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Infectious Disease Risks of Xenotransplantation: 

Infectious Disease Risks of Xenotransplantation 1. Exogenous viruses • Examples • Control measures Endogenous viruses (PERV) • Biology • Risk assessment • Experience to date • Control measures

Nature of the Infectious Disease Risk: 

Nature of the Infectious Disease Risk To the recipient: • Given reasonable control measures, infectious disease risk should be less than for allotransplantation. • Clearly acceptable in many transplant settings. To the public: • Risk derives from remote possibility that a new infectious agent or novel transmission mode will be inadvertently created. •Potential seriousness of the consequences, and examples from recent history require us to give it full consideration.

Examples of Emerging Viruses : 

Examples of Emerging Viruses Virus Species of origin Important events Canine parvovirus Cat 1. Mutations that allow efficient infection of dogs. 2. Rapid worldwide transmission Ebola ?? 1. Cross-species transmission 2. Local transmission only (so far) HIV Chimpanzee 1. Cross-species transmission 2. Access to transmission chain Subgroup J avian Chicken 1. Acquisition of new env gene by leukosis virus recombination with end. virus 2. Needle-borne and vertical transmission

Exogenous Viruses of Pigs: 

Exogenous Viruses of Pigs 1. Known agents of zoonotic infection D. Onions et al. 2000 Xenotransplantation. 7:143-55.

Exogenous Viruses of Pigs: 

Exogenous Viruses of Pigs Capable of infecting human cells, or other indication of zoonotic risk D. Onions et al. 2000 Xenotransplantation. 7:143-55.

Exogenous Viruses of Pigs: 

Exogenous Viruses of Pigs 3. Possibly oncogenic due to potential for aborted replication D. Onions et al. 2000 Xenotransplantation. 7:143-55.

Exogenous Viruses of Pigs: 

Exogenous Viruses of Pigs 4. Possibility of change in pathogenicity or host range. D. Onions et al. 2000 Xenotransplantation. 7:143-55.

Exogenous Viruses of Pigs: 

Exogenous Viruses of Pigs 5. Indicators of breaches in biosecurity or risk to animals D. Onions et al. 2000 Xenotransplantation. 7:143-55.

Exogenous Virus Infections: Control Measures : 

Exogenous Virus Infections: Control Measures 1. Derive breeding stock by hysterotomy, brief maintenance under gnotobiotic conditions, maintain under positive pressure, pest-free, sterilized feed and water, etc. 2. Health screening of staff. 3. Close monitoring for infections of concern: • Serology. • PCR necessary in some cases. • Many viruses of concern do not have adequate tests available at present.

Exogenous viruses: Conclusions: 

Exogenous viruses: Conclusions 1. More than 60 infectious agents of pigs are of concern for one reason or another. 2. Considerable challenges in detection and control remain. 3. All in all, however, risks of infection with viruses of this sort are considered to be less than with currently used allotransplant technology, given appropriate control measures.

Endogenous viruses: Biology: 

Endogenous viruses: Biology 1. Found as proviruses in all vertebrate species examined as a significant fraction of the DNA (we may have more proviruses than genes). 2. Derived by infection of the germ line with exogenous retrovirus. 3. Many (but not all) known groups of exogenous retroviruses have endogenous relatives. 4. Some confer protection against infection with related viruses. 5. Many are old and defective, but some of those found in some species including pigs, cats, mice and baboons, can yield infectious virus.

Gammaretroviruses (Mammalian C-Type Viruses) : 

Gammaretroviruses (Mammalian C-Type Viruses) 1. Simple retroviruses that include murine leukemia virus (MLV), feline leukemia virus (FeLV), gibbon ape leukemia virus (GALV), reticuloendotheliosis virus (REV), and many others. 2. Cause a wide variety of diseases, including malignancies, immunodeficiencies, and others in lab and natural settings. 3. Have endogenous relatives in the genomes of all mammals and many other vertebrates. 4. Most are transmitted vertically, from mother to offspring, and can infect only newborns. 5. Viruses derived from endogenous proviruses are generally non-pathogenic and replicate slowly, but some can become pathogenic by recombination (with related viruses) and mutation.

Porcine Endogenous Viruses (PERV’s): 

Porcine Endogenous Viruses (PERV’s) 1. Are gammaretroviruses closely related to MLV, FeLV, etc. 2. Are present as multiple (>20) proviruses in all breeds of pig examined. 3. Are highly polymorphic in location among different breeds (individuals?) 4. Are expressed as infectious virus from many cell lines and primary cultures. 5. Can be divided into 3 groups (A, B, C) by host range and sequence. 6. Two of these (A and B) infect human cells relatively well. 7. Do not replicate to high titers (compared to MLV, FeLV) in culture. 8. Have not been found as infectious viruses (or associated with disease) in pigs. 9. Can infect at least one species of laboratory animal.

