CD4+/CD8+ T cells, together with their T cell receptor (TCR) repertoires, were robust in hematology patients, irrespective of B cell numbers, and comparable to healthy participants. Vaccinated patients with breakthrough infections developed higher antibody responses, while T cell responses were comparable to healthy groups. COVID-19 vaccination induces robust T cell immunity in hematology patients of varying diseases and treatments irrespective of B cell numbers and antibody response.

SARS-CoV-2 breakthrough infection induces rapid memory and de novo T cell responses.

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.

Prospective comprehensive profiling of immune responses to COVID-19 vaccination in patients on zanubrutinib therapy.

Zanubrutinib-treated and treatment-naïve patients with chronic lymphocytic leukaemia (CLL) or Waldenstrom's macroglobulinaemia were recruited in this prospective study to comprehensively profile humoral and cellular immune responses to COVID-19 vaccination. Overall, 45 patients (median 72 years old) were recruited; the majority were male (71%), had CLL (76%) and were on zanubrutinib (78%). Seroconversion rates were 65% and 77% following two and three doses, respectively. CD4+ and CD8+ T-cell response rates increased with third dose. In zanubrutinib-treated patients, 86% developed either a humoral or cellular response. Patients on zanubrutinib developed substantial immune responses following two COVID-19 vaccine doses, which further improved following a third dose.

Engineered Ferritin Nanoparticle Vaccines Enable Rapid Screening of Antibody Functionalization to Boost Immune Responses.

Employing monoclonal antibodies to target vaccine antigens to different immune cells within lymph nodes where adaptive immunity is initiated can provide a mechanism to fine-tune the magnitude or the quality of immune responses. However, studying the effects of different targeting antibodies head-to-head is challenging due to the lack of a feasible method that allows rapid screening of multiple antibodies for their impact on immunogenicity. Here self-assembling ferritin nanoparticles are prepared that co-display vaccine antigens and the Fc-binding domain of Staphylococcal protein A, allowing rapid attachment of soluble antibodies to the nanoparticle surface. Using this tunable system, ten antibodies targeting different immune cell subsets are screened, with targeting to Clec9a associated with higher serum antibody titers after immunization. Immune cell targeting using ferritin nanoparticles with anti-Clec9a antibodies drives concentrated deposition of antigens within germinal centers, boosting germinal center formation and robust antibody responses. However, the capacity to augment humoral immunity is antigen-dependent, with significant boosting observed for prototypic ovalbumin immunogens but reduced effectiveness with the SARS-CoV-2 RBD. This work provides a rapid platform for screening targeting antibodies, which will accelerate mechanistic insights into optimal delivery strategies for nanoparticle-based vaccines to maximize protective immunity.

Nice and slow make the germinal centers go: measured and escalating antigen delivery enhance durability and quality of humoral immune responses against HIV-1.

A recently published article has confirmed that a novel immunization method of sustained and escalating antigen delivery augments the magnitude, quality and durability of humoral immune responses.

SARS-CoV-2-specific T cell memory with common TCRαß motifs is established in unvaccinated children who seroconvert after infection.

As the establishment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell memory in children remains largely unexplored, we recruited convalescent COVID-19 children and adults to define their circulating memory SARS-CoV-2-specific CD4+ and CD8+ T cells prior to vaccination. We analyzed epitope-specific T cells directly ex vivo using seven HLA class I and class II tetramers presenting SARS-CoV-2 epitopes, together with Spike-specific B cells. Unvaccinated children who seroconverted had comparable Spike-specific but lower ORF1a- and N-specific memory T cell responses compared with adults. This agreed with our TCR sequencing data showing reduced clonal expansion in children. A strong stem cell memory phenotype and common T cell receptor motifs were detected within tetramer-specific T cells in seroconverted children. Conversely, children who did not seroconvert had tetramer-specific T cells of predominantly naive phenotypes and diverse TCRαß repertoires. Our study demonstrates the generation of SARS-CoV-2-specific T cell memory with common TCRαß motifs in unvaccinated seroconverted children after their first virus encounter.

The magnitude and timing of recalled immunity after breakthrough infection is shaped by SARS-CoV-2 variants.

Vaccination against SARS-CoV-2 protects from infection and improves clinical outcomes in breakthrough infections, likely reflecting residual vaccine-elicited immunity and recall of immunological memory. Here, we define the early kinetics of spike-specific humoral and cellular immunity after vaccination of seropositive individuals and after Delta or Omicron breakthrough infection in vaccinated individuals. Early longitudinal sampling revealed the timing and magnitude of recall, with the phenotypic activation of B cells preceding an increase in neutralizing antibody titers. While vaccination of seropositive individuals resulted in robust recall of humoral and T cell immunity, recall of vaccine-elicited responses was delayed and variable in magnitude during breakthrough infections and depended on the infecting variant of concern. While the delayed kinetics of immune recall provides a potential mechanism for the lack of early control of viral replication, the recall of antibodies coincided with viral clearance and likely underpins the protective effects of vaccination against severe COVID-19.

