Analysis of the composition of samples of Babesia bovis and the influence of different environmental conditions on genetically distinct subpopulations.

A recombinant DNA probe specific for a tandemly repeated sequence located within the BoVA1 gene of Babesia bovis was used to analyse 10 independent samples of B. bovis. Twelve different alleles of the BoVA1 gene and flanking regions were identified in the 18 different subpopulations analysed. Most samples of B. bovis originally derived from single animals contained more than one genetically distinct subpopulation. However, only one population of parasites was identified in samples of the Ka line used in Australia from 1979 until 1990 as the live attenuated vaccine strain. In contrast, the replacement attenuated vaccine line, Ta, contained two genetically distinct subpopulations of parasites. Changes in the ratios of subpopulations of parasites were identified during attenuation and under different culture conditions. Batch-to-batch variation in the composition of doses of the live attenuated vaccine may lead to differences in efficacy and in severity of the infection associated with vaccination.     

Constitutively dead, conditionally live HIV-1 genomes. Ex vivo implications for a live virus vaccine

An effective vaccine against AIDS is unlikely to be available for many years. As we approach two decades since the first identification of human immunodeficiency virus, type 1 (HIV-1), currently, only one subunit vaccine candidate has reached phase 3 of clinical trials. The subunit approach has been criticized for its inability to elicit effectively cytotoxic T-lymphocyte (CTL) response, which is felt by many to be needed for protection against HIV-1 infection. In subhuman primates, a live attenuated simian immunodeficiency virus (SIV) vaccine candidate, capable of inducing CTL, has been found to confer prophylactic immunity sufficient to prevent simian AIDS. Because replication competent (live) attenuated viruses could over time revert to virulence, such a live attenuated approach has largely been dismissed for HIV-1. Here, we describe the creation of constitutively dead conditionally live (CDCL) HIV-1 genomes. These genomes are constitutively defective for the Tat/TAR axis and are conditionally dependent on tetracycline for attenuated replication with robust expression of viral antigens. Our results suggest that CDCL genomes merit consideration as safer "live" attenuated HIV-1 vaccine candidates.     

Molecular cloning and sequencing of the merozoite surface antigen 2 gene from Plasmodium falciparum strain FCC-1/HN and expression of the gene in mycobacteria

Strain bacillus Calmette-Guerin (BCG) of Mycobacterium bovis has been used as a live bacterial vaccine to immunize more than 3 billion people against tuberculosis. In an attempt to use this vaccine strain as a vehicle for protective antigens, the gene encoding merozoite surface antigen 2 (MSA2) was amplified from strain FCC-1/HN Plasmodium falciparum genome, sequenced, and expressed in M. bovis BCG under the control of an expression cassette carrying the promoter of heat shock protein 70 (HSP70) from Mycobacterium tuberculosis. The recombinant shuttle plasmid pBCG/MSA2 was introduced into mycobacteria by electroporation, and the recombinant mycobacteria harboring pBCG/MSA2 could be induced by heating to express MSA2; the molecular mass of recombinant MSA2 was about 31 kDa. This first report of expression of the full-length P. falciparum MSA2 gene in BCG provides evidence for use of the HSP70 promoter in expressing a foreign gene in BCG and in development of BCG as a multivalent vectoral vaccine for malaria.     

Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG

Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18 kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.     

Development of vaccines to control bovine tuberculosis in cattle and relationship to vaccine development for other intracellular pathogens

Vaccination of cattle against bovine tuberculosis could be an important strategy for the control of disease either where there is a wildlife reservoir of Mycobacterium bovis infection or in developing countries where it is not economically feasible to implement a 'test and slaughter' control program. Advances in the understanding of protective immune responses to M. bovis infection in cattle and the advent of new molecular biological techniques, coupled with the sequencing of the M. bovis genome have provided opportunities for the rational development of improved tuberculosis vaccines. A number of new tuberculosis vaccines including attenuated M. bovis strains, killed mycobacteria, protein and DNA vaccines are under development and many are being assessed in cattle. Recent results have revealed several promising vaccine candidates and vaccination strategies. Ways of distinguishing between vaccinated and infected cattle are becoming available and the possibility of new approaches to the eradication of tuberculosis from domestic livestock is discussed. Similarities between the mechanisms of protective immunity against M. bovis and against other intracellular parasites continue to be found and discoveries from vaccine studies on bovine tuberculosis may provide helpful insights into requirements for vaccines against other intracellular pathogens.     

