Issues associated with residual cell-substrate DNA in viral vaccines

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Biologicals 37 (2009) 190e195 www.elsevier.com/locate/biologicals

Issues associated with residual cell-substrate DNA in viral vaccines Li Sheng-Fowler, Andrew M. Lewis Jr., Keith Peden* Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drugs Administration, Bethesda, MD 20892, USA Received 2 February 2009; accepted 2 February 2009

Abstract The presence of some residual cellular DNA derived from the production-cell substrate in viral vaccines is inevitable. Whether this DNA represents a safety concern, particularly if the cell substrate is derived from a tumor or is tumorigenic, is unknown. DNA has two biological activities that need to be considered. First, DNA can be oncogenic; second, DNA can be infectious. As part of our studies to assess the risk of residual cell-substrate DNA in viral vaccines, we have established assays that can quantify the biological activities of DNA. From data obtained using these assays, we have estimated the risk of an oncogenic or an infectious event from DNA. Because these estimates were derived from the most sensitive assays identified so far, they likely represent worst-case estimates. In addition, methods that inactivate the biological activities of DNA can be assessed and estimations of risk reduction by these treatments can be made. In this paper, we discuss our approaches to address potential safety issues associated with residual cellular DNA from neoplastic cell substrates in viral vaccines, summarize the development of assays to quantify the oncogenic and infectivity activities of DNA, and discuss methods to reduce the biological activities of DNA. Published by Elsevier Ltd on behalf of The International Association for Biologicals. Keywords: Oncogenic DNA; Infectious DNA; Risk evaluation

1. Introduction: potential concerns associated with DNA The variety of cell substrates that have been used for the manufacture of viral vaccines licensed in the United States is limited to primary cells of avian or monkey origin, to the diploid cell lines (formerly termed diploid cell strains [1]) WI-38, MRC5, and FRhL-2, and to one continuous cell line, the VERO line (derived from African green monkey kidney cells) [2]. While these cell substrates have produced vaccines of proven safety and efficacy, it is increasingly apparent that this repertoire is insufficient for the production of the next generation of viral vaccines, such as those against HIV/AIDS, against emerging infectious diseases (e.g., SARS), and against agents of bioterrorism. In addition, the potential of a pandemic influenza outbreak caused by influenza viruses that either cannot be propagated to high titers in eggs or that are pathogenic for * Corresponding author. Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drugs Administration, Building 29A, Room 3D08, CBER, FDA, 29 Lincoln Drive, Bethesda, MD 20892, USA. Tel.: þ1 301 827 1708; fax: þ1 301 496 1810. E-mail address: [email protected] (K. Peden).

chickens, such as H5N1 avian influenza viruses, has prompted additional cell substrates to be evaluated for influenza vaccines, such as the Madin-Darby canine kidney cell line [3e5]. So far, many of the new mammalian cell substrates that are being evaluated for viral vaccine manufacture are considered to be neoplastic, since they have been immortalized by various mechanisms, and some are tumorigenic. The fear that components derived from the production-cell substrate could induce cancer in vaccine recipients was one of the reasons that the Armed Forces Epidemiological Board recommended in 1954 against the use of tumorigenic cells or cells derived from human tumors for the manufacture of vaccines for human use [6,7]. That this recommendation remained for over 40 years was due at least in part to the inability to evaluate this concern scientifically, which was due first to the inability to identify the risk factors and second, once these risk factors were identified, to a lack of assays capable of quantifying the risk posed by these factors. It should be pointed out that this manuscript discusses potential risks associated with use of novel highly tumorigenic neoplastic cells for the manufacture of viral vaccines, and any discussion about other types of cell substrates or products is beyond its scope.

