Immunology and molecular biology of tropical infectious diseases

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Immunology and Molecular Biology of Tropical Infectious Diseases J.R, David and D.A, Ham Since it is not possible to summarize reports from all 48 speakers at the Second Annual Meeting of the International Centers for Tropical Disease Research, this review is selective, emphasizing talks on protective antigens, cytokines and the immune response and presentation of antigens for developing vaccines. Highlights in several other areas will be mentioned briefly. Interleukin I 0

The importance of interleukin 10 (IL-10) in suppressing the immune re sponse in Leishmania was brought up by several speakers. Following stimulation with Leishmania antigen, lymphocytes from patients with acute visceral leishmaniasis produced IL-10, but failed to do so after therapy (Steve Reed, Seattle Biomedical Research Institute, Seattle, WA, USA; Edgar Carvalho, Federal University of Bahia Medical School, Salvador, Bahia, Brazil; and Richard Pearson, University of Virginia Medical School, Charlottesville, VA, USA). The antigen-induced proliferation, diminished in lymphocytes from acute patients, was markedly enhanced by anti IL-10 (SR and EC). Some cloned L. braziliensis induced both THI and TH2 lymphokine mRNAs, whereas the Leishmania homologue of elongation initiation factor stimulated only THI mRNA (SR). Patients exhibiting delayed hypersensitivity failed to produce IL-10 (RP). In relation to IL-10, Donald Ham (Harvard School of Public Health, Boston, MA, USA) discussed the role of certain oligosaccharides on the protective immune response. Antibodies to lacto-N-fucopentaose (LNFIII, found on schistosome eggs) were present in highly protective sera. LNFIII contains the Lewis × trisaccharide, the ligand for P-selectins. LNFIII was shown to strongly stimulate B cells to proliferate and produce IL-10 (which inhibits THI cells), thus switching the immune response from THI to TH2. As LNFIII or structurally similar sugars are also on the surface of Trypanosomo cruzi, HIV gpl20 and certain tumour cells, this 0 1993 Elsevier Science Pubhshers Ltd, I LJK)

mechanism may be used by microorganisms and tumor cells to subvert the protective immune response, and it should be amenable to attack. Interleukin 12

Interleukin 12 (IL-12), a cytokine which stimulates natural killer (NK) cells and T cells to produce IFN-~/ appears to be critical in THI differentiation and immunity to Leishmonia. Phillip Scott (University of Pennsylvania, Philadelphia, PA, USA) reported that spleens from L. major-infected C3H/HeN resistant mice made more IL-12 than cells from BALB/c susceptible mice. Further, the levels of IL-12 correlated directly with those of interferon gamma (IFN-~) in the cultures, and that anti-lL-12 monoclonal antibodies decreased the level of IFN-~. Finding that injecting IL-12 with soluble Leishrnania antigen (SLA) blocked IL-4 production and increased IFN-'y in BALB/c mice, he vaccinated BALB/c mice with SLA and IL-12 and showed that, following challenge, they were markedly protected, similar to C3HNeH mice at eight weeks. Fred Heinzel (Case Western Reserve University, Cleveland, OH, USA) reported that BALB/c mice injected IP with IL-12 for the first seven days of infection showed 80% protection. This protection was partially blocked by antiIFN-~y. Infected macrophages show no mRNA for IL-12, but do if stimulated with LPS. Sergio Romagnani (Instituto di Clinica Medica III, Firenze, Italy) reported that IL-12 and IFN-~ favor Tnl differentiation of human T cells. Indeed, IL-12 can change the profile of established TH2 clones to produce IFN~/. Thus, IL-12 appears to play an important role in THI differentiation and activity and may be useful in vaccine development. Reverse Genetics

Adrian Hill (University of Oxford, UK) described reverse genetics as a

novel route to identify protective antigens in infectious organisms. Studies of a large case-control study in the Gambia showed a significant association between HLA class I molecule HLA-B53 and resistance to severe malaria, suggesting that HLA class I restricted cytotoxic T lymphocytes (CTLs) were playing a role. To test this prediction, analysis of self-peptides eluted from HLA-B53 indicated a binding motif of a proline at position 2 and hydrophobic amino acid at position 9. Synthesized peptides corresponding to this motif from four pre-e~throcytic antigens were tested for binding to HLA-B53. Although all bound with high affinity, only one from liver-stagespecific-antigen- I (LSA-I) was the target of CTL in Gambians exposed to malaria. This epitope appears to be conserved in wild-type isolates from the Oambia. This approach can be applied to infectious diseases for which protective HLA associations have been identified and can probably be extended to HLA Class II. Malaria Vaccine

Other studies on vaccine development focused on specific modes of immunization and specific antigens. Ruth Hussenzweig (New York University Medical Center, New York, NY, USA) found that immunizing mice with a recombinant influenza virus containing the cytotoxic T-cell epitope of the circumsporozoite (CS) protein conferred good protection against sporozoite challenge, 60% being completely protected. The protective response was CD8+ T-cell dependent. Further, Multiple Antigen Peptides (MAPs) based on the B- and T-cell epitopes of the CS protein of Plasmodium falci parum were given with alum to mice. Another group got the MAPs with complete Freund's adjuvant. Both groups showed similarly high levels of anti-sporozoite antibody. Accordingly, these MAPs are being constructed for human trials. Stephen Hoffman (Naval Medical Research Institute, Rockville,

