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Serogroup B meningococcus (MenB) is a leading cause of meningitis and sepsis across the world and vaccination is the most effective way to protect against this disease. 4CMenB is a multi-component vaccine against MenB, which is now licensed for use in subjects >2 months of age in several countries. In this study, we describe the development and use of an ad hoc protein microarray to study the immune response induced by the three major 4CMenB antigenic components (fHbp, NHBA and NadA) in individual sera from vaccinated infants, adolescents and adults. The resulting 4CMenB protein antigen fingerprinting allowed the identification of specific human antibody repertoire correlating with the bactericidal response elicited in each subject. This work represents an example of epitope mapping of the immune response induced by a multicomponent vaccine in different age groups with the identification of protective signatures. It shows the high flexibility of this microarray based methodology in terms of high-throughput information and minimal volume of biological samples needed., 4CMenB is an approved multi-component vaccine against Serogroup B meningococcus. Here the authors develop a protein microarray for three major 4CMenB antigenic components (fHbp, NHBA and NadA) and describe antibody repertoires in sera from vaccinated infants, adolescents and adults correlating with bactericidal response.

Nowadays, in scientific fields such as Structural Biology or Vaccinology, there is an increasing need of fast, effective and reproducible gene cloning and expression processes. Consequently, the implementation of robotic platforms enabling the automation of protocols is becoming a pressing demand. The main goal of our study was to set up a robotic platform devoted to the high-throughput automation of the polymerase incomplete primer extension cloning method, and to evaluate its efficiency compared to that achieved manually, by selecting a set of bacterial genes that were processed either in the automated platform (330) or manually (94). Here we show that we successfully set up a platform able to complete, with high efficiency, a wide range of molecular biology and biochemical steps. 329 gene targets (99 %) were effectively amplified using the automated procedure and 286 (87 %) of these PCR products were successfully cloned in expression vectors, with cloning success rates being higher for the automated protocols respect to the manual procedure (93.6 and 74.5 %, respectively).

Both active and passive immunization strategies against Staphylococcus aureus have thus far failed to show efficacy in humans. With the attempt to develop an effective S. aureus vaccine, we selected five conserved antigens known to have different roles in S. aureus pathogenesis. They include the secreted factors α-hemolysin (Hla), ess extracellular A (EsxA), and ess extracellular B (EsxB) and the two surface proteins ferric hydroxamate uptake D2 and conserved staphylococcal antigen 1A. The combined vaccine antigens formulated with aluminum hydroxide induced antibodies with opsonophagocytic and functional activities and provided consistent protection in four mouse models when challenged with a panel of epidemiologically relevant S. aureus strains. The importance of antibodies in protection was demonstrated by passive transfer experiments. Furthermore, when formulated with a toll-like receptor 7-dependent (TLR7) agonist recently designed and developed in our laboratories (SMIP.7-10) adsorbed to alum, the five antigens provided close to 100% protection against four different staphylococcal strains. The new formulation induced not only high antibody titers but also a Th1 skewed immune response as judged by antibody isotype and cytokine profiles. In addition, low frequencies of IL-17-secreting T cells were also observed. Altogether, our data demonstrate that the rational selection of mixtures of conserved antigens combined with Th1/Th17 adjuvants can lead to promising vaccine formulations against S. aureus.

Natural immunity against obligate and/or facultative intracellular pathogens is usually mediated by both humoral and cellular immunity. The identification of those antigens stimulating both arms of the immune system is instrumental for vaccine discovery. Although high-throughput technologies have been applied for the discovery of antibody-inducing antigens, few examples of their application for T-cell antigens have been reported. We describe how the compilation of the immunome, here defined as the pool of immunogenic antigens inducing T- and B-cell responses in vivo, can lead to vaccine candidates against Chlamydia trachomatis . We selected 120 C. trachomatis proteins and assessed their immunogenicity using two parallel high-throughput approaches. Protein arrays were generated and screened with sera from C. trachomatis -infected patients to identify antibody-inducing antigens. Splenocytes from C. trachomatis -infected mice were stimulated with 79 proteins, and the frequency of antigen-specific CD4 + /IFN-γ + T cells was analyzed by flow cytometry. We identified 21 antibody-inducing antigens, 16 CD4 + /IFN-γ + –inducing antigens, and five antigens eliciting both types of responses. Assessment of their protective activity in a mouse model of Chlamydia muridarum lung infection led to the identification of seven antigens conferring partial protection when administered with LTK63/CpG adjuvant. Protection was largely the result of cellular immunity as assessed by CD4 + T-cell depletion. The seven antigens provided robust additive protection when combined in four-antigen combinations. This study paves the way for the development of an effective anti- Chlamydia vaccine and provides a general approach for the discovery of vaccines against other intracellular pathogens.

