SARS-CoV-2 Breakthrough Infections According to the Immune Response Elicited after mRNA Third Dose Vaccination in COVID-19-Naïve Hospital Personnel.

BACKGROUND: Vaccine-induced SARS-CoV-2-anti-spike antibody (anti-S/RBD) titers are often used as a marker of immune protection and to anticipate the risk of breakthrough infections, although no clear cut-off is available. We describe the incidence of SARS-CoV-2 vaccine breakthrough infections in COVID-19-free personnel of our hospital, according to B- and T-cell immune response elicited one month after mRNA third dose vaccination. METHODS: The study included 487 individuals for whom data on anti-S/RBD were available. Neutralizing antibody titers (nAbsT) against the ancestral Whuan SARS-CoV-2, and the BA.1 Omicron variant, and SARS-CoV-2 T-cell specific response were measured in subsets of 197 (40.5%), 159 (32.6%), and 127 (26.1%) individuals, respectively. RESULTS: On a total of 92,063 days of observation, 204 participants (42%) had SARS-CoV-2 infection. No significant differences in the probability of SARS-CoV-2 infection for different levels of anti-S/RBD, nAbsT, Omicron nAbsT, or SARS-CoV-2 T cell specific response, and no protective thresholds for infection were found. CONCLUSIONS: Routine testing for vaccine-induced humoral immune response to SARS-CoV-2 is not recommended if measured as parameters of 'protective immunity' from SARS-CoV-2 after vaccination. Whether these findings apply to new Omicron-specific bivalent vaccines is going to be evaluated.

Biomimetic nanoplasmonic sensor for rapid evaluation of neutralizing SARS-CoV-2 monoclonal antibodies as antiviral therapy.

Monoclonal antibody (mAb) therapy is one of the most promising immunotherapies that have shown the potential to prevent or neutralize the effects of COVID-19 in patients at very early stages, with a few formulations recently approved by the European and American medicine agencies. However, a main bottleneck for their general implementation resides in the time-consuming, laborious, and highly-specialized techniques employed for the manufacturing and assessing of these therapies, excessively increasing their prices and delaying their administration to the patients. We propose a biomimetic nanoplasmonic biosensor as a novel analytical technique for the screening and evaluation of COVID-19 mAb therapies in a simpler, faster, and reliable manner. By creating an artificial cell membrane on the plasmonic sensor surface, our label-free sensing approach enables real-time monitoring of virus-cell interactions as well as direct analysis of antibody blocking effects in only 15 min assay time. We have achieved detection limits in the 102 TCID50/mL range for the study of SARS-CoV-2 viruses, which allows to perform neutralization assays by only employing a low-volume sample with common viral loads. We have demonstrated the accuracy of the biosensor for the evaluation of two different neutralizing antibodies targeting both Delta and Omicron variants of SARS-CoV-2, with half maximal inhibitory concentrations (IC50) determined in the ng/mL range. Our user-friendly and reliable technology could be employed in biomedical and pharmaceutical laboratories to accelerate, cheapen, and simplify the development of effective immunotherapies for COVID-19 and other serious infectious diseases or cancer.

Viral load decrease in SARS-CoV-2 BA.1 and BA.2 Omicron sublineages infection after treatment with monoclonal antibodies and direct antiviral agents.

The efficacy on the Omicron variant of the approved early coronavirus disease-2019 (COVID-19) therapies, especially monoclonal antibodies, has been challenged by in vitro neutralization data, while data on in vivo antiviral activity are lacking. We assessed potential decrease from Day 1 to Day 7 viral load (VL) in nasopharyngeal swabs of outpatients receiving Sotrovimab, Molnupiravir, Remdesivir, or Nirmatrelvir/ritonavir for mild-to-moderate COVID-19 due to sublineages BA.1 or BA.2, and average treatment effect by weighted marginal linear regression models. A total of 521 patients (378 BA.1 [73%], 143 [27%] BA.2) received treatments (Sotrovimab 202, Molnupiravir 117, Nirmatrelvir/ritonavir 84, and Remdesivir 118): median age 66 years, 90% vaccinated, median time from symptoms onset 3 days. Day 1 mean VL was 4.12 log2 (4.16 for BA.1 and 4.01 for BA.2). The adjusted analysis showed that Nirmatrelvir/ritonavir significantly reduced VL compared to all the other drugs, except versus Molnupiravir in BA.2. Molnupiravir was superior to Remdesivir in both BA.1 and BA.2, and to Sotrovimab in BA.2. Sotrovimab had better activity than Remdesivir only against BA.1. Nirmatrelvir/ritonavir showed the greatest antiviral activity against Omicron variant, comparable to Molnupiravir only in the BA.2 subgroup. VL decrease could be a valuable surrogate of drug activity in the context of the high prevalence of vaccinated people and low probability of hospital admission.

