GLI1+ perivascular, renal, progenitor cells: The likely source of spontaneous neoplasia that created the AGMK1-9T7 cell line.

The AGMK1-9T7 cell line has been used to study neoplasia in tissue culture. By passage in cell culture, these cells evolved to become tumorigenic and metastatic in immunodeficient mice at passage 40. Of the 20 x 106 kidney cells originally plated, less than 2% formed the colonies that evolved to create this cell line. These cells could be the progeny of some type of kidney progenitor cells. To characterize these cells, we documented their renal lineage by their expression of PAX-2 and MIOX, detected by indirect immunofluorescence. These cells assessed by flow-cytometry expressed high levels of CD44, CD73, CD105, Sca-1, and GLI1 across all passages tested; these markers have been reported to be expressed by renal progenitor cells. The expression of GLI1 was confirmed by immunofluorescence and western blot analysis. Cells from passages 13 to 23 possessed the ability to differentiate into adipocytes, osteoblasts, and chondrocytes; after passage 23, their ability to form these cell types was lost. These data indicate that the cells that formed the AGMK1-9T7 cell line were GLI1+ perivascular, kidney, progenitor cells.

Evaluating the sensitivity of newborn rats and newborn hamsters to oncogenic DNA.

To evaluate the risk of residual cellular DNA in vaccines manufactured in tumorigenic cell lines, we have been establishing in vivo assays to quantify the oncogenic activity of DNA. We had generated three oncogene-expression plasmids: pMSV-T24-H-ras, which expresses activated H-ras; pMSV-c-myc, which expresses c-myc; and pMSV-T24-H-ras/MSV-c-myc, which expresses both oncogenes. Tumors were induced in mice by pMSV-T24-H-ras plus pMSV-c-myc or by pMSV-T24-H-ras/MSV-c-myc. Because newborn hamsters and newborn rats have been recommended for oncogenicity testing of the DNA from tumorigenic mammalian cell-substrates used for vaccine production, we evaluated their sensitivity. Newborn hamsters and rats were inoculated with different doses of pMSV-T24-H-ras/MSV-c-myc to determine their sensitivity to tumor induction and with the single-oncogene-expression plasmids to determine whether single oncogenes could induce tumors. Newborn rats were more sensitive than newborn hamsters, and activated H-ras but not c-myc induced tumors in newborns of both rodent species. DNA from four cell lines established from tumors induced by pMSV-T24-H-ras/MSV-c-myc was inoculated into newborn rats. Because no tumors were induced by this cellular DNA, which should be optimal as it contains both oncogenes linked and present in several copies, we conclude that available in vivo models are not sensitive enough to detect the oncogenicity of cellular DNA.

The AGMK1-9T7 cell model of neoplasia: Evolution of DNA copy-number aberrations and miRNA expression during transition from normal to metastatic cancer cells.

To study neoplasia in tissue culture, cell lines representing the evolution of normal cells to tumor cells are needed. To produce such cells, we developed the AGMK1-9T7 cell line, established cell banks at 10-passage intervals, and characterized their biological properties. Here we examine the evolution of chromosomal DNA copy-number aberrations and miRNA expression in this cell line from passage 1 to the acquisition of a tumorigenic phenotype at passage 40. We demonstrated the use of a human microarray platform for DNA copy-number profiling of AGMK1-9T7 cells using knowledge of synteny to 'recode' data from human chromosome coordinates to those of the African green monkey. This approach revealed the accumulation of DNA copy-number gains and losses in AGMK1-9T7 cells from passage 3 to passage 40, which spans the period in which neoplastic transformation occurred. These alterations occurred in the sequences of genes regulating DNA copy-number imbalance of several genes that regulate endothelial cell angiogenesis, survival, migration, and proliferation. Regarding miRNA expression, 195 miRNAs were up- or down-regulated at passage 1 at levels that appear to be biologically relevant (i.e., log2 fold change >2.0 (q<0.05)). At passage 10, the number of up/down-regulated miRNAs fell to 63; this number increased to 93 at passage 40. Principal-component analysis grouped these miRNAs into 3 clusters; miRNAs in sub-clusters of these groups could be correlated with initiation, promotion, and progression, stages that have been described for neoplastic development. Thirty-four of the AGMK1-9T7 miRNAs have been associated with these stages in human cancer. Based on these data, we propose that the evolution of AGMK1-9T7 cells represents a detailed model of neoplasia in vitro.

