Technical Review: Testing Novel Minimized Pan-Coronavirus (CoV) Vaccines in Feline Immunodeficiency Virus-Infected Cats With or Without Feline CoV Serotype-1 (FCoV1) Coinfection

Full Title: Pilot Studies Testing Novel Minimized Pan-Coronavirus (CoV) Vaccines in Feline Immunodeficiency Virus-Infected Cats With or Without Feline CoV Serotype-1 (FCoV1) Coinfection and in Specific-Pathogen-Free Cats Against Pathogenic FCoV2

Authors:Pranaw Sinha, Marco B. Prevedello, Ananta P. Arukha, Valentina Stevenson, Karen F. Keisling, Taylor G. Nycum, Nina M. Beam, Elise D. Barras, Bikash Sahay, and Janet K. Yamamoto

Affiliations: University of Florida, Gainsville, Florida, USA

Background Introduction

Feline coronaviruses (FCoVs) exist primarily as two serotypes: Serotype-1 (FCoV1) and Serotype-2 (FCoV2). FCoV1 is more prevalent but difficult to culture than FCoV2, though both can mutate into the lethal Feline Infectious Peritonitis Virus (FIPV). The study addresses the lack of effective vaccines that cross-protect against FCoV1, as current commercial options are based on the easier to culture FCoV2. By leveraging comparative virology, the authors explore a "minimized" pan-coronavirus vaccine strategy. This approach utilizes pDNA (plasmid DNA) encapsulated in lipid nanoparticles (LNPs), incorporating conserved epitopes shared between FCoV and SARS-CoV-2 (SCoV2) to induce broad-spectrum immunity.

(A) The vaccination schedule of two FIV-infected cats (FCoV1-free/FIV-infected HOK and FCoV1/FIV-coinfected HOL) is shown with the vaccine dose in μg, vaccine type, and vaccination route as either subcutaneous (SC) or intramuscular (IM). The serum (blue arrow), blood (light-blue arrow) for PBMC and plasma, feces (green arrow), and tissues (red arrow) are collected at the time points shown with the designated arrow. The samples from unvaccinated FCoV1/FIV-coinfected cat HON and unvaccinated FCoV1- free/FIV-infected cat HOO were collected at the same time as those collected from vaccinated cats. (B) The XbAbs to FIPV2-WV for vaccinated cats HOK and HOL at 1:50 dilution are shown for serum/plasma collected at 1, 3, and 6 months post-FCoV1 infection (6 mpi), which occurred before vaccination. Both HOK and HOL were single housed at 2.5 mpi. HALO Quantitative Image displayed the strong bands with an arrow for one or more strips at 24–74% intensity and the weak bands with a white arrowhead at 10–17% intensity (HOL S2, FCoV+ UG4 NC). (C) The serum/plasma of all four cats were evaluated for the bAbs to SCoV2 RBD (top) and FCoV1 RBD (bottom) at 1:50 and 1:500 dilutions for vaccinated cats HOK and HOL and 1:100 dilution for unvaccinated cats HON and HOO. The HALO intensity values of all strong bands without arrowheads are 30–89% intensity for FCoV1 RBD and 17–31% for SCoV2 RBD. The weak bands with a white arrowhead are 13–16% intensity. The immunoblot strips have the brightness and contrast intensities of 8% and 0% for (B), respectively, and both 5% for (C).

Materials and Methodology

The researchers used a sequential pilot approach involving both in vitro validation and in vivo trials on a feline model. First, novel minimized B-cell epitope constructs were optimized using AI-based homology. These constructs were then transfected into feline Fc9 cells, with expression verified via flow cytometry. Finally, the vaccine was evaluated in cats separated into various experimental groups across two pilot studies. For the first study, cats with chronic FIV were separated into an unvaccinated control (with/without FCoV1) and two experimental vaccinated groups: one with FCoV1 coinfection, and one without. For the second study, four specific-pathogen-free (SPF) cats received a sequential vaccination regimen with three variations of the vaccine over several months, while three SPF cats (two with empty LNPs and one with no vaccine) served as a control. In this second study, all cats were challenged with a high dose of heterologous FIPV2-UCD2 to assess immune response.

Results

In Vitro Validation

The minimized vaccine constructs demonstrated robust expression in feline embryonic-9 (Fc9) cells. The SCoV2-fHR recombinant construct achieved the highest GFP expression at 32.2%, followed closely by the pooled FCoV CTL epitope chains at 27.5% and FCoV1-Ori at 26.2%. The FCoV1-sRBD construct showed the lowest expression at 20.0%. These expression levels confirmed that the strategic deletion of adverse motifs (neurotoxin, ICAM-1, and bullous pemphigus regions) did not compromise protein production or cellular processing. The inclusion of stem helix (SH), transmembrane (TM), and cytoplasmic tail (CT) sequences not only maintained protein stability but also enhanced presentation on cellular membranes, mimicking natural spike expression during viral infection.

