Wild-type SARS-CoV-2 vaccine antigenic sin shapes poor cross-neutralization of BA.4/5/2.75 subvariants in breakthrough BA.2 infections – Nature Communications

Study design

Compared to BA.2, BA.4/5 carries 4 mutations in the spike protein (Del69-70, L452R, F486V and R493Q) and BA.2.75 has 11 changes (S24L, Ins25-27, K147E, W152R, F157L, I210V, G257S, D339H, G446S, N460K and R493Q) (Fig. 1a). In this study, we constructed two pseudotyped viruses BA.4/5 and BA.2.75 based on our previous established methods for the Pango A (wild-type, WT) and BA.2 lines17,18,19. We then measured neutralizing antibody titers in plasma samples obtained from 34 individuals with breakthrough BA.2 infections (Fig. 1b, Supplementary Fig. 1, Supplementary Tables 1 and 2). All participants were confirmed RT-PCR positive and admitted to Shenzhen Third People’s Hospital during the BA.2 epidemic wave in Shenzhen in March 2022. Seventeen received two doses of inactivated SARS-CoV-2 vaccines and another 17 individuals received a homologous third booster vaccination . We collected their plasma samples at a very early stage of BA.2 infection (visit 1, within 2 days after RT-PCR positive diagnosis) and during the convalescent period (visit 2, 81 days after diagnosis). Therefore, a total of 68 plasma samples were evaluated for neutralization against 4 types of SARS-CoV-2 pseudoviruses carrying different spike proteins of the WT, BA.2, BA.4/5 and BA.2.75 subvariants (Supplementary Fig. 2 and Table 3). In this study, all reported neutralization results represented neutralization of pseudotyped virus.

Giant. 1: SARS-CoV-2 Micron subvariants and BA.2 breakthrough infection cohorts included in this study.
Figure 1

AND Mutations located in the spike protein have been identified in Omicron subvariants including BA.2, BA.4/5 and BA.2.75. Amino acid residues consistent with WT SARS-CoV-2 are highlighted in gray and mutations in Omicron subvariants are highlighted in orange. b Schematic overview of study design. Blood samples from individuals infected with breakthrough BA.2 who were previously immunized with 2-dose or 3-dose inactivated vaccines were collected early in the infection (visit 1, up to 2 days after diagnosis) and during the convalescence period (visit 2, 81 days after diagnosis).

Cross-neutralization against BA.4/5 and BA.2.75 subvariants in breakthrough BA.2 infections

As shown in Fig. 2a, the 2-dose inactivated vaccination produced a low level of antibody memory against SARS-CoV-2 approximately 260 days after the last immunization. Only 11.8% (2/17) of plasma neutralized the WT virus, whose 50% inhibitory dilution (ID50) was more than the 1:20 dilution. All plasma lost their neutralizing activities against the tested Omicron subvariants (BA.2, BA.4/5 and BA.2.75). In contrast, plasma in the 3-dose group maintained a relatively higher level of neutralization (70.6% positive rate for WT, 41.2% for BA.2, 23.5% for BA.4/5, and 23.5% for BA .2.75). Whole plasma neutralization in the 2-dose and 3-dose groups was rescued and enhanced by BA.2 breakthrough infection (Fig. 2b). Of note, there were only minor differences in neutralizing activities against SARS-CoV-2 between the 2-dose and 3-dose vaccination groups.

Giant. 2: Neutralization of SARS-CoV-2 Omicron Subvariant by Plasma from Inactivated Vaccine Recipients Infected with BA.2.
figure 2

