Limited Protection Conferred by a DNA Vaccine against a Lethal Pseudorabies Virus Infection at Day 5 Postvaccination

Abstract

No pseudorabies virus (PRV)-specific neutralizing or immunoglobulin G1-type antibodies were detected in sera 5 days after injection of a DNA vaccine against PRV infection in pigs. PRV-stimulated peripheral blood mononuclear cells produced gamma interferon mRNA in vitro. Two out of five pigs recovered from lethal PRV infection without attenuation of nasal viral excretion.

Infection of swine with the swine alpha herpesvirus pseudorabies virus (PRV) causes Aujeszky's disease, a serious illness with high morbidity and mortality leading to severe losses in the pig industry. Various vaccination strategies based on modified live or inactivated vaccines are employed to control Aujeszky's disease. DNA vaccination provides an alternative to conventional vaccination and is reported to efficiently protect swine, mice, or rabbits against PRV infection 21 to 28 days after plasmid injection (8-10, 14, 18, 30). These ideal experimental conditions are not usually encountered in practice, since it is not known when vaccinated animals will be exposed to viral pathogens. Pigs can be infected soon after vaccination.

The capacity of conventional inactivated or live attenuated vaccines to induce early protection has been described in several animal models of infection. For example, pigs are protected against classical swine fever virus (CSFV) infection 6 days after vaccination (25). Cattle are protected against bovine herpesvirus-1 (BHV-1) infection 3 to 5 days after vaccination (2, 16, 19, 26) and against foot-and-mouth disease virus infection 7 days after vaccination (12, 22). Early protection has also been described for DNA vaccination against viral hemorrhagic septicemia in rainbow trout (20, 24).

We investigated the ability of a single DNA vaccine injection to induce early immune and protective responses against PRV infection in swine. As reported above, different animals, including pigs, were protected against various viral infections 3 to 7 days after vaccination. We selected the mean minimal period (5 days) needed to confer protection after vaccination in these different studies. The DNA vaccine consisted of the 3 plasmids individually encoding PRV-gB, -gC and -gD, already used successfully in our laboratory (5, 8). Groups of 5 or 6 specific-pathogen-free pigs, housed and treated in accordance with local regulations (Direction des Services Vétérinaires des Côtes d'Armor, France), were injected in the neck with 600 μg of DNA vaccine or empty pcDNA3 5 or 21 days before challenge with 10⁶ 50% tissue culture infective doses (TCID₅₀) of the virulent NIA3 PRV strain (kindly provided by J.C. Audonnet, Merial, Lyon, France). Pigs were 11 weeks old, with a mean weight of 38.3 ± 6.2 kg, when challenged.

Titers of serum anti-PRV immunoglobulin G1 (IgG1)-type antibodies, markers of a humoral immune response (23), and neutralizing antibodies (NAb) were determined as previously described (5, 7, 8). These antibodies were not detected 5 days after vaccination and were produced at low levels 7 days after PRV challenge in the group vaccinated 5 days before PRV challenge (Fig. 1 and 2). In comparison, specific anti-PRV IgG1 and NAb (titers of 1:3 and 1:6) were produced in all pigs and in 2 out of 6 pigs, respectively (Fig. 1 and 2). The levels of antibodies increased after PRV challenge.

FIG. 1.

Anti-PRV IgG1 subclass in serum. The anti-PRV IgG1 subclass was determined by indirect enzyme-linked immunosorbent assay using microtitration plates coated with PRV antigen. The mean titers, including standard error bars, for each group of pigs are expressed in the log₁₀ of the highest serum dilution giving an optical density value of more than three times the optical density of a pool of control serum from unvaccinated pigs. *, P < 0.05; **, P < 0.01, calculated with the nonparametric Mann-Whitney test; #, no survivor.

FIG. 2.

PRV-specific NAb in serum. The seroneutralization assay was performed on a PRV-infected swine kidney cell line. The mean titers, including standard error bars, for each group of pigs are expressed as the highest serum dilution inhibiting the cytopathogenic effect in 50% of PRV-infected PK15 cell line cultures. *, P < 0.05; **, P < 0.01, calculated with the nonparametric Mann-Whitney test; #, no survivor.

Levels of porcine gamma interferon (IFN-γ) mRNA, a key mediator for cytotoxic T cells (25, 31), and interleukin-4 (IL-4) mRNA, known to favor a B-cell response (31), were determined in PRV-restimulated peripheral blood mononuclear cells (PBMCs) by quantitative reverse transcriptase PCR as previously described (5, 7). IFN-γ mRNA was produced 5 and 21 days after DNA vaccine injection (Fig. 3A) and was strongly increased in both groups 7 days after challenge. IL-4 mRNA was not detected 5 days after DNA vaccine injection, but low levels were produced at 21 days (Fig. 3B). PRV-stimulated PBMCs from animals of both vaccinated groups produced similar levels of IL-4 mRNA 7 days after PRV challenge (Fig. 3B).

