Biosafety Issues in Laboratory Research and in the Use of Gene7cally Modified Microorganisms (e.g. LVV)
Dimitri Sossai PhD Director Preven1on and Ptotec1on Department IRCCS San Mar1no IST Luca Nelli, RBP Biosafety Consultant, Siena Dimitri Sossai Luca Nelli June 2016 I plan to talk about….
§  Defini1ons §  Elements of Biorisk Assessment/Management §  Biosafety considera1on on the use of GMMs -­‐ LVV §  Conclusion Dimitri Sossai Luca Nelli June 2016 Dimitri Sossai – Luca Nelli 269 Dimitri Sossai Luca Nelli June 2016 Defini8ons….
§  Biohazard. poten1al source of harm caused by biological agents or toxins (from CWA 15793:2011) §  Biological agents. any microorganism including those which have been gene1cally modified, cell cultures and endoparasites, which may be able to provoke any infec1on, allergy or toxicity in humans, animals or plants (adapted from EU Direc1ve 2000/54/EC) Dimitri Sossai Luca Nelli June 2016 Defini8ons….
§  Biorisk combina1on of the probability of occurrence of harm and the severity of that harm where the source of harm is a biological agent or toxin (adapted from ISO/IEC Guide 51:1999) The source of harm may be an uninten1onal exposure, accidental release or loss, the_, misuse, diversion, unauthorized access or inten1onal unauthorized release. Dimitri Sossai Luca Nelli June 2016 Defini8ons….
Contained use or biocontainment is a “keep-­‐in concept” keep-­‐out
keep-­‐in
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Protec8on of product
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Protec8on of staff and environment
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Prevent cross-­‐contamina8on
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Prevent the shedding
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Flow: from clean to dirty
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Flow: from dirty to clean
In an Animal Facility the Biocontainment Procedures and the Animal Care and Sossai uca Nelli June 2016 Experiments procedures can Dimitri have cLonflicts, due to the different aim… What is Biosafety?
§  Describes the containment principles, technologies and prac1ces that are implemented to prevent the uninten1onal exposure to biological agents and toxins, or their accidental release §  Measures employed when handling biohazardous materials to avoid infec1ng oneself, others or the environment, achieved through; ü Administra1ve Controls ü Engineering Controls ü Personal Protec1ve Equipment ü Prac1ces and Procedures Dimitri Sossai Luca Nelli June 2016 “ Risk Group is not a synonym for Biosafety Level ”
While the Risk Group classifica1on is based on the microbiology and epidemiology of agents, Biosafety Level corresponds to the facili1es, equipment, prac1ces and procedures for safe conduct of work with an agent. Assessing a GMM: §  doesn’t mean assigning the GMM to a Biological Risk Group, but §  means: to iden1fy/establish the Biosafety Level (or contained use) needed for use it Dimitri Sossai Luca Nelli June 2016 Defini8ons….
§  biological agent any microorganism including those which have been gene1cally modified, cell cultures and endoparasites, which may be able to provoke any infec1on, allergy or toxicity in humans, animals or plants (adapted from EU Direc1ve 2000/54/EC) §  ‘micro-­‐organism’ means any microbiological en1ty, cellular or non-­‐cellular, capable of replica1on or of transferring gene1c material, including viruses, viroids, and animal and plant cells in culture (from Direc1ve 2009/41/EC) §  ‘geneGcally modified micro-­‐organism’ (GMM) means a micro-­‐organism in which the gene1c material has been altered in a way that does not occur naturally by ma1ng and/or natural recombina1on (from Direc1ve 2009/41/EC) Dimitri Sossai Luca Nelli June 2016 A simple example….
The video shows what happens normally and rou1nely in the lab (in BSC or at the bench): “opening a vial. Por1oning of a solu1on in another vial. Using pipefe and 1p”. What's not percep1ble and visible to the eye, it's actually present. The UV light shows contamina1on of the glove (fingers) Dimitri Sossai Luca Nelli June 2016 A simple example….
