Immunogenicity Evaluation of Modified Adenovirus Vaccines Expressing Porcine Circovirus Type 2 Capsid Protein in Pigs

Abstract

Porcine circovirus type 2 (PCV2) adenovirus vaccine has been reported, but strong immune responses induced by adenovirus vector can decrease vaccine efficacy. To reduce the immunogenicity of adenovirus proteins, in previous study, we constructed the PCV2 adenovirus vaccine either modified with human cytomegalovirus first intron (Intron A) and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to increase the expression of Cap, or coexpressed porcine tumor necrosis factor-related activate protein (CD40L) and granulocyte macrophage colony-stimulating factor (GMCSF) to improve the immunogenicity of PCV2 Cap adenovirus vaccine. All these vaccines were evaluated in mice. In the present study, the protective immune responses of Intron A/WPRE-modified recombinant adenovirus Ad-A-C-W and CD40L/GMCSF-modified recombinant adenovirus Ad-CD40L-Cap-GMCSF were evaluated in pigs. Enzyme-linked immunosorbent assay and virus neutralization assay showed that both Ad-A-C-W and Ad-CD40L-Cap-GMCSF could induce a higher specific antibody and neutralizing antibody than Ad-Cap (p < 0.05). Lymphocyte proliferation assay and cytokine release assay showed that Ad-A-C-W and Ad-CD40L-Cap-GMCSF induced a stronger cellular immune response than Ad-Cap. The PCV2 challenge experiment showed that viral loads of Ad-A-C-W-vaccinated group and Ad-CD40L-Cap-GMCSF-vaccinated group were lower than Ad-Cap vaccinated group (p < 0.05) after pigs were oronasally challenged with 5 × 10⁵ TCID₅₀ PCV2. Autopsy and histopathological examination showed that no obvious clinical and microscopic lesions were observed in groups Ad-Cap, Ad-A-C-W, and Ad-CD40L-Cap-GMCSF. Taken together, the results demonstrated that two modified recombinant adenovirus vaccines (Ad-A-C-W and Ad-CD40L-Cap-GMCSF) induced stronger humoral and cellular immune responses and provided better protection than unmodified adenovirus Ad-Cap. Therefore, Ad-A-C-W and Ad-CD40L-Cap-GMCSF would be used as potential vaccines for prevention and control of PCV2 infection.

Introduction

Porcine circovirus type 2 (PCV2), a main pathogen of porcine circovirus- associated disease (PCVAD), results in great economic losses in pig industry (20). Capsid protein (Cap) is the main immunogen of PCV2 and contains type-specific antigenic epitopes, which is usually applied to develop vaccines (13,17,23). Current vaccine strategies include inactivated virus vaccine (22), live vector engineering vaccine (11), DNA vaccine (12), chimeric virus of PCV1-PCV2 (9), and expression of PCV2 Cap in Pichia pastoris (26). Although some commercial PCV2 vaccines have been available and play certain roles in preventing PCVAD, the efficacy of these vaccines still needs to be further improved for eradicating PCVAD in the field completely (2).

The adenovirus expression system, one of the most widely used viral expression systems, shows more advantages than other systems, such as easily constructing, higher efficiency of gene transfer, higher titers, and inducing humoral, mucosal, and cellular immune responses together (3,8,10,14,17,24). However, strong immune responses induced by adenovirus proteins (mainly penton and hexon proteins) result in poor efficacy (32). Although the adenovirus vector system expressing PCV2 Cap has been studied, the vaccine efficacy is limited (27,28). So, it is necessary to reduce the immunogenicity of adenovirus itself. In the previous study, we have verified that either inserting largest intron of the human cytomegalovirus (Intron A) and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) into adenovirus vector, or coexpression of CD40 ligand (CD40L) and granulocyte macrophage colony-stimulating factor (GMCSF) can increase adenovirus vector vaccine efficacy in mice (15,16).

In this study, the immune responses of recombinant adenoviruses Ad-A-C-W and Ad-CD40L-Cap-GMCSF were further evaluated in pigs. The results demonstrated that two kinds of recombinant adenovirus vaccines could induce stronger humoral and cellular immune responses and provide better protective efficacy than unmodified adenovirus Ad-Cap.

Materials and Methods

Viruses and cell lines

The AdEasy™ adenovirus vector system derived from human adenovirus serotype 5 was bought from Agilent Technologies (La Jolla). Recombinant adenoviruses Ad-Cap, Ad-A-C-W, Ad-CD40L-Cap-GMCSF and empty vector adenovirus Ad-blank (human adenovirus serotype 5) were cultured and propagated in human embryonic kidney 293 cells (HEK293) (ATCC, CRL-1573) as previously described (15,16), and viral titers of these adenoviruses were all about 10¹⁰ TCID₅₀/mL. Porcine kidney 15 cells (PK15), which were PCV1-free, were used to propagate PCV2 (GenBank No. EU366323).

