Two major groups of neutralizing anti-gp120 antibodies exist in HIV-infected individuals. Evidence for epitope diversity around the CD4 attachment site.

The aim of this study was to dissect neutralizing anti-gp120 antibody populations in seropositive asymptomatic individuals. Murine anti-Id mAb were raised against polyclonal affinity-purified human anti-gp120 antibodies. These anti-Id mAb were used to fractionate anti-gp120 antibodies from a pool of HIV-positive sera into idiotypically distinct anti-gp120 antibody (Id+Ab) preparations. Immunochemical and neutralization studies indicated that all Id+Ab that neutralized HIV-1 in vitro interacted with either the V3 loop or the CD4 attachment site of gp120. The V3-specific Id+Ab neutralized HIV-1 in a strain-restricted manner. Id+Ab specific for the CD4 attachment site exhibited different spectra of neutralizing activities against multiple strains of HIV-1. This finding indicates that multiple, antigenically diverse epitopes reside around the CD4 attachment site of gp120. Significantly, depletion of the Id+Ab from affinity-purified total anti-gp120 antibodies abrogated most of the neutralizing activities of these antibodies, suggesting that neutralizing anti-gp120 antibodies consist of two major specificities, either to the V3 region or to the CD4 attachment site. The understanding of specificities and neutralizing activities of different anti-gp120 antibodies in seropositive healthy individuals will be helpful for designing effective vaccines and immunotherapeutic strategies for AIDS.     

Antiidiotypic antibody mimicry of a bluetongue virus neutralizing antigen.

Syngeneic antiidiotypic (Anti-Id) mAb were produced to the Bluetongue virus (BTV) serotype 17 neutralizing mAb. Three anti-Id mAb had the characteristics of an internal image of the Id as demonstrated by 1) the internal image anti-Id mAb were capable of mimicking BTV by blocking the neutralizing activity of the idiotypic neutralizing mAb and 2) the anti-Id mAb bound specifically to the surface of BTV susceptible cells in indirect binding experiments as determined by immunocytochemistry and flow cytometric analysis. The potential application of these internal image anti-Id mAb in this arbovirus system is discussed.     

VH-related idiotopes detected by site-directed mutagenesis. A study induced by the failure to find CD4 anti-idiotypic antibodies mimicking the cellular receptor of HIV.

The function of the CD4 cell surface protein as coreceptor on T helper lymphocytes and as receptor for HIV makes this glycoprotein a prime target for an immune intervention with mAb. A detailed understanding of the structural determinants on the therapeutic CD4 mAb that are involved in Ag binding or are recognized by anti-idiotypic mAb (anti-Id) may be important for designing antibodies with optimal therapeutic efficacy. Seven anti-Id raised against the CD4 mAb M-T310 were selected from a large panel with the intention to obtain CD4 mimicking structures with specificity for HIV gp120. The selected anti-Id did not react with other CD4-specific mAb cross-blocking M-T310. Among these, mAb M-T404, although having the same L chain as M-T310 and a VH region sequence differing only at 14 amino acid positions, was not recognized by the anti-Id. M-T310 H chain complexed with the J558L L chain reacted with all anti-Id, thus demonstrating that the recognized idiotopes are located within the VH region. To identify the idiotopes of M-T310 seen by the anti-Id, variants of M-T404 containing one or more of the M-T310-derived substitutions were generated by oligonucleotide-directed mutagenesis. The reactivity pattern of the mutant proteins with the anti-Id demonstrated that the idiotopes reside within the complementarity determining region (CDR) 2 and CDR3 loops of the VH region. A major idiotope was defined by a single amino acid in CDR2 that was recognized by three anti-Id, whereas the four other anti-Id reacted with determinants of CDR3. Although the performed amino acid substitutions did influence the Id recognition, Ag binding was not significantly affected, suggesting that none of the anti-Id can be considered as a mimicry of the CD4 Ag.     

Neutralizing monoclonal antibodies against human immunodeficiency virus type 2 gp120.

