Antigenic Mimics Research Articles

Selection of HIV-Specific Immunogenic Epitopes by Screening Random Peptide Libraries with HIV-1-Positive Sera

Efforts to develop a protective HIV-1 vaccine have been hindered by difficulties in identifying epitopes capable of inducing broad neutralizing Ab responses. In fact, the high mutation rate occurring in HIV-1 envelope proteins and the complex structure of gp120 as an oligomer associated with gp41 result in a high degree of antigenic polymorphism. To overcome these obstacles, we screened random peptide libraries using sera from HIV-infected subjects to identify antigenic and immunogenic mimics of HIV-1 epitopes. After extensive counterscreening with HIV-negative sera, we isolated peptides specifically recognized by Abs from HIV-1-infected individuals. These peptides behaved as antigenic mimics of linear or conformational HIV-1 epitopes generated in vivo in infected subjects. Consistent with these findings, sera of simian HIV-infected monkeys also recognized the HIV-specific epitopes. The selected peptides were immunogenic in mice, where they elicited HIV-specific Abs that effectively neutralized HIV-1 isolates. These results demonstrate that pools of HIV-1 mimotopes can be selected from combinatorial peptide libraries by taking advantage of the HIV-specific Ab repertoire induced by the natural infection.

Dual specificity of a human neutralizing monoclonal antibody, specific for the V3 loop of GP120 (HIV-1)

Neutralizing antibodies specific for the third variable (V3) domain of gp120, the HIV-1 surface envelope protein, appear early in infection. However, they are usually highly specific for the priming isolate. To identify potential mimotopes of the V3 domain, we have screened a hexapeptide phage library with a human neutralizing mAb, mAb 268, specific for the V3 loop of the viral MN isolate. We have identified two groups of sequences. Within the first group, sequence 268-1 reproduces the linear epitope identified using a conventional epitope mapping approach. The sequence 268-1, H L G P G R, corresponds to amino acids 315–320, localized in the highly conserved tip of the V3 loop. A second group of sequences was identified, including sequence 268-2, K A I H R I. Partial homology with a more variable region of the V3 loop can be found. Using synthetic peptides, we demonstrated that peptides, 268-1 and 268-2, both interact with the same binding site as the V3 region on the 268 mAb. Moreover, both peptides can inhibit the interaction of the 268 mAb with the original immunogen, gp120MN. Peptide 268-1 can compete with peptide 268-2, albeit poorly, for binding of the 268 mAb. When injected into rabbits, KLH conjugated peptide 268-2 elicited antibodies that interact specifically with the initial immunogen gp120MN. These data suggest that peptide 268-2 is both an antigenic and immunogenic mimic of the natural antigen, gp120MN.

The Varicella-Zoster Virus and Multiple Sclerosis

This article is a review of the evidence suggesting a unique relationship between the varicella-zoster virus (as a possible antigen or antigen mimic) and multiple sclerosis (MS). Both MS and varicella have increased prevalences in temperate zones and both are rare in countries closer to the equator. Migration studies suggest an infectious agent acquired prior to age 14 plays a role in the risk of subsequent MS. Hutterites, who educate their children at home, have less varicella, MS, and herpes zoster than their neighbors and have the appropriate reduced varicella-zoster seropositivity matching these clinical observations. Paradoxically, patients with MS report more herpes zoster, and at an earlier age and more often, than a group of non-MS patients.

Do chickens immunized with bacterial immunoglobulin G-binding proteins produce rheumatoid factor-like antibodies?

An association between the production of rheumatoid factor (RF)-like antibodies in animals immunized with bacterial immunoglobulin (Ig)G-binding proteins has been noted. Three potential explanations have been proposed: (1) altered host IgG due to binding of the immunogen; (2) B-cell superantigenic properties of the binding proteins; and (3) idiotype-anti-idiotype response leading to an antibody which acts as an antigen mimic. In order to distinguish among these possibilities, it is necessary to carry out studies in animals whose IgG does not react with the IgG-binding protein immunogen. Consequently, we have determined the effects of immunizing chickens with a purified group C streptococcal IgG-binding protein, FcRc, a bacterium expressing this protein, and appropriate control immunogens. The results of these studies provided evidence for production of specific antibodies to FcRc in groups of chickens immunized with either the pure protein or bacteria expressing the protein. No significant association with production of RF-like antibodies was noted, favouring the altered IgG-binding explanation for the association between RF-like antibodies and immunization with the bacterial IgG-binding proteins.

