Cellular & Molecular Immunology

The WHO has declared SARS-CoV-2 outbreak a public health emergency of international concern. However, to date, there was hardly any study in characterizing the immune responses, especially adaptive immune responses to SARS-CoV-2 infection. In this study, we collected blood from COVID-19 patients who have recently become virus-free and therefore were discharged, and analyzed their SARS-CoV-2-specific antibody and T cell responses. We observed SARS-CoV-2-specific humoral and cellular immunity in the patients. Both were detected in newly discharged patients, suggesting both participate in immune-mediated protection to viral infection. However, follow-up patients (2 weeks post discharge) exhibited high titers of IgG antibodies, but with low levels of virus-specific T cells, suggesting that they may enter a quiescent state. Our work has thus provided a basis for further analysis of protective immunity to SARS-CoV-2, and understanding the pathogenesis of COVID-19, especially in the severe cases. It has also implications in designing an effective vaccine to protect and treat SARS-CoV-2 infection.

The COVID-19 pandemic has underscored the importance of understanding the intricate mechanisms of the humoral immune response to SARS-CoV-2. This study aimed to elucidate the diversity and specificity of antibodies generated from convalescent COVID-19 patients by isolating and characterizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 spike protein. Employing cutting-edge technologies, including single-cell analysis and fluorescence-activated cell sorting, we successfully isolated live memory B cells secreting IgG antibodies from the peripheral blood of convalescent patients. A total of 17 mAbs were generated, encompassing various heavy and light variable genes, with only a few common between patients. In vitro assays demonstrated varying degrees of inhibition against wild-type and Omicron strains, highlighting discrepancies between ACE2 competition and actual neutralization capacity. Bio-layer interferometry and in silico docking analyses revealed unique binding motifs and mechanisms of action, with notable differences in neutralization abilities based on epitope specificity. Furthermore, animal experiments using K18-hACE2 transgenic mice demonstrated the therapeutic potential of these mAbs in preventing SARS-CoV-2 infection. This study provides novel insights into the humoral immune response to SARS-CoV-2 and highlights the importance of patient-derived mAbs as therapeutic agents for COVID-19 treatment and prevention.

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current SARS-CoV-2 vaccines are based on spike (S) protein, S1 subunit, or receptor-binding domain (RBD) of prototype strain. Emergence of several novel SARS-CoV-2 variants has raised concern about potential immune escape. In this study, we performed an immunogenicity comparison of ancestral RBD, S1, and S ectodomain trimer (S-trimer) antigens and tested the efficacy of these prototype vaccines against the circulating variants, especially B.1.617 that has been linked to Indias current COVID-19 surge. We found that RBD and S-trimer proteins could induce significantly higher neutralizing antibody titers than S1 protein. For the three vaccines, the neutralizing titers decreased over time, but still remained high for at least five months after immunization. Importantly, the three prototype vaccines were still effective in neutralizing the variants of concern, although B.1.351 and B.1.617.1 lineages showed varying degrees of reduction in neutralization by the immune sera. The vaccines-induced sera were shown to block receptor binding and inhibit S protein-mediated membrane fusion. In addition, the immune sera did not promote antibody-dependent enhancement (ADE) in vitro. Our work provides valuable information for development of SARS-CoV-2 subunit vaccines and also supports the continued use of ancestral RBD or S-based vaccines to fight the COVID-19 epidemic.

The effectiveness of the prototypic SARS-CoV-2 vaccine largely decreased overtime against the emerging virus strains, necessitating the universal vaccine development. The most abundant structural membrane (M) protein is highly conserved in amino acid sequence, which arouses our research interests in developing a universal immunogen based on it. Serological analysis showed that IgG responses specific to its N-terminal peptides can be strongly detected in many serum samples from both convalescent patients and vaccinees receiving inactivated vaccines, indicating the potential existence of human B-cell epitopes in reactive peptides. Microneutralization assays showed that the N-terminal peptide S2M2-30-specific hyperimmune serum was capable of cross-neutralizing the authentic viruses including wild-type HKU-001a, B.1.617.2/Delta, and Omicron subvariant BQ.1.1, and synergized with RBD-specific serum in reinforcing antiviral activities. Strong S2M2-30-specific immunities elicited in hACE2-transgenic mice could effectively inhibit B.1.1.7/Alpha (UK) infections. Our results suggest the potentiality of conserved M peptides as vaccine targets for conferring cross-protections against sarbecoviruses.

