Protection against Borreliella burgdorferi infection mediated by a synthetically engineered DNA vaccine.

Lyme disease is the most common vector-borne disease in North America. The etiological agent is the spirochete Borreliella burgdorferi, transmitted to mammalian hosts by the Ixodes tick. In recent years there has been an increase in the number of cases of Lyme disease. Currently, there is no vaccine on the market for human use. We describe the development of a novel synthetically engineered DNA vaccine, pLD1 targeting the outer-surface protein A (OspA) of Borreliella burgdorferi. Immunization of C3 H/HeN mice with pLD1 elicits robust humoral and cellular immune responses that confer complete protection against a live Borreliella burgdorferi bacterial challenge. We also assessed intradermal (ID) delivery of pLD1 in Hartley guinea pigs, demonstrating the induction of robust and durable humoral immunity that lasts at least 1 year. We provide evidence of the potency of pLD1 by showing that antibodies targeting the OspA epitopes which have been associated with protection are prominently raised in the immunized guinea pigs. The described study provides the basis for the advancement of pDL1 as a potential vaccine for Lyme disease control.

Preparation of pseudotyped lentiviral vectors resistant to inactivation by serum complement.

INTRODUCTION: A major obstacle to in vivo delivery of lentivirus or other retroviral vectors is their lability in the presence of serum. In vivo, these viral particles are rapidly destroyed by nonspecific complement-mediated degradation mechanisms. The eventual effective use of retroviral vectors for in vivo gene delivery would be greatly facilitated by the development of methods to protect the viral particles from such degradation. This protocol describes methods for the production of complement-stabilized lentiviral vectors either by pseudotyping the viral particles with a fusion envelope protein containing the complement-regulatory protein CD55 (decay accelerating factor, DAF) or by coassembly with the native DAF protein. An in vitro serum inactivation assay is also described.

Factors associated with induced chronic inflammation in mdx skeletal muscle cause posttranslational stabilization and augmentation of extrasynaptic sarcolemmal utrophin.

Chronic inflammation in tibialis anterior muscles of mdx mice was produced by a single injection of a recombinant adenovirus vector (AV) expressing an immunogenic beta-galactosidase (beta-gal). In regions of intense beta-gal staining, mononuclear infiltrates abounded, and muscle fibers showed strong extrasynaptic utrophin immunostaining, restoration of dystrophin-associated protein complex, and a marked reduction of the prevalence of centronucleation. Immunoblot analysis confirmed an increase of endogenous utrophin without an increase of the mRNA of the major muscle isoform utrA. Significantly better maximal tetanic force values were demonstrated in the inflammatory versus control mdx muscles. The resistance to lengthening contraction- induced damage was also significantly increased in the former. In muscles of mice lacking TNF-alpha gene, AV vector did not induce inflammation and extrajunctional utrophin increase did not occur. In the inflammatory mdx muscles, proteolytic activity of calcium-activated calpain was reduced, and in mdx myotubes in vitro, incubation with NO donors also reduced calpain-mediated utrophin proteolysis. Since utrophin was shown to be a natural substrate of calpain and known inhibitors of calpain in cultured mdx myotubes increased utrophin levels, the above results were consistent with the following conclusions: (1) extrasynaptic utrophin increase is mainly responsible for the antidystrophic effect; (2) extrasynaptic utrophin increase is a result of posttranscriptional mechanism(s) related to proinflammatory factors; and (3) reduction of endogenous muscle calpain activity by inflammatory cytokines has an important role in the stabilization and increase of the extrasynaptic utrophin.

Baculovirus GP64-pseudotyped HIV-based lentivirus vectors are stabilized against complement inactivation by codisplay of decay accelerating factor (DAF) or of a GP64-DAF fusion protein.

The eventual development of efficient gene transfer vectors for in vivo gene delivery will require the development of a number of important new technologies such as stabilization of vectors against protective mechanisms that destroy or otherwise inactivate foreign infectious agents like gene transfer vectors. It is known that the baculovirus envelope protein GP64 of Autographa californica nucleopolyhedrovirus can efficiently pseudotype lentivirus vectors and that modified forms of the baculovirus envelope protein GP64 can also assemble efficiently into baculovirus particles to display functional foreign proteins on the baculovirus surface. In the present study we have combined these techniques to prepare HIV-based lentivirus vectors pseudotyped with GP64 envelope protein and coexpressing a fusion protein of GP64 with the complement-regulatory, decay accelerating factor (DAF, CD55). In addition, we have also prepared GP64-pseudotyped vectors in the presence of a DAF expression plasmid to allow the incorporation of DAF protein into viral particles. Our results demonstrate both the efficient expression and the high-titer production of GP64/GP64-DAF and GP64/DAF-pseudotyped particles and their stability against inactivation by human and nonhuman primate serum.

