Identification and Characterization of Epithelial Cell-Derived Dense Bodies Produced upon Cytomegalovirus Infection.

Dense bodies (DB) are complex, noninfectious particles produced during CMVinfection containing envelope and tegument proteins that may be ideal candidates as vaccines. Although DB were previously described in fibroblasts, no evidence of DB formation has been shown after propagating CMV in epithelial cells. In the present study, both fibroblast MRC-5 and epithelial ARPE-19 cells were used to study DB production during CMV infection. We demonstrate the formation of epithelial cell-derived DB, mostly located as cytoplasmic inclusions in the perinuclear area of the infected cell. DB were gradient-purified, and the nature of the viral particles was confirmed using CMV-specific immunelabeling. Epithelial cell-derived DB had higher density and more homogeneous size (200-300 nm) compared to fibroblast-derived DB (100-600 nm).In agreement with previous results characterizing DB from CMV-infected fibroblasts, the pp65 tegument protein was predominant in the epithelial cell-derived DB. Our results also suggest that epithelial cells had more CMV capsids in the cytoplasm and had spherical bodies compatible with nucleus condensation (pyknosis) in cells undergoing apoptosis that were not detected in MRC-5 infected cells at the tested time post-infection. Our results demonstrate the formation of DB in CMV-infected ARPE-19 epithelial cells that may be suitable candidate to develop a multiprotein vaccine with antigenic properties similar to that of the virions while not including the viral genome.

Corrigendum: Brief Research Report: Virus-Specific Humoral Immunity at Admission Predicts the Development of Respiratory Failure in Unvaccinated SARS-CoV-2 Patients.

[This corrects the article DOI: 10.3389/fimmu.2022.878812.].

Brief Research Report: Virus-Specific Humoral Immunity at Admission Predicts the Development of Respiratory Failure in Unvaccinated SARS-CoV-2 Patients.

Introduction: There is robust evidence indicating that the SARS-CoV-2-specific humoral response is associated with protection against severe disease. However, relatively little data exist regarding how the humoral immune response at the time of hospital admission correlates with disease severity in unimmunized patients. Our goal was toidentify variables of the humoral response that could potentially serve as prognostic markers for COVID-19 progressionin unvaccinated SARS-CoV-2 patients. Methods: A prospective cross-sectional study was carried out in a cohort of 160 unimmunized, adult COVID-19 patients from the Hospital Universitario 12Octubre. Participants were classified into four clinical groups based on disease severity: non-survivors with respiratory failure (RF), RF survivors, patients requiring oxygen therapy and those not receiving oxygen therapy. Serum samples were taken on admission and IgM, IgG, IgG subclass antibody titers were determined by ELISA, and neutralizing antibody titersusing a surrogate neutralization assay. The differences in the antibody titers between groups and the association between the clinical and analytical characteristics of the patients and the antibody titers were analyzed. Results: Patients that developed RF and survived had IgM titers that were 2-fold higher than non-survivors (p = 0.001), higher levels of total IgG than those who developed RF and succumbed to infection (p< 0.001), and than patients who required oxygen therapy (p< 0.05), and had 5-fold higher IgG1 titers than RF non-survivors (p< 0.001) and those who needed oxygen therapy (p< 0.001), and 2-fold higher than patients that did not require oxygen therapy during admission (p< 0.05). In contrast, RF non-survivorshad the lowest neutralizing antibodylevels, which were significantly lower compared those with RF that survived (p = 0.03). A positive correlation was found between IgM, total IgG, IgG1 and IgG3 titers and neutralizing antibody titers in the total cohort (p ≤ 0.0036). Conclusions: We demonstrate that patients with RF that survived infection had significantly higher IgM, IgG, IgG1 and neutralizing titers compared to patients with RF that succumb to infection, suggesting that using humoral response variables could be used as a prognostic marker for guiding the clinical management of unimmunized patients admitted to the hospital for SARS-CoV-2 infection.

Isolation of Functional SARS-CoV-2 Antigen-Specific T-Cells with Specific Viral Cytotoxic Activity for Adoptive Therapy of COVID-19.

