ABSTRACT
Biolistic vaccination using gene gun is developed as a safer tool for delivery of DNA vaccines, a technique that combines high vaccine efficiency with lower antigen dosage and lower cost per vaccine dose. In this study, we compared the protective responses in mice after delivering the Brugia malayi abundant larval transcript-2 (BmALT-2) DNA vaccine using the conventional intradermal approach or with the needleless gene gun delivery approach. BmALT-2 is a leading vaccine candidate against B. malayi, a lymphatic filarial parasite of human. After optimizing the DNA dose and gene gun parameters for delivery into mouse skin, groups of mice were biolistically vaccinated with 5 µg of BmALT-2pVAX. Groups of mice vaccinated intradermally with 5 µg or 100 µg of BmALT-2pVAX was used for comparison of vaccine efficacy. Results demonstrated that gene gun vaccination with 5 µg of BmALT-2pVAX conferred significant protection against challenge infection that was comparable to the degree of protection conferred by intradermal vaccination with 100 µg of BmALT-2pVAX. This observation was further supported by an in vitro antibody dependent cellular cytotoxicity (ADCC) assay. Analysis of the immune response showed that the gene gun vaccination predominantly induced an IgG1 antibody response and significantly high Th2 cytokine response (IL-4) from spleen cells compared to intradermal BmALT-2 DNA delivery that induced predominantly an IgG2a and Th1 cytokine response (IFN-γ, IL-12 and TNF-α). These findings show that host protective responses could be achieved with 20 fold decrease in DNA dose using a gene gun and could prove to be an efficient delivery method in BmALT-2 DNA vaccination against lymphatic filariasis.
Subject(s)
Antigens, Helminth/immunology , Biolistics/methods , Elephantiasis, Filarial/prevention & control , Recombinant Proteins/immunology , Vaccines, DNA/immunology , Vaccines/immunology , Animals , Antibodies, Helminth/immunology , Antigens, Helminth/genetics , COS Cells , Chlorocebus aethiops , DNA, Helminth/immunology , Immunoglobulin G/immunology , Injections, Intradermal , Interferon-gamma/immunology , Interleukin-12/immunology , Interleukin-4/immunology , Mice , Mice, Inbred BALB C , Recombinant Proteins/genetics , Tumor Necrosis Factor-alpha/immunologyABSTRACT
Previous studies have indicated a long-duration of effect of in vivo pertussis toxin (PTX) on morphine analgesia in the mouse. However, the time-course of potency changes in morphine analgesia as determined in dose-response studies and biochemical correlates of PTX treatment have not been reported to date. Therefore, in the present studies the effects of in vivo PTX on morphine analgesia ED50 and PTX-catalyzed incorporation of [32P]-ADP-ribose and synapsin content in mouse spinal cord were examined. Mice were injected IT & ICV with saline or PTX (total dose = 0.2 microg) and tested for systemic morphine analgesia (tail-flick) 1, 10, 16 & 40 days later. There was no significant decrease in morphine potency 1 day following PTX treatment, whereas PTX produced a significant decrease in morphine potency at 10, 16 & 40 days. Concurrent decreases in the incorporation of [32P]-ADP-ribose in spinal cord by PTX were observed on days 10, 16 & 40. No changes were observed in synapsin content which suggests that the effect was not nonspecific. This study indicates that in vivo PTX produces co-ordinate long-lasting effects in both functional (analgesia) and biochemical (Gi/o-proteins) assays.
Subject(s)
Adenosine Diphosphate Ribose/metabolism , Analgesics, Opioid/pharmacology , GTP-Binding Proteins/metabolism , Morphine/pharmacology , Pertussis Toxin , Virulence Factors, Bordetella/pharmacology , Analysis of Variance , Animals , Catalysis , Drug Evaluation, Preclinical , Male , Mice , Synapsins/metabolism , Time FactorsABSTRACT
A step-by-step protocol for assaying cAMP is presented. This method is based on the standard binding protein assay that is available commercially. However, using the present procedure, the per tube cost is dramatically reduced. In the current protocol, four different binding proteins are compared for their ability to bind cAMP. The source of all reagents is noted as well as necessary precautions for insuring reliable assays. A simple tissue preparation method is outlined for assaying cAMP in brain. The utility of the assay is illustrated by demonstrating the effect of forskolin on cAMP in mouse striatal tissue.
