Safety and immunogenicity of an oral, replicating adenovirus serotype 4 vector vaccine for H5N1 influenza: a randomised, double-blind, placebo-controlled, phase 1 study

Summary Background Replication-competent virus vector vaccines might have advantages compared with non-replicating vector vaccines. We tested the safety and immunogenicity of an oral adenovirus serotype 4 vector vaccine candidate (Ad4-H5-Vtn) expressing the haemagglutinin from an avian influenza A H5N1 virus. Methods We did this phase 1 study at four sites in the USA. We used a computer-generated randomisation list (block size eight, stratified by site) to assign healthy volunteers aged 18–40 years to receive one of five doses of Ad4-H5-Vtn (107 viral particles [VP], 108 VP, 109 VP, 1010 VP, 1011 VP) or placebo (3:1). Vaccine or placebo was given on three occasions, about 56 days apart. Participants, investigators, and study-site personnel were masked to assignment throughout the study. Subsequently, volunteers received a boost dose with 90 μg of an inactivated parenteral H5N1 vaccine. Primary immunogenicity endpoints were seroconversion by haemagglutination-inhibition (HAI), defined as a four-times rise compared with baseline titre, and HAI geometric mean titre (GMT). We solicited symptoms of reactogenicity daily for 7 days after each vaccination and recorded symptoms that persisted beyond 7 days as adverse events. Primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01006798. Findings We enrolled 166 participants (125 vaccine; 41 placebo) between Oct 19, 2009, and Sept 9, 2010. HAI responses were low: 13 of 123 vaccinees (11%, 95% CI 6–17) and three of 41 placebo recipients (7%, 2–20) seroconverted. HAI GMT was 6 (95% CI 5–7) for vaccinees, and 5 (5–6) for placebo recipients. However, when inactivated H5N1 vaccine became available, one H5N1 boost was offered to all participants. In this substudy, HAI seroconversion occurred in 19 of 19 participants in the 1011 VP cohort (100%; 95% CI 82–100) and eight of 22 placebo recipients (36%; 17–59); 17 of 19 participants in the 1011 VP cohort (89%; 67–99) achieved seroprotection compared with four of 22 placebo recipients (18%; 5–40); GMT was 135 (89–205) with 1011 VP, compared with 13 (7–21) with placebo. The cumulative frequency of abdominal pain, diarrhoea, and nasal congestion after all three vaccinations was significantly higher in vaccinees than placebo recipients (21 [16·8%] of 125 vs one [2·4%] of 41, p=0·017; 24 [19·2%] of 125 vs two [4·9%] of 41, p=0·027; 41 [32·8%] of 125 vs six [14·6%] of 41, p=0·028; respectively). No serious treatment-related adverse events occurred. Interpretation Oral Ad4 vector priming might enhance the efficacy of poorly immunogenic vaccines such as H5N1. Funding Wellcome Trust Foundation, PaxVax.

