Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections (preprint)

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin’s efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight significantly reduces the viral load in the trachea. Our results show that the inhibition of cathepsins, a protein family of the host organism, is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections. An intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets.

Zoonotic potential of a novel bat morbillivirus (preprint)

Bats are significant reservoir hosts for many viruses with zoonotic potential 1 . SARS-CoV-2, Ebola virus, and Nipah virus are examples of such viruses that have caused deadly epidemics and pandemics when spilled over from bats into human and animal populations 2,3 . Careful surveillance of viruses in bats is critical for identifying potential zoonotic pathogens. However, metagenomic surveys in bats often do not result in full-length viral sequences that can be used to regenerate such viruses for targeted characterization 4 . Here, we identify and characterize a novel morbillivirus from a vespertilionid bat species ( Myotis riparius ) in Brazil, which we term myotis bat morbillivirus (MBaMV). There are 7 species of morbilliviruses including measles virus (MeV), canine distemper virus (CDV) and rinderpest virus (RPV) 5 . All morbilliviruses cause severe disease in their natural hosts 6–10 , and pathogenicity is largely determined by species specific expression of canonical morbillivirus receptors, CD150/SLAMF1 11 and NECTIN4 12 . MBaMV used Myotis spp CD150 much better than human and dog CD150 in fusion assays. We confirmed this using live MBaMV that was rescued by reverse genetics. Surprisingly, MBaMV replicated efficiently in primary human myeloid but not lymphoid cells. Furthermore, MBaMV replicated in human epithelial cells and used human NECTIN4 almost as well as MeV. Our results demonstrate the unusual ability of MBaMV to infect and replicate in some human cells that are critical for MeV pathogenesis and transmission. This raises the specter of zoonotic transmission of a bat morbillivirus.

Immunization with SARS-CoV-2 nucleocapsid protein triggers a pulmonary immune response in rats (preprint)

ABSTRACT The SARS-CoV-2 pandemic have been affecting millions of people worldwide, since the beginning of 2020. COVID-19 can cause a wide range of clinical symptoms, which varies from asymptomatic presentation to severe respiratory insufficiency, exacerbation of immune response, disseminated microthrombosis and multiple organ failure, which may lead to dead. Due to the rapid spread of SARS-CoV-2, the development of vaccines to minimize COVID-19 severity in the world population is imperious. One of the employed techniques to produce vaccines against emerging viruses is the synthesis of recombinant proteins, which can be used as immunizing agents. Based on the exposed, the aim of the present study was to verify the systemic and immunological effects of IM administration of recombinant Nucleocapsid protein (NP), derived from SARS-CoV-2 and produced by this research group, in 2 different strains of rats ( Rattus norvegicus ); Wistar and Lewis. For this purpose, experimental animals received 4 injections of NP, once a week, and were submitted to biochemical and histological analysis. Our results showed that NP inoculations were safe for the animals, which presented no clinical symptoms of worrying side effects, nor laboratorial alterations in the main biochemical and histological parameters, suggesting the absence of toxicity induced by NP. Moreover, NP injections successfully triggered the production of specific anti-SARS-CoV-2 IgG antibodies by both Wistar and Lewis rats, showing the sensitization to have been well sufficient for the immunization of these strains of rats. Additionally, we observed the local lung activation of the Bronchus-Associated Lymphoid Tissue (BALT) of rats in the NP groups, suggesting that NP elicits specific lung immune response. Although pre-clinical and clinical studies are still required, our data support the recombinant NP produced by this research group as a potential immunizing agent for massive vaccination, and may represent advantages upon other recombinant proteins, since it seems to induce specific pulmonary protection.

Beneficial effects of a mouthwash containing an antiviral phthalocyanine derivative on the length of hospital stay for COVID-19: Randomised trial (preprint)

Background: The risk of contamination and dissemination by SARS-CoV-2 has a strong link with nasal, oral and pharyngeal cavities. Recently, our research group observed the promising performance of an anionic phthalocyanine derivative (APD) used in a mouthwash protocol without photoexcitation; this protocol improved the general clinical condition of patients infected with SARS-CoV-2. Methods: The present two-arm study evaluated in vitro the antiviral activity and cytotoxicity of APD. Additionally, a triple-blind randomized controlled trial was conducted with 41 hospitalized patients who tested positive for COVID-19. All the included patients received World Health Organization standard care hospital treatment (non-intensive care) plus active mouthwash (experimental group AM/n=20) or nonactive mouthwash (control group NAM/n=21). The adjunct mouthwash intervention protocol used in both groups consisted one-minute gargling/rinsing / 5 times/day until hospital discharge. Groups were compared considering age, number of comorbidities, duration of symptoms prior admission and length of hospital stay (LOS). The associations between group and sex, age range, presence of comorbidities, admission to Intensive care unit (ICU) and death were also evaluated. Results: The in vitro evaluation demonstrated that APD compound was highly effective for reduction of SARS-CoV-2 viral load in the 1.0 mg/mL (99.96%) to 0.125 mg/mL (92.65%) range without causing cytotoxicity. Regarding the clinical trial, the median LOS of the AM group was significantly shortened (4 days) compared with that of the NAM group (7 days) (p=0.0314). Additionally, gargling/rinsing with APD was very helpful in reducing the severity of symptoms (no ICU care was needed) compared to not gargling/rinsing with APD (28.6% of the patients in the NAM group needed ICU care, and 50% of this ICU subgroup passed way, p=0.0207). Conclusions: This study indicated that the mechanical action of the protocol involving mouthwash containing a compound with antiviral effects against SARS-CoV-2 may reduce the symptoms of the patients and the spread of infection. The use of APD in a mouthwash as an adjuvant the hospital COVID-19 treatment presented no contraindication and reduced the hospital stay period. Trial Registration: The clinical study was registered at REBEC - Brazilian Clinical Trial Register (RBR-58ftdj) in 10/28/2020.

