[Relationship between serum brain-derived neurotrophic factor and clinical stage and dysmenorrhoea of enodmetriosis].

Objective: To explore the association between serum brain-derived neurotrophic factor and clinical stage and dysmenorrhoea of endometriosis. Methods: A total of 82 patients were studied with laparoscopically diagnosed endometriosis between June 2017 and June 2019, and 75 healthy women with reproductive age were selected as the control group during the same period. The endometriosis patients were scored by visual analogue scale(VAS)according to their preoperative dysmenorrhoea.And endometriosis was staged and scored according to the score of Revised American Fertility Society(r-AFS).Enzyme-linked immunosorbent assay (ELISA) was used to determine preoperative BDNF level in serum, and the correlation between BDNF level with clinical stage as well as dysmenorrhea of endometriosis were analysed. Results: The serum BDNF level in endometriosis patients was (1 082±43) ng/L, significantly higher than that in the normal control [(649±30) ng/L], there was statistical difference between the two groups(P<0.001). The BDNF expression in patients with r-AFS stage Ⅲ-Ⅳ was higher than that in patients with Ⅰ-Ⅱ stage [(1 164±389) ng/L vs (791±218)ng/L, P<0.001]. BDNF level in serum was closely correlated with the degree of dysmenorrhea (r=0.682), and the BDNF level in patients with moderate or severe dysmenorrhea was significantly higher than that in patients without dysmenorrhea and patients with mild dysmenorrhea [(1 292±43) ng/L vs(718±36) ng/L, P<0.001]. Conclusions: The serum BDNF level in endometriosis patients is positively correlated with clinical stage and dysmenorrhea.

Oral bisphosphonate use and lung cancer incidence among postmenopausal women.

Background: Bisphosphonates are common medications for the treatment of osteoporosis in older populations. Several studies, including the Women's Health Initiative (WHI), have found inverse associations of bisphosphonate use with risk of breast and endometrial cancer, but little is known about its association with other common malignancies. The objective of this study was to evaluate the association of bisphosphonate use on the incidence of lung cancer in the WHI. Patients and methods: The association between oral bisphosphonate use and lung cancer risk was examined in 151 432 postmenopausal women enrolled into the WHI in 1993-1998. At baseline and during follow-up, participants completed an inventory of regularly used medications including bisphosphonates. Results: After a mean follow-up of 13.3 years, 2511 women were diagnosed with incident lung cancer. There was no evidence of a difference in lung cancer incidence between oral bisphosphonate users and never users (adjusted hazard ratio = 0.91; 95% confidence intervals, 0.80-1.04; P = 0.16). However, an inverse association was observed among those who were never smokers (hazard ratio = 0.57, 95% confidence interval, 0.39-0.84; P < 0.01). Conclusion: In this large prospective cohort of postmenopausal women, oral bisphosphonate use was associated with significantly lower lung cancer risk among never smokers, suggesting bisphosphonates may have a protective effect against lung cancer. Additional studies are needed to confirm our findings.

Innate immunity drives xenobiotic-induced murine autoimmune cholangitis.

Although primary biliary cirrhosis (PBC) is considered a model autoimmune disease, it has not responded therapeutically to traditional immunosuppressive agents. In addition, PBC may recur following liver transplantation, despite the absence of major histocompatibility complex (MHC) matching, in sharp contrast to the well-known paradigm of MHC restriction. We have suggested previously that invariant natural killer T (iNK T) cells are critical to the initiation of PBC. In this study we have taken advantage of our ability to induce autoimmune cholangitis with 2-octynoic acid, a common component of cosmetics, conjugated to bovine serum albumin (2-OA-BSA), and studied the natural history of pathology in mice genetically deleted for CD4 or CD8 following immunization with 2-OA-BSA in the presence or absence of α-galactosylceramide (α-GalCer). In particular, we address whether autoimmune cholangitis can be induced in the absence of traditional CD4 and CD8 responses. We report herein that CD4 and CD8 knock-out mice immunized with 2-OA-BSA/PBS or 2-OA-BSA/α-GalCer develop anti-mitochondrial antibodies (AMAs), portal infiltrates and fibrosis. Indeed, our data suggest that the innate immunity is critical for immunopathology and that the pathology is exacerbated in the presence of α-GalCer. In conclusion, these data provide not only an explanation for the recurrence of PBC following liver transplantation in the absence of MHC compatibility, but also suggest that effective therapies for PBC must include blocking of both innate and adaptive pathways.

