Release Date:  December 15, 1998

RFA:  HL-99-003


National Heart, Lung, and Blood Institute

Letter of Intent Receipt Date:  January 11, 1999
Application Receipt Date:  February 12, 1999



The Division of Blood Diseases and Resources invites grant applications for
research on the development of assay methods for the detection of CJD.  The study
of CJD and other Transmissible Spongiform Encephalopathies (TSE) has been
hampered seriously by the lack of a satisfactory assay system.  The ultimate goal
of this program is the development of an assay to screen donated blood and donors
of organs or tissues, although other uses of the technique are probable. 
Approaches that might be considered for mass screening for the hallmark abnormal
isoform (PRP-res) or other potential markers for CJD are serologic, virologic,
physical or physico-chemical.


The Public Health Service (PHS) is committed to achieving the health promotion
and disease prevention objectives of Healthy People 2000, a PHS-led national
activity for setting priority areas.  This Request for Applications (RFA),
Development of Assay Methods for CJD, is related to the priority area of
immunization and infectious diseases.  Potential applicants may obtain a copy of
"Healthy People 2000" (Full Report: Stock No. 017-001-00474-0 or Summary Report:
Stock No. 017-001-00473-1) through the Superintendent of Documents, Government
Printing Office, Washington, DC 20402-9325 (telephone 202-512-1800) or at


Applications may be submitted by domestic and foreign for-profit and non-profit
organizations, public and private, such as universities, colleges, hospitals,
laboratories, units of State or local governments, and eligible agencies of the
Federal government.  Racial/ethnic minority individuals, women, and persons with
disabilities are encouraged to apply as Principal Investigators.  All current
policies and requirements that govern the research grant programs of the National
Institutes of Health (NIH) will apply to grants awarded under this RFA.


This RFA will use the NIH individual research project grant (R01) mechanism of
support. However, specific application instructions have been modified to reflect
"MODULAR GRANT" and "JUST-IN-TIME" streamlining efforts being examined by the
NIH.  The modular grant concept establishes specific modules in which direct
costs may be requested as well as a maximum level for requested budgets.  Only
limited budgetary information is required under this approach.  The just-in-time
concept allows applicants to submit certain information only when there is a
possibility for an award.  It is anticipated that these changes will reduce the
administrative burden for the applicants, reviewers and Institute staff.

For this RFA, funds must be requested in $25,000 direct cost modules and a
maximum of eight modules, $200,000 direct costs per year may be requested.  For
subcontracts, the $200,000 includes the direct costs and facilities and
administrative costs. A feature of the modular grant concept is that no
escalation is provided for future years, and all anticipated expenses for all
years of the project must be included within the number of modules being
requested.  Only limited budget information will be required and any budget
adjustments made by the Initial Review Group will be in modules of $25,000. 
Instructions for completing the Biographical Sketch have also been modified.  In
addition, Other Support information and the application Checklist page are not
required as part of the initial application.  If there is a possibility for an
award, necessary budget, Other Support and Checklist information will be
requested by NHLBI staff following the initial review.  The APPLICATION
PROCEDURES section of this RFA provides specific details of modifications to
standard PHS 398 application kit instructions.  Responsibility for the planning,
direction, and execution of the proposed project will be solely that of the
applicant.  It is anticipated that support for this program will begin in
September 1999.  Administrative adjustments in the project period and/or amounts
may be required at the time of the award.

This RFA is a one-time solicitation.  Future unsolicited competing continuation
applications will compete with all investigator-initiated applications and be
reviewed according to the customary peer review procedures.


It is anticipated that for fiscal year 1999, approximately $1.2 million total
costs will be available for the first year of support for this initiative.  The
award of grants pursuant to this RFA is contingent upon receipt of such funds for
this purpose.  It is anticipated that approximately four to six new grants will
be awarded under this program for up to four years of support.  The specific
number to be funded will, however, depend on the merit and scope of the
applications received and on the availability of funds.  Direct costs will be
awarded in modules of $25,000, less any overlap or other necessary administrative
adjustments.  Facilities and Administrative costs  will be awarded based on the
negotiated rates.