Despite several decades of intensive studies, no vaccines againstChlamydia trachomatis, an intracellular pathogen causing serious ocular and urogenital diseases, are available yet. Infection-induced immunity in both animal models and humans strongly supports the notion that for a vaccine to be effective a strong CD4+Th1 immune response should be induced. In the course of our vaccine screening program based on the selection of chlamydial proteins eliciting cell-mediated immunity, we have found that CT043, a protein annotated as hypothetical, induces CD4+Th1 cells both in chlamydia-infected mice and in human patients with diagnosedC. trachomatisgenital infection. DNA priming/protein boost immunization with CT043 results in a 2.6-log inclusion-forming unit reduction in the murine lung infection model. Sequence analysis of CT043 fromC. trachomatishuman isolates belonging to the most representative genital serovars revealed a high degree of conservation, suggesting that this antigen could provide cross-serotype protection. Therefore, CT043 is a promising vaccine candidate againstC. trachomatisinfection.

LytM proteins belong to a family of bacterial metalloproteases. In Gram-negative bacteria, LytM factors are mainly reported to have a direct effect on cell division by influencing cleavage and remodeling of peptidoglycan. In this study, mining nontypeable Haemophilus influenzae (NTHI) genomes, three highly conserved open reading frames (ORFs) containing a LytM domain were identified, and the proteins encoded by the ORFs were named YebA, EnvC, and NlpD on the basis of their homology with the Escherichia coli proteins. Immunoblotting and confocal analysis showed that while NTHI NlpD is exposed on the bacterial surface, YebA and EnvC reside in the periplasm. NTHI ΔyebA and ΔnlpD deletion mutants revealed an aberrant division phenotype characterized by an altered cell architecture and extensive membrane blebbing. The morphology of the ΔenvC deletion mutant was identical to that of the wild-type strain, but it showed a drastic reduction of periplasmic proteins, including the chaperones HtrA, SurA, and Skp, and an accumulation of β-barrel-containing outer membrane proteins comprising the autotransporters Hap, IgA serine protease, and HMW2A, as observed by proteomic analysis. These data suggest that EnvC may influence the bacterial surface protein repertoire by facilitating the passage of the periplasmic chaperones through the peptidoglycan layer to the close vicinity of the inner face of the outer membrane. This hypothesis was further corroborated by the fact that an NTHI envC defective strain had an impaired capacity to adhere to epithelial cells and to form biofilm. Notably, this strain also showed a reduced serum resistance. These results suggest that LytM factors are not only important components of cell division but they may also influence NTHI physiology and pathogenesis by affecting membrane composition., IMPORTANCE Nontypeable Haemophilus influenzae (NTHI) is an opportunistic pathogen that colonizes the human nasopharynx and can cause serious infections in children (acute otitis media) and adults (chronic obstructive pulmonary disease). Several virulence factors are well studied, but the complete scenario of NTHI pathogenesis is still unclear. We identified and characterized three NTHI LytM factors homologous to the Escherichia coli LytM proteins. Although LytM factors are reported to play a crucial role in the cell division process, in NTHI they are also involved in other bacterial functions. In particular, YebA and NlpD are fundamental for membrane stability: indeed, their absence causes an increased release of outer membrane vesicles (OMVs). On the other hand, our data suggest that EnvC could directly or indirectly affect peptidoglycan permeability and consequently, bacterial periplasmic and outer membrane protein distribution. Interestingly, by modulating the surface composition of virulence determinants, EnvC also has an impact on NTHI pathogenesis.