Three-dimensional structure of a human Fab with high affinity for tetanus toxoid

Background: The wide range of antibody specificity and affinity results from the differing shapes and chemical compositions of their binding sites. These shapes range from discrete grooves in antibodies elicited by linear oligomers of nucleotides and carbohydrates to shallow depressions or flat surfaces for accommodation of proteins, peptides and large organic compounds. Objectives: To determine the Fab structure of a high-affinity human antitoxin antibody. To explore structural features which enable the antibody to bind to intact tetanus toxoid, peptides derived from the sequence of the natural immunogen and antigenic mimics identified by combinatorial chemistry. To explain why this Fab shows a remarkable tendency to produce crystals consistently diffracting to d spacings of 1.7–1.8 Å. To use this information to engineer a strong tendency to crystallize into the design of other Fabs. Study design: The protein was crystallized in hanging or sitting drops by a microseeding technique in polyethylene glycol (PEG) 8000. Crystals were subjected to X-ray analysis and the three-dimensional structure of the Fab was determined by the molecular replacement method. Interactive computer graphics were employed to fit models to electron density maps, survey the structure in multiple views and discover the crystal packing motif of the protein. Results: Exceptionally large single crystals of this protein have been obtained, one measuring 5×3×2 mm (l×w×d). The latter was cut into six irregular pieces, each retaining the features of the original in diffracting to high resolution (1.8 Å) with little decay in the X-ray beam. In an individual Fab, the active site is relatively flat and it seems likely that the protein antigen and derivative peptides are tightly held on the outer surface without significant penetration into the interior. There is no free space to accommodate even a dipeptide between VH and VL. One of the unique features of the B7-15A2 Fab is a large aliphatic ridge dominating the center of the active site. The CDR3 of the H chain contributes significantly to this ridge, as well as to adjoining regions projected to be important for the docking of the antigen. Both the ease of crystallization and the favorable diffraction properties are mainly attributable to the tight packing of the protein molecules in the crystal lattice. Discussion: The B7-15A2 active site provides a stable and well defined platform for high affinity docking of proteins, peptides and their mimotopes. The advantages for future developments are suggested by the analysis of the crystal properties. It should be possible to incorporate the features promoting crystallization, close packing and resistance to radiation damage into engineered human antibodies without altering the desired specificities and affinities of their active sites.

Selection of antigenic and immunogenic mimics of hepatitis C virus using sera from patients.

Using sera from hepatitis C virus (HCV)-infected patients and noninfected subjects to screen random peptide libraries displayed on phage, we selected peptides specifically reacting with sera from infected patients. These phage- borne peptides were shown to mimic distinct HCV determinants. They detected in all cases the presence of anti-HCV Abs in a large panel of patients' sera, thus demonstrating the high sensitivity of the selected peptides as diagnostic markers. In addition, this diagnostic approach allowed a detailed characterization of the individual humoral response to viral infection. Phage-displayed HCV mimics were substitutes for the authentic HCV epitopes in inducing a strong specific response against HCV when used as immunogens in mice. These results support the search for HCV mimics with the potential to elicit a protective immune response as leads for the development of a mimotope-based vaccine against viral infection.

6] Immunization with phage-displayed mimotopes

Phage-displayed mimotopes (phagotopes) are selected from random peptide libraries, because they bind to specific antibodies, thus behaving as antigenic mimics that resemble to some extent the original antigen. If the positive image of the antigen (Ag) these mimotopes provide is accurate enough to induce the production of specific antibodies (Ab) in animals, the mimotopes prove to be suitable substitute immunogens, totally distinct from the original antigen (that raised the antibodies used to identify the phagotopes). At present, the only feasible way of assessing this capability is the active immunization of experimental animals. Filamentous phages are known to be excellent immunogens; de la Cruz and collaborators first proposed using filamentous phage as immunological carriers, by cloning short peptides derived from the malaria circumsporozoite at the N terminus of pIII. Greenwood et al . cloned malaria peptides at the N terminus of the major coat protein VIII and Minenkova et al. cloned an antigenic determinant of human immunodeficiency virus (HIV) gag protein fused to pVIII and used the recombinant phage to successfully immunize rabbits. Similarly, when Willis et al. immunized mice with malaria peptides fused to pVIII, they not only obtained an antibody response, but also demonstrated that the response in mice is T-cell dependent and undergoes class switching from immunoglobulin M (IgM) to IgG. Mimotopes, selected by using either hepatitis B virus surface antigen (HBsAg)-specific monoclonal antibodies or human sera, have been used to induce HBsAg-specific antibodies in both mice and rabbits.

