Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection.

Elevated expression of heat-shock proteins (HSPs) can benefit a microbial pathogen struggling to penetrate host defenses during infection, but at the same time might provide a crucial signal alerting the host immune system to its presence. To determine which of these effects predominate, we constructed a mutant strain of Mycobacterium tuberculosis that constitutively overexpresses Hsp70 proteins. Although the mutant was fully virulent in the initial stage of infection, it was significantly impaired in its ability to persist during the subsequent chronic phase. Induction of microbial genes encoding HSPs might provide a novel strategy to boost the immune response of individuals with latent tuberculosis infection.

Transmission blocking immunity in Plasmodium vivax malaria: antibodies raised against a peptide block parasite development in the mosquito vector.

One approach towards the development of a vaccine against malaria is to immunize against the parasite sexual stages that mediate transmission of the parasite from man to mosquito. Antibodies against these stages, ingested with the blood meal, inhibit the parasite development in the mosquito vector, constituting "transmission blocking immunity." Most epitopes involved in transmission-blocking immunity depend on the tertiary conformational structure of surface antigens. However, one of the transmission-blocking monoclonal antibodies we have raised against Plasmodium vivax reacts with a linear epitope on both asexual stages and gametes. This monoclonal antibody (A12) is capable of totally blocking development of the parasite in the mosquito host when tested in membrane feeding assays with gametocytes from P. vivax-infected patients. Immune screening of a P. vivax lambda gt11 genomic expression library with A12 led to the isolation of a clone to which was mapped the six-amino acid epitope recognized by A12. Antisera raised in mice against a 12-mer synthetic peptide containing this epitope coupled to bovine serum albumin not only had high titers of antipeptide antibodies as measured by enzyme-linked immunosorbent assay, but in addition recognized the same 24- and 57-kD parasite components as A12 on Western blots and reacted with the parasite by immunofluorescence. When tested in membrane feeding assays, these antibodies have significant suppressive effects on parasite development in the mosquito.

Characterisation of the dihydrofolate reductase-thymidylate synthetase gene from human malaria parasites highly resistant to pyrimethamine.

To investigate the genetic basis of drug resistance in human malaria parasites, we have sequenced the entire dihydrofolate reductase thymidylate synthetase DHFR-TS bifunctional gene from the highly pyrimethamine-resistant K1 isolate of Plasmodium falciparum. The protein is predicted to consist of 607 amino acids (aa), (71,685 Da), with an N-terminal methionine encoded by the second start codon of the open reading frame. Compared to the sequence from drug-sensitive parasites, there are two nucleotide changes in the coding region which bring about a substitution of Arg for Cys at aa position 59 and Asn for Thr at aa position 108. Both changes occur in regions of the DHFR domain involved in inhibitor and cofactor binding and are hence strongly implicated in drug resistance. The gene is present as a single copy in both K1 and drug-sensitive FCR3 isolates, and is assigned to chromosome 4. Codon usage follows the pattern observed in that of malarial surface antigen genes, with the exception fo codons corresponding to Val and Pro. The Asn and Lys contents of the predicted protein are exceptionally high, these residues being particularly concentrated in the DHFR and junction domains.

Cloning and characterisation of a gene from Plasmodium vivax and P. knowlesi: homology with valine-tRNA synthetase.

We have previously described a lambdagt11 clone detected by immune screening with a monoclonal antibody (mAb) A12. This mAb is capable of completely blocking Plasmodium vivax transmission in the mosquito vector. An epitope recognised by A12 was mapped to six amino acids (aa) within the translated sequence of this clone. Here, we describe the complete sequence of the gene within which we mapped this epitope. Surprisingly, the translated sequence of the full-length open reading frame shows homology with that of valine-tRNA synthetases (Val-tRS) from other organisms. DNA cross-hybridisation with several of these species was observed by Southern blot. In addition, the corresponding gene has been obtained from the closely related simian malaria parasite, P. knowlesi. The two aa sequences show 66% identity and yet are very divergent from other Val-tRS sequences, apart from conserved blocks related to functional activity. Multiple sequence alignments reflect this dichotomy, as do predicted differences in antigenicity.

Polymorphism of the alleles of the merozoite surface antigens MSA1 and MSA2 in Plasmodium falciparum wild isolates from Colombia.

The degree of polymorphism and the allelic distribution of 2 major Plasmodium falciparum merozoite surface antigens (MSA1 and MSA2) have been analysed in clinical isolates from Colombia. DNA was prepared directly from patients' blood and used in PCR reactions to amplify block 2 of MSA1 and the central region from MSA2. Thirty one samples were analysed and a marked degree of length polymorphism was detected, especially for MSA2. A high proportion of multiple bands was also observed, most probably resulting from mixed infections. Allele-specific oligonucleotides were used to type both alleles. For MSA1, 26 out of 31 clinical isolates were of the RO33 type, 15 were MAD20 and three were typed as KI. When the MSA2 allele was analysed, 7 isolates hybridised with a CAMP specific probe and 6 hybridised strongly with an FC27-derived oligonucleotide. Two samples, which showed multiple bands, hybridised with both probes. Interestingly, in 14 out of 27 isolates the MSA2 allele remained unassigned by the specific probes. Five of these were cloned and their DNA sequenced; these sequences are discussed.

