Assessment: Microorganisms and Materials Containing Recombinant DNA Srisin Khusmith Professor, Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University Committee, Technical Biosafety Committee, BIOTEC, National Science and Technology Development Agency Chair of MU-IBC, Mahidol University Faculty of Tropical Medicine, Mahidol Univeristy June 16-17, 2015 1 Biohazardous Materials Infectious microorganisms (bacteria, viruses, fungi, parasites, prions, rickettsia, etc.) affecting humans and animals Toxin agents Prions Recombinant DNA (viral vectors, gene therapy, cloning) Diagnostic (clinical) specimens eg blood, serum, secretions, or body fluids etc. Human and non-human primate cells, cell culture (primary and immortalized), tissues, stem cells blood (whole blood or any components) and body fluids Animal or plant cells, cell cultures, fluids, tissues or derived wastes which may contain pathogens 2 Laboratory Acquired Infections (LAI) Bacterial: 76% from clinical labs 8% from research labs Exposure: 60% acquired from inhalation Virus 16% from clinical labs 70% from research labs 32% from animal related activities Laboratory Acquired Infections (LAI) Only 20% causative or defined event 80% of which are caused by human factors 20% are caused by equipment failure Top 4 accidents resulting in infection Spillages & splashes Needle and syringe Sharp object, broken glass Bite or scratch from animals or ecto-parasites Working Safely with Biological Materials, Central Michigan University 3 http://www.weizmann.ac.il/safety/bio2.html 1
Bio Assessment Principles What Are the Key Factors Needed to Conduct a Assessment for Infectious Microorganisms? There is always risk! The risk must be identified The risk is evaluated The risk must be measured Plan to minimize the risk What factors define likelihood? What factors define consequences? What are the consequences of disease from accidental exposure? What are the Agent factors? What are host range of agent/ vector? What are the vectors (viral vector) / host / product? What are the laboratory factors? Assessment Factors Should Be Considered in A Assessment for Pathogen To determine the level of containment to handle a biohazardous agent based on the following: Pathogenicity- the ability of an organism to cause disease. Virulence - the severity of disease. Transmission route (route of exposure): Inhalation: droplets or droplet nuclei (upper or lower respiratory tract) (eg M. tuberculosis) Ingestion: gastrointestinal tract (eg S. typhi Contact: mucous membrane/ eye/ percutaneous, through wound eg Staphylococcus Direct injection: blood borne contact eg HIV-virus, Hepatitis B,C Parenteral Inoculation (needle stick) Source: http://www.austrac.gov.au/elearning_amlctf_programcourse/mod4/module_4_risk_16.html 8 8 2
Factors Should Be Considered in A Assessment for Pathogen Infectious dose: the dose required to cause infection in humans or animals eg. M. tuberculosis (low) ; Francicella tularensis etc. Agent stability: survival in environment or otherwise prolonged viability (spore formation) eg. Rickettsia, B. anthracis Host range Availability of effective preventive measure (eg. vaccines) Availability of effective treatment (eg. antibiotics) Antibiotic resistance eg MDR-TB Whether the pathogen is indigenous to the country 9 Where are Blood-borne Pathogens Found? Human blood: blood, blood component, blood product Other Potentially infectious materials (OPIM): Unfixed human tissue or organ, Primary human tissue culture, Primary human cell lines (capability=20-70 passages), Human cell line (unlimited) Organ cultures Human body fluid eg. semen, vaginal secretion, CSF, synovial fluid, pleural fluid, peritoneal fluid, amniotic fluid, saliva etc. Infected experimental animals 9 10 Contact with another person s blood or bodily fluid that may contain blood Mucous membranes: eyes, mouth, nose Non-intact skin Blood Borne Pathogen: Transmission Potential Contaminated sharps/needles Facts for HBV, HCV and HIV HBV/HCV can live on a dry surface for at least 7 days. You have a 6 to 30% greater chance of contracting HBV/HCV from a needle stick versus a 1% chance of contracting HIV from a needle stick. There IS a vaccine for HBV. There is NO vaccine for Hepatitis C. Medical personnel have 0.4% chance of exposure to HIV from a needle stick. There is NO vaccine for HIV 3