Antibody Response in Seropositive Multiple Sclerosis Patients Vaccinated with Attenuated Live Varicella Zoster Virus

To determine the safety and effectiveness of live attenuated varicella zoster virus (VZV) vaccine (OKA/Merck) on 50 patients with chronic progressive multiple sclerosis (MS), based on the hypothesis that VZV might be the antigen or antigen mimic of MS plus the fact that repeated high antigen doses have produced 'antigen paralysis' in experimental allergic encephalomyelitis mice. Fifty patients were randomly selected without controls. They were assessed clinically at entry and on four other occasions over 14 months. Enhanced cranial magnetic resonance imaging (MRI) was performed at entry and at six and 12 months post entry. All were vaccinated after initial assessment and again six weeks later. All clinical and laboratory assessments were performed at the Health Sciences Centre, Winnipeg, in the out-patient department. All MRI examinations were performed at the St Boniface General Hospital, Winnipeg, Manitoba. Both are tertiary care hospitals. Fifty randomly selected patients with chronic progressive MS, age 18 to 60 years, and a disability status scale of 2.0 or greater were included. Forty-five patients completed the study. Two vaccinations with attenuated live VZV six weeks apart. All patients were VZV seropositive at entry and all showed an increased antibody level following vaccination. No one was harmed by the vaccine. There may have been some changes in the MS of 15 patients. It may be reasonable and safe to challenge the process of MS using large doses of the immunogenic proteins of the VZV to induce 'immune paralysis'.

Isolation of antigenic mimics of MDR1-P-glycoprotein by phage-displayed peptide libraries.

To identify an MC57 epitope which is more efficiently expressed on inactivated forms of P-glycoprotein we utilized peptide libraries displayed on filamentous phage. Using this technology, we selected specific phage clones blocking the binding of the murine monoclonal (MAb) MC57 with live human multi-drug-resistant (MDR) cells, and sequenced and analyzed their DNA. The results we obtained indicate that MAb MC57 epitope could be formed by 2 regions localized on the predicted fourth and sixth extracellular loops of the current 12-transmembrane-domain model predicted for MDR1-P-glycoprotein. Surprisingly, a third region, defined by residues 800-807 of the MDR1-P-glycoprotein sequence and postulated to be intracellular, was also identified as a putative part of the MC57 epitope. This finding adds weight to the interesting hypothesis that a P-glycoprotein structure different from the current model may exist.

Recognition by human sera and immunogenicity of HBsAg mimotopes selected from an M13 phage display library

We used two mouse monoclonal antibodies (mAb) specific for the human hepatitis B virus surface antigen (HBsAg) to screen a random peptide library of 15 amino-acid residues displayed as a fusion to protein III of filamentous phage M 13. By a combination of affinity selection, immuno-screening and ELISA techniques, we selected peptides that are recognized by the anti-HBsAg mAb and show aa similarity with the natural antigen. The selected phage-displayed epitopes (phagotopes) behave as antigenic mimics of HBsAg. One phagotope is specifically recognized by human sera from HBsAg-immunized individuals, pointing to the possible use of phagotopes as markers to detect the presence of specific Ab in the serum. The same phagotope also elicits Ab directed against HBsAg in mice, indicating that mAb-selected phagotopes can also be immunogenic mimics of the natural antigen. These findings demonstrate that it is possible to identify disease-specific epitopes that can be used as diagnostic reagents and as leads for the development of acellular vaccines.

A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera.

A strategy to identify disease-specific epitopes from phage-displayed random peptide libraries using human sera is described. Peptides on phage (phagotopes) that react with antibodies present in patient sera are purified from > 10(7) different sequences by affinity selection and immunological screening of plaques. Disease-specific phagotopes can be identified out of this pool through an 'antigen independent' procedure which avails itself only of patient and normal human sera. Using this strategy, we have selected antigenic mimics (mimotopes) of two different epitopes from the human hepatitis B virus envelope protein (HBsAg). We could show that a humoral response to these mimotopes is widespread in the immunized population, suggesting that the strategy identifies phagotopes that have a potential role as diagnostic reagents. Immunization of mice with the selected phagotopes elicited a strong specific response against the HBsAg. These results open new inroads into disease-related epitope discovery and provide the potential for vaccine development without a requirement for the use of, or even information about, the aetiological agent or its antigens.

Cytotoxic T lymphocytes show HLA-C-restricted recognition of EBV-bearing cells and allorecognition of HLA class I molecules presenting self-peptides.

Human CTL have been isolated that show self-restricted recognition of autologous lymphoblastoid cell lines and allorecognition. The lymphoblastoid cell line ligand most likely used a peptide that is expressed in EBV-bearing cells when the virus enters the lytic cycle. This peptide is presented to CD8+ CTL by HLA-Cw7 molecules. The allogeneic ligand recognized on non-EBV-infected cells is composed of a class I glycoprotein and a naturally selected self-peptide. In previous studies we demonstrated that this ligand is determined by two MHC-linked genes: one gene encodes the allogeneic class I molecule whereas the other controls the self-peptide. Despite the use of different peptides and different class I molecules, seemingly equivalent structures are formed that enable these two ligands to function as antigenic mimics of each other. CTL with the same patterns of dual specificity could be isolated from four unrelated donors, indicating that HLA-Cw7 is frequently involved in self-restricted recognition of EBV-harboring cells. Such CTL could help not only to contain lytic virus during a primary infection but also may be maintained life-long to eliminate cells in which reactivated virus appears.