Severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) serology in the vaccination era and post booster vaccination

Abstract

Background

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has caused over 6 million deaths world-wide. In the pre-vaccination era, we noted a 5·3% SARS-CoV-2 IgG antibody positivity rate in 81,624 subjects.

Methods

Utilizing assays for serum SARS-CoV-2 spike (S) protein antibody (Roche) and neutralizing antibody (Diazyme), both >90% IgG, we measured antibodies in 13,189 subjects in the post-vaccination era, and in 69 subjects before and 60 days after booster vaccination.

Results

In 2021, in 10,267 subjects, 25·0% had negative S protein levels (<0.80 U/L), 24·4% had low positive levels (0.80-250 U/L), and 50·7% had high positive levels (>250 U/L). Median neutralizing antibody levels were 1·16 and 2·06 AU/mL in the low and high positive groups, respectively. In 2022, we evaluated 2,016 subjects where samples were diluted 1:100 if S protein antibody levels were >250 U/L. Median S protein and neutralizing antibody levels were 2,065 U/L (86.3% positivity) and 2·68 AU/mL (68.0% positivity), respectively. Antibody levels were also measured in 69 subjects before and 60 days after receiving SARS-CoV-2 booster vaccinations. Treatment resulted in a 15-fold increase in S protein antibody levels from 1,010 to 17,236 U/L, and a 6-fold increase in neutralizing antibody from 1·51 to 12·51 AU/mL in neutralizing antibody levels, respectively (both P<0.00001), with a wide variability in response.

Conclusions

Our data indicate that by early 2022 86% of subjects had positive SARS-CoV-2 S protein antibody levels, and that these levels and neutralizing antibody levels were increased 15-fold and 6-fold, respectively, 60 days after SARS-Cov-2 booster vaccination.

1. Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes coronavirus disease 2019 (COVID-19) [1,2]. While many infected subjects may remain asymptomatic, others often develop significant symptoms with fever, cough, fatigue, body aches, nausea, vomiting, diarrhea, and loss of taste and smell [1]. Moreover, some subjects develop severe pneumonia, respiratory failure, and marked inflammation which can lead to prolonged hospitalizations requiring ventilator support or death [1], [2], [3]. These latter outcomes are especially common in the elderly, the immunocompromised, and those with cardiovascular disease, diabetes and/or obesity [1], [2], [3]. Characteristic findings in SARS-CoV-2 positive patients requiring hospitalization are the presence of markedly elevated levels of the serum inflammatory markers C reactive protein ≥10 mg/L and interleukin-6 ≥10 pg/mL, as well as elevated antibody levels [3]. Treatments with steroids, monoclonal antibodies, and oral anti-viral medication have been shown to efficacious in treating symptomatic SARS-CoV-2 infected patients, especially if the treatment is given early in the course of the disease [1,4,5].

The infection, likely of horse-shoe bat origin, appears to have been first reported and spread to humans in Wuhan, China in late December of 2019 and then rapidly spread worldwide [6,7]. As of June 1^st, 2022, SARS-CoV-2 had caused over 530 million reported cases and over 6.3 million reported deaths worldwide, with the largest numbers being in the United States (over 85 million cases and over 1 million deaths) [8]. Moreover, while there has been widespread SARS-CoV-2 testing in the United States using nasal swabs via the polymerase chain reaction and more recently antigen testing with rapid tests, such testing can only provide information about recent infection in the past 3-4 weeks. Serology testing, on the other hand, can identify subjects who have been infected or vaccinated and have generated an immune response over many months [2,3,9,10]. Despite the introduction of effective vaccines at the end of 2020, especially mRNA vaccines from Pfizer-BioNTech and Moderna, reported cases and deaths have continued, especially with the emergence of new more transmissible genetic SARS-CoV-2 variants [10], [11], [12], [13], [14], [15], [16]. Neutralization of the newer variants after the primary two-dose regimen of the mRNA vaccines was lower than that of the original virus but increased substantially after a booster dose of the mRNA vaccine [16]. The Center for Disease Control (CDC) is currently recommending updated bivalent boosters that protect against both the original virus that causes SARS-CoV-2 infection and the newer variants [17]. Like the H1N1 virus, SARS-CoV-2 has now become embedded in the world-wide population and will undoubtedly require annual updated booster vaccination.

