* FILE Co?( AD-A REPORT DOCUMENTATION :J.. PAGE. Naval Medical Research (If applicable) Naval Medical Command

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1 * FILE Co?( AD-A REPORT DOCUMENTATION It. RESTRICTIVE PAGE MARKI;GS Unclassified 2a. SECURITY CLA S1FICATION AUTHORITY -- 3 DISTRIBUTIO,nAVAILAaILITY OF REPORT Approved for public release; 2. DECLASSIFIC-ATION/DOWNGRADING SCHEDULE distribution is unlimited 4 PERFORMING ORGANIZATION REPORT NUMBER(S) S. MONITORING ORGANIZATION RFPORT NUMaER(S) NMR I a. NAME OF PERFORMING ORGANIZATION 6b OFFICE SYMBOL 7a. NAME OF MONITORING ORGANIZATION Naval Medical Research (If applicable) Naval Medical Command 6. ADDRESS (City, State, and ZIP Code) 7b. ADDRESS (City, State, and ZIP Code) Bethesda, Maryland Department of the Navy Washington, D.C a. NAME OF FUNDING/SPONSORING 8b. OFFICE SYMBOL 9. PROCUR, EMENT INSTRUMENT IDENTIFICATION NUMBER ORGANIZATION Naval Medical (If applicable) Research, and Development Command 8c. ADDRESS (Ciry, State, ard ZIP Code) 10. SOURCE OF FUNDING NUMBERS Bethesda, Maryland PROGRAM PROJECT I TASK IWORK UNIT ELEMENT NO. NO. NO. ACCES10ON NO 63807A 3M4R9107.D808 A DA3l 569 I1. TITLE (Incluce Security Classification) Plasmodium falciparam-infected anopheles stephensi inconsistentl\ transmit malaria to humans J 12. PERSONAL AUTOR(S) Rickman LS, Jones TR, Long GW, Paparello S, Schneider I, Paul CF, Beaudoin RL, Hoffn,di SL a. TYPE OF REPORT9 r 13b. TIME COVERED 14. DATE OF REPORT (YearMonth.Day) 115. PAGE COUNT iournal article FROM DAO O 99 REO T (er5otd y 16. SUPPLEMPAENTARY NOTATION Reprinted from: American Journal of Tropical Medicine and Hygiene 1990 Vol.43(5) pp. 90-1?0 7. COSATI CODES 18. SUBJECT TERMS (Continue on reverse if necesary and identify by block number) FIELD GROUP SUBGROUP Plasmodium talciparum, human malaria, anopheles stephensi, sporo oites, human volunteers, infectivity, gametocyte cultures, NF54 -trin, 3D7 cl1drie.!1$. A55TRAL4onttue reverse if necessary and identify by block number) :J I. 20. DiSTRISUTIONIAVALABILITY OF ABSTRACT 21. ABSTRACT SECURITY CLASSIFICATION (2L;CLASSIFIEOJPJrA'dTED U SAME AS RPT. 0 OtIC USERS Unclassified 22j. JrAME OF RESPONSIBLE INDIVIDUAL 22b. TELEPHONE (Include Area Code) I 22c. OFFICE SY '1iOL Phyllis Blum, Information Services Division isd/adnini 00 FORM 1473, 84 MAR 83 APR ecition may oe used until exnausted. SECURITY CLASSIFICATION OF TWIS PAGE All other editions are obsolete. UNCLAS SI FT ED

2 Im J 1-p W.ed itire 43(5) pp (90-120) 1-opn r : 4,0 b Thc.- mcncan S-.ct, oi Tiopicai Med,cire andi H'gien PLASMODIM FALCIPA RLUM-INFECTED ANOPHELES STEPHENSI INCONSISTENTLY TRANSMIT MALARIA TO HUMANS LELAND S. RICKMAN, TREVOR R. JONES. GARY W. LONG, SCOTT PAPARELLO. IMOGENE SCHNEIDER, CHRISTOPHER F. PAUL, RICHARD L. BEAUDOIN, AND STEPHEN L. HOFFMAN.Vational Naval Medical Center. Bethesda. Maryland: Naval Medical Research Institute. Bethesda. MarYland: "alter Reed Army Institute of Research, Washington, DC.lbstraci. Malaria was transmitted to only 5 of 10 volunteers bitten by 1-2,4nopheles stephensi carrying sporozoites of the 3D7 clone of the NF54 strain of Plasmodium falciparumn in their salivary glands. Parasites were detectable by culture in blooe taken 7-10 days follow.ag exposure and by thick blood film days after exposure. Infectivity did not correlate with the numbers of sporozoites in the salivary glands. Studies which preceded the first trials of ma- vaccine trials can be designed to maximize the laria sporozoite vaccines showed that the bites possibility of demonstrating protective immuof 5 Anopheles stephensi infected with Plasmo- nity. dium,1alciparum reproducibly infected volun- MATERIALS AND METHODS teers.' When immunized volunteers were bitten by 5 infected mosquitoes within 1 hr. there Subjects was a significant delay in the onset of parasitem- Ter adult males, 25-39,e - of ag, were ia. but only 2 of 23 immnnized ;'olurnteers did., at ofag, - not eomey infected ithblod svogelp not become infected with blood stage parasites.' ites. d cruited NalMeilRsarhIttue( from the active duty military staff RIan of the, In rodent malaria models, antibody-induced Naval Medical Research Institute (NMRI) and protective immunity can be overcome by in-the protocol was approved by institutional review creasing the sporozoite inoculum. 