substrate, KPL; Gaithersburg, MD). Spots were enumerated with a computer-assisted ELISPOT reader formatted with image capture and counting software (C.T.L.; Shaker Heights, OH). Vaccine induced responses were scored positive if they exceeded 80 net spots/10 6 cells, after subtracting the no-antigen background, and were >4-fold above the pre-vaccine response.
Ad4-H5-Vtn Polymerase Chain Reaction (PCR): PCR testing was designed at PaxVax, and transferred to Focus Diagnostics who performed the assays. The presence of Ad4-H5-Vtn in clinical specimens (throat swabs, rectal swabs and blood serum samples) was detected by a specific real time PCR method with confirmation of a positive result by both the culture and detection of Ad4-H5-Vtn in A549 cells. PaxVax designed primers and a Taqman probe that are specific to the boundary between Ad4 and the 5' H5-Vtn transgene and supplied them to Focus Diagnostics. The Focus method utilized automated extraction of DNA using the Roche MagNA Pure LC System and real time PCR using Taqman chemistry with the appropriate controls including the use of an internal positive amplification control (IPC) is included with each specimen. Any PCR positive samples were further tested by culture on A549 cells and, following visual detection of cytopathic effects (CPE), were fixed and the presence of H5-Vtn detected in cells using an antibody to the hemagglutinin of A/VietNam/1194/2004. Secondary staining with FITC allows the visual detection of infected cells. In approximately 65% of specimens positive by PCR, the Ad4-H5-Vtn virus was identified in this cell culture method.
Statistical methods Safety and immunogenicity were prospectively specified as co-primary objectives. Safety endpoints included solicited signs and symptoms of reactogenicity reported for 7 days post vaccination on a diary card; unsolicited adverse events; and PCR analyses of rectal, throat and blood samples for evidence of excreted or systemic vaccine virus. Household contacts (HHCs) were monitored at regular intervals for evidence of Ad4-H5-Vtn transmission through PCR analyses of rectal and throat samples, and by serum Ad4 microneutralization assay. Adverse events were also collected for HHCs. Two primary immunogenicity endpoints were pre-specified: seroconversion by H5N1 HAI, defined by a 4-fold rise over the pre-vaccination titer, and HAI geometric mean titer (GMT). Additional endpoints included serum H5N1 microneutralization and H5N1 HA-specific ELISA; cellular immune response measured by ELISPOT; serum Ad4 antibody microneutralization; and vaccine "take" defined as either detection of Ad4-H5-Vtn in a rectal sample or Ad4 seroconversion.
The sample size of 24 vaccine recipients per treatment group was determined by the HAI seroconversion endpoint. Specifically, power to detect a two-fold difference in the seroconversion rate between any two treatment groups was estimated at 76% assuming use of a two-sided Fisher's exact test with p=0·05, and assuming the rate in the better performing group was 80%.
Subjects were enrolled in ascending dose cohorts and randomized in 3:1 ratio to either Ad4-H5-Vtn vaccine or placebo. At each site an independent, unblinded pharmacist dispensed either vaccine or placebo according to a computer-generated randomization list with a block size of 8. Subjects, HHCs, investigators, study site personnel, and medical monitors were blinded to subjects' treatment assignment throughout treatment and follow-up. Unblinded administration of the boost vaccination was offered to both vaccine and placebo recipients. Postrandomization stratification was used for analyses of the impact of baseline Ad4 seropositivity.
Pre-boost reactogenicity rates were calculated from the percentage of subjects who reported a specific sign or symptom at least once following any of the three vaccinations with Ad4-H5-Vtn or placebo. The denominator following the first, second, and third vaccinations was the number of subjects who received the first, second, or third vaccination, respectively. Severity rates were determined by the highest severity reported by a subject for a particular sign or symptom. Confidence intervals for percentages were calculated by the Clopper-Pearson method, and pairwise comparisons between percentages were made using Fisher's exact test. Post-boost reactogenicity rates were calculated from the subset of individuals who received the boost vaccination.
Pre-boost rates for vaccine take, seroconversion by either H5N1 or Ad4 antibodies, and cellular immune response were calculated on cumulative basis where the denominator was the number of subjects with a valid baseline measurement for a particular assay and at least one valid post-vaccination result. The denominator for post-boost rates was the number of subjects who had at least one pre-boost measurement and a valid post-boost result.
Confidence intervals for the geometric mean were estimated by log-transforming the data, constructing intervals based on the t distribution in the log domain, and back-transforming the resulting interval to the original scale. Pairwise comparisons of continuous endpoints were made using either t-tests performed on log-transformed data or Wilcoxon tests.
Analyses were specified in a statistical analysis plan. Analyses and sample size estimation were performed using SAS 9·2, S-Plus 7·0, StatXact 8, and nQuery Advisor 5·0. Severity and potential relationship to vaccine were assessed by blinded investigators at clinical sites. A grade 3 event was considered significant and something that prevented daily activity. There were no grade 4 or 5 events. Events designated as related to vaccine were considered by an investigator to be at least possibly related. ID is a unique pseudo-identifier assigned to each individual in the table.

Safety
'    For each category of reactogenicity, "Any" refers to the percent (95% CI) of subjects who reported that symptom at least once following any of the three vaccinations with Ad4-H5-Vtn or placebo. For "Mild," "Moderate," and "Severe", a subject is counted once at the highest severity level he or she reports. The group comprised of all Ad4-H5-Vtn recipients reported significantly more abdominal pain (p=0·017)*, diarrhea (p=0·027) †, and incidences of nasal congestion/runny nose (p=0·028) ‡ than placebo recipients. A vaccinee is counted once within each category if he/she reports reactogenicity at least once in that category. All subjects who received the boost vaccination completed diary cards; percentages are calculated from the number of subjects in each treatment group. *p=0.042. Both placebo recipients who reported chills graded the symptoms as mild. †p=0.020. All 17 Ad4-H5-Vtn recipients who reported injection site pain graded it as mild.