NK-lysin peptides ameliorate viral encephalopathy and retinopathy disease signs and provide partial protection against nodavirus infection in European sea bass.

Antimicrobial peptides (AMP) comprise a wide range of small molecules with direct antibacterial activity and immunostimulatory role and are proposed as promising substitutes of the antibiotics. Additionally, they also exert a role against other pathogens such as viruses and fungi less evaluated. NK-lysin, a human granulysin orthologue, possess a double function, taking part in the innate immunity as AMP and also as direct effector in the cell-mediated cytotoxic (CMC) response. This molecule is suggested as a pivotal molecule involved in the defence upon nervous necrosis virus (NNV), an epizootic virus provoking serious problems in welfare and health status in Asian and Mediterranean fish destined to human consumption. Having proved that NK-lysin derived peptides (NKLPs) have a direct antiviral activity against NNV in vitro, we aimed to evaluate their potential use as a prophylactic treatment for European sea bass (Dicentrarchus labrax), one of the most susceptible cultured-fish species. Thus, intramuscular injection of synthetic NKLPs resulted in a very low transcriptional response of some innate and adaptive immune markers. However, the injection of NKLPs ameliorated disease signs and increased fish survival upon challenge with pathogenic NNV. Although NKLPs showed promising results in treatments against NNV, more efforts are needed to understand their mechanisms of action and their applicability to the aquaculture industry.

VA-MENGOC-BC Vaccination Induces Serum and Mucosal Anti Neisseria gonorrhoeae Immune Responses and Reduces the Incidence of Gonorrhea.

BACKGROUND: Overall, there are over 30 different sexually transmitted infections with Neisseria gonorrhoeae being the third most frequent with a reported 78 million cases per year. Gonococcal infection causes genital inflammation, which can be a risk factor for others sexually transmitted infections, particularly human immunodeficiency virus. Gonorrhea is a treatable disease, but recently an increase in antibiotic resistance has been of concern. There are currently no vaccines available. However, parenteral vaccination with anti N. meningitidis serogroup B vaccine has been reported to decrease the incidence of gonococcal burden in New Zealand and in Cuba despite the fact that parenteral vaccination is not deemed to induce mucosal IgA. Here we explore possible mechanisms of protection against gonococcal infection through parenteral meningococcal B vaccination. METHODS: Ninety-two serum, saliva and oropharyngeal swabs samples of young adults (healthy and Neisseria carriers) of the internal higher school were obtained. They have been vaccinated with VA-MENGOC-BC (MBV) during their infancy and boosted with a third dose during this study. Serum and saliva samples were analyzed by ELISA and Western blot to measured IgG and IgA antibodies against N. meningitidis and N. gonorrhoeae antigens. N. meningitidis carriers were determined by standard microbiologic test. In addition, we reviewed epidemiologic data for N. meningitidis and N. gonorrhoeae infections in Cuba. RESULTS: Epidemiologic data show the influence of MBV over gonorrhea incidence suggesting to be dependent of sexual arrival age of vaccines but not over syphilis. Laboratorial data permit the detection of 70 and 22 noncarriers and carriers of N. meningitidis, respectively. Serum anti-MBV antigens (PL) responses were boosted by a third dose and were independent of carriage stages, but saliva anti-PL IgA responses were only present and were significant induced in carriers subjects. Carriers boosted with a third dose of MBV induced similar antigonococcal and -PL saliva IgA and serum IgG responses; meanwhile, serum antigonococcal IgG was significantly lower. In saliva, at least 2 gonococcal antigens were identified by Western blot. Finally, gonococcal-specific mucosal IgA antibody responses, in addition to the serum IgG antibodies, might contributed to the reduction of the incidence of N. gonorrhoeae. We hypothesize that this might have contributed to the observed reductions of the incidence of N. gonorrhoeae. CONCLUSION: These results suggest a mechanism for the influence of a Proteoliposome-based meningococcal BC vaccine on gonococcal incidence.

Fortetropin inhibits disuse muscle atrophy in dogs after tibial plateau leveling osteotomy.

