DETECÇÃO DE ANTICORPOS NEUTRALIZANTES CONTRA AS VARIANTES DELTA, GAMA E OMICRON APÓS IMUNIZAÇÃO POR CORONAVAC E BOOSTER COM PFIZER

Introdução A vacinação é uma ferramenta essencial para o controle da infecção por SARS-CoV-2 e da pandemia de COVID-19. O surgimento de novas variantes genéticas do vírus SARS-CoV-2 nos trouxe a questão se há diferencial capacidade neutralizante dos anticorpos quanto às variantes de preocupação (VOCs). Objetivo Nosso estudo se dirigiu a avaliar a capacidade neutralizante dos anticorpos de indivíduos imunizados com a vacina CoronaVac e dose de reforço com Pfizer contra as variantes Gama, Delta e Omicron. Método Amostras de soro foram obtidas de 41 profissionais da saúde da Faculdade de Medicina da USP, sem infecção prévia por SARS-CoV-2 no esquema vacinal CoronaVac (2 doses) seguido de dose booster com vacina Pfizer. Os níveis de anticorpos neutralizantes para as variantes Gama, Delta e Omicron foram avaliados 32 e 186 dias após a segunda dose da vacina. Também avaliamos a atividade neutralizante dos anticorpos contra a variante Omicron em 39 dos indivíduos após 62 dias de imunização de reforço, com a vacina Pfizer. Os títulos de anticorpos foram obtidos pelo Teste de Neutralização Viral (VNT) e observação de efeito citopático. Resultados A neutralização por anticorpos contra as variantes Gama, Delta e Omicron foi de 78%, 65.9% e 58.5% respectivamente, após uma média de 32 dias após a segunda dose por CoronaVac. Houve uma diminuição na frequência de anticorpos neutralizantes para 17.1%, 24.4% e 2.4% contra as variantes Gama, Delta e Omicron, respectivamente, após, em média 186 dias das duas doses da vacina CoronaVac. A dose booster com a vacina Pfizer foi capaz de induzir a produção de anticorpos neutralizantes contra a variante Omicron em 87.2% dos indivíduos avaliados. Conclusão Os indivíduos vacinados com CoronaVac apresentaram uma queda nítida de anticorpos neutralizantes contra as 3 variantes de SARS-CoV-2 analisadas após 186 dias da imunização por 2 doses. A dose de reforço com Pfizer induziu a produção de anticorpos neutralizantes contra a variante Omicron na maior parte dos indivíduos avaliados (87.2%), 60 dias após imunização. Não houve diferença significativa na frequência de anticorpos neutralizantes entre as variantes analisadas.

Absence of neutralizing antibodies against the Omicron SARS-CoV-2 variant in convalescent sera from individuals infected with the ancestral SARS-CoV-2 virus or its Gamma variant.

OBJECTIVES: The aim of the present study was to evaluate if neutralizing antibody responses induced by infection with the SARS-CoV-2 strain that was dominant at the beginning of the pandemic or by the Gamma variant was effective against the Omicron variant. METHODS: Convalescent sera from 109 individuals, never exposed to a SARS-CoV-2 vaccine, who had mild or moderate symptoms not requiring hospitalization following either a documented SARS-CoV-2 ancestral strain infection or a Gamma variant infection, were assayed for in vitro neutralizing antibody activity against their original strains and the Omicron variant. RESULTS: Following an infection with the ancestral strain, 56 (93.3%), 45 (77.6%) and 1 (1.7%) serum sample were positive for neutralizing antibodies against the ancestral, Gamma variant, and Omicron variant, respectively. After infection with the Gamma variant, 43 (87.8%) and 2 (4.1%) sera were positive for neutralizing antibodies against the Gamma and Omicron variants, respectively. CONCLUSIONS: Neutralizing antibodies generated following mild or moderate infection with the SARS-CoV-2 ancestral strain or the Gamma variant are not protective against the Omicron variant.

Generation of neutralizing antibodies against Omicron, Gamma and Delta SARS-CoV-2 variants following CoronaVac vaccination.

Vaccination is a fundamental tool to prevent SARS-CoV-2 infection and to limit the COVID-19 pandemic. The emergence of SARS-CoV-2 variants with multiple mutations has raised serious concerns about the ability of neutralizing antibody responses elicited by prior vaccination to effectively combat these variants. The neutralizing capacity against the Gamma, Delta and Omicron variants of sera from individuals immunized with the CoronaVac vaccine remains incompletely determined. The present study evaluated 41 health care workers at the Faculdade de Medicina of the Universidade de Sao Paulo, in Sao Paulo, Brazil, naive to previous SARS- CoV-2 infection, who were vaccinated with two doses of the CoronaVac SARS-CoV-2 vaccine 28 days apart. Neutralizing antibody levels against the Gamma, Delta, and Omicron variants were measured at 32 and 186 days after the second vaccination. We also measured neutralizing antibodies against Omicron in 34 of these individuals following a subsequent booster immunization with the Pfizer vaccine. Quantification of neutralizing antibodies was performed using the Cytopathic Effect-based Virus Neutralization test. Neutralization antibody activity against the Gamma, Delta and Omicron variants was observed in 78.0%, 65.9% and 58.5% of serum samples, respectively, obtained at a mean of 32 days after the second immunization. This decreased to 17.1%, 24.4% and 2.4% of sera having activity against Delta, Gamma and Omicron, respectively, at 186 days post-vaccination. The median neutralizing antibody titers at 32 days were 1:40, 1:20 and 1:20 against Gamma, Delta and Omicron, respectively, and decreased to an undetectable median level against all variants at the later time. A booster immunization with the Pfizer vaccine elicited neutralizing antibodies against Omicron in 85% of subjects tested 60 days after vaccination. We conclude that two doses of the CoronaVac vaccine results in limited protection of short duration against the Gamma, Delta and Omicron SARS-CoV-2 variants. A booster dose with the Pfizer vaccine induced antibody neutralizing activity against Omicron in most patients which was measurable 60 days after the booster.

