Baculovirus Expression of Feline Panleukopenia Virus Capsid Protein and Induces Robust Immune Responses in Cats

doi:10.21203/rs.3.rs-3569093/v1

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Baculovirus Expression of Feline Panleukopenia Virus Capsid Protein and Induces Robust Immune Responses in Cats

https://doi.org/10.21203/rs.3.rs-3569093/v1

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Background

Feline panleukopenia (FP) is a highly contagious, often fatal viral disease that suffered by almost all members of the family Felidae. Feline panleukopenia virus (FPLV) is the major etiological pathogen of FP, which is a single-stranded, non-enveloped DNA virus. The VP2 protein is the major protective antigen of FPLV and be considered as a promising candidate for developing a novel subunit vaccine to immunoprophylaxis of FP.

Results

FPLV-VLPs was created by expressing VP2 protein in a baculovirus expression vector system (BEVS), and it showed higher antigenicity and higher hemagglutination (up to 1:2¹⁴⁾. Cats vaccinated with the FPLV-VLP vaccine produced a higher level of anti-FPLV HI antibodies (1:2¹⁶) and were 100% protected from challenge with virulent FPLV.

Methods

In this study, the gene encoding FPLV VP2 (GenBank OP491797) was codon optimized and obtained by gene splicing by overlap extension PCR(SOE PCR). Then, the optimized VP2 sequence was ligated to the pFastBac-I vector through homologous recombination technology. Finally, VP2 protein was expressed in a baculovirus expression vector system (BEVS) and it self-assemble into virus-like particles after treated with 100 mM NaHCO₃solution.

Conclusion

In summary, FPLV-VLPs with higer antigenicity and higher hemagglutination (up to 1:2¹⁴), was created by expressing VP2 protein in a baculovirus expression vector system (BEVS). Then, a novel a novel pet vaccine against FP with a higher effectiveness and safety profile was developed in this study. Cats vaccinated with the FPLV-VLP vaccine produced a higher level of anti-FPLV HI antibodies (1:2¹⁶) and were 100% protected from challenge with virulent FPLV. In subsequent applications, the FPLV-VLP vaccine can be used as a single vaccine or in combination with other vaccines to vaccinate cats for immunoprophylaxis against FPs.

In the past decade, China’s pet industry has rapidly developed. According to the White Paper on China's Pet Industry (2022), the number of pet cats raised in China has reached approximately 65 million, with an estimated market size of nearly 600 billion yuan by 2023. Therefore, diseases in cats must be prevented and controlled.

Feline panleukopenia (FP) is the most important disease in cats, once known as feline distemper [1, 2], feline infectious enteritis [3-5] or feline ataxia [6], is a highly contagious viral disease with high morbidity and mortality, especially in kittens or unvaccinated cats. Vaccinating cats is a cost-effective way to prevent and control FP. So far, approximately 10 vaccines, including inactivated vaccine or attenuated live vaccine, have been developed for immunoprophylaxis against FP worldwide. But, recent widespread outbreaks of FP have raised speculations that the effectiveness of vaccines may have decreased (Barrs, 2019; Diakoudi et al., 2022; Lloret, 2016; Pandey, 2022) based on the following knowledge: viral strains of the vaccines were collected over 50 years ago, and variation in the viruses have occurred since the strains were collected [7-10].

Feline panleukopenia virus (FPLV), the major aetiologic pathogen of FP, is a single-stranded, nonenveloped DNA virus with two open reading frames (ORFs). The structural protein VP2, encoded by the right ORF, is the major protective antigen that stimulates the host to produce neutralizing antibodies, and it is generally considered a promising candidate for the FPLV novel subunit vaccine [7, 11].

Virus-like particles (VLPs) refer to particles that self-assemble due to the expression of one or more capsid proteins [12], which can mimic the organization and conformation of authentic native viruses but lack viral genomes. Attributed to these properties, VLPs become a safe, economical, potent vaccine candidate of novel vaccine [13, 14]. The baculovirus expression system (BES) plays a key role in the self-assembly and release of VLPs [15-18] due to its capacity for large-scale cultivation and posttranslational modifications (PTMs) compared to other expression systems.

