Congenital cytomegalovirus infection is the leading cause of sensorineural hearing loss in the U.S. CMV vaccines developed to date do not protect the majority of women of childbearing age from primary CMV infection. Insufficient vaccine-induced epithelial entry neutralizing activity may be the reason for poor performance of these vaccines. CMV entry into endothelial and epithelial but not fibroblast cells requires the virion envelope complex gH/gL/UL128-131. Since current vaccines do not target this complex, epithelial entry mediators UL128-131 are attractive subunit CMV vaccine candidates, since they should target mucosal immunity. The mucosal immune response, specifically salivary epithelial entry neutralizing activity, has not been previously described. This report demonstrates that salivas from CMV seropositive children under two, adolescents, and Towne vaccine recipients do not have epithelial or fibroblast neutralizing activity. Epithelial but not fibroblast neutralizing activity was identified in half of the salivas from CMV seropositive adults tested. This activity correlated with the level of serum neutralizing activity, suggesting that salivary neutralizing activity results from passively transferred serum IgG. Furthermore, this report describes three highly immune individuals with serum and saliva neutralizing titers two- to four-fold above average. These individuals also have UL130 antibodies detectable in western blot assays. This is the first report of antibodies by western blot in CMV seropositive sera to UL128, UL130, or UL131. To determine the feasibility of UL128-131 as vaccine candidates both peptide and DNA vaccines were tested in animal models. Rabbit anti-peptide sera from UL130 and UL131 vaccinated animals induced epithelial entry neutralizing activity similar to that found following natural infection. Mixing anti-peptide UL130 and UL131 sera neutralized CMV infection of epithelial cells at titers higher than natural infection. DNA vaccination with these proteins was not as successful but based on DNA vaccination of mice UL130 is the most immunogenic of the three proteins. These data support further development of UL130 as a CMV vaccine. Future vaccines, including the vaccine candidates described in this report, should strive to induce levels of immunity seen in the three highly immune individuals, specifically serum epithelial neutralizing titers >1:7,000 and saliva epithelial neutralizing titers >1:20.

Cytomegalovirus (CMV) infections remain a significant problem in congenitally infected infants and immunocompromised individuals. Modest antiviral activities of currently approved drugs coupled with dose-limiting toxicities restrict effectiveness and promote development of resistance. The potential for ribonucleotide reductase (RR) inhibitors hydroxyurea (HU), Didox, and Trimidox to synergize, through reduction of nucleotide pools, with the deoxynucleotide analog Ganciclovir (GCV) was examined. A yield reduction assay that utilizes luciferase expressed by a recombinant virus as a surrogate measure of viral infectious units was developed and used to determine effective dose ranges for each drug. RR inhibitors exhibited intrinsic anti-CMV activities on their own with IC50 values well below toxic levels. Moreover, RR inhibitors significantly synergized with GCV. These findings provide a rationale for exploration of RR inhibitors and deoxynucleotide analogs in anti-CMV combination therapy.

