Proliferative response of peripheral blood lymphocytes to mitogens in the owl monkey Aotus nancymae

The new world primate Aotus sp. has been recommended by the World Health Organization as a model for evaluation of malaria vaccine candidates, given its susceptibility to experimental infection with the human malaria parasites Plasmodium falciparum and P. vivax. The present study examined the in vitro proliferative response of peripheral blood mononuclear cells (PBMCs) isolated from Aotus monkeys, utilizing a wide range of mitogens. Results presented herein demonstrate that the in vitro proliferative response of PBMCs from the Aotus sp. is quite variable from monkey to monkey for each of the mitogens assessed. PBMCs from the Aotus monkey exhibited a delayed kinetic proliferative response and, particularly, a different sensitivity to proliferation in response to various concentrations of Phytohemagglutinin-P and favin lectins, the phorbol ester Phorbol myristate acetate and the calcium ionophore ionomycin. Altogether, our findings are consistent with the conclusion that the in vitro proliferative response of PBMCs from the Aotus differ in their activation requirements compared with PBMCs from humans.     

The Plasmodium vivax rhoptry-associated protein 1

Rhoptries are cellular organelles localized at the apical pole of apicomplexan parasites. Their content is rich in lipids and proteins that are released during target cell invasion. Plasmodium falciparum rhoptry-associated protein 1 (RAP1) has been the most widely studied among this parasite species' rhoptry proteins and is considered to be a good anti-malarial vaccine candidate since it displays little polymorphism and induces antibodies in infected humans. Monoclonal antibodies directed against RAP1 are also able to inhibit target cell invasion in vitro and protection against P. falciparum experimental challenge is induced when non-human primates are immunized with this protein expressed in its recombinant form. This study describes identifying and characterizing RAP1 in Plasmodium vivax, the most widespread parasite species causing malaria in humans, producing more than 80 million infections yearly, mainly in Asia and Latin America. This new protein is encoded by a two-exon gene, is proteolytically processed in a similar manner to its falciparum homologue and, as observed by microscopy, the immunofluorescence pattern displayed is suggestive of its rhoptry localization. Further studies evaluating P. vivax RAP1 protective efficacy in non-human primates should be carried out taking into account the relevance that its P. falciparum homologue has as an anti-malarial vaccine candidate.     

Reactivity of human T-lymphocyte-specific antibodies with peripheral blood mononuclear cells and spleen of Aotus azarae ssp. boliviensis (owl monkey)

Aotus monkeys offer one of the few models that can be used for the evaluation of the immunogenicity and efficacy of new vaccine candidates against the human malarias, Plasmodium falciparum and Plasmodium vivax. However, the tools available for evaluation of the immune responses in these New World primates are still limited. In the present study, a previously selected set of monoclonal antibodies that were raised against human T cell determinants and were reactive with at least one other primate species was investigated for its reactivity with Aotus lymphocytes using FACS analysis, indirect immunofluorescence (IFA) and immunohistochemistry. From a panel of 19 mAb, six were found to react consistently with Aotus lymphocytes using FACS analysis. Further evaluation of the mAb using IFA confirmed these findings. Analysis of the selected mAb on spleen sections of Aotus monkeys identified one anti-CD4 and one anti-CD8 mAb that can be used for immunohistochemical studies. The set of mAb identified in this study can be used for the detection of various T lymphocyte markers in peripheral blood and in tissues of Aotus monkeys. Together with data published by others, mAb are now identified for detection of six different markers of Aotus T lymphocytes. These mAb are very valuable for the characterisation of immune responses after vaccination and infection in the Aotus malaria models.     

