key: cord-0814266-jps70ao5 authors: Teresa Valenzuela, María; Urquidi, Cinthya; Rodriguez, Nicolás; Castillo, Luis; Fernández, Jorge; Ramírez, Eugenio title: Development of neutralizing antibody responses against SARS‐CoV‐2 in COVID‐19 patients date: 2021-03-25 journal: J Med Virol DOI: 10.1002/jmv.26939 sha: 63db6f97500895515a63cc15d6f158f4a0d38582 doc_id: 814266 cord_uid: jps70ao5 The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and coronavirus disease 2019 (COVID‐19) are new global problems. The understanding of the host immune response in COVID‐19 and its implications in the development of therapeutic agents are new challenges. Here, we evaluated the development of immunoglobulin G (IgG) and neutralizing (Nt) antibodies in symptomatic hospitalized COVID‐19 patients. We followed up 117 COVID‐19 confirmed patients from a reference health center for COVID‐19 during the epidemic in Santiago de Chile. One and two sequential blood samples from 117 to 68 cases were, respectively, obtained to evaluate the immune response. Immunofluorescence and neutralization assays in Vero E6 cells with a Chilean SARS‐CoV‐2 strain were performed. Out of the 68 patients, 44% were women and 56% men, and the most frequent comorbidities were hypertension (47.7%) and diabetes (27.4%). The most frequent symptoms or signs related to COVID‐19 were dyspnea, cough, fever, myalgia, and headache. In all the study population, 76.1% and 60.7% of patients were positive for IgG and Nt antibodies in the first blood sample. All patients except one were positive for IgG and Nt antibodies in the second sample. IgG and Nt antibodies positivity increased significantly according to the disease evolution periods. Higher Nt antibody titers were observed in the first sample in patients under 60 years of age. Obese and diabetic patients had no increase in Nt antibodies, unlike normal weight and diabetes‐free patients. Both hypertensive and normotensive patients showed a significant increase in Nt antibodies. These results show an early and robust immune response against SARS‐CoV‐2 infection during severe COVID‐19. has an R 0 × 2.5 and a median incubation of 5 days, with some symptoms similar to a flu-like disease. 1 The infecting period has been reported to begin 2 days before the onset of symptoms and extends for 8 days after the onset of the disease. 2, 3 The infecting period of subjects with severe forms of COVID-19 or those with some immune system compromise has not yet been established with certainty. An important feature of COVID-19 is that a larger number of cases are oligoasymptomatic or asymptomatic, making it difficult to detect total patients and control transmission. Antibody production is early, within 7 days of onset of symptoms, 40% of patients may have immunoglobulin G (IgG)-class antibodies; at 2 weeks, virtually all patients produce them. 4 The immune system first produces IgM-type antibodies, with faster and less potent activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Later IgGs are produced, more potent and specific to the viral infection. IgM antibodies are produced from Days 5 to 7 after the onset of symptoms in COVID-19, and it is closely followed by the production of IgG. In most cases studied IgM levels disappear within a few weeks, while IgG levels persist for at least three months. 5 Recently, a longitudinal study that evaluated changes in antibody levels in people recovered from COVID- 19 , concluded that they did not decline after four months of diagnosis. 4 Neutralizing (Nt) antibodies are key to the elimination of viruses that cause various infections and are recognized for their critical role in the protective immune response against invasive viral infections. Their activity is triggered against proteins located on the surface of viruses, which they join to block the infection. In the face of this new SARS-CoV-2 infection, there is a lack of experience if these antibodies are sufficient to induce effective and lasting immunity. [5] [6] [7] Studies conducted so far in SARS-CoV-2 indicate that Nt appear about 2 weeks after infection begins, and that their maximum activity would occur between 4 and 6 weeks. Nevertheless, it has not been confirmed whether all patients generate Nt antibodies, if their titers are related to the severity of the infection, or whether their neutralization levels are always sufficient to induce protection, as their concentrations are variable and not detected in all patients. There are still questions regarding the function of Nt antibodies and their role in controlling infection and symptoms of the disease, or whether after recovering from the disease and generated the antibodies it is possible to re-infect with SARS-CoV-2. 