ABSTRACT
Certain aspects of the immunogenicity and effectiveness of the messenger ribonucleic acid (mRNA) vaccines (mRNA-1273 and BNT162b2) developed in response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic are still uncharacterized. Serum or plasma samples from healthy donor recipients of either vaccine (BNT162b2 n = 53, mRNA-1273 n = 49; age 23–67), and individuals naturally infected with SARS-CoV-2 (n = 106; age 18–82) were collected 0–2 months post-infection or 1- and 4 months after second dose of vaccination. Anti-Spike antibody levels and avidity were measured via an enzyme-linked immunosorbent assay (ELISA). Overall, vaccination induced higher circulating anti-Spike protein immunoglobulin G (IgG) antibody levels and avidity compared to infection at similar time intervals. Both vaccines produced similar anti-Spike IgG concentrations at 1 month, while mRNA-1273 demonstrated significantly higher circulating antibody concentrations after 4 months. mRNA-1273 induced significantly higher avidity at month 1 compared to BNT162b2 across all age groups. However, the 23–34 age group was the only group to maintain statistical significance by 4 months. Male BNT162b2 recipients were approaching statistically significant lower anti-Spike IgG avidity compared to females by month 4. These findings demonstrate enhanced anti-Spike IgG levels and avidity following vaccination compared to natural infection. In addition, the mRNA-1273 vaccine induced higher antibody levels by 4 months compared to BNT162b2.
KEYWORDS: Avidity, IgG, SARS-CoV-2, serology, vaccines, infection, Spike
Introduction
The 2019 SARS-CoV-2 coronavirus was novel and highly transmissible to humans, producing a range of manifestations from asymptomatic to severe respiratory stress, sometimes with multi-system pathology.1,2 The first licensed SARS-CoV-2 vaccines were produced by Pfizer (BNT162b2) and Moderna (mRNA-1273); both are directed at the SARS-CoV-2 Spike protein, employ RNA platforms, and are administered intra-muscularly.3 However, there are differences between the two, most notably the proprietary mRNA constructs, and the mRNA-1273 dose delivers more than 3 times the mRNA load compared to the BNT162b2 formulation.4–6 Both mRNA vaccines proved to be safe and effective against the initial viral strain, with vaccine efficacy preventing SARS-CoV-2 illness caused by early strains by over 90% at 6 months post-vaccination, but declining after 6 months.7–10
Many studies of mRNA vaccines have focused on the magnitude and durability of antibody response following vaccination.11–14 However, the strength of antibody binding to neutralizing antibody epitopes (avidity) could have an important role in protection against infection and disease.15,16 In light of the likely role of antibody avidity in protection against infection,17–19 this study sought to evaluate and compare avidity following natural infection or vaccination of healthy individuals. Humoral immune responses (antibody concentrations and avidity) to SARS-CoV-2 Spike protein were measured in individuals with evidence of natural infection and in uninfected vaccine recipients of BNT162b2 and mRNA-1273 (≤4 months after second dose). Additionally, the influence of participant age and sex on the magnitude of antibody response and the strength of antibody binding to the viral Spike protein were evaluated.
Results
Sample demographic characteristics
Anti-SARS-CoV-2 antibody and antibody avidity levels were studied in the sera and plasma of individuals with natural infection (n = 106) and in healthy recipients of the BNT162b2 (n = 53) or mRNA-1273 vaccine (n = 49). Sex and age demographics of participants are listed in Table 1. The number of females and males in each group tested was similar, with a slightly higher number of females in both groups (infected: 57 females, 49 males; vaccinated: 56 females, 46 males; p = .89). Sera and plasma from participants who were naturally infected were collected 0–2 months after symptom onset (mean = 32.8 days, male; 33.3 days, female).
Table 1.