§  This condi1on is not a problem or an extraordinary situa1on. It’s normal. And the gloves are donned for protec1ng scien1st. §  But the ques1on is: what’s your behaviour a_er that opera1on?… a_er pipekng… a_er injec1on lab animal?…. do you remove and replace gloves? Do you disinfect effec1vely its surface?..... or…. do you con1nue your ac1vi1es touching other objectes and surfaces with the same gloves?…. The issue remain even using a double pair of gloves Dimitri Sossai Luca Nelli June 2016 Dimitri Sossai Luca Nelli June 2016 Dimitri Sossai Luca Nelli June 2016 Biosafety: una disciplina DLgs 81/08 Biosafety & C. “2000/54/EC” Luca Nelli – Dimitr Sossai 35 Defini8ons….
§  ‘Contained use’ means any ac1vity in which micro-­‐ organisms are gene1cally modified or in which such GMMs are cultured, stored, transported, destroyed, disposed of or used in any other way, and for which specific containment measures are used to limit their contact with, and to provide a high level of safety for, the general popula1on and the environment (from Direc1ve 2009/41/EC) Contained uses of GMMs should be classified in rela1on to the risks they present to human health and the environment. Such classifica1on should be in line with interna1onal Biosafety prac1ce and based on an assessment of the risk. -­‐ BSL1 BSL2 BSL3 BSL4 Dimitri Sossai Luca Nelli June 2016 + Biosafety Level-­‐1 Concepts of Biosafety
Biosafety Level-­‐1 (BSL-­‐1 or ABSL-­‐1) •  Well characterized agents •  Agents not known to cause disease (in healthy human adults; now healthy immunocompetent adults) •  Prophylac1c treatment available •  Open bench procedures •  Animals in open cage system or open environment (outdoors) •  Good laboratory prac1ces Risk Group 1 Agents
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E.coli K-­‐12 Transgenic Plants Plasmids Fungi Mold Yeast BSL-­‐1 Prac8ces
•  Bench-­‐top work allowed •  Daily Decontamina1on •  Manual pipekng •  Required Handwashing •  Red bag waste •  Bio cabinet not required (unless crea1ng aerosols) •  2˚ containment Risk Group 2 Agents
•  Human or Primate Cells •  Herpes Simplex Virus •  Replica1on Incompetent Afenuated Human Immunodeficiency Virus •  Pa1ent specimens BSL-­‐2 Prac8ces Concepts of Biosafety
PracGces & Procedures •  Agents associated w/ human disease •  Treatment for disease available •  Agent poses moderate hazard to personnel and environment •  Direct contact or exposure •  Percutaneous exposure •  Scratch, Puncture, Needle s1ck •  Mucus membrane exposure •  Eyes, Mouth, open cut BSL-­‐2 Prac8ces
•  Limited access to lab when work in progress •  Daily decontamina1on •  Mechanical pipekng •  Labcoat, safety glasses and gloves required •  Red bag & sharps containers required BSL-­‐2 Prac8ces (con’t)
•  Biohaz. Sign posted at entrance to lab •  Label all equipment (incubators, freezers, etc.) •  TC room – nega1ve air flow •  Documented training •  Baseline serology or pre-­‐
vaccina1on may be required Risk Group 3 Agents
•  Human Immunodeficiency Virus •  Mycobacterium tuberculosis •  Coxiella burne1i Biosafety Level 3 Working in High Containment
Biosafety Level-­‐3 (BSL-­‐3 or ABSL-­‐3) •  Indigenous or exo1c agents •  Aerosol transmission •  Serious health effects •  Treatment may or may not exist BSL-­‐3 Prac8ces
•  Public access NOT permifed •  Daily decontamina1on a_er spill and upon comple1on of experiment •  Autoclave required and waste is disposed at the end of day •  Required foot ac1vated handwashing sink and controls •  No sharps unless absolutely necessary BSL-­‐3 Prac8ces (con’t)
•  Aerosol minimiza1on procedures required •  Wrap around disposable clothing is required. Specialized equipment may be required depending upon procedures •  Biohaz. Signs and labels posted •  Air flow from low hazard to high hazard “Pressure Mapping” BSL-­‐3 Prac8ces (con’t)
