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REPORT NUMBER lB

DEVELOPMENTr OF SEROLOGIC ASSAYS FOR THE DIAGNOSISOF NEW WOKRLD LEISHMANIASIS

ANNUAL REPORT (DECEMBER 1, 1 982-NOVEMBER 30, 1983)

U Ronald L. Anthony, Ph.D.Department of Pathology

II-

i January 31, 1984

C1 US ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMlANDFort Detrick, Frederick, Maryland 21701

Contract No. DAMD 17-83-C-3031

University of Maryland School of Medicine

Baltimore, Maryland 21201

Approved for public release; distribution unlimited

The findings in this report are not to be construed as an officialdocument of the Army' position unless so designated by another authorizeddocument.

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Development of Serologic Assays for Annualthe Diagnosis of New World 12-1-82 to 11-30-83Leishmaniasis 6. PERFORMING ORG. REPORT NUMBER

7. AUTHOR(a) 8. CONTRACT OR GRANT NUMBER(&)

Ronald L. Anthony

9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT, PROJECT. TASKAREA & WORK UNIT NUMBERS

Department of Pathology

University of Maryand School of MedicineBaltimore, Maryland 21201 DAMD 17-83-C-3031

1I. CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATE

US Army Medical Research and Development Command January 31, 1984Fort Detrick, Frederick, Maryland 21701 IS. NUMBER OF PAGES

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Approved for public release: distribution unlimited

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LeishmeniaMonoclonal antibodiesELISA

20. ABSTRACT (Cntiwe aim revee si if necery and identilty by block namber)

Two-hundred eighty-nine monoclonal antibodies have been generated to surfaceand Intracellular antigens of various isolates of New World Leishmania.Concentrated efforts have been focused upon using these monoclonals to detectantigenic differences between Leishmania mexicana mexicana and Leishmaniabraziliensis panamanensis. Indirect immunofluorescent antibody assays havebeen used to demonstrate qualitative differences; enzyme linked immunosorbentassays have been used to demonstrate quantitative differences. We appear to

00 , 1473 mno or Io VSIs OML&T3

SECURITY CLASStFICATION f'F THNf Phi(F r na'. FM....%-

have at least 6 monoclonal antibodies w?*ch can distinguish between air-driedpromastigotes of the two species. Additional monoclonals react with bothspecies but the intensity of reactivity is often significantly different. Infact, we now have some degree of confidence in our ability to differentiateL.m. mexicana from L.b. panamanensis on the basis of the quantitativedifferences.

li

REPORT NO. lB

DEVELOPMENT OF SEROLOGIC ASSAYS FOR THE DIAGNOSISOF NEW WORLD LEISHMANIASIS

ANNUAL REPORT (DECEMBER 1, 1982-NOVEMBER 30, 1983)

Ronald L. Anthony, Ph.D., Department of Pathology

January 31, 1984

US Army Medical Research and Development CommandFort Detrick, Frederick, Maryland 21701

Contract No. DAMD 17-83-C-3031

University of Maryland School of MedicineBaltimore, Maryland 21201

2

Forward:

In conducting the research described is this report, the investigatoradhered to the *Guide for Care and Use of Laboratory Animals,* prepared by theCommittee on Care and use of Laboratory Animals of the Institute of LaboratoryAnimal Resources, National Research Council (DHEW Publication No. (NIH) 78-23,Revised 1978.

3

Summary:

Objectives for the initial year of the contract DAMD 17-83-C-3031 in-cluded:

(a) establishment of in-house cultures of leishmania promastigotesrepresentative of a panel of WRAIR isolates.

(b) preparation of monoclonal antibodies to specific antigens of thoseisolates.

(c) identification and characterization of species-specific,strain-specific and stage-specific antigens.

In vitro cultures of 12 WRAIR isolates of New World Leishmania,thought to be representative of 4 species and 6 sub-species, were establishedin our laboratory. Adequate stocks of most isolates are now being stored inour liquid nitrogen facility.

Monoclonal hybridoma antibodies have been generated to surface andintracellular antigens of 7 isolates. Most of these monoclonals have beenisotyped and they have been classified on the basis of their site ofreactivity, by indirect immunfluorescence (IFA), with fixed promastigotes.One antibody, to a surface antigen of Leishmania mexicana mexicana (222B)stationary promastigotes has been used to develop an unlabeledperoxidase-anti-peroxidase technique for localization of antigen byimmunoelectron microscopy.

