Plasmodium falciparum: Ingested anti-sporozoite antibodies affect sporogony in Anopheles stephensi mosquitoes

EXPERIMENTAL

PARASITOLOGY

66, 171-182(1988)

Plasmodium falciparum: Ingested Anti-sporozoite Antibodies Affect Sporogony in Anopheles stephensi Mosquitoes JEFFERSON A. VAUGHAN,* VIRGILIO Do ROSARIO,? PAMELA LELAND,~ ANGELA ADJEPONG,? JULIE LIGHT,? GILLIAN R. WOOLLETT,~ MICHAEL R. HOLLINGDALE,~ AND ABDU F. AZAD* *Department of Microbiology & Immunology, University of Maryland Maryland 21201, U.S.A., and fBiomedica1 Research Institute, Rockville, Maryland, U.S.A. 20852

School of Medicine, Baltimore, 12111 Parklawn Drive,

(Accepted for publication 28 December 1987) VAUGHAN, J. A., HOLLINGDALE,

Do

ROSARIO, V., LELAND, P., ADJEPONG, A., LIGHT, J., WOOLLETT, M. R., AND AZAD, A. F. 1988. Plasmodium falciparum: Ingested anti-sporozoite antibodies affect sporogeny in Anopheles stephensi mosquitoes. Experimental Parasitology 66, 171-182. In endemic areas, malaria-infected mosquitoes may feed upon humans who possess antibodies against malaria sporozoites. Therefore, we examined the effect that ingested anti-sporozoite antibodies have upon Plasmodium falciparum sporogony within Anopheles stephensi mosquitoes. Anti-sporozoite antibodies (IgG) traversed the midgut into the hemocoel within 3 hr following ingestion and, depending upon the titer, persisted for 6-24 hr. When fed to infected A. stephensi at 12 days postinfection (pi.), anti-sporozoite antibodies bound to sporozoites in the hemocoel, but not to sporozoites residing in the salivary glands of the same mosquitoes. Anti-sporozoite antibodies also bound to developing oocysts when fed to infected A. stephensi at 5 days p.i. Oocysts in mosquitoes that had been fed anti-sporozoite antibodies on Day 5 p.i. produced significantly more sporozoites than did oocysts in nonimmune-fed (Day 5 p.i.) mosquitoes. In addition, the sporozoites from Day 5 immune-fed mosquitoes were significantly more infective to cultured human hepatoma cells than were sporozoites from nonimmune-fed controls. Use of hetereologous immune feedings at Day 5 p.i. did not result in an enhanced production of sporozoites, suggesting that enhancement is related to the specificity of the antibody and is not merely a nutritional effect. 0 1988 Academic Press, Inc. Malaria; Protozoa; Plasmodium falciparum; INDEX DESCRIPTORS AND ABBREVIATIONS: Sporozoite; Oocyst; Anopheles stephensi, Anti-sporozoite antibody; Anti-R3 2 tet3 2 antibody; Indirect immunofluorescent assay (IFA); Enzyme-linked immunosorbent assay (ELISA); Postinfection (p.i.); Fluorescein isothiocyanate conjugate (FITC).

G. R.,

INTRODUCTION

Malarial infections are initiated when an infected Anopheles mosquito, during the act of biting, injects sporozoites into a susceptible host. After repeated exposure to the bites of infected mosquitoes, humans within malaria-endemic regions develop antibodies against sporozoites (Hoffman et al. 1986; Nardin et al. 1979; Nussenzweig and Nussenzweig 1985). Such antibodies have been shown to decrease in vitro infectivity of sporozoites to hepatoma cells (Hollingdale et al. 1984). In areas of the world where a portion of the human popu-

lation possesses antibodies against sporozoites, anophelines may frequently ingest anti-sporozoite antibodies in a bloodmeal. Ingested antibodies may, in turn, influence the parasite’s development within infected vectors. For example, anti-gamete antibodies have been shown to prevent gamete fertilization in the midgut of mosquitoes, aborting further parasite development within the vector (Carter and Chen 1976; Carter et al. 1984; Kashal et al. 1983). Recently, it has been shown that certain monoclonal antibodies against Plasmodium vivax gametes as well as some polyclonal 171 0014-4894188 $3.00 Copyright 6 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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VAUGHANETAL.

human sera, when fed to mosquitoes with gametocytes, inhibited gamete fertilization at high antibody titers but enhanced fertilization at low titers (Mendis et al. 1987). In the case of anti-gamete antibodies, the antibody-parasite interaction takes place in the lumen of the mosquito midgut as the antibodies are ingested along with the infective bloodmeal (i.e., gametocytes). The interaction of antibodies against oocysts and sporozoites is more complicated since both these lifestages reside outside the gut lumen of the mosquito. In order for ingested antibodies to bind to oocysts or sporozoites, antibodies must pass from the bloodmeal through the gut and into the hemocoel and, in so doing, retain their biological activity within the hemolymph. The passage of ingested host antibodies from the gut into the hemocoel of arthropod vectors has been reported previously for ticks (Ackerman et al. 1981; Ben-Yakir et al. 1986; Fujisaki et al. 1984), fleas (Azad and Emala 1987), and sarcophagid flies (Schlein et al. 1976). In this report, we examine the passage of ingested antibodies and the effect that such antibodies have upon P. falciparum oocyst production of sporozoites in Anopheles stephensi mosquitoes. MATERIALS