OBJECTIVE: To determine if a commercial myostatin reducer (Fortetropin®) would inhibit disuse muscle atrophy in dogs after a tibial plateau leveling osteotomy. DESIGN: A prospective randomized, double-blinded, placebo-controlled clinical trial. ANIMALS: One hundred client-owned dogs presenting for surgical correction of cranial cruciate ligament rupture by tibial plateau leveling osteotomy. PROCEDURES: Patients were randomly assigned into the Fortetropin® or placebo group and clients were instructed to add the assigned supplement to the dog's normal diet once daily for twelve weeks. Enrolled patients had ultrasound measurements of muscle thickness, tape measure measurements of thigh circumference, serum myostatin level assays, and static stance analysis evaluated at weeks 0, 8, and 12. RESULTS: From week 0 to week 8, there was no change for thigh circumference in the Fortetropin® group for the affected limb (-0.54cm, P = 0.31), but a significant decrease in thigh circumference for the placebo group (-1.21cm, P = 0.03). There was no significant change in serum myostatin levels of dogs in the Fortetropin® group at any time point (P>0.05), while there was a significant rise of serum myostatin levels of dogs in placebo group during the period of forced exercise restriction (week 0 to week 8; +2,892 pg/ml, P = 0.02). The percent of body weight supported by the affected limb increased in dogs treated with Fortetropin® (+7.0%, P<0.01) and the placebo group (+4.9%, P<0.01) at the end of the period of forced exercise restriction. The difference in weight bearing between the Fortetropin® and placebo groups was not statistically significant (P = 0.10). CONCLUSION: Dogs receiving Fortetropin® had a similar increase in stance force on the affected limb, no significant increase in serum myostatin levels, and no significant reduction in thigh circumference at the end of the period of forced exercise restriction compared to the placebo. These findings support the feeding of Fortetropin® to prevent disuse muscle atrophy in canine patients undergoing a tibial plateau leveling osteotomy.

Efficient treatment of Parkinson's disease using ultrasonography-guided rhFGF20 proteoliposomes.

Fibroblast growth factor-20 (FGF20) is a paracrine member of the FGF family that is preferentially expressed in the substantia nigra pars compacta (SNpc). Previous studies have demonstrated that FGF20 enhances the survival of dopaminergic neurons suggesting the potential use of FGF20 to treat Parkinson's disease (PD). However, the reduced solubility of the bacterial recombinant human FGF20 (rhFGF20) and the absence of efficient strategies to transport rhFGF20 across the blood-brain barrier (BBB) have halted its clinical application. In the present study, we have examined the efficiency of fuzing a small ubiquitin-related modifier (SUMO) to rhFGF20 to enhance its soluble expression and further investigated the efficacy of FUS-guided, rhFGF20-liposome transport across the BBB. We also examined the bioavailability and behavioral improvement in a 6-hydroxydopamine-lesioned rat model of PD following 2 weeks' FUS-liposomal combinatorial treatment. Our results showed that, in contrast with rhFGF20 or LIP-FGF20, the FUS-LIP-rhFGF20 treatment could significantly improve the apomorphine-induced rotations by protecting against the loss of dopaminergic neurons in the SNpc. Our Results suggest that our combinatorial method would help overcome key challenges that hinder the currently available methods for the use of rhFGF20 in PD treatment.

The influence of different peptide combinations to increase the immunogenicity of the Gonadotrophin Releasing Hormone Vaccine for prostate cancer treatment.

PURPOSE: Therapeutic vaccines, specifically the Gonadotrophin Releasing Hormone (GnRH) vaccine, are considered an additional therapeutic option for advanced stage prostate cancer. Our work showed amplification of the immune response when combining two peptides with and without the Very Small Size Proteoliposomes (VSSP). VSSP is a potent adjuvant for dendritic cells activation and Th1 differentiation. as enhanced immune response. METHODS: The test was carried out in Copenhagen rats as animal model. RESULTST: The use of both peptides and their combination with VSSP generated a potentiation of the immune response statistically superior, in term of generating anti GnRH antibody and effects on target organs, when it was compared with the effects which occurs with independent peptides and with and without the VSSP. These results can find application in the development of GnRH vaccine candidates and in peptide based vaccine strategies. CONCLUSIONS: Immunization with the peptide combination enhances the immune response when mixed with the VSSPs.