FIG. 3.

IFN-γ and IL-4 relative gene expression in PRV-stimulated PBMCs. The relative quantities of cytokine mRNA were determined by relative quantitative reverse transcriptase PCR using β-actin gene expression as the housekeeping gene. The mean relative quantities of cytokine mRNA ± standard deviations are shown. (A) Mean relative IFN-γ mRNA quantities. (B) Mean relative IL-4 mRNA quantities. *, P < 0.05, calculated with the nonparametric Mann-Whitney test; #, no survivor.

The IFN-γ results suggest that a cellular immune response against PRV is detected as early as 5 days after DNA vaccination. This was also reported 5 days after immunization of cattle against BHV-1 (32) and 6 days after vaccination of pigs against CSFV (25). The levels of IL-4 and PRV antibodies indicate that a humoral immune response was not observed 5 days postvaccination and required between 5 and 21 days to develop.

All pigs injected with empty pcDNA3 died after PRV challenge, lost weight between the day of challenge and day of death, developed high fever as early as day 2 postchallenge, and presented mild to severe nervous symptoms (scores were 0, 1, and 2 for no, mild, or severe symptoms, respectively) (Table 1). The levels of viral excretion, determined from swabs of nasal fluid samples as previously described (17, 27), were around 6 and 7.5 log₁₀ TCID₅₀/g of nasal swab 2 and 5 days post-PRV challenge, respectively (Table 1). The observed severity of the PRV challenge in the nonvaccinated groups may explain why all pigs in the group vaccinated 21 days before PRV challenge presented mild to severe nervous symptoms and why 2 out of 6 pigs succumbed (Table 1). In these conditions, DNA vaccination 5 days before PRV challenge permitted the survival of 2 out of 5 pigs but did not reduce high fever at day 2 or nasal viral excretion, contrary to the results for the group vaccinated 21 days before challenge (Table 1).

TABLE 1.

Clinical parameters and levels of nasal excretion of PRV following vaccination and PRV challenge^a

DNA vaccine or control for each group	Day postvaccination	No. of survivors/total no. in group	Mean relative daily wt (kg/100 kg/ day ± SD)b	Mean body temp at day 2 (°C ± SD)	Nervous symptoms at day 5 (mean score ± SD)	Nasal excretion of PRV (log10 TCID50/g nasal fluid ± SD)
						Day 2	Day 5
PRV-pcDNA3	21	4/6	−3.2 ± 0.6	40.0 ± 0.5**	1.7 ± 0.5	4.4 ± 0.5*	5.8 ± 0.3*
PRV-pcDNA3	5	2/5	−3.6 ± 0.7	40.5 ± 0.7	0.8 ± 1.0	5.9 ± 0.4	7.3 ± 0.5
Empty pcDNA3	21	0/6	−3.7 ± 0.5	41.0 ± 0.2**	1.0 ± 1.2	6.5 ± 0.5	7.4 ± 0.3
Empty pcDNA3	5	0/6	−3.6 ± 0.7	40.9 ± 0.3	1.3 ± 1.0	6.0 ± 0.3	7.4 ± 0.3
No injection, no PRV challenge		5/5	NA	39.3 ± 0.3	0.0	0.0	0.0

^aSD, standard deviation; NA, not applicable; *, P < 0.05; **, P < 0.01, calculated with the nonparametric Mann-Whitney test.

^bFor dead pigs, between day of challenge and day of death.

In conclusion, a severe PRV challenge (10⁶ TCID₅₀), which provoked the death of all the unvaccinated pigs, was partially controlled in the group vaccinated 5 days before challenge, when a cellular, but not humoral, immune response was present. Cell-mediated immunity is important for early protection against PRV infection (28, 33) and CSFV infection (25) in swine, against BHV-1 infection in cattle (15, 26), and against viral hemorrhagic septicemia infection in fish (1). Protection was increased when both cellular and humoral immune responses were detected (i.e., 21 days after DNA vaccination). In fact, NAb play an important role in clinical protection against PRV infection (6, 21) but cannot confer full protection (11, 13). The highly virulent PRV challenge may have affected the described levels of protection. An additional assay with a less-severe PRV challenge (3, 4, 29) and a group injected with an attenuated PRV vaccine (4) to evaluate challenge severity would be useful to evaluate the protective potential of DNA vaccine at day 5 under conditions closer to natural infections in pigs.