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NCI-FREDERICK

INSTITUTIONAL BIOSAFETY COMMITTEE

Minutes

February 20, 2007

NCI-Frederick

The NCI-Frederick Institutional Biosafety Committee was convened at
12:01 p.m. in the Building 426 Training Room with the following members
in attendance:

Ms. Theresa Duley, Secretary Dr. Henry Hearn

Dr. Randall Morin, Chair Dr. David Garfinkel

Ms. Alberta Peugeot Dr. Mike Baseler

Dr. Bruce Crise Dr. Stephen Hughes

Ms. Dianna Boissey Dr. Stephen Creekmore

Dr. Dan McVicar

Members not in attendance: Mr. Lucien Winegar, Dr. Jeanne Herring, Dr.
Melinda Hollingshead

Others in attendance: Ms. Cara Leitch, Dr. Scott Keimig, Dr. Robert
Thomas, Dr. Barbara Felber, Dr. George Pavlakis, Dr. Antonio Valentin,
Mr. Anthony Welch, Dr. Dexter Poon, Dr. Ray Harris, Dr. Robin Dewar, Mr.
Mohammed Tauseef Rehman

**[Pavlakis & Felber]{.underline}**

Dr. Pavlakis and Dr. Felber attended the IBC meeting, distributed a
comprehensive packet of information containing their proposed responses
to the IBC members comments, and took the time to explain each line item
and how the issues pertinent to their laboratory would be addressed to
comply with IBC and regulatory requirements.

\- All responses to IBC questions are provided in blue font.

\- All safety items are addressed to include knowledge, training, the
practice of handling the lowest possible amount of infectious agents,
and procedures put in place to minimize accidents and mitigate risks.

\- Attachments are included for floor plans of both BSL2\* and BSL2
areas.

\- Avoidance of recombination was addressed in conjunction with never
using two different viruses during transfections or use of infected cell
lines.

\- Only pieces of viral proteins will be manipulated, so if
recombination events were to occur, there is a negligible risk that a
full-length virus would be produced.

\- Materials are synthesized in vitro, so sequence homology is not
present.

\- Sharps use has been modified. There will be no use of scalpels or
needles in the BSL2\* lab- Blunt nosed forceps and scissors will be used
for tissue mixing and mincing.

\- Dr. Valentin and Dr. Alicia are the only employees who will perform
in-vitro work.

\- Scalpels will only be used to cut bands for acrylamide and agarose
gels, where no infectious material is involved. Employees will wear cut
resistant gloves also when using scalpels.

\- Single use needles will be used with anti-retrovirals to access
vials, but will not be used with any infectious material/DNA.

\- Aerosol control/containment is mitigated while working in a BSL-2\*
facility including a biological safety cabinet with proper Personal
Protective Equipment.

\- Limited quantities of full-length molecular clones are used on a rare
occasion (1-2x/year) and no sharps are permitted for use with this
material.

\- No high-speed centrifugation occurs. Infectious cells are spun in a
clinical centrifuge in closed containers outside of BSC. Containers are
opened in the BSC and decontaminated prior to removal outside of BSC.

\- Co-Principle Investigators are acceptable for these registrations, so
long as each PI identifies their own employees, who will work with each
specific material, and who will be permitted to work in the BSL-2\* lab.

\- SEB is less than 5 mg, so it is not considered a Select Agent.

\- The HBV vaccine is encouraged -- Dr. Pavlakis and Dr. Felber believe
everyone has received the vaccination, although this is a confidential
matter between the employee and OHS. HIV medical surveillance is also
made available and employees can be tested on a voluntary basis.

\- The IBC will vote on the proposed review and comment period deadline
and Ms. Bell will go over comments with Drs. Pavlakis and Felber and
Terry Jones to integrate final comments and modifications into the form.

\- Drs. Pavlakis and Felber hold a lot of common meetings with their
staff to discuss issues pertinent to the work and health and safety.

\- The IBC agreed that 2 weeks would be necessary for the IBC to perform
their review of the plan documents. Furthermore, an additional 2 weeks
immediately following the IBC review would be granted for Drs. Pavlakis
and Felber to integrate comments into the registration documents with
the assistance of Ms. Bell.

\- The final documents will be in place and complete by the March 20 IBC
meeting.

**[Wiltrout (06-70)]{.underline}**

\- This study involves using the same packaging gene with a replication
competent lentivirus. Appropriate mitigation measures are clearly
outlined in the registration document. Barrier methods are in place and
there is no potential for mixing or combining harmful agents. There are
no modifications requested for this registration.

