· Web viewContaminated liquid waste: Will be either autoclaved or diluted to 10% bleach before disposal. Contaminated solid waste: Disposable solid waste will be placed in the biohazard

Approval Date: 1/10/12 Expiration date: 2/15/15 Protocol Number: 1202-70118

Institutional Biosafety Committee (IBC) PROTOCOL SUBMISSION FORM

Submit the completed application by email to [email protected] with “IBC-Application” in the subject line.

Section A: TITLE AND PERSONNELTitle of the study: Development of Attenuated Shigella Vaccines

Submission date: 1/10/2012 Is this a new submission? YESPrincipal investigator*: Joe Smith Position/title: Assistant ProfessorDepartment: Microbiology and Immunology School: School of Medicine

Email: [email protected] Phone: 988-0000 Emergency phone: 504-123-4567

Names of co-investigators or collaborators Title: Email: Phone:Bunsen Honeydew Associate Professor [email protected] 8-0000

Name other personnel under your supervision working on this project **Assistant Beaker Laboratory technician [email protected] 8-0000

*Must be a Tulane Investigator ** If needed, add more names at the end of Section G

Section B: TYPE OF RESEARCHThe research described in this application involves: (select all that apply)

1. Recombinant DNA/RNA Yes No If “YES” complete Sections A, B, C, D,G and H of this application

2. Infectious/Pathogenic microorganisms or toxins Yes No If “YES” complete Sections A , B,C,E, G and H of this application

3. Select Agents (see appendix 1 in the IBC web page) Yes No If “YES” complete Sections A, B,C, E, F, G and H of this application

Select Agents are certain microorganisms or toxins that HHS and/or USDA consider to have the potential to pose a severe threat to human, animal or plant health. A list of these agents can be found in: http://www.selectagents.gov/Select%20Agents%20and%20Toxins.html

4. Animals Yes No If “YES” Species: RodentsIACUC approval number:10000099

5. Transgenic Animals Yes No

6. Humans Yes No If “YES” provide IRB approval number:

IBC Office ([email protected])Internal address: Mail Box TW-5Phone: (504) 988-0300; Fax: (504) 988-0370 Version December 2011 Page 1 of 13

http://www.selectagents.gov/Select%20Agents%20and%20Toxins.html

http://www.selectagents.gov/Select%20Agents%20and%20Toxins.html

mailto:[email protected]

SECTION C: GENERAL INFORMATION

PERFORMANCE SITESResearch location(s)--check all that apply:

Tulane University- St. Charles Campus Tulane University- School of Medicine Tulane University- School of Public Health and Tropical Medicine Tulane National Primate Research Center Other location(s). Specify :

Building and Room Number(s) where research will be conducted (provide buildings/room numbers for all experiments detailed in this application), and biocontainment level of that room.

Building: Room No: Biocontainment level for this laboratory

Experiment (ie. in vitro work, animal infection)

SOM 43009 BSL2 In vitro work

SOM vivarium BSL2-N (animals) In vivo work: animal immunization & euthanasia

Does this project involve work to be done by collaborators at other institutions?Name of the institution(s):

Yes No

If ‘YES’, did the institution grant approvals for this project? IBC

IACUC IRB

Don’t know Don’t know Don’t know

Yes Yes Yes

No No No

SECTION C-2: PROJECT OVERVIEW

1. Provide a description of the work to be conducted in this project. Briefly explain in language understandable to a layperson the aim of this research and its importance to human or animal health, the advancement of knowledge, or the benefit to society.

Members of the genus Shigella are bacterial enteropathogens which cause bacillary dysentery and bloody diarrhea. The incidence of shigellosis in the US is between 15000-25000 infected people a year. The infections are usually self-limited but they can cause serious consequences in children and malnourished individuals. Effective vaccines against Shigella are not currently available. The aim of this research is to develop potential vaccines against shigellosis, based on attenuated Shigella flexneri mutants. Our hypothesis is that Shigella attenuated mutants can induce long-lasting immunological protection without causing disease. Mutated strains of S. flexneri will be constructed as described below and will be tested for immunogenicity in mice.

