Vaccine 26 (2008) 2890–2898

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A double-blind, placebo-controlled study of the safety and immunogenicity oflive, oral type 4 and type 7 adenovirus vaccines in adults�

Arthur Lyonsa,∗, Jenice Longfieldb, Robert Kuschnera, Timothy Straighta, Leonard Binna,Jitvimol Seriwatanaa, Raven Reitstetterb, Irma B. Frohc, David Craft c, Kevin McNabbd,Kevin Russell e, David Metzgare, Alan Liss f, Xiao Sung, Andrew Towleh, Wellington Suna

a

Department of Virus Diseases, Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Silver Spring, MD, USAb University of Texas Health Science Center at San Antonio, San Antonio, TX, USAc Department of Pathology, Walter Reed Army Medical Center, Washington, DC, USAd Department of Pathology, Brooke Army Medical Center, Fort Sam Houston, TX, USAe Naval Respiratory Disease Laboratory, Naval Health Research Center, San Diego, CA, USAf Department of Health and Human Services, Washington, DC, USAg Merck & Co., Inc., CBARDS Upper Gwynedd, PA, USA

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h VaccGen International LLC, Larchmont, NY, USA

a r t i c l e i n f o

Article history:Received 12 October 2007Received in revised form 14 March 2008Accepted 20 March 2008Available online 10 April 2008

Keywords:Adenovirus

a b s t r a c t

Adenovirus serotypes 4 (A(ARD) in US military recassociated ARD, are no lodouble-blind, placebo-conufacturer to assess their stogether in a single dosewere then observed for 8

VaccineLiveOralVirusType 4Type 7

(33%), cough (33%), sore throa(13%) and diarrhea (13%). Nonevirus fecal shedding was 7–21(GMT 23.3) while 63% seroconvwere safe and induced a good iefficacy are in progress.

1. Introduction

Adenoviruses have been the most important cause of febrileacute respiratory disease (ARD) in US military recruit populations.They were first implicated as a cause of ARD in 1953 in a case at FortLeonard Wood, MO [1]. Since then, adenovirus types 4 (ADV-4) and7 (ADV-7) and occasionally types 3, 11, 14 and 21 have been identi-fied as important causes of ARD [2] among military recruits acrossall uniformed services in the United States, resulting in loss of train-ing due to significant morbidity, and in rare instances, mortality [3].Prior to the introduction of live, oral adenovirus vaccines in 1971,

� The views expressed herein are those of the authors and do not necessarilyrepresent those of the Department of Defense or the Department of the Army.

∗ Corresponding author. Tel.: +1 301 319 9021; fax: +1 301 319 9661.E-mail address: arthur.lyons@na.amedd.army.mil (A. Lyons).

0264-410X/$ – see front matter. Published by Elsevier Ltd.doi:10.1016/j.vaccine.2008.03.037

) and 7 (ADV-7) are important causes of febrile acute respiratory diseasePreviously licensed vaccines, which effectively controlled adenovirus-available. In the Fall of 2004 we conducted this Phase 1 randomized,

ed trial of the live, oral ADV-4 and ADV-7 vaccines made by a new man-and immunogenicity. The adenovirus vaccines were administered orallyrty subjects. Twenty eight additional subjects received placebo. Subjectsks. The most commonly reported adverse events were nasal congestiont (27%), headache (20%), abdominal pain (17%), arthralgia (13%), nauseaof these rates differed significantly from placebo. The duration of vaccine

days. Seventy three percent of vaccine recipients seroconverted to ADV-4erted to ADV-7 (GMT 51.1) by Day 28. The new ADV-4 and ADV-7 vaccinesmmune response in the study population. Expanded trials for safety and

Published by Elsevier Ltd.

adenovirus accounted for more than 67% of all ARD in basic train-ing and caused the hospitalization of as many as 20% of militaryrecruits and 90% of pneumonias requiring hospitalizations [4–8].Outbreaks in the civilian population, though unusual, have alsooccurred [9–13].

In the 1960s, the Department of Defense (DoD) and the NationalInstitutes of Health (NIH) contracted Wyeth Laboratories to pro-duce oral, live, enteric coated vaccine tablets against adenovirusserotypes 4 and 7. These vaccines were unique in that they pro-duced a selective, asymptomatic infection of the gastrointestinaltract with production of serum antibody, without producing a sys-temic infection or viral cross interference [14,15]. These vaccineswere safe [16–19] and effective [20–27] and were used extensivelyin military basic training centers from 1971 until 1996. In 1996,the manufacturer, Wyeth, discontinued production. When vacci-nation ceased, adenovirus-associated ARD returned to pre-vaccinelevels and outbreaks of adenovirus-associated ARD became com-

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monplace once again among basic training posts across all services[28–35].

The loss of the adenovirus vaccines and the subsequent surgeof adenovirus-associated ARD prompted a DoD effort to restore theadenovirus vaccines. Cost-effectiveness analyses [36,37] revealedsubstantial case prevention and cost savings with reinstatement ofseasonal or year-round vaccination schedules. Duramed Research, asubsidiary of Barr Laboratories, was contracted by the DoD in 2001to produce the new ADV-4 and ADV-7 vaccines. This paper reportson the results of a Phase 1 study of the new manufacturer’s oral,live, enteric-coated adenovirus vaccines.

2. Methods and materials

2.1. Subjects

A sample size of 60 eligible subjects was the target for the study.Subjects were recruited from the 91W (Combat Medic) School at theU.S. Army Medical Department Center and School (AMEDDC&S),Fort Sam Houston, Texas in the Fall of 2004. The 91W training is8 weeks long during which the student companies (150–200 stu-dents each) are housed in barracks with minimal contact with theoutside community. Each participating subject provided informedconsent. Inclusion criteria included age 18–40 years and robusthealth as evidenced by review of medical history, physical exam-ination and screening laboratory tests. Female subjects had to beof non-childbearing potential or not be pregnant prior to vaccina-tion and must have agreed not to become pregnant or be nursingan infant during the study. Because the 91W training takes placeimmediately after basic combat training it was anticipated thata significant number of the students would have been previouslyexposed to ADV-4 based on DoD surveillance data. A preliminaryseroprevalence survey among 91W student blood donors at FortSam Houston confirmed that only 11% were seronegative for ADV-4(authors’ unpublished data). Only subjects seronegative for ADV-4 or ADV-7 or both on screening serum microneutralization testswere eligible for enrollment.