Classification of Infectious Microorganism by Group Group 1 Group 2 Group 3 Group 4 no or low individual and community risk moderate individual risk, low community risk high individual risk, low community risk high individual and community risk A microorganism that is unlikely to cause human disease or animal disease eg. non-pathogenic organisms, non-pathogenic E. coli-k12, B. subtilis; well established human cell line- history of safe etc. A pathogen that can cause human or animal disease but is unlikely to be a serious hazard to laboratory workers, the community, livestock or the environment. Laboratory exposures may cause serious infection, but effective treatment and preventative measures are available and the risk of spread of infection is limited. Eg. Bacillus cereus, Clostridium spp, Campylobacter, Most wild type E. coli eg E.coli O157, Hepatitis B virus, Adeno virus, CMV, Vaccinia virus, human blood & blood products, clinical samples etc. A pathogen that usually causes serious human or animal disease but does not ordinarily spread from one infected individual to another. Effective treatment and preventive measures are available. Serious respiratory agents eg. Bacillus anthrasis, Mycobacterium tuberculosis, Rickettsia spp, HIV, Yersenia pestis (resistant strains), prion etc. A pathogen that usually causes serious human or animal disease and that can be readily transmitted from one individual to another, directly or indirectly. Effective treatment and preventive measures are not usually available. Eg. Ebola virus, Marburg virus, Crimean-Congo hemorrhagic fever WHO Laboratory Biosafety Manual 3rd Edition (2004) Fungal Agents vs BSL Infections are not communicable, but require common exposure from a point source. Some fungi have dimorphic: yeast form and mold form Yeast forms may be present in the tissues of infected animals and in clinical specimens, identifying isolates, and processing animal tissues. Mold form cultures containing infectious conidia or spore may pose a hazard of aerosol exposure. BSL-2 and ABSL-2 practices, containment equipment, and facilities are recommended for activities with clinical materials, animal tissues, yeast-form cultures, and infected animals eg. Crytococcus neoformans, yeast form of Blastomyces dermatitidis. BSL-3 practices, containment equipment, and facilities are required for or propagating and manipulating sporulating mold-form cultures eg. Blastomyces dermatitidis, eg. Coccidioides spp, Histoplasma capsulatum etc. BMBL 5th Edition (2010) External factors to be considered in a risk assessment include. Titer/volume of material used - titer may increase several orders of magnitude compared to levels in clinical samples, upon culturing. What are the laboratory factors? (Lab procedures with risk of exposure: e.g., potential for splashes, vortexing, centrifugation, sharps, needles or injection, animals; skills and training level of investigators. Health status of investigator - e.g., immune state, pregnancy, vaccination status. 15 Assessment: Large Volumes and High Concentrations Scaling up a process can change BSL-1, bench-top experiment to BSL-2, PPE required experiment Small or bench scale: < 10 liters Large scale: > 10 liters High Concentrations: 10 3 versus 10 10 have different requirements http://ehs.unc.edu/training/self_study/bsl2_dla m/container.php?page=66&x=15&y=13 16 4
management Appropriate Biosafety Level Agent Group + Assessment = Appropriate Biosafety Level of activity same agent can have different containment levels Protection should match the risk Relation of Group to Biosafety Level, Practices and Equipment Group Group 1 Group 2 Group 3 Group 4 Biosafety Level Basic Biosafety Level 1 Basic - Biosafety Level 2 Containment - Biosafety Level 3 Maximum containment Biosafety Level 4 Lab Type Lab Practices Safety Equipment Basic teaching, research Primary health services; diagnostic services, research Special diagnostic services, research Dangerous pathogen units WHO Laboratory Biosafety Manual 3rd Edition (2004) GMT GMT plus protective clothing, biohazard sign As Level 2 plus special clothing controlled access directional airflow As Level 3 plus airlock