Demonstration of Antibodies Against SARS-CoV-2,Neutralizing or Binding,in Seroconversion Panels After mRNA-1273,BNT-162b2,and Ad26.COV2.S Vaccine Administration

Abstract

Vaccines are an important tool in combating the COVID-19 pandemic. Two mRNA vaccines (mRNA-1273 and BNT-162b2) and an adenovirus vector vaccine (Ad26.COV2.S) were among the first vaccines to be approved by global regulatory authorities. The aim of this observational study was to characterize the levels and time course of the generation of anti-SARS-CoV-2 spike protein antibodies after vaccination with 3 different vaccines and the neutralizing activity of these antibodies. Seroconversion panels were generated from blood samples collected before and after vaccination with 3 COVID-19 vaccines: mRNA-1273, BNT-162b2, and Ad26.COV2.S. The seroconversion panels were tested for antibody activity by chemiluminescent immunoassay or enzyme-linked immunosorbent assay (ELISA), and 1 panel was tested for neutralization activity in a pseudovirus assay. Participants positive for anti-SARS-CoV-2 antibodies before vaccination (18.6%) had a higher response to the first dose than participants who tested negative. For 2-dose vaccines, older participants showed a lower response to the first dose than younger participants. All participants showed positive responses after the second vaccine. For the adenovirus vector vaccine, 2 participants did not generate antibody responses after vaccination. Four participants were negative at 2 weeks but positive at 2 months. Pseudovirus neutralization showed good correlation with antibody activity (correlation coefficient = 0.78, P < .0001). Antibody responses in participants over 45 years old tended to be less robust. Participants that had been infected with SARS-CoV-2 and had antibodies prior to vaccination showed a more robust response to initial vaccination. Older participants (>45 years) showed less robust responses to both types of vaccine. All participants receiving full mRNA vaccination showed positive antibody responses. Some participants receiving the adenovirus vaccine did not respond. Antibody responses correlated well with neutralization activity. Seroconversion panels can be useful in the development of antibody assays and in investigating their effectiveness against new SARS-CoV-2 variants.

Introduction

Since the start of the SARS-CoV-2 pandemic, nearly 30 pharmaceutical companies, research institutes, and academic laboratories worldwide have been working toward the development of effective vaccines against COVID-19. These efforts have taken various approaches including mRNA, DNA, protein subunits, and attenuated or nonreplicating viruses. ¹ Among the vaccines utilizing mRNA and nanotechnology, mRNA-1273 (Spikevax, Moderna) and BNT-162b2 (Comirnaty, Pfizer/BioNTech) were the first approved in the United States.^2,3

Within 1 year of the emergence of the SARS-CoV-2 virus, these 2 novel and effective mRNA vaccines became available under emergency use authorization (EUA) by the US Food and Drug Administration (FDA). The BNT162b2 vaccine contains 30 μg of SARS-CoV-2 full-length spike (prefusion conformation) mRNA administered as 2 doses 3 weeks apart. An additional dose has been added as part of the primary vaccination series in immunocompromised individuals.^4,5 The mRNA-1273 vaccine 100 μg of prefusion-stabilized spike glycoprotein mRNA was given as 2 doses 4 weeks apart.^5,6

The BNT-162b2 vaccine was first utilized under an EUA beginning December 11, 2020, ⁷ and became the first COVID-19 vaccine approved by the FDA on August 23, 2021. ² It was originally approved for the prevention of COVID-19 in individuals 16 years of age and older but is now approved for those 12 years and older. In addition, the BNT-162b2 vaccine is available for use under an EUA for children ages 6 months to 11 years and as 2 booster doses in immunocompromised individuals, people over 65, individuals between 16 and 64 who are at high risk of severe COVID-19, and person with high environmental exposure to SARS-CoV-2, which puts them at risk for severe COVID-19. ⁸

The FDA issued an EUA for the mRNA-1273 vaccine for the prevention of COVID-19 in individuals over 18 years of age on December 18, 2020. As with the EUA for BNT-162b2, the EUA for mRNA-1273 was amended in August 2021 to allow the administration of an additional dose to certain immunocompromised individuals. ⁹ The mRNA vaccine was approved for those 18 years or older on January 31, 2022. ³

The Ad26.COV2.S vaccine (Janssen, Johnson & Johnson) uses a recombinant, replication-incompetent human adenovirus type 26 vector. This vector encodes for a full SARS-CoV-2 spike protein in a stabilized prefusion conformation. A single dose of Ad26.COV2.S protected vaccinated individuals against symptomatic and asymptomatic SARS-CoV-2 infection. For those who did get infected, the vaccine was effective in preventing severe disease including hospitalization and death. ¹⁰ The Ad26COV2.S vaccine was authorized under an EUA in February 2021. The EUA was amended to allow for a booster dose of the vaccine in October 2021. ¹¹

Infection with SARS-CoV-2 or vaccination with an effective vaccine initiates an immune response, which includes the production of antibodies that bind to viral proteins, ie, binding antibodies. ¹² Not all binding antibodies can block cellular entry and/or replication by the SARS-CoV-2 virus. The subpopulation of antibodies that can block these processes is called neutralizing antibodies (NAbs). It is unknown when in the course of infection or after vaccination NAbs are produced or if they are produced from the onset of antibody formation. While most infected or vaccinated individuals produce binding antibodies in response to SARS-CoV-2 infection, not all will develop NAbs to SARS-CoV-2. ¹³

Most SARS-CoV-2 infections induce a response in the adaptive immune system. ¹⁴ NAbs that prevent the virus from binding to the host cell receptor, angiotensin-converting enzyme 2 (ACE2), are essential for this immune response. NAbs target the receptor-binding domain (RBD) of the viral spike protein and thereby inhibit ACE2 binding.^15,16 These NAbs may be of therapeutic value through treatments such as convalescent plasma ¹⁷ and hyperimmune globulins. ¹⁸ In addition, they can serve as structural templates for informing vaccine development. Consequently, there is a need for assays that reliably and efficiently identify the most potent NAbs against ancestral and major variant strains of SARS-CoV-2. The gold standard to detect NAbs is a virus neutralization test conducted on live cells, which can be performed with live virus or pseudoviruses. ¹⁹ The level of NAbs that confers immunity to infection/reinfection with SARS-CoV-2 has yet to be elucidated. ²⁰

Seroconversion panels are a series of blood samples collected before and after the development of antibodies in response to viral infection or vaccination. The observational studies in this paper describe the characterization of seroconversion panels from donors vaccinated for COVID-19 with the 3 vaccines available in the United States at the time the samples were collected: BNT-162b2, mRNA-1273, and Ad26.COV2.S.

Materials and Methods

Seroconversion Panel Collection

The seroconversion panels described in this paper were derived from samples collected at a hospital in Tennessee (USA) from volunteer donors. The donors provided informed consent, and their samples were collected under an approved Institutional Review Board (IRB) protocol ([1149706-4] Diagnostic QC and Pre-Clinical Sample Collection Project: Ballad Health System Institutional Review Board [IRB #00003204], Johnson City, TN, USA). These studies were conducted in compliance with all applicable regulatory guidelines and published in accordance with STROBE guidelines. ²¹ The seroconversion panels were comprised of serum samples collected before and after administration of the COVID-19 vaccines. This study characterizes the appearance of anti-SARS-CoV-2 antibodies after vaccine administration. In compliance with US Centers for Disease Control and Prevention (CDC) guidelines, participants were required to be symptom-free at the time of vaccination. In addition, as recommended by the CDC, participants were questioned about having a positive COVID-19 test or exposure to a person with COVID-19 in the last 14 days. Participants who answered positively to either of these questions were not vaccinated at that time or included in the study.

Four seroconversion panels were analyzed in this study: 1 mRNA-1273 (Spikevax™, Moderna, Cambridge, MA, USA), n = 15 donors; 2 BNT-162b2 (Comirnaty®, BioNTech, Mainz, Germany/Pfizer, New York, NY, USA), n = 15 donors for each panel; and 1 Ad26.COV2.S (Janssen/Johnson & Johnson, Beerse, Belgium), n = 14 donors. For mRNA-based vaccine (mRNA-1273 and BNT-162b2) panels, samples (20 mL) were collected prior to the first vaccination (objective target ≤ 2 days: sample 1), prior to the second vaccination (objective target ≤ 2 days: sample 2), and after the second vaccination (objective target 13-15 days; sample 3). For Ad26.COV2.S, samples (20 mL) were collected on vaccination day (prior to the injection: sample 1), 2 weeks after vaccination (14 days: sample 2), and 2 months after vaccination (59-62 days: sample 3). Samples were collected as plasma (in the presence of potassium ethylenediaminetetraacetic acid (EDTA) and/or serum (in serum-separating tubes).

