Host receptor-targeted therapeutic approach to counter pathogenic New World mammarenavirus infections

Five New World mammarenaviruses (NWMs) cause life-threatening hemorrhagic fever (HF). Cellular entry by these viruses is mediated by human transferrin receptor 1 (hTfR1). Here, we demonstrate that an antibody (ch128.1/IgG1) which binds the apical domain of hTfR1, potently inhibits infection of attenuated and pathogenic NWMs in vitro. Computational docking of the antibody Fab crystal structure onto the known structure of hTfR1 shows an overlapping receptor-binding region shared by the Fab and the viral envelope glycoprotein GP1 subunit that binds hTfR1, and we demonstrate competitive inhibition of NWM GP1 binding by ch128.1/IgG1 as the principal mechanism of action. Importantly, ch128.1/IgG1 protects hTfR1-expressing transgenic mice against lethal NWM challenge. Additionally, the antibody is well-tolerated and only partially reduces ferritin uptake. Our findings provide the basis for the development of a novel, host receptor-targeted antibody therapeutic broadly applicable to the treatment of HF of NWM etiology.


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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. For in vitro inhibition studies ( Fig. 1), sample sizes were determined based on accepted standards of 3-4 biological replicates per experimental group. For the biolayer interferometry experiments (Fig. 2), no sample size calculations were performed to power the experiment, and no statistical methods were used to predetermine sample size. For the in vivo efficacy studies (Figs. 4 and 5), the number of mice per group was based on power analysis performed using commonly accepted values for type I error (0.05) and power (80%). For the progenitor assays ( Fig. 6 and Supplementary Fig. 1), no sample size calculations were performed to power the experiment, and no statistical methods were used to predetermine sample size. The experiment was conducted according to the manufacturer's recommendations. The assay is normally performed with duplicates or triplicates so our use of quadruplicates is sufficient. This experiment was performed three times using BMMC from different donors. For the flow cytometry experiment (Fig. 7), no sample size calculations were performed to power the experiment, and no statistical methods were used to predetermine sample size. Flow cytometry assays are not performed in replicates due to the high number of acquisition events collected per sample (we collected data for 10,000 events, which is common for simple binding studies that were performed). For the imaging flow cytometry experiments (Figs. 8 and 9), no sample size calculations were performed to power the experiment, and no statistical methods were used to predetermine sample size. Flow cytometry assays (including imaging studies) are not performed in replicates due to the high number of acquisition events collected per sample (we collected data for 10,000 events, which is common for this type of imaging study). Multiple images are obtained for each event (brightfield and fluorescent images) so the amount of data collected is very large.
No data were excluded from any of the analyses Inhibitory activity of the ch128.1 antibody was reproduced versus 2 strains of Junin virus (Fig. 1). For the biolayer interferometry assessment, the experiment was performed twice with similar results (Fig. 2). Efficacy of the ch128.1 and mutant antibodies was reproduced in a second mouse efficacy experiment (Figs. 4 and 5). The progenitor assay was performed 3 times with 3 different BMMC donors with reproducible results ( Fig. 6 and Supplementary Fig. 1). The standard flow cytometric study with MM.1S cells (Fig. 7) was performed twice with reproducible outcomes. Imaging flow cytometry experiments (Figs. 8 and 9) were performed one time at the 37 degree C incubation temperature. Repeat trials were performed at room temperature with similar results. Because the imaging flow cytometry assay was performed to confirm the standard flow cytometry data (Fig. 7), and to show the internalization of H-Ft or transferrin in addition to just binding, multiple independent trials were not deemed necessary.
For the in vitro inhibition (Fig. 1) and biolayer interferometry (Fig. 2) experiments, sample allocation was not randomized because the results were quantitative and did not require subjective judgment or interpretation. For in vivo efficacy studies, 3-week-old mice were sorted by sex (covariate) prior to infection so that all treatment groups consisted of equivalent percentages of males and females. For the progenitor study (Fig. 6), sample allocation was not randomized because BMMC are from the same donor for all conditions tested and the results were quantitative and did not require subjective judgment or interpretation. For the standard flow cytometry experiment (Fig. 7), sample randomization is not applicable because a single cell population of erythroblasts was used. Additionally, the results were quantitative and did not require subjective judgment or interpretation. For the imaging flow cytometry assay (Figs. 8 and 9), sample randomization is not applicable because a single cell line (MM.S1) was used. Additionally, the results were quantitative and did not require subjective judgment or interpretation.
Investigators at Utah State University were not blinded for studies with the pathogenic JUNV Romero strain due to logistical reasons and limited personnel vaccinated with Candid#1 (requirement to work with the virus). Additionally, the results were quantitative and did not require subjective judgment or interpretation. The investigators at Harvard Medical School were not blinded during experiments or to outcome assessment because the results were quantitative and did not require subjective judgment or interpretation. Blinding is also not typically used in the field for similar biolayer interferometry-based competition assays. Researchers at UCLA were not blinded for the progenitor or flow cytometry studies. Counting of colonies for the progenitor assay was not blinded, but was performed by one person who is trained on the identification of such colonies. Blinding was not deemed necessary and is not typically used for flow cytometry studies as the The antibodies targeting TfR1 (ch128.1/IgG1, ch128.1/IgG1 mutant and monoclonal 128.1), as well as the isotype negative control (anti-dansyl IgG1), were produced in murine hybridoma or myeloma cells grown in roller bottles and purified using affinity chromatography. They were validated using BCA assay to assess concentration and SDS-PAGE under non-reducing and reducing conditions to assess the molecular weight and proper assembly of light and heavy chains. In addition, binding to antigen and species specificity was assessed by ELISA and flow cytometry. See the Methods section of the paper for citations.