Immunogenic Polypeptides Research Articles

Strategy to Configure Multi-epitope Recombinant Immunogens with Weightage on Proinflamatory Response using SARS-CoV-2 Spike Glycoprotein (S-protein) and RNA-dependent RNA Polymerase (RdRp) as Model Targets

Development of a suitable recombinant peptide vaccine against pathogens requires designing of effective immunogenic polypeptide taking various aspects and complexity of immune-response into consideration. Implementing SARS-CoV-2 spike glycoprotein (S-protein) and RNA-dependent RNA polymerase (RdRp) as model targets, in this study, we outline and assess a strategy for in silico recombinant vaccine designing. After mapping the linear B-cell epitopes and MHC1-binding T-cell epitopes six epitopes were sorted from each of the proteins on the basis of extent of residue-conservancy among three types of coronaviruses namely SARS-CoV-2, SARS-CoV and MERS-CoV. Each of the selected epitopes were profiled for their pro-inflammatory potential through molecular docking analysis with surface bound Toll-like receptors, namely TLR2, TLR4 and TLR5. Based on a custom scoring function, the epitopes were ranked for highest and least pro-inflammatory potential. Segments of Spike and RdRp harboring such epitopes were combined using linkers to design immunogenic recombinant polypeptide. Antigenicity and allergenicity of each of the combination was scored; and the best fitting one was docked against TLR2, TLR4 and TLR5 for assessing pro-inflammatory potential. Codon optimization and in silico cloning in expression vector indicated that the designed peptide can be satisfactorily expressed in bacteria, reinforcing the viability of the strategy in identification and designing of potential immunogens.

Personal Neoantigens From Patients With NSCLC Induce Efficient Antitumor Responses.

ObjectiveTo develop a neoantigen-targeted personalized cancer treatment for non-small cell lung cancer (NSCLC), neoantigens were obtained from collected human lung cancer samples, and the utility of neoantigen and neoantigen-reactive T cells (NRTs) was assessed.MethodsTumor specimens from three patients with NSCLC were obtained and analyzed by whole-exome sequencing, and neoantigens were predicted accordingly. Dendritic cells and T lymphocytes were isolated, NRTs were elicited and IFN-γ ELISPOT tests were conducted. HLA-A2.1/Kb transgenic mice were immunized with peptides from HLA-A*02:01+patient with high immunogenicity, and NRTs were subjected to IFN-γ, IL-2 and TNF-α ELISPOT as well as time-resolved fluorescence assay for cytotoxicity assays to verify the immunogenicity in vitro. The HLA-A*02:01+lung cancer cell line was transfected with minigene and inoculated into the flanks of C57BL/6nu/nu mice and the NRTs induced by the immunogenic polypeptides from autologous HLA-A2.1/Kb transgenic mice were adoptively transfused to verify their immunogenicity in vivo.ResultsMultiple putative mutation-associated neoantigens with strong affinity for HLA were selected from each patient. Immunogenic neoantigen were identified in all three NSCLC patients, the potency of ACAD8-T105I, BCAR1-G23V and PLCG1-M425L as effective neoantigen to active T cells in suppressing tumor growth was further proven both in vitro and in vivo using HLA-A2.1/Kb transgenic mice and tumor-bearing mouse models.ConclusionNeoantigens with strong immunogenicity can be screened from NSCLC patients through the whole-exome sequencing of patient specimens and machine-learning-based neoantigen predictions. NRTs shown efficient antitumor responses in transgenic mice and tumor-bearing mouse models. Our results indicate that the development of neoantigen-based personalized immunotherapies in NSCLC is possible.PrecisNeoantigens with strong immunogenicity were screened from NSCLC patients. This research provides evidence suggesting that neoantigen-based therapy might serve as feasible treatment for NSCLC.

