Anti-SARS-CoV-2 Omicron BA.5 RBD antibody titers generated by the protein subunit vaccine Abdala in breast milk (2024)

This study involved 104 puerperal women aged between 15 and 41 years. Fewer of the participants included in groups A and B had at least one NCD. The most common NCD were arterial hypertension and asthma. Nulliparity predominated in the members of both groups (Table 1).

IgA and IgG antibodies against RBDs of Wuhan-Hu-1 and Omicron BA.5 of SARS-CoV-2 were detected in all breast milk samples. The homemade ELISA was considered valid to reveal specific signals of the antibodies that recognize both RBDs, since the inhibition percentages in the specificity assay were greater than 99.8% in the upper half of the sample mixture dilution range, and remained above 92% in the other half of the range (Table 2).

The antibody titers against RBD-Wuhan-Hu-1 were always significantly higher than those against Omicron BA.5, regardless of the post-vaccination time (Fig. 1). The table adjacent to Fig. 1 shows that the confidence intervals (95%) of the difference of the titer mean logarithms did not include zero, and it confirmed the significant differences between titers against both RBD sequences.

In all samples, IgA titers decreased from the fifth to the ninth week post-vaccination. Five weeks after vaccination, IgA anti-RBDOmic titers were lower by 9.86% compared to RBDWuh. After 9 weeks, IgA anti-RBDOmic titers were lower by 7.91% in respect to RBDWuh.

All samples presented IgG anti-RBDWuh and anti-RBDOmic. Unlike the observed reduction in IgA titers from the fifth to the ninth week after vaccination, IgG titers increased, on average, more than 6-fold in samples collected 9 weeks post-vaccination. Antibody titers against RBDWuh were also significantly higher than against RBDOmic (Fig. 1). In the fifth week, the IgG titers against RBDWuh were 3.64% higher than against RBDOmic; in the ninth week, an increase of 24% was detected in the IgG anti-RBDWuh titers compared to anti-RBDOmic.

When the samples were organized into two independent subgroups, according to the health status of the participants, the antibody responses between the subgroups were significantly different (Fig. 2). IgA and IgG titers against both RBDs were lower in the subgroup of participants with NCD. The significant differences in the antibody response to both RBDs were confirmed by not including zero in the confidence intervals informed in the table adjacent to Fig. 2.

According to the Pearson's test, there was a moderate negative correlation between the parity of the study participants at the beginning of vaccination and the IgA and IgG antibody responses against RBDWuh and RBDOmic. The correlations were significant in breast milk samples collected at 5- and 9-weeks post-vaccination (Table 3).

The COVID-19 protein subunit vaccine Abdala generates anti-RBD antibody responses, of the IgA and IgG isotypes, in breast milk; not only against the RBD with which it was designed, the Wuhan-Hu-1 RBD, but against the Omicron BA.5, one of the last declared SARS-CoV-2 VOCs.

The detection of anti-RBD antibodies in all samples from groups A and B does not mean that the ELISA revealed non-specific A492nm signals, due to interference from any component of the assay. The specificity of the ELISA was demonstrated in a wide range of dilutions of the sample mixture with anti-RBD antibodies, in the presence of an antigen excess in solution (15 μg/mL of RBD-Wuhan-Hu-1 or RBD-Omicron BA.5). The inhibition percentages were 100% at almost half of the dilution points, including the highest, 1:100, which was the dilution applied to the samples in the screening of anti-RBD antibodies. The antigen–antibody reaction in solution allowed the recognition of epitopes that are not exposed when the antigen is coating the solid phase of the plate. A 100% inhibition rate means that all specific anti-RBD antibodies in the sample mixture reacted with the epitope conformations of the antigen, during the incubation in solution, and that no free antibodies remained to bind to the RBD coating the plate; consequently, the A492nm signals, adjusted with blank signal, were zero. Therefore, no other component of the ELISA gave rise to signals unrelated to the detection of specific antibodies.

In the breast milk samples evaluated in the present study, the anti-RBDWuh IgA and IgG antibody responses generated by Abdala, and their potential neutralization activity, had already been described.18 But these samples are also valuable to demonstrate that the vaccine could generate a specific antibody response against the Omicron BA.5 subvariant, since they were obtained between September 30 and November 25, 2021, and Omicron variant was declared VOC on November 26 of that year.2 Accordingly, the Omicron BA.5 subvariant was not yet circulating in Cuba at the time of sampling for the study.

Omicron BA.5 RBD contains mutations that make it very different from the Wuhan-Hu-1 RBD that Abdala contains.6,7 However, this vaccine is capable of generating antibodies in breast milk that recognize Omicron BA.5. One of the reasons for this antibody response could be related to the glycosylation in the RBD of Abdala, which is expressed in the methylotrophic yeast P. pastoris. N-glycans from P. pastoris are often hypermannosylated,19 and mannosylation enhances the activation of antigen-presenting cells, such as dendritic cells and macrophages, and increases immunogenicity relative to their non-glycosylated counterparts.20 Moreover, the 2 N-glycosylation sites in RBD of Abdala are at 331 and 343. Liu and Wilson21 have described that most residues in N343 are more conserved compared to others, and this site is exposed regardless of whether the RBD is up, down, or in intermediate configurations. Glycosylation modulates the conformation or exposure of peptide epitopes, as a result of intramolecular carbohydrate-protein interactions.22 Therefore, the impact of glycosylation of Abdala RBD could be, in addition to the immunogenicity increasing, on the exposure of epitopes that are conserved in pandemic variants, such as Omicron BA.5, and that enables the response of specific antibodies against them.

