Viral safety of biological products came to my attention many years ago when I worked within different technical groups of WHO. In the middle of the years 1980's as a virologist at the Pasteur Institute, I became more involved in the area of biological safety since colleagues from French and European regulatory systems asked me to help them.

Transmission of infectious agents by pharmaceutical has a long and rich history but I will try to mention here only a few significant examples in order to illustrate that every technical progress, that allowed a new generation of biologicals, was marked by tragic iatrogenic accidents. The term "iatrogen", that is, a disease induced by a medical procedure or a treatment, was used for the first time in the 1920's to designate the treatment of the neurological phase of syphilis by malaria initiated by the Austrian clinician Wagner von Jaureg. The plasmodium parasite was deliberately inoculated to the patients in order to provoke a high fever access capable of destroying the treponema of the syphilis. This treatment was called ""iatrogenic therapy" and Wagner von Jaureg received in 1927 the Nobel Prize.

Tissues and fluids of human and animal origin and plant extracts were probably the first medicines used by man. Tribal or religious practices led then to illnesses such as Kuru, a form of spongiform encephalopathy described by Gajdusek, which persisted in Papua New Guinea until recently.

Biologicals are a large category of medicinal products and their classification is shown in the slide 1. The risk of viral transmission by different category of biologicals is presented in the slide 2 and 2bis.

Louis Pasteur, developing the empirical observation of Jenner, created the basis of a new industry of vaccines, that used as starting material human and animal pathogenic micro-organisms. For this reason it became evident that vaccines brought, in association to their enormous benefic effect, a high risk of adverse reactions.

Severe accidents of transmission of infectious agents were reported from the early period of vaccination on. The first was linked to the Jennerian era of small pox immunization, when vaccinia material from human origin was used for vaccination by passages from arm to arm. The most spectacular accident occurred in 1861 in Rialta, Italy where 46 children and 20 nurses vaccinated with human material were contaminated with syphilis. It is very probable that during the 19th century the practice of small-pox vaccination with human vaccinia contributed significantly to the spread of syphilis.

Historically, the fortuitous spread of tuberculosis by BCG vaccine, became well known under the name of Lübeck (Germany) accident. Between December 1929 and 30 April 1930, 251 infants, in Lübeck, received, during the first 10 days of life, three doses of BCG vaccine administered by mouth. Out from the vaccinated children, 72 died of tuberculosis in the next 2-5 months, 135 developed a clinical tuberculosis but recovered and 44 became tuberculin-positive but remained well. In parallel, none of the 161 unvaccinated children, born during the same period, died of tuberculosis in the subsequent three years. This tragedy created a strong emotion of the scientific community and accounted for a number of international studies performed by well-known bacteriologists. Although it has been clear that the vaccine prepared in Lübeck in the laboratory of Dr. Georg Deycke, an experienced bacteriologist and a tuberculosis expert, contained virulent tuberculosis bacilli, it was not possible to determine the circumstances in which the BCG vaccine strain has been contaminated.

I refer in some details to this tragedy, because the Lübeck accident led to a trial, which, to the best of my knowledge was the first time a iatrogenic disease induced by a biological product, in this case a live attenuated bacterial vaccine, had legal consequences. The trial was long, since it took one year and a half, and the great British microbiologist Graham ? Wilson said that "The long-drawn-out spectacle of the trial resembled ancient Greek tragedy, played between the doctors and the fates pursuing its way relentlessly to its climax of horror and death".

Two defendants were found guilty of manslaughter by negligence in 68 cases and injury by negligence in 131 and a sentence imprisonment was passed and two other were acquitted. If the Lübeck accident was the first that ended with a trial, unfortunately it was not the last.

The list of iatrogenic accidents induced by vaccines is too long to be detailed here, but the place occupied by adverse reactions to viral vaccines was important and some of them are mentioned in slides 3 and 4.

If we examine chronologically the accidents induced vaccines we can observe that most of them occurred when a new generation of products became available. This is particularly true for viral vaccines, because the iatrogenic accidents usually paralleled a new cell substrate used for vaccine manufacturing. This was at the origin of the debate around the cell substrate safety used for the production of viral vaccines.

An extensive review of iatrogenic accidents generated by immuno-prophylactic products can be found in the book published by Sir Graham Wilson in 1966 ("The Hazard of Immunization").

The accidents provoked by vaccines have today only an historical interest. Thanks to the progress of scientific knowledge and therefore to a better risk-benefit analysis, and to the implementation of severe national and international requirements for manufacturing and control, the vaccines are today a safe category of biologicals.

The invention of a replacement therapy, pioneered in 1921 to 1922 by Banting and Best, was a major step forward in Twentieth Century medicine. These Canadian scientists identified the function of insulin in carbohydrate metabolism and demonstrated that the hormone extracted from animals was able to compensate for the pancreatic deficiency. As the same time, Landsteiner was identifying the blood groups, providing a scientific basis for transfusional medicine and a boosting blood transfusion in the 1920's. The beginning of the replacement therapy were followed, after the Second World War, by other important developments such as Cohn's alcoholic fractionation of plasma.

A look back to the 1980's shows that the AIDS pandemic surprised the medical world and before we were able to identify its causal agent, thousands of transfused patients and a large part of hemophiliac population was heavily contaminated. This tragedy will probably remain the major event that marked the public health, in the second half of our century.

After the transmission of HIV in the mid 1980's, governmental agencies in the USA and Europe, in cooperation with the industry, made a considerable effort to develop new regulations for the manufacturing and control of blood derivatives. Despite the remarkable progresses accomplished in the field of viral safety of blood and its derivatives, some iatrogenic transmission occurred until recently (Slide 5- data transmitted by PEI).

The story of blood viruses is not yet finished since new viruses are described, but their role as human pathogens is not yet well established. Nevertheless, the increased vigilance for the viral safety of plasma derivatives is justified, since they will continue to be used in the next years. At the present time, for conceptual, technical and commercial reasons, it is not possible to replace them with products obtained by other technologies, such as recombinant DNA.

Biologicals obtained recently from animal cell culture represent now a large category of products, including highly purified recombinant proteins or monoclonal antibodies. Although so far they were not involved in transmission of viruses (touch wood!) they are not exempted of risk.

These categories of biologicals are produced on large bio-reactors in media containing ingredients from animal origin (such calf sera) or from human origin (such as albumin). However, even in the absence of media ingredients from animal origin, viral contamination with a murine parvovirus has been reported.

The operator can be also the source of contamination as was the case for a rhinovirus present in a culture medium used for the production of a hepatitis A vaccine. Some recent data on the contamination in process, are presented in slide 6.

The transmission of CJD by pituitary growth hormone raised numerous and complex problems. When revisiting this tragic accident, we must consider what was rightly so stated by George Craig in the paper he published recently in the New-York Review of Books: "Our view of the past can never be a permanent one. Revision is a part of the historical process, made inevitable by the passage of the time and the change perspective that comes with it and the accumulation of new knowledge". In other terms, the knowledge we have today on CJD and its mode of transmission was not available in the early 1960's when the risk of hGH was evaluated and accepted in the treatment of hypophysary deficiency.

During the 1960's and 1970's important progresses were accomplished in the field of molecule separation and protein purification. These achievements made possible the preparation of a new generation of medicinal products, mainly toxoids and vaccines, cytokines and hormones. With hindsight, it is clear that the development of pituitary GH extracted from pituitary glands collected from cadavers in autopsy rooms, brought to the fore its clinical benefit without a rigorous assessment of the potential risk. In short, the power and the simplicity of the technology used to prepare the hormone in a form quite pure created the impression that the risk, if it existed at all, was extremely small.

Since the start of the 1920's when CJD was described in Germany, this disease remained for forty years a rare clinical curiosity, and archetypal degenerative disease of CNS occurring to elder persons. The situation changed in a spectacular manner when Gajdusek described, in 1957, Kuru disease and later, in 1965-66, when the team of Gajdusek transmitted the disease to chimpanzees. However, while the transmission of CJD to non-human primates by intra-cerebral inoculation was accepted until the appearance of the first identified cases of CJD transmitted by GH, the peripheral routes of transmission were matter of debate. We have not enough time to review in details this iatrogenic transmission and therefore I will comment only on the most significant factors that contributed to the transmission of CJD prions to patient treated with pituitary GH.

The frequency of CJD transmitted by hGH is presented on slide 7 (data of December 1998). From this table, it can be noted that in all situations, CJD was transmitted with a low frequency, a common circumstance for the iatrogenic transmission of the disease.

In slides 8, 9, 10 and 11 are presented some factors that contributed to the transmission of CJD.

The conditions in which the glands were collected from autopsy halls facilitated the cross contamination of hypophyses. Moreover, the presence of infectious material in the SNC of patients apparently healthy augmented the number of infected glands that were, probably, introduced into the pool used for manufacturing hGH.

One important notion to understand the low frequency of transmission is the existence of sub-infectious doses of TSE agents. Kimberline showed, in the case of scrapie, that the amount of infectious doses needed to transmit the disease by the peripheral route is much more higher than from the intra-cerebral inoculation. As mentioned in slide 10, at difference of conventional viruses, the repeated exposure to sub-infectious doses of a TSE agent cannot elicit an immunological response.

The low concentration of the infectious agent in the final product (Poisson distribution) is a situation encountered not only for CJD transmission by hGH, but also for BSE ("low exposure dose"- Kimberline).

