In vivo cytotoxic T-lymphocyte induction may take place via CD8 T helper lymphocytes

Immunization of mice with peptide constructs, consisting of a determinant recognized by T cytotoxic cells colinearly linked to a determinant recognized by T helper cells (TDc-TDh) was able to induce cytotoxic T lymphocytes in vivo. Interestingly, this induction could be achieved in the absence of adjuvant in non-depleted as well as in CD4(+)-cell-depleted BALB/c mice. In the latter case, induction took place simultaneously with the activation of CD8+ T helper cells specific for a TDh contained within the sequence of the TDc RIQRGPGRAFVTIGK from the immunodominant V3 loop of HIV1 gp120. The possible implications of these findings in HIV infection and AIDS disease are discussed

Antitumor effect of allogenic fibroblasts engineered to express Fas ligand (FasL)

Fas ligand is a type II transmembrane protein which can induce apoptosis in Fas-expressing cells. Recent reports indicate that expression of FasL in transplanted cells may cause graft rejection and, on the other hand, tumor cells may lose their tumorigenicity when they are engineered to express FasL. These effects could be related to recruitment of neutrophils by FasL with activation of their cytotoxic machinery. In this study we investigated the antitumor effect of allogenic fibroblasts engineered to express FasL. Fibroblasts engineered to express FasL (PA317/FasL) did not exert toxic effects on transformed liver cell line (BNL) or colon cancer cell line (CT26) in vitro, but they could abrogate their tumorigenicity in vivo. Histological examination of the site of implantation of BNL cells mixed with PA317/FasL revealed massive infiltration of polymorphonuclear neutrophils and mononuclear cells. A specific immune protective effect was observed in animals primed with a mixture of BNL or CT26 and PA317/FasL cells. Rechallenge with tumor cells 14 or 100 days after priming resulted in protection of 100 or 50% of animals, respectively. This protective effect was due to CD8+ cells since depletion of CD8+ led to tumor formation. In addition, treatment of pre-established BNL tumors with a subcutaneous injection of BNL and PA317/FasL cell mixture at a distant site caused significant inhibition of tumor growth. These data demonstrate that allogenic cells engineered with FasL are able to abolish tumor growth and induce specific protective immunity when they are mixed with neoplastic cells

Poly(Anhydride) Nanoparticles Act as Active Th1 Adjuvants through Toll-Like Receptor Exploitation

The mechanisms that underlie the potent Th1-adjuvant capacity of poly(methyl vinyl ether-co-maleic anhydride) nanoparticles (NPs) were investigated. Traditionally, polymer NPs have been considered delivery systems that promote a closer interaction between antigen and antigen-presenting cells (APCs). Our results revealed that poly(anhydride) NPs also act as agonists of various Toll-like receptors (TLRs) (TLR2, -4, and -5), triggering a Th1-profile cytokine release (gamma interferon [IFN- ], 478 pg/ml versus 39.6 pg/ml from negative control; interleukin-12 [IL-12], 40 pg/ml versus 7.2 pg/ml from negative control) and, after incubation with dendritic cells, inducing a 2.5- to 3.5-fold increase of CD54 and CD86 costimulatory molecule expression. Furthermore, in vivo studies suggest that NPs actively elicit a CD8 T-cell response. Immunization with empty NPs resulted in a significant delay in the mean survival date (from day 7 until day 23 postchallenge) and a protection level of 30% after challenge against a lethal dose of Salmonella enterica serovar Enteritidis. Taken together, our results provide a better understanding of how NPs act as active Th1 adjuvants in immunoprophylaxis and immunotherapy through TLR exploitation

Induction of cytotoxic T lymphocytes in mice against the principal neutralizing domain of HIV-1 by immunization with an engineered T-cytotoxic-T-helper synthetic peptide construct

Peptide constructs were engineered by colinear synthesis of two short synthetic peptide determinants; a determinant recognized by T helper cells (TDh) and a determinant recognized by T cytotoxic cells (TDc). Three types of constructs were synthesized: TDc-TDh, TDh-TDc, and TDh-KK-TDc, where KK are two lysine residues. In vivo immunization with free construct induced cytolytic lymphocytes (CTL) only in the case of TDc-TDh. However, immunization with spleen cells to which these constructs had been internalized by hypertonic shock, induced CTL activity in all three cases. No CTL could be induced after immunization with free TDc in either protocol. These results indicate that cell internalization of the construct might be essential for CTL induction, and also, that "help" from the TDh seems to be required

