A total of 13 eight-week-old cats free of feline calicivirus (FCV), feline parvovirus (FPV), FHV-1, and Feline coronavirus (FCoV) were randomly divided into three groups. Each cat was kept in a separate cage, with different groups separated into distinct spaces. The animal studies were approved by the Committee on the Ethics of Animal Experiments of the National Research Center for Veterinary Medicine (Permit Number: 202307001) and conducted in conformity with the Guide for the Care and Use of Animals in Research of the People’s Republic of China.

Feline kidney cells (F81 cells) were maintained in Roswell Park Memorial Institute (RPMI) 1,640 medium (Gibco, Carlsbad, CA, United States), supplemented with 8% fetal bovine serum (Cegrogen Biotech, Germany), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37°C with 5% CO₂. The FHV-1 strain 64 was isolated from the eyelid and nasal swab suspension of FHV-1 infected cats in Henan Province in 2016. The eyelid and nasal swab suspension were inoculated on F81 cells for virus isolation as previously described (18). When CPE were observed in 80% of the cells, cultures were harvested and the resultant virus stocks were stored at −80°C and named as FHV-1 strain 64. Cats infected with FHV-1 strain 64 presented with ocular and nasal discharge, conjunctival congestion, and sneezing (data not shown). The serial passage was used to attenuate the FHV-1 strain 64, and the MLV vaccine was prepared by attenuating the FHV-1 strain 64 (10^6.0 TCID₅₀/mL).

The experimental schedule is illustrated in Figure 1A. The cats were randomly divided into three groups: (1) Unvaccinated controls, n = 5, (2) vaccine immunization group, n = 5, (3) Mock group, n = 3; based on the immunization schedule of the vaccine in the published article, cats were immunized subcutaneously (SC) on days 0 and 21 (19, 20). Groups 1 and 2 were challenged with intranasal FHV-1 64 on day 35, and the mock group was inoculated with RPMI 1640 medium. Clinical symptoms and body temperatures were monitored daily, and venous blood was collected on days 0, 21, and 35 to detect the virus-neutralizing (VN) antibody and IgG levels. Serum cytokine levels were detected in venous blood collected on days 0, 21, 35, 42, and 49, and serum was stored at −20°C for further testing. On day 49, peripheral blood was collected to isolate lymphocytes, after which the cats were humanely euthanized.

The serum was analyzed for FHV-1 VN antibodies by heat-inactivating it at 56°C for 30 min, followed by a two-fold serial dilution ranging from 1:2 to 1:256 according to the previous method (21). A 200 TCID₅₀ dose of FHV-1 was mixed with the serum sample in a volume ratio 1:1, incubated at 37°C for 1 h, and then added F81 cells (2 × 10⁴ cells/100 μL). After a 4-day incubation at 37°C with 5% CO₂, antibody titers were measured by CPE and expressed as the reciprocal of the highest dilution at which infection of the F81 cells was inhibited in 50% of the culture wells.

The IgG antibodies in the serum were detected 14 days after the second immunization using an indirect enzyme-linked immunosorbent assay (ELISA) according to the previous method (17). Microtiter plates (96-well) were coated with lab-stored, purified FHV-1 (0.25 μg/well) and incubated overnight at 4°C. The plates were then blocked with a blocking buffer for 24 h at 4°C. Serum samples were heat-inactivated at 56°C for 30 min and pre-diluted (1:2,000) with phosphate buffer saline (PBS). The samples were then incubated at 37°C for 1 h, followed by incubation with HRP-conjugated goat anti-feline IgG (1,2000) at 37°C for 30 min. Tetramethyl benzidine (TMB) was added to each well, and then the reaction was stopped with 2 M H₂SO₄. The absorbance was measured at an optical density (OD) of 450 nm using a microplate reader (BioTek, Vermont, United States).

Clinical scores were obtained by trained observers masked to the vaccine groups from day 0 to 14 days after the FHV-1 challenge (day 35), as described in previous studies (22, 23). Two observers recorded signs for 30 min per group each morning. Clinical signs included fever, conjunctivitis, blepharospasm, ocular discharge, sneezing, and nasal discharge. The eyelids, turbinates, tracheas, and lungs of cats were harvested 14 days after the FHV-1 challenge. All tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned before hematoxylin and eosin staining (H&E). Monoclonal antibody 5H8 against FHV-1 gD and goat anti-mouse IgG conjugated to HRP (ThermoFisher Scientific, Waltham, MA, United States) were used for immunohistochemical (IHC) assays. Stained sections were visualized using a microscope (Leica DM2500, Leica Microsystems, Germany).

