Virus-like Particle Vaccine for Feline Panleukopenia: Immunogenicity and Protective Efficacy in Cats

Abstract

Background/objectives: Feline panleukopenia, caused by FPV, is a highly contagious disease in cats. Current vaccines face challenges including complex production, high cost, and safety risks. Developing safer, more efficient alternatives is crucial. This study aimed to produce FPV virus-like particles (VLPs) using a recombinant baculovirus system expressing the VP2 gene and evaluate their immunogenicity and protective efficacy in cats.

Methods: Sf9 insect cells were infected with recombinant baculovirus to express VP2 protein. The VP2 protein was purified using ultrafiltration and size-exclusion chromatography (SEC). Dynamic light scattering (DLS) and transmission electron microscopy (TEM) confirmed the assembly of VLPs. Twenty healthy cats were randomly divided into four groups; three groups received different doses (5 μg, 15 μg, and 45 μg) of FPV VLP vaccine, while the fourth group served as the control group immunized with PBS. Blood samples were collected on day 21 to measure hemagglutination inhibition (HI) and virus-neutralizing (VN) antibody responses. Cats in the 15 μg dose group were challenged with virulent FPV strain 708 on day 21, and clinical signs and white blood cell counts were monitored for 10 days.

Results: Immunized cats exhibited significantly higher HI and VN antibody titers compared to controls. After challenge, vaccinated cats showed no clinical signs of disease, and their white blood cell counts remained stable. In contrast, control cats developed severe symptoms and experienced significant leukopenia.

Conclusions: The FPV VLP vaccine generated in this study are highly immunogenic and provide effective protection against virulent FPV challenge, demonstrating their potential as a safer vaccine candidate for feline panleukopenia.

Keywords: feline parvovirus; vaccine; virus-like particles (VLPs).

Figure 1

Generation of recombinant baculovirus and identification of VP2 proteins expressed in Sf-9 cells. (A,B) Infection with recombinant baculovirus alters the morphology of Sf9 cells. (A) Sf9 cells infected with recombinant baculovirus for 72 h, and (B) Sf9 control cells were observed under a light microscope (magnification, 100×). (C,D) Indirect fluorescence assay (IFA) of VP2 protein expressed in Sf9 cells. (C) Sf9 Cells infected with the recombinant baculovirus for 72 h and (D) Sf9 control cells were identified by IFA and observed under a fluorescence microscope (magnification at 100×). (E,F) SDS-PAGE and Western blot analysis of VP2 protein expression. (E) 12%SDS-PAGE and Coomassie Brilliant Blue staining and (F) Western blot analysis. Lane 1, pre-stained protein markers; lane 2, Sf9 control cells; lane 3, Sf9 cells infected with recombinant baculovirus. The red arrows indicate the FPV VP2 protein. (G,H) hemagglutination (HA) activity of FPV VP2. HA titers were detected using 1% pig erythrocytes. (G) Sf9 cells infected with recombinant baculovirus for 72 h, and (H) Sf9 control cells were observed.

Figure 2

Purification and characterization of FPV virus-like particles (VLPs). (A) Gel filtration chromatography profile of FPV VLPs using a Hi Load 16/60 Superdex 200-pg column. (B) 12%SDS-PAGE and Coomassie Brilliant Blue staining analysis of proteins obtained from gel filtration chromatography. The red arrows indicate the FPV VP2 protein. (C) Size-exclusion chromatography (SEC) profile of purified FPV VLPs, showing the main peak corresponding to nanoparticle formation. (D) Particle size distribution of FPV VLPs as determined by dynamic light scattering (DLS), with an average diameter of approximately 25 nm. (E) Transmission electron microscopy (TEM) image of FPV VLPs, negatively stained with 3% phosphotungstic acid, showing spherical particles with uniform morphology and a diameter of approximately 25 nm. Scale bar = 200 nm. The black arrows indicate FPV VLPs.

Figure 3

Immunogenicity induced by FPV VLPs. (A) The hemagglutination inhibition (HI) titer in the serum of cats from Groups 1 to 4 was determined using HI assay with 1% pig erythrocytes. (B) Virus-neutralizing antibody titers were measured by IFA and expressed as the reciprocal of the highest dilution at which infection of the F81 cells was inhibited in 50% of the culture wells. Blue represents Group 1 (45 μg VLPs immunization), yellow represents Group 2 (15 μg VLPs), green represents Group 3 (5 μg VLPs), and gray represents Group 4 (PBS control). All groups contained an equal number of cats (n = 5 per group). The horizontal lines represent the group means, the error bars indicate the standard deviation, and the gray dashed line represents the 1:40 HI antibody titer.

Figure 4

Protective efficacy of FPV VLPs. (A,B) Severe vomiting and diarrhea were observed in the PBS control group after challenge. (C) Body temperature monitoring after challenge, showing the range of maximum and minimum values within the group. (D) Survival rate after viral challenge. (E) White blood cell count on day 6 post-challenge. (F) Intestinal tissues (jejunum) were analyzed by hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) assays. The arrows indicate villous atrophy and shortening of the jejunum, as well as hemorrhage/congestion in H&E staining; The arrows point to FPV positivity in IHC.