Beta Burst-Driven Adaptive Deep Brain Stimulation Improves Gait Impairment and Freezing of Gait in Parkinson's Disease.

Background: Freezing of gait (FOG) is a debilitating symptom of Parkinson's disease (PD) that is often refractory to medication. Pathological prolonged beta bursts within the subthalamic nucleus (STN) are associated with both worse impairment and freezing behavior in PD, which are improved with deep brain stimulation (DBS). The goal of the current study was to investigate the feasibility, safety, and tolerability of beta burst-driven adaptive DBS (aDBS) for FOG in PD. Methods: Seven individuals with PD were implanted with the investigational Summit™ RC+S DBS system (Medtronic, PLC) with leads placed bilaterally in the STN. A PC-in-the-loop architecture was used to adjust stimulation amplitude in real-time based on the observed beta burst durations in the STN. Participants performed either a harnessed stepping-in-place task or a free walking turning and barrier course, as well as clinical motor assessments and instrumented measures of bradykinesia, OFF stimulation, on aDBS, continuous DBS (cDBS), or random intermittent DBS (iDBS). Results: Beta burst driven aDBS was successfully implemented and deemed safe and tolerable in all seven participants. Gait metrics such as overall percent time freezing and mean peak shank angular velocity improved from OFF to aDBS and showed similar efficacy as cDBS. Similar improvements were also seen for overall clinical motor impairment, including tremor, as well as quantitative metrics of bradykinesia. Conclusion: Beta burst driven adaptive DBS was feasible, safe, and tolerable in individuals with PD with gait impairment and FOG.

Preclinical studies of a PARP targeted, Meitner-Auger emitting, theranostic radiopharmaceutical for metastatic ovarian cancer.

Advanced ovarian cancer currently has few therapeutic options. Poly(ADP-ribose) polymerase (PARP) inhibitors bind to nuclear PARP and trap the protein-inhibitor complex to DNA. This work investigates a theranostic PARP inhibitor for targeted radiopharmaceutical therapy of ovarian cancer in vitro and PET imaging of healthy mice in vivo. METHODS: [77Br]RD1 was synthesized and assessed for pharmacokinetics and cytotoxicity in human and murine ovarian cancer cell lines. [76Br]RD1 biodistribution and organ uptake in healthy mice were quantified through longitudinal PET/CT imaging and ex vivo radioactivity measurements. Organ-level dosimetry following [76/77Br]RD1 administration was calculated using RAPID, an in-house platform for absorbed dose in mice, and OLINDA for equivalent and effective dose in human. RESULTS: The maximum specific binding (Bmax), equilibrium dissociation constant (Kd), and nonspecific binding slope (NS) were calculated for each cell line. These values were used to calculate the cell specific activity uptake for cell viability studies. The half maximal effective concentration (EC50) was measured as 0.17 (95 % CI: 0.13-0.24) nM and 0.46 (0.13-0.24) nM for PARP(+) and PARP(-) expressing cell lines, respectively. The EC50 was 0.27 (0.21-0.36) nM and 0.30 (0.22-0.41) nM for BRCA1(-) and BRCA1(+) expressing cell lines, respectively. When measuring the EC50 as a function of cellular activity uptake and nuclear dose, the EC50 ranges from 0.020 to 0.039 Bq/cell and 3.3-9.2 Gy, respectively. Excretion through the hepatobiliary and renal pathways were observed in mice, with liver uptake of 2.3 ± 0.4 %ID/g after 48 h, contributing to estimated absorbed dose values in mice of 19.3 ± 0.3 mGy/MBq and 290 ± 10 mGy/MBq for [77Br]RD1 and [76Br]RD1, respectively. CONCLUSION: [77Br]RD1 cytotoxicity was dependent on PARP expression and independent of BRCA1 status. The in vitro results suggest that [77Br]RD1 cytotoxicity is driven by the targeted Meitner-Auger electron (MAe) radiotherapeutic effect of the agent. Further studies investigating the theranostic potential, organ dose, and tumor uptake of [76/77Br]RD1 are warranted.

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.

Pulmonary gas exchange in the morbidly obese.

