RESISTANCE TO ANTIVIRALS TARGETED TO HUMAN IMMUNODEFICIENCY VIRUS

NIH GUIDE, Volume 21, Number 44, December 11, 1992



PA NUMBER:  PA-93-31



P.T. 34



Keywords:

  AIDS 

  Antivirals 

  Drug Resistance+ 

  Screening of Drugs/Agents 



National Institute of Allergy and Infectious Diseases



Application Receipt Dates:  January 2, May 1, September 1, 1993



PURPOSE



This Program Announcement (PA) is designed to stimulate research to:

(1) elucidate mechanisms of Human Immunodeficiency Virus (HIV) drug

resistance, (2) determine the effects of viral drug resistance in

lentivirus pathogenesis, (3) identify improved methods to screen for

drug resistant HIV and animal lentivirus variants, and (4) design and

evaluate novel therapies for treating or preventing drug resistance,

utilizing in vitro and animal model systems as applicable.



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 PA,

Resistance to Antivirals Targeted to HIV, is related to the priority

area of HIV infection.  Potential applicants may obtain a copy of

"Healthy People 2000" (Full Report:  Stock No. 017-001-00474-0) or

"Healthy People 2000: (Summary Report:  Stock No. 017-001-00473-1)

through the Superintendent of Documents, Government Printing Office,

Washington, DC 20402-9325 (telephone 202-783-3238).



ELIGIBILITY REQUIREMENTS



Applications may be submitted by domestic, 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.  Foreign

institutions are not eligible for First Independent Research Support

and Transition (FIRST) (R29) awards.  Applications from minority

individuals and women are encouraged.



MECHANISM OF SUPPORT



This PA solicits R01 and R29 applications.  While no funds have been

set aside specifically for supporting applications submitted in

response to this PA, the NIAID regards additional quality research in

this area to be of high programmatic priority.  The total project

period for awards made in response to this PA will not exceed four

years for domestic institutions and three years for foreign

institutions.  If an R29 application is submitted, three reference

letters are required to be submitted with the application and stapled

to the face page of the original application.



RESEARCH OBJECTIVES



Background



The Developmental Therapeutics Branch (DTB), Basic Research and

Development Program (BRDP), Division of AIDS (DAIDS), National

Institute of Allergy and Infectious Diseases (NIAID) supports basic and

applied research grants leading to the discovery of new therapies for

the treatment of HIV infection and the opportunistic infections (OIs)

associated with AIDS.  This goal is accomplished through:  (1)

management of investigator-initiated research for HIV; (2) fostering of

innovative, multidisciplinary efforts leading to the discovery and

design of new anti-HIV and anti-OI therapies through the National

Cooperative Drug Discovery Group programs for treatment of HIV

infection (NCDDG-HIV) and for treatment of the OIs associated with AIDS

(NCDDG-OI); and (3) preclinical drug development contract resources to

confirm activity of anti-HIV and anti-OI compounds, and to evaluate

combination and immune-based therapies in vitro and in animal models.

The DTB released a Request for Applications (RFA) entitled "Drug

Resistance and the Human Immunodeficiency Virus" in 1989.  Applications

funded in response to that RFA focused predominantly on methodologies

for detecting HIV variants with reduced susceptibility to antivirals.



During the past few years, our understanding of the emergence of

drug-resistant HIV has increased dramatically.  For example, diminished

in vitro susceptibility of HIV clinical isolates from patients

undergoing treatment with AZT for as few as six to twelve months is

well documented.  Associated with this phenotype of reduced

susceptibility to AZT is a characteristic pattern of genetic

alterations at codons 41, 67, 70, 215, and 219 of reverse transcriptase

(RT) that appear to emerge in a temporal fashion conferring

progressively increasing resistance.  The complex interplay among these

mutations as evidenced by (1) the disappearance of the codon 70

mutation in sequential clinical isolates that show increased resistance

and (2) the stability of the mutation at codon 215 in isolates from

patients switched to ddI therapy that have reverted to AZT

susceptibility is not well understood.  The precise mechanism by which

these mutations confer the resistant phenotype in cell culture remains

unknown.



