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1.
J Immunol Methods ; 360(1-2): 1-9, 2010 Aug 31.
Article in English | MEDLINE | ID: mdl-20542039

ABSTRACT

The objective of this study was to produce and evaluate the immunogenic potential of a recombinant HLA-class I antigen linked to dextran. The HLA-A*0201 heavy chain and beta2 microglobulin were cloned by PCR amplification of overlapping oligonucleotides and produced in E. coli. These were assembled with a CMV binding peptide motif, the HLA complex was biotinylated and bound by streptavidin coated dextran at a ratio of 24 HLA to 1 dextran molecule (termed Dextramer). Allostimulation of human PBMC in vitro and in vivo immunization of Balb c mice with the HLA-A*0201 construct elicited CD4+ and CD8+ T cell proliferative responses, IgG specific antibodies in mice and in human T cell proliferation and APOBEC3G mRNA. These adaptive and innate immune responses induced by a novel recombinant HLA construct in human cells and mice suggest their application as a potential vaccine candidate against HIV infection.


Subject(s)
AIDS Vaccines , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Dextrans/pharmacology , HLA-A Antigens/metabolism , Recombinant Proteins/pharmacology , Adaptive Immunity/drug effects , Animals , Antibody Formation/drug effects , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cell Proliferation/drug effects , Cells, Cultured , Cloning, Molecular , Dextrans/genetics , Dextrans/metabolism , HLA-A Antigens/genetics , HLA-A Antigens/pharmacology , HLA-A2 Antigen , Humans , Immunity, Innate/drug effects , Immunization , Immunoglobulin G/blood , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
2.
Vaccine ; 22(9-10): 1188-98, 2004 Mar 12.
Article in English | MEDLINE | ID: mdl-15003647

ABSTRACT

Plasmodium falciparum malaria is a major cause of morbidity and mortality worldwide. To evaluate the efficacy of a possible vaccine antigen against P. falciparum infection, a fusion protein, derived from P. falciparum Glutamate-rich protein (GLURP) genetically coupled to P. falciparum Merozoite surface protein 3 (MSP3) was produced in Lactococcus lactis as a secreted recombinant GLURP-MSP3 fusion protein. The hybrid protein was purified to homogeneity by ion exchange and hydrophobic-interaction chromatography and its composition was verified by MALDI MS, SDS/PAGE and Western blotting with antibodies against antigenic components of GLURP and MSP3. Mice immunized with the hybrid protein produced higher levels of both GLURP- and MSP3-specific antibodies than mice immunized with either GLURP, MSP3 or a mix of both. The protective potential of the hybrid protein was also demonstrated by in vitro parasite-growth inhibition of mouse anti-GLURP-MSP3 IgG antibodies in a monocyte-dependent manner. These results indicate that the GLURP-MSP3 hybrid could be a valuable strategy for future P. falciparum vaccine development.


Subject(s)
Antibodies, Protozoan/biosynthesis , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Lactococcus lactis/metabolism , Plasmodium falciparum/immunology , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Animals , Antibodies, Protozoan/analysis , Antigens, Protozoan/chemistry , Chromatography, High Pressure Liquid , Enzyme-Linked Immunosorbent Assay , Female , Fermentation , Fluorescent Antibody Technique, Indirect , Immunoglobulin G/analysis , Immunoglobulin G/immunology , Lactococcus lactis/genetics , Mice , Mice, Inbred BALB C , Plasmids/genetics , Protozoan Proteins/chemistry , Recombinant Fusion Proteins/chemistry , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Vaccines, Synthetic/biosynthesis , Vaccines, Synthetic/immunology
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