entry, shower exit, special waste disposal None; open bench Biosafety work, sink, eye shower Open bench plus BSC for potential aerosols BSC and/or other primary devices for all activities Class III BSC, or positive pressure suits in conjunction with Class II BSCs, double-ended autoclave (through the wall), filtered air Asessment Cells, Tissues and Cell Lines The origin of the cells or tissues (species and tissue type) The source (recently isolated or wellcharacterized). Potential Harzadous Human Cell Lines Potential laboratory hazards associated with human cells and tissues include Blood borne pathogens HBV, HIV, HCV, EBV, HPV and CMV M. tuberculosis that may be present in human lung tissue. Other primate cells and tissues also present risks to laboratory workers. Cells immortalized with viral agents such as SV-40, EBV adenovirus or HPV, as well as cells carrying viral genomic material also present potential hazards to laboratory workers. Tumorigenic human cells also are potential hazards as a result of self-inoculation. There has been one reported case of development of a tumor from an accidental needle-stick. Laboratory workers should never handle autologous cells or tissues, blood, lymphoid and neural tissues should always be considered potentially hazardous 19 20 5
Primary Cell Culture/Cell Lines vs BSL BMBL 5 th edition (CDC) recommended that human and other primate cells should be handled using BSL2 practice and containment. All work should be performed in a BSC Primary cultures of insect or mammalian cells from a source where infection status is UNKNOWN (or known to be infected) must be treated as though they are contaminated with infectious agents and utilize BSL-2 practices and procedures. All primary human cell cultures (explants) and subsequent in vitro passages fall under the Blood borne pathogen standard (BSL2) Human cell lines are considered to be potentially infectious unless the specific cell line has been characterized to be free of hepatitis viruses, HIV, Epstein-Barr virus, human papilloma viruses and other recognized blood borne pathogens (BSL2) 21 Primary Cell Culture/Cell Lines vs BSL Transformed mammalian cell lines Group 2 -HeLa (Human - contains papovavirus) Any injections with human cell lines into animals will be conducted in a BSC and handled at Animal Biosafety Level 2 (ABSL-2). When in doubt, all human, primate, mammalian, or insect cell lines obtained from an outside source (e.g. repositories such as the American Type Culture Collection, other institutions, or investigators) must be treated using biosafety levels used by that source. 22 Research Involving Recombinant DNA Molecules Molecules constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell Synthetic DNA segments, likely to yield a potentially harmful polynucleotide or polypeptide, are considered equivalent to their natural DNA counterpart. Examples: Human gene transfer Cloning plasmids Viral or bacterial vectors Transgenic animals Genetically modified cell lines RISK ASSESSMENT: Recombinant DNA The risk assessment for recombinant DNA should include: Source of the DNA to be transferred (host) Ability of vector to survive outside the laboratory (vector) Interaction between transferred gene and host (product) When assessing the risk group and containment level for a genetic engineered protocol, if one of the components is potentially hazardous, a risk level appropriate to the known hazard is assigned. 24 6
Factors Should Be Considered in a Assessment for Recombinant Research What are the vectors (viral vector) / host / product? 1. Host /recipient system: Intrinsic characteristics of host/recipient (risk group) Source of DNA, nature of insert gene - Is the gene or sequence (including synthetic) from RG-2, RG-3, or RG-4 agent, biological toxin, or select agent? - Is there any risks associated w/ the gene or sequence such as: up-regulation/silencing expression, regain of function, oncogenes, virulence factors, toxins, or expanded host range? - Does the gene or sequence change sensitivity to antibiotics, herbicides, pesticides, or insecticides that would be used to control the host? WHO Laboratory Biosafety Manual 3rd Edition (2004); Section III D of the NIH Guidelines Factors Should Be Considered in a