2.2. Vaccine

The ADV-4 strain used in the vaccine was initially provided toWyeth Laboratories by Dr. Robert Chanock on 15 April 1965. Thisstrain was originally isolated from a throat swab specimen in WI-26

cells of a US Marine recruit with ARD at Camp Lejeune, NC. It wascarried through five additional passages in WI-38 cells at Wyeth. In2000, the Wyeth Primary Seed (Lot 089, passage 12) was passagedtwice in WI-38 cells prior to production of a new Adenovirus type 4Virus Bank (Lot 1096.01) at passage level 15. The GMP lot for vaccineproduction was at virus passage 16 in WI-38 cells.

The ADV-7 strain used in the vaccine was isolated on HEK cellsand transferred (HEK passage 2) to Wyeth Laboratories from theNIH on 25 September 1964. The strain was originally isolated froma rectal swab of a US Marine recruit with ARD at Camp Lejeune, NCon 28 August 1964. Wyeth subsequently passaged the virus on HEKcells (HEK p3-5) and then continued to passage on WI-38 cells toproduce the Wyeth Working Seed (Lot 85, passage 13). In 2000, theWyeth lot 85 viruses were passaged twice in WI-38 cells and a newadenovirus type 7 virus bank (Lot 1097.02) produced at passage 16.The GMP lot for the vaccine production was at virus passage 17.

Both vaccine viruses underwent additional passage in order toprovide enough seed virus for production of the vaccine lots. TheSeed Lot System was used and master and production seeds pro-duced. These seeds are rigorously tested for quality assurance, andonce the lots have passed, then additional vaccine lots never more

(2008) 2890–2898 2891

than 1–2 passages from the master seeds are produced. In this way,testing of bulk vaccine lots is reduced.

Each enteric-coated tablet (ECT) contains live adenovirus, eithertype 4 or 7, at a potency of no less than 32,000 tissue-culture infec-tive doses (4.5 log10 TCID50) as per the package insert of the Wyethvaccines. In previous studies, 4.2–4.7 log10 TCID50 of virus in thevaccine resulted in nearly all vaccine recipients becoming infected[15]. In addition to virus, the tablets contain materials added inprocessing, components arising from the growth and maintenancemedia for virus. There are no antibiotics in the preparations. Thefollowing Quality Control Standards and Requirements specifica-tions were achieved prior to administering the vaccine: physicalappearance, disintegration, infectivity titer, residual solvents, ani-mal safety studies, and virus identification. The type 4 tablets werewhite in appearance. The type 7 outer coating had an added yellowdye (FD&C No. 6 lake dye) giving an off-white appearance to thetablet. The placebo tablets were lactose pills, manufactured to thesame size, shape and appearance as the vaccine tablets describedabove.

2.3. Study design

This study was a randomized, double-blind, placebo-controlledsingle-center trial conducted in the Fall of 2004. Because of thetime requirements for the antibody assays and the large numberof subjects that had to be screened to meet eligibility require-ments, screening took place up to 42 days prior to the day ofvaccination. To eliminate any coercion, prospective volunteers werebriefed on the specifics of the study by a military study investi-gator dressed in civilian clothes, with no other members of thevolunteers’ chain of command present, after which any questionswere answered by a study investigator with a civilian ombudsmanpresent, without other investigators or volunteers’ chain of com-mand present in the same room. The subjects then reviewed andsigned the informed consent document. The investigators deter-mined each subject’s eligibility for the study by conducting aninterview reviewing the inclusion/exclusion criteria with each sub-ject, and performing a screening physical examination. Blood wasdrawn for HBsAg, HCV, HIV, creatinine (Cr), alanine aminotrans-ferase (ALT), complete blood count (CBC), and adenovirus 4 and 7serologies. All female subjects underwent a urine pregnancy test.Any identified abnormalities in medical history/physical exami-nation or screening labs considered clinically significant in theopinion of the investigators led to disqualification of the subject.

During the time period from consent and screening through vacci-nation and the duration of the study, all subjects were engagedin their 91W training at the AMEDDC&S, Fort Sam Houston,TX.

On vaccination day (Day 0), subjects who met all eligibility crite-ria were randomized to receive either the ADV-4 or ADV-7 vaccinetablets together or two placebo tablets. Subjects were interviewedand examined for evidence of acute illness on the day of vaccinationprior to study test article administration under direct observation.

During the first 7 days of the study, all subjects were asked tocomplete a daily symptom diary. On Days 7, 14, 21, 28, and 56 of thestudy, subjects returned for scheduled follow-up visits with studypersonnel. At these visits (1) diaries were reviewed and turnedin (Day 7), (2) adverse events (AE) were reviewed, (3) symptom-directed physical exams were done, including measurement of vitalsigns, (4) blood was drawn for safety evaluation, serology (exceptDay 21), and viremia, (5) throat swab and stool or rectal swab (doneonly if subject was not able to submit a stool sample) specimenswere collected for virus isolation, and (6) all female subjects under-went urine pregnancy testing. A Day 180 contact was made witheach subject to ascertain any additional reports of serious adverse

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events (SAE) or any other information related to the subject’s par-ticipation in the initial 56-day study.

