I, / AD-Ao l; 565 BIOMEDICAL RESEARCH INST ;;e oils VACCINE DEVELOPMENT (U) SEP 79 N A STIREWALT F A LEWIS N0001 e 76 C Qjfl UNCLASSIF!FD NI i r i N I I J I
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- - - - -r - - 4// 1 Li/ i(f 1 ( / Officp Q Naval Research 914-76-C p146 I Task No NR 204-005 Contract *QN J Annual Report No 4 Schistosome Materials for Vaccine Development M A /Stirewalt nd (F A Lewis J / D ID Reproduction in whole or in part is permitted C 1 for any purpose of the United States Goverment i zsmisurioi STATEMENT A Approved for public xeie s$ Distributios Unlimited 1 11/ io 01 96 - - - - -
-- - i, I $i Curnv I A S S S S r A T I O,, ar ls % r A r, Nhn Iaim P RE O( T DOCUM I TATIGN P GE eov1 I I( H( NUMUIN S iit 1 4 TItI I ( id 3wemt I IIrrI i ( (I i i T i N ( i (l RN I Kt CIPILN1 AGt IUN NC) Annu a1 VACCINE DEVI LOPMENT P 30 Sep 1978 1 Oct 1979 S P & N f l I N G 0MG M I P O N T N U M P E N S C O NT NA C T O N G M A N T N U M S ( M ( Ø ONR N00014 76 C 0146 10 PO G q A M i ML PN3ECT T&S PIMP OMMiN OMUAN I l A T I C ) N NAM E ANO A0DI C$S 4 I T Pc or KIPO NT S PNOO CovC ro SCHISTOSOME MATERIALS FOR M A Stirowalt and F A Lewis AA LU NUMULfl \merican Foundation for Biological Researct 12111 Parkiawn Drive 1ockvi1le yiry1and 20852 U COsT MOILlN OFFICE NA ME A ND A DOMEIS rocurement Contract Officer ) f f i c e of Nava l Research ( 4 4 3 ) t h e Nay Wash i iq tpn 1 D C Dept of 203 6 0 MEPOAT 0* 11 IT SIiMIILMOP P A G E S I i- I*ON ItOHI$G AGE NCy NAM L S A O OMESS( a I dus,nl S,a C enhiino OSSS s) NR 204 005 IS IS 24 5ep 1979 ICCUHI TY Cl ASS (1 All p rlj Unclasa iliad 1 I O IC LAssIr g IATION $Cst Ou ( O0WNGNADI NO - 14 O ttplie utl ON STA T EMENT (1 Al Npf l ) Standard Distribution Lists IT O1STNIPUT,O N STATEMEN T (of IS SU P P L I M I N T A M Y NOTES IS r, K EY 101105 (COnIMs A Srac, d ) 20 U dpu rli (, p $t e*, asd II,,e& d gdil U) y bic* rnb) Schistosoma mansoni; trematode; cercarise; worms ; penetration enzymes; rotifer inhibition ; optima l maintenance conditions nffiw SO A $ I MAC T ((?, Idl SI not ll id bp Sleek *b ij I ni$ I emperature and food being controlled at optima l levels, the presrnce of rotifers in the snail colonies was the most important facor in parasite maintenance Rotifer infestation of schistosomenfoctcd snails was highly inimical to pdrasite development With otifor control, large numbers of schistosomos and products of ost-shistosome interaction were produced on a weekly schedule,imits of variability in production of parasites were set for the 9t, % *n AbisnL DO,, 1473 I O I T IO N C Y I NCç$ l$ OIOl(tt u N 0102 I 014 O1 P $ICWIITY CLkSSIPICA Ios OP ISIS PA GE (NT ø DSP SRPISSØ - -
, - - -, - - BACKGROUND The Immunoparasitology Department at the Naval Medical Research Institute (NMRI) is involved in research centered primarily on the development of effective vaccines against several parasitic diseases Immunological research in one such disease, schistosomiasis, is particularly difficult due to the limited quantity of schistosoma] materials available to most laboratories It has been the objective of this contract to supply large quantities of schistosomal materials to investigators at NMRI and the Biomedical Research Institute (BRI) to help realize the goal for the development of an effective vaccine against schistosomiasis The variàus materials provided include adult schistosomes, eggs, cercariae, schistosomules, cercarial penetration enzymes, and vertebrate and invertebrate host serum and tissues H : 7 2 : I & pt Ia! -,- -
