Guidance for Industry 

Clinical Data Needed to Support the 

Licensure of Trivalent Inactivated 

Influenza Vaccines 

DRAFT GUIDANCE 

This guidance document is for comment purposes only. 

Submit comments on this draft guidance by the date provided in the Federal Register notice 

announcing the availability of the draft guidance. Submit written comments to the Division of 

Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, 

Rockville, MD 20852. Submit electronic comments to http://www.fda.gov/dockets/ecomments. 

You should identify all comments with the docket number listed in the notice of availability that 

publishes in the Federal Register. 

Additional copies of this draft guidance are available from the Office of Communication, 

Training and Manufacturers Assistance (HFM-40), 1401 Rockville Pike, Suite 200N, Rockville, 

MD 20852-1448, or by calling 1-800-835-4709 or 301-827-1800, or from the Internet at 

http://www.fda.gov/cber/guidelines.htm. 

For questions on the content of this guidance, contact Antonia Geber, M.D., or Donna Chandler, 

Ph.D., at 301-827-3070. 

U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Biologics Evaluation and Research 

March 2006 
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Table of Contents 

I. INTRODUCTION............................................................................................................. 1

II. BACKGROUND ............................................................................................................... 2

III. CLINICAL DATA TO SUPPORT THE LICENSURE OF TRIVALENT 

INACTIVATED INFLUENZA VACCINES.................................................................. 3

A. Traditional Approval of a BLA for a New Trivalent Inactivated Influenza 

Vaccine ................................................................................................................... 3

1. Effectiveness ............................................................................................... 3 

2. Additional Studies to Support the Effectiveness of the Vaccine in 

Populations Not Included in the Clinical Efficacy Study ........................... 4 

3. Safety .......................................................................................................... 5 

B. Accelerated Approval of a BLA for a New Trivalent Inactivated Influenza 

Vaccine ................................................................................................................... 6

1. Effectiveness ............................................................................................... 7 

2. Safety .......................................................................................................... 9 

3. Post-marketing Confirmatory Studies......................................................... 9 

C. Additional Considerations.................................................................................... 9

1. Types of Influenza Vaccines....................................................................... 9 

2. Clinical Lot Consistency............................................................................. 9 

3. Adjuvanted Trivalent Inactivated Influenza Vaccines.............................. 10 

IV. REFERENCES................................................................................................................ 11

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Guidance for Industry 

Clinical Data Needed to Support the Licensure of Trivalent 

Inactivated Influenza Vaccines 

current thinking on this topic. 

does not operate to bind FDA or the public. 

If you want to 

If you cannot identify the 

This draft guidance, when finalized, will represent the Food and Drug Administration’s (FDA’s) 

It does not create or confer any rights for or on any person and 

 You can use an alternative approach if the 

approach satisfies the requirements of the applicable statutes and regulations. 

discuss an alternative approach, contact the appropriate FDA staff.

appropriate FDA staff, call the appropriate number listed on the title page of this guidance. 

I. INTRODUCTION 

This document is intended to provide to you, sponsors of trivalent inactivated influenza vaccines, 

guidance on clinical development approaches to support a Biologics License Application (BLA). 

Currently two classes of trivalent vaccines are licensed in the United States, “split virus” 

inactivated trivalent vaccines and a live attenuated trivalent vaccine. In this document we, FDA, 

summarize clinical development approaches to facilitate and expedite the licensure of new “split 

virus” trivalent inactivated influenza vaccines, and we address both traditional and accelerated 

approval. These approaches are also applicable to vaccines made with other manufacturing 

processes; e.g., whole virus inactivated, cell-culture derived inactivated, recombinant 

hemagglutinin-based protein, and adjuvanted influenza vaccines. This document does not 

address live attenuated influenza vaccines or influenza vaccines that do not contain a 

hemagglutinin component. 

This document does not address the nonclinical or early clinical development of investigational 

vaccines. Successful evaluations of nonclinical and early clinical development are important 

steps before proceeding with additional clinical development (Ref 1). This document also does 

not address the chemistry, manufacturing, control, or inspection of the manufacturing facility 

needed for licensure. These aspects of the license application are addressed in the guidance 

document entitled, “Guidance for Industry: Content and Format of Chemistry, Manufacturing, 

and Controls Information and Establishment Description Information for a Vaccine or Related 

Product” dated January 19991

 (64 Federal Register 518, January 5, 1999). Applicants may 

contact the Center for Biologics Evaluation and Research (CBER) for additional information 

about these aspects of vaccine development. 