SARS-CoV-2 recombinant proteins stimulate distinct cellular and humoral immune response profiles in samples from COVID-19 convalescent patients.

OBJECTIVES: In this preliminary study we investigated cellular and humoral immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in blood samples from 14 recovered coronavirus disease 2019 (COVID-19) patients and compared them to those in samples from 12 uninfected/unvaccinated volunteers. METHODS: Cellular immunity was assessed by intracellular detection of IFN-γ in CD3+ T lymphocytes after stimulation with SARS-CoV-2 spike (S1), nucleocapsid (NC), or receptor-binding domain (RBD) recombinant proteins or overlapping peptide pools covering the sequence of SARS-CoV-2 spike, membrane and nucleocapsid regions. The humoral response was examined by ELISAs and/or chemiluminescence assays for the presence of serum IgG antibodies directed to SARS-CoV-2 proteins. RESULTS: We observed differences between humoral and cellular immune profiles in response to stimulation with the same proteins. Assays of IgG antibodies directed to SARS-CoV-2 NC, RBD and S1/S2 recombinant proteins were able to differentiate convalescent from uninfected/unvaccinated groups. Cellular immune responses to SARS-CoV-2 protein stimuli did not exhibit a specific response, as T cells from both individuals with no history of contact with SARS-CoV-2 and from recovered donors were able to produce IFN-γ. CONCLUSIONS: Determination of the cellular immune response to stimulation with a pool of SARS-CoV-2 peptides but not with SARS-CoV-2 proteins is able to distinguish convalescent individuals from unexposed individuals. Regarding the humoral immune response, the screening for serum IgG antibodies directed to SARS-CoV-2 proteins has been shown to be specific for the response of recovered individuals.

Torquetenovirus in saliva: A potential biomarker for SARS-CoV-2 infection?

Torquetenovirus (TTV) is present in biological fluids from healthy individuals and measurement of its titer is used to assess immune status in individuals with chronic infections and after transplants. We assessed if the titer of TTV in saliva varied with the presence of SARS-CoV-2 in the nasopharynx and could be a marker of COVID-19 status. Saliva from 91 individuals positive for SARS-CoV-2 in nasal-oropharyngeal samples, and from 126 individuals who were SARS-CoV-2-negative, all with mild respiratory symptoms, were analyzed. Both groups were similar in age, gender, symptom duration and time after symptom initiation when saliva was collected. Titers of TTV and SARS-CoV-2 were assessed by gene amplification. Loss of smell (p = 0.0001) and fever (p = 0.0186) were more prevalent in SARS-CoV-2-positive individuals, while sore throat (p = 0.0001), fatigue (p = 0.0037) and diarrhea (p = 0.0475) were more frequent in the SARS-CoV-2 negative group. The saliva TTV and nasal-oropharyngeal SARS-CoV-2 titers were correlated (p = 0.0085). The TTV level decreased as symptoms resolved in the SARS-CoV-2 infected group (p = 0.0285) but remained unchanged in the SARS-CoV-2 negative controls. In SARS-CoV-2 positive subjects who provided 2-4 saliva samples and in which TTV was initially present, the TTV titer always decreased over time as symptoms resolved. We propose that sequential TTV measurement in saliva is potentially useful to assess the likelihood of symptom resolution in SARS-CoV-2-positive individuals and to predict prognosis.

SARS-Cov-2 seroprevalence and risk factors among health care workers: Estimating the risk of COVID-19 dedicated units.

We evaluated the seroprevalence of SARS-CoV-2 and risk factors among 1,996 oligo/asymptomatic health care workers. The seroprevalence was 5.5% and risk factors associated with being infected with SARS-CoV-2 was professional category of cleaning (adj odds ratio 2.22, 95% confidence interval: 1.12-4.44, P: .023) and male gender (adj odds ratio: 1.54, 95% confidence interval: 1.03-2.32, P: .035).Working at dedicated COVID-19 units (high-risk group) was not an independent risk factor for seropositivity.

Decontamination and re-use of surgical masks and respirators during the COVID-19 pandemic.

