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G F Mitchell

Publications and source records attributed to G F Mitchell.

At least 109 records · Page 6Linked to original sources
PubMed1986

Molecular vaccines against parasites.

Prophylactic vaccines can be expected to be one of the major practical outputs of parasitology research. Various groups within Australia have pursued the vaccine objective for several years, with particular emphasis on blood-stage falciparum malaria in man, intestinal helminths of sheep and cattle, cutaneous myiasis (blowfly strike) in sheep, cysticercosis in sheep and cattle, bovine babesiosis, and cattle ticks. Other vaccine programmes are concerned with giardiasis, filariasis, toxoplasmosis, fascioliasis, coccidiosis in poultry, cutaneous leishmaniasis and schistosomiasis japonica. For many years, the only available vaccine against a parasite in Australia has been the attenuated Babesia bovis vaccine produced by the Tick Fever Research Centre of the Queensland Department of Primary Industries. Strategies for achieving molecular vaccines are generally similar within the various research groups. They involve analysis of the immunology and immunochemistry of a model or in-vitro system; development of functional monoclonal antibodies; analysis of antibody specificities in clinically and/or functionally defined polyclonal sera; screening of cDNA or genomic expression libraries; peptide synthesis; identification of an appropriate vaccination schedule involving adjuvants or new recombinant DNA-based antigen delivery systems. Outlined below are five of the major vaccine programmes.

Journal Article↗
PubMed1986

Immunochemical analysis of Taenia taeniaeformis antigens expressed in Escherichia coli.

Previously we reported the isolation of several Escherichia coli clones expressing fragments of Taenia taeniaeformis antigens as beta-galactosidase fused proteins (Bowtell, Saint, Rickard & Mitchell, 1984). Here we describe the isolation of additional antigen-expressing clones from a larval cDNA library and the assignment of these clones to 7 antigen families. These were isolated with a polyspecific rabbit antiserum raised to the oncosphere. Since this serum was capable of reacting with a large number of antigens, it was important to develop techniques for rapidly determining the identity of the native T. taeniaeformis molecule corresponding to a cloned antigen gene. These included active immunization of rabbits with fused proteins and several techniques involving affinity purification on immobilized fused proteins. The reactivity of the antigen-positive clones with sera from humans infected with related parasites was also assessed. Finally, immunization of mice with several fused proteins failed to protect against subsequent infection, although antigens previously identified as candidate host-protective antigens (Bowtell, Mitchell, Anders, Lightowlers & Rickard, 1983) have yet to be identified in the expression library.

Animals↗
PubMed1986

Resistance of 129/J mice to Schistosoma mansoni infection.

Mice of the strain 129/J bred at this Institute (WEHI 129/J) were exposed once or repeatedly to cercariae of Schistosoma mansoni and worm burdens determined. In keeping with reports of S. japonicum infection in these mice, a high level of resistance was recorded. Radio-labelled extracted antigens of S. mansoni adult worms were reacted with sera from exposed 129/J mice and infected BALB/c mice. Immunoprecipitated antigens preferentially recognized by antibodies in 129/J mice include proteins of Mr 99,000 and 21,000. Whether responses to these antigens are a cause or a consequence of resistance remains to be established.

Animals↗
PubMed1986

Mr 26,000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione S-transferase.

Mice of the inbred strain 129/J bred at this Institute (WEHI 129/J) are relatively resistant to chronic infection with the parasitic helminth Schistosoma japonicum. In contrast to more permissive mouse strains such as BALB/c, the WEHI 129/J mice are high responders to a Mr 26,000 adult worm antigen designated Sj26. Cloned cDNAs corresponding to Sj26 have been identified in a S. japonicum phage lambda gt11 amp3 expression library, and their nucleotide sequences have been deduced. The predicted amino acid sequence of the antigen specified by these cDNAs shows striking homology with class mu isozymes of mammalian glutathione S-transferases (RX:glutathione R-transferase, EC 2.5.1.18). Extracts of adult worms contain glutathione S-transferase activity, and affinity chromatography of enzyme activity on glutathione columns leads to the purification of a Mr 26,000 molecule that comigrates with Sj26. Although vaccination studies in mice with a beta-galactosidase-Sj26 fusion protein from Escherichia coli are encouraging, more immunogenic preparations of the antigen are likely to be required to establish the utility of Sj26 as a model vaccine.

Amino Acid Sequence↗
PubMed1986

The glycoconjugate derived from a Leishmania major receptor for macrophages is a suppressogenic, disease-promoting antigen in murine cutaneous leishmaniasis.

