Piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. In vitro antiviral activity evaluation against Zika and Dengue viruses.

Since 2011 Direct Acting antivirals (DAAs) drugs targeting different non-structural (NS) viral proteins (NS3, NS5A or NS5B inhibitors) have been approved for clinical use in HCV therapies. However, currently there are not licensed therapeutics to treat Flavivirus infections and the only licensed DENV vaccine, Dengvaxia, is restricted to patients with preexisting DENV immunity. Similarly to NS5 polymerase, the NS3 catalytic region is evolutionarily conserved among the Flaviviridae family sharing strong structural similarity with other proteases belonging to this family and therefore is an attractive target for the development of pan-flavivirus therapeutics. In this work we present a library of 34 piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. The library was developed through a privileged structures-based design and then biologically screened using a live virus phenotypic assay to determine the half-maximal inhibitor concentration (IC50) of each compound against ZIKV and DENV. Two lead compounds, 42 and 44, with promising broad-spectrum activity against ZIKV (IC50 6.6 µM and 1.9 µM respectively) and DENV (IC50 6.7 µM and 1.4 µM respectively) and a good security profile were identified. Besides, molecular docking calculations were performed to provide insights about key interactions with residues in NS3 proteases' active sites.

Maraviroc as a potential HIV-1 latency-reversing agent in cell line models and ex vivo CD4 T cells.

Recent studies have suggested that the CCR5 antagonist maraviroc (MVC) may exert an HIV-1 latency reversal effect. This study aimed at defining MVC-mediated induction of HIV-1 in three cell line latency models and in ex vivo CD4 T cells from six patients with suppressed viraemia. HIV-1 induction was evaluated in TZM-bl cells by measuring HIV-1 LTR-driven luciferase expression, and in ACH-2 and U1 latently infected cell lines by measuring cell-free (CFR) and cell-associated (CAR) HIV-1 RNA by qPCR. NF-κB p65 was quantified in nuclear extracts by immunodetection. In ex vivo CD4 T cells, CAR, CFR and cell-associated DNA (CAD) were quantified at baseline and 1-7-14 days post-induction (T1, T7, T14). At T7 and T14, the infectivity of the CD4 T cells co-cultured with MOLT-4/CCR5 target cells was evaluated in the TZM-bl assay (TZA). Results were expressed as fold activation (FA) with respect to untreated cells. No LTR activation was observed in TZM-bl cells at any MVC concentration. NF-κB activation was only modestly upregulated (1.6±0.4) in TZM-bl cells with 5 µM MVC. Significant FA of HIV-1 expression was only detected at 80 µM MVC, namely on HIV-1 CFR in U1 (3.1±0.9; P=0.034) and ACH-2 cells (3.9±1.4; P=0.037). CFR was only weakly stimulated at 20 µM in ACH-2 (1.7±1.0 FA) cells and at 5 µM in U1 cells (1.9±0.5 FA). Although no consistent pattern of MVC-mediated activation was observed in ex vivo experiments, substantial FA values were detected sparsely on individual samples with different parameters. Notably, in one sample, MVC stimulated all parameters at T7 (2.3±0.2 CAD, 6.8±3.7 CAR, 18.7±16.7 CFR, 7.3±0.2 TZA). In conclusion, MVC variably induces HIV-1 production in some cell line models not previously used to test its latency reversal potential. In ex vivo CD4 T cells, MVC may exert patient-specific HIV-1 induction; however, clinically relevant patterns, if any, remain to be defined.

In vitro cross-resistance to doravirine in a panel of HIV-1 clones harbouring multiple NNRTI resistance mutations.