SARS- CoV-2 is an enveloped single-stranded RNA virus that is considered to possess the largest viral RNA genome of any RNA virus. This genome is covered with a nucleocapsid (N) protein embedded in a phospholipid bilayer. This bilayer also contains the spike glycoprotein (S), hemagglutinin-esterase (HE), membrane protein (M), and envelope provide (E) and provides the virus with a crown-like or corona shape [1,6,7,11]. Following SARS-CoV-2 infection, anti-viral antibodies are produced in response to the spike (S) and nucleocapsid (N) proteins and are commonly measured for serological testing [[1], [2], [3],[9], [10], [11]]. The virus has a receptor binding domain on the tip of the spike protein which binds to the angiotensin converting enzyme receptor-2 on cell surfaces. Once bound to the receptor, the virus enters the cell and causes the cell to rapidly replicate the virus [11]. With regard to antibody response, IgM antibodies levels rise first as an initial T-cell independent humoral response to viral entry usually within 5-7 days of infection [1]. Viral antigen presentation to T cells leads to IgG production within 10-18 days after infection, and these antibodies usually persist for at least 6 months. Neutralizing antibodies are mainly of the IgG class [1,3,[9], [10], [11]].

Once the SARS-CoV-2 sequence was published two companies, BioNTech of Mainz, Germany and Moderna of Cambridge, MA began development of mRNA vaccines [6,7] and it was available from December of 2020. mRNA vaccines teaches our cells to make a protein or even just a piece of a protein that then triggers an immune response. Clinical trials with these vaccines have demonstrated marked efficacy in prevention of infection and hospitalization, as well as induction of antibody response [12], [13], [14], [15], [16]. In the United States there are four vaccines that are currently available and have either received Food and Drug Administration (FDA) approval or emergency use authorization [17,18] though at the time of this study we only had 2 mRNA vaccines and one viral vector vaccine. Viral vector vaccines use a harmless, modified version of a different virus that helps cells recognize and fight SARS-CoV-2. Since the origin of the pandemic, multiple new SARS-CoV-2 variants have emerged that are more resistant to antibodies [19].There are now monovalent and bivalent booster recommendations in the United States [17]. Much remains to be learned regarding the immune response to SARS- CoV-2 and vaccine- induced protective immunity.

Fabricius and colleagues have found that cellular and serological immune responses, including neutralizing capacity against variants of concern, were significantly stronger with mRNA vaccines as compared to levels found in COVID-19-convalescent individuals or vaccinated individuals receiving vector vaccines [20]. Immunizations with mRNA vaccines have been shown to stimulate the production of total and neutralizing antibodies and IFN-γ responses in 100% of vaccinated individuals investigated [20], [21], [22], [23]. Understanding the immunogenic antigens of the virus and the natural immune response is instrumental in developing effective vaccines, however given the challenges in resources, manufacturing and distribution globally, it is important to also study the nature of immune response and duration of immune response following these vaccines so effective and therapeutic strategies can be developed. Now that we have authorized vaccines for COVID-19, it is important to understand how long antibodies circulate, and if age gender and health conditions affect this humoral immunity. It has been reported that COVID-19-neutralizing antibodies predict disease severity and survival [23,24].

The purpose of the present study was to ascertain the prevalence of positive SARS-CoV-2 spike protein antibodies and neutralizing antibodies in the general population in the post-vaccination era, as well as to study the increase in these antibodies about two months after a booster injection of a SARS-CoV-2 vaccine.

