Development of Detection Antibody Targeting the Linear Epitope in SARS-CoV-2 Nucleocapsid Protein with Ultra-High Sensitivity.

The COVID-19 pandemic caused by SARS-CoV-2 highlighted the importance of reliable detection methods for disease control and surveillance. Optimizing detection antibodies by rational screening antigens would improve the sensitivity and specificity of antibody-based detection methods such as colloidal gold immunochromatography. In this study, we screened three peptide antigens with conserved sequences in the N protein of SARS-CoV-2 using bioinformatical and structural biological analyses. Antibodies that specifically recognize these peptides were prepared. The epitope of the peptide that had the highest binding affinity with its antibody was located on the surface of the N protein, which was favorable for antibody binding. Using the optimal antibody that can recognize this epitope, we developed colloidal gold immunochromatography, which can detect the N protein at 10 pg/mL. Importantly, this antibody could effectively recognize both the natural peptide antigen and mutated peptide antigen in the N protein, showing the feasibility of being applied in the large-scale population testing of SARS-CoV-2. Our study provides a platform with reference significance for the rational screening of detection antibodies with high sensitivity, specificity, and reliability for SARS-CoV-2 and other pathogens.

Near-infrared responsive gold nanorods for highly sensitive colorimetric and photothermal lateral flow immuno-detection of SARS-CoV-2.

The highly infectious characteristics of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlight the necessity of sensitive and rapid nucleocapsid (N) protein-based antigen testing for early triage and epidemic management. In this study, a colorimetric and photothermal dual-mode lateral flow immunoassay (LFIA) platform for the rapid and sensitive detection of the SARS-CoV-2 N protein was developed based on gold nanorods (GNRs), which possessed tunable local surface plasma resonance (LSPR) absorption peaks from UV-visible to near-infrared (NIR). The LSPR peak was adjusted to match the NIR emission laser 808 nm by controlling the length-to-diameter ratio, which could maximize the photothermal conversion efficiency and achieve photothermal detection signal amplification. Qualitative detection of SARS-CoV-2 N protein was achieved by observing the strip color, and the limit of detection was 2 ng mL-1, while that for photothermal detection was 0.096 ng mL-1. Artificial saliva samples spiked with the N protein were analyzed with the recoveries ranging from 84.38% to 107.72%. The intra-assay and inter-assay coefficients of variation were 6.76% and 10.39%, respectively. We further evaluated the reliability of this platform by detecting 40 clinical samples collected from nasal swabs, and the results matched well with that of nucleic acid detection (87.5%). This method shows great promise in early disease diagnosis and screening.

Nucleocapsid and Spike Protein-Based Anti-SARS-CoV-2 Assay Performance in the Minority and Rural Coronavirus Insights Study: Characteristics of Socioeconomically Disadvantaged Populations with Health Disparities.

BACKGROUND: COVID-19 has had a devastating impact on Black, Hispanic, and other underserved, disadvantaged populations. Here anti-SARS-CoV-2 tests are characterized in disadvantaged patients to examine equivalence in US populations. METHODS: Underserved participant adults (age > 18 years) were enrolled before the availability of SARS-CoV-2 vaccines in Federal Qualified Health Centers in California, Florida, Louisiana, Illinois, and Ohio and contributed samples to the Minority and Rural Coronavirus Insights Study (MRCIS). A subset coined the MRCIS SARS-CoV-2 Antibody Cohort of 2365 participants was tested with the Roche Anti-SARS-CoV-2 assay (Cobas e601). Five hundred ninety-five of these were also tested with the Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 IgG assay (VITROS-5600); 1770 were also tested with the Abbott ARCHITECT SARS-CoV-2 IgG assay (ARCHITECT-2000). Assay-specific cutoffs classified negative/positive results. RESULTS: Eight point four percent (199/2365) of the MRCIS SARS-CoV-2 Antibody Cohort was SARS-CoV-2 RNA positive at enrollment. Agreement between the Ortho/Roche and the Abbott/Roche antibody testing did not vary by enrollment RNA status. The Ortho (anti-spike protein) vs Roche (anti-nucleocapsid protein) comparison agreed substantially: kappa = 0.63 (95% CI: 0.57-0.69); overall agreement, 83%. However, agreement was even better for the Abbott vs Roche assays (both anti-nucleocapsid protein tests): kappa = 0.85 (95% CI: 0.81-0.87); overall agreement, 95%. Anti-SARS-CoV-2 comparisons stratified by demographic criteria demonstrated no significant variability in agreement by sex, race/ethnicity, or age. CONCLUSIONS: Analytical agreement is 96.4% for anti-spike-protein vs anti-nucleocapsid-protein comparisons. Physiologically, seroreversion of anti-nucleocapsid reactivity after infection occurred in the disadvantaged population similarly to general populations. No anti-SARS-CoV-2 assays included demonstrated a clinically significant difference due to the demographics of the disadvantaged MRCIS SARS-CoV-2 Antibody Cohort.