OBJECTIVES: Doravirine is a recently licensed HIV-1 NNRTI with improved efficacy, pharmacokinetics and safety profile compared with efavirenz and limited cross-resistance with rilpivirine and etravirine. In this in vitro study, cross-resistance to doravirine was analysed in a representative panel of NNRTI-resistant clones. METHODS: In vitro phenotypic susceptibility to doravirine was assessed in 10 clinically derived infectious clones with intermediate- to high-level resistance to rilpivirine, etravirine, efavirenz and nevirapine, and in NL4-3 site-directed mutants harbouring K103N, Y181C, M230L or K103N/Y181C NNRTI mutations. RESULTS: Although none of the infectious clones harboured any of the major doravirine resistance-associated mutations (RAMs) included in the IAS-USA reference list, doravirine fold change (FC) values were comparable to or higher than those calculated for other NNRTIs, particularly etravirine and rilpivirine. As expected, single NNRTI mutations K103N and Y181C did not impair doravirine susceptibility (FC 1.4 and 1.8, respectively), while reduced activity was observed with the single M230L or double K103N/Y181C mutations (FC 7.6 and 4.9, respectively). Median FC values increased significantly with increasing numbers of NNRTI RAMs (P = 0.005) and were >10 in 4/4 and 1/4 clones harbouring four and three NNRTI RAMs, respectively. FC values correlated well with predicted susceptibility as inferred by Stanford HIV Drug Resistance Database (HIVdb) and ANRS algorithms (both P < 0.001). CONCLUSIONS: Substantial cross-resistance to doravirine was detected in NNRTI-resistant viruses harbouring complex mutational patterns, even in the absence of major IAS-USA doravirine RAMs. Therefore, based on the simple IAS-USA reference list, doravirine resistance may be underestimated in viruses harbouring multiple NNRTI mutations.

Sofosbuvir Selects for Drug-Resistant Amino Acid Variants in the Zika Virus RNA-Dependent RNA-Polymerase Complex In Vitro.

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure significantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40-80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half- maximal inhibitory concentration (IC50) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC50 shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofosbuvir as a possible lead for anti-ZIKV drug development.

Comparable In Vitro Activities of Second-Generation HIV-1 Integrase Strand Transfer Inhibitors (INSTIs) on HIV-1 Clinical Isolates with INSTI Resistance Mutations.

Second-generation HIV-1 integrase strand transfer inhibitors (INSTIs) dolutegravir (DTG), bictegravir (BIC), and cabotegravir (CAB) showed a high genetic barrier to resistance and limited cross-resistance with first-generation INSTIs raltegravir (RAL) and elvitegravir (EVG). In this study, DTG, BIC, and CAB demonstrated a comparable activity on a panel of INSTI-resistant strains isolated from patients exposed to RAL, EVG, and/or DTG, with a significantly reduced susceptibility only with the pathway Q148H/K/R plus one to two additional INSTI mutations.

The HIV-1 reverse transcriptase E138A natural polymorphism decreases the genetic barrier to resistance to etravirine in vitro.

OBJECTIVES: The HIV-1 reverse transcriptase (RT) natural polymorphism E138A is included among the mutations with a minor impact on response to etravirine. However, the interpretation of E138A on etravirine susceptibility is not consistent across different genotypic resistance algorithms. The aim of the study was to investigate the effect of E138A on the genetic barrier to resistance to etravirine in vitro. METHODS: A panel of 20 clinically derived recombinant viruses (10 with WT 138E and 10 with 138A, all without any other resistance mutation) were cultured in the presence of increasing etravirine concentrations and analysed for genotypic changes at virus breakthrough. Parallel experiments were conducted with 138E/A/G/K/Q NL4-3-based clones. RESULTS: In the NL4-3 background, codon 138 changes increased etravirine resistance in the following order: Q > K > A > G > E. The 138A viruses were less susceptible to etravirine compared with the 138E viruses [median (IQR) fold change, 1.8 (1.5-2.8) versus 1.3 (0.8-1.8); P = 0.026], overcame etravirine pressure earlier [HR (95% CI) for viral outgrowth with 138A, 5.48 (2.95-28.24); P < 0.001] and grew at higher drug concentrations [median (IQR), 1350 (1350-1350) versus 0 (0-1350) nM; P = 0.005]. A variety of etravirine resistance-related mutations and changes in the RT connection and RNase H domains accumulated without any consistent pattern depending on baseline codon 138. CONCLUSIONS: E138A can contribute to reduced response to etravirine through a decreased genetic barrier to resistance. In vitro drug resistance selection is a valuable complement to define the full potential of low-level resistance mutations.

Neutralizing activity of African lineage Zika virus immune sera towards Asian lineage.

