key: cord-0684903-otays6at
authors: Ozgocer, Tuba; Dagli, Şeyda N.; Ceylan, Mehmet R.; Disli, Faruk; Ucar, Cihat; Yildiz, Sedat
title: Analysis of long‐term antibody response in COVID‐19 patients by symptoms grade, gender, age, BMI, and medication
date: 2021-11-18
journal: J Med Virol
DOI: 10.1002/jmv.27452
sha: c2b4933f0ac3eb2487083ad335c1565e481b53c7
doc_id: 684903
cord_uid: otays6at

The first aim of the study was to analyze the change in antibody titer at 15‐day intervals until 60 days postsymptom onset (PSO). The second aim was to analyze the relationship between antibody titer and symptom grade, gender, age, body mass index (BMI), medications, vitamin supplements, and herbal therapies. Blood samples were collected from 43 patients (5 mild, 21 moderate, 17 severe diseases), 18 women (41.9%), and 25 men (58.1%), on 15, 30, 45, and 60 days PSO after COVID‐19 infection. The serum antibody titers were determined by measuring the COVID‐19 immunoglobulin G (IgG) antibodies by enzyme‐linked immunoassay (ELISA). Associations between the duration of symptoms, demographic and clinical parameters, medications and vitamins used, and herbal therapies were evaluated by interviewing the participants. Within the first 15 days of illness, 81.4% of the patients were positive. From Day 45 PSO, seropositivity was 89.5%. The anti‐SARS‐CoV‐2 antibody titers were statistically higher in men than women at all times (p < 0.01). Antibody titer was higher in older participants compared to younger participants (p < 0.02). Plaquenil or favipiravir use did not affect antibody response (p > 0.05). Men had a higher fever (p = 0.006), shortness of breath (p = 0.004), and chest pain (p = 0.03) than women. We found powerful antibody response by 60 days PSO, as well as higher antibody response and severity of symptoms in the men gender. Data also showed that SARS‐CoV‐2 antibodies are higher in individuals with older age, whereas BMI, concomitant chronic disease, and medications had no effect on antibody titers.

understanding of the development of humoral immunity and antibody resistance to the infection response of exposed individuals is required. Recent studies have offered suggestions to improve the therapeutic use of immune plasma for the acquisition of protective immunity against COVID-19. 4 Although neutralizing antibodies against S-protein may be protective, it has been stated that antibodies against other viral proteins are not functional. 5 A strong antibody response to COVID-19 will significantly reduce the number of virions capable of infecting cells expressing the ACE2 receptor; therefore, studies on antibody responses to COVID-19 are a priority issue for prophylaxis and treatment. In addition, determining the antibody levels of asymptomatic patients and detecting viral load carriers play important roles in preventing transmission.

After SARS-CoV-1 and Middle East respiratory syndrome (MERS) infections, IgG levels can be detected for at least 2 years and up to 17 years. 6, 7 With some exceptions, humoral immunity developed against COVID-19 is likely to have a protective effect for at least a year. 8, 9 Few studies with small numbers of participants have analyzed antibody levels at 35 or 19 days after COVID-19 infections. [10] [11] [12] However, antibody production after infection or vaccination is nonlinear and cannot be predicted from early time points. Therefore, longer term follow-up studies are needed.

The clinical course of COVID-19 infection may be asymptomatic, or it may involve moderate or severe symptoms. Respiratory failure requiring mechanical ventilation along with changes in different tissues and organs, such as multiorgan dysfunction outside the lung, can be seen in severe cases. 13 Information on the modulation of disease severity by antibodies and the durability of antibody responses following infection is limited and conflicting. For example, COVID-19 specific antibodies were reported to be stable for about 82 days in one study, 14 but elsewhere, they were found to decrease 2-3 months after infection. Moreover, lower antibody responses have been reported in patients with mild symptoms of COVID-19. 11, 15 At present, little is known about the adaptive immune response of COVID-19 antigenicity, whether the infection protects against reinfection, or whether the medications and the severity of symptoms increase the antibody response. More data are needed to evaluate the efficacy of antibody levels and their association with symptoms. In this study, we aimed to analyze the antibody titer response at 15, 30, 45, and 60 days PSO in a group of patients with COVID-19. Our second goal was to analyze how the antibody titer changed in terms of age, gender, body mass index (BMI), symptom grade, and medications.

