Pediatric SARS-CoV-2 infection can give rise to a range of clinical presentations, from asymptomatic or mild cases to severe pulmonary COVID-19, and to multisystem inflammatory syndrome in children (MIS-C). The latter is characterized by hyperinflammation and involvement of multiple organs. Although various aspects of antibody responses to pediatric SARS-CoV-2 infection have been reported, there has been limited research on the parallel antibody responses to both viral and self-antigens. We examined whether clinical phenotypes were linked to particular antiviral antibody and autoantibody profiles. By using custom arrays, we discovered that all manifestations of SARS-CoV-2 infection were linked to increased autoantibody production when compared to uninfected subjects, suggesting that pediatric SARS-CoV-2 infection may predispose to immune dysregulation. We observed subtle differences in autoantibody patterns among infection groups, with some autoantibodies being more associated with mild symptoms and others linked to severe disease manifestations. In particular, subsets of subjects with MIS-C and/or severe COVID-19 exhibited elevated autoreactive antibody responses against thyroperoxidase, IL-13, and IFN-epsilon, although differences across clinical groups did not reach statistical significance. When we compared subjects with MIS-C to those with severe COVID-19, we noted differences in the abundance of IgG (primarily IgG1), but no differences in Fc-mediated effector functions. Our study shows that the antibody repertoire in children varies with the clinical presentation of SARS-CoV-2. Moreover, MIS-C may be linked to abnormal antibody function, indicating that this syndrome-and potentially other post-acute sequelae of SARS-CoV-2 infection-could be related to antibody dysfunction.

Pediatric SARS-CoV-2 infection results in clinical presentations ranging from asymptomatic/mild infection to severe pulmonary COVID-19, to Multisystem Inflammatory Syndrome in Children (MIS-C), characterized by hyperinflammation and multi-organ involvement. While various aspects of antibody responses to pediatric SARS-CoV-2 infection manifestations have been reported, parallel studies of antibody responses to viral and self-antigens are understudied. We tested whether clinical presentations of increasing severity corresponded to different antiviral antibody and autoantibody signatures. Using custom arrays, we found that, relative to uninfected subjects, all SARS-CoV-2 infection manifestations were associated with increased autoantibody production, suggesting pediatric SARS-CoV-2 infection as a risk factor for autoimmune complications. Subtle differences were seen in autoantibody patterns among infection groups, with some autoantibodies more associated with mild manifestations and others with severe ones. When we compared MIS-C and severe COVID-19 subjects, we found differences in IgG (mostly IgG1) abundance but not in Fc-mediated effector functions. Thus, MIS-C may be associated with abnormal antibody function, suggesting that this syndrome, and perhaps other post-acute sequelae of SARS-CoV-2 infection, may be associated with antibody dysfunction. Our study shows that the antibody repertoire varies with clinical presentation of SARS-CoV-2 in children and its analysis may help understand long COVID pathogenesis.

OBJECTIVES:To achieve global TB control, more sensitive and user-friendly diagnostic tools for tuberculosis infection (TBI) are necessary, as it is a potential transmission reservoir. VIDASⓇ TB-IGRA (bioMérieux) is a fully automated assay recently developed. We report here the results of a global, multicenter, cross-sectional, prospective study to evaluate the diagnostic accuracy of the assay. METHODS:Patients with TB disease (n=200) or participants at varying levels of TB exposure risk (n=1460; mixed TB-exposure risk population) were tested with both the VIDASⓇ TB-IGRA and the QuantiFERONⓇ-TB Gold Plus (QFTⓇ-Plus, QIAGEN). RESULTS:In culture-confirmed TB cases, VIDASⓇ TB-IGRA had a sensitivity significantly higher than QFTⓇ-Plus (97.5% vs 80.7%, P<0.0001). Specificity evaluated in blood donors from a low-prevalence country (n=125) was high for both VIDASⓇ TB-IGRA and QFTⓇ-Plus (97.6% [93.1-99.5] vs 95.2% [89.8-98.2]; P=0.083), respectively. In the whole mixed TB-exposure risk population, negative (NPA) and positive percent agreement (PPA) were 90.1% (1097/1217) and 92.1% (223/242), respectively. However, regression analyses revealed that VIDASⓇ TB-IGRA correlated better with the TB-exposure risk gradient than QFTⓇ‑Plus. CONCLUSIONS:Compared with QFTⓇ-Plus, VIDASⓇ TB-IGRA was significantly more sensitive without a reduction in specificity, and it correlated better with an exposure gradient, suggesting that it is a valuable tool for TBI diagnosis.

