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New strategies for the development of oral vaccines : vectors based on non-pathogenic commensal bacteria, synthetic peptides and lipophilic adjuvants

Objective

* to identify linear neutralizing and protective B cell epitopes of the measles virus; * to define and study the presentation of T cell epitopes of the measles virus F, H, and NP proteins; * to study the immune response to Pam3Cys-lipopeptide conjugates; * construction of recombinant S. gordonii expressing MV-H and MV-F and to study their antigenicity and immunogenicity.
Mucosal model. An animal model was developed to study mucosal immunizations against a challenge with rodent adapted measles virus (MV).
Linear B cell epitopes of the MV. Using a large panel of MV monoclonal antibodies, a large array of peptides and phage-display libraries two new domains involved in the neutralization in vitro and protection in vivo against a lethal challenge of a rodent adapted MV in an animal model were identified. Peptides were designed for the induction of protection.
T cell epitopes of the MV nucleoprotein, hemagglutinin and fusion protein: aspects of antigen presentation. Analysis of the interactions between a peptide and the MHC II/TCR complex revealed that amino acids of the ragged tail of the class II-restricted peptides were important for T cell activation, suggesting that the antigen presenting cell can modulate the clonality of the T cell response by trimming the flanking sequences of the T cell epitope. Two T cell hybridomas were compared which recognized the same core epitope but differed in their sensitivity to the flanking sequences. Processing of NP in the presence or absence of a mAb, whose binding site corresponds to the T cell epitope, modulated the flanking sequences of the nested set of peptides corresponding to their epitope. Dominant and subdominant T cell epitopes were identified in the animal model, by studying T cell hybridomas and by direct immunization with peptides. Peptides other than the immunodominant ones were able to prime T cells against the virus. A `hierarchy of immunodominance' was established among different T cell epitopes. Co-immunization strategies using admixed T cell epitopes for simplified immunization with non-conjugated T and B cell epitopes were investigated.
Lipophilic adjuvants. A number of different lipophilic adjuvants based on conjugates including (i) different Pam3Cys derivatives, (ii) polyethylene glycol and peptides corresponding to (iii) T and (iv) B cell epitopes were synthesized. In a series of immunization experiments, constructs were identified which gave an optimal immune response to a peptide as B cell epitope.
Analysis of MHC I/II peptide interactions using peptide libraries. The impact of every amino acid in each sequence position of undecapeptide amides on the binding to the class II molecule HLA-DRB1*0101 was investigated using peptides libraries of different degrees of complexity in a binding competition assay with a fluorescence labeled DR1-specific peptide. From the activity pattern of the undecapeptide library strong and weak DR1-binders were designed. Octapeptide sublibraries in the positional scanning format were used to study the effects of the individual amino acids in the different positions of octapeptides. The contribution of every single amino acid side chain to MHC class 1 binding and to cytotoxic T cell activity was shown to be influenced by other amino acids in the sequence.
Simultaneous synthesis of free and immobilized peptides. To optimize peptide synthesis, a polymeric support suitable for solid-phase peptide synthesis was functionalized with two different anchor groups for simultaneous preparation of free and immobilized peptides.
Immunogenicity of recombinant S. gordonii. Chromosomal integration of the M6-MV constructs was obtained using the recombinant plasmid pSMB55. The F1 and F2 polypeptides were expressed in different recombinant strains, and also the N-and C-terminal portions of H were expressed separately. The recombinant bacteria expressing the F or H protein were recognized by MV-specific human and rabbit sera. The immunogenicity of the constructs was confirmed whether Freund's complete adjuvant or Pam3Cys was used.
MAJOR SCIENTIFIC BREAKTHROUGHS:
* Identification of linear neutralizing epitopes which are involved in the protection in a rodent model. * Identification of a role for the ragged tail of MHC class II restricted peptides. * Description of a new mechanism by which antigen presenting cells can modulate the clonality of a T cell response. * Lipophilic constructs designed for optimal immune response. * Peptide libraries were used to investigate contributions of individual amino acids of T cell epitopes to MHC class I/II binding. * Expression of MV-H/F in the oral commensal S. gordonii. * Confirmation of antigenicity and immunogenicity of these constructs.

Call for proposal

Data not available

Coordinator

LABORATOIRE NATIONAL DE SANTE
Address
Rue Auguste Lumiere 20A
1950 Luxembourg
Luxembourg
 

Participants (2)

THE UNIVERSITY OF TUEBINGEN
Germany
Address
Auf Der Morgenstelle 18
72076 Tuebingen
 
UNIVERSITA' DEGLI STUDI DI SIENA
Italy
Address
Via Banchi Di Sotto 55
Siena