Antitumor immunity and cellular cancer therapies

The identification of tumor specific antigens has provided important advance in tumor immunology. It is now established that specific cytotoxic T lymphocytes (CTL) and natural killer cells infiltrate tumor tissues and are effector cells able to control tumor growth. However, such a natural antitumor immunity has limited effects in cancer patients. Failure of host defenses against tumor is consecutive to several mechanisms which are becoming targets to design new immunotherapeutic approaches. CTL are critical components of the immune response to human tumors and induction of strong CTL responses is the goal of most current vaccine strategies. Effectiveness of cytokine therapy, cancer vaccines and injection of cells improving cellular immunity have been established in tumor grafted murine models. Clinical trials are underway. To day, interest is particularly focused on cell therapy: injected cells are either "ready to use" effector cells (lymphocytes) or antigen presenting cells able to induce a protective immune reaction in vivo (dendritic cells). The challenge ahead lie in the careful optimization of the most promising strategies in clinical situation.     

HIV-1 p24 DNA和P24蛋白联合免疫小鼠的效果

DNA疫苗能够诱导机体产生特异的细胞免疫和体液免疫反应,在肿瘤和感染性疾病的疫苗开发中显示出巨大的潜能。以HIV-1核心蛋白P24为抗原基因,构建pVAX1-p24 DNA,经Western blotting和动物活体成像检测证明,pVAX1 DNA携带的外源基因可以在293T 细胞和小鼠肌肉组织有效表达。采用不同的免疫策略免疫BALB/c小鼠 (DNA/DNA,DNA/Protein),实验结果表明：pVAX1-p24单独免疫BALB/c小鼠,可诱导明显的体液免疫及细胞免疫反应;pVAX1-p24与P24蛋白联合免疫诱导的体液免疫反应高于pVAX1-p24单独免疫,所获得的抗体滴度是单独免疫的7.3~8.0倍,但细胞免疫反应则不及单独免疫组。研究结果表明采取不同的免疫策略可以诱导产生不同的免疫反应,根据具体情况调整免疫策略将获得更好的免疫效果。这些研究为艾滋病疫苗的研发提供了实验依据。     

Strategies for designing vaccines eliciting Th1 responses in humans

There is currently a major interest in designing vaccines capable of eliciting strong cellular immune responses. The induction of cytotoxic and Th1 helper cellular responses is for example highly desirable for vaccines targeting either chronic infectious diseases or cancers (therapeutic vaccines). Similarly, Th1 vaccines would be useful in redirecting inappropriate antigen-specific immune responses in patients with autoimmune diseases and allergies. Importantly, emerging technologies and a better understanding of the physiology of immune responses offer new avenues to rationally design such vaccines. Approaches based on the identification and selection of immunogens containing T cell epitopes can be used, together with epitope-enhancement strategies, to increase binding to MHC, or to improve recognition by T cell receptor complexes. Optimized immunogens can subsequently be presented to the immune system with appropriate vectors allowing to target professional antigen-presenting cells, such as dendritic cells. Such antigen presentation platforms can be used alone or in association, as part of mixed immunization regimens (heterologous prime-boosts), in order to elicit broad immune responses. The rational design of Th1 adjuvants can also benefit from our better understanding of the nature of proinflammatory signals leading to the initiation of both innate and adaptive immune effector mechanisms. Candidate Th1 vaccines (or components such as vectors or adjuvants) will have to be tested in exploratory clinical studies, implying a need for new assays and methods allowing to assess in a qualitative and quantitative manner low-frequency T cell responses in humans.     

