Biomedical subjects

R Mark Buller

Publications and source records attributed to R Mark Buller.

7 recordsLinked to original sources

PubMed2007

Poxvirus-encoded gamma interferon binding protein dampens the host immune response to infection.

Ectromelia virus (ECTV), a natural mouse pathogen and the causative agent of mousepox, is closely related to variola virus (VARV), which causes smallpox in humans. Mousepox is an excellent surrogate small-animal model for smallpox. Both ECTV and VARV encode a multitude of host response modifiers that target components of the immune system and that are thought to contribute to the high mortality rates associated with infection. Like VARV, ECTV encodes a protein homologous to the ectodomain of the host gamma interferon (IFN-gamma) receptor 1. We generated an IFN-gamma binding protein (IFN-gammabp) deletion mutant of ECTV to study the role of viral IFN-gammabp (vIFN-gammabp) in host-virus interaction and also to elucidate the contribution of this molecule to the outcome of infection. Our data show that the absence of vIFN-gammabp does not affect virus replication per se but does have a profound effect on virus replication and pathogenesis in mice. BALB/c mice, which are normally susceptible to infection with ECTV, were able to control replication of the mutant virus and survive infection. Absence of vIFN-gammabp from ECTV allowed the generation of an effective host immune response that was otherwise diminished by this viral protein. Mice infected with a vIFN-gammabp deletion mutant virus, designated ECTV-IFN-gammabp(Delta), produced increased levels of IFN-gamma and generated robust cell-mediated and antibody responses. Using several strains of mice that exhibit differential degrees of resistance to mousepox, we show that recovery or death from ECTV infection is determined by a balance between the host's ability to produce IFN-gamma and the virus' ability to dampen its effects.

Animals↗

PubMed2006

Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase.

Vaccinia virus DNA polymerase catalyzes duplex-by-duplex DNA joining reactions in vitro and many features of these recombination reactions are reprised in vivo. This can explain the intimate linkage between virus replication and genetic recombination. However, it is unclear why these apparently ordinary polymerases exhibit this unusual catalytic capacity. In this study, we have used different substrates to perform a detailed investigation of the mechanism of duplex-by-duplex recombination catalyzed by vaccinia DNA polymerase. When homologous, blunt-ended linear duplex substrates are incubated with vaccinia polymerase, in the presence of Mg2+ and dNTPs, the appearance of joint molecules is preceded by the exposure of complementary single-stranded sequences by the proofreading exonuclease. These intermediates anneal to form a population of joint molecules containing hybrid regions flanked by nicks, 1-5 nt gaps, and/or short overhangs. The products are relatively resistant to exonuclease (and polymerase) activity and thus accumulate in joining reactions. Surface plasmon resonance (SPR) measurements showed the enzyme has a relative binding affinity favoring blunt-ended duplexes over molecules bearing 3'-recessed gaps. Recombinant duplexes are the least favored ligands. These data suggest that a particular combination of otherwise ordinary enzymatic and DNA-binding properties, enable poxvirus DNA polymerases to promote duplex joining reactions.

Catalysis↗

PubMed2006

Oral 1-O-octadecyl-2-O-benzyl-sn-glycero-3-cidofovir targets the lung and is effective against a lethal respiratory challenge with ectromelia virus in mice.

Hexadecyloxypropyl-cidofovir (HDP-CDV) has been shown to be orally active against lethal infection with orthopoxviruses including, mousepox, cowpox, vaccinia and rabbitpox. The alkoxyalkyl group provides oral absorption and reduces greatly the amount of drug reaching the kidney, the site of CDV's dose limiting toxicity. However, the amount of HDP-CDV detected in lung, an important site of early poxvirus replication, is low and the reduction of viral titers in surviving animals is reduced moderately compared with the liver where poxvirus titers are virtually undetectable. We synthesized a novel glycerol ester of CDV, 1-O-octadecyl-2-O-benzyl-sn-glycero-3-CDV (ODBG-CDV), and compared its oral pharmacokinetics with that of HDP-CDV. Surprisingly, ODBG-CDV levels in lung are much higher and liver levels are reduced, suggesting that the compound is transported in small intestinal lymph instead the portal vein. ODBG-CDV has excellent in vitro activity in cells infected with ectromelia virus (ECTV). In mice infected with a lethal aerosol or intranasal challenge of ECTV, HDP-CDV and ODBG-CDV are equally effective in preventing death from disease. Other drugs esterified to 1-O-octadecyl-2-O-benzyl-sn-glycerol or 1-O-octadecyl-2-O-benzyl-sn-glycerol-3-phosphate may provide lung targeting for treatment of microbial or neoplastic diseases while reducing first pass removal by the liver during oral absorption.