As previously described, ²² the mRNA-1273 panel was made up of undiluted, unpreserved serum samples collected from 15 participants between December 22, 2020, and February 25, 2021. The donors were healthy adults (21-76 years old) who received the prescribed course of mRNA-1273 vaccines (2 injections of 100 µg—objective target 28 days apart). There were 10 female and 5 male donors, and all were white/Caucasian.

The 2 BNT-162b2 panels were collected at different times. The first panel (BNT-162b2 panel 1) was collected between February 19, 2021, and April 23, 2021. The second panel (BNT-162b2 panel 2) was collected between May 25, 2021, and September 24, 2021. The BNT-162b2 panels were either undiluted, unpreserved serum or EDTA-treated plasma collected from 15 donors each. These donors were healthy adults 36 to 70 years old for the first panel and 29 to 67 years old for the second panel. The participants for both panels received the approved vaccination regimen (2 injections of BNT-162b2 30 µg—objective target 21 days apart). There were 7 female and 8 male donors in panel 1 and 9 female and 6 male donors in panel 2. All the donors were white/Caucasian.

The Ad26.COV2.S panel was undiluted, unpreserved serum collected from 14 donors between March 7, 2021, and September 24, 2021. These donors were between 31 and 64 years of age. There were equal numbers (n = 7) of female and male donors, and all were white/Caucasian. These donors received the approved regimen of a single injection (0.5 mL) of the Ad26.COV2.S vaccine.

All samples were stored at -20 °C until analyzed. Prior to analysis, samples were thawed at room temperature and mixed by inversion. The seroconversion panels used in this study are commercially available as sets of 42 or 45 × 1 mL frozen samples (14 or 15 vials [1 mL] × 3 collection points; Access Biologicals, Vista, CA, USA).

Chemiluminescent Immunoassay and Enzyme-Linked Immunosorbent Assay

The samples in the seroconversion panels were tested for the presence of anti-SARS-CoV-2 antibodies using chemiluminescent immunoassays (CLIA: Liaison SARS-CoV-2 IgG assay, Diasorin, Inc., Saluggia, Italy; EUA approved) and enzyme-linked immunosorbent assays (ELISA: Progenika anti-SARS-CoV-2 IgG Kit, Progenika Biopharma, Derio, Bizkaia, Spain; CE-IVD-certified immunoassay). These assays were performed following the directions provided by the manufacturers. The Diasorin assay utilizes antigens to the S1 and S2 spike protein IgG while the Progenika assay utilizes only antigens to S1 IgG. The specific sequence antigens are not disclosed. Since the assays were developed independently, they are assumed to be nonidentical.

Manufacturer data showed the CLIA had 98.5% specificity (95% confidence interval (CI) 97.5%-99.2%) in samples from 1000 blood donors. Concordance of the CLIA with a plaque reduction neutralization was 97.8% (95% CI 94.4%-99.1%) on negative samples and 94.4% on positive samples (95% CI 88.8%-97.2%). ²³ For the ELISA, the manufacturer showed the negative percent agreement was 99% (95% CI 97%-99%) in 480 serum samples from individual donors, and the positive percent agreement was 97% (95% CI 90%-99%) for 65 samples from patients with confirmed SARS-CoV-2 (by reverse transcription - polymerase chain reaction (RT-PCR) collected 15 or more days after symptoms onset. ²⁴

Neutralizing Antibody Determination

Cell Line

HEK293T cells (presumably of female origin) overexpressing wild-type human ACE-2 (Integral Molecular, USA) were used as target for SARS-CoV-2 spike expressing pseudovirus infection. Cells were maintained in T75 flasks with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1 μg/mL puromycin.

Pseudovirus Generation

Pseudoviruses (human immunodeficiency virus (HIV) reporter) expressing the SARS-CoV-2 S protein and luciferase were constructed using the defective HIV plasmid, pNL4-3.Luc.R-.E-. The plasmid was obtained from the NIH AIDS Reagent Program. ²⁵ Expi293F cells were transfected using ExpiFectamine293 Reagent (Thermo Fisher Scientific, Waltham, MA, USA) with pNL4-3.Luc.R-.E- and SARS-CoV-2.SctΔ19 (Wuhan, G614 or B.1.1.7), at an 8:1 ratio, respectively. Control pseudoviruses were generated by replacing the S protein expression plasmid with a vesicular stomatitis virus (VSV)-G protein expression plasmid as previously reported. ²⁶ Supernatants were harvested 48 hours after transfection, filtered at 0.45 μm, frozen, and titrated on HEK293T cells overexpressing wild-type human ACE-2.