ABSTRACT This preliminary study compares VIDAS TB-IGRA (bioMérieux, Marcy-l'Etoile, France) with the established QuantiFERON-TB Gold Plus (QFT-Plus) (Qiagen, Hilden, Germany) to evaluate diagnostic performance for the diagnosis of individuals infected with Mycobacterium tuberculosis complex (latent infection and disease). The study was multi-center and performed between 2 October 2019 and 4 February 2020. Participants were divided into tuberculosis (TB) disease, high-risk, and low-risk populations. The confirmed TB disease population included 104 patients. The high-risk population included 162 individuals with flagged risk factors on a questionnaire but without objective clinical confirmation of TB. The low-risk population included 117 healthy blood donors from the French National Blood Bank. Positive and negative percent agreement (PPA and NPA) were determined between the VIDAS TB-IGRA and QFT-Plus. In the TB disease population, sensitivity was measured against bacterial culture and PCR. The VIDAS TB-IGRA produced fewer indeterminate results than the QFT-Plus (1/104 vs 23/104) in the TB disease population and exhibited a sensitivity of 95.0% against bacterial culture. Furthermore, a 98.2% PPA was obtained in comparison to QFT-Plus. In the low-risk population, the VIDAS TB-IGRA demonstrated high specificity (94.9%) and a strong NPA (98.2%) compared to QFT-Plus. In the high-risk population, the VIDAS TB-IGRA exhibited a strong PPA (94.4%) with the QFT-Plus. A lower NPA was observed (85.2%) compared to QFT-Plus, which may be due to a higher sensitivity demonstrated in the TB disease population. The fully automated VIDAS TB-IGRA is a promising aid in the diagnosis of individuals infected with Mycobacterium tuberculosis (latent infection and active disease). It exhibits higher sensitivity while maintaining specificity and produces fewer indeterminate interpretations than QFT-Plus. Its easy-to-use, single-patient format may lead to increased TB testing to aid in the adequate diagnosis and management of the disease. IMPORTANCE This study presents a comprehensive evaluation of the VIDAS TB-IGRA diagnostic test. This test is compared with the established QuantiFERON-TB Gold Plus to assess its effectiveness in diagnosing both latent and active tuberculosis (TB) infections. The study highlights the VIDAS TB-IGRA’s higher sensitivity, fewer indeterminate results, and robust performance across different patient populations, including those with confirmed TB disease, high-risk, and low-risk groups. The findings suggest that the VIDAS TB-IGRA could enhance TB diagnosis and management, offering a fully automated, easy-to-use solution that reduces human error and result variability.

With waning immunity and vaccine hesitancy, the COVID-19 pandemic continues to pose risks. A live microbial consortium (OL-1) with bacteria containing potentially cross-reactive antigens (CRAGs) stimulates anti-SARS-CoV-2 immune responses in vitro/vivo. We evaluated OL-1’s efficacy in enhancing anti-SARS-CoV-2 immunity in unvaccinated, previously infected adults. We conducted a pilot, parallel-group, triple-blind randomized controlled trial in 2021–2022 involving 52 generally healthy adults ages 18–60, unvaccinated against COVID-19, with SARS-CoV-2 infection ≥ 4 months prior. Participants received 21 days of either standard-dose OL-1, high-dose OL-1, or placebo. The primary outcome was change in plasma anti-SARS-CoV-2 IgG titers from baseline to Day 21. Secondary efficacy outcomes included changes through Day 42, interferon gamma (IFNg) release from stimulated peripheral blood mononuclear cells, and new SARS-CoV-2 infections. Safety was assessed through adverse events. Significant increases in plasma IgG levels were observed by Day 42 in the standard-dose OL-1 group (n = 17) compared to placebo (n = 18) (p = 0.02). No significant changes were observed in the high-dose group (n = 17). Marginal increases in IFNg release were observed in standard-dose recipients after stimulation with CD4+-specific CRAG and SARS-CoV-2 peptides and TLR7 ligands; only changes post-TLR7 ligand stimulation were significant. No new SARS-CoV-2 infections were detected. The most common adverse events overall were mild gastrointestinal symptoms; headaches were more frequent in OL-1 recipients. The live microbial consortium OL-1 was well-tolerated and associated with slightly increased anti-SARS-CoV-2 IgG levels in previously infected, unvaccinated adults at standard, but not high, dosage. Further research should confirm these findings and their clinical implications in larger populations. This study was registered on ClinicalTrials.gov (NCT04847349) on April 14, 2021.

