Safety Evaluation Of DNA Vaccine Against Infectious Canine Hepatitis

Objective Infectious canine hepatitis virus (ICHV) is the causative agent of infectious canine hepatitis (ICH) that is a common acute infectious disease. The mainly host for this virus is the puppy of no more than one years old. Mortality has been reaching to 40%. The best way to prevent ICH is to improve the management, healthy measure and immunization using vaccine.Currently, vaccinations with attenuated vaccine provide an effective approach in preventing disease. Besides, there are other vaccines such as the bigeminy vaccine of ICH and Canine parvoviral enteritis, the composition vaccine of canine distemper, Minute virus, parainfluenza, Coronavirus and ICHV. The experiment carried out by Willianms in 1991 brought on the concept of DNA vaccine is named nucleic acid vaccine as well. DNA vaccines consist of the gene for foreign antigen, which is delivered into host cells and expressed corresponding protein using the cells'transcription and translation machinery. The protein can elicit specific immune response to cure and prevent disease. Recently, much attention has been paid on DNA vaccine because it can bring revolution to cure and prevent disease. Amounts of DNA vaccines such as influenza, tuberculosis, malaria and so on have been given approval to perform clinical trial by FDA.Despite the potential applications in many fields, there are still some concerns on the safety of DNA vaccine as followed:(1) The injected DNA vaccine may integrate into the host cell genome. Integration is by definition insertional mutagenesis and has the potential to activate oncogenes.(2) Long-term expression of foreign antigen may lead to some disadvantage theoretic including immune tolerance, autoallergic disease, anaphylactic response and so on.(3) The potential threat of DNA vaccines on the environment, involving mostly the gene residue, transmission and transfer after the plasmid DNA was uptaken by. These safety problems are essential for DNA vaccines research. Although so far there is no side effect found in animal model, more detailed experimental observations are still in need to completely exclude the possible fatalness theoretically.We found that vaccination of BALB/c mice with the DNA vaccine pVAX1-CpG-Loop alone resulted in the following consequences: High-level specific antibody (IgG) against ICHV was induced; T cell activation was elicited; and neutralizing antibodies were detectable in immunized mice.The protection of DNA vaccine pVAX1-CpG-Loop against Infectious canine hepatitis virus constructed by our laboratory has been verified. The safety of the DNA vaccine will be further evaluated in this study.Methods1 Extracted eukaryotic recombinant plasmid abundantlyDH5αthat contained the eukaryotic recombinant plasmid pVAX1-CpG-Loop was cultivated for 36h by shake culture method. Then the sediment of DH5αwas collected by centrifugalization and recombinant plasmids were extracted by SDS-alkaline lyses. The purity and concentration of the extracted recombinant plasmid were determined by grating spectrophotometer.2 Immunization of miceA total of 112 BALB/c mice (four animals for each time point), 56 females and 56 males, evaluated in the study were 6–8 weeks old and obtained from the Animal Facilities of Hebei Medical University. They were maintained under standard laboratory conditions. The mice were divided into high dosage group (200μg /mouse, i.m), low dosage group (100μg each, i.m), coimmunization group (intramuscular injection 100μg, hypodermic injection 50μg, nasal instillation 50μg) as well as control group (PBS 200μl each, i.m), and injected with DNA vaccine or PBS respectively at an interval of two weeks for three times.3 The safety evaluation of immunized miceGeneral safety: During this study the animals were observed twice a day for general wellbeing. Mortality and clinical signs were followed and any deviations from normal were recorded. The body weight was measured every week. The blood of mice was collected after 4 weeks and 24 weeks of the final vaccination, respectively. The hematology of mice was investigated after 4 weeks of the last vaccination. Biochemistry of blood including AST and ALT was observed after 4 weeks and half year later of the last immunization.The distribution and persistence of the DNA vaccine: Total RNA was isolated from the tissues, gonads (ovaries or testes), heart, liver, spleen, lung, kidney, as well as muscle of injection site, on 7 days, 15 days and 30 days after the last immunization. PCR was performed after reverse transcription. Genome DNA was extracted after 1 week, 8 weeks, 12 weeks of the last vaccination, and then PCR was carried out.The influence to F1: The male and female mice treated with the same dosage were made brood, 7 pairs each group, to investigate conception and the influence to generation F1. The total cellular DNA of spleen and liver respectively was extracted from offspring after boring 2 weeks to monitor genomic integration.Histological analysis: The heart, liver, spleen, lungs, kidney, as well as muscle of injection site, were collected after 4 weeks and 6 months of the last vaccination respectively to carry out paraffin sections in ordinary procedures.Results1 General safetyNo significant differences were observed between the vaccination and control groups in the examination of hematology, mortality and clinical signs were not observed either. In contrast to the control group, there was no significant difference among the tested groups on body weight. AST increased significantly in the experiment mice at 4 weeks after the last vaccination, but it recovered after half year later, however, no conspicuous difference on ALT among all the tested mice was recorded.Distribution and persistence of pVAX1-CpG-Loop plasmidTo identify the distribution and persistence of pVAX1-CpG-Loop plasmid, Loop gene expression was assessed by RT-PCR. Multiple tissue samples of mice immunized with pVAX1-CpG-Loop were collected for distribution detection of the plasmid. On day 7 after immunization, the result of RT-PCR revealed that the transcription of Loop persisted in liver, spleen, as well as the injection site of muscle. The expression of the plasmid message was still observed in liver and muscle on day 15. However the transcription of Loop can not be detected in all the tissues collected on day 30. No transcription of Loop was detected in the tissue from the PBS group. On week 1 after inoculation, PCR analysis of tissue genome DNA from high and low groups demonstrated that the plasmid presented at liver, spleen, kidney muscle, as well as liver, spleen, kidney, lung, and muscle of coimmunization groups. In addition to muscle of injection site, plasmid DNA was not detected in any tissue on week 8. And on week 12, the plasmid DNA could not be found from the muscle of injection site either.Liver and kidney lesion in immunized miceTo assess histological changes, the heart, liver, spleen, lung, kidney, as well as muscle of injection site, were collected after 4 weeks and half year immunization, and then paraffin sections were prepared. The histological lesions were found in liver and kidney of immunized mice, including leukom- onocytes in liver and kidney, and hydropic degeneration of liver after 4 weeks immunization. However the chronic inflammation and lesions turned out to be recovered half year later. No significant changes were detected in other organs or in any collected tissues of control groups.The influence to generation F1To explore the influence to conception and generation F1, the male and female mice treated with the same dosage were made brood. No obvious differences were observed between the vaccination groups and control in the amounts and the weight of offspring(P>0.05). The total cellular DNA of spleen and liver was extracted from offspring after boring 2 weeks respectively, and then PCR was performed. No implication was detected, which indicated no genomic integration.Conclusions1 The results demonstrated that no obvious difference was detected in the examination of weight, and hematology, the influence to generation F1 was not observed either.2 The result of influence to F1 revealed the DNA vaccine against Infectious canine hepatitis was safe to reproductive system as well as generation F1.3 No amplification was detected in any collected tissue in 12 week after last immunization, which indicated no integration.4 Compared with the control, there was a conspicuous difference on AST among all the tested mice after 4 weeks, but it recovered half year later.5 The result of distribution, which correlated with the immunization approach, indicated there was no Loop gene in the reproductive tissues.6 Lesions were observed in liver and spleen tissues of the immunized mice, but it recovered half year later, which demonstrated that the damage was temple.