Sequence and diversity of MHC <Emphasis Type="Italic">DQA</Emphasis> and<Emphasis Type="Italic"> DQB</Emphasis> genes of the owl monkey<Emphasis Type="Italic"> Aotus nancymaae</Emphasis>

The New World primate Aotus nancymaae has been recommended by the World Health Organization (WHO) as a model for evaluation of malaria vaccine candidates, given its susceptibility to experimental infection with the human malaria parasites Plasmodium falciparum and Plasmodium vivax. We present here the nucleotide sequences of the complete cDNA of MHC-DQA1 and of the polymorphic exon 2 segments of MHC-DQB1/DQB2. In a group of three nonrelated animals captured in the wild, five alleles of MHC-DQA1 could be identified. They all belong to one lineage, namely Aona-DQA1*27. This lineage has not been described in any other New World monkey species studied. In a group of 19 unrelated animals, 14 Aona-DQB1 alleles could be identified which are grouped into the two lineages Aona-DQB1*22 and Aona-DQB1*23. These lineages have been described previously in the common marmoset and cotton-top tamarin. In addition, two Aona-DQB2 sequences could be identified which are highly similar to HLA-DQB2 sequences. Essential amino acid residues contributing to MHC DQ peptide binding pockets number 1 and 4 are conserved or semi-conserved between HLA-DQ and Aona-DQ molecules, indicating a capacity to bind similar peptide repertoires. These results fully support the use of Aotus monkeys as an animal model for evaluation of future subunit vaccine candidates.     

Plasmodium falciparum infection and exoerythrocytic development in mice with chimeric human livers

The exoerythrocytic stage of Plasmodium falciparum has remained a difficult phase of the parasite life-cycle to study. The host and tissue specificity of the parasite requires the experimental infection of humans or non-human primates and subsequent surgical recovery of parasite-infected liver tissue to analyze this stage of the parasites development. This type of study is impossible in humans due to obvious ethical considerations and the cost and complexity in working with primate models has precluded their use for extensive studies of the exoerythrocytic stage. In this study we assessed, for the first time, the use of transgenic, chimeric mice containing functioning human hepatocytes as an alternative for modeling the in vivo interaction of P. falciparum parasites and human hepatocytes. Infection of these mice with P. falciparum sporozoites produced morphologically and antigenically mature liver stage schizonts containing merozoites capable of invading human red blood cells. Additionally, using microdissection, highly enriched P. falciparum liver stage parasites essentially free of hepatocyte contamination, were recovered for molecular studies. Our results establish a stable murine model for P. falciparum that will have a wide utility for assessing the biology of the parasite, potential anti-malarial chemotherapeutic agents and vaccine design.     

New World monkey efficacy trials for malaria vaccine development: critical path or detour?

Neither GMP malaria antigens nor GMP vaccines have been compared for efficacy in monkeys and humans. It is too risky to base categorical (go/no go) development decisions on results obtained using partially characterized (non-GMP) antigens, adjuvants that are too toxic for human use or unvalidated primate models. Such practices will lead to serious errors (e.g. failure to identify and stop flawed efforts, rejection of effective vaccine strategies) and unjustifiable delays. Successful malaria vaccine development will emphasize definitive field trials in populations at risk of malaria to define and improve vaccine efficacy.     

Structural and functional characterisation of the Toll like receptor 9 of Aotus nancymaae, a non-human primate model for malaria vaccine development

In the absence of suitable rodent animal models for Plasmodium falciparum malaria, the efficacy testing of asexual blood-stage vaccine candidates in Aotus nancymaae represents a tool to select between different formulations before conducting expensive human clinical trials. CpG oligonucleotides (ODN) specifically promote the production of pro-inflammatory and Th1-type cytokines and they enhance the immunogenicity of co-administered antigens. Toll like receptor 9 (TLR-9) binds directly and sequence-specifically to single-stranded un-methylated CpG-DNA mediating the biological effects of CpG ODN. We cloned and functionally characterised the TLR-9 cDNA of A. nancymaae. The cDNA encompassed 3,099 bp predicted to code for 1,032 amino acid residues. Results of homology searches to human TLR-9 suggested that the receptor is 93 and 94% identical at the nucleotide and amino acid sequence levels, respectively. Stimulation of splenocytes of A. nancymaae with CpG ODN resulted in proliferative responses in all animals analysed. FACS analysis of cultures incubated with CpG ODN 2006 indicated that the B cell marker CD20 was up-regulated consistent with B cell activation. The high level of sequence conservation of Aona-TLR-9 reinforces the suitability of A. nancymaae as animal model for malaria subunit vaccine development.The nucleotide sequence has been submitted to the GenBank nucleotide sequence database under the accession number AY788894.     