CJD is a slow degenerative disease of the central nervous system characterized
by motor dysfunction, progressive dementia, and vacuolar degeneration of the
brain.  A protease-resistant protein (PRP-res) or prion is the hallmark of the
transmissible spongiform encephalopathies (TSE) to which CJD belongs.  This
protein is an isoform of a normal membrane protein (PRP-sen) that has undergone
a marked conformational change.  PRP-res appears able to recruit normal molecules
to the abnormal shape.  Some believe that PRP-res is the cause of TSE; others
maintain that it is a result of infection by the true agent(s), despite repeated
failure to detect nucleic acid-containing material.  At the very least, it
appears that this abnormal protein is a marker of TSE.  The recent demonstration
of an association between an abnormal spinal fluid protein, 14-3-3, and
clinically manifested TSE shows that other markers may exist.  A spinal fluid
test is not practicable as a test for pre-symptomatic individuals or for
screening large number of individuals (e.g., blood donors), but its discovery
suggests that a search for other markers could be fruitful.  PRP-res has been
found in several tissues in man and animals with TSE (e.g., CJD, scrapie in sheep
and goats, bovine spongiform encephalopathy, also referred to as "mad cow
disease"); most notably in brain, lymph nodes, spleen and circulating platelets. 
Peripheral blood lymphocytes are in continuous circulation through lymphatics,
lymph nodes, and peripheral blood.  Hence, it is a tenable hypothesis that small
amounts of PRP-res are also present in circulating lymphocytes.

Cases of CJD occur worldwide at a rate of one to two cases per million population
per year.  From 1979 through 1994, CJD was recorded as a cause in 3,642 deaths
in the United States, with a stable incidence.  The disease is rare, but
invariably fatal, and has been proven to be associated with a transmissible
agent.  Cases arise spontaneously at low frequency for unknown reasons (sporadic
CJD), perhaps acquired by external exposure to the etiologic agent, or may arise
spontaneously at higher frequency in persons with certain genetic mutations
(familial CJD).  The disease can also be iatrogenic, i.e., transmitted between
humans by transplanted corneas and cadaver dura mater grafts from affected
individuals, by use of contaminated EEG electrodes, by certain neurological
procedures, and by injections of human growth hormone (HGH).  The incubation
period between inoculation and disease is long (for US HGH cases, the median
incubation period was 14 years; in Europe it was 18-20 years), making it
difficult to detect new sources of infection.

Some animal TSEs appear to be transmitted through the food chain.  For example,
the epidemic of bovine spongiform encephalopathy (BSE) in the United Kingdom (UK)
appears to have been caused by the practice of grinding together material left
over after slaughtering and butchering beef and using that material as a high
protein feedstock.  Banning that practice may have begun to control the epidemic
in livestock.  A new variant of CJD (nv-CJD) has been described in more than 25
patients in the UK and is apparently related to the BSE epidemic.  Whether or not
this came from humans eating infected beef is not yet clear.  Nv-CJD may
represent a jump across the species barrier, which is generally uncommon for TSE. 
The discovery of nv-CJD has heightened the concern about transmissibility by
blood because the BSE agent(s) appears to be in higher concentrations in lymph
tissues than classical CJD.

Recent reports of 37 US donors who had made a number of blood donations between
1983 and 1997 before they were diagnosed with CJD stimulated concern about
possible transmission by blood components or plasma protein derivatives.  These
reports led to precautionary product quarantines, withdrawals and recalls, and
the adoption an FDA suggestion for CJD-related donor deferral criteria for both
the plasma industry and whole blood sector.  This loss of usable product is
partially responsible for severe shortages of protein derivatives, most notably
immune globulins and à-1 protease inhibitor.  An April 1998 poll conducted by the
Immune Deficiency Foundation revealed that 87 percent of the physicians surveyed
reported serious problems in the previous six months obtaining immune globulin
for their patients.

