Luis Angel Murillo

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.

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.

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.

Sequence of the cry11Bb11 gene from Bacillus thuringiensis subsp. medellin and toxicity analysis of its encoded protein

The nucleotide sequence of the cry11Bb1 gene from Bacillus thuringiensis subsp. medellin was determined. The corresponding protein has a deduced molecular mass of 88.2 kDa, and is 60.9% and 83% identical to the proteins Cry11Aa1 and Cry11Ba1, respectively. The Cry11Bb1 protein contains five repetitive blocks of 16 amino acids at the C terminal part. It is highly toxic to first instar laboratory reared Aedes aegypti, Anopheles albimanus and Culex quinquefasciatus larvae.

A specific T-cell receptor genotype preference in the immune response to a synthetic Plasmodium falciparum malaria vaccine

Summary In recent studies with 63 and 122 volunteers vaccinated with the SPf 66 synthetic malaria vaccine, specific antibody patterns were classified as high or low responders. Using the Polymerase Chain Reaction (PCR), a specific and selective preference was shown for the Vβ arrangement of the T‐cell receptor in the high responder group involving the Vβ‐8 gene. The low responder group showed the rearrangement of a different set of genes, and a particular association with Vβ‐10.

Identification, cloning, and sequencing of different cytokine genes in four species of owl monkey.

Abstract. Non-human primates could prove to be suitable models for the study of infectious diseases such as malaria, tuberculosis, and hepatitis; the molecules of their immune systems are in the process of being fully characterized. Due to the relevance of cytokines in the modulation of the immune response, a molecular analysis of these proteins in non-human primates from the Aotus genus was carried out. Peripheral blood mononuclear cells from four species of Aotus monkey were obtained and their mRNAs for interleukin-2 (IL-2), IL-4, IL-6, IL-10, interferon-γ (IFN), and tumor necrosis factor (TNF)-α were characterized. This study shows a high degree of conservation between nucleotide and amino acid sequences of cytokines from different Aotus species and those from humans. The TNF-α molecules were identical in amino acid sequences for both.

Molecular analysis of HLA DR4-/β1 gene in malaria vaccinees. Typing and subtyping by PCR technique and oligonucleotides

Summary The combination of the PCR technique and the synthetic oligonucleotides has proved to be a useful tool in the molecular analysis of HLA class II genes, allowing recognition of as little as a single nucleotide modification in the sequence of the gene. The molecules encoded by these genes have been associated with genetic control of the immune response and with susceptibility to certain diseases. Studies carried out in our laboratory have shown three patterns of humoral immune response in the human volunteers vaccinated with the synthetic protein SPf 66: high, intermediate and low responders. Approximately 73‐3% of the low responders were serologically typed as HLA DR4 and 42% as DQw6. These results moved us to look for a subtype (Dw) correlation between the DR4 positive individuals and the different humoral immune response patterns. Using oligo‐typing methods after previous amplification of the DR4 B1 exon, we subtyped 20 DR4 volunteers, classified as high, intermediate and low responders. We did not find any direct association between the HLA DR4 Dw special subtype in the high or low responders immunized with the SPf 66 vaccine.

Identification of a differentially expressed mRNA in axenic Leishmania panamensis amastigotes

Differential display technique was applied in order to identify transcripts which are present in axenic amastigotes but not in promastigotes of the Leishmania panamensis parasites. One of them was cloned and the sequence reveals an open reading frame of 364 amino acids (approximately 40 kDa). The deduced protein is homologous to the serine/threonine protein kinases and specially to the mitogen activates protein kinases from eukaryotic species. Southern blot analysis suggest that this transcript, named lpmkh, is present in the genome of the parasite as a single copy gene. These results could imply that lpmkh could be involved in the differentiation process or the preservation of amastigotes in axenic conditions.

Leishmania panamensis: a 44bp deletion in gp63 gene is found in cDNA and genomic libraries.

Leishmanolysin (EC 3.4.24.36) (gp63) is a zincmetalloprotease (ND Rawlings et al. 1995 Meth-ods Enzymol 248 : 183-228) abundantly expressedin the promastigote form of Leishmania parasites,where it is attached to the plasma membrane by aglycosylphosphatidylinositol (GPI) anchor (PSchneider et al. 1990 J Biol Chem 265: 16955-16964, TJ Salvatore et al. 1992 Annu Rev Microbiol46 : 65-94). It has been suggested that gp63 be-comes active at multiple steps during the processof macrophage invasion and its function seems tobe required for the parasite’s intraphagolysosomalsurvival (GD Rusell et al. 1989 Immunol Today10: 328-333, AR Miller et al. 1990 Mol BiochemParasitol 39: 267-274, A Brittingham et al. 1996J Immunol 155 : 3102-3111). The protein is encodedby genes present in multiple copies that are clus-tered in two to three tandem repeats usually lo-cated on a single chromosome (JR Webb et al. 1991Mol Biochem Parasitol 48 : 173-184, T Hanekempet al. 1991 Mol Biochem Parasitol 48 : 27-38) andwhich are highly conserved throughout the genus(LL Button et al. 1988 J Exp Med 167: 724-729,HB Steinkraus 1993 Mol Biochem Parasitol 62:173-186, SC Roberts et al. 1993 Mol BiochemParasitol 62 : 157-172, E Medina-Acosta et al. 1993Mol Biochem Parasitol 57: 31-46).The gp63 familiy is constituted by a group ofproteins with different molecular weights depend-ing upon the species. The importance of the majorsurface glycoprotein of Leishmania promastigotes,gp63, in the binding of promastigotes to macroph-ages has been inferred largely due to its abundance,surface localization, and proteolytic activity (JBouvier et al. 1985 J Biol Chem 260 : 15504-15509,R Etges et al. 1986 J Biol Chem 261: 9098-9101,CS Chang 1986 Proc Natl Acad Sci USA 83 : 100-104, Brittingham loc. cit.).In the present work, we report for the first timethe molecular characterization of gp63 gene in L.panamensis, which is the most prevalent Leishma-nia species in Colombia, and also the presence ofcopies containing deletions of this gene either oncDNA or DNA material.In order to identify the gp63 gene from L.panamensis, an internal 940 bp long probe wasgenerated by PCR, using two consensus primersdesigned from the alignment of all Leishmaniagp63 nucleotide sequences reported to date. Lpan1:5’TACGTCGCCTCGGTGCCGA3’ and Lpan2:5´GCACCTGGACGCTGTACG3’ primers weresynthesized by the solid phase phosphito-triestermethod. This probe (U62634) was radiolabelledand used for hybridization assays and libraryscreening.A cDNA library was constructed from L.panamensis infective clone M/HOM/PA/71/LS/74stationary phase promastigotes. cDNA was syn-thesized using a cDNA synthesis kit (Amersham)and ligated into the Eco RI site of bacteriophageλgt11. Several clones were isolated and sequencedeither by manual (Sequenase v.2.0; United StatesBiochemical) or automatic (Applied Biosystems373; Perkin Elmer) sequencing using both DNAstrands. Of those, Lp63c1, represents a 2648-ntlong cDNA sequence that is homologous to othergp63 sequences and includes the 1728 nucleotidescoding region, 79 nt of the 5’ untranslated region,and 841 nt of the 3’ untranslated region (Figure).However, this clone exhibits a 44 bp deletion 26 ntdownstream to the start codon causing a frame-shift compared to other previously described gp63genes from Leishmania spp.In order to detect the presence of this gp63 formin L. panamensis genome, we built a Sal I genomiclibrary containing restriction fragments rangingfrom 2.8-3.2 kb in size into the pMOS vector