AND METHODS Parasites. Plasmodium falciparum (NF-54) gametocytes were produced as described by Ponnudurai et al. (1982). To infect mosquitoes, gametocyte cultures at ca. 5% gametocytemia were diluted 1:l with normal human sera and administered to Anopheles stephensi mosquitoes (3-5 days posteclosion) via glass membrane feeders. Mosquitoes were maintained at 26 C, 75% RH. Immune sera. Three different P. falciparumimmune bloodsources were used: rat anti-sporozoite, rabbit anti-R32tet,,, and human anti-sporozoite. (A) Female Lewis white rats were immunized against whole P. falciparum sporozoites by anesthetizing the rats and then allowing ca. 150 P. falciparum-infected A. stephensi mosquitoes (1417 days p.i.) to feed upon these rats. Rats were subjected to three infectious feedings at weekly intervals and, prior to use, sera had IFA titers > 1:5000against P. falciparum sporozoites. Sera were characterized for binding activity to the repeat region of the circumsporozoite protein by eluting

sera samples over R32-leucine-arginine (R32LA) covalently linked to Afft-gel beads (Bio-Rad, Richmond, CA). Intact and R32-depleted sera were compared for their respective binding activity to P. falciparum sporozoite extracts and to R32LA with ELISA. The majority of binding activity in the rat sera was found to be against the repeat region of the circumsporozoite protein. (B) A recombinant construct of the repeating portion of the P. falciparum circumsporozoite protein (i.e., R32tet,,; Young et al. 1985) was used to immunize a New Zealand white rabbit. The rabbit received six subcutaneous injections of the construct at ca. monthly intervals, using Freund’s complete adjuvant for the primary injection (350 pg R32tet,,) and Freund’s incomplete adjuvant for the boosts (100 p,g R32tet,,). Two weeks after the fifth injection, serum had an IFA titer of 1:lOOOagainst methanol-fixed P. falciparum sporozoites and was used in antibody passage studies below. Two weeks after the sixth injection, serum was collected for use in sporogony studies. The IgG fraction was partially purified from this serum by ammonium sulfate precipitation and subsequent dialysis against phosphate-buffered saline, then reconstituted to the original serum volume with nonimmune human serum. Prior to use, the serum had an IFA titer of 1:4000. (C) Pooled human sera from malariaendemic areas in Kenya (provided to us by Drs. N. P. Dinh and G. Campbell, Centers for Disease Control) with an IFA titer 5 1:lOOOwere partially purified as above for use in sporogony studies. Control sera. Nonimmune sera consisted of either partially purified sera from normal rabbits or humans or complete blood from normal rats. Heterologous immune bloodsources consisted of pooled monkey sera against P. cynomolgi sporozoites with IFA titers 3 1:4000 (provided by Dr. A. Cochrane, New York University) and rats immunized against Rickettsia typhi (IFA 2 1:SOOO) (Azad and Emala 1987). All control sera had negative IFA reactivity to P. falciparum sporozoites. Antibody administration. Rabbit, human, and monkey sera were mixed (1: 1) with washed uninfected human erythrocytes and fed to infected mosquitoes via membrane feeders. Rat antibodies were administered via live (anesthetized) host. Antibody passage through mosquito midgut. Uninfected A. stephensi mosquitoes were used to examine antibody passage. Two different immune sera were examined: rat anti-sporozoite and rabbit anti-R32tet,, antibodies. In each trial, one group of mosquitoes was fed an immune bloodmeal while a control group was fed a nonimmune bloodmeal. Immediately after feeding and at selected intervals thereafter, ca. 15 mosquitoes were removed from each of the two groups, chilled, and washed in saline and hemolymph was collected. Two different methods of collection were used. In initial trials, mosquitoes were laid upon their sides

Plasmodiumfalciparum:

ANTIBODY

adjacent to a drop (20 ~1) of buffered saline, the legs were excised at the trochanter-femoral joint, and the coxae were gently pulled away from the thorax to the point where the exoskeleton ripped but the connecting muscles remained intact. With gently pressure applied to the thorax, a miniature current was created in the dissecting fluid in close proximity to the coxae with a swirling motion of forceps. This “flushing” action aided in drawing out hemolymph from the thorax. Later trials utilized the more efficient method of hemocoel perfusion. In this method, small perforations were made in the pleura of abdominal segments 7 and 8. The mosquito was then restrained via light vaccuum onto the bent point of a 20-gauge syringe needle. Saline solution was introduced into the thorax at the membranous area adjacent to the mesothoracic spiracle with a fine-tipped glass needle mounted in a micromanipulator. The hemocoel was flushed with ca. 5 ~1 saline and the perfusate exuding from the abdominal incisions was collected in a lo-p1 microcapillary tube. Hemolymph samples were either frozen at - 70 C for later assay or spotted unto a glass microscope slide to air-dry. During collection, it was crucial that the digestive tract remain intact so as not to contaminate hemolymph samples with gut contents. Dissections performed on mosquitoes fed a sucrose solution containing methylene blue prior to hemolymph collection demonstrated that neither of these techniques ruptured the digestive tract of the mosquito. Following hemolymph collection, midguts were excised and the ingested blood was emptied into a drop (ca. 20 ~1) of saline. Midgut contents for each time interval were pooled, immediately frozen on dry ice, and stored at - 70 C. Hemolymph and gut samples were assayed for the presence of antibodies with IFA techniques using appropriate antigen slides and FITC conjugates to host w. Antibody binding in vivo. Antibody binding was examined at two different stages of parasitic development: oocysts and hemocoel sporozoites. To examine in vivo binding of ingested host antibodies to oocysts, P. falciparum-infected A. stephensi were used at a time in the sporogonic cycle when oocysts were developing (i.e., Day 5 p.i.). These mosquitoes were fed upon a sporozoite-immune rat. Three hours after feeding, mosquitoes were quick frozen in embedding compound (O.C.T., Miles Laboratories) and sagittal sections (4-6 pm) were prepared using a cryostat. Frozen sections were stained with FITC-labeled anti-rat IgG and examined under uv microscopy. To examine binding of ingested anti-sporozoite antibodies to hemocoel sporozoites, P. fulciparum-infected A. stephensi were fed on an immune rat at a time when sporozoites were being released into the hemocoel from mature oocysts and initiating salivary gland invasion (i.e., Day 12, p.i). A control group of infected mosquitoes was fed upon a nonimmune rat. At intervals of 3, 6, 24, and 48 hr

