The recombinant Klebsiella pneumoniae outer membrane protein OmpA has carrier properties for conjugated antigenic peptides

Abstract

Klebsiella pneumoniae OmpA, the 40-kDa major protein of the outer membrane, was cloned and expressed in Escherichia coli. The recombinant protein was produced intracellularly in E. coli as inclusion bodies. Fusion of a short peptide to the N-terminus of native P40 facilitated high-level expression of the recombinant protein. Purified recombinant P40 was analyzed to verify purity and structural integrity. The molecular mass of purified recombinant P40 determined by electrospray mass spectrometry was 37 061 Da, in agreement with the theoretical mass deduced from the DNA sequence. Specific proliferation of recombinant-P40-primed murine lymph node cells in response to recombinant P40 stimulation in vitro indicated the presence of a T-cell epitope on recombinant P40. The induction of high serum antibody titers to a synthetic peptide derived from the attachment protein G of the respiratory syncytial virus when chemically coupled to recombinant P40 indicated that the protein had potent carrier properties. OmpA [20, 21] have been demonstrated to mediate expression of gram-negative bacteria, present at about 10 5 copies/cell. It is believed to occur in a monomeric form in its native state. A of peptides on the surface of live enterobacterial vectors. OmpC, the outer-membrane protein complex of Neisseria meningitidis, typical feature of OmpA is that it can be modified by heat: the mobility of Escherichia coli OmpA on SDS/PAGE decreases has been shown to be effective in humans as a conjugate vaccine with Haemophilus influenzae, pneumococcal and meningococcal when it is heated in the presence of SDS [1]. OmpA is highly conserved among gram-negative bacteria and is thought to con-capsular polysaccharides [22Ϫ25]. Other clinically useful carrier proteins have been derived from bacteria: diphteria and tetanus sist of two domains. The N terminus, including amino acid residues 1Ϫ170, forms a membrane-spanning domain and crosses toxoids are both successfully used in conjugated H. influenzae vaccines to transform the capsular polysaccharide, which is a Tthe outer membrane eight times in antiparallel β-strands, leading to a typical amphiphilic β-barrel [2Ϫ4]. The C-terminal moiety independent antigen, into a T-dependent antigen [26]. KeywordsIn this paper we describe the expression and production in of the protein is thought to be periplasmic [5]. The protein seems to be multifunctional. In addition to non-physiological functions, E. coli and purification of Klebsiella pneumoniae OmpA [27].Using various analytical criteria, the purity and structural integsuch as serving as a receptor for phages and colicins [6], it serves as a mediator in F-factor-dependent conjugation [7]. It is rity of the protein were evaluated. We demonstrate the presence of at least one T-cell epitope on the molecule and its potential also required for the structural integrity of the outer membrane and the generation of normal cell shape [8]. The capacity to use as a carrier protein for conjugated peptides. The immunological carrier properties of the recombin...

Cancer Vaccine Design: A Novel Bacterial Adjuvant for Peptide-Specific CTL Induction

“…As previously described for proteosomes, which are preparations of Omp of meningococci (8), we hypothesize that P40/peptide complexes are also formed by means of hydrophobic interactions of the peptides with the membrane-spanning domain of the protein P40. Finally, the fact that an exogenous helper epitope (such as TT) is not required in the immunizing mixture might be explained by the presence of at least one CD4 T cell epitope in the amino acid sequence of P40 (9), allowing this protein to act as both an adjuvant and a carrier protein. In this context, the observation that preimmunization with P40 does not compromise subsequent immune responses to P40-associated peptides represents an improvement compared with classic carrier proteins, such as TT, which have been shown to elicit epitope suppression (23,24).…”

“…Acetonitrile was of HPLC grade. K. pneumoniae P40 was expressed as a recombinant protein in Escherichia coli, purified as previously described (9,18), and will be referred to as P40 thereafter. Purification of P40 was designed in apyrogen conditions.…”

“…Therefore, we asked whether the 40-kDa major Omp (referred to as P40 hereafter) of Klebsellia pneumoniae, which contains at least one helper epitope (9) can be used to target antigenic peptides to the immune system and elicit a specific CTL response. Peptides from two melanoma-associated differentiation Ags, Melan-A/MART-1 (Melan-A) (10, 11) and tyrosinase-related protein 2 (TRP-2) (12) were used, namely 1) the HLA-A*0201-restricted decapeptide Melan-A 26 -35 (EAAGIG ILTV) substituted at position 2 (Melan-A 26 -35 A27L peptide analog), which is more immunogenic than the parental peptide both in vitro in human and in vivo in experimental models (13,14), and 2) the TRP-2 T cell epitope mapping to residues 181-188 (VYD-FFVWL) and presented in association with the mouse K b class I molecule (12).…”

Targeting of Nasal Mucosa-Associated Antigen-Presenting Cells In Vivo with an Outer Membrane Protein A Derived fromKlebsiellapneumoniae

“…This OmpA displays carrier-related properties for peptides and polysaccharides (8,27) when administered by the parenteral route. The aim of this study was to determine the efficacy and mechanism of action of this carrier molecule following nasal administration using, as antigen models, the pure B-cell epitope G5 and the G2Na protein, both derived from the respiratory syncytial virus subgroup A (RSV-A) G protein.…”

Complement Dependent Amplification of the Innate Response to a Cognate Microbial Ligand by the Long Pentraxin PTX3

“…Recombinant KpOmpA was expressed and purified by Pierre Fabre as described (20). Cobra venom factor (CVF) was obtained from Quidel.…”