Towards the development of wheat extruded snacks with reduced gluten toxicity

The negative effects of gluten on human health are becoming a serious problem for the development of new foods. Gluten-free or very low-gluten labeling is an important factor in FMCG food marketing. An alternative to a gluten-free diet is the use of enzymatic hydrolysis to break down the immunogenic polypeptides of the gliadin and glutenin fractions. Gluten polypeptides that are resistant to gastrointestinal digestion can be hydrolyzed by prolyl endoproteases from Flavobacterium meningosepticum, Sphingomonas capsulate and Myxococcus Xanthus, or enzyme preparations from Aspergillus niger or Aspergillus oryzae. In many studies, the ability of high-temperature short-term extrusion to modify molecular structure of gluten and its fractions and improve enzymatic access to the biopolymers is noted. A method for integrating biocatalysis and extrusion processes to reduce or neutralize the immunogenic effect of gluten epitopes and to obtain extruded ready-to-eat snacks is proposed. The process flow chart includes the use of extrusion as a pretreatment stage for enzymatic hydrolysis of wheat and as a stage of thermal and shaping processing of extruded hydrolyzed wheat into ready-to-eat snacks.

Electrochemical aptasensor using optimized surface chemistry for the detection of Mycobacterium tuberculosis secreted protein MPT64 in human serum

Tuberculosis (TB) remains one of the leading causes of mortality worldwide. There is a great need for the development of diagnostic tests, which are reliable, sensitive, stable, and low cost to enable early diagnosis of TB in communities with scarce resources. This study reports the optimization and evaluation of a synthetic receptor, an aptamer, for the detection of the secreted protein MPT64, which is a highly immunogenic polypeptide of Mycobacterium tuberculosis, a causative agent of TB. The study investigates combinatorial effects of an aptamer linker and a co-adsorbent onto a gold electrode for optimal binding efficiency and reduced non-specific interactions for label-free detection of MPT64 using electrochemical impedance spectroscopy. Two types of co-adsorbents and two types of aptamer linkers were studied and high specificity and sensitivity to MPT64 was observed for a surface prepared with a thiol PEGylated aptamer HS-(CH2)6-OP(O)2O-(CH2CH2O)6-TTTTT-aptamer and 6-mercaptohexanol in a ratio of 1:100. The developed aptamer-based sensor was successfully used with spiked human serum sample with a limit of detection of 81 pM This work demonstrates the use of the MPT64 aptamer as a lower cost, more sustainable and stable alternative of antibodies for the development of point-of-care TB biosensors decreasing the detection time from several days or hours to thirty minutes.

Immunolocalization and immunodetection of the excretory/secretory (ES) antigens of Fasciola gigantica.

The digenetic trematode Fasciola gigantica is a parasite of great agricultural and economic importance. Along with Fasciola hepatica, F. gigantica incurs huge economic losses to the agricultural sector. Because of unavailability of an effective and commercial vaccine, the earliest diagnosis of the disease is the only way to control the disease. The conventional coprological techniques are able to detect the disease only after the parasites get matured and starts releasing their eggs with the faeces of host, therefore prepatent infection remain undiagnosed. The alternative method is by serological tests that uses circulatory antigens. Despite high sensitivity, their reliability is quite low because of the common antigens shared between different helminth parasites. To overcome this, investigation was shifted to identify the copro-antigens which could be more sensitive and reliable. In the present study, we tried to identify some of the immunodominant proteins from the Excretory Secretory (ES) product of F. gigantica which can be further characterized and used for early detection of infection and also as drug and vaccine candidates. The ES products of F. gigantica were collected and used for raising the polyclonal antibody in rabbit. The polypeptide profile was generated as well as immunogenic polypeptides were identified. The Source of ES antigen was immunolocalized using confocal microscopy and dot blot assay was performed to diagnose field infection. The polypeptide profile of ES products revealed a total of 24 polypeptides out of which 12 immunogenic polypeptides were identified by western blotting. Confocal micrographs showed the immunolocalization of antigens in the intestinal caecae, vitalline glands, gonads as well as in the tegument of the worm. The dot blot assay confirmed the utility of ES products for the detection of field infection. Subsequently, cross reactivity was found negative with Gigantocotyle explanatum; an amphitome parasite of same habitat. However, the cross reactivity with other helminths needs to be worked out.