Background Non-typeable Haemophilus influenzae (NTHi) is a Gram negative microorganism residing in the human nasopharyngeal mucosa and occasionally causing infections of both middle ear and lower respiratory airways. A broadly protective vaccine against NTHi has been a long-unmet medical need, as the high genetic variability of this bacterium has posed great challenges. Results In this study, we developed a robust serum bactericidal assay (SBA) to optimize the selection of protective antigens against NTHi. SBA takes advantage of the complement-mediated lysis of bacterial cells and is a key in vitro method for measuring the functional activity of antibodies. As a proof of concept, we assessed the bactericidal activity of antibodies directed against antigens known to elicit a protective response, including protein D used as carrier protein in the Synflorix pneumococcal polysaccharide conjugate vaccine. Prior to SBA screening, the accessibility of antigens to antibodies and the capacity of the latter to induce C3 complement deposition was verified by flow cytometry. Using baby rabbit serum as a source of complement, the proposed assay not only confirmed the bactericidal activity of the antibodies against the selected vaccine candidates, but also showed a significant reproducibility. Conclusions Considering the rapidity and cost-effectiveness of this novel SBA protocol, we conclude that it is likely to become an important tool to prove the capability of antibodies directed against recombinant antigens to induce NTHi in vitro killing and to both select new protective vaccine candidates, and predict vaccine efficacy. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0420-x) contains supplementary material, which is available to authorized users.

Chlamydia are intracellular bacteria associated to serious human disease. A vaccine has proved difficult to obtain so far, and current opinions agree that multi-antigen combinations may be required to induce optimal protective responses. In order to identify new potential vaccine candidates, we recently screened the Chlamydia pneumoniae (Cpn) genome and described 53 recombinant proteins which elicited antibodies binding to purified Cpn cells. We now report that six proteins in this group can also induce in vitro neutralizing antibodies. Antibody specificity for the corresponding antigens was assessed by immunoblot analysis of 2DE Cpn protein maps. Furthermore, four of the six in vitro neutralizing antigens (Pmp2, Pmp10, OmpH-like and enolase) could inhibit Cpn dissemination in a hamster model. The results show that these Cpn proteins are immunoaccessible in infectious EBs, and recommend further investigation on their value as vaccine components.

The age-related and T cell-independent immunological properties of most capsular polysaccharides limit their use as vaccines, especially in children under 2 years of age. To overcome these limitations, polysaccharide antigens have been successfully conjugated to a variety of carrier proteins, such as diphtheria toxoid or tetanus toxoid (TT) and the diphtheria mutant (CRM197) to produce very successful glycoconjugate vaccines. The increasing demand for new conjugate vaccines requires the availability of additional carriers providing high and long-lasting T helper cell immunity. Here we describe the design and construction of three recombinant carrier proteins (N6, N10, N19) constituted by strings of 6, 10 or 19 human CD4(+) T cell epitopes from various pathogen-derived antigens, including TT and proteins from Plasmodium falciparum, influenza virus and hepatitis B virus. Each of these epitopes is defined as universal in that it binds to many human MHC class II molecules. When conjugated to Haemophilus influenzae type b (Hib) oligosaccharide, these carriers elicit a potent anti-Hib antibody response in mice. In the case of the N19-Hib conjugate, this response is at least as good as that observed with CRM197-Hib, a conjugate vaccine currently used for mass immunization. We also show that some of the universal epitopes constituting the recombinant carriers are specifically recognized by two human in vitro systems, suggesting that T cell memory is provided by the selected epitopes. The data indicate that rationally designed recombinant polyepitope proteins represent excellent candidates for the development and clinical testing of new conjugate vaccines.

Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudomembranous colitis. While the role of toxins in pathogenesis has been extensively described, the contribution of surface determinants to intestinal colonization is still poorly understood. We focused our study on a novel member of the MSCRAMM family, named CbpA (Collagen binding protein A), for its adhesive properties towards collagen. We demonstrate that CbpA, which carries an LPXTG-like cell wall anchoring domain, is expressed on the bacterial surface of C. difficile and that the recombinant protein binds at high affinity to collagens I and V (apparent Kd in the order of 10(-9 ) M). These findings were validated by confocal microscopy studies showing the colocalization of the protein with type I and V collagen fibres produced by human fibroblasts and mouse intestinal tissues. However, the collagen binding activity of the wild-type C. difficile 630 strain was indistinguishable to the cbpA knock-out strain. To overcome this apparent clostridial adherence redundancy, we engineered a Lactococcus lactis strain for the heterologous expression of CbpA. When exposed on the surface of L. lactis, CbpA significantly enhances the ability of the bacterium to interact with collagen and to adhere to ECM-producing cells. The binding activity of L. lactis-CbpA strain was prevented by an antiserum raised against CbpA, demonstrating the specificity of the interaction. These results suggest that CbpA is a newsurface-exposed adhesin contributing to the C. difficile interaction with the host.

Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudomembranous colitis. While the role of toxins in pathogenesis has been extensively described, the contribution of surface determinants to intestinal colonization is still poorly understood. We focused our study on a novel member of the MSCRAMM family, named CbpA (Collagen binding protein A), for its adhesive properties towards collagen. We demonstrate that CbpA, which carries an LPXTG-like cell wall anchoring domain, is expressed on the bacterial surface of C. difficile and that the recombinant protein binds at high affinity to collagens I and V (apparent Kd in the order of 10(-9 ) M). These findings were validated by confocal microscopy studies showing the colocalization of the protein with type I and V collagen fibres produced by human fibroblasts and mouse intestinal tissues. However, the collagen binding activity of the wild-type C. difficile 630 strain was indistinguishable to the cbpA knock-out strain. To overcome this apparent clostridial adherence redundancy, we engineered a Lactococcus lactis strain for the heterologous expression of CbpA. When exposed on the surface of L. lactis, CbpA significantly enhances the ability of the bacterium to interact with collagen and to adhere to ECM-producing cells. The binding activity of L. lactis-CbpA strain was prevented by an antiserum raised against CbpA, demonstrating the specificity of the interaction. These results suggest that CbpA is a newsurface-exposed adhesin contributing to the C. difficile interaction with the host.

We present an interdisciplinary approach that, by incorporating a range of experimental and computational techniques, allows the identification and characterization of functional/immunogenic domains. This approach has been applied to ArtJ, an arginine-binding protein whose orthologs in Chlamydiae trachomatis (CT ArtJ) and pneumoniae (CPn ArtJ) are shown to have different immunogenic properties despite a high sequence similarity (60% identity). We have solved the crystallographic structures of CT ArtJ and CPn ArtJ, which are found to display a type II transporter fold organized in two α-β domains with the arginine-binding region at their interface. Although ArtJ is considered to belong to the periplasm, we found that both domains contain regions exposed on the bacterial surface. Moreover, we show that recombinant ArtJ binds to epithelial cells in vitro, suggesting a role for ArtJ in host-cell adhesion during Chlamydia infection. Experimental epitope mapping and computational analysis of physicochemical determinants of antibody recognition revealed that immunogenic epitopes reside mainly in the terminal (D1) domain of both CPn and CT ArtJ, whereas the surface properties of the respective binding-prone regions appear sufficiently different to assume divergent immunogenic behavior. Neutralization assays revealed that sera raised against CPn ArtJ D1 partially reduce both CPn and CT infectivity in vitro, suggesting that functional antibodies directed against this domain may potentially impair chlamydial infectivity. These findings suggest that the approach presented here, combining functional and structure-based analyses of evolutionary-related antigens can be a valuable tool for the identification of cross-species immunogenic epitopes for vaccine development.

N19, a string of human universal CD4 T-cell epitopes from various pathogen-derived antigens, was shown to exert a stronger carrier effect than CRM197 for the induction of anti-group CNeisseria meningitidiscapsular polysaccharide (MenC), after immunization of mice with various dosages of N19-MenC or CRM-MenC conjugate vaccines. After two immunizations, the N19-based construct induced anti-MenC antibody and protective bactericidal antibody titers higher than those induced by three doses of the CRM-MenC conjugate and required lower amounts of conjugate. N19-based conjugates are superior to CRM-based conjugates to induce protective immune responses to MenC conjugates.

We report that intraperitoneal injection of Chlamydia pneumoniae purified elementary bodies (EBs) in the hamster causes a systemic infection allowing the isolation of viable chlamydiae from several organs for several days post-infection (p.i.). In particular, spleen infection occurred up to Day 7 p.i. in 100% of animals. Systemic infection probably occurs via macrophages as intraperitoneally injected chlamydiae which are taken up by the hamster macrophages remain viable and can infect in vitro cell cultures. Immunization of 18 hamsters with heat-inactivated purified EBs, completely protected the spleens of 16 animals and substantially reduced infection levels in the remaining two. This model, therefore, provides a robust screening tool for the assessment of the protective activity of potential vaccine candidates. In a pilot study on five recombinant antigens recently described as EB surface proteins, three gave results undistinguishable from non-immunized, or mock-immunized controls; however two antigens, derived, respectively, from the product of the lcrE gene (a component of the putative TTSS of C. pneumoniae) and the product of Cpn0498 open reading frame, proved to be capable of inducing protective immune responses.