EFFECT

ON SPOROGONY

173

postfeeding, 10 mosquitoes were selected at random from each group. Hemolymph was collected by perfusion and spotted individually onto a microscope slide, air-dried, and fixed in methanol. Following hemolymph collection, the salivary glands from the same mosquitoes were excised, washed, crushed onto a slide, air-dried, and methanol-fixed. Preparations were stained with FITC-conjugated second antibody to rat IgG to discern whether recovered sporozoites had rat anti-sporozoite antibodies on their surfaces. lntruthoracic inoculation of antibody. Rabbit antiR32tet,, serum (IFA titer 5 1:lOOO)was injected intrathoracically (0.1 ~1) into P. falciparum-infected A. stephensi (17 days p.i.) to examine passage and binding of anti-sporozoite antibodies through mosquito salivary glands. At intervals of 1, 3, 6, and 18 hr postinoculation, salivary glands were dissected out intact and washed twice in sterile saline to remove exogenous serum. Washed glands from each time interval were pooled, crushed unto a glass slide, and stained with FITC-conjugated anti-rabbit IgG for detection of anti-R32tet,, antibody-coated sporozoites. In addition, hemolymph samples were collected and stained at 1 and 3 hr postinoculation. Effect on sporogony. Separate experiments were conducted using rabbit anti-R32tet,,, rat antisporozoite, and human anti-sporozoite antibodies. In each experiment, groups of infected mosquitoes (ca. 500 per cage) were fed at 5 days p.i. (early oocyst development) on either immune or nonimmune (control) bloodsources. On Day 14 pi., sporozoite infections were quantified. Initial pilot trials utilized single large pools of 50 mosquitoes to compare salivary gland infections between immune-fed vs. nonimmune-fed mosquitoes. Salivary glands were excised and triturated and sporozoite counts were performed using a hemacytometer. Harvested sporozoites were then tested for their infectivity to cultured Hep G-2 human hepatoma cells (Hollingdale er al. 1984). In later trials, salivary gland sporozoites counts were performed on four pools of 10 mosquitoes each, for statistical purposes. Rates of mosquito infection (%), as well as mean numbers of oocysts per infected midgut, were determined by staining excised midguts with mercurochrome and examining them under phase-contrast microscopy. Oocyst counts were performed at Day 8 p.i. (n = 10-30) and then again at Day 14 p.i. (n = 40) on the same mosquitoes used for salivary gland sporozoite counts. Oocyst diameters were measured with an ocular micrometer. In certain experiments, comparative counts were made on sporozoites from the hemocoel (four pools of perfused hemolymph from two mosquitoes each), whole mosquito bodies (one pool of 40 mosquitoes each) (Ozaki et al. 1984), and salivary glands (four pools of 10 mosquitoes each). Data analysis. Mean values for sporozoite and oocyst counts within each experiment were compared

174

VAUGHANETAL.

between immune-fed and nonimmune-fed groups with Student’s t test. Size frequency data on oocyst diameters were compared using contingency tables and x2 probability distributions. The sporozoite yield per oocyst for each group was calculated as follows. An “expected” number of oocysts on Day 14 pi. was obtained by multiplying the mean number of oocysts per infected midgut on Day 8 p.i. by the infection rate. The mean number of oocysts actually observed on Day 14 pi. was then subtracted from the calculated expected value. In our experiments, the expected value proved almost invariably to be larger than the observed value, yielding a positive number. The assumption of this model was that missing oocysts (i.e., expected minus observed) represented oocysts that had released sporozoites and subsequently atrophied, becoming undetectable. Data from our laboratory support the validity of this assumption. The number of sporozoites observed on Day 14 p.i. was then divided by the number of oocysts missing to arrive at an estimate of the number of sporozoites produced per oocyst. Separate calculations were performed for each replicate within a group to obtain a mean value for that group and means were compared between immune-fed vs. nonimmune-fed groups with Student’s t test.

