Immune evasion after SARS-CoV-2 Omicron BA.5 and XBB.1.9 endemic observed from Guangdong Province, China from 2022 to 2023

Abstract

Background

From 2022 to 2023, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused by Omicron variants spread rapidly in Guangdong Province, resulting in over 80% of the population being infected.

Results

To investigate the levels of neutralizing antibodies (NAbs) in individuals following the rapid pandemic and to evaluate the cross-protection against currently circulating variants of SARS-CoV-2 in China, neutralization assay and magnetic particle chemiluminescence method were used to test the 117 serum samples from individuals who had recovered 4 weeks post-infection. The results indicated that the levels of NAbs against prototype and Omicron variants BA.5 were significantly higher than those against Omicron variants BQ.1, XBB.1.1, XBB.1.9, XBB.1.16 and EG.5, regardless of whether the infection was primary or secondary.

Conclusions

The cross-protection provided by NAbs induced by prototype and Omicron BA.5 variants was limited when challenged by BQ.1, XBB.1.1, XBB.1.9, XBB.1.16 and EG.5 variants. This indicates that we should pay more attention to the risk of multiple infection from any novel Omicron variants that may emerge in the near future.

Background

Since its first report at the end of 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly around the world and undergone continuous evolution [1]. Wild type (WT) is the earliest discovered and prevalent SARS-CoV-2 strain, forms new variants through random mutations in its genome during the epidemic process. The World Health Organization (WHO) classifies novel SARS-CoV-2 variants into two categories based on the public health risks caused by the virus strains: variant of concern (VOC) and variant of interest (VOI) [2], [3]. There are five types of VOC variants recognized by WHO, including Alpha (B.1.1.7), first discovered in southeast England [4]; Beta (B.1.351), first discovered in South Africa [5]; Gamma (P.1), first discovered in Brazil [6]; Delta (B.1.617.2), first discovered in India [7]; and Omicron (B.1.1.529), first discovered in South Africa [8].Since November 2021, Omicron variant strains, such as BA.2, BA.5, BQ.1, XBB.1, JN.1 and KP.2 have quickly become the predominant strains globally [9]. Compared with other variants, the Omicron variant has attracted significant attention due to its greater antigenic changes, stronger infectivity, higher infection rates, and immune evasion capabilities [10].

In April 2023, Annabel et al. reported a national prevalence of SARS-CoV-2 antibodies in primary and secondary school children in England [11]. They found a high seroprevalence rate of SARS-CoV-2 in both primary and secondary school students, with approximately 3-fold higher seroprevalence than confirmed infections in unvaccinated children. In late 2022, coronavirus disease 2019 (COVID-19) caused by Omicron variants of SARS-CoV-2 spread rapidly throughout China, particularly in Guangdong Province (approximately two weeks) [12, 13]. Considering the high coverage of COVID-19 vaccines in China [13], the prevention strategies for COVID-19 had shifted from whole population-wide to focus on key and high-risk populations [14].

The archived data of COVID-19 vaccine has proved their safety and effectiveness, significantly contributing to preventing virus transmission and reducing disease and death. However, the immune protection provided by vaccination is less durable. The emerging variants of SARS-CoV-2 have shown stronger immune escape and transmission abilities, making breakthrough and secondary infections to be the most significant challenge to public. This study was designed to investigate the neutralizing antibody (NAb) levels in individuals following the first wave of a rapid pandemic and subsequent sporadic infection since the end of 2022, aiming to evaluate the cross-protection against the latest circulating variants of SARS-CoV-2 in China.

Materials and methods

Serum samples

From December 2022 to July 2023, a total of 117 serum samples were collected from public health workers (adults) in Guangdong Province who had recovered from their first or secondary SARS-CoV-2 infection at least 4 weeks prior to sample collection.

