[Show abstract][Hide abstract] ABSTRACT: Immunization of volunteers under chloroquine prophylaxis by bites of Plasmodium falciparum sporozoite (PfSPZ)-infected mosquitoes induces > 90% protection against controlled human malaria infection (CHMI). We studied intradermal immunization with cryopreserved, infectious PfSPZ in volunteers taking chloroquine (PfSPZ chemoprophylaxis vaccine [CVac]). Vaccine groups 1 and 3 received 3× monthly immunizations with 7.5 × 10(4) PfSPZ. Control groups 2 and 4 received normal saline. Groups 1 and 2 underwent CHMI (#1) by mosquito bite 60 days after the third immunization. Groups 3 and 4 were boosted 168 days after the third immunization and underwent CHMI (#2) 137 days later. Vaccinees (11/20, 55%) and controls (6/10, 60%) had the same percentage of mild to moderate solicited adverse events. After CHMI #1, 8/10 vaccinees (group 1) and 5/5 controls (group 2) became parasitemic by microscopy; the two negatives were positive by quantitative real-time polymerase chain reaction (qPCR). After CHMI #2, all vaccinees in group 3 and controls in group 4 were parasitemic by qPCR. Vaccinees showed weak antibody and no detectable cellular immune responses. Intradermal immunization with up to 3 × 10(5) PfSPZ-CVac was safe, but induced only minimal immune responses and no sterile protection against Pf CHMI.
Full-text · Article · Dec 2015 · The American journal of tropical medicine and hygiene
[Show abstract][Hide abstract] ABSTRACT: Background
Plasmodium falciparum is transmitted from person to person by Anopheles mosquitoes after completing its sexual reproductive cycle within the infected mosquito. An efficacious vaccine holds the potential to interrupt development of the parasite in the mosquito leading to control and possibly eradication of malaria. A multi-component, R0.10C, was developed comprising P. falciparum glutamate-rich protein (R0) fused in frame to a correctly folded fragment of Pfs48/45 (10C). Here, a series of novel adjuvants were screened for their ability to elicit transmission-blocking (TB) antibodies.
The recombinant fusion protein R0.10C was produced in Lactococcus lactis and purified by affinity-chromatography on a monoclonal antibody (mAb 85RF45.1) against a major epitope for TB antibodies (epitope 1) harboured on R0.10C. Immune-purified R0.10C was mixed with a series of adjuvants and tested in mice and rats.
In general, all R0.10C formulations elicited high levels of antibodies recognizing native Pfs48/45 in macrogametes/zygotes. TB activity of anti-R0.10C antisera was assessed in the standard membrane-feeding assay (SMFA). Potency of different adjuvant/R0.10C combinations was tested in mice and rats using aluminium hydroxide (Alum), Alum with micellar and emulsion formulations of a synthetic TLR4 agonist, Glucopyranosyl Lipid Adjuvant (GLA), stable emulsion (SE)/GLA, AbISCO-100 and Freund’s adjuvant (as reference). All formulations produced high antibody titres recognizing the native Pfs48/45 protein in macrogametes/zygotes. Interestingly, the GLA-Alum combination adjuvant was the most potent inducer of TB antibodies based on serum collected after two immunizations. In agreement with previous observations, biological activity in the SMFA correlated well with the level of anti-Pfs48/45 antibodies.
The combined data provide a strong basis for entering the next phase of clinical grade R0.10C production and testing.
[Show abstract][Hide abstract] ABSTRACT: Background:
Sterile protection in >90% of volunteers against homologous Plasmodium falciparum infection has been achieved only using the controlled human malaria infection (CHMI) model. This efficient model involves whole parasite immunizations under chloroquine prophylaxis (CPS-immunization), requiring only 30-45 mosquitoes bites infected with P. falciparum-sporozoites. Given the large diversity of P. falciparum parasites, it is essential to assess protection against heterologous parasite strains.
In an open-label follow-up study, 16 volunteers previously CPS-immunized and challenged with P. falciparum NF54 (West-Africa) in a dose de-escalation and challenge trial were re-challenged with clone NF135.C10 (Cambodia) at 14 months after the last immunization (NCT01660854).
