An Equal Opportunity Provider and Employer

VETERINARY SERVICES MEMORANDUM 800.202

TO: Veterinary Services Leadership Team

Directors, Center for Veterinary Biologics

Biologics Licensees, Permittees, and Applicants

FROM: Jack Shere

Deputy Administrator

SUBJECT: General Licensing Considerations: Efficacy Studies for Prophylactic 

 and Therapeutic Biologics

I. PURPOSE

General licensing considerations provide guidance to licensees, permittees, and 

applicants concerning the submission of documents to support an application for a U.S. 

Veterinary Biological Product License or U.S. Veterinary Biological Product Permit for 

Distribution and Sale, according to title 9, Code of Federal Regulations (9 CFR), parts 

102.5 and 104.5. This memorandum addresses basic principles for conducting efficacy 

studies for prophylactic and therapeutic biological products.

II. REPLACEMENT

This memorandum replaces Veterinary Services (VS) Memorandum No. 800.202 dated 

October 27, 2014.

III. BACKGROUND

Veterinary biological products work via immunological mechanisms to prevent, treat, or 

diagnose animal disease. This memorandum addresses prophylactic and therapeutic 

products. Prophylactic products, intended to prevent or control the occurrence of disease, 

include vaccines, bacterins, and toxoids. Therapeutic products, such as antitoxins, 

hyperimmune serum, or immunostimulants, are intended to treat existing conditions.

Efficacy is the capacity to produce a desired or intended result. The ability of a 

biological product to perform as indicated on product labeling must be adequately 

demonstrated prior to issuance of a USDA license or permit.

IV. GUIDELINES

“General Licensing Considerations: Efficacy Studies for Prophylactic and Therapeutic 

Biologicals” is appended to this memorandum.

V. IMPLEMENTATION/APPLICABILITY

This guidance is applicable upon publication of this memo.

Animal and Plant

Health Inspection

Service

Veterinary Services

1400 Independence 

Ave, SW

Washington, DC

20250
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General Licensing Considerations

Efficacy Studies for Prophylactic and Therapeutic Biologicals

1. Introduction

1.1 Products. This document applies to prophylactic products, herein called vaccines, and 

therapeutic products. Diagnostic products are discussed in Veterinary Services (VS) 

Memorandum No. 800.73.

1.2 Efficacy. Efficacy is the direct effect of a medical intervention on an individual subject.

The effect of an intervention program in the population is often termed effectiveness. The 

concept of effectiveness includes both direct effects and the indirect effects of the 

intervention at the herd or population level. Herd immunity, which protects 

nonvaccinated individuals in a group by reducing disease transmission through the 

vaccinated individuals, is an example of an indirect effect. Vaccine efficacy may be 

isolated from effectiveness by design or analysis.

1.3 Design. The preferred design for animal vaccine efficacy studies is the prospective, 

placebo-controlled, randomized, and double-blinded vaccination-challenge trial. In such 

studies, each subject receives the same exposure to the virulent pathogen by active 

challenge. By design, challenge studies aim to isolate the direct effect of the vaccine on 

individual subjects. Other types of studies, such as those relying on natural exposure, may 

be considered where warranted. Immunotherapeutic trials should aim to compare the 

responses of product- and placebo-treated subjects that have the existing condition.

Deviations from these design features should be noted and justified in the study protocol.

1.4 Protocols. Applicants are strongly encouraged to submit detailed efficacy study protocols 

to the Center for Veterinary Biologics (CVB) for review and comment at least 60 days 

prior to the initiation of a study. The CVB may arrange with the applicant to observe the 

study at selected times. If protocols are highly similar to previously reviewed studies, it is 

helpful to cite the prior protocol and list the differences.

Succeeding sections of this document list important considerations for the materials, 

methods, and criteria of efficacy studies. Protocols of proposed efficacy studies and final 

reports should address these considerations in addition to those noted in the General 

Licensing Considerations for Study Practices and Documentation (see VS Memorandum 

No. 800.200).

2. Materials

2.1 Experimental product. The applicant is responsible for establishing the validity of the 

experimental product used to demonstrate efficacy. The experimental product must 

accurately represent the product that will be produced once a product license is granted.

Describe in detail its composition, including antigenic mass. Give its potency and state 
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the potency test method. Provide the passage level from Master Seed and Master Cell 

Stock from which each antigen lot in the product was produced.