Enhanced In Vitro and In Vivo Potency of a T Cell Epitope in the Ebola Virus Glycoprotein Following Amino Acid Replacement at HLA-A*02:01 Binding Positions.

Studies on Ebola virus disease (EVD) survivors and clinical studies on Ebola virus (EBOV) vaccine candidates have pinpointed the importance of a strong antibody response in protection and survival from EBOV infection. However, little is known about the T cell responses to EBOV or EBOV vaccines. We used HLA-A*02:01 (HLA-A2) transgenic mice to study HLA-A2-specific T cell responses elicited following vaccination with EBOV glycoprotein (EBOV-GP) presented with three different systems: (i) recombinant protein (rEBOV-GP), (ii) vesicular stomatitis replication-competent recombinant virus (VSV-EBOV-GP), and (iii) modified vaccinia Ankara virus recombinant (MVA-EBOV-GP). T cells from immunized animals were analyzed using peptide pools representing the entire GP region and individual peptides. Regardless of the vaccine formulation, we identified a minimal 9mer epitope containing an HLA-A2 motif (FLDPATTS), which was confirmed through HLA-A2 binding affinity and immunization studies. Using binding prediction software, we identified substitutions surrounding position 9 (S9V, P10V, and Q11V) that predicted enhanced binding to the HLA-A2 molecule. This enhanced binding was confirmed through in vitro binding studies and enhanced potency was shown with in vivo immunization studies using the enhanced sequences and the wild-type sequence. Of note, in silico studies predicted the enhanced 9mer epitope carrying the S9V substitution as the best overall HLA-A2 epitope for the full-length EBOV-GP. These results suggest that EBOV-GP-S9V and EBOV-GP-P10V represent more potent in vivo immunogens. Identification and enhancement of EBOV-specific human HLA epitopes could lead to the development of tools and reagents to induce more robust T cell responses in human subjects. IMPORTANCE Vaccine efficacy and immunity to viral infection are often measured by neutralizing antibody titers. T cells are specialized subsets of immune cells with antiviral activity, but this response is variable and difficult to track. We showed that the HLA-A2-specific T cell response to the Ebola virus glycoprotein can be enhanced significantly by a single residue substitution designed to improve an epitope binding affinity to one of the most frequent MHC alleles in the human population. This strategy could be applied to improve T cell responses to Ebola vaccines designed to elicit antibodies and adapted to target MHC alleles of populations in regions where endemic infections, like Ebola virus disease, are still causing outbreaks with concerning pandemic potential.

Regulatory Considerations on the Development of mRNA Vaccines.

Developing traditional viral vaccines for infectious diseases usually takes years, as these are usually produced either by chemical inactivation of the virus or attenuation of the pathogen, processes that can take considerable time to validate and also require the live pathogen. With the advent of nucleic-acid vaccines (DNA and mRNA), the time to vaccine design and production is considerably shortened, since once the platform has been established, all that is required is the sequence of the antigen gene, its synthesis and insertion into an appropriate expression vector; importantly, no infectious virus is required. mRNA vaccines have some advantages over DNA vaccines, such as expression in non-dividing cells and the absence of the perceived risk of integration into host genome. Also, generally lower doses are required to induce the immune response. Based on experience in recent clinical trials, mRNA-based vaccines are a promising novel platform that might be useful for the development of vaccines against emerging pandemic infectious diseases. This chapter discusses some of the specific issues that mRNA vaccines raise with respect to production, quality, safety and efficacy, and how they have been addressed so as to allow their evaluation in clinical trials.

Development of mRNA Vaccines: Scientific and Regulatory Issues.

