Neutralizing Antibodies to Adenovirus Serotype 5 Vaccine Vectors Are Directed Primarily against the Adenovirus Hexon Protein

Human serum samples

Serum samples from clinical diagnostic specimens were obtained from healthy adults from the United States (n = 59), Haiti (n = 67), Botswana (n = 57), Zambia (n = 29), and South Africa (n = 59) without patient identifiers. Studies using these samples were approved by our Institutional Review Board.

Virus neutralization assay

Ad-specific NAb responses were assessed by luciferase-based virus neutralization assays essentially as described (25). A549 human lung carcinoma cells were plated at a density of 1 × 10⁴ cells per well in 96-well plates and infected with E1-deleted, replication-incompetent rAd-Luciferase reporter constructs at a multiplicity of infection of 500 with 2-fold serial dilutions of serum in 200-μl reaction volumes. Following a 24-h incubation, luciferase activity in the cells was measured using the Steady-Glo Luciferase Reagent System (Promega). Ninety percent neutralization titers were defined as the maximum serum dilution that neutralized 90% of luciferase activity.

Mice and immunizations

Six- to 8-wk-old BALB/c or C57BL/6 mice were purchased from Charles River Laboratories. For rAd immunizations, mice were injected i.m. with varying doses of E1-deleted, replication-incompetent rAd vectors in 100 μl of sterile PBS. All animal studies were approved by our Institutional Animal Care and Use Committee.

Adoptive transfers

To study the inhibitory effects of Ad5-specific NAbs, adoptive transfer studies were performed essentially as described (9). Mice were preimmunized with capsid chimeric rAd5/rAd35 vectors, and purified IgG was prepared from 2 ml of serum pooled from eight mice using a Protein A Ab Purification kit (Sigma-Aldrich) and dialyzed into 5 ml of endotoxin-free Dulbecco’s PBS (Invitrogen Life Technologies) before use in adoptive transfer studies. These IgG preparations had similar concentrations of 2–3 mg/ml total IgG (Bio-Rad). A volume of 500 μl of purified IgG was then adoptively transferred to naive recipient mice by tail vein injection. On the day following adoptive transfer, recipient mice were immunized with 10⁸ virus particles (vp) of rAd5-Env.

Western blots

Western blots were performed to characterize Ad-specific NAbs in purified IgG preparations. A total of 10⁸ vp of rAd5 or rAd35 was electrophoresed by SDS-PAGE using a 7.5% resolving gel (Bio-Rad) and transferred to nitrocellulose membranes for Western blotting. Membranes were blocked overnight in TTBS (0.05% Tween 20, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl) containing 2.5% nonfat dry milk. Membranes were then incubated with purified mouse IgG at a 1/100 dilution for 2 h in TTBS containing 0.5% milk, washed three times with TTBS containing 0.5% milk, and incubated with ImmunoPure peroxidase-conjugated goat anti-mouse IgG secondary Ab (Pierce) at a 1/20,000 dilution for 1 h in TTBS containing 0.5% milk. Samples were detected with the SuperSignal West Pico Mouse IgG Detection kit (Pierce) following the manufacturer’s protocol and exposed to BioMax ML film (Kodak) for 2 min.

Tetramer staining

Tetrameric H-2D^d complexes folded around the dominant HIV-1 IIIB V3 loop P18 epitope peptide (P18-I10; RGPGRAFVTI) (26) were prepared and used to stain P18-specific CD8⁺ T lymphocytes from BALB/c mice essentially as described (27, 28). Mouse blood was collected in RPMI 1640 containing 40 U/ml heparin. Following lysis of the RBCs, 0.1 μg of PE-labeled D^d/P18 tetramer in conjunction with allophycocyanin-labeled anti-CD8α mAb (Ly-2; Caltag) was used to stain P18-specific CD8⁺ T lymphocytes. The cells were washed in PBS containing 2% FBS and fixed in 0.5 ml of PBS containing 1.5% paraformaldehyde. Samples were analyzed by two-color flow cytometry on a FACSCalibur (BD Biosciences). Gated CD8⁺ T lymphocytes were examined for staining with the D^d/P18 tetramer. CD8⁺ T lymphocytes from naive mice were used as negative controls and exhibited <0.1% tetramer staining. For C57BL/6 mice immunized with vectors expressing SIV Gag, we used tetrameric H-2D^b complexes folded around the dominant SIVmac239 Gag AL11 epitope peptide (AAVKNWMTQTL) (8).

