Hyaluronan is a natural and effective immunological adjuvant for protein-based vaccines

One of the main goals of vaccine research is the development of adjuvants that can enhance immune responses and are both safe and biocompatible. We explored the application of the natural polymer hyaluronan (HA) as a promising immunological adjuvant for protein-based vaccines. Chemical conjugation of HA to antigens strongly increased their immunogenicity, reduced booster requirements, and allowed antigen dose sparing. HA-based bioconjugates stimulated robust and long-lasting humoral responses without the addition of other immunostimulatory compounds and proved highly efficient when compared to other adjuvants. Due to its intrinsic biocompatibility, HA allowed the exploitation of different injection routes and did not induce inflammation at the inoculation site. This polymer promoted rapid translocation of the antigen to draining lymph nodes, thus facilitating encounters with antigen-presenting cells. Overall, HA can be regarded as an effective and biocompatible adjuvant to be exploited for the design of a wide variety of vaccines.     

IL-12 is a potent neonatal vaccine adjuvant

Neonatal animals show generally poor responsiveness to foreign antigens and are known to display polarized expression of Th2-like cytokines and antibody responses. We now report that newborn mice display a reduction in peripheral expression of the Th1-inducing cytokine, IL-12. Attempts to overcome this decrease by immunization and treatment with IL-12 within 24 h of birth resulted in elevated levels of IFN-γ and IL-10 mRNA in the spleens of mice compared to animals exposed to antigen only. Moreover, such animals showed dramatic enhancement of IgG2a and IgG2b antibody levels upon adult challenge compared to mice primed with antigen alone. These effects appeared to be due to induction of neonatal B cell memory. IgG1 antibody levels, a measure of Th2 activity, were unaffected or even somewhat enhanced by neonatal IL-12 treatment. Taken together, these results provide evidence that IL-12 administration induces a Th1-like cytokine response in newborns and causes priming for heightened memory antibody responses in vivo. Our findings suggest the use of IL-12 as a vaccine adjuvant in neonates for inducing protection against common childhood pathogens.     

Evaluation of properties of chitosan as an adjuvant for inactivated influenza vaccines administered parenterally

Studies on mice showed that chitosan as an adjuvant for H5 inactivated influenza vaccines administered intramuscularly enhances significantly antibody titers and protective efficiency not only against homologous influenza viruses, but also against drift variants. Chitosan adjuvanted vaccines induced high antibody titers after a single immunization and with a low dose of antigen. High antibody titers remained for at least 6 months. Chitosan adjuvanted vaccine stored at 4°C preserves its adjuvant properties for at least 8 months. Chitosan stimulates proliferative and cytotoxic activity of splenic mononuclear leukocytes in mice and promotes an increase in the numbers of CD3, CD3/NK, I‐AK (MHC II), and H‐2Db (MHC I) cells. After intramuscular immunization, chitosan did not induce IgE antibodies and antibodies against chitosan itself. Chitosan is a promising adjuvant candidate for inactivated influenza vaccines administered parenterally. J. Med. Virol. 81:494–506, 2009. © 2009 Wiley‐Liss, Inc.     

In vivo preclinical efficacy of a PDLLA/PGA porous copolymer for dental application

This study aimed to analyze surface morphology and physical-chemical properties of a copolymer of polylactic/polyglycolic acid (Fisiograft, Ghimas SpA, Casalecchio di Reno, Italy) by scanning electron microscopy (SEM), porosimetry, and rheological analysis. Then the material was implanted in vivo to test its efficacy at promoting bone healing and new bone formation in postextraction sockets. Under general anaesthesia, sockets were created in 12 minipigs and then randomly filled with the porous copolymer in SPONGE or GEL form and compared with commercial BioOss (Geistlich Biomaterials) and Biocoral (Inoteb, France). At 15, 30, and 60 days from surgery, the newly formed trabecular bone quality was evaluated by means of histology and histomorphometry. The SEM and rheological analyses performed on GEL showed a surface microporosity and a rheological shear thinning behavior, whereas the SPONGE porosimetric measurements revealed larger pores. At 15 days, the new bone regrowth was observed in all treated sockets but appeared immature, as the trabeculae were very dense and thin. At 30 days, GEL and SPONGE were degraded, and the sockets were filled with bone that, in terms of bone volume fraction, trabecular number, and separation, was not statistically different from normal bone.     

