A Gamma-adapted subunit vaccine induces broadly neutralizing antibodies against SARS-CoV-2 variants and protects mice from infection.

In the context of continuous emergence of SARS-CoV-2 variants of concern (VOCs), one strategy to prevent the severe outcomes of COVID-19 is developing safe and effective broad-spectrum vaccines. Here, we present preclinical studies of a RBD vaccine derived from the Gamma SARS-CoV-2 variant adjuvanted with Alum. The Gamma-adapted RBD vaccine is more immunogenic than the Ancestral RBD vaccine in terms of inducing broader neutralizing antibodies. The Gamma RBD presents more immunogenic B-cell restricted epitopes and induces a higher proportion of specific-B cells and plasmablasts than the Ancestral RBD version. The Gamma-adapted vaccine induces antigen specific T cell immune responses and confers protection against Ancestral and Omicron BA.5 SARS-CoV-2 challenge in mice. Moreover, the Gamma RBD vaccine induces higher and broader neutralizing antibody activity than homologous booster vaccination in mice previously primed with different SARS-CoV-2 vaccine platforms. Our study indicates that the adjuvanted Gamma RBD vaccine is highly immunogenic and a broad-spectrum vaccine candidate.

Longitudinal Follow-Up of the Immunity to SARS-CoV-2 in Health Care Workers in Argentina: Persistence of Humoral Response and Neutralizing Capacity after Sputnik V Vaccination.

SARS-CoV-2 vaccine protection has encountered waning of immune response and breakthrough infections. The hybrid immune response generated by the combination of vaccination and infection was shown to offer higher and broader protection. Here, we present a seroprevalence study of anti-SARS-CoV-2 spike/RBD IgG in 1,121 health care workers immunized with Sputnik V and a follow-up of humoral response at 2 and 24 weeks postvaccination (wpv), including neutralizing antibody response (NAT) against ancestral, Gamma, and Delta variants. The first seroprevalence study showed that among 122 individuals with one dose, 90.2% were seropositive versus 99.7% seropositivity among volunteers with the complete two-dose regimen. At 24 wpv, 98.7% of the volunteers remained seropositive, although antibody levels decreased. IgG levels and NAT were higher in individuals that had acquired COVID-19 previous to vaccination than in naive individuals at 2 and 24 wpv. Antibody levels dropped over time in both groups. In contrast, IgG levels and NAT increased after vaccine breakthrough infection. At 2 wpv, 35/40 naive individuals had detectable NAT against SARS-CoV-2 Gamma and 6/40 against Delta. In turn, 8/9 previously infected individuals developed a neutralizing response against SARS-CoV-2 Gamma and 4/9 against Delta variants. NAT against variants followed a trajectory similar to NAT against ancestral SARS-CoV-2, and breakthrough infection led to an increase in NAT and complete seroconversion against variants. In conclusion, Sputnik V-induced humoral response persisted at 6 months postvaccination, and hybrid immunity induced higher levels of anti-S/RBD antibodies and NAT in previously exposed individuals, boosted the response after vaccination, and conferred wider breadth of protection. IMPORTANCE Since December 2020, Argentina has begun a mass vaccination program. The first vaccine available in our country was Sputnik V, which has been approved for use in 71 countries with a total population of 4 billion people. Despite all the available information, there are fewer published studies on the response induced by Sputnik V vaccination compared to that of other vaccines. Although the global political context has paralyzed the verification by the WHO of the efficacy of this vaccine, our work aims to add new clear and necessary evidence to Sputnik V performance. Our results contribute to general knowledge of the humoral immune response developed by vaccines based on viral vector technology, highlighting the higher immune protection conferred by hybrid immunity and reinforcing the importance of completing vaccination schedules and booster doses to maintain adequate antibody levels.

A Novel Bacterial Protease Inhibitor Adjuvant in RBD-Based COVID-19 Vaccine Formulations Containing Alum Increases Neutralizing Antibodies, Specific Germinal Center B Cells and Confers Protection Against SARS-CoV-2 Infection in Mice.

In this work, we evaluated recombinant receptor binding domain (RBD)-based vaccine formulation prototypes with potential for further clinical development. We assessed different formulations containing RBD plus alum, AddaS03, AddaVax, or the combination of alum and U-Omp19: a novel Brucella spp. protease inhibitor vaccine adjuvant. Results show that the vaccine formulation composed of U-Omp19 and alum as adjuvants has a better performance: it significantly increased mucosal and systemic neutralizing antibodies in comparison to antigen plus alum, AddaVax, or AddaS03. Antibodies induced with the formulation containing U-Omp19 and alum not only increased their neutralization capacity against the ancestral virus but also cross-neutralized alpha, lambda, and gamma variants with similar potency. Furthermore, the addition of U-Omp19 to alum vaccine formulation increased the frequency of RBD-specific geminal center B cells and plasmablasts. Additionally, U-Omp19+alum formulation induced RBD-specific Th1 and CD8+ T-cell responses in spleens and lungs. Finally, this vaccine formulation conferred protection against an intranasal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge of K18-hACE2 mice.