2.4. Randomization

At the vaccination day visit (Day 0), subjects were randomizedto receive either vaccine or placebo tablets in a 1:1 ratio using ablock size of four and stratified by serotype. Randomization wasstratified to ensure that (1) at least 15 subjects in each treatmentgroup (vaccine and placebo) were seronegative to ADV-4 at baselineand (2) at least 15 subjects in each treatment group (vaccine andplacebo) were seronegative to ADV-7 at baseline (Day 0). A subject,who was seronegative at baseline to both ADV-4 and ADV-7, whenrandomized, was counted as satisfying the criterion for the 15 sub-jects per treatment group criterion for both the ADV-4 stratum andthe ADV-7 stratum.

2.5. Specimen collection and analysis

Throat swabs and rectal swabs were collected using the BBLCulturette system, placing the swab in Remel M4 transport media(Remel, Lanexa, KS), frozen to −70 ◦C and transported to the lab-oratory in sterile containers. Stool specimens were collected instool specimen cups, processed by placing 1 gm stool into 9 mLHBS/penicillin/streptomycin/BSA solution (resulting in a 10% sus-pension), frozen to −70 ◦C and then transported to the virologylab for isolation and identification. Blood specimens were collectedon each scheduled visits and processed and sent for routine clin-ical tests, viral isolation and adenoviral serology. All specimenswere stored at −70 ◦C. Adenoviral serology (neutralizing antibody)and cultures were performed by the Adenovirus Clinical Labora-tory, Department of Virus Diseases, Walter Reed Army Institute ofResearch (WRAIR). Routine clinical laboratory tests were done bya CAP-certified central clinical laboratory. ARD viral culture andPCR analyses were done by the Naval Respiratory Disease Lab-oratory (NRDL), San Diego, CA. Stool viral culture and isolationwere done at the Department of Pathology, WRAIR, Washington,DC. The handling of all specimens occurred in class 2 laminarflow hoods to protect both the personnel and integrity of thesamples.

2.6. Laboratory

2.6.1. Virus isolation and identification

Virus culture procedures to detect the presence of virus in blood,

stool and throat used A549 human lung carcinoma cell lines aspreviously described [38,39]. Specimens demonstrating cytopathiceffect (CPE) were stained by direct immunofluorescent technique(D3 DFA, Diagnostic Hybrids Inc., Athens, OH), followed by neu-tralization tests to identify virus serotype [40]. PCR was done onisolated virus to determine vaccine or wild-type.

2.6.2. Serological assaysA modification of the colorimetric microneutralization test orig-

inally described by Crawford-Miksza and Schnurr [41] was used.One hundred TCID50 (±0.7 log) of virus was the challenge virusdose. Negative and positive human and animal homologous ref-erence antisera were employed in each test to identify the virusand as sensitivity control. Serum specimens from Days 0, 7, 14and 28 from each subject were tested concurrently in the sameassay.

2.6.3. ADV PCRPCR was performed as previously described for ADV types 3, 7

and 21 [42], species-specific (A-F) [43], subgroup C [44], and ADV

(2008) 2890–2898

type 4 [45]. Acceptable specimens had total nucleic acid extractedfrom oropharyngeal swabs using the QIAmp® DNA 96 blood kit(Qiagen Inc., Valencia, CA). Detection of amplified products wasdone by agarose gel electrophoresis with staining by ethidiumbromide (Sigma, St. Louis, MO), and was recorded with a digi-tal microscopy documentation system (Kodak Digital Science DC120; Kodak, Rochester, NY). To evaluate the possible presence ofinhibitors in throat swab samples collected and stored in viral trans-port media, PCR was performed before and after DNA extraction byusing the QIAmp blood kit. From either the eluted DNA or the unex-tracted sample (in viral transport media), 10 �L of template wasused for PCR. Each test included negative (water) and positive (dilu-tions of stock ADV-4 or ADV-7) extraction and PCR controls. Assayswere accepted only when all controls gave the expected results. Thetechnician performing the PCR’s was blinded to specimen cultureresults.

Primers developed at the NRDL were used to distinguish wild-type adenovirus infections from vaccine type. Two primer pairsfor the wild circulating strain of ADV type 4, and 2 primerpairs for the vaccine strain were developed targeting identicalpositions of the hexon gene. Total nucleic acid extraction wasperformed as described above. PCR was then performed understandard conditions after optimization of the annealing temper-ature. Optimization and validations were performed using controlsincluding the vaccine strain itself, well-characterized prototypestrains, and strains identified as “vaccine” or “wild-type” throughhexon sequencing [46]. A wide variety of negative controls includ-ing common commensals and non-ADV-4 respiratory pathogenswere also included, and specificity was demonstrated. To be des-ignated a “wild-strain”, a positive PCR result was required for bothwild-type primer sets, with simultaneous negative results to bothvaccine–strain primer sets.

2.7. ARD surveillance

Active and passive surveillance for cases of ARD among subjectswas done during the study. Subjects were issued a study identifica-tion card, and their military identification cards were marked witha blue dot. ARD cases were managed per the medical facility’s stan-dard operating procedure (SOP) for febrile illness; in addition, sixthroat swabs (three for adenoviral isolation and identification, threefor bacterial isolation and identification) were taken. The throatswabs were placed in Remel M4 media, frozen to −70 ◦C, and sent to

the NRDL, San Diego, CA, for isolation, identification and serotyping.Active surveillance was done at each scheduled study visit. All sub-jects with fever detected at these follow-up visits were evaluated bya study investigator with a medical history, physical examination,throat swabs and serology, and, if indicated, referred for furthermedical management at the post hospital.

2.8. Safety analysis

All subjects who received test article were included in the safetyanalysis. Adverse events (AEs) reported from study Day 0 to 180served as the principal means to evaluate safety. Before Day 56, AEswere captured during the regularly scheduled site visits on the AEcase report forms, from the 7-day symptom diary, and from the ARDsurveillance described above. The diary solicited for severity (one-unnoticed, two-little effect on daily activities or three-large effecton activities) of each of 10 specified symptoms. Each reported eventwas classified and presented according to the MedDRA system. Seri-ous adverse events (SAEs) and AEs with 5% or more occurrence ratewere tabulated. CBC, ALT, Cr, and blood chemistries were obtainedon each scheduled visit.