The global research and development of mRNA vaccines have been prodigious over the past decade, and the work in this field has been stimulated by the urgent need for rapid development of vaccines in response to an emergent disease such as the current COVID-19 pandemic. Nevertheless, there remain gaps in our understanding of the mechanism of action of mRNA vaccines, as well as their long-term performance in areas such as safety and efficacy. This paper reviews the technologies and processes used for developing mRNA prophylactic vaccines, the current status of vaccine development, and discusses the immune responses induced by mRNA vaccines. It also discusses important issues with regard to the evaluation of mRNA vaccines from regulatory perspectives. Setting global norms and standards for biologicals including vaccines to assure their quality, safety and efficacy has been a WHO mandate and a core function for more than 70 years. New initiatives are ongoing at WHO to arrive at a broad consensus to formulate international guidance on the manufacture and quality control, as well as nonclinical and clinical evaluation of mRNA vaccines, which is deemed necessary to facilitate international convergence of manufacturing and regulatory practices and provide support to National Regulatory Authorities in WHO member states.

The science of vaccine safety: Summary of meeting at Wellcome Trust.

Vaccines are everywhere hugely successful but are also under attack. The reason for the latter is the perception by some people that vaccines are unsafe. However that may be, vaccine safety, life any other scientific subject, must be constantly studied. It was from this point of view that a meeting was organized at the Wellcome Trust in London in May 2019 to assess some aspects of vaccine safety as subjects for scientific study. The objective of the meeting was to assess what is known beyond reasonable doubt and conversely what areas need additional studies. Although the meeting could not cover all aspects of vaccine safety science, many of the most important issues were addressed by a group of about 30 experts to determine what is already known and what additional studies are merited to assess the safety of the vaccines currently in use. The meeting began with reviews of the current situation in different parts of the world, followed by reviews of specific controversial areas, including the incidence of certain conditions after vaccination and the safety of certain vaccine components. Lastly, information about the human papillomavirus vaccine was considered because its safety has been particularly challenged by vaccine opponents. The following is a summary of the meeting findings. In addition to this summary, the meeting organizers will explore opportunities to perform studies that would enlarge knowledge of vaccine safety.

Responsiveness to basement membrane extract as a possible trait for tumorigenicity characterization.

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.

Development of a micro-neutralization assay for ebolaviruses using a replication-competent vesicular stomatitis hybrid virus and a quantitative PCR readout.

Development of vaccines against highly pathogenic viruses that could also be used as agents of bioterrorism is both a public health issue and a national security priority. Methods that can quantify neutralizing antibodies will likely be crucial in demonstrating vaccine effectiveness, as most licensed viral vaccines are effective due to their capacity to elicit neutralizing antibodies. Assays to determine whether antibodies are neutralizing traditionally involve infectious virus, and the assay most commonly used is the plaque-reduction neutralization test (PRNT). However, when the virus is highly pathogenic, this assay must be done under the appropriate level of containment; for tier one select agents, such as Ebola virus (EBOV), it is performed under Biological Safety Level 4 (BSL-4) conditions. Developing high-throughput neutralization assays for these viruses that can be done in standard BSL-2 laboratories should facilitate vaccine development. Our approach is to use a replication-competent hybrid virus whose genome carries the envelope gene from the pathogenic virus on the genetic backbone of a non-pathogenic virus, such as vesicular stomatitis virus (VSV). We have generated hybrid VSVs carrying the envelope genes for several species of ebolavirus. The readout for infectivity is a one-step reverse transcriptase quantitative PCR (RT-qPCR), an approach that we have used for other viruses that allows robustness and adaptability to automation. Using this method, we have shown that neutralization can be assessed within 6-16h after infection. Importantly, the titers obtained in our assay with two characterized antibodies were in agreement with titers obtained in other assays. Finally, although in this paper we describe the VSV platform to quantify neutralizing antibodies to ebolaviruses, the platform should be directly applicable to any virus whose envelope is compatible with VSV biology.

RT-qPCR-based microneutralization assay for human cytomegalovirus using fibroblasts and epithelial cells.

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 AD169(wt131), was obtained. AD169 and AD169(wt131) 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.

A mouse strain defective in both T cells and NK cells has enhanced sensitivity to tumor induction by plasmid DNA expressing both activated H-Ras and c-Myc.

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.

MicroRNAs as potential biomarkers for VERO cell tumorigenicity.

