MECHANISMS OF HIV-1 TRAFFICKING IN THE CNS

Release Date:  January 18, 2000

RFA:  MH-00-010

National Institute of Mental Health
National Institute of Neurological Disorders and Stroke

Letter of Intent Receipt Date:  April 24, 2000
Application Receipt Date:       May 24, 2000

THIS RFA USES "MODULAR GRANT" AND "JUST-IN-TIME" CONCEPTS.  THIS RFA INCLUDES 
DETAILED MODIFICATIONS TO STANDARD APPLICATION INSTRUCTIONS THAT MUST BE USED 
WHEN PREPARING AN APPLICATION IN RESPONSE TO THIS RFA.

PURPOSE

HIV-1 infection of central nervous system (CNS) tissues results in neurological 
and neuropsychiatric abnormalities in a significant number of patients.  HIV-1 
localization in CNS parenchyma and CSF has been well documented.  Advances in 
highly active anti-retroviral therapy (HAART) have resulted in significant 
reductions in viral load in peripheral as well as CNS tissues in many patients.  
However, the presence of the blood-brain barrier (BBB) precludes efficient drug 
penetration into the CNS compartment and thus current therapies may not 
eradicate latent viral reservoirs in the brain.  Therefore, the CNS may serve as 
an important viral reservoir for re-infection of peripheral tissues subsequent 
to viral clearing in response to effective treatment.  The BBB serves as a 
critical gatekeeper for regulating HIV-1 transit into and out of the CNS.  It is 
therefore imperative to understand the mechanisms of viral passage through the 
blood-brain barrier in order to develop strategies for blocking early infection 
of this compartment as well as subsequent reinfection of peripheral tissues.

This RFA solicits applications that will examine potential pathways and 
mechanisms for HIV-1 entry into and out of the CNS through the BBB.  In vivo and 
in vitro models may be proposed to study the involvement of leukocyte and 
monocyte trafficking, proinflammatory cytokines, chemokines, chemokine 
receptors, adhesion molecules, viral proteins (Tat, Vpr, Nef, gp120), adsorptive 
endocytosis, transcytosis, changes in BBB permeability, excitotoxic damage to 
endothelial cells, glial and endothelial apoptosis, and infection of endothelial 
cells or glial cells in promoting HIV-1 entry into the brain.  The study of 
interactions of viral and host factors may lead to an improved understanding of 
the mechanism of establishment of CNS viral reservoirs and approaches to prevent 
passage of HIV-1 through the blood-brain barrier.

HEALTHY PEOPLE 2000

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), 
Mechanisms of HIV-1 Trafficking in the CNS, is related to the priority areas of 
HIV-1 infection and Mental Health and Mental Disorders.  Potential applicants 
may obtain a copy of "Healthy People 2000" at 
http://odphp.osophs.dhhs.gov/pubs/hp2000/

ELIGIBILITY REQUIREMENTS

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 and 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.

MECHANISM OF SUPPORT

This RFA will use the National Institutes of Health (NIH) research project grant 
(R01) award mechanism. Responsibility for the planning, direction, and execution 
of the proposed project will be solely that of the applicant.  The total project 
period for an application submitted in response to this RFA may not exceed 5 
years.  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.  
The anticipated award date is September 29, 2000.

For all applications requesting up to $250,000 direct costs per year, specific 
application instructions have been modified to reflect  MODULAR GRANT  and 
 JUST-IN-TIME  streamlining efforts being undertaken at NIH.  More detailed 
information about modular grant applications, including a sample budget 
narrative justification pages and a sample biographical sketch, is available via 
the Internet at: http://grants.nih.gov/grants/funding/modular/modular.htm.  
Applications that request more than $250,000 in any year must use the standard 
PHS 398 (rev. 4/98) application instructions.