Animal experiments

Thirty-five commercial 28-day-old Yorkshire pigs without the PCV2 antibody were randomly divided into seven groups each with five pigs and housed in seven separate rooms. All pigs used in the experiment were confirmed to be free of porcine parvovirus, porcine reproductive and respiratory syndrome virus, and classical swine fever virus by routine serological testing. Pig experiments were approved by the Animal Ethics Committee of North West Agriculture and Forestry University. Groups 1–3 were immunized intramuscularly with 10¹⁰ TCID₅₀ of Ad-Cap, Ad-A-C-W, and Ad-CD40L-Cap-GMCSF, respectively; group 4 was vaccinated intramuscularly with commercial inactivated vaccine (inactivated virus of PCV2b [SH Strain]) according to the manufacturer's instruction (Qianyuanhao, Nanjing, China); group 5 was immunized intramuscularly with 10¹⁰ TCID₅₀ of empty vector adenovirus Ad-blank (human adenovirus serotype 5); group 6 was vaccinated with phosphate-buffered saline (PBS) (nonvaccinated but challenged group); group 7 was used as untreated control (nonvaccinated and nonchallenged group). Booster immunization was conducted on 14 days postimmunization (dpi) under the same condition. On 28 dpi, groups 1–6 were challenged oronasally with 5 × 10⁵ TCID₅₀ PCV2 per pig. All animals were monitored for 63 days after challenge. Rectal temperatures and clinical signs were detected weekly. Serum samples of each group were collected on 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91 dpi for enzyme-linked immunosorbent assay (ELISA) and virus neutralization assay (VN). On 28 dpi, swine peripheral blood lymphocytes were used for further lymphocyte proliferation assay and cytokine (IFN-γ and IL-13) release assay. On 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91 dpi, serum samples were collected for detecting viremia by quantitative real-time PCR (Q-PCR). On 91 dpi, all animals were euthanized, and superficial inguinal lymph nodes and lungs were collected for histopathological examination.

Enzyme-linked immunosorbent assay

Sera of five pigs from each group were collected from the precava on 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91 dpi for detecting specific antibody by using commercial PCV2 antibody ELISA detection kits (Yuanye Biotechnology Co., Shanghai, China) according to the manufacturer's instruction.

Virus neutralization assay

The PCV2 neutralizing antibody was tested by VN. Briefly, serum samples were diluted in twofold, starting with 1:2. Then, 100 TCID₅₀ PCV2 was added and incubated for 1 h at 37°C. Then, the serum–virus mixture was added into PK15 as previously described (5). After 72 h, PK15 cells were fixed with cold absolute ethanol at 4°C for 15 min, incubated with the rabbit anti-PCV2 antibody, and stained with FITC-conjugated goat anti-rabbit IgG (Boshide, Wuhan, China). Titers were conducted as the reciprocal of the last serum dilution at 80% or greater than 80% fluorescent focus reduction by the fluorescent microscope.

Lymphocyte proliferation assay

On 28 dpi, lymphocytes were aseptically separated from the swine peripheral blood with pig lymphocyte separation medium (Dakewe, Shenzhen, China) following the manufacturer's instruction. Lymphocytes were seeded into a 96-well plate and stimulated for 68 h with concanavalin A (ConA, 5 μg/mL; Sigma, Los Angeles) as positive control, Cap protein (20 μg/mL) as the stimulant, and RPMI-1640 as negative control. Then, 20 μL of MTT (3-[4, 5-dimethylthiazol-2-y]-2, 5-diphenyltetrasodium bromide tetrazolium, Sigma, 5 mg/mL in PBS) was added per well with further incubation for 4 h. Dimethyl sulfoxide was added 100 μL per well to stop the reactions. The OD570 nm was detected and the stimulation index (SI) was determined as follows: SI = mean OD of Cap-stimulated cells/mean OD of unstimulated cells.

Cytokine release assay

Lymphocytes were separated aseptically from the swine peripheral blood of five pigs from each group as above on 28 dpi. Then, the lymphocytes were seeded into a 96-well plate with 2 × 10⁵ cells per well. RPMI-1640 alone and 2 μg Cap protein were added into each well and the supernatants were collected 72 h later. Titers of IFN-γ and IL-13 were detected by pig IFN-γ and pig IL-13 ELISA Kits (ExCell Bio, Shanghai, China) following standard procedures. The concentrations of IFN-γ and IL-13 in the supernatants were calculated by the standard curves.

Detection of viremia in blood

On 28 dpi, groups 1–6 were challenged with 5 × 10⁵ TCID₅₀ PCV2. Following challenge, all pigs were monitored daily for clinical signs, including sneezing, lethargy, and coughing. Serum samples were collected on 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91 dpi. Virus DNA was extracted from serum samples and detected by Q-PCR established previously (29). Briefly, PCV2 genomic DNA was extracted by DNA extraction kits (Axygen, Hangzhou, China). The concentrations of plasmid containing PCV2 genomic DNA were determined by the NanoDrop 2000 Spectrophotometer (Thermo Scientific). Copy number was calculated using the following formula: amount (copies/μL) = 6.02 × 10²³ (copies/mol) × concentration (g/μL)/MW (g/mol). The viral copy number in samples per μL was tested by real-time PCR with serially diluted plasmids from 10² to 10⁸ copies/μL. Real-time PCR was performed with primers (forward 5′-AAGGGCTGGGTTATGGTATGT-3′ and reverse 5′-CGCTGGAGAAGGAAAAATGG-3′). Fluorescence data were recorded in real time with an IQ5 multicolor real-time PCR detection system (Bio-Rad). The viral loads were determined as the mean of the logarithmic DNA copy number divided by mL (Lg copies/mL). The sensitivity of Q-PCR used in the study was 1,000 copies/mL.