Genetic and phylogenetic studies indicated that Zika virus (ZIKV) has evolved into 2 major lineages, the African and Asian. However, ZIKV has been described as a single serotype. This study aimed at assessing the cross-neutralization between ZIKV African and Asian lineages strains. Sixthy-five samples collected in 2007 and 30 samples collected from the same subjects in 2011/2012 in West Africa and positive to neutralizing antibody against ZIKV MR-766 strain (African lineage) were tested against ZIKV H/PF/2013 strain (Asian lineage) by microneutralization assay. All samples showing neutralizing antibodies against MR-766 strain showed also neutralizing activity against H/PF/2013 strain, although with lower titers. This is consistent with about 120 amino acid differences between the two strains. Despite differences in the magnitude of neutralizing activity against different ZIKV strains, all samples showed neutralizing antibody titers considered to be protective.

Proviral location affects cognate peptide-induced virus production and immune recognition of HIV-1-infected T cell clones.

BACKGROUNDHIV-1-infected CD4+ T cells contribute to latent reservoir persistence by proliferating while avoiding immune recognition. Integration features of intact proviruses in elite controllers (ECs) and people on long-term therapy suggest that proviruses in specific chromosomal locations can evade immune surveillance. However, direct evidence of this mechanism is missing.METHODSIn this case report, we characterized integration sites and full genome sequences of expanded T cell clones in an EC before and after chemoradiation. We identified the cognate peptide of infected clones to investigate cell proliferation and virus production induced by T cell activation, and susceptibility to autologous CD8+ T cells.RESULTSThe proviral landscape was dominated by 2 large clones with replication-competent proviruses integrated into zinc finger (ZNF) genes (ZNF470 and ZNF721) in locations previously associated with deeper latency. A third nearly intact provirus, with a stop codon in Pol, was integrated into an intergenic site. Upon stimulation with cognate Gag peptides, infected clones proliferated extensively and produced virus, but the provirus in ZNF721 was 200-fold less inducible. While autologous CD8+ T cells decreased the proliferation of cells carrying the intergenic provirus, they had no effect on cells with the provirus in the ZNF721 gene.CONCLUSIONSWe provide direct evidence that upon activation of infected clones by cognate antigen, the lower inducibility of intact proviruses in ZNF genes can result in immune evasion and persistence.FUNDINGOffice of the NIH Director and National Institute of Dental & Craniofacial Research; NIAID, NIH; Johns Hopkins University Center for AIDS Research.

Clonally expanded HIV-1 proviruses with 5'-leader defects can give rise to nonsuppressible residual viremia.

BackgroundAntiretroviral therapy (ART) halts HIV-1 replication, decreasing viremia to below the detection limit of clinical assays. However, some individuals experience persistent nonsuppressible viremia (NSV) originating from CD4+ T cell clones carrying infectious proviruses. Defective proviruses represent over 90% of all proviruses persisting during ART and can express viral genes, but whether they can cause NSV and complicate ART management is unknown.MethodsWe undertook an in-depth characterization of proviruses causing NSV in 4 study participants with optimal adherence and no drug resistance. We investigated the impact of the observed defects on 5'-leader RNA properties, virus infectivity, and gene expression. Integration-site specific assays were used to track these proviruses over time and among cell subsets.ResultsClones carrying proviruses with 5'-leader defects can cause persistent NSV up to approximately 103 copies/mL. These proviruses had small, often identical deletions or point mutations involving the major splicing donor (MSD) site and showed partially reduced RNA dimerization and nucleocapsid binding. Nevertheless, they were inducible and produced noninfectious virions containing viral RNA, but lacking envelope.ConclusionThese findings show that proviruses with 5'-leader defects in CD4+ T cell clones can give rise to NSV, affecting clinical care. Sequencing of the 5'-leader can help in understanding failure to completely suppress viremia.FundingOffice of the NIH Director and National Institute of Dental and Craniofacial Research, NIH; Howard Hughes Medical Institute; Johns Hopkins University Center for AIDS Research; National Institute for Allergy and Infectious Diseases (NIAID), NIH, to the PAVE, BEAT-HIV, and DARE Martin Delaney collaboratories.