FUNDS AVAILABLE

The NIMH intends to commit approximately $1,000,000 in FY 2000 to fund 3-5 new 
and/or competitive continuation grants in response to this RFA.  The NINDS 
intends to commit approximately $500,000 in FY 2000 to fund 2-3 new and/or 
competitive continuation grants in response to this RFA.  An applicant may 
request a project period of up to 5 years.  Because the nature and scope of the 
research proposed may vary, it is anticipated that the size of each award will 
also vary.  It is expected that the direct costs will be awarded in modules of 
$25,000, however program and grants management adjustments may be necessary 
prior to this award.  Although the financial plans of the Institute provide 
support for this program, awards pursuant to this RFA are contingent upon the 
availability of funds and the receipt of a sufficient number of applications of 
outstanding scientific and technical merit.  At this time, it is not known if 
competing renewal applications will be accepted and/or if this RFA will be 
reissued.

RESEARCH OBJECTIVES

Background

It is well established that HIV-1 can penetrate the central nervous system 
(CNS), frequently very soon after infection.  One of the consequences of HIV-1 
infection is HIV-1-associated dementia (HAD) which occurs to varying degrees in 
up to 20-30 percent of infected individuals.  HAD consists of a spectrum of 
progressive cognitive, motor, and behavioral impairments in the absence of HIV-
1-related opportunistic infection or malignancy.  The neuropathogenesis of human 
immunodeficiency virus (HIV-1)-associated dementia has remained elusive, despite 
identification of HIV-1 as the causal agent.  Although a number of contributing 
factors have been identified, the series of events that culminate in motor and 
cognitive impairments following HIV-1 infection of the CNS are still not known.  
Only with better understanding of neuropathogenesis will the opportunity to 
interrupt progression and to design better treatments for HAD be realized.

HIV-1 entry into the CNS is believed to occur primarily through trafficking of 
HIV-1 infected monocyte/macrophages or T-cells through the blood-brain barrier. 
Multiple chemokines, proinflammatory cytokines and adhesion molecules 
orchestrate trafficking of lymphocytes and monocytes.  There is evidence that 
viral proteins such as Tat modulates the expression of chemokines, chemokine 
receptors as well as adhesion molecules (VCAM-1, ICAM-1, VLA-4, LFA-1) on 
monocytes as well as cells lining the blood-brain barrier(endothelial cells and 
astrocytes). Proinflammatory cytokines (TNF-alpha, IL-1) induced by virus 
infection or viral proteins regulate adhesion and transendothelial migration of 
inflammatory cells. HIV-1 viral proteins (Tat and Nef) demonstrate chemotactic 
properties and promote leukocyte recruitment into the CNS.  The mechanisms of 
action of viral proteins through transcriptional regulation or modulation of 
signalling pathways involved in controlling expression of adhesion molecules, 
chemokines and cytokines are areas of potential interest.  Studies on the role 
of astrocytes are also highly relevant because they contribute to maintaining 
the integrity of the blood-brain barrier as well as induction of various 
molecules involved in leukocyte trafficking.  Astrocyte-endothelial interactions 
as well as astrocyte factors may be critically involved in transendothelial 
migration of HIV-1 infected cells into and out of the CNS.

Another potential mechanism of HIV-1 passage is through infection of endothelial 
cells lining the blood-brain barrier.  The role of endothelial cell infection 
and viral replication in this cell type as a mechanism for seeding the brain is 
highly controversial.  While some investigators have demonstrated infection of 
brain-derived endothelial cells using human as well as SIV models others have 
been unable to do so.  If the endothelial cells can be infected, further studies 
on the host and viral molecular determinants regulating this process are 
warranted.

A third and less explored possibility is the role of adsorptive endocytosis and 
transcytosis through the endothelial cells as an mechanism for HIV-1 entry into 
the CNS.  Studies with gp120 have suggested a role for adsorptive endocytosis in 
viral trafficking.  However a definite role for this mechanism in whole virus 
entry has not been established.