Cutting Edge: SARS-CoV-2 Infection Induces Robust Germinal Center Activity in the Human Tonsil.

Understanding the generation of immunity to SARS-CoV-2 in lymphoid tissues draining the site of infection has implications for immunity to SARS-CoV-2. We performed tonsil biopsies under local anesthesia in 19 subjects who had recovered from SARS-CoV-2 infection 24-225 d previously. The biopsies yielded >3 million cells for flow cytometric analysis in 17 subjects. Total and SARS-CoV-2 spike-specific germinal center B cells, and T follicular helper cells, were readily detectable in human tonsils early after SARS-CoV-2 infection, as assessed by flow cytometry. Responses were higher in samples within 2 mo of infection but still detectable in some subjects out to 7 mo following infection. We conclude the tonsils are a secondary lymphoid organ that develop germinal center responses to SARS-CoV-2 infection and could play a role in the long-term development of immunity.

Establishment and recall of SARS-CoV-2 spike epitope-specific CD4+ T cell memory.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination elicit CD4+ T cell responses to the spike protein, including circulating follicular helper T (cTFH) cells that correlate with neutralizing antibodies. Using a novel HLA-DRB1*15:01/S751 tetramer to track spike-specific CD4+ T cells, we show that primary infection or vaccination induces robust S751-specific CXCR5- and cTFH cell memory responses. Secondary exposure induced recall of CD4+ T cells with a transitory CXCR3+ phenotype, and drove expansion of cTFH cells transiently expressing ICOS, CD38 and PD-1. In both contexts, cells exhibited a restricted T cell antigen receptor repertoire, including a highly public clonotype and considerable clonotypic overlap between CXCR5- and cTFH populations. Following a third vaccine dose, the rapid re-expansion of spike-specific CD4+ T cells contrasted with the comparatively delayed increase in antibody titers. Overall, we demonstrate that stable pools of cTFH and memory CD4+ T cells established by infection and/or vaccination are efficiently recalled upon antigen reexposure and may contribute to long-term protection against SARS-CoV-2.

Lung-resident memory B cells established after pulmonary influenza infection display distinct transcriptional and phenotypic profiles.

Recent studies have established that memory B cells, largely thought to be circulatory in the blood, can take up long-term residency in inflamed tissues, analogous to widely described tissue-resident T cells. The dynamics of recruitment and retention of memory B cells to tissues and their immunological purpose remains unclear. Here, we characterized tissue-resident memory B cells (BRM) that are stably maintained in the lungs of mice after pulmonary influenza infection. Influenza-specific BRM were localized within inducible bronchus-associated lymphoid tissues (iBALTs) and displayed transcriptional signatures distinct from classical memory B cells in the blood or spleen while showing partial overlap with memory B cells in lung-draining lymph nodes. We identified lung-resident markers, including elevated expression of CXCR3, CCR6, and CD69, on hemagglutinin (HA)- and nucleoprotein (NP)-specific lung BRM. We found that CCR6 facilitates increased recruitment and/or retention of BRM in lungs and differentiation into antibody-secreting cells upon recall. Although expression of CXCR3 and CCR6 was comparable in total and influenza-specific memory B cells isolated across tissues of human donors, CD69 expression was higher in memory B cells from lung and draining lymph nodes of human organ donors relative to splenic and PBMC-derived populations, indicating that mechanisms underpinning BRM localization may be evolutionarily conserved. Last, we demonstrate that human memory B cells in lungs are transcriptionally distinct to populations in lung-draining lymph nodes or PBMCs. These data suggest that BRM may constitute a discrete component of B cell immunity, positioned at the lung mucosa for rapid humoral response against respiratory viral infections.

Poor protective potential of influenza nucleoprotein antibodies despite wide prevalence.

Humans are exposed to influenza virus through periodic infections. Due to these repeated exposures, human populations commonly have elevated antibody titers targeting the conserved internal influenza virus nucleoprotein (NP). Despite the presence of anti-NP antibodies, humans are acutely susceptible to drifted influenza viruses with antigenically different surface proteins and the protective potential of human NP antibodies is unclear. In this study, high levels of anti-NP antibody and NP-specific B cells were detected in both adult humans and influenza-infected mice, confirming that NP is a major target of humoral immunity. Through sorting single B cells from influenza-exposed human adults, we generated a panel of 11 anti-NP monoclonal antibodies (mAbs). The majority of anti-NP human mAbs generated were capable of engaging cellular Fc receptors and bound NP on the surface of influenza-infected cell lines in vitro, suggesting that anti-NP mAbs have the potential to mediate downstream Fc effector functions such as antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis. However, human anti-NP mAbs were not protective in vivo when passively transferred into a murine influenza challenge model. Future in vivo studies examining the synergistic effect of anti-NP mAbs infused with other influenza-specific mAbs are warranted.