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MD, USA) has been immunizing mice with naked DNA, using a vector with a CMV promoter that has been successful for other systems. Schistosome Vaccine

Mette Strand (Johns Hopkins University School of Medicine, Baltimore, MD, USA) vaccinated mice against Schistosoma mansoni using a recombinant 62 kDa protein, a portion of a 200 kDa protein (related to myosin) identified by sera from mice that had been made highly resistant by exposure to irradiated cercariae. Mice vaccinated three times with antigen in proteosomes exhibited 42-83% protection. The level of protection in rats was 94-97%. Protection in baboons ranged from 0-54% with mean of 28%. Of special interest, there was a direct correlation, p = 0.967, between the antibody titer to the 62 kDa protein and percent reduction in worm burden. The antibody was IgG~. No antifecundity effect was seen. Bernadette L. Ramirez (Research Institute for Tropical Medicine, Manila, The Philippines) presenting results on vaccination studies in mice with S. japonicum, reported that paramyosin given several times without adjuvant gave 73% protection, whereas glutathione S-transferase (GST) was not protective. Improved Diagnostics

Exciting new developments in improved diagnostics were presented. Janice Kolberg (Chiton Corporation, Emeryville, CA, USA) reported a method to detect DNA or RNA by signal amplification using branched DNA and a chemiluminescent detection system. Clinical samples are placed directly into microtiter plates; the 'handson' time is four hours, and the results are quantitative. Qualitative results can be obtained by exposure to photographic film. The assay has been used

Organizing

to detect a number of viruses including HIV and is being developed for Mycobacterium tuberculosis. John Chan (Albert Einstein College of Medicine, Bronx, MY, USA) reported on a luciferase phage assay for the diagnosis of resistant tuberculosis. Mycobacterium tuberculosis is infected by a specific recombinant mycobacteriophage containing the luciferase gene (FFlux) and can be detected in one to two hours. If M. tuberculosis have been previously treated with drugs for 48 hours, the mycobacteriophage will only enter living bacteria. Thus, they have great potential for rapidly (in 48 h) identifying drug resistance in mycobacteria. Polymerase Chain Reaction and Diagnostics

There were several reports on improved diagnosis by PCR. Thomas Wellems (Laboratory for Malaria Research, NIAID, NIH, Bethesda, MD, USA) having identified a single mutation in the P. falciparum dihydrofolate reductase gene (DHFR) associated with pyrimethamine resistance and an additional mutation for cycloguanil (two inhibitors of DHFR), constructed primers to detect drug resistance to these two drugs by PCR. When applied to P. falciparum isolates from Rondonia, a site of epidemic malaria in Brazil, most of the isolates showed the pyrimethamine resistant phenotype but none showed the cycloguanil phenotype. PCR diagnosis was useful in surveying reinfection of black flies for the Onchocerciasis Control Program in Africa (Thomas Unnash, University of Alabama at Birmingham, AL, USA), the diagnosis of post-treatment re-lapse in visceral leishmaniasis (Jorge Arevelo, Instituto de Medicina Tropical Alexander von Humboldt, Lima, Peru), and in the diagnosis of cutaneous leishmaniasis (Kristen Weigle, University of North Carolina, Chapel Hill, NC, USA) and tuberculosis (Robert Barker, Harvard School of Public Health, Boston, MA, USA).

a Meeting?

Immunodiagnosis

Reports on improvements using immunodiagnosis for clinical samples included the simple detection of Crypto sporidium and Giardia by monoclonal antibodies using immunofluorescence (Charles Sterling, University of Arizona, Tucson, AZ, USA) differentiating pathogenic from nonpathogenic Entamoeba histotytica by monoclonal antibodybased enzyme-linked immunosorbent assay (ELISA) (William Petri, University of Virginia, Charlottesville, VA, USA), the use of the FAST~ELISA for field work (Victor Tsang, Center of Disease Control and Prevention, Atlanta, AL, USA), and the detection of fecal lactoferrin by latex agglutination for the diagnosis of inflammatory diarrhea (Richard Guerrant, University of Virginia School of Medicine, Charlottesville, VA, USA). Transfection of Malaria

A report not directly related to the main theme of the program needs to be mentioned. This is the work by Dyann Wirth, Kamini Mendis and associates (Harvard School of Public Health, Boston, MA, USA) on the first successful transfection of the malaria parasite. Firefly luciferase was transiently expressed under the control of pgp28 in the sexual stage of Plasmodium galinacium. Acknowledgements The Second Annual Meeting of the International Centers for Tropical Disease Research of the National Institute of Allergy and Infectious Disease was held 28-30 April 1993, at the National Institutes of Health in Bethesda, MD, USA, with an emphasis on vaccine development and new technologies for diagnosis. John R. David and Donald A. Ham are at the Department of Tropical Public Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA.

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