Sequence and diversity of DRB genes of Aotus nancymaae, a primate model for human malaria parasites

 The New World primate Aotus nancymaae is susceptible to infection with the human malaria parasite Plasmodium falciparum and Plasmodium vivax and has therefore been recommended by the World Health Organization as a model for evaluation of malaria vaccine candidates. We present here a first step in the molecular characterization of the major histocompatibility complex (MHC) class II DRB genes of Aotus nancymaae (owl monkey or night monkey) by nucleotide sequence analysis of the polymorphic exon 2 segments. In a group of 15 nonrelated animals captivated in the wild, 34 MHC DRB alleles could be identified. Six allelic lineages were detected, two of them having human counterparts, while two other lineages have not been described in any other New World monkey species studied. As in the common marmoset, the diversity of DRB alleles appears to have arisen largely by point mutations in the β-pleated sheets and by frequent exchange of fixed sequence motifs in the α-helical portion. Pairs of alleles differing only at amino acid position b86 by an exchange of valine to glycine are present in Aotus, as in humans. Essential amino acid residues contributing to MHC DR peptide binding pockets number 1 and 4 are conserved or semiconserved between HLA-DR and Aona-DRB molecules, indicating a capacity to bind similar peptide repertoires. These results support fully our using Aotus monkeys as an animal model for evaluation of future subunit vaccine candidates. Received: 10 August 1999 / Revised: 11 October 1999     

Reference strand conformational analysis (RSCA) is a valuable tool in identifying MHC-DRB sequences in three species of Aotus monkeys

The Aotus monkey has been of great value in the pre-clinical study of malaria vaccine candidates. Several components of this primate’s immune system have been studied and they display great similarity to their human counterparts. Cloning and sequencing studies have revealed extensive sequence polymorphisms in Aotus MHC-DRB with very high similarities to several human allelic lineages, grouping at least nine distinct MHC-DRB lineages. As the efficacy of peptide vaccines in this animal model may be strongly influenced by exon 2 MHC-DRB polymorphism, the availability of a reliable and rapid MHC-DRB typing method for three species of Aotus (Aotus nancymaae, Aotus vociferans and Aotus nigriceps) is necessary. Reference strand conformational analysis (RSCA) was used here for differentiating the distinctive Aotus MHC-DRB sequences’ mobility using five fluorescently labelled references proved to be very useful for resolving closely related sequences, establishing the number of sequences transcribed in a particular monkey and their identity. The RSCA method’s reliability in terms of identifying Aotus MHC-DRB sequences will facilitate evaluating individual responsiveness to vaccines and prompt studies associating susceptibility/resistance to infectious agents or auto-immune disease, for which Aotus monkeys may be considered to be an appropriate animal model.     

Vaccines against malaria

There is no licenced vaccine against any human parasitic disease and Plasmodium falciparum malaria, a major cause of infectious mortality, presents a great challenge to vaccine developers. This has led to the assessment of a wide variety of approaches to malaria vaccine design and development, assisted by the availability of a safe challenge model for small-scale efficacy testing of vaccine candidates. Malaria vaccine development has been at the forefront of assessing many new vaccine technologies including novel adjuvants, vectored prime-boost regimes and the concept of community vaccination to block malaria transmission. Most current vaccine candidates target a single stage of the parasite's life cycle and vaccines against the early pre-erythrocytic stages have shown most success. A protein in adjuvant vaccine, working through antibodies against sporozoites, and viral vector vaccines targeting the intracellular liver-stage parasite with cellular immunity show partial efficacy in humans, and the anti-sporozoite vaccine is currently in phase III trials. However, a more effective malaria vaccine suitable for widespread cost-effective deployment is likely to require a multi-component vaccine targeting more than one life cycle stage. The most attractive near-term approach to develop such a product is to combine existing partially effective pre-erythrocytic vaccine candidates.     

