EXPERIhlENTAL
PARASITOLOGY
41, 167-171
(1977)
Plasmodium vivax: Correlation of Circumsporozoite Precipitation (CSP) Reaction with Sporozoite-Induced Protective Immunity in Man AND DAVID F. CLYDE l
VINCENT C. MCCARTHY International
Health
Program, Baltimore,
University Maryland
of Maryland School of Medicine, 21201, U.S.A.
(Accepted for publication
21 April 1976)
MCCARTHY, VINCENT C., AND CLYDE, DAVID F. 1977. Plasmodium vivax: Correlation of circumsporozoite precipitation (CSP) reaction with sporozoite-induced protective immunity in man. Experimental Parasitology 41, 167-171. A volunteer was immunized with X-irradiated sporozoites of Plasmodium vivax against a nonirradiated, infective sporozoite challenge of the same species and strain. Shortly before his first successful challenge, his serum was found to contain anti-sporozoite antibodies. Subsequent immunizations and challenges with another strain demonstrated that his protective immunity lasted 3-5 months and could be restored or boosted by challenge with a small number of nonirradiated, infective sporozoites. His CSP serum reaction paralleled his level of protective immunity. INDEX DESCRIPTORS: Plasmodium vivax; Sporozoa; Malaria; Anopheles stephensi; Diptera; X-irradiated sporozoites; Human malarial immunity; Circumsporozoite precipitation reaction.
INTRODUCTION
The circumsporozoite precipitation reaction was first described in an Anopheles stephensS’lasmodium berghei-rodent test system by Vanderberg et al. (1969). They noted that many sporozoites, incubated in immune serum, ejected a tail-like precipitate: Later work by Nussenzweig et al. (1969) and by Spitahly and Nussenzweig ( 1973) demonstrated the immunological specificity of this reaction. More recently Nussenzweig et al. (1973) and Nussenzweig and Chen (1974) examined the resl”‘ll”c of Plawwdium fulcipurum and Piasmotliurn vivux sporozoites to immune nonhuman sera. The CSP reaction of P. fulciparum sporo1 Present address: Department of Tropical Medicine and Medical Parasitology, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, Louisiana 70112, U.S.A.
zoites in immune human serum was first observed by Clyde et al. (1973a, b). A similar reaction took place when P. vivax sporozoites were placed in immune human serum (Clyde et al. 1975); normal serum was nonreactive, as were sera from individu’als with high levels of immunity to the blood stage of the parasite. There was no cross-reaction of vivax or falciparum sporozoites with heterologous immune human sera. It was noted that the CSP reaction always accompanied the development of protective immunity in man, but the duration of this anti-sporozoite reaction and of the immunity itself remained unknown. In order to elucidate these points the following study was undertaken. MATERIALS
AND
~~ETHODS
Volunteers. This investigation was conducted in strict accordance with the guide107
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0014 4894
168
MCCARTHY AND CLYDE
lines provided by medical ethics committees of the University ‘of Maryland and the supporting agency. Details of volunteer selection and treatment have been described in this Journal (McCarthy and Clyde 1973) and more recently by Clyde et al. ( 1975). W.K. (the principal participant) at the time of this investigation was a 26-yearold Caucasian of northern European stock. Prior to this study he had not been exposed to malaria. Parasite strains. Two strains of Plasmo&urn &ax were employed in this study, the Chesson from New Guinea and the El Salvador (Gue. ), These strains are antigenitally similar in CSP reactions and convey reciprocal cross-immunity to infective challenge (Clyde et al. 1975). Transmission and immunization. Anopheles stephensi, reared in our insectary, were used to transmit both normal and irradiated sporozoites. For immunizing purposes infected mosquitoes were X-irradiated in order