2. Methods

2.1. Neutralizing antibody chemiluminescence assay

The SARS-CoV-2 neutralizing antibody assay utilized was obtained from Diazyme Laboratories (catalog number DZ901A, Poway, CA). This assay is a competitive chemiluminescence immunoassay based on the specific interaction between the SARS-CoV-2 spike protein receptor binding domain (RBD) and the human angiotensin-converting enzyme 2 receptor (hACE2) on the surface of host cells. The assay and its validation with a cell-based assay has been previously described [3,25,26]. In the absence of SARS-CoV-2 neutralizing antibodies, hACE2 and RBD form complexes that generate a high chemiluminescent signal (measured in relative light units, RLU). In the presence of SARS-CoV-2 neutralizing antibodies originating from human serum or plasma, the interaction between hACE2 and RBD is compromised; and the chemiluminescent signal is reduced in a dose-dependent manner. The assay has been validated with a cell-based assay as previously described [27]. The assay was documented to have no interfering substances and to be specific for SARS-CoV-2. The assay showed excellent correlation with the cell-based SARS-CoV-2 Reporter Neutralizing Antibody Assay. Serum samples with neutralizing antibody values ≥2·60 AU/mL all showed >98·0% inhibition of viral infection in cell-based assay validation studies [25,26]. In our laboratory this assay was found to have within- and between-run coefficients of variation of <4·0%, with a positive value being ≥1·0 AU/mL and a linear range up to 30 AU/mL [3].

2.2. Anti-SARS-CoV-2 S protein antibody assay

The anti-SARS-CoV-2 S protein antibody electrochemiluminescence immunoassay utilized was obtained from Roche Diagnostics (Indianapolis, IN) as described [[28], [29]]. The assay has received Food and Drug Administration Emergency Use Authorization for the qualitative and semiquantitative detection of antibodies to the SARS‑CoV‑2 S protein RBD in human serum and plasma. The antigens within the assay reagents capture predominantly anti‑SARS‑CoV‑2 IgG (about 92%), but also some anti‑SARS‑CoV‑2 IgA and IgM [29]. The analytical measuring interval was found to be from 0·40 units (U)/mL to 250 U/L, with positive values being ≥0·80 U/L. The negative percent agreement rate was 99·98% using 5991 serum samples obtained prior to October 1^st, 2019. The positive percent agreement rate was 96·6 % utilizing 1485 plasma samples from 331 symptomatic SARS-CoV-2 RNA positive patients [29]. The within and between run coefficients of variation of the assay were found to be <3.0% for positive samples with values ≥0·80 U/mL. In 2022 we also began to dilute samples 1:100 if values were >250 U/L, allowing us to measure values up to 25,000 U/L as recommended by the manufacturer [29].

2.3. Human subjects

During 2021, we measured SARS-CoV-2 neutralizing antibody and SARS-CoV-2 S protein antibody levels in serum from a total of 11,124 subjects (41·8% male, 58·2% female), and of these 25·5% were under age 45 years, 37·8% were 45-65 years of age, and 36·3% were >65 years of age (see Table 1 ). We did a further analysis of antibody positivity rates on a subset of 10,267 of these subjects (see Table 2 ). In the first quarter of 2022 we did a further analysis on serum samples obtained from 2,179 subjects (39·7% male, 60·3% female) and in this latter analysis, all samples that had S protein antibody levels >250 U/L were diluted 1:100 so that values up to 25,000 U/L could be reported. These samples were all submitted by healthcare providers from around the United States for the measurements of SARS-CoV-2 S protein antibody and neutralizing antibody levels. For this analysis, all subjects were anonymized.

Table 1.

