Resources For Research on Onchocerca volvulus

by John Donelson

Resource materials that are readily available

cDNA and genomic DNA libraries. Perhaps the easiest O. volvulus materials to obtain are recombinant DNA reagents. The American Type Culture Collection (ATCC) maintains four independent O. volvulus cDNA libraries constructed from poly(A)+RNA of adult female parasites and intrauterine microfilariae (1). These gt11 libraries are filed under ATCC #37509 and can be ordered by sending $50 ($75 for overseas institutions) to: ATCC, 12301 Parklawn Drive, Rockville, Maryland 20852, USA; phone #1-800-638-6597; FAX #301-231-5826.

Several O. volvulus genomic DNA libraries have also been constructed using different restriction enzymes. A EMBL3 library of partial SaußA-fragments (2) is available upon request by contacting John Donelson, Dept. of Biochemistry, University of Iowa, Iowa City, Iowa, 52224, USA; phone #319-335-7889, FAX #319-335-6764. A gt11 library of EcoR1 fragments (3) has been prepared by Fran Perler, New England Biolabs, 32 Tozer Road, Beverly, Massachusetts 01915, USA; phone #508-927-5054, FAX #508-921-1350.

A ZAP cDNA library made from total RNA of infective L3 larvae of O. volvulus is also available from ATCC and is filed under ATCC #37711. Although several specific cDNA clones have been identified in this L3 cDNA library, including those encoding actin (2) and OV33 (J. Donelson, unpublished), the library has not been very useful for immunoscreening, probably because a large proportion of the clones contain small segments of the rRNA sequences. At this time the use of this library can only be recommended for those cases where the other libraries are not warranted.

An L3 cDNA library in gt11 from the related organism Brugia pahangi is available from the lab of Jim McKerrow, Dept. of Pathology, University of California at San Francisco, San Francisco, California 94143, USA; phone #415-476-2940, FAX #415-750-6947.

Additional O. volvulus genomic or cDNA libraries have been constructed by several labs and they are probably available by contacting the individual labs. Finally, cDNA or genomic DNA clones encoding more than fifty proteins of O. volvulus, or containing unique O. volvulus DNA sequences, have been reported in the literature, at conferences, or as described elsewhere in the Greene sheet. Most of these DNA clones can be obtained by speaking softly and nicely to the appropriate investigators.

Expression plasmids. A number of laboratories have successfully produced O. volvulus antigens in E. coli using one of two expression plasmid systems. In one system, pioneered by New England Biolabs, the foreign DNA sequence is ligated adjacent to the gene for a maltose binding protein so that a resultant fusion protein can be purified on an amylose column (see the New England Biolabs catalog). The other system is similar in that the foreign DNA is inserted downstream of a gene for glutathione-S-transferase (originally derived from Schistosoma japonicum, of all places). In this case, the resultant fusion protein can be purified on a glutathione column (see the Pharmacia P-L Biochemicals catalog). A bewildering variety of additional expression systems is available for producing foreign proteins in both prokaryote and eukaryote cells. Many of these systems are excellent, but their description is too involved for this column

Research materials that can be accessed with moderate effort

Vaccine test sites for O. volvulus antigens. Two programs are supported by the Edna McConnell Clark Foundation (EMCF) to examine potential immune responses to O. volvulus antigens. David Abraham's lab (Dept. of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA; phone #215-955-8917, FAX #215-955-7771) implants into vaccinated mice small chambers that are permeable to serum proteins and cells but not to multicellular organisms. RichardLucius' lab (Fachgebiet Parasitologie, Universitat Hohenheim, Pstfach 70 05 62, 700 Stuttgart 70, Germany; phone #49 711-459-2275; FAX #49 711-459-2276) studies experimental jirds infected with A. viteae, another filarial parasite.

Antigen localization. Rocky Tuan's lab (Dept. of Orthopedic Research, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, Pennsylvania 19107, USA; phone #215-955-5479, FAX #215-955-2177) is studying the immunohistochemical and electron microscopy patterns of antigens entering into the vaccine trials.

Details of any of these experimental systems can be obtained from the respective labs. Investigators who wish to submit antigens for testing in the vaccine trials should contact Cathy Steel, Laboratory of Parasitic Diseases, Bldg. 4, Rm. 126, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, USA; phone #301-496-5398, FAX #301-480-3757.

Research materials that are somewhat difficult to acquire

Infective L3 larvae. The EMCF has been funding both Milan Trpis at Johns Hopkins University and Fred Prince/Sara Lustigman at the New York Blood center (see News Briefs) to collect O. volvulus L3 larvae from infected black flies in Liberia, West Africa. The larvae are stored in liquid nitrogen and periodically transported to the United States. Unfortunately, the number of L3's currently available is extremely small (about 5000). Future supply is uncertain because both groups have been forced out of Liberia (see Brief Updates, this issue). However, both groups are currently investigating other sites in Africa. When supplies 'pick up', researchers with valid, important needs for L3's can send for an L3 request form from Cathy Steel, (address above).

In summary, the paucity of parasite material is indeed a problem for laboratory-based research on onchocerciasis. This paucity need not dampen the investigator's enthusiasm, however. Rather, it should be viewed as a stimulus for imaginative, creative approaches to collaborate on the many questions surrounding this helminth's ability to flourish in the midst of the human immune response.

References

1. Donelson, J. E., et. al. (1988). Mol. Biochem. Parasitol. 31:241-250.
2. Zeng, W. and Donelson, J. E. (1992). Mol. Biochem. Parasitol. 55:207-216.
3. Perler, F. and Karam, M. (1986). Mol. Biochem. Parasitol. 21:171-178.

Editor's note: Future resources are dependent upon all investigators in the field of onchocerciasis. In the spirit of collaboration which now exists among scientists in this field, we encourage anyone who has available resource(s) or has a need for a particular resource, to write to Cathy Steel, Editor; The Greene Sheet. If the response is good, the Greene Sheet (beginning with the next issue) will publish a "Resource Bulletin Board" to stimulate contacts among investigators.