Manuel E. Patarroyo

Vaccination with SPf66, a chemically synthesised vaccine, against Plasmodium falciparum malaria in Colombia

Preclinical and clinical studies have established the safety and immunogenicity of the chemically synthesised SPf66 malaria vaccine. The present study is a phase III randomised, double-blind, placebo-controlled, efficacy trial completed in La Tola, Colombia. 1548 volunteers over one year of age received three doses of either the vaccine (n = 738) or placebo (n = 810). Active and passive case detection methods were used to document clinical episodes of malaria among the study population. The follow-up period began one month after the third dose and lasted for one year. 168 and 297 episodes of Plasmodium falciparum malaria were documented in the SPf66 group and the placebo group, respectively; this corresponds to a crude protective efficacy of 38.8%. Incidence rates for first or only P falciparum malarial episodes were 22.3% per annum among the vaccinee group and 33.5% among the placebo group (RR = 1.5; 95% Cl 1.23, 1.84). Therefore, the protective efficacy of SPf66 against first or only episodes was 33.6% (95% Cl 18.8, 45.7), being highest in children aged 1-4 years (77%) and adults older than 45 years (67%). The estimated protective efficacy against second episodes was 50.5% (95% Cl 12.9-71.9). Our study shows that the chemically synthesised SPf66 malaria vaccine is safe, immunogenic, and protective against P falciparum malaria in semi-immune populations subject to natural challenge.

Association of a B-cell alloantigen with susceptibility to rheumatic fever

THE possibility that genetic factors determine susceptibility to rheumatic fever following infection with Group A streptococci was suggested by epidemiological and family studies1. However, with the exception of a slightly increased frequency of Lewis group secretors2, the identification of a genetic marker in the patient group has been unsuccessful. In particular, the distribution of HLA-A and B allotypes did not reveal any consistent difference from controls3–5. We report here that a novel B-cell alloantigen has been found at a significantly increased frequency among patients with rheumatic fever, providing further evidence for the value of B-cell alloantisera as reagents for the demonstration of disease associations with genetic determinants relevant to the immune system. This alloantigen was of particular interest because it was not found to be associated with any recognised HLA-D locus determinant.

Identification of Plasmodium falciparum MSP-1 peptides able to bind to human red blood cells.

To determine amino acid sequences of the Plasmodium falciparum MSP‐1 protein that interact with red blood cell membranes in a specific receptor‐ligand interaction, 78 sequential peptides, 20 amino acids long and spanning the entire length of the molecule, were synthesized and analysed with a specific binding assay developed for this purpose. Results show that peptides based on conserved and dimorphic regions of MSP‐1, interact with human red blood cells (RBCs). This interaction occurs predominantly with peptides contained within the MSP‐1 proteolytic fragments of 83 kDa, 38 kDa, 33 kDa and 19 kDa. Affinity constants of these peptides were between 140 and 250 nM. Peptide‐RBC binding post enzyme treatment showed that the RBC receptors are not sialic acid dependent and appear to be proteic in nature. Some of these peptides inhibited merozoite invasion of RBCs yet did not inhibit intra‐erthrocytic development. These peptides, in conjunction with those from other merozoite surface proteins, may be used to rationally design a second generation of synthetic peptide‐based malaria vaccines.

T cell recognition and therapeutic effect of a phosphorylated synthetic peptide of the 70K snRNP protein administered in MRL/lpr mice

Modifications of self antigens that occur during apoptosis might be involved in the generation of neo‐antigens, which can break tolerance and induce autoimmunity. We have previously identifiedan epitope at residues 131–151 of the U1‐70K snRNP protein, recognized by IgG antibodies and CD4+ T cells from at least two strains of lupus mice. With the aim of investigating the possible role of phosphorylation on the antigenicity of peptide 131–151 and to gain a better understanding of how this peptide can drive autoimmune response, we synthesized two peptides phosphorylated on Ser137 and 140, respectively. We show here that peptide P140 phosphorylated on Ser140 is recognized by both CD4+ T cells and antibodies from MRL/lpr mice. Furthermore, intravenous administration to lupus‐prone MRL/lpr mice of P140 in saline (but not of the non‐phosphorylated peptide) decreased proteinuria and anti‐DNA antibody production, and significantly prolonged survival of treated mice. We further demonstrated that P140 is recognized by antibodies from lupus patients and binds to various HLA DR molecules, offering new hope for manipulating T cell response in humans.

