Unique properties of tissue-resident memory T cells in the lungs: implications for COVID-19 and other respiratory diseases.

Tissue-resident memory T (TRM) cells were originally identified as a tissue-sequestered population of memory T cells that show lifelong persistence in non-lymphoid organs. That definition has slowly evolved with the documentation of TRM cells having variable terms of tissue residency combined with a capacity to return to the wider circulation. Nonetheless, reductionist experiments have identified an archetypical population of TRM cells showing intrinsic permanent residency in a wide range of non-lymphoid organs, with one notable exception: the lungs. Despite the fact that memory T cells generated during a respiratory infection are maintained in the circulation, local TRM cell numbers in the lung decline concomitantly with a decay in T cell-mediated protection. This Perspective describes the mechanisms that underpin long-term T cell lodgement in non-lymphoid tissues and explains why residency is transient for select TRM cell subsets. In doing so, it highlights the unusual nature of memory T cell egress from the lungs and speculates on the broader disease implications of this process, especially during infection with SARS-CoV-2.

Longitudinal Analysis of Memory T-Cell Responses in Survivors of Middle East Respiratory Syndrome.

BACKGROUND: Middle East respiratory syndrome (MERS) is a highly lethal respiratory disease caused by a zoonotic betacoronavirus. The development of effective vaccines and control measures requires a thorough understanding of the immune response to this viral infection. METHODS: We investigated cellular immune responses up to 5 years after infection in a cohort of 59 MERS survivors by performing enzyme-linked immunospot assay and intracellular cytokine staining after stimulation of peripheral blood mononuclear cells with synthetic viral peptides. RESULTS: Memory T-cell responses were detected in 82%, 75%, 69%, 64%, and 64% of MERS survivors from 1-5 years post-infection, respectively. Although the frequency of virus-specific interferon gamma (IFN-γ)-secreting T cells tended to be higher in moderately/severely ill patients than in mildly ill patients during the early period of follow-up, there was no significant difference among the different clinical severity groups across all time points. While both CD4+ and CD8+ T cells were involved in memory T-cell responses, CD4+ T cells persisted slightly longer than CD8+ T cells. Both memory CD4+ and CD8+ T cells recognized the E/M/N proteins better than the S protein and maintained their polyfunctionality throughout the period examined. Memory T-cell responses correlated positively with antibody responses during the initial 3-4 years but not with maximum viral loads at any time point. CONCLUSIONS: These findings advance our understanding of the dynamics of virus-specific memory T-cell immunity after MERS-coronavirus infection, which is relevant to the development of effective T cell-based vaccines.

Limited induction of polyfunctional lung-resident memory T cells against SARS-CoV-2 by mRNA vaccination compared to infection.

Resident memory T cells (TRM) present at the respiratory tract may be essential to enhance early SARS-CoV-2 viral clearance, thus limiting viral infection and disease. While long-term antigen-specific TRM are detectable beyond 11 months in the lung of convalescent COVID-19 patients, it is unknown if mRNA vaccination encoding for the SARS-CoV-2 S-protein can induce this frontline protection. Here we show that the frequency of CD4+ T cells secreting IFNγ in response to S-peptides is variable but overall similar in the lung of mRNA-vaccinated patients compared to convalescent-infected patients. However, in vaccinated patients, lung responses present less frequently a TRM phenotype compared to convalescent infected individuals and polyfunctional CD107a+ IFNγ+ TRM are virtually absent in vaccinated patients. These data indicate that mRNA vaccination induces specific T cell responses to SARS-CoV-2 in the lung parenchyma, although to a limited extend. It remains to be determined whether these vaccine-induced responses contribute to overall COVID-19 control.

Persistence of spike-specific immune responses in BNT162b2-vaccinated donors and generation of rapid ex-vivo T cells expansion protocol for adoptive immunotherapy: A pilot study.

