Loop Epitope Research Articles

The Chemokine SDF-1/CXCL12 Binds to and Signals through the Orphan Receptor RDC1 in T Lymphocytes

Combined phylogenetic and chromosomal location studies suggest that the orphan receptor RDC1 is related to CXC chemokine receptors. RDC1 provides a co-receptor function for a restricted number of human immunodeficiency virus (HIV) isolates, in particular for the CXCR4-using HIV-2 ROD strain. Here we show that CXCL12, the only known natural ligand for CXCR4, binds to and signals through RDC1. We demonstrate that RDC1 is expressed in T lymphocytes and that CXCL12-promoted chemotaxis is inhibited by an anti-RDC1 monoclonal antibody. Concomitant blockade of RDC1 and CXCR4 produced additive inhibitory effects in CXCL12-induced T cell migration. Furthermore, we provide evidence that interaction of CXCL12 with RDC1 is specific, saturable, and of high affinity (apparent KD approximately 0.4 nM). In CXCR4-negative cells expressing RDC1, CXCL12 promotes internalization of the receptor and chemotactic signals through RDC1. Collectively, our data indicate that RDC1, which we propose to rename as CXCR7, is a receptor for CXCL12.

Analysis of a 17-amino acid residue, virus-neutralizing microantibody

The antibody-binding site, through which an antibody binds to its epitope, is a complex structure formed by the folding together of six complementarity-determining regions (CDRs). However, certain peptides derived from CDR sequences retain antibody specificity and function; these are know as microantibodies (MicroAbs). For example, the F58 MicroAb is a 17 residue, cyclized peptide (CDLIYYDYEEDYYFDYC) derived from CDR-H3 of F58, an IgG1 specific for the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). Both MicroAb and IgG recognize the same epitope in the V3 loop and, despite its small size, the MicroAb neutralizes the infectivity of HIV-1 IIIB only 32-fold less efficiently on a molar basis. The advantage of MicroAbs is that their small size facilitates structure-function analysis. Here, the F58 MicroAb was investigated using alanine scanning, mass spectroscopy and surface plasmon resonance. Neutralization of infectious IIIB was generally more sensitive to alanine substitution than binding to soluble gp120. There appeared to be a division of function within the MicroAb, with some residues involved in antigen binding (alanine substitution of 11D, 12Y or 13Y abrogated both binding and neutralization), whereas others were concerned solely with neutralization (substitution of 3L, 8Y or 14F abrogated neutralization, but not binding). The MicroAb is predominantly beta-sheet and has strong conformational constraints that are probably essential for activity. The MicroAb and soluble gp120 formed a 1 : 1 complex, with an association rate that was threefold greater than that with IgG and a faster dissociation rate. Its equilibrium dissociation constant is 37.5-fold greater than that of IgG, in line with neutralization data. This study demonstrates how MicroAbs can make a useful contribution to the understanding of antigen-antibody interactions.

Identification of a Linear Peptide Recognized by Monoclonal Antibody 2D7 Capable of Generating CCR5-Specific Antibodies with Human Immunodeficiency Virus-Neutralizing Activity

CCR5 is the major coreceptor for human immunodeficiency virus (HIV) infection. The murine monoclonal antibody (MAb) 2D7, which recognizes a conformation-dependent epitope in the second extracellular loop of CCR5, is one of the most potent inhibitors of R5 virus cell entry. However, attempts to humanize 2D7 for in vivo human use have been unsuccessful so far. A filamentous phage library expressing random peptides was used to identify a peptide mimitope that is recognized by MAb 2D7. A synthetic peptide containing this sequence (2D7-2SK) bound to MAb 2D7 with high affinity and reversed its HIV type 1 (HIV-1) fusion inhibitory activity. The peptide contains sequence homologies to two distal regions of the second extracellular loop of human CCR5, both of which are required for MAb 2D7 binding. Rabbit anti-2D7-mimitope antibodies competed with MAb 2D7 for binding to the 2D7-2SK peptide in Biacore biosensor testing. Importantly, the rabbit anti-2D7-2SK antibodies bound to CCR5 on cells and specifically inhibited R5 (but not X4) envelope-mediated syncytium formation. These antibodies also neutralized infection of human peripheral blood mononuclear cells with R5 HIV isolates comparably to MAb 2D7. In summary, we have identified a novel peptide that closely mimics the MAb 2D7 epitope on CCR5. This peptide could be included as a potential vaccine candidate or to isolate 2D7-like human antibodies as entry inhibitors for R5 viruses.