- METHODOLOGY A Puerto Rican strain of Schisto oma mansc,ni was maintained in Biomphalaria glabrata snails and Swiss albino mice Six 20- gallon aquaria, supporting pproximate1y 1000 snails each, pro vided uninfected snails Approximately 300 snails (5-7mm dia ) were collected each week and exposed individually with 6-8 miracida When requested, snails were exposed to 1 miracidium each for development of single-sex schistosomal infections Miracidia were derived from livers of 8-week infected mice A constant Supply of approximately 1000 infected snails were maintained for production of cercariae From these infected snails, from 1 to 3 million cercariae were collected 4 days per week and processed as needed or used for experimental work For the production of adult parasites and eggs two hundred and fifty Swiss mice were exposed percutaneously weekly to 250-270 cercariae each Adult worms were perfused from these mice at 7 weeks post infection, and eggs isolated from the livers Schjstosomules, the postpenetration stage of cercariae, were recovered from ear skin of mice exposed in vivo to cercariae Schiatosontules were also collected after cercaria] penetration of dried rat abdominal epidermis, or by the Colley syringe shear technique Fifty to 100 ml of secreted enzyme solution were harvested two to four days each week from cercariae stimulated to secrete in a temperature gradient over skin surface lipid or the active fraction, linolenic acid Total protein (Lowry method) and enzyme activity against azocofl (dye-coupled collagen) were established spectrophotometrically for each collection 3 -- -
RESULTS AND DISCUSSION The list of investigators at NMRI and BRI for whom schistosomal materials were supplied by the contract is given in Appendix 1 Each week the materials supplied consisted of: 8 to 12 million cercariae ; 20 to 25 thousand adult worms ; 600,000 to 900,000 schistosornules, the numbers adjusted to demand ; frozen irradiated schistosoinules; about 200ml of preacetabular gland enzyme secretion ; and excretions and secretions of cercariae as requested Maintenance of this large scale life cycle of S mansoni allowed investigations into the nature of the observed natural fluctuations encountered in cercarial harvests and infectivity we noticed that cercarial production decreased during infestation of the snail colony with rotifers of the genera Philodina and Rotifer A dramatic decrease in cercarial production per snail (Figure 1) was noted, as well as an inhibition of normal cercarial activity The cercariae which did emerge from the affected snails were lethar9ic, accumulated on the bottom surface of the dish, and had tightly curled tail furci In sui nary, the provision of schistosoma] materials on a large scale requires close control of the conditions known to be important for optimum parasite development These conditions include maintaining constant temperature of infected snails, systematic feeding, reduction of rotifer infestation as much as practical, etc Fluctuations in cercaria] and adult worm production persist even when controlling these known optimal conditions it is important there fore to continue investigating details of the schistosome life cycle in order to supply materials in quantity for vaccine development 4
-,,- -- - - -,- - - SIGNIFICANT ACCOMPLISHMENTS 1) Established natural flut tuations of S mansoni life cycle under our conditions 2) Compared the results of 1 miracidium vs 8-10 miracidia snail exposures in terms of: (a) patency of infection in snails (b) deaths of snails (c) cercarial production (d) cercarial infectivity 3) Demonstrated that rotifer-infestation of schistosome-jnfected snails reduced cercaria] production 5
- - Appendix 1 List of Investigators Supplied with Schistosomal Materials on Contract ONR N000]4-76-c-0l46 NAVAL MEDICAL RESEARCH INSTITUTE Dr W E Vannier Dr K D Murrel] D D A Dean Dr A H Smith Dr P Minard Dr D WTaylor Dr P Coulis Dr V Schins3cj Dr H Stek Dr C H Dorsey Dr A Attallah BIOMEDICAL RESEARC H Dr M A Stirewalt Dr F A Lewis Dr E Hayunga Dr C Cousin INSTITUTE -6- - - -
