Ejaj Ahmad

Development, characterization and efficacy of niosomal diallyl disulfide in treatment of disseminated murine candidiasis

UNLABELLED In the current study, a novel niosome based formulation of diallyl disulfide (DADS) was evaluated for its potential to treat disseminated candidiasis in mouse model. Among various non-ionic surfactants tested, niosome formulation prepared using Span 80 was found to be most efficient in the entrapment of DADS. The DADS loaded niosomes had size dimensions in the range of 140 ± 30 nm with zeta potential of -30.67 ± 4.5. Liver/kidney function tests as well as histopathologic studies suggested that noisome-based DADS formulations are safe at the dose investigated. When administered to Candida albicans infected animals, the DADS bearing niosomal formulation cleared the fungal burden and increased their survival much efficiently than its free form. FROM THE CLINICAL EDITOR In this study, a novel niosomal formulation of the antifungal DADS was utilized in a murine candidiasis model, resulting in more efficient fungal clearance compared to the free formulation.

Ether lipid vesicle-based antigens impart protection against experimental listeriosis

Background Incidence of food-borne infections from Listeria monocytogenes, a parasite that has adapted intracellular residence to avoid antibody onslaught, has increased dramatically in the past few years. The apparent lack of an effective vaccine that is capable of evoking the desired cytotoxic T cell response to obliterate this intracellular pathogen has encouraged the investigation of alternate prophylactic strategies. It should also be noted that Archaebacteria (Archae) lipid-based adjuvants enhance the efficacy of subunit vaccines. In the present study, the adjuvant properties of archaeosomes (liposomes prepared from total polar lipids of archaebacteria, Halobacterium salinarum) combined with immunogenic culture supernatant antigens of L. monocytogenes have been exploited in designing a vaccine candidate against experimental listeriosis in murine model. Methods Archaeosome-entrapped secretory protein antigens (SAgs) of L. monocytogenes were evaluated for their immunological responses and tendency to deplete bacterial burden in BALB/c mice challenged with sublethal listerial infection. Various immunological studies involving cytokine profiling, lymphocyte proliferation assay, detection of various surface markers (by flowcytometric analysis), and antibody isotypes (by enzyme-linked immunosorbent assay) were used for establishing the vaccine potential of archaeosome-entrapped secretory proteins. Results Immunization schedule involving archaeosome-encapsulated SAgs resulted in upregulation of Th1 cytokine production along with boosted memory in BALB/c mice. It also showed protective effect by reducing listerial burden in various vital organs (liver and spleen) of the infected mice. However, the soluble form of the antigens (SAgs) and their physical mixture with sham (empty) archaeosomes, besides showing feeble Th1 response, were unable to protect the animals against virulent listerial infection. Conclusion On the basis of the evidence provided by the current data, it is inferred that archaeosome-entrapped SAgs formulation not only enhances cytotoxic T cell response but also helps in the clearance of pathogens and thereby increases the survival of the immunized animals.

Plasma beads loaded with Candida albicans cytosolic proteins impart protection against the fungal infection in BALB/c mice

The development of a prophylactic vaccine against systemic candidiasis, employing Candida albicans cytosolic proteins (Cp) as antigen and fibrin cross-linked plasma beads as an antigen bearing dual delivery system is described. Groups of mice were administered either with free Cp, or Cp entrapped in plasma beads, Cp entrapped in liposomes or liposome encapsulated Cp further entrapped in plasma beads. Humoral immunity was studied by measuring the anti-Cp antibody titers in the sera of the immunized animals. Induction of cell-mediated immunity was assessed by delayed type hypersensitivity (DTH), NO production, up-regulation of co-stimulatory molecules viz. CD80, CD86 on APCs on one hand and T-cells proliferation as well as induction of IFN-γ and IL-4 on the other. The efficacy of various vaccine formulations in protecting mice against a lethal challenge with C. albicans, was assessed by determining animal survival rate and fungal burden in the systemic circulation and vital organs. Among various Cp-based vaccines investigated, the preparation containing liposomized Cp entrapped in plasma beads imparted superior protection in the immunized mice as compared to other antigens delivery systems.