Cloning of a Partial cDNA for Rat Interleukin-12 (IL-12) and Analysis of IL-12 Expression In Vivo

Experimental models of autoimmunity in the rat may feature selective activation of either the Th1 or Th2 subset of helper T cells. Interleukin-12 (IL-12) is a key cytokine in the development of Th1 responses. In order to study IL-12 in the rat we used polymerase chain reaction (PCR) primers based on murine IL-12 to amplify a partial cDNA from rat tissue. The product was cloned and sequenced: it shows 94% nucleotide identity with the murine gene and 94% identity of predicted amino acid sequence. Primers based on the rat IL-12 sequence were used to analyse IL-12 expression in vivo using semi-quantitative PCR. We studied RNA from lymphoid tissues of two rat strains which differ in their response to mercuric chloride (HgCl₂): Brown Norway (BN) rats develop autoimmunity with a predominant Th2 response; Lewis rats are resistant. Interleukin-12 expression was higher in Lewis than BN, and higher in spleen than lymph node. After HgCl₂, IL-12 expression increased in BN towards the time when the autoimmune response autoregulates. Variation in baseline levels of IL-12 expression may account for the Th2 predisposition of BN rats compared to Lewis rats; IL-12 may play a role in the autoregulation of the Th2 response induced by HgCl₂     

In vivo evaluation of an oral delivery system for P-gp substrates based on thiolated chitosan

Recently, thiolated polymers, so called thiomers, have been reported to modulate drug absorption by inhibition of intestinal P-glycoprotein (P-gp). The aim of the present study was to provide a proof-of-principle for a delivery system based on thiolated chitosan in vivo in rats, using rhodamine-123 (Rho-123) as representative P-gp substrate. In vitro, the permeation enhancing effect of unmodified chitosan, chitosan-4 thiobutylamidine (Ch-TBA) and the combination of Ch-TBA with reduced glutathione (GSH) was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to buffer only, Rho-123 transport in presence of 0.5% (w/v) chitosan, 0.5% (w/v) Ch-TBA and the combination of 0.5% (w/v) Ch-TBA/0.5% (w/v) GSH, was 1.8-fold, 2.6-fold, 3.8-fold improved, respectively. Furthermore, enteric-coated tablets based on unmodified chitosan or Ch-TBA/GSH, were investigated in vivo. In rats, the Ch-TBA/GSH tablets increased the area under the plasma concentration time curve (AUC0-12) of Rho-123 by 217% in comparison to buffer control and by 58% in comparison to unmodified chitosan. This in vivo study showed that a delivery system based on thiolated chitosan significantly increased the oral bioavailability of P-gp substrate Rho-123.     

Enhancing the immunogenicity of inactivated polio vaccines with chitosan used as an adjuvant

Addition of chitosan to inactivated trivalent polio vaccine or inactivated preparations of attenuated poliomyelitis viruses (Sabin strains) significantly increases immunogenicity of these inactivated poliomyelitis virus preparations. High neutralizing antibody titers are detected after two immunizations of mice and a single immunization of rats, as well as when the antigen dose was reduced by 4 times. Addition of chitosan as an adjuvant significantly induces cellular immunity.     

In vitro and in vivo evaluation of mucoadhensiveness of chitosan/cellulose acetate multimicrospheres

Chitosan/cellulose acetate multimicrospheres (CCAM) with or without ranitidine (RT) were prepared by the method of W/O/W emulsion with no toxic reagents and had the size interval of 200-280 microm. The angles of repose were only a little more than 30 degrees and the maximum angles of one-plane-critical-stability (OPCS phi) were about 20 degrees . The CCAM had good suspension ability for the tapped density of CCAM was less than 0.127g/mL. The pH value affected the swelling ability of CCAM and the relative humidity had little effect on the characteristics of CCAM when it was not more than 75%. The CCAM system had good effect on the controlled release of RT in vitro and the release rate was almost 60% during 48 h. Furthermore the release of RT was not affected by pH value of release medium. The mucoadhesive tests showed that CCAM could retain in gastrointestinal tract for an extended period of time. There were 53.7% of CCAM remained in stomach after administered for 2(1/2) h and 98.9% of CCAM remained in stomach and small intestine after administered for 3(1/2) h. These results suggest that CCAM is a useful dosage form targeting the gastric mucosa or prolonging gastric residence time as a multiple-unit mucoadhesive system.     

