Mycobacterial heat shock protein 65 (mbHSP65)-induced atherosclerosis: Preventive oral tolerization and definition of atheroprotective and atherogenic mbHSP65 peptides.

OBJECTIVE: The aim of this study was to identify atherogenic and atheroprotective peptides of bacterial HSP60 [taking mycobacterial HSP65 (mbHSP65) as a potent paradigmatic representative] that could be used as candidates for an orally applied tolerizing vaccine against atherosclerosis. METHODS: ApoE(-/-) mice were immunized with mbHSP65 protein or peptides, given mbHSP65 orally and then kept either on chow or high cholesterol diet. Atherosclerosis was assessed by en face and immunohistological analysis. Anti-HSP autoantibodies were detected by ELISA. The number and in vitro suppressive function of splenic and lymph node regulatory T cells (Tregs) were analyzed by flow cytometry. Specific T cell reactivity against mbHSP65 protein or peptides was assessed by proliferation assay. RESULTS: Decreased lesion size was accompanied by (a) increased splenic Treg numbers; (b) increased interleukin (IL)-10 mRNA levels in the aorta; (c) increased levels of anti-mbHSP65 and anti-mouse HSP60 antibodies pointing to pro-eukaryotic HSP60 humoral crossreaction, not curtailed by oral tolerization; (d) most importantly, we identified and functionally characterized novel atherogenic and atheroprotective mbHSP65 epitopes. CONCLUSION: Atheroprotective mbHSP65 peptides may be considered as potential candidates for the development of a tolerizing vaccine to prevent and treat atherosclerosis, while keeping protective immunity to non-atherogenic domains of mbHSP65 intact.

Considerations for non-clinical safety studies of therapeutic peptide vaccines.

Guidelines for non-clinical studies of prophylactic vaccines against infectious diseases have been published widely, but similar guidelines for therapeutic vaccines, and especially therapeutic peptide vaccines, have yet to be established. The approach to non-clinical safety studies required for therapeutic vaccines differs from that for prophylactic vaccines due to differences in the risk-benefit balance and the mechanisms of action. We propose the following guidelines for non-clinical safety studies for therapeutic peptide vaccines. (i) Since the main safety concern is related to the immune response that might occur at normal sites that express a target antigen, identification of these possible target sites using in silico human expression data is important. (ii) Due to the strong dependence on HLA, it is not feasible to replicate immune responses in animals. Thus, the required non-clinical safety studies are characterized as those detecting off-target toxicity rather than on-target toxicity.

Repetitive immunization breaks tolerance to type XVII collagen and leads to bullous pemphigoid in mice.

Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease of the elderly associated with considerable morbidity and mortality. As unspecific immunosuppressants are still the mainstay of BP therapy, several animal models, based on the passive transfer of autoantibodies or immune cells, have been developed to obtain a better understanding of the pathogenesis of BP and evaluate novel therapeutic interventions. We describe in this study an experimental model inducing BP by immunization of immunocompetent mice with a recombinant form of the immunodominant 15th noncollagenous domain of murine BP180 (type XVII collagen). The homologous noncollagenous 16A domain of human BP180 has previously been identified as an immunodominant region in human BP. Immunization of female SJL/J mice with the murine peptide led to clinical disease within 14 wk in 56% of mice. In contrast, none of the other strains developed blisters despite the presence of autoantibodies. The clinical disease manifested for at least 8 wk without further manipulation. This novel immunization-induced model reflects key immunopathological characteristics of human BP, including binding of complement-fixing autoantibodies along the dermal-epidermal junction, elevated total IgE serum levels, and infiltration of skin lesions with eosinophilic granulocytes. The use of immunocompetent mice and the induction of sustained clinical disease not requiring additional interventions make this immunization-induced mouse model most suitable to further explore the pathogenesis of BP and novel therapeutic interventions for this and other autoantibody-mediated diseases.

Phase I trial of personalized peptide vaccination for cytokine-refractory metastatic renal cell carcinoma patients.