•  Bench top work not permifed •  Documented training and personnel competency cer1fica1on (for BSL-­‐3 procedures) •  Baseline serology •  Spills – report immediately and treat accordingly •  Vaccina1ons/post exposure protocols and SOP’s, Biosafety Manual, Biosafety Officer Biosafety Level-­‐4 Working in High Containment
Biosafety Level-­‐4 •  Builds on BSL-­‐3/ ABSL-­‐3 prac1ces •  Maximum containment facili1es •  Pressurized Containment Suite •  BSL-­‐3 + Class III Biosafety Cabinet •  Chemical decontamina1on showers •  Liquid effluent collec1on / decontamina1on •  No BSL-­‐4 labs exist at UCSD Biosafety Level 4
•  Lassa Fever Virus •  Ebola Hemmorrhagic Fever Virus •  Marburg Virus •  Herpes B Virus Biosafety Concepts Working in High Containment
Biosafety Level-­‐4 (BSL-­‐4 or ABSL-­‐4) •  Dangerous/exo1c agents •  Life threatening disease •  Aerosol transmission •  Agents of unknown risk of transmission or health affects •  No known treatment General Good Lab Technique
•  Hygienic Prac1ces •  No Smoking, Ea1ng, Applying cosme1cs, lip balm, contacts •  Wash hands a_er procedures •  Decontaminate lab bench before and a_er work Dimitri Sossai Luca Nelli June 2016 Biohazardous Materials ▪  Human and Primate Cells, Cell Culture, Tissues, and Body Fluids ▪  Brain Tissue from Demented Pa1ents ▪  Animals that are Poten1al Reservoirs of Zoono1c Diseases ▪  Viral Vectors ▪  Conven1onal Agents ▪  Recombinant DNA or GMM ▪  Anatomical Specimens and Tissues ▪  Unconven1onal Agents – dual use agents Luca Nelli – Dimitri Sossai 20 DOSI INFETTANTI Luca Nelli – Dimitri Sossai 93 Caraferis1che strufurali Caraferis1che procedurali e comportamentali Luca Nelli -­‐ 19/22 Gen 2016 Corso BSL3 -­‐ S.Mar1no 15 What is a laboratory acquired infec1on (LAI)? An infecGon resulGng from laboratory work (Sulkin and Pike 1951) Luca Nelli – Dimitri Sossai 7 Informazioni dalle LAI Laboratory Acquired Infec1ons (LAI) le LAI piu’ comuni riportate Luca Nelli – Dimitri Sossai da Harding e B. Byer, 2006 modif. 4 FROM 2000 TO MORE RECENTLY REPORTED LABORATORY-­‐ACQUIRED INFECTIONS OR RELATED STUDIES ▪  Fiori PL, Mastrandrea S, Rappelli P, Cappuccinelli P. Brucella abortus infecGon acquired in microbiology laboratories. J Clin Microbiol 2000; 38(5): 2005-­‐2006. ▪  Boutet R, Stuart JM, Kaczmarski EB, Gray SJ, Jones DM, Andrews N. Risk of laboratory-­‐acquired meningococcal disease. J Hosp Infect. 2001; 49(4): 282-­‐284. ▪  Laboratory-­‐acquired meningococcal disease. Morbidity and Mortality Weekly Report, Centers for Disease Control and Preven1on. February 22, 2002 ▪  Shapiro DS, Schwartz DR. Exposure of laboratory workers to Francisella tularensis despite a bioterrorism procedure. J Clin Microbiol. 2002; 40(6):2278-­‐2281. ▪  Suspected Cutaneous Anthrax in a Laboratory Worker (Texas), Morbidity and Mortality Weekly Report, Centers for Disease Control and Preven1on. April 5, 2002 ▪  Laboratory-­‐Acquired West Nile Virus InfecGons (USA), Morbidity and Mortality Weekly Report, Centers for Disease Control and Preven1on. December 20, 2002 ▪  Gosbell IB, Mercer JL, Neville SA. Laboratory-­‐acquired EMRSA-­‐15 infecGon. J Hosp. Infec1on. 2003; 54: 324-­‐325. ▪  Moussatché N, Tuyama M, Kato SE, Castro AP, Njaine B, Peralta RH, Peralta JM, Damaso CR, Barroso PF. Accidental infec1on of laboratory worker with vaccinia virus. Emerging Infec1ous Diseases. 2003; 9(6): 724-­‐726 ▪  Laboratory-­‐Acquired vaccinia infecGon. Public Health Agency of Canada, August 1, 2003 ▪  Mempel M, Isa G, Klugbauer N, Meyer H, Wildi G, Ring J, Hofmann F, Hofmann H. Laboratory acquired infec1on with recombinant vaccinia virus containing an immunomodula1ng construct. J Invest Dermatol. 