Vaccination-infection interval determines cross-neutralization potency to SARS-CoV-2 Omicron after breakthrough infection by other variants.

Background: The immune profile against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has dramatically diversified due to a complex combination of exposure to vaccines and infection by various lineages/variants, likely generating a heterogeneity in protective immunity in a given population. To further complicate this, the Omicron variant, with numerous spike mutations, has emerged. These circumstances have created the need to assess the potential of immune evasion by Omicron in individuals with various immune histories.
Methods: The neutralization susceptibility of the variants, including Omicron and their ancestors, was comparably assessed using a panel of plasma/serum derived from individuals with divergent immune histories. Blood samples were collected from either mRNA vaccinees or from those who suffered from breakthrough infections of Alpha/Delta with multiple time intervals following vaccination. Findings: Omicron was highly resistant to neutralization in fully vaccinated individuals without a history of breakthrough infections. In contrast, robust cross-neutralization against Omicron was induced in vaccinees that experienced breakthrough infections. The time interval between vaccination and infection, rather than the variant types of infection, was significantly correlated with the magnitude and potency of Omicron-neutralizing antibodies. Conclusions: Immune histories with breakthrough infections can overcome the resistance to infection by Omicron, with the vaccination-infection interval being the key determinant of the magnitude and breadth of neutralization. The diverse exposure history in each individual warrants a tailored and cautious approach to understanding population immunity against Omicron and future variants. Funding: This study was supported by grants from the Japan Agency for Medical Research and Development (AMED).

Introduction

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to cause significant morbidity and mortality globally. Since the first detection of a new SARS-CoV-2 variant belonging to the Pango lineage B.1.1.529 in South Africa, it has spread rapidly worldwide, especially in African countries. , The World Health Organization (WHO) classified SARS-CoV-2 variant B.1.1.529 as a variant of concern (VOC), due to possible changes in viral characteristics, and designated it as “Omicron” (WHO, https://www.who.int/). The Omicron variant is characterized by approximately 30 amino-acid mutations, three small deletions, and one insertion in the spike protein compared with the vaccine strain. Among these, 15 mutations are located in the receptor-binding domain (RBD), which induces antibody evasion. Compared with previous VOCs, the Omicron variant contains a larger number of mutations in its spikes, which can dramatically alter its infectivity and immune-evasion capabilities compared with any other variant to date, raising a serious global public health concern.3, 4, 5

A retrospective study using routine epidemiological surveillance data taken from several countries demonstrated that the Omicron variant was associated with a substantial ability to evade immunity from prior infection.6, 7, 8 In addition, a number of studies based on sera collected from vaccinated or convalescent patients have reported that the Omicron variant evades neutralization. , , , In contrast, it has also been reported that booster immunization with an mRNA vaccine significantly increases the serum neutralizing potency against the Omicron variant11, 12, 13 and also provides a significant increase in protection against mild and severe disease, , indicating that an enhancement of pre-existing immunity induced by ancestral virus antigens could overcome the antigenic shift of the Omicron variant and confer cross-protection against it. Moreover, it has been reported that sera from individuals who experienced COVID-19-vaccine breakthrough infections have improved cross-neutralization potency against SARS-CoV-2 variants, especially Delta-variant breakthrough infections. Currently, the cumulative number of COVID-19 cases, seroprevalence of SARS-CoV-2, types of vaccines available, primary vaccination coverage, booster vaccine availability, and number of COVID-19-vaccine breakthrough infections vary in different regions of the world, and the immunity status of populations in each region is also diverse. To properly assess the risk of the spread of the Omicron variant in each region, it is necessary to understand the ability of the variant to evade immunity induced in various settings. Therefore, there is a need to better understand the susceptibility of the Omicron variant to neutralizing antibodies elicited by other variant breakthrough infections, as well as sera from vaccine recipients, to thoroughly assess the risk of immune evasion by the Omicron variant in populations with diverse immune histories to SARS-CoV-2. Here, we determined the neutralization susceptibility of the Omicron variant to antibodies from COVID-19 mRNA vaccine recipients with or without breakthrough infections and compared it with the ancestral SARS-CoV-2 lineage A virus, D614G, and Beta and Delta variants.

