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 (IC 50 = 0.5–5.3 μM) 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 (IC 50 = 0.5 μM, 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.

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Objectives : Our aim was to describe the longitudinal evolution of neutralizing antibody titres (NtAb) in three different cohorts of healthcare workers including vaccinated individuals with and without a previous SARS-CoV-2 infection and previously infected unvaccinated subjects. 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 HCW and after about 2 months (N_60), 10 months (N_300) and 13 months (N_390) from natural infection in unvaccinated HCW. NtAb was measured by authentic virus neutralization with a SARS-CoV-2 B.1 isolate circulating in Italy at HCW enrolment. Results : Sixty-two HCW were enrolled. NtAb were comparable in infected HCW with no or mild disease at all the study points. NtAb of uninfected HCW were significantly lower with respect to those of previously infected subjects at V1_20, V2_20 and V2_90. The median NtAb fold decrease from V2_20 to V2_90 was higher in the uninfected subjects with respect to subjects with mild infection (6.26 vs 2.58, p=0.03) and to asymptomatic HCW (6.26 vs 3.67, p=0.022). The median Nabt at N_390 was significantly lower with respect to N_60, p=0.007). Conclusions : In uninfected subjects 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 to previously infected, vaccinated subjects. Although at low level Nabt are still at detectable level after 13 months in two third of previously infected and not vaccinated subjects.

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Objectives To measure SARS-CoV-2 neutralizing antibody (NtAb) titres in previously infected or uninfected healthcare workers (HCW) who received one or two doses of BNT162b2 mRNA COVID-19 vaccine. Methods NtAbs were titrated as dose inhibiting 50% virus replication (ID 50 ) by live virus microneutralization. Forty-one HCW recovering from mild or asymptomatic infection were evaluated on the day of first-dose vaccination (T1_inf) and 21 days later (T2_inf). Sixteen uninfected HCW were evaluated 20 days after the first dose (T2_uninf) and 20 days after the second dose of vaccine (T3_inf). Results At T2_inf, but not at T1_inf, there was a significant correlation between days from diagnosis (median [IQR] 313 [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). Likewise, there was a significant increase in NtAb titres at T3_uninf with respect to 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.

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The described phenotypic assay can be adopted to evaluate the antiviral activity of licensed and investigational HIV-1 drugs targeting any of the three HIV-1 enzymes.

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