In vitro and in vivo evaluation of the chitosan/Tur composite film for wound healing applications

We have developed tourmaline/chitosan (Tur/CS) composite films for wound healing applications. The characteristics of composite films were studied by optical microscope, infrared spectra and X-ray diffraction. Tur particles were uniformly distributed in the CS film and the crystal structure of CS was not remarkably changed except the decrease of crystallinity. The influence of Tur on wound healing applications was characterized by modulating Tur concentrations in the Tur/CS composite film prepared by loading Tur powder into CS matrix with different proportion (0, 1/40 and 1/10). Then L929 cells were co-cultured on the composite films to access the cytotoxicity in vitro. Tur concentrations strongly influenced cell process extension. Tur/CS composite film with 1/40 mass ratio could promote the cell adhesion and proliferation. Fewer and shorter processes were observed at high Tur density. When the composite films were transplanted on porcine full-thickness burn wounds, histological results demonstrated that the Tur/CS group with 1/40 mass ratio had a significantly higher number of newly-formed and mature blood vessels, and fastest regeneration of dermis. Based on the observed facts these films can be tailored for their potential utilization in wound healing and skin tissue engineering applications.     

In vivo evaluation of a metronidazole-containing gel for the adjuvant treatment of chronic periodontitis: preliminary results

The present study developed an experimental metronidazole-based gel and evaluated its efficacy for the adjuvant treatment of chronic periodontitis. Sixteen patients were randomly allocated into two groups of eight subjects according to the following proposed treatments: (1) scaling and root planing (active control) or (2) scaling and root planing and direct periodontal intrapocket application of 15% metronidazole-based gel in two sites (≥5 mm in depth) (experimental group). Potential changes in the subgingival microbiota were assessed using a DNA Checkerboard method at three proposed times: baseline and following 7 or 30 days of drug administration. High-performance liquid chromatography (HPLC) monitored metronidazole concentrations in the crevicular fluid during treatment. The metronidazole experimental group presented lower bacterial counts than the control group at the three evaluated times (p<0.01 for baseline, p<0.001 for 7 or 30 days) when the target species were analyzed as a pool of bacteria. Samples revealed significantly lower counts 7 days after drug administration compared with baseline or after 30 days (p<0.05). HPLC analysis detected gel 1 h after application. The metronidazole-based gel significantly decreased the total bacterial count at the three evaluated times. Periodontopathogenic species were not different after gel administration.     

In silico identification of novel protective VSG antigens expressed by Trypanosoma brucei and an effort for designing a highly immunogenic DNA vaccine using IL-12 as adjuvant

African trypanosomiasis continues to be a major health problem, with more adults dying from this disease world-wide. As the sequence diversity of Trypanosoma brucei is extreme, with VSGs having 15-25% identity with most other VSGs, hence it displays a huge diversity of adaptations and host specificities. Therefore the need for an improved vaccine has become an international priority. The highly conserved and specific epitopes acting as both CD8+ and CD4+ T-cell epitopes (FLINKKPAL and FTALCTLAA) were predicted from large bunch of VSGs of T. brucei. Besides, some other potential epitopes with very high affinity for MHC I and II molecules were also determined while taking consideration on the most common HLA in the general population which accounts for major ethnicities. The vaccine candidates were found to be effective even for non-african populations as predicted by population coverage analysis. Hence the migrating travelers acting as a spread means of the infection can probably also be treated successfully after injection of such a multiepitopic vaccine. Exploiting the immunoinformatics approaches, we designed a potential vaccine by using the consensus epitopic sequence of 388 VSG proteins of T. brucei and performed in silico cloning of multiepitopic antigenic DNA sequence in pBI-CMV1 vector. Moreover, various techniques like codon adaptation, CpG optimization, removal of self recognized epitopes, use of adjuvant and co-injection with plasmids expressing immune-stimulatory molecules were implemented to enhance the immunogenicity of the proposed in silico vaccine.     

In vivo evaluation of a new system for 3D computerized angiography

A new system has been designed and built to validate the concept of 3D computerized angiography (CA). This system can acquire a set of 2D digital subtracted angiography images while rotating around a patient and then, using these images, reconstruct a 3D representation of the opacified vasculature. The design principles and main characteristics of the system are described, with special attention paid to data processing aspects. An initial in vivo evaluation of this system performed on anaesthetized animals and human volunteers is presented. The influence on the quality of the 3D reconstruction of different factors such as volume resolution, estimation method, source trajectory and number of projections is discussed.     