Inflammatory bowel disease (IBD) refers to a group of multifactorial and chronic inflammation affecting intestinal tract. Based on the pathogenic mechanisms, IBD mainly comprised of two categories: ulcerative colitis (UC) and Crohns disease (CD). In this study, we established a dextran sulfate sodium (DSS)-induced colitis model to mimic UC. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) as therapeutic vectors have shown regenerating effect of damaged tissues. Here, intravenous administration of MSC-EVs ameliorated IBD symptoms including gaining weight, reducing disease activity index and restoring colon length. In addition, the protective effect of MSC-EVs were demonstrated by repairing colon mucosa and reducing the infiltration of macrophages in submucosa. Collectively, MSC-EV shows a therapeutic potential for IBD.

Seroconversion appeared early after COVID-19 onset, and convalescent sera therapy benefit some critical patients. However, neutralizing antibody (nAb) in convalescents is largely unknown. We found that 97.01% (65/67) of COVID-19 convalescents maintained IgG antibodies with high binding and avidity to SARS-CoV-2 spike subunits S1 and S2, and 95.52% (64/67) had neutralization activity against SARS-CoV-2 pesudovirus, one month after discharge (median ID50, 2.75; IQR, 2.34-3.08). Some sera exhibited cross-neutralization against SARS-CoV (76.12%), MERS-CoV (17.91%), or both (10.45%). Interestingly, individuals recovered from severe disease (severe group) had nAbs with binding and neutralization titers higher than non-severe group. Severe group appeared a rapid increase of lymphocytes and a high proportion of circulating CXCR3+ Tfh cells. Interestingly, the later were spike-specific and positively correlated with SARS-CoV-2 nAb titers. All subjects had no autoimmunity. Our findings provide novel insights into nAb responses in COVID-19 convalescents and facilitate treatment and vaccine development for SARS-CoV-2 infection.

BackgroundThe longevity of the immune response against SARS-CoV-2 is currently debated. We thus profiled the serum anti-SARS-CoV-2 antibody levels and virus specific memory B- and T-cell responses over time in convalescent COVID-19 patients. MethodsA cohort of COVID-19 patients from the Lombardy region in Italy who experienced mild to critical disease and Swedish volunteers with mild symptoms, were tested for the presence of elevated anti-spike and anti-receptor binding domain antibody levels over a period of eight months. In addition, specific memory B- and T-cell responses were tested in selected patient samples. ResultsAnti-SARS-CoV-2 antibodies were present in 85% samples collected within 4 weeks after onset of symptoms in COVID-19 patients. Levels of specific IgM or IgA antibodies declined after 1 month while levels of specific IgG antibodies remained stable up to 6 months after diagnosis. Anti-SARS-CoV-2 IgG antibodies were still present, though at a significantly lower level, in 80% samples collected at 6-8 months after symptom onset. SARS-CoV-2-specific memory B- and T-cell responses were developed in vast majority of the patients tested, regardless of disease severity, and remained detectable up to 6-8 months after infection. ConclusionsAlthough the serum levels of anti-SARS-CoV-2 IgG antibodies started to decline, virus-specific T and/or memory B cell responses increased with time and maintained during the study period (6-8 months after infection). FundingEuropean Unions Horizon 2020 research and innovation programme (ATAC), the Italian Ministry of Health, CIMED, the Swedish Research Council and the China Scholarship Council.

Knowledge of the host immune response after natural SARS-CoV-2 infection is essential for informing directions of vaccination and epidemiological control strategies against COVID-19. In this study, thirty-four COVID-19 patients were enrolled with 244 serial blood specimens (38.1% after hospital discharge) collected to explore the chronological evolution of neutralizing (NAb), total (TAb), IgM, IgG and IgA antibody in parallel. IgG titers reached a peak later (approximately 35 days postonset) than those of Nab, Ab, IgM and IgA (20[~]25 days postonset). After peaking, IgM levels declined with an estimated average half-life of 10.36 days, which was more rapid than those of IgA (51.25 days) and IgG (177.39 days). Based on these half-life data, we estimate that the median times for IgM, IgA and IgG to become seronegative are 4.59 (IQR 4.12-5.03), 7.78 (IQR 6.71-9.16) and 42.72 (IQR 33.75-47.96) months post disease onset. The relative contribution of IgM to NAb was higher than that of IgG (standardized {beta} regression coefficient: 0.53 vs 0.48), so the rapid decline in NAb may be attributed to the rapid decay of IgM in acute phase. However, the relative contribution of IgG to NAb increased and that of IgM further decreased after 6 weeks postonset. Its assumed that the decline rate of NAb might slow down to the same level as that of IgG over time. This study suggests that SARS-CoV-2 infection induces robust neutralizing and binding antibody responses in patients and that humoral immunity against SARS-CoV-2 acquired by infection may persist for a relatively long time.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to infect people globally. The increased COVID-19 cases and no licensed vaccines highlight the need to develop safe and effective vaccines against SARS-CoV-2 infection. Multiple vaccines candidates are under pre-clinical or clinical trails with different strengths and weaknesses. Here we developed a pilot scale production of a recombinant subunit vaccine (RBD-Fc Vacc) with the Receptor Binding Domain of SARS-CoV-2 S protein fused with the Fc domain of human IgG1. RBD-Fc Vacc induced SARS-CoV-2 specific neutralizing antibodies in non-human primates and human ACE2 transgenic mice. The antibodies induced in macaca fascicularis neutralized three divergent SARS-CoV2 strains, suggesting a broader neutralizing ability. Three times immunizations protected Macaca fascicularis (20ug or 40ug per dose) and mice (10ug or 20ug per dose) from SARS-CoV-2 infection respectively. These data support clinical development of SARS-CoV-2 vaccines for humans. RBD-Fc Vacc is currently being assessed in randomized controlled phase 1/II human clinical trails. SummaryThis study confirms protective efficacy of a SARS-CoV-2 RBD-Fc subunit vaccine.

