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Protection of Humans against Malaria by Immunization with Radiation-Attenuated Plasmodium falciparum Sporozoites

  1. Stephen L. Hoffman1a,
  2. Lucy M. L. Goh3,a,
  3. Thomas C. Luke4,
  4. Imogene Schneider2,
  5. Thong P. Le1,a,
  6. Denise L. Doolan1,5,
  7. John Sacci1,6,
  8. Patricia de la Vega1,6,
  9. Megan Dowler2,
  10. Chris Paul1,
  11. Daniel M. Gordon2,
  12. Jose A. Stoute2,a,
  13. L. W. Preston Church1,a,
  14. Martha Sedegah1,6,
  15. D. Gray Heppner2,
  16. W. Ripley Ballou2,a and
  17. Thomas L. Richie1
  1. 1Malaria Program, Naval Medical Research Center, Silver Spring
  2. 2Department of Immunology and Entomology, Walter Reed Army Institute of Research, Silver Spring
  3. 3Henry M. Jackson Foundation, Rockville
  4. 4National Naval Medical Center, Bethesda
  5. 5Department of Molecular Microbiology and Immunology, School of Hygiene and Public Health, Johns Hopkins University, Baltimore
  6. 6Department of Microbiology, University of Maryland School of Medicine, Baltimore
  1. Correspondence: Dr. Stephen L. Hoffman, Biologics, Celera Genomics, 45 W. Gude Dr., Rockville, MD 20850 (stephen.hoffman{at}celera.com)

  1. Presented in part: 48th annual meeting, American Society of Tropical Medicine and Hygiene, Washington, DC, 1999 (abstract 801).

  • ↵a Present affiliations: Celera Genomics, Rockville, Maryland (S.L.H.); Pediatric Specialty Center, Monroe, Louisiana (L.M.L.G); Department of Medicine, St. Luke's Hospital, Bethlehem, Pennsylvania (T.P.L.); US Army Medical Research Unit, Nairobi, Kenya (J.A.S); Division of Infectious Diseases, Medical University of South Carolina, Charleston (L.W.P.C.); and MedImmune, Gaithersburg, Maryland (W.R.B.).

 
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Abstract

During 1989–1999, 11 volunteers were immunized by the bites of 1001–2927 irradiated mosquitoes harboring infectious sporozoites of Plasmodium falciparum (Pf) strain NF54 or clone 3D7/NF54. Ten volunteers were first challenged by the bites of Pf-infected mosquitoes 2–9 weeks after the last immunization, and all were protected. A volunteer challenged 10 weeks after the last immunization was not protected. Five previously protected volunteers were rechallenged 23–42 weeks after a secondary immunization, and 4 were protected. Two volunteers were protected when rechallenged with a heterologous Pf strain (7G8). In total, there was protection in 24 of 26 challenges. These results expand published findings demonstrating that immunization by exposure to thousands of mosquitoes carrying radiation-attenuated Pf sporozoites is safe and well tolerated and elicits strain-transcendent protective immunity that persists for at least 42 weeks.

In 1967, Nussenzweig et al. [1] reported that immunizing mice with radiation-attenuated Plasmodium berghei sporozoites protected them against challenge with fully infectious sporozoites. These rodent studies provided the impetus for human studies, and, during the 1970s, Clyde and colleagues [2–5], Rieckmann and colleagues [6–8], and McCarthy and Clyde [9] established that immunizing human volunteers with the bites of irradiated mosquitoes carrying P. falciparum (Pf) or P. vivax (Pv) sporozoites in their salivary glands could protect humans against challenge with fully infectious Pf or Pv sporozoites. These studies demonstrated that a malaria vaccine offering sterile protective immunity was possible. However, it has been considered to be clinically and logistically impractical to immunize large numbers of susceptible persons with the irradiated sporozoite vaccine, because the sporozoites must be delivered alive either by the bite of infected mosquitoes or, potentially, by intravenous injection, as is done with mice.

Therefore, many scientists have focused on understanding the clinical requirements for this protective immunity, the immune mechanisms responsible for the protection, and the antigenic targets of these protective immune responses and on developing vaccine delivery systems that induce such protection. Much of this basic work, carried out in P. berghei and P. yoelii rodent model systems, has yielded important insights into irradiated sporozoite vaccine-induced protection and has led to the development of a number of candidate vaccines (reviewed in [10–12]). In fact, the results from rodent and human model systems are strikingly concordant.

To better delineate the clinical characteristics and requirements for protecting humans with the irradiated sporozoite vaccine, to assess the protective immune responses elicited in humans, and to identify the antigens and epitopes on the proteins that elicited immune responses in humans, in 1989 we began immunizing volunteers with gamma radiation-attenuated Pf sporozoites. Preliminary clinical results and extensive immunologic assay results from these studies have been published [13–19]. These immunologic studies, combined with those of others [20–26], have increased our understanding of the immunologic responses in humans immunized with radiation-attenuated Pf sporozoites. Here we report the results of 10 years' clinical experience with these immunizations and challenges and compare our results with those of published clinical reports of human immunization with irradiated Plasmodium sporozoites.

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Methods

Preparation of sporozoite-infected mosquitoes. The Pf asexual and sexual erythrocytec-stage parasites were grown in normal human erythrocytes, using standard culture medium containing 10% heat-inactivated (56°C, 30 min) normal human serum. All erythrocytes and sera were obtained from donors who were at low risk for both hepatitis and human immunodeficiency virus (HIV) infection and whose serum did not contain hepatitis B surface antigen or antibodies to hepatitis C, Treponema pallidum, or HIV. Blood and serum for culture were purchased (Interstate Blood Bank), and each shipment carried a certificate of analysis certifying that the blood products were negative or nonreactive for the above pathogens.