1

 See http://www.fda.gov/cber/gdlns/cmcvacc.pdf. 

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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. 

The use of the word should in FDA’s guidances means that something is suggested or 

recommended, but not required. 

II. BACKGROUND 

Influenza viruses are enveloped ribonucleic acid viruses belonging to the family of 

Orthomyxoviridae and are divided into three distinct types on the basis of antigenic differences 

of internal structural proteins (Ref. 2). Two influenza types, Type A and B, are responsible for 

yearly epidemic outbreaks of respiratory illness in humans and are further classified based on the 

structure of two major external glycoproteins, hemagglutinin (HA) and neuraminidase (NA). 

Type B viruses, which are largely restricted to the human host, have a single HA and NA 

subtype. In contrast, 15 HA and 9 NA Type A influenza subtypes have been identified to date. 

Type A strains infect a wide variety of avian and mammalian species. 

Type A and B influenza variant strains emerge as a result of frequent antigenic change, 

principally from mutations in the HA and NA glycoproteins. These epidemic variants may result 

from one of two mechanisms. They may emerge as a result of selective point mutations in the 

viral genome (Refs. 3 and 4). Other epidemic variants may evolve from reassortment between 

two co-circulating strains (Refs. 5 and 6). 

Since 1977, influenza A viruses (subtype H1N1), influenza A viruses (subtype H3N2), and 

influenza B viruses have been in global circulation. The current U.S. licensed inactivated 

trivalent vaccines are formulated to prevent influenza illness caused by these influenza viruses. 

Because of the frequent emergence of new influenza variant strains, the antigenic composition of 

influenza vaccines needs to be evaluated yearly, and the trivalent inactivated influenza vaccines d

are reformulated almost every year. The immune response elicited by previous vaccination may 

no longer be protective against new variants. Annual vaccination is indicated for persons who 

wish to be protected from influenza illness and is recommended for people who are at high risk 

for complications of influenza illness (Ref. 7). 

The Centers for Disease Control and Prevention’s (CDC’s) Advisory Committee on 

Immunization Practices (ACIP) has expanded the recommendations for receipt of influenza 

vaccination to include additional at risk populations, such as pregnant women, persons 50 years 

of age and older, and children 6 to 23 months of age (Refs. 8, 9, and 10). The increased demand 

for influenza vaccines resulting from the broader recommendations, coupled with the withdrawal 

from the U.S. market by several influenza vaccine manufacturers over the same time period and 

the recurrent instances where some manufacturers were unable to provide influenza vaccines to 

the market due to manufacturing problems, has led to shortages or delays in the availability of 

influenza vaccine over the past several seasons. These shortages highlighted the need for 

increased availability of influenza vaccines from multiple manufacturers. In addition the 

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availability of adequate supplies of licensed trivalent inactivated influenza vaccines from 

multiple manufacturers will be of value in responding to the emergence of a new pandemic 

influenza strain. 

III. CLINICAL DATA TO SUPPORT THE LICENSURE OF TRIVALENT 

INACTIVATED INFLUENZA VACCINES 

Licensure of trivalent inactivated influenza vaccine may be sought through either traditional or 

accelerated pathways. This section provides recommendations for clinical data to support 

traditional and accelerated license approvals for new trivalent inactivated influenza vaccines. 

CBER has prepared similar draft guidance for pandemic influenza vaccines. For an opportunity 

to comment, please refer to CBER's draft guidance, “Guidance for Industry: Clinical Data 

Needed to Support the Licensure of Pandemic Influenza Vaccines” dated March 2006. 

A. Traditional Approval of a BLA for a New Trivalent Inactivated Influenza 

Vaccine 

Biological products are licensed under the authority of section 351 of the Public Health 

Service Act (PHS Act) (42 U.S.C. 262). Under section 351 of the PHS Act, BLAs are 

approved only upon a showing that the product is “safe, pure and potent,” and that the 

manufacturing facility meets standards designed to assure that the biological product 

“continues to be safe, pure, and potent.” In previously issued guidance entitled, 

“Guidance for Industry: Providing Clinical Evidence of Effectiveness for Human Drug 

and Biological Products” dated May 1998 (section II.A.), FDA stated, “Potency has long 

been interpreted to include effectiveness (21 CFR 600.3(s)). In 1972, FDA initiated a 

review of the safety and effectiveness of all previously licensed biologics. The Agency 

stated then that proof of effectiveness would consist of controlled clinical investigations 

as defined in the provision for ‘adequate and well-controlled studies’ for new drugs (21 

CFR 314.126), unless waived as not applicable to the biological product or essential to 

the validity of the study when an alternative method is adequate to substantiate 

effectiveness (21 CFR 601.25(d)(2)).” 

