Feasibility of direct venous inoculation of the radiation-attenuated Plasmodium falciparum whole sporozoite vaccine in children and infants in Siaya, western Kenya.

PfSPZ Vaccine, composed of radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum sporozoites, is administered by direct venous inoculation (DVI) for maximal efficacy against malaria. A critical issue for advancing vaccines that are administered intravenously is the ability to efficiently administer them across multiple age groups. As part of a pediatric safety, immunogenicity, and efficacy trial in western Kenya, we evaluated the feasibility and tolerability of DVI, including ease of venous access, injection time, and crying during the procedure across age groups. Part 1 was an age de-escalation, dose escalation trial in children aged 13 months-5 years and infants aged 5-12 months; part 2 was a vaccine efficacy trial including only infants, using the most skilled injectors from part 1. Injectors could use a vein viewer, if needed. A total of 1222 injections (target 0.5 mL) were initiated by DVI in 511 participants (36 were 5-9-year-olds, 65 were 13-59-month-olds, and 410 infants). The complete volume was injected in 1185/1222 (97.0%) vaccinations, 1083/1185 (91.4%) achieved with the first DVI. 474/511 (92.8%) participants received only complete injections, 27/511 (5.3%) received at least one partial injection (<0.5 mL), and in 10/511 (2.0%) venous access was not obtained. The rate of complete injections by single DVI for infants improved from 77.1% in part 1 to 92.8% in part 2. No crying occurred in 51/59 (86.4%) vaccinations in 5-9-year-olds, 25/86 (29.1%) vaccinations in 13-59-month-olds and 172/1067 (16.1%) vaccinations in infants. Mean administration time ranged from 2.6 to 4.6 minutes and was longer for younger age groups. These data show that vaccination by DVI was feasible and well tolerated in infants and children in this rural hospital in western Kenya, when performed by skilled injectors. We also report that shipping and storage in liquid nitrogen vapor phase was simple and efficient. (Clinicaltrials.gov NCT02687373).

Live attenuated malaria vaccine designed to protect through hepatic CD8⁺ T cell immunity.

Our goal is to develop a vaccine that sustainably prevents Plasmodium falciparum (Pf) malaria in ≥80% of recipients. Pf sporozoites (PfSPZ) administered by mosquito bites are the only immunogens shown to induce such protection in humans. Such protection is thought to be mediated by CD8(+) T cells in the liver that secrete interferon-γ (IFN-γ). We report that purified irradiated PfSPZ administered to 80 volunteers by needle inoculation in the skin was safe, but suboptimally immunogenic and protective. Animal studies demonstrated that intravenous immunization was critical for inducing a high frequency of PfSPZ-specific CD8(+), IFN-γ-producing T cells in the liver (nonhuman primates, mice) and conferring protection (mice). Our results suggest that intravenous administration of this vaccine will lead to the prevention of infection with Pf malaria.

Report of a consultation on the optimization of clinical challenge trials for evaluation of candidate blood stage malaria vaccines, 18-19 March 2009, Bethesda, MD, USA.

Development and optimization of first generation malaria vaccine candidates has been facilitated by the existence of a well-established Plasmodium falciparum clinical challenge model in which infectious sporozoites are administered to human subjects via mosquito bite. While ideal for testing pre-erythrocytic stage vaccines, some researchers believe that the sporozoite challenge model is less appropriate for testing blood stage vaccines. Here we report a consultation, co-sponsored by PATH MVI, USAID, EMVI and WHO, where scientists from all institutions globally that have conducted such clinical challenges in recent years and representatives from regulatory agencies and funding agencies met to discuss clinical malaria challenge models. Participants discussed strengthening and harmonizing the sporozoite challenge model and considered the pros and cons of further developing a blood stage challenge possibly better suited for evaluating the efficacy of blood stage vaccines. This report summarizes major findings and recommendations, including an update on the Plasmodium vivax clinical challenge model, the prospects for performing experimental challenge trials in malaria endemic countries and an update on clinical safety data. While the focus of the meeting was on the optimization of clinical challenge models for evaluation of blood stage candidate malaria vaccines, many of the considerations are relevant for the application of challenge trials to other purposes.

Viral vectors in malaria vaccine development.

