Long-lasting neutralizing antibody responses in SARS-CoV-2 seropositive individuals are robustly boosted by immunization with the CoronaVac and BNT162b2 vaccines.

The durability of circulating neutralizing antibody (nAb) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and their boosting by vaccination remains to be defined. We show that outpatient and hospitalized SARS-CoV-2 seropositive individuals mount a robust neutralizing antibody (nAb) response that peaks at days 23 and 27 post-symptom onset, respectively. Although nAb titers remained higher in hospitalized patients, both study groups showed long-lasting nAb responses that can persist for up to 12 months after natural infection. These nAb responses in previously seropositive individuals can be significantly boosted through immunization with two doses of the CoronaVac (Sinovac) or one dose of the BNT162b2 (BioNTech/Pfizer) vaccines, suggesting a substantial induction of B cell memory responses. Noteworthy, three obese previously seropositive individuals failed to mount a booster response upon vaccination, warranting further studies in this population. Immunization of naïve individuals with two doses of the CoronaVac vaccine or one dose of the BNT162b2 vaccine elicited similar levels of nAbs compared to seropositive individuals 4.2 to 13.3 months post-infection with SARS-CoV-2. Thus, this preliminary evidence suggests that both, seropositive and naïve individuals, require two doses of CoronaVac to ensure the induction of robust nAb titers.

The durability of circulating neutralizing antibody (nAb) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination has become a central question during the current pandemic to determine correlates of protection against disease. Current evidence shows that SARS-CoV-2 spike-specific antibodies decline over time but remain detectable up to 8 months post-symptoms onset[1]. However, additional longitudinal data are needed to characterize the medium- and long-term protective antibody dynamics, starting from the acute phase of disease of patients with mild and moderate/severe outcome, and to determine their nAb memory responses upon immunization with different vaccines currently in use.

We enrolled 74 individuals (overall mean age 44 years [range 14 to 83, >60 23%]), of whom 37 were outpatient (mild disease, mean age 37 years [range 14 to 66]) and 37 were hospitalized (moderate and severe disease, mean age 51 years [range 16 to 83]) with a confirmed SARS-CoV-2 quantitative RT-PCR test (Suppl. Table 1). These individuals were followed longitudinally to determine nAb response for up to one year from the onset of symptoms (demographic and baseline characteristics of the patients are summarized in Suppl. Table 1; samples were collected between 2 to 414 days after the onset of symptoms).

Regardless of disease severity, infected individuals developed robust nAb responses during the first month. These responses declined over time but were sustained for up to 12 months (Fig. 1A, B), as determined with a microneutralization assay based on a recombinant vesicular stomatitis virus carrying a SARS-CoV-2 spike protein that showed strong correlation (Pearson’s r = 0.86, R2 = 0.75, P< 0.001) with authentic SARS-CoV-2 microneutralization (Fig. S1). We performed kinetic analyses with samples from 41 individuals that were sampled weekly during the first month from symptom onset (Fig. 1A and Fig. S2A–B). In agreement with previous reports, hospitalized individuals had significantly higher neutralization titers as compared to outpatients (Fig. S2C), with peak average nAb responses at day 23 and at day 27 post-symptom onset, respectively (Fig. 1A and Fig. S2A–B). We included longitudinal samples for all participants and performed a nAb titer time decay analysis starting from the respective peak average responses. Fitting our nAb data to a one-phase decay model, the initial decay half time was 42 days (95% CI : 2.21 to 362.4) for outpatients and 84 days (95% CI : 1.7 to indeterminate) for hospitalized individuals, and when we used a continuous decay fit, the half time was 225 (95% CI : 121 to 1,648) and 195 (95% CI : 120 to 535) days for these groups, respectively (Fig. 1B). None of the individuals in the study had evidence of re-infections. Hence, albeit the nAb titers remained higher in hospitalized patients than in the outpatients, both study groups showed long-lasting responses of circulating antibodies after natural infection.

Figure 1.

Neutralizing antibody responses to SARS-CoV-2 in seropositive and naïve individuals before and after CoronaVac or BNT162b2 vaccination.

(Panels A to D) The half-maximum inhibitory concentration (IC50) of sera was determined by microneutralization assay of recombinant vesicular stomatitis virus carrying SARS-CoV-2 spike protein (rVSV-SARS2-S). (Panel A) Neutralizing antibody (nAb) titers (IC50) from 15 outpatients (57 samples; grey circles) and 26 hospitalized (84 samples; red circles) at 2 to 36 days post-symptom onset. Second order polynomial (quadratic) curve fitting was used to establish the days at which peak titers occurred (Ymax). (Panel B) Longitudinal nAb titers from 36 outpatients (66 samples) and 31 hospitalized (44 samples) taken from day 27 (outpatients) or day 23 (hospitalized) until day 414 post-symptom onset. One-phase decay fit is indicated as a bold line, while continuous decay fit is shown with the thinner line in red and gray for the corresponding patient group. (Panel C) nAb titers from 13 outpatient (26 samples) or 14 hospitalized (28 samples) individuals immunized with one or two doses of CoronaVac (24 participants) or one or two doses of BNT162b2 (3 participants) vaccines. (Panel D) nAb titers from naïve individuals after the first and second dose of CoronaVac (11 participants) or BNT162b2 (10 participants) vaccines, compared to seropositive individuals who were not vaccinated (26 participants) or received one dose (8 samples) or two doses (20 samples) of the indicated vaccines. Geometric means with 95% confidence intervals are shown. Circles, non-vaccinated; squares, vaccinated with CoronaVac; triangles, vaccinated with BNT162b2. Dashed line indicates the limit of detection (LOD) of the microneutralization assay. Statistics were performed using unpaired two-tailed Mann-Whitney test. ***P<0.001; ****P<0.0001; ns, non-significant.