Mouse cytomegalovirus (MCMV), a β-herpesvirus, encodes the m145 family of glycoproteins. Several members of this family are predicted to adopt the MHC-I fold although their amino acid sequences exhibit less than 30% identity to classical MHC-I (MHC-Ia) proteins. Our aim was to determine how related the viral proteins are to MHCIa and characterize them in terms of cellular expression, structure and function. We studied the cellular localization of FLAG-tagged m17, M37, m145, m151, m152, m153 and m155 in transfected mouse fibroblasts. Flow cytometry analysis of transfected cells showed that M37, m145, m151 and m153 localize predominantly to the cell surface, whereas the majority of m17, m152 and m155 remain inside the cell. MHC-Ia proteins require assembly with β2-microglobulin (β2m) and peptide for stable cell surface expression. Transient transfection studies with β2m- or transporter associated with antigen (TAP)-deficient cell lines revealed that M37, m145, m151 and m153 could be expressed stably at the cell surface in the absence of β2m or TAP expression. To generate protein material for crystallization screening we evaluated both bacterial and insect cell expression systems. Although most m145 family members could be expressed in bacteria in insoluble inclusion bodies, none of the proteins could be accurately refolded. Since M37, m151 and m153 are cell surface molecules with the potential to bind receptors on host cells, we focused our structure determination efforts on them and evaluated their expression in Drosophila S2 insect cells. The extracellular domains of all three proteins expressed at significant levels, however, m151 tended to aggregate and precipitate over time. M37 and m153 were stable and could easily be purified to homogeneity. Size exclusion chromatography and SDS-PAGE analysis of m153 suggested that it forms a non-covalent homodimer. Analytical ultracentrifugation experiments confirmed this observation and provided an estimated molecular mass of 78.8 kDa. Enzymatic and mass spectrometry analyses showed that insect-expressed m153 is highly glycosylated. We tested a wide range of crystallization conditions for m153. It formed very fragile crystals and after optimization we obtained several that diffracted to 2.3 Å. To determine the structure of m153, we prepared a seleno-methionine derivative in insect cells, collected data on a single crystal and solved the phases by the single anomalous dispersion method. The m153 model was refined at 2.4 Å resolution to final Rcryst and Rfree of 23% and 27.9%, respectively. The m153 homodimer is formed by two MHC-I-like heavy chains, each consisting of two α-helices arranged on a platform of seven β-strands and an Ig-like α3 domain. The monomers are arranged “head-to-tail”, with the α1α2 platform domain of one chain interacting with the Ig-like α3 domain of the other. The α1 and α2 helices are closely juxtaposed and do not form a peptide binding groove. Three N-linked carbohydrate residues were visualized in the crystal structure. Major deviations from the MHC-I fold include an extended N-terminus, which originates next to the α3 domain, and an elongated α2 helix (designated H2b) that reaches down towards the α3 domain. In addition, m153 has two unique disulfide bonds, one between strands of the platform domain and another that links the extended N-terminus to the H2b helix. Both unique disulfide bonds were verified by mass spectrometry. The canonical Ig-fold disulfide bond is present in the α3 domain. Alanine mutation of four amino acids involved in interface hydrogen bonds abolished m153 dimer formation, validating the dimer interface visualized in the structure. The crystal structure of m153, together with the recently reported m157 structure, confirms the MHC-I fold for the MCMV m145 family and highlights shared structural features in the m145 family. We have demonstrated dimerization of full-length m153 in mammalian cells by bimolecular fluorescence complementation and co-immunoprecipitation studies. Further, we have shown that m153 is expressed at the surface of MCMV-infected cells at early times after infection. To initiate a search for host ligands of m153, we generated a reporter cell line by introducing an m153-human zeta chain fusion protein into 43.1 cells that contain an NFAT-driven GFP construct. While a variety of mouse cell lines were unable to stimulate the m153-reporter cells, coculture with mouse splenocytes specifically induced GFP production in m153-reporters but not in the parental or control reporter cell lines. We identified conventional CD11c+ and plasmacytoid dendritic cells (DCs) as the most potent m153-reporter cell stimulating populations in the spleen. The stimulation was shown to be m153-specific, dose- and cell contact-dependent. DCs derived from bonemarrow cultures also potently stimulated the m153-reporter cells. Macrophages and NK cells exhibited weaker stimulation of the reporter cells, indicating lower levels of ligand or that only small subsets of the cells express a ligand. DCs from several mouse strains, but not from the rat, stimulated m153-reporter cells. We evaluated DC surface phenotype and migratory capacity after coculture with m153-reporter cells or on m153-coated plates, but could not detect any changes induced specifically by the presence of m153.

No description available.