The mosquito species was Anopheles stephensi. Colonies of A. stephensi have been maintained at the Biomedical Research Institute/Naval Medical Research Center (NMRC) and the Walter Reed Army Institute for Research (WRAIR) for several decades. To date, no adventitious agent of disease has been detected within these mosquito colonies.

About 16–20 days before a volunteer immunization, female mosquitoes from a secure insectary were infected with gametocytes of the PfNF54 strain or the 3D7 clone of NF54 [27], as described elsewhere [13, 28–31]. One hour before immunization, the female mosquitoes were exposed to 15,000 rad of gamma radiation from a 60Co or 137Cs source. This radiation dose is believed to be sufficient to attenuate the sporozoites, allowing them to enter hepatic cells and undergo partial development but preventing them from developing into mature liver-stage schizonts, and thereby eliminating their ability to infect erythrocytes [2–5, 13–15].

Study subjects. Healthy adult male volunteers aged 18-50 years were recruited from the US Navy, Army, and Public Health Service. Volunteers were enrolled if they met previously described eligibility criteria [13]. Due to the long-term duration of this study, approval was obtained from the institutional review boards before immunizing or rechallenging volunteers who passed age 50 years in the latter part of the study. The longest duration of participation was 10 years for 2 volunteers. Volunteer identification numbers, listed in table 1, are not consecutive, because the numbers were sequentially assigned at screening, and some potential volunteers did not meet eligibility requirements and were not enrolled.

Immunization of volunteers. For the purposes of this study, we defined 2 categories of immunizations: a primary immunization series and secondary immunizations. Primary immunizations were defined as occurring before the first challenge. Secondary immunizations were those after the first challenge. Sporozoites were administered to volunteers via the bites of hundreds of irradiated female anopheline mosquitoes, by a process described earlier [13]. Of the mosquitoes taking a blood meal, 50 were dissected to estimate the percentage of mosquitoes having a sporozoite gland score ≥2 (usually 50%–75%, as described elsewhere [28]). This percentage was multiplied by the total number of mosquitoes taking a blood meal, to calculate the number of immunizing bites. A physician with emergency medical drugs and equipment was present during the immunization. Volunteers were observed for 30 min after each immunization for immediate adverse reactions to the mosquito bites.

Over the 10 years of our studies, volunteers were exposed to the bites of irradiated mosquitoes on 165 occasions, and no breakthrough blood-stage infections were detected. After immunization, blood smears were not routinely done. Volunteers were instructed to contact study physicians should fever (oral temperature ≥38°C), headache, chill, myalgia, or malaise develop at any time within 6–28 days after an immunization session—allowing timely diagnosis and treatment without risking the onset of severe disease.

Immunizations were done no more than once every 2 weeks. The goal for the primary immunization series (i.e., all bites occurring before first challenge) was to immunize volunteers with > 1000 bites from irradiated mosquitoes having a sporozoite gland score ≥ 2 before their first challenge. However, 1 of the early volunteers in the study was first challenged after only 606 immunizing bites. Volunteers averaged 9 immunizations with a mean of 1071 immunizing mosquito bites per volunteer over 9–10 months before their first challenge. After the initial challenge, subsequent secondary immunizations with additional batches of irradiated mosquitoes were done to maintain or regain sterile protective immunity.

Challenge of immunized volunteers. Immunized volunteers were challenged with infectious mosquitoes to ascertain the development of protective immunity, since there were no surrogate markers for protection. The challenge method has been described elsewhere [28–30]. Volunteers were closely monitored by study physicians and were instructed to report fever (oral temperature ≥ 38°C), headache, chill, myalgia, malaise, or other medical concerns. All volunteers were monitored daily with thick blood smears, beginning 7 days after challenge and continuing until day 28, and then were monitored weekly for 4 weeks if they became parasitemic or for 8 weeks if they did not become parasitemic. Parasitemic volunteers were treated with a standard course of chloroquine for chloroquinesensitive Pf strains; for the chloroquine-resistant Pf strain (7G8), a standard course of mefloquine was given. Repeated challenges were performed on immunized volunteers to assess the duration of protection and to study the effects of secondary immunizations. The 3 Pf strains or clones used for challenges were NF54, the 3D7 clone of NF54, and the 7G8 clone of IMTM22Brazil [31]. We consider NF54 and the 3D7 clone of NF54 to be heterologous to the 7G8 clone, for reasons that include the following: First, 3D7 was cloned from NF54, which was isolated from a Dutch person who lived near the Amsterdam airport. It is thought that the parasite came from West Africa. 7G8 was cloned from the IMTM-22 isolate from Brazil. Second, NF54 and 3D7 are sensitive to chloroquine, and 7G8 is resistant to chloroquine. Third, there are ≥ 6 known variations in T cell epitopes on the Pf circumsporozoite protein (n = 3) and sporozoite surface protein 2 (n = 3) between the 3D7 and 7G8 sequences. A volunteer was considered to be protected if parasites were never detected in the bloodstream during follow-up.

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Results

Safety and Tolerability and Reactions to Immunizations

In our experience with 165 separate mosquito immunizations in 13 volunteers, 12 of whom were challenged, the procedure was well tolerated, without an unexpected adverse event. No volunteer withdrew from the study due to complications resulting from mosquito immunization or from the malaria challenge. Table 1 shows data on the cumulative number of exposures to immunizing mosquitoes, the total number of immunizing mosquitoes to which each volunteer was exposed, and the number of days from first to last exposure. During the feeding, volunteers usually reported some mild discomfort, but none requested cessation of exposure due to discomfort. After removal of the mosquito containers, a depressed ring corresponding to the container's circumference was visible and palpable on the volunteer's epidermis secondary to pressure contact between the container and skin. The epidermis enclosed by this ring usually had a mild erythematous hue studded by focal moderately erythematous papules. However, some persons were notably less reactive to the immunizations and displayed only a few mildly erythematous papules.

