Background:HIV-1 sub-subtype A6 is predominant in Eastern Europe and was associated with increased risk of treatment failure with the long-acting cabotegravir plus rilpivirine regimen. In this study, we aimed to evaluate the in vitro susceptibility and the genetic barrier to resistance to INSTI in recombinant viruses harboring clinically derived A6 integrase coding regions. Methods:We generated 23 NL4-3 strain-based recombinant viruses harboring clinically derived integrase coding region. We measured their susceptibility to second-generation INSTIs dolutegravir, bictegravir, and cabotegravir in a TZM-bl cell-based phenotypic assay. The genetic barrier to resistance was evaluated by exposing MT-2 cell cultures infected with 4 A6 integrase recombinant viruses, as well as the NL4-3 and HXB2 subtype B reference strains. Results:All 23 recombinant viruses generated with clinically derived A6 integrase displayed full susceptibility to dolutegravir, bictegravir, and cabotegravir, showing median (interquartile range) fold-change values of 1.2 (0.9-1.5), 1.1 (0.7-1.5), and 0.9 (0.6-1.1), respectively. Of 4 A6 viruses assessed for their genetic barrier to resistance in vitro, only 1 showed emerging integrase mutations E138K or Q148R at subinhibitory concentrations of dolutegravir or cabotegravir, respectively. Conclusions:These data suggest that sub-subtype A6 integrase has full susceptibility and largely maintains a high genetic barrier to resistance to second-generation integrase strand transfer inhibitors.

Viruses and host cells are intricately connected through a shared “chemical language” that may trace back to the prebiotic chemistry of early Earth. In this study, we present an innovative platform for antiviral exploration inspired by this primordial chemical framework. By “doping” the formamide-based prebiotic chemistry model with orotic acid derivatives, we generated complex, non-natural chemical mixtures capable of disrupting the replication of multiple viruses with minimal or no toxicity for eukaryotic cells. This strategy underscores the potential of an evolution-inspired approach in antiviral discovery, offering a novel avenue for identifying new agents with unconventional mechanisms that might elude traditional discovery methods.

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.

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 mu M and 1.9 mu M respectively) and DENV (IC50 6.7 mu M and 1.4 mu M 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.

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.

Fostemsavir, a prodrug of the active compound temsavir, has been recently approved as the first-in-class HIV-1 gp120-directed attachment inhibitor for salvage therapy in subjects with MDR HIV-1. Since the drug target is highly polymorphic, a key concern is whether different HIV-1 lineages vary in their natural susceptibility to temsavir. While subjects enrolled in Phase II and III studies mostly harboured HIV-1 subtypes B and C,1,2 the phenotypic susceptibility of different HIV-1 subtypes to temsavir has been analysed in vitro in a preliminary study and more recently in a larger survey including more than 1300 isolates.3,4 However, due to the large number of classified HIV-1 subtypes and circulating recombinant forms (CRFs) and the over-representation of B and C subtypes also in the in vitro studies, some lineages remain to be tested, including CRF02_AG, which is the most prevalent non-B subtype in Italy.5 To fill...

Inhibition of DDX3X expression or activity reduces proliferation in cells from various tumor tissues, in particular in breast cancer, and its expression often correlates to tumor aggressiveness. This makes DDX3X a prominent candidate for the design of drugs for novel personalized therapeutic strategies. Starting from an in silico drug discovery approach, a group of molecules has been selected by molecular docking at the RNA binding site of DDX3X. Here, the most promising among them, FHP01, was evaluated in breast cancer preclinical models. Specifically, FHP01 exhibited very effective antiproliferative and killing activity against different breast cancer cell types, among which those from triple-negative breast cancer (TNBC). Interestingly, FHP01 also inhibited WNT signaling, a key tumorigenic pathway already correlated to DDX3X functions in breast cancer model cell lines. Ultimately, FHP01 also caused a significant reduction, in vivo, in the growth of MDA MB 231-derived TNBC xenograft models. Importantly, FHP01 showed good bioavailability and no toxicity on normal peripheral blood mononuclear cells in vitro and on several mouse tissues in vivo. Overall, our data suggest that the use of FHP01 and its related compounds may represent a novel therapeutic approach with high potential against breast cancer, including the triple-negative subtype usually correlated to the most unfavorable outcomes because of the lack of available targeted therapies.

