SARS-CoV-2 exhibits a high infectivity capacity; the new disease reached the mark of 65.8 million cases worldwide, being observed more than 1,5 million deaths7. Brazil is entering through the second wave of COVID-19, and some restrictions are being imposed. However, the number of cases is still climbing. In the absence of vaccines and a useful serological assay to trial even asymptomatic people, SARS-CoV-2 infection remains a significant problem affecting Brazil. Although qPCR to detect the viral genome is a gold standard diagnostic assay, additional screening methods that detect the presence of SARS-Cov-2 infection, including serological tests, can be highly advantageous to ensure timely diagnosis in a pandemic 31. Several studies have reported the profile of the antibody response to SARS-CoV-2, which includes the broad clinical spectrum of COVID-19. Many aspects of the humoral immune response in COVID-19 remain obscure 44,45−47, particularly concerning clinical utilities of serological testing in symptomatic and hospitalized patients.
In this attempt, we evaluated the IgG response in hospitalized patients with mild and severe clinical manifestation, showing RBD-specific IgG antibody responses begins 4 days after symptoms onset, reaching the plateau at 15 days. The average time to detect IgG antibodies against SARS-CoV-2 based on RBD IgG-ELISA was similar to what was found in other studies, regardless of the serological assay and SARS-CoV-2 antigen used 44,45−47. Our in-house ELISA's high immunoreactivity was observed with 100% specificity and 94.3% of sensibility, suggesting that RDB IgG-ELISA could be used to assess SARS-CoV-2 infection in patients 15 day-post onsets of symptoms. Wolf at. al; 2020 observed that hospitalized COVID-19 patients showed higher levels of antibodies when compared to outpatients cases43. A previous report demonstrated that hospitalized COVID-19 patients showed higher levels of antibodies than outpatients cases43, suggesting an association between illness severity and antibody production.
Interestingly, in our study, survivors who developed the severe form of illness displayed higher anti-RBD IgG levels compared to patients with mild disease, also lighting that clinical presentation of disease may produce substantial differences in IgG responses. For this purpose, we also investigated the anti-RBD IgG levels in recovered patients compared to those who died. Although no statistical differences were observed, a slightly lower anti-RBD IgG level was observed in patients who died compared to the recovered ones. The divergence observed in our study may be due to differences in the number of serum samples analyzed and the time of blood collection in our study since a limited number of samples was obtained from critically ill patients who quickly progressed to death by COVID-19.
Besides the comorbidities observed in patients who died, obesity was more frequently observed in our study. The high percentage of obese patients who died with COVID-19 is in concordance with previous studies 48,49. Obesity is a factor that directly associated chronic activation of innate immune system cells and consequent local and systemic inflammation50. B cells from obese patients express leptin induced-activation markers (TNF-α, TLR4, micro-RNAs) that correlate reduced B-cell functions 49,51,52. Therefore, obesity and COVID-19 share common elements of the inflammatory process (and possibly also metabolic disturbances), exacerbating SARS-CoV-2 infection in the obese 53, leading these individuals to severe COVID-19, even to death.
In the present study, high levels of anti-RBD IgG were detected in both groups of patients who died or survived. However, these findings are not enough to support the hypothesis that these individuals displayed either an extended-lasting protective humoral response against SARS-CoV-2 or neutralizing antibodies in convalescent plasma. Our study has some limitations in this context since it was not possible to assess the presence and neutralizing antibodies. Interestingly, a possible association between SARS-CoV-2 spike antibody avidity with neutralizing IgG titter, as a potential screening parameter for identifying optimal convalescent plasma donors was proposed42. Likewise, high-avidity antibodies toward another virus capable of blocking receptor binding were protective and promoted virus neutralization54, indicating that antibody avidity maturation could be, at least in part, associated with the production of protective neutralizing antibodies in viral infections. Although our study has limited data on temporal dynamics (< 45 days) to correlates SARS-CoV-2 antibody avidity with the illness severity, it was observed that the majority of patients who had symptoms showed low avidity. Our data is by what was reported to SARS-CoV-2 infection with low IgG antibody avidity during the 50 days after symptoms onset 42, however, evaluating antibodies against nucleoprotein55 , and to SARS-CoV-2 anti-spike and anti-nucleoprotein, that reached the avidity peak at 21 after days of symptoms onset, during a study period median of 45days42 .
