Transient and durable T cell reactivity after COVID-19.

This study analyzed whole blood samples (n = 56) retrieved from 30 patients at 1 to 21 (median 9) mo after verified COVID-19 to determine the polarity and duration of antigen-specific T cell reactivity against severe acute respiratory syndrome coronavirus 2-derived antigens. Multimeric peptides spanning the entire nucleocapsid protein triggered strikingly synchronous formation of interleukin (IL)-4, IL-12, IL-13, and IL-17 ex vivo until ∼70 d after confirmed infection, whereafter this reactivity was no longer inducible. In contrast, levels of nucleocapsid-induced IL-2 and interferon-γ remained stable and highly correlated at 3 to 21 mo after infection. Similar cytokine dynamics were observed in unvaccinated, convalescent patients using whole-blood samples stimulated with peptides spanning the N-terminal portion of the spike 1 protein. These results unravel two phases of T cell reactivity following natural COVID-19: an early, synchronous response indicating transient presence of multipolar, antigen-specific T helper (TH) cells followed by an equally synchronous and durable TH1-like reactivity reflecting long-lasting T cell memory.

B cells promote CD8 T cell primary and memory responses to subunit vaccines.

The relationship between B cells and CD4 T cells has been carefully studied, revealing a collaborative effort in which B cells promote the activation, differentiation, and expansion of CD4 T cells while the so-called "helper" cells provide signals to B cells, influencing their class switching and fate. Interactions between B cells and CD8 T cells are not as well studied, although CD8 T cells exhibit an accelerated contraction after certain infections in B-cell-deficient mice. Here, we find that B cells significantly enhance primary CD8 T cell responses after vaccination. Moreover, memory CD8 numbers and function are impaired in B-cell-deficient animals, leading to increased susceptibility to bacterial challenge. We also show that interleukin-27 production by B cells contributes to their impact on primary, but not memory, CD8 responses. Better understanding of the interactions between CD8 T cells and B cells may aid in the design of more effective future vaccine strategies.

Follicular T cells optimize the germinal center response to SARS-CoV-2 protein vaccination in mice.

Follicular helper T (Tfh) cells promote, whereas follicular regulatory T (Tfr) cells restrain, germinal center (GC) reactions. However, the precise roles of these cells in the complex GC reaction remain poorly understood. Here, we perturb Tfh or Tfr cells after SARS-CoV-2 spike protein vaccination in mice. We find that Tfh cells promote the frequency and somatic hypermutation (SHM) of Spike-specific GC B cells and regulate clonal diversity. Tfr cells similarly control SHM and clonal diversity in the GC but do so by limiting clonal competition. In addition, deletion of Tfh or Tfr cells during primary vaccination results in changes in SHM after vaccine boosting. Aged mice, which have altered Tfh and Tfr cells, have lower GC responses, presenting a bimodal distribution of SHM. Together, these data demonstrate that GC responses to SARS-CoV-2 spike protein vaccines require a fine balance of positive and negative follicular T cell help to optimize humoral immunity.

T-Helper Cell Subset Response Is a Determining Factor in COVID-19 Progression.

The immune response type organized against viral infection is determinant in the prognosis of some infections. This work has aimed to study Th polarization in acute COVID-19 and its possible association with the outcome through an observational prospective study. Fifty-eight COVID-19 patients were recruited in the Medicine Department of the hospital "12 de Octubre," 55 patients remaining after losses to follow-up. Four groups were established according to maximum degree of disease progression. T-helper cell percentages and phenotypes, analyzed by flow cytometer, and serum cytokines levels, analyzed by Luminex, were evaluated when the microbiological diagnosis (acute phase) of the disease was obtained. Our study found a significant reduction of %Th1 and %Th17 cells with higher activated %Th2 cells in the COVID-19 patients compared with reference population. A higher percent of senescent Th2 cells was found in the patients who died than in those who survived. Senescent Th2 cell percentage was an independent risk factor for death (OR: 13.88) accompanied by the numbers of total lymphocytes (OR: 0.15) with an AUC of 0.879. COVID-19 patients showed a profile of pro-inflammatory serum cytokines compared to controls, with higher levels of IL-2, IL-6, IL-15, and IP-10. IL-10 and IL-13 were also elevated in patients compared to controls. Patients who did not survive presented significantly higher levels of IL-15 than those who recovered. No significant differences were observed according to disease progression groups. The study has shown that increased levels of IL-15 and a high Th2 response are associated with a fatal outcome of the disease.

