Trevor R. Jones

Synthetic oligodeoxynucleotides containing CpG motifs enhance immunogenicity of a peptide malaria vaccine in Aotus monkeys

Synthetic peptide and recombinant protein vaccines are optimally immunogenic when delivered with an effective adjuvant. Candidate vaccines currently insufficiently immunogenic may induce a protective immunity if they could be delivered with more effective adjuvants. For example, immunogens that induce promising responses when administered to mice with complete and incomplete Freunds adjuvants perform less well in primate animal models where complete Freunds adjuvant is not used. We report the use of synthetic oligodeoxynucleotides containing CpG motifs, the sequences of which are based on immunostimulatory bacterial DNA sequences, to enhance the immune response in Aotus monkeys to a synthetic peptide malaria vaccine. Monkeys were immunized with the synthetic peptide PADRE 45, a synthetic peptide containing amino acid sequences derived from the circumsporozoite protein (CSP) from Plasmodium falciparum, and delivered in an emulsion of saline and Montanide 720, a mannide oleate in oil solution, that also contained one of three oligodeoxynucleotides. The animals receiving oligodeoxynucleotides containing either three or four CpG motifs produced antibodies that bound a recombinant CSP as measured in ELISA, and reacted with P. falciparum sporozoites in a sporozoite immunofluorescent test. These responses were significantly greater than those seen in animals receiving the oligodeoxynucleotide without CpG motifs. These data indicate that oligodeoxynucleotides containing CpG motifs improve immunogenicity of peptide immunogens in non-human primates, and may be immunopotentiators useful in humans.

Protection of Aotus Monkeys by Plasmodium falciparum EBA-175 Region II DNA Prime—Protein Boost Immunization Regimen

Aotus monkeys received 4 doses of Plasmodium falciparum EBA-175 region II vaccine as plasmid DNA (Dv-Dv) or recombinant protein in adjuvant (Pv-Pv) or as 3 doses of DNA and 1 dose of protein (Dv-Pv). After 3 doses, antibody titers were approximately 10(4) in DNA-immunized monkeys and 10(6) in protein-immunized monkeys. A fourth dose did not significantly boost antibody responses in the Dv-Dv only or Pv-Pv only groups, but titers were boosted to approximately 10(6) in monkeys in the Dv-Pv group. Four weeks after the last immunization, the animals were challenged with 10(4) P. falciparum-parasitized erythrocytes. Peak levels of parasitemia were lower in the 16 monkeys that received region II-containing plasmids or proteins than in the 16 controls (geometric mean: 194,178 and 410,110 parasites/microL, respectively; P=.013, Students t test). Three of 4 monkeys in the Dv-Pv group did not require treatment. These data demonstrate that immunization with EBA-175 region II induces a significant antiparasite effect in vivo.

Needle-free Biojector injection of a dengue virus type 1 DNA vaccine with human immunostimulatory sequences and the GM-CSF gene increases immunogenicity and protection from virus challenge in Aotus monkeys

A dengue-1 DNA vaccine containing sequences encoding premembrane and envelope proteins (DIME) was previously shown to elicit virus neutralizing antibodies in rhesus and Aotus monkeys, and the primates were partially protected from viremia upon challenge. To increase the neutralizing antibody levels and subsequent protection from virus challenge, four strategies were evaluated: (a) coimmunization with a plasmid expressing Aotus GM-CSF gene; (b) coimmunization with a plasmid containing human immunostimulatory sequences (ISS); (c) coimmunization with both the GM-CSF gene and ISS; and (d) delivery of vaccine using the needle-free Biojector system. Vaccination with the mixed formulation containing DIME, GM-CSF gene, and ISS, by either needle injection or Biojector, led to neutralizing antibody titers that were stable for up to 6 months after vaccination. Furthermore, 6 of 7 monkeys (85%), and 7 of 8 monkeys (87%) receiving this formulation were completely protected from viremia when challenged 1 and 6 months after vaccination, respectively. This is a significant improvement compared to our previous study in which one of three monkeys (33%) receiving just the DIME vaccine was completely protected from viremia at 6 months after immunization.

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.