Experimental animal data suggest that blood may be infectious. Blood from mice
ill with a mouse-adapted human CJD agent was separated into components, the
plasma fractionated and blood and protein derivatives injected intracerebrally
into other mice.  All components and most protein fractions  transmitted the
disease; it seemed that cryoprecipitate was more infectious and albumin less
infectious than other components and derivatives, which were not detectably
different.  Hamster studies with plasma "spiked" with infectious brain tissue and
fractionated showed some transmission with fraction V, but at a low level.  In
a single experiment, blood from hamsters infected with hamster-adapted scrapie
transmitted disease to one of 22 recipients.  Full units of human blood from
patients with CJD have failed to transmit the infection to chimpanzees, although
these animals are still being observed years after inoculation.  Studies with
animal agents suggest that infectivity established by the intravenous route is
logs lower than by the intracerebral route.  Current assay procedures employing
experimental animals which require months to years to obtain end points may not
be sensitive enough to detect low levels of infectivity.  Nevertheless,
intravenous infectivity is probably much lower than via the intracerebral route,
perhaps unmeasurable, but not zero.  The discovery of nv-CJD in UK and the
apparent greater presence of PRP-res in lymph nodes of patients with nv-CJD has
raised the possibility that this type of CJD may be more likely transmissible by
blood components or protein derivatives.  Studies with animal models are
incomplete as yet.

Conversely, epidemiological data do not support transmission of CJD via blood
transfusion in humans, either in the US and Germany with classical CJD or in the
UK with nv-CJD.  Furthermore, there have been no verified cases of CJD in
patients with hemophilia, even among those treated for nearly 20 years
exclusively with cryoprecipitate from a donor pool that contained several
individuals who later developed CJD themselves.  "Look back" from more than 20
of the 37 US donors later known to have developed CJD detected no cases of the
disease in blood recipients.

Studies of CJD and other TSE's have been seriously hampered by existing assay
systems which are slow in yielding results (months or years), insensitive and
require intracerebral injection of test material into experimental animals.  The
infectivity of brain and other tissues has been assessed by intracerebral
injections using rodents (time from inoculation to end point determination -
three months to a year), small primates (squirrel monkeys þ two years) or
chimpanzees (three-five years).  These cumbersome biological assay systems make
studies slow, tedious and difficult to plan and design.  It is very difficult to
determine in a timely manner the "clearance" or "partitioning" of CJD agent(s)
between fractions during plasma fractionation. Furthermore, the  relative
infectivity of material injected intracerebrally compared to intravenous
injections is difficult to determine with any precision.

A Special Emphasis Panel on CJD and Blood Transfusion, recently convened by the
NHLBI, concluded that "an unqualified and irreducible risk of exposure to CJD
through blood and blood products does exist."  They also agreed that the lack of
a rapid sensitive and specific test for TSE infectivity was holding back progress
in the study and control of CJD.


The purpose of this solicitation is to encourage established investigators with
expertise in basic biochemistry, protein chemistry, physical chemistry, virology,
immunology and molecular biology to develop serologic, virologic or physical
methods to detect  markers of TSE pre-clinical infection.  The ultimate goal of
this program is the development of an assay to screen blood and tissue donors for
the presence of the CJD agent.  One strategy might be the development of
monoclonal antibodies (Mab) specific to the conformation of PRP-res.  Such a Mab
could be utilized in an enzyme-linked immunosorbant assay (ELISA or EIA) for the
detection of PRP-res.  Physical or physico-chemical techniques might also be
developed that would detect the abnormal conformation of PRP-res with sufficient
specificity and sensitivity to be useful.  There is evidence that the infectious
agent(s) for CJD are present in plasma and lymph nodes, that B lymphocytes may
be important in enabling the agent(s) to reach the brain and that PRP-res is
present in platelets.  Accordingly, any one of these peripheral blood components
could represent a source for the assay.  Another strategy might focus on the
identification of TSE-associated  foreign molecules in white blood cells that
could be of value in developing a test.  If the etiologic agent of CJD is a slow
virus, peripheral lymphocytes may carry other viral-specific molecule(s) amenable
to detection, either directly with nucleic acid amplification methods, or through
serologic assay with the proper monoclonal/polyclonal antibodies.  Applications
should provide sufficient preliminary information from the literature, personal
research or other sources to support the procedure(s) proposed.

These assays, if developed, could help determine if CJD is truly transmitted by
blood and blood products.  It could form the basis of a blood/tissue donor
screening test, and additionally, it could provide a diagnostic test for
neurologists, since there is no way of detecting disease in the preclinical
state.  These tests could also be useful for testing TSE in animals, especially
in domestic animals used for human consumption.


