PHENOTYPIC CHARACTERIZATION OF NOVEL ANTIVIRALS FOR THE TREATMENT OF MULTIDRUG RESISTANT HIV-1 AND EMERGING VIRUSES

Abstract Phenotypic characterization of novel antivirals for the treatment of multidrug resistant HIV-1 and emerging viruses Doctoral Research School of Medical Biotechnologies – Cycle XXXV Supervisor: Maurizio Zazzi; Candidate: Federica Giammarino Background The need for new antiviral drugs has increased overtime due to the worldwide circulation of different viruses together with the increased frequency and diversity of new outbreaks. The ideal option for a prompt response against both emerging and re-emerging viruses is represented by the use and the development of direct acting antiviral agents. During my PhD I was involved in several projects focused on the evaluation of the antiviral activity of licensed and investigational antiviral drugs against Human Immunodeficiency (HIV-1), West Nile (WNV), Dengue (DENV) and SARS-CoV-2 viruses. Results and discussion Doravirine The antiviral activity of the NNRTI doravirine was evaluated against viruses harbouring different patterns of NNRTI resistance mutations in two studies. Globally, our data confirmed that the antiviral activity of doravirine may be compromised by the presence of multiple NNRTI resistance mutations, even in the absence of specific doravirine mutations. A third study was focused on the role of the natural polymorphism of the reverse transcriptase V106I. Our results indicate that it minimally affects the susceptibility to doravirine in clinical isolates and that it does not impact the genetic barrier to resistance as compared to reference wild-type virus, while viruses including the NNRTI resistant mutation V106A or V106M rapidly showed viral breakthrough under doravirine pressure due to the reduced susceptibility. Islatravir Our study confirmed the decrease of susceptibility of the investigational NRTTI islatravir due to the presence of M184V mutation. The clinical impact of NRTI mutations in the activity of islatravir has still to be defined and the threshold of fold-change values associated to reduced activity in vivo remains to be established. Ibalizumab The combinatorial activity of ibalizumab together with other antivirals, both approved and investigational, was evaluated through a newly developed cell-based assay consisting in the infection of the MOLT4-R5 cell line with the wild-type strains NL4-3 and AD8, and by the analysis of the results using the innovative software SynergyFinderPlus. Our data suggest that ibalizumab positively interacts with other antivirals with possible synergistic effects in select cases. Further studies are needed to determine the impact of Env variability and viral tropism in combination with other entry inhibitors. Development of a Cell-Based Immunodetection Assay for Simultaneous Screening of Antiviral Compounds Inhibiting Zika and Dengue Virus Replication An easy-to-perform and fast flavivirus immunodetection assay (IA) was developed to determine antiviral activity of promising compounds against ZIKV and DENV. The system, validated with references compounds against both viruses, was able to distinguish between the inhibitory effect of molecules targeting the early and the post-budding phase of viral replication cycle. Evaluation of sofosbuvir activity and resistance profile against West Nile virus in vitro Since the activity of sofosbuvir has been documented against different flaviviruses, we investigated whether it may exert an activity also against WNV. In both cell-based and enzymatic assays sofosbuvir was able to inhibit WNV replication in the low micromolar range. Moreover, in vitro selection and molecular docking experiments indicated that HCV and WNV share a similar sofosbuvir resistance pattern. ORIGINALE CHEMIAE in Antiviral Strategy - Origin and Modernization of Multi-Component Chemistry as a Source of Innovative Broad Spectrum Antiviral Strategy The “ORIGINALE CHEMIAE in Antiviral Strategy” project aims to identify promising broad-spectrum antivirals by taking advantage of the Multi-Component Chemistry strategy. Following the synthetization of molecules, their antiviral activity was determined in in vitro standardized virus-cell systems against DENV, WNV, HIV-1 and SARS-CoV-2. We identified eight molecules able to inhibit at least one of the viruses tested. However, their low selectivity indexes indicate the need to further improve the design of these molecules to increase the antiviral activity and/or reduce the cell toxicity in order to identify candidates for preclinical testing in animal models. Monoclonal antibodies and antivirals vs. SARS-CoV-2 After the development of a quantitative live-virus microneutralization assay, we evaluated the efficacy of licensed monoclonal Antibodies (mAbs) and the antiviral drugs remdesivir, nirmaltrevir and molnupiravir against different circulating SARS-CoV-2 variants. Our results showed that these drugs, contrary to the mAbs, retained activity against all tested variants. Conclusions A continuous challenge for public health is represented by the control of viral infections. Both vaccines and antiviral drugs may synergistically help to reduce the spread and the fatality of acute viral diseases and chronic infections. All the studies described in this thesis emphasize the role of the laboratory of virology within all the steps of the in vitro investigation of antiviral drugs, from the identification of molecules endowed with antiviral activity to the definition of the mechanism of action.