1. Effectiveness 

As discussed above, demonstration of effectiveness against influenza illness in an 

adequate and well-controlled clinical study would support licensure of a new trivalent 

inactivated influenza vaccine. In this document, a clinical endpoint efficacy study 

refers to a clinical trial in which influenza illness is assessed as the primary endpoint. 

The study design should take into account the following parameters: 

a. The study population should be carefully considered. A placebo-controlled 

clinical efficacy study conducted in a population that is not at increased risk for 

complications from influenza would allow for a precise estimation of clinical 

effectiveness against influenza illness (absolute efficacy). The ACIP usually lists, 

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at least annually, those persons who are at increased risk for influenza 

complications; we will rely on that list (Ref. 11). Alternatively, a population at 

increased risk for influenza illness complications may be studied, but an adequate 

sample size should be used to demonstrate non-inferiority of the new vaccine to a 

U.S. licensed product with regard to clinical effectiveness. 

b. The case definition for influenza illness should be prospectively defined. 

Inclusion of culture confirmation, viral typing and antigenic characterization in 

the case definition increases the specificity. The increased specificity allows for a 

more precise estimate of vaccine effectiveness and would likely reduce the 

sample size needed to assess effectiveness. Additionally, culture confirmation 

would facilitate interpretation of study results in the event that circulating 

influenza strains do not match antigen components contained within the vaccine. 

An analysis of whether the immune response elicited by the vaccine correlates 

with protection against influenza illness will depend upon the use of a specific 

case definition (e.g., culture confirmation of influenza). 

c. Study sample size calculations should be based on estimates of vaccine 

effectiveness and influenza attack rates. The study should be powered to assess 

the lower bound of the 95% confidence interval (CI) of vaccine effectiveness, 

anticipated to be substantially above zero (e.g., in the range of 40 to 45%). 

d. Immunogenicity evaluations in a substantial number of study participants are 

important elements of the study design. Characterization of the immune response 

elicited post-vaccination in the clinical endpoint efficacy study may allow for 

extrapolating the effectiveness to other populations if they have an immune 

response to vaccination comparable to that observed in the clinical endpoint 

efficacy study. Furthermore, immune response data collected in the course of a 

prospectively designed clinical endpoint efficacy study may lead to the 

establishment of an immune correlate of protection. Such a correlate could 

greatly facilitate future influenza vaccine development. 

2. Additional Studies to Support the Effectiveness of the Vaccine in Populations Not 

Included in the Clinical Efficacy Study 

Some populations who are at increased risk for complications from influenza 

vaccination (e.g., individuals 6 to 23 months of age and those 65 years of age and 

older) may not have been included in the clinical endpoint efficacy study because of 

the challenges in conducting a comparative efficacy study. Effectiveness studies in 

these populations can be based on appropriate immunogenicity endpoints. 

a. Immunogenicity bridging studies can be conducted to compare the immune 

response observed in the clinical endpoint efficacy study to that elicited in other 

populations. Appropriate endpoints may be the hemagglutination inhibition (HI) 

antibody responses to each of the three viral strains contained within the vaccine. 

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Studies should be adequately powered to assess the following co-primary 

endpoints: 1) geometric mean titer (GMT), and 2) rates of seroconversion, 

defined as a four-fold rise in antibody titer post-vaccination. (See 

recommendations for these six endpoints outlined in section III.B.1.a.). While 

this approach may expand the use of the new vaccine in additional populations, an 

important consideration is that immune responses in the very young and the 

elderly might be lower than those observed in healthy adults enrolled in a 

placebo-controlled clinical efficacy study. Additionally, changes to the annual 

formulation of the vaccine might complicate the design of such studies. 

Identification of an immune correlate of protection during the course of a clinical 

endpoint efficacy study may facilitate the design and interpretation of such 

bridging studies. 

b. Alternatively, non-inferiority immunogenicity studies comparing a new 

vaccine to a U.S. licensed vaccine may support the use of the new vaccine in 

populations not included in the clinical endpoint efficacy study. Studies should 

be adequately powered to assess the co-primary endpoints for HI antibodies to 

each viral strain contained in the vaccine: 1) GMT, and 2) seroconversion rates 

(as outlined in section III.B.1.a.). 

