Absence of antibody responses to SARS-CoV-2 N protein in COVID-19 vaccine breakthrough cases

Abstract

Understanding the risk factors for breakthrough coronavirus disease 2019 (COVID-19) (BC19) is critical to inform policy. Herein, we assessed Delta (Lineage B.1.617.2) variant-specific effectiveness of the BNT162b2 (Pfizer) vaccine and characterized Delta-driven BC19 cases (fully vaccinated individuals who get infected) with known-time-since-vaccination. In this longitudinal prospective study (January 21-October 30, 2021), 90 naïve and 15 convalescent individuals were enrolled at the initiation of vaccination. Samples from 27 unvaccinated individuals with previous laboratory-confirmed COVID-19 diagnosis were collected at a single time point. Longitudinal serology profile (antibodies against severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] S and N proteins) and live-virus-based neutralization capacities were assessed while controlling for age. Sex, age, history of reactions to the COVID-19 vaccine, and viral neutralization capacities were identified as significant risk factors for breakthrough COVID-19. At 8 months postvaccination, male sex, individuals ⩾65 years of age, and individuals who experienced noticeable side effects with the COVID-19 vaccine were at 5.47 (p-value = 0.0102), 4.33 (p-value = 0.0236), and 4.95 (p-value = 0.0159) fold greater risk of BC19 as compared to their peers, respectively. Importantly, every five-fold increase in viral neutralization capacities (by live-virus-based assays) was significantly associated with ~4-fold reduction in the risk occurrence of breakthrough COVID-19 (p-value = 0.045). Vaccine boosting remarkably increased these viral neutralization capacities by 16.22-fold (p- value = 0.0005), supporting the importance of the BNT162b2 booster in efforts to control the incursion of future variants into the population at large. Strikingly, BC19 cases exhibited a delayed/absent antibody response to the N protein, suggesting limited exposure to the virus. Since antibodies against N protein are widely used to evaluate the extent of virus spread in communities, our finding has important implications on the utility of existing serological diagnostic and surveillance for COVID-19.

Keywords: COVID-19 breakthrough; SARS-CoV-2; and N protein; booster.

Figure 1.

Study design and sampling time points. Participants included naïve and convalescent individuals given either two or three doses of Pfizer vaccine; a convalescent individual given the J&J vaccine; and unvaccinated convalescent individuals. Viral RNA, antibodies against S or N proteins, and inhibition capacities against the viral receptor-binding domain (RBD) of SARS-CoV-2 were evaluated at T1 (2 weeks post the first dose) and T2 (2 weeks post the second dose) in individual participants. At T3 (24 weeks post the first dose or ~5 months postvaccination) and T4 (36 weeks post the first dose or ~8 months postvaccination), viral RNA, antibodies against S or N proteins of SARS-CoV-2, and neutralizing antibodies were evaluated by live-virus-based microneutralization assay. Since the Delta variant emerged (around T3), we evaluated neutralization capacities against the basal B.1 virus (original SARS-CoV-2) and the Delta variant. The Delta variant became the dominant variant ( ⩾ 99%) as most of our participants were reaching T4, thus, we mainly focused on the Delta variant at this time point. Participants gradually received BNT162b2 booster shots after T3 until the endpoint of our study.

Figure 2.

Study groups. Study participants were stratified into groups according to their vaccination and infection status. After T2 individuals are considered fully vaccinated and asymptomatic infection detected at T3 or T4 is thus referred to as “Breakthrough infection” (n = 5; 4 at T3 and n = 1 at T4). Individuals, who were infected after T2, and developed COVID-19, were considered as “COVID-19 Breakthrough” (black throughout figures, n = 16; 5 at T3 and n = 11 after T3). Participants who received a single BNT162b2 booster shot are shown in dark blue. Participants were stratified into four main groups: 1. Individuals vaccinated with no history/evidence of natural exposure before and during the study period: Vac_naive group (red throughout figures, n = 59). 2. individuals with no history of infection at the time of vaccination, but who were found to be either infected or had evidence of infection (antibodies to viral N protein) at any time throughout the study (i.e. T1, T2, T3, or T4): Vac_infect group (light blue in subsequent figures, n = 31). This group includes partially vaccinated and infected individuals (asymptomatic infection at T1 and T2 – referred to as part_vac_infect), and fully vaccinated and infected individuals (breakthrough infection and COVID-19 breakthrough at T3 and T4 – referred to as full_vac_infect). 3. Vaccinated individuals with a history of natural infection prior to the initiation of vaccination: Con_vac group (orange throughout figures, n = 15). 4. Unvaccinated individuals with previous laboratory-confirmed COVID-19 diagnosis (enrolled 8–16 months postinfection): Con_unvac group (green throughout figures, n = 27). Not all participants provided specimens at all time points. Therefore, the number of available data points for each analysis at each time point is annotated in all figures.

