Huali Jin

Induction of regulatory T cells by physiological level estrogen.

Naturally occurring CD4+CD25+ regulatory T cells (Treg) exert an important role in mediating maternal tolerance to the fetus during pregnancy, and this effect might be regulated via maternal estrogen secretion. Although estrogen concentration in the pharmaceutical range has been shown to drive expansion of CD4+CD25+ Treg cells, little is known about how and through what mechanisms E2 within the physiological concentration range of pregnancy affects this expansion. Using in vivo and in vitro mouse models in these experiments, we observed that E2 at physiological doses not only expanded Treg cell in different tissues but also increased expression of the Foxp3 gene, a hallmark for CD4+CD25+ Treg cell function, and the IL‐10 gene as well. Importantly, our results demonstrate that E2, at physiological doses, stimulated the conversion of CD4+CD25− T cells into CD4+CD25+ T cells which exhibited enhanced Foxp3 and IL‐10 expression in vitro. Such converted CD4+CD25+ T cells had similar regulatory function as naturally occurring Treg cells, as demonstrated by their ability to suppress naïve T cell proliferation in a mixed lymphocyte reaction. We also found that the estrogen receptor (ER) exist in the CD4+CD25− T cells and the conversion of CD4+CD25− T cells into CD4+CD25+ T cells stimulated by E2 could be inhibited by ICI182,780, a specific inhibitor of ER(s). This supports that E2 may directly act on CD4+CD25− T cells via ER(s). We conclude that E2 is a potential physiological regulatory factor for the peripheral development of CD4+CD25+ Treg cells during the implantation period in mice. J. Cell. Physiol. 214: 456–464, 2008.

Induction of Th1 type response by DNA vaccinations with N, M, and E genes against SARS-CoV in mice

Abstract Vaccination against the SARS-CoV infection is an attractive means to control the spread of viruses in public. In this study, we employed a DNA vaccine technology with the levamisole, our newly discovered chemical adjuvant, to generate Th1 type of response. To avoid the enhancement antibody issue, genes encoding the nucleocapsid, membrane, and envelope protein of SARS-CoV were cloned and their expressions in mammalian cells were determined. After the intramuscular introduction into animals, we observed that the constructs of the E, M, and N genes could induce high levels of specific antibodies, T cell proliferations, IFN-γ, DTH responses, and in vivo cytotoxic T cells activities specifically against SARS-CoV antigens. The highest immune responses were generated by the construct encoding the nucleocapsid protein. The results suggest that the N, M, and E genes could be used as the targets to prevent SARS-CoV infection in the DNA vaccine development.

The adjuvant effects of co-stimulatory molecules on cellular and memory responses to HBsAg DNA vaccination

Because DNA vaccines on their own tend to induce weak immune responses in humans, adjuvant methods are needed in order to improve their efficacy. The co‐stimulatory molecules 4‐1BBL, OX40L, and CD70 have been shown to induce strong T cell activities; therefore, in this study, we investigated whether they may be used as molecular adjuvants for a hepatitis B surface antigen (HBsAg) DNA vaccine (pcDS2) in eliciting strong cellular and memory responses. Compared to mice immunized with pcDS2 alone, addition of the co‐stimulatory molecules increased T cell proliferation and an HBsAg‐specific antibody response that was marked with a higher ratio of IgG2a/IgG1. Importantly, pcDS2 plus these co‐stimulatory molecules elicited a higher level of IFN‐γ and IL‐4 in CD4+ T cells and a higher level of IFN‐γ in CD8+ T cells. In addition, a significantly robust antigen‐specific cytotoxic T lymphocyte (CTL) response and the production of long‐term memory CD8+ T cells were also observed in the groups immunized with pcDS2 plus 4‐1BBL, OX40L, or CD70. Consistently, as late as 100 days after immunization, upregulated expressions of BCL‐2, Spi2A, IL‐7Ra, and IL‐15Ra were still observed in mice immunized with pcDS2 plus these co‐stimulatory molecules, suggesting the generation of memory T cells in these groups. Together, these results suggest that the co‐stimulatory molecules 4‐1BBL, OX40L, or CD70 can enhance the immunogenicity of HBsAg DNA vaccines, resulting in strong humoral, cellular, and memory responses. This approach may lead to an effective therapeutic vaccine for chronic hepatitis B virus (HBV) infection. Copyright