Chemobacterial Synthesis of a Sialyl-Tn Cyclopeptide Vaccine Candidate.

A conjugatable form of the tumour-associated carbohydrate antigen sialyl-Tn (Neu5Ac-α-2,6-GalNAc) was efficiently produced in Escherichia coli. Metabolically engineered E. coli strains overexpressing the 6-sialyltransferase gene of Photobacterium sp. and CMP-Neu5Ac synthetase genes of Neisseria meningitidis were cultivated at high density in the presence of GalNAc-α-propargyl as the exogenous acceptor. The target disaccharides, which were produced on the scale of several hundreds of milligrams, were then conjugated by using copper(I)-catalysed azide-alkyne cycloaddition click chemistry to a fully synthetic and immunogenic scaffold with the aim to create a candidate anticancer vaccine. Four sialyl-Tn epitopes were introduced on the upper face of an azido-functionalised multivalent cyclopeptide scaffold, the lower face of which was previously modified by an immunogenic polypeptide, PADRE. The ability of the resulting glycoconjugate to interact with oncofoetal sialyl-Tn monoclonal antibodies was confirmed in ELISA assays.

C3d regulates immune checkpoint blockade and enhances antitumor immunity.

Despite expression of immunogenic polypeptides, tumors escape immune surveillance by engaging T cell checkpoint regulators and expanding Tregs, among other mechanisms. What orchestrates these controls is unknown. We report that free C3d, a fragment of the third component of complement, inside tumor cells - or associated with irradiated tumor cells and unattached to antigen - recruits, accelerates, and amplifies antitumor T cell responses, allowing immunity to reverse or even to prevent tumor growth. C3d enhances antitumor immunity independently of B cells, NK cells, or antibodies, but it does so by increasing tumor infiltrating CD8+ lymphocytes, by depleting Tregs, and by suppressing expression of programmed cell death protein 1 (PD-1) by T cells. These properties of C3d appear specific for the tumor and dependent on complement receptor 2, and they incur no obvious systemic toxicity. The heretofore unrecognized properties of free C3d suggest that protein might determine the effectiveness of immune surveillance and that increasing availability of the protein might prove advantageous in the treatment or prevention of cancer and premalignant conditions.

Autoimmunity against a defective ribosomal insulin gene product in type 1 diabetes.

Identification of epitopes that are recognized by diabetogenic T cells and cause selective beta cell destruction in type 1 diabetes (T1D) has focused on peptides originating from native beta cell proteins. Translational errors represent a major potential source of antigenic peptides to which central immune tolerance is lacking. Here, we describe an alternative open reading frame within human insulin mRNA encoding a highly immunogenic polypeptide that is targeted by T cells in T1D patients. We show that cytotoxic T cells directed against the N-terminal peptide of this nonconventional product are present in the circulation of individuals diagnosed with T1D, and we provide direct evidence that such CD8+ T cells are capable of killing human beta cells and thereby may be diabetogenic. This study reveals a new source of nonconventional polypeptides that act as self-epitopes in clinical autoimmune disease.

Survey of Epstein Barr Virus (EBV) Immunogenic Proteins and their Epitopes: Implications for Vaccine Preparation