Safety of a proteoliposome from Neisseria meningitides as adjuvant for a house dust mite allergy vaccine.

The proteoliposome (PL) of Neisseria meningitidis serogroup B has been reported as a safe and potent vaccine adjuvant, inducing a TH1-skewed response. The present study describes a pre-clinical safety evaluation of an allergy therapeutic vaccine candidate based on purified allergens from Dermatophagoides siboney house dust mite and PL as adjuvant, both components adsorbed onto aluminum hydroxide gel. Two separate studies of acute toxicity evaluation were performed in mice and rabbits, and two repeat-dose studies were conducted in non-sensitized and allergen-sensitized Balb/c mice, respectively. The study in sensitized mice intends to model a therapeutic setting. Aerosolized allergen challenge was used in both settings to model natural respiratory exposure. In the therapeutic setting, mice were administered with three doses containing 2 µg allergen at weekly intervals [subcutaneous route] and subsequently challenged with aerosolized allergen for 6 consecutive days. Parameters of general toxicity effects were assessed via measures of behavior, body weight, food and water consumption, and macroscopic evaluation of organs. Histological examination of organs and the injection site was performed. Potential immunotoxicity effects at the systemic level were assessed by blood eosinophil counting and serum allergen specific IgE by ELISA The vaccine did not produce general or functional toxic effects of significance, at a dose up to 100 µg allergen per kg body weight. An expected local reaction at the injection site was observed, which could be attributed mostly to the immunological effect of aluminum hydroxide. The models implemented here suggest an acceptable safety profile of this vaccine for testing in clinical trials of allergy immunotherapy.

The Effects of Fortetropin Supplementation on Body Composition, Strength, and Power in Humans and Mechanism of Action in a Rodent Model.

OBJECTIVE: The purpose of this study was to investigate the effects of Fortetropin on skeletal muscle growth and strength in resistance-trained individuals and to investigate the anabolic and catabolic signaling effects using human and rodent models. METHODS: In the rodent model, male Wistar rats (250 g) were gavage fed with either 1.2 ml of tap water control (CTL) or 0.26 g Fortetropin for 8 days. Then rats participated in a unilateral plantarflexion exercise bout. Nonexercised and exercised limbs were harvested at 180 minutes following and analyzed for gene and protein expression relative to mammalian target of rapamycin (mTOR) and ubiquitin signaling. For the human model, 45 (of whom 37 completed the study), resistance-trained college-aged males were divided equally into 3 groups receiving a placebo macronutrient matched control, 6.6 or 19.8 g of Fortetropin supplementation during 12 weeks of resistance training. Lean mass, muscle thickness, and lower and upper body strength were measured before and after 12 weeks of training. RESULTS: The human study results indicated a Group × Time effect (p ≤ 0.05) for lean mass in which the 6.6 g (+1.7 kg) and 19.8 g (+1.68 kg) but not placebo (+0.6 kg) groups increased lean mass. Similarly, there was a Group × Time effect for muscle thickness (p ≤ 0.05), which increased in the experimental groups only. All groups increased equally in bench press and leg press strength. In the rodent model, a main effect for exercise (p ≤ 0.05) in which the control plus exercise but not Fortetropin plus exercise increased both ubiquitin monomer protein expression and polyubiquitination. mTOR signaling was elevated to a greater extent in the Fortetropin exercising conditions as indicated by greater phosphorylation status of 4EBP1, rp6, and p70S6K for both exercising conditions. CONCLUSIONS: Fortetropin supplementation increases lean body mass (LBM) and decreases markers of protein breakdown while simultaneously increasing mTOR signaling.

Protective capacity of proteoliposomes from Mycobacterium bovis BCG in a mouse model of tuberculosis.

