D. Grosenbach - Academia.edu (original) (raw)

Papers by D. Grosenbach

Cancer research, 2001

Several different vaccine strategies have been evaluated and combined in an attempt to amplify T-... more Several different vaccine strategies have been evaluated and combined in an attempt to amplify T-cell responses toward induction of antitumor immunity. The model tumor antigen used was carcinoembryonic antigen (CEA). While initial T-cell activation studies were conducted in conventional mice, combined vaccine strategy studies and antitumor studies were conducted in transgenic mice in which CEA is expressed in normal gastrointestinal tissue and CEA protein is found in sera. The studies reported here demonstrate: (a) A recombinant avipox (fowlpox, rF) vector expressing the signal 1 (CEA) and the B7-1 costimulatory molecule transgenes (designated rF-CEA/B7-1) is more potent in inducing CEA-specific T-cell responses than rF-CEA; one administration of recombinant fowlpox vector expressing CEA and three different costimulatory molecule transgenes (B7-1, ICAM-1, LFA-3, designated rF-CEA/TRICOM) was more potent in inducing CEA-specific T-cell responses than four vaccinations with rF-CEA or ...

The Journal of Immunology, 2005

The avidity of Ag-specific CTL is a critical determinant for clearing viral infection and elimina... more The avidity of Ag-specific CTL is a critical determinant for clearing viral infection and eliminating tumor. Although previous studies have demonstrated that vaccines using enhanced costimulation will enhance the level and avidity of Ag-specific T cells from naive mice, there are conflicting data about the effects of vaccines using enhanced costimulation (vector or dendritic cell based) on the survival of memory T cells. In this study we have first extended previous observations that primary vaccination with a recombinant vaccinia virus (rV-) expressing a model Ag (LacZ) and a triad of T cell costimulatory molecules (B7-1, ICAM-1, and LFA-3 (designated TRICOM)) enhances the level and avidity of T cells from naive vaccinated C57BL/6 (Thy1.2) mice. Adoptive transfer of Thy1.1 memory CD8 ؉ T cells into naive Thy1.2 C57BL/6 mice was followed by booster vaccinations with a recombinant fowlpox (rF-)-expressing LacZ rF-LacZ) or booster vaccinations with rF-LacZ/TRICOM. Analysis of levels of ␤-galactosidase tetramer-positive T cells and functional assays (IFN-␥ expression and lytic activity) determined that booster vaccinations with rF-LacZ/TRICOM were superior to booster vaccinations with rF-LacZ in terms of both maintenance and enhanced avidity of memory CD8 ؉ T cells. Antitumor experiments using a self-Ag (carcinoembryonic Ag (CEA) vaccines in CEA transgenic mice bearing CEA-expressing tumors) also demonstrated that the use of booster vaccinations with vaccines bearing enhanced costimulatory capacity had superior antitumor effects. These studies thus have implications in the design of more effective vaccine strategies. 3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; ␤-gal, ␤-galactosidase; PCC, pigeon cytochrome c; rV-, recombinant vaccinia virus; rF-, recombinant fowlpox virus; CEA, carcinoembryonic Ag; Tg, transgenic; TRI-COM, triad of costimulatory molecules (B7-1, ICAM-1, and LFA-3).

Developments in biologicals, 2004

Journal of virology, 1998

Vaccinia virus encodes a 37-kDa palmitylated protein (p37) that is required for envelopment, tran... more Vaccinia virus encodes a 37-kDa palmitylated protein (p37) that is required for envelopment, translocation, and cell-to-cell spread of virions. We have analyzed the biological significance of the palmitate modification by constructing a recombinant vaccinia virus that expresses a nonpalmitylated p37 and comparing its biological activity to that of the wild-type virus. The mutant virus is inefficient at cell-to-cell spread and does not produce or release enveloped virions, although it produces normal amounts of nonenveloped virions. Furthermore, the mutant virus is not able to nucleate actin to propel itself through and out of the cell, a function requiring the indirect participation of p37. The deficiency in protein function appears to result from a lack of appropriate targeting to the membranes of the trans-Golgi network (TGN) which leaves p37 soluble in the cytoplasm. We conclude that the palmitate moiety is necessary for targeting or anchoring p37 to the TGN membrane, where, alon...

Frontiers in bioscience : a journal and virtual library, Jan 22, 1998

Posttranslational processing of vaccinia virus proteins has proven to be a common mechanism for e... more Posttranslational processing of vaccinia virus proteins has proven to be a common mechanism for exerting regulatory control of function or targeting to subcellular and/or subviral structures. Fatty acylation, most commonly observed as the addition of myristate or palmitate, occurs on numerous vaccinia proteins and affects each in a distinct manner. Labeling of vaccinia-infected cells with tritiated myristic or palmitic acids demonstrates that vaccinia encodes at least six myristylproteins and six palmitylproteins. Where investigated, each of these proteins have been demonstrated to play important roles in the virus life cycle. Likewise, in each case studied, the fatty acyl modification greatly influences the function and/or biological activity of the protein.