In vitro Evaluation of Antiviral Efficacy of a Standardized Hydroalcoholic Extract of Poplar Type Propolis Against SARS-CoV-2.

Except for specific vaccines and monoclonal antibodies, effective prophylactic or post-exposure therapeutic treatments are currently limited for COVID-19. Propolis, a honeybee's product, has been suggested as a potential candidate for treatment of COVID-19 for its immunomodulatory properties and for its powerful activity against various types of viruses, including common coronaviruses. However, direct evidence regarding the antiviral activities of this product still remains poorly documented. VERO E6 and CALU3 cell lines were infected with SARS-CoV-2 and cultured in the presence of 12.5 or 25 µg/ml of a standardized Hydroalcoholic Extract acronym (sHEP) of Eurasian poplar type propolis and analyzed for viral RNA transcription, for cell damage by optical and electron microscopy, and for virus infectivity by viral titration at 2, 24, 48, and 72 h post-infection. The three main components of sHEP, caffeic acid phenethyl ester, galangin, and pinocembrin, were tested for the antiviral power, either alone or in combination. On both cell lines, sHEP showed significant effects mainly on CALU3 up to 48 h, i.e., some protection from cytopathic effects and consistent reduction of infected cell number, fewer viral particles inside cellular vesicles, reduction of viral titration in supernatants, dramatic drop of N gene negative sense RNA synthesis, and lower concentration of E gene RNA in cell extracts. Interestingly, pre-treatment of cells with sHEP before virus inoculation induced these same effects described previously and was not able to block virus entry. When used in combination, the three main constituents of sHEP showed antiviral activity at the same levels of sHEP. sHEP has a remarkable ability to hinder the replication of SARS-CoV-2, to limit new cycles of infection, and to protect host cells against the cytopathic effect, albeit with rather variable results. However, sHEP do not block the virus entry into the cells. The antiviral activity observed with the three main components of sHEP used in combination highlights that the mechanism underlying the antiviral activity of sHEP is probably the result of a synergistic effect. These data add further emphasis on the possible therapeutic role of this special honeybee's product as an adjuvant to official treatments of COVID-19 patients for its direct antiviral activity.

Comparative analysis of the neutralizing activity against SARS-CoV-2 Wuhan-Hu-1 strain and variants of concern: Performance evaluation of a pseudovirus-based neutralization assay.

Objectives: Emergence of new variants of SARS-CoV-2 might affect vaccine efficacy. Therefore, assessing the capacity of sera to neutralize variants of concern (VOCs) in BSL-2 conditions will help evaluating the immune status of population following vaccination or infection. Methods: Pseudotyped viruses bearing SARS-CoV-2 spike protein from Wuhan-Hu-1/D614G strains (wild type, WT), B.1.617.2 (Delta), or B.1.1.529 (Omicron) VOCs were generated to assess the neutralizing antibodies (nAbs) activity by a pseudovirus-based neutralization assay (PVNA). PVNA performance was assessed in comparison to the micro-neutralization test (MNT) based on live viruses. Sera collected from COVID-19 convalescents and vaccinees receiving mRNA (BNT16b2 or mRNA-1273) or viral vector (AZD1222 or Ad26.COV2.S) vaccines were used to measure nAbs elicited by two-dose BNT16b2, mRNA-1273, AZD1222 or one-dose Ad26.CO2.S, at different times from completed vaccination, ~ 1.5 month and ~ 4-6 months. Sera from pre-pandemic and unvaccinated individuals were analyzed as controls. Neutralizing activity following booster vaccinations against VOCs was also determined. Results: PVNA titers correlated with the gold standard MNT assay, validating the reliability of PVNA. Sera analyzed late from the second dose showed a reduced neutralization activity compared to sera collected earlier. Ad26.CO2.S vaccination led to very low or absent nAbs. Neutralization of Delta and Omicron BA.1 VOCs showed significant reduction of nAbs respect to WT strain. Importantly, booster doses enhanced Omicron BA.1 nAbs, with persistent levels at 3 months from boosting. Conclusions: PVNA is a reliable tool for assessing anti-SARS-CoV-2 nAbs helping the establishment of a correlate of protection and the management of vaccination strategies.