Table 1.
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Table 1.

Summary of immunization and challenge studies at the Naval Medical Research Center and the Walter Reed Army Institute for Research, 1989–1999.

A mild generalized swelling of the skin within the confines of this ring developed in most volunteers, with the papules coalescing into a confluent erythematous plaque within minutes to an hour after the immunization. One volunteer, upon each immunization, developed focal wheals within the confines of the ring that then coalesced. In this subject, a 2-cm flare extended beyond the limits of the ring. The focal swelling was not associated with loss of function in the immunized arm and resolved within 24–72 h. No volunteer developed any symptom or sign suggestive of a systemic anaphylactic reaction.

A tightly circumscribed erythematous papular rash characterized the immunization sites in some persons after 72 h and completely resolved in 7–10 days. Weeping, pustules, crusting, or other evidence of a secondary bacterial infection at the immunization sites did not occur. After resolution of the erythematous papular rash, the immunization sites of some volunteers had mildly hyperpigmented macules. However, within a few weeks, the immunization site in these persons was indistinguishable from adjoining skin.

The evening after some immunization sessions, a few volunteers reported a mild headache and/or malaise that completely resolved by the following morning. Volunteers did not report other constitutional symptoms and never modified their activities or lost work secondary to the reactions they experienced.

Protection

Protection against first challenge with homologous Pf strain after primary immunization with > 1000 Pf-irradiated sporozoites. Over the past 10 years at the NMRC/WRAIR, 11 white male volunteers, enrolled at ages 19–44 years (mean, 36), were challenged with infective bites from 5 mosquitoes infected with a homologous Pf strain after receiving > 1000 immunizing bites from Pf-infected mosquitoes exposed to 15,000 rad of gamma radiation (table 1). In this primary immunization series, the volunteers received a mean of 9 immunizations, a mean of 123 immunizing bites per immunization, and 1001–1163 immunizing bites before their first challenge. Ten volunteers were first challenged 2–9 weeks after their last primary immunization, and all were protected. One volunteer (table 1, no. 15) was first challenged 10 weeks after his last primary immunization and was not protected. A 12th volunteer (table 1, no. 9) served as an infectivity control before completing 9 primary immunizations and was not challenged.

At the University of Maryland [20, 21], 3 volunteers (table 2, subjects 6–8) were challenged with infective bites from 5 mosquitoes infected with the CVD1 clone of Pf NF54 strain after receiving > 1000 immunizing bites from Pf-infected mosquitoes exposed to 17,000–24,710 rad (mean, 20,820). In the primary immunization series, the volunteers received 19 immunizations, a mean of 86 immunizing bites per immunization, and 1563–1681 immunizing bites before their first challenge. These volunteers were first challenged 3 weeks after their last primary immunization, and all were protected.

Table 2.
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Table 2.

Summary of immunizations and challenges with homologous Plasmodium falciparum (Pf) strain at the Universitv of Marvland during the late 1980s and earlv 1990s [20, 21].

On the basis of combined data from these institutions, all 13 volunteers challenged 2–9 weeks after receiving > 1000 immunizing bites in a primary immunization series were protected. One volunteer (no. 15), challenged 10 weeks after receiving 1008 immunizing bites in his primary immunization series, was not protected. None of the earlier reports by Clyde and colleagues [2–5] or Rieckmann and colleagues [6–8] included volunteers who had received > 1000 bites by infected irradiated mosquitoes before first challenge.

Reduced protection against first challenge with homologous Pf strain after primary immunization with < 1000 Pf-irradiated sporozoites. At both the NMRC/WRAIR and the University of Maryland, the level of protection appeared to be less for volunteers who received < 1000 primary immunizing bites. At the NMRC/WRAIR, 1 volunteer (table 1, no. 1) was challenged 2 weeks after receiving 606 immunizing bites from mosquitoes irradiated at 15,000 rad and was not protected. In this case, the lack of protection was probably due to an insufficient number of immunizing bites.

At the University of Maryland, 2 volunteers (table 2, subjects 4 and 5) underwent a homologous challenge 5 weeks after receiving 625 and 715 immunizing bites from Pf NF54 straininfected mosquitoes irradiated with 20,000–27,000 rad (mean, 23,610) and were not protected. The lack of protection in these 2 volunteers likely resulted from an insufficient number of immunizing bites but could have been due to overattenuation of the sporozoites by excessive radiation [32].

In the studies done by Clyde and colleagues [2–5], Rieckmann and colleagues [6–8], and McCarthy and Clyde [9] (table 3), all volunteers were first challenged after receiving < 1000 immunizing bites from mosquitoes irradiated with a minimum of 12,000 or 15,000 rad. Five volunteers (EN, RP, GZ, LA, and DS) were challenged 2 weeks after receiving 379–954 primary immunizing bites before first challenge with a homologous strain. Three of the 5 were protected when challenged with 7–14 infective bites from nonirradiated mosquitoes, including the 2 with the most primary immunizing bites (440 and 954; LA and DS, respectively). Four additional volunteers were challenged ∼2 weeks after receiving < 200 primary immunizing bites and were not protected. These persons are not described in table 3. The lack of protection in these volunteers was probably the result of an insufficient number of immunizing bites.

Table 3.
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Table 3.

Summary of immunization and challenge studies done in the 1970s, excluding persons who received < 200 immunizing bites [2–9].

