Safety and Immunogenicity of a Baculovirus- Expressed Hemagglutinin Influenza Vaccine a Randomized Controlled Trial

PRELIMINARY COMMUNICATION

Safety and Immunogenicity of a Baculovirus- Expressed Hemagglutinin Influenza Vaccine A Randomized Controlled Trial

John J. Treanor, MD Context A high priority in vaccine research is the development of influenza vaccines Gilbert M. Schiff, MD that do not use embryonated eggs as the substrate for vaccine production. Frederick G. Hayden, MD Objective To determine the dose-related safety, immunogenicity, and protective efficacy of an experimental trivalent influenza virus hemagglutinin (rHA0) vaccine pro- Rebecca C. Brady, MD duced in insect cells using recombinant baculoviruses. C. Mhorag Hay, MD Design, Setting, and Participants Randomized, double-blind, placebo-controlled Anthony L. Meyer, BS clinical trial at 3 US academic medical centers during the 2004-2005 influenza season Jeanne Holden-Wiltse, MPH among 460 healthy adults without high-risk indications for influenza vaccine. Hua Liang, PhD Interventions Participants were randomly assigned to receive a single injection of saline placebo (n=154); 75 µg of an rHA0 vaccine containing 15 µg of hemagglutinin Adam Gilbert, PhD from influenza A/New Caledonia/20/99(H1N1) and influenza B/Jiangsu/10/03 virus Manon Cox, PhD and 45 µg of hemagglutinin from influenza A/Wyoming/3/03(H3N2) virus (n=153); or 135 µg of rHA0 containing 45 µg of hemagglutinin each from all 3 components LL CURRENTLY LICENSED IN- (n=153). Serum samples were taken before and 30 days following immunization. fluenza vaccines in the United Main Outcome Measures Primary safety end points were the rates and severity of States are produced in em- solicited and unsolicited adverse events. Primary immunogenicity end points were the bryonated hen’s eggs. There rates of 4-fold or greater increases in serum hemagglutinin inhibition antibody to each areA several well-recognized disadvan- of the 3 vaccine strains before and 28 days after inoculation. The prespecified primary tages to the use of eggs as the sub- efficacy end point was culture-documented influenza illness, defined as development strate for influenza vaccine. Eggs re- of influenza-like illness associated with influenza virus on a nasopharyngeal swab. quire specialized manufacturing Results Rates of local and systemic adverse effects were low, and the rates of systemic facilities and could be difficult to scale adverse effects were not different in either vaccine group than in the placebo group. He- up rapidly in response to an emerging magglutinin inhibition antibody responses to the H1 component were seen in 3% of pla- need such as a pandemic. It is usually cebo, 51% of 75-µg vaccine, and 67% of 135-µg vaccine recipients, while responses to necessary to adapt candidate vaccine vi- B were seen in 4% of placebo, 65% of 75-µg vaccine, and 92% of 135-µg vaccine recipients. Responses to the H3 component occurred in 11% of placebo, 81% of 75-µg vac- ruses for high-yield growth in eggs, a cine, and 77% of 135-µg vaccine recipients. Influenza infections in the study population process that can be time consuming, is were due to influenza B and A(H3N2), and influenza A infections were A/California/7/2004– not always successful, and can select re- like viruses, an antigenically drifted strain. Seven cases of culture-confirmed CDC-defined ceptor variants that may have subop- influenza-like illness occurred in 153 placebo recipients (4.6%) compared with 2 cases (1.3%) timal immunogenicity.1 In addition, ag- in 150 recipients of 75 µg of vaccine, and 0 cases in recipients of 135 µg of vaccine. ricultural diseases that affect chicken Conclusions In this study, a trivalent rHA0 vaccine was safe and immunogenic in a flocks, and that might be an impor- healthy adult population. Preliminary evidence of protection against a drifted influenza tant issue in a pandemic due to an avian A(H3N2) virus was obtained, but the sample size was small. Inclusion of a neuraminidase influenza virus strain, could easily dis- component did not appear to be required for protection. rupt the supply of eggs for vaccine Trial Registration clinicaltrials.gov Identifier: NCT00328107 manufacturing. Therefore, develop- JAMA. 2007;297:1577-1582 www.jama.com ment of alternative substrates for influenza vaccine production2 has been identified as a high-priority objective. (HA)usingrecombinantDNAtechniques. Author Affiliations are listed at the end of this article. One potential alternative method for In this study, we evaluated an experimen- Corresponding Author: John J. Treanor, MD, Univer- sity ofRochester Medical Center, Room 3-630, 601 productionofinfluenzavaccineisexpres- tal influenza vaccine consisting of recom- Elmwood Ave, Rochester, NY 14642 (John_Treanor sion of the influenza virus hemagglutinin binant HA expressed in insect cells by a @urmc.rochester.edu).