In vivo performance of a muscle-powered drive system for implantable blood pumps

A unique biomechanical implant has been developed to convert muscle power into hydraulic energy for the purpose of driving an implanted blood pump. This device, called a muscle energy converter (MEC), is designed to attach to the humeral insertion of the latissimus dorsi (LD) muscle, so that stimulated contractions cause a rotary cam to compress a fluid-filled bellows. Here we report results from the latest in a series of canine implant trials where the MEC was connected to an adjustable pressure load to measure power output and assess long-term function. Full-length (2 cm) actuator strokes were maintained for a period of 1 month with no discernable discomfort to the animal. Load conditions were cycled periodically to measure stroke work capacity and pressure production. The peak driveline pressure recorded in this experiment was 1743 mm Hg. Steady state power generation was measured to 478 +/- 21 mJ/stroke (mean +/- SD) with stroke work levels reaching 785 mJ in one test. Normal left and right ventricular stroke work levels in dogs this size (35 kg) are 700 and 150 mJ, respectively. These data confirm that MEC/LD power levels--maintained in tandem with an appropriate cardiac assist device--are sufficient to provide significant long-term circulatory support. Further testing, however, is still needed to demonstrate the long-term stability of this drive system.     

Comparison of different HER2/neu vaccines in adjuvant breast cancer trials: implications for dosing of peptide vaccines

We have performed multiple adjuvant clinical trials using immunogenic peptides from the HER2/neu protein (AE37/E75/GP2) plus (GM-CSF) given intradermally to breast cancer patients. Four trials were performed with similar dose-escalation design with increasing doses of peptide (AE37/E75/GP2) and varying amounts of GM-CSF. Dose reductions (DRs) were made for significant local and/or systemic toxicity by decreasing GM-CSF for subsequent inoculations. Ex vivo and in vivo immunologic responses were used to compare groups. Of 132 patients, 39 required DR (30 for robust local reactions [DR-L]). DR patients, particularly DR-L, had greater immune responses both ex vivo and in vivo. Postvaccine delayed-type hypersensitivity in DR-L patients compared with all others was larger for E75 (p = 0.001), AE37 (p = 0.077) and GP2 (p = 0.076). All three peptide vaccines were safe and well-tolerated. These findings have led to a clinically relevant optimal vaccine dosing strategy, which may be applicable to other peptide-based cancer vaccines.     

In vivo clearance of an intracellular bacterium, Francisella tularensis LVS, is dependent on the p40 subunit of interleukin-12 (IL-12) but not on IL-12 p70

To determine the role of interleukin-12 (IL-12) in primary and secondary immunity to a model intracellular bacterium, we have comprehensively evaluated infection with Francisella tularensis LVS in three murine models of IL-12 deficiency. Mice lacking the p40 protein of IL-12 (p40 knockout [KO] mice) and mice treated in vivo with neutralizing anti-IL-12 antibodies survived large doses of primary and secondary LVS infection but never cleared bacteria and exhibited a chronic infection. In dramatic contrast, mice lacking the p35 protein (p35 KO mice) of heterodimeric IL-12 readily survived large doses of primary sublethal LVS infection as well as maximal secondary lethal challenge, with only a slight delay in clearance of bacteria. LVS-immune wild-type (WT) lymphocytes produced large amounts of gamma interferon (IFN-gamma), but p35 KO and p40 KO lymphocytes produced much less; nonetheless, similar amounts of NO were found in all cultures containing immune lymphocytes, and all immune lymphocytes were equally capable of controlling intracellular growth of LVS in vitro. Purified CD4(+) and CD8(+) T cells from both WT and p40 KO mice controlled intracellular growth, even though T cells from WT mice produced much more IFN-gamma than those from p40 KO mice, and p40 KO T cells did not adopt a Th2 phenotype. Thus, while IL-12 p70 stimulation of IFN-gamma production may be important for bacteriostasis, IL-12 p70 is not necessary for appropriate development of LVS-immune T cells that are capable of controlling intracellular bacterial growth and for clearance of primary or secondary LVS infection. Instead, an additional mechanism dependent on the IL-12 p40 protein, either alone or in another complex such as the newly discovered heterodimer IL-23, appears to be responsible for actual clearance of this intracellular bacterium.     

IFN-gamma and IL-12 plasmid DNAs as vaccine adjuvant in a murine model of grass allergy.