Topical application of a peptide inhibitor of transforming growth factor-beta1 ameliorates bleomycin-induced skin fibrosis

Transforming growth factor-beta (TGF-beta) plays a crucial role in the pathogenesis of skin fibrotic diseases. Systemic TGF-beta inhibitors effectively inhibit fibrosis in different animal models; however, systemic inhibition of TGF-beta raises important safety issues because of the pleiotropic physiological effects of this factor. In this study, we have investigated whether topical application of P144 (a peptide inhibitor of TGF-beta1) ameliorates skin fibrosis in a well-characterized model of human scleroderma. C3H mice received daily subcutaneous injections of bleomycin for 4 wk, and were treated daily with either a lipogel containing P144 or control vehicle. Topical application of P144 significantly reduced skin fibrosis and soluble collagen content. Most importantly, in mice with established fibrosis, topical treatment with P144 lipogel for 2 wk significantly decreased skin fibrosis and soluble collagen content. Immunohistochemical studies in P144-treated mice revealed a remarkable suppression of connective tissue growth factor expression, fibroblast SMAD2/3 phosphorylation, and alpha-smooth muscle actin positive myofibroblast development, whereas mast cell and mononuclear cell infiltration was not modified. These data suggest that topical application of P144, a peptide inhibitor of TGF-beta1, is a feasible strategy to treat pathological skin scarring and skin fibrotic diseases for which there is no specific therapy

Enhancement of CD4 and CD8 immunity by anti-CD137 (4-1BB) monoclonal antibodies during hepatitis C vaccination with recombinant adenovirus

The induction of protective or therapeutic cellular immunity against hepatitis C virus (HCV) is a difficult goal. In a previous work we showed that immunization with a recombinant adenovirus encoding HCV-NS3 (RAdNS3) could partially protect mice from challenge with a vaccinia virus encoding HCV antigens. We sought to investigate whether systemic administration of an immunostimulatory monoclonal antibody directed against the lymphocyte surface molecule CD137 could enhance the immunity elicited by RAdNS3. It was found that treatment with anti-CD137 mAb after the administration of a suboptimal dose of RAdNS3 enhanced cytotoxic and T helper cell responses against HCV NS3. Importantly, the ability of RAdNS3 to induce protective immunity against challenge with a recombinant vaccinia virus expressing HCV proteins was markedly augmented. Thus, combination of immunostimulatory anti-CD137 mAb with recombinant adenoviruses expressing HCV proteins might be useful in strategies of immunization against HCV

Regression of colon cancer and induction of antitumor immunity by intratumoral injection of adenovirus expressing interleukin-12

Interleukin-12 (IL-12) has been shown to possess potent immunoregulatory and antitumoral effects. We have evaluated the anti-oncogenic potential and the mechanisms of the antitumoral effect of in vivo adenovirus-mediated transfer of IL-12 gene in a murine model of colon cancer. AdCMVIL-12 was constructed to permit coordinated production of p40 and p35 subunits of IL-12 gene to obtain the maximum IL-12 bioactivity. Infection of murine colon cancer CT-26 cells in vitro with AdCMVIL-12 resulted in the production of high levels of IL-12. In vivo gene therapy of colon cancer nodules by intratumoral injection of AdCMVIL-12 induced a local increase in IL-12 and interferon-gamma levels and a complete regression of the tumor in 26 of 34 (76%) mice. Tumor disappeared between days 7 and 10 after vector administration. The antitumoral effect was mediated by CD8+ T cells and was associated with the generation of cytotoxic T lymphocytes against colon cancer cells. Animals that eliminated the tumor were protected against a second administration of neoplastic cells. Treatment with AdCMVIL-12 of one tumor nodule also caused regression of established tumors at distant sites. These data demonstrate that AdCMVIL-12 is a useful therapeutic tool for established colon cancer in mice and should be considered for application in humans

Induction of gp120-specific protective immune responses by genetic vaccination with linear polyethylenimine-plasmid complex