Design and Optimization of Quinazoline Derivatives: New Non-nucleoside Inhibitors of Bovine Viral Diarrhea Virus.

Bovine viral diarrhea virus (BVDV) belongs to the Pestivirus genus (Flaviviridae). In spite of the availability of vaccines, the virus is still causing substantial financial losses to the livestock industry. In this context, the use of antiviral agents could be an alternative strategy to control and reduce viral infections. The viral RNA-dependent RNA polymerase (RdRp) is essential for the replication of the viral genome and constitutes an attractive target for the identification of antiviral compounds. In a previous work, we have identified potential molecules that dock into an allosteric binding pocket of BVDV RdRp via a structure-based virtual screening approach. One of them, N-(2-morpholinoethyl)-2-phenylquinazolin-4-amine [1, 50% effective concentration (EC50) = 9.7 ± 0.5 µM], was selected to perform different chemical modifications. Among 24 derivatives synthesized, eight of them showed considerable antiviral activity. Molecular modeling of the most active compounds showed that they bind to a pocket located in the fingers and thumb domains in BVDV RdRp, which is different from that identified for other non-nucleoside inhibitors (NNIs) such as thiosemicarbazone (TSC). We selected compound 2-[4-(2-phenylquinazolin-4-yl)piperazin-1-yl]ethanol (1.9; EC50 = 1.7 ± 0.4 µM) for further analysis. Compound 1.9 was found to inhibit the in vitro replication of TSC-resistant BVDV variants, which carry the N264D mutation in the RdRp. In addition, 1.9 presented adequate solubility in different media and a high-stability profile in murine and bovine plasma.

Identification of potent bovine viral diarrhea virus inhibitors by a structure-based virtual screening approach.

Bovine viral diarrhea virus (BVDV) is a pestivirus whose infection in cattle is globally distributed. The use of antivirals could complement vaccination as a tool of control and reduce economic losses. The RNA-dependent RNA polymerase (RdRp) of the virus is essential for its genome replication and constitutes an attractive target for the identification of antivirals. With the aim of obtaining selective BVDV inhibitors, the crystal structure of BVDV RdRp was used to perform a virtual screening. Approximately 15,000 small molecules from commercial and in-house databases were evaluated and several structurally different compounds were tested in vitro for antiviral activity. Interestingly, of twelve evaluated compounds, five were active and displayed EC50 values in the sub and low-micromolar range. Time of drug addition experiment and measured intracellular BVDV RNA showed that compound 7 act during RNA synthesis. Molecular Dynamics and MM/PBSA calculation were done to characterize the interaction of the most active compounds with RdRp, which will allow future ligand optimization. These studies highlight the use of in silico screening to identify a new class of BVDV inhibitors.

Synthesis, antiviral evaluation and molecular docking studies of N4-aryl substituted/unsubstituted thiosemicarbazones derived from 1-indanones as potent anti-bovine viral diarrhea virus agents.

A series of N4-arylsubstituted thiosemicarbazones derived from 1-indanones and a set of compounds lacking such substitution in the N4 position of the thiosemicarbazone moiety were synthesized and evaluated for their anti-bovine viral diarrhea virus (BVDV) activity. Among these, derivatives 2 and 15 displayed high activity (EC50=2.7±0.4 and 0.7±0.1µM, respectively) as inhibitors of BVDV replication. Novel key structural features related to the anti-BVDV activity were identified by structure-activity relationship (SAR) analysis. In a previous study, the thiosemicarbazone of 5,6-dimethoxy-1-indanone (5,6-TSC) was characterized as a non-nucleoside inhibitor (NNI) of the BVDV RNA-dependent RNA polymerase. In the present work, cross-resistance assays were performed with the most active compounds. Such studies were carried out on 5,6-TSC resistant BVDV (BVDV-TSCr T1) carrying mutations in the viral polymerase. This BVDV mutant was also resistant to compound 15. Molecular docking studies and MM/PBSA calculations were performed to assess the most active derivatives at the 5,6-TSC viral polymerase binding site. The differences in the interaction pattern and the binding affinity of derivative 15 either to the wild type or BVDV-TSCr T1 polymerase were key factors to define the mode of action of this compound.

Complexation of a 1-Indanone Thiosemicarbazone with Hydroxypropyl-ß-Cyclodextrin Enhances Its Activity Against a Hepatitis C Virus Surrogate Model.

The current standard of care of the infection by hepatitis C virus (HCV) is effective in a limited number of patients and the high cost hinders therapy affordability and compliance. In this context, the research of new direct-acting antiviral agents (DAAs) for a more effective and long-lasting therapy is an urgent need and an area of active investigation. In an effort to develop novel DAAs, a series of 1-indanone thiosemicarbazones (TSCs) was synthesized and fully characterized. However, the high self-aggregation tendency and extremely poor aqueous solubility of these antiviral candidates often preclude their reliable biological evaluation in vitro. To maintain constant TSC concentrations over the biological assays, different TSC/cyclodextrin complexes were produced. In the present work, we report for the first time the cytotoxicity and antiviral activity of 5,6-dimethoxy TSC inclusion complexes with hydroxypropyl-ß-cyclodextrin on bovine viral diarrhea virus (BVDV) as HCV surrogate model. Results showed a potent suppression of the virus replication, with greater activity for the inclusion complexes than the free compound.