Further observations on immunization of sheep against Schistosoma mansoni and S. bovis using irradiation-attenuated schistosomula of homologous and heterologous species.

This paper describes further characteristics of the immunization of sheep against schistosomes using live, irradiation schistosomula. Sheep immunized with a non-virulent strain of Schistosoma mattheei were protected against a more virulent strain of the same species for over a year. As there was no evidence that the irradiated parasites were able to persist this long, it was concluded that the vaccine had induced a sterile resistance. Heterologous vaccination, using irradiated S. mattheei schistosomula to immunize against S. bovis or irradiated S. mansoni schistosomula to immunize against S. mattheei, failed to induce any protection.     

减毒鼠伤寒沙门氏菌全长hpaA基因工程菌的构建

为构建表达HpaA蛋白的重组减毒鼠伤寒沙门氏菌 ,并探讨以减毒鼠伤寒沙门氏菌为载体构建H .pylori疫苗株的意义 ,应用PCR法从H .pylori基因组DNA中扩增 783bp的hpaA基因 ,经酶切 连接反应将其克隆入原核表达质粒pTrc99A的NcoⅠ SalⅠ位点 ,并进行了核苷酸序列测定。重组质粒转化减毒鼠伤寒沙门氏菌SL3 2 6 1 ,提取重组菌质粒 ,PCR和酶切鉴定 ,筛选阳性克隆。用SDS PAGE电泳和Westernblot进行HpaA表达分析和鉴定 ,用薄层扫描分析HpaA含量。重组菌C5 7BL 6小鼠喂灌 ,分批两d和 1 0d后处死小鼠 ,取脾和末段回肠进行细菌培养 ,挑菌落提质粒鉴定。结果表明 ,经PCR和酶切证实 ,构建了含 783bphpaA基因的重组原核表达质粒 ,并将后者成功转化了减毒鼠伤寒沙门氏菌。重组菌能表达约3 0kDHpaA蛋白 ,重组HpaA量约占全菌体蛋白量的 3 8 9% ,Westernblot证实其有免疫反应性。小鼠重组菌喂灌两d或 1 0d后 ,脾和末段回肠均发现携目的基因的菌落。这些结果提示 ,构建了表达H .pyloriHpaA的重组减毒…     

Search for Babesia bovis vaccine candidates

Babesia bovis is a tick-borne apicomplexan pathogen that remains an important constrain for the development of cattle industries worldwide. Effective control can be achieved by vaccination with live attenuated forms of the parasite, but they have several drawbacks and thus the development of alternative subunit vaccines, either based in recombinant versions of full size proteins or in recombinant or synthetic peptides containing combinations of protective B-cell and T-cell epitopes is needed. Our current strategies for the identification of vaccine candidate antigens include the identification of functionally relevant antigens, bioinformatics, and comparative genomics using the recently sequenced B. bovis genome. These led us to the functional and immunological characterization of members of the VMSA gene family, a group of well conserved putative cysteine and serine proteases, and to the definition of a surface exposed B-cell epitope present in the Merozoite Surface Antigen-2c. Work in progress is focused in defining additional epitopes, and to determine whether they are neutralization-sensitive. These approaches might unravel useful vaccine candidates for B. bovis, and will increase our understanding of the pathogenicity mechanisms of these and related hemoparasites.     

Use of helper T cells to identify potential vaccine antigens of Babesia bovis

Babesia bovis is an economically important hemoprotozoon parasite o f cattle that is prevalent in many, tropical and subtropical regions o f the world. Effective vaccines against this tick-transmitted parasite consist o f live organisms attenuated by passage through splenectomized calves. However, the nature o f acquired resistance to challenge infection with heterologous isolates of this parasite has not been clearly defined. Unsuccessful attempts to select protective antigens have relied upon the use of antibodies to identify immunodominant proteins. In this review, Wendy Brown and Allison Rice-Ficht discuss the limitations of this approach and the rationale behind using helper T cells to select potential vaccine antigens.     

Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG

A recombinant (r-) Salmonella typhimurium aroA vaccine that secretes the naturally secreted protein of Mycobacterium bovis strain BCG, Ag85B, by means of the HlyB/HlyD/TolC export machinery (termed p30 in the following) was constructed. In contrast to r-S. typhimurium control, oral vaccination of mice with the r-S. typhimurium p30 construct induced partial protection against an intravenous challenge with the intracellular pathogen Mycobacterium tuberculosis, resulting in similar vaccine efficacy comparable to that of the systemically administered attenuated M. bovis BCG strain. The immune response induced by r-S. typhimurium p30 was accompanied by augmented interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) levels produced by restimulated splenocytes. These data suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated r-S. typhimurium as carrier is capable of inducing an immune response against mycobacterial antigens.     

The species specificity of immunity generated by live whole organism immunisation with erythrocytic and pre-erythrocytic stages of rodent malaria parasites and implications for vaccine development

A promising strategy for the development of a malaria vaccine involves the use of attenuated whole parasites, as these present a greater repertoire of antigens to the immune system than subunit vaccines. The complexity of the malaria parasite's life cycle offers multiple stages on which to base an attenuated whole organism vaccine. An important consideration in the design and employment of such vaccines is the diversity of the parasites that are infective to humans. The most valuable vaccine would be one that was effective against multiple species/strains of malaria parasite. Here we compare the species specificity of pre-erythrocytic and erythrocytic whole organism vaccination using live parasites with anti-malarial drug attenuation. The cross-stage protection afforded by each vaccination strategy, and the possibility that immunity against one stage may be abrogated by exposure to other stages of both homologous and heterologous parasites was also assessed. The rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium vinckei lentum are to address these questions, as they offer the widest possible genetic distance between sub-species of malaria parasites infectious to rodents. It was found that both erythrocytic and pre-erythrocytic stage immunity generated by live, attenuated parasite vaccination have species-specific components, with pre-erythrocytic stage immunity offering a much broader pan-species protection. We show that the protection achieved following sporozoite inoculation with concurrent mefloquine treatment is almost entirely dependent of CD8(+) T-cells. Evidence is presented for cross-stage protection between erythrocytic and pre-erythrocytic stage vaccination. Finally, it is shown that, with these species, an erythrocytic stage infection of either a homologous or heterologous species following immunisation with pre-erythrocytic stages does not abrogate this immunity. This is the first direct comparison of the specificity and efficacy of erythrocytic and pre-erythrocytic stage whole organism vaccination strategies utilising the same parasite species pair.     

Improving vaccines against tuberculosis

Tuberculosis remains a major cause of mortality and physical and economic deprivation worldwide. There have been significant recent advances in our understanding of the Mycobacterium tuberculosis genome, mycobacterial genetics and the host determinants of protective immunity. Nevertheless, the challenge is to harness this information to develop a more effective vaccine than BCG, the attenuated strain of Mycobacterium bovis derived by Calmette and Guérin nearly 90 years ago. Some of the limitations of BCG include the waning of the protective immunity with time, reduced effectiveness against pulmonary tuberculosis compared to disseminated disease, and the problems of a live vaccine in immuno-compromised subjects. Two broad approaches to vaccine development are being pursued. New live vaccines include either attenuated strains of Mycobacterium tuberculosis produced by random mutagenesis or targeted deletion of putative virulence factors, or by genetic manipulation of BCG to express new antigens or cytokines. The second approach utilizes non-viable subunit vaccines to deliver immunodominant mycobacterial antigens. Both protein and DNA vaccines induce partial protection against experimental tuberculosis infection in mice, however, their efficacy has generally been equivalent to or less than that of BCG. The comparative effects of cytokine adjuvants and vaccines targeting antigen presenting cells on enhancing protection will be discussed. Coimmunization with plasmid interleukin-12 and a DNA vaccine expressing Antigen 85B, a major secreted protein, was as protective as BCG. The combination of priming with DNA-85B and boosting with BCG was superior to BCG alone. Therefore it is possible to achieve a greater level of protection against tuberculosis than with BCG, and this highlights the potential for new tuberculosis vaccines in humans.     