2. Succinctly describe in logical and chronological order the experiments that will be conducted.

We will develop between 3-5 attenuated mutants of Shigella as explained in Section D-2. The mutations will be confirmed by DNA sequencing and Western Blot analysis (by testing for lack of expression of the proteins encoded by the mutated genes).These bacterial mutants will be grown in the laboratory, centrifuged and adjusted for concentration; mice will be orally vaccinated with 2 doses (between 1x10^(3) and 1x10^(6)). We will vaccinate animals 2 times at monthly intervals. Four weeks after the final immunization animals are euthanized and samples collected for immunological analysis.


SECTION D:Recombinant DNA Research Questionnaire.

Check 'YES' or 'NO' to the following questions. For any items checked 'YES', include a thorough description of the work in the following page .The relevant section of the NIH Guidelines is referenced for each question (refer to the NIH Guidelines* for more information).

1. Does your project include deliberate transfer of a drug resistance trait to microorganisms that are not known to acquire the trait naturally (Section III-A*)?

1a. If “YES”, could such a transfer compromise the use of the drug to control disease agents in humans, veterinary medicine, or agriculture?

Yes

Yes

No

No

2. Does your project include cloning toxin molecules with an LD50 of less than 100 nanograms per kilogram body weight (Section III-B*)? Yes No

3. Does your project include experiments using Risk Group 2, Risk Group 3, Risk Group 4, or Select Agents as host-vector systems (Section III-D-1*)? Yes No

4. Does your project include experiments in which DNA from Risk Group 2, Risk Group 3, Risk Group 4 , or Restricted Agents is cloned into nonpathogenic prokaryotic or lower eukaryotic host-vector systems (Section III-D-2*)?

Yes No

5. Does your project include 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 (Section III-D-3*)?

Yes No

6. Does your project include experiments involving genetically engineered plants (Section III-D-5, III-E-2*)? Yes No

7. Does your project include experiments involving more than 10 liters of culture (Section III-D-6*)? Yes No

8. Does your project include experiments involving the formation of recombinant DNA molecules containing two-thirds or less of the genome of any eukaryotic virus (Section III-E-1*)?

Yes No

9. Does your project include experiments involving viable rDNA-modified microorganisms tested on animals (Section III-D-4, III-E-3*)? If “YES” answer question 2 on the next page.

Yes No

10. Does your project include experiments involving whole animals in which the animal’s genome has been altered by introduction of DNA into the germ line (i.e. transgenic animals) (Section III-D-4, III-E-3*) .If “YES” answer questions 2 and 3 on the next page.

Yes No

11. Does your project include experiments involving the deliberate transfer of recombinant DNA, or DNA or RNA derived from recombinant DNA, into one or more human research participants (Section III-C*)?*). If “YES”, answer question 4 on the next page.

Yes No

* The NIH Guidelines are found in this link: http://oba.od.nih.gov/rdna/nih_guidelines_oba.html Updated information about required levels of Biosafety are found in the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL) http://www.cdc.gov/biosafety/publications/bmbl5/


http://www.cdc.gov/biosafety/publications/bmbl5/

http://oba.od.nih.gov/rdna/nih_guidelines_oba.html

SECTION D-2:Recombinant DNA Research Information

1. Provide succinct explanations for items checked ‘YES’ in questions 1-8 of the Recombinant DNA Research Questionnaire in the previous page (include the question number).

Question 1.To develop attenuated mutants of S. flexneri we will insert a Kanamycin containing casette into specific loci in the Shigella bacterial chromosome. We have received (from Dr. R. Toodtoo, Emory University) three recombinant suicide plasmids derived from pXZTY. The plasmids carry the Shigella genes xxx1, xxx2, and xxx3 with a Kan casette inserted in the middle of the gene. We will create at least three mutated Shigella strains by transforming wild type S. flexneri with these plasmids.

Question 1a. Kanamycin is not used in the clinic to treat shigellosis (neither in the US nor in other countries); therefore, transfer of a Kan. gene into Shigella does not compromise the way Shigella infections are controlled. Unintended dissemination of Kanamycin-resistant Shigella strains will be avoided by handling the strains under strict BSL2 containment.

Question 3. Shigella is a RG2 organisms and will be used as a host of plasmids pXZTY1, pXZTY2 and pXZTY3,which carry the mutated genes and Kan cassettes.

