Investigating the SARS-COV-2 specific antibody response in convalescent pediatric COVID-19 patients

Abstract

The novel coronavirus SARS-CoV-2, has emerged in Wuhan, China at the end of 2019 and it quickly spread worldwide, causing the disease now known as COVID-19. WHO declared COVID-19 a pandemic on March 11, 2020 and Turkey reported its first confirmed case on the same day. Within a few months, COVID-19 caused millions of confirmed cases and deaths worldwide. Coronaviruses belong to the Nidovirales order are the largest known RNA virus family with a large genome. They are commonly divided into four genera; alpha-, beta-, delta- and gammacoronaviruses. There are some human coronaviruses under the Alphacoronavirus genus, that cause mild, flu- like disease. However, there are some coronaviruses in the Betacoronavirus genus like SARS-CoV, MERS-CoV and their recent member SARS-CoV-2, that cause severe respiratory syndromes in humans and caused disastrous epidemics. After its rapid, global spread, SARS-CoV-2 was isolated from airway epithelial cells and its genome was sequenced in order to place it into its appropriate spot in the phylogenetic tree. Since this novel coronavirus was causing severe acute respiratory syndrome and showed genetic similarities to original SARS-CoV, it was named as SARS-CoV-2 and confirmed as the new member of Betacoronavirus genus in January 2020. SARS-CoV-2 genome consists of 10 open reading frames (ORFs) which is similar to SARS and MERS-CoV genomes. First ORF is responsible for the non-structural proteins' (NSPs) synthesis while the other ORFs are responsible for the synthesis of four major structural proteins of the virus; spike (S), membrane (M), nucleocapsid (N), envelope (E). Spike protein of the virus is responsible for the binding and entry of the virus into the host cell. Spike protein has two major domains; S1 and S2; receptor binding domain (RBD) which is responsible for the ACE2 receptor binding is located in the S1 domain. Nucleocapsid protein of the virus is responsible for the viral RNA packaging and virion assembly. The virus enters into host cells either via direct ACE2 interaction or TMPRSS2 interaction. Either way, two cleavage events are required for the proper binding and internalization of the virus into host cell. COVID-19, the disease that SARS-CoV-2 causes, can be clinically presented in many forms, from asymptomatic to death. Generally, the disease is characterized by severe acute respiratory syndrome while affecting multiple organs. After initial infection, the incubation period of the virus is generally 4-5 days but symptomatic patients start to show symptoms generally around 11,5 days. The most commonly observed symptoms are dry cough, fever, shortness of breath, muscle and joint pains, diarrhea, nausea, headache etc. To date, many vaccines by different companies have been approved by FDA but still new, reliable treatment options are searched, since current vaccination status of the world population is not enough for herd immunity protection. B cells and antibody production is crucial for the immune system protection against many diseases. B cells can be either activated via help from T cells or independently from T cell help. After their activation they go through multiple maturation steps that begin in the bone marrow and completed in the spleen or lymph nodes. Through this maturation steps, B cells are "educated" not to react to self-antigens. B cells produce proteins called antibodies or immunoglobulins which can either serve as B cell receptors or secreted into extracellular space to bind to foreign antigens to neutralize them or tag them for other immune cells to destroy. There are different types of immunoglobulins and differentiation from one immunoglobulin type to another occurs during the B cell development with a distinct event called the immunoglobulin class switching. During class switching events, V, D, and J segments of immunoglobulin heavy chains and V and J segments of Ig light chains go under several DNA cleavage and repair events, that enables nearly an endless possible combination of different segments. This event eventually results in an extensive antibody repertoire that is highly specific to many, various antigens. This recombination event also determines the antibody type via the selected Fc region; IgM, IgD, IgG, IgA, and IgE. IgG type has four subtypes; IgG1, 2, 3 and 4. Immune responses to COVID-19 varies greatly among individuals; from asymptomatic to pneumonia cases with ARDS and sometimes death. This severity is generally associated with high pro-inflammatory responses such as elevated serum interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNFα); inflammatory chemokines, lymphopenia, cytokine storm, coagulopathy in vessels. However, it has been observed that children were less affected by COVID-19 and mostly asymptomatic when infected. Even though children present milder disease outcomes compared to older adults, the dysregulated immune response in children after the COVID-19, can result with a life-threatening condition named as multisystem inflammatory syndrome in children (MIS-C) which develops after the children have recovered from COVID-19, potentially 4-6 weeks after infection. When the antibody responses of children and adults were compared, it was observed that less amount of children produced N-IgG antibody compared to adults, and S-IgG antibody. It was shown that the primary antibody produced against SARS-CoV-2 was mainly S-IgG and low levels of S-IgM, both in MIS-C and non-MIS-C patients. With the subclasses of IgG, IgG1 and IgG3 were commonly observed in higher levels compared to IgG2 and IgG4 since they are the known primary responders in viral infections. The main aim of this study was to investigate how children's antibody responses change throughout a long period of time, up to nine months after the children recovers from COVID-19, how different disease severities and age differences affect antibody responses and fill the gaps in knowledge of how immune system reacts to SARS-CoV- 2 in children. In order to investigate this, blood samples were collected from convalescent pediatric patients at different time points after they are recovered from COVID-19; 1st, 3rd, 6th and 9th months. Plasma from these blood samples were collected. To investigate the SARS-CoV-2 spike and nucleocapsid-specific antibody levels in blood plasma, homemade indirect ELISA were developed and optimized with high sensitivity and specificity. Our findings showed that S-IgM levels generally low and similar to those of healthy control group. In the contrary, N-IgG levels were similar to S-IgG levels at the first month post-COVID and were high, however, after six months, N-IgG levels decrease significantly. Unlike N-IgG levels, high S-IgG levels seemed to persist longer than N-IgG, where no significant decrease during the nine- month period have been observed. Although, the antibody responses were slightly higher in moderate and severe patients compared to the mild patients, there were no significant differences. Nevertheless, a significant decrease of N-IgG responses after six months has been observed in mild patients, which suggests that N-IgG levels might be persisting longer in moderate and severe patients. Another important finding was that pediatric patients between the ages of 10-14 had lower antibody responses compared to children between the ages of 5-9 and this difference was significant in N-IgG levels and in S-IgM levels of moderate + severe patients. The second aim of this study was to optimize a possible alternative method for indirect ELISA called the cytometric multiplex bead assay for the detection of IgG subtypes against SARS-CoV-2. We have successfully cytometric bead assay for multiplex use and were able to detect SARS- CoV-2-specific total IgG and IgG subtypes. This method would be a promising alternative for indirect ELISA since it enables detection of multiple antibodies against multiple antigens in one tube and also is more rapid compared to ELISA. We have tested our multiplex cytometric bead assay with a single patient and a healthy control who have given valid results with indirect ELISA and high levels of both spike and nucleocapsid-specific IgG1 and IgG3 compared to IgG2 and IgG4 was observed in patient sample. These results were concordant with the current literature. Thus, it was decided to test all patient and healthy control samples with this assay to detect their SARS-CoV-2 specific IgG subtype levels. This study presented that our homemade indirect ELISA is highly sensitive and specific for the detection of SARS-CoV-2 specific antibodies. Our results show that children produce high amounts of N-IgG and S-IgG against SARS-CoV-2 where S- IgG persists longer in the blood compared to N-IgG after they are recovered from COVID-19. Additionally, moderate to severe patients had slightly higher antibody levels compared to mild pediatric patients. In conclusion, this study provides an overview of how antibody responses of pediatric patients change according to their age, disease severities and how much time has passed since they are recovered from COVID-19.