Diversity and selection in Babesia bovis and their impact on vaccine use

In the past few years the prospect of a recombinant vaccine effective against the cattle hoemoparosite Babesia bovis has almost become a reality. However, in Australia, vaccination with live parasites has been practised since before the turn of the century and it has recently been proposed that selection of parasites resistant to immunity induced by the B. bovis line Ka (since 1979 the only component of the live attenuated vaccine) may have occurred. Brian Dalrymple examines the evidence for and against this proposal and discusses examples of strain diversity and variation and their effect on the long-term viability of defined attenuated and recombinant vaccines.     

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.     

New tuberculosis vaccines based on attenuated strains of the Mycobacterium tuberculosis complex

The world urgently needs a better tuberculosis vaccine. Bacille Calmette-Guerin (BCG), an attenuated strain of Mycobacterium bovis, has been very widely used as a vaccine for many years but has had no major effect on reducing the incidence of tuberculosis. A number of alternative living and non-living vaccines are being investigated. Live vaccine candidates include genetically modified forms of BCG, genetically attenuated strains of the Mycobacterium tuberculosis complex and genetically engineered vaccinia virus and Salmonella strains. Non-living vaccine candidates include killed mycobacterial species, protein subunits and DNA vaccines. One requirement for acceptance of any new vaccine will be a favourable comparison of the protection it induces relative to BCG in a range of animal models, some of which may need further development. Molecular genetic techniques are now available that enable production of live attenuated strains of the M. tuberculosis complex with vaccine potential. In the first of two broadly different approaches that are being used, large numbers of mutants are produced by transposon mutagenesis or illegitimate recombination and are screened for properties that correlate with attenuation. In the second approach, putative genes that may be required for virulence are identified and subsequently inactivated by allelic exchange. In both approaches, mutants that are attenuated need to be identified and subsequently tested for their vaccine efficacy in animal models. Many mutants of the M. tuberculosis complex have now been produced and the vaccine properties of a substantial number will be assessed in the next 3 years.     

Genome composition analysis of the reassortant influenza viruses used in seasonal and pandemic live attenuated influenza vaccine

The cold-adapted, temperature sensitive and attenuated influenza master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/ 60/69 were used to generate the vaccine viruses to be included in live attenuated influenza vaccine. These vaccine viruses typically are 6:2 reassortant viruses containing the surface antigens hemagglutinin and neuraminidase of current wild type influenza A and influenza B viruses with the gene segments encoding the internal viral proteins, and conferring the cold-adapted, temperature sensitive and attenuated phenotype, being inherited from the master donor viruses. The 6:2 reassortant viruses were selected from co-infections between master donor virus and wild type viruses that theoretically may yield as many as 256 combinations of gene segments and thus 256 genetically different viruses. As the time to generate and isolate vaccine viruses is limited and because only 6:2 reassortant viruses are allowed as vaccine viruses, screening needs to be both rapid and unambiguous. The screening of the reassortant viruses by RT-PCRs using master donor virus and wild type virus specific primer sets was described to select both influenza A and influenza B 6:2 reassortant viruses to be used in seasonal and pandemic live attenuated vaccine.     

Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis.

The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.     

Babesia bovis: molecular and biological characteristics of cloned parasite lines

An in vivo limiting dilution technique was used to produce several Babesia bovis cloned lines with which to study the basis of virulence and immunogenicity in this parasite. DNA hybridization using a cloned DNA fragment from the BabR locus demonstrated that the cloned lines were a more restricted genetic population than the parent strain. Biosynthetic labeling and immunoprecipitation studies indicated that the cloned lines differed from each other and from the parentals in the expression of a small number of polypeptides and antigens. Animal trials with three of the lines demonstrated that the parental line contains both virulent and avirulent parasite populations, at least three of which are not tick transmissible, and that while the lines do provide significant protection against heterologous challenge, they may not give as effective protection as the parental line. These experiments demonstrated the existence of subpopulations with distinctive molecular and biological properties, providing evidence that the attenuation process is based on the selection of preexisting parasite subpopulations combined with the ability of these parasites to vary genetically.     

Babesia bovis: analysis of and preliminary vaccination studies with a defined infected erythrocyte membrane binding antigen.

Murine monoclonal antibodies (MAB) were produced against the 'beta' fraction of Babesia bovis. A MAB, W11C5, selected on the criterion of its staining of erythrocytes infected with B. bovis, was purified. The antigen identified by MAB W11C5 was extracted from B. bovis infected erythrocytes by affinity chromatography and used in a vaccination trial to test its vaccine efficacy against homologous B. bovis infection in splenectomized calves. The vaccinated group showed significantly different parasitaemias from the control group and it was concluded that the B. bovis antigen 11C5 induced a protective immune response when used as a vaccine. This antigen should be synthesized using recombinant DNA techniques to determine its efficacy and suitability as a commercial vaccine against B. bovis infection.     