Anti-SARS-CoV-2 antibody levels in the general population in 2021 by age groups#

Parameter	<45 years	45-65 years	>65 years	P Value
All (n = 11,124)	n=2,837 (25.5%)	n=4,205 (37.8%)	n=4,039 (36.3%)
Spike protein antibodies, U/L	70.6 (0.40-250.0)	244.0 (0.40-250.0)	250.0 (72.7-250.0) ***
Neutralizing antibodies, AU/mL	1.18 (1.00-1.74)	1.31 (1.0-2.09)	1.45 (1.03-2.26) ***
Men, n = 4,649 (41.8%)	n=1,108 (23.8%)	n=1,710, 36.8%)	n=1,802, 38.8%)
Spike protein antibodies, U/L	67.3 (0.40-250.0)	237.0 (0.50-250.0)	250.0 (68.5-250.0) ***
Neutralizing antibodies, AU/mL	1.15 (1.00-1.74)	1.33 (1.00-2.12)	1.33 (1.00-2.10) ***
Women, n = 6,475 (58.2%)	n=1,729 (26.7%)	n=2,495 (38.5%)	n=2,237 (34.6%)
Spike protein antibodies, U/L	72.5 (0.40-250.0)	245.1 (0.40-250.0)	250.0 (77.5-250.0) ***
Neutralizing antibodies, AU/mL	1.21 (1.00-1.74)	1.32 (1.02-2.08)	1.50 (1.07-2.36) ***

# Median values ± interquartile range, *** P<0.00001 for trend; positive results for spike protein antibody and neutralizing antibody are ≥0.80 U/L and ≥1.0 AU/mL, respectively, while maximal values are 30.0 AU/mL and 250.0 U/L, respectively.

Table 2.

Anti-SARS-CoV-2 levels in the general population in 2021 by age groups

Spike Protein Antibody Category	<0.80 U/L (Negative)	0.80-250.0 U/L (Positive)	>250.0 U/L (Very Positive)	P Value
All, n = 10,267	n=2,562 (25.0%)	n=2,498 (24.3%)	n=5,207 (50.7%)
Spike protein antibodies, U/L	<0.80	76.80 (31.2-153.0)	>250.0
Neutralizing antibodies, AU/mL	<1.00	1.16 (1.00-1.36)	2.08 (1.53-3.75) ***
Men, n = 4,262 (45.3%)	n=1,018 (23.9%)	n=1,088, 25.5%)	n=2,156 (50.6%)
Spike protein antibodies, U/L	<0.80	78.00 (32.1-159.3)	>250.0
Neutralizing antibodies, AU/mL	<1.00	1.13 (1.00-1.34)	2.10 (1.51-4.50) ***
Women (n = 6,005)	n=1,544 (25.7%)	n=1,410 (23.5%)	n=3,051 (50.8%)
Spike protein antibodies, U/L	<0.80	74.5 (30.1-146.8)	>250.0
Neutralizing antibodies, AU/mL	<1.00	1.18 (1.00-1.38)	2.06 (1.56-3.36) ***

# Median values ± interquartile range, *** P<0.00001 for trend

This type of research is exempted from requirement for human institutional review board (IRB) approval as per exemption 4, as listed at https://grants.nih.gov/policy/ humansubjects.htm and at the open education resource (OER) website for research involving human subjects. This exemption “involves the collection or study of data or specimens if publicly available or recorded such that subjects cannot be identified”. We had this designation and our research reviewed by the Advarra Institutional Review Board (Columbia, MD). They determined that this research met the criteria for exemption from institutional review board review under 45 CFR 46.104(d)” and, therefore, ruled that this research did not require IRB approval.

In addition, following protocol approval for pre- and post-booster (60 days) serum serology studies from the Trinity Health of New England Institutional Review Board, we enrolled 69 subjects (mean age 53·3 ± 14.1 years, 78·3% female, 21·7% male) who had received two doses of either the Pfizer/BioNTech SARS-CoV-2 mRNA vaccine (n =32, 46·4%) or the Moderna SARS-CoV-2 mRNA vaccine (n = 34; 49·3%) or one dose of the COVID-19 vaccine Ad26.CPV2.S developed by Johnson & Johnson (n = 3; 4·3%).  Men and women were included if they were ≥18-90 years of age, were previously vaccinated, and were scheduled to receive a booster dose of vaccine.  The booster vaccine could either be the Pfizer/BioNTech or the Moderna mRNA vaccine.  Subjects were not required to receive a booster vaccine that was developed by the same company as their initial vaccine. Men and women who were <18 or >90 years of age, had never received a SARS-CoV-2 vaccine, and/or were not scheduled for a booster vaccine were excluded. Subjects were studied 1-7 days before and a mean (± SD)56 ± 7 days after they received a SARS-CoV-2 booster vaccination. All demographic and laboratory data were de-identified prior to analysis. Information on these latter study subjects in provided in Table 4.