Demographics of the study participants. The number of females and males in each group tested was similar (infected = 57 female, 49 male; vaccinated = 56 female, 46 male; p = 0.89). Serum and plasma from participants, who were naturally infected was collected 0-2 months after symptom onset (mean days = 32.8 male; 33.3 female). Antibody and avidity levels were measured in vaccinated individuals at month 1 (n = 102) and month 4 (n = 102) after the second dose of vaccine.
| Participants | SARS-CoV-2 Natural Infection | BNT162b2 | mRNA-1273 |
|---|---|---|---|
| Number (n) | 106 | 53 | 49 |
| Age (years) | |||
| Mean (SD) | 45.1 (13.9) | 46.9 (12.1) | 44.6 (12.2) |
| Max, Min |
82.0, 18.0 |
66.0, 23.0 |
67.0, 23.0 |
| Gender, n (%) | |||
| Female | 57 (53.8) | 30 (56.6) | 26 (53.1) |
| Male |
49 (46.2) |
23 (43.4) |
23 (46.9) |
| *Sample Collection Details | |||
| Days after symptom onset | |||
| Female, Mean (SD) | 33.3 (13.6) | N/A | N/A |
| Male, Mean (SD) | 32.8 (14.2) | N/A | N/A |
| Days after receiving primary vaccination series at 1 month (SD): 4 months (SD) | |||
| Female, Mean (SD) | N/A | 23.8 (10.9): 115.4 (11.8) | 20.6 (7.0): 114.3 (7.1) |
| Male, Mean (SD) | N/A | 24.0 (11.3): 114.9 (11.7) | 19.3 (9.9): 113.4 (13.6) |
*Mean collection day for the natural infection group represents days after symptom onset, and the mean collection day for the vaccinated groups represent days following the second dose of vaccine. N/A- Not Applicable.
Antibody and avidity levels were measured in vaccinated individuals at month 1 (n = 102) and month 4 (n = 102) after the second dose of vaccine. For the month 1 serum samples, the mean collection day was 23.9 ± 11.0 days for BNT162b2 recipients and 20.0 ± 8.4 days for mRNA-1273 recipients (males and females combined). Forty-nine participants in the mRNA-1273 and 53 in the BNT162b2 group continued in the study through a second blood collection. For month 4, the mean collection day was 115.2 ± 11.7 days for BNT162b2 and 113.9 ± 10.5 days for mRNA-1273 recipients (males and females combined).
Antibody responses to SARS-CoV-2 Spike following natural infection or vaccination
Antibody levels were measured via standardized ELISA (Figure 1a). A chaotrope ELISA was used to evaluate the avidity of serum and plasma IgG antibodies for SARS-CoV-2 Spike (Figure 1b). Both natural infection and vaccination induced significant antibody levels with a wide range of avidity to SARS-CoV-2 Spike (Figure 1a,b).
Figure 1.
Anti-SARS-CoV-2 Spike IgG levels and avidity in blood from vaccine recipients were compared to naturally infected individuals. Serum and plasma samples were collected from study participants with confirmed SARS-CoV-2 infection or 1 and 4 months after the approved vaccination regimens for BNT162b2 or mRNA-1273. (a) Anti-SARS-CoV-2 spike IgG (BAU/mL), (b) Anti-SARS-CoV-2 spike IgG avidity (M). Left, natural infection; center, mRNA-1273 1 and 4 months after vaccination; right, BNT162b2 1 and 4 months after vaccination. Geometric means and 95% confidence intervals are listed above each time point.
Anti-SARS-CoV-2 IgG levels were significantly higher in vaccinated individuals (mRNA-1273 and BNT162b2 combined) compared to infected participants, 3469 BAU/mL (Geometric mean of both vaccines at month 1; 95% CI, 2960–4066 BAU/mL) compared to 353 (Geometric mean of natural infection; 95% CI, 290–429), respectively (p < .0001). Additionally, vaccine recipients (mRNA-1273 and BNT162b2 combined) exhibited statistically higher geometric mean avidity levels than those detected in sera or plasma from naturally infected participants; 4.1 M (95% CI, 4.0–4.3 M) as compared to 2.9 M (95% CI, 2.8–3.1 M), respectively, p< .0001.
