Nicanor Obaldia

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.

Malaria DNA vaccines in Aotus monkeys

In preparation for the development of DNA vaccines designed to produce protective antibodies against Plasmodium falciparum antigens (Ag), we conducted studies to optimize antibody responses in Aotus monkeys after immunization with the P. yoelli circumsporozoite (CSP) DNA vaccine. We demonstrate in Aotus monkeys that an intradermal route of immunization with a PyCSP plasmid DNA vaccine generates antibody responses equivalent to a multiple antigen peptide/adjuvant based vaccine, and that these data support the use of the intradermal route for initial studies of the efficacy of DNA vaccines in inducing protective antibodies against P. falciparum antigens in Aotus monkeys.

Antimalarial Activities of New Pyrrolo[3,2-f]Quinazoline-1,3-Diamine Derivatives

ABSTRACT WR227825 is an antimalarial pyrroloquinazolinediamine derivative with a high potency but a low therapeutic index. A series of carbamate, carboxamide, succinimide, and alkylamine derivatives of WR227825 were prepared to search for compounds with an improved therapeutic index. The new acetamides and imide showed potent cell growth inhibition against four clones of Plasmodium falciparum (D-6, RCS, W-2, and TM91C235), with a 50% inhibitory concentration of ∼0.01 ng/ml, and were highly active against Plasmodium berghei, with 100% cure at doses from <0.1 mg/kg of body weight to 220 mg/kg. The carbamates and alkyl derivatives, however, showed weak activity against Plasmodium falciparum cell growth but were highly efficacious in tests against P. berghei by the Thompson test. The best compounds, bis-ethylcarbamate (compound 2a) and tetra-acetamide (3a) derivatives, further demonstrated high potency against the sporozoite Plasmodium yoelii in mice and P. falciparum and Plasmodium vivax in aotus monkeys. Against the AMRU-1 strain of P. vivax, which has four dihydrofolate reductase mutations and is highly resistant to antifolates, tetra-acetamide 3a cured the monkeys at doses of 1 and 3 mg/kg. Compound 2a cured only one out of two monkeys at 3 mg/kg. The results indicated that the new derivatives 2a and 3a not only have retained/improved the antimalarial efficacy of the parent compound WR227825 but also were less toxic to the animals used in the tests.

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.

Clonal Outbreak of Plasmodium falciparum Infection in Eastern Panama

Identifying the source of resurgent parasites is paramount to a strategic, successful intervention for malaria elimination. Although the malaria incidence in Panama is low, a recent outbreak resulted in a 6-fold increase in reported cases. We hypothesized that parasites sampled from this epidemic might be related and exhibit a clonal population structure. We tested the genetic relatedness of parasites, using informative single-nucleotide polymorphisms and drug resistance loci. We found that parasites were clustered into 3 clonal subpopulations and were related to parasites from Colombia. Two clusters of Panamanian parasites shared identical drug resistance haplotypes, and all clusters shared a chloroquine-resistance genotype matching the pfcrt haplotype of Colombian origin. Our findings suggest these resurgent parasite populations are highly clonal and that the high clonality likely resulted from epidemic expansion of imported or vestigial cases. Malaria outbreak investigations that use genetic tools can illuminate potential sources of epidemic malaria and guide strategies to prevent further resurgence in areas where malaria has been eliminated.

Clonal outbreak of Plasmodium falciparum in eastern Panama

Identifying the source of resurgent parasites is paramount to a strategic, successful intervention for malaria elimination. Although the malaria incidence in Panama is low, a recent outbreak resulted in a 6-fold increase in reported cases. We hypothesized that parasites sampled from this epidemic might be related and exhibit a clonal population structure. We tested the genetic relatedness of parasites, using informative single-nucleotide polymorphisms and drug resistance loci. We found that parasites were clustered into 3 clonal subpopulations and were related to parasites from Colombia. Two clusters of Panamanian parasites shared identical drug resistance haplotypes, and all clusters shared a chloroquine-resistance genotype matching the pfcrt haplotype of Colombian origin. Our findings suggest these resurgent parasite populations are highly clonal and that the high clonality likely resulted from epidemic expansion of imported or vestigial cases. Malaria outbreak investigations that use genetic tools can illuminate potential sources of epidemic malaria and guide strategies to prevent further resurgence in areas where malaria has been eliminated.