Figure 3.

Vaccinated individuals exhibited robust antibody responses after two doses of Pfizer vaccine. The capacity to inhibit RBD–ACE2 interaction and the titers of antibodies to S protein in (a) Vac_naive, (b) partially vaccinated and infected individuals (part_vac_infect), (c) fully vaccinated and infected (full_vac_infect), and (d) vaccinated with history of infection at vaccination (Con_vac) groups were assayed. Lines connect longitudinal samples from the same individual over the sampling times as indicated. Significance was determined using two-sided Wilcoxon matched-pairs signed-rank for comparison of the RBD–ACE2 inhibition capacities between two groups. Two-sided Kruskal–Wallis tests followed by a two-stage-step-up Benjamini, Krieger, and Yekutieli false discovery rate method were used to compare the titer of anti-S protein antibodies between three groups. p-values ⩽ 0.05 were considered statistically significant.

Figure 4.

Reduced neutralizing capacity against SARS-CoV-2 Delta variant at 5 months postvaccination and in unvaccinated convalescent individuals. (a) Correlation between neutralization capacities (presented as EC50: half-maximal effective concentration) against B.1-isolate and Delta variant as evidenced by the Spearman correlation coefficient. (b) Neutralization capacities against B.1-isolate (top panels) and Delta variant (bottom panels) in older Vac_naive individuals as compared to younger Vac_naive individuals. Significance was determined using two-sided Mann–Whitney U tests. (c) Age-adjusted neutralization capacities against Delta (filled circles) compared to those against B.1-isolate (open circles). #: Significance was determined using the two-sided Kruskal–Wallis test followed by a two-stage-step-up Benjamini, Krieger, and Yekutieli false discovery rate method for multiple comparison correction. &: The rank ANCOVA was used to evaluate neutralization capacities across study groups, while controlling for the confounding effect of age. The rank ANCOVA results were presented at F-distribution with p-value. The rank ANCOVA was followed by two-sided Dunnett’s post hoc tests to correct multiple comparisons.

Figure 5.

Significantly lower neutralization capacities prior to infection in breakthrough COVID-19 cases. (a) Neutralization capacities against Delta variant (EC50) prior to the occurrence of COVID-19 breakthrough between breakthrough COVID-19 cases and Vac_naive individuals. Significance was determined using the Mann–Whitney U test. (b) Plot of risk factors for the occurrence of breakthrough COVID-19 among, using two-sided Fisher’s exact test; *The association of neutralization capacities against Delta variant (EC50) and the risk of the occurrence of breakthrough COVID-19 among vaccinated naïve individuals, including 16 BC19 cases and 74 non-COVID-19 vaccinated individuals: 59 cases of vac_naive group, 10 cases of part_vac_infect group, five breakthrough infection cases – 4 at T3 and 1 at T4 (referred Figure 2), was evaluated using multivariable logistic regression with History of reaction to COVID-19 vaccine, sex, and age (⩾ 65 versus < 65) as covariants. The vertical line represents an odds ratio of 1. Odds ratio with 95% confidence interval.

Figure 6.

Enhancement of humoral immune responses against Delta after the BNT162b2 booster. (a) Neutralization capacities against Delta variant at T3 compared to those at T4. (b, c) Neutralization capacities pre-and post booster (2–4 weeks post booster) against Delta variant (b) and B.1-isolate (c). (d) Ratio of neutralization capacities pre- over post booster for Delta variant (black dots) and B.1-isolate (solid circles) at T4. Significance was determined using two-sided Wilcoxon matched-pairs signed-rank test.

Figure 7.

Absent or delayed and muted antibody responses to the viral N protein among BC19 cases. (a) Scatter plots of neutralization capacities pre-and postinfection at different days postsymptom onset (dpo). (b) Ratio of neutralization capacities against B.1-isolate over Delta variant postinfection as compared to the most recent time prior to the infection. Significance was determined using the Mann–Whitney U test. (c) Kinetics of antibodies to N protein at days pre-and postsymptom onset; the gray area represents the positive cut-off. Samples were analyzed as triplicates or duplicates from at least three independent experiments.