Study population demographics and vaccine safety.
Baseline demographics are shown in . In VRC 304, 56% of subjects were women, 80% were white, and 13% were African-American, and the mean age at the time of enrollment was 39.6 years. In VRC 305, 46% were women, 73% were white, and 11% were African-American, and the mean age at the time of enrollment was 36.9 years. In both studies, 93% of subjects reported their highest education level as either college or an advanced degree.
Baseline characteristics of subjects
In VRC 304, 15 subjects in group 1 received three injections of a 1-mg dose, and one subject was lost to follow-up after receiving all study injections and moving out of the area. Two subjects in group 2 had to discontinue vaccinations after two immunizations because of the need for a systemic corticosteroid (unrelated to vaccination), and two subjects were lost to follow-up in this group. In the placebo group, one participant withdrew after one study injection. One subject in the placebo group completed all visits except week 32 due to moving from the area. The other 40 subjects completed all study visits through week 32 safety monitoring. In VRC 305, nine subjects were withdrawn from the vaccination schedule: two in group 1, three in group 2, one in group 3, and three in group 4. The reasons related to intercurrent illness or a need for a contraindicated medication in five subjects and noncompliance with protocol schedule or lost to follow-up in three subjects. In VRC 305, 80% of the subjects completed all vaccinations, and 91% completed the protocol through week 32 safety monitoring ().
VRC 304 and VRC 305 study diagram. Study enrollment, allocation, follow-up, and analysis are shown for all subjects screened and enrolled/randomized. These details are shown for VRC 304 on the left and for VRC 305 on the right.
In both studies, the vaccine was well tolerated, and there were no serious adverse events related to vaccine in either study. Adverse events assessed as related to i.d. vaccination in VRC 305 included mild, superficial skin lesion that healed without sequelae in 13/43 (30.2%) subjects and mild “itchiness” (pruritis) reported by 9/43 (20.9%) subjects. Solicited reactogenicity categorized by group is presented in and . Across all three groups in VRC 304, the worst severity of local reactogenicity was reported as none by 15.6%, mild by 82.2%, and moderate by 2.2% of subjects with the moderate symptom (pain at the injection site) being reported by a placebo recipient. The worst severity of systemic reactogenicity was reported as none by 40%, mild by 53.3%, and moderate by 4.4% of subjects, with the moderate symptoms being reported in the 1-mg and placebo groups. One placebo subject did not return any diary cards and therefore has missing reactogenicity data. Across all four groups in VRC 305, the worst severity of local reactogenicity was reported as none by 23.3% and mild by 76.7% of subjects. Overall, across both studies, the worst severity of systemic reactogenicity was reported as none by 34.9%, mild by 58.1%, and moderate by 7% of subjects. In addition, there was no apparent trend toward increasing reactogenicity with sequential dosing, even at the highest dose in either study (see Tables S1 and S2 in the supplemental material). There was no severe reactogenicity in either study.
Local reactogenicity by group
Systemic reactogenicity by group
Influenza virus-specific antibody responses.
The primary time point for immune assessment in both studies was 4 weeks after the third injection, at week 12 of the study. All subjects were negative for H5 antibodies by HAI assay, ELISA, and neutralizing antibody (NT) assay at baseline. At week 12, the frequency of positive HAI and neutralization responses were slightly higher in subjects who received 4 mg i.m. than in other groups ( and ). When we compared the routes of administration, 1 mg i.d. in VRC 305 (delivered as a divided dose in the same deltoid or divided between each deltoid) induced a higher frequency of response and higher ELISA titers () than 1 mg i.m. in VRC 304. This route effect on the 1-mg dose was not seen for HAI or neutralization. The 0.5-mg dose induced negligible HAI and neutralization responses, and none of these responses reached positivity criteria ( and ). There were no significant differences seen by any assay when comparing 1 mg delivered as a divided dose with both injections given in the same deltoid compared to a divided dose with a single 0.5-mg injection given in each deltoid.
Fig 2 Magnitude of H5 specific antibody responses. The responses determined by HAI assay (A), ELISA (B), and neutralization assay (i.e., the ID80) (C) are shown for all subjects (nonresponders and responders) by group in VRC 304 and VRC 305. Statistical differences (more ...) Influenza virus specific T-cell responses.
Antigen specific T-cell responses were assessed by ELISPOT assay and ICS at week 12 (). The rate of ELISPOT-positive response was the greatest, at 50%, in groups receiving 1 mg i.m., 4 mg i.m., and 1 mg using Bioject i.d. (two arms). The rate of CD8 (ICS) response was generally low, with the highest frequency of response seen in the 4-mg i.m. group at 17%. H5-specific CD4 T-cell responses were detected by ICS more frequently than H5-specific CD8 T cell responses. The rates of CD4 (ICS) response were highest at 67 and 79% in subjects who received 4 and 1 mg i.m., respectively, and were 50 and 63% in subjects who received 1 mg i.d. by Biojector (one arm and two arms, respectively). The pattern of differences suggests a dose effect and overall T cell responses were more consistent among the groups in the VRC 304 study (i.m. route) than in the VRC 305 study (i.d. route).
Frequency of H5 T cell responses. The percent response rates are shown for both the VRC 304 and VRC 305 studies as determined by T cell assay (ELISPOT and ICS for CD4 and ICS for CD8) for each dose group.