Polymeric nanoparticles and nanomicelles of hydroxychloroquine co-loaded with azithromycin potentiate anti-SARS-CoV-2 effect.

The outbreak of coronavirus (COVID-19) has put the world in an unprecedented scenario. To reestablish the world routine as promote the effective treatment of this disease, the world is looking for new (and old) drug that can efficiently kill the virus. In this study, we have developed two nanosystems: polymeric nanoparticles and nanomicelles-based on hydroxychloroquine and azithromycin. The nanosystem was fully characterized by AFM and DLS techniques. Also, the nanosystems were radiolabeled with 99mTc and pulmonary applied (installation) in vivo to evaluate the biological behavior. The toxicity of both nanosystem were evaluated in primary cells (FGH). Finally, both nanosystems were evaluated in vitro against the SARS-CoV-2. The results demonstrated that the methodology used to produce the nanomicelles and the nanoparticle was efficient, the characterization showed a nanoparticle with a spherical shape and a medium size of 390 nm and a nanomicelle also with a spherical shape and a medium size of 602 nm. The nanomicelles were more efficient (~ 70%) against SARS-CoV-2 than the nanoparticles. The radiolabeling process with 99mTc was efficient (> 95%) in both nanosystems and the pulmonary application demonstrated to be a viable route for both nanosystems with a local retention time of approximately, 24 h. None of the nanosystems showed cytotoxic effect on FGH cells, even in high doses, corroborating the safety of both nanosystems. Thus, claiming the benefits of the nanotechnology, especially with regard the reduced adverse we believe that the use of nanosystems for COVID-19 treatment can be an optimized choice. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40097-022-00476-3.

Robust specific RBD responses and neutralizing antibodies after ChAdOx1 nCoV-19 and CoronaVac vaccination in SARS-CoV-2- seropositive individuals.

Background: The pandemic unleashed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 500 million people worldwide and caused more than 6 million deaths. Cellular and humoral immunity induced by infection or immunization are key factors in controlling the viral burden and avoiding the recurrence of coronavirus disease. The duration and effectiveness of immunity after infection is relevant to pandemic policy interventions, including the timing of vaccine boosters. Objectives: We sought to evaluate longitudinal binding and functional antibodies against SARS-CoV-2 receptor-binding domain in police officers and health care workers with a history of coronavirus disease 2019 and compare with SARS-CoV-2-naive individuals after vaccination with adenovirus-based ChAdOx1 nCoV-19 (AstraZeneca-Fiocruz) or the inactivated CoronaVac vaccine (Sinovac-Butantan Institute). Methods: A total of 208 participants were vaccinated. Of these, 126 (60.57%) received the ChAdOx1 nCoV-19 vaccine and 82 (39.42%) received the CoronaVac vaccine. Prevaccination and postvaccination blood was collected, and the amount of anti-SARS-CoV-2 IgG and the neutralizing ability of the antibodies to block the interaction between angiotensin-converting enzyme 2 and receptor-binding domain were determined. Results: Subjects with preexisting SARS-CoV-2 immunity and who received a single dose of ChAdOx1 nCoV-19 or CoronaVac have similar or superior antibody levels when compared with levels in seronegative individuals even after 2 doses of the vaccine. Neutralizing antibody titers of seropositive individuals were higher with a single dose of either ChAdOx1 nCoV-19 or CoronaVac compared with those of seronegative individuals. After 2 doses, both groups reached a plateau response. Conclusions: Our data reinforce the importance of vaccine boosters to increase specific binding and neutralizing SARS-CoV-2 antibodies.

The third vaccine dose significantly reduces susceptibility to the B.1.1.529 (Omicron) SARS-CoV-2 variant.