AND, b Comparison of plasma neutralizing activities in different groups against WT SARS-CoV-2 and Omicron subvariants at visit 1 (early stage of infection) (AND) and visit 2 (recovery period) (b). C, d Neutralization titers against WT SARS-CoV-2 and Omicron subvariants in individuals infected with breakthrough BA.2 with 2 doses (C) and 3-dose (d) inactivated vaccines. E, F Enhancement of neutralization effects against WT SARS-CoV-2 and Omicron subvariants by BA.2 breakthrough infection at 2 doses (E) and 3-dose (F) group. G Complex change in enhanced neutralization of WT SARS-CoV-2 and Omicron subvariants by breakthrough BA.2 infection. h Correlation analysis between ID50 values ​​against WT SARS-CoV-2 at visit 1 and fold change of increased neutralization in 34 individuals infected with breakthrough BA.2. A perfect fit correlation line has been included in the graph. Non-parametric Spearman correlation coefficients (R) and statistically significant P value has been provided. id50 values ​​are means of at least two independent experiments. Data are presented as geometric mean ± standard deviation (SD). Sample size, geometric mean, fold change and significance of difference were indicated at the top. “-” represents a decreased value and “+” represents an increased value. Statistical significance was performed using a two-tailed unpaired Wilcoxon test in (AND, b), two-tailed Kruskal-Wallis test with paired Wilcoxon multiple comparison test in (C, d, G), and two-tailed paired Wilcoxon test in (E, F). ****, P < 0.0001; ***, P< 0.001; **, P< 0.01; *, P< 0.05; ns, not significant. Dotted horizontal line in (af) indicates the limit of detection (1:20 dilution) for the neutralization test. Non-neutralization data are set as 20 for analysis and visualization. id50 indicates 50% inhibitory dilution. GMT indicates the geometric mean titer. FC indicates folding change. Source data and accurate Pvalues ​​are provided as a source data set.

To better understand the differential antibody leakage of BA.4/5 and BA.2.75 subvariants, we rearranged these neutralization results by different groups to make a direct comparison. As shown in Fig. 2c, in the 2-dose breakthrough group, geometric mean titers (GMT) of plasma nAbs against BA.2, BA.4/5, and BA.2.75 were reduced by 2.2-fold, 4.1-fold, and 14, respectively. 7 times compared to WT. GMTs against BA.4/5 and BA.2.75 were reduced by 1.8 times and 6.6 times, respectively, compared to the values ​​against BA.2. Compared to BA.4/5, GMT against BA.2.75 was significantly reduced by 3.6 times. A similar decreasing trend was also observed in the 3-dose breakthrough group (Fig. 2d), with reductions of 2.9-fold (BA.2), 5.4-fold (BA.4/5) and 9.7-fold (BA.2.75) . compared to WT. GMT against BA.2.75 was also the lowest of all SARS-CoV-2 pseudoviruses tested. Together, these results indicated that the current immune barrier established by vaccination or vaccination plus breakthrough BA.2 infection would be severely compromised by recently emerging Omicron subvariants.

Of particular interest, we noted that the potentiating effects of neutralization against WT SARS-CoV-2 and Omicron subvariants and between the 2- and 3-dose groups are significant. In subjects vaccinated with 2 doses, the GMTs of plasma nAbs against WT, BA.2, BA.4/5, and BA.2.75 were increased 36.0-fold, 17.8-fold, 9.8-fold, and 2.7-fold after BA.2 breakthrough infection (Fig. 2e). In contrast, the increased GMT folds were 21.4-fold, 17.5-fold, 12.4-fold, and 7.0-fold against WT, BA.2, BA.4/5, and BA.2.75 in subjects vaccinated with 3 doses, respectively. (Fig. 2f). Finally, we analyzed the differential enhancement of BA.2 breakthrough infection against WT SARS-CoV-2 and Omicron subvariants. As shown in Fig. 2g, increased neutralization against WT was greatest in the 2-dose group, followed by BA.2, BA.4/5 and BA.2.75. However, this difference was apparently reduced in the 3-dose breakthrough treatment group. In addition, the increase in neutralizing activities against WT virus was negatively correlated with ID50 values ​​when BA.2 infection occurred (Fig. 2h). Most of the plasma lost neutralization against BA.2, BA.4/5 and BA.2.75 at visit 1, whose ID50 values ​​were uniformly set to 20 for analysis and visualization. Thus, a similar correlation analysis was not performed for the Omicron variants. These findings indicated that BA.2 breakthrough infection induced mainly cross-linked nAbs against WT virus and weakly induced Omicron-specific nAbs in recipients of the prototype vaccine.

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