Monoclonal antibody directed to the PilQ -PilA DSL region in Pseudomonas aeruginosa improves survival of infected mice with antibiotic combination.

The infections caused by Pseudomonas aeruginosa are related to high mortality and morbidity in critically ill patients because of multidrug resistance. Thus, we performed the efficacy of the monoclonal antibody (mAb) against PilQ -PilA DSL region (QA) in combination with antibiotics in a model of P. aeruginosa infection. In the present study, three clinically applicable antibiotics (levofloxacin, ceftazidime and gentamicin) and the anti-QA mAb were utilized for treatment of P. aeruginosa sepsis in mice. Reliably, in comparison with other treatment groups (antibody or antibiotic administration), the combination of antibiotic and anti-QA mAb essentially enhanced the survival of mice infected with P. aeruginosa PAO1. This synergistic effect was due to improved bactericidal effect, which prevented bacterial dissemination to different organs. Consequently, the antibiotic and anti-QA mAb combination gives a new effective strategy for the treatment of P. aeruginosa sepsis, particularly when large numbers of exceptionally virulent strains are present.

Detecting Receptor Tyrosine Kinase ROR1 Using a Developed Anti-ROR1 Polyclonal Antibody.

Receptor tyrosine kinase ROR1 has been introduced as an interesting prognostic cancer marker in histopathology. The aim of this study was to produce a polyclonal antibody (PAb) against recombinant human ROR1 protein to be used as a tool for investigation of ROR1 expression in human cancer tissue blocks. The extracellular part of human ROR1 recombinant protein was expressed using pET-28b(+) plasmid in Escherichia coli Bl21(DE3) host. The recombinant ROR1, as a candidate immunogen, was purified and injected to a New Zealand rabbit. Followed by raising the titration of antibody, polyclonal anti-ROR1 antibody was purified through affinity chromatography column. After determining the purity of PAb anti-ROR1, its specific reactivity was assessed through various assessments. Flow cytometry analysis showed that PAb anti-ROR1 specifically recognizes ROR1 molecule in a number of positive and negative cell lines. Results obtained from detection of ROR1 in paraffin-embedded breast adenocarcinoma tissue blocks (n = 11) also demonstrated that PAb anti-ROR1 can effectively be used in immunohistochemistry. In conclusion, the developed anti-ROR1 PAb can be used as a tool for determining the prognostic value of ROR1 in histopathology of cancer tissues.

Antibody Response to Human Extracellular HER2 Subdomain Proteins in Mice.

BACKGROUND: In addition to passive immunotherapy using anti-HER2 monoclonal antibodies, active immunotherapy via HER2 targeting is an interesting approach to inducing specific anti-tumor immune responses. We have recently reported the immunogenicity of HER2 subdomains following DNA immunization and HER2 protein boosting. In the present study, we evaluated the immunogenicity of different HER2 extracellular subdomains for the induction of anti-HER2 antibody response in BALB/c mice. OBJECTIVE: To investigate and characterize antibody responses to human recombinant proteins of HER2 extracellular subdomains in immunized mice. METHODS: Four subdomains of HER2 extracellular domain were expressed in E.coli; subsequently, purified recombinant proteins were intraperitoneally injected in BALB/c mice with Freund's adjuvant. The anti-HER2 antibody response was detected by ELISA, immunoblotting and flow cytometry. RESULTS: All the four HER2 subdomains along with the full extracellular domain (fECD) were able to induce specific anti-HER2 antibodies. Although anti-HER2 subdomains antibodies could not react with eukaryotic recombinant fECD protein by ELISA, they were able to recognize this protein by immunoblotting under both reduced and non-reduced conditions. Furthermore, only the sera of mice immunized with fECD protein could recognize native HER2 on HER2 overexpressing tumor cells (>99%) by flow cytometry. Moreover, fECD immunized mice sera inhibited the proliferation of tumor cells by XTT assay. CONCLUSION: The prokaryotic recombinant proteins of HER2 extracellular subdomains are immunogenic, yet the induced specific antibodies do not react with the native HER2 protein due to the paucity of post-translation modifications and /or distortion of the native conformation of isolated HER2 extracellular subdomains which might be potentially effective for induction of cell mediated immune response against HER2.

Restricted antibody response to Bordetella pertussis filamentous hemagglutinin induced by whole-cell and acellular pertussis vaccines.

BACKGROUND: Filamentous hemagglutinin (FHA) is a principal virulence factor, an important immunogenic antigen of Bordetella pertussis, and a major component of many acellular pertussis vaccines. In the present study, the human antibody response to different regions of FHA was determined in healthy children and adults vaccinated with either whole-cell or acellular pertussis vaccines. METHODS: To define the immunodominant regions of FHA, four overlapping recombinant fragments were expressed and produced in Escherichia coli and then purified by His-tagged based affinity chromatography. Two groups comprising healthy preschool children (n = 50) and adults (n = 26) were vaccinated with a single dose of commercial whole-cell and acellular DTaP vaccines, respectively. An antigen-based ELISA was applied to measure serum levels of anti-FHA antibody to both native and recombinant proteins in vaccinated volunteers. RESULTS: In both groups of vaccinated individuals, the anti-FHA antibody response was mainly directed against epitopes located within a fragment of FHA spanning amino acid residues 1877-2250 of the mature FHA molecule (p < 0.001). No or little antibody was detected against the other recombinant segments of FHA. CONCLUSION: Our results suggest that the human antibody response to FHA is directed to an immunodominant region located within residues 1877-2250 of the FHA molecule. Characterization and epitope mapping of the major components of acellular pertussis vaccine and future modifications in vaccine formulation may improve its efficacy and protectivity.

Production and characterization of recombinant light chain and carboxyterminal heavy chain fragments of tetanus toxin.

BACKGROUND: Light chain (LC) and heavy chain carboxyterminal subdomain (HCC) fragments are the most important parts of tetanus neurotoxin (TeNT) which play key roles in toxicity and binding of TeNT, respectively. In the present study, these two fragments were cloned and expressed in a prokaryotic system and their identity was confirmed using anti-TeNT specific polyclonal and monoclonal antibodies. METHODS: LC and HCC gene segments were amplified from Clostridium tetani genomic DNA by PCR, cloned into pET28b(+) cloning vector and transformed in Escherichia coli (E. coli) BL21(DE3) expression host. Recombinant proteins were then purified through His-tag using Nickel-based chromatography and characterized by SDS-PAGE, Western blotting and ELISA techniques. RESULTS: Recombinant light chain and HCC fragments were successfully cloned and expressed in (E. coli) BL21 (DE3). Optimization of the induction protocol resulted in production of high levels of HCC (~35% of total bacterial protein) and to lesser extends of LC (~5%). Reactivity of the His-tag purified proteins with specific polyclonal and monoclonal antibodies confirmed their renatured structure and identity. CONCLUSION: Our results indicate successful cloning and production of recombinant LC and HCC fragments of TeNT. These two recombinant proteins are potentially useful tools for screening and monitoring of anti-TeNT antibody response and vaccine production.