人乳头瘤病毒(HPV)58型DNA疫苗免疫原性的初步分析

为了检测HPV 58型不同L1基因的DNA疫苗的免疫原性,以pcDNA3.1为载体分别构建含HFV 58型不同L1基因的DNA疫苗,命名为L1h、L1h△c、L1S、L1SM和L1wt.用免疫印迹法检测各DNA疫苗的体外表达情况;各重组质粒与pcDNA3.1-h58L2和pcDNA3.1-GFP共转染293FT细胞,检测其形成假病毒的能力;并将各DNA疫苗肌肉注射免疫小鼠,利用假病毒中和实验检测中和抗体水平,用ELISPOT检测细胞免疫情况.结果显示,本实验成功构建了五种DNA疫苗,L1h△c的体外表达量最高,L1S和L1SM的表达量次之,L1wt没有表达;重组质粒L1S能够形成假病毒,而其他四种重组质粒均不能形成假病毒.L1S和L1h均可在小鼠体内诱导中和抗体,但L1S诱导的中和抗体的平均滴度为1:6 400,明显高于L1h诱导的中和抗体水平(平均滴度为1:48),而其他疫苗在小鼠体内未产生中和抗体.对五种疫苗均未检测出特异性的细胞免疫反应.结果提示,体外能够组装成假病毒的DNA疫苗在免疫动物后可诱导高滴度的中和抗体,为今后DNA疫苗的筛选提供参考.     

Persistence of circulating memory B cell clones with potential for dengue virus disease enhancement for decades following infection

Symptomatic dengue virus infection ranges in disease severity from an influenza-like illness to life-threatening shock. One model of the mechanism underlying severe disease proposes that weakly neutralizing, dengue serotype cross-reactive antibodies induced during a primary infection facilitate virus entry into Fc receptor-bearing cells during a subsequent secondary infection, increasing viral replication and the release of cytokines and vasoactive mediators, culminating in shock. This process has been termed antibody-dependent enhancement of infection and has significantly hindered vaccine development. Much of our understanding of this process has come from studies using mouse monoclonal antibodies (MAbs); however, antibody responses in mice typically exhibit less complexity than those in humans. A better understanding of the humoral immune response to natural dengue virus infection in humans is sorely needed. Using a high-efficiency human hybridoma technology, we isolated 37 hybridomas secreting human MAbs to dengue viruses from 12 subjects years or even decades following primary or secondary infection. The majority of the human antibodies recovered were broadly cross-reactive, directed against either envelope or premembrane proteins, and capable of enhancement of infection in vitro; few exhibited serotype-specific binding or potent neutralizing activity. Memory B cells encoding enhancing antibodies predominated in the circulation, even two or more decades following infection. Mapping the epitopes and activity of naturally occurring dengue antibodies should prove valuable in determining whether the enhancing and neutralizing activity of antibodies can be separated. Such principles could be used in the rational design of vaccines that enhance the induction of neutralizing antibodies, while lowering the risk of dengue shock syndrome.     

Development of artificial vaccines against HIV using defined epitopes.

HIV may not follow the paradigm that has been used successfully for developing most viral vaccines, namely, that the best vaccine is the one that most closely mimics natural infection. This approach is based on the premise that natural infection leads to long-lasting protective immunity, which may not be applicable to HIV. Also, some immune responses elicited by infection with HIV may enhance infection or contribute to the development of immune deficiency. To overcome these problems, an artificial vaccine could be constructed using only antigenic epitopes that elicit neutralizing antibodies, helper T cells, and CD8+ cytotoxic T cells, and avoiding epitopes that elicit deleterious responses. Progress has been made in identifying all three of these types of epitopes, in characterizing their activity in animals, and in demonstrating that at least two of these can be linked to induce neutralizing antibodies without a carrier. Methods have also been developed to induce cytotoxic T cells. It is therefore feasible to construct an artificial vaccine for HIV that should be safer and more effective than a natural whole viral or subunit vaccine.     

Induction of unnatural immunity: prospects for a broadly protective universal influenza vaccine

The immune system normally responds to influenza virus by making neutralizing antibodies to regions of the viral spike, the hemagglutinin, that vary year to year. This natural response protects against circulating subtypes but necessitates production of new vaccines annually. Newer vaccine approaches have succeeded in eliciting broadly neutralizing antibodies to highly conserved yet vulnerable regions of the hemagglutinin and suggest potential pathways for the development of universal influenza vaccines.     