to attenuate the infectivity to man of their sporozoites which, however, retained their antigenicity. The technique is similar to that described by Vanderberg et al. (1970) in a P. berghei system. Mosquito infectivity was assessed by demonstration of sporozoites in dissected mosquito salivary glands, the density having to be estimated on the scale I+ to 4+ for lack of a more accurate method, and by the results of exposure of unprotected participants to infected nonirradiated mosquitoes. When the immunized volunteer was exposed to infected, nonirradiated mosquitoes in order to test his immune state, the paired (split-fed) method was generally used, the test subject being partially fed on by the same mosquitoes as the control. While one mosquito is capable of transmitting the infection to both men, at least six were used in each challenge in order to ensure the validity of the test. Our method of X-irradiation of the infected mosquitoes has been described previously (Clyde et al. 1973b). The mini-
mum dosage for this work was 15,000 rad (mean dosage, 17,500). The presence of anti-sporozoite antibodies was determined by means of the circumsporozoite precipitation (CSP) reaction. Aliquots of whole serum (or dilutions in Hanks’ salt base medium 199) from the participant were mixed with vivax sporozoites and the reactions read with phase contrast microscopy. RESULTS
The details and results of the immunization series on W.K. and CSP reactions are summarized in Tables I and II and are set forth sequentially by series in the following paragraphs. Series I On Days 0, 3, and 6 W.K. was exposed to infected, irradiated A. stephensi. A total of 728 infected mosquitoes fed. Challenge with seven infected, nonirradiated mosquitoes on Day 13 resulted in W.K. becoming patent on D,ay 28 (1 day earlier than his control partner). Immediate treatment with chloroquine and primaquine permitted only 2 days of parasitemia. The El Salvador (Gue.) strain was used in all the immunizations, the challenge, and the CSP tests of Days 7, 13, and 20. The Chesson strain was used in the remaining CSP tests. CSP reactions remained negative against all serum drawn between Day 1 and Day 108 (the start of the next series). Series Zl On Days 108, 111, 114, and 118 a total of 1251 X-irradiated, infected A. stephensi fed on W.K. Challenge with six infected, nonirradiated mosquitoes on Day 123 resulted in W.K.‘s control partner becoming patent on Day 136. W.K. did not contract vivax malaria. Chesson vivax sporozoites were used in this series.
&.YmOdiU?7Z
ViVUX:
PRECIPITINS
TABLE
AND
I
Series I and II : Immunizations, CSP Reactions, and Challenges W. K. against Plasmodium vivax Strain of parmite 0 1 3 6 7 13 1X
20 27-54 108 108 111
111 114 114 118 118 122 123 132 139 146 13.5 181 188
Gue. Chesson Gue. We. Gue. Gue. (iue. Gue. and Chesson Chesson Chesson Chesson Chesson Chesson Chesson Chesson Chesson Chesson Cheason Chesson Chesson Chesson Chesson Chesson Chesson Chesson
169
PROTECTION
of
Type of test Immunization CSP Immunization Immunization CSP CSP Challenge CSP CSP (weekly) Immunization CSP Immunization CSP Immunization CSP Immunization CSP CSP Challenge CSP CSP CSP CSP CM CSP
W.K.‘s serum from Day 111 was negative, however, serum drawn on Day 114 gave positive reactions through a 1 in 20 dilution. Serum from Day 118 gave a similar result but sera drawn on Day 122 (1 day prior to challenge) and Day 132 were positive at a 1 in 40 dilution. In the weeks following, reactivity of the serum specimens gradually declined and by Day 180, the serum was no longer reactive, Series Ill No additional immunization was attempted. Instead, W.K. and his control partner were challenged with 12 mosquitoes infected with Chesson strain on Day 276. Both men became patent on Day 287. Chloroquine-prim,aquine therapy was started immediately and W.K. showed ouly 1 day of patency.
