Antiviral Product Development--Conducting and Submitting Virology Studies to the Agency

Administrative guidance, not enacted legislation. This captured publisher edition preserves the listed document and attachments; later developments are not independently consolidated.

Guidance for Industry
Antiviral Product
Development — Conducting
and Submitting Virology
Studies to the Agency
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Drug Evaluation and Research (CDER)
June 2006
Clinical Antimicrobial

Guidance for Industry
Antiviral Product
Development — Conducting
and Submitting Virology
Studies to the Agency
Additional copies are available from:
Office of Training and Communications
Division of Drug Information, HFD-240
Center for Drug Evaluation and Research
Food and Drug Administration
5600 Fishers Lane
Rockville, MD 20857
(Tel) 301-827-4573
http://www.fda.gov/cder/guidance/index.htm
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Drug Evaluation and Research (CDER)
June 2006
Clinical Antimicrobial

TABLE OF CONTENTS
I. 	INTRODUCTION............................................................................................................. 1
II. 	BACKGROUND ............................................................................................................... 2
III. 	NONCLINICAL VIROLOGY STUDIES ...................................................................... 3
A. 	Mechanism of Action Studies........................................................................................................ 3
B. 	Antiviral Activity ........................................................................................................................... 4
1. Antiviral Activity in Vitro................................................................................................................. 4
2. Antiviral Activity in Vitro in the Presence of Serum Proteins ......................................................... 6
3. Inhibitory Quotient .......................................................................................................................... 7
4. Antiviral Activity in Vivo.................................................................................................................. 7
C. 	Cytotoxicity and Therapeutic Indexes ......................................................................................... 7
D. 	In Vitro Combination Activity Analysis ...................................................................................... 8
E. 	Resistance ....................................................................................................................................... 8
1. Selection of Resistant Virus in Vitro ................................................................................................ 8
2. Genotypic Analysis......................................................................................................................... 10
3. Phenotypic Analysis ....................................................................................................................... 10
4. Cross-Resistance............................................................................................................................ 11
IV. 	PROPOSAL FOR MONITORING RESISTANCE DEVELOPMENT .................... 11
V. 	VIROLOGY STUDY REPORTS.................................................................................. 13
VI. 	SUMMARY ..................................................................................................................... 14

Contains Nonbinding Recommendations
Guidance for Industry1
Antiviral Product Development — Conducting and Submitting
Virology Studies to the Agency
This guidance represents the Food and Drug Administration’s (FDA’s) current thinking on this topic. It
does not create or confer any rights for or on any person and does not operate to bind FDA or the public.
You can use an alternative approach if the approach satisfies the requirements of the applicable statutes
and regulations. If you want to discuss an alternative approach, contact the FDA staff responsible for
implementing this guidance. If you cannot identify the appropriate FDA staff, call the appropriate
number listed on the title page of this guidance.
I. 	INTRODUCTION
The purpose of this guidance is to assist sponsors in the development of antiviral drugs and
biological products (i.e., therapeutic proteins and monoclonal antibodies) from the initial pre-
IND through the new drug application (NDA) and postmarketing stages. This guidance should
serve as a starting point for understanding what nonclinical and clinical virology data are
important to support the submission of an investigational new drug application (IND), NDA, or
biologics license application (BLA) for approval of an antiviral product. This guidance focuses
on nonclinical and clinical virology study reports and makes recommendations for collecting and
submitting resistance data to the Food and Drug Administration (FDA). Nonclinical and clinical
virology study reports, based on collected data, are essential for the FDA’s review of antiviral
drug investigational and marketing applications. Specific topics discussed in this guidance
include:
• 	Defining the mechanism of action
• 	Establishing specific antiviral activity of the investigational product
• 	Assessing the potential for antagonism of other antiviral products that might be used in
combination with the investigational product
• 	Providing data on the development of viral resistance to the investigational product
• 	Providing data that identify cross-resistance to approved antiviral products having the
same target
FDA’s guidance documents, including this guidance, do not establish legally enforceable
responsibilities. Instead, guidances describe the FDA’s current thinking on a topic and should be
viewed only as recommendations, unless specific regulatory or statutory requirements are cited.
1 This guidance has been prepared by the Division of Antiviral Products in the Center for Drug Evaluation and
Research (CDER) at the Food and Drug Administration.
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The use of the word should in FDA guidances means that something is suggested or
recommended, but not required.
