Neutralizing antibody responses to SARS-CoV-2 Omicron variants: Post six months following two-dose & three-dose vaccination of ChAdOx1 nCoV-19 or BBV152

Globally, COVID-19 cases were on the rise along with reported mortality in March–April 20231. Over 12,591 new SARS-CoV-2 infections were recorded in India on April 21, 2023, with a daily positivity rate of 5.64 per cent and 40 deaths. The World Health Organization (WHO) also reported an increase in COVID-19 cases (437%) and deaths (114%) in India towards the end of March 202323 and 712 sequences of Omicron variant XBB.1.16 from 21 countries4. In India, the majority of sequences were from the XBB.1, XBB.1.16, BA.2.10 and BA.2.75 lineages5 which were responsible for sudden rise in number of cases.

The diverse Omicron sub-lineages emerging with immune escape potential, and decreased vaccine efficacy is a global concern67. Furthermore, Omicron variants are known to be highly transmissible and cause an unexpected increase in breakthrough and re-infections891011.

Reports on waning immune response after primary immunization led to the recommendation of a booster dose1213. An additional precautionary dose (third or booster) was introduced on 10 January 2022. As of 21 April 2023, about 228 million individuals were vaccinated, majorly with the homologous booster regimen with Covishield/Covaxin14. Although the primary immunization coverage in India was >90 per cent, the uptake of booster dose remained at 30 per cent of the eligible population. The primary reason for decreased acceptance for the third dose seems to be the reduced severity of infections in terms of hospitalizations and deaths. India experienced at least three peaks of COVID-19 transmission after the introduction of COVID-19 vaccination: April-June 2021, January-march 2022 and March-April 2023.15 This has created a significant pool of people with hybrid immunity. Currently, in India, two major groups of COVID-19-vaccinated populations exist: the first category comprises individuals who have completed their primary immunization with two doses and have been infected with SARS-CoV-2 one or more times, whereas the second category is of individuals who have taken a precutionry/booster dose (received three doses) and have been infected with SARS-CoV-2 one or multiple times. Limited studies have documented the comparative dynamics of antibody responses in these two groups16171819202122232425. Hence, we undertook a comparative analysis of neutralizing antibody responses among the above two vaccinated population groups, against SARS-CoV-2 variants circulating recently.

Material & Methods

Study participants: The study was conducted in two different cohorts of COVID-19-vaccinated individuals. The first cohort comprised 88 individuals (44 each in Covaxin and Covishield groups) whose sample collection was done between January 2023 - February 2023 and had completed their primary immunization at least six months before sample collection (six months to one year) and had not received any booster. These samples were collected from individuals at the Institute of Genomics and Integrative Biology (IGIB), Council of Scientific & Industrial Research (CSIR), New Delhi as part of continued efforts for CSIR-Cohort232425. This cohort had undergone three previous phases of national serological surveys and was well characterized for vaccination history. The second cohort comprised individuals whose samples were collected between September 2022 and January 2023 and had been vaccinated with a third dose (n=102) six months prior to sample collection. Samples from the booster cohort were collected from Indian Council of Medical Research (ICMR) institutes present across India. The Institutional Human Ethics Committees of the ICMR-National Institute of Virology, Pune, and IGIB, New Delhi approved this study.

Isolation of SARS-CoV-2 strains: Isolation of BF.7, BA.5.2, BQ.1 and XBB.1 variants was performed with in vitro and in vivo approaches as described earlier by Yadav et al26. In brief, hamsters were inoculated intranasally with 100 µl of nasal swab/throat swabs collected from COVID-19-positive cases. After the third day post infection, tissue specimen suspension (lungs, NT) was screened using real-time reverse transcription–polymerase chain reaction (RT-PCR). In addition, Vero CCL-81 cells were inoculated with hamster specimens positive for SARS-CoV-2.

Hamster specimens and virus isolates were confirmed by next-generation sequencing (NGS) along with retrieval of the complete genome of the Omicron variants27. In brief, the RNA was extracted and quantified using Qubit® 2.0. cDNA synthesis was done using the first strand and second synthesis kit, and ribosomal RNA was depleted by the NEBNextrRNA Depletion kit (NEB, MA, USA), followed by the preparation of RNA libraries with the TruSeq Stranded Total RNA library preparation kit (Illumina, USA). Using the normalized Illumina NGS platform, quantification of the amplified RNA library was done. Subsequently, with the CLC Genomics Workbench v20.0.4 (QIAGEN, Aarhus, Denmark) the retrieved genomes were analyzed.