Cell lines

Vero-E6 and Vero cells are both sensitive cell lines to SARS-CoV-2. Vero-E6 cells are a clone isolated from Vero cell lines. Compared with Vero cells, Vero-E6 cells exhibit enhanced viral proliferation capabilities and a broader viral host range. Vero-E6 cells demonstrate increased sensitive to SARS-CoV-2, and more pronounced cytopathic effects. Therefore, Vero-E6 cells were selected as experimental cells in this study.

Virus

Six clinical isolates of SARS-CoV-2 were used: the prototype strain (2020XN4276) and six Omicron variant strains, including BA.5 (GDPCC 2.00303), BQ.1 (GDPCC 2.01502), XBB.1.1 (GDPCC 2.01503), XBB.19 (GDPCC 2.01543), XBB.1.16 (GDPCC 2.01541), and EG.5 (GDPCC 2.02228). These isolates were obtained from the Biosafety Level 3 Laboratory (BSL-3) of the Guangdong Provincial Center for Disease Control and Prevention (GDCDC) and were preserved by the Guangdong Provincial Center for the Collection of Pathogenic Microorganisms (Viruses) Infectious from Humans. All strains were temporarily stored at -70℃ and stored in liquid nitrogen for long-term storage.

Virus titration

Each SARS-CoV-2 strain was inoculated into a 75cm² cell culture flask (Corning^®, Sigma-Aldrich) containing Vero-E6 cells (2 × 10⁵ cells/ml) and incubated at 37 ± 1℃ with 5% CO₂. The cytopathic effect (CPE) was monitored since the third day. The virus suspension was harvested once 75% CPE was observed, followed by centrifugation at 1000 ×g for 10 min and stored at -70 °C.

The collected virus suspension was diluted in a 10-fold gradient, and inoculated into a 96-cell plate containing Vero-E6 cells (2 × 10⁵ cells/ml). The culture conditions were maintained as described previously, and the CPE of each well was recorded on day six. The TCID₅₀ of each virus strain was calculated using the Reed-Munch method [15].

Neutralization experiment

The serum samples were inactivated at 56℃ for 30 min and then diluted from 1:4 to 1:1024 using a 4-fold dilution series. Each 100 TCID₅₀/50µL virus suspension was mixed with an equal volume of diluted serum and incubated at 37 ± 1℃ with 5% CO₂ for 2 h. Subsequently, each mixture was transferred to new wells containing Vero-E6 cells (2.0 × 10⁵ cells/ml), and incubated at 37 ± 1℃ with 5% CO₂ for 5–7 days. Negative and positive serum controls, as well as the virus, were included for quality control. The CPE of each cell well was recorded, and the titer of neutralizing antibodies was calculated using the Reed-Munch method [15].

Magnetic particle chemiluminescence method

IgG antibodies in serum were detected using a commercially available SARS-CoV-2 IgG antibody detection kit (magnetic particle chemiluminescence method; Autobio, Ltd., Shanghai, China) according to the manufacturer’s instructions. The tests were performed on an automated magnetic chemiluminescence analyzer (Axceed 260, Bioscience) following the manufacturer’s guidelines. All the tests are conducted under strict bio-safety conditions. Each serum sample was evaluated for antibody titer once. The antibody level is expressed as the measured chemiluminescence value divided by the cutoff value (S/CO), where the cutoff value is defined by the receiver operating characteristic curve.

Statistical analysis

Statistical analyses were performed using IBM SPSS Statistics 25 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism for the calculation of mean values for each data point related to authentic virus neutralization assessments. The NAb titers of prototype virus and Omicron sub-lineages of SARS-CoV-2 were log2-transformed prior to analysis, and were compared using Mann-Whitney test. The correlation between the NAb titers of the prototype virus and IgG antibodies levels was assessed using Spearman’s rank correlation coefficient. The significance level was set at α = 0.05, with a p-value of less than 0.05 indicating a statistically significant difference.

