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Characterization of an insect cell-derived Theileria parva sporozoite vaccine antigen and immunogenicity in cattle.

ABSTRACT: Previous data showed that six out of a group of nine cattle inoculated with NS1-p67, a recombinant form of a 67-kDa Theileria parva sporozoite surface protein, were immune to East Coast fever. This bacterially expressed antigen encoded all 709 amino acid residues of p67 fused to the C-terminal end of 87 residues derived from NS1, a structural protein of influenza virus, and a linker DNA sequence. NS1-p67 lacked reactivity with TpM 12, a monoclonal antibody to native p67, and had an estimated molecular mass of 110 kDa, as opposed to the calculated mass of 85,000 Da. We have used the baculovirus expression system in an attempt to express this parasite protein in a native form and thereby increase the protective capacity of the antigen. However, Spodoptera frugiperda SF21AE cells infected with recombinant virus expressed p67 as a 100-kDa molecule. The host cells exhibited a limited capacity to glycosylate this molecule to a 110-kDa form, and p67 was not exported to the surface membrane. TpM 12 did not bind to these recombinant forms but, at time points late during viral infection, reacted with a molecule of about 70 kDa. Since the bulk of insect cell-derived p67 was not expressed in an appropriate form, we tested the immunogenicity of these partially processed recombinant p67 forms in cattle. Two groups of three cattle were inoculated with antigen formulated either with saponin or Freund's adjuvant. As seen previously with NS1-p67, all animals developed high levels of anti-p67 antibodies that neutralized sporozoite infectivity in vitro, but antigen-specific T-cell proliferative responses were not detected in peripheral blood. Given the caveat of the small number of cattle analyzed, insect cell-derived p67 does not appear to be superior to NS1-p67 as an immunogen, and the latter remains the molecule of choice for the development of vaccines against East Coast fever.

SUBMITTER: Nene V

PROVIDER: S-EPMC173023 | biostudies-other | 1995 Feb

REPOSITORIES: biostudies-other

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Characterization of an insect cell-derived Theileria parva sporozoite vaccine antigen and immunogenicity in cattle.

Nene V V Inumaru S S McKeever D D Morzaria S S Shaw M M Musoke A A

Infection and immunity 19950201 2

Previous data showed that six out of a group of nine cattle inoculated with NS1-p67, a recombinant form of a 67-kDa Theileria parva sporozoite surface protein, were immune to East Coast fever. This bacterially expressed antigen encoded all 709 amino acid residues of p67 fused to the C-terminal end of 87 residues derived from NS1, a structural protein of influenza virus, and a linker DNA sequence. NS1-p67 lacked reactivity with TpM 12, a monoclonal antibody to native p67, and had an estimated mol ...[more]

PMID: 7822014

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Project description:Delivery of various forms of recombinant Theileria parva sporozoite antigen (p67) has been shown to elicit antibody responses in cattle capable of providing protection against East Coast fever, the clinical disease caused by T. parva. Previous formulations of full-length and shorter recombinant versions of p67 derived from bacteria, insect, and mammalian cell systems are expressed in non-native and highly unstable forms. The stable expression of full-length recombinant p67 in mammalian cells has never been described and has remained especially elusive. In this study, p67 was expressed in human-derived cells as a full-length, membrane-linked protein and as a secreted form by omission of the putative transmembrane domain. The recombinant protein expressed in this system yielded primarily two products based on Western immunoblot analysis, including one at the expected size of 67 kDa, and one with a higher than expected molecular weight. Through treatment with PNGase F, our data indicate that the larger product of this mammalian cell-expressed recombinant p67 cannot be attributed to glycosylation. By increasing the denaturing conditions, we determined that the larger sized mammalian cell-expressed recombinant p67 product is likely a dimeric aggregate of the protein. Both forms of this recombinant p67 reacted with a monoclonal antibody to the p67 molecule, which reacts with the native sporozoite. Additionally, through this work we developed multiple mammalian cell lines, including both human and bovine-derived cell lines, transduced by a lentiviral vector, that are constitutively able to express a stable, secreted form of p67 for use in immunization, diagnostics, or in vitro assays. The recombinant p67 developed in this system is immunogenic in goats and cattle based on ELISA and flow cytometric analysis. The development of a mammalian cell system that expresses full-length p67 in a stable form as described here is expected to optimize p67-based immunization.

Project description:Theileria parva is a protozoan parasite transmitted by the brown-eared ticks, Rhipicephalus appendiculatus and Rhipicephalus zambeziensis. Buffaloes are the parasite's ancestral host, with cattle being the most recent host. The parasite has two transmission modes namely, cattle-cattle and buffalo-cattle transmission. Cattle-cattle T. parva transmission causes East Coast fever (ECF) and January disease syndromes. Buffalo to cattle transmission causes Corridor disease. Knowledge on the genetic diversity of South African T. parva populations will assist in determining its origin, evolution and identify any cattle-cattle transmitted strains. To achieve this, genomic DNA of blood and in vitro culture material infected with South African isolates (8160, 8301, 8200, 9620, 9656, 9679, Johnston, KNP2, HL3, KNP102, 9574, and 9581) were extracted and paired-end whole genome sequencing using Illumina HiSeq 2500 was performed. East and southern African sample data (Chitongo Z2, Katete B2, Kiambu Z464/C12, Mandali Z22H10, Entebbe, Nyakizu, Katumba, Buffalo LAWR, and Buffalo Z5E5) was also added for comparative purposes. Data was analyzed using BWA and SAMtools variant calling with the T. parva Muguga genome sequence used as a reference. Buffalo-derived strains had higher genetic diversity, with twice the number of variants compared to cattle-derived strains, confirming that buffaloes are ancestral reservoir hosts of T. parva. Host specific SNPs, however, could not be identified among the selected 74 gene sequences. Phylogenetically, strains tended to cluster by host with South African buffalo-derived strains clustering with buffalo-derived strains. Among the buffalo-derived strains, South African strains were genetically divergent from other buffalo-derived strains indicating possible geographic sub-structuring. Geographic sub- structuring was also observed within South Africa strains. The knowledge generated from this study indicates that to date, ECF is not circulating in buffalo from South Africa. It also shows that T. parva has historically been present in buffalo from South Africa before the introduction of ECF and was not introduced into buffalo during the ECF epidemic.

Dataset Information

Characterization of an insect cell-derived Theileria parva sporozoite vaccine antigen and immunogenicity in cattle.

Publications

Characterization of an insect cell-derived Theileria parva sporozoite vaccine antigen and immunogenicity in cattle.

Similar Datasets

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