The main coronavirus disease 2019 (COVID-19) vaccine formulations used today are mainly based on the wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein as an antigen. However, new virus variants capable of escaping neutralization activity of serum antibodies elicited in vaccinated individuals have emerged. The Omicron (B.1.1.529) variant caused epidemics in regions of the world in which most of the population has been vaccinated. In this study, we aimed to understand what determines individual's susceptibility to Omicron in a scenario of extensive vaccination. For that purpose, we collected nasopharynx swab (n = 286) and blood samples (n = 239) from flu-like symptomatic patients, as well as their vaccination history against COVID-19. We computed the data regarding vaccine history, COVID-19 diagnosis, COVID-19 serology, and viral genome sequencing to evaluate their impact on the number of infections. As main results, we showed that vaccination in general did not reduce the number of individuals infected by Omicron, even with an increased immune response found among vaccinated, noninfected individuals. Nonetheless, we found that individuals who received the third vaccine dose showed significantly reduced susceptibility to Omicron infections. A relevant evidence that support this finding was the higher virus neutralization capacity of serum samples of most patients who received the third vaccine dose. In summary, this study shows that boosting immune responses after a third vaccine dose reduces susceptibility to COVID-19 caused by the Omicron variant. Results presented in this study are useful for future formulations of COVID-19 vaccination policies.

The epitranscriptome of Vero cells infected with SARS-CoV-2 assessed by direct RNA sequencing reveals m6A pattern changes and DRACH motif biases in viral and cellular RNAs.

The epitranscriptomics of the SARS-CoV-2 infected cell reveals its response to viral replication. Among various types of RNA nucleotide modifications, the m6A is the most common and is involved in several crucial processes of RNA intracellular location, maturation, half-life and translatability. This epitranscriptome contains a mixture of viral RNAs and cellular transcripts. In a previous study we presented the analysis of the SARS-CoV-2 RNA m6A methylation based on direct RNA sequencing and characterized DRACH motif mutations in different viral lineages. Here we present the analysis of the m6A transcript methylation of Vero cells (derived from African Green Monkeys) and Calu-3 cells (human) upon infection by SARS-CoV-2 using direct RNA sequencing data. Analysis of these data by nonparametric statistics and two computational methods (m6anet and EpiNano) show that m6A levels are higher in RNAs of infected cells. Functional enrichment analysis reveals increased m6A methylation of transcripts involved in translation, peptide and amine metabolism. This analysis allowed the identification of differentially methylated transcripts and m6A unique sites in the infected cell transcripts. Results here presented indicate that the cell response to viral infection not only changes the levels of mRNAs, as previously shown, but also its epitranscriptional pattern. Also, transcriptome-wide analysis shows strong nucleotide biases in DRACH motifs of cellular transcripts, both in Vero and Calu-3 cells, which use the signature GGACU whereas in viral RNAs the signature is GAACU. We hypothesize that the differences of DRACH motif biases, might force the convergent evolution of the viral genome resulting in better adaptation to target sequence preferences of writer, reader and eraser enzymes. To our knowledge, this is the first report on m6A epitranscriptome of the SARS-CoV-2 infected Vero cells by direct RNA sequencing, which is the sensu stricto RNA-seq.

SARS-CoV-2 infection and replication kinetics in different human cell types: The role of autophagy, cellular metabolism and ACE2 expression.

AIMS: This study evaluated SARS-CoV-2 replication in human cell lines derived from various tissues and investigated molecular mechanisms related to viral infection susceptibility and replication. MAIN METHODS: SARS-CoV-2 replication in BEAS-2B and A549 (respiratory tract), HEK-293 T (kidney), HuH7 (liver), SH-SY5Y (brain), MCF7 (breast), Huvec (endothelial) and Caco-2 (intestine) was evaluated by RT-qPCR. Concomitantly, expression levels of ACE2 (Angiotensin Converting Enzyme) and TMPRSS2 were assessed through RT-qPCR and western blot. Proteins related to autophagy and mitochondrial metabolism were monitored in uninfected cells to characterize the cellular metabolism of each cell line. The effect of ACE2 overexpression on viral replication in pulmonary cells was also investigated. KEY FINDINGS: Our data show that HuH7, Caco-2 and MCF7 presented a higher viral load compared to the other cell lines. The increased susceptibility to SARS-CoV-2 infection seems to be associated not only with the differential levels of proteins intrinsically related to energetic metabolism, such as ATP synthase, citrate synthase, COX and NDUFS2 but also with the considerably higher TMPRSS2 mRNA expression. The two least susceptible cell types, BEAS-2B and A549, showed drastically increased SARS-CoV-2 replication capacity when ACE2 was overexpressed. These modified cell lines are relevant for studying SARS-CoV-2 replication in vitro. SIGNIFICANCE: Our data not only reinforce that TMPRSS2 expression and cellular energy metabolism are important molecular mechanisms for SARS-CoV-2 infection and replication, but also indicate that HuH7, MCF7 and Caco-2 are suitable models for mechanistic studies of COVID-19. Moreover, pulmonary cells overexpressing ACE2 can be used to understand mechanisms associated with SARS-CoV-2 replication.

