Small-Angle Neutron Scattering Characterization of Monoclonal Antibody Conformations and Interactions at High Concentrations

Small-angle neutron scattering (SANS) is used to probe the solution structure of two protein therapeutics (monoclonal antibodies 1 and 2 (MAb1 and MAb2)) and their protein-protein interaction (PPI) at high concentrations. These MAbs differ by small sequence alterations in the complementarity-determining region but show very large differences in solution viscosity. The analyses of SANS patterns as a function of different solution conditions suggest that the average intramolecular structure of both MAbs in solution is not significantly altered over the studied protein concentrations and experimental conditions. Even though a strong repulsive interaction is expected for both MAbs due to their net charges and low solvent ionic strength, analysis of the SANS data shows that the effective PPI for MAb1 is dominated by a very strong attraction at small volume fraction that becomes negligible at large concentrations. The MAb1 PPI cannot be modeled simply by a spherically symmetric central forces model. It is proposed that an anisotropic attraction strongly affects the local interprotein structure and leads to an anomalously large viscosity of concentrated MAb1 solutions. Conversely, MAb2 displays a repulsive interaction potential throughout the concentration series probed and a comparatively small solution viscosity.     

Production and characterization of monoclonal antibodies to the hemocytes of mud crab, Scylla serrata

Monoclonal antibodies (MAbs) to hemocytes of mud crab, Scylla serrata, were produced by immunizing mice with formalin-fixed hemocytes. Of the six MAbs produced, two (MAb 1D11 and MAb 1A2) reacted specifically with hemocyte proteins in western blot. MAb 1A2 showed strong immunofluorescent reaction with granular hemocytes and a weak reaction with semigranular cells. However, hyaline cells did not show any reaction. The MAbs also showed strong cross-reactivity with the hemocytes of different crab species but not with other crustaceans. The MAbs produced can be used as a marker for granular hemocytes of crabs.     

On the long-range attraction between proteins due to nonadsorbing polysaccharide

The effect of a nonadsorbing polysaccharide (dextran) on the structure factor of a solution of lysozyme was studied using small-angle neutron scattering (SANS) experiments. By choosing the appropriate water/deuterium ratio as solvent, we made the scattering signal from dextran invisible for the SANS measurements. Dextran induces a weak long-range attraction between the lysozyme molecules. This attraction is described using a depletion interaction potential from theory for two spheres in an ideal polymer solution. Incorporation of the theory in a mean-spherical approximation shows that the wave vector below which the structure factor increases depends on the polymer size. The theoretical prediction is in fair agreement with the measured structure factor of lysozyme, as affected by nonadsorbing dextran.     

Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.     

Time-resolved fluoroimmunoassay of potato virus M with monoclonal antibodies

Mouse monoclonal antibodies (MAbs) specific for potato virus M (PVM) were prepared and the properties of three of them were studied. MAb M4C1 is IgG2b, it binds with high affinity to PVM coat protein, to purified virus preparations and recognises PVM in infected potato leaves and tubers. MAb M6D5 is IgG2a and also reacts with PVM coat protein, purified PVM and with PVM in potato leaf and tuber extracts. In double-antibody sandwich ELISA (DAS ELISA) MAbs M4C1 and M6D5 reacted with all 17 PVM isolates tested. MAb M7 is IgG2b and recognises PVM only in indirect dot ELISA on nitrocellulose filters and viral coat protein on Western blots. MAbs against PVM were used as capture antibodies and europium-labelled MAbs as conjugates in time-resolved fluoroimmunoassay (EuTRFIA). The standard EuTRFIA curve of PVM detection is approximately linear over a range of PVM concentrations from 0.5 ng/ml to 1000 ng/ml. The lowest PVM concentration detectable in EuTRFIA was 0.5 ng/ml and correspondingly 6 ng/ml in DAS ELISA. The use of the europium chelate label allows PVM detection in potato leaf and tuber sap at dilutions greater than 10^--⁴ with very low background fluorescence. EuTRFIA with MAbs, with either one or two incubations is about 10–20 times more sensitive for PVM detection than is DAS ELISA. PVM and PVX, mixed with healthy potato tuber sap, were simultaneously tested in a single sample at concentrations lower than 10 ng/ml by double-label TRFIA using europium-labelled MAbs to PVM and samarium-labelled MAbs to PVX.     