The Epstein-Barr virus (Human herpesvirus 4) encodes approximately 80 proteins, from which 15 possess at least 90 antigenic epitopes. Many of them stimulate the T cell receptors (TCR), but a few interact with the B cell receptors (BCR). Activation of B-cells and subsequent antibody production has not only been related to at least 3 envelope glycoproteins (mostly gp350) but also to latency associated membrane proteins (LMPs). The majority of EBV epitopes (over 80) inducing either cytotoxic and/or helper T lymphocytes were located on non-structural and/or latency associated polypeptides. The former interaction mediated by CD8plus/T cells is restricted by the HLA I molecules, predominantly of HLA-A subclass. In acute infectious mononucleosis (IM) patients (about 40 %) a considerable proportion of HLA B8 restricted CTL reactivity is directed against a single peptide (RAKFKQLL) of transactivator protein BZLF1/Zta. The EBV vaccines designed so far fall into two categories: those preventing any kind of infection (including prophylaxis of EBVassociated malignancies) and those designed for therapeutic purposes (to be used in subjects already infected). Preventive vaccines protecting against acute disease (such as IM) contain, as a rule, the gp350 polypeptide(s) encoded by the BLLF1 gene. Vaccines destined for tumor prevention rather consist of peptides derived from latency associated nuclear proteins (EBNA 2, 3 and 6) and/or from oncogenic latent membrane proteins (LMP1/LMP2a). Whereas the former generates antibodies preventing virus entry, the latter would potentiate the cell mediated response. In addition to recently described and purified individual recombinant immunogenic EBV polypeptides and/or their mixes, new perspectives were opened by construction of random overlapping strongly immunogenic scrambled polypeptide(s). Further novel approaches are based on carefully selected antigenic peptides (oligopeptides) coming from both, structural as well as non-structural or latencyassociated proteins bound to suitable carriers. Any constructs based on latency-associated proteins might be useful either for immunoprophylactic therapy following bone marrow and/or heart transplantations or for the prevention of EBVrelated tumors such as lymphomas and nasopharyngeal carcinoma. Due to the growing importance of the selected immunogenic epitopes as future vaccine components, at least the half of them has been patented not only as the natural amino acid sequence but also in different variations.

O6.06 * PERIPHERAL T CELL RESPONSES IN GLIOBLASTOMA PATIENTS ARE ASSOCIATED WITH AN IMPROVED SURVIVAL

BACKGROUND: Spontaneous anti-tumor T cell responses have been detected in several tumor types. However, not only in glioblastoma it is unclear if such immune responses influence patient outcome. MATERIAL AND METHODS: To detect such immune responses, occurence and frequencies of memory T-cells were determined in the peripheral blood of GBM patients applying the IFN gamma ELISPOT assay and using corresponding tumor lysates (n = 106) or a set of immunogenic polypeptides (n = 32). Extent of resection was assessed by either intraoperative or early postoperative MRI. In addition, intratumoral infiltration of different T-cell subpopulations at the time of first diagnosis was analyzed by immunohistochemistry (n = 20) and compared to the occurrence of spontaneous immune responses. RESULTS: T-cell responses against autologous tumor lysate were detected in 61/106 patients. Subsequent survival analysis concentrating on IDH1wt glioblastoma revealed a significant difference towards an improved survival for patients with a spontaneous immune response (p = 0.0251). Most interestingly, when stratifying for the extent of surgery those patients, who received a complete tumor resection and showed a spontaneous T cell response, survived significantly longer (p = 0.0002). In contrast, in subtotally resected patients the immune response did not improve survival. In line with these observations, occurrence of memory T cell responses against a set of known GBM-associated antigens (TTR, MAGE A3, S100A9) also trended to a better survival in those GBM patients who responded to all three antigens. Finally, increased frequency of intratumoral T cell infiltrates was also associated with the occurrence of spontaneous immune responses and an improved survival. CONCLUSIONS: The present analysis for the first time depicts on several levels a significant influence of the patient's immune system on the course of the disease in GBM patients. In this regard the most crucial prerequisite seems to be the MRI-based complete tumor resection. Most likely surgical removal of the immunosuppressive tumor environment allows for the beneficial reactivation of pre-existing immune responses.