Monoclonal antibodies (MAbs) were obtained by immunizing mice with synthetic peptides corresponding to the third variable (V3) or the third conserved (C3) domain of the external envelope protein (gp120) of human immunodeficiency virus type 2 (HIV-2ROD). One MAb, designated B2C, which was raised against V3 peptide NKI26, bound to the surface of HIV-2-infected cells but not to their uninfected counterparts. B2C was capable of neutralizing cell-free and cell-associated virus infection in an isolate-specific fashion. The antibody-binding epitope was mapped to a 6-amino-acid peptide in the V3 variable domain which had the core sequence His-Tyr-Gln. Two MAbs, 2H1B and 2F19C, which were raised against the C3 peptide TND27 reacted with gp120 of HIV-2ROD in a Western immunoblot assay. The C3 epitopes recognized by these two MAbs appeared inaccessible because of their poor reactivity in a surface immunofluorescence assay. Although partial inhibition of syncytium formation was observed in the presence of the anti-C3 MAbs, their neutralizing activity appeared weak. Finally, the effects of these MAbs against CD4-gp120 binding were assessed. Partial inhibition of CD4-gp120 binding was observed in the presence of high concentrations of B2C. On the other hand, no inhibition of CD4-gp120 binding was observed in the presence of anti-C3 MAbs. Since complete neutralization could be achieved at a concentration corresponding to that of partial binding inhibition by B2C, some different mechanisms may be involved in the B2C-mediated neutralization. These results, taken together, indicated that analogous to the function of the V3 region of HIV-1, the V3 region of HIV-2ROD contained at least a type-specific fusion-inhibiting neutralizing epitope. In this respect, the V3 sequence of HIV-2 may be a useful target in an animal model for HIV vaccine development.     

Anti-idiotypic antibody response to monoclonal anti-CD4 preparations in nonhuman primate species.

A series of mouse monoclonal anti-CD4 preparations was characterized for the ability to recognize overlapping epitopes on CD4 and to inhibit HIV/simian immunodeficiency virus (SIV) syncytium formation. Based on this characterization, mAb able to recognize CD4 epitopes overlapping the HIV binding site were selected and used to immunize nonhuman primates to elicit the production of specific anti-Id antibodies. Five baboons and five rhesus monkeys were immunized with either individual or a cocktail consisting of several monoclonal anti-CD4 preparations. All the nonhuman primates produced specific anti-Id that recognized either private or cross-reactive Id depending on the monoclonal anti-CD4 used to generate the anti-Id response. Inhibition assays were performed to ascertain the ability of: 1) soluble CD4 to inhibit the Id-anti-Id reaction and 2) the various anti-Id to inhibit the CD4-monoclonal anti-CD4 reaction. These studies demonstrated that some of the anti-Id recognized a cross-reactive Id that was associated with the Ag-combining site. In addition, some of the anti-Id weakly recognized SIV gp120 by Western blot analysis. These studies may be useful in designing experiments that may lead to a better understanding of the CD4-HIV gp120 interaction and to the production of Id and/or anti-Id reagents that might be used to manipulate this virus-receptor interaction.     

Paratope- and framework-related cross-reactive idiotopes on anti-acetylcholine receptor antibodies

Cross-reactive idiotopes are a possible target for therapeutical interventions in autoimmune diseases. To investigate their role in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG) we analyzed the Id of rat anti-AChR mAb 6, 35, 61, 65 and a control myeloma protein IR27. Anti-Id 6, 35, 61, 65 bound in a direct binding assay with various affinity to all rat anti-AChR mAb that were tested. Anti-Id IR27 recognized none of the anti-AChR mAb. The specificity of these crossreactions was confirmed by inhibition studies with anti-AChR mAb and two control rat myeloma proteins (IR27 and IR241). In addition, the Id expression on mAb D6, a mouse anti-human AChR mAb was recognized by anti-Id 6, 35, and 65. Anti-Id, except anti-Id IR27, bound to affinity purified IgG from the sera of rats with EAMG, but not to preimmune Lewis IgG. These results suggest extensive sharing of idiotopes among anti-AChR mAb, which are also present in EAMG serum. Anti-AChR mAb against the main immunogenic region (6, 35, 65) from different rat strains, shared at least one paratope-related cross-reactive idiotopes. In the view of the fact that anti-main immunogenic region antibodies might form a predominant fraction of the polyclonal response against AChR, it is conceivable that an anti-Id recognizing these antibodies could have therapeutical applications as for example a selective immune absorbent or in immunotoxin therapy.     