Sample collection from participants: A total of 5 ml of blood was collected from each of the participants in two-dose and three-dose cohorts for antibody estimation and neutralization assays. Throat swabs/nasal swabs were taken in viral transport medium for symptomatic individuals any time after receipt of the third dose and screened for SARS-CoV-2 by RT-PCR. History of known COVID-19 infections before or after vaccination was sought from the study participants.

Assessment of SARS-CoV-2 spike (S1)-RBD IgG antibodies: Anti-S1-RBD immunoglobulin G antibodies in the sera of all participants were determined using a two-step SARS-CoV-2 IgG II Quant assay. The analytical range of the assay is 21-40,000 AU/ml, and the cut-off for positive is ≥50 AU/ml.

Micro-neutralization assay: The assay was conducted as reported earlier26 using Vero CCL-81 adapted Indian Omicron isolates: XBB.1, BA.5.2, BQ.1 and BF.7. Serum samples were inactivated by incubating at 56°C for 1 h, followed by serial dilution, first in 1:10 ratio, and by four-fold dilution. A neutralizing mixture was prepared by adding an equal volume of diluted samples and two log TCID50 Omicron isolates. Followed by one hour of incubation at 37°C, the Vero CCL-81 monolayer was infected by neutralization mixture in 96-well plates. The plates were incubated for four days at 37°C with 5 per cent CO₂.

Virus control (100 TCID50 of the virus without antibody) of respective variants, positive control (anti-SARS-CoV-2 IgG antibody) and negative control (normal non-immune serum) were used as assay controls. The 50 per cent of the virus neutralization observed at the uppermost serum dilution was used to calculate the neutralizing titre. The neutralization potential of sera of all individuals against SARS-CoV-2 strains used in this study was determined using a positive cut-off value ≥1:2028.

Data analysis: Data were analyzed using GraphPad Prism 9.5 statistical software (Dotmatics, Boston, USA; https://www.graphpad.com/company). Geometric mean titres (GMT) for NAb were calculated. Within-SARS-CoV-2 variant NAb response assessment and comparison of NAb responses among two groups was assessed by the Paired t-test (P<0.05).

Results

A pictorial summary of nucleotide and amino acid mutations in the sequence of the lineage of Omicron sub-variants (XBB.1, BA.5.2, BQ.1 and BF.7) retrieved from clinical specimens, in vivo (hamster model) and adapted in vitro (Vero CCL-81 cells) isolates, is represented in heat map (Fig. 1).

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Fig. 1

The diagrammatic presentation of nucleotide and amino acid mutations in the sequence of XBB.1, BA.5.2, BQ.1 and BF.7 retrieved from clinical specimens, in vivo (hamster model) and in vitro (Vero CCL-81 cells) isolates.

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The two-dose cohort comprised individuals who had completed their primary immunization and comprised 88 individuals with equal numbers (44 each) having received either Covishield only or Covaxin only. This cohort had 46 men and 42 women with a median age of 29 and 28 yr (age range of 21–61 yr), respectively.

The three-dose group comprised 102 participants who got three doses of homologous SARS-CoV-2 vaccine; Covaxin was administered to 56 people and Covishield to 46 people. There were 78 men and 24 women with a median age of 44 and 39 yr (age range of 20–66 yr), respectively. A total of 11 out of 88 participants in the two-dose cohort and 30 out of 102 participants in the booster-dose cohort had breakthrough infections observed in the last six months. The overall characteristics of the study participants and infection history are represented in Table.

SARS-CoV-2 spike (S1-RBD) immunoglobulin G antibody assessment indicated persistence of detectable levels of S1-RBD IgG titres among all the individuals in both cohorts. The mean S1-RBD IgG antibody concentration was 2893 AU/ml in individuals receiving two doses (range: 159–40000 AU/ml) and 7594 AU/ml (range: 106–40000 AU/ml) in individuals receiving three doses. Furthermore, in individuals from the three-dose group, the geometric mean S1 RBD IgG titres rose to 4668 AU/ml when compared to that in two-dose cohort (GMT-1760 AU/ml) (Fig. 2).