The application of gene expression microarray technology to kinetoplastid research

Protozoan parasites in the order Kinetoplastida cause severe disease primarily in tropical and subtropical areas. Vaccines to control these diseases have shown some promise, but none are in active clinical use. Drug treatments are available for all of the acute infections, but the emergence of resistance and an unresponsive chronic phase are current problems. Rapid advances in genomic technology open the possibility of discovering new genes that can contribute to vaccine initiatives or serve as targets for development of new drugs. The DNA microarray is a genomic technology, which is being applied to new gene discovery in kinetoplastid parasites. Both cDNA and genomic microarrays for Leishmania major have identified a number of new genes that are expressed in a stage-specific fashion and preliminary results from a L. donovani genomic microarray also demonstrated new gene discovery. A microarray of Trypanosoma brucei genomic fragments identified new genes whose expression differs between the insect borne stage and the human infectious stage of the parasite. The next few years, building on this foundational work, should witness the most exciting stage as microarrays are applied to questions such as the basis of drug resistance, post kala azar dermal leishmaniasis, the regulation of differentiation to infectious stages, linking coordinately regulated pathways of genes and development of genetically defined parasites that may have potential as live attenuated vaccines.     

The Effect of Vaccination on the Evolution and Population Dynamics of Avian Paramyxovirus-1

Newcastle Disease Virus (NDV) is a pathogenic strain of avian paramyxovirus (aPMV-1) that is among the most serious of disease threats to the poultry industry worldwide. Viral diversity is high in aPMV-1; eight genotypes are recognized based on phylogenetic reconstruction of gene sequences. Modified live vaccines have been developed to decrease the economic losses caused by this virus. Vaccines derived from avirulent genotype II strains were developed in the 1950s and are in use globally, whereas Australian strains belonging to genotype I were developed as vaccines in the 1970s and are used mainly in Asia. In this study, we evaluated the consequences of attenuated live virus vaccination on the evolution of aPMV-1 genotypes. There was phylogenetic incongruence among trees based on individual genes and complete coding region of 54 full length aPMV-1 genomes, suggesting that recombinant sequences were present in the data set. Subsequently, five recombinant genomes were identified, four of which contained sequences from either genotype I or II. The population history of vaccine-related genotype II strains was distinct from other aPMV-1 genotypes; genotype II emerged in the late 19^th century and is evolving more slowly than other genotypes, which emerged in the 1960s. Despite vaccination efforts, genotype II viruses have experienced constant population growth to the present. In contrast, other contemporary genotypes showed population declines in the late 1990s. Additionally, genotype I and II viruses, which are circulating in the presence of homotypic vaccine pressure, have unique selection profiles compared to nonvaccine-related strains. Collectively, these data show that vaccination with live attenuated viruses has changed the evolution of aPMV-1 by maintaining a large effective population size of a vaccine-related genotype, allowing for coinfection and recombination of vaccine and wild type strains, and by applying unique selective pressures on viral glycoproteins.     

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.     

Designing blood-stage vaccines against Babesia bovis and B. bigemina.

The tick-transmitted apicomplexan parasites Babesia bovis and B. bigemina cause significant disease in cattle in many tropical and temperate areas of the world. These parasites present a challenge for vaccine development, and yet provide a system for studying the pathogenesis, mechanisms of protective immunity and regulation of host immune responses associated with intraerythrocytic protozoan parasites in a non-rodent species. In this article, Wendy Brown and Guy Palmer review strategies for identifying candidate vaccine antigens of B. bovis and B. bigemina and for priming immune responses to evoke strain crossprotective immunity.     

Identification of protein biomarkers of attenuation and immunogenicity of centrin or p27 gene deleted live vaccine candidates of Leishmania against visceral leishmaniasis

Currently, no licensed vaccine is available for human visceral leishmaniasis (VL), a fatal disease caused by the protozoan parasite Leishmania donovani. Two of our live attenuated L. donovani vaccine candidates, either deleted for Centrin1 (LdCen1^−/−) or p27 gene (Ldp27^−/−), that display reduced growth in macrophages were studied to be safe, immunogenic and protective against VL in various animal models. This report involves the identification of differentially expressed proteins, their related pathways and its underlying mechanism in the intracellular stage of these parasites, using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) methods. Out of 50–60 proteins, found to be differentially expressed in these mutant parasites, 36 were found to be common in both the parasites. Such proteins mainly belong to the functional categories viz. metabolic enzymes, chaperones and stress proteins, proteins involved in translation, processing and transport and proteins involved in nucleic acid processing. Proteins known to be host protective, like Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cytochrome c, calreticulin and those responsible for inducing immune response, namely tubulins, DEAD box RNA helicases, HSP70 and tryparedoxin, have been detected to be modulated in these parasites. Such proteins could be predicted as biomarkers, with further scope of study for their role in growth attenuation.SignificanceThis study aims at predicting proteomic biomarkers of Leishmania parasite growth attenuation, that have immunomodulatory role in the disease leishmaniasis. Advanced studies could be helpful in establishing the role of these identified proteins in parasitic virulence and to predict the host interaction at molecular level. Also, these proteins could be exploited as attenuation markers during the development of genetically modified live attenuated parasites as vaccine candidates. These could be cross validated in varied species of Leishmania and other tyrpanosomatids for similar response towards identifying them as universal biomarkers of attenuation.     