Prepare the experimental product:

2.1.1 In accordance with the filed Outline of Production. Cite the applicable Outline 

approval date in the study report. If an Outline has not yet been filed at the time the 

experimental product is made for an efficacy study, or if changes in manufacturing 

have occurred since the last Outline was filed, ensure that the study report contains 

sufficient detail on the manufacture of the experimental product to support a 

subsequently filed Outline.

2.1.2 In licensed production facilities in accordance with filed facility documents. If 

prepared in research facilities, establish that the experimental product fully 

represents the product that will be prepared in production facilities. Validation of 

production may be required after scale-up.

2.1.3 At or below minimum potency provided in the Outline of Production for the

antigen under study and any other product antigens that may provide crossprotection (e.g., bovine viral diarrhea virus (BVDV) type 1 when demonstrating

efficacy against BVDV type 2).

The potency of the efficacy serial is used in determining the specifications for the

potency of post-license serials at release into the marketplace and throughout

dating.

2.1.4 At the highest passage from the Master Seed and Master Cell Stock allowed by the 

Outline of Production. Generally, the 5th passage from the Master Seed and the 

20th passage from the Master Cell Stock are the highest allowed unless a higher 

passage is justified.

2.2 Placebo. State the composition of the placebo or active control treatment. Placebos may

be “product matched” to contain all constituents of a vaccine except the antigen of 

interest and cross-reacting antigens (preferred), or they may be immunologically inert 

(e.g., normal saline). Justify the type of placebo or active control selected.

2.3 Challenge. State the source, composition, and quantity/concentration of the challenge 

material. In general, the challenge material must be a single pathogenic agent. In those 

rare cases where co-challenge with multiple agents may be scientifically justified, contact 

the CVB for further guidance.
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3. Methods

3.1 Subjects.

3.1.1 Signalment. Specify the age, breed, sex, source, and other distinguishing 

features of the animals to be used in the study. Describe how they represent 

the target population in which the product is likely to be used after licensure.

3.1.2 Enrollment criteria. List the inclusion/exclusion criteria for enrollment into 

the study. Generally, subjects should be immunologically naïve to the agent 

under study; exceptions must be justified. For therapeutic trials, it may be 

prudent to consider the stage/severity of the existing disease and any prior 

treatments.

3.1.3 Removal criteria. Once enrolled, animals should be removed from the study 

only with ample justification. If certain scenarios are a material possibility 

(e.g., removing an injured animal for humane euthanasia), describe removal 

procedures.

3.1.4 Identification. Unless otherwise justified, identify each animal uniquely.

Individual animal identification typically is not required for fish or large-scale 

poultry studies where observations are summarized at the level of the 

experimental unit, which is typically the housing unit or tank.

3.1.5 Environment. State how the subjects will be grouped, housed, and managed in 

each phase of the study. A floor or site plan often facilitates regulatory review 

of protocols and reports. Specify whether different treatment groups are in 

contact or separated.

3.1.6 Auxiliary procedures. Indicate any concomitant treatments or procedures to be 

done during the study. Where it is plausible that these procedures might bias 

study conclusions, justify their use.

3.2 Group assignment and treatment allocation.

3.2.1 Experimental unit. Identify the experimental unit. The experimental unit is the 

individual animal or the smallest group of animals that may be randomly 

assigned to a distinct treatment, where treatment means a unique set of applied 

conditions (e.g., vaccination) and environmental conditions. Indicate any 

clustering or grouping of units.

Housing factors may impact the designation of the experimental unit for 

statistical analysis. For example, if it is necessary to house vaccine and 

placebo groups separately rather than commingle all treatment groups, the 

housing unit becomes the experimental unit. It then becomes necessary to 

include replicate housing units for each treatment group.
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Include enough units for the study to produce sufficiently precise estimates of 

efficacy. Although not required, sample size calculations based on

information from pilot studies and other sources may help in planning the 

study. 

3.2.2 Group assignment. Describe the randomization structure and method of 

randomly assigning subjects to treatment groups. Design the study so that 

randomization takes into account features affecting the independence of 

observations or the confounding of effects. For example, blocking by subject 

characteristics such as antibody titer, age, weight, litter, or parity may be 

important. If so, include the blocking plan. Or, if subjects are naturally 

clustered, indicate whether treatment allocation or sample selection is within 

or between clusters.