ditional studies using affinity-purified FITC-conjugated antisera to rat IgG (Fab) and rat IgG (Fc) fragments (Cappel Laboratories) indicated the presence of intact antibodies in the hemolymph. Sagittal sections of immune-fed mosquitoes at Day 5 p.i. indicated that ingested rat antibodies against whole sporozoites traversed the midgut epithelium and bound to developing oocysts (Fig. 1). Sections were counterstained with Giemsa and examined under light microscopy to confirm that the fluorescing structures were indeed oocysts. Ingested rat antibodies also bound to sporozoites in the hemocoel of infected mosquitoes (Day 12 p.i.) within 3 hr following ingestion (Fig. 2A). At 6 hr postfeed, the pattern of immunofluorescence on hemocoel sporozoites was more patchy in its distribution (Fig. 2B). At 24 hr postfeed, antibodycoated sporozoites in the hemolymph were fewer in number, immunofluorescence was RESULTS very faint, and many of the sporozoites reAntibody passage and binding. Ingested covered did not possess antibody on their antibody passed relatively rapidly (~3 hr surfaces. By 48 hr, none of the sporozoites after ingestion) from the bloodmeal into the in the hemocoel of immune-fed mosquitoes had antibody on their surfaces. In contrast, hemocoel of uninfected Anopheles antibody-coated sporozoites were never restephensi mosquitoes (Table I). Antibody persisted longer in the gut than in the he- covered from the salivary glands of immolymph. Persistance of antibody in the mune-fed mosquitoes, despite the fact that hemolymph was greater at a higher titer (up salivary gland invasion was taking place to 18 hr at IFA titer of 15000) than at a during this time as evidenced by the prolower titer (6 hr at IFA titer of 1: 1000). Ad- gressively increasing numbers of sporozoTABLE I Passage of Anti-Plasmodium falciparum Antibodies (IgG) from the Bloodmeal into the Hemocoel of Uninfected Anopheles stephensi Mosquitoes as Determined by Indirect Fluorescent Antibody Assays” Rat anti-sporozoite (IFA titer > 15000)

Rabbit Anti-R32tet,, (IFA P 1:lOOO)

Time posffeeding

Bloodmeal

Hemolymph

Bloodmeal

Oh

+

-

+

-

3 hr 6hr 18 hr 24 hr 48 hr

+ + + + +

+ + + -

+ + +

+ +

Hemolymph

-

a Rat anti-whole sporozoite antibodies were administered via live (anesthetized) host. Rabbit anti-R32tet,, antibodies were administered via membrane feeder.

Plasmodiumfalciparum:

ANTIBODY

EFFECT ON SPOROGONY

FIG. 1. Sagittal section of Plasmodium falciparum-infected Anopheles stephensi midgut prepared 3 hr after mosquito fed upon a sporozoite-immune rat (IFA titer 2 15000). Oocysts were 5 days old. Preparation was stained with FITC-conjugated goat anti-rat IgG. This demonstrates that ingested anti-sporozoite antibodies bind to developing oocysts.

ite5i recovered from the salivary glands of bot.h nonimmune- and immune-fed mosquitoe S. 7To confirm that antibody in the he-

molymph was not able to traverse the salivary glands, rabbit anti-R32tet3, serum \was injected directly into the thoraces of infected mosquitoes. None of the sporozoi ites

176

VAUGHAN

recovered from salivary glands (1, 3,6, and 18 hr post-inoculation) were positively stained with FITC anti-rabbit IgG conjugate whereas sporozoites recovered from the hemolymph at 3 and 6 hr were brightly stained. To ensure that the immune serum did indeed have activity against salivary gland sporozoites, slides were rinsed and the same serum used in the inoculations was incubated with the gland samples and standard IFA procedures were performed using the same conjugate. This resulted in moderate to strong immunofluorescence, indicating that antibodies in the serum, when applied to exposed sporozoites released from salivary glands by trituration, had excellent binding activity. Thus, antibody that was either ingested or injected into the hemocoel bound to sporozoites within the hemocoel but not to sporozoites residing in the salivary glands, demonstrating that mosquito salivary glands are impervious to the passage of antibody. Effect on sporogony. When rat antisporozoite or rabbit anti-R32tet,, antibodies were fed to Plasmodium falciparuminfected mosquitoes at Day 5 p.i., the numbers of salivary gland sporozoites in the anti-sporozoite-fed and the anti-R32tet,,fed mosquitoes were 2-3 times greater than those of corresponding controls. In addition, the infectivity of salivary gland sporozoites to Hep G-2 human hepatoma cells was increased significantly over that of sporozoites from nonimmune-fed mosquitoes (t test, P < 0.05, Table II). To determine if the increase in salivary gland sporozoite numbers resulted from an increase in the sporozoites’ capacity to invade mosquito salivary glands or from an overall increase in sporozoite production, more de-

ET AL.

tailed studies were conducted. In two separate experiments (Table III), mosquitoes that fed on sporozoite-immune rats at Day 5 p.i. had more sporozoites in their salivary glands, hemocoel, and within whole body homogenates than did nonimmune-fed controls. This indicates that increased numbers of salivary gland sporozoites were due to an overall increase in sporozoite production, rather than (or in addition to) an increase in sporozoite infectivity to salivary glands. In subsequent trials where mosquito midguts were examined on Day 8 pi., no significant differences were detected in the mean numbers of oocysts per infected mosquito between Day 5 immune-fed and Day 5 nonimmune-fed groups. Moreover, the oocysts from immune-fed mosquitoes were larger in diameter (Z = 31.3 pm) than those from nonimmune-fed controls (X = 27.5 pm) (x2 = 36.9; df = 9). This implied that enhanced sporozoite production may have resulted from enhanced numbers of sporozoites per oocyst rather than enhanced numbers of oocysts per mosquito. In view of this, subsequent experiments included oocyst sampling on Day 8 p.i. and estimates for the sporozoite yield per oocyst were calculated. Applying these calculations to experiments with a sporozoiteimmune rat as the immune bloodsource, there were significant increases in the estimated sporozoite yield per oocyst for immune-fed mosquitoes as compared to nonimmune-fed controls (Table IV). Incorporation of oocyst data from Day 8 p.i. provided for a more in-depth analysis. For instance, the model illustrated the occurrence of a five- to sixfold enhancement of sporozoite production when calculated on a per oocyst basis, compared to only a twofold enhance-