A COVID-19 peptide vaccine for the induction of SARS-CoV-2 T cell immunity.

T cell immunity is central for the control of viral infections. CoVac-1 is a peptide-based vaccine candidate, composed of SARS-CoV-2 T cell epitopes derived from various viral proteins1,2, combined with the Toll-like receptor 1/2 agonist XS15 emulsified in Montanide ISA51 VG, aiming to induce profound SARS-CoV-2 T cell immunity to combat COVID-19. Here we conducted a phase I open-label trial, recruiting 36 participants aged 18-80 years, who received a single subcutaneous CoVac-1 vaccination. The primary end point was safety analysed until day 56. Immunogenicity in terms of CoVac-1-induced T cell response was analysed as the main secondary end point until day 28 and in the follow-up until month 3. No serious adverse events and no grade 4 adverse events were observed. Expected local granuloma formation was observed in all study participants, whereas systemic reactogenicity was absent or mild. SARS-CoV-2-specific T cell responses targeting multiple vaccine peptides were induced in all study participants, mediated by multifunctional T helper 1 CD4+ and CD8+ T cells. CoVac-1-induced IFNγ T cell responses persisted in the follow-up analyses and surpassed those detected after SARS-CoV-2 infection as well as after vaccination with approved vaccines. Furthermore, vaccine-induced T cell responses were unaffected by current SARS-CoV-2 variants of concern. Together, CoVac-1 showed a favourable safety profile and induced broad, potent and variant of concern-independent T cell responses, supporting the presently ongoing evaluation in a phase II trial for patients with B cell or antibody deficiency.

COVID-19 genetic risk variants are associated with expression of multiple genes in diverse immune cell types.

Common genetic polymorphisms associated with COVID-19 illness can be utilized for discovering molecular pathways and cell types driving disease pathogenesis. Given the importance of immune cells in the pathogenesis of COVID-19 illness, here we assessed the effects of COVID-19-risk variants on gene expression in a wide range of immune cell types. Transcriptome-wide association study and colocalization analysis revealed putative causal genes and the specific immune cell types where gene expression is most influenced by COVID-19-risk variants. Notable examples include OAS1 in non-classical monocytes, DTX1 in B cells, IL10RB in NK cells, CXCR6 in follicular helper T cells, CCR9 in regulatory T cells and ARL17A in TH2 cells. By analysis of transposase accessible chromatin and H3K27ac-based chromatin-interaction maps of immune cell types, we prioritized potentially functional COVID-19-risk variants. Our study highlights the potential of COVID-19 genetic risk variants to impact the function of diverse immune cell types and influence severe disease manifestations.

Sterilizing Immunity against COVID-19: Developing Helper T cells I and II activating vaccines is imperative.

Six months after the publication of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) sequence, a record number of vaccine candidates were listed, and quite a number of them have since been approved for emergency use against the novel coronavirus disease 2019 (COVID-19). This unprecedented pharmaceutical feat did not only show commitment, creativity and collaboration of the scientific community, but also provided a swift solution that prevented global healthcare system breakdown. Notwithstanding, the available data show that most of the approved COVID-19 vaccines protect only a proportion of recipients against severe disease but do not prevent clinical manifestation of COVID-19. There is therefore the need to probe further to establish whether these vaccines can induce sterilizing immunity, otherwise, COVID-19 vaccination would have to become a regular phenomenon. The emergence of SARS-CoV-2 variants could further affect the capability of the available COVID-19 vaccines to prevent infection and protect recipients from a severe form of the disease. These notwithstanding, data about which vaccine(s), if any, can confer sterilizing immunity are unavailable. Here, we discuss the immune responses to viral infection with emphasis on COVID-19, and the specific adaptive immune response to SARS-CoV-2 and how it can be harnessed to develop COVID-19 vaccines capable of conferring sterilizing immunity. We further propose factors that could be considered in the development of COVID-19 vaccines capable of stimulating sterilizing immunity. Also, an old, but effective vaccine development technology that can be applied in the development of COVID-19 vaccines with sterilizing immunity potential is reviewed.

Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.

CCR2 Regulates Vaccine-Induced Mucosal T-Cell Memory to Influenza A Virus.