Familial Alzheimer's Disease Presenilin 1 Mutations Cause Alterations in the Conformation of Presenilin and Interactions with Amyloid Precursor Protein

Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, an enzymatic activity that cleaves amyloid beta (Abeta) from the amyloid precursor protein (APP). More than 100 mutations spread throughout the PS1 molecule are linked to autosomal dominant familial Alzheimer's disease (FAD). All of these mutations lead to a similar phenotype: an increased ratio of Abeta42 to Abeta40, increased plaque deposition, and early age of onset. We use a recently developed microscopy approach, fluorescence lifetime imaging microscopy, to monitor the relative molecular distance between PS1 N and C termini in intact cells. We show that FAD-linked missense mutations located near the N and C termini, in the mid-region of PS1, and the exon 9 deletion mutation all change the spatial relationship between PS1 N and C termini in a similar way, increasing proximity of the two epitopes. This effect is opposite of that observed by treatment with Abeta42-lowering nonsteroidal anti-inflammatory drugs (NSAIDs) (Lleo et al., 2004b). Accordingly, treatment of M146L PS1-overexpressing neurons with high-dose NSAIDs somewhat offsets the conformational change associated with the mutation. Moreover, by monitoring the relative distance between a PS1 loop epitope and the APP C terminus, we demonstrate that the FAD PS1 mutations are also associated with a consistent change in the configuration of the PS1-APP complex. The nonpathogenic E318G PS1 polymorphism had no effect on PS1 N terminus-C terminus proximity or PS1-APP interactions. We propose that the conformational change we observed may therefore provide a shared molecular mechanism for FAD pathogenesis caused by a wide range of PS1 mutations.

Subapical Localization of the Dopamine D3 Receptor in Proximal Tubules of the Rat Kidney

The dopamine D3 receptor (D3R), intensively studied in neuroscience, also plays an important role in the regulation of renal and cardiovascular function. In contrast to functional findings, less information is available on its localization in the kidney. Neither RT-PCR studies nor radioligand binding assays are suitable to selectively determine the distribution of renal D3R at the level of cellular or even subcellular structures. We studied the renal D3R distribution in Sprague-Dawley rats by a polyclonal antiserum directed against an epitope in the third intracytoplasmic loop. D3R immunoreactivity was detected by indirect immunofluorescence and confocal laser scanning microscopy. D3R staining was confined to the renal cortex and occurred in proximal convoluted tubules near or in direct connection with the urinary pole of the glomeruli. The fluorescent spots were restricted to the subapical portion of the proximal tubular cells. Double staining with the F-actin marker phalloidin revealed a localization of the D3R below the brush border region. However, staining by anti-beta1/beta2-adaptins, recognizing clathrin-coated compartments, did not correspond to the distribution of the D3R signal. This is the first description of a D3R accumulation in a cytoplasmic pool in the kidney, probably corresponding to a recycling mechanism or storage compartment.

Different immune response of mice immunized with conjugates containing multiple copies of either consensus or mixotope versions of the V3 loop peptide from human immunodeficiency virus type 1.