A general approach to isolating Plasmodium falciparum genes using non-redundant oligonucleotides inferred from protein sequences of other organisms.

We have constructed a number of oligonucleotide probes and tested their utility in identifying various genes in Plasmodium falciparum. The probe sequences were based on known conserved regions of proteins from other organisms, coupled with an analysis of the codon usage of the parasite. By using long single oligonucleotides, we have successfully isolated the DHFR-TS gene, two actin genes and two tubulin genes from the K1 (Thailand) isolate of P. falciparum. We compare these single probes to multiply-redundant short oligonucleotide probes and to heterologous probes. We also present a detailed quantitative analysis of optimal probe design, and of how this approach can best be implemented as a general method of isolating plasmodial genes.

Plasmodium vivax:recombination between potential allelic types of the merozoite surface protein MSP1 in parasites isolated from patients.

The merozoite surface protein MSP1, which is one of the most promising candidates for a malaria vaccine directed against erythrocytic stages, has been shown to be polymorphic in different malarial species. Characterization of the Plasmodium vivax MSP1 gene (Pv200) in two strains (Belem and Salvador-1) revealed the existence of several polymorphic regions. One of these regions has been examined here in primary parasite isolates obtained from patients in Sri Lanka. Oligonucleotide primers hybridizing to conserved parts of the gene on either side of a polymorphic region were used to amplify DNA from 22 isolates. Sequence analysis of the amplified portion of the MSP1 gene in five patients showed the existence of three types of polymorphic regions. Two were almost identical either to that of the Belem or to that of the Salvador-1 strain. The third polymorphic type appeared to have resulted from recombination between the two others. This recombination event took place inside a repeated part of the sequence.

Evaluation of human antimycobacterial immunity using recombinant reporter mycobacteria.

A novel in vitro whole blood model was developed to study human antimycobacterial immunity. Recombinant reporter mycobacteria were used to enumerate the bacteria, and interactions between host immune cells and mycobacteria were studied using whole blood rather than cell fractions. The ability of healthy tuberculin-positive and tuberculin-negative individuals to restrict mycobacterial growth was compared. Growth of luminescent mycobacteria was significantly lower in blood samples of tuberculin-positive individuals than in blood samples of tuberculin-negative individuals (P=.005). Restricted mycobacterial growth was associated with significantly higher production of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma (P=.01 and.004, respectively). Inhibition of the TNF-alpha and IFN-gamma response pathways by neutralizing monoclonal antibodies increased mycobacterial growth in whole blood. This model is the first functional assay in which individual variations in cell-mediated immunity are shown to correlate with differences in ability to control mycobacterial growth. It provides a new tool for studying human mycobactericidal mechanisms and, potentially, for the evaluation of improved vaccines.

Assessment of immunity to mycobacterial infection with luciferase reporter constructs.

Protective immunity to mycobacterial infection is incompletely understood but probably involves the coordinated interaction of multiple cell types and cytokines. With the aim of developing assays that might provide a surrogate measure of protective immunity, we have investigated the use of recombinant mycobacteria carrying luciferase reporter enzymes to assess the effectiveness of antimycobacterial immunity in model systems. Measurement of luminescence was shown to provide a rapid and simple alternative to the counting of CFU as a means of monitoring mycobacterial viability. We describe optimization of a luciferase reporter strain of Mycobacterium tuberculosis and demonstrate its application for the study of mycobacterial interactions with host cells in tissue culture and the rapid assessment of vaccine efficacy in a murine model.

Contribution of Th1 and Th2 cells to protection and pathology in experimental models of granulomatous lung disease.

Mice that had received adoptive transfer of DO11.10 TCR transgenic T cells polarized toward a Th1 or a Th2 phenotype were challenged with Ag-coated beads or with recombinant Mycobacterium tuberculosis expressing the OVA determinant. The resulting bead-induced pulmonary granulomas reflected the phenotype of the adoptively transferred T cells, with the Th2 cells promoting a fibrotic reaction. Mice receiving Th1 cells mounted an epitope-specific protective response to challenge with recombinant M. tuberculosis. Th2 recipients were characterized by enhanced weight loss and lung fibrosis during acute high-dose infection. The combination of TCR transgenic T cells and epitope-tagged mycobacteria provides a novel experimental model for investigation of the pathogenesis of tuberculosis.