Background: Detection and treatment of individuals with presumed latent tuberculosis (TB) infection (i.e., excluding active disease; LTBI) is imperative to achieve global TB control, as they represent a potential transmission reservoir. However, more sensitive and user–friendly diagnostic tools are needed. Methods: We evaluated the accuracy for TB infection detection of the new VIDAS® TB–IGRA (bioMérieux), a fully automated, single tube (thus eliminating the need for batch testing) overnight incubation assay, compared to the QuantiFERON®–TB Gold Plus (QFT–Plus, QIAGEN), in a global multi–centre cross–sectional study ([NCT04048018][1]) that included patients with TB disease (n=200) or participants at varying levels of TB exposure (n=1460; mixed exposure–risk–population). Results: VIDAS® TB–IGRA identified TB disease with greater sensitivity than QFT–Plus (97.5% vs. 80.7%, P<0.01%), and yielding significantly fewer false–negatives (2.5% vs. 17.5%; P<0.01%) and indeterminate results (1.0% vs. 9.5%; P=0.02%). In the mixed exposure–risk–population, negative (NPA) and positive percent agreement (PPA) were 90.1% (1097/1217) and 92.1% (223/242), respectively. PPA increased with TB–exposure risk (up to 95.7% for high–risk participants), whereas NPA decreased (starting from 96.9% for low–risk participants). Regression analyses revealed that VIDAS® TB–IGRA had a better fit with the risk–exposure gradient than the QFT‑Plus. Specificity in extremely low TB–exposure risk participants (n = 125) was high for both VIDAS® TB–IGRA and QFT–Plus (97.6% vs. 95.2%; P=8.33%). Conclusions: VIDAS® TB–IGRA displayed greater sensitivity than QFT–Plus, had a lower indeterminate rate, correlated better with an exposure gradient, and was highly specific, suggesting that it is a potentially valuable tool for the diagnosis of LTBI. ### Competing Interest Statement All authors had financial support from bioMérieux for performing this study. ### Clinical Trial NCT04048018 ### Funding Statement This study was funded by bioMérieux ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The following gave ethical approval for this work: - The French National Ethics committee (CPP, ref. 2019-A00998-49) - The UK Health Research Authority (Wales Research Ethics Committee 7, REC refs: 19/WA/0284 and 19/WA/0285) - The INMI "L. Spallanzani" (Rome, Italy) Ethics Committee (approval n°35/2019) - The "Comité de Ética en Investigación del Hospital General de Mexicali" (Mexico, ref. 02-01-HGMXL/FMED-UABC-2019-08-29-254) - In South Africa: - Pharma ethics refs. 190822774 and 190822777 - University of Cape Town Human Research Ethics Committee ref. 840/2019 - In USA: - Western Institutional Review Board (WIRB, tracking numbers: 20191965; 20192037; 20192039) - The Rutgers University Institutional Review Board (Refs: Pro2019001840 and Pro2019001936) - The Stanford University Institutional Review Board (eProtocol number: 53485) - The University of Illinois at Chicago Institutional Review Board (research protocol number: 2019-1145) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT04048018&atom=%2Fmedrxiv%2Fearly%2F2024%2F07%2F05%2F2024.07.03.24309158.atom