Exposures in early life: associations with DNA promoter methylation in breast tumors.

There is evidence that epigenetic changes occur early in breast carcinogenesis. We hypothesized that early-life exposures associated with breast cancer would be associated with epigenetic alterations in breast tumors. In particular, we examined DNA methylation patterns in breast tumors in association with several early-life exposures in a population-based case-control study. Promoter methylation of E-cadherin, p16 and RAR-ß2 genes was assessed in archived tumor blocks from 803 cases with real-time methylation-specific PCR. Unconditional logistic regression was used for case-case comparisons of those with and without promoter methylation. We found no differences in the prevalence of DNA methylation of the individual genes by age at menarche, age at first live birth and weight at age 20. In case-case comparisons of premenopausal breast cancer, lower birth weight was associated with increased likelihood of E-cadherin promoter methylation (OR = 2.79, 95% CI, 1.15-6.82, for ⩽2.5 v. 2.6-2.9 kg); higher adult height with RAR-ß2 methylation (OR = 3.34, 95% CI, 1.19-9.39, for ⩾1.65 v. <1.60 m); and not having been breastfed with p16 methylation (OR = 2.75, 95% CI, 1.14-6.62). Among postmenopausal breast cancers, birth order was associated with increased likelihood of p16 promoter methylation. Being other than first in the birth order was inversely associated with likelihood of ⩾1 of the three genes being methylated for premenopausal breast cancers, but positively associated with methylation in postmenopausal women. These results suggest that there may be alterations in methylation associated with early-life exposures that persist into adulthood and affect breast cancer risk.

Use of RNA interference to modulate liver adenoma development in a murine model transgenic for hepatitis B virus.

Chronic hepatitis B virus (HBV) infection is closely related to the development of severe liver complications, including hepatocellular carcinoma. In previous studies, we reported that in vivo long-term HBV suppression in transgenic mice can be achieved without apparent toxicity by short hairpin RNA sequentially delivered using adeno-associated viral (AAV) vectors of different serotypes. Our goal herein was to address the clinical utility of this delivery system and, in particular, to determine whether RNA interference (RNAi) and its ability to induce long-term HBV suppression will modulate the development of HBV-associated liver pathology. As a model system, we used a unique HBV transgenic mouse model, containing a 1.3 times over length of the HBV genome, on the ICR mouse background. These transgenic mice produce high serum HBV titers comparable with human chronic HBV patients, and, importantly, manifest characteristic HBV-associated pathology, including progressive hepatocellular injury and the development of hepatocellular adenoma. Using this system, we injected animals with AAV vectors expressing either HBV-specific or a control luciferase-specific short hairpin RNA and followed animals for a total of 18 months. We report herein that AAV-mediated RNAi therapy profoundly inhibits HBV replication and gene expression, with a significant reduction in hepatic regeneration, liver enzymes and, importantly, the appearance of liver adenomas. Indeed, the therapeutic effect of RNAi correlated with the reduction in HBV titers. Our data demonstrate that appropriately designed RNAi therapy has the potential to prevent formation of HBV-associated hepatocellular adenoma.

Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA.

RNA interference (RNAi) was reported to block hepatitis B virus (HBV) gene expression and replication in vitro and in vivo. However, it remains a technical challenge for RNAi-based therapy to achieve long-term and complete inhibition effects in chronic HBV infection, which presumably requires more extensive and uniform transduction of the whole infected hepatocytes. To increase the in vivo transfection efficiency in liver, we used a double-stranded adeno-associated virus 8-pseudotyped vector (dsAAV2/8) to deliver shRNA. HBV transgenic mice were used as an animal model to evaluate the inhibition effects of the RNAi-based gene therapy. A single administration of dsAAV2/8 vector, carrying HBV-specific shRNA, effectively suppressed the steady level of HBV protein, mRNA and replicative DNA in liver of HBV transgenic mice, leading to up to 2-3 log(10) decrease in HBV load in the circulation. Significant HBV suppression sustained for at least 120 days after vector administration. The therapeutic effect of shRNA was target sequence dependent and did not involve activation of interferon. These results underscore the potential for developing RNAi-based therapy by dsAAV2/8 vector to treat HBV chronic infection, and possibly other persistent liver infections as well.

