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 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: 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: 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: 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: The application is also available at 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 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: - 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: 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: 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: 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: 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: 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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