Longitudinal Follow-Up of the Immunity to SARS-CoV-2 in Health Care Workers in Argentina: Persistence of Humoral Response and Neutralizing Capacity after Sputnik V Vaccination

ABSTRACT SARS-CoV-2 vaccine protection has encountered waning of immune response and breakthrough infections. The hybrid immune response generated by the combination of vaccination and infection was shown to offer higher and broader protection. Here, we present a seroprevalence study of anti-SARS-CoV-2 spike/RBD IgG in 1,121 health care workers immunized with Sputnik V and a follow-up of humoral response at 2 and 24 weeks postvaccination (wpv), including neutralizing antibody response (NAT) against ancestral, Gamma, and Delta variants. The first seroprevalence study showed that among 122 individuals with one dose, 90.2% were seropositive versus 99.7% seropositivity among volunteers with the complete two-dose regimen. At 24 wpv, 98.7% of the volunteers remained seropositive, although antibody levels decreased. IgG levels and NAT were higher in individuals that had acquired COVID-19 previous to vaccination than in naive individuals at 2 and 24 wpv. Antibody levels dropped over time in both groups. In contrast, IgG levels and NAT increased after vaccine breakthrough infection. At 2 wpv, 35/40 naive individuals had detectable NAT against SARS-CoV-2 Gamma and 6/40 against Delta. In turn, 8/9 previously infected individuals developed a neutralizing response against SARS-CoV-2 Gamma and 4/9 against Delta variants. NAT against variants followed a trajectory similar to NAT against ancestral SARS-CoV-2, and breakthrough infection led to an increase in NAT and complete seroconversion against variants. In conclusion, Sputnik V-induced humoral response persisted at 6 months postvaccination, and hybrid immunity induced higher levels of anti-S/RBD antibodies and NAT in previously exposed individuals, boosted the response after vaccination, and conferred wider breadth of protection. IMPORTANCE Since December 2020, Argentina has begun a mass vaccination program. The first vaccine available in our country was Sputnik V, which has been approved for use in 71 countries with a total population of 4 billion people. Despite all the available information, there are fewer published studies on the response induced by Sputnik V vaccination compared to that of other vaccines. Although the global political context has paralyzed the verification by the WHO of the efficacy of this vaccine, our work aims to add new clear and necessary evidence to Sputnik V performance. Our results contribute to general knowledge of the humoral immune response developed by vaccines based on viral vector technology, highlighting the higher immune protection conferred by hybrid immunity and reinforcing the importance of completing vaccination schedules and booster doses to maintain adequate antibody levels.


RESULTS
Study design. A total of 1,121 health care workers vaccinated with the Sputnik V vaccine in Argentina between December 2020 and March 2021 at Hospital Provincial del Centenario, Rosario, Santa Fe, were enrolled in this study. Participants' ages ranged from 21 to 78 years (median 42 years), and 725 (64.7%) were females. Vaccination with Sputnik V consisted of two doses with an interval between doses of 21 days. We carried out a first seroprevalence study between January and March 2021 in 1,121 individuals who were immunized with at least one dose of Sputnik V: 122 individuals were tested 2 weeks after the first dose, and 999 individuals were tested after the second dose, 732 of which were tested 2 weeks after completing the vaccination schedule (2 wpv). After 6 months (24 wpv), a new seroprevalence analysis was performed in 538 fully vaccinated volunteers. Among them, 380 individuals, who had been also initially tested at 2wpv, were selected for further analysis. To analyze humoral response in the context of hybrid immunity, this cohort was differentiated into individuals: (i) without a diagnosis of COVID-19 prior or during the study period (n = 271) (non-COVID, naive); (ii) with confirmed COVID-19 diagnosis prior to the start of the vaccination schedule (COVID-PreV) (n = 74); and (iii) with breakthrough infections (confirmed COVID-19 diagnosis between 2 and 24 wpv, COVID-PostV) (n = 35). COVID-19 diagnosis was confirmed by antigen tests or PCR. From this cohort of 380 individuals, 49 anti-S/RBD IgG seropositive individuals were selected to assess the neutralizing antibody response both at 2 and 24 wpv (Fig. 1).