Babesia bovis: evidence for selection of subpopulations during attenuation

DNA probes were used to detect variation in subpopulations of virulent and serially passaged Babesia bovis. Two distinct patterns were evident after hybridization to genomic DNA; the first was a basic profile typical of virulent B. bovis and the second, a more variable array, was characteristic of B. bovis after various stages of attenuation. Tick transmission of avirulent B. bovis causes reversion to the virulent genomic pattern, suggesting that selective enrichment of a small residual subpopulation caused reversion to a virulent profile of subpopulations. Certain genomic fragments, predominant in either virulent or avirulent parasite forms, are putative "markers" or actual elements responsible for these biological characteristics.     

Differential Response of the Cynomolgus Macaque Gut Microbiota to Shigella Infection

Little is known about the role of gut microbiota in response to live oral vaccines against enteric pathogens. We examined the effect of immunization with an oral live-attenuated Shigella dysenteriae 1 vaccine and challenge with wild-type S. dysenteriae 1 on the fecal microbiota of cynomolgus macaques using 16 S rRNA analysis of fecal samples. Multi-dimensional cluster analysis identified different bacterial community types within macaques from geographically distinct locations. The fecal microbiota of Mauritian macaques, observed to be genetically distinct, harbored a high-diversity community and responded differently to Shigella immunization, as well as challenge compared to the microbiota in non-Mauritian macaques. While both macaque populations exhibited anti-Shigella antibody responses, clinical shigellosis was observed only among non-Mauritian macaques. These studies highlight the importance of further investigation into the possible protective role of the microbiota against enteric pathogens and consideration of host genetic backgrounds in conducting vaccine studies.     

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.     

鸡球虫入侵机理、免疫学性质及基因工程在疫苗研制上的应用

鸡球虫病是严重危害养禽业的一种寄生虫病。长期以来主要以化学药物进行防治。但由于球虫抗药性和药物残留等问题日益严重,使得新药开发和应用受到限制。在免疫学中强毒活苗应用广泛但生产费用昂贵,从而促使我们转向对重组亚单位疫苗的研究。不同种类的球虫有较为严格的寄生部位。且不同发育阶段的球虫其免疫原性和抗原构成也有很大差异。在真核寄生生物中,由于受到宿主免疫系统的限制,选用鸡球虫免疫保护性抗原做重组疫苗目前还没有很大突破,因此我们需要采用新方法发现新抗原,有效防治鸡球虫病。     

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.     

Natural communities of Achromatium oxaliferum comprise genetically, morphologically, and ecologically distinct subpopulations.

The diversity and ecology of natural communities of the uncultivated bacterium Achromatium oxaliferum were studied by use of culture-independent approaches. 16S rRNA gene sequences were PCR amplified from DNA extracted from highly purified preparations of cells that were morphologically identified as A. oxaliferum present in freshwater sediments from three locations in northern England (Rydal Water, Jenny Dam, Hell Kettles). Cloning and sequence analysis of the PCR-amplified 16S rRNA genes revealed that multiple related but divergent sequences were routinely obtained from the A. oxaliferum communities present in all the sediments examined. Whole-cell in situ hybridization with combinations of fluorescence-labelled oligonucleotide probes revealed that the divergent sequences recovered from purified A. oxaliferum cells corresponded to genetically distinct Achromatium subpopulations. Analysis of the cell size distribution of the genetically distinct subpopulations demonstrated that each was also morphologically distinct. Furthermore, there was a high degree of endemism in the Achromatium sequences recovered from different sediments; identical sequences were never recovered from different sampling locations. In addition to ecological differences that were apparent between Achromatium communities from different freshwater sediments, the distribution of different subpopulations of Achromatium in relation to sediment redox profiles indicated that the genetically and morphologically distinct organisms that coexisted in a single sediment were also ecologically distinct and were adapted to different redox conditions. This result suggests that Achromatium populations have undergone adaptive radiation and that the divergent Achromatium species occupy different niches in the sediments which they inhabit.