Results

High resistance of SARS-CoV-2 Omicron variant to neutralizing antibodies from mRNA vaccinees

First, we tested the in vitro neutralization activity of plasma samples obtained from individuals vaccinated twice with BNT162b2 (Pfizer/BioNTech mRNA vaccine) against SARS-CoV-2 Pango lineage A (as a reference for ancestral strains), D614G (B.1), Beta variant (B.1.351), Delta variant (B.1.617.2), and Omicron variant (B.1.1.529) by vesicular stomatitis virus (VSV) pseudovirus-based and live-virus neutralization assays. Twenty healthcare workers who received two doses of mRNA vaccine were enrolled for blood donation at early (median = 31.5 days) and late (median = 150.5 days) time points after the second dose (Table S1). All volunteers were confirmed to be negative for anti-nucleocapsid antibodies in the pre-vaccinated plasma. Both VSV pseudovirus-based and live-virus neutralization assays with plasma samples from fully vaccinated individuals demonstrated a significant reduction in neutralization activity against the Omicron variant at early and late time points compared with the ancestral virus (Figure 1 ). Almost all samples lost all neutralizing activity against the Omicron variant in both VSV pseudovirus-based and live-virus neutralization assays, and the reduction in neutralization for the Omicron variant was greater than 18.5, or approximately 8-fold, at the early time point and 5.4, or 3.0-fold, at the late time point by VSV pseudovirus or live-virus neutralization assay, respectively, which was greater than that for the Beta variant, which has the most pronounced in vitro escape phenotype to date, or for the Delta variant (Figure 1).

Figure 1

High resistance of SARS-CoV-2 Omicron variant to neutralizing antibodies from mRNA vaccinees

(A and B) Neutralization titers (NTs) of early vaccinee serums against variants of SARS-CoV-2 pseudoviruses (A) and live viruses (B).

(C and D) NTs of late vaccinee serums against variants of SARS-CoV-2 pseudoviruses (C) and live viruses (D).

Data from the same serum are connected with lines, and the mean ± 95% confidence interval (CI) of each serum titer is presented. The titers between the ancestral virus and variants were compared using one-way ANOVA with Dunnett’s test; ∗∗∗∗p < 0.0001. Fold reductions are indicated above columns in statistically significant cases.

Cross-neutralization of the Omicron variant by sera from individuals with mRNA-vaccine breakthrough Alpha- or Delta-variant infections

While sera from individuals who completed two doses of mRNA vaccination showed only limited neutralizing activity, breakthrough infections after full vaccination induced elevated immune responses and cross-neutralization potency against SARS-CoV-2 variants. To investigate the neutralizing activity against the Omicron variant using sera from individuals who had mRNA-vaccine breakthrough infections due to non-Omicron variants, convalescent sera (obtained 10–22 days after infection with the Alpha or Delta variant; Table S1) were examined in VSV pseudovirus-based and live-virus neutralization assays. In the pseudovirus-based neutralization assay, neutralizing activity against the Delta variant was elevated compared with that against the ancestral virus and was particularly pronounced in those with breakthrough infection with the Delta variant (Figures 2A and 2C). In contrast, the neutralizing activity against the Beta or Omicron variant was markedly lower than that against the ancestral virus in the VSV pseudovirus-based neutralization assay, with a 3.8- or 9.7-fold decrease for the Beta or Omicron variant, respectively (Figure 2A). In the live-virus neutralization assay, the neutralizing activity of the two isolates of the Omicron variant was also reduced by approximately 10-fold compared with that against the ancestral virus (Figure 2B). In contrast to the sera of mRNA vaccinees without breakthrough infections, we detected neutralizing activity against the Omicron variant in most sera of individuals with breakthrough infections in both assays, and there were large individual variations in the degree of reduction in neutralizing activity compared with the ancestral virus. Notably, some sera from individuals with breakthrough infections showed high cross-neutralizing activity and neutralized the Omicron variant to a level comparable to that of the other variants. In addition, the cross-neutralizing activity against the Omicron variant tended to be higher in sera from individuals with Delta breakthrough infections than that from Alpha breakthrough infections (Figures 2C and 2D). Notably, breakthrough infection with the Delta variant also induced antibodies with higher neutralizing activity against the Delta and Beta variants than the Alpha breakthrough infection did (Figure 2C). However, the Alpha breakthrough infection did not show any advantage over the Delta breakthrough infection even in the induction of neutralizing antibodies against the Alpha variant (Figure 2D). These observations indicated that the Delta breakthrough infection is more beneficial than the Alpha breakthrough infection for the induction of cross-neutralizing antibodies to other variants, including the Omicron variant. In the breakthrough cases included in this study, there was variation in the interval between vaccination and breakthrough infection, as epidemics caused by the Alpha variant and those caused by the Delta variant occurred during different periods. Specifically, half of the breakthrough infections due to the Delta variant, which occurred later than the Alpha epidemic, occurred >60 days after vaccination (Figure 2E).