II. 	BACKGROUND
The recommendations in this guidance are based on the antiviral product review experience of
the Division of Antiviral Products and input from pharmaceutical sponsors and the scientific
community. Because of the experience, history, and lessons learned with HIV-1 studies, this
guidance employs studies commonly used to evaluate HIV-1 products as a paradigm for studies
of products to treat other viruses. Although assays and model systems vary with different
viruses, many of the principles in this guidance can be applied to antiviral products in
development for the treatment of other viral infections (e.g., hepatitis B virus, hepatitis C virus,
herpes simplex virus, varicella zoster virus, influenza virus, rhinovirus, cytomegalovirus, and
human papillomavirus). Since the field of virology is dynamic and continually evolving, this
guidance will be revised as new information accumulates and as circumstances warrant.
Sample formats have been developed to assist sponsors in providing resistance data to the
Agency (see the stand-alone documents accompanying this guidance). The sample formats are
being provided as stand-alone documents to help sponsors provide the appropriate information to
the Agency. These sample formats will be updated as needed, and additional formats for other
viruses may be provided.
Guidances on the overall organization of INDs and NDAs can be found at
www.fda.gov/cder/regulatory/applications/default.htm. Sponsors are encouraged to contact the
division early in the development of an investigational antiviral product to facilitate the review
and approval process. To assist prospective sponsors, the FDA accepts submissions in an
abbreviated IND format (i.e., pre-INDs) for review and comment. Pre-INDs are especially
useful in instances when sponsors are unfamiliar with the process for evaluating investigational
products in humans. Information about submitting pre-INDs to the division (“Getting Started
with the Pre-IND Process”) can be found at www.fda.gov/cder/ode4/preind/getting.htm. The
FDA accepts electronic submissions to expedite the review process. FDA Web sites can be
consulted for information on electronic application submission.
Sponsors are advised to consult the division for additional guidance on the development of
investigational products against orthopoxviruses, influenza virus, severe acute respiratory
syndrome (SARS) virus, or other emerging infections. For antiviral products that treat smallpox
and other orthopoxviruses, related information can be found in the draft guidance for industry
Vaccinia Virus — Developing Drugs to Mitigate Complications from Smallpox Vaccination.2
Sponsors should give careful attention to observing all provisions of the Select Agent rule3 and
other applicable governmental and institutional biosafety and biosecurity provisions.
2 When final, this guidance will represent the FDA’s current thinking on this topic. For the most recent version of a
guidance, check the CDER guidance Web page at http://www.fda.gov/cder/guidance/index.htm.
3 See 42 CFR part 73 and http://www.cdc.gov/od/sap/sitemap.htm.
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III. 	NONCLINICAL VIROLOGY STUDIES
Nonclinical virology studies aid in the evaluation of the safety and efficacy of an investigational
product before it is tested in humans. Recommended studies can identify the mechanism of
action, help establish specific antiviral activity of the product in a model system, and provide
data on the development of viral resistance to the investigational product. Additionally, co
administration of an investigational product with other products approved for the same indication
is often necessary to treat viral infections in clinical settings. In these cases, it is desirable to
have in vitro combination activity studies designed to identify possible negative interactions on
antiviral activity (i.e., antagonism) of the investigational product with other antiviral products.
As more antiviral products are developed to treat particular viral diseases, cross-resistance (i.e.,
viral resistance to one product causing resistance to more than one product within that drug
class) can become a major issue in clinical settings. Therefore, from a scientific perspective, the
following information is critical in the development of antiviral products:
• 	Determining the antiviral activity of an investigational product against relevant viruses
resistant to other approved products with the same target molecule or complex
• 	Determining the antiviral activity of approved products against viruses resistant to an
investigational product with the same target molecule or complex
We recommend conducting nonclinical studies (i.e., mechanism of action, antiviral activity in
vitro, cytotoxicity and therapeutic indexes, and effects of serum protein binding on antiviral
activity) before the initiation of phase 1 clinical studies. When developing products for viruses
where in vitro infection systems exist, sponsors should complete in vitro drug combination
activity studies of the investigational product with other approved products against the same
virus (i.e., all approved and available investigational products that target the same protein and at
least one representative product from each of the other existent drug classes) before the initiation
of clinical trials that will examine the efficacy of the investigational product in combination with
other antiviral products. Furthermore, we recommend examining the in vitro selection of
resistant viruses to the investigational product, the phenotypic and genotypic characterization of
resistant viruses, and cross-resistance before initiation of clinical studies in patients infected with
a particular virus. Complete study reports on nonclinical and clinical virology studies can be
submitted to the FDA upon their completion and need not be held until submission of the NDA.
Specific details of each of the nonclinical studies recommended by the division are discussed in
the following sections.
A. 	Mechanism of Action Studies
Mechanism of action studies should be conducted before the initiation of phase 1 clinical studies.