The Front Cover shows bithiazole derivatives acting as broad-spectrum antiviral agents (BSAAs) by targeting human host cells. These molecules block the replication of human rhinoviruses (hRVs) and Zika virus (ZIKV) via inhibition of the intracellular protein PI4KIIIβ while the inhibition of SARS-CoV-2 entry and replication seems to be connected with the modulation of an additional target. Cover design by Marco Radi. More information can be found in the Communication by Maria Grazia Martina, Marco Radi et al.

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.

Objectives: This study aimed to describe the longitudinal evolution of neutralizing antibody titres (NtAb) in three different cohorts of healthcare workers (HCWs), including vaccinated HCWs with and without a previous SARS-CoV-2 infection and previously infected unvaccinated HCWs. COVID-19 was mild or asymptomatic in those experiencing infection. Methods: NtAb was tested before BNT162b2 mRNA COVID-19 vaccine (V0), 20±2 days after the first dose (V1_20), 20±3 days (V2_20) and 90±2 days (V2_90) after the second dose in vaccinated HCWs and after about 2 months (N_60), 10 months (N_300) and 13 months (N_390) from natural infection in unvaccinated HCWs. NtAb were measured by authentic virus neutralization with a SARS-CoV-2 B.1 isolate circulating in Italy at HCW enrolment. Results: Sixty-two HCWs were enrolled. NtAb were comparable in infected HCWs with no or mild disease at all the study points. NtAb of uninfected HCWs were significantly lower with respect to those of previously infected HCWs at V1_20, V2_20 and V2_90. The median NtAb fold decrease from V2_20 to V2_90 was higher in the uninfected HCWs with respect to those with mild infection (6.26 vs 2.58, p=0.03) and to asymptomatic HCWs (6.26 vs 3.67, p=0.022). The median Nabt at N_390 was significantly lower than at N_60 (p=0.007). Conclusions: In uninfected HCWs completing the two-dose vaccine schedule, a third mRNA vaccine dose is a reasonable option to counteract the substantial NtAb decline occurring at a significantly higher rate compared with previously infected, vaccinated HCWs. Although low, Nabt were still at a detectable level after 13 months in two-thirds of previously infected and unvaccinated HCWs.

Over half a century since the description of the first antiviral drug, "old" re-emerging viruses and "new" emerging viruses still represent a serious threat to global health. Their high mutation rate and rapid selection of resistance toward common antiviral drugs, together with the increasing number of co-infections, make the war against viruses quite challenging. Herein we report a host-targeted approach, based on the inhibition of the lipid kinase PI4KIIIβ, as a promising strategy for inhibiting the replication of multiple viruses hijacking this protein. We show that bithiazole inhibitors of PI4KIIIβ block the replication of human rhinoviruses (hRV), Zika virus (ZIKV) and SARS-CoV-2 at low micromolar and sub-micromolar concentrations. However, while the anti-hRV/ZIKV activity can be directly linked to PI4KIIIβ inhibition, the role of PI4KIIIβ in SARS-CoV-2 entry/replication is debated.

