Bacterial and Parasitic Diseases Section is engaged in research activities in the following two major areas: 1. Development of nucleic acid based tests for the screening of donor blood for contaminating pathogenic bacteria and malaria parasites: In the US, transfusion transmitted bacterial infections leading to septicemia, and parasitic infections primarily from malaria have been always a matter of concern. A blood screening test that could simultaneously detect the presence of these contaminating pathogens would further increase the safety of the nation's blood supply. Recent data suggest that nucleic acid based tests (NAT) can be used to detect infectious agents in blood and blood products. These tests potentially offer sensitive, reliable, and practical methods to screen blood for infectious agents. We are developing PCR-based detection assays, 5'-nuclease Taqman PCR assay and microarray DNA tests for high-through put pathogen screening of individual or pooled blood samples. Our recent data suggests that both PCR test and TaqMan assay can be used for the detection of bacteria and malaria parasites in blood samples and platelets. We have also developed a DNA microarray test for sensitive, species-specific identification of malaria parasites. The NAT technology is being further developed as a sensitive high-throughput method for screening of donor blood for the presence of contaminating pathogens. 2. Malaria Research. We are conducting malaria research in the following areas: -Determination of the mechanism of protective immunity induced by a recombinant malaria vaccine. -Multiple Antigen Paptide Vaccine against Plasmodium falciparum malaria. -Understanding malaria pathogenesis in murine malaria Plasmodium berghei. -Centrins of Plasmodium: Understanding cells division in malaria parasite. -Genomics approach for the identification of novel protective malarial antigens. Synthesis, Characterization, and Immunogenicity of Multiple Antigen Peptide Vaccines Based on Antigens from P. Falciparum malaria. Given the emerging difficulties with malaria drug resistance and vector control, as well as the persistent lack of an effective vaccine, new malaria vaccine development strategies are needed. We used a novel methodology to synthesize and fully characterize multiple antigen peptide conjugates (MAPs) containing protective epitopes from Plasmodium falciparum and evaluated their immunogenicity in four different strains of mice. A di-epitope MAP (T3-T1) containing two T-cell epitopes of liver stage antigen-1 (LSA-1), a di-epitope MAP containing T-cell epitopes from LSA-1 and from merozoite surface protein-1 (MSP-1), and a tri-epitope MAP (T3-CS-T1) containing T3-T1 and a potent B-cell epitope from the circumsporozoite protein (CSP) central repeat region were tested in this study. Mice of all four strains produced peptide specific antibodies, however the magnitude of the humoral response showed marked genetic variation between the different strains of mice. Anti-MAP antibodies recognized stage-specific proteins on the malaria parasites in an immunofluoresence assay (IFA). In addition, serum from hybrid BALB/cJ x A/J CAF1 mice that had been immunized with the tri-epitope MAP T3-CS-T1 successfully inhibited the malaria sporozoite invasion of hepatoma cells in vitro. Spleen cells from immunized mice also showed a genetically restricted cellular immune response when stimulated with the immunogen in vitro. We have synthesized new MAP and their immunogenicity testing in mice is in progress. The results obtained from these ongoing studies will determine whether these MAP deserve further testing in primates. Development of nucleic acid based tests to detect infectious agents in donor blood and blood components. Transfusion-induced sepsis is a major, often fatal adverse event resulting from bacterial contamination of blood products, particularly platelets. Nucleic acid tests (NAT) may prove to be useful in detecting contaminated blood products. By DNA sequence homology, we identified a set of universal primers on the 16S ribosomal RNA gene conserved in 22 different Gram-negative and Gram-positive bacterial species, many of which have been implicated in the 78 fatal cases of transfusion-induced sepsis reported to the FDA between 1976 and 1998 (Gram-negatives: Klebsiella, Serratia, Salmonella, Enterobacter, E. coli, Peudomonas, P. mirabilis; Gram-positives: Bacillus, Streptococcus, S. epidermidis, S. aureus). Using our set of universal primers in a single-step PCR, we detected as few as 5 colony-forming units of E. coli or S. aureus in 1-2 microliter of human blood. Test sensitivity may be improved in a two-step nested PCR. The use of these primers in the TaqMan? assay or the DNA microarray system may prove sufficiently sensitive and specific for these NAT to be useful as additional blood screening tools. Development of TaqMan? assay, and DNA microarray test for bacterial detection is in progress. Malaria is distributed worldwide, and is attributed to 300 to 500 million clinical cases, and over 1 million deaths each year. In the USA, an average of 3 cases malaria each year are transfusion-transmitted, accounting for 0.25 cases per million units of blood collected. About 50,000 individuals each year are deferred from donating blood because of prior infection or potential exposure to malaria parasites during travel to endemic regions. Most deferred travelers do not have malaria and would otherwise be suitable blood donors. At present, malaria is diagnosed by direct demonstration of parasites in thick blood smears-- a technically challenging and time consuming technique. Using the 18S ribosomal RNA gene as amplification target, we developed a PCR, a TaqMan assay, and a DNA microarray test for species-specific detection of all 4 human Plasmodium species. In spiking experiments, using a two-step nested PCR and TaqMan assay, we detected a single malaria parasite present in 1ml of whole human blood, a greater than 200-fold increase in sensitivity compared with conventional blood smears. Any of these nucleic acid based detection tests described here has the potential to be further developed as a sensitive high-throughput method for improved clinical diagnosis of malaria, and screening of donor blood. Understanding the mechanism of anti-malaria immunity in mice induced by a recombinant sub-unit vaccine. Data obtained from experimental malaria models show that immunization with irradiated malaria sporozoites or repeated infections and drug cure with asexual stage parasites protect the host against live parasite infection. Furthermore, the fact that adults in malaria endemic areas develop immunity against clinical disease suggests that it is feasible to develop a vaccine against malaria parasite. However, the immune mechanism(s) that provide protection against parasite infection is not clearly understood. It is the belief of many malaria vaccine researchers that understanding the mechanism of protective immunity is critical to develop an effective anti-malaria vaccine. We have recently developed a recombinant sub-unit vaccine that when delivered in an effective adjuvant protects mice against Plasmodium yoelii sprorozoite challenge. The murine malarias are excellent models to elucidate the complex immune mechanisms where both antibody and cellular responses contribute to immunity. Infection with murine malaria P. yoelii causes a non-lethal infection in mice. Previous work by us and by other laboratories has clearly defined the genetic background of mice that is found to most suitable for P. yoelii-challenge studies. In the immunization-challenge experiments, we have found that vaccination with the recombinant P. yoelii circumsporozoite protein when delivered in Montanide ISA 51 and murine CpG ODN induced complete protection against sporozoite challenge in 80% of immunized mice. In vivo depletions of CD4+ and CD8+ T and IFN-gamma and IL-12 cytokines had no effect on protective immunity suggesting that immunity was complex and probably medaited by both antibodies and T cells. Further experiments to discern the precise mechanism of this vaccine induced protective immunity are being performed in normal immunized mice and by immunizations in T cell, B cell, IFN-gamma, and IL-12 knockout mice. This project incorporates FY2002 projects 1Z01BP005019-02, 1Z01BP005022-01, and 1Z01BP005024-01.