Autoantibodies, hallmarks of autoimmune disorders, have been described in individuals who have had COVID-19. We evaluated for autoantibodies in children after SARS-CoV-2 infection, including children who developed multisystem inflammatory syndrome in children (MIS-C). We used a custom 77-plex bead-based protein array to test plasma from 95 subjects 0-21 years old representing three diagnostic groups (27 asymptomatic/mild SARS-CoV-2 infection, 32 severe COVID-19, and 36 MIS-C) for IgG antibodies found in classical autoimmune diseases, antiviral antibodies including SARS-CoV-2 nucleocapsid and receptor-binding domain, and anti-cytokine antibodies. Samples were obtained 0-600 days after infection onset. The average mean fluorescence intensity (MFI) for each antigen was calculated using samples from healthy children (HC) 4-17 years old who have not had COVID-19 or diagnoses of autoimmune diseases. Antibodies were considered positive if the MFI was >5 SD above the average MFI of HC and >3000 MFI units. We found that children with current or prior SARS-CoV-2 infection had more autoantibodies than HC. IgG autoantibodies against ACE2 and fibrillarin were the most common autoantibodies in the asymptomatic/mild (63%, 19%) and severe COVID-19 groups (22%, 13%) and were also present in children with MIS-C (25%, 14%). Additionally, anti-interferon, especially anti-IFN-epsilon antibodies, and anti-interleukin antibodies were present in the plasma of children with acute severe COVID-19. Collectively, we found a high prevalence of autoantibodies in children who have had COVID-19 or MIS-C. These profiles can inform future efforts for surveillance of specific antigens and autoimmune diseases in children who have been infected by SARS-CoV-2.

BACKGROUND:Protection from severe disease and hospitalization by SARS-CoV-2 vaccination has been amply demonstrated by real-world data. However, the rapidly evolving pandemic raises new concerns. One pertains efficacy of adenoviral vector-based vaccines, particularly the single-dose Ad26.COV2.S, relative to mRNA vaccines. MAIN BODY:We investigated the immunogenicity of Ad26.COV2.S and mRNA vaccines in 33 subjects vaccinated with either vaccine class 5 months earlier on average. After controlling for the time since vaccination, Spike-binding antibody and neutralizing antibody levels were higher in the mRNA-vaccinated subjects, while no significant differences in antigen-specific B cell and T cell responses were observed between the two groups. CONCLUSIONS:A dichotomy exists between the humoral and cellular responses elicited by the two vaccine classes. Testing only for humoral responses to compare the durability of SARS-CoV-2 vaccine-induced responses, as typically performed for public health and research purposes, is insufficient.

Importance: Accurate diagnosis of tuberculosis (TB) infection can be achieved with interferon gamma ; release assays. Their performance can be improved by utilizing fully automated, single-patient formats. Objective: Establish clinical thresholds for a new interferon gamma; release assay, the VIDAS(R) TB-IGRA, and compare diagnostic performance in detecting tuberculosis infection and disease with the established QuantiFERON-TB Gold Plus (QFT-Plus). Design: Preliminary diagnostic performance study (October 2nd, 2019 to February 20th, 2020). Setting: Multicenter. Participants: Participants were divided into TB disease, high-risk, and low-risk populations. The confirmed TB disease population included 107 patients. The high-risk population included 162 individuals with flagged risk factors on a questionnaire but without objective clinical confirmation of TB. The Low-risk population included 117 healthy blood donors from the French National Blood Bank. Exposures: Tuberculosis. Main Outcomes and Measures: Positive and negative percent agreement (PPA, NPA) were determined between the VIDAS(R) TB-IGRA and QFT-Plus. In the TB disease and low-risk populations, sensitivity was also measured against bacterial culture and PCR. Results: The VIDAS(R) TB-IGRA produced fewer indeterminate results than the QFT-Plus (1/107 vs. 23/107) in the TB disease population. One analysis included indeterminate results as false negatives (94 positives and 10 false negatives vs. 56 positives and 48 false negatives), and the VIDAS(R) TB-IGRA exhibited higher sensitivity than the QFT-Plus (90.4% vs. 53.8%) (p<0.0001). Another analysis excluded indeterminate results (76 positives and 4 false negatives vs. 55 positives and 25 false negatives), and the VIDAS(R) TB-IGRA again exhibited higher sensitivity than the QFT-Plus (95.0% vs. 68.8%) (p<0.0001). A 98.2% PPA was calculated between the two tests with this dataset. In the high-risk population, the VIDAS(R) TB-IGRA exhibited a strong PPA (94.4%) with the QFT-Plus. However, a lower than expected NPA was observed (85.2%). In the low-risk population, the VIDAS(R) TB-IGRA demonstrated high specificity (94.9%) and a strong NPA (98.2%) with the QFT-Plus. Conclusions and Relevance: The fully automated VIDAS(R) TB-IGRA is a promising diagnostic test for both TB infection and disease. It exhibits higher sensitivity while maintaining specificity and produces fewer indeterminate interpretations. Its easy-to-use, single-patient format may lead to increased TB testing to help with the worldwide eradication of the disease.

