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.

TopoBuilding precision vaccines: To induce strong and targeted neutralizing antibody (nAb) responses against vaccine targets, one strategy has been to use computationally designed immunogens. However, the structural complexity of many known neutralization epitopes has posed a major challenge for the design of accurate epitope mimetics. Sesterhenn et al. created a protein design algorithm called TopoBuilder to design scaffolds for irregular and discontinuous neutralization epitopes. As a proof of principle, the authors generated epitope-focused immunogens based on the prefusion conformation of the respiratory syncytial virus (RSV) fusion protein. When these immunogens were used to vaccinate mice and nonhuman primates in RSV infection models, they generated targeted nAb responses to RSV and boosted site-specific nAb responses in heterologous prime-boost vaccination schemes. Science , this issue p. eaay5051

Sexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell. The sperm-restricted single-pass transmembrane protein HAP2-GCS1 has been postulated to function in membrane merger. Its presence in the major eukaryotic taxa-animals, plants, and protists (including important human pathogens like Plasmodium)-suggests that many eukaryotic organisms share a common gamete fusion mechanism. Here, we report combined bioinformatic, biochemical, mutational, and X-ray crystallographic studies on the unicellular alga Chlamydomonas reinhardtii HAP2 that reveal homology to class II viral membrane fusion proteins. We further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion. These results demonstrate that HAP2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion, indicating a way to block Plasmodium transmission and highlighting the impact of virus-cell genetic exchanges on the evolution of eukaryotic life.

HAPLESS2 (HAP2) is a broadly conserved, gamete-expressed transmembrane protein that was shown recently to be structurally homologous to viral class II fusion proteins, which initiate fusion with host cells via insertion of fusion loops into the host membrane. However, the functional conformation of the HAP2 fusion loops has remained unknown, as the reported X-ray structure of Chlamydomonas reinhardtii HAP2 lacked this critical region. Here, we report a structure-guided alignment that reveals diversification of the proposed HAP2 fusion loops. Representative crystal structures show that in flowering plants, HAP2 has a single prominent fusion loop projecting an amphipathic helix at its apex, while in trypanosomes, three small nonpolar loops of HAP2 are poised to interact with the target membrane. A detailed structure-function analysis of the Arabidopsis HAP2 amphipathic fusion helix defines key residues that are essential for membrane insertion and for gamete fusion. Our study suggests that HAP2 may have evolved multiple modes of membrane insertion to accommodate the diversity of membrane environments it has encountered during eukaryotic evolution. The fusion of gamete plasma membranes is the fundamental cellular event that brings two parental cells together to form a new individual, yet we know surprisingly little about this process at the molecular level. HAPLESS 2 (HAP2) is a conserved sperm plasma membrane protein that is essential for gamete fusion in a diverse array of eukaryotes. It was recently shown to share a common ancestor with viral proteins that drive fusion of the viral envelope with host membranes, but its mechanism of action remained elusive, since the reported structure did not resolve the proposed membrane interaction surface. Here, we report two new HAP2 structures revealing that HAP2 has evolved diverse membrane interaction surfaces. In the flowering plants, HAP2 uses an amphipathic helix that presents nonpolar residues to the target membrane; in trypanosomes, the membrane interaction surface comprises three shallow nonpolar loops.

Infections with viral pathogens are widespread and can cause a variety of different diseases. In-depth knowledge about viral triggers initiating an immune response is necessary to decipher viral pathogenesis. Inflammasomes, as part of the innate immune system, can be activated by viral pathogens. However, viral structural components responsible for inflammasome activation remain largely unknown. Here we analyzed glycoproteins derived from SARS-CoV-1/2, HCMV and HCV, required for viral entry and fusion, as potential triggers of NLRP3 inflammasome activation and pyroptosis in THP-1 macrophages. All tested glycoproteins were able to potently induce NLRP3 inflammasome activation, indicated by ASC-SPECK formation and secretion of cleaved IL-1β. Lytic cell death via gasdermin D (GSDMD), pore formation, and pyroptosis are required for IL-1β release. As a hallmark of pyroptosis, we were able to detect cleavage of GSDMD and, correspondingly, cell death in THP-1 macrophages. CRISPR-Cas9 knockout of NLRP3 and GSDMD in THP-1 macrophages confirmed and strongly support the evidence that viral glycoproteins can act as innate immunity triggers. With our study, we decipher key mechanisms of viral pathogenesis by showing that viral glycoproteins potently induce innate immune responses. These insights could be beneficial in vaccine development and provide new impulses for the investigation of vaccine-induced innate immunity.