Functional, immunological and three-dimensional analysis of chemically synthesised sporozoite peptides as components of a fully-effective antimalarial vaccine.

Our ongoing search for a fully-effective vaccine against the Plasmodium falciparum parasite (causing the most lethal form of human malaria) has been focused on identifying and characterising proteins' amino acid sequences (high activity binding peptides or HABPs) involved in parasite invasion of red blood cells (RBC) by the merozoite and hepatocytes by the sporozoite. Many such merozoite HABPs have been recognised and molecularly and structurally characterised; however, native HABPs are immunologically silent since they do not induce any immune response or protection against P. falciparum malaria infection and they have to be structurally modified to allow them to fit perfectly into immune system molecules. A deeply structural analysis of these conserved merozoite HABPs and their modified analogues has led to rules or principles becoming recognised for constructing a logical and rational methodology for a minimal subunit-based, multi-epitope, multi-stage, chemically-synthesised vaccine. The same in-depth analysis of the most relevant sporozoite proteins involved in sporozoite cell-traversal and hepatocyte invasion as well as the hepatic stage is shown here. Specifically modifying these HABPs has resulted in a new set of potential pre-erythrocyte targets which are able to induce high, longlasting antibody titres in Aotus monkeys, against their corresponding recombinant proteins and the complete parasite native molecules. This review shows how these rules may be applied against the first stage of parasite invasion (i.e. the sporozoite) to mount the first line of defence against the malarial parasite, which may indeed be the most effective one. Our results strongly support including some of these modified sporozoite HABPs in combination with the previously-described modified merozoite HABPs for obtaining the aforementioned fully-protective, multiepitope, multi-stage, minimal subunit-based, chemically-synthesized, antimalarial vaccine.

Immunological and structural characterization of an epitope from the Trypanosoma cruzi KMP-11 protein.

The K1 peptide is an HLA-A*0201-restricted cytotoxic epitope derived from the Trypanosoma cruzi KMP-11 protein, this being the etiological agent of Chagas' disease. This work describes the K1 peptide's secondary structure and its recognition by sera from chagasic patients. Circular dichroism and NMR spectroscopy analysis revealed that the K1 peptide adopts an alpha-helical conformation. Fifty-six percent of individuals had anti-K1 and 86% anti-KMP-11 antibodies by ELISA in the chronic Chagas' group and 28 and 68% in the indeterminate Chagas' group, respectively. By contrast, no reactivity was observed in sera from healthy individuals and tuberculosis patients. Antibody response subclass specificity to the K1 peptide was IgG1 and IgG3. Taken together these results support the idea that the K1 peptide acts as a B-cell-inducer epitope during Chagas' disease.

Based on HLA-DR beta1* allele binding specificities, striking differences in distance and TCR Contacting Residue Orientation can be observed in modified protection-inducing malarial synthetic peptides.

An anti-malarial vaccine is urgently needed, especially against P. falciparum which causes 2 to 3 million deaths each year, mostly in Sub-Saharan African children. This vaccine should contain molecules from the parasite's different developmental stages due to the parasite's remarkable complexity and genetic variability. The first approach using synthetic peptides from different parasite stage molecules (the SPf66 malaria vaccine) conferred limited protective efficacy in Aotus monkeys and in large field-trials carried out in different parts of the world SPf66 contains red blood cell (RBC) binding merozoite peptides for which immune responses against them are genetically controlled by HLA-DR region. Therefore, a systematic search of conserved high activity binding peptides (HABP) was undertaken aimed at using them as immunogens. However, these peptides were poorly immunogenic and had poor protection-inducing capacity against experimental challenge with a P. falciparum strain highly infective for Aotus monkeys an experimental model with an immune system quite similar to humans. Modifications were thus made to key residues to render them immunogenic and protection-inducing. These native and modified HABPs' three-dimensional structure was determined by (1)H-NMR studies and their ability in forming stable Major Histocompatibility Class II - peptide (MHCII-peptide) complexes was correlated with their ability to bind in vitro to purified HLA-DR beta1* molecules. Our experimental data suggests a correlation between modified HABPs' three-dimensional structure, HLA-DR beta1* binding preferences and their protection-inducing capacity in monkeys. Furthermore, the data presented here indicates that a synthetic peptide vaccine's three-dimensional structural features dictate both HLA-DR beta1* allele binding preference (imposing genetic restriction on the immune response) and on these vaccines' protection-inducing value. Basic knowledge of a parasite's functionally active peptides, their 3D structure and their interaction for forming the MHC II- peptide-TCR complex will thus contribute towards designing fully effective multi-component, multi-stage subunit-based malarial vaccines.