Coronavirus disease 2019 (COVID-19) expresses a wide spectrum of disease severity. We investigated the profile of IgG and IgG subclass antibody responses to SARS- CoV-2 in Tunisian patients with COVID-19 according to disease severity (86 patients with severe disease and 63 with mild to moderate disease). Two in house developed ELISA with excellent performance were used to test for antibodies to the nucleocapsid (N) protein and the receptor-binding domain of the spike antigen (S-RBD) of SARS-CoV-2. IgG, IgG1 and IgG3 antibodies were significantly higher in patients with severe disease compared to non-severe disease. Antibodies to S-RBD or the N protein were dominated by IgG1 and IgG3 or IgG1/IgG3 and IgG2 subclasses respectively. In patients with severe disease, IgG antibodies appearance to S-RBD was delayed compared to the N protein. IgG subclass imbalance may reflect the pathophysiology of COVID-19 and may herald disease aggravation. This study brings information on the immune responses to SARS-CoV-2 in North African patients and completes the picture drawn on COVID-19 in different African populations and worldwide.

COVID-19 is caused by a newly identified coronavirus, SARS-CoV-2, and has become a pandemic around the world. The illustration of the immune responses against SARS-CoV-2 is urgently needed for understanding the pathogenesis of the disease and its vaccine development. CD8+ T cells are critical for virus clearance and induce long lasting protection in the host. Here we identified specific HLA-A2 restricted T cell epitopes in the spike protein of SARS-CoV-2. Seven epitope peptides (n-Sp1, 2, 6, 7, 11, 13, 14) were confirmed to bind with HLA-A2 and potentially be presented by antigen presenting cells to induce host immune responses. Tetramers containing these peptides could interact with specific CD8+ T cells from convalescent COVID-19 patients, and one dominant epitope (n-Sp1) was defined. In addition, these epitopes could activate and generate epitope-specific T cells in vitro, and those activated T cells showed cytotoxicity to target cells. Meanwhile, all these epitopes exhibited high frequency of variations. Among them, n-Sp1 epitope variation 5L>F significantly decreased the proportion of specific T cell activation; n-Sp1 epitope 8L>V variant showed significantly reduced binding to HLA-A2 and decreased the proportion of n-Sp1-specific CD8+ T cell, which potentially contributes to the immune escape of SAR-CoV-2.

BackgroundCombined antibiotic regimens have caused problems such as increasing antimicrobial resistance to H. pylori and intestinal flora disturbance. Vaccination is a great alternative approach, but also faces the limited immune response induced by monovalent vaccines. Therefore, the development of multi-epitope vaccines is promising immunotherapy to control H. pylori infection. ObjectiveTo develop a multi-epitope vaccine and evaluate its therapeutic efficacy against H. pylori infection. Materials and MethodsThe B and T cell epitopes from UreB, FlaA, AlpB, SabA, and HpaA were linked for producing 2 multi-epitope vaccines (CTB-S3 and CTB-S5) by a structural evaluation based on computer-aided design. The abilities to produce antigen-specific antibodies and neutralizing antibodies of CTB-S3 and CTB-S5 were evaluated in BALB/c mice. After that, their therapeutic efficacy was explored in H. pylori-infected mice. ResultsCTB-S3 or CTB-S5 could induce high levels of specific antibodies against UreB, FlaA, AlpB, SabA, HpaA, and neutralizing antibodies against H. pylori urease and adhesion. Also, oral therapeutic immunization with CTB-S3 or CTB-S5 could decrease H. pylori colonization and reduce stomach damage; the protection was correlated with H. pylori-specific IgG, SIgA antibodies, and CD4+ T cell immune response. ConclusionsOur study developed a multi-epitope vaccine based on a computer-aided design. The CTB-S3 and CTB-S5 vaccines may be promising therapeutic candidate vaccines against H. pylori infection and provide a reference for vaccine design of other pathogens.