Biology of PERV's: Distribution and Infectivity: 

Biology of PERV's: Distribution and Infectivity 1. Low level viremia can be detected in some pigs. 2. At least 6 proviruses are common to all pigs, but their infectivity is unknown. 3. Efficient isolation requires cocultivation with pig (ST IOWA) cells. 4. Virus isolated by induction of lymphocytes increases infectivity for human cells on passage through them (adaptation, or loss of less infectious virus).

Distribution of PERV Proviruses among Different Breeds of Pig : 

Distribution of PERV Proviruses among Different Breeds of Pig PERV-A PERV-B LxD MP M P MP M P LxD Le Tissier P, Stoye JP, Takeuchi Y, Patience C, Weiss RA. 1997. Two sets of human-tropic pig retrovirus. Nature. 1997 Oct 16;389:681-682.

Risks of PERV Infection: 

Risks of PERV Infection 1. Unknown (i.e., unquantified). 2. Risk to recipient requires spreading infection, pathogenesis, and is almost certainly as acceptable as in allotransplantation in many circumstances. 3. Of greater concern is the remote possibility of also generating a virus that can be transmitted from one individual to another. 4. Most likely varies with nature of transplant (cellular, whole organ, extracorporeal), due to differences in size, chimerism, time, extent of immunosuppression, etc. 5. Likely to be reduced by natural immunity (e.g. against galactose alpha(1-3)-galactose), at least for one round of infection, but measures to circumvent this immune response will also increase infection risk. 6. Never goes away! 1/x * unquantified risk = unquantified risk

Possible Adverse Events Following Transplantation with PERV-containing Cells (The Stoye Scale) : 

Possible Adverse Events Following Transplantation with PERV-containing Cells (The Stoye Scale) 1. Expression of infectious virus Very likely 2. Localized infection of host cells Likely 3. Spreading infection in the host Unlikely 4. Persistent viremia Very unlikely 5. Disease (e.g. lymphoma, “AIDS”) Very, very unlikely 6. Transmission to close contacts Very, very, very unlikely 7. Spreading, epidemic transmission Very, very, very, very unlikely

Testing for PERV : 

Testing for PERV 1. Tests include serology (western blot for p30); RT PCR of plasma, PCR of lymphocytes, PCR-based RT assays. 2. Use pig-specific PCR (mitochondrial or centromeric DNA to reveal microchimerism). 3. Evidence for infection by virus or following transplant obtained in 2 animal models. 4. Testing of patient samples to date negative.

Infection of Guinea Pigs with PERV: 

Infection of Guinea Pigs with PERV 1. 8 animals infected twice with 106 virions (?103-104 infectious units?) human cell-grown PERV B. 2. All became PERV antibody and DNA positive (4000-70,000 proviruses/106 cells in the spleen), but no RNA could be detected. 3. Suggests transient spread of virus and at least one cycle of replication within host, but that infection was then suppressed. D. Onions, FDA Presentation, January, 2000

PERV Infection Following Xenotransplantation in Mice: 

PERV Infection Following Xenotransplantation in Mice 1. Immunodeficient mice transplanted with 5000-8000 pig islets at various sites. 2. Various organs examined by PCR for PERV and pig mitochondrial DNA at various time after transplant. 3. Evidence found for both infection and microchimerism in a number of organs as late as 8 weeks post transplant. Van der Laan et al. 2000. Nature 407: 90-94.

Lack of PERV Infection in Humans Exposed to Pig Tissue : 

Lack of PERV Infection in Humans Exposed to Pig Tissue 160 patients exposed to pig tissue (or cells) up to 12 years previously. PBMC’s examined for PERV proviral DNA (by PCR) or antibody. 3. No evidence for PERV infection could be found, although viral DNA persisted in pig cells up to 8 years after treatment (microchimerism). Paradis et al.. Science. 1999. 285:1236-1241.

What Can be Done to Minimize Risk of PERV Infection?: 

What Can be Done to Minimize Risk of PERV Infection? 1. Monitor recipients for evidence of infection or associated disease. (Requires development of suitable PCR/serologic tests.) 2. Minimize their ability to transmit infection. (Defer from blood donation, counsel regarding transmission, etc.) ? 5. Identify active proviruses in donor animals and remove by selective    breeding or knockout. (Complex, time-consuming, expensive.) ?? 3. Monitor contacts (ethical and practical problems.) ??? 4. Treat for infection. (Very few antivirals active vs PERV. How do you        test? Approve? Balance risks? Would have to be for life.) ???? 6. Use transgene to suppress PERV expression (We don’t know how to do this.)

Slide23: 

Factors to Consider in Estimating Risk On the one hand... 1. Viruses like PERV do not infect adult animals very well, and are generally transmitted vertically. 2. Endogenous viruses (including PERV) replicate poorly and have low pathogenicity. 3. No evidence for human infection despite millennia of close pig-human contact (Butchering, etc.). 4. No evidence for infection following xenotransplantation. On the other hand... 1. There are a number of notable exceptions. 2. Can be restored by recombination and/or mutation, in some models. 3. Xenotransplantation is a novel kind of close contact, unlike all others. 4. Are we looking in the right place? Infection of at least some experimental animals has been seen.

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