Fibrin matrices: The versatile therapeutic delivery systems

Fibrin sealants, that have been employed for over a century by surgeons to stop post surgery bleeding, are finding novel applications in the controlled delivery of antibiotics and several other therapeutics. Fibrinogen can be easily purified from blood plasma and converted by thrombolysis to fibrin that undergoes spontaneous aggregation to form insoluble clot. During the gelling, fibrin can be formulated into films, clots, threads, microbeads, nanoconstructs and nanoparticles. Whole plasma clots in the form of beads and microparticles can also be prepared by activating endogenous thrombin, for possible drug delivery. Fibrin formulations offer remarkable scope for controlling the porosity as well as in vivo degradability and hence the release of the associated therapeutics. Binding/covalent-linking of therapeutics to the fibrin matrix, crosslinking of the matrix with bifunctional reagents and coentrapment of protease inhibitors have been successful in regulating both in vitro and in vivo release of the therapeutics. The release rates can also be remarkably lowered by preentrapment of therapeutics in insoluble particles like liposomes or by anchoring them to the matrix via molecules that bind them as well as fibrin.

Efficacy of Cell Wall-Deficient Spheroplasts Against Experimental Murine Listeriosis

Various strategies adapted to develop an efficient vaccine against foodborne pathogen, Listeria monocytogenes, have met with little success. Spheroplasts (bacterial cell devoid of cell wall) are likely to undergo membrane–membrane fusion, leading to the delivery of their content to the cytosol of antigen‐presenting cells, thus facilitating MHC class I antigen processing and presentation. In this study, we evaluated the prophylactic potential of Listeria spheroplast‐based vaccine against experimental murine listeriosis in comparison with heat‐killed Listeria (HKL) and archaeosome‐entrapped Listeria whole‐cell protein (LWCP). Compared with HKL, the spheroplast‐based vaccine was found to evoke better Th1 response as exhibited by the presence of type 1 cytokines in the host (interferon‐γ and IL‐12) and a high IgG2a/IgG1 ratio. Robust lympho‐proliferative efficacy was apparent in both spheroplast‐immunized and archaeosome‐entrapped LWCP‐immunized groups. The upregulation of costimulatory and effector memory markers upon immunization with spheroplasts was found to be at par with that evoked by archaeosome‐entrapped LWCP‐immunized group. Central memory response in gated CD8+ T cell was much higher in spheroplast‐immunized animals when compared with archaeosome‐entrapped LWCP group. The data presented here clearly demonstrate that spheroplasts evoked a robust immune response and offer better prophylactic potential against L. monocytogenes.

Vaccine potential of plasma bead-based dual antigen delivery system against experimental murine candidiasis

The development of prophylactic anti-candidal vaccine comprising the Candida albicans cytosolic proteins (Cp) as antigen and plasma beads (PB) prepared from plasma as sustained delivery system, is described. The immune-prophylactic potential of various PBs-based dual antigen delivery systems, co-entrapping Cp pre-entrapped in PLGA microspheres were tested in the murine model. Induction of cell mediated immunity was measured by assaying DTH and NO production as well as in vitro proliferation of lymphocytes derived from the immunized animals. Expression of surface markers on APCs (CD80, CD86) and T-cells (CD4+, CD8+) was also evaluated. Humoral immune response was studied by measuring circulating anti-Cp antibodies and their subclasses. When the prophylactic efficacy of the vaccines was tested in mice challenged with virulent C. albicans, the PB-based formulation (PB-PLGA-Cp vaccine) was found to be most effective in the generation of desirable immune response, in terms of suppression of fungal load and facilitating the survival of the immunized animals.