The critical role that antibody responses to the V3 loop epitope play in human immunodeficiency virus type 1 (HIV-1) neutralization has caused this peptide to be used in many HIV-1 vaccine candidates. To enhance cross-reactivity toward several V3 sequences, a database of 50 peptides of the V3 region from HIV-1 subtype A was used to design both a consensus peptide and a combinatorial peptide (mixotope) library representative of these sequences. The two immunogens (consensus and mixotope) were incorporated into multiple antigen peptide (MAP) constructions, conjugated to a recombinant surface antigen from hepatitis B virus (HbsAg) carrier protein, and inoculated to mice in combination with a C4 (CD4-binding) peptide MAP construction, also conjugated to HBsAg. The respective responses and cross-reactivity to several V3 loop sequences of both types of immunogens were compared. Mice inoculated with the V3 consensus-MAP-HBsAg + C4-MAP-HBsAg mixture elicited higher antibody responses than those given the V3 mixotope-MAP-HBsAg + C4-MAP-HBsAg mixture. In addition, pooled serum from the first group of immunogens analyzed at dilution 1:100 had higher cross-reactivity against V3 peptides on cellulose membranes than those from mice given the combinatorial immunogen. Fine epitope mapping of both consensus and C4 peptide by the spot synthesis technique showed that sera of the first group strongly recognized both sequences in their entirety, whereas mice immunized with the mixotope library recognized only the N-terminal region of V3. These results seem to suggest that the V3 consensus peptide is superior to the combinatorial strategy in inducing potent and cross-reactive responses to HIV.

Efficient CD8 + T cell response to the HIV-env V3 loop epitope from multiple virus isolates by a DNA prime/vaccinia virus boost (rWR and rMVA strains) immunization regime and enhancement by the cytokine IFN-γ

The cytotoxic T-lymphocyte response (CTL) has been shown to be determinant in the clearance of many viral infections and hence, vaccine candidates against AIDS are designed to enhance this arm of the immune system. In this study, we have analyzed the antigen specific immune responses triggered in mice by different combinations of vaccine vehicles expressing the multiepitope polypeptide TAB13. This chimeric protein contains the V3 region of the gp120 from eight different HIV-1 isolates and was efficiently expressed by a DNA vector (DNA-TAB), and also by vaccinia virus recombinants (rVV) based either on the attenuated modified vaccinia virus Ankara (MVA-TAB) or Western Reserve (VV-TAB) strains. Inoculation of a DNA-TAB vector in priming followed by a booster with VV-TAB or MVA-TAB induces a humoral immune response against TAB13 protein and efficiently enhanced the CD8 + T cell response against V3 epitopes from HIV-1 isolates LR150, MN, and IIIB in comparison with animals immunized with two doses of DNA-TAB. A protocol that incorporates a DNA vector expressing IFN-γ (DNA-IFN-γ) with DNA-TAB in the priming, followed by a booster with MVA-TAB, triggered the highest values of specific CD8 + T cell response. By examining the cytokine pattern, the immune response induced by these vaccination approaches was predominantly of Th-1 type. These findings establish safe strategies for the enhanced generation of T cell mediated immunity to HIV-1 that can benefit in the design of an effective vaccine against AIDS.

Formation of polyomavirus-like particles with different VP1 molecules that bind the urokinase plasminogen activator receptor.

Icosahedral virus-like particles formed by the self-assembly of polyomavirus capsid proteins (Py-VLPs) can serve as useful nanostructures for delivering nucleic acids, proteins, and pharmaceuticals into animal cells and tissues. Four predominant surface-exposed loops in the VP1 structure offer potential sites to display sequences that might contribute new targeting specificities. Introduction into each of these loops of sequences derived from the amino-terminal fragment of urokinase plasminogen activator (uPA) or a related phage display peptide reduced the solubility of VP1 molecules when expressed in insect cells, and insertions into the EF loop reduced VP1 solubility least. Coexpression in insect cells of the uPA-VP1 molecules and VP1 containing a FLAG epitope in the HI loop permitted the formation of heterotypic Py-VLPs containing uPA-VP1 and FLAG-VP1. These heterotypic VLPs bound to uPAR on the surfaces of animal cells. Heterotypic Py-VLPs containing ligands for multiple cell surface receptors should be useful for targeting specific cells and tissues.