In this Journal, the emergence of novel variants with potential to escape vaccine-induced immunity has received commentary.1Tang J.W. Tambyah P.A. Hui D.S. Emergence of a new SARS-CoV-2 variant in the UK.J Infect. 2021; 82: e27-e28Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOC) and variants of interest (VOI) are challenging the management of the evolving pandemic across countries. The VOI labelled as Eta (WHO Classification),2Pereira F. Tosta S. Lima M.M. Reboredo de Oliveira da Silva L. Nardy V.B. Gómez M.K.A. et al.Genomic surveillance activities unveil the introduction of the SARS-CoV-2 B1.525 variant of interest in Brazil: case report.J Med Virol. 2021; (Epub ahead of print)Crossref Scopus (20) Google Scholar combines relevant spike mutations detected in several VOC, such as the same 3 deletions of the Alpha lineage (69del, 70del, 144del), the E484K mutation found in the Gamma and Beta lineages as well as in some Alpha isolates and the ubiquitous D614G. In addition, three mutations (A67V, Q677H and F888L) are unique to Eta variant and it is currently unknown whether they favor escape from natural or vaccine induced immunity to the wild type lineage (B.1), as shown for other variants.3Janik E. Niemcewicz M. Podogrocki M. Majsterek I. Bijak M. The Emerging Concern and Interest SARS CoV-2 Variants.Pathogens. 2021; 10: 633Crossref PubMed Scopus (69) Google Scholar To test this hypothesis, we measured the serum neutralizing antibody (NtAb) response to Eta variant, as well as to other viral variants, in a cohort of health care workers (HCWs) including both previously infected (n = 15) and uninfected individuals (n = 15) vaccinated with two doses of the BNT162b2 COVID-19 mRNA vaccine. The study was approved by the Ethics Committee of the University of Milan (protocol n. 23/21) and conducted in compliance with Good Clinical Practice guidelines and the Declaration of Helsinki. The previously infected group was tested at baseline (T0inf) and 17±6 days after receiving the second vaccine dose (T2inf); the uninfected HCWs were tested 18±4 days after the second dose vaccination (T2uninf). The infected group had median age [IQR] of 38 (31–52) years, included 8 females and was infected during the first wave of the pandemic. The uninfected group had a median age of 38 (29–59) years with 11 females. NtAb titers were determined by a microneutralization live virus assay performed in VERO E6 cells using the quantification of cell viability as readout system, as previously described.4Vicenti I. Gatti F. Scaggiante R. Boccuto A. Zago D. Basso M. et al.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.Int J Infect Dis. 2021; 108: 176-178Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar NtAb titers were expressed as median (IQR) and were defined as the reciprocal value of the sample dilution that showed a 50% protection of virus-induced cytopathic effect (ID50). Sera with ID50 titres ≥10 were defined as SARS-CoV-2 neutralizing, while sera with ID50 <10 were defined as negative and scored as 5 for statistical analysis. Fifteen, 14 and 11 individuals at T0inf, T2inf and T2uninf, respectively, had also a quantitative anti-spike protein Ab determination, performed by the SARS-CoV-2 IgG II Quant assay (Abbott). The viral isolates used in the microneutralization live virus assay were sequenced by NGS and the full-lenght SARS-CoV-2 genome was submitted to GISAID (http://gisaid.org/) to assign the right variant (Accession numbers: EPI_ISL_2,472,896, EPI_ISL_1,085,167, EPI_ISL_2,472,918 and EPI_ISL_2,472,916 for the wild type, Alpha, Gamma and Eta variants, respectively). Statistical analyses were performed using IBM SPSS Statistics, version 20. The non-parametric Friedman test and Wilcoxon Signed Rank Sum test was used to analyze changes in paired data. The non-parametric Mann-Whitney test was used to compare unpaired data. Spearman analysis was used to measure the correlation between NtAb titres against the different variants. In previously infected HCWs, NtAb titres to all viral variants significantly increased after vaccination (mean T2inf/T0inf ratio 119±66; p<0.001). Notably, 2 to 12 subjects, depending on the reference virus, were negative at T0inf but