Dr. Creekmore made a motion to approve the registration as written, Dr.
Garfinkel seconded and all were in favor.

**[Poon & Harris (07-08)]{.underline}**

\- The vector proposed for use is conditionally replication competent
and is designed for use in humans, which raises important issues.

\- Is the actual virus stock being received or is it possible for the
source provider to isolate intact DNA from the viral stock and/or
provide only plasmids?

\- Clarify that either the DNA or viral stock will be directly sequenced
before handling. Unambiguous results are necessary so PCR or blots may
not be sufficient and are not recommended.

\- There are potential hazards with HSV production, given a biologically
active cytokine present in replication competent HSV. Be sure these are
sufficiently addressed with appropriate mitigation.

\- Acknowledge that infection is possible in an immune competent
individual

\- Clarify and acknowledge that the material will not replicate in the
Central Nervous System like normal HSV, but can spread from person to
person.

\- Provide a statement to address the potential advantage of the vector
in use retaining TK, which would enable treatment to be administered to
someone accidentally exposed to the vector with an anti-herpetic
medication. Provide an assurance, in accordance with OHS, that a
Post-Exposure Prophylaxis protocol is in place for both regular business
hours as well as for evenings and weekends, and that OHS does maintain
anti-herpetic medication on-site.

\- There was a recommendation to request some extra PCR on other HSV
projects, to ensure the integrity of the material in use.

\- A recommendation was made to use outside labs for sequencing plasmid
DNAs, to ensure the proper material is being received.

\- Confirm if PCRs will also be done to check for wild-type.

\- Verify the lab is dedicated to Herpes work.

\- Need statement in paperwork explaining how they are keeping
adenovirus and HSV separate. Is there a potential risk for cross
contamination between projects or agents? If so, how will this be
mitigated?

Dr. Hughes made a motion to conditionally approve pending receipt of the
requested modifications above, Dr. Crise seconded and all were in favor.
Dr. Creekmore abstained from the vote.

**[Anderson (07-02)]{.underline}**

\- There were no comments from the lead reviewers to request
modifications.

\- No virus or retroviral vectors are being used in this proposed
research.

Dr. Garfinkel made a motion to approve the registration as written, Dr.
Baseler seconded and all were in favor.

**[Dewar (07-05)]{.underline}**

\- Is there additional information available from the source providers
at CDC regarding the viral material and the known infectivity?

    -Are CDC/USDA permits required?

    -Would this or any of the other items be considered a select agent?

\- In Part A1, \"sufficiently high titer\" should be more clearly
defined.

\- Should the Puerto Rico strain mentioned in B3 also be identified in
C1a?  Ensure the numbers match between sections B and C
(A/Vietnam/1203/04 versus /2004 for clarification.

\- Are the MDCK\'s the only cell lines that will be used?

\- The current SOP is sufficient but minimal information is provided
regarding spills and validation of clean-up procedures.

\- Individuals w/ flu-like symptoms cannot go into lab.

**Dr. Crise made a motion to conditionally approve pending receipt of
the requested modifications above, Dr. Creekmore seconded and all were
in favor. Dr. Baseler abstained from the vote.**

**[Young (07-03)]{.underline}**

\- According to the registration, cell lines are considered infectious
for two passages. Why only two? Human cell lines should always be
handled as \"Other potentially infectious material (OPIM)\".

\- It is noted that two different vectors are in use: pLKO (not tat
responsive) and pGypsy (tat responsive). There is a technical issue in
that pGypsy may not work with the packaging gene specified for use.

\- How will the work with the two vectors be kept separate?

\- Isn\'t it possible that the HIV envelope could be produced? Why or
Why not?

\- B5f1: Clarify this statement regarding the potential for
recombination with natural virus. The absence of the HIV envelope makes
it safe but in B5f1 the answer should be \"NO\". HIV does not recombine
with endogenous retroviruses. Clarify there is no HIV involved with this
work, and that there is no potential for recombination.

\- Provide the technical information regarding safety features and
requirements for packaging lines. There are underlying safety features
inherent to the packaging systems that are critical to the overall
safety of this work.