Assessment for Recombinant Research 2. Vector system Intrinsic characteristics (risk group) Replication competence Residual viral gene expression Expression vectors: Increased risk over vector alone 3 End product/gene product effects: Expression of a foreign gene Possible introduction or increase of virulence Toxicity (Gene code for toxin) Increased ability to evade immune system Allergenicity Pharmaceutical activity eg. antibiotic resistance WHO Laboratory Biosafety Manual 3rd Edition (2004); Section III D of the NIH Guidelines Viral Vector Considerations 1. Route of Transmission Blood-borne Lentivirus Direct contact Vaccinia Respiratory Adenovirus, Adeno-associated virus (>150 pfu intranasal) Influenza virus (790 pfu (nasopharyncheal) Viral Vector Considerations 2. Host Range Based on human pathogens Replication incompetent eg Lentivirus Potential for oncogenesis through insertional mutagenesis eg Lentivirus Viral gene products in vector Non-pathogenic viruses Adeno-associated virus Based on non-human pathogens Reduced pathogenicity (Vaccinia, Avipox) Non-pathogenic (Baculovirus) Tropism and host range Change in cell type or species affected WHO Laboratory Biosafety Manual 3rd Edition (2004) WHO Laboratory Biosafety Manual 3rd Edition (2004) 7
3. Contamination Viral Vector Considerations Viral vectors, e.g. adenovirus may be contaminated with replication-competent viruses, generated by rare spontaneous recombination events in the propagating cell lines, or may derive from insufficient purification. These vectors should be handled at the same biosafety level as the parent adenovirus from which they are derived. WHO Laboratory Biosafety Manual 3rd Edition (2004) Classification of Recombinant DNA Activities by Group Group 1 Group 2 Group 3 Group 4 no or low individual and community risk moderate individual risk, low community risk high individual risk, low community risk high individual and community risk -rdna activities holding no or a negligible risk eg. rdna activities using such non-pathogenic organisms as hosts for the expression of genes eg E. coli K-12, Saccharomyces cerevisiae, vector from commercial supplier, molecular technique which did not involve organism eg. PCR, northern &southern blotting except DNA coding for oncogenes etc. -recombinant DNA activities using viral vector systems such as Adenoviruses and some Retroviral vectors, particularly Lentiviral vectors, and expression of recombinant DNA in BSL-2 organisms. Recombinant DNA activities using genetic material from BSL- 3 organisms or such organisms as host cells, cell lines modified using DNA from Group 3, cell lines contains a toxin with an LD50 of less than 100 nanograms per kg/kg body weight Recombinant DNA activities using genetic material from BSL-3 organisms or such organisms as host cells, cell lines modified using DNA from Group 4 WHO Laboratory Biosafety Manual 3rd Edition (2004) Experiments that are exempt but still require registration: 1. Experiments that use synthetic nucleic acids that can neither replicate nor generate nucleic acids capable of replicating in any living cell; are not designed to integrate into DNA, and do not poduce a toxin that is lethal for vertebrates at an LD50 of <100 ng/kg body weight. 2. Cloning of all other DNA in E. coli K12 Host-Vector systems, or derivatives of E. coli K-12; Saccharomyces Host-Vector Systems eg. S. Cerevisiae and S. uvarum ; and B. subtilis or Bacillus subtilis or Bacillus licheniformis Host-Vector Systems host-vector systems (with the exception of DNA from Group 3 or 4 pathogens). 3. Introduction into cultured cells of any recombinant DNA containing less than half of a eukaryotic viral genome (with the exception of Group 3 or 4 pathogens). 4. Breeding experiments to generate transgenic rodents that may be housed under BSL1,with the exception of those listed in Section III- E. Section III-F NIH Guidelines 31 Experiments that require IBC approval PRIOR to initiation Experiments using RG 2-4 agents as host/vector system Deliberate transfer of a drug resistance trait to microorganisms that are not known to acquire the trait naturally if such acquisition could compromise the use of the drug to control disease. Use of viral vectors (e.g. lentivirus, adenovirus, pox virus, vectors for gene delivery to living systems) III-B-1: Experiments involving the cloning of toxin molecules with LD50 of less than 100 ng per kg body weight Experiments involving the use of infectious DNA or RNA viruses or defective DNA or RNA viruses in the presence of helper virus in tissue culture systems Transfer of rdna to human research participants 32 Section III-A,B,C, D NIH Guidelines 8