Neutralization Assays

Neutralization assays were performed in duplicate. Briefly, 200× the median tissue culture infectious dose (TCID50) of pseudovirus was preincubated with 3-fold serial dilutions (1/60-1/14,580) of heat-inactivated plasma samples in Nunc 96-well cell culture plates (Thermo Fisher Scientific) for 1 hour at 37 °C. Then 2 × 10⁴ HEK293T/hACE2 cells treated with diethylaminoethyl (DEAE)-Dextran (Sigma-Aldrich, St. Louis, MO, USA) were added. Results were read after 48 hours using an EnSight Multimode Plate Reader and BriteLite Plus Luciferase reagent (Perkin Elmer, Waltham, MA, USA).

The results were normalized, and the ID50 (the reciprocal dilution inhibiting 50% of the infection) was calculated by plotting the log of plasma dilution versus response and fitting to a 4-parameter equation in Prism 8.4.3 (GraphPad Software, San Diego, CA, USA). This neutralization assay has been previously validated in a large subset of samples. ¹²

Statistical Analyses

A kappa correlation test (JMP software 16.0, Cary, NC, USA) was conducted to compare qualitative values between CLIA and ELISA assays. Furthermore, a Pearson correlation test (JMP software) was utilized to compare quantitative values between the neutralization and ELISA assays.

In order to assess the normality of data distribution, both the Shapiro–Wilk test and the Anderson–Darling test were employed. These tests were executed using JMP software 16.0, revealing a nonnormal distribution of the data (results not displayed).

For the purpose of comparing antibody responses among seroconversion panels and differentiating between groups of naïve and convalescent participants, quantitative variables were subjected to Mann–Whitney and Kruskal–Wallis tests (rank sums) due to the nonnormal distribution of the data. Kruskal–Wallis tests were performed for comparisons involving multiple groups (eg, mRNA-1273 vs BNT162b2 vs Ad26.COV2.S panels), while the Mann–Whitney test was chosen for comparisons between 2 groups (eg, naïve vs convalescent participants) (JMP software 16.0).

Results

mRNA-1273 (Moderna) Seroconversion Panel

As described in the previously published study, ²² the prevaccination samples in the mRNA-1273 panel showed positive values for 2 participants suggesting that they had been previously infected with SARS-CoV-2 (participants 5 and 15). The postvaccination samples collected from these participants after the first dose showed the largest antibody responses in the panel. After the first dose of mRNA-1273, samples from 13 of the 15 participants showed positive antibody response by CLIA and 14 of 15 by ELISA. The 2 participants who tested negative after the first dose of vaccine were the oldest participants in the panel (>70 years old: participants 3 and 4). After the second dose, samples for all 15 of the participants were positive for anti-SARS-CoV-2 antibodies (in both assays). Overall, there was good agreement between the results of the CLIA and ELISA assays (kappa coefficient = 0.947). The value of the equivocal range was eliminated to perform this statistical test.

In this study, the postvaccination second dose samples from the mRNA-1273 panel were tested for neutralizing antibodies. The samples from all participants were positive for neutralizing antibodies (Figure 1). When the NAb results were compared with the ELISA antibody detection results, a positive correlation was observed between both analyses (correlation coefficient = 0.78 and P < .0001).

Figure 1.

Scatter diagram illustrating the comparison between the neutralizing assay and ELISA for measuring antibody titers in 15 samples collected from participants who received 2 doses of the mRNA-1273 SARS-CoV-2 vaccine. The results of neutralizing antibody titers are expressed as IC50 reciprocal dilution values, while binding antibody (ELISA) titers are expressed as S/CO. Responses with IC50 reciprocal dilution ≥ 250 and S/CO ≥ 1.1 were considered positive. Pearson's correlation coefficient (r²) was determined to be 0.78 (P < .0001). Abbreviations: ELISA, enzyme-linked immunosorbent assay; S/CO, signal-to-cutoff values.