In order to demonstrate the feasibility of preparing clinical-grade SARS-CoV-2-specific T-cells from convalescent donors and the ability of these cells to neutralize the virus in vitro, we used blood collected from two COVID-19 convalescent donors (before and after vaccination) that was stimulated with specific SARS-CoV-2 peptides followed by automated T-cell isolation using the CliniMacs Prodigy medical device. To determine cytotoxic activity, HEK 293T cells were transfected to express the SARS-CoV-2 M protein, mimicking SARS-CoV-2 infection. We were able to quickly and efficiently isolate SARS-CoV-2-specific T lymphocytes from both donors before and after they received the Pfizer-BioNTech vaccine. Althoughbefore vaccination, the final product contained up to 7.42% and 30.19% of IFN-γ+ CD3+ T-cells from donor 1 and donor 2, respectively, we observed an enrichment of the IFN-γ+ CD3+ T-cells after vaccination, reaching 70.47% and 42.59%, respectively. At pre-vaccination, the isolated SARS-CoV-2-specific T-cells exhibited cytotoxic activity that was significantly higher than that of unstimulated controls (donor 2: 15.41%, p-value 3.27 × 10-3). The cytotoxic activity of the isolated SARS-CoV-2-specific T-cells also significantly increased after vaccination (donor 1: 32.71%, p-value 1.44 × 10-5; donor 2: 33.38%, p-value 3.13 × 10-6). In conclusion, we demonstrated that SARS-CoV-2-specific T-cells can quickly and efficiently be stimulated from the blood of convalescent donors using SARS-CoV-2-specific peptides followed by automated isolation. Vaccinated convalescent donors have a higher percentage of SARS-CoV-2-specific T-cells and may be more suitable as donors. Although further studies are needed to assess the clinical utility of the functional isolated SARS-CoV-2-specific T-cells in patients, previous studies using the same stimulation and isolation methods applied to other pathologies support this idea.

Discordance Between SARS-CoV-2-specific Cell-mediated and Antibody Responses Elicited by mRNA-1273 Vaccine in Kidney and Liver Transplant Recipients.

Severe acute respiratory syndrome coronavirus 2-specific cell-mediated immunity (SARS-CoV-2-CMI) elicited by mRNA-based vaccines in solid organ transplant (SOT) recipients and its correlation with antibody responses remain poorly characterized. METHODS: We included 44 (28 kidney, 14 liver, and 2 double organ) recipients who received the full series of the mRNA-1273 vaccine. SARS-CoV-2-CMI was evaluated at baseline, before the second dose, and at 2 wk after completion of vaccination by an ELISpot-based interferon-γ FluoroSpot assay using overlapping peptides covering the S1 domain. SARS-CoV-2 immunoglobulin G seroconversion and serum neutralizing activity against the spike protein were assessed at the same points by commercial ELISA and an angiotensin-converting enzyme-2/spike antibody inhibition method, respectively. Postvaccination SARS-CoV-2-CMI was compared with 28 healthcare workers who received the BNT162b2 vaccine. RESULTS: Positive SARS-CoV-2-CMI increased from 6.8% at baseline to 23.3% after the first mRNA-1273 dose and 59.5% after the completion of vaccination (P < 0.0001). Lower rates were observed for immunoglobulin G seroconversion (2.3%, 18.6%, and 57.1%, respectively) and neutralizing activity (2.3%, 11.6%, and 31.0%). There was a modest correlation between neutralizing titers and the magnitude of SARS-CoV-2-CMI (Spearman's rho: 0.375; P = 0.015). Fifteen recipients (35.7%) mounted SARS-CoV-2-CMI without detectable neutralizing activity, whereas 3 (7.1%) did the opposite, yielding poor categorical agreement (Kappa statistic: 0.201). Rates of positive SARS-CoV-2-CMI among SOT recipients were significantly decreased compared with nontransplant controls (82.1% and 100.0% after the first dose and completion of vaccination, respectively; P < 0.0001). Kidney transplantation, the use of tacrolimus and prednisone, and the number of immunosuppressive agents were associated with lower cell-mediated responses. Results remained unchanged when 3 recipients with prevaccination SARS-CoV-2-CMI were excluded. CONCLUSIONS: Two-thirds of SOT recipients mounted SARS-CoV-2-CMI following vaccination with mRNA-1273. Notable discordance was observed between vaccine-induced cell-mediated and neutralizing humoral immunities. Future studies should determine whether these patients with incomplete responses are effectively protected.