**Dr. McVicar made a motion to defer approval pending receipt of the
requested modifications above, Dr. Baseler seconded and all were in
favor. A subcommittee of two IBC members will reconsider the responses
and be responsible for issuing final approval.**

**[Renewals]{.underline}**

**[Daar (07-04)]{.underline}**

\- Second Part B - B1 seems to contradict B5. B1 mentions the vector
pCS2+ and sCMV, but B5 states no viruses, promoters, of vectors are
used. Then all of the subparts of B5 should be answered.

\- Standardize what face protection will be used (glasses with side
shields, goggles, face shields, etc).

\- Clarify the details regarding protein expression in B8b and B8b1. If
there is no hazard with expression of the foreign protein, then this
should be answered \"No\"

\- Review the document overall for clarity and consistency.

**Dr. Creekmore made a motion to conditionally approve pending receipt
of the requested modifications above, Dr. Garfinkel seconded and all
were in favor.**

**[Hurwitz (07-06)]{.underline}**

\- Identify the vectors involved. Explain what the vectors do and why is
this safe to work with? 

\- Confirm this is a non-commercial sequence regarding the homemade
vector to be received from a colleague. The vector safety features
should be mentioned as well as the annotation for the HIV vector
sequence.

\- Mice are likely to shed E. coli -- how will the hazards be addressed
and how will animal handlers be adequately informed and trained to
accommodate these risks.

\- A2 describes four different activities that should be sorted out as
follows:

 

Part 1: Viral vectors

Part 2: Plasmids

Part 3: Pertussis toxin

Part 4: E. coli

 

\- A3: This question should be answered to address the experiments as
follows:

> \- Parts 1 and 2 from A2 should be grouped together to describe
> genetic modifications used with amphotropic viruses and mammalian
> cells.

\- Part 3 - Pertussis toxin should be described in a separate
registration.

\- Part 4 - E. Coli should also be described in a separate
registration. 

\- It currently appears from the responses in the paperwork that each of
these studies are independent from one another and involve separate ACUC
protocols as well. 

\- Parts 1, 2, and 4 should address replication competence and
mobilizable elements with respect to the use of VSV-g, MLV based vectors
in cells and amphotropic virus.  

\- For centrifugation, provide some assurance that the centrifuge will
be opened in a BSC and give a description of decontamination measures
for the centrifuge between experiments. 

\- A4: Are sharps used in the laboratory or only in the animal facility
for injections? 

\- A6a1 answers are insufficient: The PI should clearly identify primary
and secondary containment that is sealed and leak proof. How are the
centrifuge tubes sealed? 

[Part B, I]{.underline}

-This first section B should ideally address information pertinent to
Parts 1 and 2 from A2. Address the hazards associated with MLV, and
identify which ASP applies to each Part B.

\- B5g1: More specific information is needed. 

# Part B, II

\- This second section B should be expanded to address specifics
regarding the E. coli.  

\- B4 should provide more detailed information. 

\- Amphotropic replication competent strain of e.coli -- describe the
mechanisms in place to work with this material safely.

\- Mice are likely to shed E. coli. How will the hazards be addressed,
and how will animal handlers be adequately informed and trained to
accommodate these risks.

**Ms. Bell made a motion to defer approval pending receipt of the
revised registrations, Dr. Crise seconded and all were in favor.**

**[Wolff (07-09)]{.underline}**

\- A6a1 should be answered.

## Part B, I

\- Why is B5c "no" -- There is a strain of virus involved, even if it is
the original strain.

\- B5j should be answered

\- B6a -- How can there be no risks? Accidental personnel injection,
virus spills, and recombination in the mouse resulting in human
infection are all risks.

## Part B, II

\- B5a should be answered.

\- B5a -- what is EvI1?

\- B5f -- Is recombination in the mouse possible? C5a suggests that it
is.

\- How will the vectors be kept separated?

\- Part E of the form should be completed.

Ms. Bell made a motion to defer approval pending receipt of a revised
registration, Dr. McVicar seconded and all were in favor.

**[Amendments]{.underline}**

**[Wang (06-96)]{.underline}**

\- Integrate the amendment into the registration document since it has
not yet been reviewed and approved.

\- Are cell lines being sorted? It appears this is done in Bethesda. How
is this being done safely?

\- A6a1: Clarify what work will be done at NCI-Frederick, and what
material will be transferred, to where, to whom, and for what purpose.

\- How does the transport of potentially hazardous materials occur
safely?

\- The use of HuH1 -- produces HBs antigen "but there is no viral
replication confirmed"- please clarify.

