Inhibition of HIV protease by monoclonal antibodies.

The protease of HIV plays a critical role in the maturation of the infectious particles of the virus. The enzyme has therefore been extensively studied with the objective of developing therapeutics that inhibit viral proliferation. We have produced monoclonal antibodies specific for the HIV-1 protease, and selected those that inhibit enzyme function for use as probes to study the enzyme's activity and as an eventual aid for the development of potential inhibitors targeted to regions other than the active site. We have characterized two such mAbs, F11.2.32 and 1696, which have inhibition constants in the low nanomolar range and which recognize epitopes from different regions of the protease. The crystal structures of the two antibodies, both in the free state as well as complexes with peptide fragments corresponding to their respective epitopes, have been solved. The structural analyses, taken together with other functional data on the antibodies, suggest mechanisms of protease inhibition by these antibodies.

Structural and functional characterization of a monoclonal antibody specific for the preS1 region of hepatitis B virus.

The monoclonal antibody 5a19, raised against the ay serotype of hepatitis B virus, binds to the segment of the preS1 region comprising residues 37-43, which is implicated in attachment of the virus to hepatocytes. The dissociation constant, derived from kinetic studies using surface plasmon resonance techniques, is in the low nanomolar range. The nucleotide sequence of the variable domains has been determined and the corresponding germ-line genes have been identified. The three-dimensional structure of the Fab fragment has been determined by X-ray crystallography to 2.6 A resolution.

Structure of the Fab fragment from F124, a monoclonal antibody specific for hepatitis B surface antigen.

The crystal structure of the Fab fragment from the monoclonal anti-preS2 antibody F124 (IgG1,kappa) has been solved by molecular replacement and refined at 3.0 A resolution. The Fab crystallizes with two independent molecules in the asymmetric unit. F124 recognizes an epitope contained within the preS2 segment between residues 120 and 132 of the surface antigen of hepatitis B virus. The antibody shows a high affinity for the glycan N-linked to Asn123, but it also cross-reacts with the non-glycosylated peptide fragment 120-132. Although crystallization was performed in the presence of an eightfold excess of the cross-reactive peptide, no evidence for the ligand was found in the antigen-binding site, which is close to a neighbouring molecule in the crystal lattice. The antigen-binding site has a groove-like topology which is modulated with pocket-like cavities. It is characterized by a large number of tyrosine and aspartate residues. The importance of germ-line mutations at the binding site is discussed.

Crystal structure of an anti-carbohydrate antibody directed against Vibrio cholerae O1 in complex with antigen: molecular basis for serotype specificity.

The crystal structure of the murine Fab S-20-4 from a protective anti-cholera Ab specific for the lipopolysaccharide Ag of the Ogawa serotype has been determined in its unliganded form and in complex with synthetic fragments of the Ogawa O-specific polysaccharide (O-SP). The upstream terminal O-SP monosaccharide is shown to be the primary antigenic determinant. Additional perosamine residues protrude outwards from the Ab surface and contribute only marginally to the binding affinity and specificity. A complementary water-excluding hydrophobic interface and five Ab-Ag hydrogen bonds are crucial for carbohydrate recognition. The structure reported here explains the serotype specificity of anti-Ogawa Abs and provides a rational basis toward the development of a synthetic carbohydrate-based anti-cholera vaccine.

Lack of significant differences in association rates and affinities of antibodies from short-term and long-term responses to hen egg lysozyme.

The affinities (Ka) and association rate constants (kon) of 23 mouse (BALB/c) anti-lysozyme mAbs obtained after short and prolonged immunizations have been measured by plasmon resonance techniques. The affinities for the 23 Abs, measured using their Fab, range from Ka = 1.1 x 10(7) to 1.4 x 10(10) M-1. There is no significant correlation between time or dose of immunization and affinity or association rates, indicating no time- or dose-dependent maturation of the response within the doses and times that were explored. IgMs are produced early and late in the response, with intrinsic affinities <10(5) M-1. Two independently derived mAbs, D44.1 (short term) and F10.6.6 (from a longer term response), result from identical or nearly identical somatic recombination events of germline gene segments. F10.6.6 has more mutations and a higher affinity constant (Ka = 1.4 x 10(10) M-1) than D44.1 (Ka = 1.1 x 10(7) M-1). Although higher affinities may result from an accumulation of mutations, they do not correlate with the length and dose of immunogenic challenge.

Inhibition of the HIV-1 and HIV-2 proteases by a monoclonal antibody.

The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates. This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity.

Sequence analysis of a monoclonal antibody specific for the preS2 region of hepatitis B surface antigen, and the cloning, expression and characterisation of its single-chain Fv construction.

The nucleotide sequence of the monoclonal antibody F124, specific for the preS2 region of the surface antigen of hepatitis B virus, has been determined and an single-chain Fv fragment (scFv) recombinant construction has been cloned and expressed into the periplasmic region of Escherichia coli. The recombinant antibody fragment contains a (Gly4Ser)3 linker connecting the C-terminus of the heavy chain variable region (V(L)) domain to the N-terminus of the light chain variable region (V(L)) domain. A 23-residue peptide segment, containing a c-myc marker for immunochemical detection of the scFv and hexahistidine tag to facilitate its purification, was added C-terminal to the V(L) domain. The scFv mimics the antibody in binding to the native antigen in the form of recombinant hepatitis B surface antigen (HBsAg) particles as well as to peptide fragments carrying the viral epitope.

