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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.

Highlights • Tumorigenicity assays can be time-consuming and results can be equivocal. • Basement membrane extract (BME) is known to enhance tumorigenicity. • The tumorigenicity of Vero (≤p258) and ARPE-19 cells were not enhanced by BME. • BME enhanced the tumorigenicity of HeLa, 293, MDCK, and transformed Vero cells. • BME may be a useful reagent for characterizing vaccine cell substrates., Immortalized cell lines used to produce vaccines are expected to be described in terms of their tumorigenicity. However, current in vivo tumorigenicity assays can be time-consuming and results can be equivocal, especially for weakly tumorigenic cells. Basement membrane extract (BME) derived from the Engelbreth-Holm-Swarm mouse tumor, such as Matrigel and Cultrex, consists of laminin, collagen IV, entactin, heparan sulfate, and proteoglycans, as well as biologically active peptides and growth factors. For nearly three decades, BME has been used in cancer research to enhance tumorigenicity assays (both tumor “take” as well as tumor growth are substantially improved). We assessed the feasibility of using BME to facilitate the evaluation of vaccine cell substrate tumorigenicity. Vero cells (WHO 10-87) were serially passaged and banked at every ten passages beginning with p140; for the present study, low-passage Vero cells (Vero LP, originating from cells banked at p140) and high-passage Vero cells (Vero HP, originating from cells banked at p250) were used. In addition, Vero TPX2 and Vero NM1, cell lines established from tumors formed in nude mice by Vero HP cells, as well as other cell lines relevant to vaccine production (HeLa, MDCK, 293, and ARPE-19), were assessed. Female adult athymic nude mice were injected subcutaneously with cells in the absence or presence of BME. We observed that the tumorigenicity of ARPE-19 cells as well as Vero cells below passage 258 (Vero LP and Vero HP; previously characterized as non-tumorigenic or weakly tumorigenic, respectively) was not enhanced by BME. In contrast, BME shortened the latency and decreased the tumor-producing cell dose of HeLa, 293, and MDCK cells as well as the tumorigenic Vero derivatives TPX2 and NM1. Thus, responsiveness to BME may reflect the status of the neoplastic process and possibly serve as a useful trait for better defining the tumorigenic phenotype of cells.

Human cytomegalovirus (HCMV) is a major cause of disability in congenitally infected infants and in the immunosuppressed. There is currently no licensed prophylactic HCMV vaccine. The HCMV envelope glycoprotein B (gB) is considered a major vaccine target antigen based on its critical role in mediating viral-host cell fusion and thus viral entry. The natural conformation of HCMV gB within the viral envelope is a trimer, but there has been no reported success in producing a recombinant trimeric gB suitable for vaccine use. Phase II clinical trials of a monomeric recombinant gB protein demonstrated 50% efficacy in preventing HCMV infection in seronegative women of reproductive age, and in reducing viremia in solid organ transplantation recipients. We now report the production of a uniformly trimeric recombinant HCMV gB protein in Chinese ovary cells, as demonstrated by Western blot analysis under modified non-reducing conditions and size exclusion chromatography with multi-angle scattering. Immunization of mice with trimeric HCMV gB induced up to 11-fold higher serum titers of total gB-specific IgG relative to monomeric HCMV gB using Alum + CpG as adjuvants. Further, trimeric HCMV gB elicited 50-fold higher complement-independent and 20-fold higher complement-dependent HCMV neutralizing titers compared to monomeric HCMV gB using the fibroblast cell line, MRC-5, and up to 6-fold higher complement-independent and –dependent HCMV neutralizing titers using the epithelial cell line, ARPE-19. The markedly enhanced HCMV neutralizing activity in response to trimeric HCMV gB was also observed using an additional four distinct clinical HCMV isolates. These data support a role for trimeric HCMV gB as an important component for clinical testing of a prophylactic HCMV vaccine.

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.

Early insulin signaling events were examined in a novel cell-free assay utilizing subcellular fractions derived from 3T3-L1 adipocytes. The following cellular processes were observed in vitro in a manner dependent on insulin, time of incubation, and exogenous ATP: 1) autophosphorylation and activation of the insulin receptor; 2) tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1); 3) association of tyrosine-phosphorylated IRS-1 with phosphoinositide 3-kinase; 4) activation of the kinase Akt via its phosphorylation on Thr-308 and Ser-473; and 5) phosphorylation of glycogen synthase kinase-3 by activated Akt. The activation of Akt in vitro was abolished in the presence of the phosphoinositide 3-kinase inhibitor, wortmannin, thus recapitulating the most notable regulatory feature of Akt observed in vivo. Evidence is presented indicating that the critical spatial compartmentalization of signaling molecules necessary for efficient signal transduction is likely to be preserved in the cell-free system. Additionally, data are provided demonstrating that full Akt activation in this system is dependent on plasma membrane-associated IRS-1, cannot be mediated by robust cytosol-specific tyrosine phosphorylation of IRS-1, and occurs in the complete absence of detectable IRS-2 phosphorylation in the cytosol and plasma membrane.