1045-1056/09/$36.00 Published by Elsevier Ltd on behalf of The International Association for Biologicals. doi:10.1016/j.biologicals.2009.02.015

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2. Approaches to determine risk of cellular DNA Our general approach to risk assessment has been described as a Defined Risks Approach (DRA) [8]. The algorithm that CBER proposed for the DRA consists of five steps. 1. Identifying the possible risk event; 2. Estimating or determining the frequency with which the risk event might occur or has been observed to occur either in nature or under experimental conditions; 3. Estimating the possible frequency of the risk event per dose of vaccine; 4. Developing and determining the sensitivity (with respect to the limits of the assay’s ability to detect the risk event) of one or more assays that can be use to detect the risk event; 5. Developing and validating one or more processes that can be used to establish a product-specific safety factor to a defined level or to determine the level at which current technology can be used to establish a safety factor/limit. [Acceptable safety factors depend on the seriousness of the risk event. In evaluating viral vaccines made in highly tumorigenic cells, the US FDA Vaccines and Related Biological Products Advisory Committee (VRBPAC) has deemed a demonstrated risk of <1 in 107 to be acceptable with respect to residual cellular DNA.] Our approach to evaluate the risk of cell-substrate DNA from highly tumorigenic cells is as follows. 1. Develop sensitive and quantitative assays to measure the biological activities of DNA. 2. Use data from the most sensitive assay to estimate risks for a particular event; thus, estimates of risk would be conservative, since they should represent a worstcase situation. 3. Use assays to quantify the amount of reduction in biological activity afforded by a particular treatment (e.g., nuclease digestion, chemical inactivation). 4. Use these data to estimate safety factors for a product with respect to the residual cell-substrate DNA in that product. 3. The biological activities of DNA DNA can have several measurable biological activities. These include an oncogenic activity and an infectivity activity. Although not related to cell-substrate issues, DNA can also have a transforming activity in bacterial and fungal systems. While the above DNA activities are mediated through products derived from gene expression (proteins, microRNA), another biological activity of DNA that does not require gene expression is the immunomodulatory activity of DNA itself [9,10]. While this immunomodulatory activity can be measured in vivo, the amounts of DNA required for this effect are not typically present in vaccines. Therefore, based on current perceptions, the biological activities of DNA that need to be considered for vaccine safety are the oncogenic activity and infectivity activity. 4. The oncogenic activity of DNA With the identification of oncogenes in the genomes of cells [11,12], the demonstration that certain activated cellular oncogenes were able to transform primary cells in vitro to neoplastic cells [13e15], and that some of these transformed

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cells were able to form tumors in vivo [13,16], one scientifically based mechanism for the concern over the transfer of cancerinducing agents from vaccine to recipient was identified, viz., through DNA capable of expressing activated oncogenes. By analogy with viruses that integrate into the host genome, a second mechanism for DNA-induced oncogenesis has been proposed, viz., through the integration of DNA. There are several ways that the integration of DNA could have oncogenic consequences. If DNA integrated next to a dominant proto-oncogene such as c-myc and increased its expression level or activated its expression inappropriately, then this could result in the oncogenic conversion of a normal cell to one with a neoplastic phenotype. If DNA integration occurred in a tumor-suppressor gene, such as the p53 gene or the RB gene, resulting in the functional inactivation of that gene, then this cell might, over time, become neoplastic through loss of heterozygosity by acquiring additional inactivating mutations in the other allele. Both of these mechanisms have been seen following retrovirus infection in both avian and mammalian systems. For example, activation of a proto-oncogene that resulted in leukemia has been observed following infection of chickens with avian leukosis virus [17] and of mice with Moloney murine leukemia virus [18,19], and the tumorsuppressor gene p53 can be inactivated following retrovirus insertion [20,21]. Clearly, therefore, integration of a retroviral proviral genome can result in oncogenic events. The issue, though, is whether the frequency of integration of exogenous DNA is high enough to be of concern. Although this is not an easy question to answer experimentally with cellular DNA due to its sequence heterogeneity and genomic complexity, data derived from studies with DNA vaccines, which have less complex genomes, suggest that integration of exogenous DNA is an extremely low frequency event [22,23] and thus the frequency of integration at a particular site will be correspondingly lower. Estimates have been made of the probability of integration of DNA at a site that would result in the activation of a cellular oncogene at approximately 1010 and for two independent events, as would be required to inactivate both alleles of a tumor-suppressor gene in a single cell, at 1019 [24,25]. A second consideration for DNA integration is whether the DNA from a tumorigenic cell would be any more of a concern than DNA of a normal diploid cell, since the differences in sequences between the two cell types are minimal, and integration with DNA is not site specific but occurs through illegitimate recombination [26e31]. As such, the risk of DNA from an integration standpoint should be similar for all cellular DNAs regardless of the phenotype of the cell from which it was derived. Since amounts of DNA vaccines in the milligram range have been approved for clinical evaluation, it is difficult to imagine that the smaller quantities of residual cell-substrate DNA present in viral vaccines would pose a significant risk due to integration [32]. In conclusion, the major oncogenic concern with respect to DNA is considered to be via the introduction of a dominant activated oncogene.