The effects of IL-6 and TNF-α as molecular adjuvants on immune responses to FMDV and maturation of dendritic cells by DNA vaccination

Various approaches have been developed to improve efficacy of DNA vaccination, such as the use of plasmid expressing cytokine as a molecular adjuvant. In this study, we investigated whether co-inoculation of a construct expressing either IL-6 or TNF-alpha as the molecular adjuvant with FMDV DNA vaccine, pcD-VP1, can increase immune responses. Compared to the group immunized with pcD-VP1 alone, the co-inoculation with either molecular adjuvant induced a higher ratio of IgG2a/IgG1, higher levels of expression of IFN-gamma in CD4+ and CD8+ T cells, IL-4 in CD4+ T cells, and in vivo antigen-specific cytotoxic response. Both adjuvants induced maturation of dendritic cells, suggesting a correlation between the initiating innate response and subsequent activating adaptive immune responses. Together, the results demonstrate that IL-6 and TNF-alpha used as molecular adjuvants can enhance the antigen-specific cell-mediated responses elicited by VP1 DNA vaccine.

Interleukin-15 enhance DNA vaccine elicited mucosal and systemic immunity against foot and mouth disease virus

Aerosol transmission of foot and mouth disease virus (FMDV) is believed to be an important route of infection. Induction of mucosal response is thought to be effective way against such infection. Various approaches have been developed including the use of molecules adjuvant and polymers delivery for the mucosal delivery of DNA vaccine. In this study, using low molecular weight chitosan as a delivery vehicle, we investigated whether co-administration intranasally of the FMDV DNA vaccine, pcD-VP1 and a construct expressing IL-15 as the molecular adjuvant can enhance mucosal and systemic immune responses in animals. Compared to the group intranasally immunized with pcD-VP1 alone, the group immunized with the molecular adjuvant not only was induced higher level of mucosal sIgA but also serum IgG. Interestingly, intranasal delivery of the IL-15 construct with pcD-VP1 significantly enhanced the cell-mediated immunity (CMI) compared to the pcD-VP1 alone, as evidenced by the higher level of antigen-specific T-cell proliferation, cytotoxic T lymphocyte (CTL) response and higher expressions of IFN-gamma in both CD4+ and CD8+ T cells inform the spleen and mucosal sites. Consistently, IL-15 as adjuvant provided higher level of FMDV neutralizing antibody against FMDV and high secretions of IgA producing cells in mucosal tissues. Taken together, the results demonstrated that intranasal delivery of IL-15 as a mucosal adjuvant can enhance the antigen-specific mucosal and systemic immune responses, which may provide a protection against the FMDV initial infection.

Levamisole is a potential facilitator for the activation of Th1 responses of the subunit HBV vaccination.

Chemical compounds activating innate responses may present potential adjuvants for the vaccine development. Levamisole (LMS), demonstrated as a potent adjuvant for DNA and viral killed vaccines in our previous studies, may activate such responses. To confirm this notion, LMS combined with the recombinant HBsAg (rHBsAg) was investigated. Compared to the vaccination with rHBsAg alone, LMS could up-regulate the expressions of TLR7&8, MyD88, IRF7 and their downstream pro-inflammatory cytokines including IFN-alpha and TNF-alpha, which promote DCs activation. Strikingly, we find that the combination of LMS and alum adjuvant synergistically enhances immunogenicity of rHBsAg and leads to a robust cell-mediated response demonstrated by the higher level of IgG2a/IgG1, T cell proliferation, and importantly, a high level of antigen-specific CTL and IFN-gamma production within these activated CD8(+) T cells. The achieved robust responses are at a comparative level with CpG+alum used as a positive control adjuvant in mice. The combination of LMS+alum with rHBsAg may provide a cost-effective, safe, and effective therapy to treat those individuals chronically infected by HBV, since antigen-specific cellular immunity is implicated for the clearance of HBV chronic infection.