Regenerative capacity of the dystrophic (mdx) diaphragm after induced injury.

Duchenne muscular dystrophy is characterized by myofiber necrosis, muscle replacement by connective tissue, and crippling weakness. Although the mdx mouse also lacks dystrophin, most muscles show little myofiber loss or functional impairment. An exception is the mdx diaphragm, which is phenotypically similar to the human disease. Here we tested the hypothesis that the mdx diaphragm has a defective regenerative response to necrotic injury, which could account for its severe phenotype. Massive necrosis was induced in mdx and wild-type (C57BL10) mouse diaphragms in vivo by topical application of notexin, which destroys mature myofibers while leaving myogenic precursor satellite cells intact. At 4 h after acute exposure to notexin, >90% of diaphragm myofibers in both wild-type and mdx mice demonstrated pathological sarcolemmal leakiness, and there was a complete loss of isometric force-generating capacity. Both groups of mice showed strong expression of embryonic myosin within the diaphragm at 5 days, which was largely extinguished by 20 days after injury. At 60 days postinjury, wild-type diaphragms exhibited a persistent loss ( approximately 25%) of isometric force-generating capacity, associated with a trend toward increased connective tissue infiltration. In contrast, mdx diaphragms achieved complete functional recovery of force generation to noninjured values, and there was no increase in muscle connective tissue over baseline. These data argue against any loss of intrinsic regenerative capacity within the mdx diaphragm, despite characteristic features of major dystrophic pathology being present. Our findings support the concept that significant latent regenerative capacity resides within dystrophic muscles, which could potentially be exploited for therapeutic purposes.

Ligand-modified vesicular stomatitis virus glycoprotein displays a temperature-sensitive intracellular trafficking and virus assembly phenotype.

The production of potentially targetable VSV-G-pseudotyped retrovirus vectors has been hampered by inadequate understanding of the structure-function relationships of the VSV-G protein. In these studies we demonstrate assembly and production of MLV-based and HIV-1-based vector particles using VSV-G proteins modified by the insertion of a peptide ligand into a site corresponding to amino acid position 24 of the native VSV-G molecule. The inserted ligand represents the decapeptide encoding the collagen-binding domain of von Willebrand factor. We have used deconvolution microscopy to demonstrate that the modified VSV-G molecules sequester in perinuclear structures and are unavailable for assembly of infectious virus particles at the cell surface under standard tissue culture conditions at 37 degrees C. In contrast, at a lower permissive temperature of 30 degrees C, the modified VSV-G protein traffics appropriately to the cell surface and participates in useful titers. Furthermore, VSV-G-pseudotyped MLV-based and HIV-1-based vectors displaying the collagen-binding domain demonstrate a statistically significant increased attachment to a collagen matrix as indicated by an ELISA-like cell binding assay and by a focus transduction assay.

Cell surface heparan sulfate is a receptor for attachment of envelope protein-free retrovirus-like particles and VSV-G pseudotyped MLV-derived retrovirus vectors to target cells.

Non-infectious, envelope protein-free, retrovirus-like particles (VLP) derived from either Moloney murine leukemia virus (MLV) or human HIV are able to bind efficiently to, but not infect, target cells. Upon subsequent addition to the bound particles of the G protein of vesicular stomatitis virus (VSV-G), an efficient surrogate retrovirus envelope protein, the VLP are efficiently taken up by the cells to produce infection. Cell attachment of the VLP is efficiently inhibited by soluble heparin and dextran sulfate and less efficiently abrogated by several other glycosaminoglycans (GAGs) including chondroitin sulfate A and chondroitin sulfate B (dermatan sulfate), as determined by deconvolution microscopic immunodetection of the viral gag protein and by quantitative binding studies of metabolically labeled (35)S-VLP. Enzymatic digestion of heparan sulfate (HS) from the cell surface with heparinase I also reduces VLP binding. Furthermore, VLP adsorption onto several CHO cell lines variably deficient in cell surface GAG is significantly but incompletely abrogated. De-sulfated heparins are less efficient than native heparin in inhibiting the Polybrene-mediated binding of VLP, whereas growth of human cells in the presence of sodium chlorate leads to significant reduction of Polybrene-mediated VLP binding. In addition, specific inhibition of VLP binding and infectivity of mature infectious VSV-G-pseudotyped virus is observed in the presence of heparin and HS under Polybrene-free conditions. We conclude from these studies that the presence of Polybrene, the degree of sulfation of cell surface GAG, and possibly the presence of charged cell surface macromolecules create an electrostatic environment that promotes optimum binding of VLP to cells. Additionally, our results demonstrate that, in the absence of Polybrene, initial attachments of non-infectious, envelope protein-free VLP and probably mature infectious virus particles are mediated by interactions of the virus particles with cell surface heparan sulfate, and possibly with other GAG molecules.