3.2.3 Group treatment allocation. Efficacy studies usually include at least one 

group treated with the experimental product and one treated with a placebo. If, 

instead of a placebo, the control group is to be given an alternative active 

treatment or left entirely untreated, explain and justify this in the protocol.

Additional groups may be needed for other treatment regimens/doses or for 

non-treated sentinels. Clearly outline any nonparallel group configurations, 

such as factorial or cross-over type designs. If cohorts of animals are enrolled 

in stages, ensure that each treatment group is equitably represented in each 

stage.

For products intended to protect neonates by maternal antibodies from 

vaccinated dams, studies should be designed according to the proposed 

vaccination regimen.

3.2.3.1 If the recommended regimen is to vaccinate the dam for the passive 

immunization of her offspring, treatment groups should be comprised 

of adults whose responses are measured by the immunity of their 

offspring.

3.2.3.2 If the recommended regimen is to vaccinate both the dam and her 

offspring for the protection of the offspring, treatment groups should 

be comprised of adult-neonate units.

3.2.3.3 If there is more than one recommended vaccination regimen, the 

efficacy of each recommended regimen must be supported by 

appropriate treatment groups. Studies of products recommended for 

both passive and active immunization should include separate groups 

of passively and actively immunized subjects.
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3.2.4 Group proportions. Prior information may suggest the proportions for 

dividing subjects between treated and control groups to optimize the study’s 

efficiency and minimize the total number of subjects. In the absence of such 

information or codified requirements, equally sized groups are recommended.

3.3 Treatment and challenge.

3.3.1 Vaccine trials. Describe the vaccination regimen and challenge method. If the 

proposed design does not include experimental challenge, but relies on natural 

exposure or other factors, explain the reason for the design.

3.3.2 Immunotherapeutic trials. Describe the existing disease condition and 

treatment regimen.

3.4 Observations.

3.4.1 Observation times. State the timing and frequency of observations. The 

overall duration of the observation period should be sufficient to monitor all 

relevant events occurring after challenge. This period is typically based on the 

expected incubation period of the disease agent and the expected duration of 

clinical disease. The presence of clinical signs of disease at the end of the 

planned observation period may indicate the planned observation period is not 

long enough to assess the study objectives properly. All reports describing 

studies in which post-challenge observations are terminated before relevant 

disease signs are resolved should include a justification why continued 

observation would not have materially affected the conclusions drawn from 

the study.

3.4.2 Blinding. Individuals performing clinical or postmortem observations or 

laboratory analyses should be blinded (masked), so that the subject’s status in 

the study is unknown. Blinding should include at least the following two 

levels, or the protocol should justify their absence.

3.4.2.1 Masked treatment allocation. The observer does not know which 

treatment a group has received.

3.4.2.2 Masked group membership. The observer does not know the group to 

which a subject is assigned.

3.5 Verification of challenge. Provide adequate evidence that the observed disease was 

due to the challenge agent. This is of particular importance when the study measures 

clinical signs or lesions that are not pathognomonic for a particular agent.

3.6 Adverse events. Record and report all adverse events occurring during the study 

whether or not they are considered related to vaccination, treatment, or challenge. See 
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VS Memorandum No. 800.204 for additional guidance on classifying and reporting 

adverse events.

3.7 Laboratory procedures. Describe procedures for all laboratory analyses. Where 

laboratory testing outcomes are fundamentally involved in the definition of the 

primary outcome, validation reports for individual laboratory methods should be 

provided before conducting the study. Methods conducted in institutional laboratories 

accredited by the American Association of Veterinary Diagnostic Laboratories or 

other recognized quality assurance standards may be acceptable without submitting 

direct proof of validation.

3.8 Clinical outcomes. An outcome is an observation on an individual subject that 

includes the clinical event and the unit of measurement.

3.8.1 Outcome specification. Define outcomes in accordance with VS 

Memorandum No. 800.200, appendix section 2.3.6.7. Outcomes may be 

specified in terms of a case definition, severity categorization, or a natural 

scale of measurement.

3.8.1.1 Case definition. A case definition is the outcome used to study 

disease prevention. Subjects meeting the case definition are 

considered positive, whereas others are negative. Disease case 

definitions should be explicit and have a natural clinical interpretation 

as representing a case of the disease. An arbitrary dichotomization of 

disease severity is not a case definition.