FIG. 2. Antibody-coated Plasmodium falciparum sporozoites recovered from the hemocoel of stephensi mosquitoes fed on a sporozoite-immune rat (IFA > 1:SOOO)12 days following infection. Sporozoites were recovered by hemocoel perfusion, fixed in methanol, and stained with FITC-conjugated goat anti-rat IgG. (A) 3 hr after immune bloodmeal. (B) 6 hr after immune bloodmeal. (C) sporozoite clumping 3 hr after immune bloodmeal. This demonstrates that ingested anti-sporozoite antibodies cross the midgut and bind to hemocoel sporozoites.

Anopheles

Plasmodiumfalciparum:

ANTIBODY

EFFECT

ON SPOROGONY

177

178

Plasmodium

VAUGHAN

ET AL.

TABLE II Sporozoite Infectivity and Production in Anopheles stephensi Salivary Glands when Mosquitoes were Fed an Immune Bloodmeal 5 Days following Infection”

falciparum

Rat Anti-sporozoite

Rabbit Anti-R32tet,,

Treatment

Infectivity

No. gland spz./mosq.

Infectivity

No. gland spz./mosq.

Immune-fed Nonimmune-fed (Control)

139 +- 4*

16,020

86 2 4*

32,300

94 k 8

9,492

55 2 8

9,100

o On Day 14 post-infection, salivary glands from 50 mosquitoes were dissected and triturated for each group. Sporozoites were enumerated with a hemacytometer. To measure infectivity, harvested sporozoites were adjusted to 30,000 by making appropriate dilutions and incubated with Hep G-2 human hepatoma cells for 2 hr, fixed, and stained with an immunoperoxidase indicator. Infectivity values represent the mean number (n = 3) of successful sporozoite invasions. * Significantly greater than corresponding control value (t test, P < 0.05).

ment observed in numbers of sporozoites recovered from the salivary glands on Day 14 p.i. (see Table IV). Naturally acquired human antibodies to P. falciparum sporozoites also significantly increased sporozoite yield per oocyst over that in nonimmune-fed controls (Table V). Feeding mosquitoes on monkey anti-P. cynomolgi sera did not result in sporozoite enhancement (Table V). Similarly, feeding infected mosquitoes on a rat immunized against a totally unrelated parasite (i.e., anti-R. typhi, IFA titer 2 1:5000) on Day 5 p.i. did not result in an increase in sporozoite production (data not shown). This suggests that the mechanism of sporozoite enhancement is related to antibody specificity and is not just a nutritional benefit of increased immunoglobulins in the bloodmeal. DISCUSSION

Both mitotic (oocyst) and postmitotic (sporozoite) stages of the malaria parasite reside outside of the gut lumen. Therefore, if ingested antibody is to interact with these stages, it must first cross the midgut epithehum into the hemocoel. We found that antisporozoite antibodies contained within a bloodmeal traversed the midgut of Anopheles stephensi mosquitoes within 3 hr following ingestion. Once in the hemolymph, antibodies retained binding activity to antigens present on developing oocysts and

hemocoel sporozoites. This finding is significant because it demonstrates that, in at least some mosquito species, host antibody ingested in a bloodmeal has the potential for modulating parasite/pathogen development within the vector hemocoel (e.g., arboviruses). This is particularly relevant to malaria since most of the parasite’s development in the vector takes place outside of the gut lumen. In A. stephensi, ingested antibody remained bound to sporozoites in the hemocoel for up to 24 hr. However, the changing pattern of immunofluorescence over time suggested that certain dynamic changes were taking place in the hemocoel of immune-fed mosquitoes. At 3 hr postfeed (Fig. 2A), most sporozoites appeared to be completely coated with antibody but at 6 hr (Fig. 2B) the majority of sporozoites displayed a more patchy pattern of immunofluorescence, suggesting that the immune complexes were capping or perhaps shedding. At 24 hr, patchy immunofluorescence was faint and many sporozoites were free of antibody. These uncoated sporozoites may have represented either sporozoites that had completely shed their antibody coats or “new” sporozoites released from oocysts subsequent to the immune feeding. The possibility that antibody binding to hemocoel sporozoites may somehow retard sporozoite invasion in salivary glands was

Plasmodiumfalciparum:

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ANTIBODY EFFECT ON SPOROGONY TABLE III

Sporozoite Production in Anopheles stephensi Mosquitoes When Mosquitoes Were Fed Either a Sporozoite-Immune (Serum IFA Titer 3 1:5000) or Nonimmune (Control) Rat 5 Days following Infection”

Plasmodium

falciparum

Sporozoite count (per mosquito) Treatment Immune-fed Control Immune-fed Control

Salivary glands

Hemocoel

Experiment 1 33,530 Ii 13,197t 1,648 2 1,494$ 9,706 2 7,976 246 ” 402 Experiment 2 36,393 k 11,969t 804 2 366$ 10,461 * 9,161 48 2 53

