Turning immunological memory into amnesia by depletion of dividing T cells.

Immunological memory, defined as more efficient immune responses on antigen reexposure, can last for decades. The current paradigm is that memory is maintained by antigen-experienced "memory T cells" that can be long-lived quiescent or dividing. The contribution of T cell division to memory maintenance is poorly known and has important clinical implications. In this study, we directly addressed the role of dividing T cells in immunological memory maintenance by evaluating the consequences of their elimination. The specific ablation of dividing T cells was obtained by administration of ganciclovir to immune mice expressing the herpes simplex type 1 thymidine kinase suicide gene in T cells. We show that depletion of dividing T cells for 5 or 2 weeks suffices to abolish in vitro and in vivo memory responses against the male H-Y transplantation alloantigen or against lymphocytic choriomeningitis virus antigens, respectively. Similar results were obtained after the nonspecific elimination of all dividing cells by using hydroxyurea, a cytostatic toxic agent commonly used for cancer chemotherapy. This immune amnesia occurred in otherwise immunocompetent mice and despite the persistence of functional quiescent T cells displaying a "memory" phenotype. Thus, division of antigen-experienced T cells is an absolute requirement for immunological memory maintenance and the current concept of memory T cells is challenged.

Induction of protective antiviral cytotoxic T cells by a tubular structure capable of carrying large foreign sequences.

Bluetongue virus (BTV) produces large numbers of tubules during infection which are formed by a single virus coded non-structural protein, NS1. The NS1 protein has been fused with full length green fluorescent protein (GFP) and was shown to retain the capacity to form tubules when expressed in heterologous expression systems. Moreover, recombinant purified chimeric tubules were demonstrated to be internalized by macrophages and dendritic cells. The ability of such chimeric tubules to induce protective cytotoxic T lymphocytes (CTL) responses has been assessed by generating chimeric tubules carrying a single CD8(+) T cell epitope from the lymphocytic choriomeningitis virus (LCMV) nucleoprotein. These chimeric tubules were recognized by MHC class I restricted T cell hybridoma in vitro and induced in vivo strong CD8(+) class I-restricted CTL responses in immunized mice. Further, the immunized mice were protected when challenged with a lethal dose of LCMV. This is the first study that demonstrates that the virus derived tubules synthesized by a recombinant non-structural protein carrying a single viral CTL epitope could induce protective immunity against a lethal viral challenge. Since recombinant tubules carrying large inserts can be purified at a large quantity from insect cells, they have potential to develop as safe multi-CTL vaccine delivery systems.

Lyssavirus glycoproteins expressing immunologically potent foreign B cell and cytotoxic T lymphocyte epitopes as prototypes for multivalent vaccines.

Truncated and chimeric lyssavirus glycoprotein (G) genes were used to carry and express non-lyssavirus B and T cell epitopes for DNA-based immunization of mice, with the aim of developing a multivalent vaccine prototype. Truncated G (GPVIII) was composed of the C-terminal half (aa 253-503) of the Pasteur rabies virus (PV: genotype 1) G containing antigenic site III and the transmembrane and cytoplasmic domains. The chimeric G (GEBL1-PV) was composed of the N-terminal half (aa 1-250) of the European bat lyssavirus 1 (genotype 5) G containing antigenic site II linked to GPVIII. Antigenic sites II and III are involved in the induction of virus-neutralizing antibodies. The B cell epitope was the C3 neutralization epitope of the poliovirus type 1 capsid VP1 protein. The T cell epitope was the H2d MHC I-restricted epitope of the nucleoprotein of lymphocytic choriomeningitis virus (LCMV) involved in the induction of both cytotoxic T cell (CTL) production and protection against LCMV. Truncated G carrying foreign epitopes induced weak antibody production against rabies and polio viruses and provided weak protection against LCMV. In contrast, the chimeric plasmid containing various combinations of B and CTL epitopes elicited simultaneous immunological responses against both parental lyssaviruses and poliovirus and provided good protection against LCMV. The level of humoral and cellular immune responses depended on the order of the foreign epitopes inserted. Our results demonstrate that chimeric lyssavirus glycoproteins can be used not only to broaden the spectrum of protection against lyssaviruses, but also to express foreign B and CTL epitopes. The potential usefulness of chimeric lyssavirus glycoproteins for the development of multivalent vaccines against animal diseases and zoonoses, including rabies, is discussed.