280 infected, irradiated mosquitoes fed on Serum negative 280 infected, irradiated mosquitoes fed on 168 infected, irradiated mosquitoes fed on Serum negative Serum negative W. K. pat,ent Day 28, control patent, Day Serum negative Serum negative 423 infected, irradiated mosquitoes fed on Serum negative 369 infected, irradiated mosquitoes fed on Serum negative 279 infected, irradiated mosquitoes fed on Serum positive through a 1 in 20 dillltion 180 infected, irradiated mosquitoes fed on Serum positive through a 1 in 20 dilution Serum positive throllgh a 1 in 40 diMon W. K. nonpatent, cont)rol patent Day 136 Serllm posit.ive through a 1 in 40 dihttion Serum positive through a 1 in 20 dilution Serllm positive through a 1 in 5 dilution 8erum positive through a 1 in 5 dihlt,ion Serum positive undilrlted Serlun negative
Serum samples on Days 276 and sera from Days positive, as was Day 304.
W. K. W. K. W. K.
29
W. K. W. K. W. K. W. K.
from W.K. were negative 283. However, mid&ted 290 and 297 were both a 1 in 5 dilution from
Series IV On Day 307, six A. stephensi carrying infective, nonirradiated sporozoites of Chesson vivax fed on W.K. and his control. W.K. did not contract the infection. Undiluted sera from W.K. gave positive reactions on Days 311, 318, 332, 346, 375, 381, 388, and 395. Diluted serum was nonreactive. Serum from D,ay 311 was tested against the Chesson strain. In the other tests the El Salvador (Gue.) strain was used.
170
MCCARTHY
AND CLYDE
TABLE Series III,
IV, and V: CSP Reactions
Day
Strain of parasite
276 276 283 290
Chesson Chesson Chesson Chesson Chesson Chesson Chesyon Chesson Gue. Gue.
297
304 307 311 318393 398
402
Gue.
409
Gue.
410 423 430 437
Gue. Gue. Gue. Gue.
Type of test CSP Challenge CSP CSP CSP CSP Challenge CSP CSP (weekly) Challenge CSP CSP CSP CSP CSP CSP
Serbs V On Day 398 W.K. and his control were fed on by six mosquitoes carrying normal sporozoites of El Salvador (Gue. ) strain P. vivax. Again W.K. did not become patent. The control became patent on Day 414. Undiluted sera from Days 402, 409, 423, and 430 were reactive. A 1 in 5 dilution on Day 416 was weakly reactive. The last serum sample (Day 437) was nonreactive. El Salvador ( Gue. ) sporozoites were used in all tests. DISCUSSION
In Series I, immunization with X-irradiated Plasmodium viwzx sporozoites from 728 infected mosquitoes over a 6-day period did not provide W.K. with protection when he was challenged with nonirradiated infective sporozoites 1 week ‘later. Similarly, the CSP reaction did not develop during this period. The failure of this reaction to develop through Day 108 indicates that the inocula were insufficient to elicit cithcr protection or serological respo11sc.
