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Biomedical subjects

F Zavala

Publications and source records attributed to F Zavala.

At least 91 records · Page 5Linked to original sources
PubMed1988

Conformational restriction of peptidyl immunogens with covalent replacements for the hydrogen bond.

A new strategy for designing synthetic vaccines is presented. In this approach synthetic peptides are conformationally restricted by replacing putative hydrogen bonds with covalent mimics. The chemistry for substituting a hydrazone-ethane link (N-N = CH-CH2-CH2) for an (i + 4)----i hydrogen bond in a pentapeptide with alpha-helical potential is reported. Chemically shaping peptides to mimic the three-dimensional surfaces of proteins may enhance their immunogenicity. To test this strategy, a potential synthetic vaccine for malaria, Cys-(Asn-Pro-Asn-Ala)3-NH2, was conformationally restricted by replacing putative hydrogen bonds between asparagine side chains with a covalent replacement, an ethylene bridge, to give first generation chemically shaped immunogens. Antibodies to one of the shaped malarial peptides show a strong reaction with living Plasmodium falciparum sporozoites, a form of malaria which infects hundreds of millions of people yearly.

Animals↗
PubMed1988

Transport to the cell surface of a peptide sequence attached to the truncated C terminus of an N-terminally anchored integral membrane protein.

Attempts to construct hybrid proteins that are transported to the plasma membrane are frequently unsuccessful because of perturbations in polypeptide folding. In seeking to minimize this problem, we have used the less common type of integral membrane protein, which has an uncleaved signal-anchor domain and an extracellular carboxyl portion, to transport a peptide sequence of interest to the cell surface. A set of plasmids was constructed that contained the gene encoding respiratory syncytial virus glycoprotein G (RSVG) interrupted immediately after one of several proline codons by a synthetic sequence containing unique restriction endonuclease sites and a stop codon. The shortened RSVG gene was flanked by vaccinia virus DNA to permit cloning and expression in a vaccinia virus vector. An open reading frame encoding four copies of the immunodominant repeating epitope of the circumsporozoite protein of Plasmodium falciparum was inserted into the tails of the truncated RSVG genes. Recombinant vaccinia viruses were isolated and shown to express hybrid proteins that reacted with a monoclonal antibody directed to the repeating circumsporozoite epitope. Moreover, immunofluorescence studies indicated that the peptide was on the external cell surface and available to react with antibodies. Expression of the hybrid protein also occurred in rabbits inoculated with the live recombinant vaccinia virus, as demonstrated by the generation of antibodies that bound to P. falciparum sporozoites in vitro.

Animals↗
PubMed1987

Synthetic peptide vaccine confers protection against murine malaria.

A synthetic peptide, (DPPPPNPN)2D, representing a subunit of the repeat domain of the Plasmodium berghei circumsporozoite protein, was conjugated to tetanus toxoid using bisdiazobenzidine. Immunization of mice and rats with the conjugate induced high serum titers of antibodies to the parasite, and most of the animals were completely protected from malaria infection when challenged with sporozoites.

Animals↗
PubMed1987

Antigenic analysis of the repeat domain of the circumsporozoite protein of Plasmodium vivax.

In the present study we analyzed the fine specificity of mouse monoclonal and human polyclonal antibodies directed against the repeat domain of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium vivax. Five synthetic peptides, representing monomeric and dimeric repeats of this malarial antigen, were assayed for their capacity to inhibit the binding of these antibodies to a yeast-derived recombinant CS protein. The results revealed the existence of at least two distinct repeated overlapping epitopes in the CS protein of P. vivax. Furthermore, polyclonal sera contain antibodies which recognize additional determinants not represented by the synthetic repeat peptides. Some of these sera contain antibodies recognizing a region flanking the repeat domain (region I). The present findings are in contrast with the antibody response in rodents and humans to the Plasmodium falciparum CS protein, which is directed against a single repeated immunodominant epitope.