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recombinantbaculovirus(rHA0).Thisal- ration containing the purified rHA0 of word of mouth. Race/ethnicity data for all ternative avoids dependence on eggs and the A/New Caledonia/20/99(H1N1), particiapnts were collected from self- is very efficient because of the high lev- A/Wyoming/3/03(H3N2), and B/Jiangsu/ reports. Participants were compensated els of protein expression under the 10/03 influenza viruses expressed from for each visit (Ϸ$25). control of the baculovirus polyhedrin genes cloned by reverse transcriptase After screening medical history and promoter. Monovalent and bivalent polymerase chain reaction from the same physical examination to determine eligi- baculovirus-derived influenza vaccines Centers for Disease Control and Preven- bility, 10 mL of blood was collected from have been evaluated in young adults and tion (CDC)–derived vaccine seed vi- an arm vein for serologic studies, and par- in community-dwelling adults older than ruses used for the licensed TIV and for- ticipants were randomly assigned to re- 65 years.3-6 These studies found that the mulated in phosphate-buffer saline ceive a single dose of either rHA0 at 135 vaccines are well tolerated and immuno- containing 0.005% detergent without µg, rHA0 at 75 µg, or saline placebo using genic. Recently, doses of a trivalent vac- preservative. Sodium dodecyl sulfate– apermuted-blockrandomizationscheme cine ranging from 15 µg to 135 µg per polyacrylamide gel electrophoresis of the stratified by study site. Vaccine was ad- component were shown to be well toler- purified monovalent materials indi- ministered as a single intramuscular in- ated in elderly persons and to induce an- cated that hemagglutinin constitutes ap- jection in the upper deltoid. tibodyresponsesatratescomparablewith proximately 95% of the total protein. Participants measured their oral tem- or superior to the licensed trivalent vac- The experimental vaccine was formu- perature daily and maintained a diary cine.7 While a clear dose-response rela- lated at 2 different concentrations, as card for 7 days after vaccination on which tionship has been shown for the H3 com- determined by SRID. The high-dose for- they recorded local and systemic reac- ponent in both healthy adults and in mulation contained 45 µg of each com- tions graded as mild (noticeable but not elderly persons, relatively little difference ponent, for a total dose of 135 µg of rHA0 interfering with normal activities), mod- hasbeenobservedintheimmuneresponse per 0.5-mL dose based on the SRID val- erate (some interference with normal ac- to doses ranging from 15 µg to 135 µg of ues on the preblend bulk. After formu- tivities), and severe (symptom pre- the H1 and B components. However, this lation, it was determined that this dose vented normal daily activities). analysis has been confounded by a poor contained 35 µg rather than 45 µg of the Participants also returned on day 2 and correlation between the antigen content H1 component. The low-dose formula- day 7 after vaccination for review of the of the H1 and B components of rHA0 vac- tion contained 45 µg of the H3 rHA0 and diary card, concomitant medications and cines, as determined by measurement of 15 µg each of the H1 and B rHA0, for a medical history, and examination of the total protein content, compared with the total dose of 75 µg of rHA0. Placebo con- vaccination site. Participants returned ap- values determined by single radial immu- sisted of normal saline for injection. Vac- proximately 28 days after vaccination for nodiffusion(SRID),thestandardmeasure- cine and placebo were supplied in coded, review of interim medical history. In ad- ment used for trivalent inactivated vac- identical-appearing single-dose vials. dition, 10 mL of venous blood was ob- cine (TIV). tained from an arm vein for assessment Therefore, the primary objective of Clinical Study Design of antibody to influenza virus. A final the current study was to evaluate the The study was conducted as a random- study visit occurred at day 180, during relative immunogenicity of the H1 and ized, double-blind, placebo-controlled which participants underwent a physi- B components of the vaccine when for- study at 3 medical centers (University of cal examination and interval medical mulated at either 15 µg or 45 µg per Rochester, Rochester, NY; Cincinnati history. component, as determined by SRID. In Children’sHospital,Cincinnati,Ohio;and The study protocol was reviewed and addition, we used the opportunity to University of Virginia, Charlottesville) approved by the investigational review follow up participants throughout the during the 2004-2005 influenza season. boards at all 3 clinical sites, and written influenza season to obtain prelimi- Participants were healthy adults aged 18 informed consent was obtained from all nary evidence of protective efficacy in to 49 years who did not belong to high- participants prior to study entry. a healthy adult population. priority target groups for influenza vaccination as defined by