Intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2 induces neutralizing salivary IgA.

Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable "sterilizing immunity" at the mucosal level. Our study uncovers a strong temporary neutralizing mucosal component of immunity, emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus-2 spike protein and demonstrate that these IgAs mediate neutralization. RBD-targeting IgAs were found to associate with the secretory component, indicating their bona fide transcytotic origin and their polymeric multivalent nature. The mechanistic understanding of the high neutralizing activity provided by mucosal IgA, acting at the first line of defense, will advance vaccination design and surveillance principles and may point to novel treatment approaches and new routes of vaccine administration and boosting.

Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of three proteins from SARS-CoV-2.

Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and nucleocapsid (N) proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and obtained cross-link sets with an average density of one cross-link per 20 residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and interdomain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryogenic electron microscopy structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair.

APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy.

Function of heparanase in prostate tumorigenesis: potential for therapy.

PURPOSE: Heparanase is the predominant enzyme that cleaves heparan sulfate, the main polysaccharide in the extracellular matrix. Whereas the role of heparanase in sustaining the pathology of human cancer is well documented, its association with prostate carcinoma remains uncertain. Our research was undertaken to elucidate the significance of heparanase in prostate tumorigenesis and bone metastasis. EXPERIMENTAL DESIGN: We applied immunohistochemical analysis of tissue microarray, in vitro adhesion and invasion assays, as well as mouse models of intraosseous growth and spontaneous metastasis of prostate cancer, monitored by whole-body bioluminescent imaging. Electroporation-assisted administration of anti-heparanase small interfering RNA in vivo was applied as a therapeutic approach. RESULTS: We report a highly statistically significant (P < 0.0001) prevalence of heparanase overexpression in prostate carcinomas versus noncancerous tissue, as well as strong correlation between tumor grade and the extent of heparanase expression. We observed >5-fold increase in the metastatic potential of PC-3 prostate carcinoma cells engineered to overexpress heparanase. Notably, overexpression of a secreted form of the enzyme also led to a dramatic increase in intraosseous prostate tumor growth. Local in vivo silencing of heparanase resulted in a 4-fold inhibition of prostate tumor growth, representing the first successful application of anticancer therapy based on heparanase small interfering RNA and validating the potential of heparanase as a target for prostate cancer treatment. CONCLUSIONS: Heparanase directly contributes to prostate tumor growth in bone and its ability to metastasize to distant organs. Thus, anti-heparanase strategy may become an important modality in the treatment of prostate cancer patients, particularly those with bone metastases.