Anti-RBDOmic IgA and IgG antibody titers were significantly lower than anti-RBDWuh. Our results are consistent with all published studies that have evaluated the antibody response generated by COVID-19 vaccines against the SARS-CoV-2 Omicron variant. But none of them involved vaccinees with protein subunit vaccines.

Bartsch et al. showed reduced Omicron isotype-specific immunity after mRNA vaccination of pregnant women. Stable anti-spike IgG binding antibodies against WT, Alpha, Beta, Delta, and Omicron were found after vaccination with Pfizer/BioNTech BNT162b2, whereas the IgA response to the Omicron variant was significantly reduced. Moderna mRNA-1273 vaccination generated the most consistent IgG responses and highest IgA responses among pregnant women across all VOCs, but responses to the Omicron variant were again the lowest.10

The BNT162b2 vaccine administered in 3 doses, like Abdala, considerably increased the neutralization efficacy of the Omicron variant.11 Other authors confirmed the importance of administering the third dose of the vaccine to improve efficacy against Omicron.8,9 However, it would be recommended an additional booster dose in pregnant women, to ensure higher anti-RBDOmic antibody titers in breast milk that support the passive immunity against this SARS-CoV-2 variant in newborns and infants.

Another finding of this study was the significant inverse relationship between the antibody titers against both RBDs and the presence of NCD in the study participants. Arterial hypertension was the most frequent chronic disease found in the participants. Some researchers have described the decrease of antibody titers in patients with hypertension in response to COVID-19 mRNA vaccine23 and to inactivated SARS-CoV-2 vaccine,24 agreeing with the results of the present work. The reason for this behavior could be in the fact that hypertension has been related with systemic inflammation. Chronic systemic inflammation can alter tissue, organ, and cellular functions, and it lead to impaired immune function, thereby affecting the response to vaccines.25

A recent study published the immune responses to the BNT162b2 vaccine among indigenous and non-indigenous people, according to comorbidity and ethnicity. Among all participants with chronic medical conditions, lower anti-RBD serum titers were associated with the chronic diseases,26 which are also in line with our results. But other authors found no significant differences in anti-RBD antibody titers associated with comorbidities, such as hypertension or obesity, after mRNA vaccine administration.27–29 Thus, future studies are required to substantiate one or the other trend.

A moderate negative correlation was established between the IgA and IgG antibody titers and the parity at the beginning of vaccination. A moderate correlation means that there is a relationship between the parity and the antibody titers, but that there is also a lot of randomness affecting one or both variables. Or, perhaps another variable could affect both variables in question. Therefore, the direct relationship is not strong but it is certainly significant.

Parity has been associated with a significant modification of breast milk composition.30 Kaplan et al. affirmed that IgA levels in breast milk may fluctuate depending on the particular characteristics of the mother, and parity can directly influence. These authors found that the IgA concentration in colostrum of primiparous mothers (35.95 g/L) was significantly higher compared to multiparous ones (27.33 g/L).31 The inverse correlation between parity and IgA levels coincides with our results and with those informed by Weaver et al.,32 Akhter et al.,33 and Trofin et al.34 Nevertheless, full understanding the mechanisms involved in the parity-associated immune response awaits further studies.

The declining trends observed in the antibody response after vaccination with 3 doses of Abdala, due to the presence of NCD or multiparity, implies an alert that once again calls for systematic control of the health status of vulnerable pregnant women, and the strict compliance with the medical treatment, for ensuring the stability of their biochemical and physiological parameters, in order to reduce the risk of SARS-CoV-2 infection.

This is the first report of simultaneous detection, by a homemade ELISA, of antibody titers against the RBD-Wuhan-Hu-1 and the RBD-Omicron BA.5 in breast milk, collected from puerperal women vaccinated with 3 doses of a COVID-19 protein subunit vaccine, based on the sequence of the RBD-Wuhan-Hu-1. IgA and IgG antibodies are detected that bind to RBD-Omicron BA.5, despite various mutations that make it very different from RBD-Wuhan-Hu-1. The glycosylation of the Abdala RBD, expressed in P. pastoris, support the immune response against VOCs like Omicron. Health status and parity are factors involved in the response to Abdala in breast milk. Having at least one chronic disease and being multiparous could reduce the anti-RBD antibodies titers generated by the vaccine, which translates into caring for pregnant women who may be vulnerable to COVID-19 for one or both of these causes.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

M.P.B.: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing − review & editing. C.H.: Formal analysis, Methodology, Validation, Writing − review & editing. R.I.: Resources, Software. M.M. and M.S.: Resources. M.D. and D.D.: Investigation. E.P. and J.M.S.R.: Project administration, Supervision. All authors have approved the final article.

Ethical approval was granted by the Institutional Review Board from the General Hospital “Camilo Cienfuegos”, Sancti Spiritus, Cuba, on August 23, 2021.