When we combined the data from our studies, those at the University of Maryland, and the studies of Clyde et al. [2–5] and Rieckmann et al. [6–8], only 3 of the 8 volunteers challenged 2–5 weeks after receiving > 200 and < 1000 immunizing bites in primary immunization series were protected (P = .0028, Fisher's exact test, 2-tailed, > 1000 vs. < 1000 immunizing bites). If the numbers are expanded to include subjects immunized with <200 infected irradiated mosquitoes, only 3 of 12 were protected.

Protection against repeated challenges (rechallenge) with homologous Pf strain in volunteers immunized with irradiated Pf sporozoites. At the NMRC/WRAIR, 6 volunteers (table 1, subjects 4,5,10–12, and 16) were rechallenged with homologous Pf strain sporozoites 1–5 times (13 additional rechallenges with the homologous strain). These rechallenges were done 2–257 weeks after a subject's last immunization. As shown in table 1, rechallenges occurred after the initial challenge or after a previous rechallenge and either with or without secondary immunization(s). Four volunteers (subjects 4, 5, 10, and 16) underwent 7 rechallenges 2–9 weeks after receiving a secondary immunization, and all were protected. One volunteer (no. 11) was rechallenged 23 weeks after receiving a secondary immunization and was protected. Two volunteers (10 and 12) were rechallenged 36 weeks after their last primary immunization: Subject 10 was protected, but subject 12 was not. Volunteers 5 and 4, respectively, were rechallenged 41 and 42 weeks after their last secondary immunization, and both were protected. Finally, volunteer 4 was rechallenged 257 weeks (almost 5 years) after a secondary immunization and was not protected. Of note, this volunteer then received a secondary immunization of 147 bites and was protected when rechallenged 2 weeks later.

At the University of Maryland, 1 volunteer (table 2, no. 8) was rechallenged 39 weeks after receiving a secondary immunization and was protected. From the studies by Clyde et al. [2–5], Rieckmann et al. [6–8], and McCarthy and Clyde [9] (table 3), fewer interpretable data are available on the longevity of homologous Pf protection. In their studies, most volunteers received Pv challenges or rechallenges (subjects GZ, DFC, and WK), received heterologous Pf challenges or rechallenges (subjects GZ, DFC, DS, and WD), were never rechallenged with a homologous Pf strain (subject WD), received < 1000 primary immunization bites, or were unrealistically rechallenged by 45 infected nonirradiated mosquitoes (subject LA). Subject DS had a primary immunization of 954 bites and was protected when first challenged at 2 weeks and when rechallenged at 8 weeks with a homologous Pf strain. Volunteer GZ had a primary immunization of 379 bites and was protected when first challenged 2 weeks later with a homologous Pf strain. He then received an additional 810 boost immunization bites (total, 1189) and was protected when rechallenged 2 weeks later with a homologous Pf strain.

Combining the interpretable data from all these studies yielded 14 cases of protection in 15 homologous rechallenges done 2–42 weeks after the last primary immunization or the most recent secondary immunization. Of the 9 rechallenges conducted at weeks 2–9, all resulted in protection. Of the 6 rechallenges between weeks 23 and 42, 5 resulted in protection (table 4).

Table 4.
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Table 4.

Summary of all Plasmodium falciparum (Pf) challenges with 5–14 infected mosquitoes in volunteers immunized with radiation attenuated with Pf sporozoites.

Protection against challenge with heterologous Pf strains in volunteers immunized with Pf-irradiated sporozoites. At the NMRC/WRAIR, 2 volunteers (table 1, subjects 4 and 5), after primary and secondary immunization with 1310 and 1297 Pf 3D7 strain bites, respectively, were protected when challenged 2 weeks later by the heterologous Pf 7G8 clone of IMTM/Brazil. In the studies by Clyde et al. [2–5] and Rieckmann et al. [6–8],2 volunteers (table 3, subjects GZ and DFC), who received > 1000 immunizing bites, were protected when challenged by a heterologous Pf strain on 5 occasions, excluding an unrealistic challenge with 90 nonirradiated infective mosquitoes. Three subjects (DFC, DS, and WD) received < 1000 immunizing bites before challenge by a heterologous Pf strain, and 1 was protected and 2 were not on 3 occasions. Volunteer GZ underwent a heterologous Pf challenge 1 week after completing 12 immunizations (1309 bites) and underwent 2 sequential heterologous Pf rechallenges 4 and 5 weeks after a secondary immunization of 132 bites (total, 1441) and was protected on all 3 occasions. Volunteer DFC underwent heterologous Pf rechallenges 1 and 3 weeks after completing several immunizations (n = 1806 bites) and was protected on both occasions. Volunteer DFC also underwent aheterologous Pf rechallenge with 90 infected nonirradiated mosquitoes 12 weeks after completing secondary immunizations (400 additional bites; total bites, 2206) and was not protected. Three volunteers (DFC, DS, and WD) received < 1000 immunizing bites before challenge with a heterologous Pf strain on 3 occasions 2–17 weeks after their last immunization: Only WD, who received the greatest number of immunizing bites (987), was protected when challenged at 8 weeks.

In combined data from these studies, all 4 volunteers who received > 1000 immunizing bites were protected against all 7 challenges or rechallenges from 5–10 mosquitoes infected with heterologous Pf. One volunteer who received > 1000 immunizing bites was not protected against a challenge by 90 mosquitoes infected with heterologous Pf. Of the volunteers who received < 1000 immunizing bites, only 1 challenge in 3 resulted in protection. Table 5 summarizes data on heterologous challenge after > 1000 immunizing bites.

Table 5.
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Table 5.

Summary of Plasmodium falciparum (Pf) and P. vivax (Pv) challenges in volunteers who received > 1000 immunizing bites from Pf infected mosquitoes within 1 year of their last primary or secondary immunization.

