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.

Human antiviral B cell responses: Emerging lessons from hepatitis B and COVID-19.

Humoral immunity is a critical component of the coordinated response required to resolve viral infections and mediate protection following pathogen clearance or vaccination. A better understanding of factors shaping the memory B cell response will allow tailored development of efficient preventative vaccines against emerging acute viral infections, therapeutic vaccines, and immunotherapies for chronic viral infections. Here, we use recent data obtained by profiling antigen-specific B cell responses in hepatitis B as a framework to explore lessons that can be learnt from different viral infections about the diverse influences on humoral immunity. Hepatitis B provides a paradigm where successful B cell responses in resolved or vaccinated individuals can be contrasted to the failed response in chronic infection, while also exemplifying the degree to which B cell responses within infected individuals can differ to two antigens from the same virus. Drawing on studies in other human and murine infections, including emerging data from COVID-19, we consider the influence of antigen quantity and structure on the quality of the B cell response, the role of differential CD4 help, the importance of germinal center vs extrafollicular responses and the emerging concept that responses residing in non-lymphoid organs can participate in B cell memory.

An in silico deep learning approach to multi-epitope vaccine design: a SARS-CoV-2 case study.

The rampant spread of COVID-19, an infectious disease caused by SARS-CoV-2, all over the world has led to over millions of deaths, and devastated the social, financial and political entities around the world. Without an existing effective medical therapy, vaccines are urgently needed to avoid the spread of this disease. In this study, we propose an in silico deep learning approach for prediction and design of a multi-epitope vaccine (DeepVacPred). By combining the in silico immunoinformatics and deep neural network strategies, the DeepVacPred computational framework directly predicts 26 potential vaccine subunits from the available SARS-CoV-2 spike protein sequence. We further use in silico methods to investigate the linear B-cell epitopes, Cytotoxic T Lymphocytes (CTL) epitopes, Helper T Lymphocytes (HTL) epitopes in the 26 subunit candidates and identify the best 11 of them to construct a multi-epitope vaccine for SARS-CoV-2 virus. The human population coverage, antigenicity, allergenicity, toxicity, physicochemical properties and secondary structure of the designed vaccine are evaluated via state-of-the-art bioinformatic approaches, showing good quality of the designed vaccine. The 3D structure of the designed vaccine is predicted, refined and validated by in silico tools. Finally, we optimize and insert the codon sequence into a plasmid to ensure the cloning and expression efficiency. In conclusion, this proposed artificial intelligence (AI) based vaccine discovery framework accelerates the vaccine design process and constructs a 694aa multi-epitope vaccine containing 16 B-cell epitopes, 82 CTL epitopes and 89 HTL epitopes, which is promising to fight the SARS-CoV-2 viral infection and can be further evaluated in clinical studies. Moreover, we trace the RNA mutations of the SARS-CoV-2 and ensure that the designed vaccine can tackle the recent RNA mutations of the virus.

Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications.

Coronavirus disease 2019 (COVID-19) is currently a global pandemic, but human immune responses to the virus remain poorly understood. We used high-dimensional cytometry to analyze 125 COVID-19 patients and compare them with recovered and healthy individuals. Integrated analysis of ~200 immune and ~50 clinical features revealed activation of T cell and B cell subsets in a proportion of patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses reaching >30% of circulating B cells. However, another subgroup had lymphocyte activation comparable with that in uninfected individuals. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. Our analyses identified three immunotypes associated with poor clinical trajectories versus improving health. These immunotypes may have implications for the design of therapeutics and vaccines for COVID-19.

Negative Clinical Evolution in COVID-19 Patients Is Frequently Accompanied With an Increased Proportion of Undifferentiated Th Cells and a Strong Underrepresentation of the Th1 Subset.

