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Human Cytomegalovirus (HCMV) infection is widespread and can result in severe sequelae in susceptible populations. Primary HCMV infection of naïve individuals results in life-long latency characterized by frequent and sporadic reactivations. HCMV infection elicits a robust antibody response, including neutralizing antibodies that can block the infection of susceptible cells in vitro and in vivo. Thus, antibody products and vaccines hold great promise for the prevention and treatment of HCMV, but to date, most attempts to demonstrate their safety and efficacy in clinical trials have been unsuccessful. In this review we summarize publicly available data on these products and highlight new developments and approaches that could assist in successful translation of HCMV immunotherapies.

Congenital infection as well as infection of immunocompromised individuals by cytomegalovirus (CMV) can be associated with significant morbidity, mortality, and long-term adverse health outcomes. Assessment of anti-viral activity using appropriate assays is essential for ensuring safe and efficacious use of therapeutic CMV immune globulin (IG) products. In this study, we used commercial ELISA kits to compare anti-CMV antibody binding activity and avidity for lots of CMV-specific and normal IG products available in the US market. Additionally, neutralizing activity of IG products was measured against CMV strains (AD169wt131 or TB40E-GFP) in MRC-5 human fibroblasts and ARPE-19 human epithelial cells. Our data revealed that, regardless of the method, anti-CMV activity was higher in CMV IG lots we tested compared with normal IG lots; CMV binding activity was at least 4-fold higher, and neutralizing activity at least 2- and 3-fold higher for epithelial and fibroblast cells, respectively, in CMV IG lots compared with normal IG lots. Furthermore, anti-CMV activity values from all three methods (ELISA, neutralization in MRC-5 cells, and neutralization in ARPE-19 cells) were highly correlated, whereas avidity, although higher in CMV IG lots, did not correlate well with either binding or neutralizing activities.

The nucleoprotein of respiratory syncytial virus (RSV-N) is immunogenic and elicits an IgG response following infection. The RSV-N gene was cloned into a mammalian expression vector, pREN2, and the expressed luciferase-tagged protein (Ruc-N) detected anti-RSV-N-specific IgG antibodies using a high-throughput immunoprecipitation method (the luciferase immunoprecipitation system [LIPS]-N RSV assay). The specificity of the assay was evaluated using monoclonal antibodies (MAbs) and monospecific pre- and postimmunization rabbit antisera. Blood serum samples from chimpanzees and humans with proven/probable RSV infection were also tested. The pre- and postimmunization serum samples from rabbits given human metapneumovirus (HMPV) or measles virus were negative when tested by the LIPS-N RSV assay, while antisera obtained after immunization with either the RSV-A or RSV-B strain gave positive signals in a dose-dependent manner. RSV-N MAb 858-3 gave a positive signal in the LIPS-N RSV assay, while MAbs against other paramyxovirus nucleoproteins or RSV-F or RSV-G did not. Serum samples from chimpanzees simultaneously immunized with vaccinia-RSV-F and vaccinia-RSV-G recombinant viruses were negative in the LIPS-N RSV assay; however, anti-RSV-N IgG responses were detected following subsequent RSV challenge. Seven of the 12 infants who were seronegative at 9 months of age had detectable anti-RSV-N antibodies when they were retested at 15 to 18 months of age. The LIPS-N RSV assay detects specific anti-RSV-N IgG responses that may be used as a biomarker of RSV infection.