II
and Challenges
of W. K. against Plasmodium vivux Results
Serum W. K. Serum Serum Serum Serum W. K. Serum Serum W. K. Serum Serum Serum Serum Serum Serum
negative patent Day 287, control patent Day 2% negative positive undiluted positive undiluted positive through a 1 in 5 dillltion nonpatent positive undiluted positive undiluted nonpatent, control patent Day 414 positive undiluted positive undiluted positive through a 1 in 5 dilution positive undiluted positive urldiluted negative
In Series II, additional immunizations with X-irradiated sporozoites brought a quick serological response, indicating that W.K. had been sensitized by the first series and that the additional sporozoites in this series quickly elicited a positive CSP reaction. It is impossible to calculate the number of sporozoites that caused this, but it should be noted th,at, while the reaction appeared after 792 infected, X-irradiated mosquitoes in this series had fed, the actual number required might be considerably lower. The CSP level rose to 1 in 40 1 day prior to challenge, remained there for 10 days, then gradually declined, becoming negative by Day 188. Here a close correlation is evident between CSP ‘and protective immunity as W.K. successfully withstood challenge from six infected mosquitoes when his CSP was at its highest level. In Series III, W.K. was challenged with 12 infective mosquitoes and became patent 11 days later. This demonstrates that the immunity of Day I23 was no longer present and that the duration of this immunity was less than 5 months. On Day 290 (3 clays after patency) a positive CSP reaction in undiluted serum showed that the
PhmOdiUm
UiUZX: PFUXIPITINS AND PROTEXXION
of normal sporozoites from 12 was capable of restoring the anti-sporozoite antibody. In Series IV, W.K. withstood a challenge with sporozoites from six infected mosquitoes. This demonstrates that the infective inoculum on Day 276 restored not only his CSP level but also his protective immunity. His CSP reaction remained positive throughout the series, but only in undiluted scra. In Series V, 3 months after his previous csposure to infective sporozoites, W.K. was again challenged with normal sporozoites from six infcctcd mosquitoes and again remained parasite free. His CSP reaction rose to positive at a 1 in 5 dilution, then gradually declined, becoming negative only 39 days after the last challenge. From these results, it may be concluded that human immunity to P. vivax infection induced by sporozoites lasts 3-5 months and is closely related to a positive CSP reaction, and once acquired it and the CSP reaction may be maintained by the inoculation of small numbers of normal sporozoites. This last point could be of practica1 importance to immunized individuals living in an endemic area.
challcngc
mosquitoes
ACKNOWLEDGMENTS
Our appreciation is expressed to the volunteers who participated in these studies and to the Warden and staff of the Maryland House of Correction, Jessup, Maryland, for their cooperation. At the University of Maryland, invaluable assistance was provided by Dr. James E. Robinson and his staff of the Department of Radiology, who irradiated the mosquitoes, and by Drs. Richard B. Hornick, Roger M. Miller, and William E. Woodward who assisted with the care of the patients. This study was sponsored by the U. S. Army Medical Research and Development Command under Contract No. DA-49-193-MD-2740. The paper is contribution No. 1389 from the Army Malaria Research Program.
171
REFERENCES CLYDE, D. F., MCCARTHY, V. C., MILLER, R. M., AND HORNICK, R. B. 1973a. Specificity of pro-
tection of man immunized against sporozoiteinduced falciparum malaria. American ~OIC1.11~J~ of the I\lcdical Sciences 266, 398-403. CLYDE, 1). F., MCCAWTRY,V. C., MILLELI, R. M., AND Wooow.4nn, W. E. 1975. Immunization of man against falciparum and vivax malaria by use of attenuated sporozoites. American Journal
of Tropical Medicine and Hygiene 24, 397-401. CLYDE, D. F., MOST, II., MCCARTHY, V. C., AND VANDENBERG, J. P. 1973,. Immunization of man against sporozoite-induced falciparum malaria. American Journal of the AfdiCfJl Sciences 266, 16%177. MCCARTHY, V. C., ANU CLYDE, D. F. 1973. Influence of sulfalene upon gametocytogenesis of Plasmodium fnlcipcuwm and subsequent infection patterns in Anopheles stephensi. Experimentul Parasitology 33, 73-78. NUSSEXZWEIG, R. S., AND CHEN, D. 1974. Anti-
body response to sporozoites of simian and human malaria parasites: Its stage and species specificity and strain cross-reactivity. Bulletin
of the World Heulth Orgunization 50, 293-297. NUSSEN~~EIG, R. S., MONTUOHY, W., SPITALNY, G. L., AND CHEN, D. 1973. Antibodies against sporozoites of human and simian malaria produced in rats. Journd of Immunology 110, 600601. NUSSENZWEIG, R. S., VANDENBERG, J. P., MOST, II., AND OnToN, C. 1969. Specificity of protective immunity produced by x-irradiated Plasmodium herghei sporozoites. Nature (London) 222, 488-489. SPITALKY, G. L., AND NUSSENZWEIG, R. S. 1973. Plnsmodium berghei: Relationship between protective immunity and anti-sporozoite (CSP) Parasitology 33, antibody in mice. Experimental 168-178. VANDERBERG, J. P., NUSSENZWEIG, R. S., AND hfOST, H. 1969. Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. V. In vitro effects of immune serum on sporozoites. Military Medicine 134, 1183-1190. VANnERBERG, J. P., NUSSENZ~EIG, R. S., AND hfOST, H. 1970. Protective immunity produced by the bite of x-irradiated mosquitoes infected with Plasmodium berghei. Journal of Parasitology 56, 350-351.
