3.1 Adenovirus NAb Titers in Multiple International Human Populations
We determined Ad5, Ad26, Ad35, and Ad48 NAb titers in 4,381 subjects in multiple international populations from North America, South America, sub-Saharan Africa, and Southeast Asia as detailed in . Ad-specific NAb titers were determined by luciferase-based virus neutralization assays as previously described [20
]. Ad5, Ad26, and Ad35 NAb assays have been validated for use in clinical trials, whereas Ad48 NAb assays represent research assays. The vector characteristics, technical assay parameters, and assay performance and dynamic ranges were similar for the four vectors utilized in this study.
depicts the raw seroepidemiology data for each population, and shows summary data. Ad5 seroprevalence was 87.9–89.5%, 90.5%, 86.4%, and 82.2% in adults in South Africa, Kenya, Uganda, and Thailand, respectively. In contrast, Ad26, Ad35, and Ad48 seroprevalence was significantly lower than Ad5 seroprevalence in all regions studied (p<0.0001 for each population, chi-square test). Ad26 seroprevalence was 43.1–53.2%, 66.2%, 67.8%, and 54.6% in adults in South Africa, Kenya, Uganda, and Thailand, respectively. Ad35 seroprevalence was 10.6–17.8%, 14.8%, 5.4%, and 17.1% in these populations, and Ad48 seroprevalence was 13.3–24.6%, 51.0%, 50.0%, and 12.8% in these cohorts, respectively.
Neutralizing antibodies against Ad5, Ad26, Ad35 and Ad48
Ad5, Ad26, Ad35, and Ad48 NAb Titers
The majority of adults in these regions also exhibited high Ad5 NAb titers >200 (61.1–78.7%), and a substantial fraction had very high Ad5 NAb titers >1000 (25.1–46.8%). In contrast, markedly fewer individuals in these regions demonstrated high Ad26 NAb titers >200 (5.4–17.8%), Ad35 NAb titers >200 (0.4–5.0%), or Ad48 Nab titers >200 (0.9–10.8%), and only rare individuals had very high Ad26 NAb titers >1000 (0.0–3.8%), Ad35 NAb titers >1000 (0.0–1.7%), or Ad48 NAb titers >1000 (0.0–2.1%).
These data demonstrate that Ad35 seroprevalence and NAb titers were low in all regions studied. Ad48 seroprevalence was low in all regions except for East Africa. Ad26 seroprevalence was moderate in adults in the developing world, but Ad26 Nab titers proved consistently and substantially lower than Ad5 NAb titers in all regions studied. Moreover, Ad26 and Ad35 seroprevalence and NAb titers were negligible in infants (3–9 months old) and were low in children (6–12 years old) in South Africa, which represent potential ultimate target populations for an HIV-1 vaccine. NAb titers to each of these Ad serotypes appeared to be independent (data not shown).
and depict the median and geometric mean Ad NAb titers in each geographic region. Median titers with interquartile ranges (IQRs) were calculated for each population, and geometric mean titers were determined among seropositives in each population. These data confirm that Ad26, Ad35, and Ad48 NAb titers were significantly lower than Ad5 NAb titers in all regions studied (p<0.001 for each region, ANOVA with post-hoc Dunnett correction). In particular, median (IQR) Ad5 NAb titers were 391 (94–1,069), 877 (259–2,369), 505 (77–1,501), and 521 (88–1,328), and geometric mean Ad5 NAb titers were 461, 967, 598, and 669 in adults in South Africa, Kenya, Uganda, and Thailand, respectively. In contrast, median (IQR) Ad26 NAb titers were 22 (18–65), 37 (18–115), 38 (18–132), and 30 (18–134), and geometric mean Ad26 NAb titers were 67, 97, 86, and 127 in these populations, respectively. Geometric mean Ad35 NAb titers were 75, 103, 50, and 86 in these regions, and geometric mean Ad48 NAb titers were 38, 90, 87, and 54 in these populations, respectively. These data show that median Ad26, Ad35, and Ad48 NAb titers were approximately 10-fold lower than median Ad5 NAb titers in these regions in the developing world. Median Ad26, Ad35 and Ad48 NAb titers were also substantially lower than median Ad5 NAb titers in the developed world.
Descriptive statistics on neutralizing antibodies against Ad5, Ad26, Ad35 and Ad48
Median and Geometric Mean Ad NAb Titers
3.2 Impact of Low Ad26 NAb Titers on rAd26 Vaccine Immunogenicity in Rhesus Monkeys
Given the moderately common but low titer Ad26 NAbs in the developing world, we evaluated the potential impact of such Ad26 NAb titers on rAd26 vaccine vector immunogenicity in a pilot study in nonhuman primates. 12 adult rhesus monkeys were pre-immunized twice by the intranasal route with either 1011 vp replication-competent rAd26-Empty (N=6) or saline (N=6). Animals that received rAd26-Empty developed serum Ad26 NAb titers that were similar in magnitude to those found in humans in sub-Saharan Africa and Southeast Asia (median titer 102; geometric mean titer 85). Four weeks later, all animals were immunized once by the intramuscular route with 4×1010 vp of the rAd26-Gag/Pol/Env/Nef vaccine.
Env-specific antibody responses were assessed by ELISA, and Gag-, Pol-, Env-, and Nef-specific cellular immune responses were assessed by interferon-γELISPOT assays at week 2 following vaccination. As shown in , Env-specific binding antibody responses were comparable in monkeys with and without low baseline Ad26 NAb titers (p=NS, Mann-Whitney test). Similarly, as shown in , Gag-, Pol-, Env-, and Nef-specific cellular immune responses were comparable in monkeys with and without low baseline Ad26 NAb titers (p=NS, Mann-Whitney test). Humoral and cellular immune responses also proved comparable between groups at week 4 and week 12 following vaccination (data not shown).
Immunogenicity of rAd26-Gag/Pol/Env/Nef in Rhesus Monkeys
We also evaluated Ad26 NAb titers in these monkeys both before and after rAd26 vaccination. As shown in , Ad26 NAb titers proved high (range 1,157 – 16,384) in the Ad26 pre-immunized monkeys following rAd26-Gag/Pol/Env/Nef vaccination. These titers were significantly higher than those observed in naïve monkeys following vaccination (p=0.002, Mann-Whitney test) but did not detectably suppress rAd26-elicited humoral or cellular immune responses and did not alter the ratio of CD4/CD8 cellular immune responses (). These data demonstrate the dynamic range of the Ad26 NAb assay and confirm that the relatively low Ad26 NAb titers observed in human populations (–) did not simply reflect technical characteristics of the Ad26 NAb assay.