Whole bodyb 7,917 1,833 40,726 17,185

a Data. were collected on Day 14 postinfection. Salivary glands were excised and triturated in chilled RPM1 media for each group (four pools of 10 mosquitoes each). Hemocoel sporozoites were collected by hemocoel perfusion (four pools of 2 mosquitoes each). For whole body counts, mosquitoes were minced and centrifuged over glass wool columns (one pool of 40 mosquitoes each). Sporozoite counts were made with a hemacytometer. b For Experiment 1, mosquitoes were centrifuged once over glass wool. For Experiment 2, the flow-through was resuspended and centrifuged a second time; hence the apparent difference in sporozoite recovery for whole bodies between the two experiments. t Significantly greater than control value (t test, P < 0.05). f Significantly greater than control value (t test, P < 0.10).

suggested by the preponderance of large clumps of antibody-coated sporozoites recovered from the hemocoel of immune-fed mosquitoes at 3 and 6 hr postfeed (Fig. 2C). Clumping was only rarely observed with sporozoites recovered from the hemocoel of nonimmune-fed mosquitoes. In contrast to the midgut, antibodies did not pass through the salivary gland wall. Differences in permeability between these two organs may be a reflection of the different embryological origins of salivary gland (ectoderm = chitin lined) versus midgut tissue (endoderm = no chitin). Despite their presence in the hemocoel, antibodycoated sporozoites were never recovered from the salivary glands of the same mosquitoes, indicating that antibody-coated sporozoites do not carry antibody with them into the glands. Whether antibodycoated sporozoites in the hemocoel are actually inhibited from invading the salivary glands or whether they shed their antibody coats and go on to invade salivary glands has yet to be determined. Nevertheless, our data indicate that once inside the salivary

glands, sporozoites are sheltered from further activity of antibody in the hemolymph. Thus, timing of the immune bloodmeal may be of critical importance if antibody is to block sporozoite invasion of salivary glands. When ingested by Plasmodium falciparum-infected A. stephensi mosquitoes on Day 5 p.i., anti-sporozoite antibodies increased subsequent production and infectivity of sporozoites. Oocysts in immunefed mosquitoes were larger and, more importantly, they produced significantly more sporozoites than did oocysts in nonimmune-fed mosquitoes (Tables IV-V). Use of hetereologous antibodies (i.e., anti-P. cynomolgi sporozoite and anti-R. typhi) did not result in a similar enhancement suggesting that enhancement is related to the specificity of the ingested antibodies. Although the mechanism(s) by which anti-sporozoite antibodies enhance sporozoite production is not known, it is assumed that antibody binds to the oocyst in some manner in order to exert its effect. We found that ingested rat anti-sporozoite antibodies bound to 5-

180

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ET AL.

TABLE IV Infections in Anopheles stephensi Mosquitoes when Mosquitoes Were Fed Either a Sporozoite-Immune (IFA Titer > 15000) or Nonimmune (Control) Rat 5 days following Infection”

Plasmodium

falciparum

Treatment Parameters

Control

Immune

Oocyst infection rate (Day 8 p.i.) No. oocysts per infected midgut (Day 8 pi.) Expected No. oocysts per 10 mosquitoes (Day 14 p.i.) Observed No. oocysts per 10 mosquitoes (Day 14 pi.) Oocyst deficit (expected - observed) Observed No. sporozoites per 10 mosquitoes (Day 14 p.i.) Estimated No. sporozoites produced per oocyst

38% (n = 15) 6.6 2 4.4

33% (n = 15) 3.6 z!z3.8 (n = 5) 0.33 x 3.6 x 10 = 11.8 3.3 k 2.3 (n = 3) 8.5 2 2.3 (n = 3) 49,333 f 13,844 (n = 3) 5,878 2 826* (n = 3)

(n = 5)

0.38 x 6.6 x 10 = 25.1 2.5 f 3.0 (n = 4)

22.6 -+ 3.0 (n = 4) 27,567 2 24,760 (n = 3)

1,116 ? 965 (n = 3)

a Oocyst data were collected on Day 8 post-infection (p.i.). From these data, estimates were calculated for the numbers of oocysts to be expected on Day 14 p.i. On Day 14 pi., oocyst and salivary gland sporozoites within the same individuals were enumerated (four pools of 10 mosquitoes each). Sporozoite numbers for each replicate were divided by the corresponding oocyst deficit (expected - observed) to estimate the sporozoite yield per oocyst for each treatment. * Significantly greater than control value (f test, P < 0.05).

day-old oocysts (Fig. l), but the precise nature of this binding could not be determined. Ingested antibody may have bound to antigens present only on the oocyst surface or it may have penetrated the oocyst and bound to internal antigens such as newly forming circumsporozoite protein present on the oocyst plasmalemma (Posthuma et al., 1987). Such information can only be determined by electron microscopy. Because extrapolation of laboratory data to the field requires caution, we designed our experimental parameters so that they would be reasonably consistent with events that could occur in a field situation. The rationale for selecting Day 5 p.i. was based upon entomological considerations. We felt that a j-day interval between bloodmeals represented a reasonable approximation of feeding frequencies for A. stephensi mosquitoes in the field (assuming a gonadotrophic duration of 2-3 days plus several days for the gravid female to oviposit, return,

and find another host). Gametocyte cultures were diluted to achieve a low production of oocysts and thereby attain a more realistic approximation of mosquito infections occurring in the wild. We examined different immune bloodsources; the assumption being that immune rats simulated naturally acquired immunity to the whole sporozoite, immune rabbit sera more closely simulated immunity acquired by vaccine to the circumsporozoite protein, and serologically positive human sera from Kenya simulated an actual situation that could occur within a malaria-endemic area. The phenomenon of sporozoite enhancement may be a pertinent factor for malaria transmission under certain conditions. For example, enhancement would be more apt to occur in hyperendemic areas where both transmission rates and human antisporozoite antibody titers are high than in areas where mosquito transmission and antibody titers are low. It also seems unlikely