Additive effects characterize the interaction of antibodies involved in neutralization of the primary dualtropic human immunodeficiency virus type 1 isolate 89.6

Human immunodeficiency virus-type I (HIV-1) infection elicits antibodies (Abs) directed against several regions of the gp120 and gp41 envelope glycoproteins. Many of these Abs are able to neutralize T-cell-line-adapted strains (TCLA) of HIV-1, but only a few effectively neutralize primary HIV-1 isolates. The nature of HIV-1 neutralization has been carefully studied using human monoclonal Abs (MAbs), and the ability of such MAbs to act in synergy to neutralize HIV-1 has also been extensively studied. However, most synergy studies have been conducted using TCLA strains. To determine the nature of Ab interaction in HIV-1 primary isolate neutralization, a panel of 12 anti-HIV-1 human immunoglobulin G (IgG) MAbs, specific for epitopes in gp120 and gp41, were used. Initial tests showed that six of these MAbs, as well as sCD4, used individually, were able to neutralize the dualtropic primary isolate HIV-1(89.6); MAbs giving significant neutralization at 2 to 10 microg/ml included 2F5 (anti-gp41), 50-69 (anti-gp41), IgG1b12 (anti-gp120(CD4bd)), 447-52D (anti-gp120(V3)), 2G12 (anti-gp120), and 670-D (anti-gp120(C5)). For studies of reagent interaction, 16 binary combinations of reagents were tested for their ability to neutralize HIV-1(89.6). Reagent combinations tested included one neutralizing MAb with sCD4, six pairs consisting of two neutralizing MAbs, and nine pairs consisting of one neutralizing MAb with another non-neutralizing MAb. To assess the interaction of the latter type of combination, a new mathematical treatment of reagent interaction was developed since previously used methods could be used only when both reagents neutralize. Synergy was noted between sCD4 and a neutralizing anti-gp120(V3) MAb. Antagonism was noted between two pairs of anti-gp41 MAbs (one neutralizing and one non-neutralizing). All of the other 13 pairs of MAbs tested displayed only additive effects. These studies suggest that Abs rarely act in synergy to neutralize primary isolate HIV-1(89.6); many anti-HIV-1 Abs act additively to mediate this biological function.     

Tumor localization and radioimaging with mixtures of radioiodinated monoclonal antibodies directed to different colon cancer associated antigens

After demonstrating enhanced tumor cell binding with a mixture of monoclonal antibodies (MAbs) in vitro, biodistribution and immunoscintigraphy studies with 3 radioiodinated anti-colon cancer MAbs and a non-specific control MAb (MOPC) were conducted in a human colon cancer (GW-39)-hamster model system. Each of the specific MAbs, but not MOPC, demonstrated extensive tumor binding and in scintigrams affected visualization of all large tumors (>0.85 g) over background. Using single MAbs, few small tumors (0.19–0.50 g) were defined above background (0–29%). However, with combinations of these specific MAbs small tumors were more frequently defined in scintigrams (43–67%). Radioimages using higher doses of MAbs and small, younger tumors more clearly demonstrated the superiority of a MAb mixture. These results confirmed that combinations of MAbs to different antigens can detect smaller tumors with better tumor localization when compared to component MAbs used singly. This study supports the concept that tumor targeting and detection may be enhanced with appropriate mixtures of MAbs.     

Generation of Neutralizing Human Monoclonal Antibodies against Parvovirus B19 Proteins

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 μg/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 μg/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.     

Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin

We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.     

Enhancement and Neutralization of Feline Infectious Peritonitis Virus Infection in Feline Macrophages by Neutralizing Monoclonal Antibodies Recognizing Different Epitopes

The interaction between the enhancing and neutralizing activities of three monoclonal antibodies (MAbs) (5-6-2, 6-4-2 and 7-4-1) to the spike protein of feline infectious peritonitis virus (FIPV) strain 79-1146 was determined using feline macrophages. At a high MAb concentration, all of the three MAbs completely inhibited the FIPV infection at 37 C. However, two of them (6-4-2 and 7-4-1) enhanced FIPV infection when either the MAb concentration or reaction temperature was lowered. These MAbs also exerted an immediate infectivity-enhancing activity for up to 10 min of reaction and by 20 min, neutralizing activities were observed. Only MAb 5-6-2 consistently showed neutralizing activity regardless of the reaction conditions. Competition with sera from cats experimentally infected with FIPV strain 79-1146 or feline enteric coronavirus strain 79-1683 showed that the two epitopes recognized by MAb 5-6-2 and MAb 6-4-2, respectively, are also recognized by the natural host.     