The severity of SARS-CoV-2 infection has been related to uncontrolled inflammatory innate responses and impaired adaptive immune responses mostly due to exhausted T lymphocytes and lymphopenia. In this work we have characterized the nature of the lymphopenia and demonstrate a set of factors that hinder the effective control of virus infection and the activation and arming of effector cytotoxic T CD8 cells and showing signatures defining a high-risk population. We performed immune profiling of the T helper (Th) CD4+ and T CD8+ cell compartments in peripheral blood of 144 COVID-19 patients using multiparametric flow cytometry analysis. On the one hand, there was a consistent lymphopenia with an overrepresentation of non-functional T cells, with an increased percentage of naive Th cells (CD45RA+, CXCR3-, CCR4-, CCR6-, CCR10-) and persistently low frequency of markers associated with Th1, Th17, and Th1/Th17 memory-effector T cells compared to healthy donors. On the other hand, the most profound alteration affected the Th1 subset, which may explain the poor T cells responses and the persistent blood virus load. Finally, the decrease in Th1 cells may also explain the low frequency of CD4+ and CD8+ T cells that express the HLA-DR and CD38 activation markers observed in numerous patients who showed minimal or no lymphocyte activation response. We also identified the percentage of HLA-DR+CD4+ T cells, PD-1+CD+4/CD8+ T cells in blood, and the neutrophil/lymphocyte ratio as useful factors for predicting critical illness and fatal outcome in patients with confirmed COVID-19.

SARS-CoV-2 mRNA Vaccines Foster Potent Antigen-Specific Germinal Center Responses Associated with Neutralizing Antibody Generation.

The deployment of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to eradicate the coronavirus disease 2019 (COVID-19) pandemic. Many licensed vaccines confer protection by inducing long-lived plasma cells (LLPCs) and memory B cells (MBCs), cell types canonically generated during germinal center (GC) reactions. Here, we directly compared two vaccine platforms-mRNA vaccines and a recombinant protein formulated with an MF59-like adjuvant-looking for their abilities to quantitatively and qualitatively shape SARS-CoV-2-specific primary GC responses over time. We demonstrated that a single immunization with SARS-CoV-2 mRNA, but not with the recombinant protein vaccine, elicited potent SARS-CoV-2-specific GC B and T follicular helper (Tfh) cell responses as well as LLPCs and MBCs. Importantly, GC responses strongly correlated with neutralizing antibody production. mRNA vaccines more efficiently induced key regulators of the Tfh cell program and influenced the functional properties of Tfh cells. Overall, this study identifies SARS-CoV-2 mRNA vaccines as strong candidates for promoting robust GC-derived immune responses.

Deregulated cellular circuits driving immunoglobulins and complement consumption associate with the severity of COVID-19 patients.

SARS-CoV-2 infection causes an abrupt response by the host immune system, which is largely responsible for the outcome of COVID-19. We investigated whether the specific immune responses in the peripheral blood of 276 patients were associated with the severity and progression of COVID-19. At admission, dramatic lymphopenia of T, B, and NK cells is associated with severity. Conversely, the proportion of B cells, plasmablasts, circulating follicular helper T cells (cTfh) and CD56- CD16+ NK-cells increased. Regarding humoral immunity, levels of IgM, IgA, and IgG were unaffected, but when degrees of severity were considered, IgG was lower in severe patients. Compared to healthy donors, complement C3 and C4 protein levels were higher in mild and moderate, but not in severe patients, while the activation peptide of C5 (C5a) increased from the admission in every patient, regardless of their severity. Moreover, total IgG, the IgG1 and IgG3 isotypes, and C4 decreased from day 0 to day 10 in patients who were hospitalized for more than two weeks, but not in patients who were discharged earlier. Our study provides important clues to understand the immune response observed in COVID-19 patients, associating severity with an imbalanced humoral response, and identifying new targets for therapeutic intervention.

3CL hydrolase-based multiepitope peptide vaccine against SARS-CoV-2 using immunoinformatics.