BNT-162b2 (Pfizer/BioNTech) Seroconversion Panels

The results of the 2 BNT-162b2 panels collected at different times are presented together for analysis purposes (Figure 2). CLIA testing of the prevaccination samples in the BNT-162b2 panels gave the following results (Figure 2; first point): 22 negative and 8 positive tests for S protein antibodies (participants 2, 5, 15, 19, 23, 24, 25, and 30). The detection of anti-SARS-CoV-2 antibodies in these 8 participants indicates that they were infected with the virus prior to collection of the samples.

Figure 2.

Antibody responses for 2 seroconversion panels (results presented together) of 30 participants (P1-P30) before and after vaccination with 2 doses of the BNT162b2 SARS-CoV-2 vaccine (Pfizer/BioNTech). These results were obtained using a chemiluminescent immunoassay and are expressed as arbitrary units per milliliter (AU/mL). Responses ≥ 15.0 units were considered positive. The first point shows results from samples collected prior to the first vaccination. The second point shows the results from samples collected after the first vaccination and prior to the second vaccination. The third point shows the results from samples collected after the second vaccination. The dashed lines correspond to the convalescent participants, while the solid lines correspond to the naïve participants.

Samples collected after the first vaccine injection showed 27 positive and 3 negative results (Figure 2; second point). The largest antibody responses were detected in the 8 participants (2, 5, 15, 19, 23, 24, 25, and 30) who had detectable antibodies in their prevaccination samples. The sample from participant 12 also showed a high antibody response to the first vaccine injection. The prevaccination sample from this participant was close to the cutoff value for positivity. Three participants had negative sample antibody responses after the first vaccination (participants 1, 22, and 27). These participants were 59, 46, and 29 years old, respectively.

Samples collected after the second vaccine injection were positive in all 30 participants (Figure 2; third point). The lowest activity was seen in participant 27 (29 years old) 16 arbitrary units (AU)/mL just above the cutoff value of 15 AU/mL. This participant (27) was 1 of the 3 participants that tested negative after the first vaccination. Lower antibody responses were seen in other 7 participants (3, 4, 7, 14, 16, 20, and 22) compared to the rest of the participants. These participants were between 46 and 76 years old. The sample from participant 7 (60 years old) showed a lower level of antibody activity after the second vaccine dose than that seen after the first vaccine dose.

Ad26.COV2.S (Janssen, Johnson & Johnson) Seroconversion Panel

Testing of the prevaccination samples for the Ad26.COV2.S panel showed 13 negative and 1 positive anti-SARS-CoV-2 antibody responses (Figure 3; first point). The 1 positive sample indicates a previous SARS-CoV-2 infection in this participant (11) prior to the collection of the sample.

Figure 3.

Antibody responses in a seroconversion panel of 14 participants (P1-P14) before vaccination with 1 dose of the Ad26.CoV2.S SARS-CoV-2 vaccine (Janssen, Johnson & Johnson). These results were obtained using a chemiluminescent immunoassay is expressed as arbitrary units per milliliter (AU/mL). Responses ≥ 15.0 units were considered positive. The first point shows results from samples collected prior the vaccination. The second point shows the results from samples collected 2 weeks after the vaccination. The third point shows the results from samples collected 2 months after vaccination. The dashed lines correspond to the convalescent participants, while the solid lines correspond to the naïve participants.

The samples collected 2 weeks after the single-dose vaccination showed 8 positive and 6 negative antibody responses (Figure 3; second point). Samples from 12 participants showed positive responses 2 months after vaccination (Figure 3; third point). Two participants (3 and 6) showed negative antibody responses after 2 weeks and 2 months. These participants were 40 and 61 years old. The other 4 participants that showed a negative antibody response at 2 weeks but a positive response at 2 months were 31, 44, 61, and 64 years old.

Comparison of Antibody Response Between Seroconversion Panels

The data are analyzed when participants were grouped by the vaccine administered: mRNA-1273, BNT-162b2, or Ad26.COV2.S (Table 1). The prevaccine and postfirst vaccination samples showed no significant difference between the groups (P = .2018 and P = .2243, respectively). Immunological responses after the second vaccination for the mRNA-1273 and BNT-162b2 vaccines and the third sample for Ad26.COV2.S vaccine evaluated were significantly different between the groups (P = .0014); antibody responses in the Ad26.COV2.S panel are lower than those for mRNA-1273 and BNT-162b2 vaccines.

Table 1.