Figure 2

Cross-neutralization to the Omicron variant by sera from individuals who experienced mRNA-vaccine breakthrough Alpha- or Delta-variant infections

(A and B) NTs of vaccine-breakthrough-case serums against variants of SARS-CoV-2 pseudoviruses (A) and live viruses (B). Data from the same serum are connected with lines, and the mean ± 95% CI of each serum titer is presented. The titers between the ancestral virus and variants were compared using one-way ANOVA with Dunnett’s test; ∗∗∗∗p < 0.0001. Fold reductions are indicated above columns in statistically significant cases.

(C and D) Comparison of the NTs between background variant types of breakthrough infection against variants of SARS-CoV-2 pseudoviruses (C) and live viruses (D). The titers between the background variant types were compared using two-way ANOVA with Sidak’s test; ∗p < 0.05, ∗∗p < 0.01. Fold increases are indicated above columns in statistically significant cases.

(E) Timeline of sample collection in vaccine-breakthrough individuals: Alpha (n = 15) and Delta (n = 15). Timing from second vaccination to sample collection are indicated as line-connected circles. Day 0 (dotted line) indicates the day of COVID-19 diagnosis.

Positive correlation between cross-neutralization activity against SARS-CoV-2 variants and interval between vaccination and breakthrough infection

To understand the factors contributing to the high heterogeneity of cross-neutralizing activity against variants in sera from breakthrough cases, we evaluated the relationship between neutralizing activity against each variant and the time between vaccine completion and breakthrough infection in each case. Intriguingly, in both pseudovirus-based and live-virus assays, the neutralizing activity against the ancestral virus and each variant, including Omicron, increased as the time between vaccination and breakthrough infection increased (Figures 3A–3L). Notably, the degree of correlation between neutralizing activity and the vaccination-to-infection interval tended to be stronger for Beta and Omicron variants, which were antigenically shifted from the ancestral virus (Figures 3C, 3E, 3I, 3K, and 3L). These trends did not differ significantly between sera after breakthrough infection with Alpha and Delta variants, except for the neutralizing activity against the Delta variant. The neutralizing activity against the Delta variant by the sera obtained after Delta-variant breakthrough infection showed a completely different trend from that of other serum-virus combinations (Figures 3D and 3J). Considering the effect of age on the cross-neutralizing activity against SARS-CoV-2, we excluded elderly patients whose sera were obtained only with a short interval between vaccination and breakthrough infection and evaluated only sera obtained from patients younger than 60 years. We found a positive correlation between cross-neutralization activity against SARS-CoV-2 variants and the interval between vaccination and breakthrough infection, similar to the results described above (Figures S1A–S1H). In addition, to evaluate the effect of the presence or absence of symptoms on cross-neutralizing activity, we compared the neutralizing activity between subjects with and without symptoms, but there was no significant difference in the neutralizing activity for each variant between subjects with and without symptoms (Figures S1I and S1J).

Figure 3

Positive correlation between cross-neutralization activity against SARS-CoV-2 variants and interval between vaccination and breakthrough infection

(A–L) Correlation between interval from vaccination to breakthrough infection and the NTs of the serums against variants of SARS-CoV-2 pseudoviruses (A–E) and live viruses (F-L). The correlation plots represent against ancestral (A and F), D614G (B and G), Alpha (H), Beta (C and I), Delta (E and J), and Omicron (F, K, and L) pseudo- and live viruses, respectively. The regression line, Pearson correlation R value, and p value are shown.