The literature on pulmonary gas exchange at rest, during exercise, and with weight loss in the morbidly obese (body mass index or BMI > or = 40 kg m(-2)) is reviewed. Forty-one studies were found (768 subjects weighted mean = 40 years old, BMI = 48 kg m(-2)). The alveolar-to-arterial oxygen partial pressure difference (AaDO2) was large at rest in upright subjects at sea level (23, range 5-38 mmHg) while the arterial pressure of oxygen (PaO2) was low (81, range 50-95 mmHg). Arterial pressure of carbon dioxide (PaCO2) was normal. At peak exercise (162 W), gas exchange improves. Weight loss of 45 kg (BMI = -13 kg m(-2)) over 18 months is associated with an improvement in PaO2 (by 10 mmHg, range 1-23 mmHg), a reduction in AaDO2 (by 8 mmHg, range -3 to -16 mmHg), and PaCO2 (by -3 mmHg, range 3 to -14 mmHg) at rest. Every 5-6 kg reduction in weight increases PaO2 by 1 and reduces AaDO2 by 1 mmHg, respectively. Morbidly obese women have better gas exchange at rest compared with morbidly obese men which is likely due to lower waist-to-hip ratios in women than from differences in weight or BMI.

Cross-protection between attenuated Plasmodium berghei and P. yoelii sporozoites.

An attenuated Plasmodium falciparum sporozoite (PfSPZ) vaccine is under development, in part, based on studies in mice with P. berghei. We used P. berghei and P. yoelii to study vaccine-induced protection against challenge with a species of parasite different from the immunizing parasite in BALB/c mice. One-hundred percent of mice were protected against homologous challenge. Seventy-nine percent immunized with attenuated P. berghei sporozoite (PbSPZ) (six experiments) were protected against challenge with P. yoelii sporozoite (PySPZ), and 63% immunized with attenuated PySPZ (three experiments) were protected against challenge with PbSPZ. Antibodies in sera of immunized mice only recognized homologous sporozoites and could not have mediated protection against heterologous challenge. Immunization with attenuated PySPZ or PbSPZ induced CD8+ T cell-dependent protection against heterologous challenge. Immunization with attenuated PySPZ induced CD8+ T cell-dependent protection against homologous challenge. However, homologous protection induced by attenuated PbSPZ was not dependent on CD8+ or CD4+ T cells, and depletion of both populations only reduced protection by 36%. Immunization of C57BL/10 mice with PbSPZ induced CD8+ T cell-dependent protection against P. berghei, but no protection against P. yoelii. The cross-protection data in BALB/c mice support testing a human vaccine based on attenuated PfSPZ for its efficacy against P. vivax.

Mutating the anchor residues associated with MHC binding inhibits and deviates CD8+ T cell mediated protective immunity against malaria.

We investigated whether immune responses induced by immunization with plasmid DNA are restricted predominantly to immunodominant CD8+ T cell epitopes, or are raised against a breadth of epitopes including subdominant CD8+ and CD4+ T cell epitopes. Site-directed mutagenesis was used to change one or more primary anchor residues of the immunodominant CD8+ T cell epitope on the Plasmodium yoelii circumsporozoite protein, and in vivo protective efficacy and immune responses against defined PyCSP CD8+ and/or CD4+ epitopes were determined. Mutation of the P2 but not P9 or P10 anchor residues decreased protection and completely abrogated the antigen-specific CD8+ CTL activity and CD8+ dependent IFN-gamma responses to the immunodominant CD8+ epitope and overlapping CD8+/CD4+ epitope. Moreover, mutation deviated the immune response towards a CD4+ T cell IFN-gamma dependent profile, with enhanced lymphoproliferative responses to the immunodominant and subdominant CD4+ epitopes and enhanced antibody responses. Responses to the subdominant CD8+ epitope were not induced. Our data demonstrate that protective immunity induced by PyCSP DNA vaccination is directed predominantly against the single immunodominant CD8+ epitope, and that although responses can be induced against other epitopes, these are mediated by CD4+ T cells and are not capable of conferring optimal protection against challenge.

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.

Production of monoclonal antibodies against a 19-kD recombinant Plasmodium vivax MSP1 for detection of P. vivax malaria in Turkey.