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dosed. Among the remaining 58 subjects, 30 received vaccine and28 received placebo. Four subjects (three placebo and one vaccinerecipient, 7%) did not complete the study. Two were involuntarilydischarged from training, and two requested to be withdrawn fromthe study due to the number of blood-draws. A total of 32 subjects(55.2%) responded to follow-up at Day 180.

3.2. Demographics and other baseline characteristics

Subjects’ demographic information at baseline is summarizedin Table 1. Among the 58 treated subjects, 69% were Caucasian, 19%were Hispanic and 7% were African–American. Seventy six percentof the subjects were males; 24% were females. The median age was19 years. The differences between the groups were not statisticallysignificant (all P > 0.2).

3.3. Safety

3.3.1. Adverse eventsThe Phase 1 study results indicate that the vaccines were

well tolerated. During the 56 days of the study, 24/30 (80.0%)

Table 2Adverse events with 5% or more occurrence rate by body system and treatmentgroupa from Day 0 to 56—all treated patients

A. Lyons et al. / Vacc

Table 1Demographic information

Race Vaccine (NV = 30)a N (%)

African-American 3 (10%)Asian 0 (0%)Caucasian 23 (77%)Hispanic, white 0 (0%)Hispanic, non-white 4 (13%)Other 0 (0%)

GenderMale 22 (73%)Female 8 (27%)

Age (years)Mean (S.D.) 20 (2.6)Median 19Min, Max 18, 28

a Total N in cohort.

2.9. Immunogenicity analysis

2.9.1. SeroconversionFor subjects who were seronegative (neutralizing antibody titer

<1:4) to either ADV-4 or 7 at Day 0, seroconversion was defined asa fourfold or greater increase in titer after immunization (to attaina titer of at least 1:8). Seroconversion rates seen at Day 28 for ADV-4 and 7 and the corresponding 95% confidence intervals (95% CI)were calculated by treatment group. The seroconversion rates atDay 28 were the primary immunogenicity endpoints. In addition,cumulative seroconversion rates for ADV-4 and 7 across specifiedtime points through Day 56 were calculated by treatment group.The GMT’s for the groups by study days were calculated.

2.9.2. Booster responseFor subjects who were seropositive (neutralizing antibody titer

≥1:4) to either ADV-4 or 7 at Day 0, a booster response was definedas a fourfold or higher increase in the titer after immunization.Cumulative booster response rates for ADV-4 and 7 across timepoints through Day 56 were calculated.

2.9.3. Statistical analysisData were summarized in tabular format. Descriptive statistics

included the number of patients, mean, median (where appro-priate), standard deviation or standard error of the mean (S.E.),minimum and maximum. Discrete data were summarized using

frequencies and/or percentages. Ninety five percent confidenceintervals were calculated by the exact binomial method. P valueswere calculated using the Fisher’s exact test (GraphPad Software©,2005) for safety results and demographics, two-sided, comparedto placebo. Odds ratios were calculated using the WinPepi soft-ware (Compare2 module, copyright JH Abramson, November 2007,Version 4.7).

3. Results

3.1. Disposition of volunteers

A total of 969 potential subjects were briefed by company (3).Four hundred and seven (42%) gave informed consent and werescreened for eligibility. Of these, 246 (60%) were seropositive toboth ADV-4 and 7; 72 (18%) were type 4 negative and type 7 pos-itive; 74 (18%) were type 7 negative and type 4 positive; 15 (4%)were seronegative to both type 4 and type 7. The overall seropreva-lence rates for ADV-4 and ADV-7 were 79% and 78%, respectively.Sixty subjects were selected and randomized (1:1 ratio) to eithervaccine or placebo. On study Day 0, 60 subjects presented them-

(2008) 2890–2898 2893

Placebo (NP = 28)a N (%) Total (NT = 58) N (%)

1 (4%) 4 (7%)0 (0%) 0 (0%)

17 (61%) 40 (69%)2 (7%) 2 (3%)5 (18%) 9 (16%)3 (11%) 3 (5%)

22 (79%) 44 (76%)6 (21%) 14 (24%)

21 (4.0) 20 (3.3)20 1918, 36 18, 36

selves for dosing. Two of the 60 subjects had a fever and were not

Adverse event Vaccine (%) Placebo (%) Odds ratio (95% CI)b

Nasal congestion 10 (33.3) 16 (57.1) 0.38 (0.11–1.23)Dry cough 10 (33.3) 10 (35.7) 0.90 (0.27–3.05)Sore throat 8 (26.7) 8 (28.6) 0.91 (0.25–3.38)Productive cough 2 (6.7) 3 (10.7) 0.60 (0.05–5.69)Seasonal rhinitis 2 (6.7) 1 (3.6) 1.93 (0.09–117.93)Runny nose 1 (3.3) 3 (10.7) 0.29 (0.01–3.92)Wheeze 1 (3.3) 3 (10.7) 0.29 (0.01–3.92)Sneeze 0 (0.0)c 2 (7.1)c 0.29 (0.01–3.92)Abdominal pain 5 (16.7) 1 (3.6) 6.75 (0.72–321.72)Diarrhea 4 (13.3) 2 (7.1) 2.00 (0.26–23.65)Nausea 4 (13.3) 6 (21.4) 0.56 (0.10–2.76)Pneumonia 1 (3.3) 3 (10.7) 0.26 (0.00–3.62)Sinusitis 3 (10.0) 2 (7.1) 1.33 (0.14–17.14)Furuncle 2 (6.7)c 0 (0.0)c 3.00 (0.22–163.01)Arthralgia 4 (13.3)c 0 (0.0)c 5.38 (0.54–263.58)Plantar fasciitis 2 (6.7)c 0 (0.0)c 3.00 (0.22–163.01)Headache 6 (20.0) 6 (21.4) 0.92 (0.21–4.01)Pyrexia 2 (6.7) 6 (21.4) 0.26 (0.02–1.69)Increased WBC 0 (0.0)c 3 (10.7)c 0.21 (0.00–2.34)

a None statistically significant to P < 0.05.b WinPepi software (Compare2 module); copyright JH Abramson, November 2007,

Version 4.7.c Added 1.0 to % for calculation of odds ratios.