Protection against cross-species challenge. In the studies by Clyde et al. [2–5], Rieckmann et al. [6–8], and McCarthy and Clyde [9],1 volunteer (table 3, subject GZ) who had received 1309 immunizing Pf bites and had been protected against 3 Pf challenges was not protected against a Pv challenge 2 weeks after his last Pf immunization. This is the only pure cross-species challenge after > 1000 immunizing bites. A second volunteer (DFC) underwent sequential Pv and heterologous Pf challenges 1 and 2 weeks after completing a primary immunization of 838 Pf bites and was not protected on either occasion. He then received secondary immunizations totaling 968 homologous Pf bites and underwent 2 sequential heterologous Pf challenges at 1 and 3 weeks and was protected on both occasions. DFC then received 539 immunizing Pv (El Salvador strain) bites and underwent a homologous Pv challenge 2 weeks later and was protected. He then received secondary immunizations with 400 homologous Pf bites and underwent a heterologous Pf challenge 12 weeks later and was protected. After that, he underwent a heterologous Pv challenge 12 weeks after his immunization with Pv sporozoites and was protected. He had a homologous Pv challenge 26 weeks after his immunization with Pv sporozoites and was not protected. The analysis of cross-species protection in DFC is confounded by his mixed immunization with Pf and Pv sporozoites. Table 5 summarizes data on cross-species challenge.

Protection against challenge with Pv in volunteers immunized with Pv-irradiated sporozoites. Rieckmann et al. [7] reported that 3 volunteers were not protected against homologous Pv challenge after receiving < 200 immunizing bites. One volunteer (table 3, subject WK) was not protected on his first homologous challenge 1 week after 728 immunizing bites with the Pv El Salvador strain [9]. After an additional 1251 immunizing bites from the Chesson strain (cumulative bites from both strains, 1979), he underwent 3 successive Chesson strain challenges 1, 23, and 27 weeks after his last immunization. He was protected at 1 and 27 weeks but not at an intervening challenge at 23 weeks. At 40 weeks, he was protected against an El Salvador strain challenge. Volunteer DFC, whose immunizations were described in the prior paragraph, was immunized with both Pf- and Pv-irradiated sporozoites. He was protected after a homologous Pv challenge 2 weeks after and a heterologous Pv challenge 12 weeks after receiving 539 immunizing bites from irradiated Pv-infected mosquitoes but was not protected after a homologous Pv challenge at 26 weeks after a previous immunization. Again, interpretation of these results is clouded by the 2 species immunization regimen. Table 5 summarizes data on Pv immunization and Pv challenges.

Validity of challenges. At the NMRC/WRAIR, 25 infectivity control volunteers (mean, 3 control volunteers per challenge) were used for the challenges, and all control volunteers became parasitemic. Similarly, at the University of Maryland, where there were 11 infectivity control volunteers, all became parasitemic, with the exception of 1 volunteer who had limited symptoms on days 14 and 15 but had negative thick blood smears between days 5 and 30. However, his blood cultures were positive for Pf on days 21 and 30 [15]. In the reports by Clyde et al. [2–5], Rieckmann et al. [6–8], and McCarthy and Clyde [9], 28 paired infectivity control volunteers were noted, and all became parasitemic.

Breakthrough blood-stage infections. In the studies at the NMRC/WRAIR and in recent University of Maryland studies, all immunizing mosquitoes received 15,000 rad (NMRC/WRAIR) or more (University of Maryland), and no breakthrough bloodstage infections occurred. In a study by Clyde [5], 4 of 7 volunteers immunized by sporozoites irradiated with 12,000 rad developed breakthrough infections. In a study by RieckMann [8], 2 of 11 volunteers immunized by sporozoites irradiated with 12,000 rad developed breakthrough infections. In addition, subject DFC had a breakthrough infection after immunization that was thought to be secondary to sporozoites escaping attenuation with 15,000 rad [2].

In combined data from these studies, 6 (all exposed to Pf) of 18 volunteers developed malaria after exposure to infected mosquitoes irradiated with 12,000 rad, likely due to insufficient irradiation of the sporozoites. In the single breakthrough infection after mosquito exposure to 15,000 rad, there was a concern regarding the number of rads actually delivered.

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Discussion

Data from the NMRC/WRAIR studies, when combined with the results of other studies, clearly demonstrate that humans receiving > 1000 bites from Pf-infected mosquitoes irradiated with 15,000 rad are protected against primary Pf challenge for at least 9 weeks after last exposure and against rechallenges for at least 23–42 weeks. They also demonstrate that protection is not specific to the Pf strain used for immunization. No data were generated in our studies regarding cross-species protection against Pv after immunization with irradiated Pf sporozoites. Of importance, exposure to thousands of irradiated Pf-infected mosquitoes was safe and generally well tolerated.

Table 4 summarizes the results of challenges, divided by the number of immunizing bites (> 1000 vs. < 1000 bites) and the interval between immunization and challenge (≤ 10 vs. > 10 weeks). Overall, 33 of 35 challenges within 42 weeks after > 1000 immunizing bites led to protection, whereas only 5 of 15 challenges after > 378 and < 1000 immunizing bites led to protection (P < .0001, Fisher's exact test, 2-tailed). This indicates that ∼1000 bites provide essentially complete protection against sporozoite challenge and that fewer bites may or may not be associated with protection.

The number of immunization sessions did not seem to affect protection. In our studies (table 1), volunteers receiving > 1000 immunizing bites were protected regardless of whether primary immunizations took place in 5 sessions (1 volunteer), 8 sessions (4 volunteers), 9 sessions (2 volunteers), 10 sessions (2 volunteers), or 11 sessions (1 volunteer). Our single failure to protect after first challenge after > 1000 immunizing bites occurred after 10 immunization sessions. Three volunteers shown in table 2 were also protected against first challenge after receiving > 1000 immunizing bites—each of these volunteers was immunized in 19 sessions.