Traditional vaccine technologies have resulted in an impressive array of efficacious vaccines against a variety of infectious agents. However, several potentially deadly pathogens, including retroviruses and parasites, have proven less amenable to the application of traditional vaccine platforms, indicating the need for new approaches. Viral vectors represent an attractive way to deliver and present vaccine antigens that may offer advantages over traditional platforms. Due to their ability to induce strong cell-mediated immunity (CMI) in addition to antibodies, viral vectors may be suitable for infectious agents, such as malaria parasites, where potent CMI is required for protection. Poxvirus-vectored malaria vaccines have been the most extensively studied in the clinic, achieving significant reductions in liver-stage parasite burden. More recently, adenovirus-vectored malaria vaccines have entered clinical testing. The most promising approach - heterologous prime-boost regimens, in which different viral vectors are sequentially paired with each other or with DNA or recombinant protein vaccines - is now being explored, and could provide high-grade protection, if findings in animal models are translatable to humans. Significant barriers remain, however, such as pre-existing immunity to the vector particle and an unexplained safety signal observed in one trial suggesting an increased risk of HIV acquisition in volunteers with pre-existing immunity to the vector.

A DNA prime-modified vaccinia virus ankara boost vaccine encoding thrombospondin-related adhesion protein but not circumsporozoite protein partially protects healthy malaria-naive adults against Plasmodium falciparum sporozoite challenge.

The safety, immunogenicity, and efficacy of DNA and modified vaccinia virus Ankara (MVA) prime-boost regimes were assessed by using either thrombospondin-related adhesion protein (TRAP) with a multiple-epitope string ME (ME-TRAP) or the circumsporozoite protein (CS) of Plasmodium falciparum. Sixteen healthy subjects who never had malaria (malaria-naive subjects) received two priming vaccinations with DNA, followed by one boosting immunization with MVA, with either ME-TRAP or CS as the antigen. Immunogenicity was assessed by ex vivo gamma interferon (IFN-gamma) enzyme-linked immunospot assay (ELISPOT) and antibody assay. Two weeks after the final vaccination, the subjects underwent P. falciparum sporozoite challenge, with six unvaccinated controls. The vaccines were well tolerated and immunogenic, with the DDM-ME TRAP regimen producing stronger ex vivo IFN-gamma ELISPOT responses than DDM-CS. One of eight subjects receiving the DDM-ME TRAP regimen was completely protected against malaria challenge, with this group as a whole showing significant delay to parasitemia compared to controls (P = 0.045). The peak ex vivo IFN-gamma ELISPOT response in this group correlated strongly with the number of days to parasitemia (P = 0.033). No protection was observed in the DDM-CS group. Prime-boost vaccination with DNA and MVA encoding ME-TRAP but not CS resulted in partial protection against P. falciparum sporozoite challenge in the present study.

Effect on antibody and T-cell responses of mixing five GMP-produced DNA plasmids and administration with plasmid expressing GM-CSF.

One potential benefit of DNA vaccines is the capacity to elicit antibody and T-cell responses against multiple antigens at the same time by mixing plasmids expressing different proteins. A possible negative effect of such mixing is interference among plasmids regarding immunogenicity. In preparation for a clinical trial, we assessed the immunogenicity of GMP-produced plasmids encoding five Plasmodium falciparum proteins, PfCSP, PfSSP2, PfEXP1, PfLSA1, and PfLSA3, given as a mixture, or alone. The mixture induced higher levels of antibodies against whole parasites than did the individual plasmids, but was associated with a decrease in antibodies to individual P. falciparum proteins. T-cell responses were in general decreased by administration of the mixture. Immune responses to individual plasmids and mixtures were generally higher in inbred mice than in outbreds. In inbred BALB/c and C57BL/6 mice, coadministration of a plasmid expressing murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), increased antibody and T-cell responses, but in outbred CD-1 mice, coadministration of mGM-CSF was associated with a decrease in antibody responses. Such variability in data from studies in different strains of mice underscores the importance of genetic background on immune response and carefully considering the goals of any preclinical studies of vaccine mixtures planned for human trials.

Utilization of genomic sequence information to develop malaria vaccines.