2003; 120(3):356-­‐358. Luca Nelli – Dimitri Sossai 5 ▪  Günther S, Feldmann H, Geisbert T.W., et al. Management of Accidental Exposure to Ebola Virus in the Biosafety Level 4 Laboratory, Hamburg,Germany. The Journal of Infec1ous Diseases. 2011; 204 (Suppl 3): 785-­‐790. ▪  Laboratory exposure -­‐ Bacillus cereus -­‐ USA. September 14, 2011. ▪  Brifon S, van den Hurk AF, Simmons, RJ et al. Laboratory-­‐acquired Dengue virus infec1on -­‐ A case report. PLoS Negelcted Tropical Diseases. 2011; 5 (11): e1324. ▪  Inves1ga1on update: Human Salmonella Typhimurium infec1ons associated with exposure to clinical and teaching microbiology laboratories. Centers for Disease Control and Preven1on. January 17, 2012 ▪  Sayin-­‐Kutlu S, Kutlu M, Ergonul O et al. Laboratory-­‐acquired brucellosis in Turkey. Journal of Hospital Infec1on. 2012; 80: 326-­‐330. ▪  Sam IC, Karunakaran R, Kamarulzaman A et al. A large exposure to Brucella melitensis in a diagnos1c laboratory. Journal of Hospital Infec1on. 2012; 80: 321-­‐325. ▪  McCollum AM, Aus1n C, Nawrocki J, Howland J, Pryde J, Vaid A et al. Inves1ga1on of the first laboratory-­‐
acquired human cowpox virus infec1on in the United States. The Journal of Infec1ous Diseases. 2012; Advance Access Published May 9. DOI: 10.1093/infdis/jis302. ▪  Lam ST, Sammons-­‐Jackson W, Sherwood J, Ressner R. Laboratory-­‐aquired tularemia successfully treated with ciprofloxacin. Infec1ous Diseases in Clinical Prac1ce. 2012; 20 (3): 204-­‐207. ▪  Felinto de Brito ME, Andrade MS, de Almeida EL, Medeiros AC, Werkhäuser RP et al. Occupa1onally acquired american cutaneous leishmaniasis. Case Rep Dermatol Med. 2012; Epub Nov 28. DOI: 10.1155/2012/279517. Luca Nelli – Dimitri Sossai C NTINUA…. 6 VIE DI TRASMISSIONE Luca Nelli – Dimitri Sossai 92 Elizabeth (Beth) R. Griffin Beth Griffin died from exposure to the
Herpes B virus after an eye splash from an
infected primate on December 10, 1997.
Griffin was wearing a lab coat, mask, boots
and gloves but no protective goggles when she
contacted the virus.
Before her death, the only known
transmissions of the virus from monkeys to
humans occurred from a bite or scratch.
Only 40 cases of such transmissions have
been recorded since 1933.
After her death, Yerkes ordered its employees
to use eyewear for protection during tasks
previously not considered risky for
transmission of the herpes B virus.
Luca Nelli – Dimitri Sossai 100 Malcolm J. Casadaban
(1949 – 2009)
Luca Nelli – Dimitri Sossai 66 Malcolm J. Casadaban
(1949 – 2009)
Professor Malcolm Casadaban's Last Illness: • "Malcolm started having flu like symptoms on Monday, Sept. 7 (Labor Day). On September 13 he was taken to the ER at the Univ. of Chicago where he passed away about 12 hours later." (Casia Holmgren, Fiancé) • He was admifed to the University of Chicago's Emergency Room with shortness of breath on September 13, 2009, but within 12 hours had died. • A weakened strain of Yersinia pes6s was found in his blood stream. • The most likely diagnosis is sep1caemic plague, which can kill even before significant symptoms begin. • Malcolm Casadaban did not have any of the usual swellings associated with bubonic plague on his body. • No other obvious cause for death has been found. • This Yersinia pes6s strain was not previously known to cause illness in humans. Luca Nelli -­‐ 19/22 Gen 2016 Corso BSL3 -­‐ S.Mar1no 67 Malcolm J. Casadaban
(1949 – 2009)
According to a CDC report on the incident, the strain that killed Casadaban (KIM D27) had never been known to infect laboratory workers as it was an "afenuated" or weakened strain that had defec1ve genes for iron uptake. On autopsy, Casadaban was found to have undiagnosed hereditary hemochromatosis (iron overload) which likely played a role in his death. Luca Nelli – Dimitri Sossai 68 ………biorisk assessment…and management.