After challenge, the eyelid swabs were tested daily for the FHV-1 DNA. Eyelid swabs were collected into 1 mL of PBS, and viral nucleic acid was extracted from 200 μL eyelid swab samples using the Viral Nucleic Acid Extraction Kit (Geneaid Biotech Ltd., Taiwan, P.R. China). The primers for the PCR test of FHV-1 as previously described (21). Briefly, the primers used were: Forward (5′-3′): GAC GTG GTG AAT TAT C; Reverse (5′–3′): CAA CTA GAT TTC CAC CAG GA. The reaction conditions were as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30s, annealing at 58°C for 30s, elongation at 72°C for 30s, 30 cycles. Amplicons were detected on 1.5% agarose gels, with a positive reaction indicated by a 288 bp band.

The feline interferon-gamma (IFN-γ) ELISA kit (Mabtech AB, Stockholm, Sweden), feline interleukin 2 (IL-2), and IL-4 ELISA kit (R&D Systems, Minneapolis, United States), and feline IL-17A ELISA kit (MyBioSource, San Diego, CA, United States) were used to measure the levels of IFN-γ, IL-2, IL-4, and IL-17A, respectively, following the manufacturer’s instructions. Each sample was analyzed in duplicate.

The peripheral blood lymphocytes were isolated with commercial kits (Tianjin Haoyang Biological Manufacture, P.R. China) following the manufacturer’s instructions and stored in liquid nitrogen. The percentages of CD4+ and CD8+ T cells in lymphocytes were determined by flow cytometry using a staining method adapted from a previously published protocol (24). The thawed lymphocytes were washed thrice with flow cytometry staining buffer by centrifugation at 2000 rpm for 5 min each. The lymphocytes were blocked by resuspending for 10 min at 4°C in flow cytometry staining buffer containing 10 μL negative cat serum. Subsequently, a total of 1 × 10⁶cells were stained with 10 μL FITC Anti-cat CD4, RPE Anti-cat CD8, and Alexa Fluor^® 647 Anti-cat CD134 antibodies (Bio-Rad Laboratories, United States) for 30 min at 4°C in the dark. After washing, the lymphocytes were stained with Hoechst 33258 (ThermoFisher Scientific, Waltham, MA, United States) for 20 min at 4°C in the dark. After three additional washes, the lymphocytes were resuspended with a flow cytometry staining buffer and analyzed using a flow cytometer (BD Biosciences, Franklin, CA, United States).

The feline IFN-γ ELISpot kit (Mabtech AB, Stockholm, Sweden) and feline IL-2 ELISpot kit (R&D Systems, Minneapolis, United States) were used to evaluate the cell-mediated immune response induced by the FHV-1 vaccine. Lymphocytes from the cats on day 49 obtained as described previously were seeded into ELISpot plates (5 × 10⁵ live cells/well) and stimulated with FHV-1 (MOI = 0.3) for 24 h. The assay was performed according to the manufacturer’s instructions. The spot-forming cells (SFCs) were counted using an ELISpot plate reader (Cellular Technology Ltd., Shaker Heights, OH, United States). The IFN-γ or IL-2 secretory cell numbers were calculated by subtracting the values from PBS-stimulated wells from FHV-1 stimulated wells.

The total clinical scores for each cat were calculated over the post-challenge (days 0–14) study periods. A Statistical Package for the Social Sciences (version 21.0) was used to conduct Pearson correlation analysis between clinical scores post-FHV-1 challenges and detection indicators. Data were analyzed by one-way analysis of variance and an independent-sample t-test at a significance level of p < 0.05.

The immunogenicity induced by the FHV-1 modified live virus vaccine was evaluated by administering two injections, 21 days apart, to cats (Figure 1A). The results showed that the immunized cats produced significantly higher levels of IgG compared to the control group at 14 days after the second immunization (Figure 1B). Similarly, the neutralizing antibody levels were notably higher in the immunized cats than those of non-immunized cats (Figure 1C). These data demonstrate that the FHV-1 MLV vaccine effectively induced IgG and VN antibodies production against FHV-1 infection in cats.