Two out of thirteen NF54 protected volunteers previously fully protected against NF54 were also fully protected against NF135.C10, while 11/13 showed a delayed patency (median prepatent period of 10.5 days (range 9.0-15.5) versus 8.5 days in 5 malaria-naïve controls (p = 0.0005). Analysis of patency by qPCR indicated a 91 to >99% estimated reduction of liver parasite load in 7/11 partially protected subjects. Three volunteers previously not protected against NF54, were also not protected against NF135.C10.
This study shows that CPS-immunization can induce heterologous protection for a period of more than one year, which is a further impetus for clinical development of whole parasite vaccines.
[Show abstract][Hide abstract] ABSTRACT: A number of synthetic pantothenate derivatives, such as pantothenamides, are known to inhibit the growth of the human malaria parasite Plasmodium falciparum, by interfering with the parasite Coenzyme A (CoA) biosynthetic pathway. The clinical use of pantothenamides is limited by their sensitivity to breakdown by ubiquitous human pantetheinases of the vanin family.
A number of pantothenate derivatives (pantothenones) with potent and specific inhibitory activity against mammalian vanins were tested in a proliferation assay of asexual P. falciparum blood stages alone, and in combination with pantothenamides.
The vanin inhibitors were found to protect pantothenamides against breakdown by plasma vanins, thereby preserving the in vitro anti-malarial activity. Moreover, some of the vanin inhibitors showed in vitro anti-malarial activity in the low micromolar range. The most potent antimalarial in this series of compounds (RR8), was found to compete with pantothenate in a combination proliferation assay. No correlation, however, was found between anti-vanin and anti-malarial activity, nor was pantetheinase activity detected in P. falciparum extracts.
Growth inhibition is most likely due to competition with pantothenate, rather than pantetheinase inhibition. As vanin inhibitors of the pantothenone class are stable in biological fluids and are non-toxic to mammalian cells, they may represent novel pantothenate-based anti-malarials, either on their own or in combination with pantothenamides.
[Show abstract][Hide abstract] ABSTRACT: A highly efficacious pre-erythrocytic stage vaccine would be an important tool for the control and elimination of malaria but is currently unavailable. High-level protection in humans can be achieved by experimental immunization with Plasmodium falciparum sporozoites attenuated by radiation or under anti-malarial drug coverage. Immunization with genetically attenuated parasites (GAP) would be an attractive alternative approach. In this study, we present data on safety and protective efficacy using sporozoites with deletions of two genes, that is the newly identified b9 and slarp, which govern independent and critical processes for successful liver-stage development. In the rodent malaria model, PbΔb9ΔslarpGAP was completely attenuated showing no breakthrough infections while efficiently inducing high-level protection. The human PfΔb9ΔslarpGAP generated without drug resistance markers were infective to human hepatocytes in vitro and to humanized mice engrafted with human hepatocytes in vivo but completely aborted development after infection. These findings support the clinical development of a PfΔb9ΔslarpSPZ vaccine. DOI: http://dx.doi.org/10.7554/eLife.03582.001
[Show abstract][Hide abstract] ABSTRACT: Immunization of healthy volunteers with chloroquine ChemoProphylaxis and Sporozoites (CPS-CQ) efficiently and reproducibly induces dose-dependent and long-lasting protection against homologous Plasmodium falciparum challenge. Here, we studied whether chloroquine can be replaced by mefloquine, which is the only other licensed anti-malarial chemoprophylactic drug that does not affect pre-erythrocytic stages, exposure to which is considered essential for induction of protection by CPS immunization. In a double blind randomized controlled clinical trial, volunteers under either chloroquine prophylaxis (CPS-CQ, n = 5) or mefloquine prophylaxis (CPS-MQ, n = 10) received three sub-optimal CPS immunizations by bites from eight P. falciparum infected mosquitoes each, at monthly intervals. Four control volunteers received mefloquine prophylaxis and bites from uninfected mosquitoes. CPS-MQ immunization is safe and equally potent compared to CPS-CQ inducing protection in 7/10 (70%) versus 3/5 (60%) volunteers, respectively. Furthermore, specific antibody levels and cellular immune memory responses were comparable between both groups. We therefore conclude that mefloquine and chloroquine are equally effective in CPS-induced immune responses and protection.