A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test.

OBJECTIVES: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. METHODS: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. RESULTS: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). CONCLUSIONS: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.

Phosphoantigens glue butyrophilin 3A1 and 2A1 to activate Vγ9Vδ2 T cells.

In both cancer and infections, diseased cells are presented to human Vγ9Vδ2 T cells through an 'inside out' signalling process whereby structurally diverse phosphoantigen (pAg) molecules are sensed by the intracellular domain of butyrophilin BTN3A11-4. Here we show how-in both humans and alpaca-multiple pAgs function as 'molecular glues' to promote heteromeric association between the intracellular domains of BTN3A1 and the structurally similar butyrophilin BTN2A1. X-ray crystallography studies visualized that engagement of BTN3A1 with pAgs forms a composite interface for direct binding to BTN2A1, with various pAg molecules each positioned at the centre of the interface and gluing the butyrophilins with distinct affinities. Our structural insights guided mutagenesis experiments that led to disruption of the intracellular BTN3A1-BTN2A1 association, abolishing pAg-mediated Vγ9Vδ2 T cell activation. Analyses using structure-based molecular-dynamics simulations, 19F-NMR investigations, chimeric receptor engineering and direct measurement of intercellular binding force revealed how pAg-mediated BTN2A1 association drives BTN3A1 intracellular fluctuations outwards in a thermodynamically favourable manner, thereby enabling BTN3A1 to push off from the BTN2A1 ectodomain to initiate T cell receptor-mediated γδ T cell activation. Practically, we harnessed the molecular-glue model for immunotherapeutics design, demonstrating chemical principles for developing both small-molecule activators and inhibitors of human γδ T cell function.

Expression of a recombinant ASFV P30 protein and production of monoclonal antibodies.

Background: African Swine Fever (ASF) is an infectious disease that affects domestic pig and wild boar populations. The ASF Virus (ASFV) has a genome characterized by a very complex DNA (170-193 kb) that encodes for more than 200 different proteins. Among these, the highly immunogenic phosphoprotein p30 plays a fundamental role in the induction of specific antibodies. To date, the lack of a vaccine against the disease requires continuous studies to improve knowledge about the virus and the development of new tests in addition to virological ones. Aim: The aim of this work was to produce specific monoclonal antibodies (mAbs) against the p30 protein of ASFV, which could find useful applications in routine diagnostics and the implementation of new diagnostic tools. Methods: ASFV p30 encoding gene was amplified and used for the generation of the recombinant baculovirus by transfection of the Sf21 insect cells. The recombinant protein was analyzed by immunofluorescence assay, purified, and used for mice Balb-c immunization. The hybridomas obtained were cultured and screened, using an indirect Enzyme-linked Immunosorbent Assay (iELISA), in order to select clones that secrete the mAbs of interest. Results: The expression of recombinant p30 protein was assessed using direct immunofluorescence. The purified p30 protein fractions were analyzed by Coomassie gels staining confirming the presence of bands with a molecular weight of 30 kDa and used for the immunization of Balb-c mice. Six clones of pure hybridomas secreting the specific mAbs against recombinant p30 were obtained and tested in iELISA. The mAbs were also characterized by Western blot and immunofluorescence assay. The best results were obtained with the anti-p30 mAb 2B8E10 clone which showed high reactivity with both recombinant and viral p30 protein, respectively. Conclusion: In this work, a recombinant p30 protein produced in an insect cell system was purified and used to immunize Balb-c mice. Six anti-p30 mAbs-secreting hybridomas clone cells were obtained. These mAbs displayed high reactivity against the recombinant protein, but only 2B8E10 mAb showed excellent functionality against the p30 protein produced by ASFV. These results open the possibility to develop different diagnostic assays.