Enzymatic E-colicins Bind to Their Target Receptor BtuB by Presentation of a Small Binding Epitope on a Coiled-coil Scaffold

Toxins and viruses often initiate their attacks by binding to specific proteins on the surfaces of target cells. Bacterial toxins (e.g. bacteriocins) and viruses (bacteriophages) targeting Gram-negative bacteria typically bind to outer membrane proteins. Bacterial E-colicins target Escherichia coli by binding to the outer membrane cobalamin transporter BtuB. Colicins are tripartite molecules possessing receptor-binding, translocation, and toxin domains connected by long coiled-coil alpha-helices. Surprisingly, the crystal structure of colicin E3 does not possess a recognizable globular fold in its receptor-binding domain. We hypothesized that the binding epitope of enzymatic E-colicins is a short loop connecting the two alpha-helices that comprise the coiled-coil region and that this flanking coiled-coil region serves to present the loop in a binding-capable conformation. To test this hypothesis, we designed and synthesized a 34-residue peptide (E-peptide-1) corresponding to residues Ala366-Arg399 of the helix-loop-helix region of colicin E3. Cysteines placed near the ends of the peptide (I372C and A393C) enabled crosslinking for reduction of conformational entropy and formation of a peptide structure that would present the loop epitope. A fluorescent analog was also made for characterization of binding by measurement of fluorescence polarization. Our analysis shows the following. (i). E-peptide-1 is predominantly random coil in aqueous solution, but disulfide bond formation increases its alpha-helical content in both aqueous buffer and solvents that promote helix formation. (ii). Fluorescein-labeled E-peptide-1 binds to purified BtuB in a calcium-dependent manner with a Kd of 43.6 +/- 4.9 nm or 2370 +/- 670 nm in the presence or absence of calcium, respectively. (iii). In the presence of calcium, cyanocobalamin (CN-Cbl) displaces E-peptide-1 with a nanomolar inhibition constant (Ki = 78.9 +/- 5.6 nm). We conclude that the BtuB binding sites for cobalamins and enzymatic E-colicins are overlapping but inequivalent and that the distal loop and (possibly) the short alpha-helical flanking regions are sufficient for high affinity binding.

Analysis of the mechanism by which the small-molecule CCR5 antagonists SCH-351125 and SCH-350581 inhibit human immunodeficiency virus type 1 entry.

Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the consecutive interaction of the envelope glycoprotein gp120 with CD4 and a coreceptor such as CCR5 or CXCR4. The CCR5 coreceptor is used by the most commonly transmitted HIV-1 strains that often persist throughout the course of infection. Compounds targeting CCR5-mediated entry are a novel class of drugs being developed to treat HIV-1 infection. In this study, we have identified the mechanism of action of two inhibitors of CCR5 function, SCH-350581 (AD101) and SCH-351125 (SCH-C). AD101 is more potent than SCH-C at inhibiting HIV-1 replication in primary lymphocytes, as well as viral entry and gp120 binding to cell lines. Both molecules also block the binding of several anti-CCR5 monoclonal antibodies that recognize epitopes in the second extracellular loop of CCR5. Alanine mutagenesis of the transmembrane domain of CCR5 suggests that AD101 and SCH-C bind to overlapping but nonidentical sites within a putative ligand-binding cavity formed by transmembrane helices 1, 2, 3, and 7. We propose that the binding of small molecules to the transmembrane domain of CCR5 may disrupt the conformation of its extracellular domain, thereby inhibiting ligand binding to CCR5.

Localization of the GSH-dependent photolabelling site of an agosterol A analog on human MRP1.

1. Human multidrug resistance protein 1 (MRP1) is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. We recently demonstrated that glutathione (GSH) is required for the labelling of the C-terminal half of MRP1 with a photoanalog of agosterol A (azido AG-A). In this study, we further characterized the GSH-dependent photolabelling site of azido AG-A on MRP1. 2. An epitope-inserted MRP1, MRP1 1222HA, which has two hemagglutinin A (HA) epitopes in the extracellular loop between transmembrane segment (TM) 16 and TM17 of the transporter, could bind azido AG-A in a GSH-dependent manner. 3. Protease digestion of the photolabelled MRP1 1222HA, followed by immunoprecipitation with an anti-HA antibody suggested that the GSH-dependent azido AG-A photolabelling site on MRP1 resides in the region within TM14-17 and the cytoplasmic region proximate to the C-terminus of TM17. 4. Arg(1210) in human MRP2 that corresponds to Arg(1202) in human MRP1 has an important role in the transporting activity of MRP2. Therefore, we replaced the Arg residue at position 1202 of MRP1 with Gly. Whereas photolabelling of the mutant MRP1 R1202G was greatly reduced, it retained leukotriene C(4) (LTC(4)) transport activity and conferred Vincristine resistance in LLC-PK1 cells. 5. In summary, this study demonstrated that the GSH-dependent azido AG-A photolabelling site on MRP1 resides in the region within TM14-17 and the cytoplasmic region proximate to the C-terminus of TM17. The charged amino acid Arg(1202) proximate to TM helix 16 is of critical importance for the GSH-dependent photolabelling of MRP1 with azido AG-A. Arg(1202) itself or the region nearby Arg(1202) may be involved in azido AG-A photolabelling.

