Characterization of therapeutic antibody efficacy against multiple SARS-CoV-2 variants in the hamster model

Abstract

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and onset of the coronavirus disease-19 (COVID-19) pandemic led to an immediate need for therapeutic treatment options. Therapeutic antibodies were developed to fill a gap when traditional antivirals were not available. In late 2020, the United States Government undertook an effort to compare candidate therapeutic antibodies in virus neutralization assays and in the hamster model of SARS-CoV-2 infection. With the emergence of SARS-CoV-2 variants, the effort expanded to evaluate the efficacy of nearly 50 products against major variants. A subset of products was further evaluated for therapeutic efficacy in hamsters. Here we report results of the hamster studies, including pathogenicity with multiple variants, neutralization capacity of products, and efficacy testing of products against Delta and Omicron variants. These studies demonstrate the loss of efficacy of early products with variant emergence and support the use of the hamster model for evaluating therapeutics.

Keywords: Efficacy evaluation; Hamster; Natural history study therapeutic antibody; SARS-CoV-2; Variants.

Fig. 1.. In vitro neutralization profiles of each candidate of therapeutic monoclonal antibodies, cocktails, and polyclonal antibodies against live SARS-CoV-2 variants.

A. Heatmap showing the NT₅₀ value of candidate products against 18 SARS-CoV-2 variants (columns). B. Heatmap representing fold-changes of NT₅₀ relative to SARS-CoV-2 WA-01. Codes assigned to the products are listed on left (rows) and categorized as monoclonal antibodies, cocktails, and polyclonal antibodies. Rows and columns have been clustered, and NT₅₀ is plotted for actual values for fold-changes using the heatmap package in Prism 10. SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; ND, not detected; /, not tested. NT₅₀, 50% neutralization titer.

Fig. 2.. Natural history hamster study design.

A Diagrammatic representation of the study design for all natural history studies. Animals were observed, weighed, and their temperatures recorded beginning three days prior to intranasal virus exposure. Blood was collected for downstream analyses at scheduled necropsies on days 3, 7, and 10 post-exposure (six animals per day). B List of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants tested in natural history studies, with animal group information, including number of animals in each cohort, exposure dose based on back-titration of the challenge stock, and scheduled study endpoints. IN, intranasal; M/F, male/female.

Fig. 3.. Hamsters exposed to Omicron BA.1 and BA.1.1 exhibited diminished clinical disease severity and reduced viral replication in the lungs.

Average body weight changes over the duration of the experiment. A The mean of each group is shown as a solid or dotted (controls) line. B Average daily clinical scores by group. Scores (on a scale of 0–5) were driven primarily by increased respiration rates of individual animals. C Average body temperatures by group. Significant statistical differences of % weight loss (A), clinical scores (B), and body temperatures (C) of groups at each time point were calculated with an ordinary one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test performed in GraphPad Prism 10 for up to 7 days post-exposure (no data from WA-01 on day 10). Significant differences from WA-01 values are indicated with as *—p ≤ 0.05; **—p ≤ 0.01; ***—p ≤ 0.001. D–F Kinetics of respiratory viral RNA detected by RT-qPCR in oropharyngeal samples (gene copies per swab; D) from days 1–3, along with nasal turbinate samples (E) and right lung tissues (F) obtained at euthanasia at 3, 7, and/or 10 days post-exposure. G, H severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral titer in right lung (G) and nasal turbinates (H). Data for panels D–H are presented as means ± SDs. Points indicate data from individual hamsters. A non-parametric test of one-way analysis of variance (ANOVA) (Kruskal–Wallis test) was performed, followed by Dunn’s multiple comparison test to determine the statistical significance of SARS-CoV-2 viral RNA of gene copies or tissue viral titers from all variants at each different time point. Data were log-transformed prior to statistical analysis. The number of asterisks corresponds to the level of significance: *indicates a significance level of p ≤ 0.05; ** indicates a significance level of p ≤ 0.01; *** indicates a significance level of p ≤ 0.001; **** indicates a significance level of p ≤ 0.0001. SgRNA, subgenomic RNA; LLOQ, lower limit of quantitation. (I) Histopathologic changes in lung disease severity over time among hamsters exposed to SARS-CoV-2 variants. Comparison groups for semi-quantitatively histopathology scores of interstitial pneumonia severity at 3, 7, and/or 10 days post-exposure. Lung sections were examined by light microscopy and the distribution (% of area affected) of interstitial pneumonia was semi-quantitatively scored on a 0–4 scale: 0, below lower limit of perception (LLOP, estimated by model); 1, ≤25%; 2, 26–50%; 3, 51–80%; or 4, ≥81%. Data are presented as the mean histopathology scores. Points indicate data from individual hamsters. A one-way analysis of variance (ANOVA) was performed, followed by Dunnett’s multiple comparisons test in GraphPad Prism 9.3.1 to determine the statistical significance for average histopathology scores at each time point. ** indicates a significance level of p ≤ 0.01; *** indicates a significance level of p ≤ 0.001.