By recombining subcellular components of 3T3-L1 adipocytes in a test tube, early insulin signaling events dependent on phosphatidylinositol 3-kinase (PI 3-kinase) were successfully reconstituted, up to and including the phosphorylation of glycogen synthase kinase-3 by the serine/threonine kinase, Akt (Murata, H., Hresko, R.C., and Mueckler, M. (2003) J. Biol. Chem. 278, 21607-21614). Utilizing the advantages provided by a cell-free methodology, we characterized phosphoinositide-dependent kinase 2 (PDK2), the putative kinase responsible for phosphorylating Akt on Ser-473. Immunodepleting cytosolic PDK1 from an in vitro reaction containing plasma membrane and cytosol markedly inhibited insulin-stimulated phosphorylation of Akt at the PDK1 site (Thr-308) but had no effect on phosphorylation at the PDK2 site (Ser-473). In contrast, PDK2 activity was found to be highly enriched in a novel cytoskeletal subcellular fraction associated with plasma membranes. Akt isoforms 1-3 and a kinase-dead Akt1 (K179A) mutant were phosphorylated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner at Ser-473 in an in vitro reaction containing this novel adipocyte subcellular fraction. Our data indicate that this PDK2 activity is the result of a kinase distinct from PDK1 and is not due to autophosphorylation or transphosphorylation of Akt.

Although the clinical introduction of human immunodeficiency virus (HIV) protease inhibitors (PIs) has resulted in a dramatic decline in HIV-related morbidity and mortality, it is now recognized that PI therapy is associated with serious adverse metabolic effects, including peripheral lipoatrophy, increased visceral fat, hyperlipidemia, and insulin resistance. Despite increasing awareness of this metabolic syndrome, the etiology of these side effects remains obscure. This review critically examines current mechanistic hypotheses in the context of the available experimental data. To date, a single unifying explanation for this syndrome has not been confirmed. As data accumulate, it is becoming clear that PIs lack precision in their cellular targets and it is likely that many of the side effects of these drugs are due to inhibition of a number of unrelated molecules.

Retroviral protease inhibitors used as therapy for HIV-1 infection have been causally associated with serious metabolic side effects, including peripheral lipodystrophy, hyperlipidemia, insulin resistance, and in some cases, overt type 2 diabetes. The etiology of this characteristic clinical syndrome remains unknown. We demonstrate that the HIV protease inhibitor, indinavir, dramatically inhibits insulin-stimulated glucose uptake in 3T3-L1 adipocytes in a dose-dependent manner (63% inhibition observed with 100 micrometer indinavir). Indinavir treatment did not affect early insulin signaling events or the translocation of intracellular Glut1 or Glut4 glucose transporters to the cell surface. To determine whether indinavir may be directly affecting the intrinsic transport activity of glucose transporters, the Glut1 and Glut4 isoforms were heterologously expressed and analyzed in Xenopus laevis oocytes. Indinavir at 100 microm had no effect on Glut1 transport activity in Xenopus oocytes, whereas Glut4 activity was significantly inhibited (45% inhibition). Similar effects on glucose transport were observed for other HIV protease inhibitors. We conclude that HIV protease inhibitors as a class are capable of selectively inhibiting the transport function of Glut4 and that this effect may be responsible for a major iatrogenic complication frequently observed in HIV patients.

As part of safety studies to evaluate the risk of residual cellular DNA in vaccines manufactured in tumorigenic cells, we have been developing in vivo assays to detect and quantify the oncogenic activity of DNA. We generated a plasmid expressing both an activated human H-ras gene and murine c-myc gene and showed that 1 µg of this plasmid, pMSV-T24-H-ras/MSV-c-myc, was capable of inducing tumors in newborn NIH Swiss mice. However, to be able to detect the oncogenicity of dominant activated oncogenes in cellular DNA, a more sensitive system was needed. In this paper, we demonstrate that the newborn CD3 epsilon transgenic mouse, which is defective in both T-cell and NK-cell functions, can detect the oncogenic activity of 25 ng of the circular form of pMSV-T24-H-ras/MSV-c-myc. When this plasmid was inoculated as linear DNA, amounts of DNA as low as 800 pg were capable of inducing tumors. Animals were found that had multiple tumors, and these tumors were independent and likely clonal. These results demonstrate that the newborn CD3 epsilon mouse is highly sensitive for the detection of oncogenic activity of DNA. To determine whether it can detect the oncogenic activity of cellular DNA derived from four human tumor-cell lines (HeLa, A549, HT-1080, and CEM), DNA (100 µg) was inoculated into newborn CD3 epsilon mice both in the presence of 1 µg of linear pMSV-T24-H-ras/MSV-c-myc as positive control and in its absence. While tumors were induced in 100% of mice with the positive-control plasmid, no tumors were induced in mice receiving any of the tumor DNAs alone. These results demonstrate that detection of oncogenes in cellular DNA derived from four human tumor-derived cell lines in this mouse system was not possible; the results also show the importance of including a positive-control plasmid to detect inhibitory effects of the cellular DNA.