Upon initiation of the program, the NHLBI will sponsor annual meetings to
encourage the exchange of information among investigators who participate in this
program.  Travel funds for a one day meeting each year, most likely to be held
in Bethesda, Maryland, should be included in the modules.  Applicants should also
include a statement in the application indicating their willingness to
participate in such meetings.


It is the policy of the NIH that women and members of minority groups and their
subpopulations must be included in all NIH supported biomedical and behavioral
research projects involving human subjects, unless a clear and compelling
rationale and justification is provided that inclusion is inappropriate with
respect to the health of the subjects or the purpose of research.  This policy
results from the NIH Revitalization Act of 1993 (Section 492B of Public Law 103-

All investigators proposing research involving human subjects should read the
"NIH Guidelines for Inclusion of Women and Minorities as Subjects in Clinical
Research", which have been published in the Federal Register of March 28, 1994
(FR 59 14508-14513), and in the NIH GUIDE FOR GRANTS AND CONTRACTS, Volume 23,
Number 11, March 18, 1994, available on the Web at:

Investigators also may obtain copies from the program staff listed under
INQUIRIES.  Program staff may also provide additional relevant information
concerning the policy.


It is the policy of the NIH that children (i.e., individuals under the age of 21)
must be included in all human subjects research, conducted or supported by the
NIH, unless there are scientific and ethical reasons not to include them.  This
policy applies to all initial (type 1) applications submitted for receipt dates
after October 1, 1998.

All investigators proposing research involving human subjects should read the NIH
Policy and Guidelines on the Inclusion of Children as Participants in Research
Involving Human Subjects that was published in the NIH Guide for Grants and
Contracts, March 6, 1998, and is available at the following URL address:


Prospective applicants are asked to submit, by January 11, 1999, a letter of
intent that includes a descriptive title of the proposed research, the name,
address, and telephone number of the Principal Investigator, the identities of
other key personnel and participating institutions, and the number and title of
the RFA in response to which the application may be submitted.  Although the
letter of intent is not required, is not binding, and does not enter into the
review of a subsequent application, the information that it contains allows the
NHLBI staff to estimate the potential review workload and to avoid conflict of
interest in the review.

The letter of intent is to be mailed, or faxed to:

Dr. C. James Scheirer
Division of Extramural Affairs
National Heart, Lung, and Blood Institute
6701 Rockledge Drive, Room 7220, MSC 7924
Bethesda, MD  20892-7924
Telephone:  (301) 435-0266
FAX:  (301) 480-3541


The research grant applications form PHS 398 (rev. 4/98) is to be used in
applying for these grants, with the modifications noted below. These forms are
available at most institutional offices of sponsored research; from the Division
of Extramural Outreach and Information Resources, National Institutes of Health,
6701 Rockledge Drive, MSC 7910, Bethesda, MD 20892-7910, telephone (301) 710-0267, e-mail:; and on the internet at

The RFA label found in the PHS 398 (rev. 4/98) application form must be affixed
to the bottom of the face page of the application.  Failure to use this label
could result in delayed processing of the application such that it may not reach
the review committee in time for review.  In addition, the RFA title (Development
of Assay Methods for Creutzfeldt-Jakob Disease) and number (HL-99-003-B) must be
typed on line 2 of the face page of the application form and the YES box must be


Samples budgets and justifications will be provided upon request or following the
submission of a letter of intent. The total direct costs must be requested in
accordance with the program guidelines and the modifications made to the standard
PHS 398 application instructions described below:


As a reminder, Items 7a and 8a should be completed to indicate Modular Direct
Costs requested and Items 7b and 8b should reflect Total Costs (Modular Direct
plus F&A costs).  Item 7 should reflect costs for the Initial Budget Period and
item 8 should reflect costs for the Total Budget Period.


Do not complete Form Page 4 of the PHS 398 (rev 4/98).  It is not required nor
will it be accepted at the time of application.