The first case of meningitis associated with SARS-CoV-2 BA.2 variant infection with persistent viremia.

Severe neurological disorders and vascular events during COVID-19 have been described. Here, we describe the first case of a female patient infected with the SARS-CoV-2 BA.2 Omicron variant of concern with meningitis with newly diagnosed central demyelinating disease.

External quality assessment of SARS-CoV-2 serology in European expert laboratories, April 2021.

BackgroundCountries worldwide are focusing to mitigate the ongoing SARS-CoV-2 pandemic by employing public health measures. Laboratories have a key role in the control of SARS-CoV-2 transmission. Serology for SARS-CoV-2 is of critical importance to support diagnosis, define the epidemiological framework and evaluate immune responses to natural infection and vaccine administration.AimThe aim of this study was the assessment of the actual capability among laboratories involved in sero-epidemiological studies on COVID-19 in EU/EEA and EU enlargement countries to detect SARS-CoV-2 antibodies through an external quality assessment (EQA) based on proficiency testing.MethodsThe EQA panels were composed of eight different, pooled human serum samples (all collected in 2020 before the vaccine roll-out), addressing sensitivity and specificity of detection. The panels and two EU human SARS-CoV-2 serological standards were sent to 56 laboratories in 30 countries.ResultsThe overall performance of laboratories within this EQA indicated a robust ability to establish past SARS-CoV-2 infections via detection of anti-SARS-CoV-2 antibodies, with 53 of 55 laboratories using at least one test that characterised all EQA samples correctly. IgM-specific test methods provided most incorrect sample characterisations (24/208), while test methods detecting total immunoglobulin (0/119) and neutralising antibodies (2/230) performed the best. The semiquantitative assays used by the EQA participants also showed a robust performance in relation to the standards.ConclusionOur EQA showed a high capability across European reference laboratories for reliable diagnostics for SARS-CoV-2 antibody responses. Serological tests that provide robust and reliable detection of anti-SARS-CoV-2 antibodies are available.

Comparative analysis of the neutralizing activity against SARS-CoV-2 Wuhan-Hu-1 strain and variants of concern: Performance evaluation of a pseudovirus-based neutralization assay

Objectives Emergence of new variants of SARS-CoV-2 might affect vaccine efficacy. Therefore, assessing the capacity of sera to neutralize variants of concern (VOCs) in BSL-2 conditions will help evaluating the immune status of population following vaccination or infection. Methods Pseudotyped viruses bearing SARS-CoV-2 spike protein from Wuhan-Hu-1/D614G strains (wild type, WT), B.1.617.2 (Delta), or B.1.1.529 (Omicron) VOCs were generated to assess the neutralizing antibodies (nAbs) activity by a pseudovirus-based neutralization assay (PVNA). PVNA performance was assessed in comparison to the micro-neutralization test (MNT) based on live viruses. Sera collected from COVID-19 convalescents and vaccinees receiving mRNA (BNT16b2 or mRNA-1273) or viral vector (AZD1222 or Ad26.COV2.S) vaccines were used to measure nAbs elicited by two-dose BNT16b2, mRNA-1273, AZD1222 or one-dose Ad26.CO2.S, at different times from completed vaccination, ~ 1.5 month and ~ 4-6 months. Sera from pre-pandemic and unvaccinated individuals were analyzed as controls. Neutralizing activity following booster vaccinations against VOCs was also determined. Results PVNA titers correlated with the gold standard MNT assay, validating the reliability of PVNA. Sera analyzed late from the second dose showed a reduced neutralization activity compared to sera collected earlier. Ad26.CO2.S vaccination led to very low or absent nAbs. Neutralization of Delta and Omicron BA.1 VOCs showed significant reduction of nAbs respect to WT strain. Importantly, booster doses enhanced Omicron BA.1 nAbs, with persistent levels at 3 months from boosting. Conclusions PVNA is a reliable tool for assessing anti-SARS-CoV-2 nAbs helping the establishment of a correlate of protection and the management of vaccination strategies.