The present study provides the first multiepitope vaccine construct using the 3CL hydrolase protein of SARS-CoV-2. The coronavirus 3CL hydrolase (Mpro) enzyme is essential for proteolytic maturation of the virus. This study was based on immunoinformatics and structural vaccinology strategies. The design of the multiepitope vaccine was built using helper T-cell and cytotoxic T-cell epitopes from the 3CL hydrolase protein along with an adjuvant to enhance immune response; these are joined to each other by short peptide linkers. The vaccine also carries potential B-cell linear epitope regions, B-cell discontinuous epitopes, and interferon-γ-inducing epitopes. Epitopes of the constructed multiepitope vaccine were found to be antigenic, nonallergic, nontoxic, and covering large human populations worldwide. The vaccine construct was modeled, validated, and refined by different programs to achieve a high-quality three-dimensional structure. The resulting high-quality model was applied for conformational B-cell epitope selection and docking analyses with toll-like receptor-3 for understanding the capability of the vaccine to elicit an immune response. In silico cloning and codon adaptation were also performed with the pET-19b plasmid vector. The designed multiepitope peptide vaccine may prompt the development of a vaccine to control SARS-CoV-2 infection.

Remodeling of the Immune Response With Aging: Immunosenescence and Its Potential Impact on COVID-19 Immune Response.

Elderly individuals are the most susceptible to an aggressive form of coronavirus disease (COVID-19), caused by SARS-CoV-2. The remodeling of immune response that is observed among the elderly could explain, at least in part, the age gradient in lethality of COVID-19. In this review, we will discuss the phenomenon of immunosenescence, which entails changes that occur in both innate and adaptive immunity with aging. Furthermore, we will discuss inflamm-aging, a low-grade inflammatory state triggered by continuous antigenic stimulation, which may ultimately increase all-cause mortality. In general, the elderly are less capable of responding to neo-antigens, because of lower naïve T cell frequency. Furthermore, they have an expansion of memory T cells with a shrinkage of the T cell diversity repertoire. When infected by SARS-CoV-2, young people present with a milder disease as they frequently clear the virus through an efficient adaptive immune response. Indeed, antibody-secreting cells and follicular helper T cells are thought to be effectively activated in young patients that present a favorable prognosis. In contrast, the elderly are more prone to an uncontrolled activation of innate immune response that leads to cytokine release syndrome and tissue damage. The failure to trigger an effective adaptive immune response in combination with a higher pro-inflammatory tonus may explain why the elderly do not appropriately control viral replication and the potential clinical consequences triggered by a cytokine storm, endothelial injury, and disseminated organ injury. Enhancing the efficacy of the adaptive immune response may be an important issue both for infection resolution as well as for the appropriate generation of immunity upon vaccination, while inhibiting inflamm-aging will likely emerge as a potential complementary therapeutic approach in the management of patients with severe COVID-19.

Loss of Bcl-6-Expressing T Follicular Helper Cells and Germinal Centers in COVID-19.

Humoral responses in coronavirus disease 2019 (COVID-19) are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined post mortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers and a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ TFH cell differentiation together with an increase in T-bet+ TH1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+ TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections, and suggest that achieving herd immunity through natural infection may be difficult.

Contriving Multi-Epitope Subunit of Vaccine for COVID-19: Immunoinformatics Approaches.

COVID-19 has recently become the most serious threat to public health, and its prevalence has been increasing at an alarming rate. The incubation period for the virus is ~1-14 days and all age groups may be susceptible to a fatality rate of about 5.9%. COVID-19 is caused by a novel single-stranded, positive (+) sense RNA beta coronavirus. The development of a vaccine for SARS-CoV-2 is an urgent need worldwide. Immunoinformatics approaches are both cost-effective and convenient, as in silico predictions can reduce the number of experiments needed. In this study, with the aid of immunoinformatics tools, we tried to design a multi-epitope vaccine that can be used for the prevention and treatment of COVID-19. The epitopes were computed by using B cells, cytotoxic T lymphocytes (CTL), and helper T lymphocytes (HTL) base on the proteins of SARS-CoV-2. A vaccine was devised by fusing together the B cell, HTL, and CTL epitopes with linkers. To enhance the immunogenicity, the ß-defensin (45 mer) amino acid sequence, and pan-HLA DR binding epitopes (13aa) were adjoined to the N-terminal of the vaccine with the help of the EAAAK linker. To enable the intracellular delivery of the modeled vaccine, a TAT sequence (11aa) was appended to C-terminal. Linkers play vital roles in producing an extended conformation (flexibility), protein folding, and separation of functional domains, and therefore, make the protein structure more stable. The secondary and three-dimensional (3D) structure of the final vaccine was then predicted. Furthermore, the complex between the final vaccine and immune receptors (toll-like receptor-3 (TLR-3), major histocompatibility complex (MHC-I), and MHC-II) were evaluated by molecular docking. Lastly, to confirm the expression of the designed vaccine, the mRNA of the vaccine was enhanced with the aid of the Java Codon Adaptation Tool, and the secondary structure was generated from Mfold. Then we performed in silico cloning. The final vaccine requires experimental validation to determine its safety and efficacy in controlling SARS-CoV-2 infections.