Varied immunity generated in mice by DNA vaccines with large and small hepatitis delta antigens

Whether the hepatitis delta virus (HDV) DNA vaccine can induce anti-HDV antibodies has been debatable. The role of the isoprenylated motif of hepatitis delta antigens (HDAg) in the generation of immune responses following DNA-based immunization has never been studied. Plasmids p2577L, encoding large HDAg (L-HDAg), p2577S, expressing small HDAg (S-HDAg), and p25L-211S, encoding a mutant form of L-HDAg with a cysteine-to-serine mutation at codon 211, were constructed in this study. Mice were intramuscularly injected with the plasmids. The anti-HDV antibody titers, T-cell proliferation responses, T-helper responses, and HDV-specific, gamma interferon (IFN-gamma)-producing CD8(+) T cells were analyzed. Animals immunized with p2577S showed a strong anti-HDV antibody response. Conversely, only a low titer of anti-HDV antibodies was detected in mice immunized with p2577L. Epitope mapping revealed that the anti-HDV antibodies generated by p2577L vaccination hardly reacted with epitope amino acids 174 to 194, located at the C terminus of S-HDAg. All of the HDAg-encoding plasmids could induce significant T-cell proliferation responses and generate Th1 responses and HDV-specific, IFN-gamma-producing CD8(+) T cells. In conclusion, HDAg-specific antibodies definitely exist following DNA vaccination. The magnitudes of the humoral immune responses generated by L-HDAg- and S-HDAg-encoding DNA vaccines are different. The isoprenylated motif can mask epitope amino acids 174 to 195 of HDAg but does not interfere with cellular immunity following DNA-based immunization. These findings are important for the choice of a candidate HDV DNA vaccine in the future.     

Induction of antigen-specific Th1-biased immune responses by plasmid DNA in schistosoma-infected mice with a preexistent dominant Th2 immune profile

A requisite for vaccines to confer protection against intracellular infections such as Human Immunodeficiency Virus or Mycobacterium tuberculosis is their capacity to induce Th1 immune responses. However, they may fail to do so in Africa and South East Asia, where most individuals have a dominant preexistent Th2 immune profile, due to persistent helminthic parasitic infections, which may undermine any Th1 response. It is well established that DNA vaccines induce strong Th1 biased immune responses against an encoded antigen, depending on the route and mode of immunization. Here, we demonstrate that intradermal immunization with plasmid DNA encoding beta-gal (pCMV-LacZ) of Schistosoma-infected mice, with preexistent dominant Th2 immune background, induce a strong Th1 anti-beta-gal response, as opposed to immunized with beta-gal only. Importantly, the established protective Th2 immune response to schistosomes was not disrupted. These findings strongly support the possibility of using plasmid DNA as a Th1 inducing adjuvant when immunizing populations with a strong preexistent Th2 immune profile.     

Characterization of antigen-specific immune responses induced by canarypox virus vaccines

Avipoxvirus-based vectors, such as recombinant canarypox virus ALVAC, are studied extensively as delivery vehicles for vaccines against cancer and infectious diseases. Effective use of such vaccines is expected to benefit from proper understanding of the interaction between these viral vectors and the host immune system. We performed preclinical vaccination experiments in a murine tumor model to analyze the immunogenic properties of an ALVAC-based vaccine against carcinoembryonic Ag (ALVAC-CEA), a tumor-associated autoantigen commonly overexpressed in colorectal cancers. The protective CEA-specific immunity induced by this vaccine consisted of CD4(+) T cell responses with a mixed Th1/Th2 cytokine profile that were accompanied by potent humoral responses, but not by CEA-specific CD8(+) CTL immunity. In contrast, protective immunity induced by a CEA-specific DNA vaccine (DNA-CEA) consisted of Th1 and CTL responses. Modification of the ALVAC-CEA vaccine through coinjection of DNA-CEA, admixture with CpG oligodeoxynucleotides, or supplementation with additional transgenes encoding a triad of costimulatory molecules (TRICOM) did not result in induction of CEA-specific CTL responses. Even though these results suggested that ALVAC does not elicit Ag-specific CTLs, immunization with ALVAC vaccines against other Ags efficiently induced CTL responses. Our data show that the capacity of ALVAC vaccines to elicit CTL immunity against transgene-encoded Ags critically depends on the presence of highly immunogenic CTL epitopes in these Ags. This consideration needs to be taken into account with respect to the design and evaluation of vaccination strategies that use ALVAC-based vaccine.     