3. Safety 

The safety of the new vaccine should be well characterized in pre-licensure clinical 

trials. Local and systemic reactogenicity events should be well defined in all age 

groups for whom approval of the vaccine is sought. Appropriate grading scales to 

describe the severity of the adverse events should be included in the study protocol. 

Serious adverse events should be monitored and collected for all subjects throughout 

the duration of the studies. The protocol should include a clinic visit or telephone 

contact at least six months post-vaccination to ascertain additional serious adverse 

events and new onset of chronic illnesses that may have occurred in the interim. 

The total size of the safety database should depend, in part, on the range of the age 

indication being sought. It is anticipated that data will be collected in adults and in 

the pediatric population in a step-wise fashion. We assume that approval for use in 

the adult population, including the geriatric population, would be sought with the 

initial application. It is recommended that several thousand subjects receive the 

investigational vaccine in the controlled clinical trials described above, and 

assessment of safety will be available from these study participants. 

The timing of the clinical development and the size of the safety database to support 

use in the pediatric age groups warrants discussion with CBER. The amount of data 

needed for a particular manufacturer’s vaccine to support approval for use in the 

pediatric population should depend on available clinical data for that trivalent 

influenza vaccine. All sponsors have obligations to study pediatric populations as 

outlined in the Pediatric Research Equity Act of 2003. 

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B. Accelerated Approval of a BLA for a New Trivalent Inactivated Influenza 

Vaccine 

Accelerated approval may be granted for certain biological products that have been 

studied for their safety and effectiveness in treating serious or life-threatening illnesses 

and that provide meaningful therapeutic benefit over existing treatments. (See 

Accelerated Approval of Biological Products for Serious or Life-Threatening Illnesses 

(21 CFR Part 601 Subpart E)). 

Such an approval will be based on adequate and well-controlled clinical trials 

establishing that the product has an effect on a surrogate endpoint that is reasonably 

likely, based on epidemiologic, therapeutic, pathophysiologic, or other evidence, to 

predict clinical benefit (21 CFR 601.41). Approval under this section will be subject to 

the requirement that the sponsor study the biological product further, to verify and 

describe its clinical benefit, where there is uncertainty as to the relation of the surrogate 

endpoint to clinical benefit (21 CFR 601.41). Post-marketing studies must also be 

adequate and well-controlled and should be conducted with due diligence (21 CFR 

601.41). The protocols for these studies should be submitted with the original BLA. 

Marketing approval for products approved under these regulations may be withdrawn, for 

example, if the clinical study fails to verify the clinical benefit or the sponsor fails to 

perform the required post-marketing study with due diligence (21 CFR 601.43(a)(2)). 

The option to pursue an accelerated approval pathway for trivalent inactivated influenza 

vaccines is available to sponsors if a shortage of influenza vaccine exists for the U.S. 

market at the time the new vaccine is approved. We interpret the accelerated approval 

regulation, 21 CFR 601.40, as allowing accelerated approval of an influenza vaccine 

during a shortage because influenza is a serious and sometimes life-threatening illness. 

Providing prophylaxis to those who would not otherwise be immunized during a shortage 

does certainly provide a meaningful benefit over the then-existing treatments, which are 

in short supply at that time. We understand a shortage to exist when the supply of 

influenza vaccine is inadequate to immunize all persons for whom the CDC recommends 

annual vaccination. The CDC estimates that there are 185 million individuals in the 

United States for whom influenza vaccination is recommended annually (Ref. 12). 

For influenza vaccines, evaluation of an immune response elicited following receipt of 

the vaccine may serve as a surrogate endpoint that is likely to predict clinical benefit, that 

is, prevention of influenza illness and its complications. Influenza virus hemagglutinins, 

present on the viral surface, are important for cell-receptor binding. The immune 

response to the hemagglutinin as measured by the presence of serum HI antibodies is an 

important protective component following vaccination and/or infection. However, 

considerable variability can be introduced into the laboratory assay used to measure HI 

antibodies as a result of a number of factors including differences in viral strains, red 

blood cell types, and the presence of non-specific inhibitors in the assay medium. Thus, 

suitable controls and assay validation are important for interpreting HI antibody results. 

