The role of LPD-nanoparticles containing recombinant major surface glycoprotein of Leishmania (rgp63) in protection against leishmaniasis in murine model.

CONTEXT: Leishmaniasis is a major public health problem. Despite numerous attempts, yet there is no effective vaccine against human leishmaniasis, mainly due to a lack of an effective vaccine delivery system as well as adjuvant. OBJECTIVE(S): The aim of this study was to evaluate the ability of recombinant glycoprotein 63 (rgp63) as a model of Leishmania antigen, entrapped in liposome-polycation-DNA (LPD) complexes nanoparticles in inducing cell mediated immune (CMI) response and protecting against L. major in BALB/c mice. MATERIALS AND METHODS: To this end, the abundant leishmania promastigote cell surface glycoprotein, gp63, was entrapped in nano-sized LPD (CpG) particles, (LPD (CpG)-rgp63), and BALB/c mice were immunized three times with either (LPD (CpG)-rgp63) or rgp63-CpG DNA or LPD (CpG) or free rgp63 and dextrose 5%. Various parameters including footpad thickness, splenic load of L. major parasites, rgp63-binding IgGs and also cytokine levels of rgp63-reactive T lymphocytes were then compared among different vaccinated animals. RESULTS: The lowest number of parasites in spleen, the higher levels of IgG2a after challenge infection, the minimal footpad swelling and high level of IFN-γ secretion, all indicated that adjuvants and antigen-delivery systems are essential in modifying immune responses; as mice received LPD (CpG)-rgp63 induced immune response stronger than the other groups. CONCLUSIONS: This study demonstrates that LPD nanoparticle is a promising and adaptable delivery system which could be modified towards specific vaccine targets to induce a more potent immune response in combination with rgp63.

Introduction of cationic virosome derived from vesicular stomatitis virus as a novel gene delivery system for sf9 cells.

Insect-derived cell lines are used extensively to produce recombinant proteins because they are capable of performing a range of post-translational modifications. Due to their significance in biotechnological applications, various methods have been developed to transfect them. In this study, we introduce a virosome constructed from vesicular stomatitis virus (VSV) as a new delivery system for sf9 cells. We labeled these VSV virosomes by fluorescent probe Rhodamine B chloride (R18). By fluorescence microscope observation and conducting a fusion assay, we confirmed the uptake of VSV virosomes via endocytosis by sf9 cells and their fusion with the endosomal membrane. Moreover, we incubated cationic VSV virosomes with a GFP-expressing bacmid and transfected sf9 cells, after 24 h some cells expressed GFP indicating the ability of VSV virosomes to deliver heterologous DNA to these cells. This is the first report of a virosome-based delivery system introduced for an insect cell line.

In Vitro Evaluation of Influenza M2 and Leishmania major HSP70 (221-604) Chimer Protein.

BACKGROUND: Permanent antigenic variation of influenza viruses causes a major concern to develop an effective human influenza vaccine. Conserved antigens are new vaccine candidates because it is not necessary to match the prepared vaccine with circulating strains. Ion channel M2 protein is conserved among all influenza A viruses, allowing the virus to enter host cells. OBJECTIVES: To prepare an effective vaccine against influenza A viruses, a chimerical DNA plasmid encoding Influenza virus M2 protein and Leishmania major HSP70 was constructed. MATERIALS AND METHODS: Influenza A/New Caledonia/20/99 (H1N1) was inoculated into MDCK cell line and total RNA was extracted. The full length M2 gene was amplified by RT-PCR using designed specific primers, cloned into pGEM-T Easy cloning vector and completely sequenced. The M2 gene was then subcloned into the pcDNA upstream of HSP70 gene. Recombinant plasmids were transfected into COS-7 cells to evaluate protein expression. RESULTS: The recombinant plasmids were confirmed by PCR, restriction enzyme analysis and sequencing. Three dimensional structure of chimer protein was assessed using specific software. Transient protein expression in eukaryotic cells was confirmed by specific mRNA detection, indirect Immunofluorescence test and western blotting. CONCLUSIONS: M2-HSP70 chimer protein was successfully expressed in eukaryotic cells. Computational studies of chimer peptide sequence revealed that fusing HSP to the C-terminal of M2 protein does not mask the predominant epitope of M2. HSP70 is a molecular chaperon and immunostimulatory component. Genetically fusing antigens to HSPs leads to the enrichment of DNA vaccine potency. The immunogenicity of this construct with different formulation would be evaluated in further investigations.

Rabbit nasal immunization against influenza by dry-powder form of chitosan nanospheres encapsulated with influenza whole virus and adjuvants.

Influenza virus is one of the main causes of respiratory diseases in human. Although different vaccines have been produced during past decades, there is still a huge demand for a safe influenza vaccine with the ability to induce mucosal immune responses and sufficient protection, especially in elderly patients. In this study, chitosan nanospheres were employed as the drug delivery system. Influenza virus, CpG oligodeoxynucleotide (CpG ODN) and Quillaja saponins (QS) were incorporated in this nanospheric system. Three doses of dry powder nanosphere vaccine were nasally administered to rabbits on days 0, 45 and 60, followed by a final booster injection on day 75. Both humoral and cellular immune responses were investigated. Hemagglutination inhibition (HI) antibody titer was elevated in all groups compared to the control group at the end of vaccination in rabbits receiving nanospheres loaded with virus and CpG, CH(WV+CpG) (P<0.001). Rabbit serum IgG raised significantly in all the vaccinated groups, with the highest responses in CH(WV+CpG) group. CH(WV+CpG) and CH(WV) induced significant sIgA titers (P<0.001). CpG adjuvant also showed a prominent role in the stimulation and secretion of of IL-2 and IFN-γ cytokines (3 and 3.5 fold increase, respectively). Finally, as CH(WV+CpG) depicted to be effective in induction of humoral and cellular immune responses after nasal administration, this nanoparticulate adjuvant could be identified as an efficient adjuvant/delivery system for mucosal immunization against influenza virus.