Three-dimensional structure of an Fab-peptide complex: structural basis of HIV-1 protease inhibition by a monoclonal antibody.

F11.2.32, a monoclonal antibody raised against HIV-1 protease (Kd = 5 nM), which inhibits proteolytic activity of the enzyme (K(inh) = 35(+/-3)nM), has been studied by crystallographic methods. The three-dimensional structure of the complex between the Fab fragment and a synthetic peptide, spanning residues 36 to 46 of the protease, has been determined at 2.2 A resolution, and that of the Fab in the free state has been determined at 2.6 A resolution. The refined model of the complex reveals ten well-ordered residues of the peptide (P36 to P45) bound in a hydrophobic cavity at the centre of the antigen-binding site. The peptide adopts a beta hairpin-like structure in which residues P38 to P42 form a type II beta-turn conformation. An intermolecular antiparallel beta-sheet is formed between the peptide and the CDR3-H loop of the antibody; additional polar interactions occur between main-chain atoms of the peptide and hydroxyl groups from tyrosine residues protruding from CDR1-L and CDR3-H. Three water molecules, located at the antigen-antibody interface, mediate polar interactions between the peptide and the most buried hypervariable loops, CDR3-L and CDR1-H. A comparison between the free and complexed Fab fragments shows that significant conformational changes occur in the long hypervariable regions, CDR1-L and CDR3-H, upon binding the peptide. The conformation of the bound peptide, which shows no overall structural similarity to the corresponding segment in HIV-1 protease, suggests that F11.2.32 might inhibit proteolysis by distorting the native structure of the enzyme.

Preliminary crystallographic studies of an anti-HIV-1 protease antibody that inhibits enzyme activity.

F11.2.32, a monoclonal antibody directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme. The antibody cross-reacts with peptides 36-46 and 36-57 from the protease. Crystals of the Fab have been obtained both in the free state and as complexes formed with the protease peptide fragments, 36-46 and 36-57. Diffraction data have been collected for the free and complexed forms of Fab F11.2.32 and preliminary models for the crystal structures were obtained by molecular replacement.

Crystallization, preliminary X-ray diffraction study, and crystal packing of a complex between anti-hen lysozyme antibody F9.13.7 and guinea-fowl lysozyme.

The complex formed between the Fab fragment of a murine monoclonal antihen egg lysozyme antibody F9.13.7 and the heterologous antigen Guinea-fowl egg lysozyme has been crystallized by the hanging drop technique. The crystals, which diffract X-rays to 3 A resolution, belong to the monoclinic space group P2(1), with a = 83.7 A, b = 195.5 A, c = 50.2 A, beta = 108.5 degrees and have two molecules of the complex in the asymmetric unit. The three-dimensional structure has been determined from a preliminary data set to 4 A using molecular replacement techniques. The lysozyme-Fab complexes are arranged with their long molecular axes approximately parallel to the crystallographic unique axis. Fab F9.13.7 binds an antigenic determinant that partially overlaps the epitope recognized by antilysozyme antibody HyHEL10.

Three-dimensional structure of an idiotope-anti-idiotope complex.

Serologically detected antigenic determinants unique to an antibody or group of antibodies are called idiotopes. The sum of idiotopes of an antibody constitute its idiotype. Idiotypes have been intensively studied following a hypothesis for the self-regulation of the immune system through a network of idiotype-anti-idiotype interactions. Furthermore, as antigen and anti-idiotypes can competitively bind to idiotype-positive, antigen-specific antibodies, anti-idiotypes may carry an 'internal image' of the external antigen. Here we describe the structure of the complex between the monoclonal anti-lysozyme FabD1.3 and the anti-idiotopic FabE225 at 2.5 A resolution. This complex defines a private idiotope consisting of 13 amino-acid residues, mainly from the complementarity-determining regions of D1.3. Seven of these residues make contacts with the antigen, indicating a significant overlap between idiotope and antigen-combining site. Idiotopic mimicry of the external antigen is not achieved at the molecular level in this example.

Three-dimensional structure and antigen binding specificity of antibodies.

A number of specific Fab and Fv fragments and their complexes with antigens (avian lysozymes), haptens, and anti-idiotopic Fabs have been studied by immunochemical and crystallographic techniques. Antigen and antibody interact through closely complementary contacting surfaces, without major conformational changes. An idiotopic determinant of a monoclonal antibody is shown to include parts of most of its complementarity determining regions. The specificity of antigen recognition resides in the close complementarity of the antigenic determinant with the antibody combining site.

Nucleotide sequence of the VH, VL regions of an anti-idiotopic antibody reacting with a private idiotope of the anti-lysozyme D1.3 antibody.