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To date, prospectively designed studies to evaluate the effectiveness of influenza 

vaccines have not identified a specific HI antibody titer associated with protection against 

culture confirmed influenza illness. Some studies of influenza infection, including 

human challenge studies following vaccination, have suggested that HI antibody titers 

ranging from 1:15 to 1:65 may be associated with protection from illness in 50% of 

subjects and protection from illness is increased with higher titers (Refs. 13 and 14). 

Seroconversion and GMT have been used as measures of vaccine activity (Refs. 15 and 

16). 

For the purposes of accelerated approval of trivalent inactivated influenza vaccines, the 

HI antibody response may be an acceptable surrogate marker of activity that is 

reasonably likely to predict clinical benefit. 

To be considered for accelerated approval, a BLA for a new trivalent inactivated 

influenza vaccine should include results from one or more well-controlled studies 

designed to meet immunogenicity endpoints and a commitment to conduct confirmatory 

post-marketing studies during the next influenza season. Since each vaccine candidate is 

unique (e.g., particular product characteristics, manufacturing process, etc.), we 

recommend that you discuss with CBER early in development the adequacy of the 

manufacturing methods and product testing and the extent of the clinical data needed to 

license your candidate vaccine. 

1. Effectiveness 

This section describes possible approaches for establishing effectiveness based on 

immune responses under an accelerated approval. 

a. A non-inferiority immunogenicity trial of HI antibody responses to the new 

vaccine as compared to a U.S. licensed trivalent inactivated influenza vaccine 

may support an accelerated approval. The study should be adequately powered to 

assess the co-primary endpoints for HI antibodies to each viral strain contained in 

the vaccine (i.e., a total of six co-primary endpoints): 1) GMT, and 2) 

seroconversion rates. Recommendations for the co-primary endpoints include the 

following: 

• The upper bound of the two-sided 95% CI on the ratio of the GMTs 

(GMTU.S. licensed vaccine/GMTnew vaccine) should not exceed 1.5. A proposal 

for use of a different GMT ratio should be based upon the characteristics 

of the assay that will be used to assess antibody responses. 

• The upper bound of the two-sided 95% CI on the difference between the 

seroconversion rates (SeroconversionU.S. licensed vaccine - Seroconversionnew 

vaccine) should not exceed 10%. 

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b. Alternatively, a placebo-controlled immunogenicity trial in which HI antibody 

responses to the new vaccine are assessed may be supportive of accelerated 

approval if the study was adequately powered to assess the co-primary endpoints 

for HI antibodies to each viral strain contained in the vaccine: 1) seroconversion 

rates, and 2) percent of subjects achieving an HI antibody titer ≥ 1:40. A saline 

placebo may be an acceptable control if the population studied is not at increased 

risk of complications from influenza illness or if the study is conducted offseason. If a study is conducted just prior to the influenza season in populations 

who are at increased risk from influenza illness, use of a U.S. licensed influenza 

vaccine as a control may be appropriate. The purpose of the control arm in this 

type of study design, whether it is a saline-placebo or a U.S. licensed influenza 

vaccine, is primarily to provide a comparative assessment of safety, not 

effectiveness. 

For example, the following recommendations, which have been modified from 

guidelines by the currently-titled, “Committee for Medicinal Products for Human 

Use of the European Medicines Agency” (Ref. 15), may support an accelerated 

approval. 

For adults < 65 years of age and for the pediatric population: 

• The lower bound of the 95% CI for the percent of subjects achieving 

seroconversion for HI antibody should meet or exceed 40%. 

• The lower bound of the 95% CI for the percent of subjects achieving an HI 

antibody titer ≥ 1:40 should meet or exceed 70%. 

For adults ≥ 65 years of age: 

• The lower bound of the 95% CI for the percent of subjects achieving 

seroconversion for HI antibody should meet or exceed 30%. 

• The lower bound of the 95% CI for the percent of subjects achieving an HI 

antibody titer ≥ 1:40 should meet or exceed 60%. 

c. Alternative study designs that assess different endpoints and/or other immune 

responses will be reviewed by CBER and may be accepted in support of an 

accelerated approval. CBER would need to determine that the study design is 

acceptable and the proposed surrogate endpoint(s) is reasonably likely to predict 

clinical benefit. 