It is well known that BSE affected mostly dairy herds. Farms specializing in the rearing of calves for meat production did not generally use the meat meal. Calves from dairy farms, fed with bone and meat, went on to develop the illness; both calves and their mothers were fed a diet including meat meal as a protein supplement. Overall, 85% of BSE cases involved the calves of dairy cows, reared elsewhere for meat production. The percentage of animals developing the illness within a herd increased with herd size, from 3.7% in herds of less that 50 head to 41.09% in specialist dairy farms with more that 200 head of cattle. The overall frequency of BSE in animals raised on dairy farms was 13.99% and more that 85% of all farms reported at least one case of BSE (data from Kimberline).

These figures confirm that the causal agent of BSE is not very virulent, a view that is now widely accepted. Based on the low frequency of the disease within herds, Kimberline developed the notion that the animals that developed the disease had been exposed to low concentrations of prions, which he described as "low dose exposure".

When reconstituting the history of CJD transmission by pituitary growth hormone, one notes that 1985 was the crucial year, when the correlation between the cases of CJD in young patients in the USA and the UK, and the fact that they were treated with pituitary growth hormone became evident. Why was this correlation established so late? This is a long story, but personally I think that the medical community was faced with a new problem and the example that illustrated this situation is given by Paul Brown in a paper published in 1988. A young 20-year-old patient, treated with hGH, developed CJD in 1984. Medical examination in several American University medical centers produced no firm diagnosis. Moreover, in the autumn of 1984, when the patient was still alive, a diagnosis of CJD was put forward at a pediatric neurology meeting but was rejected since the subject was too young.

However, even though there is no publication, before 1985, warning clearly about the danger of CJD transmission by pituitary growth hormone, some scientists, such as Montagnier in France or Wildy in UK evoked this risk in their reports addressed to different organizations. Nevertheless, the general opinion was so much in favor of the use of these new hormones and the safety report of the product was so good, that the decision to stop was, at that time, not justified.

It is interesting to see what happened finally in 1985 when the role of GH in the transmission of the disease was recognized. The USA and UK decided to discontinue the treatment: the same decision was taken by one of the pharmaceutical companies. In USA, Paul Brown expressed clearly doubts about the possibility of eliminating completely the CJD infectious agent. In France, based on the safety report of the hormone prepared by the non-profit organization, France Hypophyse, the treatment was continued until 1987 when the first cases of the disease appeared.

The collection of pituitary glands from cadavers is another factor that facilitated transmission, given the impossibility of selecting glands in the absence of a rapid and sensitive diagnostic test. The presence in the pool of glands contaminated with CJD prions from subjects who had died in the preclinical phase of the disease increased the probability of CJD transmission.

The analysis herein implicates a putative cross contamination during the production of the hormone as the major source of transmission. The origin of this contamination was the pituitary glands contaminated with CJD prions present in the pool of hypophyses used as a source material. The high resistance of the prions to decontamination procedures facilitated cross contamination at all of the steps involved in the manufacturing process. Such contamination must have been infrequent and its level differed between batches (Slide 12).

Under the light of current knowledge, it does not require a complex analysis to recognize that the risk associated with medicines originating from the human CNS is unacceptable. However, there was no reason to take this viewpoint before 1985 and the producers of pituitary hormone were ignorant of the large risk associated with their product.

As we have already seen, the corollary of the transmission of HIV and CJD was the particular attention given by state organizations and by the pharmaceutical industry to the viral safety of biological products and to the enforcement of national and international regulations. During the 1990's, in addition to the WHO recommendations and to the monographs of the European Pharmacopoeia, which have international relevance, Japan, Europe and the United States agreed to formulate very precise consensual norms within the framework of the International Harmonization Conference. Viral safety was considered having a particular priority in the formulation of these norms. Governmental bodies now cooperate directly with scientists in both academia and industry. Groups of experts are brought together to evaluate the risk of viral transmission of every product of human or animal origin. This policy was developed as a consequence of the BSE epidemic in the United Kingdom and the necessity of limiting the possible effects on public health. Despite the considerable efforts that have been made, we must remain vigilant because the lessons of the past show that we must be circumspect with all pharmaceutical products of human or animal origin.

 

National cell line resource banks have been created to serve industry as well as academic and governmental research institutions and agencies. Industrial scientists in many cases will require fewer lines but those generally need to be expanded and reauthenticated under GMP conditions prior to use.

This presentation summarizes steps taken by the American Type Culture Collection (ATCC), an international cell resource and supply center, to provide reference cell cultures for research, production, and testing purposes. Initial cultures are provided for propagation and final re-characterization by the recipient laboratory.

Most established cell lines have been characterized by the originator and collaborators well beyond the steps essential for quality control. Specific details include, for example, phase contrast and ultrastructural morphologies; detailed cytogenetic analyses; definition of proto-oncogene, oncogene, or oncogene product presence, nature, and location; detailed evaluation of intermediate-filament proteins; and demonstration of tissue-specific antigens or production of other specific products. These characterizations obviously increase the value of each line for research and for production work. However, cell banking organizations need not attempt to repeat all these tests before distributing the stock cultures. Decisions must be made to establish the most acceptable authentication steps, consistent with maintaining the lowest possible cost, to provide a high quality cell stock. Authentication can be considered the act of confirming or verifying the identity and critical features of a specific line, whereas characterization is the definition of the many traits of the cell line, some of which may be unique and also may serve later to identify or authenticate that line specifically. Essential steps for quality control will vary with the type of cell bank constructed; such minimal descriptive data frequently will be supplemented with a much broader characterization base for each particular cell line.

SEED/MASTER CELL BANK CONCEPT

The utility of any bank of cell lines depends upon the degree of characterization of the holdings performed by the originators, the banking agency, and by other scientists. Ready availability at reasonable cost both of the lines and such data, plus the capability to track distribution of the biologicals are additional critical considerations. Figure 1 outlines steps applied to characterize and authenticate cultures provided by the deriving investigator. Progeny from the ampule or flask culture initially supplied are utilized to produce the first or "token" freeze. Cultures derived from such token material are then tested for bacterial, fungal, and mycoplasmal contamination. The species of each cell line is verified. These quality control steps are the minimum that must be performed before eventual release of a line. If warranted, the material is expanded to produce the seed and distribution stocks. Additional major quality control and characterization efforts are applied to cell populations from seed stock ampules. Test results are derived for individual lots and, therefore, refer to specific stocks. The distribution stock consists of ampules that are distributed on request to investigators. The reference seed stock, however, is retained to generate further distribution stocks as the initial distribution stock becomes depleted. The degree of characterization applied to master cell banks or master working cell banks in production facilities is generally more rigorous, although the seed stock here, like the master cell bank, is used as a reservoir to replenish depleted distribution lots over the years. By adherence to this principle, one can avoid problems associated with genetic instability, cell line selection, senescence, or transformation.

MICROBIAL AND VIRAL CONTAMINATION

Microbial and fungal contamination of cell cultures often are overt and easily observed. Still, less apparent or masked infections occur undetected. The ATCC receives cell cultures, even for the Patent Depository, that contain cryptic bacteria, yeast, filamentous fungi, or mycoplasma.

1. Bacteria and Fungi. Microscopic examination is only sufficient for the detection of gross contaminations. Even some of these cannot be detected readily by simple observations. Therefore, an extensive series of culture tests is required to provide reasonable assurance that a cell line stock or medium is free of fungi and bacteria. Details are given in one of the reprints appended (Hay, 1998).
2. Mycoplasma. Contamination of cell cultures by mycoplasma can be a much more insidious problem. Although the presence of some mycoplasma species may be apparent because of the degenerative effects induced, other mycoplasmas metabolize and proliferate actively without producing any overt morphological change in the contaminated cell line. Thus, cell culture studies relating to metabolism, surface receptors, virus-host interactions, and so forth, are certainly suspect to interpretation, if not negated in interpretation entirely, when conducted with cell lines that harbor mycoplasma. The seriousness of these problems can be documented through published data from testing services and cell culture repositories.

   Data from seven different testing laboratories on mycoplasma infection frequencies in cell lines examined is summarized as Table 1. The results indicate clearly that there is a significant problem internationally.

   Protocols for test procedures are numerous. A sensitive Polymerase Chain Reaction (PCR)-based kit is now available for the detection and identification of the common species of mycoplasma and Acholeplasma laidlawii known to infect cell cultures. A photo of a representative gel showing amplicons generated using the kit is presented as Figure 2.

   Four general recommendations can be offered to avoid mycoplasma infection. The implementation of an effective regimen to monitor cell lines for mycoplasma is one critical step. Quarantining all new untested lines and using mechanical pipetting aids are others. Most experts also strongly suggest that the use of antibiotics be eliminated when possible. Antibiotic-free systems permit overgrowth by bacteria and fungi to provide ready indication whenever a lapse in aseptic technique occurs. When the initial tissue is used, e.g., a human tumor sample, antibiotics may be employed, but after the primary population has grown out and been cryopreserved, reconstituted cells may be propagated further in antibiotic-free medium.

3. Viruses. Verification as to the absence of viruses in cell lines is recognized as a most significant problem. Industrial production for human and veterinary applications has especially stringent requirements in this regard. That virus may coexist as noncytopathic entities (e.g., with the c-type retroviruses) or in a latent form (e.g., papilloma viruses and some herpes viruses) compounds difficulties in detection. Judicious choices are necessary not only to select appropriate methods available for recognizing viruses associated with cell lines, but also to identify the offending species. The nature of the cell line resource, its users, the budget available, and the intended purposes for which the line will be needed all affect decisions on testing. More complete detail and protocols are provided in the article appended (Hay, 1998).