Introduction: The BNT162b2 mRNA-based vaccine has shown high efficacy in preventing COVID-19 infection but there are limited data on the types and persistence of the humoral and T cell responses to such a vaccine. Methods: Here, we dissect the vaccine-induced humoral and cellular responses in a cohort of six healthy recipients of two doses of this vaccine. Results and discussion: Overall, there was heterogeneity in the spike-specific humoral and cellular responses among vaccinated individuals. Interestingly, we demonstrated that anti-spike antibody levels detected by a novel simple automated assay (Jess) were strongly correlated (r=0.863, P<0.0001) with neutralizing activity; thus, providing a potential surrogate for neutralizing cell-based assays. The spike-specific T cell response was measured with a newly modified T-spot assay in which the high-homology peptide-sequences cross-reactive with other coronaviruses were removed. This response was induced in 4/6 participants after the first dose, and all six participants after the second dose, and remained detectable in 4/6 participants five months post-vaccination. We have also shown for the first time, that BNT162b2 vaccine enhanced T cell responses also against known human common viruses. In addition, we demonstrated the efficacy of a rapid ex-vivo T cell expansion protocol for spike-specific T cell expansion to be potentially used for adoptive-cell therapy in severe COVID-19, immunocompromised individuals, and other high-risk groups. There was a 9 to 13.7-fold increase in the number of expanded T cells with a significant increase of anti-spike specific response showing higher frequencies of both activation and cytotoxic markers. Interestingly, effector memory T cells were dominant in all four participants' CD8+ expanded memory T cells; CD4+ T cells were dominated by effector memory in 2/4 participants and by central memory in the remaining two participants. Moreover, we found that high frequencies of CD4+ terminally differentiated memory T cells were associated with a greater reduction of spike-specific activated CD4+ T cells. Finally, we showed that participants who had a CD4+ central memory T cell dominance expressed a high CD69 activation marker in the CD4+ activated T cells.

Shifting the Immune Memory Paradigm: Trained Immunity in Viral Infections.

Trained immunity is defined as the de facto memory characteristics induced in innate immune cells after exposure to microbial stimuli after infections or certain types of vaccines. Through epigenetic and metabolic reprogramming of innate immune cells after exposure to these stimuli, trained immunity induces an enhanced nonspecific protection by improving the inflammatory response upon restimulation with the same or different pathogens. Recent studies have increasingly shown that trained immunity can, on the one hand, be induced by exposure to viruses; on the other hand, when induced, it can also provide protection against heterologous viral infections. In this review we explore current knowledge on trained immunity and its relevance for viral infections, as well as its possible future uses.

Functional antibodies exhibit light chain coherence.

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens¹. In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1-CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens^2-22. Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes^23,24. We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.

Functional heterogeneity in the memory B-cell response.

Most vaccines induce robust antibody and memory B-cell (MBC) responses that are capable of mediating protective immunity. However, antibody titers wane following vaccination necessitating the administration of booster vaccines to maintain a protective antibody titer. MBCs are stably maintained following vaccination and can rapidly give rise to antibody-secreting cells or undergo further affinity maturation upon antigen re-encounter. Repeated antigen encounter results in the development of MBCs that encode antibodies capable of mediating broadly protective immunity against viruses such as SARS-CoV-2 and influenza. Here, we summarize emerging evidence that MBCs are a heterogeneous population composed of transcriptionally and phenotypically distinct subsets that have discrete roles in mediating protective immunity upon antigen re-encounter and examine the implications of these findings for the development of vaccines capable of eliciting broadly protective immunity.

A heterologous AZD1222 priming and BNT162b2 boosting regimen more efficiently elicits neutralizing antibodies, but not memory T cells, than the homologous BNT162b2 regimen.