A case-control study in Shanghai of fruit and vegetable intake and endometrial cancer.

In a population-based case-control study of 832 incident endometrial cancer cases and 846 frequency-matched controls among Chinese women in Shanghai, using a validated food-frequency questionnaire, dietary habits were estimated by in-person interviews. Total vegetable consumption was inversely associated with endometrial cancer risk (highest quartile vs lowest: OR=0.69, 95% CI 0.50-0.96). The risk was reduced with increasing intake of dark green/dark yellow vegetables (trend test, P=0.02), fresh legumes (trend test, P<0.01), and allium vegetables (trend test, P=0.04). Fruit consumption was unrelated to risk. These results suggest that high consumption of certain vegetables may reduce the risk of endometrial cancer.

Protective mechanisms induced by a Japanese encephalitis virus DNA vaccine: requirement for antibody but not CD8(+) cytotoxic T-cell responses.

We have previously shown that a plasmid (pE) encoding the Japanese encephalitis virus (JEV) envelope (E) protein conferred a high level of protection against a lethal viral challenge. In the present study, we used adoptive transfer experiments and gene knockout mice to demonstrate that the DNA-induced E-specific antibody alone can confer protection in the absence of cytotoxic T-lymphocyte (CTL) functions. Plasmid pE administered by either intramuscular or gene gun injection produced significant E-specific antibodies, helper T (Th)-cell proliferative responses, and CTL activities. Animals receiving suboptimal DNA vaccination produced low titers of anti-E antibodies and were only partially or not protected from viral challenge, indicating a strong correlation between anti-E antibodies and the protective capacity. This observation was confirmed by adoptive transfer experiments. Intravenous transfer of E-specific antisera but not crude or T-cell-enriched immune splenocytes to sublethally irradiated hosts conferred protection against a lethal JEV challenge. Furthermore, experiments with gene knockout mice showed that DNA vaccination did not induce anti-E titers and protective immunity in Igmu(-/-) and I-Abeta(-/-) mice, whereas in CD8alpha(-/-) mice the pE-induced antibody titers and protective rate were comparable to those produced in the wild-type mice. Taken together, these results demonstrate that the anti-E antibody is the most critical protective component in this JEV challenge model and that production of anti-E antibody by pE DNA vaccine is dependent on the presence of CD4(+) T cells but independent of CD8(+) T cells.

Suppression of immune response and protective immunity to a Japanese encephalitis virus DNA vaccine by coadministration of an IL-12-expressing plasmid.

IL-12 plays a central role in both innate and acquired immunity and has been demonstrated to potentiate the protective immunity in several experimental vaccines. However, in this study, we show that IL-12 can be detrimental to the immune responses elicited by a plasmid DNA vaccine. Coadministration of the IL-12-expressing plasmid (pIL-12) significantly suppressed the protective immunity elicited by a plasmid DNA vaccine (pE) encoding the envelope protein of Japanese encephalitis virus. This suppressive effect was associated with marked reduction of specific T cell proliferation and Ab responses. A single dose of pIL-12 treatment with plasmid pE in initial priming resulted in significant immune suppression to subsequent pE booster immunization. The pIL-12-mediated immune suppression was dose dependent and evident only when the IL-12 gene was injected either before or coincident with the pE DNA vaccine. Finally, using IFN-gamma gene-disrupted mice, we showed that the suppressive activity of the IL-12 plasmid was dependent upon endogenous production of IFN-gamma. These results demonstrate that coexpression of the IL-12 gene can sometimes produce untoward effects to immune responses, and thus its application as a vaccine adjuvant should be carefully evaluated.

Enhanced antitumor immunity by fusion of CTLA-4 to a self tumor antigen.