A conserved coronavirus epitope, critical in virus neutralization, mimicked by internal-image monoclonal anti-idiotypic antibodies.

Monoclonal antibody (MAb) 6A.C3 neutralizes transmissible gastroenteritis coronavirus (TGEV) and is specific for a conserved epitope within subsite Ac of the spike (S) glycoprotein of TGEV. Six hybridomas secreting anti-idiotypic (Ab2) MAbs specific for MAb 6A.C3 (Ab1) have been selected. All six MAbs inhibited the binding of Ab1 to TGEV and specifically cross-linked MAb1-6A.C3. Four of these hybridomas secreted gamma-type anti-idiotypic MAbs. The other two Ab2s (MAbs 9A.G3 and 9C.E11) were recognized by TGEV-specific antiserum induced in two species. This binding was inhibited by viruses of the TGEV group but not by serologically unrelated coronaviruses. These results indicate that MAb2-9A.G3 and MAb2-9C.E11 mimic an antigenic determinant present on the TGEV surface, and they were classified as beta-type ("internal-image") MAbs. TGEV-binding Ab3 antiserum was induced in 100% of mice immunized with the two beta-type MAb2s and in 25 to 50% of mice immunized with gamma-type MAb2. Both beta- and gamma-type Ab2s induced neutralizing Ab3 antibodies in mice that were mainly directed to antigenic subsite Ac of the S protein.     

An epitope of the Semliki Forest virus fusion protein exposed during virus-membrane fusion

Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic pH in the endocytic pathway. Fusion is mediated by the spike protein E1 subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion. We have characterized four monoclonal antibodies (MAbs) specific for the acid conformation of E1. These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptide. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map the binding site, we selected for virus mutants resistant to one of the MAbs, E1a-1. One virus isolate, SFV 4-2, showed reduced binding of three acid-specific MAbs including E1a-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 mutant was fully infectious, formed the E1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Decreased binding of MAb E1a-1 was observed under a wide range of assay conditions, strongly suggesting that the E1 G157R mutation directly affects the MAb binding site. These data thus localize an E1 region that is normally hidden in the neutral pH structure and becomes exposed as part of the reorganization of the spike protein to its fusion-active conformation.     

Synergistic neutralization of human immunodeficiency virus type 1 by a chimpanzee monoclonal antibody against the V2 domain of gp120 in combination with monoclonal antibodies against the V3 loop and the CD4-binding site.

Synergistic neutralization of human immunodeficiency virus type 1 (HIV-1) was observed in studies using a chimpanzee anti-V2 monoclonal antibody (MAb), C108G, in combination with anti-V3 loop and anti-CD4 binding-site (bs) MAbs of different epitope specificities. C108G paired with either of two anti-V3 loop MAbs or either of two anti-CD4 bs MAbs synergistically neutralized both the uncloned IIIB and clonal HXB2 strains of virus in H9 target cells. Synergism was quantitated by calculation of combination indices. Significant synergy with a given MAb pair was seen over a range of MAb ratios, with the optimal effect centering around the ratio at which the MAbs were equipotent for a given HIV-1 strain (on the basis of the 50% neutralization titer). In preliminary experiments with monocytotropic strains of HIV-1 in peripheral blood mononuclear cell targets, significant synergism was also observed between anti-V2-anti-V3 and anti-V2-anti-CD4 bs MAb pairs. Synergism by all MAb pairs tested was greater against heterogeneous isolates of HIV-1 (IIIB and Ba-L) than against clonal isolates (HXB2 and NLHXADA), suggesting that strain broadening may be a component of the synergism observed against the heterogeneous isolates. In addition, conformational changes in gp120 upon binding of one or both MAbs may result in increased affinity or exposure of the epitope of one or both MAbs. Finally, a three-MAb combination of C108G, an anti-V3 MAb, and an anti-CD4 bs MAb was more effective in neutralizing the HXB2 strain of HIV-1 than any of the three two-MAb combinations within this trio, as determined by the dose reduction indices of each MAb required to achieve a given level of neutralization. This is the first report of synergistic neutralization of HIV-1 by a three-MAb combination composed of MAbs directed against the three major neutralization epitope clusters in gp120. Implications for vaccine design and for immunoprophylaxis and immunotherapy with a combination of MAbs are discussed.     