CD8+ and CD4+ T cells play a crucial role in clearing viral infections. To further investigate the T cell responses induced by the FHV-1 MLV vaccine, the percentages of CD8+ and CD4+ T cells within the lymphocytes of vaccinated cats were measured using flow cytometry. Figure 2A depicts the gating strategy of CD4+ T cells and CD8+ T cells in flow cytometry. The results revealed a substantial increase in the CD4+ T cells in the immunized cats compared to the control and mock group (Figure 2B; p < 0.05). The immunized group also exhibited a significantly increased number of CD8+ T cells than the control and mock group (Figure 2C; p < 0.05). Furthermore, the mean fluorescence intensity (MFI) of CD134+ T cells was also detected as a result of CD134 being expressed on activated CD4 T cells (25). However, there was no significant increase in the MFI of CD134+ T cells in the immunized group compared to the control and mock group (Figure 2D; p > 0.05).

To assess the activation of the cellular immune response after immunization with the FHV-1 MLV vaccine, the cytokines levels of IFN-γ, IL-2, IL-4, and IL-17A in serum were detected by ELISA. Figure 3A illustrates significantly higher levels of IL-2 in the FHV-1 MLV vaccinated group than the control group (p < 0.01), with minimal IL-2 production in the control group after the FHV-1 challenge. However, no significant difference in IFN-γ and IL-17A levels was observed between the control and vaccinated groups (Figures 3B,C; p > 0.05). Furthermore, IL-4 levels were undetectable in all groups.

Based on the significant increase in percentages of CD8+ and CD4+ T cells and the elevated levels of IL-2 in the immunized group, it was hypothesized that the FHV-1 MLV vaccine may induces a Th1-type cell immune response in cats. To verify this, ELISpot assays detected significantly more SFCs secreting IFN-γ and IL-2, which are Th1-type cytokines, in the immunized groups compared to the control and mock groups (Figures 3D–G; p < 0.05). These results indicate a pronounced cellular immune response induced by the FHV-1 MLV vaccine in cats.

The protective efficacy of the FHV-1 MLV vaccine against FHV-1 was further evaluated by challenging cats with FHV-1 strain 64. As depicted in Figures 4A,B, cats in the control group exhibited severe clinical symptoms, including mouth breathing, mucoid ocular and nasal discharge, and sneezing. In contrast, vaccinated cats showed only mild clinical symptoms, and mock cats did not show any clinical symptoms. Clinical scores were evaluated as described previously (22, 23), and the results indicated a significant reduction in clinical scores in the vaccinated group compared to the control group (Table 1; Figure 4C; p < 0.05). A transient temperature increase was observed after the FHV-1 challenge in both groups, with temperatures exceeding 40°C in several control cats (Figure 4D). Moreover, as shown in Figure 4E, cats in the control group shed viral DNA in eyelid secretions for up to 2 weeks. In contrast, cats in the MLV vaccine group did not shed the virus until day 4 after infection for 5 days, and no viral shedding was detected in the mock group. These data suggest that the MLV vaccine significantly reduced clinical signs and greatly reduced FHV-1 shedding in cats.

Pathological examination 14 days post-infection revealed minimal lesions in immunized cats, while in control cats, lesions were limited to eyelids, turbinates, tracheas, and lungs, characterized by focal necrosis and desquamation of epithelial cells. Moreover, FHV-1 inclusion bodies were observed in the epithelial cells of the nasal mucosa (Figure 5A). Similarly, immunohistochemical (IHC) assays against FHV-1 showed positive staining in all control samples and negative staining in the MLV vaccine group (Figure 5B). Given that the cats in the mock group exhibited no clinical symptoms and no viral DNA was detected in their eyelid secretions, pathological examinations were not conducted on this group in consideration of animal welfare. These results indicated that the MLV vaccine confers potent protection against FHV-1 infection in cats.

Correlation analysis revealed significant negative correlations between clinical scores and VN titers (p < 0.05), IgG antibody titers (p < 0.05), IFN-γ SFC (p < 0.05), CD4 + T cells (p < 0.05), and CD8 + T cells (p < 0.05). Moderate inverse correlations were observed between clinical scores and IL-2 SFC, approaching significance (p > 0.05) (Figure 6).