Convalescent plasma for COVID-19 in hospitalised patients : an open-label, randomised clinical trial

Background: The effects of convalescent plasma (CP) therapy in hospitalised patients with coronavirus disease 2019 (COVID-19) remain uncertain. This study investigates the effect of CP on clinical improvement in these patients. Methods: This is an investigator-initiated, randomised, parallel arm, open-label, superiority clinical trial. Patients were randomly (1:1) assigned to two infusions of CP plus standard of care (SOC) or SOC alone. The primary outcome was the proportion of patients with clinical improvement 28 days after enrolment. Results: A total of 160 (80 in each arm) patients (66.3% critically ill, 33.7% severely ill) completed the trial. The median (interquartile range (IQR)) age was 60.5 (48–68) years; 58.1% were male and the median (IQR) time from symptom onset to randomisation was 10 (8–12) days. Neutralising antibody titres >1:80 were present in 133 (83.1%) patients at baseline. The proportion of patients with clinical improvement on day 28 was 61.3% in the CP+SOC group and 65.0% in the SOC group (difference −3.7%, 95% CI −18.8–11.3%). The results were similar in the severe and critically ill subgroups. There was no significant difference between CP+SOC and SOC groups in pre-specified secondary outcomes, including 28-day mortality, days alive and free of respiratory support and duration of invasive ventilatory support. Inflammatory and other laboratory marker values on days 3, 7 and 14 were similar between groups. Conclusions: CP+SOC did not result in a higher proportion of clinical improvement on day 28 in hospitalised patients with COVID-19 compared to SOC alone

Immunodominant antibody responses directed to SARS-CoV-2 hotspot mutation sites and risk of immune escape

IntroductionConsidering the likely need for the development of novel effective vaccines adapted to emerging relevant CoV-2 variants, the increasing knowledge of epitope recognition profile among convalescents and afterwards vaccinated with identification of immunodominant regions may provide important information.MethodsWe used an RBD peptide microarray to identify IgG and IgA binding regions in serum of 71 COVID-19 convalescents and 18 vaccinated individuals. ResultsWe found a set of immunodominant RBD antibody epitopes, each recognized by more than 30% of the tested cohort, that differ among the two different groups and are within conserved regions among betacoronavirus. Of those, only one peptide, P44 (S415-429), recognized by 68% of convalescents, presented IgG and IgA antibody reactivity that positively correlated with nAb titers, suggesting that this is a relevant RBD region and a potential target of IgG/IgA neutralizing activity.DiscussionThis peptide is localized within the area of contact with ACE-2 and harbors the mutation hotspot site K417 present in gamma (K417T), beta (K417N), and omicron (K417N) variants of concern. The epitope profile of vaccinated individuals differed from convalescents, with a more diverse repertoire of immunodominant peptides, recognized by more than 30% of the cohort. Noteworthy, immunodominant regions of recognition by vaccinated coincide with mutation sites at Omicron BA.1, an important variant emerging after massive vaccination. Together, our data show that immune pressure induced by dominant antibody responses may favor hotspot mutation sites and the selection of variants capable of evading humoral response

Seroprevalence of Zika (ZIKV) and Dengue (DENV) virus infection in parturients and transplacental transfer of anti-ZIKV and anti-DENV antibodies in newborns in Southeast, Brazil