Link between protein-solvent and weak protein-protein interactions gives insight into halophilic adaptation

Malate dehydrogenase (Hm MalDH) from the extreme halophile Haloarcula marismortui is a very acidic protein with extensive ion binding properties. It is a good model for the study of solvation-solubility relationships. We measured the small-angle neutron or X-ray scattering profiles of folded and stable Hm MalDH at various protein concentrations and derived the second virial coefficients A(2). In NaCl, CsCl, KF, KCl, and NaCH(3)CO(2), A(2) values are positive, indicating globally repulsive protein-protein interactions. Below 1 M MgCl(2) and MgSO(4) or above 2 M (NH(4))(2)SO(4), A(2) rapidly decreases. From structure factor modeling with DLVO (Derjaguin, Landau, Verwey, and Overbeek)-like potentials, an effective diameter of 80-82 A is found for the protein particle in solution, compatible with its structural dimensions; the effective charge of the particle is undefined because of the high salt concentration. The strong variations of the protein-protein interaction are correlated to an attractive potential whose depth evolves with the salinity but in an opposite way in Mg salts and (NH(4))(2)SO(4). A repulsive Donnan term, corresponding to counterion dissociation, and an attractive term related to previously measured preferential salt binding parameters are discussed from well-established thermodynamics considerations and qualitatively account for the behavior of the protein-protein interactions in the various solutions. Because a solvation shell with a composition different from bulk induces protein-protein attraction, molecular adaptation to high salt would be directed to allow protein-salt interactions in order to avoid water or salt enrichment at the surface of the protein and thus preserve its solubility.     

Characterisation of monoclonal antibodies to haemocyte types of scallop (Chlamys farreri)

Four monoclonal antibodies (MAbs) (1E7, 1F12, 2H5, 2C6) against haemocytes of scallop (Chlamys farreri) were produced by immunising Balb/C mice. Analysed by the indirect immunofluorescence assay test (IIFAT), immunocytochemical assay, flow cytometry (FCM) and Western-blotting, they showed specificity for more than one haemocyte type (hyalinocyte and granulocyte) and various haemocyte components of scallop. Using IIFAT to detect monolayers separated from gradient density centrifugation, the four MAbs were positive with haemocytes at different interfaces. The percentage of positive cells (percent reactivity, PR) that MAb 1E7 reacted with at the 20-30%, 30-40% and 40-50% interfaces were 43.50%, 41.25% and 60.00% respectively, PR of MAb 1F12 were 31.00%, 63.50% and 41.00%, MAb 2C6 were 11.00%, 51.00%, 77.00%, and MAb 2H5 were 20.25%, 34.75%, 38.25%. For the immunocytochemical assay, MAb 1E7 1F12 and 2H5 was positive with the cytoplasm of both hyalinocyte and granulocyte, 2C6 was positive with the membrane and cytoplasm of hyalinocyte and granulocyte. Analysed by FCM, the PR of the four MAbs (1E7, 1F12, 2H5, 2C6) with haemocytes were 54.23%, 38.56%, 56.4%, and 79.7% respectively; moreover, the PR with different haemocyte types was variable. The results of Western-blotting showed that MAb 1E7 recognised an antigen of molecular weight 200 kDa, MAb 2C6 an antigen of 60 kDa, however, MAb 1F12 reacted with antigens of 70 kDa, 60 kDa and 45 kDa. There was no protein band that MAb 2H5 detected. In conclusion, 2C6 seems to be a very promising MAb to identify and differentiate granulocytes, and the four MAbs will be used in further studies on cellular defence mechanism research.     

Viscosity study of interactions between sodium alginate and CTAB in dilute solutions at different pH values

Interactions between anionic polyelectrolyte sodium alginate and the cationic surfactant cetytrimethylammonium bromide (CTAB) have been investigated by viscosity measurement techniques. The polymer–surfactant interactions are observed between alginate and CTAB at different pH in dilute solution. The results show that the rheological response of alginate dilute solutions is sensitive to a change of pH in the low pH range. The steady shear and intrinsic viscosity measurements reveal that the strong association between alginate and CTAB by electrostatic attraction above pH 5.0. However, as the pH value of solution decrease from 5.0 to 3.0, the strong association between alginate and CTAB is affected by not only electrostatic attraction but also hydrophobic interaction.     

