Epitope mapping of Der p 2, a clinically important dust-mite allergen is the first step in designing immunotherapy hypoallergen vaccine candidates. Twenty-one single alanine mutants of Der p 2 were generated and their secondary structure was analysed using circular dichroism spectra. Only one mutant, K96A resulted in a misfolded protein. All mutants were tested for serum IgE reactivity using serum from dust mite allergic individuals by immuno dot-blots. Mutations to five residues, N10, E25, K77, K96 and E102 consistently showed reduced IgE reactions compared to wild-type Der p 2, and therefore these residues constitute the major IgE epitopes of Der p 2. Two mutants with consistent low IgE binding, K96A and E102A, were subsequently evaluated as hypoallergen candidates. IgG antibodies raised in mice against both mutants could inhibit human IgE-binding to WT Der p 2. Both mutants had intact T-cell epitopes as they were able to stimulate peripheral blood mononuclear cell proliferation similar to WT Der p 2. However, a switch in Th1:Th2 cytokine profile was not observed. In summary, we have identified the major conformational epitopes of Der p 2, and evaluated two Der p 2 hypoallergen vaccine candidates for immunotherapy.
Filamentous phage random peptide libraries were used to identify the epitopes of Burkholderia pseudomallei protease by panning against IgG polyclonal sera that exhibited protease neutralizing properties. The isolated fusion peptides presented a consensus peptide sequence, TKSMALSG, which closely resembles part of the active site sequence, 435GTSMATPHVAG445, of B. pseudomallei serine metalloprotease. By comparing the consensus sequence, TKSMALSG, with the predicted three-dimensional molecular model of B. pseudomallei serine metalloprotease, it appears that the potential antibody binding epitope was buried within the molecule. This active site was conformational whereby one continuous sub-region (SMA) was located between two discontinuous sub-regions, supplied by the flanking residues in the same polypeptide. All phages selected from the biopanning with IgG polyclonal sera showed good binding towards the polyclonal antibodies when compared to the negative control. In addition, these peptide-bearing phages showed competitive inhibition of B. pseudomallei serine metalloprotease binding to the polyclonal IgG.
A random peptide library of heptamers displayed on the surface of M13 bacteriophage was used to identify specific epitopes of antibodies in pooled sera of swine naturally infected by Nipah virus. The selected heptapeptides were aligned with protein sequences of Nipah virus and several putative epitopes were identified within the nucleocapsid protein. A total of 41 of 60 (68%) selected phage clones had inserts resembling a region with the sequence SNRTQGE, located at the C-terminal end (amino acids 503-509) of the nucleocapsid protein. The binding of antibodies in the swine and human antisera to the phage clone was inhibited by a synthetic peptide corresponding to this region. Epitopes identified by phage display are consistent with the predicted antigenic sites for the Nipah virus nucleocapsid protein. The selected phage clone used as a coating antigen discriminated swine and human Nipah virus sera-positive from sera-negative samples exhibiting characteristics, which might be attractive for diagnostic tests.
Enterovirus A71 (EV-A71) neutralization escape mutants were generated with monoclonal antibody MAB979 (Millipore). The VP2-T141I and VP1-D14N substitutions were identified. Using reverse genetics, infectious clones with these substitutions were constructed and tested by neutralization assay with immune sera from mice and humans. The N-terminus VP1-14 is more important than EF loop VP2-141 in acute human infection, mainly because it recognised IgM present in acute infection. The N-terminus VP1 could be a useful target for diagnostics and therapeutic antibodies in acute infection.
Plasmodium knowlesi can cause potentially life threatening human malaria. The Plasmodium merozoite surface protein-142 (MSP-142) is a potential target for malaria blood stage vaccine, and for diagnosis of malaria. Two epitope mapping techniques were used to identify the potential epitopes within P. knowlesi MSP-142. Nine and 14 potential epitopes were identified using overlapping synthetic peptide library and phage display library, respectively. Two regions on P. knowlesi MSP-142 (amino acid residues 37-95 and residues 240-289) were identified to be the potential dominant epitope regions. Two of the prominent epitopes, P10 (TAKDGMEYYNKMGELYKQ) and P31 (RCLLGFKEVGGKCVPASI), were evaluated using mouse model. P10- and P31-immunized mouse sera reacted with recombinant P. knowlesi MSP-142, with the IgG isotype distribution of IgG2b>IgG1>IgG2a>IgG3. Significant higher level of cytokines interferon-gamma and interleukin-2 was detected in P31-immunized mice. Both P10 and P31 could be the suitable epitope candidates to be used in malaria vaccine designs and immunodiagnostic assays, provided further evaluation is needed to validate the potential uses of these epitopes.
