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. 2022 Jul:203:105332.
doi: 10.1016/j.antiviral.2022.105332. Epub 2022 May 6.

Development of a cost-effective ovine antibody-based therapy against SARS-CoV-2 infection and contribution of antibodies specific to the spike subunit proteins

Stephen Findlay-Wilson  1 , Linda Easterbrook  1 , Sandra Smith  2 , Neville Pope  3 , Gareth Humphries  4 , Holger Schuhmann  4 , Didier Ngabo  1 , Emma Rayner  1 , Ashley David Otter  1 , Tom Coleman  1 , Bethany Hicks  1 , Victoria Anne Graham  1 , Rachel Halkerston  1 , Kostis Apostolakis  1 , Stephen Taylor  1 , Susan Fotheringham  1 , Amanda Horton  1 , Julia Anne Tree  1 , Matthew Wand  1 , Roger Hewson  1 , Stuart David Dowall  5
Affiliations

Development of a cost-effective ovine antibody-based therapy against SARS-CoV-2 infection and contribution of antibodies specific to the spike subunit proteins

Stephen Findlay-Wilson et al. Antiviral Res. 2022 Jul.

Abstract

Antibodies against SARS-CoV-2 are important to generate protective immunity, with convalescent plasma one of the first therapies approved. An alternative source of polyclonal antibodies suitable for upscaling would be more amendable to regulatory approval and widespread use. In this study, sheep were immunised with SARS-CoV-2 whole spike protein or one of the subunit proteins: S1 and S2. Once substantial antibody titres were generated, plasma was collected and samples pooled for each antigen. Non-specific antibodies were removed via affinity-purification to yield candidate products for testing in a hamster model of SARS-CoV-2 infection. Affinity-purified polyclonal antibodies to whole spike, S1 and S2 proteins were evaluated for in vitro for neutralising activity against SARS-CoV-2 Wuhan-like virus (Australia/VIC01/2020) and a recent variant of concern, B.1.1.529 BA.1 (Omicron), antibody-binding, complement fixation and phagocytosis assays were also performed. All antibody preparations demonstrated an effect against SARS-CoV-2 disease in the hamster model of challenge, with those raised against the S2 subunit providing the most promise. A rapid, cost-effective therapy for COVID-19 was developed which provides a source of highly active immunoglobulin specific to SARS-CoV-2 with multi-functional activity.

Keywords: Antibodies; COVID-19; Development; Omicron; SARS-CoV-2; Therapy.

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Conflict of interest statement

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sandra Smith and Neville Pope are employees of International Therapeutic Proteins Ltd. Gareth Humphries and Holger Schuhmann are employees of the Native Antigen Company. All other authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Binding of ovine sera to full-length recombinant SARS-CoV-2 spike protein after immunisation with glycoprotein antigens. (a) Outline of study schedule. (b) Reactivity of S1-immunised sheep, n = 3. (c) Reactivity of S2-immunised sheep, n = 3. (d) Reactivity of full-length immunised sheep, n = 6. Lines show mean values with error bars denoting standard error.
Fig. 2
Fig. 2
Antigen binding kinetics of purified and affinity-purified IgG preparations to recombinant SARS-CoV-2 glycoproteins. (a) Reactivity to whole spike protein. (b) Reactivity to S1 subunit protein. (c) Reactivity to S2 subunit protein. Lines show mean values. Solid line, purified antibodies; dashed line, affinity-purified antibodies.
Fig. 3
Fig. 3
Functional activity of affinity-purified antibodies produced against recombinant SARS-CoV-2 glycoproteins. (a) Binding to the SARS-CoV-2 RBD. Bars show mean values with error bars denoting standard error from triplicate samples. (b) Antibody-dependent complement deposition. (c) Antibody-dependent neutrophil phagocytosis. Results from a single assay are shown. (b–c) Data is calibrated to the NIBSC 20/162 SARS-CoV-2 antibody diagnostic calibrant consisting of a pool of convalescent plasma from 3 separate donors and assigned a unitage of 1000 units/ml.
Fig. 4
Fig. 4
Clinical outcomes of hamsters receiving antibody preparations after challenge with SARS-CoV-2. (a) Weight of animals. Lines show mean value with error bars denoting standard error. Dotted line shows day of challenge. Three animals in the PBS group met humane clinical endpoint as indicated by skull and crossbones symbol. (b) Maximum weight loss of animals, with line and whisker plots showing mean value and standard error. (c) Clinical score. Lines show mean values with error bars denoting standard error. . n = 6 hamsters per group.
Fig. 5
Fig. 5
Antibody levels on day of challenge and comparison to subsequent weight loss. (a) Antibody levels from sera collected on the day of challenge. Results show mean absorbance level from duplicate wells from each animal tested at a 1:100 dilution. Bar and whisker plots denote mean and standard error. (b) Comparison of animal level at time of challenge with maximal weight loss observed after challenge with SARS-CoV-2. n = 6 hamsters per groups.
Fig. 6
Fig. 6
Virology readouts of hamsters receiving ovine antibody preparations after challenge with SARS-CoV-2. (a) Quantification of live virus detected by focus-forming assay in nasal wash and pharyngeal swab samples collected 2 days post-challenge with SARS-CoV-2. Bars show mean values with error bars denoting standard error. No statistical significance between groups receiving antibodies compared to PBS control (P > 0.05). (b) Viral RNA levels in pharyngeal swabs. Bars show mean values with error bars denoting standard error. *, P < 0.05. (c) Viral RNA levels in lung tissue collected at necropsy. Open circles indicate animals which met humane clinical endpoints. Individual results shown with line and whisker plots showing mean value and standard error. *, P < 0.05.
Fig. 7
Fig. 7
Representative microscopic images of lungs and nasal cavities of hamsters receiving ovine antibodypreparations after challenge with SARS-CoV-2. Top row, lung-multifocal to patchy areas of pneumonic consolidation (arrows) (H&E); middle row, nasal cavity-inflammation and degeneration of the mucosa with variable luminal exudate (asterisks). Inset, higher power images of nasal epithelium (×800 magnification) (H&E); lower row, nasal cavity-staining for SARS-CoV-2 viral RNA in the mucosa and luminal exudate (in situ hybridisation).
Fig. 8
Fig. 8
Pathological readouts in the lungs and nasal cavities of hamsters receiving ovine antibody preparations after challenge with SARS-CoV-2. (a) Area of consolidation in the lung as a percentage. (b) Total pathology score in the lung. (c) Total pathology score in the nasal cavity. Line and whisker plots show mean value and standard error. *, P < 0.05.
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