Recombinant mid gut antigen (Bm95) as a vaccine against Indian Rhiphicephalus haemaphysaloides in Bos indicus cattle

In the present study, we report for the first time the efficacy of recombinant Bm95 mid gut antigen isolated from an Argentinean strain of Rhipicephalus microplus strain A in controlling the tick infestations in India. The synthetic gene for Bm95 optimized for expression in yeast was obtained and used to generate yeast transformants expressing Bm95 which was purified to apparent homogeneity. Liquid chromatography-mass spectrometry analysis of the purified protein confirmed its identity as Bm95. Vaccine was prepared by blending various concentrations of purified Bm95 with aluminium hydroxide as an adjuvant. lmmunogenicity studies of the vaccine in rabbits and cattle indicated that the vaccine was highly immunogenic. The efficacy studies of the vaccine was done in cattle. Naive Bos indicus cattle were vaccinated with the recombinant vaccine and were challenged with the larval, nymphal and adult forms of Rhiphicephalus haemaphysaloides. The vaccine protected the animals from larval, nymph and adult tick challenges with an efficacy of 98.7%, 84.6% and 78.9% respectively. The results obtained from the above studies clearly demonstrated the advantage and possibilities of the use of Bm95 in controlling R. haemaphysaloides infestations in the field. (C) 2010 Elsevier Ltd. All rights reserved.

Pre-clinical toxicity & immunobiological evaluation of DNA rabies vaccine & combination rabies vaccine in rhesus monkeys (Macaca mulatta)

Background & objectives: Pre-clinical toxicology evaluation of biotechnology products is a challenge to the toxicologist. The present investigation is an attempt to evaluate the safety profile of the first indigenously developed recombinant DNA anti-rabies vaccine DRV (100 mu g)] and combination rabies vaccine CRV (100 mu g DRV and 1.25 IU of cell culture-derived inactivated rabies virus vaccine)], which are intended for clinical use by intramuscular route in Rhesus monkeys. Methods: As per the regulatory requirements, the study was designed for acute (single dose - 14 days), sub-chronic (repeat dose - 28 days) and chronic (intended clinical dose - 120 days) toxicity tests using three dose levels, viz. therapeutic, average (2x therapeutic dose) and highest dose (10 x therapeutic dose) exposure in monkeys. The selection of the model i.e. monkey was based on affinity and rapid higher antibody response during the efficacy studies. An attempt was made to evaluate all parameters which included physical, physiological, clinical, haematological and histopathological profiles of all target organs, as well as Tiers I, II, III immunotoxicity parameters. Results: In acute toxicity there was no mortality in spite of exposing the monkeys to 10XDRV. In sub chronic and chronic toxicity studies there were no abnormalities in physical, physiological, neurological, clinical parameters, after administration of test compound in intended and 10 times of clinical dosage schedule of DRV and CRV under the experimental conditions. Clinical chemistry, haematology, organ weights and histopathology studies were essentially unremarkable except the presence of residual DNA in femtogram level at site of injection in animal which received 10X DRV in chronic toxicity study. No Observational Adverse Effects Level (NOAEL) of DRV is 1000 ug/dose (10 times of therapeutic dose) if administered on 0, 4, 7, 14, 28th day. Interpretation & conclusions: The information generated by this study not only draws attention to the need for national and international regulatory agencies in formulating guidelines for pre-clinical safety evaluation of biotech products but also facilitates the development of biopharmaceuticals as safe potential therapeutic agents.

A Multiantigenic DNA Vaccine That Induces Broad Hepatitis C Virus-Specific T-Cell Responses in Mice

There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising ``multiantigen'' vaccine that elicits robust CMI. IMPORTANCE Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans.

sopB of Salmonella enterica serovar Typhimurium is a potential DNA vaccine candidate in conjugation with live attenuated bacteria

The immune response against Salmonella is multi-faceted involving both the innate and the adaptive immune system. The characterization of specific Salmonella antigens inducing immune response could critically contribute to the development of epitope based vaccines for Salmonella. We have tried to identify a protective T cell epitope(s) of Salmonella, as cell mediated immunity conferred by CD8+ T cells is the most crucial subset conferring protective immunity against Salmonella. It being a proven fact that secreted proteins are better in inducing cell mediated immunity than cell surface and cytosolic antigens, we have analyzed all the genbank annotated Salmonella pathogenicity island 1 and 2 secreted proteins of Salmonella enterica serovar Typhimurium (S. typhimurium) and S. enterica serovar Typhi (S. typhi). They were subjected to BIMAS and SYFPEITHI analysis to map MHC-I and MHC-II binding epitopes. The huge profile of possible T cell epitopes obtained from the two classes of secreted proteins were tabulated and using a scoring system that considers the binding affinity and promiscuity of binding to more than one allele, SopB and SifB were chosen for experimental confirmation in murine immunization model. The entire SopB and SifB genes were cloned into DNA vaccine vectors and were administered along with live attenuated Salmonella and it was found that SopB vaccination reduced the bacterial burden of organs by about 5-fold on day 4 and day 8 after challenge with virulent Salmonella and proved to be a more efficient vaccination strategy than live attenuated bacteria alone.