Functional fine-mapping and molecular modeling of a conserved loop epitope of the measles virus hemagglutinin protein.

Neutralizing and protective monoclonal antibodies (mAbs) were used to fine-map the highly conserved hemagglutinin noose epitope (H379-410, HNE) of the measles virus. Short peptides mimicking this epitope were previously shown to induce virus-neutralizing antibodies [El Kasmi et al. (2000) J. Gen. Virol.81, 729-735]. The epitope contains three cysteine residues, two of which (Cys386 and Cys394) form a disulfide bridge critical for antibody binding. Substitution and truncation analogues revealed four residues critical for binding (Lys387, Gly388, Gln391 and Glu395) and suggested the binding motif X7C[KR]GX[AINQ]QX2CEX5 for three distinct protective mAbs. This motif was found in more than 90% of the wild-type viruses. An independent molecular model of the core epitope predicted an amphiphilic loop displaying a remarkably stable and rigid loop conformation. The three hydrophilic contact residues Lys387, Gln391 and Glu395 pointed on the virus towards the solvent-exposed side of the planar loop and the permissive hydrophobic residues Ile390, Ala392 and Leu393 towards the solvent-hidden side of the loop, precluding antibody binding. The high affinity (Kd = 7.60 nm) of the mAb BH216 for the peptide suggests a high structural resemblance of the peptide with the natural epitope and indicates that most interactions with the protein are also contributed by the peptide. Improved peptides designed on the basis of these findings induced sera that crossreacted with the native measles virus hemagglutinin protein, providing important information about a lead structure for the design of more stable antigens of a synthetic or recombinant subunit vaccine.

A 'first loop' linear epitope accessible on native hepatitis B surface antigen that persists in the face of 'second loop' immune escape.

Murine monoclonal antibodies (mAbs) were raised following immunization with native mutant hepatitis B surface antigen (HBsAg) purified from human sera. A set of antibodies binding to a linear epitope carried between residues 121 and 129 of the s region was demonstrated. These antibodies were shown by cross-competition assays to bind to a single epitope whose antigenicity was influenced by the TTP motif lying between residues 125 and 127. This first loop epitope remained accessible on the surface of HBsAg in spite of major second loop mutations abrogating the normal a conformational epitopes. The mAb and its binding region in the first loop are important diagnostically and may represent an importance immunological target, one that is stable in the face of immunologically driven escape.

Allelic genes of blood group antigens: a source of human mutations and cSNPs documented in the Blood Group Antigen Gene Mutation Database.

In this report, we analyze data assembled in the Blood Group Antigen Gene Mutation Database (www.bioc.aecom.yu.edu/bgmut/index.htm), which describes sequence information on human genes associated with expression of the various serologically-determined blood group phenotypes. The database documents 38 genetic loci and a total of 624 alleles that together encode a large repertoire of proteins and constitute 27 serologically-defined blood group systems. Analysis of sequence variation patterns across alleles of a number of genes is focused on their molecular profiles, including mutational sites and recurrence, patterns of gene rearrangements in duplicated gene families, correlation of predicted location of epitopes in extracellular loops with sites of alterations, and effects of mutations on protein expression. That information, and the relative ease of identifying individuals bearing variant alleles, has led to the proposal that genes encoding blood group antigens are an important and unique resource for studies of human DNA variation. Another focus is on mutations in regions that encode the antigenic epitopes and on their occurrence in world populations. These mutations may be viewed as coding single nucleotide polymorphisms (cSNPs). We propose that one group of these cSNPs, which are known to occur with significant frequency in all world populations, could serve as well-validated genetic markers. In addition, specific mutations in a number of "low incidence" and rare alleles could serve as cSNPs specific for a given population. The allelic frequencies of these mutations and knowledge of their world-wide occurrence add a valuable dataset to the existing cSNP pools documented in SNP databases.