Animals↗
PubMed1987

Peripheral benzodiazepines enhance the respiratory burst of macrophage-like P388D1 cells stimulated by arachidonic acid.

P388D1, a murine cell with macrophage properties, responds to exogenous arachidonic acid with superoxide anion production. This oxidative burst is enhanced by peripheral and mixed type benzodiazepines and this stimulation is specifically reversed by the peripheral antagonist PK 11195. In contrast, PK 11195 is unable to antagonize a stimulation caused by a non-benzodiazepine ligand such as the chemotactic peptide fMet-Leu-Phe. The optimal concentrations were close to 10 nM and corresponded to the affinities of the compounds for the peripheral benzodiazepine receptor detected on these cells. Compared to other tissues where peripheral benzodiazepines acted only at micromolar concentrations, the macrophage with its functional receptor appears as a privileged site of action for these molecules.

Animals↗
PubMed1987

Detection and anatomical localization of Plasmodium falciparum circumsporozoite protein and sporozoites in the afrotropical malaria vector Anopheles gambiae s.l.

Salivary glands from Anopheles gambiae s.l. collected in Burkina Faso, West Africa, were analyzed by both microscopic examination and immunoradiometric assay to determine the Plasmodium falciparum sporozoite rates. Using the same mosquito samples, the immunoassay revealed positive salivary glands with low sporozoite loads, which were frequently missed by microscopy. A closer agreement between both techniques was found using salivary glands with high sporozoite loads. We also found a number of mosquitoes with uninfected salivary glands which harbored the circumsporozoite antigen in their thoraces. In a particular village these mosquitoes represented 43.5% of all sporozoite antigen carrying specimens.

Animals↗
PubMed1987

Comparative testing of monoclonal antibodies against Plasmodium falciparum sporozoites for ELISA development.

Ten monoclonal antibodies developed against Plasmodium falciparum sporozoites at four institutions were evaluated for use in an enzyme-linked immunosorbent assay (ELISA). Four of the antibodies were eliminated because of their low sensitivity or requirement for high concentrations of capture antibody, while an additional four were rejected because they exhibited cross-reactivity with P. berghei sporozoites. Of the two remaining monoclonal antibodies, that designated 2A10 had the highest sensitivity, a requirement for lower concentrations of capture antibody, and had been tested successfully against sporozoites from a wider range of geographical areas than the others. Use of this monoclonal antibody in a standardized ELISA method gave a test ten times more sensitive than previously reported for P. falciparum sporozoites and its detection limit was less than 100 sporozoites per mosquito.

Animals↗
PubMed1986

Synthetic peptides as antigens for the detection of humoral immunity to Plasmodium falciparum sporozoites.

The presence of antibodies against P. falciparum sporozoites in humans living in malaria-endemic areas was measured using as antigen the synthetic peptide (NANP)3, which represents the immunodominant region of the circumsporozoite (CS) protein. By using a competitive binding assay it was determined that antibodies which recognize (NANP)3 do not react with a 22-Mer synthetic peptide representing a cross-reacting epitope present in an antigen (5.1) from the blood stages of the parasite. Antibodies present in human sera which react with the 5.1 peptide did not react with (NANP)3. This strongly suggests that antibodies to (NANP)3 found in sera of individuals living in endemic areas are a reflection of exposure to P. falciparum sporozoites. These results validate the use of (NANP)3 for epidemiological studies to detect and measure humoral immunity to P. falciparum sporozoites.

Amino Acid Sequence↗
PubMed1986

Monoclonal anti-gametocyte antibodies identify an antigen present in all blood stages of Plasmodium falciparum.