[Show abstract][Hide abstract] ABSTRACT: Malaria transmission blocking vaccines (TBV) directed against proteins expressed on sexual stages of Plasmodium falciparum in the mosquito midgut are considered an effective means to reduce malaria transmission. Antibodies induced by TBV block sporogonic development in the mosquito, and thus transmission to the next human host. The Pfs25 protein, expressed on the surface of gametes, zygotes and ookinetes, is one of the primary targets for TBV development. Using a plant virus-based transient expression system, we have successfully produced Pfs25 fused to a modified lichenase (LicKM) carrier in Nicotiana benthamiana, purified and characterized the protein (Pfs25-FhCMB), and evaluated this vaccine candidate in animal models for the induction of transmission blocking antibodies (TBA). Soluble Pfs25-FhCMB was expressed in plants at a high level, and induced TBA that persisted for up to 6 months post immunization in mice and rabbits. These data demonstrate the potential of the new malaria vaccine candidate and also support feasibility of expressing Plasmodium antigens in a plant-based system.
No preview · Article · Aug 2014 · Human Vaccines and Immunotherapeutics
[Show abstract][Hide abstract] ABSTRACT: Background. Immunization of healthy volunteers by bites from Plasmodium falciparum–infected mosquitoes during chloroquine chemoprophylaxis (hereafter, chemoprophylaxis and sporozoites [CPS] immunization) induces sterile protection against malaria. CPS-induced protection is mediated by immunity against pre-erythrocytic stages, presumably at least partially by cytotoxic cellular responses. We therefore aimed to investigate the association of CPS-induced cytotoxic T-cell markers with protection.
Methods. In a double-blind randomized controlled trial, we performed dose titration of CPS immunization followed by homologous challenge infection in 29 subjects. Immune responses were assessed by in vitro restimulation of peripheral blood mononuclear cells and flow cytometry.
Results. Dose-dependent complete protection was obtained in 4 of 5 volunteers after immunization with bites from 45 P. falciparum–infected mosquitoes, in 8 of 9 volunteers with bites from 30, and in 5 of 10 volunteers with bites from 15 (odds ratio [OR], 5.0; 95% confidence interval [CI], 1.5–17). Completely protected subjects had significantly higher proportions of CD4 T cells expressing the degranulation marker CD107a (OR, 8.4; 95% CI, 1.5–123; P = .011) and CD8 cells producing granzyme B (OR, 11; 95% CI, 1.9–212; P = .004) after P. falciparum restimulation.
Conclusions. These data underline the efficiency of CPS immunization to induce sterile protection and support a possible role for cytotoxic CD4 and CD8 T-cell responses in pre-erythrocytic immunity.
Clinical Trials Registration. NCT01218893.
Full-text · Article · May 2014 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: The development of drugs and vaccines to reduce malaria transmission is an important part of eradication plans. The transmission reducing activity (TRA) of these agents is currently determined in the standard membrane feeding assay (SMFA) based on subjective microscopical read-outs and with limitations in up-scaling and throughput.
Utilising a Plasmodium falciparum strain expressing the firefly luciferase protein, we present a luminescence based approach to SMFA evaluation that eliminates the requirement for mosquito dissections in favour of a simple approach where whole mosquitoes are homogenised and examined directly for luciferase activity.
Analysis of 6860 Anopheles stephensi mosquitoes across 68 experimental feeds shows that the luminescence assay was as sensitive as microscopy for infection detection. The mean luminescence intensity of individual and pooled mosquitoes accurately quantifies mean oocyst intensity and generates comparable TRA estimates. The luminescence assay presented here could increase SMFA throughput so that 10-30 experimental feeds could be evaluated in a single 96-well plate.
This new method of assessing Plasmodium infection and transmission intensity could expedite the screening of novel drug compounds, vaccine candidates and sera from malaria exposed individuals for TRA. Luminescence-based estimates of oocyst intensity in individual mosquitoes should be interpreted with caution.