2. Describe the use of animals in your experiments. Briefly explain experiments in logical, chronological order. The approval of this protocol will not be effective until IACUC approval is received.

Groups of Balb/c male mice will be orally immunized, twice (day 0 and 30), with 1x10^5 or 1x10^3 attenuated Shigella CFUs; control groups will receive the same dose of non-recombinant E. coli K-12 or saline. Four weeks after the final immunization, the mice will be euthanized and blood and tissue samples will be harvested for immunological analysis.

3. If using transgenic animals please answer the following questions:a. Are you acquiring or breeding rodents that can be safely housed under BSL1 containment?*

Yes Nob. Do these rodents (parents or progeny) contain more than 50 percent of the genome of an

exogenous eukaryotic virus from a single family?* Yes Noc. Is the transgene in these rodents (parents or progeny) under the control of a gammaretroviral long

terminal repeat (LTR)? * Yes No

*You MUST fill appendix C and submit it with this application IF you are using transgenic animals other than rodents OR your transgenic rodents need to be housed at BSL2 or higher containment OR you answered YES to one or both of the last 2 questions.

4. Describe the use of humans in your experiments. Explain experiments in logical, chronological order. The approval of this protocol will not be effective until IRB approval is received.

N/A

5. Identify the host(s) to be used (e.g., the target of gene transfer). Examples: E. coli, S. cerevisiae, human/animal cells, whole animals, humans. Provide species designations for all organisms where possible.

The host will be Shigella flexneri


6. Identify the vector(s) to be used. Examples: Bacterial plasmids, yeast vectors, mammalian cell vectors, baculoviruses, transforming viruses, etc. Provide name, source, and description of the vector, and include description of antimicrobial resistance genes.

The vector is a modified plasmid pXZTY . This vector was obtained from Dr. R. Toodtoo (Emory University). The vector was originally derived from pBR322 but it does not carry the gene encoding for resistance to ampicillin. It is a low copy number plasmid with the ColE1 origin of replication. pXZTY has 2 multiple cloning sites located upstream and downstream of the LacZ gene. It carries a Kanamycin resistance gene flanked by two FRP (recombination) sites.

7. Identify the nature of the insert DNA sequence, including the species of origin (i.e., specific gene, promoter, expressed product and function (if known). If available attach a vector and/or insert map.

The insert DNA sequences are the mutated Shigella genes xx1, xxx2 and xxx3. These genes encode the X1, X2, and X3 proteins respectively. These are outer membrane proteins (porins) that allow diffusion of macromolecules into the bacterial cytoplasm. These proteins are not toxic for animals or humans. When any of these 3 proteins is not functional, the resultant mutants cannot survive in the murine hosts for more than 96 hours. A map of the plasmid+insert is provided as an attachment at the end of this application.

8. If foreign gene product(s) will be purified, indicate which foreign gene product will be purified and briefly describe the procedures for purification, including volumes of culture

N/A

9. If replication-incompetent vectors will be used, include information about how incompetent vectors are tested for reversion mutations (e.g., endpoint dilution analysis, plaque assay, commercially obtained and tested by manufacturers).

N/A


SECTION E:Hazardous Biological Research Questionnaire

Check 'YES' or 'NO' to the following questions. For any items checked 'YES', include a thorough description of the work in the following page (question 1).1. Is agent/material a potential human, animal or plant pathogen? Human Animal Plant N/A

Yes No

2. Is agent/material a toxin? If “YES” name of the toxin (s): Toxic to (animals/humans/plants): LD50 :

Yes No

3. Do you work with quantities (i.e. cultures) larger than 1 liter? If “YES”, what is the largest volume (liters)?

Yes No

4. Do you inactivate the agent/material prior to laboratory manipulation?If “YES”, by what method?

Heat Chemical Radiation Other

Yes No

5. Do you concentrate the agent/material? If “YES”, specify method:

Centrifugation Precipitation Filtration Other Yes No

6. Do you expose live animals or humans to the agent/material? If “YES”, specify the animal species: Mice

IACUC Approval # 10000099 IRB Approval:#

Yes No

7. Does your project include experiments with Risk Group 2, Risk Group 3 or, Risk Group 4 agents? *If “YES”, which Risk Group? RG-2 RG-3 RG-4

Yes No

8. Does your experiment involve work with arthropods (i.e. insects, spiders. others)? If “YES”, provide specific details in the next page (question 1)Work with certain arthropods must conform to the guidelines set forth in the BMBL and those established by the American Committee of Medical Entomology.