BACKGROUND: Comparative analyses of SARS-CoV-2-specific immune responses elicited by diverse prime-boost regimens are required to establish efficient regimens for the control of COVID-19. METHOD: In this prospective observational cohort study, spike-specific immunoglobulin G (IgG) and neutralizing antibodies (nAbs) alongside spike-specific T-cell responses in age-matched groups of homologous BNT162b2/BNT162b2 or AZD1222/AZD1222 vaccination, heterologous AZD1222/BNT162b2 vaccination, and prior wild-type SARS-CoV-2 infection/vaccination were evaluated. RESULTS: Peak immune responses were achieved after the second vaccine dose in the naïve vaccinated groups and after the first dose in the prior infection/vaccination group. Peak titers of anti-spike IgG and nAb were significantly higher in the AZD1222/BNT162b2 vaccination and prior infection/vaccination groups than in the BNT162b2/BNT162b2 or AZD1222/AZD1222 groups. However, the frequency of interferon-γ-producing CD4+ T cells was highest in the BNT162b2/BNT162b2 vaccination group. Similar results were observed in the analysis of polyfunctional T cells. When nAb and CD4+T-cell responses against the Delta variant were analyzed, the prior infection/vaccination group exhibited higher responses than the groups of other homologous or heterologous vaccination regimens. CONCLUSION: nAbs are efficiently elicited by heterologous AZD1222/BNT162b2 vaccination, as well as prior infection/vaccination, whereas spike-specific CD4+T-cell responses are efficiently elicited by homologous BNT162b2 vaccination. Variant-recognizing immunity is more efficiently generated by prior infection/vaccination than the other homologous or heterologous vaccination regimens.

Influenza vaccination reveals sex dimorphic imprints of prior mild COVID-19.

Acute viral infections can have durable functional impacts on the immune system long after recovery, but how they affect homeostatic immune states and responses to future perturbations remain poorly understood1-4. Here we use systems immunology approaches, including longitudinal multimodal single-cell analysis (surface proteins, transcriptome and V(D)J sequences) to comparatively assess baseline immune statuses and responses to influenza vaccination in 33 healthy individuals after recovery from mild, non-hospitalized COVID-19 (mean, 151 days after diagnosis) and 40 age- and sex-matched control individuals who had never had COVID-19. At the baseline and independent of time after COVID-19, recoverees had elevated T cell activation signatures and lower expression of innate immune genes including Toll-like receptors in monocytes. Male individuals who had recovered from COVID-19 had coordinately higher innate, influenza-specific plasmablast, and antibody responses after vaccination compared with healthy male individuals and female individuals who had recovered from COVID-19, in part because male recoverees had monocytes with higher IL-15 responses early after vaccination coupled with elevated prevaccination frequencies of 'virtual memory'-like CD8+ T cells poised to produce more IFNγ after IL-15 stimulation. Moreover, the expression of the repressed innate immune genes in monocytes increased by day 1 to day 28 after vaccination in recoverees, therefore moving towards the prevaccination baseline of the healthy control individuals. By contrast, these genes decreased on day 1 and returned to the baseline by day 28 in the control individuals. Our study reveals sex-dimorphic effects of previous mild COVID-19 and suggests that viral infections in humans can establish new immunological set-points that affect future immune responses in an antigen-agnostic manner.

Role of IgM Memory B Cells and Spleen Function in COVID-19.

IgM memory B cells, are a peculiar subset of memory B cells, which probably originates in the spleen and outside germinal centers and provide a rapid line of defence against mucosal infections. Their role in counteracting COVID-19 is still elusive but, recent evidence, mainly boosted by studies on spleen function/involvement in COVID-19, seems to support the notion that this subset of memory B cells could exert a protective role against this virus, along with other coronaviruses, particularly in the acute setting of the infection, as outlined by worst clinical outcomes observed in unvaccinated patients with impaired IgM B memory response and spleen function. Herein we critically summarise the current landscape of studies on IgM memory B cells, focusing on the clinical impact of their depletion, by comparing the COVID-19-related splenic dysfunction with other hypo- and asplenic conditions and by adding recent data on follow-up studies and postulate a mechanistic explanation for their reduced numbers. The early detection of an impaired IgM memory B cell response in patients with COVID-19 may contribute to their improved care through different strategies, such as through tailored vaccine strategies, prompt hospital admission and/or administration of anti-infective treatments, thus resulting in an better prognosis, although at present management algorithms are still unavailable. Moreover, further studies with longer follow-up are needed to assess the evolution of COVID-19-associated/exacerbated immune deficit.

The BNT162b2 vaccine induces humoral and cellular immune memory to SARS-CoV-2 Wuhan strain and the Omicron variant in children 5 to 11 years of age.