Enhanced neutralizing potency index of anti-Omicron variant antibody in sera from individuals with breakthrough infections

To assess the quality of neutralizing activity by distinguishing the antibodies with a high neutralizing potency from those with a lower potency that might be present in abundance, the neutralization potency index (NPI), which represents the average of the neutralization potencies of individual antibodies, was estimated in the sera from individuals who had breakthrough infections. The immunoglobulin G (IgG) titer of antibodies against the RBD of the Omicron variant was greatly reduced by more than 40-fold compared with that of the ancestral virus (Figure 4A). Notably, as the interval between vaccination and infection increased, the IgG titer of antibodies recognizing the RBD of the ancestral virus did not change, whereas the IgG titer of antibodies against the Omicron-variant RBD increased, suggesting that IgG antibodies recognizing the Omicron RBD were readily induced as this period extended (Figure 4B). The NPI against the ancestral virus, D614G, and Alpha, Beta, Delta, and Omicron variants was then calculated by dividing the neutralization titer to each virus with the RBD IgG titer to each virus, as previously described. The NPI was calculated using the neutralizing activity determined in pseudovirus-based and live-virus assays, and the NPI calculated in both assays demonstrated a significant increase in the Omicron variant compared with the ancestral virus (Figures 4C and 4D). Furthermore, evaluation of the relationship between NPI against the Omicron variant and vaccination-to-infection interval showed that, as with neutralizing activity, NPI was positively correlated with vaccination-to-infection interval: the longer the interval, the higher the NPI and the quality of antibodies produced (Figures 4E, 4F, and 4G). These results suggest that high-quality antibodies play a role in cross-neutralization against the Omicron variant in sera from individuals with breakthrough infections.

Figure 4

Enhanced neutralizing potency index of anti-Omicron-variant antibody in breakthrough-infection sera

(A) IgG titers of vaccine-breakthrough-case serums against ancestral RBDs and RBD mutants of SARS-CoV-2 variants. Data from the same serum are connected with lines, and the mean ± 95% CI of each serum titer is presented. The titers between the ancestral virus and variants were compared using one-way ANOVA with Dunnett’s test; ∗∗∗∗p < 0.0001. Fold reductions are indicated above columns in statistically significant cases.

(B) Correlation between interval from vaccination to breakthrough infection and the IgG titers of the serums against the ancestral (gray) and Omicron (red) RBDs. The regression line, Pearson correlation R value, and p value are shown.

(C and D) Neutralization potency index (NPI) of vaccine-breakthrough-case serums against variants of SARS-CoV-2 pseudoviruses (C) and live viruses (D). The NPI to each variant was calculated by dividing the NT to each virus with RBD IgG titers to each virus. Data from the same serum are connected with lines, and the mean ± 95% CI of each serum titer is presented. The titers between the ancestral virus and variants were compared using one-way ANOVA with Dunnett’s test; ∗p < 0.05, ∗∗∗∗p < 0.0001. Fold increases are indicated above columns in statistically significant cases.

(E–G) Correlation between interval from vaccination to breakthrough infection and the NPI of the serums against ancestral and Omicron variants of SARS-CoV-2 live viruses. The correlation plots represent against ancestral (E) and Omicron (F and G) live viruses, respectively. The regression line, Pearson correlation R value, and p value are shown.

Discussion

The Omicron variant is a highly divergent SARS-CoV-2 variant with approximately 30 amino-acid mutations in the spike protein, causing a severe evasion of humoral immunity induced by vaccines and previous infections. , , , In this study, we demonstrated that sera from individuals with BNT162b2 mRNA vaccine breakthrough infection cases by the SARS-CoV-2 Alpha or Delta variant showed robust cross-neutralizing potency against the Omicron variant, even though sera from vaccinees without breakthrough infection had greatly reduced neutralizing potency against the Omicron variant. Furthermore, a longer interval between vaccination and breakthrough infection was favorable for better antibody responses against the Omicron variant in serum from individuals who had non-Omicron-variant breakthrough infections.