The large extracellular loop of human CD81, a tetraspanin mediating hepatitis C virus envelope protein E2 binding to human cells, has been crystallized in a hexagonal form. The three-dimensional structure, solved and refined at 2.6 A resolution (R-factor = 22.8%), shows that the protein adopts a dimeric assembly, based on an association interface built up by tetraspanin-conserved residues. Structural comparisons with the tertiary structure of human CD81 large extracellular loop, previously determined in a different crystal form, show marked conformational fluctuations in the molecular regions thought to be involved in binding to the viral protein, suggesting rules for recognition and assembly within the tetraspan web.

We have used DNA microarrays to follow Neisseria meningitidis serogroup B (MenB) gene regulation during interaction with human epithelial cells. Host-cell contact induced changes in the expression of 347 genes, more than 30% of which encode proteins with unknown function. The upregulated genes included transporters of iron, chloride, amino acids, and sulfate, many virulence factors, and the entire pathway of sulfur-containing amino acids. Approximately 40% of the 189 upregulated genes coded for peripherally located proteins, suggesting that cell contact promoted a substantial reorganization of the cell membrane. This was confirmed by fluorescence activated cell sorting (FACS) analysis on adhering bacteria using mouse sera against twelve adhesion-induced proteins. Of the 12 adhesion-induced surface antigens, 5 were able to induce bactericidal antibodies in mice, demonstrating that microarray technology is a valid approach for identifying new vaccine candidates and nicely complements other genome mining strategies used for vaccine discovery.

Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working withC. pneumoniaeand in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization ofC. pneumoniae, we have adopted a combined genomic-proteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization inC. pneumoniae.

Human CD81, a known receptor for hepatitis C virus envelope E2 glycoprotein, is a transmembrane protein belonging to the tetraspanin family. The crystal structure of human CD81 large extracellular domain is reported here at 1.6 A resolution. Each subunit within the homodimeric protein displays a mushroom-like structure, composed of five alpha-helices arranged in 'stalk' and 'head' subdomains. Residues known to be involved in virus binding can be mapped onto the head subdomain, providing a basis for the design of antiviral drugs and vaccines. Sequence analysis of 160 tetraspanins indicates that key structural features and the new protein fold observed in the CD81 large extracellular domain are conserved within the family. On these bases, it is proposed that tetraspanins may assemble at the cell surface into homo- and/or hetero-dimers through a conserved hydrophobic interface located in the stalk subdomain, while interacting with other liganding proteins, including hepatitis C virus E2, through the head subdomain. The topology of such interactions provides a rationale for the assembly of the so-called tetraspan-web.

B. Subtilis expression plasmids generally require a stringent Shine-Dalgarno Sequence (SDS). Site-directed-mutagenesis was explored to change the Shine-Dalgarno Sequence from AAAAATGGGG (mutant type) to AAAAAGGGGG (wild type) in recombinant plasmid PSM604. The single base substitution made the plasmid with wild SDS unstable in structure and segregation. The interaction of SDS with subtilisin leader sequence of PSM604 might be responsible for the instability of plasmid.

Infections with genital human papillomaviruses (HPV) are likely to be neutralised more efficiently if a mucosal immune response can be elicited at the viral entry site. Local IgA antibodies are highly induced when antigens are co-administered with mucosal adjuvants, such as cholera toxin (CT) and Escherichia coli heat labile enterotoxin (LT) which, however, are not expected to have wide application because of their pronounced toxicity. We have immunised mice intranasally with HPV-6b virus-like particles (VLPs) and a genetically modified LT-derived molecule with only residual toxicity, LTR72, and compared the humoral responses with those obtained following systemic immunisation with VLPs and the MF59 adjuvant. Titration of anti-HPV antibodies in sera and vaginal secretions established that LTR72 was able to elicit higher serum and mucosal IgA titers, in addition to IgG serum levels, comparable to those obtained by parenteral immunisation. These results confirm the potential of toxin-derived adjuvants and extend their use in combination with HPV antigens.

The large extracellular domain of CD81, a member of the tetraspanin family and a receptor protein for hepatitis C virus envelope E2 glycoprotein, has been expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data to 1.6 A resolution were obtained at the ID14 beamline of the European Synchrotron Radiation Facility from a flash-frozen crystal at 100 K. The crystals belong to space group P2(1), with unit-cell parameters a = 31.5, b = 77.2, c = 38.5 A, beta = 107.4 degrees, and are likely to contain two extracellular domains (2 x 99 residues) per asymmetric unit.

