Dissection of the humoral immune response toward an immunodominant epitope of HIV: a model for the analysis of antibody diversity in HIV+ individuals.

Understanding the dynamics of the humoral immune response to HIV epitopes in the presence of genetic drift and antigenic variation of the virus may reveal critical elements of protective immunity against HIV. Analysis of antibody maturation and diversity is difficult to study at the molecular level in humans. We used a combinatorial phage display peptide library to elucidate antibody diversity in HIV-infected individuals to a single immunodominant epitope in gp41. A serum sample derived from an HIV+ individual was used to screen a phage display a 12 mer cysteine-constrained loop peptide library. In doing so, we isolated mimotope-presenting phages corresponding to the immunodominant gp41 epitope CSGKLIC (residues 603-609). The mimotopes and control phages expressing epitope variants were reacted with a panel of 30 HIV+ sera. The patients showed distinct and variable recognition patterns compared with one another. Subfractions of the polyclonal sera were affinity purified and analyzed for epitope specificities. These analyses illustrated that epitope variants can be used to decipher antibody diversity. Elucidation of the plasticity of the humoral response and its polyclonality toward discrete epitopes contributes to our understanding of the antibody maturation process in individuals infected with viruses such as HIV.

The rational design of a 'type 88' genetically stable peptide display vector in the filamentous bacteriophage fd.

Filamentous bacteriophages are particularly efficient for the expression and display of combinatorial random peptides. Two phage proteins are often employed for peptide display: the infectivity protein, PIII, and the major coat protein, PVIII. The use of PVIII typically requires the expression of two pVIII genes: the wild-type and the recombinant pVIII gene, to generate mosaic phages. 'Type 88' vectors contain two pVIII genes in one phage genome. In this study a novel 'type 88' expression vector has been rationally designed and constructed. Two factors were taken into account: the insertion site and the genetic stability of the second pVIII gene. It was found that selective deletion of recombinant genes was encountered when inserts were cloned into either of the two non-coding regions of the phage genome. The deletions were independent of recA yet required a functional F-episome. Transcription was also found to be a positive factor for deletion. Taking the above into account led to the generation of a novel vector, designated fth1, which can be used to express recombinant peptides as pVIII chimeric proteins in mosaic bacteriophages. The fth1 vector is not only genetically stable but also of high copy number and produces high titers of recombinant phages.

Use of human CD4 transgenic mice for studying immunogenicity of HIV-1 envelope protein gp120.

HIV-1 envelope protein, gp120, is a major immunogenic protein of the AIDS virus. A specific feature of this protein is its interaction with the receptor protein, human CD4, an important component of the immune system. This interaction might affect the immunogenic properties of the gp 120 and modulate the immune response towards HIV. To test this hypothesis we used human CD4-transgenic mice for immunization with gp120. The dynamics of the immune response towards gp120, CD4 and other proteins was followed. The results show that the primary immune response to gp120 (two weeks) developed somewhat faster in CD4-transgenic mice versus non-transgenic mice. Both animals, however, ultimately mounted the same level of response over time. The primary immune response to gp120 when complexed with soluble CD4 before the immunization, developed similarly in both groups. The secondary immune response was earlier and markedly stronger in non-transgenic mice compared with the transgenic mice where a less efficient memory response to gp120 was observed. The ability of gp120 to directly interact with CD4+ helper lymphocytes appears to affect the humoral response towards this antigen. Moreover, these effects illustrate how viral modulation of these cells may in turn lead to potentially different states of immunological equilibrium.

Expansion of epitope cross-reactivity by anti-idiotype modulation of the primary humoral response.

The primary humoral response produces antigen-specific antibodies so to clear the initial infection, and generates a population of corresponding memory cells to prevent infection by future encounters with the same pathogen. The continuous genetic modification of a pathogen's exterior, however, is one mechanism used to evade the immune defenses of its host. Here we describe a novel means, involving anti-idiotypic antibodies, by which the host can counteract such pathogen genetic alterations by modulation of its primary humoral response. An autoimmune response against primary antibodies, Ab1's, creates anti-idiotypic antibodies (Ab2's), some of which (designated Ab2alpha) are able to bind the Ab1/antigen complex. We have discovered that binding of Ab2alpha to its corresponding Ab1 can expand Ab1's ability to bind variations of its antigen. This expanded epitope cross-reactivity is shown not only to increase the binding activity of Ab1 but also its ability to neutralize a variant infectious virus. MAb M77 is an Ab1, which is highly strain-specific for the HIV-1 envelope protein gp120(IIIB). This Ab1 can be rendered cross-reactive and neutralizing for an otherwise resistant HIV strain by its interaction with a unique anti-idiotypic Ab2alpha (GV12). Furthermore, molecular characterization of this expanded cross-reactivity was accomplished using combinatorial phage display peptide libraries.

