Multiplex antibody profiling in rhesus macaque model
The multiplex antibody assay was employed to analyze humoral immune responses (panel included seven human coronaviruses, as described in Methods) in the plasma of non-SIV and SIV-infected animals immunized with SARS-CoV-2 S-RBD protein.
Total IgG antibodies against SARS-CoV-2 S-RBD. In non-SIV-infected animals (n = 7), anti-SARS-CoV-2 S-RBD antibodies (IgG) were detectable at 2 weeks post-first dose in three macaques (Median MFI=79; IQR:32, 248) (Fig. 1A). At four weeks post-first dose, five macaques had anti-SARS-CoV-2 S-RBD antibodies (Median MFI=535; IQR:105, 1322). A strong antibody response to SARS-CoV-2 S-RBD was detectable in all macaques at 2 weeks (Median MFI=6625; IQR:3674, 9477) and 4 weeks (Median MFI=5489; IQR:3488, 8368) post-second dose immunization (Fig. 1A).
We also studied antibody responses in SIV-infected macaques (n=2) under combination antiretroviral therapy (cART) vaccinated with SARS-CoV-2 S-RBD protein (Fig. 1B). Antibodies against S-RBD were detectable at 2 weeks post-first dose (Median MFI=1042; IQR:866, 1217) and steadily increased at 2 weeks post-second dose immunization (Median MFI=10356; IQR:10063, 10650). However, the antibody responses were stable at 4 weeks (Median MFI=7571; IQR:6842, 8299) and 8 weeks (Median MFI=8157; IQR:7348, 8965) post-second dose immunization (Fig. 1B). Since the number of animals in the SIV group (n=2) was less compared to the non-SIV group (n=7), the variations in antibody levels and neutralization titer should be interpreted cautiously.
Total IgG antibodies against SARS-CoV-2 N protein. In non-SIV infected rhesus macaques immunized with adenoviral vector vaccine expressing N protein (n=3), SARS-CoV-2 N specific antibodies (IgG) were detectable at 2 weeks post-first dose immunization in all three macaques (Median MFI=693; IQR:539, 1494) (Fig. 1C). SARS-CoV-2 N-specific antibodies peaked at 2 weeks post-second dose immunization (Median MFI=9636; IQR:8929, 10212). Even though antibody levels dropped by week 6 post-second dose (Median MFI=5623; IQR:5198, 6118), the levels were significantly higher (p-value=0.0042) compared to 2 weeks post-first dose immunization (Fig. 1C). Plasma from most healthy controls (n=116) did not contain antibodies to S-RBD or N proteins (Supplementary Table S2); in two macaques the SARS-CoV-2 S-RBD and N antibodies were slightly above the baseline cut-off value.
Neutralizing antibodies against S1 or S-RBD display a broad-spectrum effect against several strains. sVNT assay was performed (described in Methods) to evaluate the neutralizing antibody titers (%-inhibition against SARS-CoV-2 wild type and nine variants) in animals immunized with S-RBD. Neutralizing antibodies were detected in two animals in the non-SIV and SIV-infected group at 2 weeks post-first dose, which further increased 4 weeks post-first dose vaccination (Fig. 1D and E). Titers for three animals in the non-SIV infected group remained low compared to other animals in this group at all tested time points post-vaccination (Fig. 1D). These animals also had the lowest total IgG at all time points post-vaccination. However, a high neutralizing antibody titer was observed at 2 weeks that persisted at 4 weeks post-second dose in both SIV and non-SIV groups (Fig. 1D and E). Neutralization capacity against the 9 SARS-CoV-2 variants was similar to the wildtype S1, and S-RBD in macaques immunized with adenoviral vector vaccine expressing S-RBD (Fig. 1F). Neutralization capacity against the beta variant was the lowest among the tested SARS-CoV-2 variants (Fig. 1F). Non-SIV infected rhesus macaques immunized with adenoviral vector vaccine expressing N protein, did not exhibit neutralization antibody titers against SARS-CoV-2 wild type or any of the tested variants.
Neutralizing antibody titer and total IgG in animals immunized with S-RBD (R2) were strongly correlated with R2 = 0.93 for the non-SIV infected group and R2 = 0.96 for the SIV-infected group (Supplementary Fig. S1 A and B respectively).
Multiplex antibody profiling in humans immunized with Pfizer and Moderna mRNA vaccines
Total IgG antibodies against SARS-CoV-2 S-RBD. Multiplex assay was employed to measure the levels of IgG against SARS-CoV-2 S-RBD in healthy adult volunteers immunized with the Pfizer (n =35) and Moderna (n=18) mRNA vaccines (Fig. 2 A and B). Plasma samples were collected at baseline, 3 weeks post-first dose (Pfizer only), 4 weeks post-first dose (Moderna only), and 4 and 26weeks post-second dose (both groups).