Journal of virology, 1997

Previous studies have shown that at least three vaccinia virus (VV) late proteins (with apparent ... more Previous studies have shown that at least three vaccinia virus (VV) late proteins (with apparent molecular asses of 37, 35, and 25 kDa) label with myristic acid. Time course labeling of VV-infected cells with [3H]myristic acid reveals at least three additional putative myristylproteins, with apparent molecular masses of 92, 17, and 14 kDa. The 25-kDa protein has previously been identified as that encoded by the L1R open reading frame, leaving the identities of the remaining proteins to be determined. Sequence analysis led to the preliminary identification of the 37-, 35-, and 17-kDa proteins as G9R, A16L, and E7R, respectively. Using synthetic oligonucleotides and PCR techniques, each of these open reading frames was amplified by using VV DNA as a template and then cloned individually into expression vectors behind T7 promoters. These plasmid constructs were then transcribed in vitro, and the resulting mRNAs were translated in wheat germ extracts and radiolabeled with either [35S]me...

Journal of virology, 1996

Gel analysis of vaccinia virus particles purified by buoyant [correction of bouyant] density demo... more Gel analysis of vaccinia virus particles purified by buoyant [correction of bouyant] density demonstrates a protein with an estimated molecular mass of 59 kDa, which is apparently restricted to the intracellular mature virion (IMV) form. Western blotting (immunoblotting) and immunoprecipitation procedures identify the protein as the vaccinia virus 4c protein, which facilitates occlusion of poxvirus particles within cowpox cytoplasmic inclusions. Western blotting procedures also identify the truncated A-type inclusion protein of vaccinia virus as a specific marker for IMV particles. Kinetic analyses of virion maturation and 4c production suggest that peak enveloped virion production occurs before peak IMV production in the virus replication cycle and that 4c production is concomitant with maturation of IMV. The implications for a distinct and evolutionarily conserved function of IMV in viral pathogenesis are discussed.

Virology, 2000

Vaccinia virus encodes at least eight proteins that incorporate label from tritiated palmitic aci... more Vaccinia virus encodes at least eight proteins that incorporate label from tritiated palmitic acid when it is added to infected cell cultures. Three of these palmitylproteins are encoded by the A33R, B5R, and F13L open reading frames and migrate by gel electrophoresis with relative molecular masses of 23-28, 42, and 37 kDa, respectively. In this report we provide evidence that the A22R and A36R open reading frames also encode palmitylproteins with apparent molecular masses of 22 and 50-55 kDa, respectively. Furthermore, the hemagglutinin protein (A56R) from the Copenhagen strain is shown to be palmitylated while the hemagglutinin protein from the WR and IHD-J strains is not. A 94-kDa VV palmitylprotein appears to be a multimeric complex composed of the B5R protein and possibly others. All vaccinia-encoded palmitylproteins are present in the membranous fraction of cells and are specific for the trans-Golgi network membrane-enveloped forms of the virus, suggesting that these proteins play a role in the envelopment and egress of virions or the infectivity of released virus.

Virology, 1999

Vaccinia virus (V V) encodes a 37-kDa envelope protein (p37) that is palmitylated on cysteine res... more Vaccinia virus (V V) encodes a 37-kDa envelope protein (p37) that is palmitylated on cysteine residues 185 and 186 of the 372-amino acid protein. We have previously reported on a loosely conserved consensus motif. Further analysis has identified a conserved consensus sequence, Hydro*AAC(C)A (Hydro* represents a hydrophobic portion of a protein determined by any one of the following: a hydrophobic sequence, a transmembrane domain 1-12 amino acids away from the modification site, or the prior addition of a hydrophobic molecule; C, palmitate acceptor cysteines; A, aliphatic residue) that is responsible for directing palmitylation of certain classes of palmitylproteins. We have analyzed the amino acid site occupancy upstream and downstream of the palmitate acceptor residues in p37 by site-directed mutagenesis and transient expression of mutated proteins in V V-infected cells. The two aliphatic alanines naturally found at positions 183 and 184 of the wild-type p37 allow for efficient palmitylation. In contrast, the replacement of leucine at position 187 with glycine increases palmitylation efficiency. The 10 amino acids immediately upstream of the palmitate acceptor site are absolutely necessary while the downstream 10 amino acids are dispensable. These results together with previous data suggests that the Hydro*AAC(C)A motif is required for efficient palmitylation of p37.