Anti-SARS-CoV-2 IgG antibody and avidity levels in serum from BNT162b2 and mRNA-1273 vaccine recipients
The mRNA-1273 vaccine induced slightly higher levels of anti-SARS-CoV-2 Spike IgG at month 1 compared to BNT162b2, with geometric mean concentrations of 3742 BAU/mL (95% CI, 2880–4861 BAU/mL) and 3234 BAU/mL (95% CI, 2666–3924 BAU/mL), respectively (p = .229) (Figure 2). By month 4, levels of anti-SARS-CoV-2 Spike antibodies declined to 1103 BAU/mL (95% CI, 890–1368 BAU/mL) in mRNA-1273 recipients and was statistically higher than in BNT162b2 recipients, who’s levels declined to 773 (95% CI, 621–963 BAU/mL); p = .0223. For either vaccine, circulating antibody levels declined substantially from 1 to 4 months post-administration of the second dose (Figures 1a and 2).
Figure 2.
Geometric mean anti-SARS-CoV-2 Spike IgG levels in sera from the vaccinated groups at month 1 versus month 4. Serum samples were collected from BNT162b2 (Blue) and mRNA-1273 (Red) vaccinees 1 and 4 months after receiving the primary vaccination series. Results presented as Geometric mean anti-SARS-CoV-2 Spike IgG binding antibody units/mL (BAU/mL) and 95% confidence intervals. BNT162b2:mRNA-1273, month 1 (p = .229); month 4 (p = .0223).
Avidity levels induced by the two vaccines increased significantly over time. At both time points, the avidity levels were higher in the mRNA-1273 vaccinated individuals compared to BNT162b2 recipients (Figure 1b). Geometric mean avidity levels for mRNA-1273 were 4.5 M (95% CI, 4.3–4.7 M) at month 1 and 5.3 M (95% CI, 5.1–5.5 M) at month 4; BNT162b2 recipients developed avidity levels of 3.8 M (95% CI, 3.6–4.0 M) at month 1 and 4.7 M (95% CI, 4.4–4.9 M) (p < .0001 for both) at month 4 (Figure 1b).
Anti-SARS-CoV-2 Spike avidity levels appeared to be largely independent of circulating antibody concentration. A weak correlation was observed between anti-SARS-CoV-2 Spike IgG levels and avidity for the two vaccines at month 1 (mRNA-1273 avidity to IgG BAU/mL:ρ = .307, p = .0318; BNT162b2 avidity to IgG BAU/mL:ρ = .276, p = .0458) (Figure 3, left). At month 4, no correlation was found between anti-SARS-CoV-2 Spike IgG antibody concentrations and avidity in BNT162b2 recipients (Pearson’s correlation, ρ = .079, p = .5715), and in mRNA-1273 recipients (Pearson’s correlation, ρ = .222, p = .1250) (Figure 3, right).
Figure 3.
Correlation of anti-SARS-CoV-2 Spike IgG levels and avidity in vaccine recipients. (Left) Serum samples collected 1 month after primary series vaccination; (Right) Serum samples collected 4 months after primary series vaccination. Red dots represent avidity level (M) plotted against anti-SARS-CoV-2 Spike IgG concentration (BAU/mL) for mRNA-1273 vaccines, while blue triangles represent BNT162b2 vaccinees’ sera. Regression lines are represented for each cohort (Red mRNA-1273, Blue BNT162b2). Pearson’s correlation, 1 month: mRNA-1273, ρ = .307 and BNT162b2, ρ = .276. Pearson’s correlation, 4 months: mRNA-1273, ρ = .222 and BNT162b2, ρ = .079.
Assessment of age and sex on avidity maturation of anti-SARS-CoV-2 Spike IgG antibodies induced by vaccination
mRNA-1273 induced higher anti-SARS-CoV-2 Spike IgG avidity levels regardless of age, with statistically significant differences between responses to the two vaccines noted for all age groups at 1 month (p23–34 = .0012; p35–55 = .0004; p>55 = .0087), and in the 23–34 age group at 4 months (p23–34 = .0017; p35–55 = .0904; p>55 = .0601) (Table 2).
Table 2.