In this study, we prepared FPLV-VLPs by using a baculovirus expression system, and the characterization and immunogenicity of FPLV-VLPs were investigated. This research may provide basic data for the development of novel FPLV vaccines.

Construction and identification of rBacmid-VP2

After overlapping extension PCR amplification, a gene fragment of approximately 2, 000 bp in size was obtained (Figure 1a, lane 1 and 2), indicated we obtained the FPLV-VP2. Then, FPLV-VP2 gene fragments were subcloned to pFastBac-I vector through homologous recombination. PCR reaction and double enzymatic digestion (BamH I and EcoR I) were used to identify the recombinant transfer plasmid pFastBac-FPLV-VP2. After a fragment of approximately 2, 200 bp in size (Figure 1B, lane 1-8), and two gene fragment, 1, 926 bp and 4, 671 bp, respectively, were obtained (figure 1C, lane 2), indicated that the recombinant transfer plasmid pFastBac-FPLV-VP2 was correctly constructed. Finally, pFastBac-VP2 was transferred into E.coli DH10Bac competent cells to obtain rBacmid-VP2. The existence of the VP2 gene in bacmid DNA was determined by PCR using PUC/M13 universal primers. After the target band, approximately 4100 bp, were succeed amplified from the multiple selected white colonies (figure 1D, lanes 3-10), suggested that the rBacmid-VP2 was successfully constructed.

Expression Analysis of Recombinant Baculovirus FPLV-VP2

SDS-PAGE and immunoblotting analysis of recombinant baculovirus FPLV-VP2-infected Sf9 cell culture are shown in Figures 2 and 3. rFPLV-VP2 protein (molecular weight ~71 kDa) was mainly present in the precipitate of infected Sf9 cells (Figure 2, lane 2 arrow indicated). Immunoblotting analysis also indicated that the expression of rFPLV-VP2 was intracellular (Figure 3, lane 2 arrows indicated).

Indirect Immunofluorescence Assay of Recombinant FPLV-VP2

Infected insect cell Sf9 with recombinant baculovirus FPLV-VP2 showed high-intensity yellow-green fluorescence (Figure 4B), while the control group cells showed no visible fluorescence (Figure 4A), indicating that recombinant FPLV-VP2 protein was expressed.

TEM

Under TEM, many protein particles that exhibit circular spherical virus-like structures with a diameter of approximately 20-30 nm, like the FPLV native virus, were found in the supernatant of NaHCO₃-treated infected Sf9 cells (Figure 5).

Antigenicity Analysis of FPLV-VLP

Feline panleukopenia virus antigen rapid test immunochromatographic strips were used to detect the seriously diluted supernatant of NaHCO₃-treated infected Sf9 cells (FPLV-VLP) (10⁰-10^-4), and an obvious reaction band was observed in some dilution ratio samples (10⁰-10^-3) (Figure 6).

The hemagglutination analysis results are shown in Figure 7. The HA titer of FPLV-VLP (P4) was as high as 1:2¹⁴.

Immunogenicity Analysis of FPLV-VLP

Body Temperature Change after Vaccination

The body temperature of all cats in the experimental group or control group showed no significant change during the 10 days post-vaccination (data not shown), indicating the safety of all the vaccines.

Change in Hemagglutination Inhibition (HI) Antibody

A fluctuating curve of hemagglutination inhibition (HI) antibody titre of all cats is shown in Figure 8.

Seven days postvaccination (dpv), no cats in any group contained hemagglutination inhibition (HI) antibodies. At 14 dpv, the HI antibody titer of cats in Group I (VLP vaccine) rapidly increased (2⁹-2¹², red ling/scatter). Cats in Group II (commercial vaccine) still cannot detect HI antibodies (2⁰, green ling/scatter). At 21 dpv, the HI antibody titer of cats in Group I continuously increased to 2¹²-2¹³. Cats in Group II start to generate HI antibodies (2⁶-2⁸); however, after sufficient the days post-vaccination (28 dpv), the HI titer of cats in Group II can also reach a higher level (2¹³).