Results

Demographic and epidemiological information

From December 2022 to January 2023, during the first wave of the infection peak, 107 serum samples were collected from public health workers (adults) in Guangdong Provincial who had recovered 4 weeks after the first wave of the COVID-19 pandemic caused by Omicron BA.5. All participants were aged between 22 and 62, with a mean age of 43 years, including 44 males and 63 females. In July 2023, during the second wave of the COVID-19 epidemic caused by Omicron XBB.1.9 in Guangdong Province, 10 participants reported first or secondary infections. Their age ranged from 31 to 57 years, with a mean age of 33 years. Among these 10 participants, there was one male and nine females.

To investigate the differences in antibody levels among populations with different immunization history, the collected serum samples from the first wave of infection were divided into three groups: the primary vaccination group, the homologous booster vaccination group and the heterologous booster vaccination group. There were 4 individuals inoculated with less than 3 doses of inactivated vaccine in the primary immunization group, 91 individuals in the homologous booster vaccination group, and 12 individuals in the heterologous booster vaccination group, which were vaccinated with recombinant novel SARS-CoV-2 vaccines (Fig. 1). In the subsequent waves, there were 3 individuals in the primary immunization group, 4 individuals in the homologous booster vaccination group, and 5 individuals in the heterologous booster vaccination group.

Fig. 1

Neutralization experiment results of seven SARS-CoV-2 variants during the first wave of the COVID-19 epidemic: (A) Neutralizing antibody levels of all samples; (B) Neutralizing antibody levels of the primary vaccination group; (C) Neutralizing antibody level of the homologous booster group; (D) Neutralizing antibody levels of the heterologous booster group

NAb titers in serum for the first wave infection against Omicron variants

For the serum samples from thee first wave of infection participants, we found that the NAb titers were significantly decreased against prototype SARS-CoV-2 compared to Omicron variants BA.5, BQ.1, XBB.1.1, XBB.1.16, XBB.1.9 and EG.5. The highest geometric mean titer (GMT) of the samples against the prototype was 280, and the GMT for BA.5 was 41 (5.80 fold, P < 0.001), XBB.1.16 was 14 (19.18 fold, P < 0.001), XBB.1.9 was 11 (23.83 fold, P < 0.001), BQ.1, XBB 1.1 and EG.5 were only 8 (BQ.1, 35.86 fold, P < 0.001; XBB.1.1, 32.67 fold, P < 0.001; EG.5, 35.62 fold, P < 0.001) (Fig. 1A).

Among the primary vaccination, homologous booster vaccination and heterologous booster vaccination group, the highest geometric mean titer (GMT) in all three groups was higher against the prototype than that for BA.5, BQ.1, XBB.1.1, XBB.1.16, XBB.1.9 and EG.5 (Fig. 1B-D). In the primary vaccination group, only one serum sample had a higher titer of neutralizing antibodies against BQ.1 than BA.5. The GMT dropped from 152 for the prototype to 38 for BA.5 (2.99 fold, P = 0.343), 11 for BQ.1 and XBB.1.9 (12.44 fold, P = 0.143), 8 for XBB.1.1 (18.00 fold, P = 0.057), 16 for XBB.1.16 (8.50 fold, P = 0.143), and 13 for EG.5 (10.30 fold, P = 0.143) (Fig. 1B). In the homologous booster vaccination group, the GMT dropped from 287 for the prototype to 45 for BA.5 (5.41 fold, P < 0.001), 15 for XBB.1.16 (18.65 fold, P < 0.001), 12 for XBB.1.9 (23.51 fold, P < 0.001), and 8 for BQ.1, XBB.1.1 and EG.5 (BQ.1, 36.55 fold, P < 0.001; XBB.1.1, 33.53 fold, P < 0.001; EG.5, 35.43 fold, P < 0.001)(Fig. 1C). In the heterologous booster vaccination group, the GMT dropped from 287 for the prototype to 23 for BA.5 (11.68 fold, P < 0.001), 6 for BQ.1 (44.20 fold, P < 0.001), 8 for XBB.1.1 and XBB.1.9 (32.86 fold, P < 0.001), 9 for XBB.1.16 (30.96 fold, P < 0.001), and 5 for EG.5 (55.95 fold, P < 0.001) (Fig. 1D). Notably, participants from the homologous booster vaccination group had the same neutralizing antibodies titers against BA.5 and BQ.1.

