The human adenovirus serotype 5 (Ad5) is used widely for applications in human gene therapy. Cellular attachment of Ad5 is mediated by binding of the carboxyl-terminal knob of its fiber coat protein to the Coxsackie adenovirus receptor (CAR) protein. However, Ad5 binding to CAR hampers the development of adenovirus vectors capable of specifically targeting (diseased) tissues or organs. Through sequence analysis and mutagenesis, a conserved receptor-binding region was identified on the side of three divergent CAR-binding knobs. The feasibility of simultaneous CAR ablation and redirection of an adenovirus to a new receptor is demonstrated.

The development of tissue-selective virus-based vectors requires a better understanding of the role of receptors in gene transfer in vivo, both to rid the vectors of their native tropism and to introduce new specificity. CAR and ␣v integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. We have constructed a set of four vectors, which individually retain the wild-type cell interactions, lack CAR binding, lack ␣v integrin binding, or lack both CAR and ␣v integrin binding. These vectors have been used to examine the roles of CAR and ␣v integrin in determining the tropism of Ad vectors in a mouse model following intrajugular or intramuscular injection. CAR was found to play a significant role in liver transduction. The absence of CAR binding alone, however, had little effect on the low level of expression from Ad in other tissues. Binding of ␣v integrins appeared to have more influence than did binding of CAR in promoting the expression in these tissues and was also found to be important in liver transduction by Ad vectors. An effect of the penton base modification was a reduction in the number of vector genomes that could be detected in several tissues. In the liver, where CAR binding is important, combining defects in CAR and ␣v integrin binding was essential to effectively reduce the high level of expression from Ad vectors. While there may be differences in Ad vector tropism among species, our results indicate that both CAR and ␣v integrins can impact vector distribution in vivo. Disruption of both CAR and ␣v integrin interactions may be critical for effectively reducing native tropism and enhancing the efficacy of specific targeting ligands in redirecting Ad vectors to target tissues.

CD20 is an antigen expressed on normal and malignant human B cells that is thought to function as a receptor during B cell activation. Here we report the isolation of a CD20‐specific cDNA clone from a lambda gt11 library using a polyclonal antiserum raised against purified CD20 antigen. Additional cDNA clones were then isolated from a lambda gt10 library. Alignment of the sequences of overlapping lambda clones reveal a single consensus sequence except for a divergence that preceded the first methionine within the open reading frame. Normal B cells and B cell lines contain a prominent 2.6 kb mRNA and a lower level of a 3.3 kb mRNA. An oligonucleotide derived from one of the divergent sequences hybridized to the 3.3 kb mRNA only, indicating that the two mRNA species are derived from an alternative splicing mechanism. The predicted amino acid sequence of CD20 reveals three major hydrophobic regions of approximately 53, 25 and 20 amino acids. CD20 lacks an NH2‐terminal signal peptide and contains a highly charged COOH‐terminal domain. Although CD20 is immunoprecipitated as a doublet of 33 and 35 kd proteins from B cells, in vitro translation of CD20 cDNA produced a single 33 kd protein that was specifically immunoprecipitated with monoclonal CD20 antibodies. CD20 was strongly phosphorylated on resting B cells after CDw40 stimulation, suggesting that CD20 may be functionally regulated by a protein kinase(s).

The structure of the Rous sarcoma virus envelope glycoprotein complex was studied by sedimentation gradient centrifugation analyses of detergent-solubilized wildtype and mutant envelope (env) gene products. These studies show that the envelope glycoprotein forms an oligomer during biosynthesis, which is most likely a trimer, and that this is the form of the complex found in virions. Our results are consistent with oligomer formation and transport out of the endoplasmic reticulum being closely linked. From analyses of mutant envelope proteins we conclude that the extracellular domain of the glycoprotein is sufficient for oligomer formation but that the transmembrane domain is required to stabilize this complex. Additional experiments suggest that interactions between external domains of the membrane-spanning, gp37 polypeptides are those most important for the formation of trimers. The significance of these observations to retroviral replication and implications for antiviral drug development are discussed.

Chikungunya virus, a mosquito-borne alphavirus, recently caused the largest epidemic ever seen for this virus. Chikungunya disease primarily manifests as a painful and debilitating arthralgia/ arthritis, and no effective drug or vaccine is currently available. Here we describe a recombinant chikungunya virus vaccine comprising a non-replicating complex adenovirus vector encoding the structural polyprotein cassette of chikungunya virus. A single immunisation with this vaccine consistently induced high titres of anti-chikungunya virus antibodies that neutralised both an old Asian isolate and a Réunion Island isolate from the recent epidemic. The vaccine also completely protected mice against viraemia and arthritic disease caused by both virus isolates.

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To create tumor-targeted Ad vectors, ablation of native CAR and integrin receptor binding is crucial to enhance the specificity of tumor transduction. Toward this aim, we have previously created base vectors in which binding to CAR (single-ablated) or to both CAR and integrins (double-ablated) has been ablated. In this study, the biodistribution of the conventional (CAR and integrin binding intact), single-ablated, and double-ablated vectors was evaluated following intraperitoneal administration. The mesothelial lining of the peritoneal organs was the principle site of CAR-dependent gene transfer by the conventional vector. Surprisingly, the single-ablated vector strongly transduced the liver parenchyma rather than the mesothelium, while the double-ablated vector did not significantly transduce the parenchyma or mesothelium. The high level of parenchymal transduction by the single-ablated vector suggested that it efficiently entered the bloodstream from the peritoneal cavity. Consistent with this hypothesis, a large proportion of active particles distributed and persisted in the bloodstream following intraperitoneal administration of either the single- or the double-ablated vector. The above results suggest that the double-ablated vector backbone may not only significantly improve targeting to cancers located in the peritoneal cavity, but may also significantly improve targeting to metastatic tumors located throughout the body by virtue of its enhanced bloodstream persistence.

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by the rapid onset of intestinal T-cell depletion that initiates the progression to AIDS. The induction of protective immunity in the intestinal mucosa therefore represents a potentially desirable feature of a preventive AIDS vaccine. In this study, we have evaluated the ability of an enteric adenovirus, recombinant adenovirus 41 (rAd41), to elicit intestinal and systemic immune responses by different immunization routes, alone or in combination with rAd5. rAd41 expressing HIV envelope (Env) protein induced cellular immune responses comparable to those of rAd5-based vectors after either a single intramuscular injection or a DNA prime/rAd boost. Oral priming with rAd41-Env followed by intramuscular boosting with rAd5-Env stimulated a more potent CD8؉ T-cell response in the small intestine than the other immunization regimens. Furthermore, the direct injection of rAd41-Env into ileum together with intramuscular rAd5-Env boosting increased Env-specific cellular immunity markedly in mucosal as well as systemic compartments. These data demonstrate that heterologous rAd41 oral or ileal priming with rAd5 intramuscular boosting elicits enhanced intestinal mucosal cellular immunity and that oral or ileal vector delivery for primary immunization facilitates the generation of mucosal immunity.