Gene therapy: Principles, practice, problems and prospects

The remarkable advances made in recombinant DNA technology over the last two decades have paved way for the use of gene transfer to treat human diseases. Several protocols have been developed for the introduction and expression of genes in humans, but the clinical efficacy has not been conclusively demonstrated in any of them. The eventual success of gene therapy for genetic and acquired disorders depends on the development of better gene transfer vectors for sustained, long term expression of foreign genes as well as a better understanding of the pathophysiology of human diseases, it is heartening to note that some of the gene therapy protocols have found other applications such as the genetic immunization or DNA vaccines, which is being heralded as the third vaccine revolution, Gene therapy is yet to become a dream come true, but the light is seen at the end of the tunnel.

Use of norethisterone and estradiol in mini doses as a contraceptive in the male : Efficacy studies in the adult male bonnet monkey (Macaca radiata)

Administration of norethisterone (NET) or NET + estradiol benzoate using an Alzet minipump or as once-a-month intramuscular injection of their depot forms, NET-enanthate (NET-EN) and estradiol valerate (E-val), resulted in azoospermia in all monkeys (n = 13) within 60 to 150 days of treatment. Although addition of depot form of testosterone (T, 20 mg/month) to the regimen restored the behavioral response typical of a normal male, it did not reverse the azoospermic state. Serum T (heightened nocturnal) levels were significantly reduced (> 85%, p < 0.001) in all the treated groups. Evidence for blockade in spermatogenesis following treatment was obtained by DNA flow cytometry. Following withdrawal of treatment, the T level was restored to normalcy within 15 days but 120 days more were required for the animals to exhibit normal sperm counts. In conclusion, the efficacy of once-a-month injection of relatively low doses of NET-EN + E-Val to bring about azoospermia in monkeys, in a relatively short time, has been demonstrated. As the results are uniform and reproducible, it appears desirable that this steroid regimen be tested in man for its contraceptive efficacy.

Prospects of riboflavin carrier protein (RCP) as an antifertility vaccine in male and female mammals

Riboflavin carrier protein (RCP) is obligatorily involved in yolk deposition of the vitamin, riboflavin, in the developing oocyte of the hen. The production of this protein is inducible by oestrogen. It is evolutionarily conserved in terms of its physicochemical, immunological and functional characteristics. It is the prime mediator of vitamin supply to the developing fetus in mammals, including primates. Passive immunoneutralization of the protein terminates pregnancy in rats. Active immunization of rats and bonnet monkeys with avian RCP prevents pregnancy without causing any adverse physiological effects of the mother in terms of her vitamin status, reproductive cycles or reproductive-endocrine profile. Denatured, linearized RCP is more effective in eliciting neutralizing antibodies capable of interfering with embryonic viability either before or during peri-implantation stages. Two defined stretches of sequential epitopes, one located at the N-terminus and the other at the C-terminus of the protein have been identified. Active immunization with either of these epitopes conjugated with diptheria toxoid curtails pregnancy in rats and monkeys. Immunohistochemical localization of RCP on ovulated oocytes and early embryos shows that the antibodies cause degeneration only of early embryos. RCP is produced intra-testicularly and becomes localized on acrosomal surface of mammalian spermatozoa. Active immunization of male rats and monkeys with denatured RCP markedly reduces fertility by impairing the fertilizing potential of spermatozoa. These findings suggest that RCP, or its defined fragments, could be a novel, first generation vaccine for regulating fertility in both the sexes.

Toward the Discovery of Vaccine Adjuvants: Coupling In Silico Screening and In Vitro Analysis of Antagonist Binding to Human and Mouse CCR4 Receptors

Background: Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses. Methodology: Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cellmigration, including that of CCR4(+) Tregs. Significance: Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.