Role of neutralizing antibodies in HIV infection.

Role of neutralizing antibodies in HIV infection.

Unique monoclonal antibody recognizing the third extracellular loop of CXCR4 induces lymphocyte agglutination and enhances human immunodeficiency virus type 1-mediated syncytium formation and productive infection.

To increase insight into the structural basis of CXCR4 utilization in human immunodeficiency virus type 1 (HIV-1) infection, a new generation of three monoclonal antibodies (MAbs) was developed in WKA rats. The A80 MAb, which binds an epitope in the third extracellular loop (ECL3) of CXCR4, has unique biologic properties that provide novel insights into CXCR4 function. This agent enhanced syncytium formation in activated human peripheral blood mononuclear cells (PBMC) infected with X4 or R5 and CEM cells infected with X4 HIV-1 strains. Exposure to A80 increased the productive infection of activated CD4(+) T cells and CEM cells with R5 and X4 viruses, respectively. This antibody uniquely induced agglutination of PBMC and CEM cells but did not activate calcium mobilization. Agglutination induced by A80 was inhibited by stromal cell-derived factor 1, T22, and phorbol 12-myristate 13-acetate but was not significantly altered by pretreatment of cells with pertussis toxin, wortmannin, or MAbs to LFA-1, ICAM-1, ICAM-2, and ICAM-3. The binding of the A145 and A120 MAbs was mapped to the N-terminal extracellular domain and a conformational epitope involving ECL1 and ECL2, respectively. Both of these MAbs inhibited HIV-1 infection and lacked the novel properties of A80. These results suggest a new role for CXCR4 in homologous lymphocyte adhesion that is ligand independent and in HIV-1 infection.

Enhanced CD8 + T cell immune response against a V3 loop multi-epitope polypeptide (TAB13) of HIV-1 Env after priming with purified fusion protein and booster with modified vaccinia virus Ankara (MVA-TAB) recombinant: a comparison of humoral and cellular immune responses with the vaccinia virus Western Reserve (WR) vector

The humoral and cytotoxic T-lymphocyte (CTL) responses have been shown to be determinant in the clearance of many viral infections and because of those characteristics, vaccine candidates against AIDS are designed to enhance both arms of the immune system. While a protocol of immunization able to confer protection in humans against HIV will have to await the results of current clinical trials, it remains important to identify protocols of immunization in animals that achieve significant levels of humoral and cellular immune responses to HIV. In this study we have carried out a comparative analysis of the immune responses elicited in mice immunized with recombinants based on the modified vaccinia virus Ankara strain (rMVA) versus the Western Reserve strain (WR) of vaccinia virus (rVV), both expressing a V3 loop multi-epitopic protein from eight different HIV isolates (TAB13). We found that during priming, rMVA elicited a two- to three-fold higher specific CD8 + T cell response than rVV. Similar enhancement was observed during priming with purified protein TAB13 followed by a booster with rMVA. The epitopes LR150, MN and IIIB, located at the ends and in the middle of the chimeric protein, were able to induce a specific CD8 + T cell response, both after priming or prime/booster with the recombinant viruses but not after prime/booster with TAB13. By examining the cytokine pattern, the immune response triggered by these vectors was of Th-1 type. Humoral immune responses were higher in animals immunized with TAB13/TAB13 or TAB13/rVV than in animals immunized with TAB13/rMVA. These findings demonstrate that during priming or in a prime/booster immunizations, rMVA is superior to rVV in the ability to enhance specific cellular responses to an HIV-1 protein, and that both humoral and cellular immune responses to theV3 loop epitope of HIV-1 Env can be obtained by priming with TAB13 followed by a booster with viral vectors.