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181

ANTIBODY EFFECT ON SPOROGONY TABLEV

Plasmodium

falciparum

Infections in Anopheles stephensi Mosquitoes after Receiving a Bloodmeal 5 Days following Infection” Treatment

Parameters Oocyst infection rate (Day 8 p.i.) No. oocysts per infected midgut (Day 8 p.i.) Expected No. oocysts per 10 mosquitoes (Day 14 p.i.) Observed No. oocysts per 10 mosquitoes (Day 14 p.i.) Oocyst deficit (expected - observed) Observed No. sporozoites per 10 mosquitoes (Day 14 pi.) Estimated No. sporozoites produced per oocyst

Nonimmune (Normal Human) 69% (n = 13) 19.7 !Z 13.1 (n = 9) 0.69 x 19.7 x 10 = 135.9 31.0 2 28.1 (n = 4) 104.9 + 28.1 (n = 4)

251,831 ? 70,141 (n = 4)

2,472 ? 590 (n = 4)

Heterologous immune (Monkey anti-P. cynomolgi) 75% (n = 16)

11.7 +- 7.3 (n = 12) 0.75 x 11.7 x 10 = 87.7 16.7 f 4.2 (n = 4) 70.9 k 4.2 (n = 4) 103,694 * 42,713 (n = 4) 1,475 * 625 (n = 4)

Immune (Human endemic) 58% (n = 12) 7.5 f 6.1 (n = 7)

0.58 x 7.5 x 10 = 43.5 16.0 * 8.5 (n = 3) 27.5 * 8.5 (n = 3) 195,700 +- 55,020 (n = 3) 7,771 ” 3,394* (n = 3)

a Immune bloodmeal consisted of partially purified human sera from Kenya with reactivity against P. fakisporozoites (IFA titer 2 1:lOOO).Heterologous immune control sera consisted of monkey sera with reactivity against P. cynomolgi sporozoites (IFA 2 1:4000) but not against P. falciparum sporozoites. Nonimmune control sera consisted of normal human sera. Sera were mixed (1: 1) with washed human erythrocytes and administered via membrane feeders. Oocyst data were collected on Day 8 postinfection (p.i.). From these data, estimates were calculated for the numbers of oocysts to be expected on Day 14 p.i. On Day 14 p.i., oocyst and salivary gland sporozoites within the same individuals were enumerated (four pools of 10 mosquitoes each). Sporozoite numbers for each replicate were divided by the corresponding oocyst deficit (expected - observed) to estimate the sporozoite yield per oocyst for each treatment. * Significantly greater than control value (t test, P < 0.05).

parum

that the enhancing effect of anti-sporozoite antibodies would directly alter infection rates (i.e., proportion of infected mosquitoes) since enhancement occurs only in mosquitoes that have already acquired infections. Rather, anti-sporozoite antibodies may increase the sporozoite dose delivered per infected mosquito bite. Since sporozoites, upon entering the human host, must contend with immune defenses (such as antibody) before reaching the liver, an increase in numbers of sporozoites inoculated may increase the numbers that succeed in invading the liver. Recent studies in Kenya (Hoffman et al. 1987) have shown that P. falciparum sporozoite transmission occurs despite the presence of antibody in the resident human population. Furthermore, studies in mice (Egan et al. 1987)

have demonstrated that high levels of antisporozoite antibodies protect only to low numbers of challenge sporozoites. Thus, the ability of malarial parasites to respond to continual antibody exposure by increasing their numbers within the mosquito vector may confer an adaptive advantage to the parasite, serving to overwhelm the immune response of the host and ensuring the parasite’s survival.

ACKNOWLEDGMENTS We thank D. Hayes and C. Sigler for technical assistance. This study was supported primarily by AID Grant DPE 6453-C-3051and in part by NIH Grant AI17828 and UNDP/World Bank/WHO special programme for Research and Training in Tropical Diseases ID-870105.

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ET AL.