PARASITOLOGY
41, 167-171
(1977)
Plasmodium vivax: Correlation of Circumsporozoite Precipitation (CSP) Reaction with Sporozoite-Induced Protective Immunity in Man AND DAVID F. CLYDE l
VINCENT C. MCCARTHY International
Health
Program, Baltimore,
University Maryland
of Maryland School of Medicine, 21201, U.S.A.
(Accepted for publication
21 April 1976)
MCCARTHY, VINCENT C., AND CLYDE, DAVID F. 1977. Plasmodium vivax: Correlation of circumsporozoite precipitation (CSP) reaction with sporozoite-induced protective immunity in man. Experimental Parasitology 41, 167-171. A volunteer was immunized with X-irradiated sporozoites of Plasmodium vivax against a nonirradiated, infective sporozoite challenge of the same species and strain. Shortly before his first successful challenge, his serum was found to contain anti-sporozoite antibodies. Subsequent immunizations and challenges with another strain demonstrated that his protective immunity lasted 3-5 months and could be restored or boosted by challenge with a small number of nonirradiated, infective sporozoites. His CSP serum reaction paralleled his level of protective immunity. INDEX DESCRIPTORS: Plasmodium vivax; Sporozoa; Malaria; Anopheles stephensi; Diptera; X-irradiated sporozoites; Human malarial immunity; Circumsporozoite precipitation reaction.
INTRODUCTION
The circumsporozoite precipitation reaction was first described in an Anopheles stephensS’lasmodium berghei-rodent test system by Vanderberg et al. (1969). They noted that many sporozoites, incubated in immune serum, ejected a tail-like precipitate: Later work by Nussenzweig et al. (1969) and by Spitahly and Nussenzweig ( 1973) demonstrated the immunological specificity of this reaction. More recently Nussenzweig et al. (1973) and Nussenzweig and Chen (1974) examined the resl”‘ll”c of Plawwdium fulcipurum and Piasmotliurn vivux sporozoites to immune nonhuman sera. The CSP reaction of P. fulciparum sporo1 Present address: Department of Tropical Medicine and Medical Parasitology, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, Louisiana 70112, U.S.A.