There are many steps in a virus life cycle that can be targeted by potential antiviral product
candidates. Products can act directly to inhibit a virus by targeting a specific viral-encoded
function (e.g., an enzyme inhibitor) or act indirectly (e.g., interferon induction of host cell
response). We recommend that mechanism of action studies:
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• 	Demonstrate the investigational product’s ability to specifically inhibit viral replication
or a virus-specific function
• 	Establish the site of the product’s action (e.g., viral replicase, protease)
Biochemical, structural, cellular, or genetic data can be presented to support the proposed
mechanism of action. Data that demonstrate the mechanism of action include, but are not limited
to, receptor binding, inhibition of enzymatic activity, X-ray crystallographic structure
determination of bound inhibitor complex, and characterization of resistance mutations in the
gene encoding the target.
A well-characterized mechanism of action is useful in predicting toxicities and in designing
studies to assess the development of resistance. A clear understanding of the mechanism of
action for an investigational antiviral product can provide insight into the regions of the viral
genome where resistance mutations could develop. These regions are not limited to the site of
action (viral-encoded target) of the investigational product but can include the enzyme substrate
or another viral-encoded protein existing in a complex with the target protein. Characterization
of the resistance mutations can provide in vivo validation of the mechanism of action studies.
The specificity of the investigational product should be demonstrated for the viral target over
cellular or host proteins, especially in those cases in which a viral enzyme has a cellular
counterpart. For example, if the investigational product targets a viral polymerase, we
recommend showing the activity of the product against the viral polymerase in comparison with
its activity against host DNA polymerases such as DNA polymerase α, β, and γ.
Immunomodulatory products raise additional issues. These products can have the potential for
unintended effects on viral replication and other adverse effects resulting from actions on the
immune system. For investigational products that act through a general immune stimulatory
mechanism, we recommend that sponsors show a reduction in antiviral activity and identify the
specific immune system components that are involved. Sponsors should consult the division for
specific advice regarding the development of immunomodulatory products for the treatment of
viral diseases as well as products with other nonviral host targets.
B. 	Antiviral Activity
1. 	Antiviral Activity in Vitro
For many human viruses, there are cell culture systems or animal hosts in which the infectious
agent can undergo a complete virus life cycle. In these cases, we recommend that the sponsor
document that the investigational product and/or its metabolites show specific, quantifiable
antiviral activity in vitro before initiating tests in humans (i.e., before initiation of phase 1
studies). It is important that these data support clinical testing in humans by providing clear
evidence of antiviral effects at drug concentrations that can be achieved in vivo with acceptable
risk-benefit. Additionally, in vitro antiviral activity and cytotoxicity assessments (see Section
III.C., Cytotoxicity and Therapeutic Indexes) using relevant cell types and virus isolates can be
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used to guide the selection of appropriate dose ranges in early clinical trials. Sponsors are
encouraged to obtain antiviral activity data using primary human target cells, if possible.
Because of viral genetic variation, the antiviral activity of the investigational product should be
examined for multiple clinical isolates and viral isolates representative of the virus population in
clinical trials. Antiviral activity evaluations that are recommended to support the development
of the investigational product include:
• 	Assessing specific antiviral activity of the investigational product against a broad range
of clinical and laboratory viral isolates including different clades, subtypes, or genotypes
• 	Evaluating the antiviral activity of the investigational product against mutant viruses that
are resistant to products with the same target molecule or complex as the investigational
product as well as a representative sample of viruses resistant to other approved products
for the same indication
We recommend determining specific antiviral activity using a quantitative assay to measure
virus replication in the presence of increasing concentrations of the product compared to
replication in the absence of the product. The effective concentration is the concentration of
product at which virus replication is inhibited by 50 percent (e.g., EC50 for cell-based assays;
IC50 for biochemical or subcellular assays). Assays that evaluate antiviral activity and
cytotoxicity include, but are not limited to, virus inactivation assays, plaque reduction assays,
cytopathic effect inhibition assays, peripheral blood mononuclear cell (PBMC) assays, and
binding and fusion assays. Other factors that can be assessed in these assays include the effect of
an increasing multiplicity of infection and the effect of pretreatment of virus or cells before
infection versus treatment post-infection. We suggest that host cell lines be low in passage
number for reproducible results.
It is important that the effective concentration be consistent with data supporting the mechanism
of action. An investigational product that inhibits virus replication at concentrations lower than
biochemical data for the proposed mechanism indicates that another target or mechanism of
inhibition may be affected. Resistance analyses can provide in vivo validation of the proposed
mechanism of action when biochemical data are not consistent with antiviral activity data. For
nucleoside or nucleotide analogs, we recommend that sponsors determine the intracellular half-
life (t1/2 ) of the triphosphate form of the active drug moiety in stationary and dividing cells from
the target tissue.