Fig. 4.. Changes in immune cell populations in peripheral blood suggests the activation of both adaptive and innate immune responses following exposure to SARS-CoV-2 and its variants.

Absolute numbers of each immune cell type for individual animals at different time points are provided. Population of B cells (A), CD4+ T cells (B), activated CD4+ T cells (C), CD8+ T cells (D), eosinophils (E), granulocytes (F), monocytes (G), and neutrophils (H) were determined within the CD45 hematopoietic cell populations. Points indicate data from individual hamsters. The mean of each group is depicted as horizontal line. Data were log-transformed prior to statistical analysis. Ordinary one-way analysis of variance (ANOVA), followed by Dunn’s multiple comparison test was performed to compare virus-exposed (all variants) animals with untreated mock-exposed controls. Significance code: *—p ≤ 0.05; **—p ≤ 0.01; ***—p ≤ 0.001. SARS-CoV-2, severe acute respiratory syndrome coronavirus-2.

Fig. 5.. Hamsters exposed to pre-Omicron variants and Omicron BA.2 exhibited a more robust humoral response than those exposed to Omicron BA.1 and BA.1.1.

Endpoint of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) S1-specific (A) and S2-specific (B) immunoglobulin G (IgG) antibody (Ab) titers in serum obtained at the indicated study days and measured by enzyme-linked immunosorbent assay (ELISA). Data are presented as means ± standard deviations. Points indicate data from individual hamsters. C. Reciprocal live-virus neutralization titers against SARS-CoV-2 WA-01 and the variants used in individual studies in serum collected at the indicated days post-exposure. Horizontal bars show the median. Points indicate data from individual hamsters. Data were log-transformed prior to statistical analysis. Ordinary one-way analysis of variance (ANOVA), followed by Dunn’s multiple comparisons test, was performed for statistics. *—p ≤ 0.05; **—p ≤ 0.01; ****—p ≤ 0.0001; ns—p > 0.05.

Fig. 6.. Therapeutic antibody efficacy study design.

A Diagrammatic representation of the study design used for all efficacy studies. Animals were treated 24 h prior to virus exposure and were otherwise assessed as indicated. Terminal blood collections with necropsies were performed on days 3 and 7, with six animals sampled per day. B Example of each experiment groups, including animal number, treatment/challenge route/dose and study endpoint. IP, intraperitoneal; IN, intranasal; N/A, not applicable.

Fig. 7.. Efficacy testing of SARS-CoV-2 isolates in hamsters.

Data obtained from studies with individual variants are listed in each column, with the specific variant and its back titer identified to the top of each column next to each other. Row A. Level of human immunoglobulin G (IgG) in systemic circulation to quantify the presence of therapeutic antibody by enzyme-linked immunosorbent assay (ELISA) from day 3 and day 7 serum samples. Row B. Body weight changes were recorded in hamsters after severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) exposure. Row C. Viral titers in day 3 lung tissue samples were determined by plaque assay. Row D. Histopathological scores of interstitial pneumonia in the lungs were recorded at 3 and 7 days post-exposure. Row E. Neutralizing antibody titers of serum collected at 3 and 7 days post-exposure were tested against SARS-CoV-2 variants. Statistical analyses were performed as described for the natural history studies. Significance code: *—p ≤ 0.05; **—p ≤ 0.01; ***—p ≤ 0.001; ****—p ≤ 0.0001. PK, pharmacokinetic; LLOQ, lower limit of quantification; FRNA₅₀, 50% fluorescence reduction neutralization assay; Ab, antibody.