- 4, CERCARIAL PRODUCTION - z 3000 4 w 2O0O 0 0 z > 1000 * AC AC AC AC AC AC AC AC AC Figure 1 Histogram comparing the average number of cercariae per snail from Rotifer CR) and Non-Rotifer (C) Biomphalaria glabrata on nine consecutive Collecting Days 7,- - -- - - -- --- -- - - - - --- --
CURRENT REPORTS AND PUBLICATIONS SUPPORTED WHOLLY OR IN PART BY SCHISTOSOME MATERIALS PRODUCED UNDER ONR CONTRACT No N00014-76-C-0146 1978 Minard, P, Dean, DA, Jacobson, RH, lannier, WE, and Murrell, K D 1978 Immunization of mice with Coba1t -60 irradiated Schistosoina mansoni cercariae Am J Trop Med & Hyg 27: 76 86 Minard, P, Dean, DA, Vannier, WE, and Murrell, KD 1978 Effect of immunization on migration of Schistosoma mansoni through lungs Am J Trop Med Hyg 27: 87-93 Dean, DA, Minard, P, Stirewalt, M A, Vannier, WE and Nurrell, KD 1978 Resistance of mice to secondary infection with Schistosoma mansoni I Comparison of bisexual and unisexua l infections Am 3 Trop Med Hyg 27: 951 956 Stirewalt, MA 1918 Quantitative collection and proteolytic activity of preacetabular gland enzyme(s) of cercariae of Schistosoma mansoni Am J Trop : Med & Hyg 27: 548 553 Dean, DA, Minard, P, Murrell, KD and Vannier, WE 1978 Resistance of mice to secondary infection with Schistosoma mansoni II Evidence for a correlation between egg deposition and worm e- limination Am 3 TropMed & Hyg 27: 957-965 Murre]1, KD, Taylor, DW, Vann ier, WE and Dean, D A 1978 Schistosoma mansoni: Analysis of surface membrane carbohydrates using lect [ns Exp Parasitol 46: 247 255 Attallah, AM, Smith, AN, Murrell, KD, F]eischer, T, Woody, J, Vannier, WE, Sche -, I, Ahmed, A and Sell, KW 1979 Characterjza tion of immunos u ssive state during Schistosorna mansoni infection 3 Inunun 122: 1413 1420 Murrell, KD, Sti ilt, MA and Lewis, PA 1979 Schistosoma mansoni: Vaccinati of mice with cryopreserved irradlited schisto somulesexpparasitol (in press) Stirewalt, MA, Lewis, FA and Murrell, K,D 1979 Schistosoma mansoni: Cryopreservatjon of schistosomules Exp Parasitol (i - presit 8-
ABSTRACTS OF PRESENTATiONS 1978 Stirewalt, M, Murrell, lcd, schistosomules of Schistosoma ical Medicine & Hy en e, i-il and Lewis, FA Cryopreservation of mansoni Amgrican Society of Troprov lfi78, Chicago, i j Lewis, FA, Campbell, DL and Stirewalt, M Murine cellular reactivity to preacetabular gland contents of Schistosoma mansoni American Society of Tropical Medicine & Hyg iene, 7-IIov 1978, Chicago, Ill Cousin, C, Stirewalt, 1 and Dorsey, C!! Comparative transformation of in vivo and in vitro schistosotnules, Program and Abstracts of the 52rTAnnual MeeETng of the American Society of Parasito1og sts, 6 il, Nov 1978, Chicago, Ill Stirewalt, M and Lewis, FA Effect of rotifers on schistosome cercariae Program and Abstracts of the 52nd Annual fleeting of the American Society of Parasitologists, 6-li Nov 1978, Chica go, Ill Murrell, KD, Clark, SS, Weaver, N, Stirewalt, M, annier, W E and Dean, DA Immunization with attenuated Schistosorna mansoni schistosomules, Fourth International Congress of Parasi tolo g ists, Warsaw, Poland, 19-26 Aug 1978 Murrell, lcd, Minard, P, Vannier, WE and Dean, D A 1978 Immunization against schistosotnes Proc Int l Conference on Schistosomiasis, Cairo, Egypt, October 18 25, 1975, Vol II, p 545 555 Dean, DA 1978 Studies on the mechanism of acquired immuni ty to Schistosoma mansoni I n t l Conference on Schistosomiasis, Cairo, Minard, P, Murrell, KD and Stirewalt, M A 1976 P e n et r a t i o n enzymes of Schistosoma mansoni cercariae Proc Int l Conference on Schistosomiasis, Cairo, Egypt, October 18-25, 1975, Vol II, p 599 602 Schinski, VD, Clutter, WG and Murrell, KD 1978 Labelled antiglobulin assays for the study of schistosome antigens and human immune responses Proc Intsl Conference on Schistosomiasis, Cairo, Egypt, October 18 25, 1975, Vol II, p 583 585 Vann ier, WE, Hussain, R and Murrell, lcd 1978 Schistosome allergens Proc Int l Conference on Schistosomiasis, Cairo, Egypt, October 18 25, 1975, Vol II, p 587 596