BACKGROUND:While the biomarkers of COVID-19 severity have been thoroughly investigated, the key biological dynamics associated with COVID-19 resolution are still insufficiently understood. MAIN BODY:We report a case of full resolution of severe COVID-19 due to convalescent plasma transfusion in a patient with underlying multiple autoimmune syndrome. Following transfusion, the patient showed fever remission, improved respiratory status, and rapidly decreased viral burden in respiratory fluids and SARS-CoV-2 RNAemia. Longitudinal unbiased proteomic analysis of plasma and single-cell transcriptomics of peripheral blood cells conducted prior to and at multiple times after convalescent plasma transfusion identified the key biological processes associated with the transition from severe disease to disease-free state. These included (i) temporally ordered upward and downward changes in plasma proteins reestablishing homeostasis and (ii) post-transfusion disappearance of a particular subset of dysfunctional monocytes characterized by hyperactivated Interferon responses and decreased TNF-α signaling. CONCLUSIONS:Monitoring specific subsets of innate immune cells in peripheral blood may provide prognostic keys in severe COVID-19. Moreover, understanding disease resolution at the molecular and cellular level should contribute to identify targets of therapeutic interventions against severe COVID-19.

While the biomarkers of COVID-19 severity have been thoroughly investigated, the key biological dynamics associated with COVID-19 resolution are still insufficiently understood. We report a case of full resolution of severe COVID-19 due to convalescent plasma transfusion. Following transfusion, the patient showed fever remission, improved respiratory status, and rapidly decreased viral burden in respiratory fluids and SARS-CoV-2 RNAemia. Longitudinal unbiased proteomic analysis of plasma and single-cell transcriptomics of peripheral blood cells conducted prior to and at multiple times after convalescent plasma transfusion identified the key biological processes associated with the transition from severe disease to disease-free state. These included (i) temporally ordered upward and downward changes in plasma proteins reestablishing homeostasis and (ii) post-transfusion disappearance of a subset of monocytes characterized by hyperactivated Interferon responses and decreased TNF-α signaling. Monitoring specific dysfunctional myeloid cell subsets in peripheral blood may provide prognostic keys in COVID-19.

Abstract Background We studied risk factors, antibodies, and symptoms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in a diverse, ambulatory population. Methods A prospective cohort (n = 831) previously undiagnosed with SARS-CoV-2 infection underwent serial testing (SARS-CoV-2 polymerase chain reaction, immunoglobulin G [IgG]) for 6 months. Results Ninety-three participants (11.2%) tested SARS-CoV-2-positive: 14 (15.1%) asymptomatic, 24 (25.8%) severely symptomatic. Healthcare workers (n = 548) were more likely to become infected (14.2% vs 5.3%; adjusted odds ratio, 2.1; 95% confidence interval, 1.4–3.3) and severely symptomatic (29.5% vs 6.7%). IgG antibodies were detected after 79% of asymptomatic infections, 89% with mild-moderate symptoms, and 96% with severe symptoms. IgG trajectories after asymptomatic infections (slow increases) differed from symptomatic infections (early peaks within 2 months). Most participants (92%) had persistent IgG responses (median 171 days). In multivariable models, IgG titers were positively associated with symptom severity, certain comorbidities, and hospital work. Dyspnea and neurologic changes (including altered smell/taste) lasted ≥ 120 days in ≥ 10% of affected participants. Prolonged symptoms (frequently more severe) corresponded to higher antibody levels. Conclusions In a prospective, ethnically diverse cohort, symptom severity correlated with the magnitude and trajectory of IgG production. Symptoms frequently persisted for many months after infection. Clinical Trials Registration. NCT04336215.