DNA shuffling and screening strategies for improving vaccine efficacy

The efficacy of vaccines can be improved by increasing their immunogenicity, broadening their crossprotective range, as well as by developing immunomodulators that can be coadministered with the vaccine antigen. One technology that can be applied to each of these aspects of vaccine development is MolecularBreeding directed molecular evolution. Essentially, this technology is used to evolve genes in vitro through an iterative process consisting of recombinant generation followed by selection of the desired recombinants. We have used DNA shuffling and screening strategies to develop and improve vaccine candidates against several infectious pathogens including Plasmodium falciparum (a common cause of severe and fatal human malaria), dengue virus, encephalitic alphaviruses such as Venezuelan, western and eastern equine encephalitis viruses (VEEV, WEEV, and EEEV, respectively), human immunodeficiency virus-1 (HIV-1), and hepatitis B virus (HBV). By recombining antigen-encoding genes from different serovar isolates, new chimeras are selected for crossreactivity; these vaccine candidates are expected to provide broader crossprotection than vaccines based on a single serovar. Furthermore, the vaccine candidates can be selected for improved immunogenicity, which would also improve their efficacy. In addition to vaccine candidates, we have applied the technology to evolve several immunomodulators that when coadministered with vaccines can improve vaccine efficacy by fine-tuning the T cell response. Thus, DNA shuffling and screening technology is a promising strategy to facilitate vaccine efficacy.     

蛋白质定向进化的研究进展及其应用前景

定向进化是改造蛋白质分子的有效新策略.它不需要了解蛋白质的空间结构,主要通过在实验室里模拟自然进化过程,采用错误倾向PCR等方法对编码蛋白质的基因进行随机突变,经DNA改组、交错延伸等技术进行体外重组,设计高通量筛选方法来选出需要的突变体.本综述了定向进化技术的发展及应用.     

Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemagglutinin,inducing specific immune responses and protection in cotton rats

Measles remains a leading cause of child mortality in developing countries. Residual maternal measles antibodies and immunologic immaturity dampen immunogenicity of the current vaccine in young infants. Because cotton rat respiratory tract is susceptible to measles virus (MV) replication after intranasal (i.n.) challenge, this model can be used to assess the efficacy of MV vaccines. Pursuing a new measles vaccine strategy that might be effective in young infants, we used attenuated Salmonella enterica serovar Typhi CVD 908-htrA and Shigella flexneri 2a CVD 1208 vaccines to deliver mucosally to cotton rats eukaryotic expression plasmid pGA3-mH and Sindbis virus-based DNA replicon pMSIN-H encoding MV hemagglutinin (H). The initial i.n. dose-response with bacterial vectors alone identified a well-tolerated dosage (1 x 10(9) to 7 x 10(9) CFU) and a volume (20 micro l) that elicited strong antivector immune responses. Animals immunized i.n. on days 0, 28, and 76 with bacterial vectors carrying DNA plasmids encoding MV H or immunized parenterally with these naked DNA vaccine plasmids developed MV plaque reduction neutralizing antibodies and proliferative responses against MV antigens. In a subsequent experiment of identical design, cotton rats were challenged with wild-type MV 1 month after the third dose of vaccine or placebo. MV titers were significantly reduced in lung tissue of animals immunized with MV DNA vaccines delivered either via bacterial live vectors or parenterally. Since attenuated serovar Typhi and S. flexneri can deliver measles DNA vaccines mucosally in cotton rats, inducing measles immune responses (including neutralizing antibodies) and protection, boosting strategies can now be evaluated in animals primed with MV DNA vaccines.     

Characterization of immune responses induced by intramuscular vaccination with DNA vaccines encoding measles virus hemagglutinin and/or fusion proteins