For some human viruses (e.g., hepatitis B and hepatitis C viruses), no satisfactory cell culture or
animal model exists, and in these cases, inhibition of an essential viral function or activity
against related viruses can be used to indicate potential activity. When no satisfactory cell
culture or animal model exists for the target human virus, it is particularly important to know
whether or not the active moiety of an antiviral product enters cells, if it has a proposed
intracellular site of action, and if the intracellular concentration correlates with biochemical
studies. Cell-based assays and host cell lines for studying viruses such as hepatitis B virus
(HBV) and hepatitis C virus (HCV) replication have advanced, but at the present time are
limited. Currently, assays that examine HBV replication include, but are not limited to:
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• 	Measuring HBV DNA polymerase activity in biochemical assays
• 	Cell culture assays with baculovirus-mediated cell transfer or transfection of HBV
genomes into human hepatoma cell lines followed by quantitative Southern blot analysis
with HBV DNA probes
• 	Cell culture assays with stably-transfected cell lines containing the HBV genome
• 	Quantitative PCR of extracellular HBV DNA
For analyses of HCV replication, replicon systems have been developed that permit studies of
viral replication and can be used to assess antiviral activity of some anti-HCV products.4,5 In
addition, recent studies indicate that cell-based systems for the replication of HCV in vitro are in
development and might be available to assess antiviral activity.6 Currently, assays that examine
HCV replication using the HCV replicon system include, but are not limited to:
• 	Measuring the level of HCV RNA by RT-PCR in HCV replicon cells
• 	Measuring HCV RNA polymerase, HCV serine protease, or reporter enzyme (e.g.,
luciferase) activity in biochemical assays
2. 	Antiviral Activity in Vitro in the Presence of Serum Proteins
Serum proteins can bind to and sequester many products and interfere with a product’s antiviral
activity. We recommend that sponsors ascertain if the investigational product is significantly
bound by serum proteins. Common methods for determining protein binding include
equilibrium dialysis, ultrafiltration methods, and fluorescence-based high throughput human
serum albumin and α-acidic glycoprotein protein binding. If the investigational product is
highly protein bound, sponsors are encouraged to examine the in vitro antiviral activity of the
investigational product in the presence of a series of dilutions of human serum up to 40 percent
(e.g., 5 percent, 10 percent, 20 percent, 40 percent). An EC50 value for 100 percent human
serum can be extrapolated from these data and the serum-adjusted EC50 values reported. In
addition, sponsors are encouraged to determine EC50 values in the presence of physiological
concentrations of α-acidic glycoprotein and human serum albumin.
4 Lohmann V, F Korner, J-O Koch, U Herian, L Theilmann, and R Bartenschlager, 1999, Replication of
Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line, Science 285:110-113.
5 Blight K, A Kolykhalov, and C Rice, 2000, Efficient Initiation of HCV RNA Replication in Cell Culture, Science
290:1972-1974.
6 Wakita T, T Pietsschmann, T Kato, T Date, M Miyamoto, A Zhao et al., 2005, Production of Infectious Hepatitis
C Virus in Tissue Culture from a Cloned Viral Genome, Nat Med. 11:791-6.
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3. 	Inhibitory Quotient
Information on plasma and intracellular product concentrations is important in assessing the
dose-response of antiviral therapy and evaluating the potential for resistance development;
therefore, it is useful to determine an inhibitory quotient (IQ). An IQ is the Cmin /serum-adjusted
EC50 value. (For more information on determining EC50 values, see Section III.B.1., Antiviral
Activity in Vitro.) We view IQ values as a useful tool integrating in vivo product concentrations
and antiviral activity for an individual product. It is a measure that characterizes the relationship
between product exposure and the susceptibility of a virus to a product. A high IQ indicates that
an effective product concentration can be achieved in a patient to inhibit the virus and minimize
the development of resistance. Since one dose may not be adequate for all patient populations,
IQ values can be used to aid in the selection of doses to further evaluate in phase 3 and phase 4
clinical studies.
4. 	Antiviral Activity in Vivo
In cases where no in vitro cell culture or replicon system has been shown to be predictive of
antiviral activity in humans, measurement of viral titers after treatment in animal model systems
can be used to assess antiviral activity of the investigational product. Analyses from animal
models include the morbidity and mortality of animals following documented infection,
histological examination of tissues, quantification of viral titers over time, isolation and
characterization of resistant isolates in animals that experience viral rebound, quantification of
viral antigens and antibodies, pharmacokinetics of the investigational product, and a description
of symptoms (e.g., neurological, weight loss).