ELISPOT

ELISPOT assays were used to assess IFN-γ production by splenocytes from BALB/c mice immunized with vectors expressing HIV-1 Env IIIB as described (7, 28). Cytokine production was assessed in response to the P18 epitope peptide or a pool of 47 overlapping 15-aa peptides derived from HIV-1 Env IIIB gp120 (Centralised Facility for AIDS Reagents, Potters Bar, U.K.). Ninety-six-well multiscreen plates (Millipore) coated overnight with 100 μl/well of 10 μg/ml rat anti-mouse IFN-γ (BD Pharmingen) in PBS were washed with endotoxin-free Dulbecco’s PBS (Invitrogen Life Technologies) containing 0.25% Tween 20 and blocked with PBS containing 5% FBS for 2 h at 37°C. The plates were washed three times with Dulbecco’s PBS containing 0.25% Tween 20, rinsed with RPMI 1640 containing 10% FBS, and incubated in triplicate with 5 × 10⁵ splenocytes per well in a 100-μl reaction volume containing 2 μg/ml peptide. For studies using the Env peptide pool, each peptide in the pool was present at 2 μg/ml. Following an 18-h incubation, the plates were washed nine times with Dulbecco’s PBS containing 0.25% Tween 20 and once with distilled water. The plates were then incubated for 2 h with 75 μl/well of 5 μg/ml biotinylated rat anti-mouse IFN-γ (BD Pharmingen), washed six times with Coulter Wash (Coulter), and incubated for 2 h with a 1/500 dilution of streptavidin-alkaline phosphatase (Southern Biotechnology Associates). Following five washes with Coulter Wash and one with PBS, the plates were developed with NBT/5-bromo-4-chloro-3-indolyl-phosphate chromogen (Pierce), stopped by washing with tap water, air dried, and read using an ELISPOT reader (Hitech Instruments).

ELISA

Serum Ab titers from immunized mice specific for HIV-1 Env or SIV Gag were measured by a direct ELISA as described (7, 28). Ninety-six well plates coated overnight with 100 μl/well of 1 μg/ml recombinant HIV-1 Env IIIB gp120 or SIV Gag p27 (Intracel) in PBS were blocked for 2 h with PBS containing 2% BSA and 0.05% Tween 20. Sera were then added in serial dilutions and incubated for 1 h. The plates were washed three times with PBS containing 0.05% Tween 20 and incubated for 1 h with a 1/2000 dilution of a peroxidase-conjugated affinity-purified rabbit anti-mouse secondary Ab (Jackson ImmunoResearch Laboratories). The plates were washed three times, developed with tetramethylbenzidine (Kirkegaard & Perry Laboratories), stopped with 1% HCl, and analyzed at 450 nm with a Dynatech MR5000 ELISA plate reader.

Statistical analyses

Statistical analyses were performed with GraphPad Prism version 4.01 (GraphPad Software). Comparisons of mean responses among groups were performed by ANOVA with Bonferroni adjustments to account for multiple comparisons. In all cases, p values of <0.05 were considered significant.

