Herd Immunity to SARS‐CoV‐2 Among the Armenian Population in the Second Half of 2022

1. Introduction

The 21st century's first coronavirus pandemic quickly became a global challenge. As of 21.01.2023, more than 743 million infections have been registered worldwide, including more than 6.7 million COVID-19 fatalities [1]. Pandemic damage to the economy has been estimated to be at least 3.3% of global GDP [2]. The Republic of Armenia (RA), as a nation, was not significantly involved in the overall pandemic. There were 445,690 confirmed cases in the entire RA, representing about 14% of the Armenian population, or 0.06% of those infected globally. There were 8,713 COVID-19 fatalities in the country. Accordingly, the mortality rate was 1.96%, which is 1.98-fold higher than the global average [3]. The coronavirus pandemic in the RA started on 03.03.2020, when the first case of COVID-19 was registered. Subsequently, the process proceeded in 6 waves (Figure 1).

During the 1st peak (weeks 27 to 32, 2020), the Alpha viral variant (B.1.1.7) dominated. During the 2nd peak (weeks 42 to 52, 2020), the Delta variant dominated, and its circulation continued until July 2021. The Omicron variant circulated from the end of 2021 until January 2022 [4]. Subsequently, a continuous change in genetic variants was noted: Kraken (XBB.1.5), Acrux (XBB.2.3), Arcturus (XBB 1.9.1), Hiperion (FL.1.5.1), Fornax (EG.5.1), Eris (BA.2.86.1), and Pirola (JN.1). Despite a wide range of successive SARS-CoV-2 genetic variants, starting from the 40th week (2022), incidence decreased to about 2,200 cases per week. By the 46th week of 2023, some stabilization of the number of infections at a level of 2,100 cases per week was noted. However, already from the 11th week of 2024, a slight increase to about 5,000 cases per week was observed (Figure 1) [1].

Starting from 01.04.2021, a vaccination campaign against SARS-CoV-2 was launched in the RA. In the initial period, the Gam-COVID-Vac vector vaccine was mainly used. Further, the range of vaccines expanded to include AZD122ChAdXo1, BNT162b2, mRNA1273, and Sinopharm BBIP [5]. It is interesting to note that after reaching 30% vaccination coverage (weeks 5–6 of 2022), a decrease in incidence was observed (Figure 1). This turned into a steady downward trend, which was replaced, in essence, by seasonal dynamics from the beginning of 2023. By this time, vaccination coverage reached almost 35%. The overview of local pandemic dynamics we present is completely consistent with previously described epidemiological mechanisms [6]. Among them, upon reaching the threshold level of collective immunity, an epidemic spontaneously dies out or, as in this case, the infection acquires a seasonal course. Hence, it can be reasonably considered that collective immunity is the central factor in protecting a social higher biological species from microbial onslaught.

Collective immunity is a multicomponent complex defense system that includes many cellular and humoral factors. Cellular factors are decisively responsible for the recognition and regulation of functions, and humoral factors are largely responsible for the executive reactions of the immune defense [7,8,9]. An essential part of the latter group are antibodies that have the ability to bind and, in one way or another, inactivate the foreign pathogen [8,9]. The purpose of the study was to assess the humoral collective immunity to SARS-CoV-2 in the Armenian population as of the end of 2022.

2. Materials and Methods

The study is part of a multi-stage implementation of scientific cooperation between the countries of Eastern Europe, Transcaucasia, and Central Asia to study collective immunity to coronavirus infection under Russian Government Order (dated 18.06.2021, No. 1658-p); Rospotrebnadzor Order (dated 09.09.2021, No. 512); and Armenian Minister of Health Order (dated 31.01.2022, No. 380-A). The organizers and implementers of the study were the Saint Petersburg Pasteur Institute (Rospotrebnadzor of Russian Federation) and the National Center for Disease Control and Prevention (Ministry of Health of Republic of Armenia).

