Adjuvance of influenza virosomes in CTL induction in vitro

The induction of cytotoxic T lymphocyte (CTL) responses is of high relevance in immunological defense against intracellular pathogens and tumor cells. While humoral immune responses are successfully induced by a number of vaccines, the activation of cellular immune responses has only been addressed more recently. The development of novel immunogens from live attenuated vaccines to subunit vaccines demands efficient and safe adjuvants to improve their immunogenicity. Importantly, there are only three adjuvants licensed for human use: aluminium salts, MF59 (microfluidized detergent stabilized oil in water emulsion) and IRIV (immunopotentiating reconstituted influenza virosomes). Aluminium salts are the most widely used adjuvants and their efficacy in enhancement of humoral responses is well documented. They are ineffective in the induction of cellular responses, whereas IRIV and MF59 might be effective, in addition to humoral responses, also in the induction of cellular responses. The aim of our group, working in the field of cancer immunotherapy, is induction of CTL specific to melanoma associated antigens. The monitoring of a clinical phase I/II trial has demonstrated increased frequencies of specific CTL in peripheral blood upon administration of antigenic epitopes encoded as minigenes with costimulatory molecules in a recombinant vaccinia virus. In the heterologous vaccination protocol adopted, however, high CTL frequencies were not sustained upon administration of the same epitopes as synthetic peptides. This pattern prompted the search for appropriate adjuvants enhancing peptide induced CTL responses. In this thesis work we focused on the in vitro characterization of immune responses elicited by influenza virosomes and on the in vitro evaluation of influenza virosome adjuvance in HLA class I restricted peptide induced CTL responses. We tested empty IRIV admixed with peptides and influenza virosomes encapsulating peptides, both produced by Pevion Biotech Ltd. Due to the low encapsulation efficiency of IRIV per se, the production of the second formulation required encapsulation of peptides into liposomes and subsequent fusion with chimeric IRIV. Thus, we characterised immune responses elicited by empty IRIV and empty chimeric IRIV fused with empty liposomes (FCIRIV). Then, we evaluated their adjuvant capacity by testing CTL induction in the presence of IRIV admixed with peptides and by peptides encapsulated in FCIRIV as compared to CTL induction by peptides in absence of influenza virosomes. For IRIV admixed with peptides we addressed induction of CTL specific for the highly immunogenic Influenza matrix 58-66 (IM58-66) and to the immunodominant melanoma
associated Melan-A/ Mart-127-35 HLA-A201 restricted epitopes. For peptides encapsulated
in FCIRIV we addressed induction of CTL specific for the L27Melan-A/Mart-126-35 HLAA0201
restricted epitope.
Our results demonstrate that all influenza virosome formulations under investigation
induce antigen triggered CD4+ T cell proliferation characterized by a T helper 1 cytokine
profile. Further dissection of CD4+ T cells identified CD4+CD45RO+ cells as proliferative
responders to IRIV stimulation and no major cell proliferation could be induced in cord
blood mononuclear cell cultures. These findings indicate that the majority of CD4+ T cells
responding to IRIV are antigen experienced. In addition, supernatants of IRIV stimulated
PBMC cultures favoured maturation of dendritic cells, as demonstrated by upregulation of
HLA-ABC, CD86 and CD83.
Both, influenza virosomes admixed with peptides or encapsulating peptides significantly
enhanced specific CTL induction, as detected by multimer staining and cytotoxicity assays.
CTL induction experiments in presence of irradiated CD4+ T cells indicated that IRIV CTL
adjuvance required CD4+ T cell activation. In addition, transwell cultures pointed to a key
role of cytokines in IRIV mediated CTL adjuvance.
In contrast to empty IRIV, FCIRIV with encapsulated peptides were characterized by CD4+
T cell independent adjuvant potential, possibly attributable to influenza virosome delivery
capacities.
Taken together, our results demonstrate that influenza virosomes are endowed with the
capacity to enhance HLA class I restricted CTL induction in vitro. Importantly, this could be
demonstrated not only for the highly immunogenic IM58-66 epitope, but also for the
melanoma associated epitopes L27Melan-A/Mart-126-35 and Melan-A/Mart-127-35.
Moreover, CTL induced by L27Melan-A/Mart-126-35 encapsulated in FCIRIV were capable of
recognizing and lysing tumor cells that constitutively express the Melan-A/Mart-1 antigen.
These in vitro findings encourage further evaluation of influenza virosome CTL adjuvance
in vivo.