Immune responses during human Schistosoma mansoni. XVII. Recognition by monoclonal anti-idiotypic antibodies of several idiotopes on a monoclonal anti-soluble schistosomal egg antigen antibody and anti-soluble schistosomal egg antigen antibodies from patients with different clinical forms of infection

A monoclonal human anti-soluble schistosomal egg Ag(SEA) antibody (E5) that stimulates anti-Id T cells and is idiotypically represented in pools of immunoaffinity-purified human anti-SEA antibodies from chronic, generally asymptomatic, intestinal (INT) patients (AM1 and AM5) was used to raise several monoclonal anti-Id: 1C2, 1C6, 4A8, 4F9, and 2A7. Cross-inhibition between these anti-Id identified distinct idiotopes on E5. Anti-SEA preparations from schistosomiasis patients (AM1, AM5, and others) were tested for their inhibition of the E5/monoclonal anti-Id reactions, in competitive ELISA. In either the E5/4A8 or E5/1C6 ELISA system, anti-SEA from INT (AM1 or AM5) or hepatointestinal (HI) (AM7) patients were able to inhibit these reactions. However, anti-SEA antibodies from acute (AM9) or hepatosplenic (HS) (AM3 or AM8) patients did not express Id that were inhibitory in these systems. These results suggest that a relatively high proportion of INT and HI anti-SEA antibodies express a dominant cross-reactive idiotope (CRI) recognized by 1C6/4A8. This CRI is also easily detected in plasmas from individual INT patients. Anti-Id 1C2 reacted strongly with an Id in AM1, AM5, or AM7, but one which also occurred, to a lesser extent, in AM3, AM8, and AM9. Monoclonal anti-Id 4F9 and 2A7 reacted weakly with idiotopes expressed by antibodies from all patients, regardless of the clinical form of their infection. These observations indicate that anti-SEA antibodies from INT and HI, but not acute or HS patients express dominant, CRI that are identified by 1C6, 4A8, or 1C2 and are also expressed on the INT-derived anti-SEA mAb E5.     

Monoclonal anti-idiotype induces protection against the cytotoxicity of the trichothecene mycotoxin T-2

An IgG1 mAb, designated HD11, specific for the trichothecene mycotoxin T-2 and capable of neutralizing its cytotoxicity was used to generate a syngeneic monoclonal anti-Id antibody. The generated anti-Id mAb, designated DE8, specifically bound to HD11 anti-T-2 mAb, and not to IgG1 mAb of irrelevant specificity or to normal mouse Ig. DE8 inhibited the binding of HD11 anti-T-2 to T-2-BSA-coated plates, whereas a control anti-Id mAb did not, suggesting recognition of an Id determinant associated with the T-2 binding site of HD11. Moreover, the binding of HD11 to DE8 and that of DE8 to HD11 were specifically inhibited by free T-2 mycotoxin. DE8 mAb was efficient in abrogating the protective effect of HD11 in the cytotoxicity of T-2 on the human epidermoid carcinoma cell line Hep-2. In vivo immunization of BALB/c mice with DE8 conjugated to KLH induced an anti-T-2 antibody titer comparable to that obtained with T-2-OVA immunization, whereas immunization with unconjugated DE8 resulted in a lower titered anti-T-2 response. Immunization with DE8-keyhole limpet or with unconjugated DE8 induced anti-T-2 antibody responses characterized by expression of "HD11-like" Id and by protection against T-2 cytotoxicity. However, the T-2-OVA-induced anti-T-2 response lacked the HD11+ Id and was only partially protective against T-2 cytotoxicity. This represents the first demonstration of the use of an anti-Id based vaccine in the in vivo induction of a protective antibody response against the cytotoxicity of a nonproteinaceous, small m.w. biologic toxin, whose very toxic nature precludes its use as the immunogen.     

Immunization with a soluble CD4-gp120 complex preferentially induces neutralizing anti-human immunodeficiency virus type 1 antibodies directed to conformation-dependent epitopes of gp120.