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2. Safety 

Safety data should be collected from subjects enrolled in pre-licensure clinical trials 

intended to support the accelerated approval of a new trivalent inactivated influenza 

vaccine. The monitoring of these subjects should follow the outline for safety 

evaluations described above for traditional approval. A total safety database large 

enough to rule out a serious adverse event that occurs at a rate of 1 in 300 may be 

adequate. The size of the pre-licensure safety database warrants discussion with 

CBER, especially for vaccines manufactured using novel processes and for 

adjuvanted vaccines. This determination may be influenced by factors such as the 

nature of the new manufacturing process and available clinical data. Safety data to 

support use in pediatric populations will also be needed and should be submitted as 

part of the BLA or as a supplement if use in a pediatric population is sought at a later 

time (see Pediatric Research Equity Act of 2003). 

3. Post-marketing Confirmatory Studies 

For the design of post-marketing studies the sponsor should refer to studies described 

in section III.A.1. on effectiveness data to support traditional approval of new 

trivalent inactivated influenza vaccines. 

C. Additional Considerations 

1. Types of Influenza Vaccines 

Currently, all three U.S. licensed trivalent inactivated influenza vaccines are 

propagated in embryonated chicken eggs, and the virus is disrupted in the 

manufacturing process yielding “split virus” inactivated vaccines. The current 

recommendations regarding clinical effectiveness and safety data to support licensure 

apply to new split virus trivalent inactivated influenza vaccines propagated in 

embryonated chicken eggs. However, they also apply to licensure of new whole virus 

inactivated, cell culture derived inactivated, recombinant derived hemagglutininbased and adjuvanted influenza vaccines. Of note, vaccines manufactured by 

processes different from those used for currently licensed vaccines in the United 

States will likely require different pre-clinical evaluations. Detailed information on 

product characteristics and manufacturing processes are needed for all new vaccines, 

regardless of their derivation (see footnote 1). 

2. Clinical Lot Consistency 

The objective of a clinical lot consistency study is the demonstration that three 

consecutively manufactured final bulk lots of vaccine elicit equivalent immune 

responses. The HI antibody assay may be used to assess the immune responses. We 

recommend a pair-wise comparison of the 95% CI on the ratio of GMTs for each 

viral strain contained in the three vaccine lots as an appropriate primary endpoint. 

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The two-sided 95% CI on the GMT ratio should not exceed 1.5. Seroconversion rates 

for the HI antibody response for the three viral strains contained in the vaccine may 

be assessed as secondary endpoints. Assessment of lot consistency may be 

incorporated in studies designed to support the accelerated approval of a new 

influenza vaccine. CBER may decide, on a case by case basis, that lot consistency 

may be evaluated and incorporated in the post-marketing commitment studies. This 

determination would be influenced by factors such as the manufacturing process used 

for the new influenza vaccine and available clinical experience. 

3. Adjuvanted Trivalent Inactivated Influenza Vaccines 

Use of an adjuvant might reduce the amount of antigen needed to elicit immune 

responses to protect against influenza illness and might enhance vaccine supply. All 

influenza vaccine products formulated with an adjuvant should be submitted as new 

products. Data supporting their approval should be submitted to a new BLA. 

• Dose and Formulation Selection 

At an early stage of development, the sponsor should demonstrate the added 

value of the adjuvant given with the antigen. Assuming that the vaccine is a 

hemagglutinin-based product, the HI antibody assay may be appropriate to 

evaluate the immune response. 

A comparative study of adjuvanted vs. non-adjuvanted vaccines should 

demonstrate that the immune response elicited by the adjuvanted antigen is 

significantly better than that elicited by the same antigen alone. Differences 

in HI antibody titer and seroconversion should be meaningful (i.e., significant 

by assessment of p-value). For sample size determination, the sponsor should 

pre-define what would constitute a meaningful difference. As an example, 

CBER may view a 0.3 log10 mean difference (same as a two-fold difference in 

GMT ratio) for the HI antibody titers and a 15% difference in seroconversion 

rates as meaningful differences. The sponsor should also justify values 

assumed for the standard deviation of the log10 HI antibody titers. The HI 

antibody titers will typically require log transformation (i.e., HI antibody titers 

converted to log10 HI antibody titers) in order to produce data that may satisfy 

the normality assumption of certain parametric statistical tests. A t-test (or 

Wilcoxon rank-sum test if the normality assumption does not hold) may be 

used to compare the mean log10 HI antibody titers, and the Fisher’s exact test 

may be used to compare the seroconversion rates. Both tests should be onesided at the 2.5% significance level. The study should be adequately powered 

to meet both analysis endpoints. Alternative analyses, or ones allowing prespecified covariate adjustment, may be acceptable and should be discussed in 

advance with CBER. 