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5. Infectivity activity of DNA A second activity of DNA from neoplastic cell substrates that needs to be addressed as a potential safety issue is its infectivity activity [8,33]. This activity arises due to the potential presence of infectious virus genomes in the cellular DNA either integrated or extrachromosomal. These genomes could be those of DNA viruses or of the proviruses of retroviruses (exogenous or endogenous), since many viral genomes are infectious in vitro, such as polyomaviruses [34e37], papillomaviruses [38e42], adenoviruses [43e46], herpesviruses [47e52], parvoviruses [53e58], and retroviruses [59e 65], or in vivo, such as polyomaviruses [66], papillomaviruses [67,68], and retroviruses [69e75]. (The main concern with respect to retroviral DNA would be due to the presence of exogenous retroviruses, since all human cells have endogenous retroviruses and to date none has been shown to be infectious.) Therefore, the risk from the infectivity activity of DNA would arise if the residual cell-substrate DNA encodes an infectious viral genome that, once inoculated, would produce an infectious virus in the transfected cell. If this virus were able to establish a productive infection in humans, the consequences would be unpredictable. For these reasons, the infectivity activity of DNA might represent more of a risk than its oncogenic activity. Possibly the most obvious case where infectious cellular DNA would be a concern would be if an inactivated human immunodeficiency virus (HIV) vaccine were produced from an infectious, replication-competent, pathogenic HIV. While inactivation of the virus would be monitored and the degree of inactivation quantified, the biological activity of the cellular DNA, which would contain the infectious provirus, would also need to be addressed. 6. Development of assays to assess the oncogenic activity of DNA in vivo Because opinions on the risk posed by residual cellular DNA in vaccines have varied from it representing no risk to it being considered to be an important risk factor [76e78], and because few studies had attempted to measure DNA oncogenicity in vivo and thus few data were available, we undertook to investigate the oncogenic activity of cellular oncogenes in an animal system with the aim of establishing a model that could be used to estimate the risk of an oncogenic event by DNA. We generated expression plasmids for the human activated T24 H-ras gene and for the murine c-myc gene. Our initial studies demonstrated that these plasmids when injected together were oncogenic in vivo, with NIH Swiss mice being more sensitive to oncogenic insult than C57BL/6, and newborn mice more sensitive than adult mice. Both oncogenes were required for tumor induction, and tumors were only induced at the highest amounts of DNA used (12.5 mg of each plasmid) [79]. Because this low efficiency of tumor induction would not permit the establishment of a practical assay, we sought ways to increase this efficiency. In the first approach, we placed both oncogenes (human activated T24 H-ras gene and the murine cmyc gene) on the same molecule, reasoning that this should