2894 A. Lyons et al. / Vaccine 26 (2008) 2890–2898

Table 3Clinical events, immunogenicity and viral cultures of all treated subjects

Subject Group Clinical event Day 0 antibody titer Post-vaccination peak antibody titer Isolate in stool culture Isolate in throat culture

ADV-4 ADV-7 ADV-4 ADV-7

103 Vaccine <4 <4 15 58 4, 7 −104 <4 <4 29 80 4, 7 −106 38 6 62 62 − −107 137 <4 211 30 7 −301 <4 8 19 420 4, 7 −303 168 143 116 246 − −305 <4 90 123 1022 4 −309 <4 <4 32 55 4, 7 −311 4 37 55 181 7 −314 <4 <4 <4 1439 7 −315 58 87 198 15,858 7 −318 <4 5 <4 9 − −319 SAEa (pneumonia) <4 19 46 637 4, 7 −321 36 1150 101 3085 − −328 <4 55 69 564 − −331 <4 1150 11 1638 4 −332 <4 27 42 5020 4, 7 −501 43 <4 199 143 7 −503 312 <4 368 106 7 −506 67 <4 77 20 7 −507b 98 <4 127 <4 − −511 28 6 166 11,066 7 −514 62 <4 98 15 7 −516 117 <4 1178 <4 − −517 724 <4 2064 <4 − −521 81 <4 127 11 7 −522 SAEa (appendicitis) 189 <4 238 12 7 −525 295 <4 290 <4 − −528 15 <4 29 <4 − −530 56 <4 159 49 7 −101 Placebo <4 <4 <4 <4 − −109 32 7 77 6 − −302 ARDc <4 31 159 62 wt 4d wt 4d

304b 116 43 127 30 − −306b 250 <4 227 <4 − −307 24 1765 23 953 − −310 <4 6 <4 5 − −312 <4 9 <4 11 − −313b <4 8 <4 8 − −317 SAEa (pneumonia) <4 <4 98 7 − wt 4d

320 <4 <4 <4322 <4 65 <4324 <4 17 <4325 273 23 102326 123 409 84329 SAEa (pneumonia) <4 583 81330 107 298 98

502 51 <4 74504 SAEa (thigh abscess) 21 <4 18505 134 <4 168508 91 <4 205515 27 20 31519 380 <4 279520 212 <4 246523 189 <4 171527 145 <4 410529 32 <4 45531 12 <4 23

a Serious adverse event.b Specimens were not collected from all time points after Day 0. −, negative.c Acute respiratory disease.d Wild-type.

vaccinees and 26/28 (92.9%) placebo recipients reported at leastone AE. Table 2 gives the incidence of AEs that occurred fromDay 0 to 56 with a 5% or greater frequency. Generally speak-ing, there were less respiratory symptoms, less pneumonia, lessfever and less WBC elevations in the vaccine group than theplacebo group. However, there was a greater incidence of abdom-

6 − −98 − −16 − −20 − −

302 − −5210 wt 4d wt 4d

252 − −

<4 − −<4 − −<4 − −<4 − −29 − −<4 − −<4 − −<4 − −<4 − −<4 − −<4 − −

inal pain, diarrhea and arthralgia reported in the vaccine groupthan in the placebo group. However, none of the differencesobserved between the vaccine and placebo groups was statisticallysignificant.

According to the symptom diaries, a greater incidence of abdom-inal cramping, abdominal pain, and diarrhea was found in the

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A. Lyons et al. / Vacc

vaccine group compared to the placebo group during the first 7days after vaccination. In contrast, fewer subjects reported nau-sea and sore throat in the vaccine group than those in the placebogroup. In addition, the vaccine group reported no fever, whereasseven cases of fever were reported in the placebo group from Day 1to 7. Overall, none of the symptoms reported in the vaccine groupwere considered as having a “large effect” on the subjects’ dailyactivities, whereas “cough”, “fever” and “headache” were reportedas having a “large effect” on the daily activities in the placebo group.None of the differences observed was statistically significant. Theabove findings were consistent with the AE reporting. There wereno notable changes in vital signs (i.e., systolic or diastolic bloodpressure, temperature, or weight) over time within or betweentreatment groups.

3.3.2. Serious adverse eventsThere were a total of five SAEs reported of which three were

reported more than 28 days after receiving the vaccination. Therewere no deaths. Of these five SAEs, three were pneumonia. Of these,one (vaccine recipient) had negative throat cultures for ADV. Theother two cases (placebo recipients) had positive throat cultures forwild-type ADV-4. No one discontinued the study due to SAEs. By theDay 180 follow-up, two additional SAEs were reported by subjectsreceiving the study medication. These were for presumptive appen-dicitis (vaccine recipient) and an MRSA right thigh abscess (placebo

recipient). The case of appendicitis was presumptive because thesubject recovered without surgery. These SAEs were felt not to berelated to the vaccines. Table 3 lists the SAEs reported in this studyalong with all treated subjects.

3.3.3. Clinical laboratory evaluationsNo notable changes were observed over the study period for

each of the safety lab parameters in the vaccine and placebo groups.