Enhanced contraceptive response by co‐immunization of DNA and protein vaccines encoding the mouse zona pellucida 3 with minimal oophoritis in mouse ovary

Zona pellucida 3 (ZP3) acts as the primary sperm receptor, induces autoantibody that can prevent oocyte fertilization and has been proposed as a vaccine candidate for contraception in humans. Due to the elicited autoreactive T cell inflammation that causes ovarian destruction, ZP3‐based vaccine with removed T epitopes from the ZP3 is considered as a preferred approach. We present here a new strategy to eliminate the T cell inflammation while retaining a high level of antibody by co‐immunization of mZP3 DNA and protein vaccines, which resulted in a higher reduction rate of fertility in this group. Histological analysis showed that there were normal follicular developments of infertile mice in the co‐immunized group; while other vaccine groups of the most infertile mice lacked mature follicles. There was a significant correlation between normal follicular development and the inhibition of T cell response in co‐immunized mice. At the same time, co‐immunization reduced the production of inflammatory cytokine, IFN‐γ, and increased the productions of IL‐10 and FoxP3 in CD4 T cells, suggesting the anti‐inflammation may be via a T regulatory function. The results indicate that co‐immunization of mZP3 DNA‐ and protein‐based vaccines can reduce fertility without interfering with the normal follicular development and present a novel strategy to develop a contraceptive vaccine in humans. Copyright

Induction of Adaptive T Regulatory Cells That Suppress the Allergic Response by Coimmunization of DNA and Protein Vaccines

Allergen-induced immediate hypersensitivity (AIH) is a health issue of significant concern. This robust inflammatory reaction is initiated by the allergen-specific T cell responsiveness. Severe lesion reactions on skin are consequential problem requiring medical treatment. Effective Ag-specific treatments or preventions are lacking. Using a rodent model of AIH induced by flea allergens, we first report that coimmunization of DNA and protein vaccines encoding the flea salivary specific Ag-1 ameliorated experimental AIH, including Ag-induced wheal formation, elevated T cell proliferation, and infiltration of lymphocytes and mast cells to the site of allergen challenge. The amelioration of AIH was directly related to the induction of a specific population of flea antigenic specific T cells exhibiting a CD4+CD25−FoxP3+ phenotype, a characteristic of regulatory T (TREG) cells. These TREG cells expressing IL-10, IFN-γ, and the transcriptional factor T-bet after Ag stimulation were driven by a tolerogenic MHC class II+/CD40low dendritic cell population that was induced by the coimmunization of DNA and protein vaccines. The tolerogenic dendritic cell could educate the naive T cells into CD4+CD25−FoxP3+ TREG cells both in vitro and in vivo. The study identified phenomenon to induce an Ag-specific tolerance via a defined Ag vaccinations and lead to the control of AIH. Exploitation of these cellular regulators and understanding their induction provides a basis for the possible development of novel therapies against allergic and related disorders in humans and animals.

The protective efficacy aganist Schistosoma japonicum infection by immunization with DNA vaccine and levamisole as adjuvant in mice

Levamisole (LMS) as an adjuvant enhances cell-mediated immunity in DNA vaccination; we investigated the efficacy and liver immunopathology alleviation of a DNA vaccine, VR1012-SjGST-32, in a LMS formulation in the murine challenge model. Compared to controls, the VR1012-SjGST-32 plus LMS can reduce worm and egg burdens, as well as, immunopathological complications associated chronic inflammation significantly in liver, which were apparently associated with Th1-type response. Together, these results suggest that the LMS as a potential Schistosome DNA vaccine adjuvant can enhance both worm killing and disease prevention, which is possibly mediated through the induction of a strong Th1-dominant environment in immunized mice.

A continuous method for the large-scale extraction of plasmid DNA by modified boiling lysis

This protocol describes a streamlined method of plasmid DNA extraction by continual thermal lysis, a modification of the basic boiling lysis technique, to simplify the processing of large volumes of Escherichia coli cultures. Fermented bacteria are harvested using a hollow fiber-membrane module and pre-treated with lysozyme prior to passing through a thermal exchange coil set at 70 °C to lyse the cells, and into a juxtaposed cooling coil on ice. The lysed and cooled bacteria are subsequently separated from the lysate by centrifugation and plasmid DNA is precipitated from the supernatant for further purification. The use of peristaltic pumps and two heating coils at constant temperature without the use of centrifugation enable the lysis process to become constant and controllable, providing a flow-through protocol for cell lysis and plasmid DNA extraction. Large volumes of bacterial cultures (20 l) can be processed in 2 h, yielding approximately 100 mg plasmid DNA l−1 culture, making this an attractive protocol for consistent and large-scale preparation of plasmid DNA.