SARS-CoV-2 Spike IgG avidity results segregated by vaccine received and age over two time points (1 month and 4 months after vaccination). Avidity was stratified by age across the two vaccines at month 1 (A) and month 4 (B). Avidity at the 23–34 y and >55 y age groups was also directly compared to the 35–55 y age group both overall and separated according to vaccine received (C).
| A. Avidity Levels 1 Month after Vaccination. |
|
23–34 years |
35–55 years |
>55 years |
|
| BNT162b2 | n | 10 | 27 | 16 | |
| GMA (95% CI) | 3.6 (3.3–3.9) | 3.9 (3.6–4.3) | 3.6 (3.3–4.0) | ||
| Median (Min, Max) | 3.5 (3.0, 4.7) | 3.6 (3.1, 6.6) | 3.6 (2.7, 5.1) | ||
| mRNA-1273 | n | 14 | 23 | 12 | |
| GMA (95% CI) | 4.5 (4.2–4.9) | 4.7 (4.4–5.0) | 4.2 (3.9–4.6) | ||
| Median (Min, Max) | 4.4 (3.7, 6.6) | 4.7 (3.1, 6.0,) | 4.2 (3.5, 5.4) | ||
| BNT162b2 vs. mRNA-1273 |
p-value |
.0012 |
.0004 |
.0087 |
|
| B. Avidity Levels 4 Months after Vaccination. |
|
23–34 years |
35–55 years |
>55 years |
|
| BNT162b2 | n | 10 | 27 | 16 | |
| GMA (95% CI) | 4.5 (4.0–5.1) | 4.9 (4.6–5.3) | 4.4 (3.9–4.8) | ||
| Median (Min, Max) | 4.7 (3.0, 5.3) | 4.8 (3.6, 7.3) | 4.4 (3.1, 6.5) | ||
| mRNA-1273 | n | 14 | 23 | 12 | |
| GMA (95% CI) | 5.5 (5.2–5.9) | 5.3 (5.0–5.5) | 5.0 (4.6–5.5) | ||
| Median (Min, Max) | 5.5 (4.5, 7.0) | 5.4 (4.1, 6.2) | 5.1 (3.8, 6.4) | ||
| BNT162b2 vs. mRNA-1273 |
p-value |
.0017 |
.0904 |
.0601 |
|
| |
1 month |
4 months |
|||
| C. Avidity Levels Compared to the 35–55 Age Group. |
|
23–34 years |
>55 years |
23–34 years |
>55 years |
| BNT162b2 | Ratio to 35–55 years (95% CI)1 | 1.04 (0.96–1.15) | 0.97 (0.83–1.07) | 1.06 (0.95–1.23) | 0.88 (0.79–1.02) |
| p-value | .4622 | .7439 | .3297 | .0808 | |
| mRNA-1273 | Ratio to 35–55 years (95% CI)1 | 1.06 (0.96–1.15) | 0.89 (0.83–0.98) | 0.96 (0.88–1.04) | 0.94 (0.87–1.05) |
| p-value | .2941 | .0143 | .3891 | .3052 | |
| Overall | Ratio to 35–55 years (95% CI)1 | 1.02 (0.93–1.13) | 0.91 (0.84–1.03) | 1.00 (0.93–1.08) | 0.91 (0.84–1.00) |
| p-value2 | .1959 | .0590 | .9816 | .0446 | |
1: Hodges–Lehmann estimate. p-value indicates result of rank-sum test vs the 35–55 years group.
2: Van Elteren’s stratified Wilcoxon test, stratified by vaccine group.
Overall, by month 4, older vaccine recipients (over 55 years of age) developed IgG antibodies to Spike protein of lower avidity than what was detected in donors in the other age groups (irrespective of vaccine received), but the difference only reached statistical significance when considering both vaccination groups together against the 35–55 age group (Table 2C; p = .0446). The 35–55 vaccinated age group trended toward the highest avidities for both months 1 and 4 (Table 2A,B), except for the mRNA-1273 23–34 age group at month 4, but none reached statistical significance. For BNT162b2, the 23–34 and >55 age group responses were similar to the 35–55 age group at month 1 and 4 (Table 2C). Age had no statistically significant impact on avidity responses in mRNA-1273 except for the >55 age group at 1 month (Table 2C; p = .0143) when compared to the 35–55 age group. However, by month 4, no statistical differences were detected in recipients of the mRNA-1273 vaccine (Table 2C).