zoites in immune human serum was first observed by Clyde et al. (1973a, b). A similar reaction took place when P. vivax sporozoites were placed in immune human serum (Clyde et al. 1975); normal serum was nonreactive, as were sera from individu’als with high levels of immunity to the blood stage of the parasite. There was no cross-reaction of vivax or falciparum sporozoites with heterologous immune human sera. It was noted that the CSP reaction always accompanied the development of protective immunity in man, but the duration of this anti-sporozoite reaction and of the immunity itself remained unknown. In order to elucidate these points the following study was undertaken. MATERIALS
AND
~~ETHODS
Volunteers. This investigation was conducted in strict accordance with the guide107
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0014 4894
168
MCCARTHY AND CLYDE
lines provided by medical ethics committees of the University ‘of Maryland and the supporting agency. Details of volunteer selection and treatment have been described in this Journal (McCarthy and Clyde 1973) and more recently by Clyde et al. ( 1975). W.K. (the principal participant) at the time of this investigation was a 26-yearold Caucasian of northern European stock. Prior to this study he had not been exposed to malaria. Parasite strains. Two strains of Plasmo&urn &ax were employed in this study, the Chesson from New Guinea and the El Salvador (Gue. ), These strains are antigenitally similar in CSP reactions and convey reciprocal cross-immunity to infective challenge (Clyde et al. 1975). Transmission and immunization. Anopheles stephensi, reared in our insectary, were used to transmit both normal and irradiated sporozoites. For immunizing purposes infected mosquitoes were X-irradiated in order to attenuate the infectivity to man of their sporozoites which, however, retained their antigenicity. The technique is similar to that described by Vanderberg et al. (1970) in a P. berghei system. Mosquito infectivity was assessed by demonstration of sporozoites in dissected mosquito salivary glands, the density having to be estimated on the scale I+ to 4+ for lack of a more accurate method, and by the results of exposure of unprotected participants to infected nonirradiated mosquitoes. When the immunized volunteer was exposed to infected, nonirradiated mosquitoes in order to test his immune state, the paired (split-fed) method was generally used, the test subject being partially fed on by the same mosquitoes as the control. While one mosquito is capable of transmitting the infection to both men, at least six were used in each challenge in order to ensure the validity of the test. Our method of X-irradiation of the infected mosquitoes has been described previously (Clyde et al. 1973b). The mini-
mum dosage for this work was 15,000 rad (mean dosage, 17,500). The presence of anti-sporozoite antibodies was determined by means of the circumsporozoite precipitation (CSP) reaction. Aliquots of whole serum (or dilutions in Hanks’ salt base medium 199) from the participant were mixed with vivax sporozoites and the reactions read with phase contrast microscopy. RESULTS
The details and results of the immunization series on W.K. and CSP reactions are summarized in Tables I and II and are set forth sequentially by series in the following paragraphs. Series I On Days 0, 3, and 6 W.K. was exposed to infected, irradiated A. stephensi. A total of 728 infected mosquitoes fed. Challenge with seven infected, nonirradiated mosquitoes on Day 13 resulted in W.K. becoming patent on D,ay 28 (1 day earlier than his control partner). Immediate treatment with chloroquine and primaquine permitted only 2 days of parasitemia. The El Salvador (Gue.) strain was used in all the immunizations, the challenge, and the CSP tests of Days 7, 13, and 20. The Chesson strain was used in the remaining CSP tests. CSP reactions remained negative against all serum drawn between Day 1 and Day 108 (the start of the next series). Series Zl On Days 108, 111, 114, and 118 a total of 1251 X-irradiated, infected A. stephensi fed on W.K. Challenge with six infected, nonirradiated mosquitoes on Day 123 resulted in W.K.‘s control partner becoming patent on Day 136. W.K. did not contract vivax malaria. Chesson vivax sporozoites were used in this series.
&.YmOdiU?7Z
ViVUX:
PRECIPITINS
TABLE
AND
I
Series I and II : Immunizations, CSP Reactions, and Challenges W. K. against Plasmodium vivax Strain of parmite 0 1 3 6 7 13 1X
20 27-54 108 108 111
111 114 114 118 118 122 123 132 139 146 13.5 181 188
Gue. Chesson Gue. We. Gue. Gue. (iue. Gue. and Chesson Chesson Chesson Chesson Chesson Chesson Chesson Chesson Chesson Chesson Cheason Chesson Chesson Chesson Chesson Chesson Chesson Chesson
169
PROTECTION
of
Type of test Immunization CSP Immunization Immunization CSP CSP Challenge CSP CSP (weekly) Immunization CSP Immunization CSP Immunization CSP Immunization CSP CSP Challenge CSP CSP CSP CSP CM CSP
W.K.‘s serum from Day 111 was negative, however, serum drawn on Day 114 gave positive reactions through a 1 in 20 dilution. Serum from Day 118 gave a similar result but sera drawn on Day 122 (1 day prior to challenge) and Day 132 were positive at a 1 in 40 dilution. In the weeks following, reactivity of the serum specimens gradually declined and by Day 180, the serum was no longer reactive, Series Ill No additional immunization was attempted. Instead, W.K. and his control partner were challenged with 12 mosquitoes infected with Chesson strain on Day 276. Both men became patent on Day 287. Chloroquine-prim,aquine therapy was started immediately and W.K. showed ouly 1 day of patency.