Recent advances in the fields of genomics, proteomics and molecular immunology offer tremendous opportunities for the development of novel interventions against public health threats, including malaria. However, there is currently no algorithm that can effectively identify the targets of protective T cell or antibody responses from genomic data. Furthermore, the identification of antigens that will stimulate the most effective immunity against the target pathogen is problematic, particularly if the genome is large. Malaria is an attractive model for the development and validation of approaches to translate genomic information to vaccine development because of the critical need for effective anti-malarial interventions and because the Plasmodium parasite is a complex multistage pathogen targeted by multiple immune responses. Sterile protective immunity can be achieved by immunization with radiation-attenuated sporozoites, and anti-disease immunity can be induced in residents in malaria-endemic areas. However, the 23 Mb Plasmodium falciparum genome encodes more than 5,300 proteins, each of which is a potential target of protective immune responses. The current generation of subunit vaccines is based on a single or few antigens and therefore might elicit too narrow a breadth of response. We are working towards the development of a new generation vaccine based on the presumption that duplicating the protection induced by the whole organism may require a vaccine nearly as complex as the organism itself. Here, we present our strategy to exploit the genomic sequence of P. falciparum for malaria vaccine development.

Onset of clinical immunity to Plasmodium falciparum among Javanese migrants to Indonesian Papua.

Onset of clinical immunity to Plasmodium falciparum occurred among Javanese migrants to Indonesian Papua. Surveillance of the 243 migrants investigated began on the day of their arrival in Indonesian Papua and continued for 33 months. Asexual parasitaemia without fever constituted objective evidence of clinical immunity. Compared with first infection, the odds ratio (OR) for not having fever at the fourth infection within 24 months was 3.2 [95% confidence interval (CI)=1.03-10.2; P=0.02]. The corresponding OR with fewer infections within 24 months was not distinguishable from 1.0. The level of the fourth parasitaemia within 24 months (N=58) was classified as 'high' or 'low' in relation to the median count at first infection (840 parasites/microl; N=187). Fourth parasitaemias that were low-but not those that were high (OR=1.8; CI=0.6-5.4; P=0.35)-were associated with dramatic protection from fever (OR=31; CI=3.5-1348; P=0.0001). Among the adult subjects, the risk of fever with low parasitaemia was significantly higher at the first infection than at the fourth (OR=12.6; CI=1.7-530; P=0.005), indicating the development of clinical immunity. A similar but less marked pattern appeared among the children investigated (OR=6.5; CI=0.8-285; P=0.06).

Malaria in a cohort of Javanese migrants to Indonesian Papua.

The epidemiology of infection by Plasmodium falciparum and P. vivax was investigated among Javanese migrants to an endemic region of Papua, Indonesia. A cohort of 243 migrants from Java was followed for malaria in a new settlement village in the endemic Armopa area of north-eastern Papua, beginning on the day each migrant arrived in the village. The subjects were monitored during home visits (three/week) and by the twice-monthly production of bloodsmears that were checked for malarial parasites. At the end of 33 months, 159 (65%) of the subjects remained under follow-up. The prevalence of parasitaemia in the village declined from 16% among those already living there when the study began in August 1996, to 5% when the study finished in June 1999. Over this period, 596 infections by P. falciparum and 723 by P. vivax occurred in the cohort, 22 and 27 of the subjects each experiencing at least six infections by P. falciparum and P. vivax, respectively. The incidence of malarial infection was higher during the first and second years post-migration (3.2 and 2.7 infections/person-year) than during the third (1.2 infections/person-year). Although the geometric mean parasite counts for P. falciparum increased over time (1209, 1478, and 1830 parasites/microl in the first, second and third years, respectively), the corresponding values for P. vivax (497, 535 and 490 parasites/microl) showed no such trend. Only one of the nine subjects who developed severe malaria (requiring intravenous quinine therapy) was a child, giving an odds ratio for a case of severe malaria being in an adult of 6.1 (P=0.08).

Primary infection by Plasmodium falciparum or P. vivax in a cohort of Javanese migrants to Indonesian Papua.