3D Bioprinted Neural-Like Tissue as a Platform to Study Neurotropism of Mouse-Adapted SARS-CoV-2.

The effects of neuroinvasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) become clinically relevant due to the numerous neurological symptoms observed in Corona Virus Disease 2019 (COVID-19) patients during infection and post-COVID syndrome or long COVID. This study reports the biofabrication of a 3D bioprinted neural-like tissue as a proof-of-concept platform for a more representative study of SARS-CoV-2 brain infection. Bioink is optimized regarding its biophysical properties and is mixed with murine neural cells to construct a 3D model of COVID-19 infection. Aiming to increase the specificity to murine cells, SARS-CoV-2 is mouse-adapted (MA-SARS-CoV-2) in vitro, in a protocol first reported here. MA-SARS-CoV-2 reveals mutations located at the Orf1a and Orf3a domains and is evolutionarily closer to the original Wuhan SARS-CoV-2 strain than SARS-CoV-2 used for adaptation. Remarkably, MA-SARS-CoV-2 shows high specificity to murine cells, which present distinct responses when cultured in 2D and 3D systems, regarding cell morphology, neuroinflammation, and virus titration. MA-SARS-CoV-2 represents a valuable tool in studies using animal models, and the 3D neural-like tissue serves as a powerful in vitro platform for modeling brain infection, contributing to the development of antivirals and new treatments for COVID-19.

Corrigendum to "Nitazoxanide superiority to placebo to treat moderate COVID-19 - A Pilot prove of concept randomized double-blind clinical trial." [EClinicalMedicine 37 (2021) 100981].

[This corrects the article DOI: 10.1016/j.eclinm.2021.100981.].

One-year surveillance of SARS-CoV-2 in wastewater from vulnerable urban communities in metropolitan São Paulo, Brazil.

The current COVID-19 pandemic has emphasized the vulnerability of communities living in the urban outskirts and informal settlements. The lack of reliable COVID-19 case data highlights the importance and application of wastewater-based epidemiology. This study aimed to monitor the COVID-19 trends in four vulnerable urban communities (slums and low-income neighborhoods) in metropolitan São Paulo by assessing the SARS-CoV-2 RNA viral load in wastewater. We analyzed 160 samples from May 2020 to June 2021 with weekly or fortnightly samplings. The samples were ultracentrifuged with glycine elution and quantified by N1/N2 SARS-CoV-2 RT-qPCR. The results of positivity were 100% (Paraisópolis, Heliópolis and Cidade Tiradentes) and 76.9% (Vila Brasilândia). The new case numbers of COVID-19, counted from the onset of symptoms, positively correlated with SARS-CoV-2 N1 viral loads from the two largest communities (p<0.001). SARS-CoV-2 infectivity was tested in Vero E6 cells after concentration with the two techniques, ultrafiltration (Centricon® Plus-70 10 kDa) and sucrose cushion ultracentrifugation, but none of the evaluated samples presented positive results. Next-generation sequencing (NGS) analysis from samples collected in March and August 2021 revealed the presence of the clade 20 J (lineage P.1) belonging to the most prevalent circulating variant in the country. Our results showed that wastewater surveillance data can be used as complementary indicators to monitor the dynamics and temporal trends of COVID-19. The infectivity test results strengthened the evidence of low risk of infection associated with SARS-CoV-2 in wastewater.

Direct RNA Sequencing Reveals SARS-CoV-2 m6A Sites and Possible Differential DRACH Motif Methylation among Variants.

