Method and diagnostic tests based on flow cytometric analysis of antigen-specific t lymphocytes

A method for in vitro immuno-diagnosis of antigen-specific T lymphocytes based on the preparation of compositions, also called stimuli, able to stimulate the T lymphocytes;

• such compositions comprising at least one among the antigens in different forms selected in the group of:
• (a) raw protein extract, (b) purified or recombinant proteins, (c) synthetic peptides and combinations of (a), (b) and (c);
• such stimuli being identified as pathogen-specific when based on antigens originating from pathogens and vaccine-specific when based on antigens originating from strains used for making vaccines they;
• said method comprising the following steps:
• i) isolation of peripheral blood mononuclear cells (PBMC) from a sample of human or animal venous blood;
• ii) preparation of at least one stimulus selected between pathogen-specific and vaccine-specific stimuli,
• iii) preparation of a negative control comprising cells cultivated in vitro in complete medium without stimuli and a positive control comprising cells cultivated in vitro in complete medium with an aspecific stimulus;
• iv) stimulation of said T-lymphocytes with the vaccine-specific or the pathogen-specific stimulus in the presence of a costimulus;
• v) incubation;
• vi) selective staining by immunofluorescence;
• vii) flow-cytometry acquisition and analysis;
• viii) measurement and characterization of the immune response.

Composition of peptides comprising at least one of following groups of peptides:

• Ortho-Poxvirus peptides from sequence 1ID84 to 85, from sequence ID86 to 87, peptides from sequence ID88 to 90, peptides from sequences ID91 to 92, peptides sequence ID93, peptides from sequence ID94 to 95, peptides from sequence ID96 to 97, peptides from sequence ID98 to 99, peptides from sequence ID100 to 101, peptides from sequence ID102 to 103;
• Anthrax (B.anthracis) peptides from sequence ID74 to 83;
• SARS coronavirus:
• peptides from sequence ID44 to 59, peptides from sequence ID45 to 46 and from ID60 to 61, peptides from sequence ID47 to 48 and from ID62 to 63, peptides from sequence ID49 to 58 and from ID64 to 73, peptides from sequence ID45 to 46;Human non-SARS Coronavirus peptides from sequence ID173 to 177,
• peptides from sequence ID178 to 182.