Background Few studies have used quantitative polymerase chain reaction (qPCR) as an approach to measure virus neutralization assay endpoints. Its lack of use may not be surprising considering that sample nucleic acid extraction and purification can be expensive, labor-intensive, and rate-limiting. Methods Virus/antibody mixtures were incubated for one hour at 37°C and then transferred to Vero cell monolayers in a 96-well plate format. At 24 (or 48) hours post-infection, we used a commercially available reagent to prepare cell lysates amenable to direct analysis by one-step SYBR Green quantitative reverse transcription PCR using primers specific for the RSV-N gene, thereby obviating the need for cumbersome RNA extraction and purification. The neutralization titer was defined as the reciprocal of the highest dilution needed to inhibit the PCR signal by 90% when compared with the mean value observed in virus control wells in the absence of neutralizing antibodies. Results We have developed a qPCR-based neutralization assay for human respiratory syncytial virus. Due to the sensitivity of qPCR in detecting virus replication, endpoints may be assessed as early as 24 hours post-infection. In addition, the dynamic range of qPCR provides a basis for the assay to be relatively robust to perturbations in input virus dose (i.e., the assay is in compliance with the Percentage Law). Conclusions This qPCR-based neutralization assay is suitable for automated high-throughput applications. In addition, our experimental approach may be generalizable for the rapid development of neutralization assays for other virus families.

Polyomaviruses are nonenveloped viruses with capsids composed primarily of 72 pentamers of the viral VP1 protein, which forms the outer shell of the capsid and binds to cell surface oligosaccharide receptors. Highly conserved VP1 proteins from closely related polyomaviruses recognize different oligosaccharides. To determine whether amino acid changes restricted to the oligosaccharide binding site are sufficient to determine receptor specificity and how changes in receptor usage affect tropism, we studied the primate polyomavirus simian virus 40 (SV40), which uses the ganglioside GM1 as a receptor that mediates cell binding and entry. Here, we used two sequential genetic screens to isolate and characterize viable SV40 mutants with mutations in the VP1 GM1 binding site. Two of these mutants were completely resistant to GM1 neutralization, were no longer stimulated by incorporation of GM1 into cell membranes, and were unable to bind to GM1 on the cell surface. In addition, these mutant viruses displayed an infection defect in monkey cells with high levels of cell surface GM1. Interestingly, one mutant infected cells with low cell surface GM1 more efficiently than wild-type virus, apparently by utilizing a different ganglioside receptor. Our results indicate that a small number of mutations in the GM1 binding site are sufficient to alter ganglioside usage and change tropism, and they suggest that VP1 divergence is driven primarily by a requirement to accommodate specific receptors. In addition, our results suggest that GM1 binding is required for vacuole formation in permissive monkey CV-1 cells. Further study of these mutants will provide new insight into polyomavirus entry, pathogenesis, and evolution.

At present, the seed viruses for the manufacture of licensed seasonal inactivated influenza vaccines in the United States are derived from primary egg isolates as a result of concerns associated with adventitious agents. According to the prevailing view, the passage of influenza viruses through eggs serves as a filtering step to remove potential contaminating viruses. We have investigated the feasibility of addressing adventitious-agent risk by subjecting influenza virus to a plaque-purification procedure using MDCK cells. SV40 and canine adenovirus-1 (representing viruses for which MDCK cells are non-permissive and permissive, respectively) were used as challenge viruses to model agents of concern that might be co-isolated along with the influenza virus. By mixing influenza virus strain A/PR/8/34 with varying amounts of each challenge virus and then performing a plaque assay for influenza virus using MDCK cells, we have attempted to determine the efficiency by which the challenge virus is removed. Our data suggest that substantial removal can be achieved even after a single round of plaque purification. If cell-derived isolates were deemed to be acceptable following a plaque-purification procedure, the manufacture of seasonal influenza vaccine would be facilitated by: (1) the expansion of the repertoire of viruses from which seed virus candidates could be generated for licensed egg-derived vaccines as well as for vaccines manufactured in mammalian cells; and (2) the mitigation of adventitious-agent risk associated with the seed virus, and hence the elimination of the need to passage seed viruses in eggs for vaccines manufactured in mammalian cells.

MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression post-transcriptionally. They play a critical role in developmental and physiological processes and have been implicated in the pathogenesis of several diseases including cancer. To identify miRNA signatures associated with different stages of neoplastic development, we examined the expression profile of 776 primate miRNAs in VERO cells (a neoplastically transformed cell line being used for the manufacture of viral vaccines), progenitor primary African green monkey kidney (pAGMK) cells, and VERO cell derivatives: spontaneously immortalized, non-tumorigenic, low-passage VERO cells (10-87 LP); tumorigenic, high-passage VERO cells (10-87 HP); and a cell line (10-87 T) derived from a 10-87 HP cell tumor xenograft in athymic nude mice. When compared with pAGMK cells, the majority of miRNAs were expressed at lower levels in 10-87 LP, 10-87 HP, and 10-87 T cells. We identified 10 up-regulated miRNAs whose level of expression correlated with VERO cell evolution from a non-tumorigenic phenotype to a tumorigenic phenotype. The overexpression of miR-376a and the polycistronic cluster of miR-376a, miR-376b and miR-376c conferred phenotypic changes to the non-tumorigenic 10-87 LP cells that mimic the tumorigenic 10-87 HP cells. Thirty percent of miRNAs that were components of the identified miRNAs in our spontaneously transformed AGMK cell model are also dysregulated in a variety of human tumors. These results may prove to be relevant to the biology of neoplastic development. In addition, one or more of these miRNAs could be biomarkers for the expression of a tumorigenic phenotype.

The use of HIV protease inhibitors (PIs) may be associated with serious adverse side effects that include fat tissue redistribution, hyperlipidemia, and insulin resistance. The etiology of this toxic metabolic syndrome (commonly referred to as 'HIV lipodystrophy syndrome') remains to be elucidated. The interpretation of available clinical data on this subject is complicated in part by the pervasiveness of potential confounding factors that cannot be easily eliminated or adequately controlled. Numerous investigators have examined the effects of PIs on cellular processes in model systems amenable to extensive experimental manipulations; the present review primarily focuses on these efforts. The ultimate goal is the unambiguous identification of discrete cellular targets being surreptitiously impacted by PIs. SREBP and Glut4 are discussed as candidate target molecules in this context. The identification of cellular factors interacting with PIs represents a necessary first step in devising rational strategies for improvement in drug therapy.

From stocks of adenovirus and poliovirus prepared in primary rhesus macaque kidney cells and dating from 1956 to 1961, the time when SV40 contaminated some poliovirus vaccine lots, we have recovered ten isolates of SV40. Of these ten isolates, based on the C-terminal region of T antigen, five novel strains of SV40 have been identified. Additionally, three pairs of isolates were found to be the same strain: one pair was strain 777, one pair was strain 776 archetype, and the third pair represented a novel strain. All strains had identical protein sequences for VP2 and VP3. There were two variants of agnoprotein and the small t antigen and three variants of VP1. These results, and those of others, suggest that a limited number of SV40 strains might exist in rhesus macaques in the United States, and thus determining the origin of the SV40 sequences detected in human tumors might be difficult.