6 In malaria en- committees at NMRI and NNMC. and written demic areas, some individuals are not exposed informed consent was obtained from each volto the bite of more than I or 2 infected mos- unteer. None of the volunteers had cardiovasquitoes per night. and may never be exposed to cular, liver, or renal function abnormalities, was 5 infected mosquitoes within 1 hr. Therefore, i taking immuno-suppressive or antimalarial has been suggested that the failure to show com- medications, had been infected with malaria. had plete protection in most volunteers previously antibodies to malaria sporozoites. blood stage immunized wi'h sporozoite vaccines may have parasites, or human imniunodeficiency virus, or been due to challenging them with an unrealist- had hepatitis B surface antigenemia. ically large sporozoite inoculuml. 6 Earlier studies disagree on the effect of the number of infective bites upon the onset of parasitemia and the Parasites, mosquitoes, and percentage of subjects ultimately infected." infection oivolunteers These studies differ from current studies in I TI-e 3D7 clone of the NF54 strain of P. falimportant aspect. In the older studies, the mos- ciparum, which is sensitive to chloroquine. pyrquitoes were infected by feeding upon gameto- imethamine, and quinine, was used in this study.' cytemic humans: the mosquitoes used in our It was maintained in culture through standard studies are infected by membrane feedings on methods.' " Mature gametocytes appeared in gametocytemic blood containing parasites that cultures days post-inoculation. Three days have been maintained in in vitro culture for sev after reaching adulthood, laboratory-reared An. oral years. This current study was designed to stephensi were infected by membrane feeding determine the fewest number of bites by infected upon gametocyte-rich cultures and were mainmosquitoes required to reliably transmit P. fal- tained during the extrinsic incubation period with ciparum of the 3D7 clone of the NF54 strain to dry sugar and water-soaked cotton balls. Sixteen all members of a study population so that future to 17 days after the bloodmeal. the mosquitoes

3 442 RI(KMAN AND OTHERS were permitted to feed on the volunteers. Each RESULTS volunteer received a bite from a mosquito which was subsequently dissected to demonstrate the infection ofvolunteers presence of blood engorgement and sporozoites in the salivary glands. The numbers of sporo- In the first experiment, 42% of the mosquitoes zoites in the salivary glands of a mosquito were had sporozoites in their salivary glands. so that graded as follows: 1-10 = 1+, = 2+, a total of 12 mosquitoes fed on the volunteers ,000 = 3 +. and > 1,000 = 4 +. A mosquito before all 5 had been exposed to an infected moswas considered possibly to have delivered spo- quito (1-4 mosquitoes/ volunteer). Since only 3 rozoites if she was engorged with blood and had of the 5 subjects developed malaria parasitemia sporozoites in her salivary glands. In the first after being bitten by I infected mosquito. a secexperiment, 5 volunteers we'e exposed to mos- ond experiment using 2 infected mosquitoes was quitoes until each subject received 1 such bite: conducted. In this experiment. 