Random 15-mer peptides displayed on filamentous phages were screened in binding studies using a Chlamydia pneumoniae-specific monoclonal antibody (RR-402) and affinity-purified, polyclonal sera from patients seropositive for C. pneumoniae infections by the microimmunofluorescence (MIF) test. One 15-mer epitope, epitope Cpnl5A (LASLCNPKPSDAPVT) was identified in both the monoclonal and polyclonal screenings, and showed higher ELISA reactivity with C. pneumoniae MIF-positive sera compared to patients with other chlamydial infections, non-chlamydial respiratory infections and normal healthy sera (MIF-negative). Interestingly, epitope Cpnl5A also showed significant (52%) amino acid sequence homology to the 56 kDa type-specific antigen of Rickettsia tsutsugamushi, a protein implicated in the virulence of this organism.
A common approach for developing diagnostic tests for influenza virus detection is the use of mouse or rabbit monoclonal and/or polyclonal antibodies against a target antigen of the virus. However, comparative mapping of the target antigen using antibodies from different animal sources has not been evaluated before. This is important because identification of antigenic determinants of the target antigen in different species plays a central role to ensure the efficiency of a diagnostic test, such as competitive ELISA or immunohistochemistry-based tests. Interest in the matrix 2 ectodomain (M2e) protein of avian influenza virus (AIV) as a candidate for a universal vaccine and also as a marker for detection of virus infection in vaccinated animals (DIVA) is the rationale for the selection of this protein for comparative mapping evaluation. This study aimed to map the epitopes of the M2e protein of avian influenza virus H5N1 using chicken, mouse and rabbit monoclonal or monospecific antibodies. Our findings revealed that rabbit antibodies (rAbs) recognized epitope 6EVETPTRN13 of the M2e, located at the N-terminal of the protein, while mouse (mAb) and chicken antibodies (cAbs) recognized epitope 10PTRNEWECK18, located at the centre region of the protein. The findings highlighted the difference between the M2e antigenic determinants recognized by different species that emphasized the importance of comparative mapping of antibody reactivity from different animals to the same antigen, especially in the case of multi-host infectious agents such as influenza. The findings are of importance for antigenic mapping, as well as diagnostic test and vaccine development.
A high incidence of sensitization to Blomia tropicalis, the predominant house dust mite species in tropical regions, is strongly associated with allergic diseases in Singapore, Malaysia, and Brazil. IgE binding to the group 5 allergen, Blo t 5, is found to be the most prevalent among all B. tropicalis allergens. The NMR structure of Blo t 5 determined represents a novel helical bundle structure consisting of three antiparallel alpha-helices. Based on the structure and sequence alignment with other known group 5 dust mite allergens, surface-exposed charged residues have been identified for site-directed mutagenesis and IgE binding assays. Four charged residues, Glu76, Asp81, Glu86, and Glu91 at around the turn region connecting helices alpha2 and alpha3 have been identified to be involved in the IgE binding. Using overlapping peptides, we have confirmed that these charged residues are located on a major putative linear IgE epitope of Blo t 5 from residues 76-91 comprising the sequence ELKRTDLNILERFNYE. Triple and quadruple mutants have been generated and found to exhibit significantly lower IgE binding and reduced responses in skin prick tests. The mutants induced similar PBMC proliferation as the wild-type protein but with reduced Th2:Th1 cytokines ratio. Mass screening on a quadruple mutant showed a 40% reduction in IgE binding in 35 of 42 sera of atopic individuals. Findings in this study further stressed the importance of surface-charged residues on IgE binding and have implications in the cross-reactivity and use of Blo t 5 mutants as a hypoallergen for immunotherapy.