The study protocol complied with the tenets of the Helsinki declaration and was approved by the institutional scientific ethics committee (Protocol#/20.15.18) . Participants were requested to provide written informed consent. In July 2020, we started screening patients who infection by COVID-19. Patients were diagnosed with COVID-19 by either COVID-19 reverse-transcriptase polymerase chain reaction (RT-PCR) (n = 40) or clinically observation (n = 3) (Table 1) . A nasopharyngeal swab was collected by standard procedures and the presence of COVID-19 was determined by RT-PCR testing.

Patients diagnosed with COVID-19 were interviewed individually and symptom onset dates were recorded. The symptom onset of the patient was accepted as Day 0. Serum was collected from peripheral venous blood samples at 15, 30, 45, and 60 days PSO, and stored at +4°C. We used a questionnaire to collect data on the participants' demographic characteristics, gender, age, BMI, medications, and symptom grades. Symptom duration of the participants was recorded in days (Table 2 ). Participants were stratified by symptom grade, age, gender, and BMI (Table 1) . These data were collected by the one-onone interviews of the specialist physician with the patients. Figure 1A ). The correlations between four antibody titers were significantly and positively correlated with each other (r > 0.775; p < 0.000). Overall symptom grade, three age quartiles, gender, and BMI classes were examined using a multigroup analysis ( Table 1) . Average of antibody responses of the greater magnitude shown in men than women; this difference (1.22 vs. 1.60 COI) was statistically significant (p = 0.003, Figure 1B ). The antibody titer was greater in older participants compared to younger participants: ≤37

versus 47-57 p = 0.02 (first AbR), p = 0.01 (third AbR and fourth AbR) (Table 3 and Figure 1C ). Differences among age classes and antibody titers were observed (p < 0.02, Table 3 ). Normal (n = 18) and overweight (n = 17) groups were compared according to BMI classes ( breath (p = 0.004) and chest pain (p = 0.03) were statistically higher in men than women.

A positive correlation was observed between the first AbR and chest pain (r = 0.305; p = 0.04). Surprisingly, first AbR, second AbR, fourth

AbR with myalgia symptom were significantly negatively correlated (p < 0.05); fourth AbR with loss of smell (r = −0.352; p = 0.02) and anxiety (r = −0.403; p = 0.01, Figure 2 ).

In the present study, we followed up patients with COVID-19 in terms of their antibody titers and its possible correlates (gender, age, BMI, medications, symptom severity, concomitant chronic disease). We found that the IgG antibody titers remained high, starting from day 15 until day 60 postsymptom onset. In addition, the antibody response was significantly correlated with myalgia, smell loss, anxiety, and chest pain, but not with other symptoms. Moreover, while age and gender affected the antibody response, BMI, medications, symptom severity, concomitant chronic disease, and medications used did not affect it.

In brief, we analyzed the antibody response to COVID-19 in 43 patients presenting with mild, moderate, and severe symptoms over a 60-day period. Of these, 89.5% of patients exhibited antigenspecific humoral responses, and the antibody titers were higher in male patients. Evidence from a recent meta-analysis indicates that the mortality rate of COVID-19 is higher among males. 16 In the first 60 days PSO, antibody titers against COVID-19 were significantly higher in males than in females. 17 However, higher antibody titers were noted in females naturally exposed to the antigens of viruses causing certain respiratory diseases. 18 Some viral infections or vaccines, such as influenza and its vaccine, may trigger stronger serologic antibody responses in males. 19 Meanwhile, in some cases, COVID-19 vaccines have been reported to elicit stronger serologic antibody and cellular immune responses in females. 20 In addition to the gender-specific differences in antibody titers, we propose that age is another factor shaping the antibody response. As such, we found higher antibody titers in older patients, and the higher frequency of fever in this group also supports our speculation. This stronger antibody response in advanced age groups may be attrib- 

Furthermore, we found that the duration of fever, shortness of breath, and chest pain was significantly longer in males than in females.