MicroRNA expression appears to capture the process of neoplastic development in vitro in the VERO line of African green monkey kidney (AGMK) cells (Teferedegne et al. PLoS One 2010;5(12):e14416). In that study, specific miRNA signatures were correlated with the transition, during serial tissue-culture passage, of low-density passaged 10-87 VERO cells from a non-tumorigenic phenotype at passage (p) 148 to a tumorigenic phenotype at p256. In the present study, six miRNAs (miR-376a, miR-654-3p, miR-543, miR-299-3p, miR-134 and miR-369-3p) were chosen from the identified signature miRNAs for evaluation of their use as potential biomarkers to track the progression of neoplastic development in VERO cells. Cells from the 10-87 VERO cell line at passage levels from p148 to p256 were inoculated into newborn and adult athymic nude mice. No tumors were observed in animals inoculated with cells from p148 to p186. In contrast, tumor incidences of 20% developed only in newborn mice that received 10-87 VERO cells at p194, p234 and p256. By qPCR profiling of the signature miRNAs of 10-87 VERO cells from these cell banks, we identified p194 as the level at which signature miRNAs elevated concurrently with the acquisition of tumorigenic phenotype with similar levels expressed beyond this passage. In wound-healing assays at 10-passage intervals between p150 to p250, the cells displayed a progressive increase in migration from p165 to p186; beginning at p194 and higher passages thereafter, the cells exhibited the highest rates of migration. By qPCR analysis, the same signature miRNAs were overexpressed with concomitant acquisition of the tumorigenic phenotype in another lineage of 10-87 VERO cells passaged independently at high density. Correlation between the passages at which the cells expressed a tumorigenic phenotype and the passages representing peaks in expression levels of signature miRNAs indicates that these miRNAs are potential biomarkers for the expression of the VERO cell tumorigenic phenotype.

Failure-to-thrive syndrome associated with tumor formation by Madin-Darby canine kidney cells in newborn nude mice.

Tumors that formed in newborn nude mice that were inoculated with 10(7) Madin-Darby canine kidney (MDCK) cells were associated with a failure-to-thrive (FTT) syndrome consisting of growth retardation, lethargy, weakness, and dehydration. Scoliosis developed in 41% of affected pups. Pups were symptomatic by week 2; severely affected pups became moribund and required euthanasia within 3 to 4 wk. Mice with FTT were classified into categories of mild, moderate, and severe disease by comparing their weight with that of age-matched normal nude mice. The MDCK-induced tumors were adenocarcinomas that invaded adjacent muscle, connective tissue, and bone; 6 of the 26 pups examined had lung metastases. The induction of FTT did not correlate with cell-line aggressiveness as estimated by histopathology or the efficiency of tumor formation (tumor-forming dose 50% endpoint range = 10(2.8) to 10(7.5)); however, tumor invasion of the paravertebral muscles likely contributed to the scoliosis noted. In contrast to the effect of MDCK cells, tumor formation observed in newborn mice inoculated with highly tumorigenic, human-tumor-derived cell lines was not associated with FTT development. We suggest that tumor formation and FTT are characteristics of these MDCK cell inocula and that FTT represents a new syndrome that may be similar to the cachexia that develops in humans with cancer or other diseases.

A neutralization assay for respiratory syncytial virus using a quantitative PCR-based endpoint assessment.

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.

Development of a neutralization assay for influenza virus using an endpoint assessment based on quantitative reverse-transcription PCR.