International seroprevalence and NAb titers to Ad5, Ad35, and Ad11

We initiated studies to assess the seroprevalence and NAb titers to Ad5 and alternate Ad serotypes in the developing world. We performed functional luciferase-based virus neutralization assays (25) using serum samples from healthy adults from the United States (n = 59), Haiti (n = 67), Botswana (n = 57), Zambia (n = 29), and South Africa (n = 59). Seroprevalence was defined as the percentage of serum samples that neutralized >90% luciferase activity at a serum dilution of 1/16 or greater. As shown in Fig. 1⇓a, the Ad5 seroprevalence was 50% in the United States. In contrast, the Ad5 seroprevalence was 82% in Haiti, 93% in Botswana, 93% in Zambia, and 88% in South Africa. These data demonstrate that the Ad5 seroprevalence was substantially higher in the developing world compared with the United States.

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FIGURE 1.

Ad5, Ad35, and Ad11 seroprevalence and NAb titers. Seroprevalence (a) and NAb titers (b) to rAd5, rAd35, and rAd11 as determined by luciferase-based virus neutralization assays. Serum samples were obtained from healthy adults in the United States (n = 59), Haiti (n = 67), Botswana (n = 57), Zambia (n = 29), and South Africa (n = 59).

As shown in Fig. 1⇑b, the median Ad5-specific NAb titers from sub-Saharan Africa were strikingly >10-fold higher than the median titers found in the United States (p < 0.001). These data extend our previous findings (13, 29) and show that Ad5-specific NAbs were nearly universal and high titer in these populations in the developing world. In contrast, the Ad35 and Ad11 seroprevalence and titers were substantially lower. These data suggest that pre-existing anti-Ad5 immunity will likely substantially suppress the immunogenicity and clinical utility of rAd5 vector-based vaccines in the developing world.

Immunodominant targets of Ad5-specific NAbs

We next used these human serum samples to determine the dominant antigenic targets of Ad5-specific NAbs in both the United States (n = 59) and sub-Saharan Africa (n = 145). Given the lack of detectable NAb cross-reactivity between Ad5 and Ad35 (8), we used capsid chimeric rAd5/rAd35 viruses expressing luciferase in virus neutralization assays. These vectors incorporated various combinations of Ad5 and Ad35 hexon, penton, and fiber proteins in the context of intact viral particles with wild-type growth kinetics (30, 31). As depicted in Table I⇓, we used the following chimeric vectors in this study: rAd5f35 (rAd5 containing the Ad35 fiber), rAd5f35p35 (rAd5 containing the Ad35 fiber and penton), and rAd35f5 (rAd35 containing the Ad5 fiber).

As shown in Fig. 2⇓a, comparable NAb titers were observed in human serum samples against rAd5, rAd5f35, and rAd5f35p35. Because the capsid of the rAd5f35p35 vector contained the Ad5 hexon but the Ad35 fiber and Ad35 penton, these data suggest that the majority of Ad5-specific NAb activity was directed against the Ad5 hexon. Lower but clearly detectable titers were also measured against rAd35f5, demonstrating that Ad5 fiber-specific NAbs were present but at 4- to 10-fold lower titers than Ad5 hexon-specific NAbs in these samples. We were not able to measure Ad5 penton-specific NAbs directly using this panel of capsid chimeric viruses, but the similar NAb titers against rAd5, rAd5f35, and rAd5f35p35 suggested that penton-specific NAbs as well as the combination of fiber- and penton-specific NAbs played at most a minor role in this neutralization. Thus, Ad5-specific NAbs were detected against multiple capsid components, but hexon-specific NAbs were substantially higher titer than fiber-specific NAbs in subjects from both the United States and sub-Saharan Africa. Binding Abs against all major capsid components were also detected by Western blots in these samples (data not shown), indicating that only a subset of binding Abs were in fact functionally relevant NAbs.

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FIGURE 2.

Immunodominant targets of Ad-specific NAbs. Serum samples from Ad5-seropositive humans (a) or rAd5- or rAd35-immunized mice (b) were assessed for NAb titers to various capsid chimeric rAd5/rAd35 viruses. NAb titers to rAd5, rAd5f35, rAd5f35p35, rAd35f5, and rAd35 were determined.