SARS-CoV-2 Infection of Airway Epithelium Triggers Pulmonary Endothelial Cell Activation and Senescence Associated with Type I IFN Production.

Airway epithelial cells represent the main target of SARS-CoV-2 replication but several pieces of evidence suggest that endothelial cells (ECs), lining pulmonary blood vessels, are key players in lung injury in COVID-19 patients. Although in vivo evidence of SARS-CoV-2 affecting the vascular endothelium exists, in vitro data are limited. In the present study, we set up an organotypic model to dissect the crosstalk between airway epithelium and pulmonary endothelial cells during SARS-CoV-2 infection. We showed that SARS-CoV-2 infected airway epithelium triggers the induction of endothelial adhesion molecules in ECs, suggesting a bystander effect of dangerous soluble signals from the infected epithelium. The endothelial activation was correlated with inflammatory cytokines (IL-1ß, IL-6, IL-8) and with the viral replication in the airway epithelium. Interestingly, SARS-CoV-2 infection determined a modulation of endothelial p21, which could be partially reversed by inhibiting IFN-ß production from ECs when co-cultured with HAE. Altogether, we demonstrated that SARS-CoV-2 infected epithelium triggers activation/senescence processes in ECs involving type I IFN-ß production, suggesting possible antiviral/damage mechanisms occurring in the endothelium.

Safety and immune response kinetics of GRAd-COV2 vaccine: phase 1 clinical trial results.

Despite the successful deployment of efficacious vaccines and therapeutics, the development of novel vaccines for SARS-CoV-2 remains a major goal to increase vaccine doses availability and accessibility for lower income setting. We report here on the kinetics of Spike-specific humoral and T-cell response in young and old volunteers over 6 months follow-up after a single intramuscular administration of GRAd-COV2, a gorilla adenoviral vector-based vaccine candidate currently in phase-2 of clinical development. At all three tested vaccine dosages, Spike binding and neutralizing antibodies were induced and substantially maintained up to 3 months, to then contract at 6 months. Potent T-cell responses were readily induced and sustained throughout the study period, with only minor decline. No major differences in immune response to GRAd-COV2 vaccination were observed in the two age cohorts. In light of its favorable safety and immunogenicity, GRAd-COV2 is a valuable candidate for further clinical development and potential addition to the COVID-19 vaccine toolbox to help fighting SARS-CoV-2 pandemic.

SARS-CoV-2 nasopharyngeal viral load in individuals infected with BA.2, compared to Alpha, Gamma, Delta and BA.1 variants: A single-center comparative analysis.

BACKGROUND: SARS-CoV-2 has evolved, leading to the emergence of new Variants Of Concern (VOCs) with significant impact on transmissibility. Although the transmission process is complex, higher nasopharyngeal viral load (NP-VL) can be considered as a proxy for greater transmissibility. OBJECTIVES: The aim of this analysis was to compare NP-VL across a set of representative VOCs observed in mildly symptomatic patients. STUDY DESIGN: Observational single-center comparative analysis of patients with early mild-to-moderate COVID-19, enrolled within the early treatment access program of Lazzaro Spallanzani Institute (March 2021-March 2022). NP-VL before drug administration was estimated through RT-PCR, based on cycle threshold values (CTs); VOCs were identified by Sanger sequencing. VOCs' average treatment effect (ATE) was estimated on the CTs fitted in the log2 scale, controlling for potential confounders. RESULTS: A total of 707 patients were included. VOCs were: 10% Alpha, 3% Gamma, 34% Delta, 34% BA.1, 19% BA.2. Mean CTs for BA.1 and BA.2 were lower than Delta and BA.1, respectively. After adjusting for calendar time, age, immunodeficiency and vaccination, CTs for Gamma were lower than those seen for Alpha and higher than Delta, for Delta were similar to BA.1, for BA.2 were lower than Delta and BA.1. CONCLUSIONS: Our analysis shows higher NP-VL of BA.2 compared to previously circulating VOCs, even after controlling for factors potentially contributing to the amount of nasopharyngeal viral RNA, included vaccination, supporting the increased transmissibility of BA.2. Further studies are necessary to clarify this mechanism and to provide guidance for public health measures.

Immunogenicity to COVID-19 mRNA vaccine third dose in people living with HIV.

