Preliminary safety assessment of CIGB-210, an investigational peptide for HIV infection.

Current human immunodeficiency virus treatments need to be periodically administered lifelong. In this study we assess the effect of repeated doses of an anti-HIV peptide drug candidate in C57BL6 strain. Two schemes of up to 15 administrations and one of 30, daily dosing for 5 days per week, all by the subcutaneous route were evaluated. Different dose concentrations of the peptide were assayed. CIGB-210 treated animals showed no symptoms or abnormal behavior as compared with placebo. All the animals gained weight during the study. Macroscopic evaluation showed no alterations in any of the organs studied. Microscopic analysis of the tissues did not show morphological changes in thymus, stomach, small and large intestines, kidney, brain, or cerebellum. The proliferative response of splenocytes and their capacity to secrete gamma interferon were not compromised by the repeated administration of CIGB-210. There were not statistically significant differences for any of the parameters evaluated during the study among treated and non-treated groups. We can conclude that CIGB-210 is well tolerated in C57BL6 mice in the dose concentration range explored and merits subsequent toxicological studies.

E2-CD154 vaccine candidate is safe and immunogenic in pregnant sows, and the maternal derived neutralizing antibodies protect piglets from classical swine fever virus challenge.

E2-CD154 subunit vaccine candidate is safe and protects swine from Classical Swine Fever (CSF). However, its safety and immunogenicity in pregnant sows, and the capacity of maternal derived neutralizing antibodies (MDNA) to protect the offspring is yet to be demonstrated. The aim of this study was to evaluate the safety and immunogenicity of E2-CD154 in pregnant sows, and the capacity of MDNA to protect the offspring. Seventeen pregnant sows were vaccinated twice with E2-CD154 in either the first or the second third of pregnancy. Pregnancy and litter parameters were compared with a control group of non-vaccinated sows. Neutralizing antibodies (NAb) were monitored. The time course of MDNA was assessed in a group of six piglets born to an E2-CD154 immunized sow, and the animals were challenged with CSFV at day 63 after birth. No local or systemic adverse effects were found. Neither abortions, nor congenital malformations, nor stillbirths were observed. All sows develop high NAb titers after the first immunization. Piglets born to an E2-CD154 vaccinated sow still showed MDNA titers of 1:100 at day 63 after birth. Five animals were negative for virus isolation after challenge, and showed neither signs of CSF, nor macroscopic lesions in the organs. The other piglet was positive for CSFV isolation, and macroscopic lesions were observed in the spleen, although no clinical signs of CSF other than fever were detected. E2-CD154 vaccine candidate was safe and immunogenic in pregnant sows, and the passive immunity transmitted to the offspring was still protective by day 63 after birth.

Dengue encephalitis-associated immunopathology in the mouse model: Implications for vaccine developers and antigens inducer of cellular immune response.

Despite the many efforts made by the scientific community in the development of vaccine candidates against dengue virus (DENV), no vaccine has been licensed up to date. Although the immunopathogenesis associated to the disease is a key factor to take into account by vaccine developers, the lack of animal models that reproduce the clinical signs of the disease has hampered the vaccine progress. Non-human primates support viral replication, but they are very expensive and do not show signs of disease. Immunocompromised mice develop viremia and some signs of the disease; however, they are not valuable for vaccine testing. Nowadays, immunocompetent mice are the most used model to evaluate the immunogenicity of vaccine candidates. These animals are resistant to DENV infection; therefore, the intracranial inoculation with neuroadapted virus, which provokes viral encephalitis, represents an alternative to evaluate the protective capacity of vaccine candidates. Previous results have demonstrated the crucial role of cellular immune response in the protection induced by the virus and vaccine candidates in this mouse encephalitis model. However, in the present work we are proposing that the magnitude of the cell-mediated immunity and the inflammatory response generated by the vaccine can modulate the survival rate after viral challenge. We observed that the intracranial challenge of naïve mice with DENV-2 induces the recruitment of immune cells that contribute to the reduction of viral load, but does not increase the survival rate. On the contrary, animals treated with cyclophosphamide, an immunosuppressive drug that affects proliferating lymphocytes, had a higher viral load but a better survival rate than untreated animals. These results suggest that the immune system is playing an immunopathogenic role in this model and the survival rate may not be a suitable endpoint in the evaluation of vaccine candidates based on antigens that induce a strong cellular immune response.

APL-2, an altered peptide ligand derived from heat-shock protein 60, induces interleukin-10 in peripheral blood mononuclear cell derived from juvenile idiopathic arthritis patients and downregulates the inflammatory response in collagen-induced arthritis model.

Juvenile idiopathic arthritis (JIA) is a heterogeneous group of diseases characterized by autoimmune arthritis of unknown cause with onset before age of 16 years. Methotrexate provides clinical benefits in JIA. For children who do not respond to methotrexate, treatment with anti-tumor necrosis factor (TNF)-α is an option. However, some patients do not respond or are intolerant to anti-TNF therapy. Induction of peripheral tolerance has long been considered a promising approach to the treatment of chronic autoimmune diseases. We aimed to evaluate the potentialities of two altered peptide ligands (APLs) derived from human heat-shock protein 60, an autoantigen involved in the pathogenesis of autoimmune arthritis, in JIA patients. Interferon (IFN)-γ, TNF-α and interleukin (IL)-10 levels were determined in ex vivo assays using peripheral blood mononuclear cells (PBMC) from these patients. Wild-type peptide and one of these APLs increased IFN-γ and TNF-α levels. Unlike, the other APLs (called APL2) increased the IL-10 level without affecting IFN-γ and TNF-α levels. On the other hand, APL2 induces a marked activation of T cells since it transforms cell cycle phase's distribution of CD4+ T cells from these patients. In addition, we evaluated the therapeutic effect of APL2 in collagen-induced arthritis model. Therapy with APL2 reduced arthritis scores and histological lesions in mice. This effect was associated to a decrease in TNF-α and IL-17 levels. These results indicate a therapeutic potentiality of APL2 for JIA.