3.8.1.2 Severity categorization. Severity categories are outcomes used to 

study reductions in disease severity. Ideally each category should 

reflect a discrete progressive disease state. Use only those categories 

that reflect sharp distinctions in severity. Excessive categories often 

undermine clinical relevance and statistical validity.

Example: The categorization dead/sick/well includes three undeniably 

discrete states. If the sick category is further subdivided into, for 

example, mild and severe disease, they may still be discrete if mild 

disease includes transient low-grade clinical signs and severe disease 

reflects serious morbidity.

3.8.1.3 Natural scales of measurement. Outcomes may be based on a 

measured quantity. The scale of measurement may be discrete, such 

as the number of days elapsed between the first and last positive 

disease observation, or relatively continuous, such as body 

temperature. Select natural scales of measurement, clearly relevant to 

clinical disease. Avoid contrived, complex scoring systems that are 

difficult to analyze and interpret because they incorporate many 

disparate types of observations.
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3.8.2 Primary outcome. Specify a single (primary) outcome on which the 

conclusion criterion for each clinical effect will be based. (For example, a 

study might be designed to study both disease prevention and pathogen 

shedding, two different types of clinical effects.) The primary outcome should 

provide the most relevant evidence directly supporting clinical efficacy. If 

clinically relevant, the primary outcome may be designed as a composite of 

more than one type of observation.

Serological responses are not usually sufficient for establishing efficacy. An 

efficacy claim based on serological data alone will be considered only when 

there is a substantial scientific basis for considering the serological test to be 

indicative of disease protection. Protocols proposing serologic outcomes, 

along with scientific documentation supporting these outcomes, should be 

submitted for review and comment by the CVB prior to initiating the study.

3.9 Conclusion criterion. State the criterion for concluding whether the study findings 

support efficacy. Conclusion criteria should be based on the size and precision of 

estimated treatment effects and their clinical relevance. Where appropriate, state the 

intended comparison of primary outcomes between treatment groups, such as by an 

estimated difference or ratio.

Study statisticians may use any scientifically sound method of comparison, but the 

two most commonly used by the CVB when evaluating submitted studies are 

prevented fraction and mitigated fraction.

3.9.1 Prevented fraction. The prevented fraction is the complement of the risk ratio 

(1 - p2/p1), where p2 is the affected fraction in the experimental product group 

and p1 is the affected fraction in the placebo group. The precision of the 

estimate is evaluated by determining the 95% confidence interval.

3.9.2 Mitigated fraction. To evaluate products that reduce the severity of disease, 

vaccine efficacy is frequently assessed by mitigated fraction (Siev, D. 2005. J 

Mod App Stat Meth, 4:500-508). Note that mitigated fraction estimates the 

probability vaccinated animals that will be less severely affected than control 

animals. Alone, however, mitigated fraction does not provide a measure of 

clinical relevance, as it does not indicate how great the expected reduction in 

severity may be. Estimates of mitigated fraction should always be 

accompanied by an estimate, on the original scale of measurement (e.g., 

duration of viremia in days), of the magnitude of the vaccine effect (e.g., the 

difference in days between treatment groups).

3.10 Data analysis. Describe the proposed method of data analysis and indicate how it is 

appropriate to the study design and nature of the data.
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3.11 Submit all data. Submit and summarize all data from each study. Submit the results 

of every study involving a product proposed for USDA licensure. This applies 

whether the results were satisfactory, the study was repeated, or the study was 

exploratory.

4. Effectiveness Indications Statements

4.1 Label claims. As of 2016, labeling for most prophylactic and some therapeutic 

products will transition to the same generic Indications statement: “This product has 

been shown to be effective for the vaccination of healthy (insert name of species) ___ 

weeks of age or older against ___.” (9 CFR 112.2(a)(5)). Users are then directed to 

productdata.aphis.usda.gov for summaries of specific studies conducted to support 

product licensure.

 

4.1.1 The data must demonstrate that the product provides a statistically significant, 

clinically relevant level of efficacy. The product may prevent clinical disease 

or reduce its clinical severity.

4.1.2 Studies showing that a vaccine merely delays the onset of disease after 

exposure to a disease agent are typically not suitable to support product 

licensure.