Os vírus Zika (ZIKV) e Dengue (DENV) são arbovírus circulantes no Brasil, que apresentam grande importância para saúde pública nacional. A capacidade de estimar a atual e futura dispersão desses vírus, depende significativamente do nosso conhecimento acerca do status sorológico em populações expostas. Atualmente, há uma escassez de dados na literatura em relação a prevalência de anticorpos anti-ZIKV e anti-DENV na população em geral e particularmente em mulheres grávidas brasileiras. A transferência transplacentária de anticorpos contra patógenos infecciosos é um importante mecanismo materno-fetal que fornece proteção ao recém-nascido (RN) nos primeiros meses de vida. Neste contexto, o presente estudo investigou a prevalência de infecção por ZIKV e DENV em parturientes, avaliando a transferência placentária de anticorpos anti-ZIKV e anti-DENV aos RNs. Dessa forma, foi realizado um estudo transversal com 601 parturientes e seus respectivos filhos recém-nascidos, atendidos no Hospital Universitário da Universidade de São Paulo (HU-USP), localizado na cidade de São Paulo, Brasil. Amostras de sangue total, soro e urina foram coletadas e armazenadas em biorrepositório. Informações sociodemográficas e características clínicas maternas e neonatais foram obtidas mediante entrevista e análise de prontuários médicos. Para avaliação da soroprevalência de infecção por ZIKV e DENV foram utilizados os ensaios de imunoabsorção enzimática (ELISA), para detecção de anticorpos IgM e IgG e os testes de neutralização viral baseado em efeito citopático (CPE-VNT) e de neutralização por redução de placas (PRNT), para detecção de anticorpos neutralizantes (nAbs). Além da investigação sorológica, os ácidos nucléicos totais das amostras biológicas foram extraídos por método semi-automatizado, seguido do RT-qPCR para o gene endógeno da RNase P e para investigação da presença do RNA viral do ZIKV e DENV. Verificou-se soroprevalência de 2,39% e 31,61% de anticorpos neutralizantes específicos contra ZIKV e DENV, respectivamente. A maior parte das gestantes apresentava imunidade aos quatro sorotipos de DENV (74,1%). Dentre as amostras com nAbs detectáveis, nenhuma amostra foi positiva para IgM anti-ZIKV e em 4,2% foram detectados anticorpos IgM anti-DENV. Todas as amostras testadas tiveram amplificação satisfatória para RNase P e foram negativas para a presença do RNA viral de ambos os vírus investigados. Ademais, foi possível determinar a efetiva passagem transplacentária (TR) de anticorpos específicos anti-ZIKV e anti-DENV de mães previamente infectadas ao respectivos filhos recém-nascidos. Níveis de IgG ZIKV e DENV específico foram significativamente maiores nas amostras do cordão umbilical do que nas respectivas amostras maternas (p<0.05). Características maternas e neonatais foram correlacionadas tanto com a soropositividade para ZIKV e DENV quanto com a efetividade de transferência de anticorpos DENV específicos aos RNs. Devido a relativa baixa soroprevalência de ZIKV e DENV na população em estudo, este cenário alerta para a susceptibilidade a infecção por estes patógenos. Assim, com o conhecimento gerado no presente estudo espera-se contribuir para um melhor entendimento da soroprevalência de ZIKV e DENV em parturientes na região investigada e do perfil de TR aos neonatos, servindo como referência para a avaliação da eficácia do transporte transplacentário de anticorpos resultantes de futuros esquemas vacinais contra ZIKV e DENV em gestantes no Brasil.Zika (ZIKV) and Dengue (DENV) viruses are arboviruses circulating in Brazil, which are of great importance for national public health. The ability to estimate the current and future spread of these viruses depends significantly on our knowledge of the serological status in exposed populations. Currently, there is few data in the literature regarding the prevalence of anti-ZIKV and anti-DENV antibodies in the general population and particularly in Brazilian pregnant women. The transplacental transfer of antibodies against infectious pathogens is an important maternal-fetal mechanism that provides protection to the newborns (NB) in the first months of life. In this context, the present study investigated the prevalence of ZIKV and DENV infection in pregnant women, evaluating the transplacental transfer of anti-ZIKV and anti-DENV antibodies to NB. Thus, a cross-sectional study was carried out with 601 pregnant women and their newborn children, attended at the University Hospital of the University of São Paulo (HU-USP), located in the city of São Paulo, Brazil. Whole blood, serum and urine samples were collected and stored in a biorepository. Sociodemographic information and maternal and neonatal clinical characteristics were obtained through interviews and analysis of medical records. To assess the seroprevalence of ZIKV and DENV infection, enzyme-linked immunosorbent assays (ELISA) were used to detect IgM and IgG antibodies and viral neutralization tests based on cytopathic effect (CPE-VNT) and plaque reduction neutralization (PRNT), for detection of neutralizing antibodies (nAbs). In addition to the serological investigation, the total nucleic acids of the biological samples were extracted by a semi-automated method, followed by RT-qPCR for an endogenous gene (RNase P) and for investigation of the presence of viral RNA of ZIKV and DENV. There was a seroprevalence of 2.39% and 31.61% of specific neutralizing antibodies against ZIKV and DENV, respectively. Most pregnant women had immunity to the four DENV serotypes (74.1%). Among the samples with detectable nAbs, no sample was positive for anti-ZIKV IgM and in 4.2% anti-DENV IgM antibodies were detected. All tested samples had satisfactory amplification for RNase P and were negative for the presence of viral RNA of the investigated viruses. Furthermore, it was possible to determine the effective transplacental transfer (TR) of specific anti-ZIKV and serotype-specific anti-DENV antibodies from previously infected mothers to the newborns. ZIKV and specific DENV IgG levels were significantly higher in the umbilical cord samples than in the respective maternal samples. Maternal and neonatal characteristics were correlated both with seropositivity for ZIKV and DENV and with the effectiveness of transferring specific DENV antibodies to newborns. Due to the relatively low seroprevalence of ZIKV and DENV in the study population, this scenario alerts to the susceptibility to infection by these pathogens. Thus, with the knowledge generated in the present study, it is expected to contribute to a better understanding of the seroprevalence of ZIKV and DENV in parturients in the region under study and the TR profile of newborns, serving as a reference for evaluating the effectiveness of transplacental transport of antibodies resulting from future vaccination schedules against ZIKV and DENV in pregnant women in Brazil