CELLULAR CROSS CONTAMINATION

Wherever cells are grown in culture, serious risk exists for the inadvertent addition and subsequent overgrowth of cells from another individual or species. One cannot rely on morphologic criteria alone to recognize specific cell lines. Data-documenting problems have been collected over the years by groups offering identification services for cell culture laboratories in the United States and elsewhere (Nelson Rees, et al., 1981; Hukku, et al., 1984, Hay, et al., 1992). Results suggest contamination frequencies of 16 to 35 percent or greater.

1. Species Verification. Species of origin can be determined for cell lines by a variety of immunological tests, by isoenzymology, and/or by cytogenetics. The indirect fluorescent antibody-staining technique is used in many laboratories to verify the species of a cell line (for details, see Hay, et al., 1992). Isozyme analyses performed on homogenates of cell lines from over 25 species have demonstrated clearly the utility for species verification by determining the mobilities of three isozyme systems–glucose-6-phosphate dehydrogenase, lactic acid dehydrogenase, and nucleoside phosphorylase. Using vertical starch gel electrophoresis, the species of origin of cell lines can be identified with a high degree of certainty. Alternatively, a standardized kit employing agarose gels and stabilized reagents may be obtained for this purpose (Innovative Chemistry, 1988).

   Karyologic techniques have long been used informatively to monitor for interspecies contamination among cell lines. In many instances, the chromosomal constitutions are so dramatically different that even cursory microscopic observations are adequate. In others, for example, in comparisons among cell lines from closely related primates, careful evaluation of banded preparations is required. Cytogenetics has the advantage of detecting even very minor contaminants, on the order of 1 percent or less in some circumstances. However, it is a time-consuming procedure and interpretation may require a high degree of skill. Consult the "Atlas of Mammalian Chromosomes" (Hsu and Benirschke, 1967-1975) for examples of conventionally stained preparations from over 550 species. Detailed protocols are available elsewhere (Hay, et al., 1992).

2. Intraspecies Cross-Contamination. With the dramatic increase in numbers of cell lines being developed, especially from human tissues, the risk of intraspecies cross-contamination rises proportionately. The problem is especially acute in laboratories in which work is in progress with the many different cell lines of human and murine origin that are available today.

Methods for verifying cell line species employing enzyme mobility studies and cytogenetics have been mentioned. Using similar technology, one can also screen for intraspecies cellular cross-contamination (Hukku, et al., 1984; Hay, et al., 1992).

The application of PCR and recombinant DNA technology, cloned DNA probes, and small microsatellite loci (2-6 bp repetitive motif) to identify and quantitate allelic polymorphisms provides additional powerful means for cell line identification. These polymorphisms can be recognized as extremely useful markers, even if they are not expressed through transcription and translation to yield structural or enzymatically active proteins. For example, Edwards, et al. (1992), demonstrated the usefulness of short tandem repeat (STR) loci in differentiating humans at the DNA level. One significant advantage of STR loci over their minisatellite cousins is their small size. This allows multiplex PCR reactions to be developed in which many loci are simultaneously examined in a single reaction. The ATCC currently employs a commercially available multiplexed STR system for routinely screening new cell line accessions for authenticity, as well as validating any subsequent distribution of an authenticated cell line (Durkin and Reid, 1998; Sajantila, et al., 1992; Hay, et al., 2000).

When authenticating a new line, it is recommended that DNA be extracted from the cell line using a traditional liquid extraction method as this tends to minimize STR artifacts that may complicate allele assignment. For validation of subsequent passages of the cell line, more expedient DNA techniques may be used, which may produce ambiguous allele assignments. Generally, comparison with the authentic DNA fingerprint easily resolves these ambiguities. The STR system utilizes fluorescent labels and an automated collection device. Typical profiling results are presented in the electropherograms shown. Figure 3 represents the data generated when analyzing two cell lines derived from the same individual. Figure 4 compares the STR profiles generated from two unrelated cell lines.

NATIONAL CELL LINE RESOURCES

National cell banks have been established to provide reference lines for use by multiple investigators. Use of such cell lines assures improved research comparability both geographically and with time. Details on the more prominent, internationally-utilized cell line repositories are provided in Table 2. While there is some overlap among these organizations in terms of culture holdings, they attempt to augment each other's strengths and are in reasonably close contact with regard to problems and new methodologies. Cell line distributions range in number from about 65,000 annually (ATCC) and 16,000 holdings (CIMR), downwards.

CONCLUSIONS

In conclusion, the overall utility of any cell line resource depends on the degree of characterization of the holdings that has been performed by the originators, the banking agency, and other individuals within the scientific community. Documenting the verification of species and identity of each cell line, when possible, is considered essential. Freedom from bacterial, fungal, and mycoplasmal infection must be assured. However, from the cell banking perspective, applying all possible characterizations to every seed or master cell stock developed is neither essential nor practical. Recipients can apply specialized assays or have additional characterizations performed commercially if necessary. At ATCC, for example, screens for particular viruses have been applied when specific program support is available for such testing. Similarly, the definition of ultrastructural, tumorigenicity, and functional traits is performed given appropriate external support and adequate rationale. The central responsibility is to produce reference stocks, authenticated and well characterized for multiple purposes, and to return to those preparations over the years for development of working stocks for distribution or other specific applications. Each replacement distribution stock requires reauthentication prior to distribution to intended users.

Figure 1. Suggested scheme for the authentication of cell lines to be added to a cell line resource. Terminology and precise group of tests applied vary somewhat depending upon the goals of the client community.

Figure 2. Second-step PCR products from eight commonly encountered Mycoplasma species and Acholyplasma Laidlawii. Photo courtesy of Dr. Charles Buck, ATCC.

Figure 3. Cell lines from same individual. Comparison of the identical STR profiles for the EBV transformed lymphoblast line CRL-5957 and the tumor line CRL-5868 derived from the same female. Upper blue tracings represent the four STR loci D5S818, D13S317, D7S820, and D16S539. The lower black tracings represent the four STR loci vWA, TH01, TPOX, and CSF1PO, as well as the amelogenin locus used for gender identification.

Figure 4. Two unrelated cell lines. Comparison of unique STR profiles for the unrelated male cell line CRL-5963 and female cell line CRL-1855. Upper blue tracings represent the four STR loci D5S818, D13S317, D7S820, and D16S539. The lower black tracings represent the four STR loci vWA, TH01, and CSF1PO, as well as the amelogenin locus used for gender identification.

 

Virus structure

Viruses can be classified taxonomically according to a variety of features. Virus structure, as visualised by electron microscopy, has been an important approach in this regard, but is not the only characteristic used for classification. Nowadays, the genetic structure and mode of replication contribute significantly to virus classification. Viruses exist in a variety of physical shapes, sizes and biochemical structure. A typical virus of vertebrates is roughly spherical in shape and between 20 and 200 nm in diameter (others may be pleomorphic and/or filamentous). Viruses have an inner core containing their genetic material (DNA or RNA), which is usually complexed with protein and often replicative enzymes. This inner core is contained within a protein shell and for many viruses - the enveloped viruses - this is additionally surrounded by a lipid membrane. The diagnostic test for the presence of an envelope is the effect of a lipid solvent, e.g., ether, on virus infectivity. Such envelopes are generally derived from a cellular membrane, typically the plasma membrane, during the maturation of the virus through the process of ‘budding’. Associated with the envelopes are viral-specific glycoproteins which have an important role in the recognition of, and entry into, the target host cell. In contrast, the non-enveloped viruses typically have their outer shell of protein arranged in an ordered and tightly packed pattern (e.g., icosahedral symmetry). The subunits of this protein shell also play an important role in initiation of virus infection.

 

Virus families

Viral species exist whose hosts include all types of living organisms from bacteria, through plants to higher mammalian species. As a rule, they are highly specific and their host range is quite narrow (the species barrier), even amongst viruses whose hosts are mammalian species. The narrow host range specificity of a virus derives mainly from its ability to infect only a specific cell type of (and even within) a species.

Virus infection involves recognition of highly specific receptors on the target host cell by the virus. For enveloped viruses, the receptor-binding molecule is a virus-encoded glycoprotein protruding from the lipid membrane. Thus, any physico-chemical damage to the viral envelope (and/or consequently to the viral receptor-binding molecule) will result in the loss of the ability of the virus to attach to and infect its target cell. This property of enveloped viruses has been used to advantage in inactivation and it is the relative ease with which a lipid envelope can be destroyed which makes these viruses much easier to inactivate than their robust protein-coated non-enveloped counterparts.

The following is a list of families of enveloped viruses and some of their more common members:

Arenaviridae (lymphocytic choriomeningitis virus)

Bunyaviridae (Haantan)

Coronaviridae (human coronavirus, murine hepatitis virus)

Filoviridae (Marburg, Ebola)

Flaviviridae (yellow fever, hepatitis C, bovine viral diarrhoea virus)

Orthomyxoviridae (influenza)

Paramyxoviridae (measles, mumps, canine distemper, parainfluenza, RSV)

Retroviridae (HIV, murine leukemia virus, avian leukosis virus)

Rhabdoviridae (rabies, vesicular stomatitis virus)

Togaviridae (Sindbis, rubella)

Hepadnaviridae (hepatitis B)

Herpesviridae (herpes, cytomegalovirus, Epstein-Barr virus)

Poxviridae (vaccinia, cowpox, orf)

The above list is not comprehensive but serves to illustrate the variety of viruses of vertebrates which exist. Even within a particular family, individual members can vary in their host range, disease and sensitivity to a particular inactivation process.