63% of the mosin the second experiment, 5 volunteers were ex- quitoes were infected. It required exposure to 16 posed to mosquitoes until each subject received mosquitoes for all 5 volunteers to have been bit- 2 such bites. ten by 2 infected mosquitoes (2-6 mosquitoes, volunteer) Parasitemia developed in 2 of the 5 subjects who received bites from 2 infected mos- Diagnosis of malaria quitoes (Table 1). Mosquitoes that fed on volunteers who did and did not become infected Twice daily, beginning on day 5 post-exposure had salivary gland infections ranging from 2+ and thereafter until they became parasitemic or to 4 + (Table I). Although the prepatent periods until day 30. volunteers were examined by an ranged from days as detected by thick investigator. Two hundred high-power fields blood film, P.falciparum was cultured from blood (1,000 x ) of thick blood fil-s -zie examined for taken on days 7-10 (Table 1): parasites were malaria. Parasite concentrations per ul blood were present in the circulation up to a week before quantitated by a modification of the method of they could be detected by thick blood film. The Earle and Perez. 7. "I Daily, beginning on day 5, earliest blood sample from each subject that blood samples from each volunteer were estab- proved to be positive by culture required 21 days lished in culture. One ml of heparinized blood of culture before parasites were detected. Culwas centrifuged and the plasma and buffy coat tures of blood samples taken on later days had removed. The cells were washed in serum-free detectable parasites after as few as 4 days in cul- RPMI Erythrocytes were resuspended in ture. medium containing 10% heat-inactivated serum and incubated in a candle jar. The medium was Clinical findings changed on alternate days and Giemsa-stained thin films were examined weekly. Results from All volunteers who developed parasitemia also culture experiments were not considered in the developed fever, chills, and myalgias: one subject clinical management of subjects. also complained of headache, and another experienced a 6 hr episodc of,.vatery dia-rhea (Tqble 2). None of the subjects developed hepato- Treatment of malaria megaly or splenomegaly. All symptoms and signs resolved within 48 hr of initiation of therapy, When parasites were detected on thick blood and only I of the subjects was sufficiently ill to film, the subject was treated with mg of require absence from duty. Subject no. I was the oral chloroquine base given over 48 hr.1 5 The only subject hospitalized. He was hospitalized volunteer and blood films were examined twice overnight and was treated only with acetamindaily until the volunteer was as)mptomatic and ophen in addition to chloroouine. His platelet 3 consecutive films were negative. Subjects who count dropped from /ul to /l 2 did not develop a blood-stage infection were giv- days after chloroquine treatment began. This en the same chloroquine regimen at day 30 post- subject also developed a mild leukopenia (3, 300 exposure. Follow-up examinations were carried WBC/u) and had pyuria ( WBC/HPF) and out 3 and 7 weeks after day 30. microscopic hematuria (5-10 RBC/HPF) for h

4 insttei'e I [N~C)NSISTENIY, rransmit MALARIA 443 TABLE I tlalaria intections in no rnal humnans sidl'ctcd to the bites 01 mrin 'ted inzosqitoes: parasiloi i ical findi nkes Initial ',I a% I M u irst da% No ot l'repatent penoid parasitemnia Parasitemnia ulture S olunteer no mrosfuiftoes ;land raze* dast parasites ili waasies..o 1iSf I N1 N I N I Ni 1 3- Ni NJ NI NJ hi ) Ni NI Ni Ni Ni NI N I NI NI Ni Ni Ni Th t.iters al from inoculation to detection ot parasitemia b\ thick hioodl film, rhe first da\ after espoure ito infected moisquitoes that P' 'mq'iarum "as present in blood established in culture ',NI- not infected. hr without evidence of renal insufficiency. None rozoite challenge. This does not provide support of the other subjects developed abnormal corn- for the hypothesis that the level of protective plete blood counts or urinalyses. Liver function immunity induced by the first generation of matests remained normal in all subjects. lania sporozoite vaccines could have been protective in a natural malarious environment but DISCUSSION was not protective in the trials because an unrealistically large, overwhelming sporozoite chal- In previous studies in which mosquitoes were lenge was used to test its protective eticacy. 