Randomized, Placebo-Controlled Trial of Atovaquone/Proguanil for the Prevention of Plasmodium falciparum or Plasmodium vivax Malaria among Migrants to Papua, Indonesia

The increasing prevalence of resistance to antimalarial drugs reduces options for malaria prophylaxis. Atovaquone/proguanil (Malarone; GlaxoSmithKline) has been >95% effective in preventing Plasmodium falciparum malaria in lifelong residents of areas of holoendemicity, but data from persons without clinical immunity or who are at risk for Plasmodium vivax malaria have not been described. We conducted a randomized, double-blinded study involving 297 people from areas of nonendemicity in Indonesia who migrated to Papua (where malaria is endemic) < or =26 months before the study period. Subjects received prophylaxis with 1 Malarone tablet (250 mg of atovaquone and 100 mg of proguanil hydrochloride; n=148) or placebo (n=149) per day for 20 weeks. Hematologic and clinical chemistry values did not change significantly. The protective efficacy of atovaquone/proguanil was 84% (95% confidence interval [CI], 44%-95%) for P. vivax malaria, 96% (95% CI, 72%-99%) for P. falciparum malaria, and 93% (95% CI, 77%-98%) overall. Atovaquone/proguanil was well tolerated, safe, and effective for the prevention of drug-resistant P. vivax and P. falciparum malaria in individuals without prior malaria exposure who migrated to Papua, Indonesia.

A DNA vaccine encoding the 42 kDa C-terminus of merozoite surface protein 1 of Plasmodium falciparum induces antibody, interferon-γ and cytotoxic T cell responses in rhesus monkeys: immuno-stimulatory effects of granulocyte macrophage-colony stimulating factor

We have constructed a DNA plasmid vaccine encoding the C-terminal 42-kDa region of the merozoite surface protein 1 (pMSP1(42)) from the 3D7 strain of Plasmodium falciparum (Pf3D7). This plasmid expressed recombinant MSP1(42) after in vitro transfection in mouse VM92 cells. Rhesus monkeys immunized with pMSP1(42) produced antibodies reactive with Pf3D7 infected erythrocytes by IFAT, and by ELISA against yeast produced MSP1(19) (yMSP1(19)). Immunization also induced antigen specific T cell responses as measured by interferon-gamma production, and by classical CTL chromium release assays. In addition, immunization with pMSP1(42) primed animals for an enhanced antibody response to a subsequent boost with the recombinant yMSP1(19). We also evaluated Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) as an adjuvant for pMSP1(42.) We tested both rhesus GM-CSF expressed from a DNA plasmid, and E. coli produced recombinant human GM-CSF. Plasmids encoding rhesus GM-CSF (prhGM-CSF) and human GM-CSF (phuGM-CSF) were constructed; these plasmids expressed bio-active recombinant GMCSF. Co-immunization with a mixture of prhGM-CSF and pMSP1(42) induced higher specific antibody responses after the first dose of plasmid, but after three doses of DNA monkeys immunized with or without prhGM-CSF had the same final antibody titers and T cell responses. In comparison, rhuGM-CSF protein did not lead to accelerated antibody production after the first DNA dose. However, antibody titers were maintained at a slightly higher level in monkeys receiving GM-CSF protein, and they had a higher response to boosting with recombinant MSP1(19). The GM-CSF plasmid or protein appears to be less potent as an adjuvant in rhesus monkeys than each is in mice, and more work is needed to determine if GM-CSF can be a useful adjuvant in DNA vaccination of primates.

Diagnosis of malaria in the field by fluorescence microscopy of QBC® capillary tubes

The diagnostic performance of commercial capillary tubes containing acridine orange dye (QBC) was compared with the standard diagnosis of malaria by microscopical examination of Giemsa-stained thick blood films (GTS) in remote field conditions. The comparison was conducted among 165 volunteers living in northeastern Irian Jaya, Indonesia, an area having hyperendemic malaria transmission. By GTS, 65 volunteers were positive for malaria, but only 49 were judged positive by QBC. Among the 100 blood films found negative by GTS, 5 were considered positive by QBC. Thus, relative to a GTS standard, the sensitivity and specificity of the QBC was 75% and 95%, respectively. The mean limit of detection for the QBC was approximately 60 parasites per microliter blood, whereas the limit of detection for GTS was 20 parasites per microliter blood. Also, a number of practical difficulties were encountered using the QBC at the field site. The QBC approach to diagnosis of malaria was less sensitive and more inconvenient than GTS under the conditions in remote Irian Jaya.