Yes No

9. Does your work involve a SELECT AGENT? (see appendix H for list of select agents)

If “YES” name the organism (s): Yes No

10. Do you work with human blood, tissues, or body fluids?If “YES” contact Tulane OEHS for guidelines on working with blood-borne pathogens.

Yes No

11. What is the recommended Biosafety Level for this agent/material? * BSL-1 BSL-2 BSL-3 BSL-4

*To identify the risk group (RG) classification of the recombinant or etiologic agent(s) and the proposed biosafety level (See the NIH Guidelines at http://oba.od.nih.gov/rdna/nih_guidelines_oba.html And the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories at: http://www.cdc.gov/biosafety/publications/bmbl5/


http://www.cdc.gov/biosafety/publications/bmbl5/

http://oba.od.nih.gov/rdna/nih_guidelines_oba.html

SECTION E-2:Hazardous Biological Research Information

1. Provide succinct explanations for items checked ‘YES’ in the Hazardous Biological Research Questionnaire in the previous page (include the question number).

Question 1. Shigella species are pathogenic in humans and are the etiological agents of bacillary dysentery.Question 5. Overnight cultures of Shigella will be centrifuged and resuspended in saline. The right concentration will be determined by spectrophotometer reading at O.D. 650 (based in pre-established growth curves).Question 6. animals will be orally immunized twice with 1x10^6 organismsQuestion 7. Shigella is a RG2 pathogen

2. Describe the key features of the agent/microorganism/toxin or material that you will use in this project, particularly as it refers to biosafety considerations. Briefly describe the pathogenesis of the disease caused by this agent in humans and/or animals.

Shigella are Gram-negative bacterial enteropathogens which are transmitted to humans by the fecal-oral route. The infectious dose can be as little as 10 organisms . Once the organisms reach the large intestine they invade epithelial cells, and kill the resident macrophages inducing a severe inflammatory reaction characterized by influx of neutrophills and intestinal tissue destruction. This tissue destruction results in cramps, and bloody diarrhea. Personnel working in this project will be instructed about the high communicability of this disease and the strikingly low infectious dose.

3. What is the source or your biohazardous agent? (Be specific: i.e. ATCC, CDC, clinical isolate, etc).

S. flexneri will be obtained obtained from ATCC (strain V9089)

4. Do you work with microorganisms of unknown identity (such as those obtained from environmental samples, animals/humans infected with unknown organisms)?

NO

5. Does import of this agent into Tulane facilities require a USDA/CDC import/transport permit?

NO

6. Will the agent be cultured/propagated at Tulane? If “YES” provide a synopsis of the procedures.

Yes, it will be propagated at Tulane. Shigella strains will be grown overnight in 10 mls of Trypticase Soy broth. Bacterial cultures will be centrifuged; bacterial pellets will be washed twice in PBS and resuspended in the amount of saline necessary to give the desired concentration. These manipulations are done at BSL2 containment.

7. Will your work be performed in a BSL3 laboratory? If “YES” detail the procedures to be done in the BSL3.

NO


Section F:Select Agent Questionnaire

All work with Select Agents must be approved by IBC before initiation. IBC protocols involving Select Agents will be subjected to yearly review.

1. Is there a vaccine available and recommended for persons handling this Select Agent?If “YES” will personnel working in this project be offered the vaccine?Expand the answer to this question under question 1 in the next page.

Yes

Yes

No

No

2. Do you plan on shipping, or transporting the select agent?If “YES” be aware that a Select Agent must be transported under the conditions described in specific Code of Federal Regulations (CFRs). Consult the Office of Biosafety for guidelines.

Yes No

3. If you are working with a toxin, indicate the largest amount that you will have in your possession at any given time.

mgs

4. What is the largest volume of culture (bacterial/viral) and approximate concentration that you will have at any given time? (i.e. 1 liter at 10x106 CFU/ml).