Evaluation of artemisone combinations in Aotus monkeys infected with Plasmodium falciparum.

ABSTRACT Artemisone (single oral dose, 10 mg/kg of body weight) cured nonimmune Aotus monkeys of their Plasmodium falciparum infections when combined with mefloquine (single oral dose, 5 and 10 mg/kg but not 2.5 mg/kg). In combination with amodiaquine (20 mg/kg/day), artemisone (10 mg/kg/day) given orally for 3 days cured all infected monkeys. Three days of treatment with artemisone (30 mg/kg/day) and clindamycin (100 mg/kg/day) was also curative.

Revising antimalarial drug policy in Central America: experience in Panama

Panama is the first country in the Central American region that has officially discarded chloroquine as a first-line drug to treat Plasmodium falciparum cases. Here we describe the clinical and molecular findings from autochthonous P. falciparum fatal cases, and the epidemiological situation that led to a change in the national antimalarial drug policy. Our results illustrate the potential pathogenicity of the strain of P. falciparum circulating in the country and provide molecular evidence of parasite resistance to chloroquine and antifolate drugs. The public health threats of these findings for the Central American region are discussed.

Long-term effect of a simple nest-box on the reproductive efficiency and other life traits of an Aotus lemurinus lemurinus monkey colony: an animal model for malaria research.

Background  The long‐term effect of a PVC pipe nest‐box on the reproductive efficiency and other life traits of an Aotus monkey‐breeding colony have not been characterized.

Determinants of low socio-economic status and risk of Plasmodium vivax malaria infection in Panama (2009–2012): a case–control study

BackgroundIdentification of risk factors is important for the establishment of malaria elimination programmes tailored to specific regions. Type of house construction had been associated with increasing risk of acquiring malaria. This study aimed at establishing the association between determinants of low socio-economic status (SES) and type of house construction with the likelihood of living in a Plasmodium vivax malarious corregimiento (smallest political division) in Panama during 2009–2012.MethodsTo determine the association between type-2 houses (build with deciduous materials) and other determinants of low SES, with living in a malarious corregimiento, this study analyzed demographic and housing census data (2010), and malaria incidence aggregated at the corregimiento level (2009–2012), using a Spearman’s non-parametric correlation test to explore for associations, followed by a case–control study and a reduced multivariate logistic regression approach for confirmation.ResultsA descriptive temporal and spatial analysis indicated that P. vivax in Panama was associated with Amerindian reservations. Moreover, this study demonstrated that a strong correlation (deleterious effect) existed between living in a malarious corregimiento and being exposed to a type-2 house (OR = > 1.0) (p < 0.001), while, it showed an inverse correlation for exposure to type-1 houses (protective effect) (build with permanent materials) (OR = < 1.0) (p < 0.001). In the same way, a significant association between exposure to type-2 houses and the outcome of living in a malarious corregimiento was found using a case–control study approach (Chi2 test = p < 0.001), that was confirmed applying a reduced multivariate logistic regression fitted model.ConclusionsThis study demonstrated that living in a P. vivax malarious corregimiento in Panama during 2009–2012 was strongly correlated with those corregimientos having a high proportion of type-2 houses. A multivariate logistic regression approach at the house and corregimiento level indicated a strong association of type-2 houses, dirt floors and illiteracy with the likelihood of living in a malarious corregimiento. It is expected that these findings will help implement a multi-sectorial approach for the elimination of malaria in poor areas of Panama where malaria is endemic, which emphasizes house improvements such as mosquito-proofing and socio-economic development.