the Advisory Com- Surveillance for Influenza METHODS mittee on Immunization Practices and Following the day 28 visit, participants Vaccine who had not received previous influenza started to complete a weekly diary to rec- The vaccine used in this study con- vaccination for the 2004-2005 season. ord influenza symptoms, and after influ- sisted of purified HA proteins pro- Women of childbearing potential had to enza was recognized in the community, duced in insect cells using a baculovi- have a negative urine pregnancy test re- participants received weekly telephone rus expression system, as previously sult at the time of randomization and be callstoreviewthediaryandascertainpres- described.7 Because the HA produced in willing to use an adequate form of con- ence or absence of respiratory illness this system is not cleaved, the resulting traception during the course of the study. symptoms. Participants were instructed product is referred to as rHA0. The vac- Participants were recruited by newspaper to return to the clinic for illness evalua- cine was formulated as a trivalent prepa- and radio advertisements, posters, and tions if they observed any acute respira-

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torytractsymptomsorfever.Duringthese ited and unsolicited adverse events. The ity end point. Assuming that from 60% illness visits, symptoms were reviewed, primaryimmunogenicityendpointswere to80%oftheparticipantsinanactivetreat- a brief physical examination was con- the rates of 4-fold or greater increases in mentgroupwouldhavea4-foldorgreater ducted, and nasopharyngeal swabs for vi- serum HAI antibody to each of the 3 vac- serum HAI antibody response to any spe- rus culture were obtained. cinestrainscomparingprevaccinationand cific strain, inclusion of 150 participants 28-daypostvaccinationsamples.Thepre- per group would have 80% power to de- Laboratory Assays specified primary efficacy end point was tect an approximately 13% to 14% differ- Serum samples were assessed for anti- culture-documentedinfluenzaillness,de- ence in response rates between study body to each of the 3 components of the fined as development of a CDC-defined groups. Although not designed primar- vaccine by hemagglutination inhibi- influenza-like illness associated with re- ilyasanefficacytrial,witha5%attackrate tion (HAI) using standard methods. covery of influenza virus from a nasopha- intheplacebogroup,thestudyhadpower Egg-grown influenza A/New Caledonia/ ryngeal swab. A CDC-defined influenza- ranging from 14% to 53% to detect vac- 20/99 and influenza A/Wyoming/ like illness was defined as the presence of cine efficacy ranging from 40% to 80%. 03/03 were obtained from the CDC, At- documentedfeverwithbodytemperature A PϽ.05 level was considered to be sta- lanta, Ga, while assays against the higher than 37.7°C (99.8°F) plus either tistically significant. The analyses were influenza B/Jiangsu/10/03 used anti- sore throat or cough. conductedusingSASsoftware,version9.1 gen prepared in Madin-Darby canine (SAS Institute Inc, Cary, NC). kidney cells from a seed virus sup- Statistical Analyses plied by the CDC. Serum samples were Rates of safety end points were based on RESULTS treated with neuraminidase (receptor- the most severe response and were evalu- A total of 460 participants were random- destroying enzyme, Denka Seiken, ated by ␹2 test. Differences between the ized, 458 were vaccinated, and 451 Tokyo, Japan) to remove nonspecific in- proportions of participants with at least (98.5%) completed all study procedures. hibitors of hemagglutination prior to a 4-fold increase in HAI antibody for each The disposition of the participants is testing and were tested in serial 2-fold pairwise treatment group comparison shown in the FIGURE. Of the 460 enrolled dilutions at an initial dilution of 1:4. Se- were tested using a ␹² test. participants, 153 were randomized to 75 rum samples with no reactivity at 1:4 The sample size for this study was cho- µg of rHA0 vaccine, 153 were random- were assigned a value of 1:2. Assays senprimarilybasedontheimmunogenic- ized to 135 µg of rHA0 vaccine, and 154 were performed using chick red blood cells (Colorado Serum Company, Den- Figure. Flow of Participants Through the Trial ver) for influenza A/New Caledonia/ 20/99 and B/Jiangsu/10/03 viruses and turkey red blood cells (Viromed Labo- 460 Participants Randomized ratories, Minnetonka, Minn) for the influenza A/Wyoming/03/03 virus. 154 Randomized to Receive 153 Randomized to Receive 153 Randomized to Receive Nasopharyngealswabsforvirusculture Placebo rHA0, 75 µg rHA0, 135 µg were stored at −70°C and shipped on dry 154 Received Placebo 151 Received Vaccine 153 Received Vaccine as Assigned as Assigned as Assigned icetoacentrallaboratory(CincinnatiChil- 2 Did Not Receive dren’s Hospital Medical Center), where Vaccine as Assigned virus isolation was performed in primary 1 Did Not Meet Entry Criteria 1 Did Not Meet Entry Criteria 2 Did Not Meet Entry Criteria rhesus monkey kidney cells (Diagnostic (Received Trivalent Influenza (Pregnant) (Received Trivalent Influenza HybridsInc,Athens,Ohio).Thepresence Vaccine) Vaccine) 2 No Day 28 Titer 1 No Day 28 Titer of influenza A or B viruses in the culture