Specificity of antibodies has been assessed by IFA and by enzyme linkedimmunosorbent assays (ELISA). We discovered that variations in methods offixation of the promastigote substrate could lead to erroneous interpretationsof specificity. However, many of the monoclonal antibodies do appear to havethe capacity to distinguish promastigotes at the species level; e.g.Leishmania braziliensis braziliensis vs Leishmania, mexicana mexicana. Moreextensive analyses of specificity using objective and quantitativemeasurements of reactivity are now in progress.

4

List of Tables

Table 1 - Description of cell fusions Pae 9

Table 2 - Description of mnoclonul antibodies Pap 0

5

.... .....

List of Figures

Figure 1 - Reactivity of monoclonal antibodies with 222B and 470 by enzymelinked immunosorbent assay. Page 13

Figure 2 - Immunoelectron microscopy of monoclonal antibody 83T-9D3-B withsurface antigen of 222B promastigotes. Page 15

6

PROGRESS REPORT:

Statement of the problem: Development and refinement of an enzyme-linkedimmunosorbent assay to detect serum antibodies against species-specific,sub-species-specific and strain-specific antigens of the New World Leishmania.

Background and approach: Several significant advances have been madetoward increasing the sensitivity of serologic assays for detection of thehumoral antibody response in New World Leishmaniasis.1 ,2 Nevertheless, ourefforts toward increasing the specificity of these assays have been dis-appointing. With the exception of an indirect immunofluorescent antibodyassay (IFA} which uses an amastigote as the substrate,3 the IFA and the ELISAcannot distinguish between anti-leishmanial antibodies and antibodies producedin response to antigenically related organisms. Since the controlling element a.of specificity in the ELISA is the antigen, a prerequisite to the developmentof a specific serodiagnostic test for New World Leishmaniasis is developmentof procedures which permit the recovery of species-specific antigens on apreparative scale. We believe that these antigens can be recovered from invitro cultures of promastigotes by using techniques of immunoadsorbent chroma-tography. The critical component of the immunoadsorbent will be a species-specific monoclonal antibody produced by a hybridoma. The hybridoma techniquepermits the generation of antibody of any desired specificity which, in turn,can be used to recovery the specific antigen from a heterogeneous mixture.

1. Roffi, J., Dedet, J., Desjeux, P., and Garre, M. Detection of circulatingantibodies in cutaneous leishmaniasis by enzyme-linked immunosorbent assay(ELISA). Am. J. Trop. Med. Hyg., 29: 183-189, 1980.

2. Anthony, R.L., Christensen, H.A., and Johnson, C.M. Microenzyme-linkedimmunosorbent assay (ELISA) for the serodiagnosis of New World leishmaniasis.Am. J. Trop. Med. Hyg., 29: 190-194, 1980.

3. Walton, B.C., Brooks, W.H., and Arjona. I. Serodiagnosis of Americanleishmaniasis by indirect fluorescent antibody test. Am. J. Trop. Med. Hyg.,21: 296-299, 1972.

7

Results and discussion: In-house cultures of 12 WRAIR isolates wereestablished during the first year of the contract period. These isolatesincluded:

Leishmania mexicana mexicana 183B, 222BLeishmania mexicana amazonensis 303Leishmania braziliensis panamanensis 390, 424, 120, 470Leishmania braziliensis braziliensis 063, 508Leishmania braziliensis peruviana 140Leishmania donovani chagasi 111, 484

The promastigotes are maintained in 150 cm tissue culture flaskscontaining 100 ml Eagles MEM with 10 mM Hepes, 20% heat inactivated new borncalf serum, penicillin, and streptomycin. The cultures are held in anup-right position at 26oc. Promastigotes, in the stationary phase of growth,are harvested weekly, washed in phosphate buffered saline (PBS) and stored at-700C. Substantial stocks of most isolates have been stored in cryoprotectionmedium in liquid nitrogen. Isolate 063 has been discarded because of heavyyeast contamination; isolate 183B was lost prior to the generation of adequateback-up stocks.

Washed promastigotes have been used to prepare substrates forimmunofluorescent microscopy. Approximately 200 slides (8 wells each) arestored at -700C for each isolate.

Extracts of promastigotes, representative of each isolate, have beenprepared by repeated freezing and thawing in PBS, followed by sonicdispersion. These extracts, standardized to contain 100 ug protein permilliliter, are used as immunogens for the BALB/c mouse and as antigens inenzyme linked immunosorbent assays (ELISA).

Fourteen successful cell fusions were performed from December 1, 1982 toNovember 30, 1983.