3.4. Wild-type ADV-4 infection among study cohort

3.4.1. Seroconversion prior to vaccinationThere was circulating wild-type ADV-4 among one of the

enrolled companies during the 42-day enrollment period, resultingin some seroconversions among the enrolled subjects prior to vac-cination. Table 4 shows the change in ADV-4 and ADV-7 serostatusin the study cohort between screening and day of vaccination, Day0. Twelve (36.4%) subjects (six vaccine and six placebo), who wereADV-4 seronegative at screening seroconverted by Day 0. Their type4 titers ranged from 4 to 273 (GMT 60). For ADV-7, there was nochange in the number of subjects, 31, who were seronegative at

Table 5Cumulative seroconversion by treatment group over time

Immunogenicity

Vaccine

Cumulative SCRa (%, CI) GMT

ADV-4Day 7 0/11c (0%, 0–28) –Day 14 6/11 (55%, 23–83) 15Day 28 8/11 (73%, 39–94) 20Day 56 8/11 (73%, 39–94) 22

ADV-7Day 7 0/17 (0%, 0–20) –Day 14 10/16 (63%, 35–85) 55Day 28 10/16 (63%, 35–85) 35Day 56 11/16 (69%, 41–89) 34

a Seroconversion rate.b Geometric mean titer.c Denominator is the number of subjects actually seronegative on Day 0 and with spec

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Table 4ADV-4 and ADV-7 Antibody status by treatment group at screening and Day 0 priorto vaccination

Antibody status Vaccine group Placebo group Total

ADV-4 ADV-7 Screening Day 0 Screening Day 0 Screening Day 0

Negative Negative 4 4 2 3 6 7Negative Positive 13 7 14 7 27 14Positive Negative 13 13 12 11 25 24Positive Positive 0 6 0 7 0 13Type 4 (−) 17 11 16 10 33 21Type 4 (+) 13 19 12 18 25 37Type 7 (−) 17 17 14 14 31 31Type 7 (+) 13 13 14 14 27 27

Sum 30 30 28 28 58 58

screening and on Day 0. Table 4 also shows the distribution of ADV-4 and ADV-7 serostatus among the two treatment groups. Due tothe circulation of wild-type ADV-4 prior to vaccination 13 subjects,6 in the vaccine group and 7 in the placebo group were random-ized even though they no longer fulfilled the serologic inclusioncriterion. The circulation of wild-type ADV-4 continued during thestudy as evidenced by three cases of adenoviral ARD in the placebogroup, two resulting in pneumonia and one detected on a sched-uled follow-up day, but not hospitalized. All three seroconverted toADV-4.

4. Immunogenicity

4.1. Seroconversion

Table 5 shows the seroconversion rates with 95% CI’s. AtDay 0, there were 21 subjects (11 vaccine and 10 placebo) whowere seronegative to ADV-4. Eight of 11 (73%) vaccine recipientsseroconverted (GMT 20) to ADV-4; 3/9 (33%) placebo recipientsseroconverted (GMT 59) by Day 28. One placebo recipient did nothave Day 28 data for the analysis. The three ADV-4 placebo recip-ients who seroconverted were all shown to have been exposed towild-type ADV-4 infection by PCR of throat swabs, while none of thevaccinees who seroconverted had adenovirus isolated from throatswabs.

At Day 0, there were 31 subjects (17 vaccine and 14 placebo) whowere seronegative to ADV-7. Ten of 16 (63%) in the vaccine group(GMT 14.4) and 0/13 in the placebo group seroconverted to ADV-7 by Day 28. One placebo and one vaccine recipient did not haveblood collection after Day 7 for the analysis. The 95% confidence

Placebo

b Cumulative SCRa (%, CI) GMTb

0/10 (0%, 0–31) –2/9 (22%, 3–60) 163/9 (30%, 7–70) 593/9 (30%, 7–70) 93

0/14 (0%, 0–23) –0/13 (0%, 0–25) –0/13 –0/13 –

imen available on specified day post-vaccination.

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intervals of the seroconversion rates were quite wide as expectedwith the small sample size.

Table 5 also shows seroconversion over time. For both vaccines,the majority of the seroconversions occurred between Day 7 and 14.For ADV-4, the cumulative seroconversion rate by Day 14 was 55%(vaccine) vs. 22.0% (placebo). For ADV-7, the rates were 58.8% (vac-cine) vs. 0% (placebo). By Day 56, the cumulative seroconversionrate for ADV-4 was 73% (vaccine) vs. 30.0% (placebo); for ADV-7,69% (vaccine) vs. 0% (placebo). The GMT peaked earlier (Day 14)and was higher in ADV-7 than in ADV-4. The GMT from wild-typeADV-4 infection in the placebo group was higher than that seenwith vaccination.

4.2. Booster response

A booster response was seen in 4 of 19 (21%) vaccinated subjectswith pre-existing ADV-4 antibody. The GMT in these four subjectswas 27, compared with 87 in the 15 (79%) seropositive subjectswithout a booster response. For ADV-7 the booster response wasseen in 9 of 13 (69%) vaccinated subjects with pre-existing ADV-7antibody. The GMT in those with booster response was 28, com-pared with 166 in the 5 (38%) without booster response.

5. Viral shedding

5.1. Throat

Throat swab specimens collected for all treated subjects atscheduled visits on Days 0, 7, 14, 21 28, and 56 were tested for thepresence of adenovirus. Only one throat swab of 334 scheduled col-lections was found to be positive for ADV-4. The positive specimencame from a placebo subject 302 on Day 7 and was found to be awild-type ADV-4 by PCR. This volunteer also seroconverted to ADV-4. Two additional positive throat culture results were found duringthe ARD workups for two placebo subjects, 317 and 329, who werehospitalized, on Day 10 and 12 for pneumonia (see Table 3). The twoADV type 4 isolates were both wild-type by PCR. These subjects alsoseroconverted to type 4 ADV. No vaccine virus was detected in thethroat swab specimens from any subjects at any time.