Tuberculosis (TB) is one of the most important causes of mortality and morbidity due to infectious diseases. BCG, the vaccine in use, is not fully protective against TB. In a previous study, we have shown that proteoliposomes (outer membrane extracts), obtained from BCG (PLBCG) were able to induce humoral immune responses against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLBCG alone or as a booster with BCG, a murine model of progressive pulmonary TB was used. Animals immunized with PLBCG adjuvanted with alum (PLBCG-Al) showed similar protection to that conferred by BCG. The group immunized with PLBCG-Al as a booster to BCG gave superior protection than BCG as evidenced by a reduction of bacterial load in lungs 2 months after infection with Mtb. Animals immunized with BCG, PLBCG-Al and this formulation as a booster of BCG, showed a significant decrease of tissue damage (percentage of pneumonic area/lung) compared with non-immunized animals. These results demonstrate that immunization with PLBCG-Al alone or as a booster to BCG induce appropriate protection against challenge with Mtb in mice and support the future evaluation of PLBCG as a promising vaccine candidate against Mtb.

Cell permeable peptide conjugated nanoerythrosomes of fasudil prolong pulmonary arterial vasodilation in PAH rats.

In this study, we tested the hypothesis that a cell permeable peptide, CARSKNKDC (CAR), conjugated nanoerythrosomes (NERs) containing fasudil, a rho-kinase (ROCK) inhibitor, produces prolonged pulmonary preferential vasodilation. CAR conjugated NERs containing fasudil were prepared by hypotonic lysis and extrusion method, and optimized for various physicochemical properties in-vitro. The formulations were then used to study the hemodynamic efficacy in a monocrotaline-induced rodent model of pulmonary arterial hypertension (PAH). CAR-NERs-Fasudil was spherical in shape with an average vesicle size and entrapment efficiency of 161.3 ± 1.37 nm and 48.81 ± 1.96%, respectively. Formulations were stable for ~3 weeks when stored at 4 °C and the drug was released in a controlled fashion for >48 h. The uptake of CAR-NERs-Fasudil by TGF-b activated pulmonary arterial smooth muscle cell was ~1.5-fold greater than the uptake of NERs-Fasudil. CAR-NERs-Fasudil inhibited ROCK activity and 5-hydroxytryptamine induced cell proliferation. In terms of reduction of pulmonary arterial pressure, intratracheal administration of CAR-NERs-Fasudil was ~2-fold more specific to the lungs compared with plain fasudil. Overall,CAR peptide grafted nanoerythrosomes offers a new platform for improving the therapeutic efficacy ofa rho-kinase inhibitor, fasudil, without affecting peripheral vasodilation.

NKLP27: a teleost NK-lysin peptide that modulates immune response, induces degradation of bacterial DNA, and inhibits bacterial and viral infection.

NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis) NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides.

Immune adjuvant effect of V. cholerae O1 derived Proteoliposome coadministered by intranasal route with Vi polysaccharide from Salmonella Typhi.

The proteoliposome derived from Vibrio cholerae O1 (PLc) is a nanoscaled structure obtained by a detergent extraction process. Intranasal (i.n) administration of PLc was immunogenic at mucosal and systemic level vs. V. cholerae; however the adjuvant potential of this structure for non-cholera antigens has not been proven yet. The aim of this work was to evaluate the effect of coadministering PLc with the Vi polysaccharide antigen (Poli Vi) of S. Typhi by the i.n route. The results showed that Poli Vi coadministered with PLc (PLc+Poli Vi) induce a higher IgA response in saliva (p<0.01) and faeces (p<0.01) than Poli Vi administered alone. Likewise, the IgG response in sera was higher in animals immunised with PLc+Poli Vi (p<0.01). Furthermore, IgG induced in sera of mice immunised with PLc+Poli Vi was similar (p>0.05) to that induced in a group of mice immunised by the parenteral route with the Cuban anti-typhoid vaccine vax-TyVi, although this vaccine did not induce a mucosal response. In conclusion, this work demonstrates that PLc can be used as a mucosal adjuvant to potentiate the immune response against a polysaccharide antigen like Poli Vi.