C. 	Cytotoxicity and Therapeutic Indexes
It is important to establish that an investigational product has antiviral activity at concentrations
that can be achieved in vivo without inducing toxic effects to cells. Furthermore, in a cell
culture model, apparent antiviral activity of an investigational product can be the result of host
cell death after exposure to the product. Cytotoxicity tests use a series of increasing
concentrations of the antiviral product to determine what concentration results in the death of 50
percent of the host cells (see also Section III.B.1., Antiviral Activity in Vitro). This value is
referred to as the median cellular cytotoxicity concentration and is identified by CC50 or CCIC50 .
The relative effectiveness of the investigational product in inhibiting viral replication compared
to inducing cell death is defined as the therapeutic or selectivity index (i.e., CC50 value/EC50
value). It is desirable to have a high therapeutic index giving maximum antiviral activity with
minimal cell toxicity. We recommend determining CC50 values in both stationary and dividing
cells from multiple relevant human cell types and tissues to ascertain the potential for cell-cycle,
species, or tissue-specific toxicities. Studies determining cytotoxicity and therapeutic indexes
should be conducted before the initiation of phase 1 clinical studies.
Because of the myelosuppressive effects of some antiviral products, we recommend assessing
the potential effects of certain investigational products (e.g., nucleoside analogs) on the growth
of human bone marrow progenitor cells in colony formation assays. Additionally, some
investigational antiviral products are potential inhibitors of cellular DNA polymerases, which are
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responsible for normal nuclear and mitochondrial DNA synthesis and repair. Sponsors should
determine IC50 values for investigational antiviral products against cellular polymerases and
show specificity for the viral target over cellular polymerases. The inhibition of human pol γ,
the enzyme responsible for mitochondrial DNA synthesis, has been linked to defects in
mitochondrial function that can lead to adverse events in humans. Therefore, it is important to
examine the effects of certain investigational products (e.g., nucleoside analogs) on human pol γ
activity and on mitochondrial toxicity (e.g., lactic acid production, mitochondrial DNA content,
mitochondrial morphology, glucose utilization).
D. 	In Vitro Combination Activity Analysis
Within an infected individual, viruses can exist as a heterogeneous population of variant viruses,
some of which may show reduced susceptibility to one or more antiviral products. Therefore, for
some viruses, administration of multiple antiviral products (e.g., three-drug combination
antiretroviral therapy against HIV-1) can be more effective than a single product in establishing
and maintaining inhibition of virus replication. However, the interactions of products are
complex and can result in antagonistic, additive, or synergistic effects with respect to antiviral
activity. For this reason, sponsors should evaluate the in vitro antiviral activity of investigational
products in two-drug combinations with other products approved for the same indication.
Specifically, combinations that should be tested include the investigational product with all
approved products that target the same protein and at least two appropriate products from each
class of products approved for the same indication. We recommend completing the in vitro drug
combination activity studies of the investigational product with approved products before
initiation of clinical trials that will evaluate the efficacy of the investigational product in
combination with other antiviral products. Often patients are infected with two or more viral
diseases (e.g., HIV and HBV or HCV); therefore, we also recommend that the in vitro antiviral
activity of antiviral products used in co-infected patients for different indications be assessed in
in vitro combination activity studies.
E. 	Resistance
1. 	Selection of Resistant Virus in Vitro
This guidance focuses on resistance to antiviral products caused by mutations in viral genomes
that result in reduced phenotypic susceptibility to a given antiviral product. Resistance, as it is
used here, is not an absolute term, but relative. We recommend that the in vitro selection of
resistant viruses to the investigational product, the phenotypic and genotypic characterization of
resistant viruses, and cross-resistance analyses be examined before initiation of clinical studies in
patients infected with the particular virus. We understand that the resistance data generated in
vitro are not necessarily predictive of clinical resistance. However, in vitro resistance selection
studies are recommended to assess the potential barrier of a target virus to develop reduced
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susceptibility (i.e., resistance) to the investigational product and to aid in designing clinical
studies.7
Selection in cell culture of virus resistant to the investigational product can provide insight into
whether the genetic threshold for resistance development is high or low. A product with a low
genetic threshold may select for resistance with only one or two mutations. In contrast, a
product with a high genetic threshold may require multiple mutations to select for resistance.
Several factors specific to the investigational product and the target virus affect the development
of resistance (e.g., product concentration). The rate of appearance of mutant viruses depends on
the rate of replication of the virus, the number of virus genomes produced, the fidelity of the
replicative machinery, and host factors. Consideration of these factors can help in designing
tests to detect resistant virus in vitro. For example, when multiple mutations are required to
develop resistance to high concentrations of the investigational product, many cell culture
systems do not produce sufficient virus titers to select resistant virus. In these instances, serial
passage of the virus in cell culture under conditions of increasing concentrations of the
investigational product can lead to the isolation of resistant virus. Sponsors are encouraged to
assess the development of resistance in vitro over the concentration range spanning the
anticipated in vivo concentration. Selection of variants resistant to the investigational product
should be repeated more than once (e.g., with different strains of wild-type, with resistant strains,
under high and low selective pressure) to determine if the same or different patterns of resistance
mutations develop, and to assess the relationship of product concentration to the genetic barrier
to resistance.