Immunity and metabolism have been viewed as separate fields, however recent evidence show that these two systems are intimately integrated, share resources and cross-regulate each other. Activated immune cells have to alter their metabolism in order to support effector functions. On the other hand, viruses are obligatory parasites that counter and exploit host pathways, including metabolism, to effectively propagate. Like immune cells, viruses have to alter the metabolic profile of infected cells in order to propagate. The regulation of metabolism in immune cells or virally infected cells has been well studied. However, the precise metabolic regulation that ensues when both immune system and viral infection in immune cells interact and compete for the limited resources and metabolic pathways available is not clear. In this thesis, I have sought to investigate the integrative process by studying the metabolic programming of macrophages infected with murine cytomegalovirus (MCMV) The central hypothesis of this thesis is that productive infection of macrophages by MCMV takes advantage of the early inflammatory metabolomic reprogramming of activated macrophages to establish infection, and modulates metabolism at late stages of infection towards fatty acid (FA) production to promote viral progeny. To study this interaction, I have analysed the temporal profile of the transcriptome and metabolome of bone marrow derived macrophages (BMDM) infected with productive (WT) and non-productive (attenuated) (MCMV) strains. This aimed to unravel the host-directed versus virus-driven metabolic alterations. I show evidence indicating that during early times of productive and non-productive MCMV infection glycolysis is, in infected BMDM, markedly increased. Furthermore, pharmacological and siRNA mediated inhibition of glycolysis resulted in attenuation of viral growth demonstrating the dependency of MCMV on this pathway. Additionally, using interferon receptor A (IFNAR) and interferon receptor A (IFNB) deficient BMDM showed that type-I interferon (IFN) signalling is essential for the early upregulation of glycolysis that was observed. In addition to the changes in glycolysis, MCMV infection alters the tricarboxylic acid (TCA) cycle in infected BMDM. Metabolomic and transcriptomic data revealed a shift from catabolic to anabolic function for the TCA to promote production of TCA intermediates. Finally, the urea cycle is also altered both on transcriptional and metabolomic level, consistent with the support of Nitric oxide (NO) production which is a hallmark metabolite in classically activated macrophages. These changes observed in the TCA cycle and glycolysis are consistent with supporting the FA elongation pathway during late time points of productive infection. Only productive MCMV infection upregulates this pathway. At the same time, pharmacological and siRNA mediated inhibition of FA elongation pathway greatly attenuates viral growth. This indicates that MCMV growth is dependenton FA elongation. The effect was very specific for the elongation and not the de novo synthesis pathway indicating that MCMV remodels FA that already in the cells. It is argued, that in agreement to known literature, MCMV uses these FA for the formation of its lipid membrane. To further investigate the dependency of MCMV on FA elongation pathways I studied additional lipids pathway associated with the former. I found that MCMV infection also upregulates the triacylglycerol formation and membrane remodelling pathways, which are dependent on FA biosynthesis and elongation. The inhibition of triacylglycerol formation and membrane remodelling pathway also attenuated MCMV growth. This indicates that apart from the formation of its lipid membrane MCMV requires FA to remodel the cellular environment. I have also explored the effects of infection on regulating lipid mediators, in particular eicosanoids. Eicosanoids are lipid signalling molecules that can act as potent inflammation modulators. Here I demonstrated that productive MCMV infection specifically increases PGE2 production in infected BMDM. Moreover, addition of PGE2 increased viral replication in infected fibroblasts in comparison to non-treated cells, while pharmacological blocking of EP4 (PGE2 receptor) rescued the phenotype. These studies reveal how MCMV advantageously use inflammatory lipid pathways to promote growth In conclusion, the data presented in this thesis support my hypothesis and provide an insight in the role of metabolism during viral infection. Evidence is provided to show that MCMV co-ops the early alterations that metabolic pathways undergo in activated macrophage, including but not limited to glycolysis, TCA cycle and urea cycle. These early changes in metabolism appear to be coupled with upregulation of FA elongation pathways and remodeling of lipids in infected cells. Finally, MCMV co-ops the function of regulatory lipids, in particular PGE2, to promote viral growth. It is further argued that MCMV productive infection dictates these fatty acid metabolism alterations in order to remodel the host cell's environment, regulate the immune system response and provide resources for its lipid membrane.

Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2005. / "A dissertation in pharmaceutical science and chemistry." Advisor: Ashim K. Mitra. Typescript. Vita. Description based on contents viewed Mar. 12, 2007; title from "catalog record" of the print edition. Includes bibliographical references (leaves 138-147). Online version of the print edition.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xiii, 127 p.; also includes graphics (some col.) Includes bibliographical references (p. 113-127). Available online via OhioLINK's ETD Center

Thesis (Ph. D.)--University of Nevada, Reno, 2003. / "December, 2003." Includes bibliographical references. Online version available on the World Wide Web.

Proefschrift Maastricht. / Lit. opg. - Met samenvatting in het Nederlands.