Measles virus (MV) hemagglutinin (MV-H) and fusion (MV-F) proteins induce plaque reduction neutralizing (PRN) antibodies and cell-mediated immune responses that protect against clinical measles. DNA vaccines that encode MV-H and MV-F are being investigated as a new generation of measles vaccine to protect infants too young to receive currently licensed attenuated measles vaccines. However, it is unclear whether DNA vaccines encoding both MV-H and MV-F act synergistically to induce stronger immunity than immunization with plasmids encoding MV-H or MV-F alone. To address this question, we generated Sindbis virus-based pSINCP DNA vaccines that encode either MV-H or MV-F alone or bicistronic or fusion system vectors that encode both MV-H and MV-F (to mimic MV infection where both MV-H and MV-F proteins are expressed by the same mammalian cell). Mice immunized with DNA vaccine encoding MV-H alone developed significantly greater PRN titers than mice immunized with bicistronic constructs. Interestingly, the presence of MV-F in the bicistronic constructs stimulated serum MV-specific immunoglobulin G of reduced avidity. By contrast, mice immunized with bicistronic constructs induced equivalent or higher levels of MV-specific gamma interferon responses than mice immunized with DNA vaccine encoding MV-H alone. These data will help guide the design of DNA-based MV vaccines to be used early in life in a heterologous prime-boost strategy.     

Development of an effective Japanese encephalitis virus-specific DNA vaccine

Intramuscular immunization with DNA vaccines has been shown to induce a broad range of immune responses and protective immunity in many animal models, but it is less effective in primates. One reason for this may be the low expression of vector-encoded antigen in cells. Here we report that the use of vaccine vector (pCJ-3) containing two regulatory elements, a chimeric intron and a bovine growth hormone (BGH) polyadenylation signal, markedly increased antigen expression both in vitro and in vivo. A positive correlation was seen between the level of expression of Japanese encephalitis virus (JEV) envelope proteins and the levels of antibodies in C3H/HeN mice. Immunization of mice with pCJ-3/ME (pCJ-3 containing the entire membrane and envelope protein genes) with or without cardiotoxin pretreatment resulted in higher antibody titers than immunization with vector containing only envelope protein and conferred full protection against infection with JEV. Electron microscopy showed that pCJ-3/ME expression resulted in the production of virus-like particles of JEV in vitro. The particles enhanced the production of higher titers of neutralizing antibodies and thus provided immunity against JEV. Consequently, the efficacy of the newly developed DNA vaccines was validated. This should pave the way to clinical trials in man.     

Evaluation of envelope vaccines derived from the South African subtype C human immunodeficiency virus type 1 TV1 strain

Human immunodeficiency virus type 1 (HIV-1) subtype C infections are on the rise in Sub-Saharan Africa and Asia. Therefore, there is a need to develop an HIV vaccine capable of eliciting broadly reactive immune responses against members of this subtype. We show here that modified HIV envelope (env) DNA vaccines derived from the South African subtype C TV1 strain are able to prime for humoral responses in rabbits and rhesus macaques. Priming rabbits with DNA plasmids encoding V2-deleted TV1 gp140 (gp140TV1DeltaV2), followed by boosting with oligomeric protein (o-gp140TV1DeltaV2) in MF59 adjuvant, elicited higher titers of env-binding and autologous neutralizing antibodies than priming with DNA vaccines encoding the full-length TV1 env (gp160) or the intact TV1 gp140. Immunization with V2-deleted subtype B SF162 env and V2-deleted TV1 env together using a multivalent vaccine approach induced high titers of oligomeric env-binding antibodies and autologous neutralizing antibodies against both the subtypes B and C vaccine strains, HIV-1 SF162 and TV1, respectively. Low-level neutralizing activity against the heterologous South African subtype C TV2 strain, as well as a small subset of viruses in a panel of 13 heterologous primary isolates, was observed in some rabbits immunized with the V2-deleted vaccines. Immunization of rhesus macaques with the V2-deleted TV1 DNA prime/protein boost also elicited high titers of env-binding antibodies and moderate titers of autologous TV1 neutralizing antibodies. The pilot-scale production of the various TV1 DNA vaccine constructs and env proteins described here should provide an initial platform upon which to improve the immunogenicity of these subtype C HIV envelope vaccines.     

Analysis of shuffled gene libraries

In vitro recombination of homologous genes (family shuffling) has been proposed as an effective search strategy for laboratory evolution of genes and proteins. Few data are available, however, on the composition of shuffled gene libraries, from which one could assess the efficiency of recombination and optimize protocols. Here, probe hybridization is used in a macroarray format to analyze chimeric DNA libraries created by DNA shuffling. Characterization of hundreds of shuffled genes encoding dioxygenases has elucidated important biases in the shuffling reaction. As expected, crossovers are favored in regions of high sequence identity. A sequence-based model of homologous recombination that captures this observed bias was formulated using the experimental results. The chimeric genes were found to show biases in the incorporation of sequences from certain parents, even before selection. Statistically different patterns of parental incorporation in genes expressing functional proteins can help to identify key sequence-function relationships.     