4.1.3 Products with additional beneficial effects other than direct disease control, 

such as the control of infectiousness through the prevention of pathogen 

shedding or a reduction in its duration, may be eligible for licensure if the size 

of the effect is clinically relevant and statistically significant. Reductions in 

the magnitude of pathogen shedding are typically not sufficient to support 

product efficacy when vaccinates still shed in appreciable amounts.

4.1.4 Prior to 2016, there were 4 tiered effectiveness claim levels. See the CVB 

website for historical details. Products licensed prior to 2016 may still have 

tiered claims on product labeling during a transition period of approximately 4 

years.

4.2 Disease form. When a microorganism is associated with more than one clinical form 

of disease, limit claims to the disease form(s) for which efficacy has been 

demonstrated, such as “respiratory form” or “reproductive form.” Use specific disease 

or syndrome names whenever applicable.

4.3 Administration regimen. Establish efficacy separately for each route (e.g.,

intramuscular, subcutaneous, intranasal, in ovo) and regimen (e.g., age and 

frequency) of administration recommended on the label.

4.4 Animal Species. Establish efficacy in each non-fish species for which the product is 

recommended. When products are recommended for multiple species of fish raised at 
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the same water temperature, demonstrate efficacy in two of the recommended 

species.

4.5 Age and susceptibility. Conduct efficacy studies in fully susceptible animals of the 

youngest age for which the product is to be recommended. (Weight may be used in 

lieu of age for aquatic species.) Minimize age variations in juvenile study subjects, as 

the designated age of the study population as a whole is based on its oldest members.

The age of the study population in efficacy studies, along with the youngest animals 

used in field safety studies (see VS Memorandum No. 800.204), determines the 

minimum age in which the product may be labeled for use. If the animal age differs 

between efficacy and safety studies, the older age applies.

If interfering levels of maternal antibody may still be present at the youngest 

recommended age, do one of the following:

4.6.1 Provide data to demonstrate efficacy of the product in the face of expected 

levels of maternal antibody, or

4.6.2 Indicate on the labeling that the product is for the vaccination of susceptible 

animals of the minimum age used in the efficacy study and indicate that the 

effect of maternal antibody on product efficacy has not been specifically 

studied.

4.6 Onset of immunity. Support with acceptable data any specific claims concerning onset 

of immunity. The onset of immunity is defined as the interval between final 

vaccination and challenge in the supporting efficacy study.

4.7 Duration of immunity. Support with acceptable studies any explicit claims for the 

duration of immunity (DOI) for any product fraction. Studies to support minimum 

DOI also may be required to support the recommended revaccination interval after 

the primary vaccination series, even when specific DOI claims are not made. This is 

determined on an agent-by-agent basis, but in general includes all “newer” agents 

(i.e., those for which biologics were first developed after ca. 2000), agents known to 

induce short-lasting immunity (e.g., influenza), and rabies virus. Data are also 

required when a revaccination interval greater than a year is proposed.

Conclusions drawn from DOI studies should be as robust as short-term efficacy 

studies, although it is understood that disease due to certain pathogens may be milder

in animals exposed at an older age. The following considerations apply:

4.7.1 To prevent the confounding effect of immunity boosted by natural exposure to 

an agent after vaccination, implement the appropriate biosecurity to protect all 

subjects from inadvertent natural exposure in the extended interval prior to 

experimental challenge. Monitor study animals for exposure at regular 

intervals between vaccination and challenge.
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4.7.2 Ideally, formulate the experimental product for DOI studies to the same 

potency used in previously approved short-term efficacy studies. If DOI is 

demonstrated using a product with greater potency, the DOI potency will be 

used as the minimum immunogenic dose for the potency specification for 

serial release.

4.8 Product lines. When there is a group of products, manufactured identically in 

composition and potency except for the total number of microbial fractions, efficacy 

is typically demonstrated with the product containing the greatest number of 

fractions. The results then may be extrapolated to the remaining products (i.e., 

breakouts/fallouts) having a subset of fractions found in the highest combination 

product. Extrapolation of conclusions to breakouts may not apply if the test product 

has a potentially immunopotentiating (e.g., gram-negative bacteria) or cross-reactive 

fraction not found in the breakout product. Extrapolations may not be made to 

products containing more fractions than the tested product because of the potential for 

immunological interference by the non-tested fractions; see VS Memorandum No. 

800.203 for further guidance.