was determined by immunofluorescence 154 Included in Safety Analysis 151 Included in Safety Analysis 153 Included in Safety Analysis using type-specific monoclonal antibod- 2 Excluded (Did Not Receive ies (Diagnostic Hybrids Inc). Influenza A Vaccine) isolates were further subtyped at Protein 151 Included in Immunogenicity 150 Included in Immunogenicity 150 Included in Immunogenicity Sciences Corp by sequence analysis of Analysis Analysis Analysis 3 Excluded 3 Excluded 3 Excluded the entire HA1 region after reverse 1 Received Trivalent 2 Did Not Receive Vaccine 2 Received Trivalent transcriptase–polymerase chain reaction Influenza Vaccine 1 Pregnant Influenza Vaccine amplificationofMadin-Darbycaninekid- 2 No Day 28 Titer 1 No Day 28 Titer

ney cell–grown virus. 153 Included in Efficacy Analysis 150 Included in Efficacy Analysis 151 Included in Efficacy Analysis 1 Excluded (Received Trivalent 3 Excluded 2 Excluded (Received Trivalent Definition of End Points Influenza Vaccine) 2 Did Not Receive Vaccine Influenza Vaccine) 1 Pregnant The primary safety end points for this study were the rates and severity of solic- rHA0 indicates recombinant hemagglutinin influenza virus vaccine.

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were randomized to placebo. There were ever, 97% of all reports of pain after rHA0 group) experienced severe adverse events 9 participants who did not complete the vaccine were rated as mild. Systemic (1 infected nevus and 1 knee injury) that study, including 1 who withdrew con- symptoms following vaccination also did were also considered to be unrelated to sent, 5 who were lost to follow-up, 2 par- not occur at significantly different rates vaccine. No participants discontinued the ticipants in the 75-µg rHA0 treatment in vaccine and placebo recipients (PϾ.07 study because of adverse events and no groupwhowererandomizedbutnotvac- for all comparisons). The most fre- participants died. cinated, and 1 who was incarcerated. The quently reported systemic symptom fol- majority of participants were white lowing vaccination was headache. The Immunogenicity (86%) and female (63%) (TABLE 1). The majority (86%) of reports of headache Serum antibody responses to vaccination mean age was 31.7 years (range, 18-49 were also rated as mild, and there was are summarized in TABLE 3. Overall, years). There were no differences with no difference in the frequency of head- amongparticipantsvaccinatedwithrHA0 respect to age, sex, or race/ethnicity be- ache between vaccine and placebo re- vaccine, the frequencies of HAI antibody tween the groups. cipients (PϾ.07). There were no re- responses (Ն4-fold increase comparing ported fevers (oral temperature Ͼ37.7°C day 0 with day 28) to influenza A/New Assessment of Vaccine Safety [Ͼ99.8°F]) following vaccination. Caledonia, B/Jiangsu, and A/Wyoming The rates and severities of local and sys- Two participants (1%) in the 135-µg were significantly greater (range, temic symptoms reported on the diary vaccine group experienced serious ad- 51%-92%) than was observed for partici- card are shown in TABLE 2. Injection of verse events that were considered to be pantsvaccinatedwithplacebo(range,3%- rHA0 vaccine was associated with local unrelated to vaccine (1 seizure related to 11%; PϽ.001). Antibody responses were injection site pain that was significantly hypoglycemia and 1 newly diagnosed seen in most participants receiving either more frequent than after saline placebo lobular carcinoma in situ). Two addi- the 75-µg or the 135-µg dose of rHA0 vac- and that was dose dependent (PϽ.001 tional participants (1 in the 75-µg vaccines.However,thefrequencyofresponses for pain; P=.04 for tenderness). How- cine group and 1 in the 135-µg vaccine to both the A/New Caledonia/99 and B/Jiangsu/03 influenza viruses were significantly higher in the group receiving Table 1. Baseline Participant Characteristics the135-µgdose(P=.003),consistentwith 135-µg the higher doses of H1 and B components Placebo 75-µg Vaccine Vaccine Overall Characteristics (n = 154) (n = 151) (n = 153) (N = 458) in the 135-µg vaccine. The frequency of Race/ethnicity, No. (%) HAIantibodyresponsetotheA/Wyoming/ White 139 (90) 126 (83) 130 (85) 395 (86) 03 (H3N2) influenza virus was not dif- Black/African American 9 (6) 12 (8) 9 (6) 30 (7) ferent between the 75-µg and 135-µg Latino/Hispanic 1 (1) 2 (1) 5 (3) 8 (2) doses,whichcontainedidenticalamounts Asian 4 (3) 10 (7) 4 (3) 18 (4) oftheH3component.Similarly,therewere American Indian/Alaskan Native 0 0 1 (1) 1 (Ͻ1) significant differences in the day 28 geo- Native Hawaiian/Pacific Islander 0 1 (1) 1 (1) 2 (Ͻ1) metricmeantiterofHAIantibodybetween Other/not stated 1 (1) 0 3 (2) 4 (1) the 75-µg and 135-µg doses for both the Male, No. (%) 65 (42) 48 (32) 57 (37) 170 (37) H1 and B components but not the H3 Age, median (range), y 32 (18-49) 32 (18-49) 30 (18-49) 31 (18-49) component.