Stability of the resistance to the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone, a non-nucleoside polymerase inhibitor of bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) is the prototype Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC) is a non-nucleoside polymerase inhibitor (NNI) of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1-5) present an N264D mutation in the NS5B gene (RdRp) whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of BVDV-TSCr T1-5 in MDBK cells in the absence of TSC to evaluate the stability of the resistance. The viral populations obtained (BVDV R1-5) remained resistant to the antiviral compound and conserved the mutations in NS5B associated with this phenotype. Along the passages, BVDV R2, R3 and R5 presented a delay in the production of cytopathic effect that correlated with a decrease in cell apoptosis and intracellular accumulation of viral RNA. The complete genome sequences that encode for NS2 to NS5B, Npro and Erns were analyzed. Additional mutations were detected in the NS5B of BVDV R1, R3 and R4. In both BVDV R2 and R3, most of the mutations found were localized in NS5A, whereas in BVDV R5, the only mutation fixed was NS5A V177A. These results suggest that mutations in NS5A could alter BVDV cytopathogenicity. In conclusion, the stability of the resistance to TSC may be due to the fixation of different compensatory mutations in each BVDV-TSCr. During their replication in a TSC-free medium, some virus populations presented a kind of interaction with the host cell that resembled a persistent infection: decreased cytopathogenicity and viral genome synthesis. This is the first report on the stability of antiviral resistance and on the evolution of NNI-resistant BVDV variants. The results obtained for BVDV-TSCr could also be applied for other NNIs.

Inhibition of bovine viral diarrhea virus RNA synthesis by thiosemicarbazone derived from 5,6-dimethoxy-1-indanone.

In the present work, we described the activity of the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone (TSC), which we previously characterized as a new compound that inhibits bovine viral diarrhea virus (BVDV) infection. We showed that TSC acts at a point of time that coincides with the onset of viral RNA synthesis and that it inhibits the activity of BVDV replication complexes (RCs). Moreover, we have selected five BVDV mutants that turned out to be highly resistant to TSC but still susceptible to ribavirin (RBV). Four of these resistant mutants carried an N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). The remaining mutant showed an A392E mutation within the same protein. Some of these mutants replicated slower than the wild-type (wt) virus in the absence of TSC, whereas others showed a partial reversion to the wt phenotype over several passages in the absence of the compound. The docking of TSC in the crystal structure of the BVDV RdRp revealed a close contact between the indane ring of the compound and several residues within the fingers domain of the enzyme, some hydrophobic contacts, and hydrogen bonds with the thiosemicarbazone group. Finally, in the mutated RdRp from resistant BVDV, these interactions with TSC could not be achieved. Interestingly, TSC inhibited BVDV replication in cell culture synergistically with RBV. In conclusion, TSC emerges as a new nonnucleoside inhibitor of BVDV RdRp that is synergistic with RBV, a feature that turns it into a potential compound to be evaluated against hepatitis C virus (HCV).

Antiviral activity of 5'-O-carbonate-2',3'-dideoxy-3'-thiacytidine prodrugs against hepatitis B virus in HepG2 2.2.15 cells.

The antiviral activities of lamivudine (3TC; 2',3'-dideoxy-3'-thiacytidine) and six 5'-O-carbonates of 3TC were determined by inhibition of hepatitis B virus (HBV) replication in HepG2 2.2.15 cells. HBV DNA in cell supernatants was quantified by real-time polymerase chain reaction (PCR). The results showed that 3TC-Etha was six times more active than 3TC and that 3TC-Buta, 3TC-Hexa and 3TC-Octa were approximately three times more active than 3TC. In contrast, 3TC-Penta and 3TC-Metha showed anti-HBV activity similar to that of the parent compound 3TC. In conclusion, 5'-O-carbonates of 3TC appear to be promising candidates as anti-HBV compounds. This modification could optimise the use of 3TC, a well-tolerated, effective and inexpensive drug, in monotherapy or combined therapy for chronic HBV infections as well as human immunodeficiency virus (HIV)/HBV co-infections.

New 1-indanone thiosemicarbazone derivatives active against BVDV.

Identification of new therapeutic agents for the treatment of viral diseases represents an area of active investigation. In an effort to develop new antiviral compounds, a series of 1-indanone thiosemicarbazone derivatives were synthesized. These derivatives were structurally characterized using several spectroscopic techniques and evaluated against bovine viral diarrhoea virus as a surrogate model for hepatitis C virus. Thiosemicarbazone 2m showed potent anti-bovine viral diarrhoea virus activity with a higher selectivity index (SI=80.29) than that of ribavirin (SI=11.64). This result determines the potentiality of these thiosemicarbazones as antiviral agents for the treatment of infections caused by other highly related members of Flaviviridae family, as hepatitis C virus.