SARS-CoV-2 Infection of Human Ovarian Cells: A Potential Negative Impact on Female Fertility.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may affect female reproductive health. Here, we investigated the potential of SARS-CoV-2 to infect the follicular microenvironment, in particular granulosa (GCs) and cumulus cells (CCs), thus providing evidence for a productive infection. GCs and CCs were recovered from women (n = 25) who underwent in vitro fertilization at the Assisted Reproductive Unit, Siena University Hospital. Follicular ovarian cells were co-cultured with SARS-CoV-2 and then analyzed by qPCR, immunofluorescence (IF), western blot (WB) and transmission electron microscopy (TEM). In addition, cell culture supernatant was used to infect VERO6 cells. We demonstrated the expression of cell host factors ACE2, TRPMSS2, BSG and CTSL, which are pivotal for the virus life cycle. Cultured GCs and CCs incubated with SARS-CoV-2 revealed productive SARS-CoV-2 infection at 24 h, 48 h and 72 h post-adsorption. Indeed, SARS-CoV-2 RNA, spike and nucleocapsid proteins were detected in GCs and CCs, and their cell culture supernatant successfully infected the standard VERO E6 cells. Finally, TEM showed full-size virions attached to the membrane and located inside the cytoplasm. This in vitro study reveals the susceptibility of human ovarian cells to SARS-CoV-2 infection, suggesting a potential detrimental effect of COVID-19 infection on female human fertility.

Comparable Post-Vaccination Decay of Neutralizing Antibody Response to Wild-Type and Delta SARS-CoV-2 Variant in Healthcare Workers Recovered from Mild or Asymptomatic Infection.

We described the long-term decay of neutralizing antibody (NtAb) to the wild-type and Delta SARS-CoV-2 variant after three antigen stimulations (mild or asymptomatic natural infection followed by two doses of the BNT162b2 mRNA vaccine after a median of 296 days) in immunocompetent healthcare workers (HCWs). Live virus microneutralization against the B.1 and Delta SARS-CoV-2 variants was performed in VERO E6 cell cultures. The median NtAb titers for B.1 and Delta were comparable and highly correlated at both 20 and 200 days after the second vaccine dose in the 23 HCWs enrolled (median age, 46 years). A small group of naturally infected unvaccinated HCWs had comparable NtAb titers for the two strains after a median follow-up of 522 days from infection diagnosis. The NtAb response to the Delta VoC appears to follow the same long-term dynamics as the wild-type response regardless of the vaccinal boost; data collected after three antigen stimulations (natural infection followed by two doses of the BNT162b2 mRNA vaccine) may be helpful for tailoring the continuous monitoring of vaccine protection against SARS-CoV-2 variants over time.

Dynamics of Total and Intact HIV-1 DNA in Virologically Suppressed Patients Switching to DTG-Based or ATV-Based Dual Therapy.

BACKGROUND: Clinical trials have demonstrated noninferior viral suppression rates of selected 2-drug regimens (2DRs) over standard 3-drug regimens (3DRs). However, the effect of simplification to 2DRs on HIV-1 reservoir remains to be fully assessed. SETTING: Retrospective analyses of samples of virologically suppressed people living with HIV remaining on the same 3DRs or switching to DTG + 3TC or ATV/r + 3TC 2DRs. METHODS: Whole blood samples were collected at enrollment and after 48 weeks. Total HIV-1 DNA (tDNA) and intact HIV-1 DNA (iDNA) were quantified by droplet digital polymerase chain reaction and intact proviral DNA assay, respectively. Statistical analysis was performed to identify associations among variables, and multiple linear regression was used to analyze potential predictors of tDNA and iDNA changes over time. RESULTS: Forty-seven individuals were switched to DTG + 3TC 2DR (N = 23) and ATV/r + 3TC 2DR (N = 24), while 18 remained on 3DRs. tDNA did not change either in the overall population or in the 3DR and 2DR groups. iDNA decreased significantly in the whole data set and in the overall 3DR and 2DR groups ( P = 0.001, P = 0.039 and P = 0.009, respectively). iDNA, but not tDNA, was inversely correlated with the time of viral suppression ( P = 0.002) and time under antiretroviral therapy ( P = 0.006). Higher nadir CD4 + T-cell counts ( P = 0.001) and lower zenith viral load ( P = 0.02) showed an association with the decrease of iDNA, but not with tDNA. CONCLUSIONS: Both tDNA and iDNA dynamics supported noninferior efficacy of 2DRs over 3DRs. iDNA could be more informative than tDNA in analyzing the dynamics of the HIV-1 reservoir under different treatment strategies.