OBJECTIVES: The coronavirus disease 2019 pandemic increased global demand for personal protective equipment (PPE) and resulted in shortages. The study evaluated the re-use of surgical masks and respirators by analysing their performance and safety before and after reprocessing using the following methods: oven, thermal drying, autoclave, and hydrogen peroxide plasma vapour. METHODS: In total, 45 surgical masks and 69 respirators were decontaminated. Visual integrity, air permeability, burst resistance, pressure differential and particulate filtration efficiency of new and decontaminated surgical masks and respirators were evaluated. In addition, 14 used respirators were analysed after work shifts before and after decontamination using reverse transcription polymerase chain reaction (RT-PCR) and viral culturing. Finally, reprocessed respirators were evaluated by users in terms of functionality and comfort. RESULTS: Oven decontamination (75 °C for 45 min) was found to be the simplest decontamination method. Physical and filtration assays indicated that all reprocessing methods were safe after one cycle. Oven decontamination maintained the characteristics of surgical masks and respirators for at least five reprocessing cycles. Viral RNA was detected by RT-PCR in two of the 14 used respirators. Four respirators submitted to viral culture were PCR-negative and culture-negative. Reprocessed respirators used in work shifts were evaluated positively by users, even after three decontamination cycles. CONCLUSION: Oven decontamination is a safe method for reprocessing surgical masks and respirators for at least five cycles, and is feasible in the hospital setting.

Nucleoprotein-based ELISA for detection of SARS-COV-2 IgG antibodies: Could an old assay be suitable for serodiagnosis of the new coronavirus?

OBJECTIVES: We evaluated the performance of a nucleoprotein-based enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies to SARS-CoV-2. METHODS: The ELISA was based on serum IgG reactivity to a 46-kDa protein derived from the recombinant SARS-CoV2 nucleoprotein. Assay sensitivity was assessed using serum samples from 134 COVID-19 confirmed cases obtained > 15 days after symptom onset. Specificity was determined by testing sera from 94 healthy controls. Cross-reactivity was evaluated with sera from 96 individuals with previous dengue or zika virus-confirmed infections, with 44 sera from individuals with confirmed infections to other respiratory viruses or with bacterial and fungal infections that cause pneumonia and with 40 sera negative for SARS-CoV-2 nucleoprotein by commercial ELISA kits. RESULTS: The majority of subjects were male and ≥ 60 years old. Assay sensitivity was 90.3 % (95 % confidence interval 84.1 %-94.2 %) and specificity was 97.9 % (92.6 %-99.4 %). There was no cross-reactivity with sera from individuals diagnosed with dengue, zika virus, influenza virus, rhinovirus, adenovirus, respiratory syncytial virus, seasonal coronavirus, Mycobacterium tuberculosis, Staphylococcus (S. aureus and coagulase-negative), Streptococcus pneumoniae, Klebsiella pneumoniae and the fungus Aspergillus fumigatus. The level of concordance of our test with results from commercial ELISA kits was 100 %. CONCLUSION: The nucleoprotein-based ELISA was specific for detection of IgG anti-nucleoprotein antibodies to SARS-CoV-2. It utilizes a frequently employed low expense assay protocol and is easier to perform than other currently available commercial SARS-CoV2 antibody detection tests.

Clinical features and natural history of the first 2073 suspected COVID-19 cases in the Corona São Caetano primary care programme: a prospective cohort study.

BACKGROUND: Despite most cases not requiring hospital care, there are limited community-based clinical data on COVID-19. METHODS: The Corona São Caetano programme is a primary care initiative providing care to all residents with COVID-19 in São Caetano do Sul, Brazil. It was designed to capture standardised clinical data on community COVID-19 cases. After triage of potentially severe cases, consecutive patients presenting to a multimedia screening platform between 13 April and 13 May 2020 were tested at home with SARS-CoV-2 reverse transcriptase (RT) PCR; positive patients were followed up for 14 days with phone calls every 2 days. RT-PCR-negative patients were offered additional SARS-CoV-2 serology testing to establish their infection status. We describe the clinical, virological and natural history features of this prospective population-based cohort. FINDINGS: Of 2073 suspected COVID-19 cases, 1583 (76.4%) were tested by RT-PCR, of whom 444 (28.0%, 95% CI 25.9 to 30.3) were positive; 604/1136 (53%) RT-PCR-negative patients underwent serology, of whom 52 (8.6%) tested SARS-CoV-2 seropositive. The most common symptoms of confirmed COVID-19 were cough, fatigue, myalgia and headache; whereas self-reported fever (OR 3.0, 95% CI 2.4 to 3.9), anosmia (OR 3.3, 95% CI 2.6 to 4.4) and ageusia (OR 2.9, 95% CI 2.3 to 3.8) were most strongly associated with a positive COVID-19 diagnosis by RT-PCR or serology. RT-PCR cycle thresholds were lower in men, older patients, those with fever and arthralgia and closer to symptom onset. The rates of hospitalisation and death among 444 RT-PCR-positive cases were 6.7% and 0.7%, respectively, with older age and obesity more frequent in the hospitalised group. CONCLUSION: COVID-19 presents in a similar way to other mild community-acquired respiratory diseases, but the presence of fever, anosmia and ageusia can assist the specific diagnosis. Most patients recovered without requiring hospitalisation with a low fatality rate compared with other hospital-based studies.