Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea, and recent outbreaks of strains with increased virulence underscore the importance of identifying novel approaches to treat and prevent relapse of Clostridium difficile-associated diarrhea (CDAD). CDAD pathology is induced by two exotoxins, toxin A and toxin B, which have been shown to be cytotoxic and, in the case of toxin A, enterotoxic. In this report we describe fully human monoclonal antibodies (HuMAbs) that neutralize these toxins and prevent disease in hamsters. Transgenic mice carrying human immunoglobulin genes were used to isolate HuMAbs that neutralize the cytotoxic effects of either toxin A or toxin B in cell-based in vitro neutralization assays. Three anti-toxin A HuMAbs (3H2, CDA1, and 1B11) could all inhibit the enterotoxicity of toxin A in mouse intestinal loops and the in vivo toxicity in a systemic mouse model. Four anti-toxin B HuMAbs (MDX-1388, 103-174, 1G10, and 2A11) could neutralize cytotoxicity in vitro, although systemic toxicity in the mouse could not be neutralized. Anti-toxin A HuMAb CDA1 and anti-toxin B HuMAb MDX-1388 were tested in the well-established hamster model of C. difficile disease. CDA1 alone resulted in a statistically significant reduction of mortality in hamsters; however, the combination treatment offered enhanced protection. Compared to controls, combination therapy reduced mortality from 100% to 45% (P < 0.0001) in the primary disease hamster model and from 78% to 32% (P < 0.0001) in the less stringent relapse model.
Virus-like particles (VLPs) present viral antigens in a native conformation and are effectively recognized by the immune system and therefore are considered as suitable and safe vaccine candidates against many viral diseases.
Here we demonstrate that chimeric VLPs containing Rift Valley fever virus (RVFV) glycoproteins GN and GC, nucleoprotein N and the gag protein of Moloney murine leukemia virus represent an effective vaccine candidate against Rift Valley fever, a deadly disease in humans and livestock. Long-lasting humoral and cellular immune responses are demonstrated in a mouse model by the analysis of neutralizing antibody titers and cytokine secretion profiles. Vaccine efficacy studies were performed in mouse and rat lethal challenge models resulting in high protection rates.
Taken together, these results demonstrate that replication-incompetent chimeric RVF VLPs are an efficient RVFV vaccine candidate.
To explore mechanisms of entry for Ebola virus (EBOV) glycoprotein (GP) pseudotyped virions, we used comparative gene analysis to identify genes whose expression correlated with viral transduction. Candidate genes were identified by using EBOV GP pseudotyped virions to transduce human tumor cell lines that had previously been characterized by cDNA microarray. Transduction profiles for each of these cell lines were generated, and a significant positive correlation was observed between RhoC expression and permissivity for EBOV vector transduction. This correlation was not specific for EBOV vector alone as RhoC also correlated highly with transduction of vesicular stomatitis virus GP (VSVG) pseudotyped vector. Levels of RhoC protein in EBOV and VSV permissive and nonpermissive cells were consistent with the cDNA gene array findings. Additionally, vector transduction was elevated in cells that expressed high levels of endogenous RhoC but not RhoA. RhoB and RhoC overexpression significantly increased EBOV GP and VSVG pseudotyped vector transduction but had minimal effect on human immunodeficiency virus (HIV) GP pseudotyped HIV or adeno-associated virus 2 vector entry, indicating that not all virus uptake was enhanced by expression of these molecules. RhoB and RhoC overexpression also significantly enhanced VSV infection. Similarly, overexpression of RhoC led to a significant increase in fusion of EBOV virus-like particles. Finally, ectopic expression of RhoC resulted in increased nonspecific endocytosis of fluorescent dextran and in formation of increased actin stress fibers compared to RhoA-transfected cells, suggesting that RhoC is enhancing macropinocytosis. In total, our studies implicate RhoB and RhoC in enhanced productive entry of some pseudovirions and suggest the involvement of actin-mediated macropinocytosis as a mechanism of uptake of EBOV GP and VSVG pseudotyped viral particles.Enveloped viruses enter cells by a variety of different pathways. Productive internalization of enveloped viruses with targeted cells is mediated through interactions of the viral glycoprotein(s) (GPs) with moieties on the surface of the cell. In general, enveloped viral entry occurs through viral adherence to the cell surface, interaction with a specific plasma membrane-associated receptor that results in a series of GP conformational changes leading to fusion of viral and cellular membranes, and delivery of the viral core particle into the cytoplasm. Fusion of the two membranes can occur at the plasma membrane or by uptake of the intact virions into endosomes with subsequent membrane fusion between the viral membrane and the lipid bilayer of the endocytic vesicle. Human immunodeficiency virus (HIV) is an example of a virus that fuses directly to the plasma membrane (5), whereas influenza virus must be internalized into acidified vesicles where the appropriate GP conformational changes can occur, mediating membrane fusion (21). Most enveloped viruses that enter through vesicles utilize a low-pH environment to mediate the necessary...
Abstract. Two 70-kD polypeptides, B3 and B4, are present in equivalent concentrations in the nucleus and cytoplasm of Xenopus oocytes. The objectives of this study were to determine if they (a) are members of the 70-kD family of heat shock proteins, and (b) recycle between the nuclear and cytoplasmic compartments. Evidence based on high-affinity binding to ATP, cross-reactivity of B3/B4-specific antibodies with rat hsc70, and a comparison of cyanogen bromide cleavage peptide maps with hsc70, verified that B3 and B4 are members of the 70-kD family of heat-shock proteins. Nuclear uptake studies were performed by microinjecting ~5I-labeled B3/B4, rat hsc70, and BSA into the cytoplasm of oocytes, and examining their subsequent intracellular distributions. By 6 h postinjection, the nuclear concentration of B3/B4 and hsc70 were ~24-fold greater than BSA controls. It was also found that B3/B4-coated gold particles as large as 120/~ in diameter were able to enter the nucleus by passing through the pores. Nuclear efflux was analyzed by microinjecting the iodinated proteins directly into the oocyte nuclei. 2 h after nuclear injection, at least 46% of the B3/B4 and 60% of the hsc70 were found in the cytoplasmic fractions, compared with <10% for the BSA controls. Cell fusion experiments, in which labeled, anucleate oocyte vegetal hemispheres were fused, under oil, with nucleate unlabeled animal hemispheres, demonstrated that cytoplasmic B3 and B4 could enter the nucleus after equilibration was reached, arguing against the existence of separate nuclear and cytoplasmic populations. Collectively, these results show that B3, B4, and rat hsc70 are transported across the nuclear envelope and recycle between the nucleus and cytoplasm.
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