Computational analysis of proteome of H5N1 avian influenza virus to define T cell epitopes with vaccine potential

The existing vaccines against influenza are based on the generation of neutralizing antibody primarily directed against surface proteins-hernagglutinin and neuraminidase. In this work, we have computationally defined conserved T cell epitopes of proteins of influenza virus H5N1 to help in the design of a vaccine with haplotype specificity for a target population. The peptides from the proteome of H5NI irus which are predicted to bind to different HLAs, do not show similarity with peptides of human proteorne and are also identified to be generated by proteolytic cleavage. These peptides could be made use of in the design of either a DNA vaccine or a subunit vaccine against V influenza. (c) 2007 Elsevier Ltd. All rights reserved.

Non-segmented negative sense RNA viruses as vectors for vaccine development

This article intends to cover two aspects of non-segmented negative sense RNA viruses. In the initial section, the strategy employed by these viruses to replicate their genomes is discussed. This would help in understanding the later section in which the use of these viruses as vaccine vectors has been discussed. For the description of the replication strategy which encompasses virus genome transcription and genome replication carried out by the same RNA dependent RNA polymerase complex, a member of the prototype rhabdovirus family - Chandipura virus has been chosen as an example to illustrate the complex nature of the two processes and their regulation. In the discussion on these viruses serving as vectors for carrying vaccine antigen genes, emphasis has been laid on describing the progress made in using the attenuated viruses as vectors and a description of the systems in which the efficiency of immune responses has been tested.

A Safe Vaccine (DV-STM-07) against Salmonella Infection Prevents Abortion and Confers Protective Immunity to the Pregnant and New Born Mice

Pregnancy is a transient immuno-compromised condition which has evolved to avoid the immune rejection of the fetus by the maternal immune system. The altered immune response of the pregnant female leads to increased susceptibility to invading pathogens, resulting in abortion and congenital defects of the fetus and a subnormal response to vaccination. Active vaccination during pregnancy may lead to abortion induced by heightened cell mediated immune response. In this study, we have administered the highly attenuated vaccine strain delta pmrG-HM-D (DV-STM-07) in female mice before the onset of pregnancy and followed the immune reaction against challenge with virulent S. Typhimurium in pregnant mice. Here we demonstrate that DV-STM-07 vaccine gives protection against Salmonella in pregnant mice and also prevents Salmonella induced abortion. This protection is conferred by directing the immune response towards Th2 activation and Th1 suppression. The low Th1 response prevents abortion. The use of live attenuated vaccine just before pregnancy carries the risk of transmission to the fetus. We have shown that this vaccine is safe as the vaccine strain is quickly eliminated from the mother and is not transmitted to the fetus. This vaccine also confers immunity to the new born mice of vaccinated mothers. Since there is no evidence of the vaccine candidate reaching the new born mice, we hypothesize that it may be due to trans-colostral transfer of protective anti-Salmonella antibodies. These results suggest that our vaccine DV-STM-07 can be very useful in preventing abortion in the pregnant individuals and confer immunity to the new born. Since there are no such vaccine candidates which can be given to the new born and to the pregnant women, this vaccine holds a very bright future to combat Salmonella induced pregnancy loss.

Dual-Drug Delivery System Based on In Situ Gel-Forming Nanosuspension of Forskolin to Enhance Antiglaucoma Efficacy

The present study was designed to improve the bioavailability of forskolin by the influence of precorneal residence time and dissolution characteristics. Nanosizing is an advanced approach to overcome the issue of poor aqueous solubility of active pharmaceutical ingredients. Forskolin nanocrystals have been successfully manufactured and stabilized by poloxamer 407. These nanocrystals have been characterized in terms of particle size by scanning electron microscopy and dynamic light scattering. By formulating Noveon AA-1 polycarbophil/poloxamer 407 platforms, at specific concentrations, it was possible to obtain a pH and thermoreversible gel with a pH(gel)/T-gel close to eye pH/temperature. The addition of forskolin nanocrystals did not alter the gelation properties of Noveon AA-1 polycarbophil/poloxamer 407 and nanocrystal properties of forskolin. The formulation was stable over a period of 6 months at room temperature. In vitro release experiments indicated that the optimized platform was able to prolong and control forskolin release for more than 5 h. The in vivo studies on dexamethasone-induced glaucomatous rabbits indicated that the intraocular pressure lowering efficacy for nanosuspension/hydrogel systems was 31% and lasted for 12 h, which is significantly better than the effect of traditional eye suspension (18%, 4-6 h). Hence, our investigations successfully prove that the pH and thermoreversible polymeric in situ gel-forming nanosuspension with ability of controlled drug release exhibits a greater potential for glaucoma therapy.