Class switch toward noninflammatory, spike-specific IgG4 antibodies after repeated SARS-CoV-2 mRNA vaccination.

RNA vaccines are efficient preventive measures to combat the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. High levels of neutralizing SARS-CoV-2 antibodies are an important component of vaccine-induced immunity. Shortly after the initial two mRNA vaccine doses, the immunoglobulin G (IgG) response mainly consists of the proinflammatory subclasses IgG1 and IgG3. Here, we report that several months after the second vaccination, SARS-CoV-2-specific antibodies were increasingly composed of noninflammatory IgG4, which were further boosted by a third mRNA vaccination and/or SARS-CoV-2 variant breakthrough infections. IgG4 antibodies among all spike-specific IgG antibodies rose, on average, from 0.04% shortly after the second vaccination to 19.27% late after the third vaccination. This induction of IgG4 antibodies was not observed after homologous or heterologous SARS-CoV-2 vaccination with adenoviral vectors. Single-cell sequencing and flow cytometry revealed substantial frequencies of IgG4-switched B cells within the spike-binding memory B cell population [median of 14.4%; interquartile range (IQR) of 6.7 to 18.1%] compared with the overall memory B cell repertoire (median of 1.3%; IQR of 0.9 to 2.2%) after three immunizations. This class switch was associated with a reduced capacity of the spike-specific antibodies to mediate antibody-dependent cellular phagocytosis and complement deposition. Because Fc-mediated effector functions are critical for antiviral immunity, these findings may have consequences for the choice and timing of vaccination regimens using mRNA vaccines, including future booster immunizations against SARS-CoV-2.

Binding of Glycans to the SARS CoV-2 Spike Protein, an Open Question: NMR Data on Binding Site Localization, Affinity, and Selectivity.

We have used NMR experiments to explore the binding of selected glycans and glycomimetics to the SARS CoV-2 spike glycoprotein (S-protein) and to its receptor binding domain (RBD). STD NMR experiments confirm the binding of sialoglycans to the S-protein of the prototypic Wuhan strain virus and yield dissociation constants in the millimolar range. The absence of STD effects for sialoglycans in the presence of the Omicron/BA.1 S-protein reflects a loss of binding as a result of S-protein evolution. Likewise, no STD effects are observed for the deletion mutant Δ143-145 of the Wuhan S-protein, thus supporting localization of the binding site in the N-terminal domain (NTD). The glycomimetics Oseltamivir and Zanamivir bind weakly to the S-protein of both virus strains. Binding of blood group antigens to the Wuhan S-protein cannot be confirmed by STD NMR. Using 1 H,15 N TROSY HSQC-based chemical shift perturbation (CSP) experiments, we excluded binding of any of the ligands studied to the RBD of the Wuhan S-protein. Our results put reported data on glycan binding into perspective and shed new light on the potential role of glycan-binding to the S-protein.

BNT162b2-boosted immune responses six months after heterologous or homologous ChAdOx1nCoV-19/BNT162b2 vaccination against COVID-19.

Heterologous prime/boost vaccination with a vector-based approach (ChAdOx-1nCov-19, ChAd) followed by an mRNA vaccine (e.g. BNT162b2, BNT) has been reported to be superior in inducing protective immunity compared to repeated application of the same vaccine. However, data comparing immunity decline after homologous and heterologous vaccination as well as effects of a third vaccine application after heterologous ChAd/BNT vaccination are lacking. Here we show longitudinal monitoring of ChAd/ChAd (n = 41) and ChAd/BNT (n = 88) vaccinated individuals and the impact of a third vaccination with BNT. The third vaccination greatly augments waning anti-spike IgG but results in only moderate increase in spike-specific CD4 + and CD8 + T cell numbers in both groups, compared to cell frequencies already present after the second vaccination in the ChAd/BNT group. More importantly, the third vaccination efficiently restores neutralizing antibody responses against the Alpha, Beta, Gamma, and Delta variants of the virus, but neutralizing activity against the B.1.1.529 (Omicron) variant remains severely impaired. In summary, inferior SARS-CoV-2 specific immune responses following homologous ChAd/ChAd vaccination can be compensated by heterologous BNT vaccination, which might influence the choice of vaccine type for subsequent vaccination boosts.