5, 6, 8, 10, 11 We aimed to evaluate the presence of IgG and Nt in symptomatic hospitalized COVID-19 patients, the subsequent development of both antibodies when they are recovered, and to identify associated clinical issues. We recruited and prospectively followed up 128 patients with a confirmed diagnosis of COVID-19 who were hospitalized at Hospital Barros Luco Trudeau (HBL) from May to August 6, 2020. HBL is a tertiary public hospital in Santiago de Chile, which was an exclusive health center for COVID-19 during the epidemic. The inclusion criteria were: (1) patients older than 18 years of age and (2) patients hospitalized, and (3) RT-PCR test positive for COVID-19. We excluded patients with high-risk multi-organic failure (diagnoses under clinical criteria) and those with a lapse of more than 5 days from hospital admission. We also excluded patients who received convalescent plasma during hospitalization for the final analysis. The protocol was approved by the Scientific-Ethics Committee South Health Service of Metropolitan Region, Chile, through the document dated May 6, 2020. Study participants were unable to directly sign informed consent, given the strict isolation measures indicated at the root of the pandemic; only the patient's proxy could be contacted via telephone and proxy signed documents. In those patients who were not in critical condition, they were asked for authorization to obtain data and blood sample, as the documents were not allowed to enter those units either. At the moment of recruitment, a trained internal medicine resident using a semi-structured questionnaire collected the following in- To evaluate the immune response against SARS-CoV-2, we obtained two blood samples from each infected patient separated by at least 18 days, first-second and third-fourth weeks of follow-up, spanning 15-25 days after symptom onset date by a certified technical professional. Hospitalization status was registered as non-ICU hospitalization, ICU, and hospital discharge. By the time of the second blood sample, the patient clinical course was evaluated as no changes or aggravated (when the patient remained hospitalized in the same unit or worsening from a non-ICU hospitalization to ICU), and favorable evolution (when the patient left the ICU to a non-ICU hospitalization or hospital discharge). Blood draw collection (3-4 ml) was performed using EDTA or heparin tubes. PBMC isolation was performed using a density gradient TERESA VALENZUELA ET AL. The positive control was a serum from a recovered COVID-19 patient, who agreed to donate blood for plasmapheresis. For immunofluorescence test, Vero E6 cells were cultured on an eightwell plate (Lab-Tek, Thermo Fisher Scientific; cat 155411; 5 × 10 4 cells/ well) with 0.3 ml minimum essential medium + 2% fetal bovine serum + 2× penicillin-streptomycin at 37°C in a 5% carbon dioxide at- We describe the study population by age groups and according to the time of the first and second blood samples. Fisher's exact t-test, analysis of variance, and the Student t test were used to compare the categorical and numerical variables between different groups. p values less than .05 were considered significant. The analysis was done in STATA (College Station, Texas) and GraphPad Prism 8.0 software (GraphPad Software). One hundred twenty-eight hospitalized COVID-19 patients were enrolled between May 10 and August 6, 2020; in whom 11 patients were excluded because they had received convalescent plasma. Of the 117 (91.4%) remaining patients, 18 patients died during the follow-up, four were transferred to another hospital outside the study hospital system, and 27 did not attend to phone calls or untraceable home address, or refused to take the second blood sample. Therefore, 68 (52.3%) patients had the second blood sample for antibodies determination. Out of the 117 patients, 48 patients were women (41%), and 69 were men (59%), with a median age of 56 years similar in both groups (p = .847). Out of the 68 patients, 44% were women and 56% men, with no age differences. Table 1 describes the study population regarding disease evolution, history of chronic diseases, and the frequency of symptoms or signs patients had diabetes and hypertension in 27.4% and 47.7% of cases, respectively. The prevalence of these chronic diseases was even significantly higher among adults aged 65 and older (50% p = .005 for diabetes; and 78.1% p < .001 for hypertension). Obesity (body mass index > 30) was present in 13.7% of the study population; nevertheless, this prevalence tended to be higher among patients younger than 40 years of age (30%, p = .007). In contrast, chronic