Distribution of the CCR5 gene 32-base pair deletion in Israeli ethnic groups.

The discovery of inhibition of HIV-1 by selected chemokines and their receptors instills hope in AIDS researchers, especially because a 32-bp deletion in the chemokine receptor CCR5 (delta32-CCR5) provides resistance to HIV infection. A recent report found that the highest delta32-CCR5 frequency is among Ashkenazi Jews (20.93%). In the present study, we have determined by PCR the allelic frequency of delta32-CCR5 in 520 individuals representing a spectrum of ethnic groups living in Israel. The samples were obtained from the Israeli National Laboratory of Genetic Diversity. Our results showed that Ashkenazi Jews, as to be expected, have the highest frequency (10.19%), yet not significantly higher than that which has been reported for whites of European decent. Other ethnic groups, North African Jews, non-Jews, Middle Eastern Jews, and Ethiopian Jews, gave allelic frequencies of 2.08, 1.35, 1.15, and 0, respectively. Thus, the delta32-CCR5 mutation is found in Jews with the same allelic frequency as that found for residents of their countries of origin. Therefore, it appears that the delta32-CCR5 allele has been introduced into Jewish communities world wide through intermarriage and genetic drift.

Prevalence of a CCR5 gene 32-bp deletion in an Israeli cohort of HIV-1-infected and uninfected hemophilia patients.

OBJECTIVE: The recently discovered connection of chemokines and their receptors to HIV pathogenesis, and the description of the 32-bp deletion in the CCR5 gene (delta 32 CCR5), led to heightened excitement and numerous reports regarding their role in HIV transmission and disease progression. The populations in most of these reports, except for one, consisted of homosexual men. Our objective was to investigate the significance of delta 32 CCR5 in hemophilia patients in Israel. STUDY DESIGN/METHODS: We have determined by polymerase chain reaction (PCR) the prevalence of delta 32 CCR5 in 34 HIV-seropositive Israeli patients with hemophilia A and compared them with a control group of 42 HIV-seronegative hemophilia patients. RESULTS: Thirteen heterozygotes were identified among the 76 hemophilia patients tested (allelic frequency, 8.5%), 5 (14.7%) among the HIV-seropositive patients, and 8 (19%) among the noninfected. CONCLUSIONS: No protective advantage to delta 32 CCR5 heterozygosity was seen as far as infection with HIV is concerned. However, a trend of a slower progression to AIDS in delta 32 CCR5 heterozygotes compared with wild-type homozygotes may be apparent, although no absolute correlation could be made.

Enhancement of human immunodeficiency virus type 1 envelope-mediated fusion by a CD4-gp120 complex-specific monoclonal antibody.

The entry of human immunodeficiency virus type 1 (HIV-1) into cells is initiated by binding of the viral glycoprotein gp120-gp41 to its cellular receptor CD4. The gp120-CD4 complex formed at the cell surface undergoes conformational changes that may allow its association with an additional membrane component(s) and the eventual formation of the fusion complex. These conformational rearrangements are accompanied by immunological changes manifested by altered reactivity with monoclonal antibodies specific for the individual components and presentation of new epitopes unique to the postbinding complex. In order to analyze the structure and function of the gp120-CD4 complex, monoclonal antibodies were generated from splenocytes of BALB/c mice immunized with soluble CD4-gp120 (IIIB) molecules (J. M. Gershoni, G. Denisova, D. Raviv, N. I. Smorodinsky, and D. Buyaner, FASEB J. 7:1185-1187 1993). One of those monoclonal antibodies, CG10, was found to be strictly complex specific. Here we demonstrate that this monoclonal antibody can significantly enhance the fusion of CD4+ cells with effector cells expressing multiple HIV-1 envelopes. Both T-cell-line-tropic and macrophage-tropic envelope-mediated cell fusion were enhanced, albeit at different optimal doses. Furthermore, infection of HeLa CD4+ (MAGI) cells by HIV-1 LAI, ELI1, and ELI2 strains was increased two- to fourfold in the presence of CG10 monoclonal antibodies, suggesting an effect on viral entry. These findings indicate the existence of a novel, conserved CD4-gp120 intermediate structure that plays an important role in HIV-1 cell fusion.

Helical epitopes determined by low-stringency antibody screening of a combinatorial peptide library.

