Humoral Immunity across the SARS-CoV-2 Spike after Sputnik V (Gam-COVID-Vac) Vaccination.

SARS-CoV-2 vaccines have contributed to attenuating the burden of the COVID-19 pandemic by promoting the development of effective immune responses, thus reducing the spread and severity of the pandemic. A clinical trial with the Sputnik-V vaccine was conducted in Venezuela from December 2020 to July 2021. The aim of this study was to explore the antibody reactivity of vaccinated individuals towards different regions of the spike protein (S). Neutralizing antibody (NAb) activity was assessed using a commercial surrogate assay, detecting NAbs against the receptor-binding domain (RBD), and a plaque reduction neutralization test. NAb levels were correlated with the reactivity of the antibodies to the spike regions over time. The presence of Abs against nucleoprotein was also determined to rule out the effect of exposure to the virus during the clinical trial in the serological response. A high serological reactivity was observed to S and specifically to S1 and the RBD. S2, although recognized with lower intensity by vaccinated individuals, was the subunit exhibiting the highest cross-reactivity in prepandemic sera. This study is in agreement with the high efficacy reported for the Sputnik V vaccine and shows that this vaccine is able to induce an immunity lasting for at least 180 days. The dissection of the Ab reactivity to different regions of S allowed us to identify the relevance of epitopes outside the RBD that are able to induce NAbs. This research may contribute to the understanding of vaccine immunity against SARS-CoV-2, which could contribute to the design of future vaccine strategies.

Neutralization of Different Variants of SARS-CoV-2 by a F(ab')2 Preparation from Sera of Horses Immunized with the Viral Receptor Binding Domain.

The Receptor Binding Domain (RBD) of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, is the functional region of the viral Spike protein (S), which is involved in cell attachment to target cells. The virus has accumulated progressively mutations in its genome, particularly in the RBD region, many of them associated with immune evasion of the host neutralizing antibodies. Some of the viral lineages derived from this evolution have been classified as Variant of Interest (VOI) or Concern (VOC). The neutralizing capacity of a F(ab')2 preparation from sera of horses immunized with viral RBD was evaluated by lytic plaque reduction assay against different SARS-CoV-2 variants. A F(ab')2 preparation of a hyperimmune serum after nine immunizations with RBD exhibited a high titer of neutralizing antibodies against the ancestral-like strain (1/18,528). A reduction in the titer of the F(ab')2 preparation was observed against the different variants tested compared to the neutralizing activity against the ancestral-like strain. The highest reduction in the neutralization titer was observed for the Omicron VOC (4.7-fold), followed by the Mu VOI (2.6), Delta VOC (1.8-fold), and Gamma VOC (1.5). Even if a progressive reduction in the neutralizing antibodies titer against the different variants evaluated was observed, the serum still exhibited a neutralizing titer against the Mu VOI and the Omicron VOC (1/7113 and 1/3918, respectively), the evaluated strains most resistant to neutralization. Therefore, the preparation retained neutralizing activity against all the strains tested.

Lasting SARS-CoV-2 specific IgG Antibody response in health care workers from Venezuela, 6 months after vaccination with Sputnik V.

BACKGROUND: Scarce information is available regarding the long-term immunogenicity of the Sputnik V vaccine. Here Sputnik V vaccinated subjects were evaluated 6 months after receiving the 2-dose prime-boost schedule. METHODS: Eighty-six hospital workers from Venezuela, 32 with a previous COVID-19 infection and 54 SARS-CoV-2 naïve subjects, were enrolled. IgG antibodies levels against the wild-type Receptor Binding Domain (RBD) were measured in an ELISA and with an in vitro ACE2-surrogate RBD binding inhibition assay at day 42 and day 180 after receiving the second dose. IgG levels were expressed in BAU/ml. Binding inhibition antibodies were expressed in IU/ml. RESULTS: On average, RBD-IgG levels decreased by approximately 50% between the two time-points in the COVID-19 naïve cohort (geometric mean concentration (GMC) 675 BAU/mL vs. 327 BAU/ml) and decreased by approximately 25% in the previously infected cohort (GMC 1209 BAU/mL vs 910 BAU/ml). Within our cohort, 94% showed a "good to excellent" neutralizing activity measured with the in vitro test 6 months after vaccination. CONCLUSIONS: The Sputnik V vaccine provided long-term and durable humoral immunity in our cohort specially if a person has been both vaccinated and had a previous infection with SARS-CoV-2.

Immunoglobulin G antibody response to the Sputnik V vaccine: previous SARS-CoV-2 seropositive individuals may need just one vaccine dose.