Long-Term Longitudinal Analysis of Neutralizing Antibody Response to Three Vaccine Doses in a Real-Life Setting of Previously SARS-CoV-2 Infected Healthcare Workers: A Model for Predicting Response to Further Vaccine Doses.

We report the time course of neutralizing antibody (NtAb) response, as measured by authentic virus neutralization, in healthcare workers (HCWs) with a mild or asymptomatic SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection diagnosed at the onset of the pandemic, with no reinfection throughout and after a three-dose schedule of the BNT162b2 mRNA vaccine with an overall follow-up of almost two years since infection. Forty-eight HCWs (median age 47 years, all immunocompetent) were evaluated: 29 (60.4%) were asymptomatic. NtAb serum was titrated at eight subsequent time points: T1 and T2 were after natural infection, T3 on the day of the first vaccine dose, T4 on the day of the second dose, T5, T6, and T7 were between the second and third dose, and T8 followed the third dose by a median of 34 days. NtAb titers at all postvaccination time points (T4 to T8) were significantly higher than all those at prevaccination time points (T1 to T3). The highest NtAb increase was following the first vaccine dose while subsequent doses did not further boost NtAb titers. However, the third vaccine dose appeared to revive waning immunity. NtAb levels were positively correlated at most time points suggesting an important role for immunogenetics.

Efficacy of Licensed Monoclonal Antibodies and Antiviral Agents against the SARS-CoV-2 Omicron Sublineages BA.1 and BA.2.

Newly emerging SARS-CoV-2 variants may escape monoclonal antibodies (mAbs) and antiviral drugs. By using live virus assays, we assessed the ex vivo inhibition of the B.1 wild-type (WT), delta and omicron BA.1 and BA.2 lineages by post-infusion sera from 40 individuals treated with bamlanivimab/etesevimab (BAM/ETE), casirivimab/imdevimab (CAS/IMD), and sotrovimab (SOT) as well as the activity of remdesivir, nirmatrelvir and molnupiravir. mAbs and drug activity were defined as the serum dilution (ID50) and drug concentration (IC50), respectively, showing 50% protection of virus-induced cytopathic effect. All pre-infusion sera were negative for SARS-CoV-2 neutralizing activity. BAM/ETE, CAS/IMD, and SOT showed activity against the WT (ID50 6295 (4355-8075) for BAM/ETE; 18,214 (16,248-21,365) for CAS/IMD; and 456 (265-592) for SOT) and the delta (14,780 (ID50 10,905-21,020) for BAM/ETE; 63,937 (47,211-79,971) for CAS/IMD; and 1103 (843-1334) for SOT). Notably, only SOT was active against BA.1 (ID50 200 (37-233)), whereas BA.2 was neutralized by CAS/IMD (ID50 174 (134-209) ID50) and SOT (ID50 20 (9-31) ID50), but not by BAM/ETE. No significant inter-variant IC50 differences were observed for molnupiravir (1.5 ± 0.1/1.5 ± 0.7/1.0 ± 0.5/0.8 ± 0.01 µM for WT/delta/BA.1/BA.2, respectively), nirmatrelvir (0.05 ± 0.02/0.06 ± 0.01/0.04 ± 0.02/0.04 ± 0.01 µM) or remdesivir (0.08 ± 0.04/0.11 ± 0.08/0.05 ± 0.04/0.08 ± 0.01 µM). Continued evolution of SARS-CoV-2 requires updating the mAbs arsenal, although antivirals have so far remained unaffected.

Cellular and Molecular Mechanisms of In Vivo and In Vitro SARS-CoV-2 Infection: A Lesson from Human Sperm.

Despite the major target of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, being the respiratory system, clinical evidence suggests that the male reproductive system may represent another viral target organ. Revealing the effect of SARS-CoV-2 infection on testis and sperm is a priority for reproductive biology, as well as for reproductive medicine. Here, we confirmed that the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is highly expressed on human testis and ejaculated sperm; moreover, we provide evidence for the expression of the co-receptors transmembrane protease/serine (TMPRSS2), Basigin (BSG), and Catepsin L (CTSL). Human sperm were readily infected, both in vivo and in vitro, by SARS-CoV-2, as demonstrated by confocal and electron microscopy. The demonstration that the seminiferous epithelium and sperm support SARS-CoV-2 viral replication suggests the possibility that the spermatogenetic process may be detrimentally affected by the virus, and at the same time, supports the need to implement safety measures and guidelines to ensure specific care in reproductive medicine.