5. Will access to the Select Agent be meticulously controlled at all times? Yes No

6. Have you submitted an agent-specific Standard Operating Procedure (SOP) with this application? (This is a requirement for approval of your protocol).

Yes No

7. Does your SOP include specific instructions to handle accidental spills and accidental personnel exposures? Yes No

8. Will personnel involved in this project receive agent-specific training, to make them aware of the risk and hazards associated with this agent, and the SOPs for accidental spills or exposures?

Who will provide the agent-specific training?

Yes No

9. Will a copy of the training certification and assessment evaluation for each person working with the agent be filed with the Biosafety Office?

Yes No

10. Are you and all the personnel involved in this project proficient in the general BSL3 SOPs written by the Biosafety Office?

Yes No


Section F-2:Select Agent Information

1. Provide relevant and detailed information to expand the answers to the questionnaire in the previous page (provide item number).

2. What agent-specific SOPs will be used in the course of these experiments? Provide name and date of the SOP (SOPs must accompany this application. The protocol will not be approved until agent specific SOPs are submitted and approved by the Biosafety Office).

3. If working with animals indicate the final fate of all animals exposed to the select agent.

4. If necessary, add additional pertinent information on the Select Agent and/or experimental procedures for this project (do not write decontamination or disposal practices in this section).


SECTION G:Biohazards, Decontamination and Training

1. Identify all known and potential hazards or risks associated with the specific biohazardous agent or with the use of recombinant DNA materials described in this application. Check all that apply and add additional information as needed.

Generation of aerosols Contaminated needles or sharps Potential spills or splashes Exposure to infected animals Exposure to blood borne pathogens Insect bites Splashes to mucosal surfaces Others: Accidental ingestion.

Unintended dissemination of new Shigella strains (the mutated strains cannot survive outside mammalian hosts for more than 2-3 hours, therefore the risk of dissemination of new strains is very low).

2. Describe the necessary facilities/equipment that will be used for all aspects of the work. (i.e. use of biosafety cabinets, aerosol-proof centrifuges, aerosolization chambers, etc.)

The bacterial cultures will be centrifuged in aerosol-proof tubes and rotorsDilutions of bacteria will be done in a Biosafety cabinetImmunization of animals will be done in the vivarium in biosafety cabinetsBSL2 guidelines will be strictly followed

3. Describe the practices for managing infectious/hazardous agent SPILLS and personnel ACCIDENTAL EXPOSURES.

Spills will be handled as per instructions in the Biosafety Office SOP Biohazard Spill Clean upBriefly, spill will be covered with paper towels andthe towels are soaked with a 70% ethanol.. Spill is allowed to soak for approximately 20 minutes before discarding materials in biohazard bag/box. Surfaces will be decontaminated with 70% ethanol. All disposable materials used for the clean up (paper towls, gloves, etc) will be discarded in a biohazard bagAll personell accidental exposures will be inmediately reported to supervisor (principal Investigator Dr. Joe Smith.

4. Indicate the personal protective equipment (PPE) required for personnel working in this project. Check all that apply and add additional information as needed.

PPE required for bench work: Lab coat Gloves Goggles Face mask Full face shield N-95 mask PAPR Tyvek coverall Shoe covers Solid front gown Head cover

PPE required for work with animals: Lab coat Gloves Goggles Face mask Full face shield N-95 mask PAPR Tyvek coverall Shoe covers Solid front gown Head cover Others:


5. Specifically describe the decontamination practices of the work area, equipment and samples*. Check all that apply and add additional information as needed.

Surfaces/equipment and or samples are thoroughly treated or cleaned with:

70% ethanol 10% Bleach Lysol Povidone/Iodine Aldehydes Quaternary ammonium compounds

Autoclave*Samples to be transported out of the lab will be treated as follows: Samples will be transported from the vivarium to room 4300 in a primary leak-proof container inside a secondary leak-proof container ( i.e.closed polypropylene tubes, placed inside a plastic tray with a lid).

Additional decontamination practices: Non disposable equipment will be autoclaved.