SPONTANEOUS IMMUNE RESPONSES IN GBM PATIENTS AFTER COMPLETE TUMOR RESECTION ARE ASSOCIATED WITH AN IMPROVED SURVIVAL

BACKGROUND: Spontaneous anti-tumor immune responses have been detected in several tumor types. However, not only in glioblastoma it is unclear if such immune responses influence patient outcome. METHODS: To detect such immune responses, occurence and frequencies of memory T-cells were determined in the peripheral blood of GBM patients applying the IFN gamma ELISPOT assay and using corresponding tumor lysates (n = 106) or a set of immunogenic polypeptides (n = 32). Extent of resection was assessed by either intraoperative or early postoperative MRI. In addition, intratumoral infiltration of different T-cell subpopulations at the time of first diagnosis was analyzed by immunohistochemistry (n = 20) and compared to the occurrence of spontaneous immune responses. RESULTS: Altogether, T-cell responses against autologous tumor lysate were detected in 61/106 patients. Subsequent survival analysis concentrating on IDH1wt glioblastoma revealed a significant difference towards an improved survival for patients with a spontaneous immune response (p = 0.0251). Most interestingly, when stratifying for the extent of surgery those patients, who got a complete tumor resection and showed a spontaneous T cell response, survived significantly longer (p = 0.0002). In contrast, in subtotally resected patients the immune response did not improve survival. In line with these observations, occurrence of memory T cell responses against a set of known GBM-associated antigens (TTR, MAGE A3, S100A9) also trended to a better survival in those GBM patients who responded to all three antigens (p = 0.0663). Finally, increased frequency of intratumoral T cell infiltrates was also associated with the occurrence of spontaneous immune responses and an improved survival (p = 0.0113). CONCLUSIONS: The present analysis for the first time depicts on several levels a significant influence of the patient's immune system on the course of the disease in GBM patients. In this regard the most crucial prerequisite seems to be the MRI-based complete tumor resection. Most likely surgical removal of the immunosuppressive tumor environment allows for the beneficial reactivation of pre-existing immune responses. SECONDARY CATEGORY: Tumor Biology.

Potential Immunogenic Polypeptides of Burkholderia pseudomallei Identified by Shotgun Expression Library and Evaluation of Their Efficacy for Serodiagnosis of Melioidosis

The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei.

The effect of antigen encapsulation in chitosan particles on uptake, activation and presentation by antigen presenting cells

Particle-based vaccine delivery systems are under exploration to enhance antigen-specific immunity against safe but poorly immunogenic polypeptide antigens. Chitosan is a promising biomaterial for antigen encapsulation and delivery due to its ability to form nano- and microparticles in mild aqueous conditions thus preserving the antigenicity of loaded polypeptides. In this study, the influence of chitosan encapsulation on antigen uptake, activation and presentation by antigen presenting cells (APCs) is explored. Fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) and ovalbumin (OVA) were used as model protein antigens and encapsulated in chitosan particles via precipitation–coacervation at loading efficiencies >89%. Formulation conditions were manipulated to create antigen-encapsulated chitosan particles (AgCPs) with discrete nominal sizes (300nm, 1μm, and 3μm). Uptake of AgCPs by dendritic cells and macrophages was found to be dependent on particle size, antigen concentration and exposure time. Flow cytometry analysis revealed that uptake of AgCPs enhanced upregulation of surface activation markers on APCs and increased the release of pro-inflammatory cytokines. Lastly, antigen-specific T cells exhibited higher proliferative responses when stimulated with APCs activated with AgCPs versus soluble antigen. These data suggest that encapsulation of antigens in chitosan particles enhances uptake, activation and presentation by APCs.

Interactome: Smart hematophagous triatomine salivary gland molecules counteract human hemostasis during meal acquisition

Human populations are constantly plagued by hematophagous insects' bites, in particular the triatomine insects that are vectors of the Trypanosoma cruzi agent in Chagas disease. The pharmacologically-active molecules present in the salivary glands of hematophagous insects are injected into the human skin to initiate acquisition of blood meals. Sets of vasodilators, anti-platelet aggregators, anti-coagulants, immunogenic polypeptides, anesthetics, odorants, antibiotics, and detoxifying molecules have been disclosed with the aid of proteomics and recombinant cDNA techniques. These molecules can provide insights about the insect-pathogen-host interactions essential for understanding the physiopathology of the insect bite. The data and information presented in this review aim for the development of new drugs to prevent insect bites and the insect-transmitted endemic of Chagas disease.