NAb titers in serum for the subsequent waves infection against Omicron variants

For the serum samples from the subsequent waves of infection participants, we found that the GMT of the samples against the prototype was 362, and the GMT for BA.5 was 119 (2.03 fold, P < 0.05), BQ.1 was 181 (1.00 fold, P = 0.121), XBB.1.1 was 104 (2.48 fold, P < 0.05), XBB.1.16 was 79 (3.59 fold, P < 0.01), XBB.1.9 was 69, and EG.5 was 64 (XBB.1.9, 4.28 fold, P < 0.01; EG.5, 4.66 fold, P < 0.01) (Fig. 2).

Fig. 2

Neutralizing antibody levels of 10 participants during the second wave of the COVID-19 epidemic

Magnetic particle chemiluminescence

In addition, the IgG antibody in the serum was detected using a commercial SARS-CoV-2 IgG antibody test kit. The positive rate of IgG antibody was 98.13% (105/107) during the second wave of the COVID-19 epidemic. The median of IgG antibody level was 130.12 (Fig. 3A). The median of IgG antibody level in the homologous booster vaccination group was the highest, at 131.52, followed by the heterologous booster vaccination group, with a median of 127.91, and the primary vacancy group, with a median of 79.72. There was a positive correlation between the IgG antibody level and the GMT against the prototype virus (r_s=0.732, P < 0.001).

Fig. 3

Immune status of the population during the first and second waves of the COVID-19 epidemic: (A) IgG antibody levels of 107 participants during the second wave of the COVID-19 epidemic; (B) IgG antibody levels of 10 participants during the second wave of the COVID-19 epidemic

During the second wave of COVID-19 epidemic, the IgG antibody of all participants was detected as positive (10/10), with a median IgG antibody level of 235.09 (Fig. 3B). There was a positive correlation between the IgG antibody level and the GMT against the prototype virus (r_s=0.748, P = 0.013).

Discussion

Previous studies have demonstrated that SARS-CoV-2 elicits a humoral immune response similar to other respiratory coronaviruses [16], [17]. Following a week of SARS-CoV-2 infection, various antibodies specifically recognizing SARS-CoV-2, including IgM, IgA, and IgG, emerge in the serum and exhibit a certain degree of neutralizing capability. After 3–4 weeks of infection, the level of neutralizing antibodies against SARS-CoV-2 reach peak. Subsequently, IgG antibody levels persist at their peak for several months, whereas IgM and IgA levels gradually decline [18,19,20,21,22,23]. This study conducted a dynamic analysis of NAbs of levels among public health workers during the recovery phase of COVID-19.

Given that Omicron variants currently dominate the global virus landscape, we selected diverse SARS-CoV-2 variants to investigate cross-protection reaction among virus strains via neutralization tests. We also evaluated whether the existing population’s immunity, derived from vaccines or past infections, can withstand the continuously evolving SARS-CoV-2 variants. Our findings reveal that public health workers were primarily infected with the Omicron BA.5 variant during the first wave of the COVID-19 epidemic, resulting in significantly higher titers of neutralizing antibodies compared to other Omicron variants (BQ.1, XBB.1.1, XBB.1.9 and XBB.1.16) (Figs. 1A and 2). During the second wave, infections were mainly caused by the Omicron XBB.1.9 variant. Notably, the neutralizing antibody titers against all Omicron variants were significantly elevated compared to those observed during the first wave. The overall trend remained consistent across both waves. Despite the population being infected with Omicron variants, the neutralizing antibody titers against the SARS-CoV-2 prototype strain were markedly higher than those against other tested Omicron variant strains, regardless of whether it was a primary or secondary infection.