3 '6 infected by feeding on gametocyte cultures. vol- The NF54 strain of P. Ifihip,win was used to unteers were challenged with 5 infected mos- challenge volunteers in previous studies. We used quitoes. - At least 45 volunteers have been ex- the 3D7 clone of the NF54 strain. The apparent posed to malaria using this method and. thus far, lower rate of infectivity of the clone cannot be (22;r22) of unimmunized control subjects explained on the basis of gland rates. In the studand 910/0(21:'23) of the immunized subjects have ies that reported gland rates. I" I mean gland rates developed malaria parasitemia. In this study. ex- ranged between I.95 and 3.8. The mean gland posure to I or 2 Infected mosquitoes led to par- rate in this study was 3.6. It is possible that this asitemnia in only 50%/ of the volunteers. These lower infectivity is a reflection of the poor in - data therefore suggest that exposure to 5 infected fectivitv of the individual sporozoites of the clone mosquitoes may not represent an enormous spo- compared to the parent strain. However, in re- FABLE 2.ta/aria intections in normial hujntans subited to the bitis o l'i'id moisqufi tis: clinical finding's Maximum D~uration of N luriteer nio Incubatiitn peiodit icmperaturel smptomsignsh I cser duration idaisi % mptoms idassi F, C. My. Ma, D I5 39 F. C. MY F.(., Mv. H NI N I NI NI NI ini NI NI NI NI F. C. My. Mla F. C. MN NI NI NI NI NI 9 NI NI NI NI NI It) NI NI NI NI N I luhtetis f-s recec,ed I bite from an infected mosqjuitio. subiects,-ill receised 2 bite% friom infected miosquitiies IDass from esxts)urc to intected mosquitoes to onset of cliitcal ssmptoms D Iegree% ---'er t hills. MN - mszalftias. Ma -malaise 1) d iatrrhea. Ii - headache. Nil not infiected

5 444 RI(KNIAN ANt) OTHERS cent studies. 3 out of 3 subjects were successfully who became inlfcted. there was a correlation beinfected by 5 bites from mosquitoes carrying spo- tween the numbers ofsporozoites in the salivary rozoites of the 3D7 clone ofnf54 (J. Egan, Wal- glands and the prepatent periods and days until ter Reed Army Institute of Research. Washing- parasites were cultured from the blood. Howton. DC. personal communication), while only ever. the salivary gland indices were equivalent I of 3 volunteers was infected when bitten by 2 in those mosquitoes that fed on the volunteers mosquitoes infected with NF54 (L. Fries. Johns who did not become infected (Table 1). The im- Hopkins University, Baltimore. MD. personal portant relationships among salivary gland incommunication). This suggests that the infectiv- dex. number and infectivity of sporozoites inity of NF54 and the 3D7 clone of the NF54 strain jected. infection rate in the human population, are similar. and prepatency period remain unclear. It may Nonetheless, it is difficult to explain the pro- be that salivary gland index will prove to be a longed picpatent period of days without poor predictor of the numbers of sporozoites ininvoking a biologic difference in the parasites. In jected. previous studies.' the prepatent period of con- More than 15 years ago, field studies indicated trols has ranged from 7 to 11 days with a median that most exposures to sporozoite-infected mosof 10 days, and parasites have been first cultured quitoes did not lead to transmission of malaria. 