Antibodies engineered with IgD specificity efficiently deliver integrated T-cell epitopes for antigen presentation by B cells.

We have developed a strategy for improving the stimulation of T cells during immune responses by constructing recombinant antibodies that enhance the delivery of antigen to antigen-presenting cells, such as B cells. These antibodies have variable regions specific for surface molecules on B cells, and a constant region with an inserted antigen. In vitro, such antibodies make B cells approximately 1000-fold more efficient at presenting antigen and stimulating specific T cells. In vivo, the antibodies turn B cells of the spleen into potent stimulators of T cells. This approach may be useful for the generation of new vaccines.     

Broadening the Heterologous Cross-Neutralizing Antibody Inducing Ability of Porcine Reproductive and Respiratory Syndrome Virus by Breeding the GP4 or M genes

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens, which causes reproductive failure in sows and respiratory disease in piglets. A major hurdle to control PRRSV is the ineffectiveness of the current vaccines to confer protection against heterologous strains. Since both GP4 and M genes of PRRSV induce neutralizing antibodies, in this study we molecularly bred PRRSV through DNA shuffling of the GP4 and M genes, separately, from six genetically different strains of PRRSV in an attempt to identify chimeras with improved heterologous cross-neutralizing capability. The shuffled GP4 and M genes libraries were each cloned into the backbone of PRRSV strain VR2385 infectious clone pIR-VR2385-CA. Three GP4-shuffled chimeras and five M-shuffled chimeras, each representing sequences from all six parental strains, were selected and further characterized in vitro and in pigs. These eight chimeric viruses showed similar levels of replication with their backbone strain VR2385 both in vitro and in vivo, indicating that the DNA shuffling of GP4 and M genes did not significantly impair the replication ability of these chimeras. Cross-neutralization test revealed that the GP4-shuffled chimera GP4TS14 induced significantly higher cross-neutralizing antibodies against heterologous strains FL-12 and NADC20, and similarly that the M-shuffled chimera MTS57 also induced significantly higher levels of cross-neutralizing antibodies against heterologous strains MN184B and NADC20, when compared with their backbone parental strain VR2385 in infected pigs. The results suggest that DNA shuffling of the GP4 or M genes from different parental viruses can broaden the cross-neutralizing antibody-inducing ability of the chimeric viruses against heterologous PRRSV strains. The study has important implications for future development of a broadly protective vaccine against PRRSV.     

Biomolecular engineering and drug development

Biomolecular engineering is a technology to create novel structures of high-value biomolecules for use in medicine and industry, through the directed alteration of proteins and/or biologically active molecules in living cells to produce a novel biometabolites as well as engineered protein itself. For the development of new drugs by biomolecular engineering, desired biomolecules have to be rationally designed based on their structure-stability/structure-activity relationship, and then screened through well-established mutation and selection program. Over the past decade, there has been significant progress in mutation and selection methodology; DNA shuffling technology mimicking natural evolution for artificial DNA recombination and phage-displayed combinatorial peptide library for rapid selection of proteins expressed from mutated genes. Bioinformatic tools including functional genomics and proteomics have been also developed for the ready access to the information related to the protein-function and genome-protein, leading to the design and identification of new drug targets. Throughout the use of an enormous amount of bioinformatic databases, many protein/peptide drugs and biometabolite molecules have been designed. The candidates of new drugs are monoclonal antibodies, vaccines, enzymes, antibiotics, therapeutic peptides, and so on. Two humanized monoclonal antibodies approved by FDA became the first line of drugs designed by biomolecular engineering approach. They are Herceptin and Synagis, for the treatment of breast cancer and pediatric respiratory syncytial viral infection, respectively. Many more newly engineered biomolecules are under developing for medicinal application. Some clinical trials for therapeutic applications are now in progress, and very positive results are already anticipated.     