The latter three families of enveloped viruses have a more complex type of envelope compared with the others, which is not necessarily derived by a simple budding process from the cell plasma membrane. Nonetheless, their envelope plays a crucial role in the infectious process and, like all enveloped viruses, they can be inactivated by methods which destroy the lipid membrane.

For completeness, the following list illustrates the more common non-enveloped virus families:

Adenoviridae (adenovirus, from a variety of animal species)

Astroviridae (astrovirus, an enteric virus, from a variety of animal species)

Caliciviridae (Norwalk virus, hepatitis E)

Papovaviridae (SV40, human papilloma (wart) virus)

Parvoviridae (B19, minute virus of mice)

Picornaviridae (poliovirus, rhinovirus, hepatitis A)

Reoviridae (reovirus, rotavirus, blue tongue)

 

Virus inactivation

There is the potential for a biological medicinal product to be contaminated by an infectious agent by virtue of the starting materials involved and/or its method of manufacture. In considering methods which could be used during manufacture to inactivate a potential viral contaminant, one has to take into account the action of the inactivation process on the biological activity of the active substance itself. A variety of approaches to achieve this have been developed over the years. The most useful generic inactivation process in use today (and for many years), is solvent/detergent (S/D) treatment.

S/D has been used in the blood fractionation industry for approximately 15 years and is very effective at inactivating enveloped viruses due to the destruction of the lipid membrane by the combined action of the lipid solvent and the detergent. In addition, S/D treatment has been shown to be innocuous to the biological activity of the various products fractionated from blood. However, solvent/detergent treatment has no effect on non-enveloped viruses and it is perhaps fortunate that the human blood-borne viruses of major concern in the fractionation industry are the enveloped viruses HIV, HBV and HCV, all of which are effectively inactivated by S/D treatment. Incidents of HIV, HBV or HCV transmission by blood products have occurred, but only with products not subjected to S/D treatment

Plasma fractionators are strongly encouraged to ensure that an effective viral inactivation step is incorporated into the manufacturing process, not only for enveloped viruses but for non-enveloped viruses also. Indeed, this is also encouraged in the production of all biological medicinal products (where possible), regardless of their origin, including the production of biotech products. Such safeguards include, in addition to S/D, other physicochemical treatments such as heat (pasteurisation or dry), pH, other chemicals, gamma-irradiation and filtration. The greater inactivation capacity of these additional processes on enveloped viruses compared to non-enveloped viruses is again by virtue of the more sensitive nature of the lipid membrane than the tightly packed, ordered array of protein sub-units which form the shell of non-enveloped viruses. For processes which may not disturb the lipid membrane itself, viral inactivation may be due to denaturation of the viral glycoproteins protruding from the envelope and which are an essential part of the viral infectious cycle. These glycoproteins tend to be much more labile than the equivalent proteins forming the shell of the non-enveloped viruses.

Viral filtration methods have been developed in recent years and are an effective approach to removing potential contaminants. These methods are generally more effective against enveloped viruses than non-enveloped viruses simply because enveloped viruses tend to be larger than the average non-enveloped virus and, as a result, easier to remove by filtration. For example, the minimum diameter of an enveloped virus is of the order 40-50 nm (and most are much larger), a particle size which can be removed with reasonable effectiveness by specialised filters, whereas many of the non-enveloped viruses are as small as 20 nm (the parvoviridae) or 30 nm (the picornaviridae), which are much more difficult to remove by filtration.

In applying a specific virucidal step during the manufacture of a biological medicinal product, it is also essential that the relevant biological activity is not compromised. In general, the virucidal steps investigated have proved to be innocuous or within acceptable limits to the quality of the biological. However, one study investigating the presence of inhibitors of Factor VIII in recipients of a preparation which had been subjected to a double viral inactivation step, found altered biological activity of the FVIII. No modification of the FVIII occurred as a result of any single viral inactivation step and it was only the combination of the two steps, S/D and pasteurisation, which were detrimental. Thus, whilst it is highly recommended that manufacturers of biological medicinal products, where possible, incorporate virus inactivation or removal steps into the manufacturing process, care has to be taken that such steps do not compromise the efficacy or safety of the medicinal product.

 

The human risk may be reduced at three different levels of the bio-pharmaceutical preparation.

A first viral reduction may be obtained from raw material screening: human plasma screening for the absence of main human pathogens, selection of healthy animals through rigourous veterinary controls, and selection of cell lines and/or cell supernatant by using in vitro and in vivo testsing.

A second reduction is usually associated to the manufacturing process since it can be able to eliminate and/or inactivate viruses. In order to evaluate this process capacity, spiking experiments need to be carried out with the more significant manufacturing steps.

My presentation will be focused on this aspect.

A third reduction may result from virological controls applied to the final product or to the concentrated bulk.

 

If we exclude gene therapy products, the viral safety strategy consists in optimizing the two first reduction ways, i.e. the raw material screening and reduction through the manufacturing process. In a large number of cases, the cumulated reduction effect of the two ways is sufficient to reach an extremely-low residual viral risk which is finally acceptable for human use.

When a too high residual viral risk is obtained (resulting usually from an non-optimal reduction capacity of the manufacturing process), controls on final product have to be introduced.

Gene therapy vectors represent a particular case for two reasons: firstly manufacturing processes are not still optimized for viral contaminant reduction (early stage of development), and secondly the product itself is usually a non-replicative virus and this characteristic needs to be controled.

Then, the viral safety of vectors is mainly supported by the raw material screening (cell banks) and by controls on the final product.

The ability of a manufacturing process to reduce a viral load has to be evaluated through rigorous strategy. In a first time, a theoretical analysis will allow to identify all the putative reduction factors of the process. Some factors are able to kill (inactivate) viruses, as high temperature, acidic / basic pH, SD treatment, UV or b irradiation, etc....

Others factors, or more precisely treatments, create a partitioning (at least two independent phases or fractions) and then a putative viral elimination if viruses and the biological active component don’t segregate in the same fraction. Examples of partitioning are protein precipitation with solvents, filtration, chromatography, ultracentrifugation, etc...

In a large number of cases, manufacturing steps are more complexe, and two, three, or more reduction parameters are simultaneously effective.

From a such theoretical analysis, two values will be deduced for each step:

- putative reduction factors for viruses,

- complexity of the spiking experiment design.

Considering these data, manufacturing steps will be selected for experimental evaluation.

Modern era vaccinology that began about 1950 was fueled by Enders' renaissance of cell culture for propagating viruses for vaccines.

1.)Its first example was that of both the live and killed poliovirus vaccines of the early 1960s. Special problems relating to safety and immunizing potency were encountered. Killed vaccine was encumbered initially by incomplete inactivation of poliovirus, uncertainty of adequate potency of individual vaccine lots, and presence of a new indigenous polyomavirus contaminant (SV₄₀) that derived from monkey kidney cells and that was incompletely inactivated in the vaccine. The SV₄₀ problem was resolved by substituting a monkey species that was free of SV₄₀ virus to provide renal tissue for cell culture. The live poliovirus vaccine, also troubled initially by SV₄₀ contamination, still retains very low residual neurotropism for man but remains the paradigm for world eradication of the polioviruses. Interference between individual polioviruses in the trivalent vaccine and coincidental natural infections in vaccine recipients may compromise vaccine efficacy.

2.)Live measles, mumps, rubella, varicella, and combined MMR vaccines, that were only a dreamy concept before 1957, required breakthrough research which related collectively to viral propagation, appropriate attenuation, safety for recipient and contact, and for achieving adequate immunogenicity with low reactogenicity. In the absence of animal models, the research was hindered by need for testing in children. Large-scale safety validation and clinical trials also needed to be carried out in children. Combined MMR vaccine, licensed in 1971, is the flagship to this day for pediatric immunization against viruses.

In contrast to the above, certain alternative measles virus vaccines encountered serious problems in routine application, as discussed below (see 4).

3.)Understanding immunopathogenesis of natural measles facilitates avoidance of and gives explanations for adverse clinical effects. Measles virus is transmitted by the respiratory route and infection at early age may be prevented by presence of maternal antibodies that disappear at different times in different individuals. An appropriate combination of humoral and cell-mediated immune responses is essential to recovery from and prevention of the disease. Natural measles, like AIDS virus, causes immunodeficiency that, in measles, may be of short- to long-term persistence and may open the way to severe opportunistic infections.

4.)An alternative (a) canine renal cell-grown vaccine, licensed in the early period, required removal from market because of excessive clinical reactions. A (b) killed measles virus vaccine gave protection of only short duration and led to severe atypical measles on subsequent infection with the wild virus in nature. A most recent explanation for this, based on animal studies, indicates an altered immune perturbation by the killed vaccine which preordains a non-protective anamnestic antibody response, with immune complex deposition and with IgE and eosinophilia. Finally (c), ordinary measles virus vaccines, used in 10 to 100 times normal infectivity titer was given in substantive numbers of 6-month old infants in developing countries under the premise that increased virus dose would overcome maternally-derived antibody. Long-term demographic studies revealed increased non-measles deaths in certain of the children where death from ordinary infections was high. The probable cause was explainable as death from measles-caused immunodeficiency that opened the way to opportunistic infections.

5.)In recent times, there have been perceived but unsubstantiated claims for vaccine-caused adverse events in children that temporally followed immunization. Recent unfounded claims for causal association between live measles vaccine and Crohn's Disease and autism have been reported. This coincides with increasing public reliance on beliefs rather than on evidence-based science in matters of health.