Elicitation of lung tissue-resident memory CD8 T cells (TRMs) is a goal of T cell-based vaccines against respiratory viral pathogens, such as influenza A virus (IAV). C-C chemokine receptor type 2 (CCR2)-dependent monocyte trafficking plays an essential role in the establishment of CD8 TRMs in lungs of IAV-infected mice. Here, we used a combination adjuvant-based subunit vaccine strategy that evokes multifaceted (TC1/TC17/TH1/TH17) IAV nucleoprotein-specific lung TRMs to determine whether CCR2 and monocyte infiltration are essential for vaccine-induced TRM development and protective immunity to IAV in lungs. Following intranasal vaccination, neutrophils, monocytes, conventional dendritic cells (DCs), and monocyte-derived dendritic cells internalized and processed vaccine antigen in lungs. We found that basic leucine zipper ATF-like transcription factor 3 (BATF3)-dependent DCs were essential for eliciting T cell responses, but CCR2 deficiency enhanced the differentiation of CD127hi, KLRG-1lo, OX40+ve CD62L+ve, and mucosally imprinted CD69+ve CD103+ve effector and memory CD8 T cells in lungs and airways of vaccinated mice. Mechanistically, increased development of lung TRMs induced by CCR2 deficiency was linked to dampened expression of T-bet but not altered TCF-1 levels or T cell receptor signaling in CD8 T cells. T1/T17 functional programming, parenchymal localization of CD8/CD4 effector and memory T cells, recall T cell responses, and protective immunity to a lethal IAV infection were unaffected in CCR2-deficient mice. Taken together, we identified a negative regulatory role for CCR2 and monocyte trafficking in mucosal imprinting and differentiation of vaccine-induced TRMs. Mechanistic insights from this study may aid the development of T-cell-based vaccines against respiratory viral pathogens, including IAV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IMPORTANCE While antibody-based immunity to influenza A virus (IAV) is type and subtype specific, lung- and airway-resident memory T cells that recognize conserved epitopes in the internal viral proteins are known to provide heterosubtypic immunity. Hence, broadly protective IAV vaccines need to elicit robust T cell memory in the respiratory tract. We have developed a combination adjuvant-based IAV nucleoprotein vaccine that elicits strong CD4 and CD8 T cell memory in lungs and protects against H1N1 and H5N1 strains of IAV. In this study, we examined the mechanisms that control vaccine-induced protective memory T cells in the respiratory tract. We found that trafficking of monocytes into lungs might limit the development of antiviral lung-resident memory T cells following intranasal vaccination. These findings suggest that strategies that limit monocyte infiltration can potentiate vaccine-induced frontline T-cell immunity to respiratory viruses, such as IAV and SARS-CoV-2.

SARS-CoV-2 Spike Impairs DNA Damage Repair and Inhibits V(D)J Recombination In Vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the coronavirus disease 2019 (COVID-19) pandemic, severely affecting public health and the global economy. Adaptive immunity plays a crucial role in fighting against SARS-CoV-2 infection and directly influences the clinical outcomes of patients. Clinical studies have indicated that patients with severe COVID-19 exhibit delayed and weak adaptive immune responses; however, the mechanism by which SARS-CoV-2 impedes adaptive immunity remains unclear. Here, by using an in vitro cell line, we report that the SARS-CoV-2 spike protein significantly inhibits DNA damage repair, which is required for effective V(D)J recombination in adaptive immunity. Mechanistically, we found that the spike protein localizes in the nucleus and inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site. Our findings reveal a potential molecular mechanism by which the spike protein might impede adaptive immunity and underscore the potential side effects of full-length spike-based vaccines.

Identification of conserved SARS-CoV-2 spike epitopes that expand public cTfh clonotypes in mild COVID-19 patients.

Adaptive immunity is a fundamental component in controlling COVID-19. In this process, follicular helper T (Tfh) cells are a subset of CD4+ T cells that mediate the production of protective antibodies; however, the SARS-CoV-2 epitopes activating Tfh cells are not well characterized. Here, we identified and crystallized TCRs of public circulating Tfh (cTfh) clonotypes that are expanded in patients who have recovered from mild symptoms. These public clonotypes recognized the SARS-CoV-2 spike (S) epitopes conserved across emerging variants. The epitope of the most prevalent cTfh clonotype, S864-882, was presented by multiple HLAs and activated T cells in most healthy donors, suggesting that this S region is a universal T cell epitope useful for booster antigen. SARS-CoV-2-specific public cTfh clonotypes also cross-reacted with specific commensal bacteria. In this study, we identified conserved SARS-CoV-2 S epitopes that activate public cTfh clonotypes associated with mild symptoms.

Dual-Antigen COVID-19 Vaccine Subcutaneous Prime Delivery With Oral Boosts Protects NHP Against SARS-CoV-2 Challenge.