DNA shuffling技术及其在基因工程疫苗中的应用

DNA shuffling技术是一项全新的体外人工进化模式,它通过基因在分子水平上的重组,再定向筛选具有预期性状的突变体,获得同时具有多个亲本基因的特征的突变基因。该文介绍DNA shuffling技术的基本原理,并列举了由该技术发展而来的新技术及其在基因工程疫苗领域的应用,展望了DNAshuffling技术的发展方向。     

Revealing the potential of DNA-based vaccination: lessons learned from the hepatitis B virus surface antigen

DNA-based vaccination is a novel technique to efficiently stimulate humoral (antibody) and cellular (T cell) immune responses to protein antigens. In DNA-based vaccination, immunogenic proteins are expressed in in vivo transfected cells of the vaccine recipients in their native conformation with correct posttranslational modifications from antigen-encoding expression plasmid DNA. This ensures the integrity of antibody-defined epitopes and supports the generation of protective (neutralizing) antibody titers. Plasmid DNA vaccination is furthermore an exceptionally potent strategy to stimulate CD8+ cytotoxic T lymphocyte (CTL) responses because antigenic peptides are efficiently generated by endogenous processing of intracellular protein antigens. These key features make DNA-based immunization an attractive strategy for prophylactic and therapeutic vaccination against extra- and intracellular pathogens. In this brief review, we summarize the current state of expression vector design, DNA delivery strategies, priming immune responses to intracellular or secreted antigens by DNA vaccines and unique advantages of DNA- versus recombinant protein-based vaccines using the hepatitis B surface antigen (HBsAg) as a model antigen.     

Perspective on molecular immunology and modulation of immune responses

Despite enormous advances made in recent years, there remain fundamental and fascinating unanswered questions in immunology. They would include the mechanism of tolerance, the biological significance of the major histocompatibility complex (MHC) restriction, generation of diversity in T cells, control of V region and isotype selection in B cells, how immune response genes work and how important their function is in man, among others. With the advent of monoclonal antibodies and recombinant DNA technologies, together with greater understanding of immunological interactions within the network and of the intracellular networks between membrane-associated receptors and cell function, a variety of immunological strategies are available for modulating immune responses in man. Some nonspecific strategies include immunomodulatory lymphokines, receptor modulation, and development of agonists and antagonists for important receptors. In addition, there is great potential for immunologically specific strategies for intervention, including the use of idiotypes and anti-idiotypes and genetic engineering of antibodies. Ultimately, there is the possibility of predicting antigenic epitopes likely to engage the cellular and humoral arms of the immune response on the basis of protein or DNA sequences. The potential of exploring these developments for new diagnostic tests and vaccines is emphasized here, as is the unpredictable importance of seemingly nonrelevant fundamental science in producing many of the tools for intervention, now and in the future.     

炭疽芽胞杆菌疫苗研究进展

炭疽芽胞杆菌引起的炭疽病死亡率非常高 ,当前的疫苗具有效力不稳定、对吸入性炭疽的保护率低、免疫程序繁琐、存在副作用等缺点。近年来人们在改造传统疫苗的同时又有一些新的发现 ,如保护性抗原 (PA)的抗体在体内可杀死芽胞 ;通过粘膜免疫能够诱导机体分泌IgA抗体 ;抗多聚谷氨酸 (γ D PGA)抗体可以同炭疽杆菌的繁殖体作用 ,从而杀死繁殖体 ;寻找到新的免疫原。DNA疫苗、活载体疫苗的出现为新一代安全、免疫程序简单、具更高保护率的疫苗奠定了基础     

Enhancing effects of the chemical adjuvant levamisole on the DNA vaccine pVIR‐P12A‐IL18‐3C

DNA‐based vaccination is an attractive alternative for overcoming the disadvantages of inactivated virus vaccines; however, DNA vaccines alone often generate only weak immune responses. In this study, the efficacy of LMS as a chemical adjuvant on a DNA vaccine (pVIR‐P12A‐IL18‐3C) encoding the P1‐2A and 3C genes of the FMDV and swine IL‐18, which provides protection against FMDV challenge, was tested. All test pigs were administered booster vaccinations 28 days after the initial inoculation, and were challenged with 1000 ID₅₀ FMDV O/NY00 20 days after the booster vaccination. Positive and negative control groups were inoculated with inactivated virus vaccine and PBS respectively. The DNA vaccine plus LMS induced greater humoral and cell‐mediated responses than the DNA vaccine alone, as evidenced by higher concentrations of neutralizing and specific anti‐FMDV antibodies, and by higher concentrations of T‐lymphocyte proliferation and IFN‐γ production, respectively. FMDV challenge revealed that the DNA vaccine plus LMS provided higher protection than the DNA vaccine alone. This study demonstrates that LMS may be useful as an adjuvant for improving the protective efficiency of DNA vaccination against FMDV in pigs.