1 from blood taken days after exposure. In More recent studies in Kenya have led to similar this study, parasites were first cultured from the conclusions (C. Oster, Walter Reed Army Medblood 7-10 days after exposure, indicating that ical Center. Washington DC. personal commuthere has been no apparent change in the time nication). Our findings also support this vicw required for the development of mature liver that while the entomologic inoculation rate reschizonts. The erythrocytic cycle ofp.alcparum flects the transmission rate of malaria. it cannot is -48 hr. and each mature blood stage P..f 1- be used to quantitate directly the transmission ciparum schizont has an average of 16 merozo- rate. Whether this is due to the failure of some ites. If 10 of these merozoites are able to suc- mosquitoes to transmit sporozoites. or the lack cessfully infect other erythrocytes. one would of infectivity of the sporozoites which are transexpect a 10-fold increase in parasitemia every 2 mitted, is unknown. A recent study indicates that, days. a 100-fold increase in 4 days, and a under laboratory conditions, the number of spofold increase in 6 days. It is difficult to explain rozoites ejected by infected mosquitoes can vary a 4-6 day delay in patency that would reflect a by 3 orders of magnitude.' 100-1,000-fold decrease in the number of par- An important finding in this study, as in earlier asites based only on the difference between 2 and studies, is that parasitemia was detected early so 5 infective bites. This is not the first description that appropriate chemotherapy could be rapidly of a strain-dependent variation in prepatency. In initiated. Due to this rapid diagnosis. symptoms a study comparing 3 P. falciparumn strains (Pan- and signs were minimal and brief ama. McLendon. and Santee-Cooper). mean pre- The criteria that predict the degree of infecpatency periods were and 9.8 days. re- tivity of mosquitoes are not known. This leaves spectively.' In another study of 60 subjects, the the vaccine developer uncertain about how many prepatency periods of the strains used varied not mosquito bites to use in a sporozoite vaccine only between strains but also over time." It re- trial. Too few bites by infected mosquitoes can mains unclear, however, whether the changes re- leave one with uninfected control subjects and a sponsible for the differences in prepatent periods loss of the ability to demonstrate protective efinvolve the injection of 100-1,000 times fewer ficacy. Too many bites by infected mosquitoes sporozoites. a great reduction in the number of may deliver a large bolus of sporozoites. thereby sporozoites reaching maturity in the liver, the overwhelming an immune response that would production of far fewer infective merozoites. or have provided protection against I bite received the prolongation of the erythrocytic cycle, in the field. Five infective bites have been shown Others have suggested that there may be a di- repeatedly to produce 100% infectivity in hurect correlation between salivary gland load and mans.' and since I or 2 bites by infected mosnumber of sporozoitei injected.' ' and that the quitoes do not produce consistent infections in short prepatent period in some studies could be volunteers, continuing to use 5 bites is reasonexplained on this basis. Among the volunteers able.

6 i.% luiea 551 SINU'oNSISI'ENTI-\ FRANSNi r MAALARIA 445.Acknowledgments: The authors thank the volunteers UI~de DF Evidence for a (3.5-day minwho selflessly joined the study and who remained de- imumcervthrocyneccyle for Plasmodium fatpendable. enthusiastic participants throughout. We also ciparumn in humans and confirmation that inthank the nurses and corpsmen 01 wards 7 West and munization.% ith a synthetic peptide h East. NNMC for their support. Roy Trimmer for representative 01 a region oi the circumsporoparasite cultures. Maryv Leef for malaria antibody as- /bite protein retards infection. J (Yin ltre/vol sass. Martha Sedegah for assistance during infe.ction ' file med: (ul of the 6olunteers. Lynn Murphy for administrative o. Davis JR. Murphy JR.Clyde DF. Baqar S. (ochisupport. Tom Coon for assistance in manuscript prep- rane AH. Zavala F. Nussenzweig RS. I1)9, Esaration, and Jose Salas for medical photography. The timate of Plasmodium, falciparumn sporotoite authors also thank John Beier and David Havnes for content of Anopheles stephensi used to challenge their veryv helpful reviews of the manuscript. human volunteers. Am J Trap tied 111g 40: file mcd: (ui) Financial