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Selection of an appropriate dose and formulation should also be guided by the 

safety profile of the formulations and regimens being studied. 

IV. REFERENCES 

1. World Health Organization. WHO guidelines on nonclinical evaluation of vaccines. Annex 1. 

WHO Technical Report Series. 2005;927:31-63. 

www.who.int/biologicals/publications/trs/areas/vaccines/nonclinical_evaluation/en/. 

2. Lamb RA, Krug RM. Orthomyxoviridae: The Viruses and Their Replication. In: Fields 

Virology, Editors-in-Chief: Knipe DM and Howley PM. 4th Edition. Philadelphia, PA: 

Lippincott Williams and Wilkins, Publishers; 2001;1487-1531. 

3. Palese P, Garcia-Sastre A. Influenza vaccines: present and future. The Journal of Clinical 

Investigation. 2002;110:9-13. 

4. Nakajima S, Nobusawa E, Nakajima K. Variation in response among individuals to antigenic 

sites on the HA protein of human influenza virus may be responsible for the emergence of 

drift strains in the human population. Virology. 2000;274:220-231. 

5. Holmes EC, Ghedin E, Miller N, Taylor J, Bao Y, St. George K, Grenfell BT, Salzberg SL, 

Fraser CM, Lipman DJ, Taubenberger JK. Whole-genome analysis of human influenza A 

virus reveals multiple persistent lineages and reassortment among recent H3N2 viruses. PLoS 

Biology. 2005;3:1579-1589. 

6. Barr IG, Komadina N, Hurt AC, Iannello P, Tomasov C, Shaw R, Durrant C, Sjogren H, 

Hampson AW. An influenza A(H3) reassortant was epidemic in Australia and New Zealand 

in 2003. Journal of Medical Virology. 2005;76:391-397. 

7. Centers for Disease Control and Prevention. Prevention and control of influenza. 

Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR 

Morbidity and Mortality Weekly Report. 2005;54(RR08):1-40. 

8. Centers for Disease Control and Prevention. Prevention and control of influenza: 

Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR 

Morbidity and Mortality Weekly Report. 1997;46(RR-9):1-25. 

9. Centers for Disease Control and Prevention. Prevention and control of influenza. 

Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR 

Morbidity and Mortality Weekly Report. 2000;49(RR-3):1-38. 

10. Centers for Disease Control and Prevention. Prevention and control of influenza. 

Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR 

Morbidity and Mortality Weekly Report. 2004;53(RR-6):1-40. 

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11. Centers for Disease Control and Prevention. Update: Influenza Vaccine Supply and 

Recommendations for Prioritization During the 2005--06 Influenza Season. MMWR 

Morbidity and Mortality Weekly Report. 2005;54(34);850:1-3. 

12. Testimony of Julie L. Gerberding, M.D., M.P.H., Director, Centers for Disease Control and 

Prevention, on “Protecting the Public’s Health: CDC Influenza Preparedness Efforts” before 

The Committee on Government Reform, U.S. House of Representatives, 2/12/2004. 

(Available at: http://www.cdc.gov/washington/testimony/In2122004200.htm). 

13. Hobson D, Curry RL, Beare AS, Ward-Gardner A. The role of serum haemagglutinationinhibiting antibody in protection against challenge infection with influenza A2 and B viruses. 

Journal of Hygiene, (Camb). 1972;70:767-777. 

14. de Jong JC, Palache AM, Beyer WEP, Rimmelzwaan GF, Boon ACM, Osterhaus ADME. 

Haemagglutination-inhibiting antibody to influenza virus. Developmental Biology (Basel). 

2003;115:63-73. 

15. Committee for Proprietary Medicinal Products (CPMP). Note for guidance on harmonisation 

of requirements for influenza vaccines. CPMP/BWP/214/96. The European Agency for the 

Evaluation of Medicinal Products (EMEA), March 1997. 

16. Treanor J, Keitel W, Belshe R, Campbell J, Schiff G, Zangwill K, Wolff M, Klimov A, 

Levandowski R, Lambert L. Evaluation of a single dose of half strength inactivated 

influenza vaccine in healthy adults. Vaccine. 2002;20:1099-1105. 

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