RESUMO
The COVID-19 pandemic and the consequent emergence of new SARS-CoV-2 variants of concern necessitates the determination of populational serum potency against the virus. Here, we standardized and validated an imaging-based method to quantify neutralizing antibodies against lentiviral particles expressing the spike glycoprotein (pseudovirus). This method was found to efficiently quantify viral titers based on ZsGreen-positive cells and detect changes in human serum neutralization capacity induced by vaccination with up to two doses of CoronaVac, Comirnaty, or Covishield vaccines. The imaging-based protocol was also used to quantify serum potency against pseudoviruses expressing spikes from Delta, Omicron BA.1.1.529, and BA.4/5. Our results revealed increases in serum potency after one and two doses of the vaccines evaluated and demonstrated that Delta and Omicron variants escape from antibody neutralization. The method presented herein represents a valuable tool for the screening of antibodies and small molecules capable of blocking viral entry and could be used to evaluate humoral immunity developed by different populations and for vaccine development.
RESUMO
SARS-CoV-2 is the etiological agent of the coronavirus disease-19 (COVID-19) and is responsible for the pandemic that started in 2020. The virus enters the host cell through the interaction of its spike glycoprotein with the angiotensin converting enzyme-2 (ACE2) on the host cell's surface. Antibodies present an important role during the infection and pathogenesis due to many reasons, including the neutralization of viruses by binding to different spike epitopes. Therefore, measuring the neutralizing antibody titers in the whole population is important for COVID-19's epidemiology. Different methods are described in the literature, and some have been used to validate the main vaccines used worldwide. In this review, we discuss the main methods used to quantify neutralizing antibody titers, their advantages and limitations, as well as new approaches to determineACE2/spike blockage by antibodies.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Anticorpos Neutralizantes , Anticorpos Antivirais , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de CoronavírusRESUMO
BACKGROUND: Adherence to antiretroviral (ARV) therapy remains a major challenge in HIV therapeutics. OBJECTIVE: To assess the adherence to ARV therapy by measuring the delay in monthly refilling of ARV drugs using pharmacy records and to correlate this with HIV plasma RNA measurements and CD4+ cell count. METHOD: Records of 170 HIV-positive patients were examined to identify HIV viral load (VL)/CD4+ results and the time interval to refill ARVs at the pharmacy. The correlation between the number of days missed to refill ARVs and plasma HIV-RNA detectability/CD4+ count was performed using the Spearman's correlation coefficient (r). RESULTS: Fewer days missed to refill ARV was positively correlated with undetectable VL and increase in CD4+ count (r = 0.407 and 0.237, respectively). Increase in adherence was correlated with longer retention in the cohort (r = 0.208). CONCLUSION: Monitoring the delay to pick up ARVs from the pharmacy can be an important and simple tool to identify patients requiring assessment of their adherence.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Antirretrovirais/uso terapêutico , Monitoramento de Medicamentos/estatística & dados numéricos , Infecções por HIV/tratamento farmacológico , Adesão à Medicação/estatística & dados numéricos , Adulto , Idoso , Brasil , Contagem de Linfócito CD4 , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Assistência Farmacêutica , Farmácias , Estudos Retrospectivos , Carga Viral , Adulto JovemRESUMO
OBJECTIVES:: Conventional imaging methods are excellent for the morphological characterization of the consequences of osteonecrosis; however, only specialized techniques have been considered useful for obtaining functional information. To explore the affinity of radiotracers for severely devascularized bone, a new mouse model of isolated femur implanted in a subcutaneous abdominal pocket was devised. To maintain animal mobility and longevity, the femur was harvested from syngeneic donors. Two technetium-99m-labeled tracers targeting angiogenesis and bone matrix were selected. METHODS:: Medronic acid and a homodimer peptide conjugated with RGDfK were radiolabeled with technetium-99m, and biodistribution was evaluated in Swiss mice. The grafted and control femurs were evaluated after 15, 30 and 60 days, including computed tomography (CT) and histological analysis. RESULTS:: Radiolabeling achieved high (>95%) radiochemical purity. The biodistribution confirmed good blood clearance 1 hour after administration. For 99mTc-hydrazinonicotinic acid (HYNIC)-E-[c(RGDfK)2, remarkable renal excretion was observed compared to 99mTc-methylene diphosphonate (MDP), but the latter, as expected, revealed higher bone uptake. The results obtained in the control femur were equal at all time points. In the implanted femur, 99mTc-HYNIC-E-[c(RGDfK)2 uptake was highest after 15 days, consistent with early angiogenesis. Regarding 99mTc-MDP in the implant, similar uptake was documented at all time points, consistent with sustained bone viability; however, the uptake was lower than that detected in the control femur, as confirmed by histology. CONCLUSIONS:: 1) Graft viability was successfully diagnosed using radiotracers in severely ischemic bone at all time points. 2) Analogously, indirect information about angiogenesis could be gathered using 999mTc-HYNIC-E-[c(RGDfK)2. 3) These techniques appear promising and warrant further studies to determine their potential clinical applications.
Assuntos
Interface Osso-Implante/fisiologia , Modelos Animais de Doenças , Compostos de Organotecnécio , Osteonecrose/fisiopatologia , Peptídeos Cíclicos , Compostos Radiofarmacêuticos , Animais , Transplante Ósseo , Difosfonatos , Feminino , Fêmur/patologia , Fêmur/fisiopatologia , Marcação por Isótopo/métodos , Camundongos , Neovascularização Fisiológica/fisiologia , Osteonecrose/patologia , Reprodutibilidade dos Testes , Fatores de Tempo , Sobrevivência de Tecidos/fisiologia , Tomografia Computadorizada por Raios XRESUMO
OBJECTIVES: Conventional imaging methods are excellent for the morphological characterization of the consequences of osteonecrosis; however, only specialized techniques have been considered useful for obtaining functional information. To explore the affinity of radiotracers for severely devascularized bone, a new mouse model of isolated femur implanted in a subcutaneous abdominal pocket was devised. To maintain animal mobility and longevity, the femur was harvested from syngeneic donors. Two technetium-99m-labeled tracers targeting angiogenesis and bone matrix were selected. METHODS: Medronic acid and a homodimer peptide conjugated with RGDfK were radiolabeled with technetium-99m, and biodistribution was evaluated in Swiss mice. The grafted and control femurs were evaluated after 15, 30 and 60 days, including computed tomography (CT) and histological analysis. RESULTS: Radiolabeling achieved high (>95%) radiochemical purity. The biodistribution confirmed good blood clearance 1 hour after administration. For 99mTc-hydrazinonicotinic acid (HYNIC)-E-[c(RGDfK)2, remarkable renal excretion was observed compared to 99mTc-methylene diphosphonate (MDP), but the latter, as expected, revealed higher bone uptake. The results obtained in the control femur were equal at all time points. In the implanted femur, 99mTc-HYNIC-E-[c(RGDfK)2 uptake was highest after 15 days, consistent with early angiogenesis. Regarding 99mTc-MDP in the implant, similar uptake was documented at all time points, consistent with sustained bone viability; however, the uptake was lower than that detected in the control femur, as confirmed by histology. CONCLUSIONS: 1) Graft viability was successfully diagnosed using radiotracers in severely ischemic bone at all time points. 2) Analogously, indirect information about angiogenesis could be gathered using 999mTc-HYNIC-E-[c(RGDfK)2. 3) These techniques appear promising and warrant further studies to determine their potential clinical applications.