INTRODUCTION: We evaluated the immunoglobulin (Ig) G antibody response against the nucleocapsid protein (NP) and the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 in a cohort of 86 individuals in Venezuela, before and after receiving the Sputnik V vaccine. METHODS: Antibody responses against NP and RBD were determined with an enzyme-linked immunosorbent assay just before, 3 weeks after the first, and 6 weeks after the second dose of the vaccine. RESULTS: Before vaccination, 59 individuals were seronegative, and 27 seropositive for NP and/or RBD. Of the seronegative cohort, 42% did not develop an IgG immune response against RBD after the first vaccine dose, but 100% had a strong IgG response after 2 doses. All seropositive individuals developed a strong IgG antibody response against RBD after the first vaccine dose, with antibody levels ∼40% higher than seronegative individuals who had received 2 doses. Previously seropositive subjects showed no significant increase in IgG antibody response against RBD after the second vaccine dose. CONCLUSIONS: We demonstrate that 2 doses of the Sputnik V vaccine triggered antibody response in all study individuals. The second Sputnik V dose had no impact on IgG response for those seropositive for SARS-CoV-2 antigens before vaccination.

Inverse Molecular Docking Study of NS3-Helicase and NS5-RNA Polymerase of Zika Virus as Possible Therapeutic Targets of Ligands Derived from Marcetia taxifolia and Its Implications to Dengue Virus.

Dengue and Zika are two mosquito-borne diseases of great impact on public health around the world in tropical and subtropical countries. DENV and ZIKV belong to the Flaviviridae family and the Flavivirus genus. Currently, there are no effective therapeutic agents to treat or prevent these pathologies. The main objective of this work was to evaluate potential inhibitors from active compounds obtained from Marcetia taxifolia by performing inverse molecular docking on ZIKV-NS3-helicase and ZIKV-NS5-RNA polymerase as targets. This computational strategy is based on renormalizing the binding scores of the compounds to these two proteins, allowing a direct comparison of the results across the proteins. The crystallographic structures of the ZIKV-NS3-helicase and ZIKV-NS5-RNA-polymerase proteins share a great similarity with DENV homologous proteins. The P-loop active site of the crystallographic structure of ZIKV-NS3-helicase presents a high percentage of homology with the four dengue serotypes. It was found that most ligands of the active compounds (5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (5DP); 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5HH); myricetin-3-O-rhamnoside (M3OR)) from Marcetia taxifolia had a better affinity for ZIKV-NS3-helicase than for ZIKV-NS5-RNA polymerase, as indicated by the negative multiple active site correction (MASC) score, except for M3RG that showed a higher affinity for ZIKV-NS5-RNA polymerase. On the other hand, the AutoDock Vina scores showed that M3OR had the highest score value (-9.60 kcal/mol) and the highest normalized score (1.13) against ZIKV-NS3-helicase. These results in silico demonstrated that the nonstructural proteins NS3-helicase and NS5-RNA polymerase, which share similar molecular structures between the selected viruses, could become therapeutic targets for some bioactive compounds derived from Marcetia taxifolia.

Omicron SARS-CoV-2 Variant Spike Protein Shows an Increased Affinity to the Human ACE2 Receptor: An In Silico Analysis.

The rise of SARS-CoV-2 variants, with changes that could be related to an increased virus pathogenicity, have received the interest of the scientific and medical community. In this study, we evaluated the changes that occurred in the viral spike of the SARS-CoV-2 Omicron variant and whether these changes modulate the interactions with the angiotensin-converting enzyme 2 (ACE2) host receptor. The mutations associated with the Omicron variant were retrieved from the GISAID and covariants.org databases, and a structural model was built using the SWISS-Model server. The interaction between the spike and the human ACE2 was evaluated using two different docking software, Zdock and Haddock. We found that the binding free energy was lower for the Omicron variant as compared to the WT spike. In addition, the Omicron spike protein showed an increased number of electrostatic interactions with ACE2 than the WT spike, especially the interactions related to charged residues. This study contributes to a better understanding of the changes in the interaction between the Omicron spike and the human host ACE2 receptor.

Unrevealing sequence and structural features of novel coronavirus using in silico approaches: The main protease as molecular target.