More recently, a decrease in in vitro sensitivity to ddI of HIV

isolates obtained from patients who changed to ddI therapy after

prolonged treatment with AZT has been reported.  An initial report

describing resistance to ddC in one patient has also appeared.  In

addition, preliminary results from clinical trials indicate that HIV

drug resistance to several non-nucleoside RT (NNRT) inhibitors, as

determined in vitro, develops rapidly in patients.  Novel mutations in

HIV RT that correlate with in vitro resistance to ddI, ddC and the NNRT

inhibitors have been identified in clinical isolates.  Introduction of

these specific mutations into the genetic background of a replication

competent, fully susceptible virus confers a resistant phenotype that

mimics the susceptibility profile of the original clinical isolates.



In summary, characteristic patterns of mutations in HIV-1 RT have been

identified at the molecular level in resistant variants and genetic

signatures appear to be distinct for each inhibitor or class of

inhibitors.  These observations notwithstanding, the exact mechanism(s)

by which the resistant phenotype is manifested in vivo via these

alterations is not known, and the clinical significance of HIV-related

drug resistance remains unknown at this time.



Research Scope



Molecular, enzymatic and biologic effects of drug resistance:  Given

the precedence of resistance to several nucleoside analogs and to NNRT

inhibitors in clinical isolates, it is expected that resistance to

inhibitors directed against other viral targets is likely to occur.

With the introduction of anti-HIV agents other than RT inhibitors into

clinical trials and the preclinical development of others, studies

involving therapeutic modalities directed against non-RT targets, e.g.,

protease, TAT, myristoylation, as well as against RT, are urgently

needed.



Crucial clinical studies to determine whether or not emergence of drug

resistant variants is correlated with disease progression are underway.

Of equal importance are studies that decipher the molecular, enzymatic

and biological aspects of HIV drug resistance that can serve as the

basis for developing therapeutic strategies to overcome or delay the

emergence of drug resistant HIV in the clinic.  While clinical

relevance awaits further corroboration, research focussed on

understanding the mechanisms underlying resistance and the biological

properties of the resistant virus that are implicated in disease

progression is encouraged.  As mentioned above, in spite of the

identification of characteristic genetic alterations conferring a drug

resistant phenotype on HIV, the precise mechanism(s) by which these

mutations mediate their enzymatic and biological impact on the

resistant phenotype is unknown.  The following areas of research are

particularly encouraged:



o  Elucidation of the basis for the apparent inability of cell-free

assays to distinguish RT purified from AZT-resistant vs. AZT-sensitive

isolates may shed additional light on this dilemma.  This is contrasted

by retention of the drug resistant phenotype in RT purified from NNRT-

resistant HIV.



o  Structure/function studies of drug resistant viral targets in the

presence and absence of drug may also contribute to delineating the

mechanisms by which genetic alterations result in loss of drug effect.



o  The biological effects the genetic alterations conferring the

resistant phenotype have on the properties of the virus in terms of its

pathogenicity and capacity to accelerate or attenuate disease

progression are not well studied.  Information is also incomplete

regarding the relationship between HIV variants capable of forming

syncytium upon cocultivation of infected patient peripheral blood

lymphocytes with MT-2 cells, viral drug resistance and clinical

progression.



o  The effect of drug resistance on viral transmission needs to be

elucidated.  If drug resistant HIV variants are transmitted, is the

frequency comparable to that of their drug sensitive counterparts?



Phenotypic and genotypic detection of drug resistance: Sensitive and

reproducible methods for (1) detection of drug resistant HIV mutants,

including primary isolates within phenotypic mixtures and (2)

quantitation of levels of drug resistance and proportion of resistant

HIV variants are urgently needed to delineate the mechanism(s) and

impact of resistance and to predict the efficacy of new therapies.

Methods for the expansion of primary HIV isolates to adequate titers

with minimal selection for specific viral variant(s) are also critical.

Recent evidence suggests that passage of clinical isolates in cell

lines may rapidly select for specific genotypes.



Specifically



o  Additional culture systems should be developed that closely model

the in vivo situation.  Currently, assays utilizing fresh peripheral

blood mononuclear cells (PBMC) have received the predominant focus.

However, variable infectability of donor cells may preclude the

standardization of this assay system.  Methods for expansion of primary

HIV isolates to adequate titers with minimal selection for specific

viral variant(s) are also critically needed.



o  Genotypic assays also suffer from certain limitations, particularly

those imposed by the possibility that the genetic signature of

resistance associated with a specific therapeutic may not yet be

comprehensive.  Technical difficulties also exist in designing

polymerase chain reaction primers that can accurately, specifically,

and reproducibly amplify the associated mutations.