Antibody E225 reacts with a private idiotope of the anti-lysozyme antibody D1.3. A complex between the Fab fragments from these BALB/c monoclonal antibodies has been crystallized and the determination of the three-dimensional structure of this idiotope-anti-idiotope complex is under way. The nucleotide VH and VL sequences of E225 presented here have been determined to provide the amino acid sequence information necessary for the interpretation of the high resolution electron density maps of the complex, obtained by X-ray crystallography. The cDNAs synthesized from the Vkappa and VH mRNAs were cloned in E. coli. Both cDNA strands were sequenced by the dideoxy termination method. The translated amino acid sequence shows that Vkappa, VH correspond to groups five (V) and II(b) of mouse immunoglobulin light and heavy chains, respectively. Sequence alignments between the complementarity determining regions of E225 and the antigenic determinant of lysozyme recognized by D1.3 do not indicate whether or not the anti-idiotopic antibody structurally mimics the external antigen.

Studies of structure and specificity of some antigen-antibody complexes.

By using X-ray diffraction and immunochemical techniques, we have exploited the use of monoclonal antibodies raised against hen egg lysozyme (HEL) to study systematically those factors responsible for the high specificity of antigen-antibody interactions. HEL was chosen for our investigations because its three-dimensional structure and immunochemistry have been well characterized and because naturally occurring sequence variants from different avian species are readily available to test the fine specificity of the antibodies. The X-ray crystal structure of a complex formed between HEL and the Fab D1.3 shows a large complementary surface with close interatomic contacts between antigen and antibody. Thus single amino acid sequence changes in heterologous antigens give antigen-antibody association constants that are several orders of magnitude smaller than that of the homologous antigen. For example, a substitution of His for Glu at position 121 in the antigen is sufficient to diminish significantly the binding between D1.3 and the variant lysozyme. The conformation of HEL when complexed to D1.3 shows no significant difference from that seen in the free molecule, and immunobinding studies with other anti-HEL antibodies suggest that this observation may be generally true for the system of monoclonal antibodies that we have studied.

Crystallization and preliminary x-ray diffraction studies of two new antigen-antibody (lysozyme-Fab) complexes.

The complexes between the Fab fragments of two monoclonal anti-lysozyme antibodies, Fab10.6.6 (high affinity) and D44.2 (lower affinity), and their specific antigen, hen egg-white lysozyme, have been crystallized. The antibodies recognize an antigenic determinant including Arg68, but differ significantly in their association constants for the antigen. Two crystalline forms were obtained for the complex with FabF10.6.6, the higher affinity antibody. One of them is monoclinic, space group P21, with unit cell dimensions a = 145.6 A, b = 78.1 A, c = 63.1 A, beta = 89.05 degrees, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 3 A making this form suitable for high-resolution X-ray diffraction studies. The second form crystallizes in the triclinic space group P1, with unit cell dimensions a = 134.0 A, b = 144.7 A, c = 98.6 A, alpha = 90.30 degrees, beta = 97.1 degrees, gamma = 90.20 degrees, consistent with the presence of 10 to 12 molecules of the complex in the unit cell. These crystals do not diffract X-rays beyond 5 A resolution. The antigen-antibody complex between FabD44.2, the lower affinity antibody, and hen egg-white lysozyme crystallizes in space group P2(1)2(1)2(1), with unit cell dimensions a = 99.7 A, b = 167.3 A, c = 84.7 A, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 2.5 A resolution.

Crystallization and preliminary X-ray diffraction studies of antigen--antibody complexes.

Monoclonal antibodies of predefined specificity have been purified and crystallized as single components or complexed with their specific antigens. The intersegmental flexibility of antibody molecules has imposed the strategy of attempting to crystallize their Fab fragments separately. Intrasegmental mobility in Fabs has rarely been an obstacle to their crystallization. The immune system, however, provides a large functional and structural diversity of antibody molecules suitable for crystallization and X-ray diffraction studies.

Antigenic mapping of the surface proteins of infectious hepatitis B virus particles.

To identify further the surface proteins of the native virus, hepatitis B virus (HBV) particles purified from HBe antigen (Ag)-positive human sera were used as immunogens to produce murine monoclonal antibody (MAb)-secreting hybridomas. The specific binding of antibodies to the HBV envelope (env) proteins was determined in indirect radioimmunoassay and by Western blot analysis. Six MAbs directed against major hepatitis B surface antigen (HBsAg) recognized conformational epitopes on S proteins (P24s/GP27s). Three preS2-specific MAbs reacted with the middle env proteins (GP33s/GP36s) in the 22 nm HBsAg spherical particles. One MAb, F222, was found to react specifically with the two very large (VL) HBV surface proteins with Mr 54K and 66K. The epitope recognized by F222 was located on the protruding N terminus which, in the assembled virus particles, was readily split off by trypsin or V8 protease treatment. The presence of these VL proteins appeared to correspond to the presence of the large env proteins (P39s/GP42s). The data described here indicate that F222 probably recognized an assembled topographic site which could be involved in virus entry into hepatocytes. Moreover, our results suggest that the preS-coded part of the HBV env proteins, which is sensitive to proteases in vitro, could be unstable in vivo and stabilized by immunoglobulins.