Table 2. Local and Systemic Symptoms Experienced in the 7 Days Following Vaccination* Placebo (n = 154) 75-µg Vaccine (n = 151) 135-µg Vaccine (n = 153)

Symptoms Mild Moderate Severe Mild Moderate Severe Mild Moderate Severe Arthralgias 7 (5) 1 (1) 0 7 (5) 2 (1) 0 7 (5) 1 (1) 0 Chills 3 (2) 0 0 2 (1) 2 (1) 0 2 (1) 2 (1) 0 Fatigue 21 (14) 7 (5) 0 21 (14) 7 (5) 0 22 (14) 3 (2) 0 Headache 48 (31) 15 (10) 0 48 (32) 4 (3) 0 53 (35) 12 (8) 0 Myalgias 16 (10) 3 (2) 0 24 (16) 2 (1) 0 28 (18) 3 (2) 0 Nausea 9 (6) 1 (1) 0 5 (3) 2 (1) 0 10 (7) 3 (2) 0 Pain 24 (16) 1 (1) 0 67 (44) 0 0 88 (58) 5 (3) 0 Sweats 6 (4) 1 (1) 0 6 (4) 1 (1) 0 2 (1) 2 (1) 1 (1) Tenderness 3 (2) 0 0 12 (8) 0 0 9 (6) 1 (1) 0 *Data are expressed as No.(%) of patients experiencing local and systemic symptoms in the 7 days following vaccination based on the most severe responses as reported on the diary cards. Severity was graded as mild (no interference with daily activities), moderate (some limitation of activity due to the symptom), or severe (the symptom prevents normal daily activity).

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Protection Against Influenza Illness Table 3. Serum Hemagglutination-Inhibiting Antibody Response to Vaccination Participants in this study were followed Placebo 75-µg Vaccine 135-µg Vaccine throughoutthesubsequentinfluenzasea- Influenza Subtype (n = 151) (n = 150) (n = 150) son with weekly telephone calls and in- A/New Caledonia/20/99(H1N1) structed to return to the study clinics for Prevaccination* 26.4 (19.9-35.0) 23.9 (18.0-31.7) 22.0 (16.6-29.2) anyacuterespiratoryillness,atwhichtime Postvaccination* 28.8 (22.8-36.4) 115.6 (91.5-146.2) 206.0 (163.0-260.5) a nasopharyngeal swab for viral culture Response, %† 3 51 67 was obtained. A total of 116 such cultures A/Wyoming/3/03(H3N2) Prevaccination* 72.8 (56.4-93.9) 65.5 (50.7-84.6) 74.2 (57.5-95.8) were obtained, 43 in the placebo group, Postvaccination* 68.9 (57.9-81.9) 933.6 (784.4-1111.2) 1028.7 (864.3-1224.5) 39 in the 75-µg vaccine group, and 34 in Response, %† 11 81 77 the 135-µg vaccine group. The primary B/Jiangsu/10/03 efficacy end point for this study was the Prevaccination* 6.1 (5.1-7.3) 6.4 (5.4-7.6) 5.5 (4.6-6.5) development of culture-confirmed influ- Postvaccination* 5.7 (4.7-6.9) 33.4 (27.6-40.4) 74.9 (61.9-90.6) enza illness meeting the influenza-like ill- Response, %† 4 65 92 ness case definition of the CDC; ie, pres- *Day 0 (prevaccination) and day 28 (postvaccination) geometric mean titers (95% confidence intervals). ence of fever higher than 37.7°C (99.8°F) †Four-fold or greater increase. and sore throat, cough, or both. There were a total of 13 positive cul- disease. The safety data generated in this 135-µg dose (45 µg of each component) tures for influenza in the study popula- study are consistent with the safety pro- wasmoreimmunogenicandmightbepre- tion, of which 10 were influenza A (all file observed in previous studies of rHA0 dicted to provide greater or longer-lasting of which were confirmed as influenza vaccine.3-8 These vaccines have been well protection. Therefore, further develop- H3) and 3 were influenza B. Sequence tolerated at all doses administered and mentoftherHA0vaccineshoulduseafor- analysis of these viruses further re- are associated with low rates of local re- mulation of 45 µg per component, and fu- vealed that all of the 10 H3N2 isolates actions. Because of widespread short- ture studies should directly compare the were A/California/7/2004–like. Of these ages of licensed TIV in the United States safety and immunogenicity of this dose 13 cases, 9 (69%) occurred in individu- in 2004-2005, we were unable to per- with that of TIV. A major advantage of the als meeting the CDC influenza illness form a direct comparison of trivalent rHA0approachisthatthesedosesarewell case definition. The rates of culture- rHA0 vaccines and TIV. In other stud- within the production capacity of the sys- positive influenza illness, the prespeci- ies of TIV in healthy adult populations, temataneconomicallyandlogisticallyfea- fied primary efficacy end