kidney disease and cancer prevalences were lower in the study population, with no difference between age groups ( Table 1 ). The most frequent symptoms or signs related to COVID-19 were dyspnea (82.3% of the cases), cough (64.1%), fever (47%), myalgia (34.9%), and headache (21.4%). There was no significant difference in signs or symptoms between age groups, although fever and cough tended to be more frequent in adults younger than 40 years; meanwhile, headache in adults among 40-60 years of age (Table 1) . Additionally, 6.8% of the patients presented a compromised general state at the time of recruitment. We recruited patients at different stages in the evolution of their disease. Thus, the mean time between the date of onset of sign or symptoms and the first blood sample date was 11 days with a standard deviation (SD) of 5 days, similarly in men and women. Henceforth, we present antibody results according to three disease T A B L E 1 Characterization of the study population and history of chronic diseases and frequency of symptoms and signs related to COVID-19 by groups of age a in 117 SARS-CoV-2 infected patients Of the total number of patients studied (n = 117), paired plasma samples were obtained in 68 infected patients (53.1%), 38 were men (55.9%) and 30 (44.1%) were women. She was admitted to the hospital in May 12th with a history of one week of dyspnea of minimal efforts, no fever, cough or expectoration. The diagnosis was confirmed by positive RT-PCR at admission, and an X-ray Computed Tomography was performed that showed multifocal pneumonia. The first sample was taken on 14 May, that is 9 days after the onset of symptoms. She did not require treatment in intensive care, and she was discharged from the hospital in May 16th. At the time of the second sample on May 28th, she was asymptomatic. Little is currently known about the relationship between the specific immune response against SARS-CoV-2 and COVID-19 severity. The specific immune response against the virus in SARS-CoV-2 infected individuals is controlled by the coordinated action of the three fundamental branches of adaptive immunity: the Nt antibody response and CD4 + and CD8 + T lymphocytes. 12 In this study we reported the immune response of IgG and Nt in T A B L E 3 Neutralizing and IgG antibody titer in the study population and stratified by age groups, clinical course, days from symptoms onset and chronic disease 19, 20 Many studies have shown that nutrient dysregulation in obese individuals impairs the immune response to infections. We are grateful to Agencia Nacional de Investigación y Desarrollo de Chile (ANID), which supported this study (Grant COVID0557). The authors declare that there is no conflict of interests. The peer review history for this article is available at https://publons. com/publon/10.1002/jmv.26939 The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Eugenio Ramírez http://orcid.org/0000-0002-5299-7928 The incubation period of coronavirus disease 2019 (COVID-19) from publicly reported confirmed cases: estimation and application Virological assessment of hospitalized patients with COVID-2019 Contact tracing assessment of COVID-19 transmission dynamics in Taiwan and risk at different exposure periods before and after symptom onset Humoral immune response to SARS-CoV-2 in Iceland Rapid generation of neutralizing antibody responses in COVID-19 patients Dynamics of anti-SARS-Cov-2 IgM and IgG antibodies among COVID-19 patients What to do next to control the 2019-nCoV epidemic? Antibody responses to SARS-CoV-2 in patients with COVID-19 Deployment of convalescent plasma for the prevention and treatment of COVID-19 Neutralizing antibody responses to severe acute respiratory syndrome coronavirus 2 in coronavirus disease 2019 inpatients and convalescent patients Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity Antibody responses to SARS-CoV-2 in patients with novel coronavirus disease 2019 Severe acute respiratory syndrome coronavirus 2−specific antibody responses in coronavirus disease 2019 patients Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study Evaluating the association of clinical characteristics with neutralizing antibody levels in patients who have recovered from mild COVID-19 in Treatment of 5 critically Ill patients with COVID-19 with convalescent plasma Neutralizing antibodies correlate with protection from SARS-CoV-2 in humans during a fishery vessel outbreak with a high attack rate Individuals with obesity and COVID-19: a global perspective on the epidemiology and biological relationships Obesity is a risk factor for developing critical condition in COVID-19 patients: a systematic review and meta-analysis