Breadth of the CD4+ T cell response to Anaplasma marginale VirB9-1, VirB9-2 and VirB10 and MHC class II DR and DQ restriction elements

Abstract

MHC class II molecules influence antigen-specific CD4+ T-lymphocyte responses primed by immunization and infection. CD4+ T-cell responses are important for controlling infection by many bacterial pathogens including Anaplasma marginale, and are observed in cattle immunized with the protective A. marginale outer membrane (OM) vaccine. Immunogenic proteins that comprise the protective OM vaccine include type IV secretion system (T4SS) proteins VirB9-1, VirB9-2, and VirB10, candidates for inclusion in a multi-epitope vaccine. Our goal was to determine the breadth of the VirB9-1, VirB9-2, and VirB10 T-cell response and MHC class II restriction elements in six cattle with different MHC class II haplotypes, defined by DRB3, DQA, and DQB allele combinations for each animal. Overlapping peptides spanning each T4SS protein were tested in T-cell proliferation assays with autologous antigen presenting cells (APC) and artificial APC expressing combinations of bovine DR and DQ molecules. Twenty immunostimulatory peptides were identified; three representing two or more epitopes in VirB9-1, ten representing eight or more epitopes in VirB9-2, and seven representing seven or more epitopes in VirB10. Of eight DRA/DRB3 molecules, four presented 15 peptides, which was biased as DRA/DRB3*1201 presented ten and DRA/DRB3*1101 presented four peptides. Four DQA/DQB molecules composed of two intrahaplotype and two interhaplotype pairs presented seven peptides, of which five were uniquely presented by DQ molecules. In addition,three functional mixed isotype (DQA/DRB3) restriction elements were identified. The immunogenicity and broad MHC class II presentation of multiple VirB9-1, VirB9-2, and VirB10 peptide epitopes justify their testing as a multi-epitope vaccine against A. marginale.

A nano particle vector comprised of poly lactic-co-glycolic acid and monophosphoryl lipid A and recombinant Mycobacterium avium subsp paratuberculosis peptides stimulate a pro-immune profile in bovine macrophages

“…This afforded an unique opportunity to examine the functionality of MoMΦ in a genetically defined context with a source of autologous cells where experiments could be duplicated as needed (Morse et al . ). A control peptide, peptide 8 of the VirB9‐1 protein had been identified which is not recognized by the MHC II allomorph expressed by the DRB3 haplotype *1101.…”

“…As a proof of concept, to show that the PLGA/MPLA NPs loaded with antigen could stimulate CD4 T cells, we used T cells from a calf that had been previously immunized with A. marginale OM expressing MHC II DRB3 haplotype *1101, which was shown to respond to peptide 5 of VirB9-2, but not peptide 8 of VirB9-1 (Morse et al 2012). PLGA/MPLA NP were loaded with each peptide (as described above) and tested with free peptide in a proliferation assay using a short-term T-cell line, started from PBMC stimulated with OM for 1 week and rested with APC without antigen for 1 week.…”

Nanoparticle-Based Delivery of Anaplasma marginale Membrane Proteins; VirB9-1 and VirB10 Produced in the Pichia pastoris Expression System

“…Therefore, it is desirable to express soluble VirB9-1 and VirB10 proteins. VirB9-1 and VirB10 have been previously expressed in bacteria using FLAG-tag or His-tag technologies, however both yielded insoluble proteins likely due to their intrinsic properties as outer membrane proteins [11,13]. This issue has been addressed in the current study, by producing soluble VirB9-1 and VirB10 using the yeast P. pastoris expression system.…”

“…To date expression of VirB9-1 and VirB10 has been reported using the FLAG-tag (a polypeptide protein tag) or His-tag systems, resulting in insoluble products presumably due to their intrinsic properties as membrane proteins [11,13]. Recently, the methylotrophic Pichia pastoris has rapidly become a highly successful system for the expression of heterologous proteins and is considered faster, easier, and less expensive than insect or mammalian protein expression systems [14,15,16].…”

Role of a Novel Human Leukocyte Antigen-DQA1*01:02;DRB1*15:01 Mixed Isotype Heterodimer in the Pathogenesis of “Humanized” Multiple Sclerosis-like Disease

“…Lotteau et al (32,33) identified the existence of mixed isotype DR␣-DQ␤ hybrid dimers in B-cell lines but could not identify any DQ␣-DR␤ mixed isotype dimers. Recently, mixed DQ␣-DR␤ isotype hybrid molecules (BoLA-DQA/DRB3) that were functional in presenting viral peptide to CD4 ϩ T-cells have been reported in cattle immunized with Anaplasma marginale vaccine (52).…”