The idiotypic determinant (Id) of the immunoglobulin expressed by a B-cell malignancy can serve as an effective tumor-specific antigen but is only weakly immunogenic. This study demonstrates that the immunogenicity of the tumor Id protein can be dramatically increased by directing it to antigen-presenting cells (APCs). Cytotoxic T-lymphocyte antigen 4 (CTLA-4) present on activated T cells has a strong binding affinity to both B7-1 and B7-2 molecules, which are primarily expressed on APCs. After construction of a fusion protein consisting of Id and CTLA-4 (Id-CTLA4), mice immunized with the fusion protein induced high titers of Id-specific antibody and T-cell proliferative responses without adjuvants and were protected from lethal tumor challenge. The Id-CTLA4 fusion protein was so potent that even low doses (down to 0.1 microg) of the immunogen were able to elicit strong antibody responses. By using an Id-CTLA4 mutant protein, the ability to bind B7 molecules on APCs was shown to be required for the enhanced immunogenicity of Id-CTLA4. These findings demonstrate that fusing CTLA-4 to a potential tumor antigen represents an effective approach to prime antitumor immunities in vivo and may be applicable to the design of vaccines for a variety of other diseases. (Blood. 2000;96:3663-3670)

DNA-Based immunization produces Th1 immune responses to hepatitis delta virus in a mouse model.

Hepatitis delta virus (HDV) superinfection is one of the major causes of fulminant hepatitis in endemic areas of hepatitis B virus (HBV) infection. Currently, there is no effective treatment or vaccine against HDV superinfection. DNA-based immunization is a promising antiviral strategy to prevent or treat persistent viral infections. In this study, we investigated the immunological effects of DNA vaccines against HDV in BALB/c mice. Plasmid (pD) encoding large hepatitis D antigen (L-HDAg), or plasmid (pS/pD) coexpressing hepatitis B surface antigen (HBsAg) and L-HDAg, were injected into mice intramuscularly. The seroconversion rate, anti-HBs levels, anti-HDV titers, T-cell proliferation responses, and T-helper (Th)-release cytokine profiles were analyzed. Mice immunized with plasmids, pS/pD or pD, produced low, but significant, titers of anti-HDV antibodies. In contrast, pS/pD induced much stronger anti-HBs titers in the immunized animals. Interestingly, splenic lymphocytes derived from pS/pD-inoculated mice demonstrated significant proliferation responses to recombinant HBsAg and HDAg, and resulted in a Th1-like immune response as suggested by the production of interferon gamma (INF-gamma) and interleukin-2 (IL-2), but not IL-4. The splenic lymphocyte derived from the pD-inoculated mice showed a similar Th1 response to the stimulation of HDAg, but not to HBsAg. In conclusion, our results suggest that DNA vaccines against HDV can induce significant cellular immune responses with a Th1 preference. HBV and HDV coimmunization can be performed by DNA vaccines. These results are promising for the future development of prophylactic and therapeutic HDV vaccines.

Limitations of in vivo IL-12 supplementation strategies to induce Th1 early life responses to model viral and bacterial vaccine antigens.

The limited induction of Th1 and cytotoxic immune responses is regarded as the main reason for the increased susceptibility to intracellular microorganisms in early life. Recently, in vitro IL-12 supplementation was shown to enhance the limited IFN-gamma release of measles-specific infant T cells. Using a series of IL-12 delivery systems, we show here that in vivo IL-12 supplementation may enhance early life murine Th1 responses to two model vaccine antigens, measles virus hemagglutinin and tetanus toxin peptide. However, this required multiple repeat injections of recombinant rIL-12, which were poorly tolerated in young mice. Local IL-12 delivery by an IL-12 expressing canarypox vector proved safe but failed to modulate vaccine responses. An IL-12 DNA plasmid or a CD40L DNA plasmid efficiently enhanced neonatal Th1 responses to measles hemagglutinin DNA vaccine. However, both plasmids only enhanced Th1 responses to DNA and not to peptide, protein, or live viral vaccines. Thus, inducing adult-like Th1 responses may be achieved in vivo by inducing (CD40L) or substituting for (IL-12 supplementation) optimal activation of neonatal APC. However, these immunomodulatory effects appear limited to certain antigen-presentation approaches and may not be broadly applicable to vaccines.

Screening of protective antigens of Japanese encephalitis virus by DNA immunization: a comparative study with conventional viral vaccines.