CIGB-247: a VEGF-based therapeutic vaccine that reduces experimental and spontaneous lung metastasis of C57Bl/6 and BALB/c mouse tumors.

CIGB-247 is a novel cancer therapeutic vaccine that uses a mutated form of human VEGF as antigen. Being metastatic disease the most dramatic factor of tumor biology affecting patient survival and cure, preclinical evaluation of the impact of CIGB-247 vaccination on experimental metastasis mouse models is highly relevant, and constitutes the focus of this work. CIGB-247 was administered in a weekly schedule known to effectively reduce primary tumor growth. The vaccine was tested in experimental and spontaneous metastasis models of colon (CT26), lung (3LL-D122) and breast (F3II) carcinomas growing in C57Bl/6 or BALB/c mice. Primary tumor growth parameters, metastatic counts, and/or animal survival were recorded. Histology and specific humoral and cellular responses to the vaccine were evaluated. As compared to control groups, CIGB-247 vaccination significantly reduced the number and size of metastatic tumor foci in lungs after intravenous inoculation of CT26 and 3LL-D122 tumor cells. Spontaneous lung dissemination from 3LL-D122 and F3II breast tumor cells implanted in the footpad, or subcutaneously, was also reduced by immunization with CIGB-247. The vaccine elicited in both mouse strains antibodies specific for human and murine VEGF that effectively blocked the interaction of VEGF with VEGF receptor 2. Differing from other experimental reports that describe the use of VEGF for active tumor immunotherapy, CIGB-247 elicited a specific cellular response, measured both by a DTH increment and the induction of spleen cells cytotoxic to syngeneic tumor cells producing murine VEGF. In summary our results reinforce the potential of CIGB-247 vaccination to reduce both tumor growth and the number and size of tumor metastasis in lungs, the latter both after direct inoculations of cells in the blood stream, or as part of primary tumor progression in immunocompetent mice.

Antigen dose escalation study of a VEGF-based therapeutic cancer vaccine in non human primates.

CIGB-247 is a cancer therapeutic, based on recombinant modified human vascular endothelial growth factor (VEGF) as antigen, in combination with the oil free adjuvant VSSP (very small sized proteoliposomes of Neisseria meningitidis outer membrane). Our previous experimental studies in mice with CIGB-247 have shown that the vaccine has both anti-tumoral and anti-metastatic activity, and produces both antibodies that block VEGF-VEGF receptor interaction, and a specific T-cell cytotoxic response against tumor cells. CIGB-247, with an antigen dose of 100 µg, has been characterized by an excellent safety profile in mice, rats, rabbits, and non human primates. In this article we extend the immunogenicity and safety studies of CIGB-247 in non human primates, scaling the antigen dose from 100 µg to 200 and 400 µg/vaccination. Our results indicate that such dose escalation did not affect animal behavior, clinical status, and blood parameters and biochemistry. Also, vaccination did not interfere with skin deep skin wound healing. Anti-VEGF IgG antibodies and specific T-cell mediated responses were documented at all three studied doses. Antigen dose apparently did not determine differences in maximum antibody titer during the 8 weekly immunization induction phase, or the subsequent increase in antibodies seen for monthly boosters delivered afterwards. Higher antigen doses had a positive influence in antibody titer maintenance, after cessation of immunizations. Boosters were important to achieve maximum antibody VEGF blocking activity, and specific T-cell responses in all individuals. Purified IgG from CIGB-247 immunized monkey sera was able to impair proliferation and formation of capillary-like structures in Matrigel, for HMEC cells in culture. Altogether, these results support the further clinical development of the CIGB-247 therapeutic cancer vaccine, and inform on the potential mechanisms involved in its effect.

Immunogenicity and some safety features of a VEGF-based cancer therapeutic vaccine in rats, rabbits and non-human primates.

We have developed a cancer vaccine candidate (hereafter denominated CIGB-247), based on recombinant modified human vascular endothelial growth factor (VEGF) as antigen, and the adjuvant VSSP (very small sized proteoliposomes of Neisseria meningitidis outer membrane). In mice, previous work of our group had shown that vaccination with CIGB-247 extended tumor-take time, slowed tumor growth, and increased animal survival. Immunization elicited anti-human and murine VEGF-neutralizing antibodies, and spleen cells of vaccinated mice are cytotoxic in vitro to tumor cells that produce VEGF. We have now tested the immunogenicity of CIGB-247 in Wistar rats, New Zealand White rabbits and the non-human primate Chlorocebus aethiops sabaeus. Using weekly, biweekly and biweekly plus montanide immunization schemes, all three species develop antigen-specific IgG antibodies that can block the interaction of VEGF and VEGF receptor 2 in an ELISA assay. Antibody titers decline after vaccination stops, but can be boosted with new immunizations. In monkeys, DTH and direct cell cytotoxicity experiments suggest that specific T-cell responses are elicited by vaccination. Immunization with CIGB-247 had no effect on normal behavior, hematology, blood biochemistry and histology of critical organs, in the tested animals. Skin deep wound healing was not affected in vaccinated rats and monkeys.