TLR5-dependent immunogenicity of a recombinant fusion protein containing an immunodominant epitope of malarial circumsporozoite protein and the FliC flagellin of Salmonella Typhimurium

Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC) protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS) protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210) was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.

An additional ORF on meloe cDNA encodes a new melanoma antigen, MELOE-2, recognized by melanoma-specific T cells in the HLA-A2 context.

We characterized a new melanoma antigen derived from one of the multiple open reading frames (ORFs) of the meloe transcript. The meloe gene is overexpressed in melanomas as compared to other cancer cell lines and normal tissues. The corresponding transcript is rather unusual, in that it does not contain a long unique ORF but multiple short ORFs. We recently characterized a tumor epitope derived from a polypeptide (MELOE-1) encoded by the ORF(1230-1370) and involved in relapse prevention of melanoma patients treated with autologous tumor infiltrating lymphocytes (TIL). Here we show that the ORF(285-404) encodes a polypeptide called MELOE-2 that also generated a HLA-A2 epitope recognized by a melanoma-specific T cell clone derived from the same TIL population from which we derived the MELOE-1-specific T cell clone. We also showed that HLA-A2 melanoma cells were spontaneously recognized by the MELOE-2-specific T cell clone, and we detected the presence of MELOE-2 reactive T cells in another TIL population infused to a patient who remained relapse-free after TIL treatment. These results demonstrate that translation of meloe transcript in melanoma cells can produce at least two immunogenic polypeptides, MELOE-1 and MELOE-2, from two distinct ORFs that could be relevant target for melanoma immunotherapy.

Discovery of new Mycoplasma pneumoniae antigens by use of a whole-genome lambda display library

Mycoplasma pneumoniae is the leading cause of atypical pneumonia in children and young adults. Bacterial colonization can occur in both the upper and the lower respiratory tracts and take place both endemically and epidemically worldwide. Characteristically, the infection is chronic in onset and recovery and both humoral and cell-mediated mechanisms are involved in the response to bacterial colonization. To identify bacterial proteins recognized by host antibody responses, a whole-genome M. pneumoniae library was created and displayed on lambda bacteriophage. The challenge of such a library with sera from individuals hospitalized for mycoplasmal pneumonia allowed the identification of a panel of recombinant bacteriophages carrying B-cell epitopes. Among the already known M. pneumoniae B-cell antigens, our results confirmed the immunogenicity of P1 and P30 adhesins. Also, the data presented in this study localized, within their sequences, the immunodominant epitopes recognized by human immunoglobulins. Furthermore, library screening allowed the identification of four novel immunogenic polypeptides, respectively, encoded by fragments of the MPN152, MPN426, MPN456 and MPN-500 open reading frames, highlighting and further confirming the potential of lambda display technology in antigen and epitope discovery.

Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display

The identification of immunogenic polypeptides of pathogens is helpful for the development of diagnostic assays and therapeutic applications like vaccines. Routinely, these proteins are identified by two-dimensional polyacrylamide gel electrophoresis and Western blot using convalescent serum, followed by mass spectrometry. This technology, however, is limited, because low or differentially expressed proteins, e.g. dependent on pathogen-host interaction, cannot be identified. In this work, we developed and improved a M13 genomic phage display-based method for the selection of immunogenic polypeptides of Mycoplasma hyopneumoniae, a pathogen causing porcine enzootic pneumonia. The fragmented genome of M. hyopneumoniae was cloned into a phage display vector, and the genomic library was packaged using the helperphage Hyperphage to enrich open reading frames (ORFs). Afterwards, the phage display library was screened by panning using convalescent serum. The analysis of individual phage clones resulted in the identification of five genes encoding immunogenic proteins, only two of which had been previously identified and described as immunogenic. This M13 genomic phage display, directly combining ORF enrichment and the presentation of the corresponding polypeptide on the phage surface, complements proteome-based methods for the identification of immunogenic polypeptides and is particularly well suited for the use in mycoplasma species.