New proteoliposome vaccine formulation from N. meningitidis serogroup B, without aluminum hydroxide, retains its antimeningococcal protectogenic potential as well as Th-1 adjuvant capacity.

Proteoliposomes purified from the Outer Membrane of Neisseria meningitidis B, have been successfully used as core for adjuvants and vaccine formulations. We have tried to increase their structural definition and to conserve their efficacy and stability avoiding the addition of the aluminum hydroxide to the final formulation. Liposomal particle systems were prepared from components of defined molecular structure, such as a Neisseria meningitidis B protein complex, extracted and purified without forming vesicle structures. Liposomes were prepared from a mixture of dioleoyl phosphatidyl serine and cholesterol, using the classical dehydration-rehydration method. Transmission Electron Microscopy (TEM) was used to characterize the liposomes. BALB/c mice were used for animal testing procedures. Analysis of specific IgG response, serum bactericidal activity as well as DTH reaction was carried out. Isolation and purification of mRNA and real-time PCR, was performed to determine the dominating Th lymphokine pattern. The new antimeningococcal formulation without aluminum hydroxide prepared with components of defined molecular structure assembled itself into Neoproteoliposomes (NPL) ranging from 50 to 70 nm in diameter. The extraction and purification of selected membrane proteins to provide the antigen for this new formulation (PD-Tp), as well as the NPL-formulation favors a Th1 response pattern, suggested by the higher percentages of DTH, increased expression of proinflamatory lymphokine mRNAs when administered by intramuscular and intranasal routes. It stimulates a systemic bactericidal antibody response against Neisseria meningitidis B and immunologic memory similar to the Cuban VA-MENGOC-BC vaccine, even at lower dosages and is less reactogenic at the injection site in comparison with the formulation with aluminum hydroxide. This new adjuvant formulation could be applicable to the development of new and improved vaccines against meningococcal disease, and eventually as modulators of the immune response against other diseases.

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition.

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.

NGcGM3/VSSP vaccine as treatment for melanoma patients.

Gangliosides are glycosphingolipids that are present in the plasma membranes of vertebrates and are involved in multiple cellular processes. In the Center of Molecular Immunology an NGcGM3 ganglioside based vaccine has been developed and is conceptualized as a targeted therapy in cancer. NGcGM3/VSSP vaccine had been used as treatment of metastatic melanoma patients and had showed to be safe and immunogenic. The treatment improved antitumoral response or maintain the response obtained with previous onco-specific treatment as chemotherapy. The results indicate that the vaccine improved overall survival of metastatic melanoma patients after first line-chemotherapy. The clinical trial ongoing currently will allow corroborating these results.

Biodegradable long circulating cellular carrier for antimalarial drug pyrimethamine.

OBJECTIVE: The objective of the present study was to develop targeted engineered nanoerythrosomes based intravenous formulation of antimalarial drug pyrimethamine. MATERIAL AND METHODS: The nanoerythrosomes formulation was developed by sonication method and optimized for effective drug loading at variable drug concentration, surface morphology, viscosity and sedimentation volume. RESULTS: The in vitro drug release of formulated product was found to be delayed after 8 hours, having good stability at 4 ± 1°C and showing controlled in vivo release. Tissue distribution studies showed higher accumulation of drug in the liver (18.71 ± 1.4 µg/ml) (P < 0.05) at 1 hour in case of pyrimethamine-loaded nanoerythrosomes as compared to that in free drug (12.82 ± 0.7 µg/ml). Higher amount of drug, i.e. 14.18 ± 0.9 µg/ml (P < 0.05), was found after 24 hours in the liver in case of pyrimethamine-loaded nanoerythrosomes as compared to free drug concentration of 9.72 ± 0.5 µg/ml). DISCUSSION: Data showed that developed pyrimethamine-loaded nanoerythrosomes hold promise for targeting and controlling the release of drug and for improving treatment of malaria when they are combined with rapid acting antimalarials such as artemisinin. CONCLUSION: A decrease in the concentration of pyrimethamine in kidneys and lungs after 24 hours was observed as compared to that observed after 1 hour, showing no or little involvement of these organs in the clearance of drug-loaded nanoerythrosomes.