Full-text · Article · May 2014 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Effective control and eventual eradication of malaria drives the imperative need for clinical development of a malaria vaccine. Asexual parasite forms are responsible for clinical disease and death while apathogenic gametocytes are responsible for transmission from man to mosquito. Vaccines that combine antigens from both stages may provide direct protection and indirect benefit by reducing the force of infection. We constructed a chimeric antigen composed of a fragment of the Plasmodium falciparum (Pf) glutamate-rich protein fused in frame to a correctly folded fragment of Pfs48/45. The chimera was produced in Lactococcus lactis and induced robust antibody responses in rodents to the individual components. Specific antibodies showed strong transmission blocking activity against multiple Pf-strains in the standard membrane feeding assay and functional activity against asexual stages in the antibody dependent cellular inhibition assay. The combined data provide a strong rationale for entering the next phase of clinical grade production and testing.
[Show abstract][Hide abstract] ABSTRACT: Mosquito feeding assays are important in evaluations of malaria transmission-reducing interventions. The proportion of mosquitoes with midgut oocysts is commonly used as an outcome measure, but in natural low intensity infections the effect of oocyst non-rupture on mosquito infectivity is unclear. By identifying ruptured as well as intact oocysts, we show that in low intensity P. falciparum infections i) 66.7-96.7% of infected mosquitoes experienced oocyst rupture between 11-21 days post-infection, ii) oocyst rupture led invariably to sporozoite release, iii) oocyst rupture led to salivary gland infections in 97.8% of mosquitoes, and iv) 1250 (IQR 313-2400) salivary gland sporozoites were found per ruptured oocyst. These data show that infectivity can be predicted with reasonable certainty from oocyst prevalence in low intensity infections. High throughput methods for detecting infection in whole mosquitoes showed that 18s PCR but not circumsporozoite ELISA gave a reliable approximation of mosquito infection rates on day 7 post-infection.
Full-text · Article · Dec 2013 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: Malaria transmission blocking vaccines (TBVs) are considered an effective means to control and eventually eliminate malaria. The Pfs25 protein, expressed predominantly on the surface of the sexual and sporogonic stages of Plasmodium falciparum including gametes, zygotes and ookinetes, is one of the primary targets for TBV. It has been demonstrated that plants are an effective, highly scalable system for the production of recombinant proteins, including virus-like particles (VLPs). We engineered VLPs (Pfs25-CP VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein (CP) and produced these non-enveloped hybrid VLPs in Nicotiana benthamiana plants using a Tobacco mosaic virus-based 'launch' vector. Purified Pfs25-CP VLPs were highly consistent in size (19.3±2.4 nm in diameter) with an estimated 20-30% incorporation of Pfs25 onto the VLP surface. Immunization of mice with one or two doses of Pfs25-CP VLPs plus Alhydrogel® induced serum antibodies with complete transmission blocking activity through the 6 month study period. These results support the evaluation of Pfs25-CP VLP as a potential TBV candidate and the feasibility of the 'launch' vector technology for the production of VLP-based recombinant vaccines against infectious diseases.
[Show abstract][Hide abstract] ABSTRACT: There is much evidence that some pathogens manipulate the behaviour of their mosquito hosts to enhance pathogen transmission. However, it is unknown whether this phenomenon exists in the interaction of Anopheles gambiae sensu stricto with the malaria parasite, Plasmodium falciparum - one of the most important interactions in the context of humanity, with malaria causing over 200 million human cases and over 770 thousand deaths each year. Here we demonstrate, for the first time, that infection with P. falciparum causes alterations in behavioural responses to host-derived olfactory stimuli in host-seeking female An. gambiae s.s. mosquitoes. In behavioural experiments we showed that P. falciparum-infected An. gambiae mosquitoes were significantly more attracted to human odors than uninfected mosquitoes. Both P. falciparum-infected and uninfected mosquitoes landed significantly more on a substrate emanating human skin odor compared to a clean substrate. However, significantly more infected mosquitoes landed and probed on a substrate emanating human skin odor than uninfected mosquitoes. This is the first demonstration of a change of An. gambiae behaviour in response to olfactory stimuli caused by infection with P. falciparum. The results of our study provide vital information that could be used to provide better predictions of how malaria is transmitted from human being to human being by An. gambiae s.s. females. Additionally, it highlights the urgent need to investigate this interaction further to determine the olfactory mechanisms that underlie the differential behavioural responses. In doing so, new attractive compounds could be identified which could be used to develop improved mosquito traps for surveillance or trapping programmes that may even specifically target P. falciparum-infected An. gambiae s.s. females.