NMR Experiments Provide Insights into Ligand-Binding to the SARS-CoV-2 Spike Protein Receptor-Binding Domain.

We have used chemical shift perturbation (CSP) and saturation transfer difference (STD) NMR experiments to identify and characterize the binding of selected ligands to the receptor-binding domain (RBD) of the spike glycoprotein (S-protein) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We also subjected full-length S-protein to STD NMR experiments, allowing correlations with RBD-based results. CSPs reveal the binding sites for heparin and fondaparinux, and affinities were measured using CSP titrations. We then show that α-2,3-sialyllactose binds to the S-protein but not to the RBD. Finally, combined CSP and STD NMR experiments show that lifitegrast, a compound used for the treatment of dry eye, binds to the linoleic acid (LA) binding pocket with a dissociation constant in the µM range. This is an interesting finding, as lifitegrast lends itself well as a blueprint for medicinal chemistry, eventually furnishing novel entry inhibitors targeting the highly conserved LA binding site.

Intranasal Delivery of MVA Vector Vaccine Induces Effective Pulmonary Immunity Against SARS-CoV-2 in Rodents.

Antigen-specific tissue-resident memory T cells (Trms) and neutralizing IgA antibodies provide the most effective protection of the lungs from viral infections. To induce those essential components of lung immunity against SARS-CoV-2, we tested various immunization protocols involving intranasal delivery of a novel Modified Vaccinia virus Ankara (MVA)-SARS-2-spike vaccine candidate. We show that a single intranasal MVA-SARS-CoV-2-S application in mice strongly induced pulmonary spike-specific CD8+ T cells, albeit restricted production of neutralizing antibodies. In prime-boost protocols, intranasal booster vaccine delivery proved to be crucial for a massive expansion of systemic and lung tissue-resident spike-specific CD8+ T cells and the development of Th1 - but not Th2 - CD4+ T cells. Likewise, very high titers of IgG and IgA anti-spike antibodies were present in serum and broncho-alveolar lavages that possessed high virus neutralization capacities to all current SARS-CoV-2 variants of concern. Importantly, the MVA-SARS-2-spike vaccine applied in intramuscular priming and intranasal boosting treatment regimen completely protected hamsters from developing SARS-CoV-2 lung infection and pathology. Together, these results identify intramuscular priming followed by respiratory tract boosting with MVA-SARS-2-S as a promising approach for the induction of local, respiratory as well as systemic immune responses suited to protect from SARS-CoV-2 infections.

Immune responses against SARS-CoV-2 variants after heterologous and homologous ChAdOx1 nCoV-19/BNT162b2 vaccination.

Currently approved viral vector-based and mRNA-based vaccine approaches against coronavirus disease 2019 (COVID-19) consider only homologous prime-boost vaccination. After reports of thromboembolic events, several European governments recommended using AstraZeneca's ChAdOx1-nCov-19 (ChAd) only in individuals older than 60 years, leaving millions of already ChAd-primed individuals with the decision to receive either a second shot of ChAd or a heterologous boost with mRNA-based vaccines. However, such combinations have not been tested so far. We used Hannover Medical School's COVID-19 Contact Study cohort of healthcare professionals to monitor ChAd-primed immune responses before and 3 weeks after booster with ChAd (n = 32) or BioNTech/Pfizer's BNT162b2 (n = 55). Although both vaccines boosted prime-induced immunity, BNT162b2 induced significantly higher frequencies of spike-specific CD4+ and CD8+ T cells and, in particular, high titers of neutralizing antibodies against the B.1.1.7, B.1.351 and P.1 variants of concern of severe acute respiratory syndrome coronavirus 2.