Proceedings of the National Academy of Sciences, 2010

The threat of smallpox as a bioweapon and the emerging threat of human monkeypox, among other pox... more The threat of smallpox as a bioweapon and the emerging threat of human monkeypox, among other poxviral diseases, highlight the need for effective poxvirus countermeasures. ST-246, which targets the F13L protein in vaccinia virus and its homologs in other orthopoxvirus species, provides full protection from lethal poxviral disease in numerous animal models and seems to be safe in humans. All previous evaluations of ST-246 efficacy have been in immunocompetent animals. However, the risk of severe poxviral disease is greater in immunodeficient hosts. Here we report on the efficacy of ST-246 in preventing or treating lethal poxviral disease in immunodeficient mice. After lethal challenge with the Western Reserve strain of vaccinia, Nude, SCID, and J H knockout mice additionally depleted of CD4 + and CD8 + T cells were not fully protected by ST-246, although survival was significantly extended. However, CD4 + T cell deficient, CD8 + T cell deficient, J H knockout, and J H knockout mice also deficient for CD4 + or CD8 + T cells survived lethal challenge when treated with ST-246 starting on the day of challenge. Delaying treatment until 72 h after infection reduced ST-246 efficacy in some models but provided full protection from lethal challenge in most. These findings suggest that ST-246 may be effective in controlling smallpox or other pathogenic orthopoxviruses in some immunodeficient human populations for whom the vaccine is contraindicated. antiviral | immunodeficiency | ST-246 | smallpox | orthopoxvirus

Molecular Microbiology, 1997

Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole call... more Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole called an inclusion. Chlamydia psittaci (strain GPIC) produces a 39 kDa protein (IncA) that is localized to the inclusion membrane. While IncA is present as a single 39 kDa species in purified reticulate bodies, two additional higher M r forms are found in C. psittaci -infected cells. This finding suggested that IncA may be post-translationally modified in the host cell. Here we present evidence that IncA is a serine/threonine phosphoprotein that is phosphorylated by host cell enzymes. This conclusion is supported by the following experimental findings: (i) treatment of infected cells with inhibitors of host cell phosphatases or kinases altered the electrophoretic migration pattern of IncA; (ii) treatment with calf intestinal alkaline phosphatase eliminated the multiple-banding pattern of IncA, leaving only the protein band with the lowest relative molecular weight;

Journal of Virology, 2008

The potential threat of smallpox use in a bioterrorist attack has heightened the need to develop ... more The potential threat of smallpox use in a bioterrorist attack has heightened the need to develop an effective smallpox vaccine for immunization of the general public. Vaccination with the current smallpox vaccine, Dryvax, produces protective immunity but may result in adverse reactions for some vaccinees. A subunit vaccine composed of protective vaccinia virus proteins should avoid the complications arising from live-virus vaccination and thus provide a safer alternative smallpox vaccine. In this study, we assessed the protective efficacy and immunogenicity of a multisubunit vaccine composed of the A27L and D8L proteins from the intracellular mature virus (IMV) form and the B5R protein from the extracellular enveloped virus (EEV) form of vaccinia virus. BALB/c mice were immunized with Escherichia coli-produced A27L, D8L, and B5R proteins in an adjuvant consisting of monophosphoryl lipid A and trehalose dicorynomycolate or in TiterMax Gold adjuvant. Following immunization, mice were either sacrificed for analysis of immune responses or lethally challenged by intranasal inoculation with vaccinia virus strain Western Reserve. We observed that three immunizations either with A27L, D8L, and B5R or with the A27L and B5R proteins alone induced potent neutralizing antibody responses and provided complete protection against lethal vaccinia virus challenge.

JNCI Journal of the National Cancer Institute, 2000

Background: Activation and proliferation of T cells are essential for a successful cellular immun... more Background: Activation and proliferation of T cells are essential for a successful cellular immune response to an antigen. Antigen-presenting cells (APCs) activate T cells through a two-signal mechanism. The first signal is antigen specific and causes T cells to enter the cell cycle. The second signal involves a costimulatory molecule that interacts with a ligand on the T-cell surface and leads to T-cell cytokine production and their proliferation. Dendritic cells express several costimulatory molecules and are believed to be the most potent APCs.

Journal of General Virology, 1995

The fungal metabolite Brefeldin A was found to inhibit the production of the infectious enveloped... more The fungal metabolite Brefeldin A was found to inhibit the production of the infectious enveloped form of vaccinia virus, although production of the infectious intracellular form was not affected. Electron microscopic analysis and caesium chloride density centrifugation of progeny virions indicates that the drug block is not due to retention and accumulation of enveloped virions within the cell. Biochemical analysis of the candidate envelopment receptor for vaccinia virus, viral protein P37, shows that the drug has no discernible effect on palmitylation of this protein and does not prevent or alter its association with intracellular membranes. This suggests that P37 may not in fact be the receptor on intracellular membranes for vaccinia virus envelopment, and leaves open the question of what function this molecule performs in the envelopment process.