Luca Nelli –Dimitri Sossai 281 Assessing all the possible exposure scenarious. Microbiology and epidemiology of a biological agent (WT or GMM) are not the only informaGon to consider. «Natural» Trasmission Routes vs «Synthe1c» Trasmission Routes due to the work process HIV (and derived LVV) is not trasmifed «naturally» by air…… but…. what about your protocol? •  are there any steps that can produce e.g. areosols in your protocol? What is the worst scenario? •  are PPEs provided sufficient to protect form exposures? •  etc... Dimitri Sossai Nelli RJune 2016 Iden1fing the Luca real isks in order to Manage them Risk of exposure….
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Agent involved Route of exposure Dura1on of exposure Volume involved in exposure Concentra1on of agent at 1me of exposure Primary containment used (e.g. BSC) PPE worn etc.. Dimitri Sossai Luca Nelli June 2016 Main Legisla8on around GMMs….
Direc1ve 2009/41/EC «contained use of GMM» Dimitri Sossai Luca Nelli June 2016 Decreto Legisla1vo (Law 206/01 «contained use of GMM» DM 25/Nov/2001 «risk assessment of GMM» each GMM must be:
§  ASSESSED (for exposure risk to lab operators, people, environment) – Biorisk Assessment §  ASSIGNED to a specific contained use level (or biosafety level) – Biorisk Managment §  NOTIFIED to the Authori1es (based on local legista1on) §  USED in a No1fied and Authorized Primises (Lab Facility, Animal Facility, etc) Dimitri Sossai Luca Nelli June 2016 No8fica8on requirements for ac8vi8es involving GMMs Dimitri Luca Nelli June 2016 and general health and safety issues – HSE UK
form: The SACGM Compendium of guidance
- Part S1:ossai Introduction
to the
legislation
Biological Agent (GMM or WT) for having a safe use and a suitable biocontainment….. the star1ng point should be, always, Risk Assessment Dimitri Sossai Luca Nelli June 2016 What are viral vectors?….
Viruses engineered to deliver foreign gene1c material (transgene) to cells Many viral vectors deliver the gene1c material into the host cells but not into the host genome where the virus replicates (unless replica1on incompetent) Retroviral and len1viral vectors deliver gene1c transgenes into the host chromosomes Dimitri Sossai Luca Nelli June 2016 Risk Assessment
While assessing the risk of experiments involving viral vectors, the following factors should be taken in considera1on: §  Agent risk group §  Cell tropism §  Nature of Transgene §  Mutagenesis §  Safety features §  Environmental stability §  ….Protocols , procedures, work-­‐flows Dimitri Sossai Luca Nelli June 2016 Risk Assessment….risk group….
§  A viral vector par1cle has genes removed from the genome of a WT parent virus to create space for the gene of interest (transgene) and to increase safety. This mostly renders vectors replica1on-­‐incompetent §  Although unlikely a viral vector may regain the deleted genes and revert to its original form §  Therefore it is important to be aware of the risk group classifica1on of the parent virus from which the vector originated §  Infec1ous agents are acategorized in different risk gorup (1-­‐4) based on their rela1ve risk to healthy adult human. Dimitri Sossai Luca Nelli June 2016 Risk Assessment….Cell tropism….
§  Viral vectors have natural host cell popula1ons that they can infect most efficiently §  For expanding the range of cells suscep1ble to trasduc1on by a viral vector, many VV have been developed in which surface proteins have been replaced by proteins from other viruses (PSEUDOTYPING) §  The HIV glycoprotein (GP120) only binds to cells with CD4 receptors. HIV based viral vectors usually have their envelope pseudotyped: VSV-­‐G (Vescicular Stoma11s Virus glycoprotein G) has been used since it binds to receptors that are present in all type of cells of any species §  Based on cell tropism VV can be classified… ECOTROPIC AMPHOTROPIC PANTROPIC Infect murine cells ONLY (mouse and rats) Infect mammalian cells, including human cells Infect any type of cells of any species (e.g. VSV-­‐G) Dimitri Sossai Luca Nelli June 2016 HAZARD Risk Assessment…. Nature of Transgene
Basically any gene whcih can significantly alter the cell cycle when over-­‐expressed ia s gene of concern. These could include: §  Oncogenes or tumor suppressors §  Growth regulators §  Targets having important cellular func1ons §  Targets focused on the host-­‐immune system §  Transgenes without known targets carry unknown risks Dimitri Sossai Luca Nelli June 2016 Risk Assessment…. Safety Features….