Histopathological examination

The gross lesions of lungs and lymph nodes were detected after necropsy. Superficial inguinal lymph nodes and lungs were removed and fixed in 10% buffered formalin. The fixed samples were dehydrated and embedded in paraffin wax. Consecutive 5-μm-thick sections were cut and processed for hematoxylin and eosin staining (HE). Microscopic pathological lesions were conducted as − (normal), + (mild), ++ (moderate), and +++ (severe) as previously described (28).

Statistical analyses

All data were treated as the mean ± standard error of mean (SEM). The ELISA, VN, lymphocyte proliferation assay, cytokine release assay, and Q-PCR results were assessed by one-way analysis of variance (ANOVA) and the Student–Newman–Keuls (SNK) test; temperature was assessed by two-way repeated measures ANOVA and the SNK test. All statistical analyses were conducted with SPSS 19.0 software (SPSS, Inc., Chicago, IL). A value of p < 0.05 was considered as a significant statistical difference.

Results

Temperature detection

Rectal temperatures of all pigs were detected weekly since the first immunization. As shown in Table 1, temperatures of all groups were normal before 28 dpi. However, temperatures of Ad-blank group and PBS group were significantly higher than the Ad-Cap-vaccinated group, Ad-A-C-W-vaccinated group, Ad-CD40L-Cap-GMCSF-vaccinated group, and SH-strain group from 35 to 56 dpi (p < 0.05), and the mean temperatures of both the Ad-blank group and PBS group exceeded 39.5°C and even reached 40°C at a certain time point. The temperatures of Ad-blank group and PBS group gradually decreased since 63 dpi and got back within 39.5°C on 77 dpi. Pigs of Ad-Cap-vaccinated group, Ad-A-C-W-vaccinated group, Ad-CD40L-Cap-GMCSF-vaccinated group, SH-strain group, and untreated group kept normal temperature (38°C–39.5°C) after challenge.

Table 1.

Temperature Detection of Recombinant Adenoviruses of Immunized Pigs

	Groups
Days	Untreated control	PBS	Ad-blank	Ad-Cap	Ad-A-C-W	Ad-CD40L-Cap-GMCSF	SH-strain
0	39.08 ± 0.07	39.10 ± 0.02	38.84 ± 0.16	39.20 ± 0.07	39.14 ± 0.06	39.12 ± 0.16	39.16 ± 0.15
7	38.58 ± 0.13	39.16 ± 0.04	38.82 ± 0.14	38.58 ± 0.20	38.80 ± 0.10	38.68 ± 0.12	38.8 ± 0.29
14	38.74 ± 0.07	39.08 ± 0.13	39.10 ± 0.17	38.96 ± 0.21	38.62 ± 0.10	38.62 ± 0.12	38.72 ± 0.26
21	38.90 ± 0.08	38.80 ± 0.05	38.88 ± 0.22	39.08 ± 0.14	38.84 ± 0.11	38.56 ± 0.16	38.94 ± 0.02
28	39.00 ± 0.20	38.60 ± 0.10	38.64 ± 0.07	38.80 ± 0.22	38.94 ± 0.18	38.74 ± 0.21	38.6 ± 0.18
35	38.92 ± 0.15a	39.90 ± 0.05b	39.74 ± 0.02b	38.82 ± 0.02a	39.04 ± 0.07a	38.58 ± 0.13a	38.78 ± 0.13a
42	38.76 ± 0.16a	40.02 ± 0.13b	40.05 ± 0.05b	38.82 ± 0.10a	38.76 ± 0.07a	39.02 ± 0.04a	38.90 ± 0.17a
49	38.46 ± 0.17a	39.92 ± 0.07b	39.86 ± 0.07b	38.82 ± 0.41a	38.94 ± 0.13a	38.92 ± 0.13a	38.62 ± 0.17a
56	38.60 ± 0.10a	39.90 ± 0.04b	40.00 ± 0.21b	38.50 ± 0.62a	38.96 ± 0.06a	39.18 ± 0.02a	38.70 ± 0.12a
63	38.98 ± 0.09a/b	39.88 ± 0.13b	39.76 ± 0.04b	38.54 ± 0.59a	39.24 ± 0.07a/b	39.32 ± 0.04a/b	38.92 ± 0.24a/b
70	38.96 ± 0.06a/b	39.80 ± 0.05c	38.98 ± 0.04a/b	38.26 ± 0.46a	39.18 ± 0.13b/c	38.86 ± 0.04a/b	39.2 ± 0.12b/c
77	38.92 ± 0.18	39.20 ± 0.13	38.86 ± 0.03	39.02 ± 0.18	38.80 ± 0.04	38.90 ± 0.09	38.92 ± 0.15
84	38.92 ± 0.22	39.06 ± 0.02	38.78 ± 0.05	38.80 ± 0.10	39.00 ± 0.24	38.54 ± 0.19	39.10 ± 0.19
91	39.12 ± 0.12	39.24 ± 0.07	38.72 ± 0.12	39.06 ± 0.10	38.84 ± 0.06	38.94 ± 0.18	38.88 ± 0.22

Rectal temperatures of different groups were detected weekly after immunization. Data are presented as the mean ± SEM. Treatments with different lowercase letters (a–c) indicate statistically significant differences between the indicated groups (p < 0.05).