The Proportion of Long-term Response to Anti-N IgG Antibody after 12 Months for COVID-19 Subclinical Infections and a Longitudinal Survey for COVID-19 Subclinical Infections in 2021.

Objective To examine the continuation of antibody prevalence status after 12 months and background factors in antibody-positive subjects following asymptomatic infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods We initially determined the SARS-CoV-2 anti-nucleocapsid protein immunoglobulin G (anti-N IgG) antibody prevalence in 1,603 patients, doctors, and nurses at 65 medical institutions in Kanagawa Prefecture, Japan. We then obtained consent from 33 of the 39 subjects who tested positive and performed follow-up for 12 months. Results Follow-up for up to 12 months showed that a long-term response of the anti-N IgG antibody could be detected in 6 of the 33 participants (18.2%). The proportions with hypertension, using an angiotensin-receptor blocker, and without a drinking habit were higher among the participants with a long-term anti-N IgG antibody response for up to 12 months than among those without a long-term antibody response. Conclusions The proportion of individuals with subclinical COVID-19 who continuously had a positive result for the anti-N IgG antibody at 12 months was low.

Longitudinal SARS-CoV-2 Nucleocapsid Antibody Kinetics, Seroreversion, and Implications for Seroepidemiologic Studies.

Given widespread use of spike antibody in generating coronavirus disease vaccines, SARS-CoV-2 nucleocapsid antibodies are increasingly used to indicate previous infection in serologic surveys. However, longitudinal kinetics and seroreversion are poorly defined. We found substantial seroreversion of nucleocapsid total immunoglobulin, underscoring the need to account for seroreversion in seroepidemiologic studies.

Ena/VASP Protein-Mediated Actin Polymerization Contributes to Naïve CD8+ T Cell Activation and Expansion by Promoting T Cell-APC Interactions In Vivo.

Naïve T cell activation in secondary lymphoid organs such as lymph nodes (LNs) occurs upon recognition of cognate antigen presented by antigen presenting cells (APCs). T cell activation requires cytoskeleton rearrangement and sustained interactions with APCs. Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are a family of cytoskeletal effector proteins responsible for actin polymerization and are frequently found at the leading edge of motile cells. Ena/VASP proteins have been implicated in motility and adhesion in various cell types, but their role in primary T cell interstitial motility and activation has not been explored. Our goal was to determine the contribution of Ena/VASP proteins to T cell-APC interactions, T cell activation, and T cell expansion in vivo. Our results showed that naïve T cells from Ena/VASP-deficient mice have a significant reduction in antigen-specific T cell accumulation following Listeria monocytogenes infection. The kinetics of T cell expansion impairment were further confirmed in Ena/VASP-deficient T cells stimulated via dendritic cell immunization. To investigate the cause of this T cell expansion defect, we analyzed T cell-APC interactions in vivo by two-photon microscopy and observed fewer Ena/VASP-deficient naïve T cells interacting with APCs in LNs during priming. We also determined that Ena/VASP-deficient T cells formed conjugates with significantly less actin polymerization at the T cell-APC synapse, and that these conjugates were less stable than their WT counterparts. Finally, we found that Ena/VASP-deficient T cells have less LFA-1 polarized to the T cell-APC synapse. Thus, we conclude that Ena/VASP proteins contribute to T cell actin remodeling during T cell-APC interactions, which promotes the initiation of stable T cell conjugates during APC scanning. Therefore, Ena/VASP proteins are required for efficient activation and expansion of T cells in vivo.

On the caveats of a multiplex test for SARS-CoV-2 to detect seroconversion after infection or vaccination.

