| Herpes simplex virus (HSV) type 1 and type 2 are ubiquitous human pathogens. Usually they result in lifelong latent infection punctuated by recurrences that may lead to clinical lesions or asymptomatic shedding. HSV-1 is a leading cause of virus-induced corneal disease. Clinically, HSV-1 infection can be manifested as herpes simplex keratitis (HSK), herpes labialis, encephalitis, etc. HSK is one of the most common ocular infectious diseases, and in fact, it is the leading cause of infectious corneal blindness as a result of viral reactivation leading to stromal keratitis and scarring. Yet, despite notable efforts, it has not been successfully preventive or therapeutic vaccines or drugs available. Although antiviral chemical drugs do play roles in the early treatments after HSV infection, they loss their gradually in the following re-infections as a result of virus drug-resistance mutation and the abusage of Steroid Hormone. Therefore a prophylactic HSV-1 vaccine aimed at preventing disease and ultimately infection is likely to be more effective at reducing the global burden of HSV disease than antiviral therapy.DNA immunization is an important vaccination strategy that has many characters desirable for an ideal vaccine, including induction of broad immune responses (humoral and cellular), long-lasting immunity and simple and cheap production. This technique is being explored as a vaccination strategy against a variety of infectious diseases, autoimmune diseases and cancers. The first generation of DNA-immunization experiments haveshown that delivery of DNA constructs encoding a specific immunogen into the host could elicit effective immune responses in vivo in a safe and well-tolerated manner in various model systems. However the potency of DNA vaccines is limited by their inability to amplify and spread in vivo. More efficacious and specific immune responses against the target pathogen are required in order to enhance its clinical utility. One strategy is the use of molecular adjuvants. Molecular or genetic adjuvants are different from the traditional adjuvants in that they consist of gene-expression constructs encoding immunologically important molecules, such as cytokines, chemokines and co-stimulatory molecules. Previous reports have shown that co-administration of genetic adjuvant constructs with immunogen constructs can modulate antigen-specific immune responses.Interleukine (IL)-18, first designed as an interferon -γ(IFN-γ)-inducing factor, is a recently identified cytokine of the T helper 1 (Th1) type. It has been known to induce IFN-γproduction by both CD4+ T cell and nature killer (NK) cells and to stimulate na?ve T cells to promote the development of Th1 cells. Since IFN-γis one of the most important cytokines that contributes to host defence, a cytokine capable of up-regulating IFN-γshould also play a key role in host defence. Indeed, IL-18 plays a critical role in the eradication of various pathogens.In this study, we tested the immune modulatory as well as vaccine effect of using IL-18 expression plasmids as genetic adjuvants to enhance gB DNA vaccine-induced preventive immune responses in a mouse HSV-1 challenge model.1. The induced immune responses after DNA vaccine immunization Four-to six–week-old male KunM mice were used in this study. Twodays before DNA inoculation, the quadriceps muscles were injected with 100μl 25% sucrose to enhance subsequent DNA absorption. For DNA inoculation, 100μg of each DNA construct in saline was injected into the same region of the muscles as the sucrose injection. Groups of mice (n=7) were immunized with 100μg pgB alone or in combination with 100μg pIL-18 at 0, 2, 4 weeks; pcDNA3-immunized mice were used as a negative control. Serum samples from the mice were collected by retro-orbital bleeding for IgG titer assay by standard ELISA; Neutralizing assay was carried out for neutralizing antibody titer determination. At the same time, mice were sacrificed, and the splenocytes were isolated for splenocytes proliferative assay. DTH response was also detected by ear pinna swelling test. The obtained results are as follows:1) To determine whether co-injection of IL-18 expression plasmid (pIL-18) with gB DNA vaccine (pgB) could influence the humoral immune response against gB, sera obtained 2 weeks after the final DNA inoculation were tested by ELISA. When pIL-18 was co-injected ,the geometric mean titre was increased to about 5700, significantly highter than the group immunized with pgB alone, in which group the mean titre was only about 4200 (P<0.05).2) intradermal injection of HSV antigen in the pinnae resulted in a significant DTH response in mice immunized with pgB compared with negative control. The DTH response was further increased in the pgB+pIL-18 group significantly higher than that in the group than received pgB alone (P<0.05).3) Both pgB alone and pgB+pIL-18 enhanced the spelenocytes proliferation compared with the pcDNA3 alone immunization. Thestimulation indexes of pgB alone and pgB+pIL-18 group are 3.065±0.2963 and 4.422±0.3933, respectively, as compared with 0.978±0.1099 in pcDNA3 alone immunization group.2. DNA vaccine protection effect after HSV-1 corneal challengeTwo weeks after the final immunization, mice were challenged with SM44 strain of HSV-1 via the cornea, which was scarified in a"="with a 12-gauge needle. 5ul of the virus containing 1×106pfu/ml was instilled into the conjunctival sac. Clinical evaluation of viral infection challenge was carried out as described previously. Briefly, every day from day1 to day 9 after the viral challenge, the same observer examined the eyes with a slit-lamp biomicroscope and scored the severity of epithelial lesions by the following criteria: (0), no epithelial lesion or punctuate epithelial erosion; (1), stellate keratitis or residue of dendritic keratitis; (2), dendritic keratitis occupying less than a quarter of the cornea;(3),dendritic keratitis occupying a quarter to half of the cornea;(4),dendritic keratitis extending over more than half of the cornea; 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. When the mice were co-injected with pIL-18, the epithelial lesions were further reduced and the recovery time was also shortened significantly (P<0.05).Thirty days after the virus challenge, mice were sacrificed and the trigeminal ganglions were removed for co-culture with vero cell to reactivate the latent viruses. The result showed that DNA vaccine immunization significantly decreased the viral load in trigeminal ganglioncompared to the negative control group. IL-18 co-administrations with gB had no abvious increase effect.In conclusion, the date presented here suggest that IL-18 expression plasmids co-administration with gB DNA vaccine could efficiently increase the antigen specific humoral responses and cellular immune responses, manifested as increase of gB-specific IgG production, gB-specific neutralizing antibody titer, antigen-specific lymphocyte proliferation and the delayed-type hypersensitivity response. |
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