The aim of this clinical trial was to investigate the toxicity and immunological responses of personalized peptide vaccination for cytokine-refractory metastatic renal cell carcinoma patients. Patients were confirmed to be human leukocyte antigen (HLA)-A24 or HLA-A2 positive and had histologically confirmed renal cell carcinoma. Ten patients were enrolled in the present study. The peptides to be administered were determined based on the presence of peptide-specific cytotoxic T lymphocyte precursors in peripheral blood mononuclear cells (PBMC) and peptide-specific IgG in the plasma of cancer patients. Patients received subcutaneous injections of four different peptides (3 mg/peptide) every 2 weeks. Vaccinations were well tolerated without any major adverse events. A minimal increase in peptide-specific interferon-gamma production in postvaccination PBMC was observed, regardless of higher levels of cytotoxic T lymphocyte activity in prevaccination PBMC. In contrast, an increase in peptide-specific IgG levels of postvaccination (sixth) plasma was observed in the majority of patients. After progression, five patients received interleukin-2 therapy and continuous vaccination, with survival of 31, 25, 23, 17, and 15 months, but interleukin-2 did not impede humoral responses boosted by the vaccination. These results encourage further clinical trials of personalized peptide vaccinations.

Vaccination: role in metastatic melanoma.

Based on the poor impact on overall survival obtained by systemic chemotherapy in metastatic melanoma and the identification of many melanoma antigens recognized by T cells, in the last decade many efforts have been devoted to the development of active specific immunotherapy as a promising systemic treatment for this neoplastic disease. A number of Phase I-II clinical trials have been performed with different vaccination approaches that included whole tumor cells, antigen peptides, antigen-pulsed dendritic cells, recombinant viruses, plasmids or naked DNA, and heat-shock proteins. Despite some promising immunological and clinical results obtained in these studies, melanoma-specific vaccines have altogether failed to prove their efficacy in the few large Phase III randomized clinical trials performed. Nonetheless, the possibility of activating the human immune system to recognize and destroy tumor cells remains a challenging investigative field, considering that the new knowledge of the intricate cellular and molecular mechanisms that regulate the immune function and tumor-host interactions may allow the development of new clinically relevant melanoma vaccination strategies.

Pertussis toxin (PTX) B subunit and the nontoxic PTX mutant PT9K/129G inhibit Tat-induced TGF-beta production by NK cells and TGF-beta-mediated NK cell apoptosis.

We show that the pertussis toxin B oligomer (PTX-B), and the PTX mutant PT9K/129G, which is safely administered in vivo, inhibit both transcription and secretion of TGF-beta elicited by HIV-1 Tat in NK cells. Tat-induced TGF-beta mRNA synthesis is also blocked by the ERK1 inhibitor PD98059, suggesting that ERK1 is needed for TGF-beta production. Moreover, Tat strongly activates the c-Jun component of the multimolecular complex AP-1, whereas TGF-beta triggers c-Fos and c-Jun. Of note, treatment of NK cells with PTX-B or PT9K/129G inhibits Tat- and TGF-beta-induced activation of AP-1. TGF-beta enhances starvation-induced NK cell apoptosis, significantly reduces transcription of the antiapoptotic protein Bcl-2, and inhibits Akt phosphorylation induced by oligomerization of the triggering NK cell receptor NKG2D. All these TGF-beta-mediated effects are prevented by PTX-B or PT9K/129G through a PI3K-dependent mechanism, as demonstrated by use of the specific PI3K inhibitor, LY294002. Finally, PTX-B and PT9K/129G up-regulate Bcl-x(L), the isoform of Bcl-x that protects cells from starvation-induced apoptosis. It is of note that in NK cells from patients with early HIV-1 infection, mRNA expression of Bcl-2 and Bcl-x(L) was consistently lower than that in healthy donors; interestingly, TGF-beta and Tat were detected in the sera of these patients. Together, these data suggest that Tat-induced TGF-beta production and the consequent NK cell failure, possibly occurring during early HIV-1 infection, may be regulated by PTX-B and PT9K/129G.

[Pseudomonas aeruginosa vaccine on the basis of antigens isolated from the supernatant of culture media K-4]. / Sinegnoinaia vaktsina na osnove antigenov, vydelennykh iz supernatanta sredy kul'tivirovaniia K-4.

The possibility of using experimental culture medium K-4 prepared on the basis of casein hydrolysate peptides with the isoelectric point 4.1 for obtaining antigens from P. aeruginosa strains was evaluated. Two antigenic fractions were isolated from the culture fluid containing extracellular slime. The study of the toxicity of the antigenic preparations revealed that one of these fractions had low toxicity for mice (the second antigenic fraction was highly toxic). The former P. aeruginosa antigenic fraction was used for obtaining pyocyanic vaccine. One vaccination dose of this vaccine contained 0.2 mg of the antigen adsorbed on aluminum hydroxide. Pyocyanic vaccine ensured the active protection of mice challenged with P. aeruginosa homologous and heterologous strains.