5.2. Stool or rectal swab

In the vaccine group, 8/30 (27%) were positive at least once for

ADV-4 fecal viral shedding over the entire study period. Of the 11subjects seronegative to ADV-4 at baseline and who received vac-cine, shedding was first detected in 7 (64%) on Day 7 and was lastdetected at Day 21. Overall, 8/11 (73%) subjects in this group shedvirus in the stool. Two ADV-4 seronegative subjects who receivedplacebo, 302 and 329, shed virus; these were the two cases ofwild-type ADV-4 ARD. None of the 19 subjects seropositive for ADV-4 at baseline and who received vaccine shed virus in the stool.As expected, those who were ADV-4 seropositive at baseline andreceived placebo, none shed virus in the stool (see Table 3).

In the vaccine group, 18/30 (60%) were positive at least once forADV-7 fecal viral shedding over the entire study period. Of the 17subjects seronegative to ADV-7 at baseline and who received vac-cine, shedding was first detected in 10 (59%) on Day 7 and noneshed virus after Day 14. Overall, 12/17 (71%) subjects in this groupshed virus in the stool. Unlike ADV-4 seven of 13 subjects (54%)who were ADV-7 seropositive at baseline and who received vac-cine shed virus. All but one of these seven subjects showed a boosterresponse, as expected with the evidence of vaccine virus replica-tion in vivo. Shedding period was between Days 7 and 14. None ofplacebo subjects shed ADV-7.

(2008) 2890–2898

Table 6Correlation between fecal viral shedding and immunogenicity in the vaccine group

Antibody status at Day 0 Fecal viral shedding Immunogenicity

Type 4 (−) 8/11 positive Same 8 seroconvertedType 4 (+) 0/19 positive 4/19 boostedType 7 (−) 12/16 positive Same 12 seroconvertedType 7 (+) 7/13 positive 9/13 boosted

Table 6 shows the correlation between fecal viral shedding andimmunogenicity in the vaccine groups. Of those eight subjects whowere ADV-4 seronegative at baseline who received the vaccinesand shed virus, all seroconverted to ADV-4. Similarly, of 12 subjectsseronegative to ADV-7 at baseline who received the vaccines andshed virus, all seroconverted to ADV-7. None who were seropositiveto ADV-4 at baseline (GMT 74) and received vaccine shed virus.In contrast, 6/13 (46%) subjects seropositive for ADV-7 at baseline(GMT 21) and received vaccine, shed virus in the stool. The GMT forthose seropositive to ADV-7, received vaccine and did not shed virusin the stool was 83. There were four subjects doubly seronegative atbaseline who received vaccine. Three shed both ADV-4 and ADV-7in their stools and seroconverted to both; the remaining shed onlyADV-7 and seroconverted only to ADV-7.

6. Viremia

Blood samples taken from all treated patients at study Days0, 7, 28 and 56 were cultured for adenovirus. No adenovirus wascultured from any of the serum samples.

7. Discussion

Adenovirus respiratory illness has re-emerged in the militaryrecruit population after cessation of vaccination in 1999. TheDuramed ADV vaccines are designed to replace the Wyeth prod-uct. The current oral vaccine virus lots were derived from the samevirus seeds with current tableting technology and otherwise dif-fer from the Wyeth product only in the absence of antibioticsand replacement of color dye in the ADV-7 vaccine tablets. TheWyeth vaccine was studied extensively and administered to sev-eral million recruits over more than 25 years and demonstrated anexcellent safety and efficacy profile. Indeed, in a review of archivedreported adverse events in the Vaccine Adverse Events ReportingSystem (VAERS) database (http://www.vaers.hhs.gov), since 1964,

only five reports of adverse events were reported in conjunctionwith the administration of live, oral adenovirus vaccines. It shouldbe noted that these adverse events were reported in conjunctionwith the concomitant receipt of other vaccines like oral polio, DTP,meningococcal, MMR, and influenza, so the individual documentedadverse event could not be definitely attributed to the adenovirusvaccines alone. The primary objectives of this Phase 1 study wereto evaluate the safety and immunogenicity of the Duramed ADVvaccines.

The Army combat medic students were selected for this trialbecause their demographics, training conditions and their mini-mum contact with the general population simulated characteristicsof military recruits in basic training, who are the target popula-tion for the oral adenovirus vaccines. In addition, medic studentshad recently completed basic training and were likely to havebeen exposed to ADV-4, the current predominant serotype circu-lating among training camps, resulting in herd immunity againstADV-4 and with less likelihood of contracting wild-type ADV-4disease. Indeed, our screening indicated a 79% ADV-4 seropreva-lence among the student population. Yet unfortunately there wasstill circulation of wild-type ADV-4 in the course of this study.

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This was somewhat unexpected given the very high seropreva-lence rate in this population. In any case the presence of wild-typeADV-4 resulted in some seroconversions among the enrolled sub-jects prior to vaccination. Twelve (36.4%) ADV-4 seronegativesubjects seroconverted to ADV-4 between screening and vaccina-tion. These wild-type ADV-4 infections were mostly asymptomatic.Later on during the study wild-type ADV-4 infection was docu-mented in three placebo subjects who developed ARD, two withpneumonia. Interestingly, no ADV-4 ARD was seen in the vaccinegroup.

Of note the seroprevalence rate for ADV-7 in the subject popu-lation was 78%, similar to ADV-4. This is consistent with previouslyobserved (61%) ADV-7 seroprevalence in recruits [47]. In contrast, aprevious 1993 serosurvey in recruits showed only 27% had ADV-7 antibody [48]. The reasons for the increase are not clear [49]since ADV-7 had not been documented as a significant cause ofARD in military recruits since 1997 [50]. Possible explanationsinclude increased ADV-7 infections in lower age groups among thegeneral population, more asymptomatic infections among militaryrecruits that were not detected by a passive surveillance of onlysymptomatic disease, or heterotypic antibody responses to ADV-7 following ADV-4 infection. There were no documented cases ofwild-type ADV-7 ARD during the study. More studies of seropreva-lence of ADV-7 in the current unvaccinated recruit population areneeded.

