Distribution of adenoviral neutralization targets detectable in serum samples from volunteers in the VRC006 trial. Data shown in Fig. 2 and 3 are summarized based on the percentage of serum samples containing antibodies directed to specific viral capsid proteins. In the naïve volunteers, vaccination generated neutralization targeted mainly to capsid proteins other than fiber alone. In the seropositive volunteers, neutralization directed to the fiber was detected broadly before vaccination, whereas the neutralization extended to fiber plus other capsid proteins after vaccination. Fiber and capsid are defined as described in the legend for Fig. 3.

Ad neutralizing titers in prevaccination serum samples from volunteers involved in the HIV vaccine trial VRC006 at week 0. Serum samples from 32 volunteers were analyzed for seropositivity to Ad5, and 12 serum samples showed titers of greater than 36. These 12 serum samples were used to neutralize Ad vectors, and their neutralizing titers to each vector were determined. Due to the limited amount of serum available, the minimum titer was set at 1:36 and the maximum was 1:26,244. The serum samples can be grouped by their neutralizing activities into two major groups: one group with neutralizing activity solely directed to Ad5 fiber (Fiber) and another group with neutralizing activity to fiber plus other Ad5 capsid proteins (Fiber+Capsid). In only serum sample number 1, neutralization required the concurrent presence of Ad5 fiber and other Ad5 capsid proteins.

Adenoviral neutralizing antibody titers in serum samples from volunteers involved in the HIV vaccine trial VRC006 at 4 weeks postvaccination. The postvaccination neutralizing titers are shown for naïve or seronegative subjects (A) and subjects with preexisting seropositivity (B) (shown in the same order as shown in Fig. 2). For volunteers with preexisting seropositivity, all serum samples showed neutralizing antibodies to both fiber and other capsid proteins, as they could neutralize rAd5, rAd5 F35, and rAd35 F5 but not rAd35 vectors. In naïve volunteers, two neutralization patterns were seen following vaccination. One group could neutralize rAd5 and rAd5 F35 but not rAd35 or rAd35 F5, showing capsid (non-fiber-mediated) neutralization; another group could neutralize rAd5, rAd5 F35, and rAd35 F5 but not rAd35, showing neutralization directed to both fiber and other capsid proteins. Capsid denotes Ad5 capsid proteins other than fiber; Fiber+Capsid indicates Ad5 fiber and other capsid proteins. The minimum titer was set at 1:36, and the maximum was 1:8,748.

Distribution of adenoviral neutralization targets detected in serum samples from volunteers in the HVTN204 trial at week 0 and its association with the vaccine response rate. (A) Seventy serum samples from 127 volunteers showed preexisting neutralizing antibodies to Ad5 (left). In the 70 positive serum samples, the percentage of sera that contained neutralizing antibodies directed to specific viral capsid proteins is shown with a bar representing the 90% confidence interval for the true percentage of responders in each category. Fiber and capsid are defined as described in the legend for Fig. 3. The distribution of preexisting anti-capsid (Ad5 F35) and anti-fiber (Ad35 F5) neutralizing antibody (Nab) titers for the 121 volunteers who participated in HVTN204 clinical trials are also indicated (right). The cutoff titer (shown as a dashed line) for Nab negatives was set at 36 for both anti-capsid (n = 96) and anti-fiber (n = 64) Nab. (B and C) The responders to the rAd5 HIV vaccine among 121 volunteers for whom valid immune response data are available were categorized according to Ad35 F5 seropositive or seronegative (positive or negative Ad5 fiber neutralizing antibodies, respectively; panel B) and Ad5 F35 seropositive or seronegative (positive or negative Ad5 capsid neutralizing antibodies, respectively; panel C). The percentage of responders with positive CD4⁺ T-cell response to EnvA or Gag is shown, with a bar representing the 90% confidence interval for the true percentage of responders in each category. ns, not significant, with P > 0.001.

Recombinant and chimeric Ads determine the specificity of neutralizing antibodies in selected subjects. (A) Wild-type and chimeric vectors based on Ad5 and Ad35 were used to analyze the neutralizing antibody activities against different viral capsid proteins. Fiber and other major capsid proteins (including hexon and penton) in wild-type Ad5 contain neutralizing epitopes targeted by human antibodies. rAd5 F35 is an Ad5-based chimeric vector that contains the fiber of Ad35 in place of Ad5 fiber. It contains all the Ad5 capsid proteins except fiber and was used to analyze specific neutralizing antibodies to Ad5 capsid proteins excluding fiber. rAd35 F5 contains only one Ad5-specific neutralizing target, the fiber. It was used to analyze specific neutralizing antibodies to Ad5 fiber. (B) Comparable transduction of A549 cells by different chimeric rAd vectors. A549 cells were transduced with rAd vectors encoding luciferase (Ad5, Ad35, Ad5 F35, and Ad35 F5) or green fluorescent protein (Control) at indicated multiples of infection viral particles (MOI). Luminescence was measured at 24 h after transduction. (C) Two examples of human serum samples from the VRC006 trial that can neutralize Ad vectors. Human serum samples at week 0 (prior to vaccination) were diluted as indicated, and their ability to neutralize adenoviral vectors was tested. Neutralization assays were performed in triplicate, and the average with standard error is shown. Serum VRC006-12 could neutralize more than 90% of the infectivity of rAd5 and rAd35 F5 but not of rAd35 and rAd5 F35, showing primarily Ad5 fiber-specific neutralization. Serum VRC006-18 could neutralize more than 90% of the infectivity of rAd5, rAd5 F35, and rAd35 F5 but not of rAd35, showing that Ad5 fiber and other capsid proteins were all targeted for neutralization.