The causative agent of COVID-19 pandemic, SARS-CoV-2, has a 29,903 bases positive-sense single-stranded RNA genome. RNAs exhibit about 150 modified bases that are essential for proper function. Among internal modified bases, the N6-methyladenosine, or m6A, is the most frequent, and is implicated in SARS-CoV-2 immune response evasion. Although the SARS-CoV-2 genome is RNA, almost all genomes sequenced thus far are, in fact, reverse transcribed complementary DNAs. This process reduces the true complexity of these viral genomes because the incorporation of dNTPs hides RNA base modifications. Here, we present an initial exploration of Nanopore direct RNA sequencing to assess the m6A residues in the SARS-CoV-2 sequences of ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10 and the 3'-untranslated region. We identified fifteen m6A methylated positions, of which, six are in ORF N. Additionally, because m6A is associated with the DRACH motif, we compared its distribution in major SARS-CoV-2 variants. Although DRACH is highly conserved among variants, we show that variants Beta and Eta have a fourth position C > U change in DRACH at 28,884b that could affect methylation. This is the first report of direct RNA sequencing of a Brazilian SARS-CoV-2 sample coupled with the identification of modified bases.

A Single Dose of a Hybrid hAdV5-Based Anti-COVID-19 Vaccine Induces a Long-Lasting Immune Response and Broad Coverage against VOC.

Most approved vaccines against COVID-19 have to be administered in a prime/boost regimen. We engineered a novel vaccine based on a chimeric human adenovirus 5 (hAdV5) vector. The vaccine (named CoroVaxG.3) is based on three pillars: (i) high expression of Spike to enhance its immunodominance by using a potent promoter and an mRNA stabilizer; (ii) enhanced infection of muscle and dendritic cells by replacing the fiber knob domain of hAdV5 by hAdV3; (iii) use of Spike stabilized in a prefusion conformation. The transduction with CoroVaxG.3-expressing Spike (D614G) dramatically enhanced the Spike expression in human muscle cells, monocytes and dendritic cells compared to CoroVaxG.5 that expressed the native fiber knob domain. A single dose of CoroVaxG.3 induced a potent humoral immunity with a balanced Th1/Th2 ratio and potent T-cell immunity, both lasting for at least 5 months. Sera from CoroVaxG.3-vaccinated mice was able to neutralize pseudoviruses expressing B.1 (wild type D614G), B.1.117 (alpha), P.1 (gamma) and B.1.617.2 (delta) Spikes, as well as an authentic P.1 SARS-CoV-2 isolate. Neutralizing antibodies did not wane even after 5 months, making this kind of vaccine a likely candidate to enter clinical trials.

Nitazoxanide superiority to placebo to treat moderate COVID-19 - A Pilot prove of concept randomized double-blind clinical trial.

BACKGROUND: The absence of specific antivirals to treat COVID-19 leads to the repositioning of candidates' drugs. Nitazoxanide (NTZ) has a broad antiviral effect. METHODS: This was a randomized, double-blind pilot clinical trial comparing NTZ 600 mg BID versus Placebo for seven days among 50 individuals (25 each arm) with SARS-COV-2 RT-PCR+ (PCR) that were hospitalized with mild respiratory insufficiency from May 20th, 2020, to September 21st, 2020 (ClinicalTrials.gov NCT04348409). Clinical and virologic endpoints and inflammatory biomarkers were evaluated. A five-point scale for disease severity (SSD) was used. FINDINGS: Two patients died in the NTZ arm compared to 6 in the placebo arm (p = 0.564). NTZ was superior to placebo when considering SSD (p < 0001), the mean time for hospital discharge (6.6 vs. 14 days, p = 0.021), and negative PCR at day 21 (p = 0.035), whereas the placebo group presented more adverse events (p = 0.04). Among adverse events likely related to the study drug, 14 were detected in the NTZ group and 22 in placebo (p = 0.24). Among the 30 adverse events unlikely related, 21 occurred in the placebo group (p = 0.04). A decrease from baseline was higher in the NTZ group for d-Dimer (p = 0.001), US-RCP (p < 0.002), TNF (p < 0.038), IL-6 (p < 0.001), IL-8 (p = 0.014), HLA DR. on CD4+ T lymphocytes (p < 0.05), CD38 in CD4+ and CD8+ T (both p < 0.05), and CD38 and HLA-DR. on CD4+ (p < 0.01). INTERPRETATION: Compared to placebo in clinical and virologic outcomes and improvement of inflammatory outcomes, the superiority of NTZ warrants further investigation of this drug for moderate COVID-19 in larger clinical trials. A higher incidence of adverse events in the placebo arm might be attributed to COVID-19 related symptoms.