Ancestral SARS-CoV-2-Driven Antibody Repertoire Diversity in an Unvaccinated Individual Correlates with Expanded Neutralization Breadth

ABSTRACT Understanding the quality of immune repertoire triggered during natural infection can provide vital clues that form the basis for development of a humoral immune response in some individuals capable of broadly neutralizing pan-SARS-CoV-2 variants. In the present study, we report variations in neutralization potential against Omicron variants of two novel neutralizing monoclonal antibodies (MAbs), THSC20.HVTR11 and THSC20.HVTR55, isolated from an unvaccinated convalescent individual that represent distinct B cell lineage origins and epitope specificity compared to five MAbs we previously reported that were isolated from the same individual. In addition, we observed neutralization of Omicron variants by plasma antibodies obtained from this particular individual postvaccination with increased magnitude. Interestingly, this observation was found to be comparable with six additional individuals who initially were also infected with ancestral SARS-CoV-2 and then received vaccines, indicating that hybrid immunity can provide robust humoral immunity likely by antibody affinity maturation. Development of a distinct antigen-specific B cell repertoire capable of producing polyclonal antibodies with distinct affinity and specificities offers the highest probability of protecting against evolving SARS-CoV-2 variants. IMPORTANCE Development of robust neutralizing antibodies in SARS-CoV-2 convalescent individuals is known; however, it varies at the population level. We isolated monoclonal antibodies from an individual infected with ancestral SARS-CoV-2 in early 2020 that not only varied in their B cell lineage origin but also varied in their capability and potency to neutralize all the known variants of concern (VOCs) and currently circulating Omicron variants. This indicated establishment of unique lineages that contributed in forming a B cell repertoire in this particular individual immediately following infection, giving rise to diverse antibody responses that could complement each other in providing a broadly neutralizing polyclonal antibody response. Individuals who were able to produce polyclonal antibody responses with higher magnitude have a higher chance of being protected from evolving SARS-CoV-2 variants.

Next, we examined the magnitude and quality of neutralizing antibodies against Omicron variants developed in the same C-03-0020 donor and compared them with antibody responses observed with individuals who were vaccinated after infection with ancestral SARS-CoV-2. The participants included in this study were members of the DBT COVID-19 consortium cohort, organized by interdisciplinary research institutes and hospitals in the National Capital Region of India. The study protocol was approved by the Institute Ethics Committees of all participating institutions. Written informed consent was obtained from all the participants who contributed biospecimens and for the clinical information collected.
As part of this case study, we first examined the magnitude of antibodies developed in the C-03-0020 donor, 20 weeks after receiving the third vaccine dose of ChAdOx1nCoV-10 (Covishield) ( Fig. 2A), for their ability to neutralize Omicron BA.1, BA.2, and BA.4. Remarkably, antibodies obtained from the follow-up visit by this individual demonstrated neutralization of Omicron BA.1, BA.2, and BA.4 with potency significantly increased by 16.06-, 11.33-, and 9.67-fold, respectively, compared to the person's baseline convalescent antibodies (Fig. 2B). To further investigate whether what we observed with this particular individual (C-03-0020) is unique or not, we examined the ability and magnitude of antibody responses developed in six additional individuals after infection with ancestral SARS-CoV-2 and after vaccination (Table S2). Notably, we observed data from the additional six individuals comparable with those we observed with the C-03-0020 donor, who received different doses of vaccines after SARS-CoV-2 "neutralizing" monoclonal antibodies reported in the CoV-AbDAb database (http://opig.stats.ox.ac.uk/webapps/covabdab/), obtained from human germ lines, were selected based on availability of both heavy and light chain sequences (11). Postfiltration, a total of 3,448 sequences were retained (including 7 MAb sequences from the present study). Variable heavy and light chain IgG amino acid sequences were aligned using MAFFT v7.453 (13). Maximum likelihood trees were constructed with IQTree (V. 1.6.12) using a custom Ab amino acid substitution model (14,15). The robustness of the tree was assessed using 1,000 ultrafast bootstrap replicates as well as 1,000 Shimodaira Hasegawa like approximate Likelihood Ratio Test (SH-aLRT) replicates. Trees were visualized and annotated using the ggtree package in R (16). (C) Pseudovirus neutralization assay. Neutralization of pseudoviruses expressing Omicron BA.1, BA.2, and BA.4 spikes by THSC20.HVTR04, THSC20.HVTR06, THSC20.HVTR11, and THSC20.HVTR26 MAbs was carried out using HeLa-ACE2 cells. LY-CoV016, REGN10933, REGN10987, and CC12.3 MAbs were used for comparison with our MAbs as known controls (17)(18)(19)(20). Values are means from duplicates, and the assay was repeated at least three times. (D) Live virus neutralization assay. Neutralization potency of THSC20.HVTR04, THSC20.HVTR06, THSC20.HVTR11, and THSC20.HVTR26 MAbs against live authentic Omicron BA.1, BA.2, and BA.5 viruses was assessed in Vero E6 cells by the focus reduction neutralization test. Each point represents mean percent neutralization at a given dose of MAb plotted on the x axis. Neutralization curves were plotted using GraphPad Prism software (v8.1.2).