point, were 7 pain at the injection site has been re- sible scale. (4.6%) of 153 placebo recipients, 2 ported in 54% to 67% of recipients,9-11 We also found that recipients of the (1.4%) of 150 recipients of the 75-µg vac- and systemic symptoms have been simi- rHA0 vaccine had reduced rates of cul- cine dose, and 0 of 151 recipients of the lar to placebo, suggesting that the triva- ture-positive CDC-defined influenza- 135-µg vaccine dose. Among the 4 posi- lent rHA0 vaccine and TIV have a simi- like illness compared with placebo re- tive cultures in individuals who did not lar safety profile. This is consistent with cipients, although the study was small. meet the CDC case definition, 1 oc- previous direct comparisons of rHA0 and When considering both vaccine groups curred in a placebo recipient, 2 oc- TIV suggesting similar rates of local re- combined, the cumulative incidence of curred in recipients of the 75-µg vac- actions between the 2 vaccines.3,4,7 culture positive CDC-defined influenza- cine, and 1 occurred in a recipient of the Both doses of the recombinant hemag- like illness was reduced by 86%. For 135-µg vaccine, so the rates of partici- glutininvaccineevaluatedinducedserum comparison, in a recently reported study pants with a positive culture associated HAI antibody responses to all 3 compo- conducted in the same influenza sea- with any acute respiratory illness were nents (H1, H3, and B) in the majority of son as was our study, the efficacy of TIV 8/153 (5.2%), 4/150 (2.7%), and 1/151 recipients. As expected, there were no dif- in healthy adults against culture- (0.7%) in placebo, 75-µg vaccine, and ferences in either the frequency of re- confirmed influenza meeting a similar 135-µg vaccine recipients, respectively. sponses or the postvaccination geomet- case definition was 77% (95% confi- ric mean titer to influenza A/Wyoming dence interval, 37%-92%)11 COMMENT (H3N2) between the 75-µg and 135-µg The majority of cases in this study were We evaluated the safety, immunogenic- doses, since both doses contain the same due to influenza A, and all of the influenza ity, and efficacy of a trivalent recombi- 45 µg of the H3 component. Significant A viruses isolated in this study that were nant hemagglutinin (rHA0) vaccine. We differences in both the frequency and the further subtyped were of the H3N2 sub- have shown that the rHA0 vaccine is well magnitudeoftheHAIresponseweredem- type,consistentwiththereportthat98.5% tolerated in healthy adults and immu- onstrated for both the H1 and B compo- of all influenza A viruses typed in the nogenic at both doses evaluated, and we nents. Although the responses to the 75- United States during the 2004-2005 sea- obtained preliminary evidence of pro- µgdoseexceededtheEuropeanUnioncri- son were H3N2.13 In addition, all of the tection against influenza infection and teria for influenza vaccine licensure,12 the influenza A(H3N2) viruses isolated from

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participants in this trial were genetically genic live influenza viruses and the at- Acknowledgment: We acknowledge the efforts of study nurses and clinical coordinators Carrie Nolan, RN, Diane similar to influenza A/California/7/2004, tendant biocontainment issues that O’Brien, RN, Pat Smith, RN, and Mary Lou Werthman, a significant antigenic variant which re- would be a particular concern for gen- RN, University of Rochester; Susan Parker, RN, and Amy 8 Hoeper, RN, University of Virginia; and Maureen Tracey, acts poorly with antiserum samples from eration of pandemic vaccines. The pre- RN, and Nicole R. Sanderfer, RN, Cincinnati Children’s persons who received the 2004-2005 for- liminary demonstration of protective ef- Hospital. These individuals were compensated for their mulation of TIV,14 as were 75% of influ- ficacy in adults provides further support activities via research contracts between Protein Sciences Corp and their respective institutions. We also thank Henk- enza A(H3N2) isolates throughout the for the development of this promising ap- Andre´ Kroon, MD, and Tam LaFleur, MS, Protein Sciences United States.13 It has been suggested that proach for prevention of seasonal and Corp, who served as project managers and the sponsor’s medical monitor (Dr Kroon); and Richard L. Ward, PhD, the neuraminidase component of vaccine pandemic influenza. Cincinnati Children’s Hospital, for laboratory supervision may be important for protection in situ- Author Affiliations: University of Rochester, Roches- (funded by a contract between Protein Sciences and Cin- ations in which there is not a close anti- ter, NY (Drs Treanor, Hay, and Liang and Ms Holden- cinnati Children’s Hospital). 