The clinical and parasitological characteristics of the first naturally acquired malarial infection have rarely been documented in humans. When 243 migrants from non-endemic Java were followed from the day of their arrival in Indonesian Papua, 217 (89%) were found to become infected with Plasmodium falciparum and/or P. vivax before they were lost to follow-up. The incidence of malarial infection in the children investigated (who were aged 6-10 years) was indistinguishable from that in the adults (aged >20 years), with 1.10 and 1.14 P. falciparum infections/person-year (relative risk=0.97; 95% confidence interval=0.72-1.29) and 1.47 and 1.49 P. vivax infections/person-year (relative risk=0.99; 95% confidence interval=0.72-1.29), respectively. During their first infections, the children had higher P. falciparum parasitaemias than the adults (with geometric means of 1318 and 759 parasites/microl, respectively; P=0.04) but similar P. vivax parasitaemias (with geometric means of 355 and 331 parasites/microl, respectively; P=0.76). At first infection, 56% of the subjects were febrile and 90% complained of symptoms. There were no differences between children and adults with respect to these two parameters, either for P. falciparum or P. vivax. These findings indicate that, with promptly diagnosed and treated uncomplicated malaria, migrant children and adults in north-eastern Indonesian Papua have an equal risk of malarial infection and of disease following their first infections with P. falciparum and P. vivax.

Induction of CD4(+) T cell-dependent CD8(+) type 1 responses in humans by a malaria DNA vaccine.

We assessed immunogenicity of a malaria DNA vaccine administered by needle i.m. or needleless jet injection [i.m. or i.m./intradermally (i.d.)] in 14 volunteers. Antigen-specific IFN-gamma responses were detected by enzyme-linked immunospot (ELISPOT) assays in all subjects to multiple 9- to 23-aa peptides containing class I and/or class II restricted epitopes, and were dependent on both CD8(+) and CD4(+) T cells. Overall, frequency of response was significantly greater after i.m. jet injection. CD8(+)-dependent cytotoxic T lymphocytes (CTL) were detected in 8/14 volunteers. Demonstration in humans of elicitation of the class I restricted IFN-gamma responses we believe necessary for protection against the liver stage of malaria parasites brings us closer to an effective malaria vaccine.

Chloroquine/doxycycline combination versus chloroquine alone, and doxycycline alone for the treatment of Plasmodium falciparum and Plasmodium vivax malaria in northeastern Irian Jaya, Indonesia.

Combination therapy is one method of overcoming the global challenge of drug-resistant Plasmodium falciparum malaria. We conducted a hospital-based 28-day in vivo test comparing chloroquine/doxycycline to chloroquine or doxycycline alone for treating P. falciparum and Plasmodium vivax malaria in Irian Jaya, Indonesia. Eighty-nine patients with uncomplicated falciparum malaria were randomized to standard dose chloroquine (n = 30), doxycycline (100 mg every 12 hours [7 days], n = 20), or chloroquine with doxycycline (n = 39); corresponding numbers for vivax malaria (n = 63) were 23, 16, 24. Endpoints were parasite sensitivity (S) or resistance (RI/RII/RIII). Of the 105 evaluable patients, chloroquine/doxycycline cured (S) 20/22 (90.9% [95% CI 78.9-100%]) patients with P. falciparum malaria; 2/22 (9.1% [0-21%]) were RIII resistant. Doxycycline cured 11/17 (64.7% [42.0-87.4%]) patients, and chloroquine 4/20 (20% [2.5-37.5%]). Against P. vivax, chloroquine/doxycycline cured (S) 12/17 (70.6% [48.9-92.2%]) patients, doxycycline 4/12 (33.3% [6.6-59.9%]), and chloroquine 5/17 (29.4% [7.7-51.1%]). Chloroquine/doxycycline was effective against P. falciparum but only modestly effective against P. vivax. These findings support the use of chloroquine/doxycycline as an inexpensive alternative to mefloquine for treating chloroquine-resistant P. falciparum but not chloroquine-resistant P. vivax in this setting.

Naturally acquired antibody responses to Plasmodium falciparum merozoite surface protein 4 in a population living in an area of endemicity in Vietnam.