This observation could be attributed to the fact that the vaccines administered to the population were developed based on the SARS-CoV-2 prototype strain. When individuals are infected with Omicron variant strains, these vaccines trigger humoral and cellular immune responses. Upon activation, antigen-specific B cells differentiate into two populations: short-lived plasma cells, which rapidly secrete antibodies with neutralizing capabilities to hinder SARS-CoV-2 dissemination in vivo, and germinal center B cells, which undergo screening, cloning, and proliferation before differentiating into long-lived plasma cells. These long-lived plasma cells, primarily residing in the bone marrow, secrete antibodies with potent neutralizing activity, maintaining optimal levels to assist the body in effectively controlling SARS-CoV-2 replication [24]. Although both processes yield memory B cells, those derived from the germinal center exhibit a prolonged lifespan and express B cell receptors with enhanced virus binding and neutralization capabilities. Together with long-lived plasma cells, they establish a robust immune memory, effectively resisting subsequent SARS-CoV-2 infections [25].

In addition to serum, IgA neutralizing antibodies are also distributed in respiratory mucosal tissues, including the nasal mucosa, which serve as entry points for viral invasion [26]. These antibodies are significant for resisting reinfection and mitigating infection symptoms. Despite the low sequence similarity between SARS-CoV-2 and other human coronaviruses, antibodies generated by prior infections still exhibit cross-reactivity, enabling them to recognize and neutralize SARS-CoV-2 to some extent, thereby resisting infection [27].

Since neutralization experiments rely on the visual interpretation of results by the experimenters, subjective differences may arise. Therefore, this study employs a commercially available detection kit for SARS-CoV-2 IgG antibodies based on magnetic particle chemiluminescence for the simultaneous analysis of serum samples. This method was developed using a double-antibody sandwich immunoassay format. Recombinant antigens containing the nucleoprotein and a peptide from the spike protein of SARS-CoV-2 were conjugated with FITC and immobilized on anti-FITC antibody-conjugated magnetic particles. Alkaline phosphatase-conjugated anti-human IgG antibodies were used as detection antibodies. As shown in Fig. 3, a positive correlation was observed between the results obtained from the neutralization detection method and those from the magnetic particle chemiluminescence method.

Currently, most SARS-CoV-2 vaccines have been designed and developed solely for the original strain. However, the continuous mutations of the virus have significantly diminished the efficacy of existing vaccine strategies against emerging variants. There is an urgent need for the development and commercialization of a universal vaccine that can induce broad-spectrum neutralizing antibodies. On one hand, enhancing immunity can to some extent improve and sustain the broad-spectrum neutralizing capability against mutant strains; sequential allogeneic vaccination has been shown to significantly bolster the neutralizing efficacy of antibodies in the body. On the other hand, investigating multiple antigenic targets from various mutant strains and developing a universal SARS-CoV-2 vaccine may better facilitate the production and maintenance of broad-spectrum neutralizing antibodies, thereby providing novel strategies and insights for establishing more robust, versatile and durable immune defenses.

Conclusions

In summary, this study identified that the NAb titers against the prototype and Omicron variants BA.5 were significantly higher than those against the Omicron variants BQ.1, XBB.1.1, XBB.1.9 and XBB.1.16, whether in cases of primary or secondary infection. The cross-protection provided by neutralizing antibodies induced by the prototype and Omicron BA.5 was limited when challenged by the BQ.1, XBB.1.1, XBB.1.9 and XBB.1.16 variants.

Additionally, this study has certain limitations, such as the small sample size of the second wave of recovered infections due to the high mobility of public health workers, which may hinder the generalizable of the results to broader populations. The findings of this study are based on specific variants at a particular point in time, and the rapid evolution of SARS-CoV-2 may result in changes in neutralization patterns that were not captured in this research. This suggests that current vaccines may need to be updated to enhance their effectiveness against emerging strains. Furthermore, this study emphasizes the risk of multiple infections with the novel Omicron variants. Continuous monitoring and ongoing research are essential for evaluating the immunity of key populations, whether derived from vaccines or past infections, against evolving strains of SARS-CoV-2.