Do not complete the categorical budget tables on page 5 of the PHS 398 (rev.
4/98) Form.  Only the requested total direct costs line for each year should be
completed based on the number of $25,000 modules being requested.  Applicants may
not request a change in the amount of each module.  A maximum of eight modules
($200,000 direct cost) per year may be requested for each R01 application. 
Applicants may request up to four years of support for this RFA.  Direct cost
budgets will remain constant throughout the life of the project (i.e., the same
number of modules requested for all budget periods).  Any necessary escalation
should be considered when determining the number of modules to be requested. 
However, in the event that the number of modules requested must change in any
future year due to the nature of the research proposed, appropriate justification
must be provided.  Total Direct Costs for the Entire Proposed Project Period
should be shown in the box provided.


o  Budget justifications should be provided under "Justifications" on Form Page
5 of the PHS 398.

o  List the names, role on the project and proposed percent effort for all
project personnel (salaried or unsalaried)and provide a narrative justification
for each person based on his/her role on the project.

o  Identify all consultants by name and organizational affiliation and describe
the services to be performed.

Provide a general narrative justification for individual categories (equipment,
supplies, etc.) required to complete the work proposed.  More detailed
justifications should be provided for high cost items.  Any large one-time
purchases, such as large equipment requests, must be accommodated within these


If collaborations or subcontracts are involved that require transfer of funds
from the grantee to other institutions, it is necessary to establish formal
subcontract agreements with each collaborating institution.  A letter of intent
from each collaborating institution should be submitted with the application. 
Only the percentage of the consortium/contractual TOTAL COSTS (direct and
facilities and administrative costs) relative to the total DIRECT COSTS of the
overall project needs to be stated at this time.  The following example should
be used to indicate the percentage cost of the consortium, "The consortium
agreement represents 27% of overall direct costs requested in the first year." 
A budget justification for the consortium should be provided as described in the
"Budget Justification" section above (no Form Page 5 required for the
consortium).  Please indicate whether the consortium will be in place for the
entire project period and identify any future year changes in the percentage
relative to the parent grant.

If there is a possibility of an award, the applicant will be requested to
identify actual direct and indirect costs for all years of the consortium. 
Please note that total subcontract costs need not be calculated in $25,000
modules. However, when subcontract funds are added to the parent grant budget,
the total direct cost amount must be included in the number of $25,000 modules


A biographical sketch is required for all key personnel, following the modified
instructions below.  Do not exceed the two-page limit for each person.

o  Complete the educational block at the top of the form page;

o  List current position(s) and those previous positions directly relevant to the

o  List selected peer-reviewed publications directly relevant to the proposed
project, with full citation;

o  The applicant has the option to provide information on research projects
completed and/or research grants participated in during the last five years that
are relevant to the proposed project.

OTHER SUPPORT - Do not complete the "Other Support" pages (Form Page 7). 
Selected other support information relevant to the proposed research may be
included in the Biographical Sketch as indicated above.  Complete Other Support
information will be requested by NHLBI staff if there is a possibility for an


No "Checklist" page is required as part of the initial application.  A completed
Checklist will be requested by NHLBI staff if there is a possibility for an

The applicant should provide the name and phone number of the individual to
contact concerning fiscal and administrative issues if additional information is
necessary following the initial review.

Applications not conforming to these guidelines will be considered unresponsive
to this RFA and will be returned without further review.

Submit a signed, typewritten original of the application and three signed,
photocopies, in one package to:

BETHESDA, MD  20892-7710
BETHESDA, MD  20817 (for express/courier service)

At the time of submission, two additional copies of the application must be sent
to Dr. C. James Scheirer, at the address listed under LETTER OF INTENT.

Applications must be received by February 12, 1999.  If an application is
received after that date, it will be returned to the applicant without review. 
The Center for Scientific Review (CSR) will not accept any application in
response to this RFA that is essentially the same as one currently pending
initial review, unless the applicant withdraws the pending application.  The CSR
will not accept any application that is essentially the same as one already
reviewed.  This does not preclude the submission of substantial revisions of
applications already reviewed, but such applications must include an introduction
addressing the previous critique.


Upon receipt, applications will be reviewed for completeness by CSR and
responsiveness by NHLBI.  Incomplete and/or unresponsive applications will be
returned to the applicant without further consideration.  Remaining applications
may be subjected to a streamlined review process by a Special Emphasis Panel
convened by NHLBI Scientific Review Office to determine their
scientific merit relative to other applications received in response to the RFA. 
The roster of reviewers for the RFA will be available on the NHLBI home page
approximately four weeks prior to the scheduled review date.  Applications
determined to be meritorious will be evaluated for scientific and technical merit
by the review committee, be discussed and receive a priority score.  All other
applications will neither be discussed nor scored.  Secondary review of the
applications will be conducted by the National Heart, Lung, and Blood Advisory
Council (NHLBAC).