Preservation of the conformation of recombinant gp120 in an adjuvant, enabling it to elicit conformation-dependent, epitope-specific, broadly neutralizing antibodies, may be critical for the development of any gp120-based human immunodeficiency virus type 1 (HIV-1) vaccine. It was hypothesized that recombinant gp120 complexed with recombinant CD4 could stabilize the conformation-dependent neutralizing epitopes and effectively deliver them to the immune system. Therefore, a soluble CD4-gp120 complex in Syntex adjuvant formulation was tested with mice for its ability to induce neutralizing anti-gp120 antibody responses. Seventeen monoclonal antibodies (MAbs) were generated and characterized. Immunochemical studies, neutralization assays, and mapping studies with gp120 mutants indicated that the 17 MAbs fell into three groups. Four of them were directed to what is probably a conformational epitope involving the C1 domain and did not possess virus-neutralizing activities. Another four MAbs bound to V3 peptide 302-321 and exhibited cross-reactive gp120 binding and relatively weak virus-neutralizing activities. These MAbs were very sensitive to amino acid substitutions, not only in the V3 regions but also in the base of the V1/V2 loop, implying a conformational constraint on the epitope. The last group of nine MAbs recognized conformation-dependent epitopes near the CD4 binding site of gp120 and inhibited the gp120-soluble CD4 interaction. Four of these nine MAbs showed broadly neutralizing activities against multiple laboratory-adapted strains of HIV-1, three of them neutralized only HIVIIIB, and the two lower-affinity MAbs did not neutralize any strain tested. Collectively, the results from this study indicate that immunization with the CD4-gp120 complex can elicit antibodies to conformationally sensitive gp120 epitopes, with some of the antibodies having broadly neutralizing activities. We suggest that immunization with CD4-gp120 complexes may be worth evaluating further for the development of an AIDS vaccine.     

Characterization of GP120 binding to CD4 and an assay that measures ability of sera to inhibit this binding

There is evidence that the initial interaction between HIV-1 and the host that is essential for infection is the specific binding of the viral envelope glycoprotein, gp120, to the CD4 molecule found on certain T cells and monocytes. Most individuals infected with HIV develop antibodies against the gp120 protein. Although in vitro treatment of CD4+ T cells with mAb to a specific epitope of the CD4 molecule (T4a) blocks virus binding, syncytia formation, and infectivity, it is unclear if antibodies to gp120 from an infected individual that can inhibit the binding of gp120 to CD4 is in any way related to the clinical course of disease. Our present study characterizes the binding of 125I-labeled rgp120 to CD4+ cells, and describes an assay system that measures a potentially relevant form of immunity to HIV infection, i.e., the blocking of HIV binding to CD4+ cells. Optimal binding conditions included a 2-h incubation at 22 degrees C, 4 x 10(6) CD4+ cells, and 1 nM gp120. The dissociation constant (KD) for gp120 binding to cell surface CD4 was 5 nM, and was inhibited by soluble CD4 and by mAb to T4a but not to T3 or T4. For the binding inhibition assay, negative controls included healthy seronegatives, seronegatives with connective tissue diseases, patients with HTLV-1 disease, and patients infected with HIV-2. In studying over 100 sera, the assay was highly sensitive (98%) and specific (100%). The majority of HIV+ sera could inhibit binding at dilutions of 1/100 to 1/1000. No correlation was noted between binding inhibition (BI) titer in this assay and clinical stage of HIV infection. In addition, there was no correlation between BI titer and HIV neutralizing activity. The BI titer was correlated with the titer of anti-gp160 (r = 0.63) and the titer of anti-gp120 (r = 0.52) antibodies determined by Western blot dilution. As with neutralizing antibodies and other forms of immune response to HIV, it is unclear what role antibody blocking of HIV binding to CD4+ cells may play in active immunity to HIV in infected individuals. This activity may prove to have some value in protection against initial HIV infection and, thus, the assay may be of use in monitoring vaccine trials.     

Structural analysis of human and macaque mAbs 2909 and 2.5B: implications for the configuration of the quaternary neutralizing epitope of HIV-1 gp120

The quaternary neutralizing epitope (QNE) of HIV-1 gp120 is preferentially expressed on the trimeric envelope spikes of intact HIV virions, and QNE-specific monoclonal antibodies (mAbs) potently neutralize HIV-1. Here, we present the crystal structures of the Fabs of human mAb 2909 and macaque mAb 2.5B. Both mAbs have long beta hairpin CDR H3 regions >20 ? in length that are each situated at the center of their respective antigen-binding sites. Computational analysis showed that the paratopes include the whole CDR H3, while additional CDR residues form shallow binding pockets. Structural modeling suggests a way to understand the configuration of QNEs and the antigen-antibody interaction for QNE mAbs. Our data will be useful in designing immunogens that may elicit potent neutralizing QNE Abs.     