15 Wiltse); Cincinnati Children’s Hospital Medical Center, genic match in the hemagglutinin. The Cincinnati, Ohio (Drs Schiff and Brady and Mr Meyer); current study suggests that it is possible University of Virginia, Charlottesville (Dr Hayden); Ock- REFERENCES ham Development Group, Cary, NC (Dr Gilbert); and to generate a substantial amount of pro- Protein Sciences Corp, Meriden, Conn (Dr Cox). 1. Katz JM, Wang M, Webster RG. Direct sequencing tection in an immunologically primed Author Contributions: Dr Treanor had full access to all of the HA gene of influenza (H3N2) reveals sequence population against influenza with a pure of the data in the study and takes responsibility for the identity with mammalian cell-grown virus. J Virol. 1990; integrity of the data and the accuracy of the data analysis. 64:1808-1811. hemagglutinin vaccine, even in the pres- Study concept and design: Treanor, Schiff, Hayden, 2. Cox MMJ. Cell-based protein vaccines for influenza. ence of significant antigenic drift. Cox. Curr Opin Mol Ther. 2005;7:24-29. Acquisition of data: Treanor, Schiff, Hayden, Brady, 3. Powers DC, Smith GE, Anderson EL, et al. Influ- The development of functional anti- Hay, Meyer. enza A virus vaccines containing purified recombi- body responses and the preliminary evi- Analysis and interpretation of data: Treanor, Hayden, nant H3 hemagglutinin are well-tolerated and in- Hay, Holden-Wiltse, Liang, Gilbert, Cox. duce protective immune responses in healthy adults. dence of protective efficacy of the vaccine Drafting of the manuscript: Treanor, Meyer, Gilbert, Cox. J Infect Dis. 1995;171:1595-1598. suggest that in adults, the minor differ- Critical revision of the manuscript for important in- 4. Treanor JJ, Betts RF, Smith GE, et al. Evaluation of ences in HA glycosylation seen in insect tellectual content: Treanor, Schiff, Hayden, Brady, Hay, a recombinant hemagglutinin expressed in insect cells Meyer, Holden-Wiltse, Liang, Cox. as an influenza vaccine in young and elderly adults. cellscomparedwithmammaliancells,the Statistical analysis: Holden-Wiltse, Liang, Gilbert. J Infect Dis. 1996;173:1467-1470. presence of HA as an uncleaved precur- Obtained funding: Treanor, Schiff, Cox. 5. Lakey DL, Treanor JJ, Betts RF, et al. Recombinant Administrative, technical, or material support: Treanor, baculovirus influenza A hemagglutinin vaccines are sor, and the lack of neuraminidase in the Schiff, Hayden, Hay, Meyer, Cox. well tolerated and immunogenic in healthy adults. rHA0 vaccine do not have a major effect Study supervision: Treanor, Schiff, Hayden, Brady. J Infect Dis. 1996;174:838-841. Financial Disclosures: Dr Treanor reports receiving 6. Powers DC, McElhaney JE, Florendo OAJ, et al. Hu- on the actual protection provided by the funding for influenza vaccine–related research from moral and cellular immune responses following vacci- vaccine. Although this study and other Sanofi Pasteur, Merck, and GlaxoSmithKline. Dr Brady nation with purified recombinant hemagglutinin from reports receiving research support from GlaxoSmithKline. influenza A (H3N2) virus. J Infect Dis. 1997;175: studiesinadultshaveshownexcellentHAI No other authors reported financial disclosures. 