Seroprevalence studies. Results of a first seroprevalence study carried out after the beginning of the vaccination campaign showed that 98.7% of the total cohort developed specific anti-S/RBD IgG antibodies ( Fig. 1). At the time, 122 individuals had received one dose of Sputnik V, and 90.2% (110/122) were seropositive. Nine hundred and ninetynine volunteers that had received the complete vaccination regimen were tested at 1 to 2 weeks after the second dose and showed 99.7% (996/999) seropositivity (Fig. 1). In particular, the 732 individuals that were analyzed at 2 weeks after completion of the vaccination schedule showed 99.9% (731/732) of seropositivity. We further assessed the response to complete vaccination by monitoring specific IgG antibodies at 24 wpv. Five hundred and thirty-eight individuals attended the second seroprevalence study, and 98.7% (531/538) were seropositive. A follow-up analysis of the humoral response after Sputnik V vaccination was performed in 380 fully vaccinated individuals that attended the study both at 2 and 24 wpv. All individuals of this subcohort were seropositive at 2 wpv, and 98.7% still presented specific IgG anti-S/RBD antibodies at 24 wpv (Fig. 1).
The analysis of anti-S/RBD antibody levels showed significantly lower levels in individuals who received one dose than in those who received two doses of Sputnik V ( Fig. 2A). On the other hand, follow-up data of 380 individuals showed that over a period of 6 months, anti-S/RBD IgG antibody levels declined significantly ( Fig. 2B and C).
Humoral response in the context of hybrid immunity. Hybrid immunity is formed in individuals receiving a COVID-19 vaccine and experiencing SARS-CoV-2 infection before or after vaccination, and it is reported to induce higher antibody levels (14,15). To evaluate humoral immune response in the context of hybrid immunity following Sputnik V vaccination, the cohort of 380 individuals was differentiated into individuals that did not acquire an infection (Non-COVID, n = 271); individuals that had acquired SARS-CoV-2 prior to vaccination (COVID-PreV, n = 74); and individuals that acquired a vaccine breakthrough infection (COVID-PostV, n = 35). At 2 wpv, geometric mean positivity index (GMPI) in COVID-PreV was significantly higher than that of Non-COVID and COVID-PostV groups, suggesting that prior infection boosted anti-S/RBD IgG antibody response (Fig. 3A). Then, in the Non-COVID and COVID-PreV groups, a 1.6-and 1.1-fold decrease in antibody levels between 2 and 24 wpv was observed, respectively, whereas, as expected, the COVID-PostV group showed a significant 1.2-fold increase in a-S/RBD IgG antibody levels (Fig. 3A).
Neutralizing humoral response against ancestral SARS-CoV-2. In order to study the neutralizing capacity against SARS-CoV-2 after Sputnik V vaccination, we selected a subgroup of 49 individuals comprising 31 Non-COVID, 9 COVID-PreV, and 9 COVID-PostV that were seropositive by COVIDAR IgG ELISA at 2 wpv ( the neutralizing capacity against ancestral SARS-CoV-2 ( Fig. 3B) indicated that 100% of individuals had detectable neutralizing antibodies at 2 wpv. At 2 wpv, neutralizing titers (NT) were similar in the two groups that had not acquired COVID-19 (Non-COVID and COVID-PostV), and significantly higher NT were obtained for previously infected individuals. At 24 wpv, 100% of the individuals exposed to infection retained neutralizing ability (COVID-PreV and COVID-PostV), whereas in the naive Non-COVID cohort, 10% of the samples fell below detectable levels. Moreover, differences between Non-COVID and COVID-PreV were still observed, with geometric mean neutralizing titers (GMNT) nearly four times lower in the Non-COVID group. Nevertheless, over time, both groups showed a decrease of nearly 2.5-fold in their NT. In turn, the COVID-PostV group showed a 5.6-fold increase in NT between 2 and 24 wpv and displayed 18 and 5 times higher NT at 24 wpv than Non-COVID and COVID-PreV groups, respectively (Fig. 3B).
Overall, neutralizing antibody response paralleled the trajectory of anti-S/RBD IgG. Neutralizing humoral response against SARS-CoV-2 VOCs. Considering the main VOCs that circulated in Argentina from January to October 2021, the neutralizing titers against SARS-CoV-2 Gamma and Delta were also assessed ( Fig. 4A and B) ( Table 1). At 2 wpv, 90% of individuals in the three groups of study had detectable neutralizing antibodies against the Gamma variant. As observed for ancestral virus, neutralizing titers against Gamma were also similar between Non-COVID and COVID-PostV, and nonstatistically significant 1.8 times higher titers were obtained for the COVID-PreV group (Fig. 4A). NT against SARS-CoV-2 Gamma dropped over time in Non-COVID and COVID-PreV groups, displaying a 2.6-and 1.9-fold decrease, respectively. Breakthrough infection led to a 9.8-fold increase in NT at 24 wpv. Indeed, this group showed the highest neutralizing antibody levels, displaying 25 and 10 times higher NT than Non-COVID and COVID-PreV groups, respectively. For SARS-CoV-2 Delta (Fig. 4B), however, most of the samples first dose, n = 122) or two doses (1 to 2 weeks after second dose, n = 999) after the beginning of the vaccination campaign was evaluated. A second seroprevalence study was carried out at 24 weeks after completion of the two-doses vaccination schedule (24 wpv) and included 538 fully vaccinated individuals from the initial cohort. Three hundred and eighty individuals with paired samples obtained both at 2 and 24 wpv were selected for further analysis and divided according to their COVID status (i) without a diagnosis of COVID-19 prior or during the study period (n = 271; Non-COVID); (ii) with confirmed COVID-19 diagnosis prior to the start of the vaccination schedule (COVID-PreV; n = 74); and (iii) with breakthrough infections (confirmed COVID-19 diagnosis between 2 and 24 wpv, COVID-PostV; n = 35). Finally, 49 of these individuals were selected for neutralizing antibodies (Nab) analysis. Persistence of Antibody Response after Vaccination mSphere presented neutralization levels below the lower dilution tested. Neutralizing antibodies were detectable in approximately 24% and 33% of individuals without previous diagnosis of COVID (Non-COVID and COVID-PostV, respectively) and in 44% of individuals with previous diagnosis (COVID-PreV). In the COVID-PostV group, exposure to the virus led to 100% positivity and to an increase in the neutralizing titers. To further study the crossneutralizing response, we focused the analysis on the Non-COVID group ( Fig. 4C and D). Neutralizing titers at 2 wpv against ancestral SARS-CoV-2 were 3.9 and 27.1 times higher than those of those against Gamma and Delta VOCs, respectively ( Fig. 4C). At 24 wpv, this difference was smaller for the Delta variant, where NT were 9.9 times lower than those against ancestral virus (Fig. 4D). As observed before, the neutralizing response and the number of positive samples (NT $ 8) against ancestral and Gamma variant decreased Persistence of Antibody Response after Vaccination mSphere between 2 and 24 wpv. For the Delta variant, NT slightly increased over time at the expense of four individuals whose neutralizing antibodies became detectable at 24 wpv. Altogether, we observed that VOCs Gamma and Delta significantly escape the neutralizing response elicited by Sputnik V vaccination and that hybrid immunity tends to confer a wider range of protection.   Correlation of anti-Spike/RBD antibody levels and neutralizing antibody titers. Correlation of anti-Spike/RBD antibody levels and neutralizing antibody titers was previously reported (6 to 12). Here, we evaluated correlation in the Non-COVID group by analyzing levels of specific antibodies and neutralizing antibodies against ancestral SARS-CoV-2. Out of the 732 samples obtained at 2 wpv, 572 fell out of the dynamic range of COVIDAR assay (PI [positivity index] . 10), including the samples selected for paired analysis of neutralization in naive individuals (n = 29). This suggests that this assay offers a limited dynamic range to discriminate samples with high antibody titers. Therefore, for the purpose of the correlation analysis, we also determined total anti-S antibody levels of the paired samples of the 29 individuals in the Non-COVID group (n = 56) using electrochemiluminescence Elecsys Anti-SARS-CoV-2 S assay (Roche) (Fig. 5). For the paired samples selected for analysis, only 31% fell in the dynamic range (PI , 10) of COVIDAR, while using Elecsys, 52% fell in the dynamic range. As a result, antibody levels determined with COVIDAR and Elecsys assays showed a modest correlation (r = 0.6071) (Fig. 5A). Next, we evaluated the correlation between NT and PI obtained by the COVIDAR IgG ELISA. We observed that using this test, anti-S/RBD IgG levels correlated with NT (r = 0.6360) (Fig. 5B). In turn, the analysis of samples tested with Elecsys Anti-SARS-CoV-2 S assay showed a robust correlation (r = 0.8177) between total anti-S/RBD and NT (Fig. 5C). Altogether, the results indicate that total IgG levels correlate with NT. Differences in the strength of correlation obtained when NT were compared with antibody levels determined using COVIDAR or Elecsys assays point to the broader dynamic range offered by Elecsys to discriminate antibody levels between samples selected for analysis.

DISCUSSION
This work presents a comprehensive 6-month follow-up study of the humoral response of a cohort of health care workers vaccinated with Sputnik V from December 2020 to March 2021. Our results add evidence to the response to vaccination with  Persistence of Antibody Response after Vaccination mSphere Sputnik V in the medium term, and report for the first time the follow-up of the neutralizing antibody response against ancestral SARS-CoV-2 and VOCs according to a differentiated analysis of naive and COVID-diagnosed individuals.
In accordance with previous reports, our results clearly reflect that the Sputnik V vaccine induces a robust humoral response against SARS-CoV-2 that is enhanced with a second dose (1,2,6,10,11,18,19). Moreover, our data confirm improved response to vaccination of previously infected individuals. Accordingly, the IgG antibody levels after a single dose of vaccine in individuals with previous infection were similar to those obtained after two doses of vaccine in naive individuals (data not shown), and after completing the vaccination schedule, anti-S/RBD IgG levels were also higher in those individuals who were previously infected. These results indicate that naturally acquired immunity is enhanced by one or two doses of vaccine (7, 11, 16, 18, 20 to 24) and reinforce what was previously reported not only for Sputnik V but also for other vaccine platforms (6, 7, 10, 16, 18 to 20, 25). Similarly, our longitudinal analysis indicates that individuals vaccinated with Sputnik V maintain robust antibody levels after 6 months, contributing additional evidence to the persistence of the humoral response induced by vaccination over time (6,7,9,10,19). Notwithstanding, we observed a decrease in antibody levels both in naive and in previously infected individuals (6,7).
Altogether, our study design revealed that hybrid immunity in the context of Sputnik V vaccination led to an enhancement of the humoral response: individuals with a previous exposure to the virus had the highest antibody response after vaccination (2 wpv) and tended to retain higher antibody levels than naive individuals even up to 6 months (24 wpv). On the other hand, individuals with a breakthrough infection after vaccination had the highest antibody levels at 24 wpv.
Analysis of the neutralizing antibody response in a subgroup of 49 individuals receiving the complete Sputnik V vaccination scheme indicated that neutralizing response against ancestral SARS-CoV-2 paralleled total IgG seroconversion and persistence, and 100% of seropositive individuals had detectable neutralizing antibodies.
Neutralizing titers were in line with anti-S/RBD levels: neutralization potency was higher in those individuals who were previously infected and decreased after 6 months in naive individuals (9, 10, 16, 18 to 20, 25). Interestingly, we also demonstrated that, although previously infected individuals maintained higher NT than naive after 6 months of vaccination, their neutralization potency also decreased. This suggests that waning of the humoral response is a common feature following infection or vaccination, and that humoral response is enhanced by a new exposure (infection or vaccine boost), as reflected here by the 5-fold increment in NT in individuals with a breakthrough infection.
Our seroprevalence study coincided with cocirculation of the Alpha, Lambda, and Gamma VOCs along with other variants. Gamma became the most prevalent from mid-April. Since June 2021, the Delta variant began to circulate, becoming the most prevalent in September of that year, being together with Gamma the main variants causing the second wave of COVID-19 in Argentina (26). Like the other first generation of COVID-19 vaccines, Sputnik V is based on the Wuhan SARS-CoV-2 spike sequence, and it was especially relevant to evaluate the cross-neutralization capacity of antibodies against SARS-CoV-2 Gamma and Delta in vaccinated individuals. As expected, the potency of neutralization against VOCs was smaller than against the ancestral virus (9,10,16,19,25). Despite this difference, 90% of individuals presented cross-neutralizing antibodies against Gamma after 2 doses of Sputnik V, regardless of whether they were previously infected or not. Noteworthy, detection of neutralizing antibodies against Delta markedly differed between naive and previously infected individuals. While 44% of individuals acquiring infection prior to vaccination presented neutralizing antibodies, less than 20% of the vaccinated individuals who were naive at 2 wpv were positive. Although the number of individuals in our study is limited, the analysis of cross-reactivity reinforces the concept that hybrid immunity generates a broader immune response (14,15). In line with this notion, after 6 months of vaccination, 100% of those individuals who had COVID-19 between 2 and 24 wpv had detectable neutralizing antibodies against both VOCs. This may be explained not only by hybrid immunity but also by the fact that these individuals may have been infected by Gamma or Delta VOCs, and infection with VOCs after vaccination may have also potentiated the immune response against ancestral virus and heterologous VOCs (25). In conclusion, vaccination reinforces and is reinforced by infection where hybrid immunity may play a key role in cross protection.
To gain insight into whether antibody levels against S or RBD, usually determined in the clinical settings, are good predictors of neutralizing response, we analyzed the correlation between specific antibody levels and neutralizing titers obtained at 2 and 24 wpv in the Non-COVID cohort. Specific antibody levels were assessed with a semiquantitative ELISA (COVIDAR IgG) and a quantitative electrochemiluminescence assay with a broader dynamic range (Elecsys Anti-SARS-CoV-2 S). The analysis indicated that specific antibody levels against S/RBD correlated with NT against ancestral virus for both semiquantitative and quantitative techniques. This correlation was previously noted in convalescent and vaccinated individuals (24, 27 to 32). For COVIDAR IgG, correlation was reported for convalescent-phase samples (33), our work being the first report showing this correlation in samples of vaccinated individuals.
Altogether, our data and previous reports indicate that the humoral response elicited by vaccination with Sputnik V shares common features with vaccines based on different platforms. Similar to mRNA vaccines, response after vaccination persisted over time despite waning of total IgG and neutralizing antibody titers. The magnitude of the drop is comparable for both adenovirus and mRNA-based vaccines and was is in the range of 2to 10-fold decrease between peaking of antibody response (2 to 3 weeks after completion of the vaccination schemes) and 6 months (24,34,35). In addition, antibody titers are induced to a similar higher extent in individuals that acquired COVID-19 prior to vaccination compared to naive individuals (24,34), and breakthrough infection boosts the antibody response for both platforms (12,14,15,36). Finally, partial escape to vaccine-induced antibodies of VOCs Gamma and Delta as assessed by the fold reduction in neutralization titers is also a common feature (25) and suggests that the continued emergence of VOCs impacts the breadth of protection conferred by vaccination.
Overall, our work provides comprehensive information regarding the response induced by Sputnik V vaccination by means of an analysis that comprised more than 1,100 individuals and the follow-up of more than 500 fully vaccinated individuals. We showed that humoral/neutralizing response persists after Sputnik V vaccination and that neutralizing capacity decreases over time if no further immunization (natural or by a vaccine boost) may occur (12), highlighting the importance of administering a complete scheme and booster doses in the medium term to keep neutralizing titers both in naive and vaccinated-infected individuals. Likewise, Sputnik V has been approved for use in 71 countries, including several countries in Latin America, and although the global political context has paralyzed the verification by the WHO of the efficacy of this vaccine, our work adds new clear and necessary evidence regarding the response mounted by Sputnik V.

MATERIALS AND METHODS
Ethics statement. This study was approved by the Ethics Committee at the Facultad de Ciencias Bioquímicas y Farmacéuticas-UNR, Rosario, Santa Fe, Argentina (res. no. 620/2021). All participants provided written informed consent prior to collection of data and specimen.
Samples and SARS-CoV-2 antibody ELISA. Blood serum samples were obtained by venipuncture and stored at 220°C. Antibodies to SARS-CoV-2 spike protein (S) and receptor binding domain (RBD) were detected using the COVIDAR IgG ELISA (Laboratorio LEMOS, S.R.L, Buenos Aires, Argentina) (33). Antibody levels were given by a positivity index (PI), which varied between 1 and 10. PI results from the ratio between the absorbance of the sample and the cutoff value of the methodology. This cutoff value was calculated following the manufacturer's instruction as (absorbance of negative control 1 0.2) Â 1.1.
To study the correlation between neutralizing capacity against SARS-CoV-2 and antibodies against S or RBD in the cohort of naive individuals, Elecsys Anti-SARS-CoV-2 S assay (Roche Diagnostics, Mannheim, Germany) was also used. This electrochemiluminescence immunoassay targets the RBD with an analytical measuring interval between 0. 4  Neutralization assay. Vero E6 cells were seeded in 96-well plates at a density of 1.5 Â 10 4 cells per well in complete DMEM and incubated 24 h at 37°C and 5% CO 2 . SARS-CoV-2 (300 TCID50) was preincubated with serially diluted sera for 1 h at 37°C starting at sera dilution 1:8. Each sera dilution was tested in duplicate. Then, virus-sera mixture was added onto Vero E6 cells in a final volume of 100 mL in DMEM 2% FBS. After 72 h at 37°C and 5% CO 2 , cultures were fixed with formaldehyde 10% at 4°C for 24 h and stained with crystal violet solution in methanol. The cytopathic effect (CPE) of the virus on the cell monolayer was assessed visually, and neutralization titer (NT) was defined as the inverse of the highest serum dilution without any CPE.
Statistical analysis. GraphPad version 8.4.2 was used for statistical analyses and generation of plots. The geometric means (GM) and 95% CI were calculated in each group of samples. Samples with NT below 8 were set at 1 to calculate GMNT and to represent them in the corresponding plots. After normality and lognormality tests, nonparametric tests were used. Paired data were analyzed by Wilcoxon matched-pair test, nonpaired data by the Mann-Whitney U test and Kruskal-Wallis test. Correlation between total antibody levels and neutralizing titers was analyzed by Spearman's r test. Statistical significance is shown with the following notations: ****, P , 0.0001; ***, P , 0.001; **, P , 0.01; *, P , 0.05; ns, not significant.
Data sharing. Data used in this study are available upon request from the corresponding author. Ethics approval. This study was approved by the Ethics Committee at the Facultad de Ciencias Bioquímicas y Farmacéuticas-UNR, Rosario, Santa Fe, Argentina (res. no. 620/2021).