The Preliminary Immunity Effect Of Herpes Simplex Virus Glycoprotein B DNA Vaccines

Herpes simplex keratitis (HSK) is caused by ocular infection withvirus 1(HSV-1).The epithelial disease is believed is to result primarily from viral toxicity. Immune effectors are pathogenically central to the second form of herpetic comeal infections-stromal keratitis. Necrotizing steomal dermatitis is the more severe form. Complications of this disorder include corneal melting, glaucoma a and irreversible comeal scarring which often leads to blindness.HSV-1 produce not only a primary infection but also latent and recurrent infections, antiviral therapy does not reduce subsequent recurrences. Similarly, treatment of recurrent infectious can decrease the severity of the disease and daily suppressive therapy can decrease both symptomatic and asymptomatic recurrence.Human HSK is usually the sequel to recurrent infection following reactivation from latency. The pathogenesis of human HSK remains poorly understood, since vaccines that effectively control HSK remain nonexistent, and antiviral therapy has limited usefulness against recurrent infection, new approaches are needed to deal with HSK. As the incidence of herpes continues to increase in the population, and as new strains of the virus resistant to chemotherapy continue to emerge, so does the need for a safe and effective vaccine.Nucleic acid vaccine (DNA vaccine) is a kind of DNA vector which can carry exogenous gene encoding immunogenic antigen. Generally, the plasmids or some viruses can be used as DNA vector .When the plasmid carrying target gene is inoculated into the body, the transfected muscular cells or antigen-presenting cells(APC) can express the immunogenic antigen in cytoplasm and present the antigen to the Th cells to induce immune response, which can protect the body from infection. More and more attentions are paid to the usage of DNA vaccine for its special immunogenicity that can induce strong cellular and humeral immune responses. Furthermore, some phaseâ…¡/â…¢clinical trials of DNA vaccines are ongoing recently. However, the potency of DNA vaccines in humans is disappointing. In addition, a large amount of plasmid DNA was required to induce immune response on the larger animals, such as rhesus macaques, chimpanzee, and so on. Therefore, it is urgent to develop new strategies in order to increase the efficacy of DNA vaccine with decreased amount of plasmid DNA.In this study ,we use three different concentration DNA vaccines to evaluate their different immune responses. The methods are :All plasmid DNA was purified from transformed Escherichia Coli DH5a,adjusted to a final concentration in the sterile PBS and stored at -20℃until used. DNA concentration was measured by absorbance at 260nm.The OD260/OD280 ratios for purified DNA was 1.8-2.0, indicating that preparation were free from protein contamination. KunM mice, aged 4 to 6 weeks, were randomly divided into4groups,receivingpcDNA-gB1(200μg/0.1ml), pcDNA-gB₂(100μg/0.1ml),pcDNA-gB₃(50μg/0.1ml)å’ŒpcDNA₃,respectively. Mice immunized with pcDNA₃ were served as the negative controls. Three days before inoculation, the quadriceps muscles were injected 100μl of a solution containing lidocaine hydrochloride to enhance subsequent DNA absorption. For DNA inoculation, 50μg/0.1ml ,100μg/0.1ml and 200μg/ 0.1ml of each DNA construct in PBS was injected into the same region of the muscle as the lidocaine injection. Mice were boosted two times with the same dose applying the same immunization schedule at weeks 2 and 4. At 7d,21d,35d,42d,49d,56d after primary immunization ,serum samples from the mice were collected for IgG titer assay by standard ELISA; Neutralizing assay was carried out for neutralizing antibody titer determination. At 42 days, mice were sacrificed ,and the splenocytes isolated for splenocytes proliferative assay. Two weeks after the final immunization ,mice were challenged with SM44 strain of HSV-1 via the comea.The results are as follow:1) Compared with injection of pcDNA₃,pgB injection significantly increased antibody titers. The specific IgG antibodies increased markedly at 21 days after primary immunization and reached a plateau at 35 days in the experiment groups. From 56 days after primary immunization, IgG antibodies begins to down. 2) Compared with the group pcDNA₃,the proliferation reaction of T cell of three pcDNA-gB groups were higher (p<0.05),especially in groups pcDNA-gB₁and pcDNA-gB₂ .On the other hand ,there is no significant difference between the groups pcDNA-gB₁ and pcDNA-gB₂(p>0.05). 3) DNA vaccine protection effect after HSV-1 corneal challenge: Epithelial lesions in control mice(immunized with pcDNA₃) reached a peak around day 2 postinfection, and gradually reduced. About 8 days after the challenge, all the epithelial lesions had recovered. However , pgB limited the lesion score to a level significantly lower than that observed in the negative control, especially in the high concentration groups(pcDNA-gB₁ and pcDNA-gB₂)In conclusion, the date presented here suggest that the HSV-1gB DNA vaccines has the ability to elicit efficacious humeral and cellular responses in vivo. Different concentration vaccines has the different effect and the effect of higher concentration vaccines is better. To the pcDNA-gB vaccines, 100μg/0.1ml is the best concentration.