2.1. Study Design and Organization

A randomized cohort study of collective immunity to coronavirus infection was conducted according to a program developed by the Federal Service for the Oversight of Consumer Protection and Welfare (Russian Federation) with the participation of the Saint Petersburg Pasteur Institute, taking into account WHO recommendations.

The organization and conduct of studies of the formed cohort of volunteers were similar to those described previously [10]. Each volunteer, or their legal representative (in the case of children), was familiarized with the goals and conditions of the upcoming study and signed a statement of informed consent. The study was organized in accordance with the provisions of the Declaration of Helsinki. It was approved by: the Ethics Committee, National Center for Disease Control and Prevention (No. 01 dated 17.02.2022); and the Ethics Committee, Saint Petersburg Pasteur Institute (protocol No. 64, dated May 26, 2020).

2.2. General Characteristics of the Volunteer Cohort

The study was conducted in Yerevan and all Armenian regions. Based on initial survey results, a cohort of 2,974 volunteers was formed, from whom blood samples were taken between November 28 and December 2, 2022. The distribution of volunteers within most adult age brackets turned out to be relatively homogeneous, both in absolute numbers and in proportional representation (Table 1). The '1-17 years old' and '18-29 years old' groups were an exception, and their representation was significantly lower than that of older groups.

Study participants represented 0.1% (95%CI: 0.10-0.11) of the Armenian population (Table 2). As expected, the largest number of volunteers was recruited in the capital, Yerevan (35% of the cohort). However, given the actual population of the city (1,098,900 as of Dec. 2022), the actual representation of volunteers there was 0.1%, which matches other regions. The share of volunteers (percentage of the overall cohort) from other regions varied from 11.1% (Gegharkunik Province) to 2.2% (Vayots Dzor Province).

As in other population-based studies, women participated more actively than men [11]. Participants were 557 male (18.7%; 95%CI: 17.4-20.2) and 2,317 female (81.3%; 95%CI: 79.8-82.6.0), respectively. Among volunteers, 983 people (33.1%; 95% CI: 31.4-34.8) had a COVID-19 history with confirmation of diagnosis by a medical institution. The cohort included representatives of twelve activity groups, including unemployed, pensioner, preschool and school age children, and students (Table 3).

The most well represented group was healthcare workers, whose share in the surveyed cohort was 47.9% (95%CI: 46.1-49.7). The smallest shares (<1.5%) were noted in the categories 'business', 'science and arts', 'students', and 'production and transport'. In some cases, due to the small number of volunteers in the categories, they were combined: preschoolers with schoolchildren; military personnel with civil servants; office workers with IT specialists, scientists with artists, and industrial workers with transportation workers (Table 3).

2.3. Organization of Laboratory Research

The presence and levels of IgG antibodies to two SARS-CoV-2 antigens were assessed in volunteer plasma: Nc and the RBD region of the S protein. A detailed description of the organization of laboratory analysis is given in our previous publication [11]. To obtain comparable results, it was fundamentally important to strictly adhere to the previously described research methodology.

2.4. Statistical Analysis

Statistical analysis was performed using Microsoft Excel 2010. Mean values ​​and confidence intervals of the proportions were calculated using the method of A. Wald and J. Wolfowitz [12] with correction according to A. Agresti and B. A. Coull [13]. Pairwise rank correlation according to Spearman was performed using the Statistica 13 package [14]. The statistical significance of differences in proportions was calculated by z-test using a special calculator [14]. For other statistical calculations, Statistica 13 was used. The significance of differences, unless otherwise stated, was assessed with a probability of p≤0.05. Graphic illustrations were made in Microsoft Excel 2010.

3. Results

3.1. SARS-CoV-2 Seroprevalence by Age

By the beginning of the study, the coronavirus pandemic had passed the 34-month mark. During this time, 445,690 COVID-19 cases were confirmed in the Republic, and almost 35% of the population was vaccinated [5]. It is worth noting that the total number of asymptomatic and/or undiagnosed cases of COVID-19 in the RA is unknown. Under these conditions, a high level of collective immunity among volunteers would be expected. The results obtained fully confirm the validity of this assumption (Figure 2).