Males and females vaccinated with mRNA-1273 had similar avidity responses over the 4 months (Figure 4a). The rate of avidity maturation in female recipients of BNT162b2 was not significantly higher than in males (Table 3); p = .1754. Male recipients of BNT162b2 developed IgG anti-spike avidity levels that tended to be lower than females that received the same vaccine; however, the difference did not reach statistical significance (p = .0607). A direct comparison of male recipients of BNT162b2 at month 4 to all other vaccine recipients at month 4 (females that received BNT162b2 and all recipients of mRNA-1273 as a group) demonstrated significantly lower avidity (p < .001) despite similar rates of avidity maturation.
Figure 4.
Maturation of the anti-SARS-CoV-2 Spike IgG avidity response to vaccination; influence of sex and vaccine formulation on avidity after vaccination. Vaccinee sera collected at months 1 and 4 after vaccination. Sera were assessed in chaotrope ELISA assays to obtain geometric mean avidity levels (M). Data are presented as geometric mean avidity levels (M), error bars represent 95% confidence intervals. Results are separated by response to vaccine (mRNA-1273 or BNT162b2), month and sex. Female vaccinees (red), males (blue). Geometric mean avidity levels (M) and 95% confidence intervals are listed above each time point.
Table 3.
Influence of sex and vaccine on geometric mean avidity levels over 4 months. The geometric mean avidity (GMA) levels were segregated by vaccine, time point and sex. Serum avidity increased significantly from months 1 to 4 in each group evaluated regardless of vaccine received. Males and females vaccinated with BNT162b2 or mRNA-1273 had similar avidity responses over the 4 months.
| Influence of Sex and Vaccine on Geometric Mean Avidity Levels Over 4 Months. | ||||||
|---|---|---|---|---|---|---|
| Vaccine | Sex at Birth | 1 Month | 4 Months | GM Fold Increase in Avidity | Sex at Birth× Time Interaction2 (p-value) |
|
| BNT162b2 | Female | n | 30 | 30 | 1.29* | .1754 |
| GMA (95% CI) | 3.8 (3.6–4.1) | 4.9 (4.6–5.3) | ||||
| Median (Q1, Q3) | 3.7 (3.4, 4.0) | 4.8 (4.5, 5.5) | ||||
| Male | n | 23 | 23 | 1.20* | ||
| GMA (95% CI) | 3.6 (3.9, 3.4) | 4.4 (4.0–4.7) | ||||
| Median (Q1, Q3) | 3.5 (3.4, 3.8) | 4.6 (3.8, 5.0) | ||||
| Female vs. Male | p-value1 | .3325 | .0607 | |||
| mRNA-1273 | Female | n | 26 | 26 | 1.20* | .3620 |
| GMA (95% CI) | 4.5 (4.2–4.8) | 5.4 (5.1–5.7) | ||||
| Median (Q1, Q3) | 4.3 (4.1, 4.9) | 5.5 (5.1, 5.9) | ||||
| Male | n | 23 | 23 | 1.14* | ||
| GMA (95% CI) | 4.6 (4.3–4.9) | 5.2 (5.0–5.5) | ||||
| Median (Q1, Q3) | 4.5 (4.2, 5.0) | 5.1 (4.9, 5.5) | ||||
| Female vs. Male | p-value1 | .7259 | .1520 | |||
*Significant increase in log avidity index, by signed-rank test (p < .0001).
1Rank-sum test.
2To test for a possible effect of sex at birth on the fold increase in avidity, a rank-sum test was applied to the 4 to 1 month change in log avidity index.
Discussion
IgG levels and avidity of IgG antibodies specific to SARS-CoV-2 Spike protein were evaluated after SARS-CoV-2 natural infection or vaccination with one of the two approved mRNA vaccines. Consistent with previous studies, the levels of anti-SARS-CoV-2 Spike IgG induced by vaccination were significantly higher than those induced by natural infection.20–23 The avidity of the anti-SARS-CoV-2 Spike IgG antibodies in the vaccinated group was also significantly higher compared to natural infection. Several studies outline specific parameters necessary to consider with in-house avidity method developments, such as viral variant choice, antigen source, serial or single serum dilution, chaotropic agent of choice and its concentration, incubation period, calculations, and interpretation of results.24,25 Our in-house assay was qualified against many of these parameters to ensure consistent and comparable results. Similar results on the avidity of anti-SARS-CoV-2 receptor-binding domain (RBD) IgG have been seen in other studies.23,26,27 Differences in IgG levels and avidity between the naturally infected and the vaccinated groups are likely due to the highly focused immune response elicited by Spike protein by vaccination compared to the more general multi-epitope response generated by infection.