External quality assessment of HIV-1 DNA quantification assays used in the clinical setting in Italy.

Total cell-associated HIV-1 DNA is a surrogate marker of the HIV-1 reservoir, however, certified systems for its quantification are not available. The Italian HIV DNA Network was launched to validate HIV-1 DNA quantification methods in use at University and Hospital labs. A quality control panel including HIV-1 DNA standards, reconstructed blood samples (RBSs) and DNA from different HIV-1 subtypes was blindly tested by 12 participating labs by quantitative real-time PCR (n = 6), droplet digital PCR (n = 3) or both (n = 3). The median 95% hit rate was 4.6 (3.7-5.5) copies per test and linearity in the tested range was excellent (R2 = 1.000 [1.000-1.000]). The median values obtained across labs were 3,370 (2,287-4,245), 445 (299-498), 59 (40-81) and 7 (6-11) HIV-1 DNA copies, for the 3,584, 448, 56 and 7-copy standards, respectively. With RBSs, measured values were within twofold with respect to the median in two thirds of cases. HIV-1 subtypes were missed (CRF01_AE by 3 labs) or underestimated by > 1 log (subtypes A, C, D, F by one lab; CRF01_AE by one lab; CRF02_AG by one lab). The overall performance was excellent with HIV-1 DNA standards, however detection of different HIV-1 subtypes must be improved.

Single-dose BNT162b2 mRNA COVID-19 vaccine significantly boosts neutralizing antibody response in health care workers recovering from asymptomatic or mild natural SARS-CoV-2 infection.

OBJECTIVES: To measure SARS-CoV-2 neutralizing antibody (NtAb) titres in previously infected or uninfected health care workers who received one or two doses of BNT162b2 mRNA COVID-19 vaccine. METHODS: NtAbs were titrated as dose-inhibiting 50% virus replication (ID50) by live virus microneutralization. We evaluated 41 health care workers recovering from mild or asymptomatic infection at first vaccination dose (T1_inf) and 21 days later (T2_inf). Sixteen uninfected health care workers were evaluated 20 days after first dose (T2_uninf) and 20 days after second vaccine dose (T3_uninf). RESULTS: At T2_inf, but not at T1_inf, there was a significant correlation between days from diagnosis (median 313, interquartile range 285-322) and NtAb levels (P = 0.011). NtAb titres increased at T2_inf with respect to T1_inf (1544 (732-2232) vs 26 (10-88), P < 0.001). Similarly, there was a significant increase in NtAb titres at T3_uninf compared with T2_uninf (183 (111-301) vs 5 (5-15), P < 0001). However, NtAb levels at T2_inf were significantly higher than those at T2_uninf and T3_uninf (P < 0.0001 for both analyses). CONCLUSIONS: A single vaccination in people with mild or asymptomatic previous infection further boosts SARS-CoV-2 humoral immunity to levels higher than those obtained by complete two-vaccination in uninfected subjects.

In Vitro Anti-SARS-CoV-2 Activity of Selected Metal Compounds and Potential Molecular Basis for Their Actions Based on Computational Study.

Metal-based drugs represent a rich source of chemical substances of potential interest for the treatment of COVID-19. To this end, we have developed a small but representative panel of nine metal compounds, including both synthesized and commercially available complexes, suitable for medical application and tested them in vitro against the SARS-CoV-2 virus. The screening revealed that three compounds from the panel, i.e., the organogold(III) compound Aubipyc, the ruthenium(III) complex KP1019, and antimony trichloride (SbCl3), are endowed with notable antiviral properties and an acceptable cytotoxicity profile. These initial findings prompted us to perform a computational study to unveil the likely molecular basis of their antiviral actions. Calculations evidenced that the metalation of nucleophile sites in SARS-CoV-2 proteins or nucleobase strands, induced by Aubipyc, SbCl3, and KP1019, is likely to occur. Remarkably, we found that only the deprotonated forms of Cys and Sec residues can react favorably with these metallodrugs. The mechanistic implications of these findings are discussed.