We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to increase the potential for MHC class II responses. The vaccine antigens are delivered by a human adenovirus serotype 5 platform, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity. Vaccination of rhesus macaques with the hAd5 S-Fusion + N-ETSD vaccine by subcutaneous prime injection followed by two oral boosts elicited neutralizing anti-S IgG and T helper cell 1-biased T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.

In-silico immunoinformatic analysis of SARS-CoV-2 virus for the development of putative vaccine construct.

COVID-19 (CoronaVirus disease 2019) is caused by the SARS-CoV-2 virus (severe acute respiratory syndrome corona virus 2). SARS-CoV-2 virus is highly contagious and affects the human respiratory tract resulting in symptoms such as high fever, body ache, cough, dysfunctions of tastebuds and smelling sense of body. The objective of the present study involves immunoinformatic analysis to predict COVID-19 protein for vaccine construct based on the genomic information SARS-CoV-2 virus. At present, as per WHO estimates, around 133 COVID-19 novel vaccines under development. Three amino acid sequences of SARS-CoV-2 were retrieved from the NCBI database for the analysis of vaccine construct. This study involves computational and immunoinformatic methods. The Immunoinformatic tools used in the present study are NetCTL server, IFN epitope server, Toxin PRED, BCPred, CTL + HTL + ADJUVANTS + LINKERS, AlgPredserver, VaxiJenserver, ProtParam to predict vaccine construct. The secondary and tertiary structure prediction is done by PSIPRED, I-TASSER, Galaxy refine, prosA + Ramachandran. Finally, docking of the vaccine constructs and ligand was done with the help of Cluspro 2.0. C-ImmSimm webserver to simulate the potential vaccine construct. The present study demonstrated three potential Vaccine constructs for the SARS-CoV-2 virus, which were docked with TLR8 (Toll-likereceptor8). Interestingly from these, all constructs one having a high potential for the inhibition effect of the SARS-CoV-2virus. Immunological simulation data shows significant elevated amount of memory B cell; also, the high response was seen in TH(Helper) and TC(cytotoxic) cell population from the vaccine construct proposed in the current study. Hence, these constructs are suitable vaccine candidates that might be useful in developing a novel vaccine.

Designing a multi-epitope vaccine candidate to combat MERS-CoV by employing an immunoinformatics approach.

Currently, no approved vaccine is available against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes severe respiratory disease. The spike glycoprotein is typically considered a suitable target for MERS-CoV vaccine candidates. A computational strategy can be used to design an antigenic vaccine against a pathogen. Therefore, we used immunoinformatics and computational approaches to design a multi-epitope vaccine that targets the spike glycoprotein of MERS-CoV. After using numerous immunoinformatics tools and applying several immune filters, a poly-epitope vaccine was constructed comprising cytotoxic T-cell lymphocyte (CTL)-, helper T-cell lymphocyte (HTL)-, and interferon-gamma (IFN-γ)-inducing epitopes. In addition, various physicochemical, allergenic, and antigenic profiles were evaluated to confirm the immunogenicity and safety of the vaccine. Molecular interactions, binding affinities, and the thermodynamic stability of the vaccine were examined through molecular docking and dynamic simulation approaches, during which we identified a stable and strong interaction with Toll-like receptors (TLRs). In silico immune simulations were performed to assess the immune-response triggering capabilities of the vaccine. This computational analysis suggested that the proposed vaccine candidate would be structurally stable and capable of generating an effective immune response to combat viral infections; however, experimental evaluations remain necessary to verify the exact safety and immunogenicity profile of this vaccine.

Antigen-dependent multistep differentiation of T follicular helper cells and its role in SARS-CoV-2 infection and vaccination.

T follicular helper (Tfh) cells play an essential role in regulating the GC reaction and, consequently, the generation of high-affinity antibodies and memory B cells. Therefore, Tfh cells are critical for potent humoral immune responses against various pathogens and their dysregulation has been linked to autoimmunity and cancer. Tfh cell differentiation is a multistep process, in which cognate interactions with different APC types, costimulatory and coinhibitory pathways, as well as cytokines are involved. However, it is still not fully understood how a subset of activated CD4+ T cells begins to express the Tfh cell-defining chemokine receptor CXCR5 during the early stage of the immune response, how some CXCR5+ pre-Tfh cells enter the B-cell follicles and mature further into GC Tfh cells, and how Tfh cells are maintained in the memory compartment. In this review, we discuss recent advances on how antigen and cognate interactions are important for Tfh cell differentiation and long-term persistence of Tfh cell memory, and how this is relevant to the current understanding of COVID-19 pathogenesis and the development of potent SARS-CoV-2 vaccines.