Plasmodium vivax malaria, which is transmitted to humans by mosquitoes, is one of the most important parasitic diseases in Turkey. The major protein on the surface of asexual erythrocytic stage merozoites of P. vivax (Pv) is 200 kD and called major merozoite surface protein-1 (PvMSP1). Polyclonal antibodies against the 19-kD C-terminal fragment of PvMSP1 (PvMSP1(19)) are protective in monkey models of P. vivax and associated with protection in field studies. In this research, monoclonal antibodies were produced against PvMSP1(19). A total of 214 IgG(1) antibody-releasing hybridomas were obtained and three monoclonal antibodies were produced (PvMSP1(19).1, PvMSP1(19).2, and PvMSP1(19).3) and selected for further study. They have now been purified from ascitic fluid on a Staphylococcus protein A affinity column. These are the first monoclonal antibodies produced against P. vivax in Turkey and the first monoclonal antibodies produced against this recombinant PvMSP1(19) in the world. The monoclonal antibodies will be used to study the epidemiology of P. vivax in patients with malaria in Turkey, and to develop better strategies for early diagnosis and treatment of the disease in our population.

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.

Chloroquine for the treatment of uncomplicated malaria in Guyana.

At a public hospital in Georgetown, Guyana, 44 patients seeking treatment for symptomatic, slide-confirmed malaria were given standard chloroquine (CQ) therapy and followed for 28 days. The patients apparently had pure infections with Plasmodium falciparum (14), P. vivax (13) or P. malariae (one), or mixed infections either of P. falciparum and P. vivax (17) or of P. falciparum, P. malariae and P. vivax (two). Each received supervised treatment with 10 mg CQ base/kg on each of days 0 and 1, and 5 mg/kg on day 2. On the day of enrollment (day 0), the patients complained of fever (100%), headache (100%), malaise (94%), myalgia (79%), nausea (67%), vertigo (49%) and vomiting (33%). Many (39%) were ill enough to confine themselves to bed. On day 4, fewer of the subjects complained of fever (15%), headache (15%), malaise (6%), myalgia (21%), nausea (6%), vertigo (24%) or vomiting (0%) despite the relatively high (>48%) risk of therapeutic failure. The cumulative incidence of parasitological failure against P. falciparum was 15% at day 4, 33% at day 7 and 48% at day 14. All of the P. vivax and P. malariae infections cleared before day 4 and none recurred by day 7. Two infections with P. vivax recurred later (on day 14 or 28) but in the presence of less than adequate, whole-blood concentrations of CQ plus desethyl-chloroquine (i.e. <100 ng/ml). Taken together, the results indicate a high risk of therapeutic failure of CQ against P. falciparum but also indicate that resistance to CQ in P. vivax occurs infrequently in Guyana.

Determining liver stage parasite burden by real time quantitative PCR as a method for evaluating pre-erythrocytic malaria vaccine efficacy.

The detection and quantitation of blood stage parasitaemia is typically used as a surrogate endpoint for estimating the efficacy of vaccines targeted against the hepatic stage, as well as the erythrocytic stage, of the parasite. However, this does not provide an adequate means of evaluating the efficacy of vaccines, which may be only partially effective at the liver-stage. This is a particular concern for effective evaluation of immune enhancement strategies for candidate pre-erythrocytic stage vaccines. Here, we have developed and validated a method for detecting and quantitating liver stage parasites, using the TaqMan fluorescent real-time quantitative PCR system (PE Applied Biosystems). This method uses TaqMan primers designed to the Plasmodium yoelii 18S rRNA gene and rodent GAPDH to amplify products from infected mouse liver cDNA. The technique is highly reproducible as demonstrated with plasmid controls and capable of efficiently quantitating liver-stage parasite burden following a range of sporozoite challenge doses in strains of mice, which differ in their susceptibility to sporozoite infection. We have further demonstrated the capacity of this technique to evaluate the efficacy of a range of pre-erythrocytic stage vaccines. Our data establish this quantitative real-time PCR assay to be a fast and reproducible way of accurately assessing liver stage parasite burden and vaccine efficacy in rodent malaria models.

Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice.