Consistent with our findings, another study has reported that antibody titers are positively associated with advanced age and prolonged fever and myalgia. 23 We also found that the loss of smell, which is a specific symptom of COVID-19, 24 was negatively correlated with antibody 

Current information on the roles of antibodies in modulating disease severity and immune response is either limited or controversial. Virusspecific antibody responses are elevated in patients with severe COVID-19. However, the effectiveness of antibodies, rather than their titer, may play a role in serological assays performed in patients who have convalesced or died. 28 Neutralizing antibodies against COVID-19 support immunity, 29 and a positive correlation between serum neutralization capacity and disease severity has been reported. 30 The reason we did not find any correlation between symptom severity and antibody titer in the present study may be the measurement of antibodies that recognize virus-like particles and not neutralizing antibodies. In another study measuring COVID-19 antibody titers using the qualitative IgG/IgM rapid test, symptom severity did not directly affect the antibody response. 31 In addition, the IgG antibody response in patients with mild-to-severe symptoms did not differ during the follow-up period (>75 days). 32 

The present study reports antibody titers over a 60-day period after the onset of COVID-19 symptoms. As the vaccination drives have started, longer follow-up is essential. The protective role of antibodies against COVID-19 remains unknown; however, we noted no decline in antibody titers within 60 days PSO. Although we did not encounter any recurrent COVID-19 cases during the follow-up period, some asymptomatic cases may have remained undetected. Of note, our findings are restricted to a specific ELISA system for recognizing antibodies against virus-like particles; therefore, these observations cannot be generalized to other ELISA systems.

There is conflicting information in the literature that the antibody response to SARS-CoV-2 is related to gender, BMI, and age. It is not known how the symptoms and the drug and vitamin supplements used in the treatment affect the antibody response. 

The study did not receive any external financial support. All authors had full access to the full data in the study and accepted the responsibility to submit for publication.

We thank Assoc. Prof. H. ÇELİK for his editing support.

Prof. Yildiz is the owner of the Y immunotek A. S. (Malatya, Turkey), which produced the QuantiCOR ELISA test. However, he was blind to the data groups and did not take part in statistical analyses. 

Data are available from the corresponding author upon reasonable request.

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals

Rapid generation of neutralizing antibody responses in COVID-19 patients

Examination of seroprevalence of coronavirus HKU1 infection with S protein-based ELISA and neutralization assay against viral spike pseudotyped virus

A systematic review of antibody mediated immunity to coronaviruses: kinetics, correlates of protection, and association with severity

Duration of serum neutralizing antibodies for SARS-CoV-2: lessons from SARS-CoV infection

Antibody responses to SARS-CoV-2: let's stick to known knowns

A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction

Antibody tests for identification of current and past infection with SARS-CoV-2. Cochrane

Antibody responses to SARS-CoV-2 in patients with COVID-19

Kinetics of SARS-CoV-2 specific IgM and IgG responses in COVID-19 patients

Extrapulmonary manifestations of COVID-19

SARS-CoV-2 infection induces robust, neutralizing antibody responses that are stable for at least three months

Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections

Male sex identified by global COVID-19 meta-analysis as a risk factor for death and ITU admission

Convergent antibody responses to SARS-CoV-2 in convalescent individuals

Correlates of severe disease in patients with 2009 pandemic influenza (H1N1) virus infection

Sexual dimorphism of humoral immunity with human vaccines

Initial observations on age, gender, BMI and hypertension in antibody responses to SARS-CoV-2 BNT162b2

Landscape of X chromosome inactivation across human tissues

Sex differences in renal angiotensin converting enzyme 2 (ACE2) activity are 17beta-oestradiol-dependent and sex chromosome-independent

Clinical predictors of donor antibody titre and correlation with recipient antibody response in a COVID-19 convalescent plasma clinical trial

Seroprevalence of SARS-CoV-2 antibodies in people with an acute loss in their sense of smell and/or taste in a community-based population in London, UK: an observational cohort study

The association between obesity and peak antibody titer response in COVID-19 infection

Evaluation of the EUROIMMUN Anti-SARS-CoV-2 ELISA assay for detection of IgA and IgG antibodies

A systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease

Distinct early serological signatures track with SARS-CoV-2 survival

DNA vaccine protection against SARS-CoV-2 in rhesus macaques

Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19

COVID-19 IgG/IgM antibody testing in

Analysis of long-term antibody response in COVID-19 patients by symptoms grade, gender, age, BMI, and medication