GMCSF, granulocyte macrophage colony-stimulating factor; PBS, phosphate-buffered saline; SEM, standard error of mean.

Humoral immune response evaluation in pigs

Specific antibody to PCV2 detection in pigs

Anti-PCV2 -specific antibody titers of different groups were evaluated on 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91 dpi by commercial ELISA kits as per the manufacturer's instruction. As shown in Table 2, specific antibody titers of Ad-Cap-vaccinated group, Ad-A-C-W-vaccinated group, Ad-CD40L-Cap-GMCSF- vaccinated group, and SH-strain group were initially detected on 14 dpi and maintained till 63 days. On 28 dpi, the specific antibody of Ad-CD40L-Cap-GMCSF-vaccinated group was higher than others (p < 0.05), Ad-A-C-W-vaccinated group was higher than Ad-Cap-vaccinated group (p < 0.05), but the Ad-A-C-W-vaccinated group was significantly lower than SH-strain group (p < 0.05). On 35, 49, 56, 63, and 70 dpi, the specific antibody of SH-strain group was significantly higher than other groups (p < 0.05), specific antibody of Ad-CD40L-Cap-GMCSF-vaccinated group was higher than Ad-Cap-vaccinated group (p < 0.05) but was lower than Ad-A-C-W-vaccinated group (p < 0.05). On 42 and 77 dpi, specific antibody of SH-strain group was higher than other groups (p < 0.05), specific antibodies of Ad-CD40L-Cap-GMCSF-vaccinated group and Ad-A-C-W-vaccinated group were not different (p > 0.05), but both were significantly higher than Ad-Cap-vaccinated group (p < 0.05). Ad-blank and PBS groups developed certain specific antibodies on 42, 49, 56, 63, and 70 dpi.

Table 2.

ELISA for PCV2-Specific Antibodies of Sera from the Pigs Immunized with Different Adenoviruses

	Groups
Days	Untreated control	PBS	Ad-blank	Ad-Cap	Ad-A-C-W	Ad-CD40L-Cap-GMCSF	SH-strain
0	0.067 ± 0.004	0.067 ± 0.001	0.074 ± 0.001	0.057 ± 0.002	0.066 ± 0.004	0.074 ± 0.002	0.064 ± 0.001
7	0.077 ± 0.004	0.076 ± 0.002	0.082 ± 0.002	0.073 ± 0.007	0.072 ± 0.004	0.084 ± 0.001	0.074 ± 0.001
14	0.074 ± 0.002a	0.077 ± 0.001a	0.076 ± 0.002a	0.192 ± 0.004b	0.214 ± 0.011c	0.187 ± 0.007b	0.259 ± 0.008d
21	0.077 ± 0.004a	0.076 ± 0.002a	0.083 ± 0.002a	0.261 ± 0.004b	0.364 ± 0.007c	0.481 ± 0.008d	0.504 ± 0.006e
28	0.079 + 0.003a	0.086 + 0.001a	0.074 + 0.002a	0.398 + 0.003b	0.568 + 0.005c	0.764 + 0.009d	0.685 + 0.005e
35	0.078 ± 0.003a	0.078 ± 0.002a	0.084 ± 0.001a	0.548 + 0.006b	0.851 + 0.015c	0.713 + 0.008d	0.935 + 0.010e
42	0.082 ± 0.002A	0.227 ± 0.007b	0.220 ± 0.007b	0.427 + 0.009c	0.665 + 0.012d	0.647 + 0.011d	0.767 + 0.009e
49	0.077 ± 0.003a	0.252 ± 0.008b	0.217 ± 0.006b	0.326 + 0.009c	0.583 + 0.005d	0.514 + 0.006e	0.665 + 0.015f
56	0.079 ± 0.004A	0.255 ± 0.008b	0.236 ± 0.001b	0.209 + 0.007b	0.440 + 0.009c	0.380 + 0.010d	0.549 + 0.009e
63	0.073 ± 0.004a	0.251 ± 0.006B	0.219 ± 0.010b	0.317 + 0.005c	0.560 + 0.013d	0.524 + 0.007e	0.657 + 0.015f
70	0.064 ± 0.001A	0.238 ± 0.008b	0.214 ± 0.007b	0.270 + 0.007c	0.357 + 0.004d	0.322 + 0.006e	0.442 + 0.008f
77	0.073 ± 0.002a	0.136 ± 0.008B	0.081 ± 0.005a	0.127 + 0.004b	0.181 + 0.005c	0.174 + 0.003c	0.227 + 0.008d
84	0.068 ± 0.002	0.086 ± 0.006	0.065 ± 0.005	0.068 ± 0.003	0.096 ± 0.009	0.096 ± 0.007	0.120 ± 0.005
91	0.064 ± 0.002	0.065 ± 0.001	0.065 ± 0.001	0.067 ± 0.002	0.081 ± 0.007	0.079 ± 0.005	0.111 ± 0.005

PCV2-specific antibodies of different groups were detected weekly after immunization. Data are presented as the mean ± SEM. Treatments with different lowercase letters (a–f) indicate statistically significant differences between the indicated groups (p < 0.05).