Structure and binding properties of a monoclonal anti-idiotypic autoantibody to anti-DNA with epibody activity.

We report the isolation and characterization of a mouse autoanti-idiotypic mAb (D7.4 IgG2a), which is directed against a major public Id (A52 IgG2b) in the murine and human autoimmune response to DNA. The natural anti-Id mAb has been produced in the course of the SLE-like disease in a female NZB/NZW F1 mouse and showed a dual specificity (epibody activity) for the public Id (A52) and for the autoantigen (DNA). The two binding activities were shown to reside in the Fab portion of the epibody and were highly specific for their respective Ag. A complete nucleotide sequence analysis of the D7.4 H and L chain V-region genes combined with computer comparisons to available Ig sequences may suggest a charge interaction between the H chain CDR3 segments of the Id and anti-Id antibodies. The D7.4 epibody may be a component of the self-binding, idiotypically connected network of natural antibodies. Alternatively, it could be elicited against the potentially pathogenic, DNA-containing immune complexes in order to facilitate their removal from the circulation of diseased individuals.     

Analysis of the cross-reactive anti-gp120 antibody population in human immunodeficiency virus-infected asymptomatic individuals.

This study was undertaken to analyze the specificity and neutralizing properties of cross-reactive anti-gp120 antibodies (Abs) in the sera of two human immunodeficiency virus (HIV)-infected asymptomatic individuals. Two panels of murine monoclonal anti-idiotype Abs (anti-id MAbs) were established against cross-reactive polyclonal anti-gp120 Abs purified from HIV+ sera by sequential affinity chromatography using gp120SF2- and gp120IIIB-Sepharose columns. These panels of anti-id MAbs were then used to affinity purify idiotype-positive (Id+) anti-gp120 Abs from HIV+ sera. The recovery of each of these Id+ Abs by purification indicated that several idiotypically distinct cross-reactive anti-gp120 Abs are present in sera over a wide range of concentrations. Immunological and biological studies showed that although all of the Id+ Abs were reactive against gp120SF2 and gp120IIIB, they exhibited unique epitope specificities and distinct neutralizing activities. Most of the Id+ Abs were directed against epitopes in the CD4 attachment site (CD4 site epitopes) of gp120 and exhibited a spectrum of broadly neutralizing activities. On the other hand, a minor population of Id+ Abs showed specificity for the V3 region of gp120 and exhibited limited cross-neutralizing activities. Together, these studies indicate that the CD4 site epitope-specific Abs are heterogeneous with respect to their clonality, neutralizing activity, and concentration in sera. This heterogeneity suggests that anti-gp120 Abs to the CD4 attachment site are developed in response to multiple overlapping epitopes present on the original virus isolate and/or epitopes on mutated variants which emerged over time.     

An IgG human monoclonal antibody that reacts with HIV-1/GP120, inhibits virus binding to cells, and neutralizes infection.

A human mAb (HmAb) termed F105 was obtained by fusion of antibody-producing EBV-transformed cells with the HMMA2.11TG/O cell line. F105 is an IgG1 kappa antibody that binds to the surfaces of cells infected with all HIV-1 strains tested: MN, RF, IIIB, and SF2, but not uninfected cells. The HmAb immunoprecipitates GP120 from all four strains. F105 does not react with denatured GP120 on Western blots, but does react with viral lysates and purified GP120 dotted onto nitrocellulose filter paper under nondenaturing conditions. rGP120 from SF2 and soluble rCD4 inhibit antibody binding to infected cells in a dose-dependent manner. F105 inhibits the binding of free, infectious virions to uninfected HT-H9 cells with 50% of maximal (100%) inhibition at approximately 1 microgram/ml. F105 inhibits infection of HT-H9 cells by 100 tissue culture infective dose 50% units of MN and IIIB strains with 50% inhibition at concentrations of HmAb readily achievable in man. It appears that the F105 HmAb reacts with a conformationally defined epitope on HIV-1/GP120 that is exposed on the free virion and is important for binding to the cell surface by the virion. The epitope, which is immunogenic in humans, appears to be within, or topographically near, the CD4-binding site. F105 and the F105 epitope are potentially useful in therapy and in the design of peptide or anti-Id based vaccines; monitoring of the expression of the Id may prove useful in evaluating immune responses in infected individuals or vaccinated volunteers.     

