# Presentation: 756099

## Laboratory of Bacterial, Parasiticand Unconventional Agents (LBPUA)Research Programs

**Report to BPAC 13 July 2006**
**Site Visit by Ad Hoc Review Committee**
**25 May 2006**
**David M. Asher, MD**
**Division of Emerging and Transfusion-Transmitted Diseases**
**Office of Blood Research and Review**
**Center for Biologics Evaluation and Research**
**United States Food and Drug Administration**

**Notes:**
Good morning.
The Laboratory of Bacterial, Parasitic and Unconventional Agents (LBPUA) was formed at the end of 1999 when a small transmissible spongiform encephalopathy (TSE) research program of four people left CBER’s Office of Vaccines to join an even smaller existing staff—a bacteriology fellow and a malaria reviewer—in the Office of Blood. With addition of Leishmania and malaria research programs the lab expanded to its current size of 18 people, including 5 principal investigators (PIs).

## Goals of LBPUA Research Programs

_**Product development and safety**_
***Leishmania*****, Chagas’ disease, malaria, TSEs:**
**candidate blood donor tests**
***Leishmania*****, malaria vaccines; TSEs, vaccine safety**
_**Other regulatory-relevant research**_
**Pathogenesis at cellular and molecular levels**
***Leishmania*****, malaria, TSEs**
_**Justification for research on pathogenesis**_
**Provides materials and information of regulatory relevance**
**Improves reviewer insight into regulatory issues**
**Maintains scientific expertise, commitment to mission**
**Usually self-supporting (supplements other funding)**

**Notes:**
LBPUA research is not restricted to reducing the risk from contaminated blood products. Some of us are also engaged in research to maintain vaccines and other biologics free of  TSE agents. Several researchers have unique expertise in parasitology not available in other CBER product offices or FDA Centers; they continue to conduct studies that may help in eventual development of vaccines for Leishmaniasis, Chagas’ disease and malaria.
Some of our efforts include research on pathogenesis of infection at cellular and molecular levels. That research is not only important in its own right; it also supports more directly applied research, maintains staff expertise, encourages continued commitment to our regulatory mission, and—incidentally—it often receives outside funding. 

## Two LBPUA Research Programs (5 PIs)

_**Parasitic and bacterial diseases**_
***Leishmania***** vaccine safety, blood donor screening assays [3 staff incl PI, R Duncan, A Selvapandyan]**
**PI: HL Nakhasi, PhD (Director, DETTD)**
***Leishmania, T. cruzi *****pathogenesis, role in blood safety**
**[3 staff incl PI]**
**PI: A Debrabant, PhD**
**Malaria***** *****pathogenesis, role in vaccine safety, role in blood safety, blood donor screening assays (transfusion-transmitted bacterial infections) [4 staff incl PI]**
**PI: S Kumar, PhD**
_**Transmissible spongiform encephalopathies**_
**Mfg safety, blood donor screening assays* [5 staff incl PI]**
**PI: D Asher, MD (Chief, LBPUA)**
*** cf. WHO Consultation September 2005: http://who.int/bloodproducts/tse/WHO%20TSE%20Guidelines%20FINAL-22%20JuneupdatedNL.pdf**
**Pathogenesis, source materials safety [3 staff incl PI]**
**PI: P Piccardo, MD**

**Notes:**
If the research programs were larger, each project in LBPUA would justify being a separate administrative as well as scientific entity. As Chief, I attempt to offer the staff regulatory and administrative support and supervision, but the committee should understand that components of LBPUA have complete scientific independence in designing and conducting research—consistent with FDA mission and policies. It is also important to note that—on the other hand—the PIs, staff scientists, technical staff and fellows in the different projects do interact cordially, productively and often.
We also depend greatly on collaborative research efforts with investigators outside the FDA. In the current situation of limited FDA resources, outside collaborations remain essential if we are to continue contributing to regulatory research in a meaningful way.
Today’s presenters are listed in the Agenda and again on this slide; I won’t repeat them, and I now surrender the lectern to Hira Nakhasi, Director of DETTD and PI for the first of LBPUA’s Leishmania projects. Thank you.

## Transmissible Spongiform Encephalopathies

**normal brain**

**spongiform degeneration**

**status spongiosus**

## LBPUA TSE Research Program: 2 Sections

**TSE Pathogenesis and Source Materials Safety**
_**(PI: P. Piccardo)**_
**Maintain human-derived/animal-derived materials TSE-free**
**TSE Manufacturing Safety Section**
_**(D. Asher)**_
**Keep manufacturing facilities/equipment TSE-free**
**Evaluate cleanup/disinfection methods for TSEs**
**Develop computerized morphometric analysis (CMA) of objective, quantifiable immunohistochemical criteria to study/diagnose TSEs: support tissue/surgical safety**
**Evaluate susceptibility of biologics cell substrates to TSE infections (NIAID-FDA Inter-Agency Agreement)****a**
**Establish US TSE Biological Reference Materials: human (and ?animal) TSE brain, other tissues, blood components****a,b**
**____________________________________________________________________________________________________________________**
**a****Joint efforts of **_**both**_** LBPUA TSE Sections      ****a,b ****Proposed project **