The data shows that the absolute majority of volunteers had Abs to both main antigens. The total share of seropositive individuals (Nc and/or RBD) in the cohort as a whole was 99% (95%CI: 98.5-99.3). The cohort was dominated by individuals seropositive for both Abs (Nc⁺RBD⁺), reaching 88.7% (95%CI: 87.5-89.1). The share of individuals who were only positive for anti-RBD Abs (Nc⁻RBD⁺) was an order of magnitude lower at 8.9% (95%CI: 7.9-10.0) on average. Regarding seronegative (Nc⁻RBD⁻), and those only seropositive for Nc (Nc⁺RBD⁻), their shares did not exceed the standard error calculated for the double positive (Nc⁺RBD⁺) group. They were 1.3% (95%CI: 0.9-1.8) and 1.0% (95%CI: 0.7-1.5), respectively.

Figure 2. Serological status by age group. Numerical values ​​are given in the table below the figure. Black vertical lines are 95% confidence intervals. Note: Nc⁻RBD⁻ is seronegative; Nc⁺RBD⁻ is seropositive (anti-Nc only); Nc⁺RBD⁺ is double seropositive (anti-Nc, anti-RBD); Nc⁻RBD⁺ is seropositive (anti-RBD only).

Figure 2. Serological status by age group. Numerical values ​​are given in the table below the figure. Black vertical lines are 95% confidence intervals. Note: Nc⁻RBD⁻ is seronegative; Nc⁺RBD⁻ is seropositive (anti-Nc only); Nc⁺RBD⁺ is double seropositive (anti-Nc, anti-RBD); Nc⁻RBD⁺ is seropositive (anti-RBD only).

3.2. Seroprevalence by Region and Activity

The Republic of Armenia is the smallest state in the Caucasus region (29,743 km²). Considering the high level of collective immunity among volunteers in all age groups, and the small geographic area involved, it would be reasonable to assume that region of residence within the RA would not have a significant impact on seroprevalence. Analysis confirmed this assumption. High seroprevalence levels had formed throughout Armenian regions, including about 90% prevalence of double-seropositive status (Nc⁺RBD⁺). This status ranged from 81.2% (95%CI: 69.5-89.9) in the Vayots Dzor region to 92.8% (95%CI: 89.4-95.3) in Gegharkunik.

Occupation is likely a significant factor determining SARS-CoV-2 infection risk and associated seroprevalence. In particular, it is believed that individuals who have frequent professional contacts with the population are most vulnerable to SARS-CoV-2 infection [15,16]. As such, assessing seroprevalence levels among individuals of different professions and activities seemed relevant. No significant differences from the average cohort value were found in any professional group.

As for the structure of immunity, it closely matched the general trends described in the age group analysis. There was a predominance of double positive (Nc⁺RBD⁺) volunteers in all groups, ranging from 82.4% (95%CI: 69.1-91.6) among business workers to 93.8% (95%CI: 88.2-97.3) among education personnel. There was a smaller, insignificant contribution to seroprevalence from monopositive RBD status (Nc⁻RBD⁺). The two remaining Nc-related categories (Nc⁺RBD⁻, Nc⁻RBD⁻) were minimal. Thus, SARS-CoV-2 seroprevalence levels among Armenian volunteers were close to maximum values. Collective immunity was not dependent on age, region of residence, or activity.

3.3. Quantitative Ab Levels (Nc, RBD) by Age

Based on the results of qualitative analysis, namely the distribution of seropositivity to the two main viral antigens, it seemed advisable to assess the quantitative structure of Ab distribution by age group. The study showed a predominance of individuals with low (32-124 BAU/ml) or medium (125-332 BAU/ml) Ab levels in all age groups (Figure 3).