The Omicron variant is spreading rapidly in regions where the population has already been immunized by vaccines or previous infections. Elucidating the underlying factors that have led to marked immune evasion in immunized populations is urgently needed to mitigate the rapid global spread of the Omicron variant. It remains unclear whether the observed rapid growth rate of the Omicron variant can be attributed to immune evasion, increased intrinsic transmissibility, or a combination of the two. A recent analysis of data estimated that the risk of reinfection with the Omicron variant was significantly higher than that with the Delta variant. In addition, several reports have shown that after the primary series (two doses) of COVID-19 vaccines, the vaccine effectiveness against infection with the Omicron variant was significantly reduced compared with that with the Delta variant. , In contrast, studies using serum samples from individuals who had been previously infected and subsequently vaccinated with the mRNA vaccine and those who had received two doses of the mRNA vaccine followed by an mRNA-vaccine booster found high levels of neutralization against the Omicron variant.11, 12, 13 Furthermore, it was also reported that vaccine effectiveness against the Omicron variant after mRNA-vaccine booster dramatically increased against SARS-CoV-2 infection. , Taken together, these results suggest that part of the waning immunity to the Omicron variant in populations immunized with the primary vaccination series or with previous infection due to other variants may be explained by a reduced serum neutralizing activity against the Omicron variant. Notably, enhanced neutralization of the Omicron variant by the ancestral-virus-based booster vaccine indicated that the spike protein of the Omicron variant still shares some neutralizing epitopes with the ancient virus. Supporting this, multiple neutralizing monoclonal antibodies have been found to overcome the antigenic shift of the Omicron variant and cross-neutralize any SARS-CoV-2 variant, as well as their ancestral strain.

In this study, we demonstrated that longer intervals between vaccination and breakthrough infection improved cross-neutralizing potency against the Omicron variant. There is emerging evidence that neutralizing antibody titer can be a correlate of protection in ancestral strains as well as for variants.20, 21, 22 COVID-19-vaccine breakthrough infections elicited robust cross-neutralizing antibody responses against several SARS-CoV-2 variants, which were largely recalled from memory B cells induced by previous vaccinations. Longer prime-boost intervals have been reported to result in higher antibody responses in both adenoviral vectors and mRNA vaccines,23, 24, 25 and mRNA vaccines induce a persistent germinal center B cell response in the draining lymph nodes at least 12 weeks after the second dose, which enables the generation of robust humoral immunity. Notably, affinity maturation of IgG antibodies to the spike-protein-conserved region, which is highly correlated with serum neutralization potency against antigenically drifted variants, persists for more than 3 months after SARS-CoV-2 infection, presumably owing to the supply of long-lived plasma cells. These observations in vaccinees and convalescents suggest that longer intervals between priming and boosting might be favorable for improving the potency and breadth of neutralizing antibodies against SARS-CoV-2 through continued affinity maturation of variant-resistant IgG antibodies expressed on memory B cells and plasma cells. Furthermore, recent studies by us and other groups also suggest that the primary vaccination series with mRNA vaccines (2 doses) induces memory B cells to produce cross-neutralizing antibodies against Omicron variants, despite limited induction of serum anti-Omicron neutralizing antibodies. ,

SARS-CoV-2 variants that emerge repeatedly with no clear seasonality expand globally and replace existing variants in a few months, making it very difficult to develop a vaccine that is fully antigenically matched to the upcoming variants, and have greatly impeded the employment of strategies similar to those used for seasonal influenza. Therefore, it is important to encourage research on vaccine development with enhanced cross-protection against not only the Omicron variant but also unidentified variants that are expected to appear in the future. This can be achieved by designing vaccine antigens with high cross-neutralizing antibody induction capacity against various variants, optimizing the vaccination interval, modifying the vaccination route, and elaborating adjuvants. Furthermore, it has been reported that booster immunization with an mRNA vaccine provides a significant increase in protection against mild and severe disease due to the Omicron-variant infection but is still less effective than protection against other variants, highlighting the need to develop next-generation vaccines.

Limitations of the study

This study had several limitations. First, the number of samples evaluated was small, and potential confounders such as disease severity may be present. Second, since sera from vaccine recipients without breakthrough infection at 6 months after vaccination had a low neutralization titer even against the ancestral virus in our assay, an accurate fold reduction in the neutralizing antibody titer could not be determined. Third, we did not include serum samples collected immediately (<10 days) after breakthrough infection. It is expected that the longer the interval between the vaccine and infection, the lower the antibody titer at the time of breakthrough infection. The possibility that reduced neutralizing activity at the time of breakthrough infection results in efficient viral replication in the upper respiratory tract, which may contribute to a better antibody response, was not evaluated in the present study. Fourth, we did not assess T cell immunity against SARS-CoV-2, including the Omicron variant, which contributes to protection when antibody titers are low in non-human primate models and may correlate with protection against severe disease. Fifth, our study does not support the idea of breakthrough infection as an option to boost one’s immunity since natural infection can cause long-term complications and be particularly dangerous for vulnerable individuals. Sixth, there was no individual who had a booster dose or experienced breakthrough infection after the booster dose. Finally, our investigation did not evaluate the actual risk of reinfection by the Omicron variant in individuals with a history of breakthrough infection.