Immune Evasion of SARS-CoV-2 Omicron Subvariants XBB.1.5, XBB.1.16 and EG.5.1 in a Cohort of Older Adults after ChAdOx1-S Vaccination and BA.4/5 Bivalent Booster

The recently emerged SARS-CoV-2 Omicron sublineages, including the BA.2-derived XBB.1.5 (Kraken), XBB.1.16 (Arcturus), and EG.5.1 (Eris), have accumulated several spike mutations that may increase immune escape, affecting vaccine effectiveness. Older adults are an understudied group at significantly increased risk of severe COVID-19. Here we report the neutralizing activities of 177 sera samples from 59 older adults, aged 62–97 years, 1 and 4 months after vaccination with a 4th dose of ChAdOx1-S (Oxford/AstraZeneca) and 3 months after a 5th dose of Comirnaty Bivalent Original/Omicron BA.4/BA.5 vaccine (Pfizer-BioNTech). The ChAdOx1-S vaccination-induced antibodies neutralized efficiently the ancestral D614G and BA.4/5 variants, but to a much lesser extent the XBB.1.5, XBB.1.16, and EG.5.1 variants. The results showed similar neutralization titers between XBB.1.16 and EG.5.1 and were lower compared to XBB.1.5. Sera from the same individuals boosted with the bivalent mRNA vaccine contained higher neutralizing antibody titers, providing a better cross-protection against Omicron XBB.1.5, XBB.1.16 and EG.5.1 variants. Previous history of infection during the epidemiological waves of BA.1/BA.2 and BA.4/BA.5, poorly enhanced neutralization activity of serum samples against XBBs and EG.5.1 variants. Our data highlight the continued immune evasion of recent Omicron subvariants and support the booster administration of BA.4/5 bivalent vaccine, as a continuous strategy of updating future vaccine booster doses to match newly emerged SARS-CoV-2 variants

Nebulized enriched heparin to treat no critical patients with Sars-Cov-2

Background: Coronavirus disease 2019 (COVID-19) is a viral respiratory disease that spreads rapidly, reaching pandemic status, causing the collapse of numerous health systems, and a strong economic and social impact. The treatment so far has not been well established and there are several clinical trials testing known drugs that have antiviral activity, due to the urgency that the global situation imposes. Drugs with specific mechanisms of action can take years to be discovered, while vaccines may also take a long time to be widely distributed while new virus variants emerge. Thus, drug repositioning has been shown to be a good strategy for defining new therapeutic approaches. Studies of the effect of enriched heparin in the replication of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) in vitro assays justify the advance for clinical tests. Methods and analysis: A phase I/II triple-blind parallel clinical trial will be conducted. Fifty participants with radiological diagnosis of grade IIA pneumonia will be selected, which will be allocated in 2 arms. Participants allocated in Group 1 (placebo) will receive nebulized 0.9% saline. Participants allocated in Group 2 (intervention) will receive nebulized enriched heparin (2.5 mg/mL 0.9% saline). Both groups will receive the respective solutions on a 4/4 hour basis, for 7 days. The main outcomes of interest will be safety (absence of serious adverse events) and efficacy (measured by the viral load). Protocols will be filled on a daily basis, ranging from day 0 (diagnosis) until day 8

Nebulized enriched heparin improves respiratory parameters in patients with COVID-19: a phase I/II randomized and triple-blind clinical trial

To evaluate the safety and the potential antiviral treatment of inhaled enriched heparin in patients with COVID-19. The specific objectives were to investigate the anticoagulation profile, antiviral and anti-inflammatory effects, and respiratory evolution of inhaled enriched heparin. We conducted a randomized, triple-blind, placebo-controlled Phase I/II clinical trial in hospitalized adults with COVID-19 receiving inhalation of enriched heparin or saline (placebo) every 4 h for 7 days. Among the 27 patients who completed the study, no changes in blood coagulation parameters were observed, indicating the safety of inhaled enriched heparin. The group receiving enriched heparin showed a significant reduction in the need for supplemental oxygen and improvement in respiratory parameters, such as the PaO2/FiO2 ratio. Inhalation of enriched heparin is shown to be safe and has also demonstrated potential therapeutic benefits for patients with COVID-19. These promising results justify the continuation of the study to the next phase, Phase II/III, to further evaluate the therapeutic efficacy of inhaled enriched heparin in the treatment of COVID-19-associated viral pneumonia. Trial registration: ClinicalTrials.gov. 08/02/2021. Identifier: NCT04743011

SARS-CoV-2 papain-like protease PLpro in complex with natural compounds reveal allosteric sites for antiviral drug design

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the host’s innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 Å and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 Å, show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, in the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections

Antiviral activity of natural phenolic compounds in complex at an allosteric site of SARS-CoV-2 papain-like protease

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host’s innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections