EVOLUTION OF HERD SARS-COV-2 HUMORAL IMMUNITY IN THE REPUBLIC OF BELARUS

Background. The course of the COVID-19 epidemic process depends on population immunity which prevents pathogen spread. Aim: to study an evolution of SARS-CoV-2 humoral immunity in the Belarusian population relative to COVID-19 pandemic dynamics. Materials and methods. The work was carried out according to a methodology for assessing herd immunity developed by Rospotrebnadzor (Russia) and the Belarusian Ministry of Health involving the St. Petersburg Pasteur Institute (SPPI) by taking into account the WHO recommendations. The study was approved by the Bioethics Committee of Belarus and the SPPI Bioethics Committee. Participant selection was carried out by questionnaire using a cloud (internet server) service. To monitor herd immunity, a cohort of 4,661 subjects (involved at all stages of seromonitoring) was formed from the total volunteer group. Study subjects were randomized into groups based on age (1-17, 18-29, 30-39, 40-49, 50-59, 60-69, 70+ years), geographic region, and occupation. For the detection of antibodies (Abs) against SARS-CoV-2 nucleocapsid (Nc) and S glycoprotein receptor-binding domain (RBD), relevant assay systems were used according to the manufacturers instructions. A four-stage study was conducted according to a unified scheme. Results. At stage 1 (pandemic month 15), herd immunity was mainly accounted for by Nc+RBD+ Ab status alone. By stage 2 (4 months later), its specific proportion decreased by 1.2-fold, whereas percentage of subjects solely bearing RBD-specific Abs increased by 1.7-fold. At stages 3 and 4 (9 and 19 months after the onset) vs. stage 2, percentage of subjects with RBD+Nc‒ decreased by 3.5%; the proportion of persons with Nc+RBD‒ Abs increased by 1.5-fold. The most important contributor in herd immunity turned out to be due to population vaccination, with coverage reaching 70% by stage 4. Among vaccines, compared with whole-virion, inactivated BIBP-CorV vaccine the Sputnik V and Sputnik Light vector were used most often. Conclusion. The evolution of herd SARS-CoV-2 humoral immunity included a series of changes in circulating Ab levels (Nc, RBD). The hybrid immunity formed helped to reduce the incidence of COVID-19 to sporadic level.


INTRODUCTION
Among the vast family of viruses that periodically cause infectious 2 illness in humans, coronaviruses did not initially attract much attention. Isolated in 3 1965 from a person with acute coryza, they have long been associated with a mild, 4 self-limiting upper respiratory tract infection. Four types of seasonal coronaviruses 5 have been known to be associated with common cold infection in humans: two alpha 6 (229E, OC43) and two beta (NL63, HKU1) [2,3]. The situation began to change 7 starting in 2002, when the first highly pathogenic strain of coronavirus emerged, 8 causing a SARS outbreak of more than 8,400 cases. Subsequently, this virus was 9 named SARS-CoV [37]. 10 Ten years later, another pathogenic Betacoronavirus representative 11 appeared in the Middle East: the causative agent of Middle East Respiratory 12 Syndrome (MERS). It caused 1,348 infections from 2012-2015, in which 479 people 13 died [4,23,37,46]. The evolution of pathogenic coronaviruses continued at the end 14 of 2019, on December 31 specifically, when a cluster of SARS patients was detected 15 in a fish market in the Chinese city of Wuhan. It was caused by a new 16 Betacoronavirus representative, SARS-CoV-2, causing a pandemic of acute 17 respiratory infection pandemic  now in its third year [32,42,45]. 18 According to statistical sources, 630,164,738 people have been infected 19 globally, including 6,577,479 deaths (as of October 14, 2022) [14,44]. A 20 characteristic feature of SARS-CoV-2 is relatively rapid viral evolution due to a 21 rather high mutational variability [19,39]. Thus, the original Wuhan viral line (2019- 22 nCoV) was replaced by a new variant: B.1.1.7 (Alpha type), first isolated on 23 September 20, 2020 in the UK [25]. In the same year, a variant, B.1.351 (Beta type) 24 [22], was identified in S. Africa (May 20). In November, a new variant, B.1.1.28 25 (P.1), was identified in Brazil, designated as the Gamma viral line [7]. Around the 26 same time, a virus was isolated in India, designated as B.1.617.2 (Delta variant) [28]. 27 The latest virus was B.1.1.529, better known as the Omicron variant, which was 28 identified in November 2021 simultaneously in S. Africa and Botswana; it circulated at least until the end of 2022 [20,24]. All of the aforementioned coronavirus variants 30 were classified as variants-of-concern (VOC) [44]. In addition to them, however, 31 there are 7 additional, less pathogenic, strains. These did not show significance in 32 the pandemic process and were quickly forced out of circulation by more pathogenic 33 representatives. 34 In the Republic of Belarus (RB), the COVID-19 pandemic turned out 35 to be less widespread than in neighboring countries, such as Russia for example [12]. 36 As of 14/10/2022, 994,037 individuals have been infected in the RB, of which 7,118 37 (0.7%) have died. In terms of incidence, the Republic ranks 65th among nations 38 globally. At the same time, like other countries globally, it was not bypassed by the 39 well-known SARS-CoV-2 lines that appeared in circulation [9]. By comparing the 40 ordering of SARS-CoV-2 variants and recorded peaks in morbidity, it was possible, 41 with some degree of probability, to predict the chronological sequence of the listed 42 variants appearing in circulation in the RB (Fig. 1). At the same time, it should be 43 noted that each subsequent viral variant caused a wave of increased incidence in the 44 RB, often 3-4 months later than peaks in many other countries. The first COVID-19 case was detected only in the 9th week of 2020, 56 that is, at the end of the 2nd month after the first case was detected in Wuhan [9]. 57 The Alpha SARS-CoV-2 variant was identified in the UK in September 2020, but 58 only in February 2021 did it start circulating in the RB. Similarly, the most 59 transmissible Omicron variant was initially isolated in S. Africa in September 2021, 60 but its rapid, albeit short-lived, spread in RB was not seen until January 2022 ( Fig.   61 1). Thus, the epidemic situation in the RB can be characterized by several features: 62 relatively low population density (45.5 km -2 ); later appearance of new SARS-CoV-63 2 genetic variants in circulation; and relatively low morbidity, not exceeding 600 64 ‱o ( Fig. 1), with mortality varying within 0.7%. 65 For comparison, in Poland (as of May 5, 2022)