At 21 dpv, all cats were challenged with 5 milliliters of virulent FPLV virus (FPLV-CC-19 cell cultures, HI=2¹⁰, TCID₅₀=10^6.5/mL) through oral administration. The challenge-protection statistics results are shown in Table 1.

The FPLV-VLP vaccine (Group I) provided 100% protection to the coats, but the commercial vaccine (Group II) only provided a 30% protection rate. All cats in the control group died at 3 days- post-challenge (dpc) or 8 dpc.

The changes in body temperature of cats during the days post-challenge are shown in Figure 9. Two-thirds (approximately 67%, Group-II, green) of the cats in the commercial group and 2/2 (100%, Control, black) of the cats in the control group showed an increase in body temperature. The body temperatures of all other cats fluctuated within a normal range (Group I, red).

Cats in group II (2/3) and the control group (2/2) showed some clinical signs associated with feline panleukopenia (FP) after virus challenge, such as depression or languishing, diarrhea or even death (Table 2). Cats in groups I or II did not show any visible clinical symptoms.

3.7 Histopathology Analysis

The histopathological examination results obtained for muscle tissue from the vaccine inoculation site are shown in Figure 10.

The muscle cells were arranged neatly and tightly, with clear cell boundaries and consistent movement. The horizontal lines of muscle cells were clear, with alternating light and dark, and no abnormalities were found in the stroma. No obvious inflammatory cell infiltration was observed in any tissue sample.

To date, many platforms have been established to develop vaccines with high efficiency and safety profiles [19], of which VLPs have attracted the most attention in the veterinary vaccine field [20-22], especially in pet vaccines. Many researchers have tried to express VP2 protein through various expression systems, such as Escherichia coli [23-25], insect cell baculovirus [7, 11, 26, 27], or even silkworm [28]. These expression systems each have their own advantages and disadvantages. Due to the naïve peculiarity of parvovirus, expressed structural proteins easily self-assemble into virus-like particles (VLPs) and are applied as vaccines.

In this study, the VP2 protein (codon optimized) of an epidemic strain of feline panleukopenia virus that was isolated in northeast China in a previous study (data not provided) was successfully expressed in an insect cell baculovirus expression system (Figure 2 and 3). The expressed VP2 protein was properly folded into virus-like particles (VLPs), as confirmed by TEM (Figure 5). FPLV-VLP shows higher antigenicity because it can react with the immunochromatographic strips of FPV (Figure 9) and a monoclonal antibody against the expressed VP2 of canine parvovirus 2. In addition, FPLV-VLP exhibits higher hemagglutination activity (up to 1:2¹⁴) compared the HA titer of the epidemic strain of FPLV (1:2⁹).

The goal of each vaccine is to induce the production of antibodies that can block viral entry. In this research, an antibody response with an HI antibody titer as high as 1:2¹² was detected within 14 dpv after primary immunization with the FPLV-VLP vaccine, which is consistent with a report by Jiao et al. [29]. The commercial vaccine began to detect HI antibodies until 21 dpv (Figure 8). The titer of HI antibody in all vaccinated cats further increased to 1:2¹⁶ at 50 dpv.

The FPLV-VLP vaccine used in this study produces a good immune effect because it can provide 100% protection to vaccinated cats when challenged with a virulent FPLV strain. However, the commercial vaccine provides only 33% protection. A possible explanation may be that the titre of HI antibodies of cats in the commercial vaccine group was at a low level (up to 1:2⁶), which cannot sufficiently provide full protection to cats.

Sarcomas are common side effects of vaccines in cats [30, 31]. Therefore, in this study, muscle tissue from the vaccine inoculation site was taken and used for histopathological examination. As expected, there were no sarcomas or histological changes in any of the cats. In addition, the temperature of all vaccinated cats was maintained within normal limits. All these results indicated that the FPLV-VLP vaccine has a high safety profile.

