In a recent study posted to the bioRxiv* preprint server, researchers confirmed that feline coronavirus (FCoV) infection induced the production of cross-reactive antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) in domestic cats, despite minimal sequence amino acid (aa) similarity between RBDs of SARS-CoV-2, and FCoV serotype 1 (FCoV1) and serotype 2 (FCoV2).
Studies have not yet reported FCoV-infected cat sera possessing cross-protective immunity with SARS-CoV-1 or SARS-CoV-2 RBDs. Based on the authors’ research laboratory report, SPF (specific pathogen-free) toms unexpectedly developed cross-protective anti-SARS-CoV-2 RBD antibodies upon mating with queens who were FCoV-infected.
About the study
In the present study, researchers performed the present study to confirm previous findings of the induction of cross-protective anti-SARS-CoV-2 RBD antibodies among FCoV-infected SPF toms.
Japanese cats were inoculated nasally/orally with FCoV2 79-1146 or FCoV1 UK-1 strains, and their serum samples were obtained after 60 days and 15 months of inoculation. Four queens [University of Georgia (UGA)-Q1, Q2, Q3, and Q4] were obtained, and mating queens produced young cats with specific pathogen-free laboratory toms (n=3). The group-housed cats (n=8) were considered unrelated to the UF (University of Florida) toms and UGA queens. SARS-CoV-2 peptides such as MB-RBD (MassBiologics-RBD) and UF-RBD and cell lines such as Fc9 (Felis catus 9), Fcwf-4 (Felis catus whole fetus-4), and Crandell feline kidney (CrFK) fibroblast were used for cell culture experiments.
CrFK cells were inoculated with the FCoV2 79-1146 strain to produce FCoV2 for in vitro analyses and FCoV2 WV (whole-virus) stock. Subsequently, transfection experiments were performed, RBD plasmids were expressed in Expi293F cells, and the RBD proteins were purified. Further, FCoV-WV and SARS-CoV-2 RBD enzyme-linked immunosorbent assays (ELISAs) were performed with overnight serum incubation. To ensure that the serum ELISA reactivity was specific to the FCoV2-WV, FCoV-RBD, or SARS-CoV-2 RBD, serum from toms (n=3) and UGA queens (n=4) were separately incubated at similar dilutions for one hour only (stringent ELISA).
Furthermore, SDS-PAGE gel analysis, immunoblot analyses, anti-FCoV2 neutralizing antibody (NAb) assays, and in vitro FCoV2 infection-blocking assays with SARS-CoV-2 RBD were performed. Cell-mediated interferon-gamma (IFNγ) immunological responses to SARS-CoV-2 RBD were evaluated. Peripheral blood mononuclear cells (PBMCs) of temporarily (4GC) and chronically (G-3) FCoV1-infected cats and two SPF cats (4GA, 2FB) were stimulated with FCoV1, FCoV2, or SCoV2 RBD.
Sequences of SARS-CoV-2 Wuhan-Hu-1 strain RBD and four strains of FCoV1 and FCoV2 were aligned by multiple sequence alignment analyses. The UGA queen cats were co-housed, and their earliest serum samples were obtained 24 days later, indicating a high likelihood of the queens being FCoV-infected and not SARS-CoV-2-infected from a SARS-CoV-2-positive caretaker given the first SARS-CoV-2 infection case was detected in Florida three months later on 2 March 2022.
All UGA queens were FCoV-2-positive, and sera from the mated toms showed weak FCoV2 seropositivity without any FCoV2 NAbs. Three of four juvenile cat serum samples showed moderate SARS-CoV-2 RBD cross-reactivity. The peak serum from UGA-Q2 and the three tom cats strongly cross-reacted with SARS-CoV-2 RBD but did not show FCoV2 spike (S) RBD cross-reactivity in the ELISA assays and immunoblotting analyses. None of the queens, toms, or kittens generated FCoV2 NAbs, indicating that the cats were FCoV1-infected, which was confirmed by immunoblotting analysis.
FCoV2 79- 1146-positive cat sera but not FCoV1 KU-2-positive sera cross-reacted strongly with SARS-CoV-2 RBD, indicating that not every FCoV1-infected cat developed cross-protective antibodies to SARS-CoV-2 RBD. The 12 amino acid sequence at the carboxyl-terminal region of SARS-CoV-2 MB RBD was similar to FCoV sequences. All eight group-housed laboratory cats unrelated to the toms tested FCoV2-positive by ELISA and immunoblot analyses. Most cats had cross-protective antibodies against SARS-CoV-2 RBD, which were preserved even after 15 months. FLA (feline leukocyte antigen)-comprising cats sustained greater cross-protective anti-SARS-CoV-2 RBD antibody titers.
Even though the four UGA queen cats, inclusive of UGAQ2, showed FCoV2 S reactivity, two of the three toms lacked FCoV2 S cross-reactivity. SARS-CoV-1 and SARS-CoV-2 RBDs showed 88% similarity and 65% identity and thus, the observation of FCoV-positive cats developing cross-protective antibodies against SARS-CoV-2 RBD despite no antibodies against SARS-CoV-1 S subunit 1 (S1) was unexpected. The findings indicated that SARS-CoV-2 RBD and FCoV2 RBD were antigenically and structurally similar. SARS-CoV-2 UF RBD at high concentrations could cross-protect against FCoV2 infections at non-toxic RBD doses of 48.0 μg/mL to 68.0 μg/mL.
Remarkably, PBMCs of FCoV1- and FCoV-2-infected cats (especially 4GC and G-3) recognized SARS-CoV-2 RBD by generating IFNγ as a response to stimulation. However, only chronically infected cats recognized FCoV1 RBD stimulation. CCoV2 (canine CoV serotype 2) RBD and FCoV2 RBD sequences showed 96% similarity and 88% identity. FCoV1 RBD and CCoV1 RBD sequences showed 81% similarity, indicative of a similar lineage with a few evolutionary modifications.
Overall, the study findings highlighted the capability of SARS-CoV-2 and FCoV2 RBDs to block FCoV2 infections in vitro and generate pan-coronavirus-targeted T-lymphocyte responses. The findings indicated that a pan-CoV vaccine could be developed against SARS-CoV-2 infections in dogs, cats, and hamsters by merging RBDs of FCoV1 [glycoprotein 52 (gp52)] and FCoV2 (gp59) with that of SARS-CoV-2 (gp40), without potentially integrating those of CCoV1 and CCoV2.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.