The Anti-SARS-CoV-2 IgG1 and IgG3 Antibody Isotypes with Limited Neutralizing Capacity against Omicron Elicited in a Latin Population a Switch toward IgG4 after Multiple Doses with the mRNA Pfizer-BioNTech Vaccine

Abstract

The aim of this study was to analyze the profiles of IgG subclasses in COVID-19 convalescent Puerto Rican subjects and compare these profiles with those of non-infected immunocompetent or immunocompromised subjects that received two or more doses of an mRNA vaccine. The most notable findings from this study are as follows: (1) Convalescent subjects that were not hospitalized developed high and long-lasting antibody responses. (2) Both IgG1 and IgG3 subclasses were more prevalent in the SARS-CoV-2-infected population, whereas IgG1 was more prevalent after vaccination. (3) Individuals that were infected and then later received two doses of an mRNA vaccine exhibited a more robust neutralizing capacity against Omicron than those that were never infected and received two doses of an mRNA vaccine. (4) A class switch toward the "anti-inflammatory" antibody isotype IgG4 was induced a few weeks after the third dose, which peaked abruptly and remained at high levels for a long period. Moreover, the high levels of IgG4 were concurrent with high neutralizing percentages against various VOCs including Omicron. (5) Subjects with IBD also produced IgG4 antibodies after the third dose, although these antibody levels had a limited effect on the neutralizing capacity. Knowing that the mRNA vaccines do not prevent infections, the Omicron subvariants have been shown to be less pathogenic, and IgG4 levels have been associated with immunotolerance and numerous negative effects, the recommendations for the successive administration of booster vaccinations to people should be revised.

Keywords: COVID-19; ELISA; IgG4; class switch; neutralizing antibody.

Figure 1

Receiver Operating Characteristic (ROC) curve and OD distribution. (A) ROC curve analysis of in-house anti-SARS-CoV-2 IgG ELISA performance including the are under curve (AUC) and 95% confidence interval 9CI). (B) OD distribution of sera/plasma from COVID-19 subjects, negative controls and from individuals with other respiratory or viral infections. (C) OD distribution of the four IgG subclasses of anti-SARS-CoV-2 IgG antibody detected in samples from COVID-19 subjects and negative control samples. **** p < 0.0001, ** p = 0.0025, ns = No significant differences.

Figure 2

OD distribution of specimens and number of positive samples from unvaccinated COVID-19 convalescent subjects. (A) OD distribution of samples from unvaccinated, convalescent COVID-19 subjects tested using in-house IgG and IgG subclass ELISAs, which were allotted into three groups (1–30 days, 31–60 days, and >60 days since the date of confirmatory SARS-CoV-2 RT-PCR testing). There were no significant differences (ns) between OD values of the different IgG isotypes regarding to the time of infection. (B) Number of seropositive samples for each IgG subclass according to the days after infection.

Figure 3

Correlation between the levels of IgG subclasses and the neutralizing activity. The OD values obtained for each IgG subclass was correlated with the surrogate virus neutralization percentage (sVNT%) obtained against (A) the wild type of strain and the VOCs (B) Alpha, (C) Delta, and (D) Omicron.

Figure 4

Levels of IgG subclasses and neutralizing capacity in serial samples from previously infected subjects that received two doses of mRNA vaccine. (A) The OD distribution of serial samples collected from previously infected subjects that received two doses of the Pfizer-BioNTech (n = 6) or (B) Moderna-1273 (n = 6) vaccines are showed. Sample 1 was collected before vaccination (baseline0, sample 2 was collected at a median of 21.5 days after the second dose, and sample 3 was collected at a median of 96 days after the second dose. Black dashed line represents the positive cut-off value for each in-house IgG subclass ELISA. (C) The average OD values for each sample from subjects that received the Pfizer-BioNTech or Moderna-1273 vaccine associated with (D) the corresponding average surrogate virus neutralization percentage (sVNT%) are showed.

Figure 5

Changes in IgG subclass levels in naive subjects that received multiple vaccinations with the Pfizer–BioNTech vaccine show a switch toward the IgG4 subclass. (A) Six samples were collected from four (n = 4) naive subjects that received multiple vaccinations with the Pfizer–BioNTech vaccine (Cohort 3a). Sample 1 was collected at baseline, sample 2 was collected at a median of 20 days after the second dose, sample 3 was collected at a median of 31 days after the third dose, sample 4 was collected at a median of 255 days after the third dose, sample 5 was collected at a median of 30 days after the bivalent vaccine, and sample 6 was collected at a median of 180 days after the bivalent vaccine. Red arrows indicate the time at which the third dose or bivalent vaccine was administered to each subject. (B) A single sample from eight (n = 8) subjects (Cohort 3b), who received three doses of the Pfizer–BioNTech vaccine, was collected at a median of 277.5 days (~9 months) following the third Pfizer–BioNTech dose and for the levels of the IgG subclasses were measured. (C) OD distribution of each IgG subclass in the samples of Cohort 3b clearly demonstrate the dominance and durability of IgG4 response in subjects that received multiple doses of the Pfizer–BioNTech vaccine. **** p < 0.0001.

Figure 6

Changes in the neutralizing capacity in specimens from naive subjects that received multiple vaccinations with the Pfizer–BioNTech vaccine. Serial samples were collected from four naive subjects (n = 4) that received multiple vaccinations and tested using a surrogate neutralization assay to quantify the viral neutralization percentage (sVNT%) against the wild-type strain and the variants of concern (VOCs) Alpha, Delta, and Omicron. Sample 1 was collected at baseline, sample 2 was collected at a median of 20 days after the second dose, sample 3 was collected at a median of 31 days after the third dose, sample 4 was collected at a median of 255 days after the third dose, sample 5 was collected at a median of 30 days after the bivalent vaccine, and sample 6 was collected at a median of 180 days after the bivalent vaccine. Black dashed line represents the positive cut-off for the sVNT% (>30%). *** p = 0.0002, **** p = 0.0001. ns = No significant differences.

Figure 7

Changes in IgG subclasses in immunocompromised subjects that received multiple vaccinations with the Pfizer–BioNTech vaccine. Four samples were collected from six subjects (n = 6) with inflammatory bowel disease (IBD), who received standard treatment with immunomodulators or biologics. Sample 1 was collected at baseline, sample 2 was collected at a median of 15 days after the second dose, sample 3 was collected at a median of 60 days after the third dose, and sample 4 was collected at a median of 180 days after the third dose (Panel A). The neutralizing capacity of these samples, measured as the surrogate virus neutralization percentages (sVNT%) against the wild-type strain (WT) and the VOCs Alpha, Delta, and Omicron, is showed in (Panel B). *** p = 0.0002. ns = No significant differences.