In this study, we evaluated the relative role of the structural and nonstructural proteins of the Japanese encephalitis virus (JEV) in inducing protective immunities and compared the results with those induced by the inactivated JEV vaccine. Several inbred and outbred mouse strains immunized with a plasmid (pE) encoding the JEV envelope protein elicited a high level of protection against a lethal JEV challenge similar to that achieved by the inactivated vaccine, whereas all the other genes tested, including those encoding the capsid protein and the nonstructural proteins NS1-2A, NS3, and NS5, were ineffective. Moreover, plasmid pE delivered by intramuscular or gene gun injections produced much stronger and longer-lasting JEV envelope-specific antibody responses than immunization of mice with the inactivated JEV vaccine did. Interestingly, intramuscular immunization of plasmid pE generated high-avidity antienvelope antibodies predominated by the immunoglobulin G2a (IgG2a) isotype similar to a sublethal live virus immunization, while gene gun DNA immunization and inactivated JEV vaccination produced antienvelope antibodies of significantly lower avidity accompanied by a higher IgG1-to-IgG2a ratio. Taken together, these results demonstrate that the JEV envelope protein represents the most critical antigen in providing protective immunity.

Sterically stabilized anti-idiotype immunoliposomes improve the therapeutic efficacy of doxorubicin in a murine B-cell lymphoma model.

A liposome containing diverse synthetic lipid derivatives of polyethylene glycol (PEG) results in smaller distribution volume and longer circulation time in blood and, thus, may improve drug targeting. The characteristics and therapeutic efficacy of immunoliposomes with similar liposomal formulation have never been studied in lymphoma models. We have developed immunoliposomes conjugated with S5A8 monoclonal antibody, an anti-idiotype antibody to 38C13 murine B-cell lymphoma, and loaded them with doxorubicin using an ammonium sulfate gradient. Purified antibodies were covalently coupled to the termini of PEG on the surface of small unilamellar liposomes. Cell binding and internalization ability of these immunoliposomes were estimated by a fluorescence assay using a pH-sensitive fluorescent dye (HPTS). The in vitro cytotoxicity of doxorubicin encapsulated in immunoliposomes was greater for idiotype-positive 38C13 cells than for the idiotype-negative variant of this cell line. In syngeneic C3H/HeN mice, doxorubicin encapsulated in immunoliposomes exhibited a long circulation time and was more effective at prolonging survival of mice bearing 38C13 tumor than non-targeted liposomal doxorubicin or free doxorubicin plus empty immunoliposomes. Our results demonstrate the superiority of targeted therapy with these immunoliposomes and its potential in lymphoma treatment.

Modulation of immune responses to DNA vaccines by codelivery of cytokine genes.

DNA vaccines containing genes for antigenic portions of viruses have recently been developed as a novel vaccination technology. Direct injection of plasmid DNA in vivo results in prolonged expression of viral proteins and may, thus, mimic the action of attenuated vaccines. An important advantage of this vaccination method is that in vivo-synthesized viral proteins can enter both major histocompatibility complex (MHC) class I and class II antigen-processing pathways to activate specific immunization. In many animal models for infectious diseases, DNA vaccines induced a broad range of immune responses, including antibody, CD8+ cytotoxic T lymphocytes (CTL) and CD4+ helper T (Th) lymphocyte responses, and protective immunity against challenge with the pathogen. The magnitude and nature of these immune responses to DNA vaccines can be further manipulated by codelivery of cytokine genes. Summarizing the many studies reported to date, we can draw conclusions regarding the adjuvant effects of these cytokine genes on DNA vaccines. Coadministration of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-2 genes induces higher antibody titers and T-cell proliferation responses than other cytokine genes tested to date. In contrast, the CTL activity is only modestly increased by the GM-CSF and IL-2 genes. The IL-12 gene polarizes the immune responses to DNA vaccines toward Th1 cell development and stimulates the strongest CTL activity. In contrast, co-injection of the IL-4 gene promotes the development of Th2 cells and increases production of antibodies, but suppresses CTL activity. Thus, the immune responses to DNA vaccines can be engineered by co-injection of an appropriate cytokine gene to favor the formation of either CTL or neutralization antibodies and, therefore, provide the best protection against a particular pathogen.