Pre- and postvaccination antibody activity after 3 anti-SARS-CoV-2 vaccines.

	Prevaccination (AU/mL)	Postvaccination 1 (AU/mL)	Postvaccination 2 (AU/mL)
mRNA-1273 (n = 15)
Mean	32.01	169.22	367.73
Median	1.00	172.00	400.00
SD	102.92	125.97	79.12
BNT-162b2 (n = 30)
Mean	28.46	174.58	334.30
Median	2.40	92.95	400.00
SD	52.74	166.27	118.96
Ad26.COV2.S (n = 14)
Mean	4.39	130.79	183.63
Median	2.42	31.35	126.25
SD	4.79	177.94	164.80
P value a	.2018	.2243	.0014

The lower and upper limits of detection for this assay were 3.8 and 400 AU/mL. Values above 400 AU/mL were recorded as 400 AU/mL. Prevaccination samples were collected for all 3 vaccine regimens. Postvaccination sample 1 was collected after the first of 2 vaccine doses and just prior to the second dose for the mRNA-1273 (target 28 days apart) and BNT-162b2 (target 21 days apart) vaccines and 2 weeks after the single dose of the Ad26.COV2.2 vaccine. Postvaccination sample 2 was collected after the second dose of the mRNA-1273 and BNT-162b2 vaccines (target 13-15 days) and 2 months after the single dose of Ad26.COV2.2.

Abbreviation: AU, arbitrary units calculated according the manufacturer's instructions.

^a

P values from Kruskal–Wallis Tests (rank sums).

Comparison of Antibody Response Between Naïve Participants Versus COVID-19 Convalescent Participants

The data were also analyzed by grouping the participants into naïve (negative prevaccination samples) and COVID-19 convalescent groups (positive prevaccination samples). Values greater than 15.0 (AU/mL) were considered positive. Values greater than the assay range (up to 400 AU/mL) were recorded as 400 AU/mL due to the upper limit of detection.

The prevaccination samples showed a significant difference between the groups (mean ± SD: naïve < 3.8 AU/mL vs convalescent 105.65 ± 110.55 AU/mL; P < .0001). Samples collected after the first vaccination showed significantly higher IgG levels (365.17 ± 85.95 AU/mL vs 111.16 ± 128.25 AU/mL; P < .0001) in convalescent participants compared to naïve participants. Immunological responses after the second vaccination for the mRNA-1273 and BNT-162b2 vaccines and the third sample for Ad26.COV2.S vaccine evaluated were also significantly different between the groups (283.32 ± 148.3 AU/mL in naïve participants and >400 AU/mL in convalescent participants; P = .006).

Discussion

Four seroconversion panels have been created by collecting serum or plasma samples from participants before and after COVID-19 vaccination. Each panel is comprised of 3 serial samples from approximately 15 vaccine recipients. Three of the panels were drawn from participants who were administered 1 of the 2-dose mRNA-based vaccines (mRNA-1273 [1 panel] and BNT-162b2 [2 panels]) and 1 panel from participants given a single dose of the replication-incompetent adenovirus-based vaccine (Ad26.COV2.S). These panels were tested using CLIA and ELISA technologies. Comparison of the CLIA and ELISA results for the mRNA-1273 panel showed good agreement between these assays. ²²

In addition, neutralization assays were conducted in this study and showed congruence between the neutralization assay and the CLIA. Neutralizing activity that blocks viral entry into cell has been shown to be responsible for antibody-mediated prevention of coronavirus infection. ²⁷ Neutralizing anti-SARS-CoV-2 antibodies have been shown to mainly target the RBD on the S1 region of the spike protein. ²⁸ When immunoassay results correlate with neutralization assays, as in this study, the simpler, less labor-intensive immunoassays may be a useful initial indirect screen for antibody neutralization activity. A similar correlation between immunoassay and neutralization results has been seen in other studies.^29,30