Administration, Oral↗

PubMed2006

Highly attenuated smallpox vaccine protects rabbits and mice against pathogenic orthopoxvirus challenge.

The possible reemergence of smallpox through bioterrorism requires the preparation of adequate stockpiles of vaccine. Dryvax, the only US-licensed vaccinia virus smallpox vaccine, has an unacceptable safety profile in the pre-event setting. LC16m8 is a Japanese-licensed attenuated vaccinia virus strain that has been safely used in over 50,000 persons. Until now, efficacy of this vaccine was unproven. Using two animal models, we show that LC16m8 and Dryvax elicit comparable humoral immune responses after a single vaccination and equivalently protect against lethal poxvirus disease. Thus, LC16m8 shows promise as a safe and effective smallpox vaccine with the potential for replacing Dryvax.

Animals↗

PubMed2006

Application of immunoproteomics to rapid cytokine detection.

Cytokines are pivotal to a balanced innate or cell-mediated immune response, and can be indicative of disease progression and/or resolution. Methods to measure key cytokines rapidly with accuracy, precision, and sensitivity are consequently important. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because of the long time (1-3 h) required to carry out the assays. An alternative, semi-quantitative approach described here, uses an immunological capture step and a mass spectral readout. The goal of the assay is speed rather than sensitivity or precision.

Animals↗

PubMed2005

Immunoproteomics.

A novel immunoproteomic assay, combining specificity of antibody with precision of mass spectral analysis is described, and a number of practical applications are presented. The assay is carried out in three steps. The first step of the assay involves antibody immobilization, using a bacterial Fc binding support. The second step is antigen capture and washing to remove non-specific binding. The third step involves analysis of the captured antigens by SELDI-TOF. The assay has many advantages in sensitivity, speed, and economy of reagents in detection of specific antigens or antibodies. In addition, under appropriate experimental conditions, semi-quantitative data may be obtained. By combining the increasing range of selective specific antibody reagents available, in part due to advances in antibody engineering technology, and the resolving power available, using mass spectrometry, immunoproteomics is a valuable technique in proteomic analysis. A number of examples of the application of this technique to analysis of biological systems are presented.

Animals↗

PubMed2004

Efficacy of oral active ether lipid analogs of cidofovir in a lethal mousepox model.

Cidofovir (CDV) is a highly effective inhibitor of orthopoxvirus replication and may be used intravenously to treat smallpox or complications arising from the smallpox vaccine under an investigational new drug application (IND). However, CDV is absorbed poorly following oral administration and is inactive orally. To improve the bioavailability of CDV, others synthesized alkoxyalkanol esters of CDV and observed >100-fold more activity than unmodified CDV against cowpox, vaccinia, and variola virus (VARV) replication. These ether lipid analogs of CDV have high oral bioavailability in mice. In this study, we compared the oral activity of CDV with the hexadecyloxypropyl (HDP)-, octadecyloxyethyl-, oleyloxypropyl-, and oleyloxyethyl-esters of CDV in a lethal, aerosol ectromelia virus (ECTV) challenge model in A/NCR mice. Octadecyloxyethyl-CDV appeared to be the most potent CDV analog as a dose regimen of 5 mg/kg started 4 h following challenge completely blocked virus replication in spleen and liver, and protected 100% of A/NCR mice, although oral, unmodified CDV was inactive. These results suggest that this family of compounds deserves further evaluation as poxvirus antiviral.

Administration, Oral↗