Treatment of B-cell lymphoma with chimeric IgG and single-chain Fv antibody-interleukin-2 fusion proteins.

Anti-idiotype (Id) antibodies (Abs) have been shown to be effective in treatment of B-cell lymphoma in animal models and in clinical trials. The combination of interleukin-2 (IL-2) can augment the therapeutic effect of anti-Id Abs. To further improve the power of the combined therapy, a monoclonal anti-Id Ab, S5A8, specifically recognizing a murine B-cell lymphoma 38C13, was genetically modified to contain the IL-2 domain and thus use the unique targeting ability of Abs to direct IL-2 to the tumor site. Two forms of the anti-Id-IL-2 fusion proteins were constructed: one configuration consisting of mouse-human chimeric IgG (chS5A8-IL-2) and the other containing only the variable light (VL) and variable heavy (VH) Ab domains covalently connected by a peptide linker (scFvS5A8-IL-2). Both forms of the anti-Id-IL-2 fusion proteins retained IL-2 biological activities and were equivalent in potentiating tumor cell lysis in vitro. In contrast, the antigen-binding ability of scFvS5A8-IL-2 was 30- to 40-fold lower than that of the bivalent chS5A8-IL-2. Pharmacokinetic analysis showed that scFvS5A8-IL-2 was eliminated about 20 times faster than chS5A8-IL-2. Finally, it was shown that chS5A8-IL-2 was very proficient in inhibiting 38C13 tumor growth in vivo, more effectively than a combined therapy with anti-Id Abs and IL-2, whereas scFvS5A8-IL-2 did not show any therapeutic effect. These results demonstrate that the anti-Id-IL-2 fusion protein represents a potent reagent for treatment for B-cell lymphoma and that the intact IgG fusion protein is far more effective than its single-chain counterpart.

Development of Th1 and Th2 populations and the nature of immune responses to hepatitis B virus DNA vaccines can be modulated by codelivery of various cytokine genes.

In this study, we provide direct evidence that the magnitude and nature of the immune response to a DNA vaccine can be differentially regulated by codelivery of various mouse cytokine genes. Mice immunized with a hepatitis B virus (HBV) DNA vaccine and the IL-12 or IFN-gamma gene exhibited a significant enhancement of Th1 cells and increased production of anti-HBV surface IgG2a Ab, as well as a marked inhibition of Th2 cells and decreased production of IgG1 Ab. In contrast, coinjection of the IL-4 gene significantly enhanced the development of specific Th2 cells and increased production of IgG1 Ab, whereas Th1 differentiation and IgG2a production were suppressed. Coinjection of the IL-2 or the granulocyte-macrophage-CSF gene enhanced the development of Th1 cells, while the development of Th2 cells was not affected, and the production of IgG1 and IgG2a Ab were both increased. The CTL activity induced by HBV DNA vaccination was most significantly enhanced by codelivery of the IL-12 or IFN-gamma gene, followed by the IL-2 or granulocyte-macrophage-CSF gene, whereas codelivery of the IL-4 gene suppressed the activity. When challenged with HBV surface Ag (HBsAg)-expressing syngeneic tumors, significant reduction of tumor growth was observed in mice that were coadministered the IL-12 gene but not the IL-4 gene. Taken together, these results demonstrate that application of a cytokine gene in a DNA vaccine formulation can influence the differentiation of Th cells as well as the nature of an immune response and may thus provide a strategy to improve its prophylactic and therapeutic efficacy.

Construction of vectors expressing bioactive heterodimeric and single-chain murine interleukin-12 for gene therapy.