A genome level survey of Burkholderia pseudomallei immunome expressed during human infection

Burkholderia pseudomallei is the etiological agent of melioidosis, a severe infectious disease of humans and animals. The role of the bacterium's proteins expressed in vivo during human melioidosis continues to remain an enigma. This study's aim was to identify B. pseudomallei target proteins that elicit the humoral immune response in infected humans. A small insert genomic expression library was constructed and immunoscreened to identify peptides that reacted exclusively with melioidosis patients’ sera. Sero-positive clones expressing immunogenic peptides were sequenced and annotated, and shown to represent 109 proteins involved in bacterial cell envelope biogenesis, cell motility and secretion, transcription, amino acid, ion and protein metabolism, energy production, DNA repair and unknown hypothetical proteins. Western blot analysis of three randomly selected full-length immunogenic polypeptides with patients’ sera verified the findings of the immunome screening. The patients’ humoral immune response to the 109 proteins suggests the induction or significant upregulation of these proteins in vivo during human infection and thus may play a role in the pathogenesis of B. pseudomallei. Identification of B. pseudomallei immunogens has shed new light on the elucidation of the bacterium's pathogenesis mechanism and disease severity. These immunogens can be further evaluated as prophylactic and serodiagnostic candidates as well as drug targets.

Structural parameterization and functional prediction of antigenic polypeptome sequences with biological activity through quantitative sequence-activity models (QSAM) by molecular electronegativity edge-distance vector (VMED).

Only from the primary structures of peptides, a new set of descriptors called the molecular electronegativity edge-distance vector (VMED) was proposed and applied to describing and characterizing the molecular structures of oligopeptides and polypeptides, based on the electronegativity of each atom or electronic charge index (ECI) of atomic clusters and the bonding distance between atom-pairs. Here, the molecular structures of antigenic polypeptides were well expressed in order to propose the automated technique for the computerized identification of helper T lymphocyte (Th) epitopes. Furthermore, a modified MED vector was proposed from the primary structures of polypeptides, based on the ECI and the relative bonding distance of the fundamental skeleton groups. The side-chains of each amino acid were here treated as a pseudo-atom. The developed VMED was easy to calculate and able to work. Some quantitative model was established for 28 immunogenic or antigenic polypeptides (AGPP) with 14 (1–14) Ad and 14 other restricted activities assigned as “1”(+) and “0”(−), respectively. The latter comprised 6 Ab(15–20), 3 Ak(21–23), 2 Ek(24–26), 2 H-2k(27 and 28) restricted sequences. Good results were obtained with 90% correct classification (only 2 wrong ones for 20 training samples) and 100% correct prediction (none wrong for 8 testing samples); while contrastively 100% correct classification (none wrong for 20 training samples) and 88% correct classification (1 wrong for 8 testing samples). Both stochastic samplings and cross validations were performed to demonstrate good performance. The described method may also be suitable for estimation and prediction of classes I and II for major histocompatibility antigen (MHC) epitope of human. It will be useful in immune identification and recognition of proteins and genes and in the design and development of subunit vaccines. Several quantitative structure activity relationship (QSAR) models were developed for various oligopeptides and polypeptides including 58 dipeptides and 31 pentapeptides with angiotensin converting enzyme (ACE) inhibition by multiple linear regression (MLR) method. In order to explain the ability to characterize molecular structure of polypeptides, a molecular modeling investigation on QSAR was performed for functional prediction of polypeptide sequences with antigenic activity and heptapeptide sequences with tachykinin activity through quantitative sequence-activity models (QSAMs) by the molecular electronegativity edge-distance vector (VMED). The results showed that VMED exhibited both excellent structural selectivity and good activity prediction. Moreover, the results showed that VMED behaved quite well for both QSAR and QSAM of poly-and oligopeptides, which exhibited both good estimation ability and prediction power, equal to or better than those reported in the previous references. Finally, a preliminary conclusion was drwan: both classical and modified MED vectors were very useful structural descriptors. Some suggestions were proposed for further studies on QSAR/QSAM of proteins in various fields.