Although Viral Vectors are occasionally created from pathogenic viruses they are modified to minimize the risk of handling them 1° genera1on 2° genera1on 3° genera1on 4° genera1on -­‐Safe + Safe Dimitri Sossai Luca Nelli June 2016 “ A Risk Group 1 (2) agent may need to be handled at BSL2 (3)”
§  The Risk Group of the parent virus from which a viral vector was derived may be the first, but NOT the ONLY factor to consider while assessing the risks §  Normally agents are handled under equivalent biosafety level of their risk group classifica1on. §  But in reality, this determina1on should be driven by professional judgment based on a risk assessment. §  Factors such as cell tropism and nature of the transgene inserted into the vector should be considered. §  But do not forget to consider exposure scenarious of the Process (work-­‐
flow) and Occupa1onal Medicine issues Dimitri Sossai Luca Nelli June 2016 What are viral vectors?….
Virus: Vehiculum that efficently transfers its gene1c material into a cell INFECTION Viral vector: Vehiculum that efficently transfers foreing gene1c material in to a cell TRANSDUCTION Viral Vector is not a Virus Biosafety is more about accidental transduc1on than about viral disease In any case the objec1ve must be AVOID EXPOSUREs preven1ng them Dimitri Sossai Luca Nelli June 2016 Consider the Chain of Infection and its possible lacks
Occupatonal
Medicine
Vaccines
…..
EQUIPMENTs
PROCEDUREs
PPEs
Dimitri Sossai Luca Nelli June 2016 LVV
Laboratory Hazards §  Direct contact with skin and mucous membranes of the eye, nose and mouth; Accidental parenteral injec1on; §  Inges1on; §  Inser1onal mutagenesis; integra1on and expression of oncogenes or poten1al oncogenes §  Hazard of aerosols exposure unknown; Dimitri Sossai Luca Nelli June 2016 LVV
Dimitri Sossai Luca Nelli June 2016 LVV
LABORATORY HAZARDS Exposure of mucus membrane (eyes, nose, mouth) Injec1on Aerosol inhala1on Direct contact with skin PPE Use of safety goggles or full face shields. Use of appropriate face mask Use of safety needles; NEVER re-­‐cap needle or remove needle from syringe Use of appropriate respiratory protec1on (e.g. FFP2-­‐ FFP3) Gloves, lab coat, closed shoes The above PPE are o_en required IN ADDITION to working in a cer1fied Biosafety Cabinet (BSC) or other primary containment device Dimitri Sossai Luca Nelli June 2016 LVV
Dimitri Sossai Luca Nelli June 2016 from University of Cincinna1 website, modif. LVV
§  Remember: replica1on deficient len1viral vectors integrate the vector into the host chromosomes §  3rd and 4th genera1on constructs unlikely to become replica1on competent with enhanced safety due to selfinac1va1ng vectors (however, consider present or future HIV infec1on) §  Replica1on deficient len1viral vectors should be regarded as single-­‐event infec1ous agents §  VSV-­‐G pseudotyping broadens the range of infected cells and increases the modes of transmission §  Len1viral (LV) risks in research sekngs primarily involve the inadvertent transduc1on of the lab worker §  These include the poten1al harmful effects of the transgene, inser1onal mutagenesis, or the ac1va1on of neighboring genes from vector integra1on or genera1on of replica1on competent len1virus (RCL) following an exis1ng or subsequent HIV infec1on §  Research regards these agents as rela1vely benign, although transgene integra1on does occur with generally unknown effects Dimitri Sossai Luca Nelli June 2016 Recommendatons for handling LV and Viral Vectors
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Follow good laboratory and hygiene prac1ces Use advanced LVV systems (3rd or 4th genera1on) Avoid mixing systems – separate in 1me and space different VV Review poten1al for replica1on competent virus Iden1fying exposure scenarios related to your specific protocol Use PPE to avoid exposures to eyes, nose and mouth Containment when possible aerosol genera1on Dimitri Sossai Luca Nelli June 2016 Containing Risks Associated with Aerosols
Aerosol Producing
Procedure
Method of Containment
Splash/Spray
biosafety cabinet, fume hood, splash shield
Vortexing
sealed tubes, biosafety cabinet
Centrifugation
sealed tubes, sealed rotor, safety cups
Homogenization