Twenty-seven of the previously seropositive individuals (mean age 45 years [range 17 to 83]) included in our longitudinally cohort study were immunized during the study period. Thus, we analyzed the nAb response in these previously infected individuals after immunization with the two main vaccines currently being used in Chile; the CoronaVac (Sinovac) vaccine based on inactivated virus or the BNT162b2 (BioNTech/Pfizer) vaccine based on spike protein-encoding messenger RNA. We compared them to nAbs titers in healthy SARS-CoV-2 naïve (seronegative) individuals immunized with two doses of either vaccine (CoronaVac, 11 participants, mean age 34 years [range 21 to 47] or BNT162b2, 10 participants, mean age 37 years [range 23 to 53]; Suppl. Table 1). The seropositive individuals were vaccinated between 4.2 to 13.3 months (average 9.9 months) after the onset of symptoms for both, the outpatient (13 participants) and hospitalized groups (14 participants; see arrows in Fig. S3). The average increase in the nAb titers was four times for outpatients (pre-vaccine mean titer = 188.9 [range 98.7 to 273.6], first dose mean titer = 666.3 [range 80.9 to 1,143]) and three times for hospitalized individuals (pre-vaccine mean titer = 8,836 [range 1.0 to 29,389], first dose mean titer = 27,562 [range = 124.7 to 91,571]) after one dose (Fig. 1C). After the second dose, the average increase was 13 times (pre-vaccine mean titer = 528.7 [range 10.9 to 1,791], mean titer = 6,597 [range 261.6 to 38,912) and 179 times (pre-vaccine mean titer = 649.1 [range 78.0 to 1,360], mean titer = 116,012 [range 31.0 to 1,000,000]), respectively (Fig. 1C). Except for four cases, all participants showed an increase in the nAb titer after receiving one or two doses of the vaccines, suggesting a significant induction of B cell memory response at 4.2 to 13.3 months after onset of symptoms. One corresponded to an outpatient (Fig. S3C, dark yellow patient) who had been vaccinated two days earlier and may therefore not have had sufficient time to mount a booster response. The other three participants (age range 29 to 63 years) were obese outpatient (Fig. S3C, light green patient) or hospitalized (Fig. S3D, grey and cyan patients) individuals for whom we only had a previous sample 5.6 to 10.7 months prior to vaccination (Fig. S3C, D), and hence no clear conclusions can be drawn about the trajectory of their nAb titers. Noteworthy, one of these participants had a marked decreased nAb titer after two doses of the vaccine (IC50 563.9 to 31.0; Fig. 1C). Due to the high prevalence of obesity in severe coronavirus disease 2019 (COVID-19)[2], further studies to monitor the induction of nAbs after vaccination in this population are required.

The induction of nAbs in seropositive individuals vaccinated with one dose as compared to the naïve individuals vaccinated with BNT162b2 was on average 18 times higher (previously infected first dose mean titer = 14,099 [range 80.91 to 91,571], naïve first dose mean titer = 791.0 [range 137.0 to 2,663]). When comparing them with naïve individuals vaccinated with one dose of CoronaVac, their nAb titers were on average 492 times higher (naïve first dose mean titer = 28.64 [range 1.0 to 71.6]). Importantly, we did not detect a statistical difference between nAb titers of seropositive individuals 0.9 to 10.1 months post-infection (mean titer = 1,860 [range 1.0 to 29,389]) with those of naïve individuals vaccinated with two doses of CoronaVac (mean titer = 1,595 [range 56.9 to 6,262]), and higher titers were observed in those individuals with two doses of BNT162b2 (mean titer = 16,648 [range 3,500 to 44,719]), suggesting the induction of robust nAbs responses in naïve individuals with both vaccines (Fig. 1D). Immunization of previously infected individuals with CoronaVac (24 participants), showed no significant differences in nAb titers after one dose or when comparing the first and second dose. Significantly increased nAb titers were only observed after both doses (P<0.01) of this vaccine (Fig. 1D), differing from vaccination of seropositive or naïve individuals with mRNA and adenovirus-based vaccines that induce high nAb titers after the first dose[3,4].

We found long lasting nAb titers that persist for over 12 months after the onset of symptoms in both, outpatient and hospitalized individuals. Neutralizing activity in seropositive individuals was boosted significantly after two doses of the CoronaVac or BNT162b2 vaccines, regardless of the time interval since the onset of COVID-19 symptoms, suggesting that infection induces a robust B-cell memory response. The correlates of protection against SARS-CoV-2 are currently unknown. However, current evidence of re-infections remains limited, and they appear to be infrequent, suggesting that natural infection provides significant protection against COVID-19 in most individuals[5]. While this is a small cohort study, our results indicate that natural infection induces long-lasting nAb responses that can be significantly boosted through vaccination, and that immunization of naïve individuals with two doses of the CoronaVac vaccine or one dose of the BNT162b2 vaccine elicit similar levels of nAbs compared to seropositive individuals 4.2 to 13.3 months post-infection with SARS-CoV-2. Our preliminary evidence suggests that both, seropositive and naïve individuals, require two doses of CoronaVac to generate a robust induction of nAb titers. Further studies to determine the long-term duration of vaccine-induced nAbs against SARS-CoV-2 are warranted.