Similarly, low antibody avidity was also observed early infection and augmented within the first month of symptom onset in SARS outbreaks 56. It is noteworthy that one-third of the patients who recovered had intermediate avidity of IgG antibodies to RDB, despite a relatively short period (weeks) post the onset of the symptoms. On the other hand, approximately one-sixth of the patients who died produced intermediate avidity antibody responses, suggesting that IgG avidity may be useful for monitoring hospitalized patients with COVID-19 in association with other serological markers. As was expected, IgG antibody avidity was low during initial infection and increased with time, although no statistical differences were observed between patients who died and recovered in the time-points post the onset of the symptoms.
The IgG1 and IgG3 subclasses represent the predominant antibody responses to several viral diseases 57,41, and recently it was also associated with the new SARS-CoV − 2 infection29,58. IgG1 and IgG3 responses are related to immune functions such as viral neutralization, opsonization, and complement activation in viral respiratory infection 41. Thus, to further analyze the antibody response, we also analyzed IgG to SARS-CoV-2 RDB in sera from patients with COVID-19. As it was expected, a robust antibody response of IgG1 and IgG3 specific to SARS-CoV-2 RDB occurred predominantly in comparison with minor IgG4 responses. Likewise, Suthar et al.; 2020 demonstrated that COVID-19 patients analysed in USA produced RBD-specific IgG1 and IgG3 early during acute infection, with no detectable IgG2 or IgG429. Similar antibody responses were also reported by Mazzini et al.; 2020 in Italy, with a strong reactivity for IgG1 and IgG3 in sera from positive patients for SARS-CoV-2 infection 58.
The comparative analysis of IgG subclasses in serum samples from COVID-19 patients who died revealed a higher level of RDB-specific IgG1 when compared to those who survived during 0–8 days after symptoms onset. However, this difference was not maintained in more advanced times of the onset of the symptoms. Although the production of RDB-specific IgG1 is consistent with activation of type 1 T helper lymphocytes (Th1) 59, this focal difference in the first week cannot be explained simply by balancing different subpopulations of T helper cells but maybe involve other factors, including sample bias, differences in individual immune responses, and/or early viral load. Also, no statistical differences were observed in IgG3 levels between patients who died and recovered. Surprisingly, we also noticed higher levels of RBD-specific IgG4 in sera from patients who died when compared to survivors in the second and third weeks of analysis. In our analysis, 55% (10 out of 18) serum samples of patients who progressed to death showed early positivity to RDB-specific IgG4 antibodies, whereas patients who recovered from COVID-19 were IgG4 negative to SARS-CoV-2 RDB in the same window of time. The IgG4 biosynthesis is known to be induced under conditions of increased IL-10 cytokine 60 having as a primary source several immune cells, including Th2 cells, regulatory T cells (Treg), or even regulatory B cells (Breg). Patients with severe COVID-19 display sustained inflammation and continued production of various anti- and pro-inflammatory cytokines (cytokine storm syndrome) 36, including the IL-10 production that may be associated with induction of IgG4 antibodies in severe COVID-19. Although substantial knowledge about the antibody response has already been generated nowadays for COVID-19, further studies are necessary to understand the role of IgG4 antibodies in COVID-19 pathophysiology.
In conclusion, the present study constitutes the effort to clarify the kinetics of IgG antibodies, avidity, and subclasses against SARS-CoV-2 RDB in symptomatic patients with COVID-19 in Brazil, highlighting the importance of IgG antibody avidity in association with IgG4 detection as a laboratory tool in the follow-up of hospitalized patients with more significant potential for life-threatening conditions in the population analyzed.