Merozoite surface protein 4 (MSP4) of Plasmodium falciparum is a glycosylphosphatidylinositol-anchored integral membrane protein that is being developed as a component of a subunit vaccine against malaria. We report here the measurement of naturally acquired antibodies to MSP4 in a population of individuals living in the Khanh-Hoa region of Vietnam, an area where malaria is highly endemic. Antibodies to MSP4 were detected in 94% of the study population at titers of 1:5,000 or greater. Two forms of recombinant MSP4 produced in either Escherichia coli or Saccharomyces cerevisiae were compared as substrates in the enzyme-linked immunosorbent assay. There was an excellent correlation between reactivity measured to either, although the yeast substrate was recognized by a higher percentage of sera. Four different regions of MSP4 were recognized by human antibodies, demonstrating that there are at least four distinct epitopes in this protein. In the carboxyl terminus, where the single epidermal growth factor-like domain is located, the reactive epitope(s) was shown to be conformation dependent, as disruption of the disulfide bonds almost completely abolished reactivity with human antibodies. The anti-MSP4 antibodies were mainly of the immunoglobulin G1 (IgG1) and IgG3 subclasses, suggesting that such antibodies may play a role in opsonization and complement-mediated lysis of free merozoites. Individuals in the study population were drug-cured and followed up for 6 months; no significant correlation was observed between the anti-MSP4 antibodies and the absence of parasitemia during the surveillance period. As a comparison, antibodies to MSP1(19), a leading vaccine candidate, were measured, and no correlation with protection was observed in these individuals. The anti-MSP1(19) antibodies were predominantly of the IgG1 isotype, in contrast to the IgG3 predominance noted for MSP4.

Antibody responses to infections with strains of Plasmodium falciparum expressing diverse forms of merozoite surface protein 2.

Individuals living in areas where Plasmodium falciparum is endemic experience numerous episodes of infection. These episodes may or may not be symptomatic, with the outcome depending on a combination of parasite and host factors, several of which are poorly understood. One factor is believed to be the particular alleles of several parasite proteins to which the host is capable of mounting protective immune responses. We report a study examining antibody responses to MSP2 in 15 semi-immune teenagers and adults living in the Khanh-Hoa area of southern-central Vietnam, where P. falciparum is highly endemic; subjects were serially infected with multiple strains of P. falciparum. The MSP2 alleles infecting these subjects were determined by nucleotide sequencing. A total of 62 MSP2 genes belonging to both dimorphic families were identified, of which 33 contained distinct alleles, with 61% of the alleles being detected once. Clear changes in the repertoire occurred between infections. Most infections contained a mixture of parasites expressing MSP2 alleles from both dimorphic families. Two examples of reinfection with a strain expressing a previously encountered allele were detected. Significant changes in antibody levels to various regions of MSP2 were detected over the course of the experiment. There was no clear relation between the infecting form of MSP2 and the ensuing antibody response. This study highlights the complexity of host-parasite relationship for this important human pathogen.

Plasmodium falciparum mdr1 mutations and in vivo chloroquine resistance in Indonesia.

Mutations in the Pfmdr1 gene are reported to be associated with chloroquine resistance in some Plasmodium falciparum isolates. A polymerase chain reaction/restriction fragment length polymorphism method was used for the detection of Pfmdr1 mutations in chloroquine-resistant field isolates of P. falciparum collected in Irian Jaya. The frequency of Pfmdr1 mutations was significantly higher in chloroquine-resistant P. falciparum parasites than background frequencies observed in the same location. The 7G8 mutation was identified in some parasites although always in a mixed genotype status. Chloroquine-resistant P. falciparum specimens were characterized using the World Health Organization 28-day criteria, supplemented by demonstrating adequate chloroquine absorption and genetic analysis.

In vivo responses to antimalarials by Plasmodium falciparum and Plasmodium vivax from isolated Gag Island off northwest Irian Jaya, Indonesia.

There is renewed interest in the rich nickel and cobalt deposits of Pulau Gag, an isolated but malarious island off the northwest coast of Irian Jaya. In preparation for an expanded workforce, an environmental assessment of malaria risk was made, focusing upon malaria prevalence in the small indigenous population, and the in vivo sensitivity of Plasmodium falciparum and P. vivax to chloroquine (CQ) and sulfadoxine/pyrimethamine (S/P), the respective first- and second-line drugs for uncomplicated malaria in Indonesia. During April-June 1997, mildly symptomatic or asymptomatic malaria infections were found in 24% of 456 native residents. Infections by P. falciparum accounted for 60% of the cases. Respective day 28 cure rates for CQ (10 mg base/kg on days 0 and 1; 5 mg/kg on day 2) in children and adults were 14% and 55% (P < 0.005). Type RII and RIII resistance characterized only 5% of the CQ failures. Re-treatment of 36 P. falciparum CQ treatment failures with S/P (25 mg/kg and 1.25 mg/kg, respectively) demonstrated rapid clearance and complete sensitivity during the 28-day follow-up period. More than 97% of the P. vivax malaria cases treated with CQ cleared parasitemia within 48 hr. Three cases of P. vivax malaria recurred between days 21 and 28, but against low drug levels in the blood. The low frequency of RII and RIII P. falciparum resistance to CQ, the complete sensitivity of this species to S/P, and the absence of CQ resistance by P. vivax are in contrast to in vivo and in vitro test results from sites on mainland Irian Jaya.