SARS-CoV-2 mRNA vaccines prevent severe COVID-19 by generating immune memory, comprising specific antibodies and memory B and T cells. Although children are at low risk of severe COVID-19, the spreading of highly transmissible variants has led to increasing in COVID-19 cases and hospitalizations also in the youngest, but vaccine coverage remains low. Immunogenicity to mRNA vaccines has not been extensively studied in children 5 to 11 years old. In particular, cellular immunity to the wild-type strain (Wuhan) and the cross-reactive response to the Omicron variant of concern has not been investigated. We assessed the humoral and cellular immune response to the SARS-CoV-2 BNT162b2 vaccine in 27 healthy children. We demonstrated that vaccination induced a potent humoral and cellular immune response in all vaccinees. By using spike-specific memory B cells as a measurable imprint of a previous infection, we found that 50% of the children had signs of a past, undiagnosed infection before vaccination. Children with pre-existent immune memory generated significantly increased levels of specific antibodies, and memory T and B cells, directed against not only the wild type virus but also the omicron variant.

Lung tissue-resident memory T cells: the gatekeeper to respiratory viral (re)-infection.

The discovery of lung tissue-resident memory T (TRM) cells and the elucidation of their function in antiviral immunity have inspired considerable efforts to leverage the power of TRM cells, in defense to the infections and reinfections by respiratory viruses. Here, we have reviewed lung TRM cell identification, molecular regulation, and function after influenza and SARS-CoV-2 infections. Furthermore, we have discussed emerging data on TRM responses induced by systemic and mucosal vaccination strategies. We hope that our current outstanding of TRM cells in this review could provide insights toward the development of vaccines capable of inducing highly efficacious mucosal TRM responses for protection against respiratory viral infections.

Early CD4+ T cell responses induced by the BNT162b2 SARS-CoV-2 mRNA vaccine predict immunological memory.

Longitudinal studies have revealed large interindividual differences in antibody responses induced by SARS-CoV-2 mRNA vaccines. Thus, we performed a comprehensive analysis of adaptive immune responses induced by three doses of the BNT162b2 SARS-CoV-2 mRNA vaccines. The responses of spike-specific CD4+ T cells, CD8+ T cells and serum IgG, and the serum neutralization capacities induced by the two vaccines declined 6 months later. The 3rd dose increased serum spike IgG and neutralizing capacities against the wild-type and Omicron spikes to higher levels than the 2nd dose, and this was supported by memory B cell responses, which gradually increased after the 2nd dose and were further enhanced by the 3rd dose. The 3rd dose moderately increased the frequencies of spike-specific CD4+ T cells, but the frequencies of spike-specific CD8+ T cells remained unchanged. T cells reactive against the Omicron spike were 1.3-fold fewer than those against the wild-type spike. The early responsiveness of spike-specific CD4+ T, circulating T follicular helper cells and circulating T peripheral helper cells correlated with memory B cell responses to the booster vaccination, and early spike-specific CD4+ T cell responses were also associated with spike-specific CD8+ T cell responses. These findings highlight the importance of evaluating cellular responses to optimize future vaccine strategies.

Mucosal immunization with a delta-inulin adjuvanted recombinant spike vaccine elicits lung-resident immune memory and protects mice against SARS-CoV-2.

Multiple SARS-CoV-2 vaccine candidates have been approved for use and have had a major impact on the COVID-19 pandemic. There remains, however, a significant need for vaccines that are safe, easily transportable and protective against infection, as well as disease. Mucosal vaccination is favored for its ability to induce immune memory at the site of infection, making it appealing for SARS-CoV-2 vaccine strategies. In this study we performed in-depth analysis of the immune responses in mice to a subunit recombinant spike protein vaccine formulated with the delta-inulin adjuvant Advax when administered intratracheally (IT), versus intramuscular delivery (IM). Both routes produced robust neutralizing antibody titers (NAb) and generated sterilizing immunity against SARS-CoV-2. IT delivery, however, produced significantly higher systemic and lung-local NAb that resisted waning up to six months post vaccination, and only IT delivery generated inducible bronchus-associated lymphoid tissue (iBALT), a site of lymphocyte antigen presentation and proliferation. This was coupled with robust and long-lasting lung tissue-resident memory CD4+ and CD8+ T cells that were not observed in IM-vaccinated mice. This study provides a detailed view of the lung-resident cellular response to IT vaccination against SARS-CoV-2 and demonstrates the importance of delivery site selection in the development of vaccine candidates.