Delivery of protein antigens to the immune system by fusion-active virosomes: a comparison with liposomes and ISCOMs

The induction of effective cellular and humoral immune responses against protein antigens is of major importance in vaccination strategies against infectious diseases and cancer. Immunization with protein alone in general does not result in efficient induction of cytotoxic T lymphocyte (CTL) and antibody responses. Numerous other immunization strategies have been explored. In this review we will discuss a number of lipid-based antigen delivery systems suitable for the induction of CTL responses. These systems comprise reconstituted virus envelopes (virosomes), liposomes, and immune-stimulating complexes (ISCOMs). We will concentrate on delivery of the protein antigen ovalbumin (OVA) since extensive studies with this antigen have been performed for all of the systems discussed, allowing direct comparison of antigen delivery efficiency. Stimulation of CTL activity requires processing of the antigen in the cytosol of antigen-presenting cells (APCs) and presentation of antigenic peptides on surface major histocompatibility class I complexes (MHC class I). In vitro, the ability of antigen delivery systems to induce MHC class I presentation indeed correlates with their capacity to deliver antigen to the cytosol of cells. This capacity appears to be less important for the induction of cytotoxic T lymphocytes in vivo. Instead, other properties of the antigen delivery system like activation of APCs and induction of T helper cells play a more prominent role. Fusion-active virosomes appear to be a very potent system for induction of CTL activity, most likely since virosomes combine efficient delivery of antigen with general stimulation of the immune system.     

Mucosal immunization with DNA vaccines

DNA vaccines can induce potent humoral and cellular immune responses in numerous animal models. Most DNA vaccines have been administered parenterally; however, more effective protection against mucosal pathogens could be achieved with mucosal immunization. This review concentrates on the use of DNA vaccines for the induction of mucosal immunity.     

Efficient Generation of Mucosal and Systemic Antigen-Specific CD8+ T-Cell Responses following Pulmonary DNA Immunization

Although mucosal CD8⁺ T-cell responses are important in combating mucosal infections, the generation of such immune responses by vaccination remains problematic. In the present study, we evaluated the ability of plasmid DNA to induce local and systemic antigen-specific CD8⁺ T-cell responses after pulmonary administration. We show that the pulmonary delivery of plasmid DNA formulated with polyethyleneimine (PEI-DNA) induced robust systemic CD8⁺ T-cell responses that were comparable in magnitude to those generated by intramuscular (i.m.) immunization. Most importantly, we observed that the pulmonary delivery of PEI-DNA elicited a 10-fold-greater antigen-specific CD8⁺ T-cell response in lungs and draining lymph nodes of mice than that of i.m. immunization. The functional evaluation of these pulmonary CD8⁺ T cells revealed that they produced type I cytokines, and pulmonary immunization with PEI-DNA induced lung-associated antigen-specific CD4⁺ T cells that produced higher levels of interleukin-2 than those induced by i.m. immunization. Pulmonary PEI-DNA immunization also induced CD8⁺ T-cell responses in the gut and vaginal mucosa. Finally, pulmonary, but not i.m., plasmid DNA vaccination protected mice from a lethal recombinant vaccinia virus challenge. These findings suggest that pulmonary PEI-DNA immunization might be a useful approach for immunizing against pulmonary pathogens and might also protect against infections initiated at other mucosal sites.Since establishing that antigen-specific CD8⁺ T-cell populations in mucosal sites may confer protection against intracellular pathogens that initiate infections at mucosal surfaces, vaccine strategies have been explored for eliciting cellular immune responses in mucosal tissues (40). Studies have been done to evaluate the immunogenicity of vaccines delivered to a variety of mucosal surfaces, including those of the nose, intestine, rectum, and vagina. These studies have shown that immunization at mucosal sites can induce larger numbers of antigen-specific CD8⁺ T cells in mucosal tissues than parenteral immunization (3).Particular attention has focused on the lungs as a target for mucosal immunization. The lungs are an important mucosal portal of entry for pathogens. They are also a readily accessed mucosal site for the delivery of immunogens that might induce diverse mucosal immune responses. Pulmonary immunization strategies have been shown to generate potent Th1 responses and protective immunity against respiratory challenge with pathogens in several animal models (4, 29, 32, 37, 38).Because of the ease of generating vaccine constructs and the ability to administer repeated inoculations of the same vector, DNA immunization remains a promising vaccination strategy for eliciting cellular immune responses. Only a limited number of studies have been done to evaluate the immunogenicity of DNA vaccines following pulmonary delivery (4, 32). Although the importance of CD8⁺ T lymphocytes in eradicating mucosal infections has been well established, it has not been determined whether pulmonary DNA immunization can induce robust functional CD8⁺ T-cell responses.In the present study, we characterized antigen-specific CD8⁺ T lymphocytes in mice induced by the noninvasive pulmonary administration of plasmid DNA complexed to the cationic polymer polyethyleneimine (PEI). We demonstrate that the delivery of a DNA vaccine to the airways can induce a high frequency of functional antigen-specific CD8⁺ T cells in both systemic and mucosal sites.     