The Covid-19 pandemic, caused by SARS-CoV-2, has resulted in over 6 million reported deaths worldwide being one of the biggest challenges the world faces today. Here we present optimizations of all steps of an enzyme-linked immunosorbent assay (ELISA)-based test to detect IgG, IgA and IgM against the trimeric spike (S) protein, receptor binding domain (RBD), and N terminal domain of the nucleocapsid (N-NTD) protein of SARS-CoV-2. We discuss how to determine specific thresholds for antibody positivity and its limitations according to the antigen used. We applied the assay to a cohort of 126 individuals from Rio de Janeiro, Brazil, consisting of 23 PCR-positive individuals and 103 individuals without a confirmed diagnosis for SARS-CoV-2 infection. To illustrate the differences in serological responses to vaccinal immunization, we applied the test in 18 individuals from our cohort before and after receiving ChAdOx-1 nCoV-19 or CoronaVac vaccines. Taken together, our results show that the test can be customized at different stages depending on its application, enabling the user to analyze different cohorts, saving time, reagents, or samples. It is also a valuable tool for elucidating the immunological consequences of new viral strains and monitoring vaccination coverage and duration of response to different immunization regimens.

Detection of SARS-CoV-2 Nucleocapsid, Spike, and Neutralizing Antibodies in Vaccinated Japanese.

Serological detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N), spike (S), and neutralizing antibodies (Abs) is commonly undertaken to evaluate the efficacy of vaccination. However, the relative efficiency of different SARS-CoV-2 Ab detection systems has not been extensively investigated. Here, we evaluated serological test systems in vaccinated Japanese. SARS-CoV-2 N, S, and neutralizing Abs in sera of 375 healthy subjects a mean 253 days after vaccination were assessed. The sensitivity of Elecsys Anti-SARS-CoV-2 S (Roche S) and Anti-SARS-CoV-2 S IgG (Fujirebio S) was 100% and 98.9%, respectively, with a specificity of 100% for both. The sensitivity of Anti-SARS-CoV-2 neutralizing Ab (MBL Neu) was 2.7%, and the specificity was 100%. Fujirebio S correlated with Roche S (rho = 0.9182, p = 3.97 × 10-152). Fujirebio S (rho = 0.1295, p = 0.0121) and Roche S (rho = 0.1232, p = 0.0170) correlated weakly with MBL Neu. However, Roche S did correlate with MBL Neu in patients with COVID-19 (rho = 0.8299, p = 1.01 × 10-12) and in healthy subjects more recently after vaccination (mean of 90 days, rho = 0.5306, p = 0.0003). Thus, the Fujirebio S and Roche S results were very similar, but neither correlated with neutralizing antibody titers by MBL Neu at a later time after vaccination.

Anti-nucleocapsid antibodies enhance the production of IL-6 induced by SARS-CoV-2 N protein.

A cytokine storm induces acute respiratory distress syndrome, the main cause of death in coronavirus disease 2019 (COVID-19) patients. However, the detailed mechanisms of cytokine induction due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unclear. To examine the cytokine production in COVID-19, we mimicked the disease in SARS-CoV-2-infected alveoli by adding the lysate of SARS-CoV-2-infected cells to cultured macrophages or induced pluripotent stem cell-derived myeloid cells. The cells secreted interleukin (IL)-6 after the addition of SARS-CoV-2-infected cell lysate. Screening of 25 SARS-CoV-2 protein-expressing plasmids revealed that the N protein-coding plasmid alone induced IL-6 production. The addition of anti-N antibody further enhanced IL-6 production, but the F(ab')2 fragment did not. Sera from COVID-19 patients also enhanced IL-6 production, and sera from patients with severer disease induced higher levels of IL-6. These results suggest that anti-N antibody promotes IL-6 production in SARS-CoV-2-infected alveoli, leading to the cytokine storm of COVID-19.

Neutralizing-antibody-independent SARS-CoV-2 control correlated with intranasal-vaccine-induced CD8+ T cell responses.