A microneutralization assay using an ELISA-based endpoint assessment (ELISA-MN) is widely used to measure the serological response to influenza virus infection and vaccination. We have developed an alternative microneutralization assay for influenza virus using a quantitative reverse transcription PCR-based endpoint assessment (qPCR-MN) in order to improve upon technical limitations associated with ELISA-MN. For qPCR-MN, infected MDCK-London cells in 96-well cell-culture plates are processed with minimal steps such that resulting samples are amenable to high-throughput analysis by downstream one-step quantitative reverse transcription PCR (qRT-PCR; SYBR Green chemistry with primers targeting a conserved region of the M1 gene of influenza A viruses). The growth curves of three recent vaccine strains demonstrated that the qRT-PCR signal detected at 6 hours post-infection reflected an amplification of at least 100-fold over input. Using ferret antisera, we have established the feasibility of measuring virus neutralization at 6 hours post-infection, a duration likely confined to a single virus-replication cycle. The neutralization titer for qPCR-MN was defined as the highest reciprocal serum dilution necessary to achieve a 90% inhibition of the qRT-PCR signal; this endpoint was found to be in agreement with ELISA-MN using the same critical reagents in each assay. qPCR-MN was robust with respect to assay duration (6 hours vs. 12 hours). In addition, qPCR-MN appeared to be compliant with the Percentage Law (i.e., virus neutralization results appear to be consistent over an input virus dose ranging from 500 to 12,000 TCID(50)). Compared with ELISA-MN, qPCR-MN might have inherent properties conducive to reducing intra- and inter-laboratory variability while affording suitability for automation and high-throughput uses. Finally, our qRT-PCR-based approach may be broadly applicable to the development of neutralization assays for a wide variety of viruses.

Mutations in the GM1 binding site of simian virus 40 VP1 alter receptor usage and cell tropism.

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.

Heterogeneity of the tumorigenic phenotype expressed by Madin-Darby canine kidney cells.

The mechanisms by which cells spontaneously immortalized in tissue culture develop the capacity to form tumors in vivo likely embody fundamental processes in neoplastic development. The evolution of Madin-Darby canine kidney (MDCK) cells from presumptively normal kidney cells to immortalized cells that become tumorigenic represents an example of neoplastic development in vitro. Studies of the mechanisms by which spontaneously immortalized cells develop the capacity to form tumors would benefit from quantitative in vivo assays. Most mechanistic correlations are evaluated by using single-dose tumor-induction experiments, which indicate only whether cells are or are not tumorigenic. Here we used quantitative tumorigenicity assays to measure dose-and time-dependent tumor development in nude mice of 3 lots of unmodified MDCK cells. The results revealed lot-to-lot variations in the tumorigenicity of MDCK cells, which were reflected by their tumor-inducing efficiency (threshold cell dose represented by mean tumor-producing dose; log(10) 50% endpoints of 5.2 for vial 1 and 4.4 for vial 2, and a tumor-producing dose of 5.8 for vial 3) and mean tumor latency (vial 1,6.6 wk; vial 2,2.9 wk; and vial 3,3.8 wk). These studies provide a reference for further characterization of the MDCK cell neoplastic phenotype and may be useful in delineating aspects of neoplastic development in vitro that determine tumor-forming capacity. Such data also are useful when considering MDCK cells as a reagent for vaccine manufacture.

Plaque purification as a method to mitigate the risk of adventitious-agent contamination in influenza vaccine virus seeds.

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.

Tumors induced in mice by direct inoculation of plasmid DNA expressing both activated H-ras and c-myc.

Vaccines contain residual DNA derived from the cells used to produce them. As part of our investigation to assess the risk of this cellular DNA, we are developing a quantitative in vivo assay to assess the oncogenicity of DNA. In an earlier study, we had generated expression plasmids for two oncogenes--human activated T24-H-ras and murine c-myc--and had shown that these two plasmids, pMSV-T24-H-ras and pMSV-c-myc, could act in concert to induce tumors in mice, although the efficiency was low. In this study, we took two approaches to increase the oncogenic efficiency: 1) both oncogene-expression cassettes were placed on the same plasmid; 2) transfection facilitators, which increase DNA uptake and expression in vitro, were tested. The dual-expression plasmid, pMSV-T24-H-ras/MSV-c-myc, is about 20-fold more efficient at tumor induction in newborn NIH Swiss mice than the separate expression plasmids, with tumors being induced with 1 microg of the dual-expression plasmid DNA. However, none of the transfection facilitators tested increased the efficiency of tumor induction. Based on these data, the dual-expression plasmid pMSV-T24-H-ras/MSV-c-myc will be used as the positive control to develop a sensitive and quantitative animal assay that can be used to assess the oncogenic activity of DNA.

Patterns of microRNA expression in non-human primate cells correlate with neoplastic development in vitro.

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.