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Formation of the volunteer cohort. A study to assess the formation and 102 progression of SARS-CoV-2 collective immunity in the Belarusian population was 103 implemented in 4 stages over 2021-22 (Fig. 1). The 1st stage was carried out (May 104 [14][15][16][17][18][19]2021) with 12929 individuals taking part in the survey. In the 2nd stage (Aug. 105 30 -Sept. 3, 2021), the number of examined individuals decreased by 28%, leaving 106 to 9269 people. By the 3rd stage (Jan. 24 -28, 2022) (Proceedings No. 64, dated May 26, 2020). 119 All volunteers included in the cohort were clinically healthy. The 120 exclusion criterion was: signs of manifest COVID-19 during the survey period. Our 121 methodology for cohort formation and examination has been exhaustively described 122 earlier [1, 27, 30,]. Over the course of four stage examination of individuals in the 123 final cohort, antibodies (Abs) to two main antigens (Ags) were determined in 124 volunteer sera according to previously described methods [29,30]. These were Abs 125 to nucleocapsid (Nc) and to S protein receptor-binding domain (RBD and J. Wolfowitz [43], with the corrections of A. Agresti and B. A. Coull [5]. 155 Correlation analysis was performed using the Spearman rank correlation method. 156 The statistical significance of differences was calculated by z-test using a 157 corresponding online calculator [38]. CoV-2 genetic variants were seen in the RB than in the rest of the world. Indeed, a 181 number of variants-of-concern were not detected in the RB (B. manifested COVID-19 [11,30], then the overall level of collective immunity could 187 exceed 60%, even without taking into account asymptomatic infections. Thus, it is 188 logical to assume that a threshold was reached; it was seemingly sufficient to reduce In the 1st stage, a significant predominance of Nc + RBD + over Nc -RBD -209 (p<0.05) was noted, mainly due to higher seropositivity among older (50-70 + ) 210 individuals (Fig. 2). The smallest proportion of seropositive individuals was noted 211 among Nc + RBD -. The share of RBD + Ncturned out to be approximately 4.5-fold 212 higher than Nc + RBD -, while being significantly lower than Nc + RBD + and Nc -RBD -213 (p<0.05), except for the group '18-39 years', where differences with Nc + RBD + were 214 not significant (Fig. 2, Table S1).  Table S1). A reason for these changes is likely expansion of vaccination coverage, 219 which by this time amounted to more than 15%. The most widely used preparation 220 during this period was Sputnik V (vector vaccine) whose mechanism leads to 221 production of RBD antigen alone. By the 3rd stage, a change in trend was noted, 222 manifested by a noticeable increase in the share of Nc + RBD + , alongside steep 223 decreases in Nc -RBDand Nc + RBD -(p<0.0001), as well as an insignificant decrease 224 in RBD + Nc - (Fig. 2, Table S1). By this time, vaccination coverage was 47% (  Republic's regional administrative entities. As such, there were no preliminary 250 grounds to expect significant regional differences in the development of the COVID-251 19 epidemic process. Our preliminary assumptions, and the results of a cross-252 sectional randomized study [30], were in good agreement with each other. 253 In the 1st stage, there was a significant predominance of Nc + RBD + and 254 Nc -RBDstatus compared with Nc + RBD -(p<0.0001) and RBD + Nc -(p<0.001) (Fig. 255 3, Table S2). Sectors: age intervals, years. Numerical values for graphs are given in Suppl. Table   263 S2. Monitoring stage is indicated above the diagrams.  Table   271 S2).