Repeated administration of cytosine-phosphorothiolated guanine-containing oligonucleotides together with peptide/protein immunization results in enhanced CTL responses with anti-tumor activity

The development of therapeutic anti-cancer vaccines designed to elicit CTL responses with anti-tumor activity has become a reality thanks to the identification of several tumor-associated Ags and their corresponding peptide T cell epitopes. However, peptide-based vaccines, in general, fail to elicit sufficiently strong CTL responses capable of producing therapeutic anti-tumor effects (i.e., prolongation of survival, tumor reduction). Here we report that repeated administration of synthetic oligonucleotides containing foreign cytosine-phosphorothiolated guanine (CpG) motifs increased 10- to 100-fold the CTL response to immunization with various synthetic peptides corresponding to well-known T cell epitopes. Moreover, repeated CpG administration allowed the induction of CTL to soluble protein even in the absence of additional adjuvant. Our results indicate that the potentiating effect of CpG in CTL responses required the participation of Th lymphocytes. Repeated CpG administration resulted in overt splenomegaly and lymphadenopathy with a significant increase in the numbers of CTL precursors and dendritic cells. Protein vaccination in combination with repeated CpG therapy was effective in delaying tumor cell growth and extending survival in mice bearing melanoma tumors. These findings support the contention that repeated administration of CpG-oligonucleotides enhances the effect of peptide and protein vaccines leading to potent anti-tumor responses, presumably through the induction of Th1 and dendritic cells, which are essential for optimal CTL responses. The immunostimulatory properties of CpG motifs may be key in inducing a consistent long term immunity to tumor-associated Ags when using peptides or proteins as T cell-inducing vaccines.     

Induction of Potent Human Immunodeficiency Virus Type 1-Specific T-Cell-Restricted Immunity by Genetically Modified Dendritic Cells

A novel technology combining replication- and integration-defective human immunodeficiency virus type 1 (HIV-1) vectors with genetically modified dendritic cells was developed in order to induce T-cell immunity. We introduced the vector into dendritic cells as a plasmid DNA using polyethylenimine as the gene delivery system, thereby circumventing the problem of obtaining viral vector expression in the absence of integration. Genetically modified dendritic cells (GMDC) presented viral epitopes efficiently, secreted interleukin 12, and primed both CD4(+) and CD8(+) HIV-specific T cells capable of producing gamma interferon and exerting potent HIV-1-specific cytotoxicity in vitro. In nonhuman primates, subcutaneously injected GMDC migrated into the draining lymph node at an unprecedentedly high rate and expressed the plasmid DNA. The animals presented a vigorous HIV-specific effector cytotoxic-T-lymphocyte (CTL) response as early as 3 weeks after a single immunization, which later developed into a memory CTL response. Interestingly, antibodies did not accompany these CTL responses, indicating that GMDC can induce a pure Th1 type of immune response. Successful induction of a broad and long-lasting HIV-specific cellular immunity is expected to control virus replication in infected individuals.     