The first field trials of the chemically synthesized malaria vaccine SPf66: safety, immunogenicity and protectivity

This paper reports the results of the first field study performed to assess the safety, immunogenicity and protectivity of the synthetic malaria vaccine SPf66 directed against the asexual blood stages of Plasmodium falciparum. Clinical and laboratory tests were performed on all volunteers prior to and after each immunization, demonstrating that no detectable alteration was induced by the immunization process. The vaccines were grouped as high, intermediate or low responders according to their antibody titres directed against the SPf66 molecule. Two of the 185 (1.08%) SPf66-vaccinated and nine of the 214 (4.20%) placebo-vaccinated volunteers developed P. falciparum malaria. The efficacy of the vaccine was calculated as 82.3% against P. falciparum and 60.6% against Plasmodium vivax.

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

Abstract 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.

Study of the safety and immunogenicity of the synthetic malaria SPf66 vaccine in children aged 1–14 years

Safety and immunogenicity tests of the SPf66 malaria vaccine have been carried out on a population of children, aged 1 to 14 years, in the town of Tumaco, Colombia. Adverse reactions measured after each vaccination were local and minimal, and observed in only a small percentage of the vaccinated children. One year later, no delayed reaction was evident. The majority of the child population developed high antibody titres against SPf66 and the degree of response did not vary with age. These induced antibodies recognize the native parasite proteins, in particular the molecules from which the amino acid sequence of this vaccine was deduced. These studies demonstrate that the SPf66 vaccine is safe and highly immunogenic for use in children greater than 1 year old.

Plasmodium falciparum AMA-1 erythrocyte binding peptides implicate AMA-1 as erythrocyte binding protein

The role of AMA-1 during merozoite invasion has not yet been determined. However, reported experimental evidence suggests that this protein can be used, in particular as erythrocyte-binding protein, since, Fab fragments against this protein are able to block merozoite invasion. Using a previously described methodology, eight peptides with high binding activity to human erythrocyte, scattered along the different domains and having around 130 nM affinity constants, were identified in the Plasmodium falciparum AMA-1 protein. Their binding activity was sialic acid independent. Some of these peptides showed homology with the erythrocyte binding domains of one of the apical organelle protein family, MAEBL, identified in rodent malarial parasites. One of these peptides shares amino acid sequence with a previously reported B-cell epitope which induces antibodies to block parasite growth. The critical residues were identified for erythrocyte binding conserved peptides 4313 (DAEVAGTQYRLPSGKCPVFG), 4321 (VVDNWEKVCPRKNLQNAKFG), 4325 (MIKSAFLPTGAFKADRYKSH) and 4337 (WGEEKRASHTTPVLMEKPYY). All conserved peptides were able to block merozoite invasion of new RBC and development, suggesting that these peptides are involved in P. falciparum invasion.

Biodegradable PLGA microspheres as a delivery system for malaria synthetic peptide SPf66

SPf66 is the first chemically synthesised vaccine to elicit a partial protective immune response against malaria. The aluminium hydroxide (alum)-adsorbed SPf66 vaccine is weakly immunogenic and of poor to moderate efficacy in humans. To investigate the possibility of improving SPf66 vaccine immunogenicity, a delivery system based on poly-D,L-lactide-co-glycolide (PLGA) microspheres was developed and the immune response induced after its subcutaneous administration into mice was evaluated. Microspheres were prepared by a solvent extraction/double emulsion (w/o/w) method and characterised for morphology, size, peptide loading, release profile and peptide integrity. The in vitro and in vivo results obtained showed that there was no apparent effect of the encapsulation procedure on SPf66 integrity and immunogenicity. The subcutaneous administration of microspheres showed a significantly higher immune response (serum IgG levels) than that obtained with alum adsorbed SPf66 and it was comparable to that of SPf66 emulsified with Freunds adjuvant (FA). These observations illustrate the potential of PLGA microspheres as a delivery system for chemically synthesised antigens.

Antigenicity of the Leishmania infantum histones H2B and H4 during canine viscerocutaneous leishmaniasis

In this study we show that sera from dogs naturally infected with Leishmania infantum contain antibodies that specifically react against the parasite H2B and H4 histones. The Leishmania H2B and the amino‐terminal region of the histone H4, expressed as fusion proteins, when confronted with sera from canine viscerocutaneous leishmaniasis (VCL) dogs, were recognized by 63% and 47%, respectively. No reactivity was detected when sera from dogs naturally infected with pathogens other than Leishmania were used. Using a collection of synthetic peptides covering the complete sequence of both proteins, we have determined that the main linear antigenic determinants are located in the amino‐terminal domains of these histones. The humoral response against histones H2B and H4 induced during canine leishmaniasis was found to be specific for Leishmania histones, since no cross‐reactivity of the VCL sera with mammal histones was observed. Also, a comparative study of the prevalence of antibodies among VCL sera against the four core histones of L. infantum was performed. Although a large heterogeneity of the humoral responses against these proteins was found, histones H2A and H3 seem to be more prevalent immunogens than histones H2B and H4 during canine natural leishmaniasis. The origin of the anti‐histone humoral response and its possible implications in the pathogenesis of Leishmania infection are discussed.