Chronic hepatitis C virus (HCV) infection occurs in about 3 percent of the world's population and is a major cause of liver disease. HCV infection is also associated with cryoglobulinemia, a B lymphocyte proliferative disorder. Virus tropism is controversial, and the mechanisms of cell entry remain unknown. The HCV envelope protein E2 binds human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Binding of E2 was mapped to the major extracellular loop of CD81. Recombinant molecules containing this loop bound HCV and antibodies that neutralize HCV infection in vivo inhibited virus binding to CD81 in vitro.

Background: Outer membrane vesicles (OMVs) are spheroid particles released by all Gram-negative bacteria as a result of the budding out of the outer membrane. Since they carry many of the bacterial surface-associated proteins and feature a potent built-in adjuvanticity, OMVs are being utilized as vaccines, some of which commercially available. Recently, methods for manipulating the protein content of OMVs have been proposed, thus making OMVs a promising platform for recombinant, multivalent vaccines development. Methods: Chlamydia muridarum DO serine protease HtrA, an antigen which stimulates strong humoral and cellular responses in mice and humans, was expressed in Escherichia coli fused to the OmpA leader sequence to deliver it to the OMV compartment. Purified OMVs carrying HtrA (CM rHtrA-OMV) were analyzed for their capacity to induce antibodies capable of neutralizing Chlamydia infection of LLC-MK2 cells in vitro . Results: CM rHtrA-OMV immunization in mice induced antibodies that neutralize Chlamydial invasion as judged by an in vitro infectivity assay. This was remarkably different from what observed with an enzymatically functional recombinant HtrA expressed in, and purified from the E. coli cytoplasm (CM rHtrA). The difference in functionality between anti-CM rHtrA and anti-CM rHtrA-OMV antibodies was associated to a different pattern of protein epitopes recognition. The epitope recognition profile of anti-CM HtrA-OMV antibodies was similar to that induced in mice during Chlamydial infection. Conclusions: When expressed in OMVs HtrA appears to assume a conformation similar to the native one and this results in the elicitation of functional immune responses. These data further support the potentiality of OMVs as vaccine platform. Keywords: neutralizing antibodies; outer membrane vesicles; Chlamydia; DO serine protease; Chlamydia trachomatis (Published: 6 May 2013) Citation: Journal of Extracellular Vesicles 2013, 2 : 20181 - http://dx.doi.org/10.3402/jev.v2i0.20181 To access the supplementary material to this article, please see Supplementary files under Article Tools online.

The recombinant hepatitis delta virus antigen was obtained as a chimaeric protein fused to the C-terminus of the phage MS2 RNA polymerase. Following induction of the temperature-sensitive promoter, two major polypeptides of about 34 kDa and 29 kDa, and two minor peptides about 21 kDa and 18 kDa, were obtained on PAGE. The 34-kDa protein was identified as the expected recombinant protein by confirming 82% of the primary structure using fast-atom-bombardment mass spectrometry. The most represented degradation product, i.e. the 29-kDa polypeptide, was also characterized by means of mass spectrometry and found to be produced by cleavage between amino acids 261 and 265. The presence of two main protein bands, with a similar difference in size, is also a typical feature of delta antigens, both extracted and recombinant, and it is considered to be derived either from heterogeneity of viral sequences, which can encode hepatitis delta antigen proteins of 195 and 214 amino acids, or from proteolysis of a single precursor. Since the data were obtained with a single viral sequence coding for 195 amino acids fused to 106 residues from MS2 polymerase, there is direct evidence that intrinsic structural properties of the protein sequence are able to cause a specific proteolysis resulting in the presence of two major forms, of which the smaller is 35-40 amino acids at the C-terminus. The recombinant protein can be used as an antigenic substitute of viral antigens both for immunoassays and for the preparation of anti-(hepatitis delta virus) antisera.

Recently, regional changes of cytokeratin patterns in human normal non-keratinized or keratinized oral mucosa have been demonstrated and the expression of individual cytokeratin polypeptides in lesions of oral mucosa has been compared with that of normal tissues. In particular, the presence of cytokeratin 19 in the suprabasal cell layers of oral epithelia has been shown to be strongly correlated with premalignancy. In the present study, we describe the results of an immunohistochemical investigation performed using a monoclonal antibody specific for cytokeratin 1 on normal oral mucosa and benign or malignant oral lesions. We show the different distribution of this polypeptide in non-neoplastic lesions from different sites of oral mucosa and describe the presence of cytokeratin 19. Our results are in agreement with the data obtained previously. In the malignant cases we demonstrate that the distribution of the two cytokeratins is characterized by complementary patterns.