The induction of IFN-gamma-secreting CD8+ T cells and neutralizing antibodies to HIV-1 are both key requirements for prevention of viral transmission and clearance of pathogenic HIV. Although DNA vaccination has been shown to induce both humoral and cellular immune responses against HIV antigens, the magnitude of the immune responses has always been disappointing. In this report, we analyze the ability of polyethylenimine (PEI)-DNA complex expressing an HIV-glycoprotein 120 (gp120) antigen (PEI-pgp120) to induce systemic CD8+ T cell and humoral responses to the gp120 antigen. The administration of PEI-plasmid complex resulted in rapid elevation of serum levels of IL-12 and IFN-gamma. Furthermore, a single administration of PEI-pgp120 complex elicits a number of gp120-specific CD8+ T cells 20 times higher than that elicited by three intramuscular injections of naked DNA. Interestingly, we found that systemic vaccination with PEI-pgp120 induced protective immune responses against both systemic and mucosal challenges with a recombinant vaccinia virus expressing a gp120 antigen. The data also demonstrated that the depletion of macrophages with liposome-encapsulated clodronate completely abolished gp120-specific cellular response. Overall, our results showed that a single administration of PEI-pgp120 complexes, eliciting strong immune responses, is an effective vaccination approach to generate protection against systemic and mucosal viral infections

Induction of cytotoxic T-cell response against hepatitis C virus structural antigens using a defective recombinant adenovirus

A replication-defective recombinant adenovirus (RAd), RAdCMV-CE1, containing core and E1 genes of hepatitis C virus (HCV) was constructed. RAdCMV-CE1 was able to express core and E1 proteins both in mice and human cells. Immunization of BALB/c mice with RAdCMV-CE1 induced a specific cytotoxic T-cell response against the two HCV proteins. This response was characterized using a panel of 60 synthetic 14- or 15-mer overlapping peptides (10 amino-acid overlap) spanning the entire sequence of these proteins. Five main epitopes were found in the core protein, four of which had been previously described either in mice or humans. One single novel epitope was found in E1. Fine mapping of this E1 determinant, showed that octamer GHRMAWDM is the minimal epitope recognized by cytotoxic T lymphocytes (CTL). The cytotoxic T-cell response was H-2d restricted, lasted for at least 100 days, and was mediated by T cells with the classic CD4-CD8+ phenotype. This work demonstrates that replication-defective recombinant adenoviruses can efficiently express HCV proteins and are able to induce an in vivo cytotoxic T-cell response against a diversity of epitopes from HCV antigens. These vectors should be taken into consideration in the design of vaccines and also as a means to stimulate specific T-cell responses in chronic HCV carriers

Therapeutic vaccination of woodchucks against chronic woodchuck hepatitis virus infection

BACKGROUND/AIMS: Therapeutic vaccination is a new approach to treat patients with chronic hepatitis B virus infection. We have used the woodchuck model to examine the efficacy and safety of this approach. METHODS: Seven woodchucks chronically infected with woodchuck hepatitis virus were immunized with surface antigen from this virus, purified from plasma, in conjunction with a peptide named FIS (encompassing amino acids 106-118: FISEAIIHVLHSR from sperm whale myoglobin), which is recognized by T helper lymphocytes. As controls, two woodchucks chronically infected with woodchuck hepatitis virus were immunized: one with FIS only and the other with surface antigen only. RESULTS: Co-immunization with surface antigen and FIS, but not with FIS or surface antigen alone, induced anti-surface antibodies in 7/7 immunized woodchucks. In the two woodchucks in which the highest titer of anti-surface antibody was elicited, severe liver damage was observed: one died of fulminant hepatitis and the other became seriously ill with hepatic injury and had to be sacrificed. CONCLUSIONS: Co-immunization of chronically infected woodchucks with surface antigen and a peptide recognized by T helper cells produces a good anti-surface antibody response. However, this strategy needs to be optimized before its implementation in humans. Although our experiments are not strictly comparable to vaccination of chronically hepatitis B virus-infected patients with recombinant or plasma-derived vaccines, we believe that precautions should be taken to avoid the risk of severe liver injury when immunizing hepatitis B virus carriers