The fourth vaccination with a non-SARS-CoV-2 variant adapted vaccine fails to increase the breadth of the humoral immune response

Abstract Escape mutations in the spike protein of SARS-CoV-2 are a major reason for Omicron breakthrough infections. After basal vaccination only very low titers of Omicron neutralizing antibodies are present. However, booster vaccinations induce higher titers against the Omicron variant. The neutralization of the Delta and Omicron variants by sera obtained 6 months after 3rd vaccination and 2 weeks or 6 months after 4th vaccination with a monovalent RNA vaccine (Spikevax) was analyzed. It was observed for the Omicron variant that 6 months after the fourth vaccination, the titer returns to the same very low neutralizing capacity as 6 months after the third vaccination. The Delta variant neutralizing capacity wanes with a comparable kinetic although the titers are higher as compared to the Omicron variant. This indicates that the fourth vaccination with a monovalent vaccine based on the ancestral isolate neither affects the kinetic of the waning nor the breadth of the humoral response

Comirnaty-Elicited and Convalescent Sera Recognize Different Spike Epitopes

Many of the approved SARS-CoV-2 vaccines are based on a stabilized variant of the spike protein. This raises the question of whether the immune response against the stabilized spike is identical to the immune response that is elicited by the native spike in the case of a SARS-CoV-2 infection. Using a peptide array-based approach, we analysed the binding of antibodies from Comirnaty-elicited, convalescent, and control sera to the peptides covering the spike protein. A total of 37 linear epitopes were identified. A total of 26 of these epitopes were almost exclusively recognized by the convalescent sera. Mapping these epitopes to the spike structures revealed that most of these 26 epitopes are masked in the pre-fusion structure. In particular, in the conserved central helix, three epitopes that are only exposed in the post-fusion conformation were identified. This indicates a higher spike-specific antibody diversity in convalescent sera. These differences could be relevant for the breadth of spike-specific immune response

The SARS-CoV-2 Variant Omicron Is Able to Escape Vaccine-Induced Humoral Immune Responses, but Is Counteracted by Booster Vaccination

The SARS-CoV-2 variant Omicron has spread world-wide and is responsible for rapid increases in infections, including in populations with high vaccination rates. Here, we analysed in the sera of vaccinated individuals the antibody binding to the receptor-binding domain (RBD) of the spike protein and the neutralization of wild-type (WT), Delta (B.1.617.2), and Omicron (B.1.1.529; BA.1) pseudotyped vectors. Although sera from individuals immunized with vector vaccines (Vaxzevria; AZ and COVID-19 Janssen, Ad26.COV2.S; J&J) were able to bind and neutralize WT and Delta, they showed only background levels towards Omicron. In contrast, mRNA (Comirnaty; BNT) or heterologous (AZ/BNT) vaccines induced weak, but detectable responses against Omicron. While RBD-binding antibody levels decreased significantly six months after full vaccination, the SARS-CoV-2 RBD-directed avidity remained constant. However, this still coincided with a significant decrease in neutralization activity against all variants. A third booster vaccination with BNT significantly increased the humoral immune responses against all tested variants, including Omicron. In conclusion, only vaccination schedules that included at least one dose of mRNA vaccine and especially an mRNA booster vaccination induced sufficient antibody levels with neutralization capacity against multiple variants, including Omicron

Quantitative and Qualitative Difference in Antibody Response against Omicron and Ancestral SARS-CoV-2 after Third and Fourth Vaccination

Waning immunity against SARS-CoV-2 and the emergence of variants, especially of the most distant variant, Omicron, affect titers of neutralizing antibodies in the sera of vaccinated individuals. Thus, two vaccinations with the mRNA vaccine BNT162b fail to induce neutralizing antibodies against the Omicron variant. A first booster vaccination increases Omicron-RBD-binding IgG and IgA and neutralizing capacity. In comparison, the Wuhan isolate titers of the Omicron variant binding antibodies are 8.5 lower. After a third vaccination, induction of Omicron-RBD- and Wuhan-RBD-binding antibodies follows the same kinetic. Five to six months after the third vaccination, there are still Omicron-RBD-binding antibodies detectable, but 35.9 percent of the analyzed sera fail to neutralize the Omicron variant, while all sera efficiently neutralize the Delta isolate. In the case of the Wuhan-RBD, a significantly larger number of stable antigen–antibody complexes is formed than in Omicron-RBD. A fourth vaccination with mRNA-1273 temporarily restores levels of Omicron-, Delta- and Wuhan-specific antibodies. Comparing different booster strategies revealed that the breadth of the immune response is not affected by the vaccination regimen. Taken together, these data indicate that booster vaccinations (third and fourth dose) increase the breadth of the immune response, but there is a qualitative difference of antibodies with respect to the stability of antigen–antibody complexes and persistence of antibody titers

Comparative Investigation of Methods for Analysis of SARS-CoV-2-Spike-Specific Antisera

In light of an increasing number of vaccinated and convalescent individuals, there is a major need for the development of robust methods for the quantification of neutralizing antibodies; although, a defined correlate of protection is still missing. Sera from hospitalized COVID-19 patients suffering or not suffering from acute respiratory distress syndrome (ARDS) were comparatively analyzed by plaque reduction neutralization test (PRNT) and pseudotype-based neutralization assays to quantify their neutralizing capacity. The two neutralization assays showed comparable data. In case of the non-ARDS sera, there was a distinct correlation between the data from the neutralization assays on the one hand, and enzyme-linked immune sorbent assay (ELISA), as well as biophysical analyses, on the other hand. As such, surface plasmon resonance (SPR)-based assays for quantification of binding antibodies or analysis of the stability of the antigen–antibody interaction and inhibition of syncytium formation, determined by cell fusion assays, were performed. In the case of ARDS sera, which are characterized by a significantly higher fraction of RBD-binding IgA antibodies, there is a clear correlation between the neutralization assays and the ELISA data. In contrast to this, a less clear correlation between the biophysical analyses on the one hand and ELISAs and neutralization assays on the other hand was observed, which might be explained by the heterogeneity of the antibodies. To conclude, for less complex immune sera—as in cases of non-ARDS sera—combinations of titer quantification by ELISA with inhibition of syncytium formation, SPR-based analysis of antibody binding, determination of the stability of the antigen–antibody complex, and competition of the RBD-ACE2 binding represent alternatives to the classic PRNT for analysis of the neutralizing potential of SARS-CoV-2-specific sera, without the requirement for a BSL3 facility