Human cytomegalovirus (HCMV) is a leading cause of congenital infection that can result in serious disabilities in affected children. To facilitate HCMV vaccine development, a microscale neutralization assay based on reverse transcription quantitative PCR (RT-qPCR) was developed to quantify HCMV-neutralizing antibodies. Our approach relies on the generation of crude lysates from virus-infected cells that are amenable to direct analysis by RT-qPCR, thereby circumventing rate-limiting procedures associated with sample RNA extraction and purification. By serial passaging of the laboratory HCMV strain AD169 in epithelial cells (ARPE-19), a revertant virus with restored epithelial cell tropism, designated AD169wt131, was obtained. AD169 and AD169wt131 were evaluated in both epithelial cells (ARPE-19) and fibroblasts (MRC-5) by one-step RT-qPCR targeting the immediate-early gene IE1 transcript of HCMV. Expression kinetics indicated that RT-qPCR assessment could be conducted as early as 6h post-infection. Human serum samples (n=30) from healthy donors were tested for HCMV-specific IgG using a commercially available ELISA and for HCMV-neutralizing activity using our RT-qPCR-based neutralization assay. In agreement with the ELISA results, higher neutralizing activity was observed in the HCMV IgG seropositive group when compared with the HCMV IgG seronegative group. In addition, HCMV IgG seropositive human sera exhibited higher neutralizing titers using epithelial cells compared with using fibroblasts (geometric mean titers of 344 and 8 in ARPE-19 cells and MRC-5 cells, respectively). Our assay was robust to variation in input virus dose. In addition, a simple lysis buffer containing a non-ionic detergent was successfully demonstrated to be a less costly alternative to commercial reagents for cell-lysate preparation. Thus, our rapid HCMV neutralization assay may be a straightforward and flexible high-throughput tool for measuring antibody responses induced by vaccination and natural infection.

Three SV40 escape mutants were identified by selection in the presence of monoclonal antibodies with neutralizing activity. The VP1 amino acid alterations in these mutants were: (1) K73 → E (in loop BC); (2) D77 → E (in loop BC); (3) K171 → R (in loop EF); and (4) Q175 → H (in loop EF). These residues are clustered in close proximity to each other on the surface of the native capsid protein, strongly suggesting that they form a conformational epitope directly recognized by the neutralizing antibody. To our knowledge, the present study represents the first experimental mapping of a neutralization epitope of a polyomavirus family member. Structural information regarding the neutralization epitope should be useful for clarifying the extent of cross-reactivity exhibited by the humoral immune response towards related primate polyomaviruses (e.g., SV40, BKV, and JCV).

Crude cell lysates are increasingly used as input for direct analysis by reverse transcription quantitative PCR (RT-qPCR), particularly for high-throughput applications. We previously demonstrated that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercial cell-lysis reagents for the preparation of RT-qPCR-ready cell lysates; addition of an exogenous RNase inhibitor (RI) to the lysis buffer was found to be unnecessary to maintain RNA stability in cell lysates either freshly prepared or previously stored frozen at −80 °C. In the present study, we have demonstrated that the stability of RNA observed in our cell lysates is due to the presence of the endogenous RI. Furthermore, we have established the generalizability and applicability of this phenomenon by evaluating lysates prepared from cell lines commonly used in virology (A549, HeLa, MDCK, and Vero). Awareness of the mechanism underlying RNA stability may engender greater confidence in generating cell lysates for RT-qPCR without relying on addition of exogenous RI (a substantial cost-saving benefit) and encourage appropriate practices for handling and storage of samples.

This report presents the ultrastructural features of a congenital epulis. The granular cells of the epulis were packed with numerous membrane bound cytoplasmic granules containing particles, small vesicles, and electron-dense materials. These granules were negative in immunohistochemical reaction for CEA (DAKO PAP KIT). Cytoplasmic organelles such as mitochondria, rough surfaced endoplasmic reticulum, and Golgi apparatus, were absent. Nuclei were markedly indented. Occasionally, banded intracellular collagen fibrils were observed within the cytoplasm. Some of these fibrils were surrounded by a limiting membrane, whereas others appeared to lie free in the cytoplasm. The collagen fibrils were also seen within a deep invagination of the cell surface. There was no basal lamina around the granular cells. Sporadically, mast cells with many granules containing lamellar formations were found between the granular cells. These observations support the idea that granular cells of the congenital epulis are derived from mesenchymal cells, probably fibroblasts.