Francisella tularensis vaccines

Francisella tularensis is the causative agent of tularaemia, a disease which occurs naturally in some countries in the northern hemisphere. Recently, there has been a high level of interest in devising vaccines against the bacterium because of the potential for it to be used as a bioterrorism agent. Previous human volunteer studies have shown that a strain of F. tularensis [the live vaccine strain (LVS)] that has been attenuated by laboratory passage is effective in humans as a vaccine against airborne disease. However, for a variety of reasons it seems unlikely that the LVS strain will be licensed for use in humans. Against this background there is an effort to devise a licensable vaccine against tularaemia. The prospects for a killed whole-cell subunit of live attenuated vaccine are reviewed. A rationally attenuated mutant seems the most likely route to a new tularaemia vaccine.     

Protective immunity against malaria liver stage after vaccination with live parasites

Despite nearly 100 years of research and control efforts, malaria remains one of the most important infectious diseases. An efficient vaccine would be a powerful to tool to reduce mortality and morbidity. Experimentally, induction of sterile immunity in humans after vaccination with attenuated sporozoites has been obtained. This observation has spurred the search for subunit vaccines that aim to reproduce this protection. As yet none of the current candidate subunit vaccines achieved complete protection reproducibly. This failure coupled to the recent advent of genetically modified Plasmodium parasites has led to a renewed interest in the use of live parasites for vaccination against malaria pre-erythrocytic stages. In this article, we review and discuss the recent developments in this field.     

Recombinant mycobacterial proteins future directions to improve protective efficacy

A large number of subunit vaccine candidates have recently been developed as alternatives to Mycobacterium bovis Bacillus Calmette-Guerin (BCG), which gives unpredictable and highly variable protection against tuberculosis. Immunological potential of various recombinant proteins against mycobacterial infections has been discussed. Further, strategies have been suggested, which include development of constructs coexpressing cytokines or regimens utilizing recombinant proteins for further improving the protective efficacy.     

Sublingual immunization with Japanese encephalitis virus vaccine effectively induces immunity through both cellular and humoral immune responses in mice

The Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis. Although there are four classes of vaccines against JEV, all of them are administered by s.c or i.m injection. Here, the effectiveness of sublingual (s.l.) administration of a JEV live‐attenuated vaccine or recombinant modified vaccinia virus Ankara (MVA) vaccine, including JEV prM/E, was investigated. The mice were immunized three times i.m. or s.c. One week after the final immunization by both s.l. and i.m. routes, the titers of IgG1 induced by the recombinant MVA vaccine were higher than those induced by the live‐attenuated vaccine, whereas the titers of IgG2a induced by the live‐attenuated vaccine were higher than those induced by the recombinant MVA vaccine. However, both vaccines induced neutralizing antibodies when given by either s.l. or i.m. routes, indicating that both vaccines induce appropriate Th1 and Th2 cell responses through the s.l. and i.m. routes. Moreover, both vaccines protected against induction of proinflammatory cytokines and focal spleen white pulp hyperplasia after viral challenge. Virus‐specific IFN‐γ⁺ CD4⁺ and CD8⁺ T cells appeared to increase in mice immunized via both s.l. and i.m. routes. Interestingly, virus‐specific IL‐17⁺ CD4⁺ T cells increased significantly only in the mice immunized via the s.l. route; however, the increased IL‐17 did not affect pathogenicity after viral challenge. These results suggest that s.l. immunization may be as useful as i.m. injection for induction of protective immune responses against JEV by both live‐attenuated and recombinant MVA vaccines.     

Recombinant viruses as tools to induce protective cellular immunity against infectious diseases.