Lymphoid cells from genetically-susceptible BALB/c mice immunized against a glycoconjugate of the protozoan parasite, Leishmania major, promote chronic cutaneous disease in BALB/c nude mice. This cell population therefore differs from cells harvested from non-immunized BALB/c mice that are known to be potent mediators of protection against cutaneous leishmaniasis in minimally-reconstituted, syngeneic nude mice. The glycoconjugate when injected into genetically-resistant C57BL/6 mice will increase the size and persistence of cutaneous lesions. Recent studies have established that the water soluble glycoconjugate is derived from a membrane-bound glycolipid that is a receptor used by the parasite in the attachment to macrophages. This glycolipid can protectively immunize mice against cutaneous leishmaniasis. Identification of a vaccinating glycolipid antigen and a suppressogenic component derived from it will greatly facilitate analysis of disease-promoting and resistance-promoting immunity in cutaneous leishmaniasis. However, the fact that a host-protective antigen contains a disease-promoting component may militate against the immediate use of this molecular vaccine in man.

Animals↗
PubMed1986

Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity.

We show that the subcellular location of foreign antigens expressed in recombinant vaccinia viruses influences their effectiveness as immunogens. Live recombinant viruses induced very poor antibody responses to a secreted repetitive plasmodial antigen (the S-antigen) in rabbits and mice. The poor response accords with epidemiological data suggesting that S-antigens are poorly immunogenic. Appending the transmembrane domain of a membrane immunoglobulin (immunoglobulin G1) to its carboxy terminus produced a hybrid S-antigen that was no longer secreted but was located on the surface of virus-infected cells. This recombinant virus elicited high antibody titers to the S-antigen. This approach will facilitate the use of live virus delivery systems to immunize against a wide range of foreign nonsurface antigens.

Amino Acid Sequence↗
PubMed1986

Sorting large numbers of clones expressing Plasmodium falciparum antigens in Escherichia coli by differential antibody screening.

We describe an approach to classifying a large number of clones expressing Plasmodium falciparum antigens in Escherichia coli by virtue of their differing reactivities with 100 human anti-malarial sera. Individual sera exhibited marked differences in the patterns of reactivity with these clones. These patterns led to the identification of sets of clones, here termed "serological families", which were shown to encode distinct P. falciparum antigens. A serological family was found to be composed of non-identical clones derived from portions of the same antigen. Using this approach six new P. falciparum antigens were identified. One of these is described in detail and is a 102 X 10(3) Mr antigen, predominantly of schizonts. Sequencing studies on four cDNA clones encoding parts of this antigen revealed blocks of hydrophilic dipeptide and tripeptide repeats and so the antigen has been termed the acidic basic repeat antigen (ABRA).

Animals↗
PubMed1985

Phenotypic diversity of cloned lines of Leishmania major promastigotes.

In vitro cultured promastigotes of virulent (V) and avirulent (A) cloned lines of Leishmania major, and the parental isolate LRC-L137, were examined with respect to morphology, cell size, growth rate, and apparent DNA content. Growth rates of all lines were comparable and both virulent (V121, LRC-L137) and avirulent parasites (A12, A52, A59) exhibited a progressive decrease in apparent DNA content with time in culture, as measured by incorporation of Hoechst Dye 33342. The four cloned lines and the parental isolate showed differences in the content of morphological variants and in the mean body length. Morphologically, there were similarities between A12 and A52 and between A59 and V121. Promastigote populations were also examined for the expression of the target antigen of a previously characterized monoclonal antibody, WIC-79.3. This antibody binds to a membrane antigen that is also present in culture supernatants of Leishmania of A1 serotype. Three different assays with culture supernatants all showed that V121, A59, and A12 were high producers with LRC-L137 and A52, low producers. Similar variation in expression of the 79.3 target antigen was detected in intact organisms of the various lines by immunofluorescence with flow cytometry. No simple correlation was found between the expression or release of the WIC-79.3 target antigen and virulence. The virulence or avirulence of all cloned lines for BALB/c mice remained stable. The data are discussed in terms of differentiation stages of L. major promastigotes and the continuing search for morphological and biochemical markers of virulence.

Animals↗
PubMed1985

Immunization with Leishmania receptor for macrophages protects mice against cutaneous leishmaniasis.