Table 4.

Post-booster vaccination antibody data

Parameter	All Subjects (n = 69)	Males (n = 15)	Females (n = 54)
Age, years (SD)	54.2 (13.8)	54.0 (11.6)	54.3 (14.4)
Sex, n (%) female	54 (78.3%)	ꟷ	ꟷ
Tested COVID-19 positive, n (%)	15 (21.7%)	4 (26.7%)	11 (20.4%)
Pre-Booster (Visit 1), n (%)
Johnson & Johnson	3 (4.3%)	0	3 (5.6%)
Moderna	34 (49.3%)	5 (33.3%)	29 (53.7%)
Pfizer/BioNTech	32 (46.4%)	10 (66.7%)	22 (40.7%)
Post-Booster (Visit 2), n (%)
Moderna	42 (60.9%)	7 (46.6%)	35 (64.8%)
Pfizer/BioNTech	27 (39.1%)	8 (53.3%)	19 (35.2%)
Anti-SARS-CoV-2 immune response*
Spike protein antibodies, U/L
Visit 1	1,010 (504-[2],068)	1,062 (345-2785)	932 (524-1876)
Visit 2	17,236 (11,916-	14,748 (10,005-	18,263 (13,559-
Fold Increase‡	25,000)† +15.4 (8.4-28.3) %	19,604)† +10.0 (6.6-30.5) %	25,000)† +16.1 (8.5-28.2) %
Neutralizing antibodies, AU/mL
Visit 1	1.51 (1.15-2.01)	1.51 (1.19-2.29)	1.49 (1.16-1.95)
Visit 2	12.86 (9.64-24.56)†	12.86 (5.55-17.62)†	13.10 (10.02-24.69)†
Fold Increase‡	+6.5 (3.2-13.0) %	+4.0 (2.8-6.0)	+7.8 (3.4-13.1)

*Data are presented as median (25^th-75^th percentile), All subjects had positive S protein levels at visit 1, and for neutralizing antibody this percentage 82.5%.

^†P <0.00001, for the comparison of visit 2 with visit 1.

^‡Median (25^th-75^th percentile) fold increase between visit 2 and visit 1 was calculated on an individual basis and summarized descriptively by visit.

2.4. Statistical analysis

All statistical analyses were performed using R software, version 3·6 (R Foundation, Vienna, Austria). Categorical variables were expressed as frequencies and percentages, while continuous variables were expressed as median values with interquartile ranges (IQR, 25^th–75^th percentile values). The statistical significance of differences between groups were assessed using non-parametric Kruskal-Wallis analysis. Spearman correlation analyses were performed to assess interrelations of biochemical variables.

3. Results

3.1. Antibody levels in the population

As shown in Table 1, in 11,124 subjects sampled in 2021, levels of both S protein and neutralizing antibody increased significantly with age, and men and women had similar values. As shown in Table 2 in a subset of 10,267 of these subjects, 25·0% had negative S protein antibody levels of <1.00 U/L while 24.3% had positive values ranging from 1·0-250·0 U/L, and 50·7% had very positive values of >250·0 U/L. These data indicated an overall positive S protein antibody rate of 75·0% during 2021. Subjects with very positive S protein values also had significantly higher neutralizing antibody levels than subjects with only positive values.