Duck Tembusu virus (DTMUV) causes substantial egg drop disease. DTMUV was first identified in China and rapidly spread to Malaysia and Thailand. The antigenicity of the DTMUV E protein has not yet been characterized. Here, we investigated antigenic sites on the E protein using the non-neutralizing monoclonal antibodies (mAbs) 1F3 and 1A5. Two minimal epitopes were mapped to (221)LD/NLPW(225) and (87)YAEYI(91) by using phage display and mutagenesis. DTMUV-positive duck sera reacted with the epitopes, thus indicating the importance of the minimal amino acids of the epitopes for antibody-epitope binding. The performance of the dot blotting assay with the corresponding positive sera indicated that YAEYI was DTMUV type-specific, whereas (221)LD/NLPW(225) was a cross-reactive epitope for West Nile virus (WNV), dengue virus (DENV), and Japanese encephalitis virus (JEV) and corresponded to conserved and variable amino acid sequences among these strains. The structure model of the E protein revealed that YAEYI and LD/NLPW were located on domain (D) II, which confirmed that DII might contain a type-specific non-neutralizing epitope. The YAEYI epitope-based antigen demonstrated its diagnostic potential by reacting with high specificity to serum samples obtained from DTMUV-infected ducks. Based on these observations, a YAEYI-based serological test could be used for DTMUV surveillance and could differentiate DTMUV infections from JEV or WNV infections. These findings provide new insights into the organization of epitopes on flavivirus E proteins that might be valuable for the development of epitope-based serological diagnostic tests for DTMUV.
In our earlier study, an immunoblot analysis using sera from febrile patients revealed that a 50-kDa band from an outer membrane protein fraction of Salmonella enterica serovar Typhi was specifically recognized only by typhoid sera and not sera from other febrile illnesses. Here, we investigated the identities of the proteins contained in the immunogenic 50-kDa band to pinpoint antigens responsible for its immunogenicity. We first used LC-MS/MS for protein identification, then used the online tool ANTIGENpro for antigenicity prediction and produced recombinant proteins of the lead antigens for validation in an enzyme-linked immunosorbent assay (ELISA). We found that proteins TolC, GlpK and SucB were specific to typhoid sera but react to antibodies differently under native and denatured conditions. This difference suggests the presence of linear and conformational epitopes on these proteins.
Chikungunya virus (CHIKV) is a mosquito-borne arbovirus which has recently re-emerged globally and poses a major threat to public health. Infection leads to severe arthralgia, and disease management remains supportive in the absence of vaccines and anti-viral interventions. The high specificities of monoclonal antibodies (mAbs) have been exploited in immunodiagnostics and immunotherapy in recent decades. In this study, eight different clones of mAbs were generated and characterised. These mAbs targeted the linear epitopes on the CHIKV E2 envelope glycoprotein, which is the major target antigen during infection. All the mAbs showed binding activity against the purified CHIKV virion or recombinant E2 when analysed by immunofluorescence, ELISA and Western blot. The epitopes of each mAb were mapped by overlapping synthetic peptide-based ELISA. The epitopes are distributed at different functional domains of E2 glycoprotein, namely at domain A, junctions of β-ribbons with domains A and B, and domain C. Alignment of mAb epitope sequences revealed that some are well-conserved within different genotypes of CHIKV, while some are identical to and likely to cross-react with the closely-related alphavirus O'nyong-nyong virus. These mAbs with their mapped epitopes are useful for the development of diagnostic or research tools, including immunofluorescence, ELISA and Western blot.
A series of 122, 9-mer overlapping peptides based on the sequence of the Salmonella typhi GroEL gene was synthesized on the surfaces of polyethylene pins and screened with monoclonal antibody to GroEL and with human sera from patients with typhoid fever and normal healthy blood donors. Three immunogenic epitopes corresponding to peptides EGQDRGYSY, YSYNKETGE and GKGTEEKEK were identified upon screening with the human sera. In addition, screening of the peptides with a monoclonal antibody to GroEL detected binding to a third peptide, KGGKGTEEK, which contains a common overlapping sequence to peptide GKGTEEKEK. Identification and definition of these epitopes will be important in delineating the biological and immunological functions of this protein and in designing better diagnostic tests and vaccines.
Helminth parasite infections are significantly impacting global health, with more than two billion infections worldwide with a high morbidity rate. The complex life cycle of the nematodes has made host immune response studies against these parasites extremely difficult. In this study, we utilized two phage antibody libraries; the immune and naïve library were used to identify single chain fragment variable (scFv) clones against a specific filarial antigen (BmR1). The V-gene analysis of isolated scFv clones will help shed light on preferential VDJ gene segment usage against the filarial BmR1 antigen in healthy and infected states. The immune library showed the usage of both lambda and kappa light chains. However, the naïve library showed preferential use of the lambda family with different amino acid distributions. The binding characteristics of the scFv clones identified from this work were analyzed by immunoassay and immunoaffinity pull down of BmR1. The work highlights the antibody gene usage pattern of a naïve and immune antibody library against the same antigen as well as the robust nature of the enriched antibodies for downstream applications.