all of them seroconverted following vaccination. As expected, the NtAb titer after vaccination was higher in the previously infected compared with the uninfected group (mean T2inf/T2uninf ratio 6 ± 2; p <0.001 (Fig. 1). Overall, median NtAb titres to the Eta variant (63 [7–323] ID50) correlated well with those to the wild type (133 [9–456]), Gamma (148 [46–988]) and Alpha (87 [5-681]) (p<0.001 for all comparisons) and high correlation was indeed observed between NtAb titres to any pair of virus variants (Fig. 2). Of note, NtAb titres to Eta variant were significantly lower with respect to those obtained for each variant (p<0.001). Anti-spike protein antibodies, as measured by enzyme immunoassay, were highly correlated with NtAb titres to B.1 (rho = 0.934), P.1 (rho = 0.914), B.1.1.7 (rho = 0.913) and B.1.525 (rho = 0.918) viruses (p<0.001 for all comparisons). Also, a significant increase was observed when comparing the anti-spike Ab median titres at T2inf and at T0inf (27,763 [18,282–46,108] vs. 1.7 [0.5–4.4]; p = 0.001).Fig. 2Spearman correlation between NtAb titres to each pair of the SARS-CoV-2 variants used in the study. Data were cumulated for all sera tested at T0inf, T2inf and T2uninf.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Overall, in our small cohort of previously infected or uninfected vaccinated-HCWs it appears that cross-neutralization among different viral variants remains substantial, following natural or artificial immunization with the wild type lineage. However, neutralization of Eta variant is significantly reduced with respect to other variants. Indeed, NtAb titres could be ranked with the definite order Gamma>wild type=Alpha>Eta. In vitro correlates of protection against the Eta variant has been investigated in uninfected vaccinated individuals only in two different works delivering inconsistent results. Indeed, Liu et al.5Liu J. Liu Y. Xia H. Zou J. Weaver S.C. Swanson K.A. et al.BNT162b2-elicited neutralization of B.1.617 and other SARS-CoV-2 variants.Nature. 2021; Google Scholar observed a modest reduction, while Zani et al.6Zani A. Caccuri F. Messali S. Bonfanti C. Caruso A. Serosurvey in BNT162b2 vaccine-elicited neutralizing antibodies against authentic B.1, B.1.1.7, B.1.351, B.1.525 and P.1 SARS-CoV-2 variants.Emerg Microbes Infect. 2021; 7: 1-6Google Scholar reported an increase in Eta variant NtAb titres with respect to the wild type variant. Of note, NtAb studies published so far have used different combination of strategies (e.g., live virus vs. pseudoparticles), viral variants, cell lines and readouts, in the absence of standardized methods and reference viral strains and neutralizing sera.7Betton M. Livrozet M. Planas D. Fayol A. Monel B. Védie B. et al.Sera neutralizing activities against SARS-CoV-2 and multiple variants six month after hospitalization for COVID-19.Clin Infect Dis. 2021; (; ciab308. Epub ahead of print. PMID: 33851216; PMCID: PMC8083257)https://doi.org/10.1093/cid/ciab308Crossref PubMed Scopus (27) Google Scholar, 8Planas D. Bruel T. Grzelak L. Guivel-Benhassine F. Staropoli I. Porrot F. Planchais C. et al.Sensitivity of infectious SARS-CoV-2 B1.1.7 and B.1.351 variants to neutralizing antibodies.Nat Med. 2021; 27: 917-924Crossref PubMed Scopus (450) Google Scholar, 9Wang P. Casner R.G. Nair M.S. Wang M. Yu J. Cerutti G. Liu L. et al.Increased resistance of SARS-CoV-2 variant P.1 to antibody neutralization.Cell Host Microbe. 2021; 29 (e4): 747-751Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 10Wang P. Nair M.S. Liu L. Iketani S. Luo Y. Guo Y. et al.Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7.Nature. 2021; 593: 130-135Crossref PubMed Scopus (1415) Google Scholar For example, the full-length sequencing of the isolates used in the assay should be always reported and submitted to public repositories. Most importantly, while NtAb studies certainly provide a solid basis to infer cross-protection among vaccines and virus variants, the in vivo correlates of in vitro data remain to be established and must be defined through accurate and continuous monitoring of vaccine induced reduction of morbidity and mortality in the context of molecular surveillance of SARS-CoV-2 lineages.