SARS-CoV-2 infected children form early immune memory responses dominated by nucleocapsid-specific CD8+ T cells and antibodies.

This is the third year of the SARS-CoV-2 pandemic, and yet most children remain unvaccinated. COVID-19 in children manifests as mostly mild or asymptomatic, however high viral titers and strong cellular and humoral responses are observed upon acute infection. It is still unclear how long these responses persist, and if they can protect from re-infection and/or disease severity. Here, we analyzed immune memory responses in a cohort of children and adults with COVID-19. Important differences between children and adults are evident in kinetics and profile of memory responses. Children develop early N-specific cytotoxic T cell responses, that rapidly expand and dominate their immune memory to the virus. Children's anti-N, but not anti-S, antibody titers increase over time. Neutralization titers correlate with N-specific antibodies and CD8+T cells. However, antibodies generated by infection do not efficiently cross-neutralize variants Gamma or Delta. Our results indicate that mechanisms that protect from disease severity are possibly different from those that protect from reinfection, bringing novel insights for pediatric vaccine design. They also underline the importance of vaccination in children, who remain at risk for COVID-19 despite having been previously infected.

Association between SARS-CoV-2 RNAemia and dysregulated immune response in acutely ill hospitalized COVID-19 patients.

Severe/critical COVID-19 is associated with immune dysregulation and plasmatic SARS-CoV-2 detection (i.e. RNAemia). We detailed the association of SARS-CoV-2 RNAemia with immune responses in COVID-19 patients at the end of the first week of disease. We enrolled patients hospitalized in acute phase of ascertained SARS-CoV-2 pneumonia, and evaluated SARS-CoV-2 RNAemia, plasmatic cytokines, activated/pro-cytolytic T-cells phenotypes, SARS-CoV-2-specific cytokine-producing T-cells (IL-2, IFN-γ, TNF-α, IL-4, IL-17A), simultaneous Th1-cytokines production (polyfunctionality) and amount (iMFI). The humoral responses were assessed with anti-S1/S2 IgG, anti-RBD total-Ig, IgM, IgA, IgG1 and IgG3, neutralization and antibody-dependent cellular cytotoxicity (ADCC). Out of 54 patients, 27 had detectable viremia (viremic). Albeit comparable age and co-morbidities, viremic more frequently required ventilatory support, with a trend to higher death. Viremic displayed higher pro-inflammatory cytokines (IFN-α, IL-6), lower activated T-cells (HLA-DR+CD38+), lower functional SARS-CoV-2-specific T-cells (IFN-γ+CD4+, TNF-α+CD8+, IL-4+CD8+, IL-2+TNF-α+CD4+, and IL-2+TNF-α+CD4+ iMFI) and SARS-CoV-2-specific Abs (anti-S IgG, anti-RBD total-Ig, IgM, IgG1, IgG3; ID50, %ADCC). These data suggest a link between SARS-CoV-2 RNAemia at the end of the first stage of disease and immune dysregulation. Whether high ab initium viral burden and/or intrinsic host factors contribute to immune dysregulation in severe COVID-19 remains to be elucidated, to further inform strategies of targeted therapeutic interventions.

Immunological memory to SARS-CoV-2 infection and COVID-19 vaccines.

Immunological memory is the basis of protective immunity provided by vaccines and previous infections. Immunological memory can develop from multiple branches of the adaptive immune system, including CD4 T cells, CD8 T cells, B cells, and long-lasting antibody responses. Extraordinary progress has been made in understanding memory to SARS-CoV-2 infection and COVID-19 vaccines, addressing development; quantitative and qualitative features of different cellular and anatomical compartments; and durability of each cellular component and antibodies. Given the sophistication of the measurements; the size of the human studies; the use of longitudinal samples and cross-sectional studies; and head-to-head comparisons between infection and vaccines or between multiple vaccines, the understanding of immune memory for 1 year to SARS-CoV-2 infection and vaccines already supersedes that of any other acute infectious disease. This knowledge may help inform public policies regarding COVID-19 and COVID-19 vaccines, as well as the scientific development of future vaccines against SARS-CoV-2 and other diseases.