Modulation of human growth hormone binding to somatogenic and lactogenic receptors by monoclonal antibodies to human growth hormone.

The relationship between the structure of human growth hormone (hGH) and the hormone-receptor interaction was investigated by studying the effects of specific monoclonal antibodies (MAbs) to hGH on the binding of [125I]hGH to rabbit liver and mouse liver microsomes. Receptor binding assays were carried out using a constant dose (1 ng) of [125I]hGH and varying concentrations of MAbs. The assay was carried out in the presence of either excess ovine prolactin for the measurement of somatogenic (SOM) binding sites, or excess bovine growth hormone for the determination of lactogenic (LAC) binding sites. Anti-hGH MAbs were found to have a whole spectrum of effects on hGH binding, including inhibitory, non-effect and enhancing activities. Enhancement of the binding of [125I]hGH to both SOM and LAC receptors was observed in liver membranes of rabbit or mouse. The observed amplified signal of [125I]hGH binding to various receptors in the presence of MAb no. 8 may be due to conformational changes which occur following MAb binding to hGH. On the other hand, most of the other MAbs caused inhibition of [125I]hGH binding. A negative correlation exists between the cross-reaction of various MAbs with the N-terminus truncated forms of hGH (Met14-hGH or Met8Leu-hGH) and their respective KD/IC50 values enabled the evaluation of the crucial role of the N-terminus region in hGH binding to both LAC and SOM receptors. MAb nos 1 and 19, which are directed towards acid residues 95-134 and the C-terminus, inhibited SOM binding more potently than LAC binding. Thus, it seems that these mid-molecule and C-terminus regions are also important in hGH binding, and that they play a role in the partial overlap of SOM and LAC binding.     

Exposed epitopes on a Trypanosoma equiperdum variant surface glycoprotein altered by point mutations.

African trypanosomes are covered by a dense protein layer that is immunologically distinct on different trypanosome isolates and is termed the variant surface glycoprotein (VSG). The different VSGs are expressed in a general order, where some VSGs appear preferentially early in infection and others only later. The exposed epitopes on a late antigen, VSG 78, of T.equiperdum were studied by the technique of monoclonal antibody (MAb) escape selection. MAbs that neutralize trypanosomes bearing VSG 78 reacted with the VSG only when it was attached to the trypanosome surface, suggesting that the most immunogenic surface epitopes are conformational. Trypanosome clones resistant to one of the MAbs yet still expressing VSG 78 or 78(20) were isolated in vitro. Two independent variants resistant to MAb H3 changed Ser192 to Arg by a single base change in the VSG gene and a variant resistant to MAb H21 had a single base change that converted Gln172 to Glu. A variant resistant to MAb H7 had several changes in the VSG gene, a gene conversion in the 5' region and an isolated mutation in codon 220 that is proposed to be responsible for the resistance phenotype. The isotypic bias of the MAbs against VSG 78 and an analysis of the natural variants that are resistant to MAb 78H21 suggest that glycosylation plays a role in the immunogenicity of these proteins. The analysis defines some of the exposed amino acid residues and demonstrates that VSG genes are altered by mutations and small gene conversions as well as replaced by large gene conversion-like events. The results provide biological data supporting the model of VSG structure obtained by crystallographic studies.     

A monoclonal antibody to the CDR-3 region of CD4 inhibits soluble CD4 binding to virions of human immunodeficiency virus type 1.

The CDR-3 region of CD4 has been proposed to be involved in the fusion reaction between human immunodeficiency virus type 1 (HIV-1) and CD4+ cells, either at a stage involving virus binding or subsequent to virus binding. Part of the evidence for this has been the observation that monoclonal antibodies (MAbs) to CDR-3 block HIV infection potently without strongly inhibiting the binding of monomeric gp120 to CD4. Here I show that, in a system using oligomeric, virion-bound gp120, a MAb to CDR-3 resembles those to CDR-2 in that it inhibits soluble CD4 binding to virions. Consequently, ternary complexes of MAb-soluble CD4-gp120 cannot be detected with CDR-2 MAbs and are detectable only at a very low level with a CDR-3 MAb, but they clearly form when a control MAb to CD4 domain 4 is used. Although not in direct conflict with previously published data on the role of CDR-3 MAbs in the inhibition of HIV-1 infection, these experiments do not support the hypothesis that the CDR-3 region is specifically involved in virus entry at a postbinding stage.