Finally the integrity of the blood brain barrier may be compromised by various 
factors associated with viral infection.  These could include changes in 
membrane permeability as a result of exposure to viral proteins (gp120, Vpr) or 
induced matrix metalloproteinases, or excitotoxic damage to BBB by nitrates, 
nitrites or glutamates.  Apoptotic cell death induced by HIV-1 proteins (Tat, 
Vpr) or other mediators may damage the endothelial cells, thus compromising the 
integrity of the BBB and allowing the passage of HIV-1 into and out of the 
brain.

As described above multiple factors are involved in regulating virus trafficking 
between CNS and peripheral compartments.  This RFA is intended to stimulate 
additional research in defining mechanisms of HIV-1 passage through the BBB.  
Examples of research include, but are not limited to the following:

o  Defining novel endothelial and leukocyte adhesion pathways involved in HIV-1 
infected leukocyte-transendothelial migration.

o  Role and characterization of CNS derived (macrophage, microglial, 
endothelial) chemokines and proinflammatory cytokines involved in regulating 
trafficking of infected cells through BBB.

o  Study of expression of chemokine receptors by CNS endothelial cells, 
astrocyte, macrophages, microglial cells and their potential impact on virus 
infection as well as response to chemokines.

o  Role of viral proteins (Tat, Vpr, Nef, gp120) in stimulating adhesion 
molecules, cytokines, chemokines and chemokine receptor expression by 
endothelial cells and astrocytes.

o  Regulation of leukocyte adhesion and transendothelial migration by viral 
proteins.

o  Chemotactic functions of HIV-1 viral proteins.

o  Development of in vitro and in vivo BBB models to study leukocyte adhesion 
and transendothelial migration.

o  Role of astrocyte-endothelial interactions in regulation of HIV-1 infected 
leukocyte trafficking.

o  Impact of astrocyte derived factors (chemokines, cytokines) in regulating 
HIV-1 infected leukocyte migration through BBB.

o  Identification of factors that control permeability (matrix 
metalloproteinases, Vpr) or damage (serum nitrates or nitrites, glutamates) the 
BBB following HIV-1 infection.

o  Role of viral proteins and induced factors in apoptosis of BBB derived cells.

o  Mechanisms of HIV-1 induced apoptosis of cells lining the BBB (endothelial 
cells and astrocytes).

o  Identification of host and viral molecular determinants that influence 
endothelial cell tropisms.

o  Role of adsorptive endocytosis and transcytosis as a mechanism for HIV-1 
passage through the BBB.

o  Isolation and molecular characterization of viral isolates that infect CNS 
endothelial cells and/or astrocytes.

o  Identification of HIV-1 envelope sequences that regulate endothelial 
tropisms.

o  Identification of endothelial receptors involved in interaction with 
endotheliotropic HIV-1 envelope sequences(if identified).

o  Effects of drugs of abuse (Cocaine, Methamphetamines) in facilitating HIV-1 
neuroinvasion through the BBB.

INCLUSION OF WOMEN, AND MINORITIES IN RESEARCH INVOLVING HUMAN SUBJECTS

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 the research.  This 
policy results from the NIH Revitalization Act of 1993 (Section 492B of Public 
Law 103-43).

All investigators proposing research involving human subjects should read the 
"NIH Guidelines for Inclusion of Women and Minorities as Subjects in Clinical 
Research," which was published in the Federal Register of March 28, 1994 (FR 59 
14508-14513) and in the NIH Guide for Grants and Contracts, Vol. 23, No. 11, 
March 18, 1994, available on the web at:
http://grants.nih.gov/grants/guide/notice-files/not94-100.html

INCLUSION OF CHILDREN AS PARTICIPANTS IN RESEARCH INVOLVING HUMAN SUBJECTS

It is the policy of 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: http://grants.nih.gov/grants/guide/notice-files/not98-024.html

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

LETTER OF INTENT

Prospective applicants are asked to submit 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 a 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 Institute staff to estimate 
the potential review workload and avoid conflict of interest in the review.