Plasmodium falciparum parasites are killed by a transition state analogue of purine nucleoside phosphorylase in a primate animal model

Plasmodium falciparum causes most of the one million annual deaths from malaria. Drug resistance is widespread and novel agents against new targets are needed to support combination-therapy approaches promoted by the World Health Organization. Plasmodium species are purine auxotrophs. Blocking purine nucleoside phosphorylase (PNP) kills cultured parasites by purine starvation. DADMe-Immucillin-G (BCX4945) is a transition state analogue of human and Plasmodium PNPs, binding with picomolar affinity. Here, we test BCX4945 in Aotus primates, an animal model for Plasmodium falciparum infections. Oral administration of BCX4945 for seven days results in parasite clearance and recrudescence in otherwise lethal infections of P. falciparum in Aotus monkeys. The molecular action of BCX4945 is demonstrated in crystal structures of human and P. falciparum PNPs. Metabolite analysis demonstrates that PNP blockade inhibits purine salvage and polyamine synthesis in the parasites. The efficacy, oral availability, chemical stability, unique mechanism of action and low toxicity of BCX4945 demonstrate potential for combination therapies with this novel antimalarial agent.     

Vaccination for vivax malaria: targeting the invaders

Among the surface-exposed antigens of the malaria parasite, those with known essential functions that can be disrupted by antibodies represent the most promising candidates for development as malaria vaccines. Two recombinant protein subunits of the Plasmodium vivax merozoite surface protein 1 have been shown to bind to reticulocytes in enzyme-linked immunosorbent assays. This article discusses the importance of such pre-clinical analyses in the validation of candidate vaccine molecules for P. vivax, given the constraints imposed by the use of primate models and the cost of producing suitable material for human trials.     

Identification and characterisation of the Plasmodium vivax rhoptry-associated protein 2

Plasmodium vivax is currently the most widespread of the four parasite species causing malaria in humans around the world. It causes more than 75 million clinical episodes per year, mainly on the Asian and American continents. Identifying new antigens to be further tested as anti-P. vivax vaccine candidates has been greatly hampered by the difficulty of maintaining this parasite cultured in vitro. Taking into account that one of the most promising vaccine candidates against Plasmodium falciparum is the rhoptry-associated protein 2, we have identified the P. falciparum rhoptry-associated protein 2 homologue in P. vivax in the present study. This protein has 400 residues, having an N-terminal 21 amino-acid stretch compatible with a signal peptide and, as occurs with its falciparum homologue, it lacks repeat sequences. The protein is expressed in asexual stage P. vivax parasites and polyclonal sera raised against this protein recognised a 46 kDa band in parasite lysate in a Western blot assay.     

Reproducible infection of intact Aotus lemurinus griseimembra monkeys by Plasmodium falciparum sporozoite inoculation

Aotus lemurinus griseimembra is considered one of the best nonhuman primate species for malarial studies because of its susceptibility to infection by Plasmodium falciparum asexual blood stages. However, reproducible transmission of infective P. falciparum sporozoites by mosquito inoculation has been difficult to achieve even in splenectomized monkeys. Characterization of an Aotus-P. falciparum cyclical transmission model has become a top priority as a result of the significant progress toward the development of preerythrocytic malaria vaccines. Herein, we describe a reproducible model developed using intact A. lemurinus griseimembra monkeys intravenously inoculated with sporozoites from a monkey-adapted P. falciparum (Santa Lucia) strain and a wild Falciparum-Cali-Colombia-4 (FCC-4) strain. Sporozoites were obtained by salivary gland dissection of laboratory-reared Anopheles albimanus mosquitoes. Parasitemia was monitored by thick-smear microscopy, parasite lactate dehydrogenase (pLDH) determination, and mosquito xenodiagnosis. The last method proved to be the most sensitive method for monitoring parasitemias. Infection with the Santa Lucia strain showed a mean prepatent period of 16 days (range 6-21 days), whereas infection with the wild FCC-4 strain resulted in a 24-day prepatent period. Mean peak parasite density was approximately 900 parasites/microliter for both parasite strains. The prepatent period, the peak of parasitemia, and the duration of patency were independent of the size of the sporozoite inoculum and the presence of spleen in the host. This model is being successfully used to test the protective efficacy of P. falciparum preerythrocytic vaccine candidates.     