Since Helicobacter pylori (H. pylori) resistance to antibiotic regimens is increased, vaccination is becoming an increasingly important alternative therapy to control H. pylori infection. UreB, FlaA, AlpB, SabA, and HpaA proteins of H. pylori were previously proved to be used as candidate vaccine antigens. Here, we developed an engineered antigen based on a recombinant chimeric protein containing a structural scaffold from UreB and B cell epitopes from FlaA, AlpB, SabA, and HpaA. The multi-epitope chimeric antigen, named MECU, could generate a broadly reactive antibody response including antigen-specific antibodies and neutralizing antibodies against H. pylori urease and adhesins. Moreover, therapeutic immunization with MECU could reduce H. pylori colonization in the stomach and protect the stomach in BALB/c mice. This study not only provides a promising immunotherapy to control H. pylori infection, but also offers a reference for antigen engineering against other pathogens.

Human genetic studies have identified defects in multiple mechanisms that predispose the risk of developing inflammatory bowel diseases (IBD), which include alterations in adaptive and innate immune responses, epithelial integrity and regulation of the intestinal mucus layer. Despite the importance of intestinal barrier integrity in the pathogenesis of IBD, essentially all current therapies modulate the immune responses. In this study, we determined the high resolution cryo-EM structure of human NXPE1, a IBD associated protein. Based on the structural homology, we identified NXPE1 as an O-acetyltransferase. Since NXPE1 is a pseudo gene in mouse, we generated knockout mouse model that lacked two of the mouse NXPE1 homologs, Nxpe2 and Nxpe4. The O-acetylation of sialic acid on red blood cells was abolished in the double knockout mice, confirming the sialic acid O-acetyltransferase function of NXPE1 family members. These findings underscore the potential of NXPE1 as a novel therapeutic target of the intestinal barrier functions for the treatment of IBD.

Cross-reactive antibodies against dengue virus are known to cause antibody-dependent enhancement (ADE) of infection or disease severity under specific conditions. In our previous study, we showed that primary immunization with the COVID-19 vaccine induces induces cross-reactive IgG causing ADE against dengue. In the present study, we investigated the influence of IgG Fc-glycosylation (analyzed by LC-MS/MS) on ADE mediated by cross-reactive IgG against dengue from IgG against SARS-CoV-2. We found a clear correlation between anti-DENV2 E IgG2 galactosylation and the ADE capacity of cross-reactive IgG against dengue in individuals vaccinated against COVID-19. IgG2 sialylation increased over time; however, it was not correlated with ADE capacity. This phenomenon was restricted to IgG2, whereas anti-DENV2 E IgG1 Fc-glycosylation remained stable after COVID-19 vaccination.

The ongoing coronavirus disease 2019 (COVID-19) pandemic is a serious threat to global public health, and imposes severe burdens on the entire human society. The severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) can cause severe respiratory illness and death. Currently, there are no specific antiviral drugs that can treat COVID-19. Several vaccines against SARS-CoV-2 are being actively developed by research groups around the world. The surface S (spike) protein and the highly expressed internal N (nucleocapsid) protein of SARS-CoV-2 are widely considered as promising candidates for vaccines. In order to guide the design of an effective vaccine, we need experimental data on these potential epitope candidates. In this study, we mapped the immunodominant (ID) sites of S protein using sera samples collected from recently discharged COVID-19 patients. The SARS-CoV-2 S protein-specific antibody levels in the sera of recovered COVID-19 patients were strongly correlated with the neutralising antibody titres. We used epitope mapping to determine the landscape of ID sites of S protein, which identified nine linearized B cell ID sites. Four out of the nine ID sites were found in the receptor-binding domain (RBD). Further analysis showed that these ID sites are potential high-affinity SARS-CoV-2 antibody binding sites. Peptides containing two out of the nine sites were tested as vaccine candidates against SARS-CoV-2 in a mouse model. We detected epitope-specific antibodies and SARS-CoV-2-neutralising activity in the immunised mice. This study for the first time provides human serological data for the design of vaccines against COVID-19.