ELISA, enzyme-linked immunosorbent assay; PCV2, porcine circovirus type 2.

Neutralizing antibody level detection in pigs

Neutralizing antibody titers were detected on 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91 dpi by VN. As shown in Table 3, neutralizing antibodies of Ad-Cap-vaccinated group, Ad-A-C-W-vaccinated group, Ad-CD40L-Cap-GMCSF-vaccinated group, and SH-strain group were initially detected on 14 dpi and maintained 63 days. On 28 dpi, neutralizing antibodies of Ad-A-C-W-vaccinated group, Ad-CD40L-Cap-GMCSF-vaccinated group, and SH-strain group were significantly higher than Ad-Cap-vaccinated group (p < 0.05), but there were no differences among them (p > 0.05). On 35 dpi, neutralizing antibody of SH-strain group was higher than other groups (p < 0.05), Ad-CD40L-Cap-GMCSF-vaccinated group was higher than Ad-Cap-vaccinated group (p < 0.05), but was not different from Ad-A-C-W-vaccinated group (p > 0.05). On 42, 49, and 63 dpi, neutralizing antibody of SH-strain group was higher than Ad-Cap-vaccinated group (p < 0.05), but was not different from Ad-CD40L-Cap-GMCSF-vaccinated group and Ad-A-C-W-vaccinated group (p > 0.05). There was no difference among Ad-Cap-vaccinated group, Ad-A-C-W-vaccinated group, and Ad-CD40L-Cap-GMCSF-vaccinated group (p > 0.05). On 56 dpi, neutralizing antibody of SH-strain group was higher than other groups (p < 0.05), there was no difference among Ad-Cap-vaccinated group, Ad-A-C-W-vaccinated group, and Ad-CD40L-Cap-GMCSF-vaccinated group (p > 0.05). On 70 dpi, neutralizing antibody of SH-strain group was higher than other groups (p < 0.05), Ad-A-C-W-vaccinated group was higher than Ad-Cap-vaccinated group and Ad-CD40L-Cap-GMCSF-vaccinated group (p < 0.05). On 77 dpi, neutralizing antibody of SH-strain group was not different from Ad-A-C-W-vaccinated group and Ad-CD40L-Cap-GMCSF-vaccinated group (p > 0.05). And there was no difference between Ad-CD40L-Cap-GMCSF-vaccinated group and Ad-Cap-vaccinated group (p > 0.05). Besides, Ad-blank and PBS groups developed certain neutralizing antibodies on 42, 49, 56, 63, 70, and 77 dpi.

Table 3.

IFA for Neutralization Activity of Sera from the Pigs Immunized with Different Adenoviruses

	Groups
Days	Untreated control	PBS	Ad-blank	Ad-Cap	Ad-A-C-W	Ad-CD40L-Cap-GMCSF	SH-strain
0	0 ± 0.0	0 ± 0.0	0 ± 0.0	0 ± 0.0	0 ± 0.0	0 ± 0.0	0 ± 0.0
7	0 ± 0.0	0 ± 0.0	0.8 ± 0.5	10.4 ± 2.4	25.6 ± 3.9	25.6 ± 3.9	44.8 ± 7.8
14	2 ± 0.0a	2 ± 0.0a	2.4 ± 0.4a	25.6 ± 3.9b	51.2 ± 7.8c	41.6 ± 9.6c	57.6 ± 6.4c
21	2.8 ± 0.5a	2.8 ± 0.5a	3.2 ± 0.5a	38.4 ± 6.4a	76.8 ± 12.8b	89.6 ± 15.7b	102.4 ± 15.7b
28	2.4 ± 0.4a	2.4 ± 0.4a	3.2 ± 0.5a	89.6 + 15.7b	153.6 + 25.6c	204.8 + 31.4c	204.8 + 31.4c
35	2 ± 0.0a	2 ± 0.0a	2 ± 0.0a	128 + 0.0b	204.8 + 31.4c	179.2 + 31.4c	256 + 0.0d
42	2.4 ± 0.4a	38. 4 ± 6.4a	41. 6 ± 9.6a	115.2 + 12.8b	179.2 + 31.4b/c	153.6 + 25.6b/c	204.8 + 31.4c
49	2 ± 0.0a	44.8 ± 7.8a/b	44.8 ± 7.8a/b	89.6 + 15.7b	128 + 0.0b/c	128 + 0.0b/c	179.2 + 31.4c
56	2 ± 0.0a	57.6 ± 6.4b	57.6 ± 6.4b	57.6 ± 6.4b	102.4 ± 15.7b	89.6 ± 15.7b	153.6 ± 25.6c
63	2 ± 0.0a	44.8 ± 7.8a/b	57.6 ± 6.4b	89.6 ± 15.7b/c	128 ± 0.0c	102.4 ± 15.7b/c	179.2 ± 31.4d
70	2 ± 0.0a	44.8 ± 7.8B	44.8 ± 7.8b	44.8 + 7.8b	89.6 + 15.7c	57.6 + 6.4b	128 + 0.0D
77	2 ± 0.0a	22.4 ± 3.9a/b	22.4 ± 3.9a/b	22.4 + 3.9a/b	57.6 + 6.4C	44.8 + 7.8b/c	89.6 + 15.7c
84	2 ± 0.0	9.6 ± 1.6	9.6 ± 1.6	9.6 ± 1.6	12.8 ± 2.0	12.8 ± 2.0	19.2 ± 3.2
91	2 ± 0.0	2 ± 0.0	2 ± 0.0	3.2 ± 0.5	5.6 ± 1.0	4.8 ± 0.8	6.4 ± 1.0

Neutralizing titers are reciprocals of the highest dilution of serum that yielded 80% reduction in virus infectivity. Data are presented as the mean ± SEM. Treatments with different lowercase letters (a–d) indicate statistically significant differences in neutralizing antibody levels between the indicated groups (p < 0.05).