In conclusion, breakthrough sera demonstrated improved cross-neutralization against the Omicron variant, and the time from vaccination to breakthrough infection was a key determinant of the magnitude and breadth of neutralizing activity against variants after breakthrough infection. These results suggest that population immunity is becoming increasingly diverse against Omicron and future variants depending on different settings, including varying degrees of exposure to existing variants, types of vaccines available, primary vaccination coverage, availability of booster vaccines, and the magnitude of COVID-19-vaccine breakthrough infections. Therefore, a tailored and cautious approach is warranted to understand the population immunity against Omicron and future variants.

STAR★Methods

Key resources table

Resource availability

Lead contact

Additional information and requests for resources and reagents should be directed to the lead contact, Tadaki Suzuki (tksuzuki@nih.go.jp).

Material availability

SARS-CoV-2 viruses in this study are available from the NIID under a material transfer agreement with the NIID, Tokyo, Japan.

Experimental models and subject details

Human subjects and sampling

The characteristics of the participants in this study are summarized in Table S1. Human plasma samples obtained from vaccinated health care workers without infection, who received two doses of BNT162b2 (Pfizer/BioNTech) mRNA vaccine, were collected on approximately day 51 and day 171 after the first vaccination, with written informed consent prior to enrollment and ethics approval by the medical research ethics committee of the National Institute of Infectious Diseases (NIID) for the use of human subjects (#1321). Blood was collected in Vacutainer CPT tubes (BD Biosciences) and centrifuged at 1800 × g for 20 min. Peripheral blood mononuclear cells (PBMCs) were suspended in plasma and harvested into conical tubes, followed by centrifugation at 300 × g for 15 min. The plasma was transferred into another conical tube, centrifuged at 800 × g for 15 min, and the supernatant transferred into another tube to completely remove PBMCs. Human serum samples obtained from breakthrough cases were also included in this study. Demographic information, vaccination status, and respiratory samples to determine the type of variant infected among breakthrough cases in this report were collected as part of the public health activity led by NIID under the Infectious Diseases Control Law and were published on the NIID website to meet statutory requirements. Sera were collected concurrently for clinical testing provided by NIID with patient consent, and neutralization assays for this report were performed using residual samples as a research activity with ethics approval by ethics approval from the medical research ethics committee of NIID for the use of human subjects (#1275) and informed consent. To examine neutralization, plasma and serum samples were heat-inactivated at 56°C for 30 min before use. The median dose interval between the first and second dose for both vaccinated uninfected individuals and breakthrough cases were 21 days. For breakthrough cases, information on sex and age was physician-reported. For vaccinated health care workers without infection, information on sex and age was self-reported. For all subjects, information on race/ancestry, ethnicity, gender, and socioeconomic status was not collected.

Ethical statement approval

All samples, protocols, and procedures were approved by the Medical Research Ethics Committee of NIID for the use of human subjects (Approval numbers 1178, 1275, 1316, and 1321).

SARS-CoV-2 virus

The SARS-CoV-2 ancestral strain WK-521 (lineage A, GISAID ID: EPI_ISL_408667), D614G strain QH-329-037 (lineage B.1, GISAID: EPI_ISL_529135), Alpha variant QK001 (lineage B.1.1.7, GISAID: EPI_ISL_9065387), Beta variant TY8-612 (lineage B.1.351, GISAID: EPI_ISL_1123289), Delta variant TY11-927 (lineage B.1.617.2, GISAID:EPI_ISL_2158617), and Omicron variant TY38-873 (lineage BA.1, GISAID: EPI_ISL_7418017) and TY38-871 (lineage BA.1.1, GISAID: EPI_ISL_7571618) were used in this study, which were isolated using VeroE6/TMPRSS2 cells at NIID with ethics approval by the medical research ethics committee of NIID for the use of human subjects (#1178). To isolate viruses belonging to the Omicron variant (TY38-873; GISAID ID: EPI_ISL_7418017, and TY38-871; GISAID: EPI_ISL_7571618), respiratory specimens, which were collected from individuals being screened at airport quarantine stations in Japan and then transferred to NIID for whole genome sequencing, were subjected to viral isolation using VeroE6/TMPRSS2 cells at NIID. The TY38-871 virus harbors an additional R346K mutation (Omicron + R346K).