SARS-CoV-2 Neutralizing Antibody Precursor Diversity
Microbiology Spectrum infection with the ancestral virus. For the C-03-0020 donor, we could obtain samples only after the person received the third vaccine dose, compared to others who received two doses of vaccines (Table S2). Additionally, we also made the following two observations: (i) antibodies elicited postvaccination from one of them (C-10-0006) neutralized all the Omicron variants (BA.1, BA.2, and BA.4) with a significantly higher magnitude (potency) and (ii) the ability of plasma antibodies obtained from other donors after vaccinations to neutralize the same Omicron variants varied-for example, antibodies elicited in donor C-03-0015 could neutralize all the Omicron variants examined in this study, while donor C-03-0008 could neutralize BA.1 and BA.2 but not BA.4 ( Fig. 2C and Table S2). Although antibodies developed in these individuals before vaccination varied in neutralizing Omicron variants, the antibody response mounted postvaccination demonstrated neutralization of the same variants with increased potency (Fig. 2C), which was comparable to that observed with C-03-0020 followup plasma samples. Very interestingly, plasma antibodies obtained from one individual  Table S2 in the supplemental material). Donor C-13-0022, infected with the ancestral SARS-CoV-2, who was included in this experiment, was not vaccinated at the time of sample collection and was used as an unvaccinated and nonbreakthrough infection control. B-UV refers to the plasma samples from baseline unvaccinated individuals, and FU-V (left panel graph) refers to follow-up vaccinated individuals postvaccination used for the neutralization assay as two groups against the pseudoviruses expressing Omicron BA.1, BA.2, and BA.4 spikes, for whom differences were found to be statistically significant (P , 0.001) with the Wilcoxon matched-pair signed-rank test. The right panel graph represents the same data as a head-to-head comparison of neutralization titers of the same individual before and after vaccination against individual BA.1, BA.2, and BA.4 variants. With a nonparametric Wilcoxon matched-pair signed-rank test, the P values were observed to be identical (P = 0.0078) across BA.1, BA.2, and BA.4 data sets. Neutralization curves were plotted using GraphPad Prism software (v8.1.2).
(C-13-0022) who was infected with the ancestral SARS-CoV-2 with no history of vaccination (and no history of being symptomatic, either) were found to confer neutralization of all the Omicron variants with very high potency ( Fig. 2C and Table S2). This could possibly be due to development of a robust and effective SARS-CoV-2-specific B cell repertoire postinfection and possibly humoral immunity boosted by likely exposure to Omicron variants. It is to be noted that we used the plasma samples of the individual C-03-0020 obtained after the third dose of the vaccine, as opposed to others in the same category who received two doses. This was because of the unavailability of the samples after the second vaccine dose due to loss of this particular donor to follow-up. Next, we compared the magnitude of the antibody response developed postvaccination in this particular individual (C-03-0020) with that of the response obtained from 16 vaccine breakthrough Omicron-infected individuals (Table S3). Interestingly, the plasma antibodies obtained from the C-03-0020 donor after receiving three doses of vaccine showed neutralization of the three Omicron variants examined with magnitude comparable to that observed with antibodies developed in individuals with Omicron-mediated breakthrough infection (BTI), which demonstrated the most potent neutralization of the same viruses (Fig. S2). This was observed in sharp contrast to that obtained from individuals who received full vaccine doses with no history of infection and reinfection (as shown in Fig. S2). This observation also indicates that the observed development of cross-neutralizing antibodies with higher magnitude following vaccine boosters was also likely due to clonal expansion of the B cell lineages developed in this individual through mutations within the antibody genes associated with affinity maturation as previously indicated elsewhere (12).
Overall, through this study, we made the following observations: (i) the diversity of antibody responses mounted in this particular individual (C-03-0020) immediately postinfection collectively overcame the mutational landscape offered by the evolving SARS-CoV-2 and (ii) while vaccination postinfection improved humoral immune responses, variability in their magnitude and potential to counter Omicron variants was possibly due to the differences in their ability to rapidly develop unique B cell repertoires postinfection associated with mounting of robust polyclonal antibody responses. In particular, it is likely that vaccination following infection or infection following vaccination each demonstrated hybrid humoral immunity of significantly higher magnitude. Similarly, the higher magnitude of humoral immune response due to vaccination following infection than of what we observed before vaccination in this particular C-03-0020 donor and also with other individuals in the same category (infection followed by vaccination) likely arose through affinity maturation of antigen-specific B cell lineages. Our study also provides evidence that identification of unique B cell germ lines such as IGVH3-30-3*01F, IGVH7-4-1*02F, IGVHV1-69*02F, and IGVH3-53*04F associated with development of SARS-CoV-2 variant cross-neutralizing antibodies THSC20.HVT04, THSC20.HVTR06, THSC20.HVTR11, and THSC20.HVTR26, respectively, can potentially inform rational approaches in developing improved vaccine immunogens that can selectively act to target B cell lineages in mounting humoral immune response not only of higher magnitude but also which can offer expanded breadth with increased potency against currently circulating variants, future SARS-CoV-2 variants, and potentially all coronaviruses.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.6 MB.
USA, for kindly providing us with the SARS-CoV-2 RBD construct used for purification of RBD protein, and the IAVI-Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA, for providing us with the REGN-10933 (casirivimab), REGN-10987 (imdevimab), and LY-CoV016 (etesevimab) monoclonal antibodies. We finally thank all the members of our laboratory, THSTI, and the Advanced Technology Platform Center (ATPC) of the Regional Center of Biotechnology for antibody sequencing, access to BLI support, and their support. We acknowledge the funding support from the Bill and Melinda Gates Foundation, Seattle, WA, USA (INV-030592), the Indian Council of Medical Research (CTU/Cohort study/17/ 10/22/2021/ECD), the Department of Biotechnology (BT/PR40401BIOBANK/03/2020), and the Research Council of Norway (project identifier [ID] 285136). J.B. is supported by the DBT-Wellcome Trust India Alliance Team Science Grant (IA/TSG/19/1/600019). P.K.G. is supported by the J C Bose fellowship from SERB.
A patent application (PCT/IB2022/057923) has been filed on the invention of the novel monoclonal antibodies.