Monitoring the burden and spread of infection with the new coronavirus SARS-CoV-2, whether within small communities or in large geographical settings, is of paramount importance for public health purposes. Serology, which detects the host antibody response to the infection, is the most appropriate tool for this task, since virus-derived markers are most reliably detected during the acute phase of infection. Here we show that our ELISA protocol, which is based on antibody binding to the Receptor Binding Domain (RBD) of the S1 subunit of the viral Spike protein expressed as a novel fusion protein, detects antibody responses to SARS-CoV-2 infection and COVID-19 vaccination. We also show that our ELISA is accurate and versatile. It compares favorably with commercial assays widely used in clinical practice to determine exposure to SARS-CoV-2. Moreover, our protocol accommodates use of various blood- and non-blood-derived biospecimens, such as breast milk, as well as dried blood obtained with microsampling cartridges that are appropriate for remote collection. As a result, our RBD-based ELISA protocols are well suited for seroepidemiology and other large-scale studies requiring parsimonious sample collection outside of healthcare settings.

Development of effective vaccines against Coronavirus Disease 2019 (COVID-19) is a global imperative. Rapid immunization of the world human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and many different vaccine approaches are being pursued to meet this task. Engineered filamentous bacteriophage (phage) have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the design, development, and initial evaluation of targeted phage-based vaccination approaches against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) by using dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. Towards a unique phage- and AAVP-based dual-display candidate approach, we first performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein for display on the recombinant major capsid coat protein pVIII. Targeted phage particles carrying one of these epitopes induced a strong and specific humoral response. In an initial experimental approach, when these targeted phage particles were further genetically engineered to simultaneously display a ligand peptide (CAKSMGDIVC) on the minor capsid protein pIII, which enables receptor-mediated transport of phage particles from the lung epithelium into the systemic circulation (termed "dual-display"), they enhanced a systemic and specific spike (S) protein-specific antibody response upon aerosolization into the lungs of mice. In a second line of investigation, we engineered targeted AAVP particles to deliver the entire S protein gene under the control of a constitutive cytomegalovirus (CMV) promoter, which induced tissue-specific transgene expression stimulating a systemic S protein-specific antibody response. As proof-of-concept preclinical experiments, we show that targeted phage- and AAVP-based particles serve as robust yet versatile enabling platforms for ligand-directed immunization and promptly yield COVID-19 vaccine prototypes for further translational development. SIGNIFICANCE:The ongoing COVID-19 global pandemic has accounted for over 2.5 million deaths and an unprecedented impact on the health of mankind worldwide. Over the past several months, while a few COVID-19 vaccines have received Emergency Use Authorization and are currently being administered to the entire human population, the demand for prompt global immunization has created enormous logistical challenges--including but not limited to supply, access, and distribution--that justify and reinforce the research for additional strategic alternatives. Phage are viruses that only infect bacteria and have been safely administered to humans as antibiotics for decades. As experimental proof-of-concept, we demonstrated that aerosol pulmonary vaccination with lung-targeted phage particles that display short epitopes of the S protein on the capsid as well as preclinical vaccination with targeted AAVP particles carrying the S protein gene elicit a systemic and specific immune response against SARS-CoV-2 in immunocompetent mice. Given that targeted phage- and AAVP-based viral particles are sturdy yet simple to genetically engineer, cost-effective for rapid large-scale production in clinical grade, and relatively stable at room temperature, such unique attributes might perhaps become additional tools towards COVID-19 vaccine design and development for immediate and future unmet needs.

Much is to be learned about the interface between immune responses to SARS-CoV-2 infection and vaccination. We monitored immune responses specific to SARS-CoV-2 Spike Receptor-Binding-Domain (RBD) in convalescent individuals for eight months after infection diagnosis and following vaccination. Over time, neutralizing antibody responses, which are predominantly RBD specific, generally decreased, while RBD-specific memory B cells persisted. RBD-specific antibody and B cell responses to vaccination were more vigorous than those elicited by infection in the same subjects or by vaccination in infection-naïve comparators. Notably, the frequencies of double negative B memory cells, which are dysfunctional and potentially pathogenic, increased in the convalescent subjects over time. Unexpectedly, this effect was reversed by vaccination. Our work identifies a novel aspect of immune dysfunction in mild/moderate COVID-19, supports the practice of offering SARS-CoV-2 vaccination regardless of infection history, and provides a potential mechanistic explanation for the vaccination-induced reduction of "Long-COVID" symptoms.