Our 11 volunteers who received > 1000 immunizing bites were first challenged at 2–10 weeks after the last immunization. The single subject first challenged at 10 weeks was not protected, whereas intervals of 2 weeks (7 volunteers), 3 weeks (2 volunteers), and 9 weeks (1 volunteer) before first challenge were all associated with protection. Repeated challenges after secondary immunization showed that protection is long lasting. In our studies (5 volunteers), and the recent University of Maryland studies (1 volunteer), 5 of 6 volunteers challenged 23–42 weeks after the most recent secondary immunization were protected. Protection was achieved at 23, 36, 39, 41, and 42 weeks (1 volunteer for each period) after secondary immunization. One failure occurred 36 weeks after the last immunization. For longer intervals, we have only a single data point: 1 volunteer challenged 257 weeks (5 years) after the last immunization who was not protected. This volunteer was treated, was given a booster immunization with 147 immunizing bites, and was protected 2 weeks after receiving this boost.

The number of mosquito bites used for challenge may also affect protection. Under natural conditions of exposure, persons are rarely bitten by > 1 infected mosquito per night. We used 5 experimental bites for all challenges. Only 50% of humans bitten by 1 or 2 Pf-infected A. stephensi mosquitoes developed parasitemia [29], whereas nearly 100% of humans bitten by 5 Pf-infected mosquitoes developed parasitemia [29, 30, 33]. In an early study [4], 1 volunteer (table 3, subject DFC) was challenged with 90 infected nonirradiated mosquitoes and was not protected, despite 2206 immunizing bites and a 12-week interval between his last immunization and challenge (table 3). This is an unrealistic challenge that we believe has little relevance to this vaccine model. Nonetheless, it suggests that dramatically increasing the challenge dose may overcome the irradiated sporozoite vaccine-induced protection.

All 7 heterologous challenges, in 4 volunteers who were challenged 1–5 weeks after their last immunization, resulted in protection (table 5). These data indicate that the irradiated sporozoite vaccine induces strain-transcendent protection, but the number of strains that have been tested is small, and exposure in the field will be with many different strains.

Interpretation of Pv data was confounded because volunteers received mixed immunizations with both Pf and Pv sporozoites before challenge. One volunteer received > 1000 immunizing Pv bites in the absence of Pf immunization and was protected in 3 of 4 Pv challenges (table 3, subject WK). With regard to crossspecies challenges, there is one clear instance of a volunteer (subject GZ) immunized only by > 1000 bites of Pf-infected irradiated mosquitoes (1309 bites) and challenged with Pv (2 weeks after the last immunization) who was not protected against Pv challenge (table 3). In a second instance, volunteer DFC was challenged with Pv after exposure to 838 Pf-infected mosquitoes and was not protected (table 3). It will be important to actually determine whether immunization with Pf- or Pv-irradiated sporozoite does or does not consistently protect against crossspecies challenge.

Protection lasted for at least 42 weeks in these studies. However, it is not known whether protection requires the ongoing presence of irradiated parasites in infected hepatocytes. A study [34] in mice treated with primaquine to eliminate hepatic-stage parasites suggested that persistence of irradiated sporozoites in hepatocytes is necessary to maintain protective immunity. If the protection resulting from the irradiated sporozoite vaccine in humans depends on the ongoing presence of attenuated hepatic-stage parasites and does not induce immunologic memory with the potential for an anamnestic response after challenge, its usefulness as a model for subunit malaria vaccine development may be limited.

We used mosquitoes exposed to 15,000 rad. Exposure of volunteers to mosquitoes that received 12,000 rad resulted in protection in some cases but was also associated with breakthrough infections, presumably due to insufficient attenuation [2, 8]. Exposure of volunteers to mosquitoes receiving > 20,000 rad may fail to protect due to overattenuation of the sporozoites. In cultured human hepatocytes, there is an inverse association between the number of sporozoites capable of penetration, limited development within the hepatocytes, and radiation dose [32]. Most investigators believe that 15,000–20,000 rad is optimal [2–5, 13–15].

The irradiated sporozoite vaccine is an excellent model for malaria vaccine development, because irradiated sporozoites enter hepatocytes and only partially develop within these cells. Thus, volunteers immunized with irradiated sporozoites do not develop clinical symptoms of malaria. They only develop immune responses against antigens expressed by irradiated sporozoites and against antigens expressed when the irradiated sporozoites partially develop within hepatocytes. They do not make immune responses against the majority of erythrocytic-stage antigens, since they are not expressed by irradiated sporozoites in liver cells. There is no progression to the sexual stage and hence no transmission of malaria. In contrast to serum and cells acquired from nonimmune persons with naturally acquired malaria, serum and cells from irradiated sporozoite-immunized volunteers can be used as specific probes for identifying protective parasite antigens expressed at the sporozoite and liver stages of the parasite life cycle.

The irradiated sporozoite vaccine provides critical data for defining mechanisms of protective immunity, defining antigenic targets of protective immunity, developing subunit malaria vaccines, developing assays that predict protective immunity, and validating reagents used to assess immune responses in clinical trials of experimental subunit vaccines. Of greatest importance, immunization of volunteers with irradiation-attenuated sporozoites demonstrates that it is feasible to develop a highly protective malaria vaccine and provides the foundation for work on developing a subunit preerythrocytic-stage malaria vaccine [35].

