Improving Stability of Measles and Mumps Vaccines by Replacing Human Serum Albumin with Amino Acids Mixes

Although bacteriocinogenic probiotics are mainly Gram-positive bacteria to date, bacteriocins produced by Gram positives usually do not inhibit enteropathogenic bacteria such as Enterobacter, Klebsiella, Proteus, or Salmonella, whereas this can be accomplished by Gram-negative bacteriocins. Because of high or undetermined pathogenicity of many Gram-negative strains, it is not easy to accept these bacteriocin producers as probiotics, especially in humans. So the development of novel bacteriocins from Gram negatives as immunostimulants against enteric pathogens have been proposed. In this study, 15 Proteus and Klebsiella isolates from the gut of marine animals such as black tiger shrimp, ornate spiny lobster, cobia and snubnose pompano exerted wide-spectrum antimicrobial and bacteriocin-like activities against foodborne, human and animal pathogens, which opens the way to their potential use as marine drugs in food, aquaculture, livestock and clinical settings. Several Proteus strains (B3.7.1, B3.10A, CT1.1, G1, N1.4 and B3.7A) were suggested to belong to novel species based on a combined approach using 16S rRNA and rpoB gene sequence analysis as well as biochemical tests. We also supported two new isolates of Proteus genomospecies 4, CT1.1 and G1, with L-rhamnose positive as the unifying characteristic. Although Proteus strains are considered as a frequent cause of urinary tract infections in humans with long-term indwelling catheters or with complicated urinary tracts, they are not usually nosocomial pathogens. Bacteriocins from Proteus, if cloned, purified and recognized to be safe, could be a promising candidate of environmentalfriendly immunostimulants against enteric pathogens. Also, it is suggested that by further assessing and deleting pathogenicity of these Proteus and Klebsiella strains or by producing genetically engineering bacteriocins they can be developed as effective and safe probiotics or immunostimulants for popular use in clinical treatment in the future. Poster Presentation Abstracts A NOVEL SALMONELLA ENTERITIDIS VACCINE CANDIDATE TO REDUCE INTERNAL EGG CONTAMINATION J. H. Lee Chonbuk National University, College of Veterinary Medicine, Jeonju, 561-756,Republic of Korea ([email protected]) Salmonella Enteritidis is a major cause of food-borne disease. Poultry-derived products, particularly meat and chicken eggs, are considered a major source of human infection with this pathogen. To evaluate the efficacy of a novel attenuated Salmonella Enteritidis (△lon△cpxR) vaccine candidate (JOL919), chickens were immunized through oral and intramuscular routes to reduce egg contamination against Salmonella Enteritidis challenge. Birds were orally immunized with JOL919 on the first day of life and were subsequently boosted in the 6th and 16th week through oral (Group B) or intramuscular (Group C) route, while control birds were unimmunized (Group A). The chickens of all groups were challenged intravenously with the virulent Salmonella Enteritidis strain in the 24th week. The immunized groups B and C showed significantly higher plasma IgG and intestinal secretory IgA levels as compared to those of the control group. The lymphocyte proliferation response and CD45+CD3+ T cell number in the peripheral blood of the B and C groups were significantly increased. In addition, the egg contamination rates were significantly lower in the group B (0%, 10.7% and 0%), and the group C (3.6%, 14.3% and 3.6%) as compared to the group A (28.6%, 42.8% and 28.6%) in the 1st, 2nd and 3rd week post challenge. All animals in the groups B and C showed lower organ lesion scores in the liver and spleen, and lower bacterial counts in the liver, spleen and ovary at the 3rd week post-challenge. These results indicate that this vaccine candidate can be an efficient tool for prevention of Salmonella infections by inducing protective humoral and cellular immune responses. In addition, this vaccine did not completely prevent egg contamination, but did appear to reduce incidence. Booster immunizations, especially via oral administration route showed an efficient protection against internal egg contamination with Salmonella Enteritidis. Day 2: Modulating the Immune system, for vaccination / Advances in overcoming co-infections Invited Speakers Abstracts DNA Vaccination Encoding CD40 Targeted to Dendritic Cells Protects Against Chronic Kidney Disease. Dr Yuan Min Wang, Senior Scientist, Senior Lecturer, University of Sydney Renal Laboratory, Centre for Kidney Research, Australia CD40-CD154 costimulatory pathway has been shown to be critical for both T and B cell activation in autoimmune disease. In this study, we assessed the effects of blocking CD40-CD154 pathway, using CD40 DNA vaccine enhanced by dendritic cell (DC) targeting, on the development of Heymann Nephritis (HN), a rat model of human membranous nephropathy. We utilized a plasmid containing the gene encoding a single-chain Fv antibody specific for the DC-restricted antigen-uptake receptor DEC205 (scDEC) and the target gene CD40 and the