Correlation of Body Mass Index (BMI), initial neutralizing antibodies (nAb), ABO group and kinetics of nAb and anti-nucleocapsid (NP) SARS-CoV-2 antibodies in convalescent plasma (CCP) donors: A longitudinal study with proposals for better quality of CCP collections. (preprint)

IntroductionA cohort of COVID-19 convalescent volunteers allowed the study of neutralizing (nAb) and ligand antibodies kinetics by providing sequential samples during a median of 100 days after onset of disease. Material and MethodsA cohort of previously RT-PCR+ve (detected by nasopharyngeal swab during the acute phase), male convalescent patients, all with mild symptoms, were enrolled on serial blood sample collection for evaluation of longitudinal nAb titers and anti-nucleocapsid (NP) antibodies (IgM, IgG and IgA). Nabs were detected by a cytopathic effect-based virus neutralization test (CPE-based VNT), carried out with SARS-CoV-2 (GenBank: MT350282) ResultsA total of 78 male volunteers provided 316 samples, spanning a total of 4820 days of study. Although only 25% of donors kept nAb titers [≥]160, after a median of 100 days after the onset of disease, there was a high probability of sustaining nAB titers [≥]160 in volunteers whose initial nAb titer was [≥]1280, weight [≥] 90kg or BMI classified as overweight or obese, evidenced by Kaplan-Meier estimates and Cox hazard regression. There was no correlation between ABO group, ABO antibody titers and persistent high nAb titers. High IgG anti-NP (S/CO [≥]5.0) is a good surrogate for detecting nAB [≥]160, defined by ROC curve (sensitivity = 90.5%; CI95% 84.5-94.7%) ConclusionSelection of CCP donors for multiple collections based on initial high nAb titers ([≥]1280) or overweight/obese (BMI) provides a simple strategy to achieve higher quality in CCP programs. High IgG anti-NP levels can also be used as surrogate markers for high nAb screening.

CORRELATION BETWEEN SARS-COV-2 ANTIBODY SCREENING BY IMMUNOASSAY AND NEUTRALIZING ANTIBODY TESTING (preprint)

BackgroundPassive antibody therapy with convalescent plasma (CP) represents a promising alternative for the treatment of SARS-CoV-2 infection. The efficacy of CP therapy has been associated with high titers of neutralizing antibodies (nAbs) in the plasma of recovered patients, but the assays for quantifying nAbs are not widely available. Our goal was to develop a strategy to predict high titers of nAbs based on the results of anti-SARS-CoV-2 immunoassays and the clinical characteristics of the CP potential donors. MethodsTwo hundred and fourteen CP donors were enrolled and tested for the presence of anti-SARS-CoV-2 antibodies using two commercial immunoassays (IA): Anti-SARS-CoV-2 ELISA IgG EUROIMMUN and Anti-SARS-CoV-2 Chemiluminescence IgG Abbott. In parallel, quantification of neutralizing antibodies (nAbs) was performed using the Cytopathic effect-based virus neutralization test (CPE-VNT). Three criteria for identifying donors with high titers of nAbs ([≥]1:160) were tested: - Criterion1: Curve ROC Method; - Criterion 2: Conditional decision tree considering only the results from the IA and -Criterion 3: Conditional decision tree including both the IA results and the clinical variables. ResultsThe performance of Abbott and EUROIMMUN immunoassays was similar referring to both S/CO and predictive value for identifying nAbs titers [≥] 1:160. Regarding the three studied criteria for identifying CP donors with high nAbs titers ([≥] 1:160): 1) Criterion 1 showed 76.1% accuracy when the S/CO cut-off of 4.65 was used, 2) Criterion 2 presented 76.1% accuracy if the S/CO [≥] 4.57 was applied and 3) Criterion 3 had 71.6% accuracy if either S/CO [≥] 4.57 or S/CO between 2.68 and 4.57 and the last COVID-19-related symptoms occurred less than 19 days from donor recruiting were used. ConclusionThe results of SARS-CoV-2 immunoassays (S/CO) can be used to predict high nAbs titers of potential CP donors. This study has proposed three different criteria for identifying donors with [≥] 1:160 nAbs titer based on either solely S/CO results or S/CO together with clinical variables, all with high efficacy and accuracy.