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Acknowledgments

We thank the Walter Reed Army Institute for Research and Biomedical Research Institute/Naval Medical Research Center entomology staffs; Dan Carucci, Ruth Nussenzweig, Victor Nussenzweig, David Clyde, Karl Rieckmann, Robert Edelman, and Deirdre Herrington for manuscript review; and, of most importance, the volunteers, for their dedication and commitment over the years.

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Footnotes

  • Written informed consent was obtained from all volunteers. All studies were conducted in accordance with guidelines established by and approved by US Navy and Army institutional review boards. The study protocol was approved by the Committee for the Protection of Human Subjects of the Naval Medical Research Center, the Office of the Special Assistant for Human Subject Protections at the Naval Bureau of Medicine and Surgery, and the Human Subjects Research Review Board of the Army Surgeon General.

  • Financial support: Military Infectious Diseases Research Program; Naval Medical Research Center (work unit 62787A 870 F 1432).

  • The opinions and assertions contained herein are those of the authors and are not to be construed as reflecting the views of the departments of the Navy or Army.

  • Reprints: Dr. Dan Carucci, Naval Medical Research Center, 503 Robert Grant Ave., Silver Spring, MD 20910-7500 (caruccid{at}nmrc.navy.mil)

  • Received August 1, 2001.
  • Revision received November 19, 2001.
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References