The letter of intent is to be sent to the program staff listed under INQUIRIES 
by April 24, 2000.

APPLICATION PROCEDURES

The research grant application form PHS 398 (rev. 4/98) is to be used in 
applying for these grants.  These forms are available at most institutional 
offices of sponsored research and 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, Email: 
[email protected].  The application is also available at 
http://grants.nih.gov/grants/funding/phs398/phs398.html

SPECIFIC APPLICATION INSTRUCTIONS FOR MODULAR GRANTS

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.  The 
research grant application form PHS 398 (rev. 4/98) is to be used in applying 
for these grants, with the modifications noted below.

BUDGET INSTRUCTIONS

Modular Grant applications will request direct costs in $25,000 modules, up to a 
total direct cost request of $250,000 per year.  (Applications that request more 
than $250,000 direct costs in any year must follow the traditional PHS 398 
application instructions.)  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:

PHS 398

FACE PAGE: Items 7a and 7b should be completed, indicating Direct Costs (in 
$25,000 increments) and Total Costs [Modular Total Direct plus Facilities and 
Administrative (F&A) costs] for the initial budget period.  Items 8a and 8b 
should be completed indicating the Direct and Total Costs for the entire 
proposed period of support.

DETAILED BUDGET FOR THE INITIAL 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.

BUDGET FOR THE ENTIRE PROPOSED PERIOD OF SUPPORT:  Do not complete the 
categorical budget table on Form Page 5 of the PHS 398.  It is not required and 
will not be accepted with the application.

o  NARRATIVE BUDGET JUSTIFICATION - Prepare a Modular Grant Budget Narrative 
page. (See http://grants.nih.gov/grants/funding/modular/modular.htm for sample 
pages.) At the top of the page, enter the total direct costs requested for each 
year.  This is not a Form page.

o  Under Personnel, List key project personnel, including their names, percent 
of effort, and roles on the project.  No individual salary information should be 
provided.  However, the applicant should use the NIH appropriation language 
salary cap and the NIH policy for graduate student compensation in developing 
the budget request.

For Consortium/Contractual costs, provide an estimate of total costs (direct 
plus facilities and administrative) for each year, each rounded to the nearest 
$1,000.  List the individuals/organizations with whom consortium or contractual 
arrangements have been made, the percent effort of key personnel, and the role 
on the project.  Indicate whether the collaborating institution is foreign or 
domestic.  The total cost for a consortium/contractual arrangement is included 
in the overall requested modular direct cost amount.  Include the Letter of 
Intent to establish a consortium.

Provide an additional narrative budget justification for any variation in the 
number of modules requested.

o  BIOGRAPHICAL SKETCH - The Biographical Sketch provides information used by  
reviewers in the assessment of each individual"s qualifications for a specific 
role in the proposed project, as well as to evaluate the overall qualifications 
of the research team.  A biographical sketch is required for all key personnel, 
following the instructions below.  No more than three pages may be used for each 
person.  A sample biographical sketch may be viewed at:  
http://grants.nih.gov/grants/funding/modular/modular.htm 

- Complete the educational block at the top of the form page,
- List position(s) and any honors,
- Provide information, including overall goals and responsibilities, on research 
projects ongoing or completed during the last three years.
- List selected peer-reviewed publications, with full citations,

o  CHECKLIST - This page should be completed and submitted with the application.  
If the F&A rate agreement has been established, indicate the type of agreement 
and the date.  All appropriate exclusions must be applied in the calculation of 
the F&A costs for the initial budget period and all future budget years.

o  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.

The RFA label available in the PHS 398 (rev. 4/98) application form must be 
affixed to the bottom of the face page of the application and must display the 
RFA number MH-00-010. A sample modified mailing label is available at: 
http://grants.nih.gov/grants/funding/phs398/label-bk.pdf.  Please note this is 
in pdf format.  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 and number, Mechanisms of HIV-1 Trafficking 
in the CNS, RFA MH-00-010, must be typed on line 2 of the face page of the 
application form and the YES box must be marked. 