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Fig. 2

Anti-S1RBD IgG antibody titres in the sera of participants in two dose and three dose cohorts post six months of vaccination for Covaxin (A) and Covishield (B) were assessed and comparatively analysed using unpaired t-test (P <0.05). Shown are the box plot for two and three doses with error bars representing minimum and maximum values, respectively, while the box represents 25th and 75th percentile from bottom to top respectively.

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In the two-dose cohort (n=88), GMTs with 95 per cent confidence interval of neutralizing antibody titres (NAbs) of the sera of Covishield participants (n=44) against B.1, BA.5.2, BF.7, BQ.1 and XBB.1 were 294, 56, 30, 28 and 17, respectively. The XBB.1 lineage showed a significant fold-reduction in NAb titres (18-fold), followed by BQ.1 (11-fold), BF.7 (10-fold) and BA.5.2 (5-fold) (P<0.05), compared to the prototype B.1 variant (Fig. 3A). However, 06, 07, 10 and 14 study participants among Covishield group had NAb titre below the assay cut-off for BA.5.2, BQ.1, BF.7 and XBB.1, respectively.

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Fig. 3

Neutralizing antibody (NAb) responses six months post two doses and three doses of SARS-CoV-2 vaccination: NAbs responses against BA 5.2, BQ.1, BF.7 and XBB.1 SARS-CoV-2 variants in comparison with B.1 prototype strain in sera collected post two doses vaccination and post three doses of Covishield (A and C) and Covaxin (B and D) vaccination. A paired t-test was used for comparative analysis among the variants, within-variant comparisons were assessed using an unpaired t-test (P *< 0.05, **<0.01, ***<0.001).

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Similarly, GMTs of NAbs of the sera of Covaxin participants (n=44) against B.1, BA.5.2, BF.7, BQ.1 and XBB.1 were 99, 15, 9, 7 and 5, respectively. The XBB.1 variant showed a higher fold-reduction in NAb titres (19-fold), followed by BQ.1 (15-fold), BF.7 (12-fold) and BA.5.2 (7-fold) (P<0.05), compared to B.1 prototype (Fig. 3B). However, 06, 11, 16, 16 and 17 study participants among Covaxin group had NAb titre below the assay cut-off for B.1, BA.5.2, BQ.1, BF.7 and XBB.1, respectively.

In the booster-dose cohort (n=102), the GMT of Covishield participants (n=46), NAb titres against B.1, BA.5.2, BF.7, BQ.1 and XBB.1 were 287, 65, 26, 27 and 14, respectively. Furthermore, the NAb titre against XBB.1, BQ.1, BF.7 and BA.5.2 decreased by 21, 11, 10 and 4 folds, respectively, in comparison to B.1 (P <0.05) (Fig. 3C).

Further analysis revealed that 02, 03, 04, 10 and 34 study participants among the Covishield group had NAb titres below the assay cut-off for B.1, BA.5.2, BQ.1, BF.7 and XBB.1, respectively.

Likewise, GMT of Covaxin participants’ (n=56) NAb titres against B.1, BA.5.2, BQ.1, BF.7 and XBB.1 were 94, 15, 7, 7 and 4, respectively. Further, the NAb titres against XBB.1, BQ.1, BF.7 and BA.5.2 decreased by 24, 13, 13 and 6 folds, respectively, in comparison to B.1 (P<0.05) (Fig. 3D). However, 23, 27, 37 and 43 study participants among the Covaxin group had NAb titre below the assay cut-off for BA.5.2, BQ.1, BF.7 and XBB.1, respectively. However, no study participants showed Nab titre below the assay cut-off for B.1 variant.

Further analysis of GMTs of NAb titres between two-dose and three-dose cohorts indicated that the levels of NAb were the highest for B.1 strain, followed by BA5.2, BQ.1, BF.7 and XBB.1, respectively, in the two groups. All individuals who received two doses and those receiving three doses (except 06 participants from the two-dose cohort and 02 participants from the three-dose cohort) had high protective level NAbs (93 and 98%) against the ancestral strain. Similarly, in the two-dose and booster-dose cohorts, 81 and 75 per cent, 74 and 70 per cent, 70 and 56 per cent and 65 and 25 per cent of individuals displayed sero-protective levels of NAbs to BA5.2, BQ.1, BF.7 and XBB.1 respectively. The percentage of sero-protectivity seemed lower against in newer lineages of Omicron, i.e. XBB.1, BF7 and BQ.1 strains, indicating a high level of neutralization escape.