Beginning in 2022 we started diluted all samples with S protein antibody values >250 U/L so that values up to 25,000 U/L could be reported. As shown in Table 3 , The median S protein antibody values was 2,065 U/L, while for neutralizing antibodies this value was 2·68 AU/L. Median values in women were significantly lower (P<0.001) than values in men. Moreover, the correlations between spike protein and neutralizing antibody levels were both highly significant (P<0.0001) at 0·75 in men and 0·76 in women. In this second study population, the percentages of all subjects (n=2,179) with positive values for S protein antibody (>0.80 U/L) for all subjects, men, and women were 86·3%, 85·0%, and 85·6%, respectively. For neutralizing antibody, the percentages of positive subjects (values >1.0 AU/mL) for all subjects, men, and women were 68·0%, 62·6%, and 68·2%, respectively.

Table 3.

Anti-SARS-CoV-2 immune response in general population in 2022

Parameter	All Subjects (n = 2,179)	Men (n = 866, 39.7%)	Women (n = 1,173, 60.3%)
Anti-SARS-CoV-2 immune response
Spike protein antibodies, U/L	2,065 (153-13,033)	2,194 (129-13,662)	1,710 (130-11,338)**
Neutralizing antibodies, AU/mL	2.68 (1.35-12.68)	2.80 (1.33-15.12)	2.52 (1.33-11.53)**

# Median values ± interquartile range (IQR). The median age and IQR was 61.0 (49.0-72.0) years. Spearman correlations between spike protein and neutralizing antibody levels were 0.75 in men and 0.76 in women, respectively, p<0.0001. The percentage of all subjects, men, and women with positive values for S protein antibody (>0.80 U/L) were 86.3%, 85.0%, and 85.6%, while for neutralizing antibody (>1.0 AU/mL) these percentages were 68.0%, 62.6%, and 68.2%. All samples with S protein antibody values ≥250 U/L were diluted 1:100 so that values of up to 25,000 U/L could be reported. **Median values in women were significantly lower (P<0.001) than values in men.

3.2. Antibody levels after booster vaccination

As part of an approved protocol, as shown in Table 4 , 69 subjects (15 men, 54 women, mean age 55, 84% Caucasian, 12% Asian, and 4% African American) had their antibody levels measured before and approximately 60 days after booster vaccination. For initial vaccination, only three of the 69 subjects received the Johnson & Johnson viral vector vaccine, while the other 66 subjects received either Moderna or Pfizer mRNA vaccines. The mean number of days between vaccination and booster vaccination was 271 days, while the mean number of days between being boosted and final antibody measurement was 58 days.

Prior to the booster vaccination, the median protein antibody level was 1,010 U/ml (range 123 - 25,000 U/L), while for neutralizing antibody, the median level was 1·51 AU/ml (range 0.99 – 16·86 AU/L). Median pre-booster S protein antibody levels in those who received the Johnson & Johnson, Moderna, and Pfizer vaccines were 261, 2,893, and 1,287 U/L, respectively, while for neutralizing antibody levels, these values were 1.057, 3·20, and 1·52 AU/ml, respectively. antibodies were measured about 60 days post booster vaccination and there was a remarkable increase in the antibody levels. Median values for S protein antibody were 17,236.00 U/ml (range 3,673 – 25,000) and for neutralizing antibody these values were 12·86 AU/ml (3·56 -29·6). The S protein antibodies in the 15 males went from 1,062 U/ml (345-2785) to 14,748 U/ml (10,004·5-19604) and in the 54 females went from 932 U/ml (548 - >25,000) to 18,856 U/ml (14,329 – 25,000). For neutralizing antibodies, median values in males went from 1·51 AU/ml (1·19 – 2·285) to 10·01 AU/ml (5·545 – 19·505), while in females values increased from 1·51 AU/ml (1·19 – 1·97) to 14·42 AU/ml (10·53 – 24·56) respectively. There was a marked variability in the increases in both S protein antibody and neutralizing antibody about 60 days after a booster vaccination as shown in Fig. 1 . For S protein antibody the median increase was 15·4- fold (range 0·1-153·1), while for neutralizing antibody the median increase was 6·5-fold (range 0·83-28·7). In our study, we did note that antibody levels were higher in those that had both been vaccinated and had been positive for SARS-CoV-2 (n=15) as compared to those that had never been positive for the virus (n=54).