ObjectivesTo 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.MethodsNtAbs 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).ResultsAt 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).ConclusionsA 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.

Background: Rapid detection and isolation of SARS-CoV-2 infections is critical to mitigate the pandemic;however, testing access across the US has been uneven and data on barriers to testing are limited. Methods: We conducted serial cross-sectional assessments of experiences around SARS-CoV-2 PCR testing in Florida, Illinois, and Maryland. We sampled ∼1000/state using an online survey from Jul 15-31 and Sep 16-Oct 15, 2020, with additional waves planned at 6-8 week intervals. At the time of surveys, there were no systematic differences in testing availability (public, private and free testing options) across these states. Participants were recruited using on online panel;demographic targets were provided to match age, sex, race/ethnicity and income distributions of each state. Participants were ≥18 years, provided consent, and resided in the study state. The survey covered demographics, symptoms, and PCR testing in the prior 2 weeks. Results: Of 3,058 persons surveyed most recently (Sep 16-Oct 15), 316 (10%) reported wanting/needing a test in the prior two weeks. Median age of participants wanting/needing a test was 36 years and 46% were female;47% self-identified as White and 57% reported working outside home. Of 316 who wanted/needed a test in the prior 2 weeks, 53% were able to get tested, of whom, 94% received results, with no significant differences by state (Figure);this was not substantially different from the proportion able to get tested in July (51%). Among those wanting/needing a test, getting tested was significantly less common among men (aOR: 0.46) and those reporting black race (aOR: 0.53) and more common in those reporting recent travel (aOR: 3.35;all p<0.05). The primary reasons for testing were desire to know status (35%) and symptoms (28%). Among those tested, 53% had to wait ≥8 days to get a result from the time they wanted/needed a test. Of those tested, 71% reported quarantining while awaiting results. An additional 146 who wanted/needed a test did not get tested;the main reasons for not testing in this group were not knowing where to go (36%) and distance/waiting time (33%);an additional 21% reported fear of being tested. Conclusion: These data reflecting similar testing barriers across three US states underscore the importance of a unified national strategy with clear messaging on who, where, when, and how to get a test, as well as improved turn-aroundtimes. As demand rises borrowing strategies from HIV such as self-testing could help overcome logistical barriers.

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.

We aimed to investigate neutralizing antibody titers (NtAbT) to the P.1 and B.1 SARS-CoV-2 variants in a cohort of healthy health care workers (HCW), including 20 previously infected individuals tested at baseline (BLinf, after a median of 298 days from diagnosis) and 21 days after receiving one vaccine dose (D1inf) and 15 uninfected subjects tested 21 days after the second-dose vaccination (D2uninf). All the subjects received BNT162b2 vaccination. D1inf NtAbT increased significantly with respect to BLinf against both B.1 and P.1 variants, with a fold-change significantly higher for P.1. D1inf NtAbT were significantly higher than D2uninf NtAbT, against B.1 and P.1. NtAbT against the two strains were highly correlated. P.1 NtAbT were significantly higher than B.1 NtAbT. This difference was significant for post-vaccination sera in infected and uninfected subjects. A single-dose BNT162b2 vaccination substantially boosted the NtAb response to both variants in the previously infected subjects. NtAb titers to B.1 and P.1 lineages were highly correlated, suggesting substantial cross-neutralization. Higher titers to the P.1 than to the B.1 strain were driven by the post-vaccination titers, highlighting that cross-neutralization can be enhanced by vaccination.