IFA, immunofluorescence assay.

Cellular immune response detection in pigs

Lymphocyte proliferative responses

Swine peripheral blood lymphocyte proliferative levels of different groups were detected on 28 dpi. As shown in Figure 1, SI of Ad-Cap-vaccinated group, Ad-A-C-W-vaccinated group, Ad-CD40L-Cap-GMCSF-vaccinated group, and SH-strain group was significantly higher than that of Ad-blank and PBS-treated pigs (p < 0.05). Ad-CD40L-Cap-GMCSF-vaccinated group and SH-strain group showed higher SI than other groups (p < 0.05), and Ad-A-C-W-vaccinated group was higher than Ad-Cap-vaccinated group (p < 0.05).

FIG. 1.

Lymphocyte proliferation levels of swine peripheral blood lymphocytes after specific stimulation. After immunization with different recombinant adenoviruses, swine peripheral blood was collected and lymphocytes were pooled for lymphocyte proliferation assay. Data are presented as the mean ± SEM. Treatments with different lowercase letters (a–d) indicate statistically significant differences in SI between the indicated groups (p < 0.05). GMCSF, granulocyte macrophage colony-stimulating factor; SEM, standard error of mean; SI, stimulation index.

Cytokine detection by ELISA

On 28 dpi, IFN-γ and IL-13 secretion levels from swine peripheral blood lymphocytes were measured to evaluate cellular immune responses. As shown in Figure 2, IFN-γ and IL-13 were higher in Ad-Cap-vaccinated group, Ad-A-C-W-vaccinated group, Ad-CD40L-Cap-GMCSF-vaccinated group, and SH-strain group than that in Ad-blank group and PBS group (p < 0.05). The Ad-CD40L-Cap-GMCSF-vaccinated group and SH-strain group showed higher IFN-γ and IL-13 levels than other groups (p < 0.05), and Ad-A-C-W-vaccinated group was higher than Ad-Cap-vaccinated group (p < 0.05).

FIG. 2.

Levels of IFN-γ and IL-13 produced from lymphocytes in immunized pigs. On 28 dpi, swine peripheral blood lymphocytes from each group were harvested, cultured in 96-well plates, and stimulated with purified Cap. IFN-γ and IL-13 production in the supernatant was analyzed by ELISA. Data represent the mean ± SEM. Treatments with different lowercase letters (a–d) indicate statistically significant differences in cytokine levels between the indicated groups (p < 0.05). ELISA, enzyme-linked immunosorbent assay.

Viral load detection in serum samples

Virus DNA extracted from swine peripheral blood of seven groups was measured on 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91 dpi. As shown in Table 4, Viremia was undetectable on 28 dpi. On 35, 42, 49, 70, 84, and 91 dpi, the viral loads of Ad-blank group and PBS group were detectable and higher than other vaccinated groups (p < 0.05). Ad-A-C-W-vaccinated group, Ad-CD40L-Cap-GMCSF-vaccinated group, and SH-strain group showed lower viral loads than Ad-Cap-vaccinated group (p < 0.05). Ad-A-C-W-vaccinated group did not differ from SH-strain group (p > 0.05), but both were lower than Ad-CD40L-Cap-GMCSF-vaccinated group (p < 0.05). On 56, 63, and 77 dpi, the viral loads of Ad-blank group and PBS group were higher than other vaccinated groups (p < 0.05). Ad-A-C-W-vaccinated group, Ad-CD40L-Cap-GMCSF-vaccinated group, and SH-strain group showed lower viral loads than Ad-Cap-vaccinated group (p < 0.05). Ad-A-C-W-vaccinated group was higher than SH-strain group (p < 0.05), but both were lower than Ad-CD40L-Cap-GMCSF-vaccinated group (p < 0.05).

Table 4.