A proteoliposome containing apolipoprotein A-I mutant (V156K) enhances rapid tumor regression activity of human origin oncolytic adenovirus in tumor-bearing zebrafish and mice.

We recently reported that the efficiency of adenoviral gene delivery and virus stability are significantly enhanced when a proteoliposome (PL) containing apolipoprotein (apo) A-I is used in an animal model. In the current study, we tested tumor removal activity of oncolytic adenovirus (Ad) using PL-containing wildtype (WT) or V156K. Oncolytic Ad with or without PL was injected into tumors of zebrafish and nude mice as a Hep3B tumor xenograft model. The V156K-PL-Ad-injected zebrafish, group showed the lowest tumor tissue volume and nucleic acids in the tumor area, whereas injection of Ad alone did not result in adequate removal of tumor activity. Reactive oxygen species (ROS) contents increased two-fold in tumor-bearing zebrafish; however, the V156K-PL-Ad injected group showed a 40% decrease in ROS levels compared to that in normal zebrafish. After reducing the tumor volume with the V156K-PL-Ad injection, the swimming pattern of the zebrafish changed to be more active and energetic. The oncolytic effect of PL-Ad containing either V156K or WT was about two-fold more enhanced in mice than that of Ad alone 34 days after the injection. Immunohistochemical analysis revealed that the PL-Ad-injected groups showed enhanced efficiency of viral delivery with elevated Ad-E1A staining and a diminished number of proliferating tumor cells. Thus, the antitumor effect of oncolytic Ad was strongly enhanced by a PL-containing apoA-I and its mutant (V156K) without causing side effects in mice and zebrafish models.

Very small size proteoliposomes (VSSP) and Montanide combination enhance the humoral immuno response in a GnRH based vaccine directed to prostate cancer.

Very small size proteoliposomes (VSSP) constitute a complex of very small size proteoliposomes that includes proteins, lipids, CpG and gangliosides tumor-associated that provides a potential target for cancer immunotherapy. This compound has been described to stimulate the humoral and cellular response, dendritic cells (DC) activation and differentiation of T-helper cells, specially, in immunocompromised patients with cancer status. This work deals with the stimulating capacity of the VSSP to reach a humoral response when they are used as a component in a peptidic vaccine based on the gonadotrophin releasing hormone (GnRH). This study was carried out in male Copenhagen rats, which were immunized with 750µg of the GnRH mimetic peptide (GnRHm1-TT) with or without the VSSP. The mixtures were always emulsified with the oil adjuvant Montanide ISA 51. The anti GnRH seroconversion analysis revealed that the group immunized with the peptide GnRHm1-TT/VSSP developed a strong anti GnRH seroconversion. These antibody levels proved to be significant superior to those reached by the use of the GnRHm1-TT peptide solely emulsified in Montanide. Post-mortem analysis on the Testosterone ablation target organs (prostate and testicles) yielded a sudden decrease in their size and weight in respect to the control group. On the other hand, the group submitted to the use of GnRHm1-TT/VSSP, showed a significant difference in the reduction of these target organs in comparison with the group only immunized with GnRHm1-TT adjuvated in Montanide ISA 51. These values turned to be of p=0.023 and p=0.009 in the prostate and testicles respectively. These findings foreground the VSSP as a useful immunopotentiator to be used as part of a GnRH based vaccine to treat prostate cancer.

Very small size proteoliposomes derived from Neisseria meningitidis: an effective adjuvant for antigen-specific cytotoxic T lymphocyte response stimulation under leukopenic conditions.