Cells, Virus Stock, Reagents, and Antibodies

Spodoptera frugiperda (Sf9) insect cells (ATCC) were maintained in suspension in Sf900-II SFM medium (Invitrogen, Waltham, CA, USA) at 28°C. Cellfectin™ II transfection reagent was purchased from Gibco (Beijing, China). The BacPAK Baculovirus Rapid Titer Kit was purchased from Takara (Dalian, China). The FPLV-CC-19 strain was isolated and identified by our laboratory. Anti-CPV-2c-VP2 monoclonal antibody was also prepared in our laboratory.

Construction of a Recombinant Bacmid-VP2

The gene encoding FPLV VP2 (GenBank OP491797) was optimized for the baculovirus-insect cell expression system (BES). Sequence optimization and synthesis were completed by Nanjing Zoonbio BioTech Co., Ltd. (Nanjing, China). The primer sequences were as follows: FPLV-VP2-F: 5’-GATTATTCATACCGTCCCACCATC

GGGCGCGGATCCGCCACCATGCTGCTGGTGAACCAGAGCCACCAG-3’. FPLV-VP2-R: 5’-GCTGATTATGATCCTCTAGTACTTCTCGACAAGCTTTTAGG

CGTAGTCAGGCACGTCGTAGGGGTAGTA-3’. To enhance VP2 display on the baculovirus, a Kozak sequence, a gp67 secretion signal peptide, and a poly-histidine (6×His) tag were added to the N-terminus, and a haemagglutinin (HA) tag was added to the C-terminus.

The VP2 fragment was subcloned and inserted into the pFastBac plasmid through homologous recombination. PCR and double enzymatic digestion (Bam H I and EcoR I) verified the construction of pFastBac-VP2. DNA sequencing confirmed the accuracy of the VP2 gene in the recombinant plasmid. Then, pFastBac-VP2 was transferred into E.coli DH10Bac competent cells to obtain rBacmid-VP2. The existence of the VP2 gene in bacmid DNA was determined by PCR using PUC/M13 universal primers.

Transfection and Protein Expression

rBacmid-VP2 was transfected into Sf9 insect cells with Cellfectin™ II transfection reagent (Gibco, USA) according to the protocol provided by the manufacturer. At 5-6 days post-transfection, the supernatant was collected, and the titer of the recombinant baculovirus (defined as P1) was determined by using a Baculovirus Titer Kit (Takara, Dalian, China). The expression of the FPLV-VP2 capsid protein in Sf9 insect cells was confirmed by SDS-PAGE and Western blotting, which used an anti-His tag monoclonal antibody as a primary antibody.

Indirect Immunofluorescence Assay (IFA)

After four rounds of subculture, Sf9 cells were harvested by centrifugation at 8, 000 ×g for 20 minutes. The cell pellets were lysed with 25 mM NaHCO₃ at 4°C for 2 hours, and then the supernatant (rpFastBac-FPLV-VP2 protein) was collected by centrifugation at 8, 000 r/minute (rotator type F-34-6-38, Eppendorf, Germany) for 20 minutes. rpFastBac-FPLV-VP2 protein was verified by an indirect immunofluorescence assay (IFA). First, Sf9 cells were infected with rpFastBac-FPLV-VP2 protein for 48 h, after which the cells were fixed with 4% paraformaldehyde for 45 minutes at RT. After 3 times washes with PBST, a blocking solution (5% BSA) was added for 60 minutes. After another 3 times washes, the homemade anti-CPV-2c monoclonal antibody (clone 5B18) was diluted (1:200), and added to the wells and incubated at 37℃ for 60 minutes.3 times of washes again, a fluorescein isothiocyanate (FITC)-labelled sheep anti-mouse IgG was added at a dilution of 1:2, 000 and incubated at 37℃ for another 45 minutes. After the last 3 time washing, results were measured with an inverted fluorescence microscope (Zeiss Axioskop-40, Germany).

Antigenicity Analysis of rFPLV-VP2 Protein

The antigenicity of rFPLV-VP2 protein (P4) was determined by the feline panleukopenia virus antigen rapid test immunochromatographic strips produced by SHANGHAI QUICKING Biotech CO., Ltd.