Quantification of PCV2 Viral Loads in Serum from the Challenged Pigs

	Groups
Days	Untreated control	PBS	Ad-blank	Ad-Cap	Ad-A-C-W	Ad-CD40L-Cap-GMCSF	SH-strain
28	ND	ND	ND	ND	ND	ND	ND
35	ND	8.03 ± 0.01a	8.25 ± 0.05a	5.91 + 0.06b	4.79 + 0.08c	5.22 + 0.13d	4.72 + 0.12c
42	ND	7.80 ± 0.07a	7.99 ± 0.10b	5.62 + 0.05c	4.59 + 0.07d	4.96 + 0.05e	4.53 + 0.04d
49	ND	7.65 ± 0.04a	7.66 ± 0.04a	5.39 + 0.06b	4.48 + 0.06c	4.87 + 0.04d	4.43 + 0.04c
56	ND	7.52 ± 0.01a	7.60 ± 0.03a	5.04 + 0.04b	4.16 + 0.03c	4.46 + 0.06d	3.96 + 0.05e
63	ND	7.32 ± 0.05a	7.39 ± 0.05b	4.96 + 0.01c	4.07 + 0.04d	4.39 + 0.03e	3.94 + 0.04f
70	ND	7.13 ± 0.03a	7.26 ± 0.04b	4.90 + 0.02c	3.91 + 0.01d	4.33 + 0.01e	3.84 + 0.03d
77	ND	6.84 ± 0.04a	7.03 ± 0.06b	4.85 + 0.04c	3.90 + 0.02d	4.22 + 0.05e	3.84 + 0.01f
84	ND	6.85 ± 0.03a	6.96 ± 0.03b	4.75 + 0.03c	3.85 + 0.02d	4.25 + 0.01e	3.78 + 0.01d
91	ND	6.99 ± 0.03a	7.02 ± 0.02b	4.66 + 0.02c	3.83 + 0.02d	4.25 + 0.01e	3.76 + 0.01d

Pigs were challenged with 5 × 10⁵ TCID₅₀ PCV2 on 28 dpi, and PCV2 viral loads were detected by Q-PCR in serum samples collected from pigs on 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91 dpi. Data are presented as the mean ± SEM. The viral loads were determined as the mean of the logarithmic DNA copy number divided by mL (Lg copies/mL). The sensitivity of Q-PCR in our study was 1,000 copies/mL. Treatments with different lowercase letters (a–f) indicate statistically significant differences in viral loads between the indicated groups (p < 0.05). ND refers to not detected.

Histopathological examination in tissues

Pigs of groups 1–6 were challenged with PCV2 on 28 dpi and all pigs were executed on 91 dpi. Superficial inguinal lymph nodes and lungs were collected to conduct histopathological examination. The microscopic lesions were observed by an optical microscope. All pigs of groups 1–6 showed PCVAD pathologic changes in different degrees. The main gross lesions of lungs showed diffuse interstitial pneumonia, swelling, volume augmented, interstitial broadening, scleroid, and varying sizes of brown area scattered on the surface. The main gross lesions of lymph nodes were swelling to varying degrees, and cut surface color of lymph nodes was incanus or kermesinus, cut surface was moist, soft, and sappiness, sometimes even bleeding. The microscopic lesions of lungs were mainly interstitial pneumonia and inflammatory cell infiltration. The alveolar septa of groups 5 and 6 were significantly incrassate and infiltrated by many inflammatory cells, but groups 1–4 were mildly incrassate. The microscopic lesions of lymph nodes showed lymphoid depletion and inflammatory cell infiltration. Lymphoid follicles of groups 5 and 6 were significantly lymphocytes-lacking and many inflammatory cells infiltration, but lymphoid follicles of groups 1–4 were structural integrity, few lymphocytes decreasing and inflammatory cells infiltration (Fig. 3). The histological lesions of lungs and lymph nodes from different groups are summarized in Table 5.

FIG. 3.

The histopathological features of lungs and lymph nodes from the challenged pigs. (A) Lung of Ad-Cap; (B) lung of Ad-A-C-W; (C) lung of Ad-CD40L-Cap-GMCSF; (D) lung of SH-strain; (E) lung of Ad-blank; (F) lung of PBS; (G) lymph node of Ad-Cap; (H) lymph node of Ad-A-C-W; (I) lymph node of Ad-CD40L-Cap-GMCSF; (J) lymph node of SH-strain; (K) lymph node of Ad-blank; (L) lymph node of PBS. Scale bar: 50 μm.

Table 5.

Comparison of Histological Lesions Found in Lungs and Lymph Nodes of Pigs in Each Group

Group/ear label		Lungs		Lymph nodes
Group	Ear label	Alveolar wall thickening	Inflammation cell infiltration	Lymphocyte deficiency	Inflammation cell infiltration
Ad-Cap	1	−	−	−	+
	2	−	−	−	−
	3	+	−	+	−
	4	−	−	−	−
	5	+	+	+	−
Ad-A-C-W	6	−	−	−	−
	7	−	−	−	−
	8	−	+	−	−
	9	−	−	+	−
	10	+	−	−	−
Ad-CD40L-Cap-GMCSF	11	+	−	−	−
	12	−	+	−	−
	13	+	−	+	+
	14	−	−	+	−
	15	−	−	−	−
SH-strain	16	−	−	−	−
	17	+	−	−	−
	18	−	+	−	−
	19	−	−	+	−
	20	+	−	−	−
Ad-blank	21	+++	++	+++	++
	22	+++	++	+++	++
	23	+++	+++	+++	+++
	24	++	++	++	++
	25	++	+++	+++	++
PBS	26	+++	+++	+++	+++
	27	+++	+++	+++	+++
	28	++	+++	+++	+++
	29	+++	+++	+++	+++
	30	+++	+++	+++	++
Untreated control	31	−	−	−	−
	32	−	−	−	−
	33	−	−	−	−
	34	−	−	−	−
	35	−	−	−	−

−, normal; +, mild lesions; ++, moderate lesions; +++, severe lesions.