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

CENTER FOR SCIENTIFIC REVIEW
NATIONAL INSTITUTES OF HEALTH
6701 ROCKLEDGE DRIVE, ROOM 1040, MSC 7710
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:

Jeymohan Joseph, Ph.D
Center for Mental Health Research on AIDS
National Institute of Mental Health
6001 Executive Boulevard, Room 6202, MSC 9619
Bethesda, MD 20892-9605

Applications must be received by May 24, 2000.  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.

REVIEW CONSIDERATIONS

Upon receipt, applications will be reviewed for completeness by the CSR and 
responsiveness by the NIMH staff.  Incomplete and/or non-responsive applications 
will be returned to the applicant without further consideration.

Applications that are complete and responsive to the RFA will be evaluated for 
scientific and technical merit by an appropriate peer review group convened by 
NIMH in accordance with the review criteria stated below.  As part of the 
initial merit review, a process will be used by the initial review group in 
which applications receive a written critique and undergo a process in which 
only those applications deemed to have the highest scientific merit, generally 
the top half of the applications under review, will be discussed, assigned a 
priority score, and receive a second level review by the National Advisory 
Council or Board.

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 comments reviewers will be asked to discuss the following aspects of the 
application 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 assigning the overall score, weighting them 
as appropriate for each application.  Note that the application does not need to 
be strong in all categories to be judged likely to have major scientific impact 
and thus deserve a high priority score.  For example, an investigator may 
propose to carry out important work that by its nature is not innovative but is 
essential to move a field forward.

(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 
field?

(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 methods?  
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?

The initial review group will also examine: the appropriateness of proposed 
project budget and duration, the adequacy of plans to include both genders, 
minorities and their subgroups, and children as appropriate for the scientific 
goals of the research and plans for the recruitment and retention of subjects, 
the provisions for the protection of human and animal subjects, and the safety 
of the research environment.

Schedule

Letter of Intent Receipt Date: April 24, 2000
Application Receipt Date: May 24, 2000
Peer Review Date: August 2000
Council Review: September 2000
Earliest Anticipated Start Date: September 29, 2000

AWARD CRITERIA

Award criteria that will be used to make award decisions include:

o  scientific merit (as determined by peer review)
o  availability of funds
o  programmatic priorities.

INQUIRIES

Inquiries concerning this RFA are encouraged.  The opportunity to clarify any 
issues or questions from potential applicants is welcome.

Direct inquiries regarding programmatic issues to:

Jeymohan Joseph, Ph.D
Center for Mental Health Research on AIDS
National Institute of Mental Health
6001 Executive Boulevard, Room 6202, MSC 9619
Bethesda, MD 20892-9605
Telephone:  (301) 443-6100
FAX:  (301) 443-9719
Email:  [email protected]

F.J. Brinley, Jr., M.D., Ph.D
Associate Director for Infection and Immunity
National Institute of Neurological Disorders and Stroke
6001 Executive Boulevard, Room 2114
Bethesda, MD 20892-9521
Telephone: (301) 496-6541
Fax: (301) 402-0302
Email: [email protected]

Direct inquiries regarding fiscal matters to:

Diana S. Trunnell
Grants Management Branch
National Institute of Mental Health
6001 Executive Boulevard, Room 6115, MSC 9605
Bethesda, MD 20892-9605
Telephone: (301) 443-2805
FAX:  (301) 443-6885
Email:  [email protected]

Dianna Jessee
Grants Management Branch
National Institute of Neurological Disorders and Stroke
6001 Executive Boulevard, Room 3621
Bethesda, MD 20892-9619
Telephone: (301) 496-9231
FAX: (301) 402-0219 
Email: [email protected]

AUTHORITY AND REGULATIONS

This program is described in the Catalog of Federal Domestic Assistance No. 
93.242 (NIMH) and 93.853 (NINDS).  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 NIH 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 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 
people.

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