The haemagglutinin activity of rFPLV-VP2 protein was tested using the haemagglutinin assay (HA). The HA test was carried out by using 96 well ‘V’ bottom microtiter plate, 25 μL of 0.1 M PBS (pH 6.4) was dispensed in well A-1 and thereafter across the rows (1-4) and columns (A-G) using 10-100 μL variable volume multichannel micropipette. Then, 25 μL of rFPLV-VP2 were put into first well of microtiter plate, set one biological replicates and control group. Then, they were serially diluted from1:2¹ to 1:2²⁰across through the first column. Next, 25 μL of PBS was dispensed all working wells and thereafter, 50 μL of 1% porcine RBC suspension was placed. The plate was agitated at 150 rpm for 2 min to ensure proper mixing of reactants. After that, the plate was put at 4℃ for 45 minutes and formation of serrated edged mat and bottom were recorded as negative and positive results (figure 7), respectively. The titer was calculated as reciprocal of the last well with agglutination.

Transmission Electron Microscopy (TEM)

The morphology of the rFPLV-VP2 protein (P4) was observed using negative staining with phosphotungstic acid. Briefly, 1 mL of rFPLV-VP2 protein and 20 μL of CaHPO₄ solution were placed into a 1.5 mL Eppendorf tube, mixed well, and stand at room temperature for 10 minutes. Then, the tube was centrifuged at 15，000 r/minutes for 15 minutes, and the sediment was dissolved with 15 μL of EDTA saturated solution to create a droplet. The droplet was dropped onto a copper grid, allowed to stand at room temperature for 20 minutes, and stained with 2% phosphotungstic acid staining solution (pH 6.8) for 1 minute. The excess staining solution was removed with filter paper, and the virus morphology was observed on a JEOL 2010 transmission electron microscope operated at an acceleration voltage of 100 kV.

Protocols for Animal Immunization and Challenge

Eight 3-4-month-old British shorthairs that were seronegative for FPLV were randomly divided into the following groups. Group I: 3 cats vaccinated with the FPLV VLP vaccine (Seppic adjuvant, 9:1); group II: 3 cats vaccinated with the commercial vaccine; and group III: 2 cats vaccinated with minimum essential medium (MEM). All cats were intramuscularly inoculated with a single dose (1.0 mL) of each sample. Blood samples were taken from all cats at 0-, 7-, 14-, 21-, 24-, 28-, 35-, 42-, and 50-days post-vaccination (dpv). The serum antibody titers against FPLV were determined by hemagglutination inhibition assay (HI).

Three weeks after vaccination, all cats were orally challenged with 5 mL of FPLV-CC-19 strain cell culture (HI=2⁹; TCID₅₀=10^6.5/mL). Continuous observation and record cats’ performances (mental state, food, and water intake, excrement shape, and rectal temperature) for 10 days post-challenge (dpc), to evaluate the immune efficacy of vaccines.

FPLV Feline Panleukopenia Virus

dpv Days Post-Vaccination

dpc Days Post-Challenge

HI Hemagglutination Inhibition

MEM Minimum Essential Medium

VLP Virus-Like Particles

TEM Transmission Electron Microscopy

Funding

This work was supported by the Science and Technology Development Plan of Jilin Province (No. 20210202099NC).

Ethics approval and consent to participate

This study was approved by the Animal Ethics Committee of Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences (approval number No.ISAPSAEC-2022-60C). Confirmed by the Animal Ethics Committee of Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, all of our methods were complied with the statement of the ARRIVE guidelines report. All methods were carried out in accordance with relevant guidelines and regulations.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

EF designed the study and performed the experiments. MY, JS and XZ calculated and statistically analyzed the experimental data. ZC, GL, CW and ZW provided support in the animal experiments. YC and SC provided experimental funding support. All the authors have read and approved the final manuscript.

Acknowledgement

Not applicable.

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Table 1 Challenge protection results obtained for different experimental groups

Groups	Vaccine	Number of Cats	Number of Diseased Cats	Protection Rate
Group I	FPLV-VLP vaccine	Three	0	100%
Group II	Commercial vaccine	Three	2	30%
Group III	Minimum essential medium	Two	2	0

No competing interests reported.

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Baculovirus Expression of Feline Panleukopenia Virus Capsid Protein and Induces Robust Immune Responses in Cats

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