Introduction of the haemagglutinin transmembrane region in the influenza virus matrix protein facilitates its incorporation into ISCOM and activation of specific CD8(+) cytotoxic T lymphocytes.
The gene encoding the influenza virus A matrix (MA) protein was cloned into the bacterial expression vector pMalC with and without the sequence encoding the transmembrane region of the haemagglutinin (HA). With the resulting recombinant proteins, immune stimulating complexes (ISCOM) were prepared. The MA protein with the hydrophobic anchor region (rMAHA) associated more efficiently with ISCOM than the unmodified MA protein (rMA). A B-lymphoblastoid cell line (B-LCL) was lysed by an autologous CD8(+) cytotoxic T lymphocyte (CTL) clone specific for the MA protein after incubation with rMAHA-ISCOM but not after incubation with rMA, rMAHA, rMA-ISCOM or empty ISCOM. The B-LCL was also lysed by the CTL clone after incubation with empty ISCOM mixed with the respective MA proteins. Incubation of ISCOM with the rMAHA protein proved to be the most efficient in this respect. Addition of the proteasome inhibitors lactacystin or clasto-lactacystin beta-lactone to the B-LCL incubated with rMAHA-ISCOM or the MA proteins mixed with empty ISCOM dramatically decreased the lysis by the CD8(+) CTL clone. These results indicate that the addition of a hydrophobic anchor to hydrophilic proteins in combination with ISCOM facilitates their entry in the MHC class I processing and presentation pathway. This may be an attractive approach for the development of subunit vaccines aiming at the induction of CTL-mediated immunity.
Voeten
J T
JT
Institute of Virology, Who National Influenza Centre, Erasmus Medical Centre Rotterdam, The Netherlands.
Rimmelzwaan
G F
GF
Nieuwkoop
N J
NJ
Lövgren-Bengtsson
K
K
Osterhaus
A D
AD
D017951:Antigen Presentation; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D015789:HLA-A2 Antigen; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D016882:ISCOMs / Q000235:genetics / Q000302:isolation & purification*; D066298:In Vitro Techniques; D009980:Influenza A virus / Q000235:genetics* / Q000276:immunology*; D007252:Influenza Vaccines / Q000235:genetics / Q000302:isolation & purification*; D008213:Lymphocyte Activation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000276:immunology*
D017931:DNA Primers; D015789:HLA-A2 Antigen; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D016882:ISCOMs; D007252:Influenza Vaccines; C031540:M-protein, influenza virus; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins