Assessment and optimization of Theileria parva sporozoite full-length p67 antigen expression in mammalian cells

Abstract

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.

Immune parameters to p67C antigen adjuvanted with ISA206VG correlate with protection against East Coast fever

“…However, p67C had several advantages over the full-length protein when expressed in E. coli , such as better stability and higher expression yields [13] , [15] . A very recent study has demonstrated stable expression of p67 without the transmembrane domain in mammalian cells [23] . It will be interesting to determine the efficacy of this antigen in vaccine trials.…”

Molecular and Antigenic Properties of Mammalian Cell-Expressed Theileria parva Antigen Tp9

“…Previous attempts to express recombinant T. parva proteins in prokaryotic or eukaryotic systems for vaccine purposes have been challenging and the process has been hindered due to protein insolubility and misfolding, among other issues (15, 16, 20). Recently, we were able to express full-length T. parva p67 antigen in mammalian cells and demonstrated that the recombinant antigen retained its immunogenic properties (21). Considering the technical and biological challenges associated with the expression of Theileria sp.…”

“…Concomitantly with the identification of novel antigens was extensive work to optimize antigen expression systems and delivery platforms for Theileria antigens. Both prokaryotic and eukaryotic systems have been tested for expression of potential T. parva vaccine antigens (15, 16, 20, 21). Codon usage, post-translational modifications, protein conformation, and solubility are a few aspects that have been considered in deciding the most suitable platform to express antigens for subunit vaccines.…”

“…Codon usage, post-translational modifications, protein conformation, and solubility are a few aspects that have been considered in deciding the most suitable platform to express antigens for subunit vaccines. Recently, we examined the antigenicity of the full-length T. parva p67 protein expressed in a mammalian system, and that work provided a basis for further studies to investigate this recombinant, mammalian expressed antigen as a subunit vaccine component to prevent T. parva (21).…”

Capacity to Elicit Cytotoxic CD8 T Cell Activity Against Mycobacterium avium subsp. paratuberculosis Is Retained in a Vaccine Candidate 35 kDa Peptide Modified for Expression in Mammalian Cells

“…Later, the NheI/SmaI cut amplicons were cloned into the shuttle vector pINT2EGFP (25) and cut with the same enzymes, generating pCMV-tPA-MMP, pCMV-tPA-MMP-5mut, and pCMV-tPA-MMP-2mut, thereby placing the tPA-MMP ORFs under the transcriptional control of the immediate early gene promoter of human cytomegalovirus (CMV), followed by the bovine growth hormone polyadenylation signal (pA). Alternatively, the NheI/SmaI cut amplicons were subcloned into the vector pEF1α-p67 (26) to put the tPA-MMP ORFs under the transcriptional control of the Human Elongation Factor 1 Alpha (EF1α) promoter followed by the bovine growth hormone pA, thus generating pEF1α-tPA-MMP, pEF1α-tPA-MMP-5mut, and pEF1α-tPA-MMP-2mut.…”