Malaria prophylaxis using azithromycin: a double-blind, placebo-controlled trial in Irian Jaya, Indonesia.

New drugs are needed for preventing drug-resistant Plasmodium falciparum malaria. The prophylactic efficacy of azithromycin against P. falciparum in malaria-immune Kenyans was 83%. We conducted a double-blind, placebo-controlled trial to determine the prophylactic efficacy of azithromycin against multidrug-resistant P. falciparum malaria and chloroquine-resistant Plasmodium vivax malaria in Indonesian adults with limited immunity. After radical cure therapy, 300 randomized subjects received azithromycin (148 subjects, 750-mg loading dose followed by 250 mg/d), placebo (77), or doxycycline (75, 100 mg/d). The end point was slide-proven parasitemia. There were 58 P. falciparum and 29 P. vivax prophylaxis failures over 20 weeks. Using incidence rates, the protective efficacy of azithromycin relative to placebo was 71.6% (95% confidence interval [CI], 50.3-83.8) against P. falciparum malaria and 98.9% (95% CI, 93.1-99.9) against P. vivax malaria. Corresponding figures for doxycycline were 96.3% (95% CI, 85.4-99.6) and 98% (95% CI, 88.0-99.9), respectively. Daily azithromycin offered excellent protection against P. vivax malaria but modest protection against P. falciparum malaria.

Chloroquine-resistant Plasmodium vivax in transmigration settlements of West Kalimantan, Indonesia.

Malariometric surveys were conducted during July 1996 in native Dayak villages and predominantly Javanese transmigration settlements in Ketapang district of West Kalimantan, Indonesia. Malaria prevalence ranged from 0.9% to 2.7% in Dayak villages and from 1% to 20% in the transmigration settlements. Plasmodium falciparum accounted for 67% of the cases among Dayaks but P. vivax was dominant among transmigrants, accounting for more than 72% of the infections. Chloroquine sensitivity/resistance was assessed by 28-day in vivo testing of uncomplicated malaria infections and measurement of chloroquine blood levels in cases where parasitemias reappeared within the 28-day test period. Resistance was based on the appearance of asexual parasites against chloroquine plus desethylchloroquine levels exceeding the minimally effective whole blood concentrations proposed for sensitive parasite strains (P. vivax, 100 ng/ml; P. falciparum, 200 ng/ml). All parasitemias cleared initially within four days of beginning supervised chloroquine therapy (25 mg base/kg over a 48-hr period), but asexual parasites reappeared within 28 days in 27 of 52 P. vivax and three of 12 P. falciparum cases. Chloroquine blood levels at the time of recurrent parasitemias revealed resistance in 12 of the 27 P. vivax cases and in one of the three P. falciparum cases. Genotypes of nine of the 12 recurrent P. vivax isolates matched with their primary isolates and ruled out reinfection. These findings establish the presence of chloroquine-resistant P. vivax on the island of Borneo. The pattern of malaria and the high frequency of chloroquine resistance by P. vivax at the West Kalimantan location may relate to demographic, ecologic, agricultural, and socioeconomic changes associated with transmigration.

Lymphocyte response to tetanus toxoid among Indonesian men immunized with tetanus-diphtheria during extended chloroquine or primaquine prophylaxis.

Immune suppression, a potential side effect of long-term chemoprophylaxis, was evaluated as part of a randomized, placebo-controlled trial that compared daily primaquine against weekly chloroquine for malaria prevention. In the last month of the year-long trial, baseline in vitro lymphoproliferative responses to tetanus toxoid were measured, and a tetanus-diphtheria (Td) immunization was administered. Proliferative responses to tetanus toxoid in each Td-immunized group increased significantly over pre-Td baselines and those of the unvaccinated control. Highest initial responses were measured in the primaquine group. The proportion of responders and the magnitude of proliferation was consistently low in the chloroquine group, and end point responses in this group were significantly below those of the placebo. These results suggest that the development and duration of the cellular response to tetanus immunization was impaired by long-term weekly chloroquine prophylaxis, while daily primaquine prophylaxis over the same time period had no inhibitory effect.