Two polypeptides of 150 and 130 kDa present in all asexual and sexual blood stages of Plasmodium falciparum have been identified with anti-gametocyte monoclonal antibodies. The apparent molecular mass of these antigens is identical in different developmental stages of the parasite and in different isolates. These antigens are released in the culture supernatant during the process of schizogony and are also detected in the sera of patients undergoing a primary P. falciparum infection. Antibodies against these antigens occur in sera of a large percentage of children and most adults living in malaria-endemic areas, suggesting that they are highly immunogenic. The anti-gametocyte monoclonal antibodies react with a synthetic peptide (Glu-Glu-Asn-Val)4, present in antigen Pf155 [Perlmann, H. et al. (1984) J. Exp. Med. 159, 1686-1704] and in the ring-infected erythrocyte surface antigen [Coppel, R.L. et al. (1984) Nature 310, 789-792], indicating that these polypeptides are closely related. In contrast, two glycophorin-binding proteins of similar molecular mass [Perkins, M.E. (1984) J. Exp. Med. 160, 788-798] appear to be entirely distinct from the presently described antigens. We failed to observe any in vitro inhibitory activity of the monoclonal antibodies on merozoite invasion and on gametocyte infectivity.

Animals↗
PubMed1986

In vivo immunomodulating activity of PK 1195, a structurally unrelated ligand for "peripheral" benzodiazepine binding sites--I. Potentiation in mice of the humoral response to sheep red blood cells.

PK 11195, an isoquinoline carboxamide compound with the highest affinity for the peripheral type benzodiazepine binding site has been shown to enhance the humoral response of mice to sheep red blood cells, a T cell dependent antigen. The compound was active when administered i.p. 1 day following immunization. The active doses ranged from 10 ng to 1 mg/kg. Such an immunomodulating activity might be related to the presence of peripheral type benzodiazepine binding sites, previously detected on the macrophage.

Adjuvants, Immunologic↗
PubMed1986

Expression of the Plasmodium knowlesi circumsporozoite antigen in Escherichia coli directed by Plasmodium bacterial-like promoter sequences.

The Plasmodium knowlesi circumsporozoite (CS) gene is expressed in Escherichia coli directly from a parasite genomic DNA fragment, using promoter and ribosome-binding site (RBS) sequences present in this fragment. Transcription of the CS gene in E. coli is directed by tandem Plasmodium bacterial-like promoter elements located within the 0.5-kb EcoRI-HindIII fragment roughly 2.5 kb 5' from the CS gene within the 11-kb EcoRI parasite genomic DNA fragment. No readthrough from vector promoters or fortuitous promotion from plasmodial A + T-rich sequences was observed. The endogenous Plasmodium promoter of the CS gene does not seem to be recognized by E. coli RNA polymerases. Two tandem E. coli-recognized promoters are relatively strong judging by their ability to drive the bacterial chloramphenicol acetyl-transferase (CAT) gene. Translation of the message must be achieved by utilising an AAGAA sequence 4 bp 5' from the ATG initiation codon as RBS.

Animals↗
PubMed1986

Field observations on the use of anti-sporozoite monoclonal antibodies for determination of infection rates in malaria vectors.

Samples of indoor-resting Anopheles gambiae s.1. from Mali and Burkina Faso (West Africa) were processed in order to compare Plasmodium falciparum sporozoite rates obtained by immunoradiometric assay (IRMA) with circumsporozoite (CS) monoclonal antibody and by microscope examination of salivary glands. The immunological method provided sporozoite rates always higher than those obtained by microscope examination. This result does not appear to be related to cross-reactions involving non-sporozoite antigens. A small fraction of IRMA-positive mosquitoes is necessarily negative by microscope, since these mosquitoes actually contain the CS antigen only in the abdomen, presumably in connection with the presence of fully mature oocysts. However, the frequency of these mosquitoes cannot explain in itself an average ratio of 1:2 between microscope and IRMA sporozoite rates. A more important source of difference appears to depend on the detection of positive mosquitoes with low sporozoite numbers which remain more frequently undetected by microscope examination. Failure of salivary gland penetration by sporozoites is also considered as a possible source of discrepancy between the two methods.

Animals↗