In order to investigate safety and immunogenicity of SARS-CoV-2 vaccine third dose in people living with HIV (PLWH), we analyze anti-RBD, microneutralization assay and IFN-γ production in 216 PLWH on ART with advanced disease (CD4 count <200 cell/mm3 and/or previous AIDS) receiving the third dose of a mRNA vaccine (BNT162b2 or mRNA-1273) after a median of 142 days from the second dose. Median age is 54 years, median CD4 nadir 45 cell/mm3 (20-122), 93% HIV-RNA < 50 c/mL. In 68% of PLWH at least one side-effect, generally mild, is recorded. Humoral response after the third dose was strong and higher than that achieved with the second dose (>2 log2 difference), especially when a heterologous combination with mRNA-1273 as third shot is used. In contrast, cell-mediated immunity remain stable. Our data support usefulness of third dose in PLWH currently receiving suppressive ART who presented with severe immune dysregulation.

SARS-CoV-2 Omicron Variant Neutralization after Third Dose Vaccination in PLWH.

The aim was to measure neutralizing antibody levels against the SARS-CoV-2 Omicron (BA.1) variant in serum samples obtained from vaccinated PLWH and healthcare workers (HCW) and compare them with those against the Wuhan-D614G (W-D614G) strain, before and after the third dose of a mRNA vaccine. We included 106 PLWH and 28 HCWs, for a total of 134 participants. Before the third dose, the proportion of participants with undetectable nAbsT against BA.1 was 88% in the PLWH low CD4 nadir group, 80% in the high nadir group and 100% in the HCW. Before the third dose, the proportion of participants with detectable nAbsT against BA.1 was 12% in the PLWH low nadir group, 20% in the high nadir group and 0% in HCW, respectively. After 2 weeks from the third dose, 89% of the PLWH in the low nadir group, 100% in the high nadir group and 96% of HCW elicited detectable nAbsT against BA.1. After the third dose, the mean log2 nAbsT against BA.1 in the HCW and PLWH with a high nadir group was lower than that seen against W-D614G (6.1 log2 (±1.8) vs. 7.9 (±1.1) and 6.4 (±1.3) vs. 8.6 (±0.8)), respectively. We found no evidence of a different level of nAbsT neutralization by BA.1 vs. W-D614G between PLWH with a high CD4 nadir and HCW (0.40 (-1.64, 2.43); p = 0.703). Interestingly, in PLWH with a low CD4 nadir, the mean log2 difference between nAbsT against BA.1 and W-D614G was smaller in those with current CD4 counts 201-500 vs. those with CD4 counts < 200 cells/mm3 (-0.80 (-1.52, -0.08); p = 0.029), suggesting that in this target population with a low CD4 nadir, current CD4 count might play a role in diversifying the level of SARS-CoV-2 neutralization.

Evidence of Maternal Antibodies Elicited by COVID-19 Vaccination in Amniotic Fluid: Report of Two Cases in Italy.

With SARS-CoV-2 infection, pregnant women may be at a high risk of severe disease and adverse perinatal outcomes. A COVID-19 vaccination campaign represents the key strategy to combat the pandemic; however, public acceptance of maternal immunization has to be improved, which may be achieved by highlighting the promising mechanism of passive immunity as a strategy for protecting newborns against SARS-CoV-2 infection. We tested the anti-SARS-CoV-2 antibody response following COVID-19 full-dose vaccination in the serum and amniotic fluid of two pregnant women who presented between April and June 2021, at the Center for the Treatment and Prevention of Infections in Pregnancy of the National Institute for Infectious Diseases "L. Spallanzani", for antenatal consultancy. Anti-SARS-CoV-2 IgG was found in residual samples of amniotic fluid collected from both women at the 18th week of gestation (63 and 131 days after the second dose's administration). Titers in amniotic fluid mirrored the levels detected in serum and were inversely linked to the time from vaccination. Our results suggest that antibodies elicited by COVID-19 vaccination can cross the placenta and reach the fetus; therefore, they may offer passive immunity at birth. It is critical to fully understand the kinetics of the maternal response to vaccination, the efficiency of IgG transfer, and the persistence of antibodies in infants to optimize maternal immunization regimens.

SARS-CoV-2 Specific Immune Response and Inflammatory Profile in Advanced HIV-Infected Persons during a COVID-19 Outbreak.