Infections by intracellular pathogens such as viruses, some bacteria and many parasites, are cleared in most cases after activation of specific T cellular immune responses that recognize foreign antigens and eliminate infected cells. Vaccines against those infectious organisms have been traditionally developed by administration of whole live attenuated or inactivated microorganisms. Nowadays, research is focused on the development of subunit vaccines, containing the most immunogenic antigens from the particular pathogen. However, when purified subunit vaccines are administered using traditional immunization protocols, the levels of cellular immunity induced are mostly low and not capable of eliciting complete protection against diseases caused by intracellular microbes. In this review, we present a promising alternative to those traditional protocols, which is the use of recombinant viruses encoding subunit vaccines as immunization tools. Recombinant viruses have several interesting features that make them extremely efficient at inducing immune responses mediated by T-lymphocytes. This cellular immunity has recently been demonstrated to be of key importance for protection against malaria and AIDS, both of which are major targets of the World Health Organization for vaccine development. Thus, this review will focus in particular on the development of new vaccination protocols against these diseases.     

表达乙型脑炎病毒E蛋白重组减毒沙门菌活载体疫苗株的构建

目的:构建表达乙型脑炎病毒(JEV)E蛋白的口服重组减毒鼠伤寒沙门菌活载体疫苗株。方法:克隆JEV E基因,将其插入表达载体pYA3341中,构建重组质粒pYA3341-E,将重组质粒电转入鼠伤寒沙门菌疫苗株X4550(缺失asd、cya、crp基因),获得重组疫苗菌株X4550(pYA3341-E);鉴定重组菌E蛋白的表达,测定重组菌的稳定性、生长曲线、安全性,以及小鼠的免疫试验和血清中和试验。结果:酶切鉴定和序列测定证实重组质粒构建成功;SDS-PAGE检测有目的蛋白条带;Western印迹证实表达的E蛋白能与猪抗JEV阳性血清特异性结合;重组菌株在体外营养选择压力下,可稳定地携带重组质粒传代繁殖,在体内可较稳定地定居于肠系膜淋巴结和脾脏;小鼠口服试验证实重组菌无毒性作用,安全可靠;小鼠口服重组菌免疫,ELISA检测产生了抗JEV抗体;中和试验表明产生的抗体具有中和活性。结论:构建了能稳定表达JEV E蛋白的口服减毒鼠伤寒沙门菌疫苗株X4550(pYA3341-E),为研究乙型脑炎口服基因工程疫苗奠定基础。     

Attenuated Bordetella pertussis BPZE1 as a live vehicle for heterologous vaccine antigens delivery through the nasal route

Whereas the great majority of the current vaccines are delivered through the parenteral route, mucosal administration has been increasingly considered for controlling infection and preventing disease. Mucosal vaccination can trigger both humoral and cell-mediated protection, not only at the targeted mucosal surface, but also systemically. In this regard, nasal vaccination has shown great potential. The live attenuated strain of Bordetella pertussis, BPZE1, is particularly attractive and promising as a nasal vaccine delivery vector of heterologous antigen vaccine candidates. BPZE1 was originally developed as a live nasal pertussis vaccine candidate, and is currently undergoing phase I clinical trial in human (http://www.child-innovac.org). Highly adapted to the human respiratory tract and offering several potential protein carriers for presentation of the heterologous antigen vaccine candidates, BPZE1 represents an appealing platform for the development of live recombinant vaccines delivered via the nasal route that would confer simultaneous protection against pertussis and the targeted infectious disease(s).     

Genetic diversity of Babesia bovis in virulent and attenuated strains

The aim of this study was to compare the genetic diversity of the single copy Bv80 gene sequences of Babesia bovis in populations of attenuated and virulent parasites. PCR/ RT-PCR followed by cloning and sequence analyses of 4 attenuated and 4 virulent strains were performed. Multiple fragments in the range of 420 to 744 bp were amplified by PCR or RT-PCR. Cloning of the PCR fragments and sequence analyses revealed the presence of mixed subpopulations in either virulent or attenuated parasites with a total of 19 variants with 12 different sequences that differed in number and type of tandem repeats. High levels of intra- and inter-strain diversity of the Bv80 gene, with the presence of mixed populations of parasites were found in both the virulent field isolates and the attenuated vaccine strains. In addition, during the attenuation process, sequence analyses showed changes in the pattern of the parasite subpopulations. Despite high polymorphism found by sequence analyses, the patterns observed and the number of repeats, order, or motifs found could not discriminate between virulent field isolates and attenuated vaccine strains of the parasite.