6.)Future viral vaccines will probably rely largely on continued development of both live and killed vaccines using the technologies of the past. Subunit hepatitis B vaccine, produced by purification from plasma or by recombinant expression of surface antigen of the virus, has given rise to general belief that subunit recombinant technologies will be a wave of the future. This belief may be questioned since there has been no encore to the recombinant hepatitis B vaccine of 1986. Subunit vaccines comprising recombinant live viral or DNA plasmid vectors have not achieved licensure to date. Oral-fed subunit vaccines produced in the tissues of transgenic plants as well as chemically synthesized vaccines are still unproved concepts. A serious deficiency of information lies in lack of determination of specific and essential B and T cell epitopes for vaccines that might be collectively assembled, in sufficient number, to overcome allelic restriction in the immune system of recipients of any particular vaccine. The future must depend on continued major breakthroughs and in understanding immunology.

7.)In some developed countries, such as the US, basic research discovery is supported by public funding. Increased complexity of the science and current requirement for establishment of feasibility before commercial development of a vaccine can begin has been marked by large gaps of basic information needed to bring commercial value to fundamental discovery. The need for a return to the public in new products and procedures deriving from taxpayer-supported research is currently under discussion. New policies are being considered that will require achievement of continuity between the basic and the applied to assure maximal benefit to the public from discovery.

Introduction

The preoccupations related to the risks of human contamination with viruses from animal sources have increased during the past years. This concern has augmented since the beginning of the epidemic of Bovine Spongiform Encephalopathy (BSE) in cattle, followed by the appearance of the linked variant form of Creutzfeldt-Jacob disease in humans. As a general rule, the analysis of the risk factors of appearance of new infections in human show that animal viruses have sometimes been involved and can be against involved in the future in human diseases. (Morse, 1995). The emergence of new infections relies on a two-step mechanism : introduction of a micro-organism in a new host, then its diffusion within the host population. In this conception of a " viral circulation ", the emergence of new viral diseases is directly function of the modification of virus-host interactions, which often corresponds to modifications of agriculture, trade, tourism, or, possibly, use of animal-derived medicinal products.

Besides the description of viruses that are well-known as zoonotic agents, we will also point out some uncertainties on the notion of species barrier and on the lack of knowledge of some of the mechanisms.

Zoonotic viruses

The main viruses known as zoonotic agents will be shown. Most of them are RNA enveloped viruses. The number of zoonotic viruses for each animal species is low, and they are responsible for a wide range of disease, from benign (Newcastle disase) to letal (rabies) diseases.

Animal viruses not known as zoonotic agents

The limits of the knowledge based on natural cases of transmission

To identify a zoonosis as such in natural conditions, it is most often necessary that the animal infection is sufficiently prevalent and/or that the human illness corresponds to identifiable symptoms, evolving as an acute disease. In these cases, the identification of human illness cases can permit to establish a relation between the human and animal infections. On the other hand, since experimental infection is indeed excluded in human, it is in practice extremely difficult to prove that a micro-organism is not a zoonotic agent. For numerous viral families, the risks of transmissibility from animal to humans are unknown. The risk assessment, when conducted, passes by the comparison of the strains isolated from human and animal, serological investigation and case-control or cohort epidemiological investigations. All these approaches endure numerous limits : the comparison of strains isolated from humans and animals rarely permits to conclude univocally on risks of transmissibility; serological surveys give information on the circulation of antigenically related viruses in animal and human populations, but these viruses can in fact belong of different biotypes.

These approaches are impossible or delayed in the case of emergent diseases, quite frequent in farm animals, and are, in practice, rarely conducted. Nevertheless, for scrapie of sheep and Bovine Leukaemia Virus (Retroviridae), they provided results in disfavor of a risk for the human health. On the other hand, coronaviruses of the cat (feline infectious peritonitis) and of the pig (Transmissible Gastroenteritis and Respiratory Coronavirus) are close to the human respiratory coronavirus 229E: nevertheless, the very few number of investigation conducted on this virus doesn't permit a satisfactory assessment of the risks of inter-species transmissibility, even though an animal coronavirus has been suggested as a factor of human nephropathies (Uzelac - Keserovic and al., 1999). Influenza viruses represent a complex situation: the aquatic birds and pigs seem to play a role of reservoir for different strains that can undergo genomic reassortments, leading to the emergence of strains pathogenic for the human. Nevertheless the natural transmission from animals to human is a rare event, whereas the transmission from human to animal (particularly pigs) appears more frequent (Wentworth and al., 1997; Zhou and al., 1996)..

The more general case of inter-species transmissions

The risks of transmission of animal viruses to human are only a particular situation of inter-species transmission. Examples in the domain of the veterinary virology should incite to be vigilant in risk assessment. For example, about ten years ago, numerous cases of serious pestivirus infection appeared in pigs, after the use of a live vaccine against Aujeszky’s disease contaminated with a pestivirus of the sheep. However, no case due to natural contamination had never been recorded in pigs, probably because of the low probability of this transmission, due to the modalities of farming of these two species (Vannier and al., 1988). One must add that the situation at one time doesn't prejudge the future situation. Again more frequently than in human populations, new variant strains or new viruses emerge regularly within the different animal species, and present modifications of organ tropism or target species. For example, a variant of the parvovirus of the cat emerged in 1978 and killed thousands of dogs after a worldwide dissemination (Osterhaus and al., 1980). The calicivirus of the hemorrhagic disease of rabbits was unknown a few years ago and is currently distributed through the whole world (Carhuman and al., 1998; Marchandeau and al., 1998). The coronavirus of the Transmissible Gastroenteritis of the pigs, an enteric virus, underwent a mutation of a few aminoacids of one of its glycoproteins that transformed its tropism and led to the emergence of a respiratory strain that spread through Europe by aerial transmission (Delmas and al., 1993; Sestak and al., 1996; Vaughn and al., 1995). Highly virulent strains of ovine and bovine pestivirus appeared during the last years, and were responsible of hemorrhagic fever, very different from the usual symptoms (Liebler and al., 1995; Pellerin and al., 1994; Weiss and al., 1994). Human viruses can also be involved : it is likely that the AIDS virus originated from an animal virus (Dixon, 1999; Gorhuman, 1998; Nathanson, 1998). Very recently, some lethal human cases have been recorded after contact with a horse which dead from a Paramyxoviridae (morbillivirus) infection, non described until then (Murray and al., 1995; Selvey and al., 1995; Williamson and al., 1998). Other paramyxoviruses of porcine origin were also recently involved in human infections (Anonymous1999a; Anonymous1999b; Anonymous1999c).

Some recent data should also incite to vigilance : the Borna virus (infecting mammals and some birds) was associated to syndromes of dementia in human (Bode and al., 1995; Deuschle and al., 1998; Ferszt and al., 1999). Finally, the recently described human hepatitis E virus could have an animal origin (Meng and al., 1998)

Is there potential risks associated with virus abortive cycles ?

Most animal viruses are unable to do a replicative cycle in human cells. It is rare that the stage where the viral cycle is blocked is perfectly known. It is likely that a lot of animal viruses don't possess the receptors able to interact with human cells in the first steps of the cycle (adsorption and penetration). Nevertheless some among them can penetrate into the cells and be blocked in early phases of the viral cycle (before the replication of their nucleic acid) or at a later stage (encapsidation, maturation...). Replication-defective viruses are besides currently a privileged tool of gene transfer in vivo. For example, the avian adenovirus type 1 (CELO) or the canine adenovirus type 1 are incapable to replicate in human cells, but they can enter in these cells and express their early genes. Some of early viral genes are potent transactivators that are able to activate viral and cellular promoters. Other early genes have complex functions and are implicated in cellular transformation because of their interactions with some cellular proteins (E1A of adenovirus or human papillomavirus E7 with the p105-RB, E1B of adénovirus and E6 of HPV6 with p53). The fact that most animal viruses undergo abortive cycles in human cells is a good guarantee that they are at low risk of inducing acute infectious disease in human, which fits well to the common experience. Nevertheless, it doesn't permit currently to disregard induction of chronic diseases. The example of the SV40 virus is classic: it establishes a lytic cycle in cells of the host species (monkey); in mouse cells, the cycle is abortive but the expression of the T antigen leads to cell transformation. In the same viral family, the bovine polyomavirus, a frequent contaminant of calf serum, is capable to transform in vitro non permissive cells, as murine cells (Parry Gardner and, 1986; Schuurhuman and al., 1991). It is necessary to insist on the fact that the described risks are potential, and don't have currently received confirmation. On the contrary, the accidental inoculation of SV40 virus to human through contaminated poliomyelitis vaccines produced in monkey primary cells, or the avian leukosis virus through the use of yellow fever vaccines produced in contaminated eggs, did not lead to any harmful consequence for the human health. However, genomic sequences related to simian SV40 virus have been discovered in human mesotheliomas (Carbon and al., 1994, Fisher and al., 1999; Hirvonen and al., 1999), which could reopen the debate.

Some molecular mechanisms of the species barrier

Different stages of host- and cell-virus interactions can be involved. We will present in the following lines some of them.

