ABSTRACT
The hepatitis B core antigen is a widely accepted carrier particle to enhance the immunogenicity of foreign epitopes. From electron cryomicroscopy, the immunodominant region between amino acid positions 79 to 81 is known to protrude from the surface of the shells. It can be replaced by heterologous sequences without interfering with the particle-forming capacity in many cases. Here we have introduced various V3 sequences of the envelope protein of different subtypes (A, B, O) of HIV-1/gp120 in order to enhance their immunogenicity and broaden the immune response against the virus. To improve purification efficiency and solubility of the E. coli-expressed hybrids, six histidine residues were fused to amino acid 156. An adjustable purification scheme was utilised including denaturation, Ni(2+)-NTA affinity chromatography and particle renaturation under high salt conditions, resulting in highly pure antigen preparations. The hybrids reacted specifically with sera of HIV-1-infected patients. They further induced an autologous, subtype-specific anti-HIV-1 antibody response superior to that of Keyhole limpet-haemocyanine-coupled peptides.
Subject(s)
HIV Envelope Protein gp120/immunology , HIV-1/classification , Hepatitis B Core Antigens/immunology , Immunodominant Epitopes/immunology , Amino Acid Sequence , Animals , Antibodies, Viral/biosynthesis , Escherichia coli , Gene Expression , HIV Envelope Protein gp120/biosynthesis , HIV Envelope Protein gp120/genetics , HIV Infections/immunology , HIV-1/immunology , Histidine , Humans , Mice , Mice, Inbred BALB C , Microscopy, Electron , Molecular Sequence Data , Peptide Fragments/biosynthesis , Peptide Fragments/genetics , Structure-Activity RelationshipABSTRACT
Experiences in the analytical application of the 2f-wavelength modulation technique for isotope selective atomic absorption spectroscopy in a graphite furnace are reported. Experimental as well as calculated results are presented, mainly for the natural lithium isotopes. Sensitivity, linearity, and (isotope) selectivity are studied by intensity modulation and wavelength modulation. High selectivities can be attained, however, on the cost of detection power. It is shown that the method enables the measurement of lithium isotope ratios larger than 2000 by absorption in a low-pressure graphite tube atomizer.
ABSTRACT
Hepatitis B virus is a major cause of human liver disease. In the case of chronic infection the virus can lead to liver cancer and cirrhosis. The virion consists of an outer envelope containing lipids of the endoplasmic reticulum and virally-encoded surface proteins. This lipoprotein shell encloses the nucleocapsid or core antigen (HBcAg), which contains the viral genome. The capsid consists of dimers of a 183-residue protein, which can be divided into an assembly (residues 1-149) and a protamin-like domain (residues 150-183), responsible for polymerization into particles and RNA packaging, respectively. Upon expression of the core gene in bacteria the products are assembled into capsids resembling those of wild type particles. A purification protocol was developed for unpolymerised (dimeric) and polymerized HBcAg by fusion of six histidine residues to a C-terminal deletion mutant of the core protein allowing the isolation of the respective antigens after denaturing Ni2+-chelate affinity chromatography and renaturing dialysis. The possible incorporation of E. coli proteins during the assembly process and the inclusion of nucleic acids can be avoided. The method might be an attractive alternative to common purification protocols of hybrid virus-like particles (VLPs) for vaccine use.
Subject(s)
Chromatography, Affinity/methods , Hepatitis B Core Antigens/isolation & purification , Cloning, Molecular , Dimerization , Escherichia coli , Hepatitis B Core Antigens/genetics , Hepatitis B Core Antigens/ultrastructure , Histidine/analysis , Histidine/chemistry , Humans , Hydrogen-Ion Concentration , Nickel , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
Recombinant peptides from Escherichia coli encoding the principal neutralizing domain (PND) and surrounding sequences of gp120 of human immunodeficiency virus type 1 (HIV-1) with a C-terminal polyhistidine tag were expressed and purified on Ni(2+)-nitrilotriacetate agarose. High yields of more than 99% pure protein were obtained. Their serological reactivity with anti-HIV-positive and -negative human sera was compared to chemically synthesized V3 loop peptides. Overall the genetic PND peptides of the HIV-1MN isolate showed higher and broader reactivity patterns (84%) with HIV-positive sera from German patients than the chemically synthesized peptides (74%). By their higher reactivity and easy way of production and purification, recombinant peptides seem to be highly preferable for the determination of antibody titers to the PND of HIV-infected patients.