Because of these current limitations and deficiencies in evaluating HIV

drug resistance, development of novel and/or validation of existing

assays to measure viral susceptibility to antivirals is/are strongly

encouraged.  For example, innovative, highly sensitive biophysical

methods recently developed may be modified and implemented to identify

alterations previously undetectable at the protein level that

correspond to mutations conferring the resistant phenotype.  Such

changes may represent markers for HIV resistance to drugs or may,

themselves, be directly responsible for the drug resistant phenotype.



Animal models of drug resistance:  Development and implementation of

relevant animal models of lentivirus infection are critically needed to

address these resistance issues.  Efforts to establish an animal model

of viral drug resistance may include development of protocols for

detection and quantitation as well as expansion of isolates, as the

problems addressed above for HIV-specific susceptibility assays are

likely to be similar.  Animal models of lentiviral infection (e.g., SIV

and FIV) of potential relevance are available.  Thus, systematic

evaluation of virus isolates from drug-treated animals may define an

animal model that parallels documented resistance in HIV-infected

patients.  Once developed, such model(s) may be exploited for assessing

drug therapy and drug resistance.



Novel strategies for circumventing HIV drug resistance:  The ultimate

goal of these studies is to design novel strategies to circumvent

and/or delay the development of viral drug resistance.  As an example,

one approach can focus on highly conserved functional domains

identified in several HIV proteins (RNA-dependent and DNA-dependent

activities, and RNaseH and RNaseD activities of RT; transactivation,

nuclear-targeting, and RNA/protein binding activities of Tat).  The

observation that these domains are spared from sequence variations

suggests that most mutations in these regions would be lethal to the

virus and, therefore, to have no or minimal consequence on drug

treatment.  This and other research objectives include, but are not

limited to:



o  Exploiting other viral targets believed to be essential for viral

survival, and thus incompatible with resistance-conferring mutations



o  Investigating combination therapies designed to inhibit more than

one distinct step in the HIV infectious cycle that may delay the

development of viral drug resistance by virtue of a multipronged action



o  Designing studies to reduce the overall rate at which HIV mutates.

While not specifically addressed in this PA, evaluation of the role of

host-mediated processes in the resistant phenotype may also provide

insight into developing new therapeutic approaches.



The rationale for any potential approach should first be tested and

analyzed in an appropriate in vitro system, if applicable.  For

example, a new therapeutic regimen may be evaluated for its effect on

in vitro emergence of resistant HIV variants.  Alternatively, a novel

therapeutic strategy may be tested for its activity against established

resistant mutants.  Subsequent evaluation of new drug strategies in

relevant animal models should yield important information on the

applicability of the proposed strategies to the clinical setting.  The

approaches outlined above are examples of potential research directions

bearing on the emergence of HIV drug resistance and are not intended to

be comprehensive.



STUDY POPULATIONS



SPECIAL INSTRUCTIONS FOR INCLUSION OF WOMEN AND MINORITIES IN CLINICAL

RESEARCH STUDIES



NIH policy is that applicants for NIH clinical research grants and

cooperative agreements will be required to include minorities and women

in study populations so that research findings can be of benefit to all

persons at risk of the disease, disorder or condition under study;

special emphasis should be placed on the need for inclusion of

minorities and women in studies of diseases, disorders and conditions

which disproportionately affect them.  This policy is intended to apply

to males and females of all ages.  If women or minorities are excluded

or inadequately represented in clinical research, particularly in

proposed population-based studies, a clear compelling rationale should

be provided.



The composition of the proposed study population must be described in

terms of gender and racial/ethnic group, together with a rationale for

its choice.  In addition, gender and racial/ethnic issues should be

addressed in developing a research design and sample size appropriate

for the scientific objectives of the study.  This information should be

included in the form PHS 398 in Items 1-4 of the Research Plan AND

summarized in Item 5, Human Subjects.  Applicants are urged to assess

carefully the feasibility of including the broadest possible

representation of minority groups.  However, NIH recognizes that it may

not be feasible or appropriate in all research projects to include

representation of the full array of United States racial/ethnic

minority populations (i.e., Native Americans (including American

Indians or Alaskan Natives), Asian/Pacific Islanders, Blacks,

Hispanics).  The rationale for studies on single minority population

groups must be provided.