Discussion

All the currently available SARS-CoV-2 vaccines are primarily developed using prototype Wuhan strain B.1. Although these vaccines have helped to reduce the disease severity, it showed lesser protection against the infection with Omicron variants. Our study highlighted significant reduction in NAb titres among two-dose and booster-dose cohorts, against Omicron lineage strains, compared to the titres against B.1 prototype strain. The persistence of binding antibodies and presence of high sero-protection against B.1 and moderate seroprotection against BA5.2 were observed compared to BQ.1 and BF.7. The presence of NAb is a surrogate marker of defence against infection; therefore, the significantly reduced NAbs responses against the currently circulating COVID-19 strains indicated immune escape, which was a matter of concern6171819. The findings of our study are in concordance with the observations reported elsewhere with a significant reduction in neutralization titre against BA.2, BA.4/5, BQ.1.1, BN.1, BQ.1, XBB.1.5 and XBB.129303132. This could be due to the waning of immunity three months post vaccination of primary two doses.

Further observation indicated that the Omicron variant showed marked neutralization escape compared to other variants of SARS-CoV-233343536. Through the structural analysis of XBB.1, Qu et al37 demonstrated mutations in the spike residue at position 486 responsible for immune suppression and dominance of XBB lineage compared to the other Omicron subvariants. The hydrophobic side chain of the amino acid F486 is a hotspot for developing a defence against the virus, while the mutation F486S makes it easier for an antibody to escape the virus.

A small number of samples per group and the non-availability of cell-mediated hybrid immunity data for correlating with antibody responses are the limitations of our study1718.

The presence of hybrid immunity may explain the higher titres of NAb observed in participants in our study. This may be due to quick recall of vaccine or infection-induced memory. The memory B-cells generated during the initial exposure of SARS-CoV-2 antigen to the immune system are quiescent cells. On re-infection or subsequent vaccination, CD4+ T-cells and T-follicular helper cells act as key factors for remembering virus-specific responses and intensification of SARS-CoV-2 memory B-cells, which lead to a higher magnitude in hybrid immunity3839.

As reported elsewhere, superior infection-neutralizing capacity are observed against all Variant of Concerns (VoCs), including omicron, after either two vaccinations in convalescents or a third vaccination or breakthrough infection of twice-vaccinated, naive individuals. These three consecutive spike antigen exposures result in an increasing neutralization capacity per anti-spike antibody unit; a similar trend was observed in our study40.

The currently prevalent hybrid immunity may provide protection by preventing severe disease with newer variant strains such as BA.5.2, BF.7 and BQ.1. However, the evolution of SARS-CoV-2 is ongoing due to immune pressure and high transmissibility. Among the Omicron sub-variants, XBB.1 showed marked neutralization escape and might have chances to expand and cause more infections. Such new variants may evade immunity and cause an alarming increase in the number of cases. However, it is critical to understand the role of cell-mediated immune response in protection against repeated infections of COVID-19. Besides, it is also important to continuously generate high-quality clinical data to understand the potential of emerging strains to cause severe disease leading to hospitalization and deaths. Care of comorbid people and adherence to COVID-appropriate behaviour, especially during the upsurge of respiratory illness, are advisable even in vaccinated individuals.

Overall, this study generated evidence for sero-protection against newer variants such as BA.5.2, BF.7 and BQ.1 through hybrid immunity of vaccination and infection in both cohorts. Persistence of NAb responses seemed comparable in two-dose and three-dose cohorts after six months of vaccination. However, clinical data and cell-mediated immunity assessment may throw more light on sustained protection against newer variants through booster doses and currently prevalent hybrid immunity. Hence, it is important to monitor the long-term protection offered by COVID-19 vaccination against immune-escape mutants.

Furthermore, as observed in our study, the percentage of sero-protectivity seems to be lower against newer lineages of Omicron, i.e. XBB.1, BF7 and BQ.1 strains, indicating a high level of neutralization escape. The possibility of developing and fast-tracking introduction of newer or tweaked COVID-19 vaccines, which could be more effective against the newer strains, should therefore be explored.