support: Naval Medical Research and De- 7Rickmnan LS. Long GW. Oberst R. Cabanban A.,,elopinent Command work unit number Sangalang R. Smith 11. Chulay JD. Hoffman SL, 63763A3M473 75ODSO8A Rapid diagnosis of malaria by acidine Xuthrs*Addrsse: LeandS. Rckmn, Uives oorange AutorsAddesss: lnd. Rckian.Unierity of staining of centrifuged parasites. Lancet file mcd: (ui) 890h2169 ('alifornia San Diego Medical Center H--208, 225 Dick- 8. Egan JE. WeberiJL. Ballou WR. Hollingdac M R. inson Street. San Diego, CA Stephen L. Hoff- Majarian WR. Gordon DM. Maloy WL. Hoffman. i revor R. Jones. Gary W. Long, Christopher F. man SL, Wirtz RA.Schneiderl.andothers Paul. and Richard L. Beaudoin. Malaria Program. Na- Efficacy of murine malaria sporozoite vaccines: val Medical Research Institute ' Washington Av- implications for human vaccine development. enue. Rockville MD Scott Paparello. Division Science 2136: file m8(,, (ui) of Infectious Diseases. National Naval Medical Center.,.By F ice F 97 bevtoso Bethesda MD Imogene Schneider. Department induced 2.lciparum nmalaria. Ai~n J Trap Mfed of Entomology. Walter Reed Army Institute of Re- 1-: search. Washington. DC Coatney GR, Cooper WC, Young MD, McLendon Reprint requests: Stephen L. Hoffman. Malaria Pro- SB Studies in human malaria.!. The protective action of sulfadiazine and sulfapyrgram. Naval Medical Research Institute Wash- azine against sporozoite-induced falciparumn ington Avenue. Rockville MD malaria. AmJ Hvg ' Jeffery GM. Young MD, Burgess RW. Eyles DE. REFERENCES1959. Early activity of sporozoite-induced Plasmodium falciparumn infections. Ann TrapMed I. Scheidr Cula 3D I CosnffTM.Hoffan 1L.. Walliker D, Quakyt IA. Wellems TE. McCutchan Ballou WR. Quakyi IA. Carter R. Trosper JH. -. TF. Szarfman A. London WT. Corcoran LM. Hockmever WT Malaria transmitted to Burkot TR. Carter R Genetic anal,,sis of humans by mosquitoes infected from cultured tehmnmlraprst lsoimfii Plasmodiurr falciparum. l. I TrrpAtd ltvi the uman maaria:6 parasite Plasmiu 6 lali- 3?5 4: ile m86: luit paum Siec ie 8; W 2.eHrington DA. Clye D. N. urphs' IR. Baqar S. 3 Burkot TR. Williams JL. Schneider In- Levine MM. do Rosario V. Hollingdale MR. letvt omsqioso'pasoim.li 19)88. -\ model for Plasmodiumn falciparum rtvt omsutesonlsoimfli sporozoite challenge and veyearly therapy of parum clones grown in vitro from the same isoparasitacinia for eflicacv studies o'f sporozotte late. Frans R Sac ltrap ted ivg % accines. Trap (ieai~'r tfed 40: tle file m83: (uit mced: tuit Earle WC. Perez M Enumeration of par- 3.Iallou WR. Hoffman SL. Sherwood JA. Holling- asites in ihe hlood of malarial patients. I Lah dale MR. Neva FA. Hockmeyer WT. (Jordon (.110./ed / D)M. Schneider 1. Witzt RA. YoungJF. and oth- 15 Hoffman SL Treatment ofinalana. Strickers, 11)87. Saliet- and efficacy of-a recombinant land GT. ed. (Vics in iropwcal niedicine and I)NA Plasmodiumn faleiparumn sporozotte vac- ies.alaria. Manutchl vol. 1. London: cine. Lancet file m?16: (ut) W. B. Saunder, -o.. I "' Pull JH. Crab B A simple epidemiological 4. Herrington DA. Clyde DF. Losonsk' 0,. Corlesia model for ev aluating the malaria inoculation rate M. Murphy JR. Davis J. Baqar S. Felix AM, and the risk of infection in infants. Bull tworld Heier EP. Gillessen D.and others Safty Health ()rian 51: file m72: tui) and immunogemecity in man of a synthetic pep tide malaria vaccine against Plasmodium falci- I7 Rosenberg R. Witzt RA, Schneider 1. Burge R. parum sporozoites. NVature 328: file An estimation of the number of malaria m86: tui) sporozoites ej* ected b,. a feeding mosquito. Frans 5. Murphy JR. Baqar S. Davis JR. Herrington DA. Rai- Soc Trap tied 1kg 84:

Infecting Anopheles stephensi With Rodent Malaria Parasites Alida Coppi & Photini Sinnis

Infecting Anopheles stephensi With Rodent Malaria Parasites Alida Coppi & Photini Sinnis A. Reagents: 1. DMEM or RPMI DMEM (4.5g/L glucose) RPMI 1640 Cellgro #MT-10-017-CM Cellgro #MT-10-040-CM 2. Giemsa