Cells

293T cells, obtained from American Type Culture Collection, were cultured in Dulbecco’s modified Eagle medium (DMEM; FUJIFILM Wako Chemical, Kanagawa, Japan) supplemented with 10% fetal bovine serum at 37°C and 5% CO2. Expi293F cells were maintained in the Expi293 expression medium (Thermo Fisher Scientific). VeroE6/TMPRSS2 cells (JCRB1819, Japanese Collection of Research Bioresources Cell Bank) were maintained in low glucose DMEM (Fujifilm) containing 10% heat-inactivated fetal bovine serum (Biowest), 1 mg/mL geneticin (Thermo Fisher Scientific), and 100 U/mL penicillin/streptomycin (Thermo Fisher Scientific) at 37°C supplied with 5% CO2.

Method details

VSV pseudovirus production

The VSV pseudovirus bearing SARS-CoV-2 spike proteins was generated as previously described. Briefly, the spike genes of the SARS-CoV-2 ancestral strain, D614G, Beta, and Delta variants were obtained from viral RNA extracted from SARS-CoV-2 strain WK-521 for ancestral strain, TY4-920 for D614G strain (lineage B.1.1, GISAID ID: EPI_ISL_2931303), TY8-612 for Beta, and TY11-927 for Delta, respectively, by RT-PCR using PrimeScript II High Fidelity One Step RT-PCR kit (Takara-Bio, Shiga, Japan). The spike gene of the SARS-CoV-2 Omicron variant was obtained from RNA extracted from nasopharyngeal swab specimens of patients infected with the SARS-CoV-2 Omicron variant (TY38-873) by RT-PCR, as described above. The cytoplasmic 19 aa-deleted SARS-CoV-2 spike genes were cloned into the CAGGS/MCS expression vector. The spike sequence in the expression plasmids was confirmed by Sanger sequencing. 293T cells transfected with expression plasmids encoding the spike genes of SARS-CoV-2 ancestral strain, D614G, Beta, Delta, and Omicron variants were infected with G-complemented VSVΔG/Luc. After 24 h, the culture supernatants containing VSV pseudoviruses were collected and stored at −80°C until use.

Recombinant RBD antigens production

Human codon-optimized sequences coding the SARS-CoV-2 RBD (amino acids: 331–529) with the following mutations were cloned into the mammalian expression vector pCAGGS: Beta, K417N/E484K/N501Y; Delta, L452R/T478K; Omicron, G339D/S371L/S373P/S375F/K417N/N440K/G446S/S477N/T478K/E484A/Q493R/G496S/Q498R/N501Y/Y505H. Recombinant Avi-tagged His-tagged proteins were produced using Expi293F cells, according to the manufacturer’s instructions (Thermo Fisher Scientific), in the presence of the secreted BirA-Flag plasmid (Addgene) and biotin. Recombinant proteins were purified from the culture supernatant using Ni-NTA agarose (Qiagen).

Electrochemiluminescence immunoassay (ECLIA)

IgG titers for variant RBDs were measured using multiplex kits (Meso Scale Discovery) according to the manufacturer’s instructions. Briefly, plates were coated with biotinylated RBDs premixed with linkers at 4°C overnight. The plates were washed with PBS supplemented with 0.05% Tween-20 and incubated with the MSD Blocker A reagent (Meso Scale Discovery) at room temperature for 1 h with shaking. Plasma samples diluted with MSD Diluent 100 (Meso Scale Discovery) were added to the plates after washing, and the plates were incubated at room temperature for 2 h with shaking. The plates were washed and incubated with sulfo-tag-conjugated anti-human IgG (Meso Scale Discovery) at room temperature for 1 h with shaking. Finally, the plates were measured for electrochemiluminescence using MESOQuickPlex SQ 120 (Meso Scale Discovery) after washing and adding MSD Gold read buffer B (Meso Scale Discovery).