Malaria: deploying a candidate vaccine (RTS,S/AS02A) for an old scourge of humankind.

Malaria is an infectious disease caused by the protist Plasmodium spp. and it currently kills more than one million people annually. The burden of malaria is concentrated in sub-Saharan Africa, India, and Southeast Asia. The parasite's resistance to commonly used anti-malarial drugs has worsened the situation in the poorest countries. The World Health Organization (WHO) estimates that more than 100 countries suffer from endemic malaria episodes. In addition to numerous control measures and treatments, several vaccines are at different research stages and trials. We have assayed RTS,S/AS02A, a pre-erythrocytic candidate vaccine that has shown promising protection levels in phase IIb trials in Mozambique. The vaccine is directed against the sporozoite form of the parasite, which is injected by the mosquito Anopheles spp. The vaccine induces a strong antibody response and stimulates Th1 cells-a subset of helper T cells that participates in cell-mediated immunity. Recent interest by international funding agencies has provided new inputs into initiatives and programs to fight malaria, which, under normal welfare and adequate social development conditions, is a curable disease.     

Plasmodium falciparum signal peptide peptidase is a promising drug target against blood stage malaria

The resistance of malaria parasites to current anti-malarial drugs is an issue of major concern globally. Recently we identified a Plasmodium falciparum cell membrane aspartyl protease, which binds to erythrocyte band 3, and is involved in merozoite invasion. Here we report the complete primary structure of P. falciparum signal peptide peptidase (PfSPP), and demonstrate that it is essential for parasite invasion and growth in human erythrocytes. Gene silencing suggests that PfSPP may be essential for parasite survival in human erythrocytes. Remarkably, mammalian signal peptide peptidase inhibitors (Z-LL)₂-ketone and L-685,458 effectively inhibited malaria parasite invasion as well as growth in human erythrocytes. In contrast, DAPT, an inhibitor of a related γ-secretase/presenilin-1, was ineffective. Thus, SPP inhibitors specific for PfSPP may function as potent anti-malarial drugs against the blood stage malaria.     

Characterisation and comparative analysis of MHC-DPA1 exon 2 in the owl monkey (Aotus nancymaae)

The Aotus nancymaae (owl monkey) is an important animal model in biomedical research, particularly for the preclinical evaluation of vaccine candidates against Plasmodium falciparum and Plasmodium vivax, which require a precisely typed major histocompatibility complex. The exon 2 from A. nancymaae MHC-DPA1 gene was characterised in order to infer its allelic diversity and evolutionary history. Aona-DPA1 shows no polymorphism and is related to other primate DPA alleles (including Catarrhini and Platyrrhini), constituting an ancient trans-specific and strongly supported lineage with different variability and selective patterns when compared to other primate-MHC-DPA1 lineages. A. nancymaae monkeys have thus a smaller MHC-DP polymorphism than MHC-DQ or MHC-DR.     

Malaria pathogenesis: a jigsaw with an increasing number of pieces

Plasmodium falciparum malaria remains as one of the most devastating global health problems of today. It is estimated that around 150 million individuals get the disease every year and of these 2-3 million die from it. Our knowledge of the mechanisms underlying the pathology has expanded greatly over the last decades, but many aspects of the molecular biology, immunology and epidemiology that govern the pathogenesis and spread of this parasite are still unclear. As new insights are gained we are also revealing a challenging biological complexity. Piecing this information together is the key to vaccine development and production of new antimalarial drugs.     

Tour de force effort for malaria vaccines

Malaria is caused by protozoan parasites of the genus Plasmodium. Four species infect humans, causing up to 500 million new infections and 2 million deaths annually. Plasmodium vivaxauses the most prevalent form of recurrent human malaria, responsible for significant morbidity throughout Asia and South America. With increased levels of drug-resistant strains and insecticide-resistant mosquito vectors, efforts to develop possible vaccines have been a major focus of malaria research. Our improved understanding of the basic biology and life cycle of Plasmodium has led to the identification and development of possible vaccine candidates and strategies.