adjuvant tetanus sequence p30 and a control plasmid without scDEC. Rats vaccinated with scDEC-CD40 were protected from developing HN. Protein nanoparticles with built-in adjuvant properties Professor Peter Burkhard, PhD, Department of Molecular and Cell Biology, The Institute of Materials Science, University of Connecticut, CT, USA We describe the use of coiled-coil domains to design a self-assembling protein nanoparticle (SAPN) with icosahedral symmetry. These SAPNs represent an ideal repetitive antigen display system for B-cell epitopes and they can further be optimized by engineering CD4 and/or CD8 epitopes in the core of the particle. The SAPNs can also incorporate flagellin molecules to efficiently stimulate the innate immune system by triggering the TLR5 pathway. We describe the use of these SAPNs as vaccines for malaria and nicotine addiction. The combined effects of genetic background and mannose-binding lectin ligands on the response to an avian coronavirus vaccination Dr Rikke Munkholm Kjærup, Dept. of Animal Science, Aarhus University, Blichers Allé Tjele, Denmark Low mannose-binding lectin (MBL) concentrations seem to be beneficial for achievement of high antibody titres after certain vaccinations, although individuals deficient in MBL have been found to be more susceptible to various infections. In this study, vaccination against the avian coronavirus infectious bronchitis virus (IBV) was used as a model vaccine. MBL ligands added to a live-attenuated IBV vaccine was shown to affect the adaptive immune response after vaccinations of two chicken lines selectively bred for high and low MBL serum concentrations. However, it remains to be shown if it correlates with protection. SMARTer Vaccines: Next-Generation Vaccinia Virus Vaccine and Therapeutic Vectors Dr Paulo H. Verardi, Ph.D., Associate Professor, University of Connecticut, Storrs, CT, USA Replication-competent vaccinia virus (VACV) vectors are widely used in the development of vaccines and therapeutics to control infectious diseases and cancer; however, as with all live vaccines, there are safety concerns. We have developed a safety mechanism for VACV based on the conditional expression of protective genes or genes essential for VACV replication, under antibiotic-controlled promoters. These “SMART” (Safety Mechanism Assisted by the Repressor of Tetracycline) vectors allow treatment of any complications that may result from VACV replication to be as simple as standard antibiotic regimens and have broad applications in the fields of vaccinology and cancer therapy. Improving the cellular immune response obtainable by adenovirus vectored vaccines Dr Peter J. Holst, Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark Adenovirus vectored vaccines are used in the most potent immunization regimens brought to clinical testing. Consequently, improving adenovirus induced immune responses further will raise the bar of the level of obtainable immunity. We have discovered that T cell adjuvants which can increase antigen presentation enhance and broaden adenovirus induced CD8+ T cell responses. Such adjuvanted vaccines have been tested using normally subdominant antigens in murine and primate models of chronic viral infections and in experimental tumor models with vaccines incorporating further T cell adjuvants. The state of HIV-1 co-infection Mr Christopher Ward, PhD Student,Innate Immune Sensors Group, Institute of Infection & Immunity, Cardiff University School of Medicine, University Hospital of Wales The introductory talk will briefly outline current understanding of major HIV-1 co-infections. Virus Related Cancer In Immunosuppressed Individuals Dr Diego Serraino, Cancer Epidemiologist, IRCCS Centro Di Riferimento Oncolologico, Italy Talk title to be confirmed Dr Sanjay Bhagani, Consultant Physician and honorary Senior Lecturer, Royal Free London Foundation Trust, UK Virus Related Cancer In Immunosuppressed Individuals Pierluca Piselli, Istituto Nazionale Malattie infettive Lazzaro Spallanzani, Rome, Italy. People with acquired immunedeficiencies following HIV infection or anti-rejection treatments after solid organ transplant are at higher risk of cancer than the respective immunecompetent population. Overall, the magnitude of such risk is about 2to 5-fold elevated, but it is particularly marked for cancers with a well established relations with oncogenic viruses (e.g., EBV and lymphoma, KSHV and Kaposi sarcoma). This presentation will present an update of the epidemiologic evidence linking acquired immunedepression and cancer risk, with a particular focus on viral-related cancers and on the relationship between degree of immune suppression, markers of infection and cancer occurrence, either in HIV infection/AIDS patients or recipients of solid organ transplants. Oral Presentation Abstracts MICRO CRYSTALLINE TYROSINE (MCT): ITS USE AS A DEPOT CARRIER IN ALLERGY IMMUNOTHERAPY, FUTURE PERSPECTIVES AND APPLICATIONS Heath, M.D1., Kramer, M.F1., Johansen, P2., Kuendig, T2., and Skinner, M.A1. 