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The structure of volunteer subpopulations in terms of seropositivity in 273 stages 3 and 4 was identical to that obtained when analyzing the data by age (Fig. 2 Table S2).  In the 1st stage, a noticeable spread in the shares of Nc -RBD -, RBD + Nc -311 and Nc + RBD + was observed (Fig. 4). When calculating the variance, the largest 312 value was noted in Nc -RBD -(81.86); the smallest was in RBD + Nc - (35.64 which can be clearly seen in Figure 2 (Stage 4). 387 Since it has been shown that neutralizing activity against SARS-CoV-388 2 is more associated with RBD Abs [34], these levels were also assessed (Fig. 6, 389 Table S5). In the 1st monitoring stage, the largest proportion of seropositive 390 volunteers carried RBD Abs at the 22.6-220.0 BAU/ml level (Fig. 6) Thus, the evolution of collective humoral immunity across the stages of for these individuals were grouped separately and designated as 'Other' (Fig. 7).  with endemic strains of coronaviruses [16]. As for the low seropositivity in the age 502 group of 18-29 years, 61.3% (95% CI: 56.4-66.1), a certain proportion of them were 503 students, among whom up to 40%, according to some data, are skeptical about the 504 idea of vaccination against SARS-CoV-2 [6,21,35]. This was probably an 505 additional reason why more than a third of volunteers of this age did not have 506 specific Abs (Table S1). the share of Nc + RBD + individuals increased by 1.5-fold to 49.5% (95% CI: 48.0-520 50.9). The growth in fully seropositive individuals was accompanied by a slight 521 decrease in the share of RBD + Ncby 3.5% and a decrease in Nc -RBDby 2-fold. 522 The outlined trend reached its greatest expression by the 4th stage. The 523 share Nc + RBD + increased to 80.0% (95% CI: 78.8-91.2), while RBD + Ncand Nc -524 RBDdecreased by 2.3 and 5.8-fold, respectively (Table S1). The described 525 processes were noted during stratification by age, region, and occupational group 526 ( Fig. 2-4, Tables S1-S3). Some minor group differences were seen leading to some 527 heterogeneity, yet the overall evolution of collective immunity was not affected in 528 any substantial way by subgroup differences.  1st stage to 77.6% in the 4th (Fig. 7). 562 Taking into account the previously noted effectiveness of vector 563 vaccines [41], the widespread use of the Sputnik family of vaccines may have 564 become a significant factor behind decreasing COVID-19 incidence in the 565 population. Another pattern confirming such a process is the inverse relationship 566 between population humoral immunity and morbidity: an increase in post-567 vaccination resistance is inevitably accompanied by a decrease in morbidity [10, 18, 568 26, 27, 30,36]. 569 The combined increase in the content of the two main antibody types 570 indicates the formation of hybrid immunity [15], usually featuring maximum 571 protection against the "aggression" of a pathogenic agent [41]. The use of a range of 572 specific vaccines made it possible to create the required level of  resistance in the population (Fig. 7). 574 In the initial period, the Gam-COVID-Vac vector vaccine (Sputnik V) 575 was mainly used, which made it possible to form a stable pool of RBD Abs, which 576 laid the foundation for the formation of hybrid immunity. In the 2nd stage, usage of 577 vector vaccines remained almost at the initial level. In the 3rd stage, usage ratios 578 were: almost 2/3 vector vaccines and 1/3 inactivated vaccine (BBIBP-CorV). In the 579 4th stage, the spectrum of vaccines was preserved, but the ratio changed due to 580 expanded usage of the Sputnik Light vaccine.  RBD+Nc-those seropositive for RBD Abs only; Nc+RBD+those seropositive for both Abs. Vertical value axis: share of the age subgroup with the indicated serological status, %. Sectors: age intervals, years. Numerical values for the graphs are given in Supp. Table S1. Monitoring stage is indicated above the diagrams.

Figure 3. Distribution of volunteer seropositivity (Nc, RBD) by region and seromonitoring stage.
Legend: Nc -RBD --SARS-CoV-2 Ab seronegative individuals; Nc + RBD -those seropositive for Nc Abs only; RBD + Nc -those seropositive for RBD Abs only; Nc + RBD +those seropositive for both Abs. Vertical value axis: share of residents with the indicated serological status, %. Sectors: age intervals, years. Numerical values for graphs are given in Suppl. Table  S2. Monitoring stage is indicated above the diagrams. Legend: Nc -RBD --SARS-CoV-2 Ab seronegative individuals; Nc + RBD -those seropositive for Nc Abs only; RBD + Nc -those seropositive for RBD Abs only; Nc + RBD +those seropositive for both Abs. Vertical value axis: share of occupational group with the indicated serological status, %. Sectors: age intervals, years. Numerical values for the graphs are given in Supp. Table S3. Monitoring stage is indicated above the diagrams.

Figure 5. Distribution of Nc Ab levels by volunteer age group and seromonitoring stage.
The vertical value axis is percentage of all seropositive individuals (normalized to 100%) in the age subgroup with the indicated serological status, %. Sectors: volunteer age intervals, years. Legend: Nc Ab quantitative levels in BAU/ml. The numerical values are given in Supp. Table S4. Monitoring stage is indicated above the diagrams.

Figure 6. Distribution of RBD Ab levels by volunteer age group and seromonitoring stage.
The vertical value axis is percentage of all seropositive individuals (normalized to 100%) in the age subgroup with the indicated serological status, %. Sectors: volunteer age intervals, years. Legend: RBD Ab quantitative levels in BAU/ml. Quantitative data are given in Supp. Table S5. Monitoring stage is indicated above the diagrams.   Table S1. Shares of seropositive and seronegative individuals by age and seromonitoring stage.