Structural characteristics correlate with immune responses induced by HIV envelope glycoprotein vaccines

AbstractHIV envelope glycoprotein (Env) is the target for inducing neutralizing antibodies. Env is present on the virus surface as a trimer, and, upon binding to CD4, a cascade of events leads to structural rearrangement exposing the co-receptor binding site and entry into the CD4+ host target cells. We have designed monomeric and trimeric Env constructs with and without deletion of the variable loop 2 (ΔV2) from SF162, a subtype B primary isolate, and performed biophysical, biochemical and immunological studies to establish a potential structure–functional relationship. We expressed these Envs in CHO cells, purified the proteins to homogeneity and performed biophysical studies to define the binding properties to CD4, structural characteristics and exposure of epitopes recognized by b12 and CD4i mAb (17B) on both full-length and mutant HIV Env proteins. Parameters evaluated include oligomerization state, number and affinity of CD4 binding sites, enthalpy and entropy of the Env–CD4 interaction and affinity for b12 and 17b mAbs. We observed one CD4 binding site per monomer and three active CD4 binding sites per trimer. A 40-fold difference in affinity of the gp120 monomer vs. the o-gp140 trimer towards CD4 was observed (Kd = 58 nM and 1.5 nM, respectively), whereas only a 2-fold difference was observed for the V2 deleted Envs (Kd of gp120ΔV2 = 19 nM, Kd of o-gp140DV2 = 9.3 nM). Monomers had 3-fold higher affinity to the mAb 17b and at least 3-fold weaker affinity to b12 compared to trimers, with gp120DV2 having the weakest affinity for b12 (Kd = 446 nM). Affinity of CD4 binding correlated with proportion of the antibodies induced against the conformational epitopes by the corresponding Envs, and changes in mAb binding correlated with the induction of antibodies directed against linear epitopes. Furthermore, biophysical analysis reveals that the V2 deletion has broad structural implications in the monomer not shared by the trimer, and these changes are reflected in the quality of the immune responses induced in rabbits. These data suggest that biophysical characteristics of HIV Env, such as affinity for CD4, and exposure of important neutralizing epitopes, such as those recognized by b12 mAb, may be important predictors of its in vivo efficacy and may serve as important surrogate markers for screening Env structures as potential vaccine candidates

Purification And Properties Of Methionine Synthase From Escherichia Coli B (cobalamine, Nitrous Oxide).

Cobalamin-dependent methionine synthase (5-methyltetrahydrofolate:homocysteine methyltransferase, E. C. 2.1.1.13) was purified to homogeneity from Escherichia coli B. Subunit and apparent molecular weights of purified enzyme were respectively 133,300 and 179,000, indicating the enzyme consisted of one polypeptide chain. The oxidation state of cobalamin in isolated enzyme was found to be cob(II)alamin, determined by EPR and visible absorbance spectra. Copper and iron were also associated with purified enzyme. Methionine synthase was irreversibly inhibited during in vitro turnover in nitrous oxide equilibrated buffer; enzyme-bound cob(I)alamin, transiently formed during turnover, reacted with nitrous oxide to generate inactive cob(II)alamin enzyme. There was spectrophotometric evidence for the partial loss of enzyme-bound cobalamin during inactivation, possibly caused by the homolytic cleavage of nitrous oxide to N(,2) and hydroxyl radical and the subsequent addition of the radical to the corrin ring. Reaction of hydroxyl radical with active center residues might also account for irreversible activity loss. The prosthetic group of inactive cob(II)alamin enzyme was methylated by S-adenosylmethionine in the presence of NADPH and two small flavoproteins from E. coli, supporting the proposed priming mechanism of inactive enzyme to the active methylcobalamin form. The EPR spectrum of cob(II)alamin enzyme was a combined spectrum of base-off cob(II)alamin and superoxo-cob(III)alamin; methylation of the cob(II)alamin resulted in loss of superoxo-cob(III)alamin signal and an overall loss of spin. The stereochemistry of the methyl transfer catalyzed by methionine synthase was determined. Transfer of the methyl group from chiral 5-methyltetrahydrofolate to homocysteine resulted in net retention of configuration of the methyl group, accompanied by about 50% racemization.Ph.D.BiochemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127907/2/8621281.pd

Biophysical characterization of antibodies with isothermal titration calorimetry

Antibodies play a key role in the immune response. Since antibodies bind antigens with high specificity and tight affinity, antibodies are an important reagent in experimental biology, assay development, biomedical research and diagnostics. Monoclonal antibodies are therapeutic drugs and used for vaccine development. Antibody engineering, biophysical characterization, and structural data have provided a deeper understanding of how antibodies function, and how to make better drugs. Isothermal titration calorimetry (ITC) is a label-free binding assay, which measures affinity, stoichiometry, and binding thermodynamics for biomolecular interactions. When thermodynamic data are used together with structural and kinetic data from other assays, a complete structure-activity-thermodynamics profile can be constructed. This review article describes ITC, and discusses several applications on how data from ITC provides insights into how antibodies function, guide antibody engineering, and aid design of new therapeutic drugs