At baseline, none of the participants had antibodies against S-RBD except for one individual in the Pfizer group. This individual had S-RBD antibodies slightly above the cut-off value but devoid of SARS-CoV-2 N antibodies throughout the study (Described in detail in the section SARS-CoV-2 N (IgG) under Results).
Antibodies against S-RBD were detectable in all the volunteers three weeks post-first dose in the Pfizer (Median MFI=2376; IQR:1838, 5795) (Fig. 2A) and four weeks post-first dose in the Moderna group (Median MFI=4110; IQR:3113, 5852) (Fig. 2B). Antibodies peaked at 4 weeks post-second dose (for Pfizer group Median MFI=8465; IQR:3278, 11016 and Moderna group Median MFI=12392; IQR:10029, 13609) and maintained at lower levels at 26 weeks post-second dose vaccination in both groups (for Pfizer group Median MFI=1526; IQR:1089, 3342 and Moderna group Median MFI=2504; IQR:2424, 4636), as reported previously [34]. Interestingly, antibody titers were waning in both groups at 26 weeks post-second dose (Fig. 2A and B). Although antibody levels dropped, these individuals remained antibody-positive throughout the study, and levels observed were similar to what was seen post-first dose, and the drop in MFI was not statistically significant compared to post-first dose levels (p-value = 0.9299 (Pfizer), p-value = 0.5242 (Moderna)).
The levels of antibodies in all three post-vaccination time points in the Moderna group were 1.5 to 2.3-fold higher (Median MFIs= 4110, 12392, 3504) than in Pfizer (Median MFI = 2376, 8465, 1526). The difference was statistically significant only at 4 weeks post-second dose (p-value 0.0004), not at 3-4 weeks post-first dose or 26 weeks post-second dose (p-values = 0.7125 and 0.8823 respectively).
Total IgG antibodies against SARS-CoV-2 N. Multiplex assay was employed to measure the levels of antibodies against N protein. At baseline, none of the participants had antibodies against N protein in both groups except one individual in the Moderna group. This individual had antibodies against N protein slightly above the cut-off, while S-RBD IgG level was below the baseline (Supplementary Fig. S2B). The same individual remained positive for N and S-RBD IgG during post-first and second dose vaccination.
Among the Pfizer group, one participant had antibodies against N and S-RBD at 4 weeks post-second dose and remained positive until 26 weeks post-second dose vaccination (Supplementary Fig. S2A). This volunteer might have been exposed to SARS-CoV-2 during the study period. Two participants in the Pfizer group became positive for SARS-CoV-2 by RT-PCR three weeks post-second dose vaccination and contained antibodies to SARS-CoV-2 N protein until 28-32 weeks post-second dose (Supplementary Fig. S2A).
Kinetics of seroconversion and role of antibody subtypes. We also measured IgM, and IgG subtypes (IgG1, IgG2, IgG3, IgG4) responses to S-RBD in plasma samples from individuals immunized with Pfizer or Moderna vaccines (Fig. 3 A-F (Pfizer); G-L (Moderna)).
Supplementary Table S3 indicates how many individuals developed detectable levels of plasma IgM, IgG (total), and subtypes against S-RBD in Pfizer and Moderna groups at each time point post-vaccination. In general, a low level of IgM antibodies was detected in both groups (Supplementary Table S3). Sixteen individuals in the Pfizer group had low levels of IgM compared to one in the Moderna group at 4 weeks post-second dose immunization. Similarly, at 26 weeks post-second dose, four individuals in the Pfizer group (Fig. 3B) had low levels of IgM compared to none in the Moderna group (Fig. 3H).
In both vaccine groups, the IgG (Total) (Fig. 3A, G) response post-first and second dose was mediated by an elevation of IgG1 (Fig. 3C, I) followed by IgG3 (Fig. 3E, K). The IgG1 (Fig. 3C, I) antibodies persisted until 26 weeks post-second dose vaccination even though the levels dropped from the previous sampling timepoint. The IgG3 (Fig. 3E, K) antibodies levels also dropped precipitously at 26 weeks post-second dose vaccination.
At 3 or 4 weeks post-first dose vaccination, 9 out of 10 individuals in the Pfizer group and 14 out of 18 individuals in the Moderna group had elevated levels of IgG1 (Supplementary Table S3). At 4 weeks post-second dose, 32 out of 35 individuals in the Pfizer group (Median MFIs = 1277; IQR: 797, 4122) and 17 out of 18 individuals in the Moderna group (Median MFIs= 5049; IQR: 167, 9216) had elevated levels of IgG1. At 26 weeks post-second dose, 33 out of 35 individuals in the Pfizer group (Median MFIs= 592; IQR: 389, 1905) and 14 out of 18 individuals in the Moderna group (Median MFIs= 1125; IQR: 60, 2967) had elevated levels of IgG1. The levels of IgG1 antibodies in all three post-vaccination time points in the Moderna group (Fig. 3C) were 1.3 to 4-fold higher (Median MFIs= 5049 (IQR 167,9216), 24544 (IQR 5449, 28067), 1125 (IQR: 60, 2967)) than the Pfizer group (Fig. 3I) (Median MFIs = 1277 (IQR 797, 4122), 14243 (IQR: 4410, 21308), 592 (IQR 389, 1905)). The results were statistically significant at 4 weeks post-second dose vaccination (p-value = <0.0001).
A low level of IgG2 was observed in most of the Pfizer group (Fig. 3D); 4 weeks post-second dose (Median MFI=75; IQR:28, 247), three individuals contained elevated levels of IgG2 (MFIs=1018, 5423,15589). Two individuals in the Moderna group (Fig. 3J) had elevated levels of IgG2 at four weeks post-first dose vaccination (MFI=2585, 7786). The IgG2 levels in the Moderna group (Median MFI=1718, IQR: 413, 3392) were significantly higher compared to the Pfizer group (Median MFI=75, IQR: 28, 247) 4 weeks post-second dose (p-value=0.0117).
Similarly, IgG3 levels in the Moderna group (Fig. 3K) were significantly higher (Median MFIs=301, IQR: 132, 564) compared to the Pfizer group (Fig. 3E) (Median MFIs=67, IQR: 44, 123) at four weeks post-first dose (p-value=0.0496). At 4 weeks post-second dose, the IgG3 levels were as follows: Pfizer group (Median MFIs=246, IQR: 112, 450) and Moderna group (Median MFIs=483, IQR: 302, 859). At 26 weeks post-second dose, IgG3 levels dropped in both groups. At this time point, 18 out of 35 individuals in the Pfizer group and 15 out of 18 individuals in the Moderna group had detectable levels of IgG3 (Supplementary Table S3).
Four individuals in the Pfizer group had detectible levels of IgG4 (Fig. 3F) at three weeks post-first dose vaccination (MFIs= 208, 1808, 4842, 158) (Supplementary Table S3). In this group, one individual at 4 weeks post-second dose (MFI=646) and 5 individuals at 26 weeks post-second dose vaccination had the IgG4 antibodies (MFIs= 585, 760, 124, 119, 1122). In the Moderna group (Fig. 3L), four individuals contained IgG4 antibodies at baseline (MFIs= 153, 5001, 175, 1341). In this group, one individual at 4 weeks post-first dose (MFI=2062), and 4 individuals at 4 weeks (MFIs= 897, 129, 564, 5797), and 2 individuals 26 weeks post-second dose (MFIs= 245, 975) vaccination contained IgG4 antibodies (Supplementary Table S3).
SARS-CoV-2 Neutralization titers. The viral neutralizing antibodies were detected in individuals immunized with Pfizer and Moderna vaccines using an sVNT assay. The MFI values against S-RBD were plotted against the % inhibition of neutralizing antibodies to SARS-CoV-2 S1 for the corresponding samples (Fig. 5 and 6). Interestingly, a strong-to-medium correlation was found between the neutralization capacity and IgG levels (Total) in the Pfizer (Fig. 5) group in all time points post-vaccination. R2 = 0.65, 0.64 and 0.71. In addition, a low correlation was noted in the neutralization capacity between IgG 1, 2, and 3 at all tested time points post-vaccination (Fig. 5). Similar to the Pfizer group, a strong-to-medium correlation was found between neutralization capacity and IgG levels (Total) in the Moderna (Fig. 6) groups at all tested time points post-vaccination. R2 = 0.75, 0.51 and 0.59. At 4 weeks post-first dose, a modest correlation was noted between neutralization capacity and the levels of IgG1 and IgG3 (R2=0.4 and 0.3, respectively) in the Moderna group. The correlation was low for the other tested time points in all IgG subtypes in the Moderna group.
Neutralizing antibodies against S1 or S-RBD display a broad-spectrum effect against several strains. The neutralization capacity of antibodies in the Moderna group (Fig. 7A) was about2-fold higher than the Pfizer group (Fig. 7B) at 4 weeks post-first dose for the wildtype SARS-CoV-2 (S1 and S-RBD). A similar trend in the neutralization capacity was noted between the Moderna and Pfizer groups for all the nine SARS-CoV-2 variants tested (1.8 to 3.7-fold). Both Pfizer and Moderna vaccines elicited the highest neutralization capacity at 4 weeks post-second dose (Fig. 7A &B). However, the neutralization capacity waned at 26 weeks post-second dose vaccination, and the reduction was more in the Pfizer group (63-81%) than in the Moderna group (51-57%) across the tested wild type and variants of SAR-CoV-2.