When characterizing the observed distribution, it is worth paying attention to the absence of significant differences in anti-Nc Ab levels between groups. Regression analysis indicated an insignificant downward trend in older age groups, which was most likely random. This is reflected in the low determination coefficient and the absence of any correlation dependence in the distribution. This likely indicates a uniform distribution of anti-Nc Abs in the population in the range 32-124 BAU/ml.

The least individuals were in the maximum Ab category (>666 BAU/ml). Within it, a tendency towards a higher share of seropositive individuals with maximum anti-Nc level was noted with age. In two older groups (50-59, 70⁺) the shares of such individuals were significantly higher than in the '30-39 year old' group (ρ=0.78; p=0.05). In contrast, the share of seronegative individuals showed a downward trend from the '1-17 year old' group to the 70⁺ group, but the trend was insignificant (ρ=-0.57; p>0.1).

Of some interest may be the distribution of volunteers with plasma anti-Nc Abs within the range 17-31 BAU/ml. In this case, a significant downward trend was noted (ρ=-0.89; p=0.01). Considering that a similar trend was noted in the group with anti-Nc titers within 32-124 BAU/ml, it can be concluded that volunteers with very low (17-31 BAU/ml) or low (32-124 BAU/ml) level are most widely represented in the age group 1-17 years. These shares gradually decrease with advancing age until the group '70⁺ years'.

The second marker analyzed was antibodies against RBD region of the S protein. Such Abs play an important protective role in the pathogenesis of infection [17]. Assessment of the quantitative distribution of anti-RBD Abs in volunteers of different ages coincided with the previous conclusion about achievement of a high level of collective immunity (Figure 4).

In all age groups, volunteers with the maximum anti-RBD IgG level (>450 BAU/ml) predominated, and their share increased with age. The trend line is described by a linear regression with an upward trend across all age groups starting from '1-17 years' until '70⁺' years with a slope (k) equal to 4. The correlation coefficient (ρ=0.94) indicates a strong relationship between age and the share of carriers with the maximum anti-RBD Ab level, and the correlation coefficient in this group was significant (p=0.001). Interestingly, in groups with medium (220.1-450.0 BAU/ml) or low (22.6-220.0 BAU/ml) Ab levels, the trend is reversed, and their slopes also decrease to -1.66 and -1.8, respectively. The ρ values ​​become negative, yet the significance of the relationships remains high (0.01 and 0.001, respectively). The share of individuals with very low anti-RBD Ab levels remained virtually at a minimum and did not depend on age.

In summarizing the data, we can make a general conclusion that the quantitative distribution of SARS-CoV-2 Abs in the COVID-19 post-pandemic period reflects some specificity. This is manifested as a high share of individuals with low or moderate anti-Nc Ab levels in the context of a high share of volunteers with the maximum level of anti-RBD Abs.

3.4. Volunteer Vaccination

Vaccination played an important role in stopping the epidemic process of coronavirus infection in the RA. By the late January - early February timeframe (2022), in other words before the study period, total vaccination coverage of the local population with coronavirus vaccines exceeded 60% according to official data. Comparison of infection and vaccination dynamics showed a significant decrease in infection rate from the moment the aforementioned threshold was reached. Thus, starting from week 4, 2022, a stable inverse correlation was revealed between the increase in vaccination coverage and the rate of decline in the number of infections (Figure 1). The Spearman correlation coefficient (ρ) for the time period from week 4 to week 44 (2022) was -0.44 (p<0.001).

Targeted and consistent COVID-19 vaccination was implemented. A sizeable portion of the population also experienced infection, either in a manifest or asymptomatic form. Together, these naturally led to the cessation of the pandemic process through achievement of almost ideal herd immunity. This was true in almost all Armenian age groups (Figure 5), and most groups featured maximal anti-RBD Ab levels (Figure 4).

A wide range of vaccines was used in the RA in 2021. We have described the logistics of their usage in detail in a previous report [18]. By the beginning of 2022, this campaign had already been completed in most of the Republic, which was reflected as a natural decrease in the rate of vaccination (Figure 5).