6. Describe the disposal practices of contaminated waste material. Provide specifics for each type of material.

Contaminated liquid waste: Will be either autoclaved or diluted to 10% bleach before disposal.Contaminated solid waste: Disposable solid waste will be placed in the biohazard autoclavable bags/boxes provided by Tulane. Carcasses of infected/exposed animals: Infected carcasses/tissues will be double bagged and placed in the JBJ vivarium carcass cold room, where vivarium personnel will decontaminate and dispose as per their standard SOPs.

Contaminated sharps: Contaminated sharps will be placed in a "sharp containers" which will be securely closed before placing it in the autoclavable boxes provided by Tulane. Others disposal practices: .

7. For Risk Group 3 agents and for agents that require BSL3 containment, briefly describe YOUR prior training or experience in this type of work.

N/A

8. Describe how personnel will be trained in the handling of the biological agents described in this application. Who is in charge of training? (The PI is responsible for keeping accurate records of personnel training).

The PI (Dr. Joe Smith) will train all personnel working in this project. Personnel will be instructed on the risks of working with infectious (wild type or mutant) Shigella strains and with recombinant DNA materials. Personnel will be trained to follow BSL2 guidelines and wear the required PPE at all times while handling Shigella strains and/or rDNA. The PI will keep records of this training.

9. If your experiments involve animals, do animal handlers need to CHANGE their normal/daily biosafety and animal handling routines? If so, please explain how.

No changes to routine veterinary care are necessary.Cages with infected animals will be properly labelled (with biohazard stickers identifying wild type Shigella or rDNA carrying Shigella as the biohazardous agents).


10. Describe any specific biosafety considerations for this agent that have NOT been addressed in the previous questions.

N/A

11. List other personnel in this project (not listed on page 1)

Name Title E-mail


SECTION H: Investigator’s Assurance

1. I confirm that all persons conducting this work at Tulane University (including students, fellows, technicians, and collaborators) have been adequately trained in good laboratory/ microbiological practices and aseptic techniques; have received instruction on the specific hazards associated with the work and are aware of the specific safety equipment, practices, and behaviors required during the course of the work and use of these facilities. I also confirm that I will keep records of training my personnel.

2. I will report to the Biological Safety Officer immediately any spill of biohazardous material, any equipment or facility failure (e.g., ventilation failure), and/or any breakdown in procedure that could result in potential exposure of laboratory personnel and/or the public to biohazardous material.

3. I confirm that any proposed changes to my work that would result in an increased level of biohazard will be reported to the IBC before the change is implemented.

4. I confirm that no work requiring IBC approval will be initiated or modified until approval is received.5. I have read and understand my responsibilities as Principal Investigator outlined in Section IV-B-7 of the NIH

Guidelines and the Tulane University Institutional Biosafety Committee (IBC) Policy Manual for the Use of Recombinant DNA, and agree to comply with these responsibilities.

6. I certify that the information provided within this application is accurate to the best of my knowledge. I also understand that, should I use the project described in this application as a basis for a funding proposal (either intramural or extramural), it is my responsibility to ensure that the description of the work in the funding proposal is identical in principle to that contained in this application.

Joe Smith 1/10/12

Typed Name of Principal Investigator Signature of Principal Investigator Date

UPON APPROVAL, YOU MUST SEND A SIGNED COPY OF THIS LAST PAGE TO THE IBC OFFICE (by Campus mail Box TW-5; Fax 8-0370; or email (scanned copy) to [email protected])

For IBC Use OnlyProtocol Number: 1202-70118 BSL Level: BSL2

Protocol Title: Development of Attenuated Shigella vaccines

Date Received: 1/10/2012 Date Approved: 2/15/12 Date Expiration: 2/15/15

Experiment Class Determination (check one)

III-A Requires IBC approval, RAC review, and NIH Director approval before initiation

III-B Requires NIH/OBA and IBC approval before initiation

III-C Requires IBC and IRB approvals and RAC review before research participant enrollment

III-D Requires IBC approval before initiation

III-E Requires IBC notice simultaneous with initiation

III-F Exempt

IBC Signature: Date: 2/15/2012


mailto:[email protected]

Documents

· Web viewContaminated liquid waste: Will be either autoclaved or diluted to 10% bleach before disposal. Contaminated solid waste: Disposable solid waste will be placed in the biohazard