In contrast to conventional vaccines, DNA and other subunit vaccines exclusively utilize host cell molecules for transcription and translation of proteins. The adenine plus thymine content of Plasmodium falciparum gene sequences (approximately 80%) is much greater than that of Homo sapiens (approximately 59%); consequently, codon usage is markedly different. We hypothesized that modifying codon usage of P. falciparum genes encoded by DNA vaccines from that used by the parasite to those resembling mammalian codon usage would lead to increased P. falciparum protein expression in vitro in mouse cells and increased antibody responses in DNA-vaccinated mice. We synthesized gene fragments encoding the receptor-binding domain of the 175-kDa P. falciparum erythrocyte-binding protein (EBA-175 region II) and the 42-kDa C-terminal processed fragment of the P. falciparum merozoite surface protein 1 (MSP-1(42)) using the most frequently occurring codon in mammals to code for each amino acid, and inserted the synthetic genes in DNA vaccine plasmids. In in vitro transient-expression assays, plasmids containing codon-optimized synthetic gene fragments (pS plasmids) showed greater than fourfold increased protein expression in mouse cells compared to those containing native gene fragments (pN plasmids). In mice immunized with 0.5, 5.0, or 50 microg of the DNA plasmids, the dose of DNA required to induce equivalent antibody titers was 10- to 100-fold lower for pS than for pN plasmids. These data demonstrate that optimizing codon usage in DNA vaccines can improve protein expression and consequently the immunogenicity of gene fragments in DNA vaccines for organisms whose codon usage differs substantially from that of mammals.

Expression of the chemokine MIG is a sensitive and predictive marker for antigen-specific, genetically restricted IFN-gamma production and IFN-gamma-secreting cells.

The evaluation of antigen-specific immune responses is critical for understanding the mechanisms of immune protection and for establishing the efficacy of candidate vaccines. Here, we describe a novel assay for IFN-gamma activity which is based on the flow cytometric detection of the chemokine, monokine induced by gamma interferon (MIG) as a sensitive and predictive measure of IFN-gamma-mediated effector function, and a surrogate marker for IFN-gamma-producing cells. Upregulation of MIG expression was demonstrated following in vitro activation of peripheral blood mononuclear cells (PBMCs) with defined CD8+ T-cell epitopes derived from influenza virus, cytomegalovirus (CMV), or Epstein-Barr virus (EBV) and was antigen-specific, genetically restricted and dependent on both CD8+ T cells and IFN-gamma. Furthermore, antigen-specific MIG expression was also demonstrated with Plasmodium falciparum circumsporozoite protein (CSP) peptides, using PBMCs from volunteers immunized with irradiated P. falciparum sporozoites. In multiple parallel experiments, the MIG assay was compared to conventional IFN-gamma ELISPOT, IFN-gamma ELISA, MIG ELISA and intracellular cytokine staining assays. The level of MIG expression was shown to be directly associated with the number of IFN-gamma spot-forming cells (SFCs) detected by ELISPOT (r2=0.94). Moreover, in all instances where cultures were considered positive by ELISPOT, a higher stimulation index was noted with the MIG assay as compared with the ELISPOT assay (on average at least threefold higher) and, in some cases, responses as detected by the MIG assay were significant, but the corresponding response as measured by ELISPOT was not significant. Finally, the flow-based MIG assay offers a number of practical and technical advantages over the ELISPOT assay. Our data validate this novel method for the detection of low as well as high levels of antigen-specific and genetically restricted IFN-gamma activity.

Randomized, parallel placebo-controlled trial of primaquine for malaria prophylaxis in Papua, Indonesia.

Malaria causes illness or death in unprotected travelers. Primaquine prevents malaria by attacking liver-stage parasites, a property distinguishing it from most chemoprophylactics and obviating 4-week postexposure dosing. A daily adult regimen of 30 mg primaquine prevented malaria caused by Plasmodium falciparum and P. vivax for 20 weeks in 95 of 97 glucose-6-phosphate dehydrogenase (G6PD)-normal Javanese transmigrants in Papua, Indonesia. In comparison, 37 of 149 subjects taking placebo in a parallel trial became parasitemic. The protective efficacy of primaquine against malaria was 93% (95% confidence interval [CI] 71%-98%); against P. falciparum it was 88% (95% CI 48%-97%), and >92% for P. vivax (95% CI >37%-99%). Primaquine was as well tolerated as placebo. Mild methemoglobinemia (mean of 3.4%) returned to normal within 2 weeks. Blood chemistry and hematological parameters revealed no evidence of toxicity. Good safety, tolerance, and efficacy, along with key advantages in dosing requirements, make primaquine an excellent drug for preventing malaria in nonpregnant, G6PD-normal travelers.