We next confirmed these findings using serum from experimentally immunized mice. BALB/c mice (n = 4/group) were immunized with 10¹⁰ vp of rAd5 or rAd35, and serum was obtained at wk 4 following immunization and was used in virus neutralization assays. As shown in Fig. 2⇑b, serum from rAd5-immunized mice had comparable NAb titers to rAd5, rAd5f35, and rAd5f35p35, but exhibited 6-fold lower NAb titers to rAd35f5, consistent with the results obtained with the human samples. As expected, serum from rAd5-immunized mice had no detectable cross-reactive NAbs against rAd35 (8). Conversely, serum from rAd35-immunized mice had comparable NAb titers to rAd35 and rAd35f5, but exhibited 5-fold lower NAb titers to rAd5f35 and rAd5f35p35. These data indicate that the immunodominant targets of Ad-specific NAbs in immunized mice mirrored those observed in humans.

Suppressive effects of low titers of Ad-specific NAbs

We next assessed the functional significance of NAbs against the major Ad5 capsid proteins by determining their capacity to suppress rAd5 vaccine immunogenicity following adoptive transfer in mice. We have previously reported that anti-Ad35 immunity did not suppress rAd5 vaccine-elicited immune responses in mice (8). Therefore, we used capsid chimeric rAd5/rAd35 viruses to elicit immune responses against specific components of the Ad5 capsid. This strategy has an advantage over using soluble proteins to induce anti-Ad5 immunity in that these chimeric viruses present the various capsid proteins to the immune system in the context of intact, infectious viral particles.

Donor mice received one injection of 10¹⁰ vp of rAd5, rAd5f35p35, rAd35f5, rAd35, or saline to elicit low NAb titers, and IgG was purified from serum after 4 wk. As shown in Fig. 3⇓a, we confirmed Ad5- and Ad35-specific neutralizing activity in these purified IgG preparations before adoptive transfer. As expected, IgG from mice immunized with rAd5 or rAd35 exhibited low serotype-specific NAb titers of 64–128 without detectable cross-reactivity. In contrast, IgG from mice immunized with the chimeric viruses exhibited NAb titers against both Ad5 and Ad35. IgG from mice that received rAd5f35p35 contained Ad5-specific NAbs that were presumably directed against the Ad5 hexon as well as low-titer Ad35-specific NAbs that were presumably directed against the Ad35 penton and fiber. Similarly, IgG from mice that received rAd35f5 contained Ad35-specific NAbs against the Ad35 hexon and penton as well as low-titer Ad5-specific NAbs that were presumably directed against the Ad5 fiber.

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FIGURE 3.

Adoptive transfer studies using low titers of Ad-specific NAbs. IgG was purified from pooled serum from BALB/c mice that received one injection of 10¹⁰ vp of rAd5, rAd5f35p35, rAd35f5, rAd35, or saline. a, The IgG preparations were assessed for NAbs to rAd5 and rAd35. The IgG preparations were then adoptively transferred into naive BALB/c mice (n = 4/group) by tail vein injection before immunization with 10⁸ vp of rAd5-Env. b, Vaccine-elicited immune responses following immunization were determined by D^d/P18 tetramer binding assays. Controls included mice that received adoptive transfer of naive IgG purified from saline-injected mice (Naive) and mice that received no adoptive transfer (None).