facilitate a single cell taking up both oncogenes. In the second approach, we evaluated both immune competent and immune incompetent mouse strains for their susceptibility to DNAinduced tumor formation. We have identified a number of strains that are more sensitive to oncogenic insult than the newborn NIH Swiss mouse. At present, data indicate that amounts of the ras/myc dual-expression plasmid down to 1 ng are capable of inducing tumors in mice (manuscript in preparation). At this level of DNA, the margin of safety from an oncogenic event from 10 ng of residual cell-substrate DNA is reduced from between 2.5  108 and 2.5  109 (estimated from our earlier data [79]) to between 104 and 105. These estimations assume a haploid genome size of 3  109 base pairs for a mammalian genome and an oncogene size of between 3000 and 30,000 base pairs. Although these revised calculations are based on results with a plasmid that expresses both oncogenes, we feel that this is justified for two reasons. First, our approach is to base estimates on the most sensitive assay system. And second, we (unpublished results) and others [80] have found that a single oncogene can induce tumors in mice. These lower estimates for the safety factor for an oncogenic event do not reach the 107 safety factors that have been considered adequate by the US FDA Vaccines and Related Biological Products Advisory Committee (VRBPAC) in 2005 with respect to residual DNA from tumorigenic cell substrates (see transcripts at: www.fda.gov/ohrms/dockets/ac/ 05/transcripts/2005-4188t1.pdf). In order to reach a safety factor of 107, methods to reduce the activity of DNA, such as nuclease digestion and/or chemical inactivation, could be incorporated into vaccine manufacture. 7. Development of assays to assess the infectivity activity of DNA in vitro As stated above, should the vaccine cell substrate contain the genome of an infectious virus either integrated or extrachromosomal, this could be a safety issue, particularly for live attenuated viral vaccines or inactivated viral vaccines manufactured from pathogenic infectious viruses. To determine what levels of DNA represented an infectivity risk, we undertook studies to ascertain the specific infectivity in vitro of the DNA of various viruses of different types. In our initial studies, we have determined the specific infectivity of the proviral DNA of the human immunodeficiency virus type 1 (HIV-1) both as a plasmid infectious clone as well as the same virus integrated into the cellular genome. An in vitro transfection/co-culture system was developed that could detect and quantify the infectivity of HIV-1 proviral DNA. This coculture system can detect the infectivity of 1 pg of an HIV-1 clone [81] and 2 mg of cellular DNA prepared from HIV-1infected cells (Sheng-Fowler et al., manuscript submitted). From the specific infectivity of HIV-1 DNA, and knowing the size of the diploid mammalian genome (6  109 base pairs) and the HIV-1 genome (10,000 base pairs), we can estimate the amount of cellular DNA containing a single provirus per cell that would correspond to 1 pg of viral DNA and produce an infection as: 1 pg O (104 O 6  109), which

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equals 6  105 pg, or 600 ng. Thus, if the amount of residual cell-substrate DNA in a product is 10 ng, then the safety factor with respect to an infectious event for cellular DNA containing an infectious viral genome is 600 ng O 10 ng, or 60. If the cell contains more than a single viral genome, then this safety factor would be reduced accordingly. As stated above, safety factors of 107 have been considered appropriate with respect to cell-substrate DNA, and thus, a safety factor of 60 or lower would be insufficient. To obtain a safety factor in the 107 range, either the level of cell-substrate DNA would need to be lowered below 10 ng, or the biological activity of the DNA would need to be reduced by nuclease digestion or chemical inactivation. Assuming that only one copy of the retroviral DNA was present, then the amount of residual cell-substrate DNA would need to be 10 fg or lower. However, if there were 100 copies of the infectious viral genome, the amount of DNA would need to be reduced to 100 ag. Reducing residual cellsubstrate DNA to these levels, even with the hardiest of viral vaccines, would likely be impractical and difficult to document. Therefore, with certain cell substrates, additional treatments of the DNA might be recommended. 8. Methods to reduce the biological activity of DNA There are three general approaches to reduce the biological activity of DNA: nuclease digestion, chemical inactivation, and irradiation. For live viral vaccines, nuclease digestion is the only method that can be used, whereas, for inactivated viral vaccines, subunit vaccines, or purified proteins, all three approaches have been used either separately or in combination. As far as we are aware, there have been no studies that have quantified the amount of inactivation of a biological activity of DNA by these treatments. Because of the sensitivity and dynamic range of the in vitro infectivity assay, we have used this assay to quantify the reduction in biological activity afforded by nuclease digestion and by b-propiolactone (BPL) treatment. Results have shown that either nuclease digestion or BPL treatment can reduce the infectivity of DNA by more than 105 folds (Sheng-Fowler et al., submitted). Current work is directed at quantifying the reduction of DNA biological activity obtained by formaldehyde and binary ethylenimine, reagents also used to inactivate viral vaccines and that also inactivate the biological activity of DNA. 9. Concluding remarks Our studies on the oncogenicity and infectivity of DNA have revealed that the levels at which both activities can be detected in sensitive in vivo or in vitro systems are lower than hitherto demonstrated. Thus, both need to be considered with respect to the potential risks associated with residual cellsubstrate DNA. Because the oncogenicity and infectivity activities were derived from sensitive assays, and because they do not take into account other factors such as that chromatin might be taken up and expressed less efficiently than naked DNA, the estimated safety factors are conservative and most likely represent worst-case scenarios. Nevertheless, because

