SIVsm Tat, Rev, and Nef1: functional characteristics of r-GV internalization on isotypes, cytokines, and intracellular degradationReport as inadecuate




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BMC Biotechnology

, 10:54

First Online: 19 July 2010Received: 15 December 2009Accepted: 19 July 2010

Abstract

BackgroundRecombinant gas vesicles r-GV from Halobacterium sp. strain SD109 expressing cassettes with different SIVsm inserts, have potential utility as an effective antigen display system for immunogen testing in vivo and for initial epitope assessments in vitro. Previous mouse model studies demonstrated immunization with r-GV expressing selected exogenous sequences elicited a prolonged immune response. Here we tested segments from three SIVsm genes tat, rev, and nef each surface displayed by r-GV. As with HIV, for SIVsm the proteins encoded by tat, rev and nef respectively serve critical and diverse functions: effects on efficient viral RNA polymerase II transcription, regulation of viral gene expression and effects on specific signaling functions through the assembly of multiprotein complexes. Humoral responses to r-GV elicited in vivo, associated changes in selected cell cytokine production following r-GV internalization, and the capacity of J774A.1 macrophage cells to degrade these internalized display-delivery particles in vitro were examined.

ResultsThe in vivo studies involving r-GV immunizations and in vitro studies of r-GV uptake by J774A.1 macrophages demonstrated: i tests for antibody isotypes in immunized mice sera showed activation and re-stimulation of memory B cells, ii during long term immune response to the epitopes, primarily the IgG1 isotype was produced, iii in vitro, macrophage degradation of r-GV containing different SIVsm inserts occurred over a period of days resulting in an inherent slow breakdown and degradation of the SIVsm peptide inserts, iv vesicle specific GvpC, a larger protein, degraded more slowly than the recombinant peptide inserts and v in vitro uptake and degradation of the r-GV populations tested was associated with SIVsm insert specific patterns for cytokines IL-10, IL-12 and IL-18.

ConclusionsTogether these findings provide new information underscoring r-GV potential. They can clearly: display various exogenous peptides, be intracellularly degraded in vitro over a period of days, affect cell cytokine levels, and retain their self-adjuvanting capacity irrespective of the specific peptide expressed within the GvpC protein. These features support the cost effective generation of vaccine components, and provide a simple, self-adjuvanting system for assessing immune visibility of and specific responses to individual pathogen peptides.

Abbreviationswt-GVwild type gas vesicles

r-GVrecombinant gas vesicles

Gvpgas vesicle protein

SIVSimian Immunodeficiency Virus

SIVsmSimian Immunodeficiency Virus infecting the sooty mangabeys

SHIVSimian-Human Immunodeficiency Virus

bpbase pair

BSABovine Serum Albumin

ODoptical density

PBSPhosphate-Buffered Saline

PCRpolymerase chain reaction

RTroom temperature

SFMserum free media

HRPhorseradish peroxidase

TMBtetramethylbenzidine-hydrogen peroxide

FITCfluorescein isothiocyanate

NFDMnon-fat dry milk

APAlkaline Phosphatase

IgImmunoglobulins

AIDSacquired immunodeficiency syndrome

PVDFPolyvinylidene fluoride membrane

FBSFetal Bovine Serum

pNPPpara-nitrophenyl phosphate

Tata 50 amino acids peptide segment encoded by the tat gene

Reva peptide segment containing 81 amino acids

Nef1a peptide segment encompassing amino acids 1-214, of the protein encoded by the nef gene.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-10-54 contains supplementary material, which is available to authorized users.

Marinko Sremac and Elizabeth S Stuart contributed equally to this work.

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Author: Marinko Sremac - Elizabeth S Stuart

Source: https://link.springer.com/article/10.1186/1472-6750-10-54







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