Fig. 1.

SARS-CoV-2 antibody response to a booster dose of a mRNA SARS-CoV-2 vaccine (n = 66). Panel A, Spike protein antibodies. Panel B, Neutralizing antibodies. Graphs show the fold difference between the serum antibody concentration (U/mL) 1-7 days before (visit 1) and 56±7 days after (visit 2) receiving a booster dose of either the Moderna or the Pfizer/BioNTech mRNA SARS-CoV-2 vaccine. The response Spike antibody response ranged from a 0.10- to 153.07-fold difference; the neutralizing antibody response from a 0.83- to 28.74-fold difference.

4. Discussion

Population surveys of seropositivity for SARS-CoV-2 have been somewhat limited in the United States. Havers and colleagues from the Centers for Disease Control reported a 5% positivity rate at 10 sites across the states in 2020 involving about 15,000 subjects [29]. We reported a similar rate over the 2020 year in over 80,000 subjects [3]. Lim and colleagues from the Centers for Disease Control published a follow-up study in late 2020 indicating a SARS-CoV-2 seropositivity rate of 23·0% in the New York City area (the highest) and 13·3% in parts of Florida, with lower rates in other locations [30]. In the data reported here we noted a 75% SARS-CoV-2 seropositivity rate for S protein antibody in 2021 in the post-vaccination era, and an 86% positivity rate in 2022. We also noted that S protein antibody levels were highly correlated with neutralizing antibody levels, but that the positivity rate for neutralizing antibody was always lower that for S protein being 68% in 2022. Moreover, in this study we used a commercially available assay that received FDA EUA for semiquantitative results. The value of this type of analysis has been documented, but only limited population data is available in the United States with regard to neutralizing antibody [31], [32], [33].

New viral variants are emerging, causing the virus to become more contagious and the new omicron variant suggests a level of immune escape leading to reinfection. On October 20^th, 2021, the FDA provided EUA for the use of a booster dose for COVID-19 vaccines in eligible populations [34]. Antibody tests can be used to assess immune response to infections and or vaccinations. SARS-CoV-2 vaccines are effective and elicit antibodies to the virus and variants in a manner that resembles natural infection [12], [13], [14], [15], [16]. Earle et al defined a correlate of protection (CoP) to assess whether antibody titers may reasonably predict efficacy using vitro neutralizing and binding antibodies of 7 vaccines, and they found a robust correlation between efficacy and neutralizing antibody titers and binding antibody titers [23]. Wheeler et al reported antibody response to four different SARS-CoV-2 antigens after first and second dose of Pfizer and Moderna mRNA vaccines [21]. Even though there were some non-responders following the first dose of vaccine there was a peak of antibody induction after the second vaccine dose with a gradual decline of antibody levels over time.

Findings have shown robust antibody response following vaccination and have found that after a single dose of COVID-19 vaccine, people with a prior COVID-19 infection (convalescent) had antibody levels similar to those without prior infection after two vaccine doses [35]. However natural immunity tends to lose its effectiveness in about 90 days [36]. Immunity and efficacy from COVID-19 vaccines and booster has been shown to last longer [37]. Since there have been several new variants of the virus, Rossler et al found that serum samples of vaccinated persons neutralized the omicron variant to a lesser extent than prior variants (alpha, beta or delta) [38]. They also found that unvaccinated convalescent subjects did not neutralize the omicron variant, although they did have cross-neutralization with other variants [39,40]. Even though studies have shown that binding and neutralizing antibodies have been shown to persist for at least 6 months post the second dose of vaccination, current recommendations are to obtain a COVID-19 booster even if you had prior infection with SARS-CoV-2 [12], [13], [14], [15], [16].