Journal of Biological Chemistry, 1997

Computer-assisted alignment of known palmitylproteins was used to identify a potential peptide mo... more Computer-assisted alignment of known palmitylproteins was used to identify a potential peptide motif, TMDX1-12AAC(C)A (TMD, transmembrane domain; X, any amino acid; C, cysteine acceptor residues; A, aliphatic residue) responsible for directing internal palmitylation of the vaccinia virus 37-kDa major envelope antigen, p37. Site-directed mutagenesis was used to confirm this motif as the site of modification and to produce a nonpalmitylated version of the p37 protein. Comparative phenotypic analysis of the wild-type and mutant p37 alleles confirmed that the p37 protein is involved in viral envelopment and egress, and suggested that attachment of the palmitate moiety was essential for correct intracellular targeting and protein function.

Artificial Cells, Blood Substitutes and Biotechnology, 2003

The activation of a T cell has been shown to require two signals via molecules present on profess... more The activation of a T cell has been shown to require two signals via molecules present on professional antigen presenting cells: signal 1, via a peptide/MHC complex, and signal 2, via a costimulatory molecule. Here, the role of three costimulatory molecules in the activation of T cells was examined. Poxvirus (vaccinia and avipox) vectors were employed because of their ability to efficiently express multiple genes. Murine cells provided with signal 1 and infected with either recombinant vaccinia or avipox vectors containing a TRIad of COstimulatory Molecules (B7-1/ICAM-1/LFA-3, designated TRICOM) induced the activation of T cells to a far greater extent than cells infected with vectors expressing any one or two costimulatory molecules. Despite this T-cell "hyperstimulation" using TRICOM vectors, no evidence of apoptosis above that seen using the B7-1 vector was observed. Results employing the TRICOM vectors were most dramatic under conditions of either low levels of first signal or low stimulator cell to T-cell ratios. Experiments employing a four-gene construct also showed that TRICOM recombinants could enhance antigen-specific T-cell responses in vivo. These studies thus demonstrate the ability of vectors to introduce three costimulatory molecules into cells, thereby activating both CD4+ and CD8+ T-cell populations to levels greater than those achieved with the use of only one or two costimulatory molecules. This new threshold of T-cell activation has broad implications in vaccine design and development. Dendritic cells infected with TRICOM vectors were found to greatly enhance naïve T-cell activation, and peptide-specific T-cell stimulation. In vivo, peptide-pulsed DCs infected with TRICOM vectors induced cytotoxic T lymphocyte activity markedly and significantly greater than peptide-pulsed DCs.

Antimicrobial Agents and Chemotherapy, 2013

Naturally occurring smallpox has been eradicated but remains a considerable threat as a biowarfar... more Naturally occurring smallpox has been eradicated but remains a considerable threat as a biowarfare/bioterrorist weapon (F. Fleck, Bull. World Health Organ. 81:917-918, 2003). While effective, the smallpox vaccine is currently not recommended for routine use in the general public due to safety concerns (http://www.bt.cdc.gov/agent/smallpox/vaccination). Safe and effective countermeasures, particularly those effective after exposure to smallpox, are needed. Currently, SIGA Technologies is developing the small-molecule oral drug, tecovirimat (previously known as ST-246), as a postexposure therapeutic treatment of orthopoxvirus disease, including smallpox. , et al., Am. J. Trop. Med. Hyg. 76:768 -773, 2007). Furthermore, in clinical trials thus far, the drug appears to be safe, with a good pharmacokinetic profile. In this study, the efficacy of tecovirimat was evaluated in both a prelesional and postlesional setting in nonhuman primates challenged intravenously with 1 ؋ 10 8 PFU of Variola virus (VARV; the causative agent of smallpox), a model for smallpox disease in humans. Following challenge, 50% of placebo-treated controls succumbed to infection, while all tecovirimat-treated animals survived regardless of whether treatment was started at 2 or 4 days postinfection. In addition, tecovirimat treatment resulted in dramatic reductions in dermal lesion counts, oropharyngeal virus shedding, and viral DNA circulating in the blood. Although clinical disease was evident in tecovirimat-treated animals, it was generally very mild and appeared to resolve earlier than in placebo-treated controls that survived infection. Tecovirimat appears to be an effective smallpox therapeutic in nonhuman primates, suggesting that it is reasonably likely to provide therapeutic benefit in smallpox-infected humans.