For the purpose of this policy, clinical research is defined as human

biomedical and behavioral studies of etiology, epidemiology, prevention

(and preventive strategies), diagnosis, or treatment of diseases,

disorders or conditions, including but not limited to clinical trials.



The usual NIH policies concerning research involving human subjects

also apply.  Basic research or clinical studies in which human tissues

cannot be identified or linked to individuals are excluded.  However,

every effort should be made to include tissues from women and

racial/ethnic minorities when it is important to apply the results of

the study broadly, and this should be addressed by applicants.



For foreign awards, the policy on inclusion of women applies fully;

since the definition of minority differs in other countries, the

applicant must discuss the relevance of research involving foreign

population groups to the United States' populations, including

minorities.



If the required information is not contained within the application,

the application will be deferred until the information is provided.



Peer review will address specifically whether the research plan in the

application conforms to these policies.  If the representation of women

or minorities in the study design is inadequate to answer the

scientific question(s) addressed AND the justification for the selected

study population is inadequate, it will be considered a scientific

weakness or deficiency in the study design and will be reflected in

assigning the priority score to the application.



All applications for clinical research submitted to NIH are required to

address these policies.  NIH funding components will not award grants

or cooperative agreements that do not comply with these policies.



APPLICATION PROCEDURES



Applications are to be submitted on grant application form PHS 398

(rev. 9/91).  Receipt dates are January 2, May 1, and September 1.



Application kits are available at most institutional offices of

sponsored research and may be obtained from the Office of Grants

Inquiries, Division of Research Grants, National Institutes of Health,

Westwood Building, Room 449, Bethesda, MD 20892, telephone

301/496-7441.  The title and number of the announcement must be typed

in Section 2a on the face page of application and the "YES" box marked.



The completed original application and five legible copies must be sent

or delivered to



Division of Research Grants

National Institutes of Health

Westwood Building, Room 240

Bethesda, MD  20892**



REVIEW PROCEDURES



Applications in response to this announcement are expected to be

assigned to the NIAID.  However, whenever there is inter-institute

programmatic overlap in the proposed research, the PHS Referral

Guidelines will prevail in the assignment of applications to the

different institutes.  Applications will be reviewed for scientific and

technical merit by study sections of the Division of Research Grants,

NIH, in accordance with the standard NIH peer review procedures.

Applications focusing on AIDS will be assigned to study sections

constituted to review AIDS-related grant applications.  Following

scientific-technical review, the applications will receive a

second-level review by an appropriate national advisory council.  For

AIDS-related applications, the earliest award date for successful

applications will be no more than six months from the receipt date.



AWARD CRITERIA



Applications will compete for available funds with all other approved

applications.  The following criteria will be considered in making

funding decisions:



o  Quality of the proposed project as determined by peer review

o  Availability of funds

o  Program balance among research areas of the announcement.



Applications from institutions that have a General Clinical Research

Center (GCRC) funded by the NIH National Center for Research Resources

may identify the GCRC as a resource for conducting the proposed

research by including a letter of agreement from either the GCRC

program director or the principal investigator.



INQUIRIES



Written and telephone inquiries are encouraged.  The opportunity to

clarify any issues or questions from potential applicants is welcome.



Direct inquiries of a scientific nature to:



Roberta Black, Ph.D.

Basic Research and Development Program, Division of AIDS

National Institute of Allergy and Infectious Diseases

Solar Building, Room 2C12

Bethesda, MD  20892

Telephone:  (301) 496-8197

FAX:  (301) 480-5703/402-3211



Direct inquiries regarding fiscal matters to:



Ms. Jane Unsworth

Chief, AIDS Grants Management Section

National Institute of Allergy and Infectious Diseases

Solar Building, Room 4B22

Bethesda, MD  20892

Telephone:  (301) 496-7075

FAX:  (301) 480-3780



AUTHORITY AND REGULATIONS



This program is described in the Catalog of Federal Domestic

Assistance, 93.856 - Microbiology and Infectious Diseases Research and

93.855 - Immunology, Allergy and Transplantation Research.  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 Part 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.



.

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