Evidence by peptide mapping that the region CD4(81-92) is involved in gp120/CD4 interaction leading to HIV infection and HIV-induced syncytium formation.

Peptide fragments of the CD4 molecule were compared in their ability to 1) inhibit CD4-dependent HIV-induced cell fusion; 2) inhibit CD4-dependent HIV infection in vitro; and 3) block gp120 envelope glycoprotein binding to CD4. Peptides from the region CD4(81-92), although inactive when underivatized, were equipotent inhibitors of CD4-dependent virus infection, cell fusion, and CD4/gp120 binding when derivatized via benzylation and acetylation. Peptides of identical chemical composition, but altered sequence and derivatization pattern that blocked gp120 binding to either CD4-positive cells or solubilized CD4, also blocked infection and fusion with similar potencies. Those that did not block gp120/CD4 interaction were also inactive in HIV-1 infection and cell fusion assays. No other peptide fragments of the CD4 molecule inhibited fusion, infection, or CD4/gp120 interaction. The peptide CD4(23-56), derived from a region of CD4 implicated in binding of CD4 antibodies that neutralize HIV infection and cell fusion, had no effect on CD4-dependent cell fusion, HIV-1 infection, or CD4/gp120 binding, but did reverse OKT4A and anti-Leu 3a blockade of gp120 binding to CD4. These data provide evidence that the 81-92 region of CD4 is directly involved in gp120 binding leading to CD4-dependent HIV infection and syncytium formation. Previous observations with structural mutants of CD4 suggest that the CDR2-homologous region of CD4 is also involved, either directly or indirectly, in binding of gp120 to CD4. The CDR2- and CDR3-like domains of CD4 may both contribute to the binding of the HIV envelope necessary for HIV-1 infection and HIV-1-induced cell fusion.     

Idiotope mapping on the variable region of an antibody clonotype produced by normal (nonmalignant) human B cells

Human anti-N-acetyl-D-glucosamine (GlcNAc) antibodies were prepared by affinity chromatography from serum of a healthy donor (MSS). They were heterogeneous but contained a unique antibody clonotype (1A) representing 7% of all anti-GlcNAc antibodies. Out of a series of monoclonal anti-idiotopic antibodies (anti-Id mAb), we identified five antibodies that bound to clonotype 1A as shown by isoelectric focusing and Western blotting. Two of them were specific for clonotype 1A (10F59 and 13F15), thus indicating its clonal origin. However, three anti-Id mAb (16F433, 16F539, and 16F812) bound to various additional portions of anti-GlcNAc antibodies of donor MSS. With the exception of one mAb, all anti-Id mAb have very similar relative affinities to clonotype 1A, so results from competition experiments between the different antibodies and between each antibody and antigen should reveal spatial relationships between the corresponding Id and between each Id and the antigen-combining site. The results show a consistent topography of Id on the V-region of clonotype 1A. Id 59, 812, and 433 were found to be arranged in one cluster (cluster I), whereas Id 15 and 539 belonged to a second cluster (cluster II). Cluster I resides completely in the antigen-combining site, whereas only Id 15 of cluster II weakly overlaps with the binding site. Our study demonstrates an analysis of spatial relationships of Id expressed on a human antibody clonotype. To our knowledge, this is the first demonstration of Id mapping on antibodies produced by a normal (nonmalignant) B cell clone that should be accessible to regulatory signals. Such analysis may contribute to a more detailed characterization of anti-Id mAb, and may provide additional information for a better understanding of their immunoregulatory effects.     

Rational design of a CD4 mimic that inhibits HIV-1 entry and exposes cryptic neutralization epitopes

The conserved surfaces of the human immunodeficiency virus (HIV)-1 envelope involved in receptor binding represent potential targets for the development of entry inhibitors and neutralizing antibodies. Using structural information on a CD4-gp120-17b antibody complex, we have designed a 27-amino acid CD4 mimic, CD4M33, that presents optimal interactions with gp120 and binds to viral particles and diverse HIV-1 envelopes with CD4-like affinity. This mini-CD4 inhibits infection of both immortalized and primary cells by HIV-1, including primary patient isolates that are generally resistant to inhibition by soluble CD4. Furthermore, CD4M33 possesses functional properties of CD4, including the ability to unmask conserved neutralization epitopes of gp120 that are cryptic on the unbound glycoprotein. CD4M33 is a prototype of inhibitors of HIV-1 entry and, in complex with envelope proteins, a potential component of vaccine formulations, or a molecular target in phage display technology to develop broad-spectrum neutralizing antibodies.     