biosafety cabinet, fume hood, splash shield
Flow cytometry
fixation or BSL2+ containment
Injection/administration
Into animals
biosafety cabinet, animal restraint
Cage cleaning
(infected animals)
biosafety cabinet, PPE
(procedures and PPE shpuld be reviewd by
BSO/HSE)
Dimitri Sossai Luca Nelli June 2016 Recommendatons for handling LV and Viral Vectors
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Use advanced LVV systems (3rd or 4th genera1on) Avoid mixing systems Review poten1al for replica1on competent virus Iden1fying exposure scenarios related to your specific protocol Use PPE to avoid exposures to eyes, nose and mouth Containment when possible aerosol genera1on Avoid sharps and glass (anyway having a sharps safe procedure) Dimitri Sossai Luca Nelli June 2016 Sharps handling (…main 8ps…)
§  Limit the use of sharps to when no other alterna1ves are available §  Keep all sharps in full view at all 1mes §  Use only Luer-­‐lock syringes and needles or units where the needle is integral to the syringe §  Implement safety engineered sharps where prac1cal (retractable needles, needle 1p shields, self-­‐sheathing scalpels, etc) §  Dispose of sharps directly, without manipula1on, in an approved sharps disposal container (i.e., do not bend, shear, break, recap, or use hands to remove needles from syringes or blades from scalpels). §  Handle broken glass or other sharps with a secondary device such as forceps or broom and dustpan-­‐ not your hands §  Do not recap needles Dimitri Sossai Luca Nelli June 2016 Recommendatons for handling LV and Viral Vectors
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Consider risk for mutagenesis or toxic proper1es of transgene Consider risk from animals treated with VV Anesthe1zing animals Consider risk of viral shedding in immunodeficient animals Dimitri Sossai Luca Nelli June 2016 Animal biosafety issues with len8viral vectors (…main 8ps…)
Dimitri Sossai Luca Nelli June 2016 from University of Cincinna1 website Animal biosafety issues with len8viral vectors (…main 8ps…)
§  For ac1vi1es conducted outside of a BSC (e.g. stereotac1c injec1on) the use of mucose membrane protec1on devices (PPE) if of extreme importance Dimitri Sossai Luca Nelli June 2016 Animal biosafety issues with len8viral vectors (…main 8ps…)
REPLICATION BLOCK IN RODENT 3 HIV-­‐1 replica1on block in mice: 1) Cell entry (circumvented by VSV-­‐G preudotyping) 2) inefficent transcipr1on of HIV sequences in rodent 3) excessive splicing of HIV-­‐1 transcripts due to the lack of Rev-­‐assisted nuclear export cell of viral mRNA Dimitri Sossai Luca Nelli June 2016 Animal biosafety issues with len8viral vectors (…main 8ps…)
POTENTIAL FOR PRODUCTION OF RCV §  Only possible during vector produc1on, but sta1s1cally extremely low (e.g. 10-­‐16) with ≥3° genera1on LVV §  Because rodents are not infected with any len1virus, recombinant between the HIV-­‐1 derived sequences of the vector and another len1virus can be excluded §  Recombina1on between len1viral and endogenous murine retroviral sequences is not possible due because of the phylogenic distance between the two genus Dimitri Sossai Luca Nelli June 2016 Animal biosafety issues with len8viral vectors (…main 8ps…)
•  A_er 24h ABSL2 to ABSL1 if injected in CNS •  A_er 72h ABSL2 to ABSL1 if i.v. Anyway do not forget the Risk Assessment content specific for your protocol Dimitri Sossai Luca Nelli June 2016 Animal biosafety issues with len8viral vectors (…main 8ps…)
§  Studies with 3rd genera1on self-­‐inac1va1ng LVV showed infec1ous LV recoverable on dry plas1c for 24 hours and in vector-­‐spiked soiled bedding for up to 72 hours §  Infec1ous virus also found at the injec1on site (tail) for up to 24 hours (afributed to vector leakage upon needle removal) §  Protocols vary on when to go to ABSL-­‐1 (usually 1-­‐7 days for non-­‐
hazardous transgenes), but usually include disinfec1ng inocula1on site Dimitri Sossai Luca Nelli June 2016 Animal biosafety issues with len8viral vectors (…main 8ps…)