[Show abstract][Hide abstract] ABSTRACT: Volunteers immunized under chloroquine chemoprophylaxis with Plasmodium falciparum sporozoites (CPS) develop complete, long-lasting protection against homologous sporozoite challenge. Chloroquine affects neither sporozoites nor liver-stages, but kills only asexual forms in erythrocytes once released from the liver into the circulation. Consequently, CPS immunization exposes the host to antigens from both preerythrocytic and blood stages, and induced immunity might target either of these stages. We therefore explored the life cycle stage specificity of CPS-induced protection. Twenty-five malaria-naïve volunteers were enrolled in a clinical trial, 15 of whom received CPS immunization. Five immunized subjects and five controls received a sporozoite challenge by mosquito bites, whereas nine immunized and five control subjects received an i.v. challenge with P. falciparum-infected erythrocytes. The latter approach completely bypasses preerythrocytic stages, enabling a direct comparison of protection against either life cycle stage. CPS-immunized subjects (13 of 14) developed anticircumsporozoite antibodies, whereas only one volunteer generated minimal titers against typical blood-stage antigens. IgG from CPS-immunized volunteers did not inhibit asexual blood-stage growth in vitro. All CPS-immunized subjects (5 of 5) were protected against sporozoite challenge. In contrast, nine of nine CPS-immunized subjects developed parasitemia after blood-stage challenge, with identical prepatent periods and blood-stage multiplication rates compared with controls. Intravenously challenged CPS-immunized subjects showed earlier fever and increased plasma concentrations of inflammatory markers D-dimer, IFN-γ, and monokine induced by IFN-γ than i.v. challenged controls. The complete lack of protection against blood-stage challenge indicates that CPS-induced protection is mediated by immunity against preerythrocytic stages. However, evidence is presented for immune recognition of P. falciparum-infected erythrocytes, suggesting memory responses unable to generate functional immunity.
Full-text · Article · Apr 2013 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: We established a new field clone of Plasmodium falciparum for use in controlled human malaria infections and vaccine studies to complement the current small portfolio of P. falciparum strains, primarily based on NF54. The Cambodian clone NF135.C10 consistently produced gametocytes and generated substantial numbers of sporozoites in Anopheles mosquitoes and diverged from NF54 parasites by genetic markers. In a controlled human malaria infection trial, 3 of 5 volunteers challenged by mosquitoes infected with NF135.C10 and 4 of 5 challenged with NF54 developed parasitemia as detected with microscopy. The 2 strains induced similar clinical signs and symptoms as well as cellular immunological responses.
Clinical Trials Registration NCT01002833.
Full-text · Article · Nov 2012 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Plasmodium falciparum is transmitted to a new host after completing its sexual cycle within a mosquito. Developing vaccines against the parasite sexual stages is a critical component in the fight against malaria. We are targeting multiple proteins of P. falciparum which are found only on the surfaces of the sexual forms of the parasite and where antibodies against these proteins have been shown to block the progression of the parasite's life cycle in the mosquito and thus block transmission to the next human host. We have successfully produced a region of the Pfs230 antigen in our plant-based transient-expression system and evaluated this vaccine candidate in an animal model. This plant-produced protein, 230CMB, is expressed at approximately 800 mg/kg in fresh whole leaf tissue and is 100% soluble. Administration of 230CMB with >90% purity induces strong immune responses in rabbits with high titers of transmission-blocking antibodies, resulting in a greater than 99% reduction in oocyst counts in the presence of complement, as determined by a standard membrane feeding assay. Our data provide a clear perspective on the clinical development of a Pfs230-based transmission-blocking malaria vaccine.
Full-text · Article · Jun 2011 · Clinical and vaccine Immunology: CVI
[Show abstract][Hide abstract] ABSTRACT: We have shown that immunity to infection with Plasmodium falciparum can be induced experimentally in malaria-naive volunteers through immunisation by bites of infected mosquitoes while simultaneously preventing disease with chloroquine prophylaxis. This immunity was associated with parasite-specific production of interferon γ and interleukin 2 by pluripotent effector memory cells in vitro. We aim to explore the persistence of protection and immune responses in the same volunteers.