  1. 1.↵
    1. Nussenzweig RS,
    2. Vanderberg J,
    3. Most H,
    4. Orton C
    . Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. Nature 1967;216:160-2.
    CrossRefMedline
  2. 2.↵
    1. Clyde DF,
    2. Most H,
    3. McCarthy VC,
    4. Vanderberg JP
    . Immunization of man against sporozoite-induced falciparum malaria. Am J Med Sci 1973;266:169-77.
    CrossRefMedlineWeb of Science
  3. 3.
    1. Clyde DF,
    2. McCarthy VC,
    3. Miller RM,
    4. Hornick RB
    . Specificity of protection of man immunized against sporozoite-induced falciparum malaria. Am J Med Sci 1973;266:398-401.
    MedlineWeb of Science
  4. 4.↵
    1. Clyde DF,
    2. McCarthy VC,
    3. Miller RM,
    4. Woodward WE
    . Immunization of man against falciparum and vivax malaria by use of attenuated sporozoites. Am J Trop Med Hyg 1975;24:397-401.
    Abstract/FREE Full Text
  5. 5.↵
    1. Clyde DF
    . Immunity to falciparum and vivax malaria induced by irradiated sporozoites: a review of the University of Maryland studies, 1971–75. Bull World Health Organ 1990;68((Suppl)):9-12.
    MedlineWeb of Science
  6. 6.↵
    1. Rieckmann KH,
    2. Carson PE,
    3. Beaudoin RL,
    4. Cassells IS,
    5. Sell KW
    . Sporozoite induced immunity in man against an Ethiopian strain of Plasmodium falciparum. Trans R Soc Trop Med Hyg 1974;68:258-9.
    CrossRefMedlineWeb of Science
  7. 7.↵
    1. Rieckmann KH,
    2. Beaudoin RL,
    3. Cassells IS,
    4. Sell DW
    . Use of attenuated sporozoites in the immunization of human volunteers against falciparum malaria. Bull World Health Organ 1979;57((Suppl 1)):261-5.
    Medline
  8. 8.↵
    1. Rieckmann KH
    . Human immunization with attenuated sporozoites. Bull World Health Organ 1990;68:13-6.
    MedlineWeb of Science
  9. 9.↵
    1. McCarthy VC,
    2. Clyde DF
    . Plasmodium vivax: correlation of circumsporozoite precipitation (CSP) reaction with sporozoite-induced protective immunity in man. Exp Parasitol 1977;41:167-71.
    CrossRefMedlineWeb of Science
  10. 10.↵
    1. Nussenzweig V,
    2. Nussenzweig RS
    . Rationale for the development of an engineered sporozoite malaria vaccine. Adv Immunol 1989;45:283-334.
    MedlineWeb of Science
  11. 11.
    1. Hoffman SL
    1. Hoffman SL,
    2. Franke ED,
    3. Hollingdale MR,
    4. Druilhe P
    . Attacking the infected hepatocyte. In: Hoffman SL, editor. Malaria vaccine development: a multi-immune response approach. Washington, DC: ASM Press; 1996. p. 35-75.
  12. 12.↵
    1. Hoffman SL
    1. Hoffman SL,
    2. Miller LH
    . Perspectives on malaria vaccine development. In: Hoffman SL, editor. Malaria vaccine development: a multi-immune response approach. Washington, DC: ASM Press; 1996. p. 1-13.
  13. 13.↵
    1. Egan JE,
    2. Hoffman SL,
    3. Haynes JD,
    4. et al
    . Humoral immune responses in volunteers immunized with irradiated Plasmodium falciparum sporozoites. Am J Trop Med Hyg 1993;49:166-73.
    Abstract/FREE Full Text
  14. 14.
    1. Malik A,
    2. Egan JE,
    3. Houghten RA,
    4. Sadoff JC,
    5. Hoffman SL
    . Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein. Proc Natl Acad Sci USA 1991;88:3300-4.
    Abstract/FREE Full Text
  15. 15.↵
    1. Wizel B,
    2. Houghten RA,
    3. Parker K,
    4. et al
    . Irradiated sporozoite vaccine induces HLA-B8-restricted cytotoxic T lymphocyte responses against two overlapping epitopes of the Plasmodium falciparum surface sporozoite protein 2. J Exp Med 1995;182:1435-45.
    Abstract/FREE Full Text
  16. 16.
    1. Wizel B,
    2. Houghten R,
    3. Church P,
    4. et al
    . HLA-A2-restricted cytotoxic T lymphocyte responses to multiple Plasmodium falciparum sporozoite surface protein 2 epitopes in sporozoite-immunized volunteers. J Immunol 1995;155:766-75.
    Abstract
  17. 17.
    1. Krzych U,
    2. Lyon JA,
    3. Jareed T,
    4. et al
    . T lymphocytes from volunteers immunized with irradiated Plasmodium falciparum sporozoites recognize liver and blood stage malaria antigens. J Immunol 1995;155:4072-7.
    Abstract
  18. 18.
    1. Doolan DL,
    2. Hoffman SL,
    3. Southwood S,
    4. et al
    . Degenerate cytotoxic T cell epitopes from P. falciparum restricted by HLA-A and HLA-B supertypes alleles. Immunity 1997;7:97-112.
    CrossRefMedlineWeb of Science
  19. 19.↵
    1. Doolan DL,
    2. Southwood S,
    3. Chesnut R,
    4. et al
    . HLA-DR-promiscuous T cell epitopes from Plasmodium falciparum pre- erythrocytic-stage antigens restricted by multiple HLA class II alleles. J Immunol 2000;165:1123-37.
    Abstract/FREE Full Text
  20. 20.↵
    1. Herrington D,
    2. Davis J,
    3. Nardin E,
    4. et al
    . Successful immunization of humans with irradiated sporozoites: humoral and cellular responses of the protected individuals. Am J Trop Med Hyg 1991;45:539-47.
    Abstract/FREE Full Text
  21. 21.↵
    1. Edelman R,
    2. Hoffman SL,
    3. Davis JR,
    4. et al
    . Long-tern persistence of sterile immunity in a volunteer immunized with X-irradiated Plasmodium falciparum sporozoites. J Infect Dis 1993;168:1066-70.
    Abstract/FREE Full Text
  22. 22.
    1. Nardin EH,
    2. Herrington DA,
    3. Davis J,
    4. et al
    . Conserved repetitive epitope recognized by CD4+ clones from a malaria-immunized volunteer. Science 1989;246:1603-6.
    Abstract/FREE Full Text
  23. 23.
    1. Nardin EH
    . T cell responses in a sporozoite-immunized human volunteer and a chimpanzee. Immunol Lett 1990;25:43-8.
    CrossRefMedlineWeb of Science
  24. 24.
    1. Nardin EH,
    2. Nussenzweig RS,
    3. Altszuler R,
    4. et al
    . Cellular and humoral immune responses to a recombinant P. falciparum CS protein in sporozoite-immunized rodents and human volunteers. Bull World Health Organ 1990;68((Suppl)):85-7.
    MedlineWeb of Science
  25. 25.
    1. Moreno A,
    2. Clavijo P,
    3. Edelman R,
    4. et al
    . Cytotoxic CD4+ T cells from a sporozoite-immunized volunteer recognize the Plasmodium falciparum CS protein. Int Immunol 1991;3:997-1003.
    Abstract/FREE Full Text
  26. 26.↵
    1. Moreno A,
    2. Clavijo P,
    3. Edelman R,
    4. et al
    . CD4' T cell clones obtained from Plasmodium falciparum sporozoite-immunized volunteers recognize polymorphic sequences of the circumsporozoite protein. J Immunol 1993;151:489-99.
    Abstract
  27. 27.↵
    1. Walliker D,
    2. Quakyi IA,
    3. Wellems TE,
    4. et al
    . Genetic analysis of the human malaria parasite Plasmodium falciparum. Science 1987;236:1661-6.
    Abstract/FREE Full Text
  28. 28.↵
    1. Chulay JD,
    2. Schneider I,
    3. Cosgriff TM,
    4. et al
    . Malaria transmitted to humans by mosquitoes infected from cultured Plasmodium falciparum. Am J Trop Med Hyg 1986;35:66-8.
    Abstract/FREE Full Text
  29. 29.↵
    1. Rickman LS,
    2. Jones TR,
    3. Long GW,
    4. et al
    . Plasmodium falciparum-infected Anopheles stephensi inconsistently transmit malaria to humans. Am J Trop Med Hyg 1990;43:441-5.
    Abstract/FREE Full Text
  30. 30.↵
    1. Hoffman SL
    . Experimental challenge of volunteers with malaria. Ann Intern Med 1997;127:233-5.
    FREE Full Text
  31. 31.↵
    1. Burkot TR,
    2. Williams JL,
    3. Schneider I
    . Infectivity to mosquitoes of Plasmodium falciparum clones grown in vitro from the same isolate. Trans R Soc Trop Med Hyg 1984;78:339-41.
    CrossRefMedlineWeb of Science
  32. 32.↵
    1. Mellouk S,
    2. Lunel F,
    3. Sedegah M,
    4. Beaudoin RL,
    5. Druilhe P
    . Protection against malaria induced by irradiated sporozoites [letter]. Lancet 1990;335:721.
    CrossRefMedlineWeb of Science
  33. 33.↵
    1. Church LW,
    2. Le TP,
    3. Bryan JP,
    4. et al
    . Clinical manifestations of Plasmodium falciparum malaria experimentally induced by mosquito challenge. J Infect Dis 1997;175:915-20.
    Abstract/FREE Full Text
  34. 34.↵
    1. Scheller LF,
    2. Azad AF
    . Maintenance of protective immunity against malaria by persistent hepatic parasites derived from irradiated sporozoites. Proc Natl Acad Sci USA 1995;92:4066-8.
    Abstract/FREE Full Text
  35. 35.↵
    1. Miller LH,
    2. Hoffman SL
    . Research toward vaccines against malaria. Nat Med 1998;4:520-4.
    CrossRefMedlineWeb of Science
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  1. J Infect Dis. (2002) 185 (8): 1155-1164. doi: 10.1086/339409
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