The Leishmania major receptor for macrophages is a lipid-containing glycoconjugate that is recognized by the monoclonal antibody WIC-79.3. When L. major promastigotes were incubated with Fab fragments of WIC-79.3 prior to injection into genetically susceptible mice, their infectivity was decreased. Fab fragments from an irrelevant control antibody of the same class had no effect. The L. major glycolipid was purified from detergent-solubilized promastigotes by affinity chromatography on immobilized WIC-79.3 and used to vaccinate mice that are genetically resistant or susceptible to disease. Genetically resistant mice could be protected totally from cutaneous disease with as little as 5 micrograms of glycolipid. A high but not absolute level of resistance was also induced in the susceptible mice, in which the disease is otherwise fatal. No protection was obtained with the carbohydrate fragment of the glycolipid alone or by injection of the glycolipid in the absence of adjuvant. Genetically susceptible mice, immunized and protected from disease as a result of multiple injections of live avirulent cloned promastigotes of L. major, produced antibodies to the glycolipid of L. major. No antibodies were detected in serum from chronically diseased mice. The data suggest that this functionally important antigen of L. major is a candidate vaccine against cutaneous leishmaniasis.

Adjuvants, Immunologic↗
PubMed1985

Interspersed blocks of repetitive and charged amino acids in a dominant immunogen of Plasmodium falciparum.

We describe an antigen of Plasmodium falciparum that is a dominant immunogen in man. The corresponding cDNA clone, Ag231, expressing this antigen in Escherichia coli reacted in an in situ colony assay with sera from up to approximately equal to 93% of 65 people living in an area in which P. falciparum is endemic. Human antibodies affinity purified on immobilized Ag231 lysates identified the corresponding parasite antigen as a polypeptide of Mr approximately equal to 300,000. It was present in schizonts and also in ring-stage trophozoites, where a speckled immunofluorescence pattern suggested an association with the erythrocyte. Its mRNA was enriched in merozoites relative to other blood stages, a distinctive property shared by a recently described antigen located on the surface of ring-infected erythrocytes, and it is encoded by a single gene having a number of allelic variants. The complete nucleotide sequence of Ag231 revealed a structural unit composed of 13 hexapeptide repeats flanked by a highly charged region containing both acidic and basic amino acids. This structural unit is itself repeated, so that blocks of repeats and charged units are interspersed along the molecule. The sequences within the repeats vary much more extensively than those in the charged units.

Amino Acid Sequence↗
PubMed1985

Antibody responses to the antigen Sj26 of Schistosoma japonicum worms that is recognized by genetically resistant 129/J mice.

Serum antibody responses of mice exposed to Philippine isolates of Schistosoma japonicum have been analysed by immunoprecipitation of exogenously radiolabelled antigens extracted from adult worms. Attention was focused on labelled protein antigens differentially recognized by sera of mice that differ genetically in their resistance status. Mice of the inbred strain 129/J can show high level resistance to first or repeated infection with S. japonicum. Even after six percutaneous administrations of 25 cercariae, approximately 50% of 129/J mice remain healthy with no or very few worms present in the portal system. Sera from 129/J mice exposed to S. japonicum consistently and differentially recognise an antigen of adult worms of mol. wt. 26,000. This antigen, termed Sj26, is not immunoprecipitated from S. mansoni adult worms by sera from resistant 129/J mice. Serum antibodies to Sj26 are present in at least some patients with a history of schistosomiasis japonica. Whether immune responses to Sj26 are involved directly in expression of resistance to S. japonicum remains to be determined. However, this antigen produced by cloned DNA in expression vectors, or isolated from adult worms, is an obvious candidate to be tested for vaccination efficacy in mice.

Animals↗
PubMed1985

A monoclonal antibody-based immunoradiometric assay for detection of circulating antigen in Bancroftian filariasis.

A monoclonal antibody designated Gib 13 has been used in an immunoradiometric assay (IRMA) to detect circulating antigen in the sera of Wuchereria bancrofti-infected subjects from an endemic area of Papua New Guinea. A clear association between the presence of patent infection and the Gib 13 target epitope in serum was established because 93% of microfilaremic individuals were antigen-positive. Moreover, there was a significant correlation between levels of serum antigen and blood microfilarial counts. Detection of circulating antigen in amicrofilaremic subjects with acute symptoms of lymphatic filariasis, and 53% of asymptomatic amicrofilaremic subjects, but not in nonendemic controls, suggests that the Gib 13 IRMA will also be of value in the diagnosis of occult filariasis. However, as in all IRMA based on detection of potentially immunogenic molecules in man, antibodies can be expected to be the major contributor to reduced sensitivity of the assay.

Adolescent↗