If the targeted virus replicates in a cell culture system, two basic methods can be employed to
isolate viruses that have reduced susceptibility to the investigational product:
• 	A high initial virus inoculum is propagated for several passages at a fixed product
concentration, using multiple cultures to test different concentrations.
• 	A low initial virus inoculum is passaged in the presence of increasing product
concentrations starting near the EC50 value for the parental virus.
Virus production is monitored by determining the genotype and phenotype of isolates throughout
the selection process to detect the outgrowth of resistant viruses.
HCV resistance to investigational products can be examined using HCV replicon systems.
Methods that select HCV resistance using HCV replicon cells include the following:
• 	HCV replicon cells are cultured at low density in the presence of neomycin and a fixed
concentration of the investigational product using multiple cultures to test different
7 For an in-depth review of resistance issues relating to HIV-1, see the draft guidance for industry Role of HIV Drug
Resistance Testing in Antiretroviral Drug Development. When final, the guidance will represent the FDA’s current
thinking on this topic. For the most recent version of a guidance, check the CDER guidance Web page at
http://www.fda.gov/cder/guidance/index.htm.
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concentrations. The cells containing resistant replicons will form colonies that are then
expanded for genotypic and phenotypic characterization.
• 	HCV replicon cells are passaged in the presence of a fixed product concentration, but in
the absence of neomycin, using multiple cultures to test different concentrations. The
HCV replicon cells from each passage are harvested and stored for phenotypic and
genotypic characterization.
2. 	Genotypic Analysis
Genotypic analysis of resistant viruses selected in vitro determines the mutations that might
contribute to reduced susceptibility to the investigational product. Identifying resistance
mutations by DNA sequence analysis of the relevant portions of the virus genome can be useful
in predicting clinical outcomes and supporting the proposed mechanism of action of the
investigational product. Sponsors should determine the entire coding sequence of the gene for
the target protein and compare the pattern of mutations leading to resistance of the
investigational product with the pattern of mutations of other products in the same class. For
larger viruses (e.g., herpesviruses, poxviruses), the relevant portions of the viral gene targeted by
the investigational product should be sequenced and analyzed for mutations that could contribute
to product resistance (e.g., marker rescue). We recommend that resistance pathways be
characterized in several genetic backgrounds (i.e., strains, subtypes, genotypes) and that isolates
be obtained throughout the selection process to identify the order in which multiple mutations
appear.
For genotypic assays, sponsors are encouraged to identify sequencing primers, state how many
bases can be read from the primer accurately, and define the sensitivity of the genotypic assay
used for detecting minority viral subpopulations. It is important that sponsors define what
percentage of the population a mutation has to represent to be detected in their genotypic assay.
3. 	Phenotypic Analysis
Phenotypic analysis determines if mutant viruses have reduced susceptibility to the
investigational product. When mutations that may be associated with resistance are identified by
genotypic analysis, the ability of each of these mutations to confer phenotypic resistance should
be evaluated in a recombinant virus system if possible (e.g., by using site-directed mutagenesis,
PCR amplification of relevant portions of virus genome to introduce these mutations into a
standard laboratory genetic background, or other suitable system). Recombinant viruses can
then be tested in vitro for susceptibility to the product to determine an EC50 value. The fold
resistant change should be calculated as the EC50 value of the isolate/EC50 value of the reference
or parental strain. Phenotypic results can be determined with any standard virus assay (e.g.,
protein assay, viral RNA assay, polymerase assay, MTT cytotoxic assay, reporter gene
expression). The shift in susceptibility (or fold resistant change) for a viral isolate should be
measured by determining the EC50 values for the isolate and comparing it to the EC50 value of a
reference (well-characterized wild-type laboratory strain) or parental virus done under the same
conditions and at the same time. The use of the EC50 value for determining shifts in
susceptibility is preferred because it can be determined with greater precision than an EC90 or
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EC95 value. The utility of a phenotypic assay depends on its sensitivity (i.e., its ability to
measure shifts in susceptibility (fold resistance change) in comparison to reference, parental
strains, or baseline clinical isolates). Calculating the fold resistant change (EC50 value of
isolate/EC50 value of reference strain) allows for comparisons among phenotypic assays.