TABLE 1

Fusion number Immunogen Hybridomas +for antibody

83B 140 183H 222B 37831 183B 1483L 470 2683M 183B 183N 222B 783P 470 2183R 470 3983S 303 183T 222B 8183U 222B 3183V 470 183Y 470 1083Z 508 19

With the exception of fusions 83T, 83U and 83V, immunogens were stationarypromastigotes, frozen (-700C) and thawed (+450C) three times and standardizedto contain 100 ug protein per ml. Promastigotes for fusions 83T and 83V werealso disrupted by sonic treatment. A membrane fraction of 2228 promastigoteswas used as the immunogen for fusion 83U.

The fifteen monoclonal antibodies to 183B represented eight distinctspecificities. An FASEB abstract of preliminary descriptions of reactivityand the use of these monoclonals to differentiate New World Leishmania speciesis appended. Since 183B is no longer available, we have discontinued ourcharacterization of these antibodies.

The monoclonal antibody to 303 reacts with a cytoplasmic component whichappears, by IFA, as sparce granules. This antibody has not beencharacterized.

The monoclonal antibody to 140 is an IgM which is reactive with a majorconstituent of the flagellum. This antibody is also strongly reactive, byELISA, with 390 and 303. We have accumulated 5000 ml of media supporting thisclone (83B7G6-A) and have recovered the immunoglobulin by precipitation insaturated ammonium sulfate. The protein concentration of our finalsolubilized antibody was 35 mg/ml. We intend to use this preparation todefine the parameters of ELISA development.

Major efforts have been directed toward discovering monoclonal antibodieswhich can distinguish L. mexicana mexicana (222b) from L. braziliensispanamanensis (470). In an earlier report (1-A, August 31, 1983) we claimedthat we had at least 12 monoclonal antibodies which were specific for 222B.This claim was based upon the failure of anti-222B monoclonals to react withacetone fixed promastigotes of 470 by IFA. However, we subsequently learnedthat the negative reactions with L.b. panamanensis promastigotes was aconsequence of prolonged fixation. When air-dried organisms were used as theIFA substrate all 12 monoclonals were equally reactive with both isolates.

9

Therefore, since September, 1983, all evaluations of antibody specificity byIFA have been made on unfixed substrates. We now have 4 monoclonals whichappear to satisfy our requirements of specificity.

TABLE 2Reactivity

Monoclonal antibody Immunogen 222B 470 508

83Z-9C7 508 - + +83V-4G4 470 - + -

83P-6B4 470 - + -

83R-8B4 470 - + +

83Z-9C7 and 83R-SB4 appear to distinguish between L. braziliensis and L.mexicana, whereas 83V-4G4 and 83P-6B4 differentiate L. braziliensispanamanensis from L. braziliensis braziliensis.

The 31 monoclonal antibodies from fusion 83U, where 222B membranes wereused as the immunogen, represented at least 5 different specificities (on thebasis of IFA patterns).

(a) linear flagellum

(b) linear flagellum and membrane

(c) A particulate flagellum and membrane

(d) C '- particulate membrane

(e) linear membrane

Two monoclonal antibodies from fusion 83U failed to react with air-driedpromastigotes of 470; 83U-2C6 (IFA pattern e) and 83U-6E6 (IFA pattern c).Eight monoclonal antibodies from fusion 83U reacted with 470 promastigotes butto a much lesser extent than was observed with 222B (1+ vs 4+).

The remaining 21 antibodies reacted equally with 222B and 470.

Although all monoclonals from fusions H,L,N,T, and Y reacted with both222B and 470 promastigotes, differences in intensity of immunofluorescencewere obvious. Thus, it was concluded that differentiation of species wouldrequire objective quantitative analyses such as those which can be made withan ELISA.

Medium supporting hybridomas from fusion 83L was tested for monoclonalantibodies using an ELISA. Antigens were saline sonicates of 222B and 470promastigotes standardized to contain 30 ug protein/ml. Each well of themicrotiter plate received 50 ul; 1.5 ug protein. When an optical density (OD)of greater than 0.200 was regarded as the lower limit of positivity (basedupon readings of less than 0.200 with IgG from related hybridomas and unfusedplasmacytoma cells) 76 hybridomas were synthesizing reactive anti-leishmanial

10

Iantibodies. Thirty-five reacted equally with 222B and 470 (difference in ODI was less than 0.100); 14 reacted more strongly with 470; 26 reacted morestrongly with 222B. Twenty-nine of these monoclonals (OD greater than 0.300for either 222B or 470) have been selected for cloning and further analyses.t

These results encouraged us to re-examine the reactivity of mnoclonalantibodies from fusion 83T, using the ELISA instead of the IFA. Medium fromunfused plasmacytomas cells (IgG-l secretors) was used as the negativecontrol. Results for the following three monoclonal antibodies are presentedin Figure 1.