Since human infection with coronavirus SARS-CoV-2 was first detected in December 2019, we are still developing an understanding of the nature and duration of protection against this infection. Most neutralizing antibodies, which are a key component of the protective response against SARS-CoV-2 infection, target the Receptor Binding Domain (RBD) of the Spike glycoprotein and critically prevent binding of the virus to the host cell receptor, and viral entry. Thus, it is of vital importance to monitor the presence of neutralizing antibodies and RBD-specific B cells that are key for rapid production of protective antibodies upon reinfection with SARS-COV2 infection. In this study, we developed a multicolor flow cytometric assay to enumerate the RBD-specific memory B cell and memory B cell subsets. We collected peripheral blood cells and plasma from 22 subjects 1–2 months since COVID-19 diagnosis (early time point - ET) and then again after 5–7 months (late time point – LT). Comparing the data collected from these two time points, we observed a significant decrease in plasma blasts and double-negative memory B cell and an increase in the IgG+ switched-memory B cells and decrease in the IgM+ switched-memory B cells at LT relative to ET. We concurrently observed a trend toward decreased anti-RBD IgG titers over time. When we tested plasma neutralizing activity employing ACE2-expressing HeLa cell lines infected with mNeonGreen(mNG)SARS-CoV-2, we also observed reduced neutralizing antibody titers over time. Thus, a correlation exists between titers of RBD-specific IgG antibody and neutralizing titers. In addition, the presence of RBD-specific B-cell memory in circulating blood is a strong indication of a durable protective response.

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), enters cells through attachment to the human angiotensin converting enzyme 2 (hACE2) via the receptor-binding domain (RBD) in the surface/spike (S) protein. Several pseudotyped viruses expressing SARS-CoV-2 S proteins are available, but many of these can only infect hACE2-overexpressing cell lines. Here, we report the use of a simple, two-plasmid, pseudotyped virus system comprising a SARS-CoV-2 spike-expressing plasmid and an HIV vector with or without vpr to investigate the SARS-CoV-2 entry event in various cell lines. When an HIV vector without vpr was used, pseudotyped SARS-CoV-2 viruses produced in the presence of fetal bovine serum (FBS) were able to infect only engineered hACE2-overexpressing cell lines, whereas viruses produced under serum-free conditions were able to infect a broader range of cells, including cells without hACE2 overexpression. When an HIV vector containing vpr was used, pseudotyped viruses were able to infect a broad spectrum of cell types regardless of whether viruses were produced in the presence or absence of FBS. Infection sensitivities of various cell types did not correlate with mRNA abundance of hACE2, TMPRSS2, or TMPRSS4. Pseudotyped SARS-CoV-2 viruses and replication-competent SARS-CoV-2 virus were equally sensitive to neutralization by an anti-spike RBD antibody in cells with high abundance of hACE2. However, the anti-spike RBD antibody did not block pseudotyped viral entry into cell lines with low abundance of hACE2. We further found that CD147 was involved in viral entry in A549 cells with low abundance of hACE2. Thus, our assay is useful for drug and antibody screening as well as for investigating cellular receptors, including hACE2, CD147, and tyrosine-protein kinase receptor UFO (AXL), for the SARS-CoV-2 entry event in various cell lines.

The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin's anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.

Several genetic studies have implicated genes that encode for components of the innate immune response in tuberculosis (TB) susceptibility. The complement system is an early player in the innate immune response and provides the host with initial protection by promoting phagocytosis of apoptotic or necrotic cells. The C1q molecule is the first component of the classical pathway that leads to the activation of complement by binding to immune complexes and is encoded by the C1Q gene cluster. We investigated variants in this region to determine its association with TB susceptibility. Five single nucleotide polymorphisms (SNPs) (rs12033074, rs631090, rs172378, rs587585, and rs665691) were genotyped using TaqMan® SNP assays in 456 TB cases and 448 healthy controls and analysed by logistic regression models. The rs587585 variant showed a significant additive allelic association where the minor G allele was found more frequently in TB cases than in controls in both the discovery (p = 0.023; OR = 1.30; 95% CI, 1.04–1.64) and validation cohort (p = 0.038; OR = 1.31; 95% CI, 1.22–1.40). In addition, we detected increased C1qA expression when comparing cases and controls (p = 0.037) and linked this to a dosage effect of the G allele, which increased C1qA expression in TB cases. This is the first study to report the association of C1Q gene polymorphisms with progression to tuberculosis.