and P. vivax sporozoites into cultured cells: An in vitro assay of protective antibodies. Journnl of ZmACKERMAN, S., CLARE, F. B., MCGILL, T. W., AND munology 132, 909-913. SONENSHINE, D. E. 1981. Passage of host serum KASHAL, E. C., CARTER, R., RENER, J., GROTENcomponents, including antibody, across the digesDORST, C. A., MILLER, L. H., AND HOWARD, R. J. tive tract of Derrnacentor variabilis. Journal of Par1983. Monoclonal antibodies against surface deterasitology 67, 737-740. minants on gametes of Plasmodium gallinaceum AZAD, A. F., AND EMALA, M. A. 1987. Suppression block transmission of malaria parasites to mosquiof Rickettsia typhi transmission in fleas maintained toes. Journal of Immunology 131, 2557-2562. on murine typhus-immune rats. American Journal MENDIS, K., PEIRIS,J. S. M., PREMAWANSA,S., UDof Tropical Medicine and Hygiene 37, 629-635. AGAMA, P. V., MUNESINGHE, Y., RANAWAKA, M., BEN-YAKIR, D., Fox, J. C., HOMER, J. T., AND CARTER, R., AND DAVID, P. H. 1987. Immune modBARKER, R. W. 1986. Quantitative studies of host ulation of parasite transmission in Plasmodium immunoglobulin G passage into the hemocoel of the vivax malaria. Anti-gamete antibodies can both ticks Amblyomma americanum and Dermacentor block and enhance transmission. In “Molecular variabilis. In “Morphology, Physiology and BehavStrategies of Parasitic Invasion” (N. Agabian, H. ioral Ecology of Ticks” (J. R. Sauer and J. A. Hair, Goodman, and N. Nogueira, Eds.), pp. 417426. Eds.), Wiley, New York. A. R. Liss, New York. CARTER,R. R., AND CHEN, D. H. 1976. Malaria trans- NARDIN, E. H., NUSSENZWEIG, R. S., MCGREGOR, mission blocked by immunization with gametes of I. A., AND BRYAN, J. H. 1979. Antibodies to sporoPlasmodium gallinaceum. Nature (London) 263, zoites: Their frequent occurrence in individuals Iiv57-60. ing in an area of hyperendemic malaria. Science 206, CARTER,R. R., MILLER, L. H., RENER,J., KAUSHAL, 597-599. D. C., KUMAR, N., GRAVES,P. M., GROTENDORST, NUSSENZWEIG, V., AND NUSSENZWEIG, R. S. 1985. C. A., GWADZ, R. W., FRENCH, C., AND WORTH, Circumsporozoite proteins of malaria parasites. Cell D. 1984. Target antigens in malaria transmission 42, 401403. blocking immunity. Philosophical Transactions of OZAKI, L. S., GWADZ, R. W.. AND GODSEN, G. N. the Royal Society of London B 307, 201-213. 1984. Simple centrifugation method for rapid sepaEGAN, J. E., WEBER, J. L., BALLOU, W. R., ration of sporozoites from mosquitoes. Journal of HOLLINGDALE, M. R., MAJARIAN, W. R., GORParasitology 70, 83 l-833. DON, D. M., MALOY, W. L., HOFFMAN, S. L., POSTHUMA, G., MEIS, J. F. G. M., VERHAVE, J. P., WIRTZ, R. A., SCHNEIDER,I., WOOLLET’T,G. R., HOLLINGDALE, M. R., PONNUDURAI, T., AND YOUNG, J. F., AND HOCKMEYER,W. T. 1987. EfIiGEUZE, H. J. 1987. Immuno-electron microscopy of cacy of murine malaria sporozoite vaccines: Implithe circumsporozoite protein of Plasmodium fulcications for human vaccine development. Science parum during oocyst development in the midgut of 236, 453456. Anopheles stephensi. In “Proceedings, 3rd ZnternaFUJISAKI, K., KAMIO, T., AND KITAOKA, S. 1984. tional Conference on Malaria and Babesiosis. AnPassage of host serum components, including antinecy, France. September 1987.” Abstract bodies specific for Theileria sergenti across the di- PONNUDURAI, T., LENSEN, A. M. W., LEEUWENgestive tract of argasid and ixodid ticks. Annuls of BERG, A. D. E. M., AND MEUSWISSEN, J. H. E. T. Tropical Medicine and Parasitology 78, 449-450. 1982. Cultivation of fertile Plasmodium falciparum HOFFMAN, S. L., OSTER, C. N., PLOWE, C. V., gametocytes in semiautomated systems. Static culWOOLLETT, G. R., BEIER, J. C., CHULEY, J. D., tures. Transactions of the Royal Society of Tropical WIRTZ, R. A., HOLLINGDALE, M. R., AND Medicine and Hygiene 76, 812-818. MUGAMBI, M. 1987. Naturally acquired antibodies SCHLEIN, Y., SPIRA, D. T., AND JACOBSON, R. L. to sporozoites do not prevent malaria: Vaccine de1976. The passage of serum immunoglobulins velopment implications. Science 237, 639-642. through the gut of Sarcophaga falculata, Pand. Annals of Tropical Medicine and Parasitology 70, 227HOFFMAN, S. L., WISTAR, R., BALLOLJ, W. R., 230. HOLLINGDALE, M. R., WIRTZ, R. A., SCHNEIDER, I., MARWOTO, H. A., AND HOCKMEYER, W. T. YOUNG, J. F., HOCKMEYER, W. T., GROSS, M., BALLOU, W. R., WIRTZ, R. A., TROSPER, J. H., BEAU1986. Immunity to malaria and naturally acquired DOIN, R. L., HOLLINGDALE, M. R., MILLER, antibodies to the circumsporozoite protein of PlusL. H., DIGGS, C. L., AND ROSENBERG, M. 1985. medium falciparum. New England Journal of MedExpression of Plasmodium falciparum circumicine 315, 601406. sporozoite proteins in Escherichiu coli for potential HOLLINGDALE, M. R., NARDIN, E. H., THARAVANJI, use in a human malaria vaccine. Science 228, 958 S., SCHWARTZ, A. L., AND NUSSENZWEIG,R. S. 962. 1984. Inhibition of entry of Plasmodium falciparum REFERENCES