Leukopenia is a severe condition resulting from both pathological processes and some treatments, like chemotherapy in cancer patients. However, the activation of the patient immune system is required for the success of immunotherapeutic strategies, as cancer vaccines. In this regard, leukopenia constitutes a major hurdle to overcome, mainly due to the impairment of cytotoxic T lymphocyte (CTL) responses. Adjuvants are basic components of vaccine formulations, which might be useful to stimulate immunity under this immunosuppressed condition. To this aim, we tested the capacity of a novel nanoparticulated complex, very small size proteoliposomes (VSSP), to promote CTL even in a leukopenic scenario. Noteworthy, we observed that a VSSP-based OVA vaccine induced a normal antigen-specific CTL response in mice rendered leukopenia by the administration of high doses of the chemotherapeutic agent cyclophosphamide (CY), while under the same conditions the OVA antigen formulated in the TLR-3 agonist polyinosinic-polycytidylic acid (P(I:C)) was ineffective. Moreover, an appropriate combination of VSSP with the P(I:C) vaccine was able to restore the CD8(+) T cell effector function in leukopenic mice. VSSP induced not only a faster repopulation of immune cells in CY-receiving animals, but also enhanced the recovery of memory T lymphocytes and myeloid dendritic cells (DCs) while simultaneously abrogated the immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs). Our results suggest that VSSP could be a particularly suitable immunomodulator to be used in CTL-promoting active immunotherapy strategies operating in severe immune compromised scenarios.

Mucosal and systemic immune responses of mice to tetanus toxoid coadministered nasally with AFCo1.

Mucosal immune responses are an early and important line of defense against pathogens. The current understanding of the mucosal immune system allows us to consider the use of nasal immunization for induction of antigen-specific immune responses at the mucosal surface and the systemic compartment. Mucosal adjuvants are key for developing novel mucosal vaccines and represent 1 approach to improving mucosal and systemic immunity. However, few mucosal vaccine adjuvants are currently approved for human use. Neisseria meningitidis B proteoliposome-derived cochleate (AFCo1 - Adjuvant Finlay Cochleate 1) has been demonstrated to be a potent mucosal adjuvant. The present work demonstrates that intranasal immunization of 3 doses of tetanus toxoid (TT) coadministered with AFCo1 in mice promotes high systemic and mucosal responses. The anti-TT IgG serum titers and the mucosal anti-TT IgA in saliva and vaginal wash were significantly higher than TT alone. The analysis of antibody subclasses showed that intranasal administration of AFCo1 + TT induced not only IgG1 but also IgG2a anti-TT antibodies at levels comparable to those obtained with TT vaccine (vax-TET). These data support the fact that AFCo1 is a potent mucosal adjuvant in nasal immunization to a coadministered protein antigen.

Inhibition of tumor-induced myeloid-derived suppressor cell function by a nanoparticulated adjuvant.

The interaction between cancer vaccine adjuvants and myeloid-derived suppressor cells (MDSCs) is currently poorly understood. Very small size proteoliposomes (VSSP) are a nanoparticulated adjuvant under investigation in clinical trials in patients with renal carcinoma, breast cancer, prostate cancer, and cervical intraepithelial neoplasia grade III. We found that VSSP adjuvant induced a significant splenomegaly due to accumulation of CD11b(+)Gr-1(+) cells. However, VSSP-derived MDSCs showed a reduced capacity to suppress both allogeneic and Ag-specific CTL response compared with that of tumor-induced MDSCs. Moreover, splenic MDSCs isolated from tumor-bearing mice treated with VSSP were phenotypically more similar to those isolated from VSSP-treated tumor-free mice and much less suppressive than tumor-induced MDSCs, both in vitro and in vivo. Furthermore, different from dendritic cell vaccination, inoculation of VSSP-based vaccine in EG.7-OVA tumor-bearing mice was sufficient to avoid tumor-induced tolerance and stimulate an immune response against OVA Ag, similar to that observed in tumor-free mice. This effect correlated with an accelerated differentiation of MDSCs into mature APCs that was promoted by VSSP. VSSP used as a cancer vaccine adjuvant might thus improve antitumor efficacy not only by stimulating a potent immune response against tumor Ags but also by reducing tumor-induced immunosuppression.