In an open-label study at the Radboud University Nijmegen Medical Centre (Nijmegen, Netherlands), from November to December, 2009, we rechallenged previously immune volunteers (28 months after immunisation) with the bites of five mosquitoes infected with P falciparum. Newly recruited malaria-naive volunteers served as infection controls. Our primary outcome was the detection of blood-stage parasitaemia by microscopy. We assessed the kinetics of parasitaemia with real-time quantitative PCR (rtPCR) and recorded clinical signs and symptoms. In-vitro production of interferon γ and interleukin 2 by effector memory T cells was studied after stimulation with sporozoites and red blood cells infected with P falciparum. Differences in cellular immune responses between the study groups were assessed with the Mann-Whitney test. This study is registered with ClinicalTrials.gov, number NCT00757887.
Four of six immune volunteers were microscopically negative after rechallenge. rtPCR-based detection of blood-stage parasites in these individuals was negative throughout follow-up. Patent parasitaemia was delayed in the remaining two immunised volunteers. In-vitro assays showed the long-term persistence of parasite-specific pluripotent effector memory T-cell responses in protected volunteers. The four protected volunteers reported several mild to moderate adverse events, of which the most commonly reported symptom was headache (one to three episodes per volunteer). The two patients with delayed patency had adverse events similar to those in the control group.
Artificially induced immunity lasts longer than generally recorded after natural exposure; providing a new avenue of research into the mechanisms of malaria immunity.
[Show abstract][Hide abstract] ABSTRACT: Malaria is a serious and sometimes fatal mosquito-borne disease caused by a protozoan parasite. Each year, it is estimated that over one million people are killed by malaria, yet the disease is preventable and treatable. Developing vaccines against the parasite is a critical component in the fight against malaria and these vaccines can target different stages of the pathogen's life cycle. We are targeting sexual stage proteins of P. falciparum which are found on the surface of the parasite reproductive cells present in the mosquito gut. Antibodies against these proteins block the progression of the parasite's life cycle in the mosquito, and thus block transmission to the next human host. Transmission blocking vaccines are essential to the malaria eradication program to ease the disease burden at the population level. We have successfully produced multiple versions of the Pfs25 antigen in a plant virus-based transient expression system and have evaluated these vaccine candidates in an animal model. The targets are expressed in plants at a high level, are soluble and most importantly, generate strong transmission blocking activity as determined by a standard membrane feeding assay. These data demonstrate the feasibility of expressing Plasmodium antigens in a plant-based system for the economic production of a transmission blocking vaccine against malaria.
[Show abstract][Hide abstract] ABSTRACT: Naturally acquired immune responses against sexual stages of P. falciparum can reduce the transmission of malaria from humans to mosquitoes. These antigens are candidate transmission-blocking vaccines but little is known about the acquisition of sexual stage immunity after exposure to gametocytes, or their longevity and functionality. We conducted a longitudinal study on functional sexual stage immune responses.
Parasitaemic individuals (n = 116) were recruited at a health centre in Lower Moshi, Tanzania. Patients presented with gametocytes (n = 16), developed circulating gametocytes by day 7 (n = 69) or between day 7 and 14 (n = 10) after treatment or did not develop gametocytes (n = 21). Serum samples were collected on the first day of gametocytaemia and 28 and 84 days post-enrolment (or d7, 28, 84 after enrolment from gametocyte-negative individuals). Antibody responses to sexual stage antigens Pfs230 and Pfs48/45 were detected in 20.7% (72/348) and 15.2% (53/348) of the samples, respectively, and were less prevalent than antibodies against asexual stage antigens MSP-1(19) (48.1%; 137/285) and AMA-1 (52.4%; 129/246)(p<0.001). The prevalence of anti-Pfs230 (p = 0.026) and anti-Pfs48/45 antibodies (p = 0.017) increased with longer duration of gametocyte exposure and had an estimated half-life of approximately 3 months. Membrane feeding experiments demonstrated a strong association between the prevalence and concentration of Pfs230 and Pfs48/45 antibodies and transmission reducing activity (TRA, p<0.01).
In a longitudinal study, anti-Pfs230 and Pfs48/45 antibodies developed rapidly after exposure to gametocytes and were strongly associated with transmission-reducing activity. Our data indicate that the extent of antigen exposure is important in eliciting functional transmission-reducing immune responses.