4. 	Cross-Resistance
Antiviral products targeting the same protein (typically products of the same drug class) may
develop mutations that lead to reduced susceptibility to one antiviral product and can result in
decreased or loss of susceptibility to other antiviral products in the same drug class. This
observation is referred to as cross-resistance. Cross-resistance is not necessarily reciprocal, so it
is important to evaluate both possibilities. For example, if virus X is resistant to drug A and drug
B, and virus Y is also resistant to drug A, virus Y may still be sensitive to drug B. We
recommend that the effectiveness of the investigational product against viruses resistant to other
approved products in the same drug class and the effectiveness of approved products against
viruses resistant to the investigational product be evaluated by phenotypic analyses.
Additionally, we recommend that cross-resistance be analyzed between drug classes in instances
where more than one drug class targets a single protein or protein complex (e.g., nucleoside
reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors
(NNRTIs), which both target the HIV-encoded reverse transcriptase). We suggest that multiple
recombinant and clinical isolates representative of the breadth of diverse mutations and
combinations of mutations known to confer reduced susceptibility to products in the same drug
class be tested for phenotypic susceptibility to the investigational product. If phenotyping is
performed in cell lines with recombinant viruses, the EC50 value should be validated with
clinical isolates.
IV. 	PROPOSAL FOR MONITORING RESISTANCE DEVELOPMENT
It is important to disseminate information about an antiviral product’s resistance profile to health
care professionals and patients so that they can make optimal treatment decisions. Furthermore,
crucial decisions in protocol design and product development plans often hinge on resistance and
cross-resistance data. Therefore, it is strongly recommended that comprehensive resistance
testing be undertaken during all phases of product development consistent with the way the
product will be used in clinical practice.
For some viruses, measurements of changes in viral concentrations are accepted endpoints to
determine clinical effectiveness of antiviral products. In these cases, assays that measure and
monitor viral load can be used. Well-characterized genotypic and phenotypic assays provide the
basis for examination of the emergence of resistant virus to investigational products and attempt
to show a relationship between viral resistance and clinical virologic failure. In addition,
phenotypic and genotypic results are used to define treatment options and predict the utility of
treating an individual with an investigational product. Genotypic analysis of viral isolates from
patients failing to respond to therapy or undergoing viral rebound can help identify mutations
that contribute to reduced susceptibility to the investigational product. In addition, we
recommend genotypic and phenotypic analyses of baseline isolates be used to determine
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response to treatment outcomes based on baseline mutations and polymorphisms and baseline
phenotypic drug susceptibilities. In cases where measurements of viral concentrations have not
been established as principal endpoints, development of assays and monitoring of resistance
emergence will be important for exploratory analyses of relationships between virologic
measurements and clinical outcomes and can assist in refining designs of future studies.
We recommend that sponsors develop and submit a plan for monitoring the development of
resistant viruses in clinical studies before the initiation of clinical studies in virus-infected
individuals. A resistance monitoring plan should include, but not be limited to:
• 	A description of the assays that will be used to monitor viral loads
• 	Viral load assay protocols and performance characteristics (if required)
• 	The genotypic and phenotypic assays that will be used
• 	Genotypic and phenotypic assay protocols and performance characteristics (if required)
• 	The methods for sample collection and storage
• 	The methods for sample handling and shipping (frozen or ambient)
• 	A description of additional resistance analyses
• 	Time points when samples for viral loads, genotypic and phenotypic assays, and other
resistance analyses will be collected (i.e., baseline, week 24, week 48, following regimen
failure or discontinuation)
The resistance monitoring plan should be included with the overall clinical development plan in
the IND.
We also recommend developing and submitting with the IND plans for genotypic and
phenotypic baseline studies and resistance substudies early in product development. We suggest
that genotypic and phenotypic analyses of baseline and post-treatment isolates be completed in a
timely manner to characterize the resistance profile of the investigational product and its cross-
resistance potential with other antiviral products. For viruses such as HBV and HCV, baseline
and post-treatment genotypic analyses are key to monitoring the development of genotypic
resistance. Comparing genotypic analyses of samples from patients exhibiting virologic
breakthrough (or rebound) with baseline samples can identify mutations associated with
resistance. The extent and type of resistance monitoring and analysis should be discussed and
agreed to with the division in advance.
Generally, in studies of treatment-experienced patients, sponsors are strongly encouraged to
collect phenotypic and genotypic data for baseline isolates from all patients and endpoint isolates
from all virologic failures and discontinuations (not suppressed). Virologic failure and
discontinuation samples should be collected when the patient is still on the study product. In
studies of treatment-naïve patients, phenotypic and genotypic data for baseline and endpoint
isolates from all virologic failures and discontinuations (not suppressed) should be obtained.