Figure 5. COVID-19 vaccination dynamics. The x-axis shows date of registration. The y-axis shows individuals who completed vaccination. Source: online portal [5].

Figure 5. COVID-19 vaccination dynamics. The x-axis shows date of registration. The y-axis shows individuals who completed vaccination. Source: online portal [5].

According to online data [5], the maximum number of people who fully completed SARS-CoV-2 vaccination occurred in the autumn of 2021. Afterwards, immunization activity consistently decreased, and it almost completely ended in April-May 2022, which is when COVID-19 incidence reached a sporadic level. At the end of 2022 during the study period, vaccination coverage in the surveyed volunteer cohort exceeded 82.9% (95%CI: 81.5-84.2) in all age groups, with the exception of children (15.5%; 95%CI: 11.6-20.4) (Figure 6).

To achieve greater representativeness, all vaccines approved in the Republic were grouped into three main categories based on production platform. The 'vector' group combined GamCOVIDVac (Russia) and AZD1222ChAdO1 (UK). The 'mRNA' group combined the BNT 162b2Vaccine (USA) and the mRNA1273 COVID-19 Vaccine (USA). The "whole virion" group combined CoviVac (Russia), CoronoVac (China), and Sinopharm BIBP (China) (Figure 7).

Whole-virion (43.5%; 95%CI: 41.6-45.4) and vector (42.3%; 95%CI: 40.4-44.2) vaccines were most often used for immunization (Figure 7). This trend was noted in all age groups. The share of mRNA vaccines was 14.2% (95%CI: 13.3-15.6), and vaccines from this platform were more often used in children and young adults (Figure 8).

The study compared the efficacy of hybrid and post-vaccination immunity in Armenian volunteers. For this purpose, COVID-19 incidence (laboratory-confirmed) was compared in two groups of vaccinated volunteers. Among those with only post-vaccination immunity (could not recall symptomatic illness during anamnesis), 17.0% (95%CI: 14.7-19.6) fell ill after vaccination, regardless of the vaccine type used. Among individuals with hybrid immunity (vaccinated after manifest COVID-19), 1.5% (95%CI: 0.6-3.5) fell ill again after vaccination. For comparison, among unvaccinated volunteers, 28.0% (95%CI: 24.4-32.0) fell ill with manifest, laboratory-confirmed coronavirus infection (Table 4). Thus, among individuals with hybrid immunity, COVID-19 incidence was 11-fold lower than among those with post-vaccination immunity, and almost 19-fold lower than among those without immunity.

In addition, quantitative indicators of immunity (anti-RBD IgG levels) were assessed in the two groups immunized with vaccine types (vector, mRNA) that initiate production of Abs to the RBD region of the S protein SARS-CoV-2. History of infection was assessed by the presence or absence of anti-Nc Abs insofar as the aforementioned vaccine types do not elicit those specific antibodies. A group of 27 volunteers had post-vaccination immunity alone as inferred from other factors: no recall of symptomatic illness as well as an absence of anti-Nc Abs. A group of 291 individuals had Abs to both RBD and Nc indicating a hybrid immune response formed as a result of infection and vaccination (Figure 9).

When comparing volunteers with hybrid immunity with those with post-vaccination immunity, the share of those with low anti-RBD level was lower (3.1%; 95%CI: 1.6-5.8 and 22.2%; 95%CI: 10.6-40.8, respectively). In the same comparison (hybrid, post-vaccination), the share of individuals with high anti-RBD level (>450 BAU/ml) was higher (68.7%; 95%CI: 63.2-73.8 and 44.4%; 95%CI: 27.6-62.7, respectively).

Thus, hybrid immunity (formed after vaccination in volunteers who had previously experienced COVID-19) was characterized by greater efficacy in protecting against symptomatic infection. It featured higher anti-RBD Ab levels compared to 'pure' post-vaccination immunity.