Given the novelty of the mRNA vaccines, correlates of protection are not known in terms of the levels of neutralizing antibodies necessary to infer protection from infection. The intent of this report is to add to the growing knowledge base of immune persistence from vaccination with either BNT162b2 or mRNA-1273, based on total anti-SARS-CoV-2 Spike IgG content and avidity, not specifically neutralizing antibody content. High correlations between binding and neutralization antibody levels have been observed for the original strain [14, 19]. Both mRNA vaccines use similar mRNA delivery systems, induce protective antibodies to the viral Spike protein, and require two doses plus boosters.4–30 Despite differences in formulation and dose, both vaccines were immunogenic in recipients regardless of sex or age and elicited comparable IgG serum concentrations to the SARS-CoV-2 Spike protein at month 1. However, a statistical difference was detected between the two vaccines by month 4. Consistent with previous reports, levels of circulating IgG antibodies to Spike protein waned over 3 months in both groups, with a slower rate of decline observed in mRNA-1273 vaccine recipients, while IgG serum avidity for SARS-CoV-2 Spike protein increased over the 3 months for both vaccine cohorts.31,32 Although the avidity response increased from month 1 to month 4 for both vaccines, the role of avidity in protection from infection or disease progression is still unknown. The literature does demonstrate a waning of antibody responses and protection against SARS-CoV-2 infection after vaccination, despite robust effectiveness against severe disease or hospitalization. Chemaitelly et al. reported that vaccine effectiveness of BNT162b2 against infection with the delta variant in vaccinees 12 years of age and older was less than 50% within 10 to 14 weeks of the primary series vaccination, and there was no significant difference between the incidence of infection in vaccinated and unvaccinated populations. However, Lin et al. evaluated the long-term effectiveness of BNT162b and mRNA-1273 against symptomatic COVID disease and found the vaccines were 94% to 96% effective 1–3 months after the second dose of vaccination, and by 8–9 months, effectiveness of BNT162b2 decreased to 66.6% while mRNA-1273 remained more than 80%.33 Moreover, there is strong evidence that both BNT162b2 and mRNA-1273 remained 75% effective against hospitalization and death for more than 6 months after vaccination34. Taken together, circulating antibody content may be a strong indicator of protection afforded by vaccination. High serum antibody levels may afford protection through direct viral neutralization or cellular effectors to prevent infection and viral propagation. The role of avidity in protection against infection and disease progression is unknown but is likely important in the antibody response. Future, larger studies, including local measurements of immunity, are warranted.
When considering both vaccines, age was a significant determinant in IgG avidity development to SARS-CoV-2 Spike. Differences were statistically significant between the 35–55 group and the >55 age group by month 4 when considering both vaccine groups together, but not between the 35–55 group and the 23–34 age group. Within the mRNA-1273 cohort, the avidity responses were not statistically significant across all age groups, except between the >55 age group and 35–55 age group at Month 1. Additional analysis described a significantly higher avidity response from mRNA-1273 vaccinees across all age groups at Month 1, but only the 23–34 age group maintained statistically significantly higher avidity responses from mRNA-1273 vaccinees at Month 4. Because the noted differences were associated with the lower inoculum BNT162b2 vaccine, these noted differences may have been dose dependent.
In this study, females generally developed higher anti-SARS-CoV-2 Spike IgG avidity from vaccination (Figure 4b,c); however, the comparisons to males and across vaccines in our study did not reach statistical significance. It will be interesting to see if later time points following vaccination highlight the same trend, as several studies have suggested that females have more pronounced innate and adaptive immune responses to viral infections, and there is a strong bias toward more severe outcomes following SARS-CoV-2 infections in men.35 Enhanced innate and adaptive immune responses of women to viral infections may also explain why they responded better to the mRNA vaccines.36
Unfortunately, at the time of this study neutralization assays were not available to correlate changes in neutralization with antibody levels or avidity. Previous results in our lab have shown a good correlation between neutralization against the original virus strain and binding assays37 in agreement with observations from other groups,38–41 suggesting that binding is a good surrogate for neutralization in this context. In addition, the remarkable increase in sera avidity and drop in circulating IgG levels following vaccination with either of the two mRNA vaccines is meaningful and reportable for comprehensive characterization of the human immune responses to mRNA vaccines, and potential value in evaluating vaccine efficacy. The potential utility of avidity assessments for evaluating vaccine efficacy is illustrated by the observation that individuals that received the BNT162b2 vaccine did not develop the quality IgG response as measured by avidity for SARS-CoV-2 Spike protein that individuals in a mRNA-1273 group did at Month 1. This trend continued with the 23–34-year-old group at Month 4. Admittedly, the sample size was relatively small, but the resultant data showed statistical differences.