In our post-vaccination study, we measured both SARS-CoV-2 S protein antibody and neutralizing antibody just prior to the booster followed by repeat measurements about 60 days post-booster vaccination. The mean number of days between completion of the first set of vaccine/vaccine and the booster vaccine was 271 days, while the mean number of days between the booster vaccination and the second blood draw was 58 days. The major goal of most vaccines is the induction of neutralizing antibodies which in turn helps to reduce disease severity [24].

In our study, about 9 months after receiving one of the three vaccine types, all subjects still had some sustained immune response with regard to both S protein antibodies and neutralizing antibodies with the highest levels being observed for the Moderna vaccine and the lowest levels for the Johnson & Johnson vaccine. All subjects received either the Moderna or the Pfizer/BioNTech mRNA vaccines as a booster vaccination. shot. Approximately 8 weeks post booster vaccination for S protein antibody the median increase was 15·4-fold (range 0·1-153·1), while for neutralizing antibody the median increase was 6·5-fold (range 0·83-28·7), indicating a marked variability in response. A limitation of our post-vaccination study is the small sample size and the fact that we did not assess cellular immunity.

Following a study by Bar-On and colleagues which showed that the rate of infection was lower by a factor of 2 following the fourth dose of a SARS-CoV-2 mRNA vaccine, on March 29^th 2022, the FDA authorized a second booster dose of either of the mRNA vaccines for older people (≥65 years of age) and certain immunocompromised individuals [41]. Even though there is evidence of waning antibody titers after any vaccination, this may not translate into to waning cellular immunity. Studies have found that neutralizing antibody titers were similar to those in individuals post booster compared to the convalescent vaccinated. Studies have also shown neutralizing activity against omicron variant is most impacted in unvaccinated, convalescent individuals and in naive individuals who acquired immunity through two mRNA COVID-19 vaccine doses [35], [36], [37], [38], [39], [40]. Our data aligns with prior studies documenting the efficacy of mRNA booster vaccines to stimulate an excellent immune response [12], [13], [14], [15], [16]. Given the sustained immune response, but the lack of clarity about the duration, postvaccination testing of antibody responses can be a vital and practical approach for following vaccinated people and or for selecting individuals who need additional boosting because of low responsiveness. Understanding the longevity of neutralizing antibodies in natural versus vaccine induced immunity is very important to the success of vaccination efforts and booster strategies. Our study focused on antibody kinetics before and after the first booster and it will be interesting to study longitudinal dynamics of neutralizing antibodies following the original booster vaccine in convalescent and naïve vaccinated individuals compared to unvaccinated convalescent plasma donors.

Conclusion

Our population studies have now documented that 86% were positive for S protein antibodies and 68% for neutralization in 2022. Even though studies have shown a gradual decline of antibodies over time, our data indicate that all vaccinated subjects had positive levels of S protein antibody and the neutralizing antibody 9 months or more following their initial vaccination. There was a significant increase in both the spike (S) protein antibodies and the neutralizing antibodies following the mRNA booster vaccination, irrespective of age or gender, with a marked variability in response. Our data indicate that serology is a valuable adjunct for assessing SARS-CoV-2 population immunity as well as post vaccination immunity, especially in the era of SARS-CoV-2 variants. To evaluate duration and efficacy of boosters, we need more studies to chart the course of antibodies and if the neutralizing antibodies are effective against the newer variants. This will help determine if boosters are required every 6 months or annually or if we need to develop new variant adapted vaccines.

Authors’ contributions

LD, EJS were instrumental in designing the protocol

LD, EJS, contributed to the literature search.

LD, LC, LD, JP were involved in the IRB approval process, securing informed consent, following study process and data collection.

LC, JP, LD were instrumental in specimen & data collection.

EJS, MRD, ML, LH were responsible for serum antibody level measurement, data collation, and data interpretation.

All authors participated in the review and editing of the manuscript.

Funding sources

This study was supported by funds from Trinity Health Of New England, a not-for-profit healthcare organization and Boston Heart Diagnostics.

Competing interests

The authors declare that they have no competing interests.

Declaration of Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.Latha Dulipsingh