Christopher P. Prior

Characterization of highly purified, inactivated HIV-1 particles isolated by anion exchange chromatography

This report characterizes inactivated, gp120 depleted, HIV-1 particles purified by an anion exchange chromatography production process. This antigen formulated with incomplete Freunds adjuvant constitutes Remune, which is being evaluated in a phase III clinical endpoint trial to determine the effect of this immune-based therapy on clinical progression of HIV-1 seropositive patients. Multiple production lots of the inactivated HIV-1 antigen strain HZ321, isolated by anion exchange chromatography, exhibit purity of > 95% by gel filtration. These findings are corroborated by thin section electron microscopy showing a homogenous field of intact particles. Analyses of the purified virus particles for protein, lipid, carbohydrate and RNA show structural retention of the envelope proteins, lipid bilayer and core components after large scale processing. The qualitative identification of at least 85% of total HIV-1 protein is determined by ELISA, Western blot, HPLC and amino acid sequencing analyses. Quantitative values are assigned to 50% of these proteins. The data confirm the presence of virally encoded proteins p6, p7, pI15, p17, p24, p32, pI39Gag, gp41, pp55Gag, p66/51, Vpr, Vif and Nef. Excellent consistency between production lots and equivalency to HIV-1 preparations purified by sucrose density gradient sedimentation has been established for protein and lipid composition, and overall purity. These findings further establish that non-viral encoded proteins and lipids are integral structural components of the intact virion and are not contaminants unique to a particular isolation method. The data confirm the presence of multicomponent antigens in the viral particles for stimulating a broad HIV-1 specific immune response. Finally, the work demonstrates that the two inactivation procedures (beta-propiolactone and gamma irradiation), which achieve efficient viral inactivation meeting US FDA guidelines, do not damage the protein antigens of the viral particles.

Production and functional characterization of a recombinant fragment of von Willebrand factor (vWF): an antagonist to platelet receptor GP Ib.

We expressed a recombinant peptide fragment (Ser445–Val733) of human von Wille-brand factor (vWF), containing the binding domain for the platelet receptor of GP Ib, in E. coli. This 33 kD peptide blocks binding of the intact vWF molecule to GP Ib in the presence of modulators. Thus, it offers potential as an antithrombotic agent. High level expression was achieved in a plasmid construct driven by the bacteriophage T7 promoter. The peptide was solubilized from inclusion bodies in strong chaotrope, then reduced and alkylated. Following purification, formulation at pH 3.5, and lyophilization, the reconstituted experimental product (RG 12986) exists as an equilibrium of monomer and dimer species. When formulated above pH 5.0, soluble aggregates are formed; these solutions have less bioactivity than RG 12986. Interestingly, the non-aggregated state of RG 12986 remains conserved following dilution and incubation with platelet-poor plasma. The overall purification/low pH formulation strategies may be applicable to other E. coli recombinant proteins having a tendency to aggregate following removal of chaotrope near physiologic pH when in a concentrated format.