Recombinant fusion proteins assembling Der p 1 and Der p 2 allergens from Dermatophagoides pteronyssinus.
BACKGROUND
Fusion proteins assembling multiple allergens can be engineered by recombinant DNA technologies in order to produce tools for diagnostic and immunotherapeutic purposes. Herein, we developed and characterized chimeras assembling Der p 1 and Der p 2 allergens as potential candidate vaccines against house dust mite allergy.


METHODS
Fusion proteins encompassing Der p 2 with either mature or proDer p 1 were expressed in Escherichia coli or Pichia pastoris. Forms with mutation in Der p 1 catalytic site were also engineered. Purified chimeras were characterized by immunoblotting, circular dichroism, disulfide bond mapping, basophil and T lymphocyte stimulation assays.


RESULTS
Four fusion proteins were expressed in E. coli as inclusion bodies, whereas only chimeras comprising proDer p 1 were obtained in yeast. All such hybrids formed polymers and aggregates, and yeast-expressed chimeras were unstable. Circular dichroism analysis performed after refolding of bacteria expressed chimeras encompassing mature Der p 1 confirmed partial folding, consistent with the occurrence of both correct and inappropriate intramolecular disulfide bonds. All fusion molecules were recognized by Der p 1- and Der p 2-specific human IgEs, monoclonal and polyclonal antibodies. Fusion proteins activate basophils from mite-allergic patients and trigger the proliferation of specific CD4+ T cells, albeit to a lower level when compared to individual allergens.


CONCLUSIONS
Production of multiple Der p 1-Der p 2 fusion proteins exhibiting partial folding and proper antigenic properties has been achieved. Nonetheless, significant solubility and stability issues currently limit the application of such chimeras for immunotherapy or diagnostic.
Bussières
Laetitia
L
Research and Development, Stallergènes, Antony, France.
Bordas-Le Floch
Véronique
V
Bulder
Ingrid
I
Chabre
Henri
H
Nony
Emmanuel
E
Lautrette
Aurélie
A
Berrouet
Christelle
C
Nguefeu
Yvette
Y
Horiot
Stéphane
S
Baron-Bodo
Véronique
V
Van Overtvelt
Laurence
L
De Conti
Anne Marie
AM
Schlegel
Anne
A
Maguet
Nicolas
N
Mouz
Nicolas
N
Lemoine
Pierrick
P
Batard
Thierry
T
Moingeon
Philippe
P
D000595:Amino Acid Sequence; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000737:chemistry / Q000276:immunology*; D060829:Arthropod Proteins; D001491:Basophils / Q000502:physiology; D003546:Cysteine Endopeptidases; D040002:Dermatophagoides pteronyssinus / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006967:Hypersensitivity / Q000628:therapy; D008213:Lymphocyte Activation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology; D014612:Vaccines / Q000276:immunology
D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; C451163:Dermatophagoides pteronyssinus antigen p 2; D011993:Recombinant Fusion Proteins; D014612:Vaccines; D003546:Cysteine Endopeptidases; C451162:Dermatophagoides pteronyssinus antigen p 1