Therefore, in treatment-naïve studies, a baseline sample should be collected and stored from all
patients for potential phenotypic and genotypic analysis of virologic failures. Additional
genotypic and phenotypic assessments and subset analyses may be appropriate depending on the
clinical study protocol or population, thus re-emphasizing the need to collect and store baseline
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samples and treatment samples throughout studies. In select cases, sponsors may propose to
collect samples at baseline and at the time of failure on a subset of treatment-naïve patients when
in vitro data indicate that acquisition of a single mutation results in a high degree of phenotypic
resistance. However, any such proposal should be discussed with the division in advance.
Virologic failures and discontinuation definitions are protocol defined. Sponsors with
investigational products can solicit advice from the division early in product development on
definitions of clinical response-failure and plans to monitor resistance in clinical studies. We
recommend that sponsors consult with the division for detailed descriptions and examples of
how to submit clinical viral resistance data. In conjunction with this guidance, we are providing
stand-alone documents to aid sponsors in submitting resistance data for HIV, HBV, HCV, and
influenza studies. These sample formats may be updated periodically and additional formats for
other viruses added as needed.
Sponsors can choose to quantify viral loads and conduct phenotypic and genotypic analyses
themselves or send samples to companies that have been certified by Clinical Laboratory
Improvement Amendments (CLIA). Proper handling procedures should be followed for
laboratory samples. If an assay is not performed according to the manufacturer’s specifications,
the assay results might not be acceptable. Sponsors are encouraged to use approved assays (if
possible) that are characterized and validated. If the assay is investigational, we recommend that
sponsors provide the performance characteristics of the assay (e.g., accuracy, precision, limits of
detection and quantification, specificity, linearity, range, robustness, stability), as well as sources
of viruses (e.g., blood, plasma), their storage and stability, and cell culture procedures. For
definitions on assay validation, refer to the guidance for industry Bioanalytical Method
Validation, the guidance for industry Antiretroviral Drugs Using Plasma HIV RNA
Measurements — Clinical Considerations for Accelerated and Traditional Approval, and the
ICH guideline for industry Q2A Text on Validation of Analytical Procedures. Assays that will
be used in clinical practice should have more extensive validation than exploratory assays that
are used for the characterization of antiviral activity and resistance of investigational products.
Commercially available assays that are routinely used should be identified, but it may not be
necessary to provide the performance characteristics. The amount and nature of validation
necessary for an assay and mechanisms of submitting assay performance characteristics (e.g.,
Data Master File) should be discussed with the division. We recommend that sponsors
consistently use the same assay for any particular analysis or measurement in phase 3 studies and
that the same assay be used for a particular patient throughout the study. Sponsors should
provide data supporting the implementation and use of any new assays that become available
during product development.
V. 	VIROLOGY STUDY REPORTS
Complete virology study reports can be extensive and should include the primary data and
derived data, the procedures used to obtain the data, and information necessary to evaluate the
data. Virology study reports should contain information on nonclinical studies, clinical antiviral
activity of the investigational product, resistance development to the investigational product in
treated patients, cross-resistance with other products in the same drug class, and baseline
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genotypic and phenotypic virologic response analyses (if applicable). The formats of virology
study reports should be similar to that of a scientific publication and typically should include the
following sections: summary, introduction, materials and methods, results, and discussion. The
methods section should describe all the protocols employed and include a description of the
statistical analyses used. Virology study reports on nonclinical and clinical studies can be
submitted upon their completion and need not be held until submission of the NDA. If the NDA
is submitted in the common technical document (CTD) format, sponsors should submit virology
study reports and datasets in Module 5, Section 5.3.5.4, Other Studies, under the specific
heading, Antiviral Reports.
VI. 	SUMMARY
This guidance identifies virology studies relevant to the development and application review of
antiviral products for the treatment of viral infections. The goal of this guidance is to stimulate
the generation of more complete analyses for antiviral products. Such analyses help provide data
that support the introduction of an investigational product into humans and provide the data
necessary for determining dose-response relationships, designing clinical trials, and selecting
appropriate patient populations. Thus, the data collected during these studies can affect the
therapeutic success of a given product.
Because nonclinical in vitro virology studies can provide useful information for the design of in
vivo studies and can help predict the development of resistant viruses in vivo, we recommend
conducting nonclinical studies before the initiation of phase 1 clinical studies. In vitro selected
resistant viruses should be analyzed carefully before initiation of studies in patients infected with
a particular virus. This guidance includes recommendations for how and when to perform
virology studies. Such information could be included in product labeling to facilitate appropriate
prescribing of antiviral products and maximize the chance for therapeutic success. To assist
sponsors in providing data from clinical resistance studies, we have developed stand-alone
documents that accompany this guidance and provide a format for submitting resistance data to
the Agency.
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