Charles Omwandho

Parasite accumulation in placenta of non-immune baboons during Plasmodium knowlesi infection

BackgroundPlacental malaria (PM) causes adverse pregnancy outcomes in the mother and her foetus. It is difficult to study PM directly in humans due to ethical challenges. This study set out to bridge this gap by determining the outcome of PM in non-immune baboons in order to develop a non-human primate model for the disease.MethodsTen pregnant baboons were acquired late in their third trimester (day 150) and randomly grouped as seven infected and three non-infected. Another group of four nulligravidae (non-pregnant) infected was also included in the analysis of clinical outcome. Malaria infection was intravenously initiated by Plasmodium knowlesi blood-stage parasites through the femoral vein on 160th day of gestation (for pregnant baboons). Peripheral smear, placental smear, haematological samples, and histological samples were collected during the study period. Median values of clinical and haematological changes were analysed using Kruskal-Wallis and Dunn’s Multiple Comparison Test. Parasitaemia profiles were analysed using Mann Whitney U test. A Spearman’s rank correlation was run to determine the relationship between the different variables of severity scores. Probability values of P <0.05 were considered significant.ResultsLevels of white blood cells increased significantly in pregnant infected (34%) than in nulligravidae infected baboons (8%). Placental parasitaemia levels was on average 19-fold higher than peripheral parasitaemia in the same animal. Infiltration of parasitized erythrocytes and inflammatory cells were also observed in baboon placenta. Malaria parasite score increased with increase in total placental damage score (rs = 0.7650, P <0.05) and inflammatory score (rs = 0.8590, P <0.05). Although the sample size was small, absence of parasitized erythrocytes in cord blood and foetal placental region suggested lack of congenital malaria in non-immune baboons.ConclusionThis study has demonstrated accumulation of parasitized red blood cells and infiltration of inflammatory cells in the placental intravillous space (IVS) of baboons that are non-immune to malaria. This is a key feature of placental falciparum malaria in humans. This presents the baboon as a new model for the characterization of malaria during pregnancy.

Polymorphisms in the Paan-AG promoter influence NF-κB binding and transcriptional activity

The human leukocyte antigen-G (HLA-G) gene encodes a protein that is highly expressed at the human maternal–fetal interface during pregnancy and may be critical to the survival of the semiallogenic fetus. A unique feature of this gene is a 13-bp deletion in the proximal promoter that renders it unresponsive to transactivation by the nuclear factor-κB (NF-κB). We previously showed that the proximal promoter of Paan-AG, the functional homologue of HLA-G in the olive baboon (Papio anubis), is intact. We cloned the promoters of two putative Paan-AG alleles (AG1 and AG2) and identified a number of regulatory elements including two κB sites. In the current study, binding and activity of the two κB elements in each putative allele were assessed by electrophoretic mobility shift and supershift assays. Functional activity was determined using luciferase reporter assays. The κB1 and κB2 elements in AG1 bound NF-κB with similar affinity. In contrast, the κB1 element of AG2 bound NF-κB with a much higher affinity than AG-1 κB1 (a 30-fold increase), whereas κB2 did not bind. Mutagenesis analysis showed that the difference in binding intensities was due to two nucleotides in the 3′ end of κB1. Similarly, failure of AG2 κB2 binding was a result of the last nucleotide in the 3′ end that differed from the consensus; mutating this nucleotide to match the consensus reestablished binding. Functional activity of the two putative alleles also differed; AG1 luciferase activity was consistently lower than that of AG2. Mutating the last two nucleotides in the 3′ end of AG1 κB1 resulted in increased luciferase activity to levels comparable to that of AG2. Overall, these results show that in vitro variations in the promoter region may influence transcription of Paan-AG.

Safety, immunogenicity, and cross-species protection of a plasmid DNA encoding Plasmodium falciparum SERA5 polypeptide, microbial epitopes and chemokine genes in mice and olive baboons.

Incorporation of biomolecular epitopes to malarial antigens should be explored in the development of strain-transcending malarial vaccines. The present study sought to determine safety, immunogenicity and cross-species efficacy ofPlasmodium falciparum serine repeat antigen 5 polypeptide co-expressed with epitopes of Bacille-Calmette Guerin (BCG), tetanus toxoid (TT) and a chemokine gene. Olive baboons and BALB/c mice were randomly assigned into vaccine and control groups. The vaccine group animals were primed and boosted twice with pIRES plasmids encoding the SERA5+ BCG+ TT alone, or with either CCL5 or CCL20 and the control group with pIRES plasmid vector backbone. Mice and baboons were challenged withP. berghei ANKA and P. knowlesi H strain parasites, respectively. Safety was determined by observing for injection sites reactogenicities, hematology and clinical chemistry. Parasitaemia and survivorship profiles were used to determine cross-species efficacy, and T cell phenotypes, Th1-, Th2-type, T-regulatory immune responses and antibody responses were assessed to determine vaccine immunogenicity. The pSeBCGTT plasmid DNA vaccines were safe and induced Th1-, Th2-type, and T-regulatory responses vaccinated animals showed enhanced CD4+ (P<0.01), CD 8+ T cells (P<0.001) activation and IgG anti-SE36 antibodies responses (P<0.001) at week 4 and 8 post vaccination compared to the control group. Vaccinated mice had a 31.45-68.69% cumulative parasite load reduction and 60% suppression in baboons (P<0.05) and enhanced survivorship (P<0.001) with no clinical signs of malaria compared to the control group. The results showed that the vaccines were safe, immunogenic and conferred partial cross-species protection.