Marie-Françoise Saron

In Vivo Induction of a High-Avidity, High-Frequency Cytotoxic T-Lymphocyte Response Is Associated with Antiviral Protective Immunity

ABSTRACT Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8+ cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8+ class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8+ T-cell epitopes, bound to 1-μm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503–7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4+T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies.

Delivery of Multiple Epitopes by Recombinant Detoxified Adenylate Cyclase of Bordetella pertussis Induces Protective Antiviral Immunity

ABSTRACT CyaA, the adenylate cyclase toxin from Bordetella pertussis, can deliver its N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells and particularly into professional antigen-presenting cells. We have previously identified within the primary structure of CyaA several permissive sites at which insertion of peptides does not alter the ability of the toxin to enter cells. This property has been exploited to design recombinant CyaA toxoids capable of delivering major histocompatibility complex (MHC) class I-restricted CD8+ T-cell epitopes into antigen-presenting cells and to induce specific CD8+cytotoxic T-lymphocyte (CTL) responses in vivo. Here we have explored the capacity of the CyaA vector carrying several different CD8+ T-cell epitopes to prime multiple CTL responses. The model vaccine consisted of a polyepitope made of three CTL epitopes from lymphocytic choriomeningitis virus (LCMV), the V3 region of human immunodeficiency virus gp120, and chicken ovalbumin, inserted at three different sites of the catalytic domain of genetically detoxified CyaA. Each of these epitopes was processed on delivery by CyaA and presented in vitro to specific T-cell hybridomas. Immunization of mice by CyaA toxoids carrying the polyepitope lead to the induction of specific CTL responses for each of the three epitopes, as well as to protection against a lethal viral challenge. Moreover, mice primed against the vector by mock CyaA or a recombinant toxoid were still able to develop strong CTL responses after subsequent immunization with a recombinant CyaA carrying a foreign CD8+ CTL epitope. These results highlight the potency of the adenylate cyclase vector for induction of protective CTL responses with multiple specificity and/or broad MHC restriction.

Chronic production of interferon in carrier mice congenitally infected with lymphocytic choriomeningitis virus

Abstract Congenital infection of mice with lymphocytic choriomeningitis virus leads to a lifelong virus carrier state. Here we provide evidence for the presence and action of interferon in such mice. The level of circulating interferon in adult carrier mice is 8–16 NIH units/ml of plasma. This interferon is acid stable and is capable of inducing 2–5A synthetase in mouse L 929 cells but not in human HeLa cells. Control, noinfected mice show less than 1 NIH unit/ml of plasma. In accord with these results, adult carrier mice have higher levels of the interferon-mediated pppA(2′p5′A)n synthetase (2–5A synthetase) in their liver and spleen than normal mice. Congenitally infected newborn mice also have higher levels of 2–5A synthetase in their liver in contrast to newborn control mice. These results in congenitally infected newborn and adult mice suggest that interferon may play a role, at least in part, in the pathogenesis of infection.

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.

Rapid enrichment of mouse natural killer cells by use of wheat germ agglutinin

The separation of mouse T and B lymphocytes by differential agglutination with wheat germ agglutinin (WGA) also enriches natural killer (NK) activity 2-7-fold. NK cells are recovered within the agglutinated cell population indicating that NK cells bind WGA. This technique can be applied to endogenous or interferon-induced NK cells.

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.

Altered cytokine genes expression by ConA-activated spleen cells from mice infected by lymphocytic choriomeningitis virus

The intravenous injection of mice with lymphocytic choriomeningitis virus (LCMV) induces a rapid and long-lasting immunodeficiency. T lymphocytes from 7-day-infected mice do not proliferate in vitro in response to ConA stimulation, do not produce IL-2 but display high affinity IL-2 receptors on their membrane. The non-coordinated regulation of these genes suggested that other cytokine-encoding genes may also be affected in their regulation. We have thus analyzed the expression of the genes encoding different cytokines transcribed during spleen cell activation by ConA. The genes encoding T lymphocyte-derived cytokines can be classified in three groups: the genes expressed similarly by normal and LCMV-cells (the p55 and the p75 chains of the IL-2 receptor [1]), the genes under expressed in LCMV-cells (IL-2, IL-3, IL-4 and IL-5) and the genes over expressed by these cells (GM-CSF and IFN-gamma). These results show that the viral infection has provoked a profound alteration of the overall regulation of the genetic program that follows T lymphocyte activation. Since T cell activation depends strictly on accessory cell-derived cytokines, we measured the level of transcription of IL-1, IL-6 and TNF-alpha; and our data show that the expression of these genes is equivalent in normal cells and in cells from LCMV-infected mice.

Evidence for the presence of T lymphocytes mediating lymphocytic choriomeningitis virus-specific delayed-type hypersensitivity in meningeal infiltrates of infected mice

Summary Meningeal infiltrates from mice infected during a period of 7 to 8 days with lymphocytic choriomeningitis virus contained T lymphocytes which mediated a virus-specific delayed-type hypersensitivity reaction upon local adoptive transfer. These preliminary results suggest that such a T-lymphocyte function may intervene in the pathogenesis of this viral infection.

Beneficial use of cyclosporin a in lymphocytic choriomeningitis virus infection in mice: Dose and kinetic effects

Summary The dose effect and the kinetics of cyclosporin A (CSA) injection in the course of lymphocytic choriomeningitis viral infection were studied. Several doses and different timings were exploited and it was found that, for maximal protection of lethally infected mice, CsA should be administered at a dose of 100 mg/kg on days 0 and 1 post-infection.

Prévention par les globulines anti-interféron de la mortalité des souris infectées par le virus de la chorioméningite lymphocytaire: Prevention of death in mice fatally infected with lymphocytic choriomeningitis virus by anti-interferon globulins

Summary Intracerebral inoculation of adult mice with lymphocytic choriomeningitis (LCM) virus is lethal, and is considered to be due to the development of sensitized T lymphocytes which are cytotoxic for virus-infected host cells. Injection of sheep anti-mouse interferon globulin into LCM virus-infected mice resulted in a 100 to 1,000-fold increase in viral titres, a 10 to 100-fold increase in serum interferon titres and the survival of virtually all of the mice. Anti-interferon globulin-treated mice also showed a focal necrosis of spleen and lymph nodes and a depletion of thymic lymphocytes. These lesions were more marked than in the organs of LCM virus-infected mice injected with normal sheep serum. The increased amounts of virus and/or interferon in mice injected with anti-interferon globulin and the marked lesions in these lymphoid organs may depress the immune response and thus be related to the survival of virus infected mice.