Objectives To determine the relative sensitivities of glucose transporter isoforms to the protease inhibitor indinavir and to determine the kinetic mechanism of indinavir-mediated Glut4 isoform inhibition. Methods The rate of 2-deoxyglucose uptake was measured in Xenopus laevis oocytes heterologously expressing mammalian Glut isoforms. 2-Deoxyglucose uptake was also measured in 3T3-L1 fibroblasts, 3T3-L1 adipocytes, and primary rat adipocytes. Results The sensitivity to inhibition by indinavir among the Glut isoforms as assayed in the X. laevis oocyte system was as follows in decreasing order: Glut4 >> Glut2 > Glut3 > Glut1 approximately Glut8. 2-Deoxyglucose uptake measurements in insulin-stimulated primary rat adipocytes indicated a non-competitive mode of transport inhibition by indinavir under zero-trans conditions with a KI of 15 microM. Conclusions Indinavir appears to be a relatively selective inhibitor of the Glut4 isoform. As the concentration required to significantly inhibit insulin-stimulated glucose uptake in primary rat adipocytes is well within the physiologic range achieved in therapy, we conclude that direct inhibition of Glut4 contributes to the insulin resistance observed in patients receiving this drug.

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.

The rate of movement of the glucose transporter isoforms Glut1 and Glut4 from the endoplasmic reticulum (ER) to the Golgi apparatus was investigated by pulse labeling and monitoring endoglycosidase H resistance in mRNA-injected Xenopus oocytes and in 3T3-L1 adipocytes, a cell line that naturally expresses both transporter isoforms. Despite their high degree of sequence identity, Glut1 and Glut4 exhibited dramatically different transit times. The t1/2 values for ER to Golgi transit for Glut1 and Glut4 were

The Glut4 glucose transporter is poorly functional compared with other glucose transporter isoforms when expressed in Xenopus oocytes. To investigate the molecular basis for this poor functionality, we compared the biosynthesis and targeting of Glut1 and Glut4 in oocytes after microinjection of the corresponding mRNAs. Both Glut1 and Glut4 were present as lower molecular weight endoglycosidase H-sensitive and higher molecular weight endoglycosidase H-resistant. Subcellular fractionation indicated that Glut1 was targeted to the plasma membrane with a 6.6-fold greater efficiency than was Glut4. Confocal immunofluorescence microscopy confirmed the relative enrichment of Glut1 in the plasma membrane and the efficient intracellular sequestration of Glut4. As in mammalian cells, the endoglycosidase H-resistant form of Glut4 was concentrated in low-density intracellular vesicles, whereas Glut1 was distributed in intracellular vesicles of higher average density. The structural basis for the differential localization of Glut1 and Glut4 was investigated by determining the plasma membrane content of a series of chimeric Glut1/Glut4 molecules. These data indicated that two distinct regions of Glut4, encompassing residues 24-132 and the COOH-terminal cytoplasmic tail, confer intracellular sequestration on the chimeric transporter molecules. At least part of the sequestration effect of the more N-terminal domain was due to the incomplete maturation of chimeras containing this region, resulting in the accumulation of lower molecular weight endoglycosidase H-sensitive and endoglycosidase H-resistant forms, whereas the COOH-terminal cytoplasmic tail conferred sequestration of fully glycosylated chimeras in a low-density intracellular membrane compartment.

The use of HIV protease inhibitors (PIs) has been associated with several metabolic changes, including lipodystrophy, hyperlipidemia, and insulin resistance. The etiology of these adverse effects remains unknown. PIs have recently been found to cause acute and reversible inhibition of GLUT4 activity in vitro. To determine the acute in vivo effects of indinavir on whole-body glucose homeostasis, glucose tolerance tests were performed on PI-naïve Wistar rats immediately after a single intravenous dose of indinavir. Glucose and insulin levels were significantly elevated in indinavir-treated versus control rats (P < 0.05) during the initial 30 min of the glucose tolerance test. Under euglycemic- hyperinsulinemic clamp conditions, indinavir treatment acutely reduced the glucose infusion rate required to maintain euglycemia by 18 and 49% at indinavir concentrations of 14 and 27 μmol/l, respectively. Muscle 2-deoxyglucose uptake was similarly reduced under these conditions. Restoration of insulin sensitivity was observed within 4 h after stopping the indinavir infusion. Indinavir did not alter the suppression of hepatic glucose output under hyperinsulinemic conditions. These data demonstrate that indinavir causes acute and reversible changes in whole-body glucose homeostasis in rats and support the contribution of GLUT4 inhibition to the development of insulin resistance in patients treated with PIs.

Using purified reaction components, a commercial monoclonal antibody (Ab) specific to RNase inhibitor (RI) was found to interfere with the activity of RI. Total RNA was mixed with a monoclonal Ab specific to either RI (clone 3F11) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNase A, RI, or a combination of the above. Following incubation for 1h at 22°C or 37°C, RNA integrity of the mixtures was assessed using microfluidics-based Bio-Rad Experion RNA electrophoresis. The addition of Ab 3F11 prevented RI from effectively inhibiting RNase A and therefore resulted in extensive RNA degradation. The data presented are associated with the research article entitled “Endogenous RNase Inhibitor Contributes to Stability of RNA in Crude Cell Lysates: Applicability to Reverse Transcription Quantitative PCR (RT-qPCR)” (Wang et al., 2016) [1].

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).