Hepatitis B virus (HBV) has killed countless lives in human history. The invention of HBV vaccines in the 20(th) century has reduced significantly the rate of the viral infection. However, currently there is no effective treatment for chronic HBV carriers. Newly emerging vaccine escape mutants and drug resistant strains have complicated the viral eradication program. The entire world is now facing a new threat of HBV and human immunodeficiency virus co-infection. Could phage display provide solutions to these life-threatening problems? This article reviews critically and comprehensively the innovative and potential applications of phage display in the development of vaccines, therapeutic agents, diagnostic reagents, as well as gene and drug delivery systems to combat HBV. The application of phage display in epitope mapping of HBV antigens is also discussed in detail. Although this review mainly focuses on HBV, the innovative applications of phage display could also be extended to other infectious diseases.
Considerable effort is directed at the development of a malaria vaccine that elicits antigen-specific T-cell responses against pre-erythrocytic antigens of Plasmodium falciparum. Genetic restriction of host T-cell responses and polymorphism of target epitopes on parasite antigens pose obstacles to the development of such a vaccine. Liver stage-specific antigen-1 (LSA-1) is a prime candidate vaccine antigen and five T-cell epitopes that are degenerately restricted by HLA molecules common in most populations have been identified on LSA-1. To define the extent of polymorphism within these T-cell epitopes, the N-terminal non-repetitive region of the LSA-1 gene from Malaysian P. falciparum field isolates was sequenced and compared with data of isolates from Brazil, Kenya and Papua New Guinea. Three of the T-cell epitopes were completely conserved while the remaining two were highly conserved in the isolates examined. Our findings underscore the potential of including these HLA-degenerate T-cell epitopes of LSA-1 in a subunit vaccine.
Hemolysin E (HlyE) is an immunogenic novel pore-forming toxin involved in the pathogenesis of typhoid fever. Thus, mapping of B-cell epitopes of Salmonella enterica serovar Typhi (S. Typhi) is critical to identify key immunogenic regions of HlyE. A random 20-mer peptide library was used for biopanning with enriched anti-HlyE polyclonal antibodies from typhoid patient sera. Bioinformatic tools were used to refine, analyze and map the enriched peptide sequences against the protein to identify the epitopes. The analysis identified both linear and conformational epitopes on the HlyE protein. The predicted linear GAAAGIVAG and conformational epitope PYSQESVLSADSQNQK were further validated against the pooled sera. The identified epitopes were then used to isolate epitope specific monoclonal antibodies by antibody phage display. Monoclonal scFv antibodies were enriched for both linear and conformational epitopes. Molecular docking was performed to elucidate the antigen-antibody interaction of the monoclonal antibodies against the epitopes on the HlyE monomer and oligomer structure. An in-depth view of the mechanistic and positional characteristics of the antibodies and epitope for HlyE was successfully accomplished by a combination of phage display and bioinformatic analysis. The predicted function and structure of the antibodies highlights the possibility of utilizing the antibodies as neutralizing agents for typhoid fever.
ProBiS is a new method to identify the binding site of protein through local structural alignment against the nonredundant Protein Data Bank (PDB), which may result in unique findings compared to the energy-based, geometry-based, and sequence-based predictors. In this work, binding sites of Hemagglutinin (HA), which is an important target for drugs and vaccines in influenza treatment, have been revisited by ProBiS. For the first time, the identification of conserved binding sites by local structural alignment across all subtypes and strains of HA available in PDB is presented. ProBiS finds three distinctive conserved sites on HA's structure (named Site 1, Site 2, and Site 3). Compared to other predictors, ProBiS is the only one that accurately defines the receptor binding site (Site 1). Apart from that, Site 2, which is located slightly above the TBHQ binding site, is proposed as a potential novel conserved target for membrane fusion inhibitor. Lastly, Site 3, located around Helix A at the stem domain and recently targeted by cross-reactive antibodies, is predicted to be conserved in the latest H7N9 China 2013 strain as well. The further exploration of these three sites provides valuable insight in optimizing the influenza drug and vaccine development.