Direct-acting antivirals are effective tools to control viral infections. SARS-CoV-2 is a coronavirus associated with the epidemiological outbreak in late 2019. Previous reports showed that HIV-1 protease inhibitors could block SARS-CoV main protease. Based on that and using an in silico approach, we evaluated SARS-CoV-2 main protease as a target for HIV-1 protease inhibitors to reveal the structural features related to their antiviral effect. Our results showed that several HIV inhibitors such as lopinavir, ritonavir, and saquinavir produce strong interaction with the active site of SARS-CoV-2 main protease. Furthermore, broad library protease inhibitors obtained from PubChem and ZINC (www.zinc.docking.org) were evaluated. Our analysis revealed 20 compounds that could be clustered into three groups based on their chemical features. Then, these structures could serve as leading compounds to develop a series of derivatives optimizing their activity against SARS-CoV-2 and other coronaviruses. Altogether, the results presented in this work contribute to gain a deep understanding of the molecular pharmacology of SARS-CoV-2 treatment and validate the use of protease inhibitors against SARS-CoV-2.

Role of changes in SARS-CoV-2 spike protein in the interaction with the human ACE2 receptor: An in silico analysis.

Many human viral diseases are a consequence of a zoonotic event. Some of the diseases caused by these zoonotic events have affected millions of people around the world, some of which have resulted in high rates of morbidity/mortality in humans. Changes in the viral proteins that function as ligands of the host receptor may promote the spillover between species. The most recent of these zoonotic events that have caused an ongoing epidemic of high magnitude is the Covid-19 epidemics caused by SARS-CoV-2. The aim of this study was to determine the mutation(s) in the sequence of the spike protein of the SARS-CoV-2 that might be favoring human to human transmission. An in silico approach was performed, and changes were detected in the S1 subunit of the receptor-binding domain of spike. The observed changes have significant effect on SARS-CoV-2 spike/ACE2 interaction and produce a reduction in the binding energy, compared to the one of the Bat-CoV to this receptor. The data presented in this study suggest a higher affinity of the SARS-Cov-2 spike protein to the human ACE2 receptor, compared to the one of Bat-CoV spike and ACE2. This could be the cause of the rapid viral spread of SARS-CoV-2 in humans.

Molecular characteristics and replication mechanism of dengue, zika and chikungunya arboviruses, and their treatments with natural extracts from plants: An updated review.

Viruses transmitted by arthropods (arboviruses) are the etiological agents of several human diseases with worldwide distribution; including dengue (DENV), zika (ZIKV), yellow fever (YFV), and chikungunya (CHIKV) viruses. These viruses are especially important in tropical and subtropical regions; where, ZIKV and CHIKV are involved in epidemics worldwide, while the DENV remains as the biggest problem in public health. Factors, such as, environmental conditions promote the distribution of vectors, deficiencies in health services, and lack of effective vaccines, guarantee the presence of these vector-borne diseases. Treatment against these viral diseases is only palliative since available therapies formulated lack to demonstrate specific antiviral activity and vaccine candidates fail to demonstrate enough effectiveness. The use of natural products, as therapeutic tools, is an ancestral practice in different cultures. According to WHO 80 % of the population of some countries from Africa and Asia depend on the use of traditional medicines to deal with some diseases. Molecular characteristics of these viruses are important in determining its cellular pathogenesis, emergence, and dispersion mechanisms, as well as for the development of new antivirals and vaccines to control strategies. In this review, we summarize the current knowledge of the molecular structure and replication mechanisms of selected arboviruses, as well as their mechanism of entry into host cells, and a brief overview about the potential targets accessed to inhibit these viruses in vitro and a summary about their treatment with natural extracts from plants.

Antiviral activity of flavonoids present in aerial parts of Marcetia taxifolia against Hepatitis B virus, Poliovirus, and Herpes Simplex Virus in vitro.

Marcetia taxifolia is a neotropical plant present in South America and it has been evaluated in several biological models due to the presence of active metabolites. Nevertheless, there is a limited quantity of studies related to the antiviral activity of the compounds present in this genus. In our work, the antiviral effect of the compounds isolated from the aerial parts of Marcetia taxifolia was evaluated against Hepatitis B virus (HBV), Herpes Simplex Virus type 1 (HSV-1), and Poliovirus type 1 (PV-1). The cytopathic effect and viral quantification by qPCR were determined as indicative of antiviral activity. Our data show that myricetin rhamnoside (MyrG), myricetin-3-α-O-ramnosil (1→6)-α-galactoside (MyrGG), 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (PMF), 5-hydroxy-3,6,7,3',4'pentamethoxyflavone (PMF-OH) had antiviral activity without cytotoxic effects. The methoxyflavones PMF and PMF-OH were the most active compounds, showing an antiviral effect against all the evaluated viruses. Computational studies showed that these compounds could interact with the Reverse Transcriptase. Altogether, these results suggest that the flavonoids (related to myricetin and methoxyflavones) are the main antiviral compounds present in the aerial parts of Marcetia taxifolia. Furthermore, our results showed that the methoxyflavones have a broad antiviral activity, which represents an opportunity to evaluate these flavonoids as lead molecules to develop new antiviral compounds.