Entrapment in plasma microparticles: A promising strategy for antigen delivery

We report the preparation of plasma microparticles (PMPs) from autologous blood plasma for sustained in vivo delivery of the entrapped antigens. The PMPs were prepared by high speed-stirring of calcium-enriched plasma, mixed with the antigen to be entrapped, in mineral oil. The preparation of PMPs did not necessitate addition of any external protein/enzyme nor special laboratory setup. Our results suggest that the PMPs release the entrapped invertase in a sustained manner both in vitro and in vivo, especially after crosslinking with glutaraldehyde. The preparations are reasonably stable to proteolysis and constitute strong candidates for eliciting immune response. Induction of humoral immune response by the PMP-entrapped invertase, as evident from the high antibody titers, was remarkable and comparable with that observed in animals receiving the antigen emulsified with Freunds Complete Adjuvant. Isotypic analysis of antibodies showed a Th1-biased immune response in animals administered uncrosslinked or crosslinked PMPs-entrapped invertase, especially after a booster dose. The analysis in animals of the group immunized with adjuvant-emulsified antigen suggested a combined Th1 and Th2 response. PMP-entrapment also caused high expression of surface markers (CD80 and CD86) on antigen presenting cells, as well as effector T-cells surface markers (CD4(+) and CD8(+) ) as revealed by FACS. The study suggests that PMPs offer remarkable promise as adjuvant-free and biocompatible vaccine delivery systems.

Effective antigen delivery via dual entrapment in erythrocytes and autologous plasma beads

Abstract Fibrin-based polymeric systems have now emerged as efficient carriers of drugs, growth factors, genes, and cells. Earlier, we have reported fibrin-based plasma beads (PB), prepared from clotted whole plasma, as an efficient system for the controlled release of entrapped therapeutics. In the present study, we investigate the dual entrapment in erythrocytes and PB, as potential particulate antigen delivery system in rabbit and mice, with yeast invertase as the model antigen. Preparations used for immunisation include- invertase entrapped in erythrocytes, the same further entrapped in PB, and crosslinked with glutaraldehyde. While route of administration of the antigen only moderately affected the antibody titres, strategies slowing its release from PB increased the antibody titres remarkably, especially after a booster. Entrapment of erythrocytes entrapped antigen in the PB and further crosslinking with glutaraldehyde also resulted in significant alterations of IgG1/IgG2a ratio, indicating a shift towards humoral response. The elicited immune response was more marked in rabbits as compared to that in mice. Considering the well-known toxicity of the adjuvant, comparable antibody titres induced by the erythrocyte-plasma bead system was encouraging in the induction of humoral immunity.

Nanoparticle-based mycosis vaccine

Many diseases that were considered major affliction of mankind in the past have been successfully eradicated with introduction of appropriate vaccine strategies. In order to expedite new challenges coming up to deal with various infectious diseases, nano-particulate-based subunit vaccines seem to be the demand of ordeal. The nano-vaccines can find better scope for the diseases that were not rampant in the semi-advanced world few years back. For example in present-day circumstances that corroborate with advancement in the field of medical sciences in terms of cancer chemotherapy, organ transplantation, therapy of autoimmune diseases, etc.; along with prevalence of altogether unheard diseases such as HIV infection, people are at risk of infliction with many more pathogens. In this regard, development of an effective prophylactic strategy against many opportunistic infections primarily caused by fungal pathogens needs better understanding of host pathogen relation and role of active immunity against pathogenic fungi. In the present study, we have tried to decipher effectiveness of a nano-sized vaccine delivery system in imparting protection against fungal pathogens.

Recent Updates on Molecular Genetic Engineering Approaches and Applications of Human Therapeutic Proteins

Therapeutic proteins are engineered proteins produced in the laboratory for pharmaceutical use. With the advent of recombinant DNA technology, the proteins can be generated in specific host cells under defined conditions. In the process of production of genetically engineered animals, the gene of interest can be added at a single cell stage to produce a cloned animal from genetically engineered cells. Several recombinant cytokines, clotting factors etc have been licensed and are currently being utilized for the treatment of cancer, infectious diseases, hemophilia, anemia, multiple sclerosis, and hepatitis B/C. Therapeutic proteins that are useful for human are successfully produced in poultry as well as in livestock animals. However, the fastest growing class of therapeutic proteins are antibodies especially monoclonal antibodies (mAb), the most important class of therapeutic protein with the potential to generate significant revolution in terms of clinical success rate. Here, we review the most recent clinical advances in the field of emerging and existing therapeutic proteins.