The main aim of this study was to describe the clinical and immunological outcomes, as well as the inflammatory profile, of patients with advanced HIV in an assisted-living facility in which an outbreak of SARS-CoV-2 occurred. SARS-CoV-2 humoral and specific T-cell response were analyzed in patients with HIV infection and COVID-19; as a secondary objective of the analysis, levels of the inflammatory markers (IL-1ß, IL-6, IL-8, and TNFα) were tested in the HIV/COVID-19 group, in HIV-positive patients without COVID-19, and in HIV-negative patients with mild/moderate COVID-19. Antibody kinetics and ability to neutralize SARS-CoV-2 were evaluated by ELISA assay, as well as the inflammatory cytokines; SARS-CoV-2 specific T-cell response was quantified by ELISpot assay. Mann-Whitney or Kruskal-Wallis tests were used for comparisons. Thirty patients were included with the following demographics: age, 57 years old (IQR, 53-62); 76% male; median HIV duration of infection, 18 years (15-29); nadir of CD4, 57/mmc (23-100) current CD4 count, 348/mmc (186-565). Furthermore, 83% had at least one comorbidity. The severity of COVID-19 was mild/moderate, and the overall mortality rate was 10% (3/30). Additionally, 90% of patients showed positive antibody titers and neutralizing activity, with a 100% positive SARS-CoV-2 specific T-cell response over time, suggesting the ability to induce an effective specific immunity. Significantly higher levels of IL-6, IL-8, and TNF-α in COVID-19 without HIV vs. HIV/COVID-19 patients (p < 0.05) were observed. HIV infection did not seem to negatively impact COVID-19-related inflammatory state and immunity. Further data are mandatory to evaluate the persistence of these immunity and its ability to expand after exposure and/or vaccination.

Retention of Neutralizing Response against SARS-CoV-2 Omicron Variant in Sputnik V-Vaccinated Individuals.

The new Omicron variant of SARS-CoV-2, first identified in November 2021, is rapidly spreading all around the world. Omicron has become the dominant variant of SARS-CoV-2. There are many ongoing studies evaluating the effectiveness of existing vaccines. Studies on the neutralizing activity of vaccinated sera against the Omicron variant are currently being carried out in many laboratories. In this study, we have shown the neutralizing activity of sera against the SARS-CoV-2 Omicron variant compared to the reference Wuhan D614G variant in individuals vaccinated with two doses of Sputnik V up to 6 months after vaccination and in individuals who experienced SARS-CoV-2 infection either before or after vaccination. As a control to our study we also measured neutralizing antibody titers in individuals vaccinated with two doses of BNT162b2. The decrease in NtAb titers to the Omicron variant was 8.1-fold for the group of Sputnik V-vaccinated individuals. When the samples were stratified for the time period after vaccination, a 7.6-fold or 8.8-fold decrease in NtAb titers was noticed after up to 3 and 3-to-6 months after vaccination. We observed a 6.7- and 5-fold decrease in Sputnik V-vaccinated individuals experiencing asymptomatic or symptomatic infection, respectively. These results highlight the observation that the decrease in NtAb to the SARS-CoV-2 Omicron variant compared to the Wuhan variant occurs for different COVID-19 vaccines in use, with some showing no neutralization at all, confirming the necessity of a third booster vaccination.

Rapid and qualitative identification of SARS-CoV-2 mutations associated with variants of concern using a multiplex RT-PCR assay coupled with melting analysis.

OBJECTIVES: Considering the spread of new genetic variants and their impact on public health, it is important to have assays that are able to rapidly detect SARS-CoV-2 variants. METHODS: We retrospectively examined 118 positive nasopharyngeal swabs, first characterized by the Sanger sequencing, using the Simplexa® SARS-CoV-2 Variants Direct assay, with the aim of evaluating the performance of the assay to detect N501Y, G496S, Q498R, Y505H, E484K, E484Q, E484A, and L452R mutations. RESULTS: A total of 111/118 nasopharyngeal swabs were in complete agreement with the Sanger sequencing, whereas the remaining seven samples were not amplified due to the low viral load. The evaluation of the ability of the assay to detect the E484Q mutation was performed using a viral isolate of the SARS-CoV-2 Kappa variant, showing concordance in 15/15 samples. Simplexa® SARS-CoV-2 Variant Direct assay was able to detect mutation pattern of Alpha, Beta, Gamma, Delta, and Omicron variants with 100% specificity and 94% sensitivity, whereas 100% sensitivity and specificity for the Kappa variant was observed. CONCLUSION: The assay can be useful to obtain faster results, contributing to a prompt surveillance of SARS-CoV-2 variants; however, it requires to be confirmed by the Sanger method, especially in the case of pattern of mutations that are different from those expected and also requires updates as new variants emerge.