A "natural" antibody response

Human serum contain antibodies that recognize some xenoantigens, in particular lipids or sugars, following contact or even without previous contact with the corresponding antigens. These antibodies may have a large specter of recognition, including virus whose envelope contains these lipids or whose glycoproteins contain these sugar residues. For example, primates of the old world (including human) don't possess the enzyme a 1-3 galactosyltransferase and, then, glycoproteins containing the disaccharide Gal(a 1-3)Gal are recognized therefore as xenoantigens. So, the animal retroviruses that incorporate these glycoproteins in their envelope are spontaneously lysed by human serum in the presence of complement (virolyse). The same virus, after replication in human cells, is not recognized by these antibodies. It is the case of Porcine Endogenous Retrovirus (PERV), which is susceptible to human serum when grown in porcine cells but is insensible after multiplication in human cells (Patience and al., 1997; Patience and al., 1998; Takeuchi and al., 1998). This notion has implications concerning virus risks associated with grafts of porcine origin in human.

Virus-cell interactions delineate the permissiveness of cells to infection

The permissiveness of cells to virus infection is dependent of many factors. We will remind some them briefly here by illustrating them by two examples.

Cell-surface virus receptor(s) permits the adsorption and the entry of the virus into the cell.

The aminopeptidase N (APN) is expressed at the apical pole of enterocytes and acts as the receptor for the TGE coronavirus of pigs, which tropism is enteric. The insertion of the APN gene in some non-permissive cells render them permissive to viral infection. This virus binds to its receptor by an interaction with the S glycoproteine (Delmas and al., 1992; Delmas and al., 1994). Expression of the pig APN in canine cells makes these cells permissive to TGE infection (Benbacer and al., 1997). It is interesting to note that the domain of interaction of the S glycoprotéine with depends APN only on some amino acids (Delmas and al., 1994), whereas coronaviruses, as a lot of RNA viruses, possess a high mutation rate.

To scale up from cells to organism, another example is the mouse, which is not susceptible to human poliovirus. Mice experimentally expressing the receptor to the poliovirus, a glycoprotein harboring sialic acid, were generated. These mice became then receptive to the poliovirus (Koike and al., 1991). Nevertheless, the tropism of the virus did not strictly parallel that of the distribution of the receptor in the organism, which demonstrates the existence of other factors of tropism (Ren Racaniello and, 1992; Zhang Racaniello and, 1997).

the presence of transcription factors necessary for viral nucleic acid transcription

The presence of the receptor is therefore not always sufficient to make cells susceptible to infection. For example, the HIV virus is not capable to achieve a complete productive cycle in mouse cells even expressing genes of the human receptors CCR5 and CD4 (Browning and al., 1997). Other steps are limiting : the Feline Immunodeficiency Virus (FIV) doesn't replicate in human cells, even after having forced its entry by direct artificial introduction of its genome in these cells. The LTR (a sequence repeated at each extremity of the genome) of the Retroviridae contains the transcriptionnal regulatory sequences of the viral genes. The replacement of the LTR of the FIV by the the early promoter of the cytomegalovirus allow for the replication of the FIV in human cells (Poeschla and al., 1998).

One will understand that numerous steps of the viral cycle, non illustrated here, can act as limiting steps in virus infection of heterologous species. Unfortunately for risk analysis, these steps are generally not identified, which limits the analysis of the efficiency of the corresponding blockage.

Conclusion

The notion of species barrier covers several aspects 1) epidemiological constraints defining a weak probability of exposition to a particular virus 2) molecular mechanisms limiting or preventing the replication of the virus in a different host. Although these mechanisms proved to be generally robust, they can be by-passed in certain circumstances linked to the modifications of ecosystems, iatrogenic conditions, increase of exposure… that can select new variants able to replicate in these new hosts. The analysis of the mechanisms of such breakthroughs can prove useful to anticipate the risks associated with some viruses and/ or epidemiological situations.

 

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Introduction

Transmissible subacute spongiform encephalopathies (TSE) are fatal human and animal neurological diseases that are characterised by a long asymptomatic incubation phase followed by a subacute clinical phase. They are caused by infection with Transmissible spongiform encephalopathy agent (TSA) also named prion which nature is still debated. Human transmissible spongiform encephalopathies are Creutzfledt-Jakob Disease (1,2), Fatal Familial Insomnia (3), Kuru (4) and Gerstmann-Sträussler-Scheinker syndrome (5). In animals, TSE are natural scrapie in sheep and goats (6), bovine spongiform encephalopathy (7), feline spongiforme encephalopathy, transmissible mink encephalopathy (8), and chronic wasting disease. The analysis of purified infectious fractions removed from infected brains are mostly if not only composed of one host-encoded protein, the prion protein (PrP), and free of any detectable specific nucleic acid (9). In animal models, the infectivity titre is always associated with a proportional accumulation of PrP. Aminoacid sequences of PrP obtained from normal and infected brains are identical, and these two proteins differ only by their biochemical and biophysical behaviour: in particular, PrP from TSE individuals resists proteinase K digestion. PrP-c is the PrP found in normal individuals; PrP-res is the proteinase K resistant PrP identifiable in infected individuals. Moreover, PrP-res accumulation is never associated with an increase of PrP messenger RNAs: this accumulation is post-translational (10). Lastly, infected organisms accumulate their own PrP-res and not the PrP-res used for infection. This data points out that infectivity is strongly associated with host-coded PrP-res. Today, it is not known if PrP-res accumulation is the TSE agent itself, or if it is a neuropathological event related to spongiosis and neuronal death, although most of the recent data support the "protein only" hypotheses. This may be a new type of disease, induced by a post-translational modified host-encoded protein.

Biochemical and biochemical properties of TSE agents.

1. Inactivation of TSE agents

Prions resist almost all the procedures generally used to inactivate conventional viruses (see J.C. Darbord, this issue). Due to their biophysical properties, prions behave differently depending upon the biophysical and biochemical characteristics of their environment. Moreover, the sensitivities to physical and biochemical disinfectants are different from one strain to another. For instance, as examples, the following procedures or compounds do not totally inactivate the scrapie agent; 1) Dry temperatures superior to 160°C during 24 hours (11) and at 360°C during 1 hour ; 2) Autoclaving at 121°C during 1 hour reduces infectivity titre of 7.5 logs (12); 3) Ultra-violet 37% inactivation dose is 42,000 Jm-2 (13); 4) 25,000 Gray of X rays only partially inactivates the agents (14); 5) No inactivation occurs after treatment with 10% formaldehyde (15); 6) Beta-propiolactone (16); 7) Sodium hypochloride 0.5% during 1 hour reduces infectivity of 4 logs (17); 8) Hydrogen peroxide 3% during one hour: 0.8 log (17); 9) these agents resist to 100% ethanol treatment.

Today, three procedures can give raise to inactivation which degree is compatible with biological safety: 1) autoclaving at 134/136 °C during 18 min; 2) treatment with sodium hydroxide 1 M during 1 hour at 20°C 3) treatment with sodium hypochloride during 1 h at 20°C (18). One should note that formaldehyde treatment before autoclaving reduces the efficacy of thermal inactivation (15).

All these inactivation data suggest that proteins play a major role in TSE agents pathogenetical properties, and that nucleic acid may not be required for TSE-infectivity.

2. Biochemical components of the infectious fractions in TSSE.

The size of the prions is estimated between 15 and 40 nm (13,14). Nevertheless, this size may have been overestimated because of the main biophysical property of this class of agents, their hydrophobicity (19); these agents may easily aggregate and therefore biased any estimation of their size by serial ultrafiltration.

The inactivation processes which have been demonstrated to be efficient against scrapie agents are those denaturing or hydrolysing protein components: treatment with high doses of proteinase K or trypsine (19), SDS, diethylpyrocarbonate, guanidium thiocyanate, urea alters infectivity (20,21,22,23,24). Procedures that interact with nucleic acids do not modify infectivity titres: nucleases, UV treatment, Zn++ hydrolysis, and psoralens (9,23,25). Therefore, one may hypothesise that prions are either constituted of proteins or harbour a small nucleic acid which is protected into a "shell". Today the "protein only" hypothesis is supported by the majority of experimental data although none of them is 100 % conclusive.

At the beginning of the eighties, S.B. Prusiner identified a 27-30 kDa protein associated with infectivity that was partially resistant to proteinase K digestion : the protease resistant protein PrP (26). The partial sequence of PrP was identified in 1985 (27). Molecular hybridisation with synthetic oligonucleotides specific of PrP-sequence showed that there is no DNA or RNA specific for PrP detectable in the infectious fractions. DNA and RNA specific for PrP are present in the infected and non-infected brains, in similar amounts. These results were crucial in demonstrating that PrP was a normal component of the host, accumulating according to a post-translational mechanism in the brains of infected individuals (28,29). PrP amount is 50 times greater in the brain than in other organs (19,22). PrP aminoacid and gene sequences have been identified: in man, PrP is a 253 aminoacid protein. This protein is associated with cell membranes by its hydrophobic C-terminal portion to which a glycosyl-phosphatidyl-inositol binds. Its half life is 5 hours for the normal PrP, and greater than 15 hours for the PrP associated with infectivity. The PrP gene is on the chromosome 20 in man (one copy per genome); it contains two exons, although a third one is now suspected; the whole coding sequence is in the same exon. PrP-mRNA is 2.1 kilobase long, and is detected in almost all the organs at various levels: brain, lung, spleen, heart, etc... (lowest expression in liver and testicle, highest expression in the central nervous system) (30). In the brain, PrP-mRNA are localised mostly in neurones, and are also detectable in astrocytes and microglial cells.