Fusion of HBsAg and prime/boosting augment Th1 and CTL responses to HCV polytope DNA vaccine

Correlation of hepatitis C virus (HCV) spontaneous resolution with Th1 and CD8⁺CTL responses during natural infection implies the potentiality of poly-CTL-epitopic HCV vaccines. We recently reported in silico design and construction of DNA vaccines (pcPOL-plasmids) harboring HCV CTL epitopes. Herein, we provide data of mice immunization by pcPOL, (encoding; core_132-142 [C], E2_405-414 [E₄], E2_614-622 [E₆] and NS3_1406-1415 [N] CD8⁺CTL epitopes as CE₄E₆N polytope) and its HBsAg-fused counterpart (pcHPOL), compared to the adjuvant-formulated (Montanide + CpG) CE₄E₆N synthetic-peptide immunization. All vaccinated groups developed different levels of cellular responses, however, only the pcHPOL-immunized mice elicited strong CTLs and IFN-γ-secreting cells that were further augmented towards a Th1 response and partial tumor protection by DNA-prime/peptide-boosting regimen. Priming with HBsAg alone could not afford its augmenting effect indicating the importance of priming by polytope itself. Hence, fusion of immunocarriers like HBsAg conjoined with DNA-prime/peptide-boost immunization regimen seems a strategy to enhance the epitope-specific immune responses towards poly-CTL-epitopic vaccines.     

Protective CD4+ and CD8+ T Cells against Influenza Virus Induced by Vaccination with Nucleoprotein DNA

DNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745–1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4⁺ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8⁺ T cells and cytokine-secreting CD4⁺ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.Cellular immune responses play an important role in protection from disease caused by infectious pathogens, such as viruses and certain bacteria (e.g., Mycobacterium tuberculosis). The specific T cells involved in conferring immunity can include both CD4⁺ and CD8⁺ T cells, often through the action of secreted cytokines and cytolytic activity, respectively. Certain types of vaccines, such as subunit proteins and whole or partially purified preparations of inactivated organisms, in general induce CD4⁺ T-cell responses but not CD8⁺ cytotoxic T lymphocytes (CTL). In contrast, live attenuated organisms and subunit proteins formulated with certain experimental adjuvants can induce both types of responses. Recently, a different approach consisting of direct immunization with plasmid DNA expression vectors (i.e., DNA vaccines) has shown promise as a viable means of inducing broad-spectrum T-cell responses. The effectiveness of DNA vaccines in animal models is likely due, at least in part, to expression of antigens in situ (35), leading to the induction of CTL (29), antibodies (3, 4, 10, 21, 22, 32), and cytokine-secreting lymphocyte responses (12, 36). During the past 5 years, many reports have been published on the immunogenicity of DNA vaccines encoding various antigens in several animal models, thereby illustrating the applicability of the technology to many pathogens (for a review, see reference 6). However, in only a few instances has the nature of the effector cells responsible for protective immunity been described (7, 16). In the present study, we have analyzed in detail the cellular immune responses induced by influenza virus nucleoprotein (NP) DNA and have established that both CD4⁺ T cells secreting Th1-type cytokines and CD8⁺ cytotoxic T cells play important effector roles in heterosubtypic protective immunity against lethal influenza virus challenge in mice.     

A novel DNA vaccine containing multiple TB-specific epitopes cast in a natural structure elicits enhanced Th1 immunity compared with BCG

Vaccination is expected to make a major contribution to the goal of eliminating tuberculosis worldwide by 2050. Because the protection afforded by the currently available tuberculosis vaccine, BCG, is insufficient, new vaccine strategies are urgently needed. Protective immunity against MTB depends on generation of a Th1-type cellular immune response characterized by secretion of IFN-γ from antigen-specific T cells. Epitope-driven vaccines are created from sub-sequences of proteins (epitopes) derived by scanning the protein sequences of pathogens and selecting epitopes with patterns of amino acids which permit binding to human MHC molecules. Guided by the crystal structure of HSP65 and its characteristics, four functional T cell epitopes elaborately elicited from ESAT-6, Ag85A, CFP-10 and Ag85B were cast into the intermediate domain of HSP65. A panel of a novel chimeric vaccine, ECANS, expressing HSP65 and combined T cell epitopes was created. Gene cloning and sequencing, DNA vaccination and humoral and cellular responses were studied. After being immunized with DNA vaccine three times, all mice injected with ECANS had specific cellular immune responses. In addition, lymphocytes obtained from the spleen of ECANS immunized mice at week eight exhibited significantly greater specific lymphocyte proliferation, IFN-γ secretion and CTL activity than those of mice that had been immunized with BCG. DNA vaccine with ECANS can successfully induce enhanced specific cellular immune response to PPD, and further study of its protective effects against Mycobacterium tuberculosis in vivo is needed.     