Pilot Study 1: Safety and Immunogenicity in FIV Cats

Two FIV-infected cats (HOK and HOL), the latter with coinfection of FCoV1, were vaccinated with escalating doses of pan-CoV vaccine-1. Administration of 25 µg and 50 µg doses caused no adverse effects, establishing a favorable safety profile at these dosages. However, the 100 µg dose elicited transient but notable adverse reactions that appeared within 24 hours and resolved completely following a single low-dose treatment with Meloxicam. No significant weight loss was recorded in vaccinated animals throughout the study period.

Both vaccinated FIV-infected cats developed clinically significant cross-neutralizing antibodies (XNAbs) to FIPV2-UCD2, despite never encountering FCoV2. Immunoblot analysis revealed that vaccinated cat HOK produced high-titer cross-reactive binding antibodies (XbAbs) to the FCoV1 RBD at 1:500 dilution, while HOL's FCoV1 RBD XbAbs were detectable at 1:50 dilution. Notably, vaccinated HOK also generated robust SCoV2 RBD-specific XbAbs, while vaccinated HOL produced negligible anti-SCoV2 RBD responses despite receiving identical vaccine formulations. The unvaccinated FCoV1-infected control cat (HON) unexpectedly developed elevated and persistent XNAbs to FIPV2 throughout the study, suggesting natural cross-reactive immunity development during chronic FCoV1 infection.

Pilot Study 2: Challenge Outcome in SPF Cats

Upon high-dose intranasal FIPV2-UCD2 challenge, all seven challenged cats became infected, as confirmed by multiple immunological and molecular markers. Fecal analysis via RT-snPCR revealed detectable viral RNA in all animals by 8 weeks post-challenge (wpc), with vaccinated cats 2FM and 2FP exhibiting a delayed shedding kinetic—showing weak or absent RdRp amplification at 8 wpc (cycle 1) that peaked by 21 wpc, approximately one week later than controls. By 23-26 wpc, all surviving cats demonstrated clearance of fecal viral load in both RT-snPCR cycles, demonstrating spontaneous viral clearance independent of vaccination status. Post-challenge immunoblot analysis revealed that all infected animals seroconverted to FIPV2, generating robust antibodies to the nucleocapsid (NC; 45 kDa) and RBD domains. Critically, three vaccinated cats and one LNP-control cat demonstrated declining S2-specific antibody titers by 27 wpc, suggesting active immune control of viral replication.

Of the four vaccinated cats, one (2FN) developed clinical disease manifesting as progressive head and neck vasculitis lesions beginning at 9 days post-challenge, with systemic signs including fever (104°F) and 25.3% weight loss by 17 days post-challenge. Despite extensive lesion investigation, immunohistochemistry and RT-PCR testing of lesion tissue proved negative for FIPV antigens and RNA, though fecal samples remained positive for FIPV2 RdRp, confirming active infection. Necropsy revealed no evidence of typical FIP-related multiorganism pathology. Among controls, two of three animals developed fatal FIP disease: LNP-control 2FO developed classic wet-form FIP with pleural effusion and multi-organ damage, while unimmunized control 2FR developed recurrent urinary tract infection complicated by FIP, manifesting as pleural effusion and multiorgan lesions. Vaccination did not prevent infection in any animal, though the three surviving vaccinated cats (2FM, 2FP, FB1) showed statistically comparable or superior disease metrics relative to controls. No evidence of vaccine-enhanced respiratory disease (VERD) or antibody-dependent enhancement (ADE) was observed.

Conclusion

Sinha and colleagues showed a promising step towards a universal coronavirus vaccine for cats. Optimized protein constructs bypassed the traditional bottleneck of culturing FCoV1, while pDNA-LNPs proved an effective vaccination platform. Complimentary strategies such as CTL priming or live-attenuated vaccine components are some possible future avenues of research. This is especially important considering the lack of sterilizing immunity under high doses of challenge virus, something which parallels limited protection against evolving SARS-CoV-2 in humans.

This paper utilized a PreciGenome NanoGenerator Flex-S along with the LipidFlex LNP formulation to develop its pDNA-LNP vaccine. Microfluidic mixing offers both excellent LNP uniformity and highly efficient encapsulation of nucleic acid, even in small doses such as those used for small animal models.

For further details, check out the link to the article here:

https://www.mdpi.com/2076-393X/13/11/1172