BACKGROUND: Plasmids encoding cytokines such as IFN-gamma and IL-12 are potential genetic adjuvants that might increase the effectiveness of allergen vaccines. OBJECTIVE: The role of plasmids expressing the cytokines IFN-gamma (pIFN-gamma) and/or IL-12 (pIL-12) as adjuvants in modulating allergic immune responses, inflammation, and asthma was investigated in a murine model of Kentucky blue grass (KBG) allergy. METHODS: Groups of naive B6D2F1 mice were vaccinated subcutaneously with KBG allergens and administered intramuscularly with pIFN-gamma, pIL-12, pIFN-gamma plus pIL-12, or a vector control. Mice were then sensitized with KBG allergens in alum (intraperitoneally) and later challenged intranasally. Mice were examined for modulation of specific immunity, prevention of the development of airway hyperresponsiveness, and inflammation. RESULTS: Mice vaccinated with cytokine plasmid adjuvants had relatively lower levels of total serum IgE and higher levels of grass allergen-specific IgG2a in comparison with control mice. The lowest IgE and highest IgG2a levels were found in mice vaccinated with the combination of pIFN-gamma and pIL-12 as an adjuvant. The vaccination of mice with both pIFN-gamma and pIL-12 as an adjuvant induced the highest level of T(H)1 cytokines, IFN-gamma, and IL-2 in comparison with mice given either of the plasmids alone. The most profound decrease in airway hyperresponsiveness and pulmonary inflammation was observed in mice receiving both pIFN-gamma and pIL-12 as an adjuvant. CONCLUSION: These results demonstrate that pIFN-gamma and pIL-12 together provide an effective adjuvant to parenteral grass allergen vaccines and show that this adjuvant can significantly enhance the effectiveness of allergen immunotherapy in human beings.     

Role of the IL-12/IL-23 system in the regulation of T-cell responses in central nervous system inflammatory demyelination

Interleukin-12 (IL-12) has long been considered essential in T-cell-mediated autoimmune diseases, including multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). This is based on the strong capacity of IL-12 to induce T-cell activation and Th1 differentiation. However, recent data have shown that the perceived central role of IL-12 in CNS inflammatory demyelination is actually due to IL-23, a closely related cytokine sharing the p40 subunit and the beta1 receptor chain with IL-12. There appear to be three different aspects of IL-12 involvement in EAE: (1) disease-promoting effects of exogenous IL-12, particularly in relapsing-remitting EAE and adoptive transfer EAE; (2) lack of IL-12 requirement in EAE pathogenesis, as indicated by studies in knockout mice; and (3) immunoregulatory effects of IL-12. Together, these observations make IL-12 a less attractive target for therapeutic intervention in MS. IL-23 neutralization may be a better candidate for therapeutic intervention, and it remains to be established whether blocking IL-23 with antibodies in adult mice will have the same effects as knocking out the IL-23p19 gene. Current clinical trials of neutralizing anti-IL-12 antibodies in other immune-mediated diseases target the p40 subunit, thereby neutralizing both IL-12 and IL-23. Thus, new experimental data are expected to have important implications for therapy of human diseases.     

Technologies for enhanced efficacy of DNA vaccines

Despite many years of research, human DNA vaccines have yet to fulfill their early promise. Over the past 15 years, multiple generations of DNA vaccines have been developed and tested in preclinical models for prophylactic and therapeutic applications in the areas of infectious disease and cancer, but have failed in the clinic. Thus, while DNA vaccines have achieved successful licensure for veterinary applications, their poor immunogenicity in humans when compared with traditional protein-based vaccines has hindered their progress. Many strategies have been attempted to improve DNA vaccine potency including use of more efficient promoters and codon optimization, addition of traditional or genetic adjuvants, electroporation, intradermal delivery and various prime-boost strategies. This review summarizes these advances in DNA vaccine technologies and attempts to answer the question of when DNA vaccines might eventually be licensed for human use.     

In vivo efficacy of albendazole againstGiardia duodenalis in mice

The antigiardial effects of albendazole were demonstrated in vivo using experimental infections ofGiardia duodenalis in mice. These results complement previous in vivo studies in which albendazole was shown to have more potent antigiardial action than the currently applied antigiardial drugs. In mice, 2–4 doses (>100 mg/kg twice daily) were required for complete inhibition of cyst excretion and full elimination of trophozoites from the small intestine. The high doses necessary in mice were not unexpected and are discussed in light of the possible pharmacokinetics of albendazole in the animal model used in this study.Dedicated to Prof. Dr. J. Eckert (Zürich) on the occasion of his 60th birthday