§  Use a restraint device §  Use a disinfectant-­‐soaked cloth to wipe away excess inoculum leaking from inocula1on site §  Conduct inocula1ons, cage changing, and other procedures in a biosafety cabinet (when possible). §  House animals in a primary containment device appropriate for the rodent species, such as a ven1lated micro-­‐isolator cage or sta1c micro-­‐isolator cage with a filter top. §  Infected animals may excrete viral vectors (refer to Risk Assessment and Risk Management established procedures) . Dimitri Sossai Luca Nelli June 2016 Recommendatons for handling LV and Viral Vectors
Consider risk for mutagenesis or toxic proper1es of transgene Consider risk from animals treated with VV Anesthe1zing animals Consider risk of viral shedding in immunodeficient animals Consider present or future risk for HIV in lab personnel along with confiden1al tes1ng (pre-­‐placement and serial tes1ng????) §  Cleaning and decontamina1on procedure § 
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Dimitri Sossai Luca Nelli June 2016 Cleaning-­‐Deac8va8ng Viruses and VV
Lipid Enveloped Viruses
(Retro, Lenti, MMLV, HIV, Herpes Simplex, Flu, Hepatitis B and C)
Ethanol solution
Quaternary Ammonium Compounds
Bleach
Autoclave
Non-Lipid Enveloped
(Adenovirus, Adeno-Associated Virus)
Bleach
Autoclave
Non-lipid Enveloped Viruses are resistant to weaker
disinfectants like ethanol and quaternary ammonium
compounds.
Bleach decomposes over time and has an approximate
half life of 2 weeks. Recommend making fresh weekly.
Corrosive for equipments and stainless steel
Liquid disinfectants must be allowed the appropriate
Dimitri Sossai Luca Nelli June 2016 contact time to be effective.
Recommendatons for handling LV and Viral Vectors
Consider risk for mutagenesis or toxic proper1es of transgene Consider risk from animals treated with VV Anesthe1zing animals Consider risk of viral shedding in immunodeficient animals Consider present or future risk for HIV in lab personnel along with confiden1al tes1ng (pre-­‐placement and serial tes1ng? – Local legisla1on?) §  Cleaning and decontamina1on procedure §  Consider an1retroviral treatment post exposure, anyway having a Post Exposure Procedure (PEP) -­‐ PRIOR to an exposure § 
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Dimitri Sossai Luca Nelli June 2016 Post exposure treatment is an essencial part of Risk Management
§  Develop specific PEP, user friendly (e.g. max 3 pages) and taking with you in case of accident §  LV vector exposures, par1cularly if associated with a hazardous transgene (e.g., an oncogene or toxin), should consider use of an an1retroviral agent §  The PEP should be developed by the Occupa1onal Health Physician and the BSO §  Planning for post-­‐exposure prophylaxis needs to be planned in advance and ini1ated quickly §  Most physicians are not familiar with len1viral vectors and need to be educated in advance regarding treatment op1ons §  Should be applied to all the staff involved: scien1sts, technicians, animal care staff Dimitri Sossai Luca Nelli June 2016 Post exposure treatment is an essencial part of Risk Management
Dimitri Sossai Luca Nelli June 2016 Recommendatons for handling LV and Viral Vectors
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Consider risk for mutagenesis or toxic proper1es of transgene Consider risk from animals treated with VV Consider risk of viral shedding in immunodeficient animals Consider present or future risk for HIV in lab personnel along with confiden1al tes1ng (pre-­‐placement and serial tes1ng? – Local legisla1on?) Cleaning and decontamina1on procedure Consider an1retroviral treatment post exposure, anyway having a Post Exposure Procedure (PEP) -­‐ PRIOR to an exposure Need to develop system to report and monitor exposures Need to proac1vely train all staff to understand poten1al risks with these agents and on ways to prevent exposures Dimitri Sossai Luca Nelli June 2016 a recent news….
……and so? Full presenta1on @: h\p://www.croiwebcasts.org/console/player/29486?mediaType=slideVideo&
Dimitri Sossai Luca Nelli June 2016 A simple example….
as well as other rou1ne lab prac1ces ( ….not specific only for BSL3/BSL2… or GMM or WT agent), what we’ve seen with this last simple example can do the difference between undergoing an exposure or not. Dimitri Sossai Luca Nelli June 2016 Many Thanks! Dimitri Sossai Luca Nelli June 2016