Review Criteria

The goals of NIH-supported research are to advance our understanding of
biological systems, improve the control of disease, and enhance health.  In the
written review, comments on the following aspects of the application will be made
in order to judge the likelihood that the proposed research will have a
substantial impact on the pursuit of these goals.  Each of these criteria will
be addressed and considered in the assignment of the overall score.

1) Significance.  Does this study address an important problem?  If the aims of
the application are achieved, how will scientific knowledge be advanced?  What
will be the effect of these studies on the concepts or methods that drive this

2) Approach.  Are the conceptual framework, design, methods, and analyses
adequately developed, well integrated, and appropriate to the aims of the
project?  Does the applicant acknowledge potential problem areas and consider
alternative tactics?

3) Innovation.  Does the project employ novel concepts, approaches or method? 
Are the aims original and innovative?  Does the project challenge existing
paradigms or develop new methodologies or technologies?

4) Investigator.  Is the investigator appropriately trained and well suited to
carry out this work?  Is the work proposed appropriate to the experience level
of the principal investigator and other researchers (if any)?

5) Environment.  Does the scientific environment in which the work will be done
contribute to the probability of success?  Do the proposed experiments take
advantage of unique features of the scientific environment or employ useful
collaborative arrangements?  Is there evidence of institutional support?

As part of the scientific and technical merit evaluation of the research plan,
reviewers will be instructed to address the adequacy of plans for including both
genders, minorities and their subgroups, and children as appropriate for the
scientific goals of the research, or justification for exclusion.

The personnel category will be reviewed for appropriate staffing based on the
requested percent effort.  The direct costs budget request will be reviewed for
consistency with the proposed methods and specific aims.  Any budgetary
adjustments recommended by the reviewers will be in $25,000 modules.  The
duration of support will be reviewed to determine if it is appropriate to ensure
successful completion of the requested scope of the project.


Applicants should be aware that, in addition to scientific merit, program
priorities and program balance, the total costs of the proposed project and the
availability of funds will be considered by NHLBI staff as well as National
Heart, Lung, and Blood Advisory Council in making funding recommendations.  In
circumstances in which applications have similar scientific merit, but vary in
cost competitiveness, NHLBI is likely to select the more cost competitive
application for funding.


Letter of Intent Receipt Date:     January 11, 1999
Application Receipt Date:          February 12, 1999
Date of Initial Review:            April 1999
Review by NHLBI Advisory Council:  September 16, 1999
Anticipated Award Date:            September 30, 1999


Inquiries concerning this RFA are encouraged.  Potential applicants may request
sample budget pages.  The opportunity to clarify any issues or questions from
potential applicants is welcome.

Direct inquiries regarding programmatic issues and requests for sample budget
pages to:

Luiz H. Barbosa, D.V.M.
Division of Blood Diseases and Resources
National Heart, Lung, and Blood Institute
6701 Rockledge Drive, Room 10146, MSC 7950
Bethesda, MD  20892-7950
Telephone:  (301) 435-0075
FAX:  (301) 480-0868

Direct inquiries regarding fiscal matters to:

Ms. Jane Davis
Grants Operations Branch
National Heart, Lung, and Blood Institute
6701 Rockledge Drive, Suite 7174, MSC 7926
Bethesda, MD  20892-7926
Telephone:  (301) 435-0166
FAX:  (301) 480-3310


This program is described in the Catalog of Federal Domestic Assistance No.
93.839.  Awards are made under authorization of the Public Health Service Act,
Title IV, Part A (Public Law 78-410, as amended by Public Law 99-158, 42 USC 241
and 285) and administered under PHS grants policies and Federal Regulations 42
CFR 52 and 45 CFR Part 74.  This program is not subject to the intergovernmental
review requirements of Executive Order 12372 or Health Systems Agency review.

The PHS strongly encourages all grant and contract recipients to provide a
smoke-free workplace and promote the non-use of all tobacco products.  In
addition, Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in
certain facilities (or in some cases, any portion of a facility) in which regular
or routine education, library, day care, health care or early childhood
development services are provided to children.  This is consistent with the PHS
mission to protect and advance the physical and mental health of the American

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