Effective vaccines are essential for the control of the coronavirus disease 2019 (COVID-19) pandemic. Currently developed vaccines inducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-antigen-specific neutralizing antibodies (NAbs) are effective, but the appearance of NAb-resistant S variant viruses is of great concern. A vaccine inducing S-independent or NAb-independent SARS-CoV-2 control may contribute to containment of these variants. Here, we investigate the efficacy of an intranasal vaccine expressing viral non-S antigens against intranasal SARS-CoV-2 challenge in cynomolgus macaques. Seven vaccinated macaques exhibit significantly reduced viral load in nasopharyngeal swabs on day 2 post-challenge compared with nine unvaccinated controls. The viral control in the absence of SARS-CoV-2-specific NAbs is significantly correlated with vaccine-induced, viral-antigen-specific CD8+ T cell responses. Our results indicate that CD8+ T cell induction by intranasal vaccination can result in NAb-independent control of SARS-CoV-2 infection, highlighting a potential of vaccine-induced CD8+ T cell responses to contribute to COVID-19 containment.

Antibodies against viral nucleo-, phospho-, and X protein contribute to serological diagnosis of fatal Borna disease virus 1 infections.

Borna disease virus 1 (BoDV-1) causes rare but often fatal encephalitis in humans. Late diagnosis prohibits an experimental therapeutic approach. Here, we report a recent case of fatal BoDV-1 infection diagnosed on day 12 after hospitalization by detection of BoDV-1 RNA in the cerebrospinal fluid. In a retrospective analysis, we detect BoDV-1 RNA 1 day after hospital admission when the cell count in the cerebrospinal fluid is still normal. We develop a new ELISA using recombinant BoDV-1 nucleoprotein, phosphoprotein, and accessory protein X to detect seroconversion on day 12. Antibody responses are also shown in seven previously confirmed cases. The individual BoDV-1 antibody profiles show variability, but the usage of three different BoDV-1 antigens results in a more sensitive diagnostic tool. Our findings demonstrate that early detection of BoDV-1 RNA in cerebrospinal fluid and the presence of antibodies against at least two different viral antigens contribute to BoDV-1 diagnosis. Physicians in endemic regions should consider BoDV-1 infection in cases of unclear encephalopathy and initiate appropriate diagnostics at an early stage.

Recognition and inhibition of SARS-CoV-2 by humoral innate immunity pattern recognition molecules.

The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in coronavirus disease 2019 (COVID-19). The present study was designed to conduct a systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRMs) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of 12 PRMs tested, the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid and spike proteins, respectively. MBL bound trimeric spike protein, including that of variants of concern (VoC), in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. Moreover, after binding to spike protein, MBL activated the lectin pathway of complement activation. Based on retention of glycosylation sites and modeling, MBL was predicted to recognize the Omicron VoC. Genetic polymorphisms at the MBL2 locus were associated with disease severity. These results suggest that selected humoral fluid-phase PRMs can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.

Emerging Role of Platelet-Endothelium Interactions in the Pathogenesis of Severe SARS-CoV-2 Infection-Associated Myocardial Injury.

Cardiovascular dysfunction and disease are common and frequently fatal complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Indeed, from early on during the SARS-CoV-2 virus pandemic it was recognized that cardiac complications may occur, even in patients with no underlying cardiac disorders, as part of the acute infection, and that these were associated with more severe disease and increased morbidity and mortality. The most common cardiac complication is acute cardiac injury, defined by significant elevation of cardiac troponins. The potential mechanisms of cardiovascular complications include direct viral myocardial injury, systemic inflammation induced by the virus, sepsis, arrhythmia, myocardial oxygen supply-demand mismatch, electrolyte abnormalities, and hypercoagulability. This review is focused on the prevalence, risk factors and clinical course of COVID-19-related myocardial injury, as well as on current data with regard to disease pathogenesis, specifically the interaction of platelets with the vascular endothelium. The latter section includes consideration of the role of SARS-CoV-2 proteins in triggering development of a generalized endotheliitis that, in turn, drives intense activation of platelets. Most prominently, SARS-CoV-2-induced endotheliitis involves interaction of the viral spike protein with endothelial angiotensin-converting enzyme 2 (ACE2) together with alternative mechanisms that involve the nucleocapsid and viroporin. In addition, the mechanisms by which activated platelets intensify endothelial activation and dysfunction, seemingly driven by release of the platelet-derived calcium-binding proteins, SA100A8 and SA100A9, are described. These events create a SARS-CoV-2-driven cycle of intravascular inflammation and coagulation, which contributes significantly to a poor clinical outcome in patients with severe disease.