Examples of Experiments That Require IBC Approval PRIOR to Initiation RG2 and RG3 Recombinant DNA Technology Experiments involving the use of infectious DNA or RNA viruses or defective DNA or RNA viruses in the presence of helper virus in tissue culture systems Transfer of rdna to human research participants eg. e.g., in vivo human gene therapy) Experiments involving viable rdna-modified microorganisms tested on whole animals. Experiments involving more than 10 liters of culture. Section III-D NIH Guidelines33 Most vaccine experiments will fall into one of the following categories: Experiments using Group 2 or Group 3 agents as Host-Vector systems. Experiments in which DNA from Group 2 or Group 3 agents is cloned into nonpathogenic prokaryotic or lower eukaryotic Host-Vector systems. Many vaccine projects, as well as other studies involving animals Experiments involving whole animal work. NIH Guidelines 34 Recombinant DNA Experiments Section III-A of the NIH Guidelines Recombinant DNA Experiments Section III-B of the NIH Guidelines Deliberate transfer of a drug resistant trait to microorganisms that could compromise medicine or agriculture in its control. Creation of a multi-drug resistant virus or bacteria that would compromise medicine, agriculture or public health, e.g., a recombinant HBV virus that was would render the current HBV vaccine series as ineffective. These experiments require Institutional Biosafety Committee Approval Before Initiation and is considered a Major Action under the NIH Guidelines. The deliberate formation of recombinant DNA containing genes for the biosynthesis of toxin molecules lethal at an LD 50 of less than 100 nanograms per kilogram of body weight. These experiments involve the potential manipulation of sequences from microbial toxins such as the Botulinum toxins, tetanus toxin, diphtheria toxin, and Shigella dysenteriae neurotoxin, e.g., creation of recombinant organism, such as E-coli K12 with a sequence to synthesize Clostridium botulinum toxin 9
Recombinant DNA Experiments Section III-B of the NIH Guidelines The deliberate transfer of recombinant DNA or RNA derived from recombinant DNA into one or more human research participants (e.g., in vivo human gene therapy) These experiments often involve in-vivo human gene therapy but may also include vaccine studies, e.g., an adenoviral mediated gene transfer experiment These experiments require Institutional Biosafety Committee and EC (IRB) Before Research Participant Enrollment Biosafety Guidelines for Infectious Microorganisms / Recombinant DNA Technology WHO Laboratory Biosafety Manual 3 rd Edition (2004) Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5 th Edition (2010) jointly published by NIH and CDC Canadian Laboratory Safety Guidelines (2004) NIH Guidelines for Research Involving Recombinant DNA Molecules (2013) Australian/New Zealand Standard Safety in laboratories Part 3: Microbiological safety and containment National Guidelines for Pathogens Guidelines from Department of Medical Science, Ministry of Public Health National Guidelines for Biotechnology 1. Biosafety Guidelines in Genetic Engineering and Biotechnology for Laboratory Work (1992) 2. Biosafety Guidelines in Genetic Engineering and Biotechnology for Field Work and Planned Release (1992) 3. Biosafety Guidelines for Field Trials of Transgenic Plants (1994) 4. Biosafety Guidelines in Safety Assessment of Genetically Modified Foods(2001) 5. Biosafety Guidelines for Contained Use of Genetically Modified Microorganisms at Pilot and Industrial Scales 6. Biosafety Guidelines for Work Related to Modern Biotechnology or Genetic Engineering (revised from 2 and 6) (2004, 2009, 2011) 2004 2009 2011 10
Package of MU Safety Guidelines Q&A Package: distributed to Faculty/Institution MU Staff /student: download from Mahidol University website (COSHEM) Thank you 11