"Small" Intestinal Immunopathology Plays a "Big" Role in Lethal Cytokine Release Syndrome, and Its Modulation by Interferon-γ, IL-17A, and a Janus Kinase Inhibitor.

Chimeric antigen receptor T cell (CART) therapy, administration of certain T cell-agonistic antibodies, immune check point inhibitors, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) and Toxic shock syndrome (TSS) caused by streptococcal as well as staphylococcal superantigens share one common complication, that is T cell-driven cytokine release syndrome (CRS) accompanied by multiple organ dysfunction (MOD). It is not understood whether the failure of a particular organ contributes more significantly to the severity of CRS. Also not known is whether a specific cytokine or signaling pathway plays a more pathogenic role in precipitating MOD compared to others. As a result, there is no specific treatment available to date for CRS, and it is managed only symptomatically to support the deteriorating organ functions and maintain the blood pressure. Therefore, we used the superantigen-induced CRS model in HLA-DR3 transgenic mice, that closely mimics human CRS, to delineate the immunopathogenesis of CRS as well as to validate a novel treatment for CRS. Using this model, we demonstrate that (i) CRS is characterized by a rapid rise in systemic levels of several Th1/Th2/Th17/Th22 type cytokines within a few hours, followed by a quick decline. (ii) Even though multiple organs are affected, small intestinal immunopathology is the major contributor to mortality in CRS. (iii) IFN-γ deficiency significantly protected from lethal CRS by attenuating small bowel pathology, whereas IL-17A deficiency significantly increased mortality by augmenting small bowel pathology. (iv) RNA sequencing of small intestinal tissues indicated that IFN-γ-STAT1-driven inflammatory pathways combined with enhanced expression of pro-apoptotic molecules as well as extracellular matrix degradation contributed to small bowel pathology in CRS. These pathways were further enhanced by IL-17A deficiency and significantly down-regulated in mice lacking IFN-γ. (v) Ruxolitinib, a selective JAK-1/2 inhibitor, attenuated SAg-induced T cell activation, cytokine production, and small bowel pathology, thereby completely protecting from lethal CRS in both WT and IL-17A deficient HLA-DR3 mice. Overall, IFN-γ-JAK-STAT-driven pathways contribute to lethal small intestinal immunopathology in T cell-driven CRS.

Humoral and circulating follicular helper T cell responses in recovered patients with COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has dramatically expedited global vaccine development efforts1-3, most targeting the viral 'spike' glycoprotein (S). S localizes on the virion surface and mediates recognition of cellular receptor angiotensin-converting enzyme 2 (ACE2)4-6. Eliciting neutralizing antibodies that block S-ACE2 interaction7-9, or indirectly prevent membrane fusion10, constitute an attractive modality for vaccine-elicited protection11. However, although prototypic S-based vaccines show promise in animal models12-14, the immunogenic properties of S in humans are poorly resolved. In this study, we characterized humoral and circulating follicular helper T cell (cTFH) immunity against spike in recovered patients with coronavirus disease 2019 (COVID-19). We found that S-specific antibodies, memory B cells and cTFH are consistently elicited after SARS-CoV-2 infection, demarking robust humoral immunity and positively associated with plasma neutralizing activity. Comparatively low frequencies of B cells or cTFH specific for the receptor binding domain of S were elicited. Notably, the phenotype of S-specific cTFH differentiated subjects with potent neutralizing responses, providing a potential biomarker of potency for S-based vaccines entering the clinic. Overall, although patients who recovered from COVID-19 displayed multiple hallmarks of effective immune recognition of S, the wide spectrum of neutralizing activity observed suggests that vaccines might require strategies to selectively target the most potent neutralizing epitopes.