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.     

Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity

There is currently a need for vaccines that stimulate cell-mediated immunity-particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)-against viral and tumor antigens. The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells (DCs). DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells. Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research. We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals. Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells. Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses.     

Immunogenicity of DNA and Recombinant Sendai Virus Vaccines Expressing the HIV-1 gag Gene

Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendal virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1( )-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens.     

增强DNA疫苗免疫应答的新进展

DNA疫苗为编码抗原蛋白的真核表达载体,注入体内后在原位表达所编码的抗原并诱导免疫应答,在预防感染、治疗自身免疫性疾病、过敏性疾病和肿瘤等疫病中有着很好的应用前景。但与灭活疫苗相比,其免疫效价还比较低。有多种策略能够增强或调节DNA疫苗诱导的免疫应答,其中,作为外源基因载体的质粒的组成及插入的有关基因均可直接或间接地影响免疫反应的效果,在构建DNA疫苗质粒时,加入细胞因子、融合信号、泛素等基因以及ISS序列,另外还可以通过设计一些对抗原提成细胞有影响的分子共注射,以及加入转移分子,都可以明显增强DNA疫苗的免疫效果,从而有利于研制更有效的DNA疫苗。     

Plasmid DNA vaccination

Plasmid DNA vaccination against tuberculosis is a very powerful and easy method for the induction of strong humoral responses, CD4+ mediated secretion of Th1 cytokines and CD8+ mediated CTL activity in mice. Tuberculosis DNA vaccines have not been assessed so far in humans, and clinical trials with DNA in general have been somewhat disappointing. However, numerous studies have reported on the potent priming capacity of plasmid DNA for Th1 and CD8+ mediated immune responses, which can be boosted subsequently by recombinant protein or recombinant pox-viruses. With respect to tuberculosis, prime/boost regimens with Mycobacterium bovis BCG vaccine are particularly promising and warrant further analysis.     

Gender differences in human immunodeficiency virus type 1-specific CD8 responses in the reproductive tract and colon following nasal peptide priming and modified vaccinia virus Ankara boosting

Induction of mucosal anti-human immunodeficiency virus type 1 (HIV-1) T-cell responses in males and females will be important for the development of a successful HIV-1 vaccine. An HIV-1 envelope peptide, DNA plasmid, and recombinant modified vaccinia virus Ankara (rMVA) expressing the H-2D(d)-restricted cytotoxic T lymphocyte P18 epitope were used as immunogens to test for their ability to prime and boost anti-HIV-1 T-cell responses at mucosal and systemic sites in BALB/c mice. We found of all prime-boost combinations tested, an HIV-1 Env peptide subunit mucosal prime followed by systemic (intradermal) boosting with rMVA yielded the maximal induction of gamma interferon (IFN-gamma) spot-forming cells in the female genital tract and colon. However, this mucosal prime-systemic rMVA boost regimen was minimally immunogenic for the induction of genital, colon, or lung anti-HIV-1 T-cell responses in male mice. We determined that a mucosal Env subunit immunization could optimally prime an rMVA boost in female but not male mice, as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tracts, colon, and lung. Defective mucosal priming in male mice could not be overcome by multiple mucosal immunizations. However, rMVA priming followed by an rMVA boost was the optimal prime-boost strategy for male mice as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tract and lung. Thus, prime-boost immunization strategies able to induce mucosal antigen-specific IFN-gamma responses were identified for male and female mice. Understanding the cellular and molecular basis of gender-determined immune responses will be important for optimizing induction of anti-HIV-1 mucosal immune responses in both males and females.