HIV-1 and GBV-C co-infection in Venezuela.

INTRODUCTION: Co-infection with GB virus C (GBV-C) in patients infected with human immunodeficiency virus 1 (HIV-1) has been associated with prolonged survival. The aim of this study was to evaluate the prevalence of GBV-C infection among HIV-1-infected patients in Venezuela, and to determine the effects of the co-infection on the levels of relevant cytokines. METHODOLOGY: Plasma samples were collected from 270 HIV-1-seronegative and 255 HIV-1-seropositive individuals. GBV-C infection was determined by RT-PCR of the NS5 region and genotyped by sequence analysis of the 5´UTR region. HIV-1 strains were characterized by sequence analysis of pol, vif, env, and nef genes. Selected cytokines were evaluated by ELISA. RESULTS: Ninety-seven of 525 (18.5%) plasma samples tested positive for GBV-C RNA. A significantly higher prevalence of GBV-C was found among HIV-1 patients compared to HIV-1-seronegative individuals (67/255, 26% versus 30/270, 11%; p < 0.001). Statistical difference was observed in the viral load between HIV-1+GBV-C+ and HIV-1+GBV-C- (p = 0.014), although no differences in CD4+ cell counts were found between both groups. TNFα concentration was higher in HIV-1+GBV-C- than in HIV-1+GBV-C+ patients (25.9 pg/mL versus 17.3 pg/mL; p = 0.02); RANTES expression levels were more variable in GBV-C co-infected patients and more frequently elevated in HIV-1 mono-infected patients compared to patients co-infected with GBV-C. CONCLUSIONS: The previously observed beneficial effect of co-infection with HIV-1 and GBV-C on disease progression is complex and might be due in part to a change in the cytokine environment. More studies are required to understand the interaction between both viruses.

Deletion, insertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patients.

BACKGROUND: Variable progression towards AIDS has been described and has been related to viral and host factors. Around 10% of the HIV-1 infected patients are slow progressors (SP), not presenting with AIDS disease signs even after more than 10 years of infection. Viral gene defects have been associated with the disease progression but more studies are still needed. METHODOLOGY: The sequence of vif and nef were analyzed for HIV-1 infecting 14 SP and 46 normal progressors (NP) patients. RESULTS: Co-circulation of a strain carrying vif deleted gene with the wild type strain was detected in an SP patient with more than 10 years of infection. Other mutations (insertion in aa 63 in one strain, two premature stop codons in another one) were found in viruses infecting two other patients. Except for the SP8 strain, which exhibited a premature stop codon in nef, no gross deletions or insertions were observed in nef genes of both NPs and SPs strains analyzed. CONCLUSIONS: Different kind of mutation: deletion, insertion and stop codon, were detected in 3/14 samples from SP, with co-circulation of a 195 bp vif deletion virus with a wild type in one of these patients. Although vif defects do not seem to be a frequent feature in SPs, this study illustrates the importance of analysing this gene, in addition to the multiple factors associated with the long-term non progression to AIDS.

Prevalence of antiretroviral drug resistance among treatment-naive and treated HIV-infected patients in Venezuela.

An in-house, low-cost method was developed to determine the genotypic resistance of immunodeficiency virus type 1 (HIV-1) isolates. All 179 Venezuelan isolates analysed belonged to subtype B. Primary drug resistance mutations were found in 11% of 63 treatment-naïve patients. The prevalence of resistance in isolates from 116 HIV-positive patients under antiretroviral treatment was 47% to protease inhibitors, 65% to nucleoside inhibitors and 38% to non-nucleoside inhibitors, respectively. Around 50% of patients in the study harboured viruses with highly reduced susceptibility to the three classical types of drugs after only five years from their initial diagnoses.

Prevalence of antiretroviral drug resistance among treatment-naive and treated HIV-infected patients in Venezuela

An in-house, low-cost method was developed to determine the genotypic resistance of immunodeficiency virus type 1 (HIV-1) isolates. All 179 Venezuelan isolates analysed belonged to subtype B. Primary drug resistance mutations were found in 11 percent of 63 treatment-naïve patients. The prevalence of resistance in isolates from 116 HIV-positive patients under antiretroviral treatment was 47 percent to protease inhibitors, 65 percent to nucleoside inhibitors and 38 percent to non-nucleoside inhibitors, respectively. Around 50 percent of patients in the study harboured viruses with highly reduced susceptibility to the three classical types of drugs after only five years from their initial diagnoses.