\- What will histology receive? What will they be doing with the
material? How will they be informed about the tumors being of human
origin and what precautions are in place as they handle tumors derived
from HCV+ patients? The histology group will also need to sign off on
their understanding the scope of their duties and how to handle the
material safely. Does histology have a questionnaire in place to
determine they type of material incoming prior to their receipt?

\- Sorting at NIH is described. How is this applicable to anything
occurring here in Frederick?

\- A5: Further describe safeguards to be put into place for those who
will be working with the cell lines. There should be specific training
and direct involvement from the PI to inform those working with these
lines of the additional hazards so the lines are not treated casually as
is common with other standard tumor lines.

\- E9 requires more detail.

**Dr. McVicar made a motion to defer approval pending receipt of the
requested modifications above, Dr. Crise seconded and all were in
favor.**

**[Tarr (06-15, 05-57)]{.underline}**

\- Confirm that this study involves making 400L of adenovirus.

\- Restate the methods for purifying the adenovirus and confirm if the
process is similar to that used for purification of DNA submitted in
previous registrations.

\- Is actual virus being made or only plasmids?

\- Clarify 400L of adenovectors is equivalent to 10^14^ pfu/L.

\- B5e: Clarify what the HEK cells are being used for.

\- Provide the GMP RCA assay.

\- Provide assurance that at no time all HIV will be present.

\- What material will be manipulated, manufactured, and analyzed. How
will these activities be conducted?

**Dr. Creekmore made a motion to defer approval pending receipt of the
requested modifications above, Dr. Garfinkel seconded and all were in
favor.**

**[Sei (06-27, P150205SSB01)]{.underline}**

-Specify what hazardous work will continue and what work will not
continue during the temporary move? 

-How will the rooms accomodate the work with each specific agent, and
how will the agents will be kept temporally and physically separated?

-The PI is to work with Dr. Kopp to identify which materials he will be
working with and how the work will be kept temporally and physically
separate from his work.

-How will safe lab practices and procedures in the interim location
continue?  Include the use of BSC\'s, Personal Protective Equipment,
entrance and gowning requirements, and immunizations required (if
applicable). Also include centrifugation, methods for avoiding aerosols,
and any other safe practice or procedures.

**Dr. Hughes made a motion to defer approval pending receipt of the
requested modifications above, Dr. Crise seconded and all were in favor.
A subcommittee of 2 IBC members will reconsider the responses and be
responsible for issuing final approval.**

**[OUTSTANDING ITEMS]{.underline}**

Discussion ensued regarding IBC registration #06-13 and the associated
SOP. The registration was conditionally approved in May 2006; however,
the IBC is still waiting for the SOP to be submitted prior to granting
final approval. Dr. Hughes made a motion recommending that Dr. Tim
Harris be notified about the SOP so that receipt of the document can be
expedited. Dr. McVicar seconded the motion and all were in favor.

The IBC requested that the IBC secretary remind the CCR-Frederick, Flow
Cytometry Core Group, that an IBC registration is necessary and should
be submitted promptly. This group has the potential to perform live
infectious cell sorting.

A revised IBC Charter and IBC Standard Operating Procedure were
redistributed after comments were received and integrated. Dr. Creekmore
made a motion to approve both documents as written, Dr. Crise seconded
and all were in favor. The Charter and SOP were officially approved at
this meeting. Both the Charter and SOP will be submitted to Dr. Reynolds
and Mr. Wheatley for their concurrence and signature.

The November 2006 minutes were distributed prior to the meeting. There
were no modifications requested. Dr. Crise made a motion to approve the
minutes as written, Dr. Garfinkel seconded and all were in favor.

Dr. Morin notified the IBC that the 5^th^ Edition of the Biosafety in
Microbiological and Biomedical Laboratories has been officially posted
on the CDC webpage. The website link will be posted on the IBC webpage
for reference and Dr. Morin will make every effort to obtain a hard copy
of the revised 5^th^ Edition for each IBC member.

The meeting was adjourned at 2:34 p.m.

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Theresa D. Bell, MPH, CBSP Ms. Cara Leitch

IBC Secretary IBC Coordinator

Biological Safety Officer, EHS Sr. Safety Specialist, EHS

APPROVED:

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\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Randall S. Morin, Dr. P.H. Date

Chairman, NCI-Frederick IBC

Director, EHS

xc: All Committee Members

Dr. Reynolds

Mr. Wheatley

Dr. Arthur

Mr. Bufter

Dr. Keimig