280 infected, irradiated mosquitoes fed on Serum negative 280 infected, irradiated mosquitoes fed on 168 infected, irradiated mosquitoes fed on Serum negative Serum negative W. K. pat,ent Day 28, control patent, Day Serum negative Serum negative 423 infected, irradiated mosquitoes fed on Serum negative 369 infected, irradiated mosquitoes fed on Serum negative 279 infected, irradiated mosquitoes fed on Serum positive through a 1 in 20 dillltion 180 infected, irradiated mosquitoes fed on Serum positive through a 1 in 20 dilution Serum positive throllgh a 1 in 40 diMon W. K. nonpatent, cont)rol patent Day 136 Serllm posit.ive through a 1 in 40 dihttion Serum positive through a 1 in 20 dilution Serllm positive through a 1 in 5 dilution 8erum positive through a 1 in 5 dihlt,ion Serum positive undilrlted Serlun negative
Serum samples on Days 276 and sera from Days positive, as was Day 304.
W. K. W. K. W. K.
29
W. K. W. K. W. K. W. K.
from W.K. were negative 283. However, mid&ted 290 and 297 were both a 1 in 5 dilution from
Series IV On Day 307, six A. stephensi carrying infective, nonirradiated sporozoites of Chesson vivax fed on W.K. and his control. W.K. did not contract the infection. Undiluted sera from W.K. gave positive reactions on Days 311, 318, 332, 346, 375, 381, 388, and 395. Diluted serum was nonreactive. Serum from D,ay 311 was tested against the Chesson strain. In the other tests the El Salvador (Gue.) strain was used.
170
MCCARTHY
AND CLYDE
TABLE Series III,
IV, and V: CSP Reactions
Day
Strain of parasite
276 276 283 290
Chesson Chesson Chesson Chesson Chesson Chesson Chesyon Chesson Gue. Gue.
297
304 307 311 318393 398
402
Gue.
409
Gue.
410 423 430 437
Gue. Gue. Gue. Gue.
Type of test CSP Challenge CSP CSP CSP CSP Challenge CSP CSP (weekly) Challenge CSP CSP CSP CSP CSP CSP
Serbs V On Day 398 W.K. and his control were fed on by six mosquitoes carrying normal sporozoites of El Salvador (Gue. ) strain P. vivax. Again W.K. did not become patent. The control became patent on Day 414. Undiluted sera from Days 402, 409, 423, and 430 were reactive. A 1 in 5 dilution on Day 416 was weakly reactive. The last serum sample (Day 437) was nonreactive. El Salvador ( Gue. ) sporozoites were used in all tests. DISCUSSION
In Series I, immunization with X-irradiated Plasmodium viwzx sporozoites from 728 infected mosquitoes over a 6-day period did not provide W.K. with protection when he was challenged with nonirradiated infective sporozoites 1 week ‘later. Similarly, the CSP reaction did not develop during this period. The failure of this reaction to develop through Day 108 indicates that the inocula were insufficient to elicit cithcr protection or serological respo11sc.