While SARS-CoV-2 vaccination of uninfected hosts is proceeding worldwide, whether and how to vaccinate those recovered from natural infection remains debatable.A single-dose mRNA vaccine administration has been reported to boost SARS-CoV-2 neutralizing antibodies (NtAb) in subjects with past infection [1,2] while the effect of a second dose in such subjects has not been documented.Within a health care worker (HCW) surveillance and vaccination program running at a SARS-CoV-2 referral hospital, we assessed the dynamics of SARS-CoV-2 NtAb in 22 uninfected and 34 previously infected individuals who completed the 2-dose schedule of the BNT162b2 mRNA vaccine.All the subjects received the second dose vaccine 3 weeks after the first dose.The subjects included in the uninfected group had no SARS-COV-2 RNA detection in the tests performed according to the hospital surveillance program and at the time of NtAb testing.In addition, all the subjects had been tested for the presence of a neutralizing antibody titer at baseline, i.e., at the time of the first vaccine administration, and confirmed as negative.NtAb titers were determined in a live virus assay in VERO E6 cells infected by a lineage B.1 isolate and defined as the reciprocal of the serum dilution decreasing virus replication by 50% (EC 50 ), as measured by the CellTiter-Glo ® Luminescent Cell Viability Assay (Promega) [3].

The worldwide circulation of different viruses coupled with the increased frequency and diversity of new outbreaks, strongly highlight the need for new antiviral drugs to quickly react against potential pandemic pathogens. Broad-spectrum antiviral agents (BSAAs) represent the ideal option for a prompt response against multiple viruses, new and re-emerging. Starting from previously identified anti-flavivirus hits, we report herein the identification of promising BSAAs by submitting the multi-target 2,6-diaminopurine chemotype to a system-oriented optimization based on phenotypic screening on cell cultures infected with different viruses. Among the synthesized compounds, 6i showed low micromolar potency against Dengue, Zika, West Nile and Influenza A viruses (IC50 = 0.5-5.3 mu M) with high selectivity index. Interestingly, 6i also inhibited SARS-CoV-2 replication in different cell lines, with higher potency on Calu-3 cells that better mimic the SARS-CoV-2 infection in vivo (IC50 = 0.5 mu M, SI = 240). The multi-target effect of 6i on flavivirus replication was also analyzed in whole cell studies (in vitro selection and immunofluorescence) and against isolated host/viral targets. (C) 2021 Elsevier Masson SAS. All rights reserved.

AbstractWe describe the time course of neutralizing antibody (NtAb) titer in a cohort of health care workers with mild or asymptomatic severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection. NtAb levels decreased over time; however, serum neutralizing activity remained detectable after a median of 7 months from SARS-CoV-2 diagnosis in the majority of cases.

Practical cell-based assays can accelerate anti-Zika (ZIKV) and anti-dengue (DENV) virus drug discovery. We developed an immunodetection assay (IA), using a pan-flaviviral monoclonal antibody recognizing a conserved envelope domain. The final protocol includes a direct virus yield reduction assay (YRA) carried out in the human Huh7 cell line, followed by transfer of the supernatant to a secondary Huh7 culture to characterize late antiviral effects. Sofosbuvir and ribavirin were used to validate the assay, while celgosivir was used to evaluate the ability to discriminate between early and late antiviral activity. In the direct YRA, at 100, 50, and 25 TCID50, sofosbuvir IC50 values were 5.0 ± 1.5, 2.7 ± 0.5, 2.5 ± 1.1 µM against ZIKV and 16.6 ± 2.8, 4.6 ± 1.4, 2.6 ± 2.2 µM against DENV; ribavirin IC50 values were 6.8 ± 4.0, 3.8 ± 0.6, 4.5 ± 1.4 µM against ZIKV and 17.3 ± 4.6, 7.6 ± 1.2, 4.1 ± 2.3 µM against DENV. Sofosbuvir and ribavirin IC50 values determined in the secondary YRA were reproducible and comparable with those obtained by direct YRA and plaque reduction assay (PRA). In agreement with the proposed mechanism of late action, celgosivir was active against DENV only in the secondary YRA (IC50 11.0 ± 1.0 µM) and in PRA (IC50 10.1 ± 1.1 µM). The assay format overcomes relevant limitations of the gold standard PRA, allowing concurrent analysis of candidate antiviral compounds against different viruses and providing preliminary information about early versus late antiviral activity.