Discussion

Many measures were taken to prevent PCVAD, and vaccination was considered to be the most effective one. Adenovirus vector system is one of the most effective virus vectors and has been used to express various proteins (4,25). Adenovirus vector system expressing PCV2 Cap protein has been studied, but has limited immune efficacy (27,28). One possible reason is that many proteins expressed by adenovirus itself could induce strong immune responses, resulting in poor efficacy (32). So it is necessary to reduce the immunogenicity of adenovirus proteins.

In previous study, we have constructed Intron A and/or WPRE-modified PCV2 adenovirus vaccines and evaluated the vaccine efficacy in mice and showed that Intron A and WPRE synergistically increase Cap expression and enhance the immune responses to Cap protein (15). Besides, we constructed CD40L and/or GMCSF-modified PCV2 adenovirus vaccines and evaluated their efficacy in mice. The results show that CD40L and GMCSF improve the immunogenicity of adenovirus vector vaccines in mice (16). Although the results of mice experiments of Ad-A-C-W and Ad-CD40L-Cap-GMCSF are impressing, the difference in body structures between mice and pigs makes it difficult to determine the efficacy of these vaccines in PCV2-infected pigs. So, in the present study, we conducted the pig experiment to evaluate the protective immune responses of Ad-A-C-W and Ad-CD40L-Cap-GMCSF.

Both humoral and cellular immune responses were evaluated in this study. Anti-PCV2 antibodies measured by ELISA and VN indicated that the humoral immunity was induced. The specific antibody and neutralizing antibody levels of Ad-A-C-W- and Ad-CD40L-Cap-GMCSF-vaccinated group were significantly higher than Ad-Cap-vaccinated group (p < 0.05), while those of Ad-CD40L-Cap-GMCSF-vaccinated group were lower than Ad-A-C-W-vaccinated group (p < 0.05), except for a certain time point. The Ad-A-C-W-vaccinated group and Ad-CD40L-Cap-GMCSF-vaccinated group elicited higher humoral immunity than Ad-Cap-vaccinated group. The possible reason for different humoral immune responses induced by Ad-A-C-W and Ad-CD40L-Cap-GMCSF was that Intron A and WPRE mainly increased the expression of Cap (30,31), but CD40L and GMCSF played an important role in the process of immune response (16).

Lymphocyte proliferation and cytokine levels are two important indexes of cellular immunity, both were detected on 28 dpi. Both Ad-A-C-W and Ad-CD40L-Cap-GMCSF could induce a stronger cellular immune response than Ad-Cap, but Ad-CD40L-Cap-GMCSF could induce a stronger cellular immune response than Ad-A-C-W in pigs. It is well known that CD40L is mainly expressed by activated CD4⁺ T cells and interacts with its receptor CD40, which plays important roles in both humoral and cellular immune responses, such as Th1 type response development (1,7), Ig type switching, antibody production (6,21), and interactions of between T cell and dendritic cell or T cell and macrophage (33). In addition to CD40L, GMCSF can stimulate neutrophil, monocyte/macrophage, and eosinophil colony formation, and enhance antibody-dependent cell-mediated cytotoxicity (18,19).

PCV2 challenge experiment showed that from 35 to 91 dpi, the viral loads of Ad-A-C-W-vaccinated group and Ad-CD40L-Cap-GMCSF-vaccinated group were lower than Ad-Cap-vaccinated group (p < 0.05). Gross lesions of lungs of group Ad-Cap, Ad-A-C-W, and Ad-CD40L-Cap-GMCSF exhibited slightly interstitial pneumonia, and lymph nodes were also slightly swelling. Microscopic examination showed no obvious lesions. The results demonstrated that recombinant adenoviruses could protect pigs from PCV2 challenge. Besides, Ad-A-C-W and Ad-CD40L-Cap-GMCSF induced better protective immune responses than Ad-Cap.

The results of pig experiment showed that the vaccine efficacy of adenoviruses Ad-A-C-W and Ad-CD40L-Cap-GMCSF was no better than the commercially inactivated vaccine. The possible reasons were as follows. The preparative techniques of commercially inactivated vaccines are mature; inactivated vaccines contain high content of antigen; there are different kinds of immunologic adjuvants for inactivated vaccines; and the whole virus particle could induce stronger immune responses than single protein, Cap. Although many researches focus on adenovirus vector vaccines, there are still some key techniques unsolved, for example, immunologic adjuvants of adenovirus vector vaccines are immature. So, to improve PCV2 adenovirus vaccine efficacy, further study must be focused on parameter optimization, such as the dose and route of delivery, vaccination procedures, and adenovirus adjuvants.

In conclusion, although the immune responses induced by Ad-A-C-W and Ad-CD40L-Cap-GMCSF were no better than inactivated vaccine, the adenovirus vector still has many advantages, for example, high transduction efficiency, wide tropism, high safety, and can infect stationary and division phase cells, making Ad-A-C-W and Ad-CD40L-Cap-GMCSF to be promising vaccine candidates to prevent and control PCV2 infection.

Footnotes

Acknowledgments

This research was supported by the Key Project of Shaanxi province Science and Technology Innovation Team (2013KCT-28), the National Natural Science Foundation of China (Nos.: 31372401, 31372411), and the Science and Technology Innovation Project in Shaanxi province (2016KTCL02-13).

Author Disclosure Statement

No competing financial interests exist.

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