The results from all the seroconversion panels illustrate several important points regarding antibody responses to vaccination against COVID-19. (1) Part of the utility of these seroconversion panels lies in the time period during which the samples were collected. These panels represent a snapshot of the immunity in a relatively naïve population that had not been extensively exposed to SARS-CoV-2 or to the vaccines. This is particularly true of the mRNA-1273 panel and the first BNT-162b2 panel which were from the earliest samples collected. The inclusive dates for sample collection were December 22, 2020, to September 24, 2021. Cumulative cases of COVID-19 in the United States ranged from 18,475,347 on December 23, 2020, to 42,521,825 on September 22, 2021. This compares to the current cumulative case estimate of 104,348,746 (April 12, 2023). ³¹ The number of individuals fully vaccinated over the sample collection period ranged from 19,349 on December 22, 2020, to 189,534,160 on September 24, 2021. The current estimate of the numbers of individuals in the United States who have complete the primary COVID-19 vaccination series is 230, 467,642 (April 11, 2023). ³²

There was a small but significant fraction (18.6%) of the general population (as measured by the participants in this study) that has been infected with SARS-CoV-2 and has antibodies to the virus prior to vaccination. These “convalescent” participants, in general, showed greater antibody response to the first vaccine injection than naïve participants. This finding is in agreement with other studies.^33,34 This greater vaccine responsiveness in convalescent individuals may translate into greater protection from infection.^35–37 (2) For the mRNA-based vaccines, the second dose raised anti-SARS-CoV-2 antibody levels in participants without a previous SARS-CoV-2 infection to the same levels as those participants who had been infected with SARS-CoV-2 and had antibodies prior to vaccination. Other studies have also shown that the antibody response is greater after the second dose of the 2-shot mRNA-based vaccine regimens. ³⁸ Initial studies with the mRNA-1273 vaccine showed that antibodies waned but were present for 6 months postvaccination. ³⁹ Subsequent studies have shown that in fully vaccinated individuals (BNT162b2, mRNA-1273, or Ad26.COV2.2), NAb activity wanes over the next 6 to 8 months.^40–42 These studies along with clinical evidence of waning vaccine effectiveness amid the emergence of less sensitive SARS-CoV-2 variants^42–45 led to recommendations for a third vaccine dose.^46,47 Real-world data now show waning vaccine effectiveness after the third dose as well^37,48 leading to recommendation for a second booster dose. ⁴⁵ (3) Antibody responses tended to be less robust in the participants over 45 years old. A decrease in humoral immunity with age has been seen with COVID-19 vaccines in other studies in healthy individuals and those with immune-suppressing diseases or undergoing immunosuppressive treatment,^49–52 although other studies report a robust response to mRNA COVID-19 vaccines in older patients (≥55 years old).^53,54

Limitations of this study include the following points. (1) The relatively small number of participants. The logistics of preparing and storing the seroconversion panels limited the number of participants that could be involved in these studies. (2) The limited time of follow-up after vaccination. Since these studies were conducted, the fading of humoral immunity over time has been documented.^39–42 The objective at the time the study was conducted was not to provide a seroconversion panel characterizing the duration of the antibody response but to create a panel that demonstrates initial antibody formation before, during, and shortly after the vaccination process. An additional study has been conducted looking at seroconversion and antibody persistence out to 6.5 months after the mRNA-1273 vaccine. ⁵⁵ (3) The study was conducted prior to widespread vaccination programs and the development of more contagious variants (eg, delta and omicron) and the advent of recurrent infections, so the effects of these factors are not captured in these seroconversion panels.

It is worth noting that a recent, small study compared booster vaccination with the recently developed bivalent booster vaccine directed at the wild-type (D614G) and omicron BA.4 to BA.5 spike proteins to booster vaccination with one of the original mRNA vaccines. This study found that the bivalent booster did not provide superior virus-neutralizing activity compared to boosting with the original monovalent vaccine. ⁵⁶ This may be due to immunologic imprinting, ie, that booster vaccines may preferentially augment immune responses to conserved antigenic sequences over immune responses to new antigenic sites contained in the variants.^56,57

Other recent studies have found greater neutralization activity and better protection against severe infection after vaccination with the bivalent vaccine compared to the monovalent vaccine.^58,59 Additional research is necessary to differentiate these possibilities, but they may indicate that the most robust and effective NAb responses are seen in naïve individuals. These naïve individuals have not been vaccinated or infected with SARS-CoV-2 as was the case with almost all the volunteers in this study.

These seroconversion panels can be useful in the development of antibody assays. Reliable detection of viral exposure and vaccine effectiveness is a critical step in gaining control of the current COVID-19 pandemic and allowing the general population to return to normal ways of life. The panels can also be a source of well-defined neutralizing antibodies (before and after vaccination and/or infection) that can be useful in investigating their effectiveness against new SARS-CoV-2 variants.