342-351. and neutralizing antibody responses to Funding/Support: Financial support for this study was 7. Treanor JJ, Schiff GM, Couch RB, et al. Dose- rHA0 vaccines,3-7 further studies in im- provided by Protein Sciences Corp, manufacturer of related safety and immunogenicity of a trivalent bacu- the vaccine. Protein Sciences Corp also funded Ock- lovirus-expressed influenzavirus hemagglutinin vac- munologically naive young children are ham Development Group, which performed the data cine in elderly adults. J Infect Dis. 2006;193:1223-1225. clearly needed. analysis and acted as the contract research organiza- 8. Treanor JJ, Wilkinson BE, Masseoud F, et al. Safety tion for the study. The study was designed jointly by and immunogenicity of a recombinant hemagglu- The use of recombinant DNA tech- the investigators and Protein Sciences Corp. tinin vaccine for H5 influenza in humans. Vaccine. niques to express proteins in cell cul- Role of the Sponsor: Protein Sciences Corp and Dr. 2001;19:1732-1737. Treanor designed the study together, with significant 9. Nichol KL, Margolis KL, Lind A, et al. Side effects ture has been a successful approach for input from Dr Gilbert. The study design was also re- associated with influenza vaccination in healthy work- generation of highly effective vaccines for viewed and modified based on comments from the US ing adults. A randomized, placebo-controlled trial. Arch the prevention of hepatitis B virus and Food and Drug Administration. Data analysis was ini- Intern Med. 1996;156:1546-1550. tially performed by Dr Gilbert at Ockham, which was 10. Treanor JJ, Campbell JD, Brady RC, et al. Rapid human papillomavirus. Among the avail- funded by Protein Sciences Corp; a second, indepen- licensure of a new, inactivated influenza vaccine in the able expression technologies, recombi- dent analysis was performed by Drs Holden-Wiltse and United States. Hum Vaccin. 2005;1:239-244. Liang, who received no compensation from Protein Sci- 11. Ohmit SE, Victor JC, Botthoff JR, et al. Preven- nant baculovirus is especially well suited ences Corp. Laboratory assays were performed in Cin- tion of antigenically drifted influenza by inactivated for production of influenza vaccine be- cinnati by Mr Meyer. Protein Sciences Corp had the op- and live attenuated vaccines. N Engl J Med. 2006;355: portunity to review and comment on the manuscript 2513-2522. cause the rapidity with which genes can prior to submission, but Dr Treanor had final respon- 12. Wood JM. Standardization of inactivated influ- be cloned and inserted into this vector sibility for writing and submitting the manuscript, with enza vaccine. In: Nicholson KG, Webster RG, Hay AJ, input from the other authors, including Dr Cox. eds. Textbook of Influenza. London, England: Black- facilitates updating the vaccine at regu- Independent Statistical Analysis: Jeanne Holden-Wiltse, well Science Ltd; 1998:333-345. lar intervals. In addition, the extraordi- MS, and Hua Liang, PhD, had access to the complete data 13. Update: influenza activity—United States and narily high yields of protein possible in set and performed independent analysis of all end points worldwide, 2004-05 season. MMWR Morb Mortal reported in this article. Except for minor typographical Wkly Rep. 2005;54:631-634. this system provide the opportunity to errors and rounding errors, their analysis was in agree- 14. Recommended composition of influenza virus vac- use much higher and potentially more ment with the analysis performed by Ockham Develop- cines for use in the 2005-2006 influenza season. Wkly ment Group; the current report presents results after all Epidemiol Rec. 2005;80:71-75. effective doses of vaccine. Expression of discrepancies were resolved. Compensation for their ef- 15. Brett IC, Johansson BE. Immunization against influ- the HA protein in insect cells using re- forts was provided by the University of Rochester. No funds enza A virus: comparison of conventional inactivated, live- to support the independent analysis were provided by attenuatedandrecombinantbaculovirusproducedpurified combinant baculovirus also avoids the either Protein Sciences Corp or by Ockham Development hemagglutinin and neuraminidase vaccines in a murine need to work with potentially patho- Group. model system. Virology. 2005;339:273-280.

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