In this descriptive Phase 1 study, the Duramed live, oral ADV-4and ADV-7 vaccines were safe and well tolerated. The overall inci-dence rates of AEs were similar between the vaccine and placebogroups. The most frequently (>10%) reported AE’s were nasal con-gestion, cough, sore throat, headache, abdominal pain, diarrhea,nausea, arthralgia and sinusitis. There was a trend towards lessnasal congestion, pneumonia, fever and WBC elevations and moreabdominal pain and diarrhea in the vaccine group. Diarrhea hadpreviously been reported in recipients of the previously licensedvaccines [19] and could be related to the oral route of the vac-cines. The abdominal pain and diarrhea only appeared within thefirst week after vaccination, was transient and mild to moder-ate in intensity. No soldiers missed any training time as a resultof taking these vaccines. The vaccine group reported no symp-toms causing large effects on daily living while subjects in theplacebo group reported large effects caused by cough, fever andheadache. This suggests that symptoms from wild-type ADV-4were more severe than vaccine-related related respiratory symp-toms. There were three hospitalizations for pneumonia within the

first 56 days; two in the placebo group diagnosed as wild-typeADV-4 by throat culture, the other in the vaccine group had a neg-ative workup for ADV-4 and ADV-7. None of the three pneumoniaswere clinically severe. Overall the similarity of occurrence of AEsin the vaccine and placebo group suggests there were no signifi-cant vaccine-attributable AEs. The safety objective of the study wasachieved.

As was the case for the previously licensed vaccines, theDuramed ADV vaccines induced a, selective, contained ADV infec-tion in the gastrointestinal tract. This infection induces serumneutralizing antibodies and is thought to protect against aden-ovirus infection of the respiratory tract. However it is possibleserum neutralizing antibody, while a surrogate marker of immunity[14,26,51], is not mechanistically responsible for protection againstadenovirus infection acquired through the respiratory mucosa. Therole of the common mucosal immune system in oral adenovirusvaccine-induced immunity should be studied. Another marker forvaccine virus gastrointestinal replication is isolation of ADV inthe stool. Administration of the Wyeth vaccines had consistentlyresulted in ADV being detected in stool samples collected up to28 days or longer after vaccination [14]. In our Phase 1 study viral

(2008) 2890–2898 2897

shedding in stool usually occurred by Day 7. Virus shed in the stoolwas clearly from the inoculum since all seronegative subjects whowere vaccinated and later shed ADV-4 or 7 or both seroconvertedto whichever virus was in the stool. No one shed virus in the stoolwithout either seroconverting or mounting a booster response tothat virus. The presence of any pre-existing serum antibody againstADV-4 (range from 1:4 to 1:724) was associated with lack of anystool shedding of ADV-4 vaccine virus, even though there weresome who mounted an antibody booster response which could bedue to a heterotypic antibody response. The baseline level of anti-body in the group without booster responses (GMT 97) was notablyhigher than the ones who boosted (GMT 27) from vaccination. Thissuggests that at the lower levels of antibody the ADV-4 vaccine viruswas able to infect and replicate enough to induce an anamnesticresponse but not able to replicate to the extent detectable by stoolshedding. At higher level of antibody there was sterile immunity atthe level of the gut. In contrast, ADV-7 vaccine virus can overcomelower levels of pre-existing antibody, of titers ranging from 1:6 to1:87, to cause significant enteric infection and stool shedding. Simi-lar to ADV-4, the level of pre-existing antibody was associated withwhether ADV-7 vaccine virus-caused booster responses. Those thatdid not boost with vaccination had GMT of 175 compared to a GMTof 24 in those who boosted from vaccination. These observations ofthe vaccine viruses support the notions that (a) detection of vaccinevirus in stool is a surrogate marker of vaccine take and (b) serumneutralizing antibody is a valid surrogate marker of protection, andthat the protective levels may differ between ADV-4 and 7.

As was found with the Wyeth vaccines under conditions of mil-itary training where the risk of vaccine virus transmission fromvaccinees to nonvaccinees was remote [14,52,53] we found no evi-dence of lateral transmission of vaccine virus to the placebo group.

The seroconversion rates at Day 28 (primary serologic end-points), were 73% for ADV-4 and 63% for ADV-7. The ADV-4 vaccineseroconversion rate may be overestimated due to circulation ofwild-type virus during the study. Previous studies of the Wyethvaccines have shown neutralizing antibody seroconversion rates inthe ranges of 74–100% for ADV-4 [14,54,55] and 76–95% for ADV-7 [19,56]. We have conducted a previous study testing the Wyethvaccines in seronegative civilian subjects and saw Day 28 serocon-version rates of 90% (GMT 13) and 93% (GMT 46) for ADV-4 and7, respectively (Kuschner, unpublished data). Because of the smallsample size of this study the CI’s around the immunogenicity esti-mates are wide; nonetheless they do include the Wyeth vaccineestimates.

In summary, given the similarity of the Duramed and Wyeth vac-cines, the safety and immunogenicity results of this study supportthe Duramed vaccines’ further evaluation in an efficacy trial. Thesafety findings of this study are consistent with the safety profilereported in the product labeling of the approved Wyeth product.The threshold level of immunity necessary within the recruit pop-ulation to prevent outbreaks of adenovirus is not clear but pastexperience has shown vaccine seroconversion rates underestimatethe effectiveness of the vaccines [24,26,51]. Because Wyeth vac-cines are no longer available for non-inferiority studies an efficacyfield trial of the Duramed vaccines is necessary and is underway.

Acknowledgements

The authors wish to thank Dr. Kathy Reape of Duramed Researchfor her review of the manuscript and her thoughtful comments, andDr. Amy Kitchen of the DVD, WRAIR for her review and manuscriptpreparation.

Financial support: US Army Medical Research and Materiel Com-mand and VaccGen International LLC.

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