Targeted isolation of diverse human protective broadly neutralizing antibodies against SARS-like viruses

The emergence of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed. Here, we utilized a targeted donor selection strategy to isolate a large panel of human broadly neutralizing antibodies (bnAbs) to sarbecoviruses. Many of these bnAbs are remarkably effective in neutralizing a diversity of sarbecoviruses and against most SARS-CoV-2 VOCs, including the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor-binding domain (RBD). Consistent with targeting of conserved sites, select RBD bnAbs exhibited protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model in vivo. These bnAbs provide new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and provide a molecular basis for effective design of pan-sarbecovirus vaccines

Relationship between tumor cell invasiveness and polyploidization.

A number of studies have shown that tumor cells fuse with other tumor and non-tumor cells. In the present study on tumor cell lines derived from glioblastoma, breast cancer, and melanoma, we estimated the frequency of fusion between tumor cells by establishing the fraction of cells with whole tumor-genome duplication in each cell line. Together with this, the capacity of the tumor cell lines to spread through a basement membrane scaffold was assessed, in order to test the hypothesis that pericellular proteolysis by enzymatic release in the spaces of intercellular contact could account for differences in the fusogenicity of tumor cells. The difference in invasiveness between the cell lines accounted for their specific amount of cells with tumor-genome duplication, which, depending on the cell line analyzed, ranged from 2% to 25% of the total cells. These results support the hypothesis that cell-to-cell invasion eliciting membrane fusion causes polyploidization in tumor cells

Relationship between invasiveness and polyploidization in tumor cells.

<p>Graph displaying a proportional ratio (r²>0.95) between the level of polyploidization in the tumor cell lines U87MG, FEMX-I, MDA-MB-231, and MA11, as expressed by the value of the gradient of the regression line, and average density of tumor cell invasion through matrigel.</p

Detection in human tumor cell lines of cells containing four-fold DNA amount.

<p>Flow cytometric analysis of cultures of the tumor lines U87MG, FEMX-I, MDA-MB-231, and MA11, at either low (top panels) or high (bottom panels) rate of proliferation, showing the distribution of cells according to DNA content, expressed as intensity of red fluorescence, and forward scatter (arbitrary units); the bar graph on top of each panel shows the percentage of cells in the G0/G1 peak (lowest DNA amount per cell), and that of cells with 2- (including cells in S phase), and 4-fold the intensity of the cells of the G0/G1 peak. The average fluorescence of each of the three populations is indicated by arrowheads. In all the cell lines, a greater number of cells with four-fold DNA quantity is present in the rapidly proliferating culture, representing 3%, 2%, 12%, and 1% of the total cells for U87MG, FEMX-I, MDA-MB-231, and MA11, respectively.</p

Relationship between cell proliferation in culture and number of tumor cells containing four-fold DNA amount.

<p>(A) Inverse relationship (r²>0.95) in U87MG, FEMX-I, MDA-MB-231, and MA11 cell lines, between the number of cells in the G0/G1 peak and that of cells carrying four-fold DNA amount, as quantitated in cultures of each cell line by flow cytometric analysis for content of DNA; both cell populations are expressed as fractions over the total number of cells analysed from each culture. (B) Lack of correlation in cultures of fibroblasts (top panel), <i>vs.</i> negative correlation in cultures of U87MG glioma cells (bottom panel), between number of cells in the G0/G1 peak and number of cells carrying four-fold DNA amount.</p

Matrigel invasion by U87MG, FEMX-I, MDA-MB-231, and MA11 tumor cell lines.

<p>Fluorescence microphotographs of representative areas of the underside of matrigel-coated membranes in transwell assays, showing that U87MG and MDA-MB-231 invade more efficiently through the matrigel, roughly by 4- and 7-fold, respectively, than MA11 and FEMX-I. The cells were fluorescently labeled with 4′,6-diamidino-2-phenylindole, appearing dark in the inverted images. Bars, 100 µm.</p

Disassembly of HIV envelope glycoprotein trimer immunogens is driven by antibodies elicited via immunization

Rationally designed protein subunit vaccines are being developed for a variety of viruses including influenza, RSV, SARS-CoV-2, and HIV. These vaccines are based on stabilized versions of the primary targets of neutralizing antibodies on the viral surface, namely, viral fusion glycoproteins. While these immunogens display the epitopes of potent neutralizing antibodies, they also present epitopes recognized by non-neutralizing or weakly neutralizing (“off-target”) antibodies. Using our recently developed electron microscopy polyclonal epitope mapping approach, we have uncovered a phenomenon wherein off-target antibodies elicited by HIV trimer subunit vaccines cause the otherwise highly stabilized trimeric proteins to degrade into cognate protomers. Further, we show that these protomers expose an expanded suite of off-target epitopes, normally occluded inside the prefusion conformation of trimer, that subsequently elicit further off-target antibody responses. Our study provides critical insights for further improvement of HIV subunit trimer vaccines for future rounds of the iterative vaccine design process.NIH (Grants UM1AI100663 and UM1AI144462)Bill and Melinda Gates Foundation (Grants OPP1115782, INV-002916, OPP1170236 and OPP1206647