Ligand Binding Characteristics of CXCR4 Incorporated into Paramagnetic Proteoliposomes

The G protein-coupled receptor CXCR4 is a coreceptor, along with CD4, for the human immunodeficiency virus type 1 (HIV-1) and has been implicated in breast cancer metastasis. We studied the binding of the HIV-1 gp120 envelope glycoprotein (gp) to CXCR4 but found that the gp120s from CXCR4-using HIV-1 strains bound nonspecifically to several cell lines lacking human CXCR4 expression. Therefore, we constructed paramagnetic proteoliposomes (CXCR4-PMPLs) containing pure, native CXCR4. CXCR4-PMPLs specifically bound the natural ligand, SDF-1alpha, and the gp120s from CXCR4-using HIV-1 strains. Conformation-dependent anti-CXCR4 antibodies and the CXCR4 antagonist AMD3100 blocked HIV-1 gp120 binding to CXCR4-PMPLs. The gp120-CXCR4 interaction was blocked by anti-gp120 antibodies directed against the third variable (V3) loop and CD4-induced epitopes, structures that have also been implicated in the binding of gp120 to the other HIV-1 coreceptor, CCR5. Compared with the binding of R5 HIV-1 gp120s to CCR5, the gp120-CXCR4 interaction exhibited a lower affinity (K(d) = 200 nm) and was dependent upon prior CD4 binding, even at low temperature. Thus, although similar regions of X4 and R5 HIV-1 gp120s appear to be involved in binding CXCR4 and CCR5, respectively, differences exist in nonspecific binding to cell surfaces, affinity for the chemokine receptor, and CD4 dependence at low temperature.

The principle of delivery of T cell epitopes to antigen-presenting cells applied to peptides from influenza virus, ovalbumin, and hen egg lysozyme: implications for peptide vaccination.

Targeting of antigens to antigen-presenting cells (APCs) increases CD4(+) T cell activation, and this observation can be exploited in the development of new vaccines. We have chosen an antigen-targeting approach in which we make recombinant antibodies (Abs) with T cell epitopes in their constant region and APC-specific variable regions. Three commonly used model epitopes, amino acids 110-120 of hemagglutinin, 323-339 of ovalbumin, and 46-61 of hen egg lysozyme, were introduced as loops in the C(H)1 domain of human IgG3. For all three epitopes, we show that the recombinant molecules are secreted from transfected cells. The epitopes are presented to specific T cells, and targeting to IgD on B cells in vitro enhances the presentation efficiency by 10(4) to 10(5) compared with the free peptide. After i.v. injection, the epitopes targeted to IgD are presented by splenic APCs to activate specific T cells, whereas little or no activation could be detected without targeting, even after the amount of antigen injected was increased 100-fold or more. Because a wide variety of T cell epitopes, in terms of both length and secondary structure, can be tolerated in loops in constant domains of Abs, the Ab constant region seems to have the intrinsic stability that is needed for this fusion molecule strategy. It might thus be possible to load the Ab with several different epitopes in loops in different domains and thereby make a targeted multisubunit vaccine.

Effect of soluble CD4 on exposure of epitopes on primary, intact, native human immunodeficiency virus type 1 virions of different genetic clades.

We have used a virus-binding assay to examine conformational changes that occur when soluble CD4 (sCD4) binds to the surface of intact, native, primary human immunodeficiency virus type 1 virions. The isolates examined belong to seven genetic clades (A to H) and are representative of syncytium-inducing and non-syncytium-inducing phenotypes. Conformational changes in epitopes in the C2, V2, V3, C5, and CD4 binding domain (CD4bd) of gp120 and the cluster I and II regions of gp41 of these viruses were examined using human monoclonal antibodies that are directed at these regions. The studies revealed that sCD4 binding causes a marked increase in exposure of epitopes in the V3 loop, irrespective of the clade or the phenotype of the virus. Sporadic increases in exposure were observed in some epitopes in the V2 region, while no changes were observed in the C2, C5, or CD4bd of gp120 or the cluster I and II regions of gp41.