Combinatorial phage display peptide libraries are routinely used to map epitopes of specific monoclonal antibodies. In this study we illustrate that these libraries can be used in the analysis of protein structure. By screening libraries at low stringency, a collection of phages can be obtained. These are characterized by the fact that they are recognized by a given monoclonal antibody yet with various affinities. Comparing the random peptides of these phages indicates the common essential residues necessary for antibody recognition. Aligning the inserts based on the detected homology has revealed structural motifs that correspond to secondary protein structures. The envelope protein of HIV-1 has been studied using this approach. A combinatorial phage display library containing a 20 mer random peptide in protein III of the filamentous phage fd-tet has been used to analyze two different monoclonal antibodies directed against gp120. Our results provide experimental evidence that indicate that the C1 domain of gp120 contains an alpha helix.

Conformational transitions in CD4 due to complexation with HIV envelope glycoprotein gp120.

The binding of the surface envelope glycoprotein gp120 to its receptor, CD4, has been well characterized and is the primary basis for the cell tropism of HIV. In this study, the interaction between recombinant soluble CD4 and native membrane-associated CD4 with gp120 is probed by the use of mAbs. Complexation of gp120 with both forms of CD4 induces conformational epitopes that can be defined with specific mAbs. CG1, CG7, and CG8 are three novel mAbs that have a distinct preference for CD4 complexed over noncomplexed with gp120. The epitopes of these unique mAbs were mapped by cross-inhibition with previously characterized mAbs to a region encompassing the CDR2 and CDR3 loops in domain 1 of CD4. Systematic analysis of CG mAbs binding to CD4 and CD4/gp120 complex delineates a region in the D1 domain of CD4 that undergoes conformational rearrangements upon gp120 binding to its receptor.

Humoral immune response to immunocomplexed HIV envelope glycoprotein 120.

To further our understanding of the nature of HIV-1 immunogenicity, we injected mice with the virus envelope protein gp120 in different configurations: free, complexed with its receptor CD4, and as an immunocomplex with a monoclonal antibody directed against the V3 loop of the protein. Analyses of the polyclonal sera, as well as of monoclonal antibodies produced in each case, allowed us to conclude that the quality of the humoral immune response depended on the complexation state of the antigen. For the free gp120 and gp120-CD4 complex the responses were directed mainly toward conformational epitopes. However, gp120 immunocomplexed with anti-V3 loop Mab produced, in addition, numerous MAbs directed toward linear epitopes. Epitopes were mapped using immunoblots of gp120 cleaved with S. aureus V8 protease and a combinatorial epitope phage-display library. It was found that some of the linear epitopes had been previously identified as T cell epitopes. These results suggest that the immunocomplexed gp120 may be particularly well taken up by antigen-presenting cells, leading to the processing of the gp120 and the efficient presentation of T cell epitopes. Thus immunocomplexation should afford a means for enhancing the immunogenicity of gp120 and improving its presentation.

Nuclear magnetic resonance (NMR) analysis of ligand receptor interactions: the cholinergic system--a model.

Elucidation of the molecular mechanisms that govern ligand-receptor recognition is essential to the rational design of specific pharmacological reagents. Whereas often the receptor and its binding site are the target of investigation, study of the ligand in its free and bound state can also reveal important information regarding this recognition process. Nuclear magnetic resonance (NMR) spectroscopy can be extremely useful for such studies. In this review, we discuss the attributes of NMR in the study of ligand receptor interactions. The cholinergic receptor and its binding to the neurotransmitter, acetylcholine, and cholinergic antagonists serve as a model system, illustrating the power of ligand analysis by NMR. The results discussed prove that the region of residues alpha 180-205 of the nicotinic acetylcholine receptor are an essential component of the cholinergic binding site and that ligand binding involves a positively charged hydrophobic motif.

Linker modification introduces useful molecular instability in a single chain antibody.

SCA 4-4-20/212, is a recombinant single chain antibody directed against fluorescein (Fl) composed of the variable light (VL) and variable heavy (VH) domains of the monoclonal antibody 4-4-20, tethered by a 14 amino acid linker. Binding of SCA 4-4-20/212 to Fl quenches its fluorescence, thus enabling the distinction between bound and free Fl. This was used to follow antibody denaturation which followed a two-step process: rapid selected and restricted denaturation followed by slow and progressive denaturation. This two-phase phenomenon might reflect selective susceptibility of the CDR loops to denaturation. Furthermore, a new SCA, SCA 4-4-20/9, was constructed by site-directed mutagenesis of SCA 4-4-20/212 using PCR methodology. SCA 4-4-20/9 was similar to SCA 4-4-20/212, but for a nine residue linker. The two SCAs were compared for Fl binding, heat stability, the effect of denaturing agents and susceptibility to proteolysis. The modification of the linker caused a general conformational rearrangement in the SCA molecule, rendering it more sensitive to denaturation and proteolysis. This molecular instability may find utility in the application of SCAs in analytical systems or as the recognition component in biosensors.