1Allergy Therapeutics, Worthing, UK 2University of Zurich Hospital, Department of Dermatology Allergen-specific immunotherapy commonly consists of administering a long-course programme of subcutaneous injections using preparations of relevant allergens (up to 50 injections / year / 3-5 yrs). Historically, the adjuvant of choice has always consisted of aluminium salt preparations (1.25 mg Al3+ per shot). Aluminium adjuvants have been consistently demonstrated to induce IgE which is clearly an unwanted and potentially adverse effect in any IgE-mediated disease, such as allergy. Since aluminium in immunotherapy is marketed and described as a depot adjuvant a suitable depot carrier/adjuvant should support the immunogenic effect of specific immunotherapy without causing unwanted side effects. In light of the growing number of toxicological considerations surrounding aluminium accumulation, its use in immunotherapy is a current hot topic of discussion between authorities, the public and academic/industrial representatives. Other endogenous and biodegradable adjuvant systems have or are being developed but difficulties in achieving regulatory approval without having extensive mode-of-action and safety studies make it costly and time-consuming to bring market. When evaluating the profile of an adjuvant for possible new applications very few adjuvants can match the extremely comprehensive cohorts that are available for aluminium adjuvants in terms of records of efficacy and safety profiles. A natural, alternative, adjuvant – Micro Crystalline Tyrosine (MCT) exists and has been marketed as a depot adjuvant, in registered allergen-specific immunotherapy products, for a number of decades. To date, >4.3 injections of MCT have been administered in Germany alone since 2005, including >1 million in children. We present the adjuvant properties of MCT in animal and human studies together with an assessment of its safe use in immunotherapy (including repeat-dose parenteral toxicity, genotoxicity, local tolerance studies and pharmacovigilance). A summary of the range of investigational data (unpublished) as well as published literature reports will be presented. An array of in vitro and in vivo studies demonstrate that MCT has ideal adjuvant properties comprising a unique adsorptive power for proteins, enhancement of IgG antibody induction with no stimulatory effect on IgE antibody level (unlike aluminium adjuvants) and action as a short-term depot adjuvant; as well as being a natural, metabolised and biodegradable alternative to aluminium. In more recent studies, results from immunized CBA mice, administered (intralymphatically) with MCT and aluminum-based allergenadjuvant preparations are presented. Here, blood was collected 2-3 weeks after the last injection and analysed for anti-PLA2/anti-OVA IgG2a, IgG1 and – IgE. In a separate rat model, radiolabelled (14C) MCT was calculated as having a half-life of approximately 48 hours at the subcutaneous injection site. The current data, its use and record in subcutaneous immunotherapy and lack of findings of toxicological concern supports the safe and effective use of MCT as a depot mediator with work ongoing to further unravel its adjuvant (Th1) potential through further mode-of-action studies across the allergy model. A proposal to apply this into a wider context using bacterial and viral candidates is currently being assessed with the possibility of using recombinant antigen +/Aluminium and +/MCT to assess and compare basic mechanistic and immunogenic profile in diseased animal models to consider the propensity of MCT as a novel adjuvant in different model systems. EVALUATION OF IMMUNE RESPONSE INDUCED BY DNA VACCINE COCKTAIL EXPRESSING COMPLETE LACK AND TSA GENES AGAINST LEISHMANIA MAJOR Fatemeh Ghaffarifar1*, Ogholniaz Jorjani,2 , Zohreh Sharifi 3, Abdolhossein Dalimi 1, Zuhair M. Hassan4, 1Parasitology and Entomology Department, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-331, Tehran I.R.Iran. 2Department of Biotechnology, Faculty of Advanced Medical Technology, Golestan University of Medical Sciences, Gorgan, Iran. 3Research Center of Iranian Blood Transfusion Organization,Tehran, I.R..Iran. 4Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, I.R.Iran Cutaneous leishmaniasis is an important disease in human. LACK and TSA as immuno-dominant antigens of Leishmania major are considered the most promising molecules for a DNA vaccine. We constructed a DNA cocktail, containing plasmids encoding LACK and TSA genes of Leishmania major and evaluated the immune response and survival rate in BALB/c mice. IgG and IFN-γ values were noticeably increased in the immunized group with DNA cocktail vaccine, which were significantly higher than those in the single-gene vaccinated and control groups (p<0.05) following the immunization and after challenging with Leishmania major. IL-4 values were decreased in all immunized groups, but only in DNA vaccine cocktail and singlegene vaccination with pc-LACK there were statistical differences with control groups (p>0.05). The immunized mice with the cocktail DNA vaccine presented a considerable reduction in diameter of lesion compared to other groups and a significant difference was observed (p<0.05) in this regard. The survival time