83T-2BI1-C:anti-cytoplasmic granules of 222B promastigotesIgG-l,knegative IFA with 222B amastigotes in VERO cellsnegative IFA with acetone fixed 470 promastigotespositive IFA with air-dried 470 promastigotes

83T-4E3-D:anti-cytoplasmic granules of 222B promastigotesIgG-l,knegative IFA with 222B amastigotes in VERO cellsnegative IFA with acetone fixed 470 promastigotespositive IFA with air-dried 470 promastigotes

83T-9D3-B:anti-surface granules of 222B promastigotesIgG-2b, kpositive IFA with 222B amastigotes in VERO cellspositive IFA with acetone fixed 470 promastigotespositive IFA with air-dried 470 promastigotespositive IFA with formalin fixed 222B promastigotesconfirmed surface reactivity by imnunoelectron microscopy (Figure 2)

I

iI

-. - -- mm m m m m m lmm m mm ~ mm ll1

Figure 1

Reactivity of anti-L. m. mexicana monoclonal antibodies (83T9D7-B eel*;83T4E3-D - ; and 83T2BII-C .- -.... ) with antigens of 222B ( 0. ) and470 ( Q ) by ELISA. Wells were coated with serial dilutions of antigensranging in concentration from 75 manograms to 20 micrograms. Mediumsupporting P3 cells served as the negative control ( ....- ). Opticaldensity was determined at 405 nan.

12

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Figure 2

Imnoelectron microscopy demonstrating reactivity of monoclonal antibody

83T9D7-B with surface antigens of 2228 promastigotes.

14

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Although similar results have been obtained with monoclonal antibodiesconcentrated with precipitation in saturated ammonium sulfate, additionalassays utilizing several preparations of antigen are required before claims ofquantitative differences can be substantiated.

16

Conclusions: Two-hundred eighty-nine monoclonal antibodies have beengenerated to surface and intracellular antigens of various isolates of NewWorld Leishmania. Concentrated efforts have been focused upon using thesemonoclonals to detect antigenic differences between Leishmania mexicanamexicana and Leishmania braziliensis panamanensis. Indirectimmunofluorescent antibody assays have been used to demonstrate qualitativedifferences; enzyme linked immunosorbent assays have been used to demonstratequantitative differences. We appear to have at least 6 monoclonal antibodieswhich can distinguish between air-dried promastigotes of the two species.Additional monoclonals react with both species but the intensity of reactivityis often significantly different. In fact, we now have some degree ofconfidence in our ability to differentiate L.m. mexicana from L.b.panamanensis on the basis of the quantitative differences.

17

emftwaumram or mW mw IzzMwrmI S11 Dy mm (wTM RW1IVY WIIN iNIW12ft AVIDI DI U lEWIS-

itJXW4CW LAUM. Krietina H. willim* Niel T.Constintinea mawinl L to Wmprsem fl 01 rthlogy.University of hrlad acaoi or rwaicine, Baltimore. Maryland21201.

A PIW)I of monoclonal antibodies ha. been ued todifferentiate uiwm strains of Iftw Wmld talheniat L.braijiaie brazillenaie, L. b. nsan!, L. b. -

a a1it nct secificities as evidIn-I by their reactivitywith viable prommatigtea, air-dried &Wl atoe fiedpromartigotae mel intracellular anestigotes. Specific site.of reactivity included the flagellum, the flagellar p~cetintracellular grmales, surface granulee, surface inmemrn, anintracellular orgenelle with a siel ad poaition cotiblewith the kinetopleet, and an antigen with a bipolar distri-bution. Strain-specif ic antiqene incluehd both a ron-granularand a granular surface ne tme a granular intracellularcopoent. The kinatopleet antigenm the bipolar subtanceere specie-pcif ic with the exception of L. b. peuiaethis strain show patterns of reactivity siiarl-a Sihim-ofthe SInm group. Mntigen of the flagellum melorI legllar pocket were como to all species ad strains ofLi , I&, a wall as to two Swiss of ftrlsmaoin.Aditinally all speciaes end straims contained at least onecomn surface antigen md one , intracellular antigen.

18

Distribution List:

CommanderU.S. Army Medical Research and Development CommandATTN: SGRD-RMSFort DetrickFrederick, Maryland 21701(2 copies)

Office of Research ManagementSGRD-4JWZ-CWalter Reed Army Institute of ResearchWashington, DC 20307(1 copy)

Patrick B. MdGreevy, CDr, MSCWalter Reed Army Institute of Research(WRAIR) Building 40Washington, DC 20012(1 copy)

19