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the safety factors with respect to either DNA oncogenicity or DNA infectivity do not reach 107 when amounts of DNA alone are considered, treatment of DNA with nucleases or chemicals, as appropriate for the vaccine, might be recommended when novel neoplastic cell substrates, such as highly tumorigenic cells or cells derived from human tumors, are to be used for viral vaccine manufacture. Finally, all decisions about the risks associated with any cell substrate need to be balanced both by the product type and the purity that the manufacturing process provides and the benefit of that product for the intended population. Acknowledgements This work summarized in this paper was supported by the National Vaccine Program Office and a contract from the Division of Microbiology and Infectious Diseases of the National Institute of Allergy and Infectious Diseases through an Interagency Agreement with CBER/FDA. We thank Phil Krause, Hana Golding, Jerry Weir, Arifa Khan, and Robin Levis for comments on the manuscript. References [1] Hayflick L. A brief history of cell substrates used for the preparation of human biologicals. Dev Biol 2001;106:5e23. [2] Plotkin S, Orenstein W, Offit P. Vaccines. 5th ed. Saunders Elsevier; 2008. [3] Robertson JS, Nicolson C, Bootman JS, Major D, Robertson EW, Wood JM. Sequence analysis of the haemagglutinin (HA) of influenza A (H1N1) viruses present in clinical material and comparison with the HA of laboratory-derived virus. J Gen Virol 1991;72:2671e7. [4] Tree JA, Richardson C, Fooks AR, Clegg JC, Looby D. Comparison of large-scale mammalian cell culture systems with egg culture for the production of influenza virus A vaccine strains. Vaccine 2001;19: 3444e50. [5] Medema JK, Meijer J, Kersten AJ, Horton R. Safety assessment of Madin Darby canine kidney cells as vaccine substrate. Dev Biol (Basel) 2006; 123:243e50 [discussion 265e6]. [6] Hilleman MR. Cells, vaccines, and the pursuit of precedent. Natl Cancer Inst Monogr 1968;29:463e9. [7] Hilleman MR. Line cell saga e an argument in favor of production of biologics in cancer cells. Adv Exp Med Biol 1979;118:47e58. [8] Lewis Jr AM, Krause P, Peden K. A defined-risks approach to the regulatory assessment of the use of neoplastic cells as substrates for viral vaccine manufacture. Dev Biol 2001;106:513e35. [9] Ishii KJ, Gursel I, Gursel M, Klinman DM. Immunotherapeutic utility of stimulatory and suppressive oligodeoxynucleotides. Curr Opin Mol Ther 2004;6:166e74. [10] Rothenfusser S, Tuma E, Wagner M, Endres S, Hartmann G. Recent advances in immunostimulatory CpG oligonucleotides. Curr Opin Mol Ther 2003;5:98e106. [11] Bishop JM, Baker B, Fujita D, McCombe P, Sheiness D, Smith K, et al. Genesis of a virus-transforming gene. Natl Cancer Inst Monogr 1978;48: 219e23. [12] Cooper GM. Cellular transforming genes. Science 1982;217:801e6. [13] Land H, Parada LF, Weinberg RA. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature 1983;304:596e602. [14] Santos E, Pulciani S, Barbacid M. Characterization of a human transforming gene isolated from T24 bladder carcinoma cells. Fed Proc 1984; 43:2280e6. [15] Bos TJ. Oncogenes and cell growth. Adv Exp Med Biol 1992;321:45e99.

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