It has been well demonstrated that interleukin-12 (IL-12) could be useful to defend against a variety of pathogens, to suppress tumor growth and metastasis, and even to be employed as an adjuvant of vaccines to enhance beneficial type 1 T helper (Th1) cell response over detrimental type 2 T helper (Th2) cell responses. To apply IL-12 genes in gene therapy such as a DNA vaccine, a pIL-12 vector was constructed that contained two cytomegalovirus (CMV) promoters to drive the expression of p35 and p40 subunits, respectively. In addition, a pscIL-12 vector was designed with a linker to fuse p35 cDNA with p40 cDNA to produce a single-chain IL-12 protein, ensuring not only that the expression of p35 and p40 subunits was equally expressed, but also that no free p40 subunits interfered with IL-12 activity. The data suggested pIL-12 could produce a rather high level of biologically active IL-12 after transfection of COS cell lines as well as C2C12 muscle cell lines, as measured by both concanavalin A blast proliferation assay and enzyme-linked immunosorbent assay. Interestingly, the pscIL-12 vector could also express a bioactive murine IL-12 fusion protein in vitro. Furthermore, in vivo functional studies also demonstrated that mice co-immunized with a pS vector expressing the major envelope protein of hepatitis B virus (HBV) and IL-12 vectors encoding native IL-12 or single-chain IL-12 fusion protein elicited higher levels of IgG2a anti-HBs antibody and of Th1-related cytokine. Because p35 and p40 genes can be expressed in a vector by using a single promoter, pscIL-12 should be useful in future applications for nucleic acid vaccination or for gene therapy against diseases.

Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2.

DNA vaccines encoding a viral protein have been shown to induce antiviral immune responses and provide protection against subsequent viral challenge. In this study, we show that the efficacy of a DNA vaccine can be greatly improved by simultaneous expression of interleukin-2 (IL-2). Plasmid vectors encoding the major (S) or middle (pre-S2 plus S) envelope proteins of hepatitis B virus (HBV) were constructed and compared for their potential to induce hepatitis B surface antigen (HBsAg)-specific immune responses with a vector encoding the middle envelope and IL-2 fusion protein or with a bicistronic vector separately encoding the middle envelope protein and IL-2. Following transfection of cells in culture with these HBV plasmid vectors, we found that the encoded major protein was secreted while the middle protein and the fusion protein were retained on the cell membrane. Despite differences in localization of the encoded antigens, plasmids encoding the major or middle proteins gave similar antibody and T-cell proliferative responses in the vaccinated animals. The use of plasmids coexpressing IL-2 and the envelope protein in the fusion or nonfusion context resulted in enhanced humoral and cellular immune responses. In addition, the vaccine efficacy in terms of dosage used in immunization was increased at least 100-fold by coexpression of IL-2. We also found that DNA vaccines coexpressing IL-2 help overcome major histocompatibility complex-linked nonresponsiveness to HBsAg vaccination. The immune responses elicited by HBV DNA vaccines were also modulated by coexpression of IL-2. When restimulated with antigen in vitro, splenocytes from mice that received plasmids coexpressing IL-2 and the envelope protein produced much stronger T helper 1 (Th1)-like responses than did those from mice that had been given injections of plasmids encoding the envelope protein alone. Coexpression of IL-2 also increased the Th2-like responses, although the increment was much less significant.

Inhibition of specific IgE response in vivo by allergen-gene transfer.

DNA immunization has been an attractive approach in altering the host immune response to antigen. To examine the utility of DNA immunization in allergic response, we examined the in vivo efficacy of an 'allergen-gene immunization' approach in the modulation of allergen-specific IgE responses in mice. Our results showed first that I.m. injection of a gene construct (pCMVD) containing an important house dust mite allergen gene (Dermatophagoides pteronyssinus group 5 allergen; Der p 5) results in the induction of Der p 5-specific IgG antibodies, but not IgE antibody. We next examined the effect of transduced allergen gene on the expression of specific IgE response in mice after i.p. challenge with recombinant Der p 5 (rDer p 5). Both vector (mock) control- and pCMVD-treated mice were i.p. sensitized with rDer p 5 at 3 weeks after injection of gene construct. Results showed that there is a 90% reduction in the level of specific IgE in pCMVD-treated mice when compared with mock-treated mice. Furthermore, the suppression of specific IgE response can be adoptively transferred with CD8+ T cells from pCMVD-treated mice and such inhibition is in an antigen-specific manner, since the level of specific IgE to an irrelevant allergen, Der p 1, remained unchanged in comparison to that of the mock-treated group. In addition, Der p 5-specific CD8+ T cells could produce high levels of IFN-gamma which probably inhibit allergen-specific IgE responses. Taken together, our results suggest that allergen-gene transfer is effective in the modulation of allergen-specific IgE responses and may provide a novel therapeutic approach.