of the immunized mice with the cocktail DNA vaccine was significantly higher than that in the other groups (p<0.05) after their being challenged with Leishmania major. The findings of this study indicated that the cocktail DNA vaccine increased the cellular response and survival rate and induced protection against infection with Leishmania in the mice. EVALUATION OF IMMUNE RESPONSE INDUCED BY DNA VACCINE CONTAIN GRA7 GENE AGAINST TOXOPLASMA GONDII Zohreh Sharifi 1*, Hossein Vazini Gheisar 2, Fatemeh Ghaffarifar3, Abdolhossein Dalimi 3, 1Research Center of Iranian Blood Transfusion Organization,Tehran, I.R..Iran. 2 Faculty of Medical Sciences, Azad University, Hamadan, I. R. Iran. 3Parasitology and Entomology Department, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-331, Tehran I.R.Iran. The severe and lethal damages of Toxoplasmosis clearly indicate the need for the development of a more effective vaccine, because until this time effective vaccines are not available. Immunization with plasmid DNA, a relatively novel technique, is a promising vaccination technique. Therefore we prepare GRA7 plasmid to use it as vaccine. This study by aim Evaluation of Immunization with plasmid encoding GRA7 and DNA Vaccine Cocktail Including Plasmids Encoding Toxoplasma gondii ROP2 and GRA7 in BALB/c mice. In this experimental study after divided BALB/c mice into 12 groups (n=10 in each group). Extract DNA from tachyzoites and performed PCR reaction on this DNA, then PCR product detected with gel electrophoresis. In next step the gen cloned in TOPO vector plasmid as a cloning vector. Results of sequencing indicate a 733 bp fragment cloned into plasmid. Then this gene sub-cloned into pcDNA3 and after transfection of eukaryotic cell(CHO) with this recombinant plasmid, expression of this gene was approved by SDS-PAGE and Western Blot. By using plasmid purification kits that allowing purification of ultrapure supercoiled plasmid DNA with high yields and Immunization carried out via IM in separate following groups three times with 3 week interval. The cytokine assay results and lymphocyte proliferation response in cocktail DNA vaccines showed that IFN-γ level were significantly higher than controls (P < 0.05), whereas IL-4 expression level in BALB/c mice immunized was lower than that in control groups(P > 0.05) and this results are conformed by MTT test. Predominance of the levels of IgG2a over IgG1 was observed in sera of the immunized mice. Furthermore, IgG2a values in cocktail DNA vaccines pcGRA7 were significantly higher than control group (P < 0.01). In contrast, IgG1 antibodies were similar between the two groups (P > 0.5). So survival time in the immune groups are significantly prolonged than control ones (P < 0.01). The immunized mice by DNA vaccine produce higher titration of IFNγ Indicated with Th1 respond which is confirmed by high level of IgG2a. These data demonstrate that the Toxoplasma gondii GRA7 is a potential vaccine candidate against toxoplasmosis. EFFECT OF INTERLEUKIN-22 ON IMMUNOGENICITY OF DNA VACCINE ENCODING TSA GENE OF LEISHMANIA MAJOR IN BALB/C MICE Hajar Ziaei-Hezarjaribi1, Fatemeh Ghaffarifar2, Abdolhossein Dalimi-Asl2, Zohreh Sharifi3, 1 PhD, Assistant Professor, Department of Parasitology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran 2 PhD, Professor, Department of Parasitology, School of Medicine, Tarbiat Modares University, Tehran, Iran 3 PhD, Professor, Department of Virology, Blood Transfusion Research Center, Tehran, Iran Background and purpose: Previous Research shows the use of plasmids containing genes TSA to be useful as vaccines for Leishmania major. Recently, the role of interleukin-22 (IL-22) in tissue repair has been demonstrated. In this research, the effect of IL-22 on encoding TSA gene of Leishmania major in BALB/c mice was assessed. Materials and methods: pcDNA3 plasmid containing the gene encoding TSA protein (pcTSA) of Leishmania major, along with the cytokine IL-22 was used. 60 BALB/c mice were divided to 4 groups of 15. Control groups received pcDNA3 and PBS and a group was vaccinated intramuscularly with the TSA gene containing plasmid. Fourth group received plasmid containing the gene for the TSA and IL-22 protein. IL-4 and interferon gamma (IFN-γ) levels (MTT test) were used to evaluate the cellular immunity and IgG2a, IgG1 and Total IgG levels [enzyme-linked immunosorbent assay (ELISA) method] to evaluate the humoral immunity. Measuring the diameter of the lesions and the age and weight of the mice was performed. Results: The simultaneous use of plasmid containing the gene encoding protein TSA and IL-22 significantly increased the mean level of IFN-γ and reduced the mean level of IL-4 compared to the other groups. While the mortality rate at 27th week after intervention was 100 % in the control group, the surveillance rates of pcTSA and pcTSA + IL-22 groups were 80%. pcTSA + IL-22 group had the highest weight in 30th week. pcTSA + IL-22 group had significantly smaller lesions compared to control and pcTSA groups. Conclusion: Results show the efficacy of pcTSA + IL-22 in improving the vaccination of cutaneous leishmaniasis.