An H1-H3 chimeric influenza virosome confers complete protection against lethal challenge with PR8 (H1N1) and X47 (H3N2) viruses in mice.

Annual health threats and economic damages caused by influenza virus are still a main concern of the World Health Organization and other health departments all over the world. An influenza virosome is a highly efficient immunomodulating carrier mimicking the natural antigen presentation pathway and has shown an excellent tolerability profile due to its biocompatibility and purity. The major purpose of this study was to construct a new chimeric virosome influenza vaccine containing hemagglutinin (HA) and neuraminidase (NA) proteins derived from the A/PR/8/1934 (H1N1) (PR8) and A/X/47 (H3N2) (X47) viruses, and to evaluate its efficacy as a vaccine candidate in mice. A single intramuscular vaccination with the chimeric virosomes provided complete protection against lethal challenge with the PR8 and X47 viruses. The chimeric virosomes induced high IgG antibody responses as well as hemagglutination inhibition (HAI) titers. HAI titers following the chimeric virosome vaccination were at the same level as the whole inactivated influenza vaccine. Mice immunized with the chimeric virosomes displayed considerably less weight loss and exhibited significantly reduced viral load in their lungs compared with the controls. The chimeric virosomes can be used as an innovative vaccine formulation to confer protection against a broad range of influenza viruses.

Determining influenza virus shedding at different time points in madin-darby canine kidney cell line.

OBJECTIVE: Monitoring of influenza virus shedding and optimization of multiplicities of infection (MOI) is important in the investigation of a virus one step growth cycle and for obtaining a high yield of virus in vaccine development and conventional basic diagnostic methods. However, eluted infectious viruses may still be present immediately after virus inoculation and when cells are washed following virus cultivation which may lead to a false positive virus infectivity assay. MATERIALS AND METHODS: In this experimental study, we investigated influenza virus progeny production in Madin-Darby canine kidney (MDCK) cells with five different MOI at determined time points. The results were analyzed by end point titration tests and immunofluorescence assay. RESULTS: Higher titers of eluted virus were observed following a high MOI inoculation of virus in cell culture. Most probably, this was the result of sialic acid residues from viral hemagglutin in proteins that were cleaved by neuraminidase glycoproteins on the surface of the influenza virus, which promoted viral spread from the host cell to the culture supernatant or during endocytosis, where viruses recycle to the cell surface by recycling endosomes which culminated in virus shedding without replication. CONCLUSION: We demonstrated that the pattern of influenza virus progeny production was dose-dependent and not uniform. This production was influenced by several factors, particularly MOI. Understanding the exact features of viral particle propagation has a major impact in producing high virus yields in the development of vaccines. Use of lower MOI (0.01) could result in accurate, precise quantitative assays in virus diagnosis and titration methods.

Expression of the influenza M2 protein in three different eukaryotic cell lines.

Current influenza virus vaccines provide protection in part by antibodies induced to the two surface glycoproteins, the hemagglutinin and the neuraminidase. As a result of the continuous antigenic drift of these glycoproteins, a frequent update of the composition of influenza vaccines is required. The search for more conserved viral epitopes which would induce protective immunity against seasonal influenza viruses and eventually also to novel pandemic influenza viruses has a long history. The ectodomain of the Influenza A Virus M2 Protein has been identified as a possible candidate immunization against influenza. The present study describes the expression of cloned M2 gene in MDCK, HeLa, and COS-7 cells, i.e., in three established eukaryotic cell lines. The expression efficiency was demonstrated by immunofluorescent staining of transfected cells by ELISA, by SDS-PAGE-, and by Western blot-analysis. High level of expression was observed in COS-7 cells. Expression in HeLa and MDCK cells was less efficient. The plasmids constructed in this study may, after modifications, be used for the production of a DNA vaccine. Alternatively the expression product could be refined and used as a purified antigen for the vaccine. Thus, the M2 recombinant protein provides an ideal product for further antigenic, biochemical, structural and functional characterization of the protein and for evaluating its potential for immunodiagnosis and in vaccine studies.

Anti-influenza Activity of a Novel Polyoxometalate Derivative (POM-4960).

There are many effective chemotherapeutic agents used in influenza disease which some of them inhibit virus replication by interfering with FluV (influenza virus) viral binding or its penetration into cell membrane. A series of polyoxometalates compounds such as POM-523 and PM-504 have been synthesized and have showed inhibitory effects on viruses. In this study we examined anti influenza activity of a novel polyoxometalate derivative (POM-4960) synthesized in the Faculty of Chemistry of Damghan University of Basic Sciences. To evaluate the anti-influenza activity of POM, following the treatment of FluV with POM at different temperatures and incubation periods, viral titer reduction was assessed by haemaglutination assay (HA). The 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine TCID50 (tissue culture infective dose) of virus, CC50 (median cytotoxic concentration) of POM, protection percentage and antiviral activity of POM in cell culture. RT-PCR and direct Immunofluorescent assays were performed to evaluate the effect of POM on viral infection and viral RNA load, respectively. POM reduced HA titer near to zero in all cell culture specimens and showed high protection against viral infection of the cells. Reduction in viral infection was confirmed by RT-PCR and Immunofluorescent staining methods. Moreover, this POM derivative has a dual (cumulative) effect on attachment and penetration inhibition compared to other POM's with just one inhibitory effect. POM-4960 could be considered as a powerful anti-influenza agent with low toxicity and high antiviral potency.