3. PrP-res is a pathological molecular isoform

Differences between the PrP isolated from normal individuals (PrP-c) and PrP isolated from infected individuals (PrP-res or PrP-sc) have been investigated. There are no differences in the sequence in aminoacids, and the secondary structure seems identical. On the other hand, the sensitivity to proteolytic enzymes is different, since normal PrP is totally degraded by proteinase K concentrations that only partially alter the pathologic isoform, the PrP-res (31). Scrapie Associated Fibrils (SAF) or prion rods are identifiable in brain homogenates from infected individuals (32). They are composed of PrP-res; antibodies raised to PrP recognise SAF, and PrP-res precipitates in rods resembling SAF in particular physicochemical conditions (19).The presence of the PrP-res is specific for TSE. Its detection is the basis for the molecular diagnosis of this disease. The kinetics of PrP-res accumulation has been studied in a number of experimental animal models: results have confirmed that it is proportional to the increase in infectivity.

The 3D structure of the PrP-c has been now determined in mouse and in hamster (33,34); the PrP molecule is composed of a globular core (aminoacids 121-231) attached to the cell membrane by a GPI anchor, and a long flexible tail (aminoacids 1- 121) that can adopt several conformations depending on the physicochemical conditions. The globular part of the molecule includes 3 alpha helices and two small beta sheets. Mainly due to aggregability, it has not been possible to obtain the 3D structure of PrP-res until now.

Cellular trafficking of the protein is now known in several cell lines (see S. Lehmann, this issue) (35,36,37) : transconformation of PrP-s into PrP-res may occur during PrP-c reinternalization through a caveolae like mechanism. This may explain why PrP-c is releasable by PIPLC treatment although PrP-res is not, due to its intracytoplaplasmic localisation.

4 The role of the PrP-c

PrP-c seems to be a neuronal protein, but at present its precise function remains unknown. In vivo data indicate that PrP is located close to synaptic proteins in the CNS. Transgenic mice which lack a PrP gene (PrP0/0) may either grow normally, without any defect in CNS functions or have alterations in long term potentiation or rapid ageing of Purkinje cells of the cerebellum (38,39,40) In TSEs, PrP-c main role is linked to genetic susceptibility. For example, transgenic mice have been grown with the hamster PrP gene: these hamster-PrP transgenic mice react as hamsters when infected with hamster scrapie (41,42) .

Human PrP gene (PRNP) is the main determinant of genetic susceptibility to TSEs [for review: (43,44,45)]. In all familial cases of familial TSEs [familial Creutzfeldt-Jakob disease cases (fCJD), Gerstmann-Straüssler-Scheinker syndrome (GSS) and Fatal Familial Insomnia (FFI)] a mutation of the PRNP gene has been evidenced:

- codon 102: Pro -> Leu, in GSS;

- codon 117: Ala -> Val, in GSS;

- codon 178: Asp -> Asn, in the familial CJD and FFI;

- codon 198: Phe -> Ser, in GSS;

- codon 200: Glu -> Lys, in the familial CJD

- repetitions (10, 11, 12, 13 or 14 times) have been indentified in the zone coding for the 5 repeated octapeptides of CJD.

These various mutations have been linked to the clinical evolution or specific neuropathological criteria, alhough it has not been possible to associate any mutation with the sporadic cases which represent the majority of Creutzfeldt-Jakob diseases (85 to 90%).

A polymorphism at codon 129 exists among the general population: valine (val) or methionine (met) could be encoded. 50% of the healthy individuals are homozygous, mainly met/met. A large and significant excess of homozygosity at the codon 129 of the PrP gene in the sporadic and iatrogenic forms of Creutzfeldt-Jakob disease (46,47,48). Aminoacid 129 is located in one of the two small B-sheets that have been evidenced in the globular domain of the PrP-c, and which may play a role in either dimerisation or initiation of the transconformation of the protein into its pathological isoform.

The role of PrP-c in prion susceptibility has been clearly demonstrated by the lack of infection of PrP transgenic mice lacking the Prnp gene (PrP0/0) (49).

Pathogenesis of prion diseases

Two major questions have to be raised in the field of prion diseases: 1) what are the mechanisms by which PrP-res induces neuronal death and gliosis that are observed in infected individuals? what are the target cells of TSE agents in periphery, before they make their neuroinvasion?

Recently, PrP-derived peptides (PrP 106-126) have been shown to exert a toxic function on neurones in culture. These peptides aggregate into fibrils similar to prion rods or SAF. This toxic effect is related to apoptosis, and occurs only if neurones express PrP-c on their membrane, and if microglial cells are present in the cell culture. This neuronal death may be related to neurotoxic factors released by glial cells. Moreover, exposure of microglial cells is capable to induce pro-inflammatory cytokine gene expression (IL-1ß and IL-6), that is comparable to what is observed in experimental mouse scrapie. Furthermore, PrP-res and Pr 106-126 are capable to induce GFAP gene overexpression by astrocytes, which mimics gliosis observed in vivo during TSE. Several experimental data have shown that PrP-res accumulation is capable to induce neuronal spongiosis and gliosis (50) only in PrP-c expressing cells. These facts might suggest that PrP-res accumulation constitutes the starting event of neurodegeneration, and is therefore the critical determinant of the neuronal death (50).

Infection of individuals by peripheral route results in a primary infection of the immune system: for example, after oral route, the presence of infectivity is detectable in the Peyer patches of infected animals soon after their inoculation (51). This infection of primary replication sites is followed by a dissemination of the agent into secondary lymphoid structures from which neuroinvasion is possible through retrograde axonal transport of the agent using peripheral nerves that innerve lymph nodes. After infection of the CNS, the infectivity develops almost exponentially until the appearance of clinical signs and death. These facts illustrate two main characteristics of TSE: 1) Certain organs of the infected individual, and particularly the brain, are highly infectious long before clinical signs appear; 2) These diseases develop without interruption and without any disappearance of the infectious agent during clinical latency. The nature of the cells involved in primary replication and in transport of prion to lymph node is not known, although elegant experimental data support the implication of B lymphocytes (52). In lymph nodes, follicular dendritic cells (FDC) are the site of TSE agent replication as assessed by immunohistochemistry: this cell type may permit TSE agent persistence, and therefore be critical for neuroinvasion.

The nature of the causative agent: current hypotheses

Any hypothesis related to TSE agent nature should take into consideration the following facts :

- TSEs have a long incubation phase, without clinical symptoms

- When started, the clinical course of the disease is slowly evolving, without remission. These diseases are always fatal;

- No inflammatory process is identifiable in both blood and cerebrospinal fluid (CSF): none of the usual immunological stigma or specific signs of chronic infections are observed in infected individuals;

- Neither immunostimulation nor immunosuppression alters the course of the disease;

- No virus or micro-organism like structure is identifiable in the brains of infected patients.

- Transmissibility can be effected by injecting ultrafiltrates of organ extracts from infected individuals: the central nervous system is by far the most infectious; so is the spleen but 104 times less than the brain.

- It is possible to demonstrate "strain specificities" suggesting the presence of an infectious agent, and not only a transmissible agent.

- Infectivity depends on the amount injected and on the route of infection: the intracerebral route (IC) is the most effective and the oral route (OR) the least effective (1 IC infectious unit = 25,000 OR infectious units).

- There is no interference with other viruses;

- There is no cytopathogenic effect in cultured cells in vitro.

- In vivo, there is no alteration of the B, T, and non-T-non-B cells (quantitative or functional).

- Today, no specific nucleic acid has been detected in association with purified infectious fractions; moreover, no non-self coded protein is associated with infectivity; this has to be taken into consideration with regard to infectious titres which may reach more than 109 infectious units per gram of brain in animal models.

- The main component of infectious fractions is PrP-res; accumulation of PrP-res is proportional to infectivity, and neutralisation of infectivity is obtained by anti-PrP antibodies.

- Familial cases are linked to PrP gene mutations

- Mice devoid of PrP are not susceptible to TSE agent infection.

Even among the most recent hypotheses, not one gives a definite and complete account of the biologic, epidemiological and clinical observations. Several hypotheses have been raised:

1) the virino hypothesis according to which the agent would consist of a nucleic acid enveloped by host proteins (accounting therefore for the lack of an immune response), 2) the unknown conventional virus,

3) the animal equivalent of plant viroid (the lack of sensitivity of TSA/Prions to nucleases is not compatible to the RNAse sensitivity of viroids),

4) Retroviruses have been also evoked as potential etiologic agents of TSE: it is well known that retrovirus may induce spongiosis in central nervous system of mouse (CasBR-E), but viral antigens are detected in infected cells, which is never observed in TSSE,

5) Other hypotheses accord a pre-eminent role to PrP as the agent or a major constituent of the infectious fractions.

- The prion hypothesis/post-translational disease hypothesis: this theory is strongly supported by the most recent molecular results. In this model, PrP-c may be converted into PrP-res by an autocatalytic process. The differences between the 3D structures of PrP-c and PrP-res are not known today; recently, it was hypothesised that transconformation occurs through direct interactions between PrP-res and PrP-c and that part of the alpha helices are transformed into beta sheet structures during this conversion process (53). Cellular factors, like chaperone molecules can participate to PrP-c transconformation (54).This transconformation of PrP could be facilitated by cellular factors (factor X) as demonstrated recently by Telling et al (54). The prion hypothesis is supported by the possibility of PrP-res to convert PrP-c into a protease resistant isoform by direct contact in an acellular experimental system (55); infectivity of this de novo transconverted PrP-res needs to be still demonstrated.

An other theory has been proposed Liautard (56): the chaperonin molecule disease. Chaperonin are proteins which role is to ensure the folding of normal proteins in the cell; several proteins (as Heat Shock Protein Hsp60) are their own chaperonin. One may suggest that PrP is its own chaperonin, and that PrP-sc are abnormally refolded proteins; in the presence of PrP-res, unfolded-PrP folding is driven into abnormal conformation. This lack of functional refolding might therefore became autocatalytic-like.