Clinical Management of Patients With B-Cell Depletion Agents to Treat or Prevent Prolonged and Severe SARS-COV-2 Infection: Defining a Treatment Pathway.

Introduction: Immunocompromised patients with B-cell depletion agents are at risk for persistence and/or severe SARS-COV-2 infection. We describe a case series of 21 COVID-19 patients under B cell depletion therapy, mostly treated with a combined therapy based on intravenous remdesevir (RDV) and steroid associated with SARS-CoV-2 monoclonal antibodies against Spike glycoprotein and/or hyper-immune convalescent plasma. Methods: This is a single-center longitudinal study. We retrospectively enrolled a total number of 21 B-cell depleted consecutive hospitalized patients with COVID-19 at the Lazzaro Spallanzani National Institute for Infectious Diseases, Rome, Italy, from November 2020 to December 2021. Demographic characteristics, medical history, clinical presentation, treatment, adverse drug reactions, and clinical and virological outcome were collected for all patients. In a subgroup, we explore immune T cells activation, T cells specific anti-SARS-COV-2 response, and neutralizing antibodies. Results: Twenty-one inpatients with B-cell depletion and SARS-COV-2 infection were enrolled. A median of 1 B cells/mm3 was detected. Eighteen patients presented hypogammaglobulinemia. All patients presented interstitial pneumonia treated with intravenous RDV and steroids. Sixteen patients were treated with monoclonal antibodies against SARS-CoV-2 Spike protein, four patients were treated with SARS-CoV-2 hyper-immune convalescent plasma infusion, and three patients received both treatments. A variable kinetic of T cell activation returning to normal levels at Day 30 after immunotherapy infusion was observed. All treated patients recovered. Conclusion: In COVID-19 immunosuppressed subjects, it is mandatory to establish a prompt, effective, and combined multi-target therapy including oxygen, antiviral, steroid, and antibody-based therapeutics, tailored to the patient's clinical needs.

Humoral and Cellular Immune Response Elicited by mRNA Vaccination Against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in People Living With Human Immunodeficiency Virus Receiving Antiretroviral Therapy Based on Current CD4 T-Lymphocyte Count.

BACKGROUND: Data on SARS-CoV-2 vaccine immunogenicity in PLWH are currently limited. Aim of the study was to investigate immunogenicity according to current CD4 T-cell count. METHODS: PLWH on ART attending a SARS-CoV-2 vaccination program, were included in a prospective immunogenicity evaluation after receiving BNT162b2 or mRNA-1273. Participants were stratified by current CD4 T-cell count (poor CD4 recovery, PCDR: <200/mm3; intermediate CD4 recovery, ICDR: 200-500/mm3; high CD4 recovery, HCDR: >500/mm3). RBD-binding IgG, SARS-CoV-2 neutralizing antibodies (nAbs) and IFN-γ release were measured. As control group, HIV-negative healthcare workers (HCWs) were used. FINDINGS: Among 166 PLWH, after 1 month from the booster dose, detectable RBD-binding IgG were elicited in 86.7% of PCDR, 100% of ICDR, 98.7% of HCDR, and a neutralizing titre ≥1:10 elicited in 70.0%, 88.2%, and 93.1%, respectively. Compared to HCDR, all immune response parameters were significantly lower in PCDR. After adjusting for confounders, current CD4 T-cell <200/mm3 significantly predicted a poor magnitude of anti-RDB, nAbs and IFN-γ response. As compared with HCWs, PCDR elicited a consistently reduced immunogenicity for all parameters, ICDR only a reduced RBD-binding antibody response, whereas HCDR elicited a comparable immune response for all parameters. CONCLUSION: Humoral and cell-mediated immune response against SARS-CoV-2 were elicited in most of PLWH, albeit significantly poorer in those with CD4 T-cell <200/mm3 versus those with >500 cell/mm3 and HIV-negative controls. A lower RBD-binding antibody response than HCWs was also observed in PLWH with CD4 T-cell 200-500/mm3, whereas immune response elicited in PLWH with a CD4 T-cell >500/mm3 was comparable to HIV-negative population.