It is generally believed that the main source of antibodies is B lymphocytes. In this study, our results first revealed that B cell-deficient mice could not produce antibodies specific for TI-Ags; however, mice with B cell deficiency could produce TD-Ag-specific antibodies, although antibody production was delayed compared with that in BALB/c mice after primary TD-Ag challenge. Subsequently, we identified that mouse lung epithelial cells could produce and secrete Ig, including IgM, IgA or IgG, which could display TD-Ag-specific antibody activity. Notably, the production of TD-Ag-specific antibodies by lung epithelial cells was found to be dependent on CD4+ T cells but not CD8+ T cells. Our findings indicate for the first time that B cells are not the only source of TD-Ag-specific antibodies but are essential for rapid TD-Ag-specific antibody production by non-B cells. This discovery may reveal a new mechanism for the production of specific antibodies.

The currently dominant variant of SARS-CoV-2 Omicron, carrying a great number of mutations, has been verified its strong capacity of immune escape in COVID-19 convalescents and vaccinated individuals. An increased risk of SARS-CoV-2 reinfection or breakthrough infection should be concerned. Here we reported higher humoral immune response elicited by Delta and Omicron variants after breaking through previous infection and cross-neutralization against VOCs, compared to the ancestral wild-type (WT) virus infection. To overcome the immune escape of Omicron, Omicron-specific vaccine was considered as a novel and potential strategy. Mouse models were used to verify whether Omicron-specific RBD subunit boost immune response by immunizing Omicron-RBD recombinant proteins. Three doses of Omicron-RBD immunization elicit comparable neutralizing antibody (NAb) titers with three doses of WT-RBD immunization, but the neutralizing activity was not cross-active. By contrast, two doses of WT-RBD with an Omicron-RBD booster increased the NAb geometric mean titers against Omicron by 9 folds. Moreover, an additional boost vaccination with Omicron-RBD protein could increase humoral immune response against both WT and current VOCs. These results suggest that the Omicron-specific subunit booster shows its advantages in the immune protection from both WT and current VOCs, and that SARS-CoV-2 vaccines administration using two or more virus lineages as antigens might improve the NAb response.

While lymphocytopenia is a common characteristic of patients infected by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the mechanisms responsible for this depletion are unclear. Through careful inspection of the spleens and lymph nodes (LNs) from six cases with postmortem examinations, we observed that SARS-CoV-2 could directly infect secondary lymphoid organs to induce cell death. Immunohistochemistry demonstrated ACE2 (angiotensin-converting enzyme 2), the potential receptor of SARS-CoV-2, expresses on tissue-resident CD169+ macrophages in spleens and LNs. Immunofluorescent staining confirmed that viral nucleocaspid protein (NP) can be found in ACE2+ cells, CD169+ macrophages, but not in CD3+ T cells or B220+ B cells in spleens and LNs. SARS-CoV-2 infection induces severe tissue damage including lymph follicle depletion, splenic nodule atrophy, histiocyte hyperplasia and lymphocyte reductions. Moreover, in situ TUNEL staining illustrated that viral infection leads to severe lymphocyte apoptosis, which might be mediated by viral antigens inducing Fas upregulation. Furthermore, SARS-CoV-2 also triggers macrophages to produce IL-6, a proinflammatory cytokine that directly promotes lymphocyte necrosis. Collectively, these results demonstrate that SARS-CoV-2 directly neutralizes human spleens and LNs through infecting tissue-resident CD169+ macrophages.

Omicron variant continues to spread all over the world. There are lots of scientific questions remaining to be answered for such a devastating variant. There are a dozen of vaccines already in clinical use. The very urgent scientific question would be whether or not these vaccines can protect Omicron variant. Here, we tested the sera from both convalescents and vaccine recipients receiving either inactivated or protein subunits vaccines (CoronaVac from Sinovac, or BBIBP-CoV from Sinopharm, or ZF2001 from Zhifei longcom) for the binding antibody titers (ELISA) and neutralization antibodies titers (pseudovirus neutralization assay). We showed that Omicron do have severe immune escape in convalescents, with 15 of 16 were negative in neutralization. By contrast, in vaccinees who received three jabs of inactivated or protein subunit vaccine, the neutralizing activity was much better preserved. Especially in the ZF2001 group with an extended period of the second and third jab (4-6 months) remains 100% positive in Omicron neutralization, with only 3.1-folds reduction in neutralizing antibody (NAb) titer. In this case, we proposed that, the multi-boost strategy with an extended interval between the second and third jab for immune maturation would be beneficial for NAb against devastating variants such as Omicron.