Further longitudinal assessments past 4 months are under way following booster vaccination to aid in the understanding of the duration and decay of antibody responses to boosters in the context of infection and both vaccines. One possible limitation of this study is the sample size and extent of possible comparisons between the naturally infected and vaccinated groups. This limitation was minimized by using samples with roughly equivalent post-symptom intervals as much as possible given limited sample availability. Another possible limitation is the lack of analysis of IgGs against other SARS-CoV-2 antigens beyond Spike, or of other antibody isotypes and subclasses. Spike protein was chosen for evaluation in this study due to the specific targeting of the Spike protein by the vaccines, and IgG was targeted due to the known neutralizing activity.3,42 However, other studies have examined other antigens and Igs with results parallel to our own.23,26,27,32 Meanwhile, although IgG levels diminish over time, this study also reveals that the quality of the IgG that remains is of high avidity. Finally, this study only analyzes the effects of two doses of either mRNA-1273 or BNT162b2 vaccine despite additional booster doses currently being administered. The effects of a third dose on antibody avidity deserve attention in future studies.
This study provides meaningful data not only on IgG levels over time after mRNA vaccination (supporting previous studies),20–23 but also on Ig avidity changes over time and differences across different vaccines and other variables. In addition, IgG levels and avidity were studied in the context of vaccination versus natural infection. These findings demonstrate enhanced IgG antibody levels and avidity to SARS-CoV-2 Spike following vaccination compared to natural infection. Individuals who received the mRNA-1273 vaccine developed serum IgG concentrations to Spike protein analogous to BNT162b2 recipients at 1-month post-vaccination, but the IgG levels persisted longer in mRNA-1273 recipients by month 4. Additionally, mRNA-1273 vaccinated participants developed higher avidity IgG antibodies to Spike protein across all age groups at Month 1. Importantly, while differences in IgG levels have previously been widely reported between the two mRNA vaccines, this study reveals that there are also differences in avidity early in the response. The biological and clinical roles of the magnitude of observed differences in immunity and vaccine effectiveness are unknown.
Materials and methods
Samples
Human serum or plasma samples (n = 106) were obtained through the Biodefense and Emerging Infections Research Resources Repository (BEI), Rutgers University, Icahn School of Medicine at Mount Sinai, Montefiore Medical Center, and Boca Biolistics, and the samples were collected under approved protocols. All natural infection samples were obtained from participants with SARS-CoV-2 infection confirmed by PCR. For the most part, there was only one sample collection per participant available.
Sera from healthy donors (n = 102) who had received two doses of either BNT162b2 (n = 53) or mRNA-1273 (n = 49) were drawn by Occupational Health Services, Frederick National Laboratory for Cancer Research, Frederick MD, under the Research Donor Protocol OH99CN046. Samples were obtained following two time points at month 1 (22.0 ± 10.0 days) and at month 4 (114.6 ± 11.1 days) following the originally recommended two doses of vaccine. The study volunteers ranged in age from 23 to 67 years.
Sample preparation
Blood collected in serum or plasma tubes was processed and frozen. Serum and plasma samples were maintained at −80°C until tested, then thawed on wet ice, aliquoted, and heat inactivated at 56°C for 30–60 min immediately prior to testing.