Heterologous prime-boost: breaking the protective immune response bottleneck of COVID-19 vaccine candidates.

COVID-19 vaccines emerging from different platforms differ in efficacy, duration of protection, and side effects. To maximize the benefits of vaccination, we explored the utility of employing a heterologous prime-boost strategy in which different combinations of the four types of leading COVID-19 vaccine candidates that are undergoing clinical trials in China were tested in a mouse model. Our results showed that sequential immunization with adenovirus vectored vaccine followed by inactivated/recombinant subunit/mRNA vaccine administration specifically increased levels of neutralizing antibodies and promoted the modulation of antibody responses to predominantly neutralizing antibodies. Moreover, a heterologous prime-boost regimen with an adenovirus vector vaccine also improved Th1-biased T cell responses. Our results provide new ideas for the development and application of COVID-19 vaccines to control the SARS-CoV-2 pandemic.

Immunogenicity of prime-boost protein subunit vaccine strategies against SARS-CoV-2 in mice and macaques.

SARS-CoV-2 vaccines are advancing into human clinical trials, with emphasis on eliciting high titres of neutralising antibodies against the viral spike (S). However, the merits of broadly targeting S versus focusing antibody onto the smaller receptor binding domain (RBD) are unclear. Here we assess prototypic S and RBD subunit vaccines in homologous or heterologous prime-boost regimens in mice and non-human primates. We find S is highly immunogenic in mice, while the comparatively poor immunogenicity of RBD is associated with limiting germinal centre and T follicular helper cell activity. Boosting S-primed mice with either S or RBD significantly augments neutralising titres, with RBD-focussing driving moderate improvement in serum neutralisation. In contrast, both S and RBD vaccines are comparably immunogenic in macaques, eliciting serological neutralising activity that generally exceed levels in convalescent humans. These studies confirm recombinant S proteins as promising vaccine candidates and highlight multiple pathways to achieving potent serological neutralisation.

Evolution of immune responses to SARS-CoV-2 in mild-moderate COVID-19.

The durability of infection-induced SARS-CoV-2 immunity has major implications for reinfection and vaccine development. Here, we show a comprehensive profile of antibody, B cell and T cell dynamics over time in a cohort of patients who have recovered from mild-moderate COVID-19. Binding and neutralising antibody responses, together with individual serum clonotypes, decay over the first 4 months post-infection. A similar decline in Spike-specific CD4+ and circulating T follicular helper frequencies occurs. By contrast, S-specific IgG+ memory B cells consistently accumulate over time, eventually comprising a substantial fraction of circulating the memory B cell pool. Modelling of the concomitant immune kinetics predicts maintenance of serological neutralising activity above a titre of 1:40 in 50% of convalescent participants to 74 days, although there is probably additive protection from B cell and T cell immunity. This study indicates that SARS-CoV-2 immunity after infection might be transiently protective at a population level. Therefore, SARS-CoV-2 vaccines might require greater immunogenicity and durability than natural infection to drive long-term protection.

Hallmarks of the aging T-cell system.

The adaptive immune system has the enormous challenge to protect the host through the generation and differentiation of pathogen-specific short-lived effector T cells while in parallel developing long-lived memory cells to control future encounters with the same pathogen. A complex regulatory network is needed to preserve a population of naïve cells over lifetime that exhibit sufficient diversity of antigen receptors to respond to new antigens, while also sustaining immune memory. In parallel, cells need to maintain their proliferative potential and the plasticity to differentiate into different functional lineages. Initial signs of waning immune competence emerge after 50 years of age, with increasing clinical relevance in the 7th-10th decade of life. Morbidity and mortality from infections increase, as drastically exemplified by the current COVID-19 pandemic. Many vaccines, such as for the influenza virus, are poorly effective to generate protective immunity in older individuals. Age-associated changes occur at the level of the T-cell population as well as the functionality of its cellular constituents. The system highly relies on the self-renewal of naïve and memory T cells, which is robust but eventually fails. Genetic and epigenetic modifications contribute to functional differences in responsiveness and differentiation potential. To some extent, these changes arise from defective maintenance; to some, they represent successful, but not universally beneficial adaptations to the aging host. Interventions that can compensate for the age-related defects and improve immune responses in older adults are increasingly within reach.