II
and Challenges
of W. K. against Plasmodium vivux Results
Serum W. K. Serum Serum Serum Serum W. K. Serum Serum W. K. Serum Serum Serum Serum Serum Serum
negative patent Day 287, control patent Day 2% negative positive undiluted positive undiluted positive through a 1 in 5 dillltion nonpatent positive undiluted positive undiluted nonpatent, control patent Day 414 positive undiluted positive undiluted positive through a 1 in 5 dilution positive undiluted positive urldiluted negative
In Series II, additional immunizations with X-irradiated sporozoites brought a quick serological response, indicating that W.K. had been sensitized by the first series and that the additional sporozoites in this series quickly elicited a positive CSP reaction. It is impossible to calculate the number of sporozoites that caused this, but it should be noted th,at, while the reaction appeared after 792 infected, X-irradiated mosquitoes in this series had fed, the actual number required might be considerably lower. The CSP level rose to 1 in 40 1 day prior to challenge, remained there for 10 days, then gradually declined, becoming negative by Day 188. Here a close correlation is evident between CSP ‘and protective immunity as W.K. successfully withstood challenge from six infected mosquitoes when his CSP was at its highest level. In Series III, W.K. was challenged with 12 infective mosquitoes and became patent 11 days later. This demonstrates that the immunity of Day I23 was no longer present and that the duration of this immunity was less than 5 months. On Day 290 (3 clays after patency) a positive CSP reaction in undiluted serum showed that the
PhmOdiUm
UiUZX: PFUXIPITINS AND PROTEXXION
of normal sporozoites from 12 was capable of restoring the anti-sporozoite antibody. In Series IV, W.K. withstood a challenge with sporozoites from six infected mosquitoes. This demonstrates that the infective inoculum on Day 276 restored not only his CSP level but also his protective immunity. His CSP reaction remained positive throughout the series, but only in undiluted scra. In Series V, 3 months after his previous csposure to infective sporozoites, W.K. was again challenged with normal sporozoites from six infcctcd mosquitoes and again remained parasite free. His CSP reaction rose to positive at a 1 in 5 dilution, then gradually declined, becoming negative only 39 days after the last challenge. From these results, it may be concluded that human immunity to P. vivax infection induced by sporozoites lasts 3-5 months and is closely related to a positive CSP reaction, and once acquired it and the CSP reaction may be maintained by the inoculation of small numbers of normal sporozoites. This last point could be of practica1 importance to immunized individuals living in an endemic area.
challcngc
mosquitoes
ACKNOWLEDGMENTS
Our appreciation is expressed to the volunteers who participated in these studies and to the Warden and staff of the Maryland House of Correction, Jessup, Maryland, for their cooperation. At the University of Maryland, invaluable assistance was provided by Dr. James E. Robinson and his staff of the Department of Radiology, who irradiated the mosquitoes, and by Drs. Richard B. Hornick, Roger M. Miller, and William E. Woodward who assisted with the care of the patients. This study was sponsored by the U. S. Army Medical Research and Development Command under Contract No. DA-49-193-MD-2740. The paper is contribution No. 1389 from the Army Malaria Research Program.
171
REFERENCES CLYDE, D. F., MCCARTHY, V. C., MILLER, R. M., AND HORNICK, R. B. 1973a. Specificity of pro-
tection of man immunized against sporozoiteinduced falciparum malaria. American ~OIC1.11~J~ of the I\lcdical Sciences 266, 398-403. CLYDE, 1). F., MCCAWTRY,V. C., MILLELI, R. M., AND Wooow.4nn, W. E. 1975. Immunization of man against falciparum and vivax malaria by use of attenuated sporozoites. American Journal
of Tropical Medicine and Hygiene 24, 397-401. CLYDE, D. F., MOST, II., MCCARTHY, V. C., AND VANDENBERG, J. P. 1973,. Immunization of man against sporozoite-induced falciparum malaria. American Journal of the AfdiCfJl Sciences 266, 16%177. MCCARTHY, V. C., ANU CLYDE, D. F. 1973. Influence of sulfalene upon gametocytogenesis of Plasmodium fnlcipcuwm and subsequent infection patterns in Anopheles stephensi. Experimentul Parasitology 33, 73-78. NUSSEXZWEIG, R. S., AND CHEN, D. 1974. Anti-
body response to sporozoites of simian and human malaria parasites: Its stage and species specificity and strain cross-reactivity. Bulletin
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