Enzyme-linked immunosorbent assays (ELISA)
ELISA assays used to quantify human serum and plasma IgG antibodies to the SARS-CoV-2 Spike protein were performed at room temperature (RT) as follows: Maxisorp 96-well plates (Thermo-Scientific Cat# 439454) were coated with recombinant SARS-CoV-2 Spike protein (SARS-CoV-2 S-2P (14–1213)-T4f-His6) sourced from the Protein Expression Laboratory at Frederick National Laboratory for Cancer Research (FNLCR) (0.15 µg/mL in PBS). After coating for a minimum of 24 h at 2–8°C, assay plates were washed with a PBS-Tween buffer and blocked with PBS-Tween 0.2% and 4% skim milk (BD, Cat# 232100) for 90 min. Heat inactivated samples were tested with appropriate in-well dilution series. Plates were incubated for 60 min with the samples at room temperature. Next, the plate was washed, and then incubated for 60 min with goat anti-human IgG enzyme horseradish peroxidase (HRP)-conjugate at room temperature. Following incubation, the plate was washed, and then developed with 3,3,’5,5’-tetramethylbenzidine (TMB) 2-component substrate (Seracare, Cat# 5120–0050). After adding 0.36 N sulfuric acid, the plate was read at 450nm and 620nm on a SpectraMax plate reader (Molecular Devices). Data analyses were performed using SoftMax Pro GxP 7.0.3. Reportable values for IgG quantitative ELISA are binding antibody units per milliliter (BAU/mL), based on a standard calibrated to the World Health Organization (WHO) International Standard.43 The lower limit of quantitation (LLOQ) of the Spike IgG assay is 10.4 BAU/mL.
Avidity enzyme-linked immunosorbent assays (chaotrope ELISA)
Avidity ELISA assays (chaotrope ELISA) are based on standard ELISA tests for IgG anti-SARS-CoV-2 Spike protein but include an additional step that exposes bound analyte (antibody) to a chaotropic agent that effectively breaks and elutes off weakly bound antibody species: a “bind and break” ELISA. Urea was the chaotropic agent used in this study due to its experimental range and minimal impact on assay plate coat integrity. ELISA assays to assess serum and plasma avidity were performed with samples that upon dilution in assay buffer produced optical densities (OD) in a standard IgG ELISA of between 0.2 and 2.0 OD units at 450nm; 1.0 was the target OD. Each assay plate tested five serum or plasma samples in duplicate. After each sample was incubated on the assay plate for 60 min at RT, the plates were washed and incubated with dilutions of urea ranging from 0 to 7 molar (M) for 15 min at RT. The plate was further developed as described for the quantitative IgG assay, continuing with the conjugate antibody. Serum and plasma avidity data are reported as geometric mean avidity levels (GMA). The avidity levels are the molar concentration of urea required to reduce the optical density of the sample to half that of untreated wells.
Statistics
Serum and plasma antibody levels and avidity were log-transformed for analysis and summarized as geometric mean concentration or geometric mean avidity (GMA). Pearson correlation analyses were performed to evaluate correlations between antibody levels and avidity results. For two-sample comparisons, statistical significance was based on a Wilcoxon rank-sum test, and the Hodges-Lehmann estimator of the fold change is reported. Paired comparisons were based on a signed-rank test. The analysis was implemented in SAS version 9.4 (SAS institute Inc., Cary, NC). p < .05 was considered significant.
Acknowledgments
The authors would like to acknowledge the scientific contributions of Leidos Biomedical Research scientific staff: Sarah Loftus, Genevieve Istas, Daisy Roy and Katrina Haynesworth for serum collection and preparation; David Fetterer for expert statistical analyses and Heidi Hempel for editorial assistance. The authors would like to thank Biodefense and Emerging Infections Research Resources Repository (BEI), Rutgers University, Icahn School of Medicine at Mount Sinai, and Montefiore Medical Center for provision of the human serum and plasma samples. We also acknowledge the Occupational Health and Safety staff that support the Research Donor Program for the Frederick National Laboratory for Cancer Research, particularly Patricia Claude, RN, for the administration of the serum collection program.
Funding Statement
This project has been funded in whole or inpart with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024. The content of this publication does not necessarily reflect the views or policies of the Department of Healthand Human Services, nor does mention of trade names, commercial products, ororganizations imply endorsement by the U.S. Government.
Disclosure statement
Thomas E Hickey owns Pfizer stock. No other potential conflict of interest was reported by the other author(s).
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