Absence of antigenic competition in Aotus monkeys immunized with Plasmodium falciparum DNA vaccines delivered as a mixture.

Aotus lemurinus lemurinus monkeys were immunized four times with one of three DNA plasmids expressing important Plasmodium falciparum blood stage vaccine candidate proteins or with a mixture containing all three vaccines. The three vaccines encoded sequences from apical merozoite antigen-1 (AMA-1), erythrocyte binding protein-175 (EBA-175) and merozoite surface protein-1 (MSP-1). Antigen-specific enzyme-linked immunosorbant assays (ELISAs) showed no significant differences in antibody titer induced to the three antigens by a single vaccine compared with the titer induced to that same antigen by the trivalent preparation. Results of immunofluorescent antibody assays against erythrocytes infected with asexual blood stage P. falciparum indicated that each of the three monovalent vaccines induced significant antibody responses to whole parasites. The trivalent vaccine mixture induced, after four immunizations, an antibody titer to whole parasites that was 3--12-fold higher than those induced by any of the single vaccines. The fourth immunization with the trivalent vaccine increased the mean antibody in IFAT by more than five-fold.

Successful Induction of CD8 T Cell-Dependent Protection Against Malaria by Sequential Immunization with DNA and Recombinant Poxvirus of Neonatal Mice Born to Immune Mothers

In some parts of Africa, 50% of deaths attributed to malaria occur in infants less than 8 mo. Thus, immunization against malaria may have to begin in the neonatal period, when neonates have maternally acquired Abs against malaria parasite proteins. Many malaria vaccines in development rely upon CD8 cells as immune effectors. Some studies indicate that neonates do not mount optimal CD8 cell responses. We report that BALB/c mice first immunized as neonates (7 days) with a Plasmodium yoelii circumsporozoite protein (PyCSP) DNA vaccine mixed with a plasmid expressing murine GM-CSF (DG) and boosted at 28 days with poxvirus expressing PyCSP were protected (93%) as well as mice immunized entirely as adults (70%). Protection was dependent on CD8 cells, and mice had excellent anti-PyCSP IFN-γ and cytotoxic T lymphocyte responses. Mice born of mothers previously exposed to P. yoelii parasites or immunized with the vaccine were protected and had excellent T cell responses. These data support assessment of this immunization strategy in neonates/young infants in areas in which malaria exacts its greatest toll.

The distribution of the intestinal parasitic diseases in the Southeast Anatolian (GAP=SEAP) region of Turkey

Objectives: The physical alterations put in place by the Southeastern Anatolia Project will undoubtedly provide a remarkable economical growth and a social development in the area. In addition, the influence that formation of dam ponds, enlargement of irrigation areas, change of product and the way of cultivation, urbanization and industrialization will have an impact on the environment. To minimize the adverse effects of this process on human beings, a Community Health Project was completed by the teams participated by Ege, Dicle, Gaziantep and Harran Universities under the Directorate of Turkish Parasitology Association and by Southeastern Anatolia Project Regional Development Administration between 2001 and 2003. Results: To identify individuals with parasite, feces samples were taken from a total of 4,470 individuals. Parasites were found in feces of 41.8% of men, 44.3% of women and 32.2% of children, 0–59xa0months old, who were included in the research and gave feces samples for parasites tests. These prevalence values indicate how widespread parasitic diseases are in the region. The high prevalence of parasitic diseases in this area is one of the causes of malnutrition in 40% of children. Parasites were detected in 44.2% of feces samples taken from rural areas and in 39.5% taken from urban areas. When the distribution of parasites detected in feces samples was studied, the most common parasites were Giardia intestinalis (18.1%), Entamoeba coli (11.8%), Ascaris lumbricoides (4.8%), Trichuris trichiura (4.5%) and Hymenolepis nana (3.9%). Distribution of parasites according to cities varied widely. The most frequently seen parasites were T. trichiura in Gaziantep; G. intestinalis in Batman, Mardin, Diyarbakır, Şırnak and Şanlıurfa; and E. coli in Siirt, Kilis and Adıyaman. Conclusions: This study is the first investigation of intestinal parasite prevalence in a large region, specifically, in this GAP region and in Turkey, in general. There is no direct relationship between irrigating the cultivation areas and diffusion of parasitic diseases because the existence of intestinal parasites mentioned above is not related to the range of irrigation of cultivation areas, but is related to factors already discussed.