The hemagglutinin envelope protein of canine distemper virus (CDV) confers cell tropism as illustrated by CDV and measles virus complementation analysis.

Measles virus (MV) and canine distemper virus (CDV) are morbilliviruses that cause acute illnesses and several persistent central nervous system infections in humans and in dogs, respectively. Characteristically, the cytopathic effect of these viruses is the formation of syncytia in permissive cells. In this study, a vaccinia virus expression system was used to express MV and CDV hemagglutinin (HA) and fusion (F) envelope proteins. We found that cotransfecting F and HA genes of MV or F and HA genes of CDV resulted in extensive syncytium formation in permissive cells while transfecting either F or HA alone did not. Similar experiments with heterologous pairs of proteins, CDV-F with MV-HA or MV-F with CDV-HA, caused significant cell fusion in both cases. These results indicate that in this expression system, cell fusion requires both F and HA; however, the functions of these proteins are interchangeable between the two types of morbilliviruses. Human-mouse somatic hybrids were used to determine the human chromosome conferring susceptibility to either MV and CDV. Of the 12 hybrids screened, none were sensitive to MV. Two of the hybrids containing human chromosome 19 formed syncytia following CDV infection. In addition, these two hybrids underwent cell fusion when cotransfected with CDV-F and CDV-HA (but not MV-F and MV-HA) glycoproteins by using the vaccinia virus expression system. To discover the viral component responsible for cell specificity, complementation experiments coexpressing CDV-HA with MV-F or CDV-F with MV-HA in the CDV-sensitive hybrids were performed. We found that syncytia were formed only in the presence of CDV-HA. These results support the idea that the HA protein is responsible for cell tropism. Furthermore, while the F protein is necessary for the fusion process, it is interchangeable with the F protein from other morbilliviruses.

Biased random mutagenesis of peptides: determination of mutation frequency by computer simulation.

Cassette mutagenesis is a method of protein engineering which generates a wide diversity of genetic variants that can be subjected to either selection or screening. As long as the target sequence to be modified is kept short (corresponding to four to six amino acids), complete combinatorial libraries can be produced. A major problem arises when longer peptides are to be engineered for desired functions. In such situations the production of a limited collection of variants can be helpful; thus, biased random mutagenesis and 'doping schemes' have been reported previously. Here we describe a computer algorithm that enables the determination of the degree of phosphoramidite contamination of nucleotide precursor reservoirs. Through simulation of biological translation, the algorithm allows the prediction of the effect of contamination levels on the number of mutations to occur for any given peptide sequence. In this study the cholinergic binding site was used as a model sequence (22 amino acids). Considerations, based on the computer program, are discussed regarding the efficient design of phage-display combinatorial libraries.

Binding of HIV-1 gp120 to an anti-V3 loop antibody reveals novel antigen-induced epitopes.

Antigen association to its corresponding binding site in the immunoglobulin molecule can elicit conformational rearrangements, generating novel epitopes termed metatopes. Such metatopes were characterized for the immunocomplex between the AIDS virus envelope protein, gp120, and M77, a mAb directed against the V3 loop. Five novel mAbs were described (GV1, GV3, GV7, GV8, and GV12). These mAbs were found to bind epitopes harbored in the M77 Fab fragment. Binding to the epitopes was shown to require the complexation of Fab with its antigen. The degree of this antigen requirement was found to be variable for the different mAbs and also for the state of IgG fragmentation. Binding of GV12 to its antigen increased the affinity of M77 for gp120. Moreover, in the presence of GV12, M77 acquired extended cross-reactivity for a second gp120 variant, namely BaL. These results could indicate a novel approach towards improving the performance of anti-HIV antibodies.

Production of linear polymers of HIV gp120-binding domains.

It has been demonstrated previously that molecular decoys of the acetylcholine receptor have therapeutic efficacy as antitoxins [Gershoni, J. and Aronheim, A. (1988) Proc. Natl Acad. Sci. USA, 85, 4087-4089], but surely a most challenging goal is to apply this approach towards the development of antiviral drugs. As viruses present multiple copies of their envelope proteins, it was proposed that polyvalent decoys could be advantageous. Here we report the design and expression of recombinant linear polymers of the HIV gp120-binding domains which are situated within the T-cell membrane protein CD4. Whereas the production of linear concatemers of CD4 variable domains is feasible, a number of conformational constraints must be considered when designing a polymeric molecule which retains biological function. Most significant is the contribution of domains flanking the binding site that apparently enable correct folding of the latter.