Adoptive transfer studies were then performed to assess the ability of these IgG preparations containing Ad-specific NAbs to suppress rAd5 vaccine immunogenicity. Groups of naive recipient mice (n = 4/group) received 500 μl of purified IgG by tail vein injection before immunization with 10⁸ vp of rAd5-Env. Env-specific CD8⁺ T lymphocyte responses against the dominant P18 epitope (RGPGRAFVTI) (26) were assessed by D^d/P18 tetramer binding assays (27, 28). As shown in Fig. 3⇑b, adoptive transfer of IgG from mice that received rAd35 or rAd35f5 (•, ▪) did not detectably suppress tetramer⁺CD8⁺ T lymphocyte responses. In contrast, adoptive transfer of IgG purified from mice that received rAd5 or rAd5f35p35 (○, ▵) substantially suppressed peak tetramer⁺CD8⁺ T lymphocyte responses by 54% (p < 0.01 comparing mean tetramer responses among groups on day 10 following immunization using ANOVA with Bonferroni adjustments). There was also a trend toward a 12–37% reduction of memory responses on day 28 following immunization. Thus, even low titers of hexon-specific NAbs effectively suppressed peak rAd5 vaccine-elicited immune responses.

Suppressive effects of high titers of Ad-specific NAbs

We next examined the suppressive effects of high titers of Ad5 hexon- and fiber-specific NAbs by repeating this experiment using donor mice that were preimmunized twice with 10¹⁰ vp of rAd5, rAd5f35p35, rAd35f5, rAd35, or saline. As shown in Fig. 4⇓a, purified IgG from these mice had Ad5- and Ad35-specific NAb titers that were ∼100-fold higher than in the previous experiment. IgG from mice that received rAd5 or rAd35 exhibited potent, serotype-specific NAb titers of 8192. IgG from mice that received rAd5f35p35 had potent Ad5-specific NAb titers that were presumably directed against the Ad5 hexon as well as lower Ad35-specific NAb titers that were presumably directed against the Ad35 fiber and penton. Similarly, IgG from mice that received rAd35f5 exhibited potent Ad35-specific NAb titers against the Ad35 hexon and penton and lower Ad5-specific NAb titers that were presumably directed against the Ad5 fiber. The clear induction of Ad5 fiber-specific NAbs following injection of rAd35f5 confirms that the Ad5 fiber protein on this virus was intact and immunogenic. Importantly, these fiber-specific NAb titers exceeded the fiber-specific NAb titers observed in humans in the developing world (Fig. 2⇑a).

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FIGURE 4.

Adoptive transfer studies using high titers of Ad-specific NAbs. IgG was purified from pooled serum from BALB/c mice that received two injections of 10¹⁰ vp of rAd5, rAd5f35p35, rAd35f5, rAd35, or saline. a, The IgG preparations were assessed for NAbs to rAd5 and rAd35. b, Western blots were performed to confirm reactivity of these IgG preparations with the 110-kDa hexon band of the expected serotype. The IgG preparations were then adoptively transferred into naive BALB/c mice (n = 4/group) by tail vein injection before immunization with 10⁸ vp of rAd5-Env. c, Serum was obtained from these mice following adoptive transfer and immediately before vaccination and assessed for NAbs to rAd5 and rAd35. d–f, Vaccine-elicited immune responses following immunization were determined by D^d/P18 tetramer binding assays (d), Env pooled peptide and P18 epitope-specific ELISPOT assays (e), and Env-specific ELISAs (f). Controls included mice that received adoptive transfer of naive IgG purified from saline-injected mice (Naive) and mice that received no adoptive transfer (None).

We then performed denaturing SDS-PAGE and Western blots to confirm that these purified IgG preparations reacted with a 110-kDa band corresponding to the hexon protein of the appropriate Ad serotype. As expected, IgG from mice that received rAd5 or rAd5f35p35 contained Ad5 hexon- but not Ad35 hexon-specific Abs (Fig. 4⇑b). Similarly, IgG from mice that received rAd35 or rAd35f5 contained Ad35 hexon- but not Ad5 hexon-specific Abs. We also detected limited cross-reactivity (<20% by gel densitometry) to a 60- to 65-kDa protein, which likely represents the IIIa assembly protein (66 kDa), the penton protein (63 kDa), or both. We were not able to detect bands corresponding to the fiber proteins on these gels, because fiber-specific NAbs recognize primarily conformational determinants (9). Bands corresponding to the fiber proteins were detected using native SDS-PAGE and similar Western blots (data not shown).