Enhancement of the immune response in rabbits to a malaria DNA vaccine by immunization with a needle-free jet device.

We compared the needle free jet device device Biojector with syringe/needle as a method to administer a DNA vaccine encoding the Plasmodium falciparum circumsporozoite protein (PfCSP) in albino rabbits. A group of three rabbits was injected by the intramuscular (IM) route using a syringe/needle combination, a second group IM with the Biojector device and a third group both IM and intradermal (ID) using the Biojector. When animals were immunized with the Biojector IM or IM/ID as compared to the syringe/needle IM, we observed 10- and 50-fold greater antibody titers, as measured by enzyme immunoassay (EIA) and indirect fluorescence antibody test (IFAT), respectively. We also observed that the Biojector conferred a greater ability to prime the immune system as compared with the needle. The subsequent boosting of all animals with a recombinant canary pox virus (ALVAC) expressing PfCSP induced significantly higher titers in both Biojector groups of rabbits as compared with the needle and naive animals. These results provided the foundation for a clinical trial using the same regime.

Incidence of symptomatic and asymptomatic Plasmodium falciparum infection following curative therapy in adult residents of northern Ghana.

Adult residents of holoendemic malaria regions in Africa have a naturally acquired immunity (NAI) to malaria that renders them more resistant to new infections, limits parasitemia, and reduces the frequency and severity of illness. Given such attributes, it is not clear how one might evaluate drug or vaccine efficacy in adults without serious confounding. To determine symptomatic and asymptomatic malaria attack rates in adults of northern Ghana, 197 men and women underwent curative therapy for any pre-existing malaria infections at the start of the high transmission (wet) season. They were monitored for first parasitemia and first clinical episode of infection by Plasmodium falciparum over a 20-week period (May-October 1996). The cumulative incidence of primary infection by P. falciparum was 0.98 and incidence density of infection was calculated to be 7.0 cases/person-year. Symptoms were reported by 19.5% of the individuals at the time of first recurrent parasitemia. Incidence of infection, parasite density, and the frequency of symptoms were comparable in males and females. The results suggest that NAI did not provide these adults with significant defense against rapid re-infection and suggest that this population-infection design could serve to demonstrate the efficacy of a drug or vaccine in preventing parasitemia.

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.

Multistage multiantigen heterologous prime boost vaccine for Plasmodium knowlesi malaria provides partial protection in rhesus macaques.

A nonhuman primate model for malaria vaccine development allowing reliable, stringent sporozoite challenge and evaluation of both cellular and antibody responses is needed. We therefore constructed a multicomponent, multistage DNA vaccine for the simian malaria species Plasmodium knowlesi including two preerythrocytic-stage antigens, the circumsporozoite protein (PkCSP) and sporozoite surface protein 2 (PkSSP2), and two blood stage antigens, apical merozoite antigen 1 (PkAMA1) and merozoite surface protein 1 (PkMSP1p42), as well as recombinant canarypox viruses encoding the four antigens (ALVAC-4). The DNA vaccine plasmids expressed the corresponding antigens in vitro and induced antiparasite antibodies in mice. Groups of four rhesus monkeys received three doses of a mixture of the four DNA vaccine plasmids and a plasmid encoding rhesus granulocyte-monocyte colony-stimulating factor, followed by boosting with a single dose of ALVAC-4. Three groups received the priming DNA doses by different routes, either by intramuscular needle injection, by intramuscular injection with a needleless injection device, the Biojector, or by a combination of intramuscular and intradermal routes by Biojector. Animals immunized by any route developed antibody responses against sporozoites and infected erythrocytes and against a recombinant PkCSP protein, as well as gamma interferon-secreting T-cell responses against peptides from PkCSP. Following challenge with 100 P. knowlesi sporozoites, 1 of 12 experimental monkeys was completely protected and the mean parasitemia in the remaining monkeys was significantly lower than that in 4 control monkeys. This model will be important in preclinical vaccine development.