Robust and Functional Immune Memory Up to 9 Months After SARS-CoV-2 Infection: A Southeast Asian Longitudinal Cohort.

The duration of humoral and cellular immune memory following SARS-CoV-2 infection in populations in least developed countries remains understudied but is key to overcome the current SARS-CoV-2 pandemic. Sixty-four Cambodian individuals with laboratory-confirmed infection with asymptomatic or mild/moderate clinical presentation were evaluated for Spike (S)-binding and neutralizing antibodies and antibody effector functions during acute phase of infection and at 6-9 months follow-up. Antigen-specific B cells, CD4+ and CD8+ T cells were characterized, and T cells were interrogated for functionality at late convalescence. Anti-S antibody titers decreased over time, but effector functions mediated by S-specific antibodies remained stable. S- and nucleocapsid (N)-specific B cells could be detected in late convalescence in the activated memory B cell compartment and are mostly IgG+. CD4+ and CD8+ T cell immune memory was maintained to S and membrane (M) protein. Asymptomatic infection resulted in decreased antibody-dependent cellular cytotoxicity (ADCC) and frequency of SARS-CoV-2-specific CD4+ T cells at late convalescence. Whereas anti-S antibodies correlated with S-specific B cells, there was no correlation between T cell response and humoral immune memory. Hence, all aspects of a protective immune response are maintained up to nine months after SARS-CoV-2 infection and in the absence of re-infection.

Immune Response in Moderate to Critical Breakthrough COVID-19 Infection After mRNA Vaccination.

SARS-CoV-2 variants of concern (VOCs) can trigger severe endemic waves and vaccine breakthrough infections (VBI). We analyzed the cellular and humoral immune response in 8 patients infected with the alpha variant, resulting in moderate to fatal COVID-19 disease manifestation, after double mRNA-based anti-SARS-CoV-2 vaccination. In contrast to the uninfected vaccinated control cohort, the diseased individuals had no detectable high-avidity spike (S)-reactive CD4+ and CD8+ T cells against the alpha variant and wild type (WT) at disease onset, whereas a robust CD4+ T-cell response against the N- and M-proteins was generated. Furthermore, a delayed alpha S-reactive high-avidity CD4+ T-cell response was mounted during disease progression. Compared to the vaccinated control donors, these patients also had lower neutralizing antibody titers against the alpha variant at disease onset. The delayed development of alpha S-specific cellular and humoral immunity upon VBI indicates reduced immunogenicity against the S-protein of the alpha VOC, while there was a higher and earlier N- and M-reactive T-cell response. Our findings do not undermine the current vaccination strategies but underline a potential need for the inclusion of VBI patients in alternative vaccination strategies and additional antigenic targets in next-generation SARS-CoV-2 vaccines.

Stratification of hospitalized COVID-19 patients into clinical severity progression groups by immuno-phenotyping and machine learning.

Quantitative or qualitative differences in immunity may drive clinical severity in COVID-19. Although longitudinal studies to record the course of immunological changes are ample, they do not necessarily predict clinical progression at the time of hospital admission. Here we show, by a machine learning approach using serum pro-inflammatory, anti-inflammatory and anti-viral cytokine and anti-SARS-CoV-2 antibody measurements as input data, that COVID-19 patients cluster into three distinct immune phenotype groups. These immune-types, determined by unsupervised hierarchical clustering that is agnostic to severity, predict clinical course. The identified immune-types do not associate with disease duration at hospital admittance, but rather reflect variations in the nature and kinetics of individual patient's immune response. Thus, our work provides an immune-type based scheme to stratify COVID-19 patients at hospital admittance into high and low risk clinical categories with distinct cytokine and antibody profiles that may guide personalized therapy.