Passive immunization with equine RBD-specific Fab protects K18-hACE2-mice against Alpha or Beta variants of SARS-CoV-2

Abstract

Emergence of variants of concern (VOC) during the COVID-19 pandemic has contributed to the decreased efficacy of therapeutic monoclonal antibody treatments for severe cases of SARS-CoV-2 infection. In addition, the cost of creating these therapeutic treatments is high, making their implementation in low- to middle-income countries devastated by the pandemic very difficult. Here, we explored the use of polyclonal EpF(ab')₂ antibodies generated through the immunization of horses with SARS-CoV-2 WA-1 RBD conjugated to HBsAg nanoparticles as a low-cost therapeutic treatment for severe cases of disease. We determined that the equine EpF(ab')₂ bind RBD and neutralize ACE2 receptor binding by virus for all VOC strains tested except Omicron. Despite its relatively quick clearance from peripheral circulation, a 100μg dose of EpF(ab')₂ was able to fully protect mice against severe disease phenotypes following intranasal SARS-CoV-2 challenge with Alpha and Beta variants. EpF(ab')₂ administration increased survival while subsequently lowering disease scores and viral RNA burden in disease-relevant tissues. No significant improvement in survival outcomes or disease scores was observed in EpF(ab')₂-treated mice challenged using the Delta variant at 10μg or 100µg doses. Overall, the data presented here provide a proof of concept for the use of EpF(ab')₂ in the prevention of severe SARS-CoV-2 infections and underscore the need for either variant-specific treatments or variant-independent therapeutics for COVID-19.

Keywords: COVID-19; EpF(ab’)2; Equine F(ab’)2; K18-hACE2 transgenic mice; SARS-CoV-2; passive immunization; polyclonal antibodies; variant of concern (VOC).

Figure 1

EpF(ab’)₂binds to RBD and neutralize RBD-hACE2 interaction. (A) Graphical representation of the mutations on each of the viral strains used in this study. Amino-acid deletions are depicted with orange crosses and point mutations with yellow squares. (B) Heat map of the ACU from ELISA detection of the binding of EpF(ab’)₂, HCP, and mRNA sera to seven peptides in the RBD region of the SARS-CoV-2 spike protein. Darker colors represent higher binding to the peptides. (C) 3D representation of the SARS-CoV-2 RBD binding to the hACE2 protein. Areas of highest binding as determined in B are highlighted in colors for EpF(ab’)₂ (yellow), HCP (blue), and mRNA (purple). (D) Heat map representing percent inhibition of RBD binding to hACE2 determined using neutralization assays at non-saturating concentrations of antibody for EpF(ab’)₂ and HCP. Darker colors represent higher binding neutralization.

Figure 2

Kinetics of EpF(ab’)₂ in serum after passive immunization in C57BL/6J. (A) Graphical representation of the timeline for EpF(ab’)₂ administration and bleeding. The timeline represents days. (B) AUC representation of EpF(ab’)₂ ELISA detection in the serum at days 1, 2, 3, and 11 in C57BL/6J mice passively immunized with 10 or 100μg of EpF(ab’)₂. Statistical analyses were performed as one-sample t-tests compared to the naïve animals (**p<0.01; ***p<0.001).

Figure 3

Treatment with EpF(ab’)₂ helps reduce disease scores and improves survival in K18-hACE2 challenged mice. Cumulative disease scores of K18-hACE2 mice infected with Alpha (A), Beta (B), and Delta (C) over time. Kaplan Meyer representation of the percentage of survival over time of mice infected with Alpha (D), Beta (E), and Delta (F). Correlations between weight loss, represented as percentage of weight at day 0, and temperature loss, represented as percentage of temperature at day 0 of K18-hACE2 mice infected with Alpha (G), Beta (H), and Delta (I). Differences in survival between each treatment group and the PBS control group were determined using a log-rank Mantel-Cox test (*p<0.05). Correlations were performed by fitting the data to a simple linear regression.

Figure 4

Kinetics of EpF(ab’)₂ in serum after passive immunization in challenged K18-hACE2 mice. AUC representation of EpF(ab’)₂ ELISA detection in the serum at days 3 and 11 in K18-hACE2 mice challenged with Alpha, Beta, and Delta, and passively immunized with either 10μg (A) or 100μg (B) of EpF(ab’)₂. Statistical analyses were performed as one-sample t-tests compared to the naïve animals (*p<0.05; ***p<0.001; ^#p<0.05; ^##p<0.01; ^&&p<0.01; and ^&&&p<0.001). Results from statistical analyses of comparisons of Alpha to naïve animals are represented with *, Beta to naïve animals with #, and Delta to naïve animals with &.

Figure 5

Viral burden in lung, nasal wash and brain detected by qPCR. qPCR detection of SARS-CoV-2 viral burden in the lung (A), nasal wash (B), and brain (C) at time of euthanasia in mice infected with Alpha, Beta, or Delta. Kruskal-Wallis with Dunn’s multiple comparisons test was used to compare viral burden in the tissue (*p<0.05; **p<0.01; ****p<0.0001).

Figure 6

SARS-CoV-2 causes chronic inflammation in the lung of infected K18-hACE2 mice. (A) Histopathological analysis of hematoxylin-eosin-stained sections of lung from non-infected and SARS-CoV-2 infected K18-hACE2 mice (Representative images at 200x magnification). Left: Characteristic features of a non-infected lung (left); Center: detection of increased numbers of mononuclear cells within the parenchyma (asterisk), surrounding blood vessels (arrowhead), and surrounding bronchi/bronchioles (arrow) in the lung of mice infected with B.1.1.7; Right: Detection of granulomatous inflammation in the lung of mice infected with B.1.351. (B) Histopathological analysis of hematoxylin-eosin stained sections of lung from SARS-CoV-2 infected K18-hACE2 mice untreated, or treated with HCP or EpF(ab’)₂ (representative images at 100x magnification). (C) Chronic histological scores from hematoxylin-eosin stained sections of lung from SARS-CoV-2 infected K18-hACE2 mice untreated, or treated with HCP or EpF(ab’)₂. \Differences between infected groups are denoted with brackets (One-way ANOVA with Tukey’s multiple comparison test). **p < 0.01, and ****p < 0.0001.