We performed adoptive transfer studies using these purified IgG preparations containing high titers of Ad5-specific NAbs. Groups of naive recipient mice (n = 4/group) received 500 μl of purified IgG before immunization with 10⁸ vp of rAd5-Env. To verify circulating NAb titers in these animals, serum was obtained after adoptive transfer and immediately before rAd5-Env vaccination. Serum NAbs in the recipient mice following adoptive transfer exhibited 4-fold lower titers compared with the titers present in the IgG preparations, as expected based on the dilutional effects of circulating volume (Fig. 4⇑c).

We assessed Env-specific cellular and humoral immune responses following rAd5-Env vaccination using tetramer binding assays, IFN-γ ELISPOT assays, and ELISAs. As depicted in Fig. 4⇑d, adoptive transfer of IgG from mice that received rAd35 or rAd35f5 (•, ▪) did not detectably suppress tetramer⁺CD8⁺ T lymphocyte responses. These data suggest that even high titers of Ad5 fiber-specific NAbs did not substantially suppress rAd5-Env vaccine immunogenicity in this system. In contrast, adoptive transfer of IgG from mice that received rAd5 or rAd5f35p35 (○, ▵) completely abrogated peak and memory tetramer⁺CD8⁺ T lymphocyte responses, demonstrating the dramatic suppressive effects of Ad5 hexon-specific NAbs (p < 0.001 comparing mean tetramer responses among groups on day 10 and day 28 following immunization). Functional pooled peptide and epitope-specific ELISPOT assays (Fig. 4⇑e) and Env-specific ELISAs (f) showed similar suppressive effects of these IgG preparations (p < 0.001). Thus, hexon-specific NAbs were substantially more effective than fiber-specific NAbs in abrogating rAd5 vaccine-elicited cellular and humoral immune responses.

To assess the generalizability of these results, we repeated this adoptive transfer study in a second experimental system. C57BL/6 mice were preimmunized twice with 10¹⁰ vp of rAd5, rAd5f35p35, rAd35f5, rAd35, or saline as above, and purified IgG was adoptively transferred to naive recipient mice before immunization with 10⁸ vp of rAd5-Gag. Ad5- and Ad35-specific NAb titers in these IgG preparations and in serum samples obtained from the mice before vaccination were comparable to those in the previous study (data not shown). Vaccine-elicited immune responses were assessed by D^b/AL11 tetramer binding assays and Gag-specific ELISAs. As shown in Fig. 5⇓, adoptive transfer of IgG from mice that received rAd35f5 showed a slight and transient trend toward reduction of tetramer⁺CD8⁺ T lymphocyte responses on day 14 following immunization. Thus, it is possible that fiber-specific NAbs may have a secondary role in suppressing vaccine immunogenicity in certain settings. However, adoptive transfer of IgG from mice that received rAd5 or rAd5f35p35 resulted in significant and durable suppression of vaccine-elicited cellular (p < 0.01) and humoral (p < 0.001) immune responses. These studies confirm our previous findings that functionally significant Ad5-specific NAbs are directed primarily against the Ad5 hexon protein.

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FIGURE 5.

Adoptive transfer studies using high titers of Ad-specific NAbs in C57BL/6 mice. IgG was purified from pooled serum from C57BL/6 mice that received two injections of 10¹⁰ vp of rAd5, rAd5f35p35, rAd35f5, rAd35, or saline. These IgG preparations were adoptively transferred into naive C57BL/6 mice (n = 4/group) by tail vein injection before immunization with 10⁸ vp of rAd5-Gag. Vaccine-elicited immune responses following immunization were determined by D^b/AL11 tetramer binding assays (a) and Gag-specific ELISAs (b). Controls included mice that received adoptive transfer of naive IgG purified from saline-injected mice (Naive) and mice that received no adoptive transfer (None).