VSV pseudovirus-based neutralization assay

Neutralization of SARS-CoV-2 pseudovirus was performed as previously described. Briefly, serially diluted plasma (five-fold serial dilution of the plasma from vaccinated participants, or eight-fold serial dilution of the plasma from breakthrough infected patients, starting at 1:10 dilution) was mixed with equal volume of the VSV pseudoviruses bearing SARS-CoV-2 spike protein and incubated at 37°C for 1 h. The mixture was then inoculated into VeroE6/TMPRSS2 cells seeded on 96 well solid white flat-bottom plates (Corning). At 24 h post-infection, the infectivity of VSV pseudovirus was assessed by measuring luciferase activity using the Bright-Glo Luciferase Assay System (Promega, Madison, WI, USA) and GloMax Navigator Microplate Luminometer (Promega). The reciprocal half-maximal inhibitory dilution for serum/plasma (ID50) is presented as the neutralization titer.

Live virus neutralization assay

Live virus neutralization assays were performed as previously described. , Briefly, plasma or serum samples were serially diluted (2-fold dilutions starting from 1:5) in high-glucose DMEM supplemented with 2% fetal bovine serum and 100 U/mL penicillin/streptomycin and were mixed with 100 TCID50 SARS-CoV-2 viruses, WK-521 (ancestral strain), QH-329-037 (D614G strain), QK001 (Alpha variant), TY8-612 (Beta variant), TY11-927 (Delta variant), TY38-873 (Omicron variant), and TY38-871 (Omicron + R346K variant), followed by incubation at 37°C for 1 h. The virus-plasma mixtures were placed on VeroE6/TMPRSS2 cells (JCRB1819) seeded in 96-well plates and cultured at 37°C with 5% CO2 for 5 days. After culturing, the cells were fixed with 20% formalin (Fujifilm Wako Pure Chemicals) and stained with crystal violet solution (Sigma-Aldrich). The neutralization titer was defined as the geometric mean of the reciprocal of the highest sample dilution that protects at least 50% of the cells from cytopathic effect from two to four multiplicate series. Assays on sera from vaccinated individuals without infection were performed in multiple independent laboratories within NIID to confirm the findings, with adjustments made using rabbit sera immunized with RBD (ancestral strain). Since sera from individuals who suffered from breakthrough infections were limited in quantity, the assay was performed once. All experiments using authentic viruses were performed in a biosafety level 3 laboratory at NIID, Japan.

Quantification and statistical analysis

Statistical analysis

Data analysis and visualization were performed using GraphPad Prism software. For statistical analysis, one-way ANOVA with Dunnett’s test and two-way ANOVA with Sidak’s test were used to compare the titers. The Pearson correlation coefficient was used to assess correlations between titers and time intervals. Statistical significance was set at p < 0.05.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains
hCoV-19/Japan/TY-WK-521/2020	National Institute of Infectious Diseases	EPI_ISL_408667
hCoV-19/Japan/QH-329-037/2020	National Institute of Infectious Diseases	EPI_ISL_529135
hCoV-19/Japan/ TY4-920/2020	National Institute of Infectious Diseases	EPI_ISL_2931303
hCoV-19/Japan/QK001/2020	National Institute of Infectious Diseases	EPI_ISL_9065387
hCoV-19/Japan/TY8-612/2021	National Institute of Infectious Diseases	EPI_ISL_1123289
hCoV-19/Japan/TY11-927-P1/2021	National Institute of Infectious Diseases	EPI_ISL_2158617
hCoV-19/Japan/TY38-873P0/2021	National Institute of Infectious Diseases	EPI_ISL_7418017
hCoV-19/Japan/TY38-871P0/2021	National Institute of Infectious Diseases	EPI_ISL_7571618
VSV pseudovirus bearing SARS-CoV-2 spike protein	31	N/A
Biological samples
Human plasma samples obtained from vaccinated health care workers	This paper	N/A
Human serum samples obtained from breakthrough cases	This paper	N/A
Chemicals, peptides, and recombinant proteins
SARS-CoV-2 RBD	17, and Kotaki et al., 2022	N/A
Critical commercial assays
Bright-Glo Luciferase Assay System	Promega	E2620
U-PLEX Assay	Meso Scale Discovery	N/A
Experimental models: Cell lines
VeroE6/TMPRSS2 cells	JCRB Cell Bank	JCRB1819
Expi293 expression system	Thermo Fisher Scientific	A29133
Software and algorithms
Graphpad Prism 9	Graphpad	N/A
Other
GroMax Navigator Microplate Luminometer	Promega	GM2000
MESOQuickPlex SQ 120	Meso Scale Discovery	AI0AA-0