Effects of CD4 synthetic peptides on HIV type I envelope glycoprotein function.

Benzylated derivatives of a peptide (CD4(81-92)) representing the CDR3-like region of CD4 were previously found to inhibit gp120 binding, HIV-1 infectivity, and syncytium formation. These results have been interpreted to indicate a role for the corresponding CD4 region in these processes. The peptide (TbYICbEbVEDQKAcEE) is the prototype of a series of similar CD4(81-92) derivatives. We report that this peptide noncompetitively inhibits binding to CD4 of both gp120 and a mAb (MAX.16H5), both of which recognize the CDR2-like region of CD4. The binding of an antibody (Leu 3a) that is directed against a different area of the D1 domain of CD4 was also inhibited. The peptide derivative inhibited both HIV-1- and HTLV-1-mediated syncytium formation in the same concentration range. Nonbenzylated cyclic and linear peptides representing the CDR3-like region of CD4 (CD4(84-101)) had only minor effects on gp120 binding which were not sequence specific. The results of this study suggest that the effects of benzylated CD4(81-92) derivatives on HIV-1 binding or fusion should not be used to reach conclusions about the function of the corresponding CD4 region.     

An idiotype shared by monoclonal antibodies to different peptides of human myelin basic protein.

A mAb of the IgG1/kappa isotype was raised against human myelin basic protein (MBP) peptide acetyl 1-9. This mAb, termed F23, reacted with human MBP and human MBP peptides acetyl 1-9, 1-14, and 1-44, but not with MBP peptides 10-19, 80-89, or 45-89. According to the guidelines of the molecular recognition theory, a complementary peptide to human MBP peptide 1-9 was synthesized and used to raise murine mAb with anti-Id activity. Two mAb anti-Id, F25F7 and F25C8, both of the IgM/kappa isotype, were selected for further study. These anti-Id reacted with F23, the mAb for which they were selected, and also reacted with another mAb, which was of the IgG1/kappa isotype and was raised to human MBP peptide 80-89. There was no reaction with another control mAb of the IgG1/kappa isotype or murine myeloma IgG1. By immunoblotting techniques, it was demonstrated that the Id on each of the mAbs to MBP peptides was located on the kappa L chain but also could be recognized in nonreduced IgG. The cross-reactive anti-Id suppressed antibody secretion of Id-producing hybridoma cells in an Id-specific manner, and kinetic studies suggest an intracellular mechanism for the suppression. These cross-reactive Id among antibodies to different MBP peptides imply that the same V region genes of kappa L chains are involved in the selection of antibodies to an autoantigen, like MBP, and may play a role in the modulation of immune responses against MBP in certain inflammatory demyelinating diseases.     

Characterization of in vitro inhibition of human immunodeficiency virus by purified recombinant CD4.

The first step in infection of human T cells with human immunodeficiency virus (HIV) is binding of viral envelope glycoprotein gp120 to its cellular receptor, CD4. The specificity of this interaction has led to the development of soluble recombinant CD4 (rCD4) as a potential antiviral and therapeutic agent. We have previously shown that crude preparations of rCD4 can indeed block infection of T cells by HIV type 1 (HIV-1). Here we present a more detailed analysis of this antiviral activity, using HIV-1 infection of the T lymphoblastoid cell line H9 as a model. Purified preparations of rCD4 blocked infection in this system at nanomolar concentrations; combined with the known affinity of the CD4-gp120 interaction, this finding suggests that the inhibition is simply due to competition for gp120 binding. As predicted, rCD4 had comparable activity against all strains of HIV-1 tested and significant activity against HIV-2. Higher concentrations of rCD4 blocked infection even after the virus had been adsorbed to the cells. These findings imply that the processes of viral adsorption and penetration require different numbers of gp120-CD4 interactions. Recombinant CD4 was able to prevent the spread of HIV infection in mixtures of uninfected and previously infected cells. Our studies support the notion that rCD4 is a potent antiviral agent, effective against a broad range of HIV-1 isolates, and demonstrate the value of purified rCD4 as an experimental tool for studying the mechanism of virus entry into cells.