## Abnormal prion protein (designated PrPSc, PrPres or PrPTSE)   derived from ubiquitous normal precursor protein (PrPC)              useful marker of TSE infections (IHC, WB, ELISAs)

**PrP****TSE**** is an N-truncated abnormally folded cleavage product (high ****β****-sheet) of normal “cellular” protein (PrP****C****),**
**relatively insoluble in detergent-salt solutions, and**
**relatively resistant to digestion with proteinase K (PK).**
**PrP****C**** is a 253-aminoacid peptide encoded by the *****PRNP***** gene on human chromosome 20.**
**Familial TSEs are linked to a number of *****PRNP***** point mutations, deletions or inserted duplications.**
**Mice lacking PrP-encoding genes **_**cannot**_** be infected with TSE agents.**
**PrP****TSE ****alone ?? = infectious TSE agent**

## Adaptation of Chen-Koski glass crush methodfor evaluation of virucides to study TSE agents

## Adaptation of Zobeley-Flechsig-Weissmann steel wire methodto evaluate TSE decontamination

**Adaptation of Zobeley-Flechsig-Weissmann steel wire method****to evaluate TSE decontamination**

## Titrations of 263K scrapie agentdried on glass slips or steel pins: ultrasonic wash in hot alkaline detergent

|  | 

Temp
x
Time | 

Log10 LD50 Reduction (Residual Infectivity) |
| --- | --- | --- |
| Glass Slip | 61oC x 90’
 | 5.3
(2.7) |
| Steel Pin | 65oC x 90’ | 5.6
(1.2) |

## 263K scrapie agent dried on steel pins (~6 log10 hamster IC LD50) “WHO” decontamination methodsplus ultrasonic cleaning in hot alkaline detergent &c

| Decontamination regimen | scrapie-infected pins/
total pins tested |
| --- | --- |
| Controls (normal brain) | 0/90 |
| NaOCl 6% 22oC 60’ | 0/36 |
| NaOH 2N 22oC 60’ | 0/36 |
| NaOH 2N 100oC 20’ | 0/37 |
| NaOH 2N SDS 3% 134oC 18’ | 0/32 |
| Proprietary phenolic  (as directed) 22oC 60’ | 0/35 |
| H20 134oC 18’ | 0/39 |

## Computerized morphometric analysis (CMA) of immunostained prion protein in scrapie hamster brains correlates well with conventional scores.

**Original images (top) transformed to CMA-thresholded (i.e., positively stained) images. Controls on left, scrapie on right**
**Good correlation between conventional visual scores (0, +, ++, +++) and CMA values expressed as % of area stained for replicate images**

## Susceptibility of biologics cell culture substratesto TSE infections

_**Artificial worst case**_
**Expose engineered human neuronal-glial cell lines overexpressing wild-type or mutant PrP to TSE agents:**
**30 serial passages, then bioassay for infectivity, various assays for PrP****TSE**
_**Actual cell substrates**_
**Expose proposed cell substrates to TSE agents:**
** ****30 serial passages, then bioassay, PrP****TSE**** assay**
**Vero**** (green monkey kidney heteroploid cells)**
**CHO**** (Chinese hamster ovary heteroploid cells)**
**HEK 293**** (human fetal kidney cells transformed with defective-adenovirus)**
**WI-38**** (? MRC-5) human diploid fibroblast cells**
**Other**** as indicated by actual biologics development (MDCK)**

## Experiments inoculating various cell lines with TSE agents, serial passaging (√ = passage completed, cells stored) 

| Cell line  | sCJD Passages
0    5    10    15    20    30 | BSE Passages
    0    5    10    15    20    30 |
| --- | --- | --- |
| SH-SY5Y
SH-SY5Y-wt
SH-SY5Y-E200K
CHO
Vero
WI38 (later MRC-5)
MDCK
HEK 293
R9ab
Mo3F4
3T3 |    √     √    √     √     √      √
   √     √    √     √     √      √
   √     √    √     √     √      √
   √     √    √     √     √      √
   √     √    √     √     √      √
 planned
planned
planned
   √     √    √     √     √      √ 
not planned
not planned |        √    √    √      √
       √    √    √      √
       √    √    √      √
       √    √    √     nd      √       √
       √    √    √     nd      √      √
       √    √    √      √       √       √
       √    √    √      √       √       √
   planned
        √    √    √     √        √      √
planned
 planned |

## Proposed TSE Blood-derived Biological Reference Reagents

| Species | Possible TSE agents |
| --- | --- |
| Rodent (mouse, hamster) | Scrapie, BSE, sCJD, vCJD |
| Sheep | Scrapie, ?BSE |
| Monkey | vCJD, ?sCJD, ?BSE |
| Chimpanzee | GSS (available), ?? other |
| Human | sCJD, fCJD (antemortem)
vCJD, other (postmortem) |