Conclusion

Several features have highlighted TSEs in the past decade. First, the scientific data that support the prion hypothesis are more and more convincing, although not 100% demonstrative. This new concept on 3D-structure protein-related pathogenicity is stimulating and me be extended to other situations of the medicine. Second, the occurrence of bovine spongiform encephalopathy in UK and at lower levels in other European countries has demonstrated the risk linked to these agents and their major economic consequences. Third, there are many consequences of the emergence of the new variant of Creutzfledt-Jakob disease. If the link between BSE is proven, it demonstrates that certain strains of animal prions are capable to contaminate humans and to induce clinical spongiform encephalopathies. Whatever the origin of the vCJD is, its emergence raises the question of a specific risk associated with tissue grafting, organ transplantation and blood transfusion. The distribution of infectivity in the organism of infected individual is then of crucial importance. Few is known on the infectivity in vCJD at the clinical stage of the disease; it has been recently reported that PrP-res could be identifiable in tonsils, in spleen and in lymph nodes (57,58) in opposite to that has been found in familial and sporadic CJD. This presence of vCJD agent in the lymphoreticular tissues raises the question of the risk associated with blood transfusion and tissue/organ transplantation. This will require special and careful investigations that will have to be extended to the inactivation spectrum of the vCJD agent in order to prevent its dissemination through the daily medical and/or surgical practice.

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58. Hill AF, Butterworth RJ, Jioner S, Jackson G, Rossor MN, Thomas DJ, Frosh A, Tolley N, Bell JE, Spencer M, King A, Al-Sarral S, Ironside JW, Lantos PL, Collinge J. Inverstigation of variant Creutzfeldt-Jakob disease and other human prion diseases with tonsil biopsy samples. Lancet 1999; 353: 183-189.

Variant Creutzfeldt-Jakob disease (vCJD) was recognised in 1996¹ and the novelty of the clinico-pathological phenotype, together with the identification of cases only in the UK, led to the hypothesis of a causal link with bovine spongiform encephalopathy (BSE). There is now compelling scientific evidence in support of this hypothesis^2,3, but the exact mechanism of transmission of the BSE agent to the human population is unproven. Cases of vCJD continue to occur in the UK and 2 cases have been identified in France and one in the Republic of Ireland, but there are a number of scientific uncertainties that preclude accurate predictions of the future numbers of cases. The possibility that there might be significant numbers of individuals incubating vCJD has led to concern about potential mechanisms of secondary transmission.

As of 31.1.2000, 52 cases of vCJD had been identified in the UK of which 24 were male and 28 female. The years of death of these cases were 1995: 3 cases, 1996: 10 cases, 1997:10 cases, 1998: 17 cases, 1999: 11 cases, 2000: 1 case. Data for deaths in 1999 is incomplete, but it is unlikely that the total will exceed 13 cases. Two cases of vCJD have been identified in France, neither of whom had visited the UK and one case has been identified in the Republic of Ireland. This case had a history of residence in the UK for a period of a few years.

The mean age at death in the 52 UK cases was 29 years (range 15-54 years) and the median duration of illness was 14 months (range 7-38 months). The clinical phenotype in vCJD is remarkably uniform in comparison to the heterogeneous clinical features in sporadic or familial CJD and diagnostic criteria for vCJD have been formulated that have a high sensitivity and specificity. Bilateral pulvinar high signal on MRI brain scan is an important component of the criteria and is likely to be of practical value in the identification of cases⁴.

Genetic analysis is available in 49/52 cases of vCJD in the UK. All cases are methionine homozygotes at codon 129 of PRNP and no mutation has been identified despite full sequencing of the open reading frame. A case-control study has not demonstrated an increased risk of vCJD in relation to diet, occupation or potential medical exposures. However it is important to stress the methodological constraints of this study, which include limited statistical power and the necessity to obtain relevant information from a surrogate witness.

Pathological information on prion protein immunostaining in lymphoreticular tissues has demonstrated positive staining in spleen, lymph node and appendix in vCJD⁵, which contrasts with negative findings in similar tissues in sporadic CJD and controls. There is a possibility that the tissue distribution of infectivity in vCJD may be distinct from other human prion diseases and this has led to concern that there may be a risk of onward transmission of vCJD through blood transfusion and perhaps surgical instruments. Measures to minimise the risks of such iatrogenic transmission have been taken or are under consideration.

References

 

Gene therapy, correction of a genetic disorder, is based on the transfer of one or several genes to target cells, by means of vectors. Up to now, vectors are divided in two major classes, non viral and viral.

In the context of gene therapy mediated by the latters, safety concerns are of major importance. Indeed, no robust elimination and / or inactivation step can be included in the manufacturing process because of the risk of impairing the therapeutic properties of the viral vector.

Hence, as for any bio-product, adventitious contamination should be carefully monitored by sensitive screening of all biologicals used during production (cell lines, culture medium, ...) 1.

Also, safety of the product itself has to be controlled as it is based on one step infectious viruses, from the manufacturing steps to ensure that no replication competent viruses (RCV) are contaminating the product, to the biohazards that may cause the use, the biodistribution and the dissemination of viral vectors. Those aspects will be the focus of this presentation.

Since the first steps of viral mediated gene therapy, production of RCV-free vector stocks have represented one of the main concern in the gene therapy field as injections of vector stocks contaminated with murine replication competent retroviruses (RCR) was shown to be harmfull to primates 2.

From the first generations of murine retroviruses-derived 3-6 and human adenovirus-derived vectors 7-9, to the AAV- 10-13, herpes- 14-18 and lentivirus-derived 19-21 vectors, almost twenty years of experience in vectorology have allowed considerable amounts of data and knowledge to be aquiered, even making HIV a promissing candidate to be used as a gene therapy vector.

Whatever the type of vectors, it is established that the presence of homologous sequences between the vector backbone carrying the transgene on one hand and with viral genes needed for the production in trans of the viral proteins (capside, enzymes, envelope) on the other hand allows recombinations events leading to production of RCVs 22-30. Improvements of structures defavoring recombination events have been developed in order to lead to almost RCV-free productions of the common vectors 12,31-36. Safe means of productions of some vectors (lentivirus, AAV) will be the focus in some of the following presentations.

Even though RCVs breakthrough now appear to be rare events, their presence in preparations of gene therapy vectors constitute potential safety risks 2,36,37 and have to be evaluted. Regulatory requirements concerning RCVs testing evolved with knowledge and with always more impressive results in production levels 38-42. As higher vector concentrations are obtained, higher sensitivity in detection techniques are needed and development of new tests are required. We will present the current techniques of testing for replication competent retroviruses (RCR) and adenoviruses (RCA) and point out the latest regulatory requirements.

Evolution in vectorology raises also some specific concerns :

- Targeting of vectors 43-45 to specific tissues imply the infection of cells that may naturally be resistant to parental wild type virus. Hence, apparition of RCV with new tropism constitute a potential safety risk 46,47.

- Increasing titers of retroviral vectors may allow encapsidation at higher levels of non-specific RNA (leading to transfer of unwanted gene 48) or of retrovirus-like elements 49-51.

 

Production of RCVs-free lots of vectors is not the only concern in regard to viral safety. In the case of ex-vivo gene transfer, safety evaluation can be performed to verify the integrity of the modified cells prior to their re-injection to the patients - if delays are suitable. In the case of direct in vivo gene transfer, safety evaluations of vector use are needed, as many adverse effects are theoretically expected or reported. Some of these risks are described below.

Toxicity : Direct injections of high amounts of viral vectors or vector producing cells may be accompanied by adverses effects. Preclinicals and clinicals studies were performed to evaluate these effects 52-58. It appears, in the case of adenoviral vectors, that there is a vector related dose-dependent toxicity mainly due to immunological responses. The death in september 1999, of a patient involved in a phase I clinical trial evaluating cytotoxicity of an OTC-adenovector (Ornithine Transcarbamylase), highlighted safety issues concerning this type of vectors 59.

Mutagenesis : The risk of mutagenesis of the target cells is a concern in the use of vectors that integrate into the host cell genome mainly retroviral vectors that need to integrate to be effective and for which integration occurs randomly.

Biodistribution : As recommended by regulatory authorities, preclinical and clinical trials have to integrate studies to determine distribution of vectors to sites other than intended therapeutic sites. This studies have to adress two issues:

- distribution of vectors to non target tissues and potential toxicity due to transgene expression in non intended tissues.

- infection of germ line cells and potential transmission to descendants 60.

Shedding : A potential concern for both patients and their environment (care personnal, family, public) rests in shedding of the vectors from the transduced cells to other organs and their possible excretion and dissemination 61. This could be obtained by complementation of defective vectors by wild type viruses 62 (this issue is particularly important when vectors are derived from human viruses), by the use of selectively replicating vectors 63,64, by injections of vector producing cells 53,65, by the use of hybrid vectors 66-68 or other systems 69 that transform the transduced cells in vector producing cells.

 

Viral gene transfer to human contains some possible harmfull aspects, for both patients and the environement. Considerable efforts have been made by scientists and industrials to produce RCV-free vector stocks. Aspects of safety related to cytotoxicity and immune reactions to vectors are largely studied in preclinical and clinical trials and vector designs of are regularly improving. Finally, with the constant progress in gene transfer efficiency and enhanced transgene expressions, effect of biodistribution, germ line transmission and vector shedding have to be carefully evaluated, always keeping in mind the balance between risk and patient benefit.

 

 

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