Optimization of capsid-incorporated antigens for a novel adenovirus vaccine approach

Abstract

Despite the many potential advantages of Ad vectors for vaccine application, the full utility of current Ad vaccines may be limited by the host anti-vector immune response. Direct incorporation of antigens into the adenovirus capsid offers a new and exciting approach for vaccination strategies; this strategy exploits the inherent antigenicity of the Ad vector. Critical to exploiting Ad in this new context is the placement of antigenic epitopes within the major Ad capsid protein, hexon. In our current study we illustrate that we have the capability to place a range of antigenic epitopes within Ad5 capsid protein hexon hypervariable regions (HVRs) 2 or 5, thus producing viable Ad virions. Our data define the maximal incorporation size at HVR2 or HVR5 as it relates to identical antigenic epitopes. In addition, this data suggests that Ad5 HVR5 is more permissive to a range of insertions. Most importantly, repeated administration of our hexon-modified viruses resulted in a secondary anti-antigen response, whereas minimal secondary effect was present after administration of Ad5 control. Our study describes antigen placement and optimization within the context of the capsid incorporation approach of Ad vaccine employment, thereby broadening this new methodology.

Figure 1

Construction of hexon modified genomes. (A) Agarose gel electrophoresis of PCR products were obtained through PCR of genomic DNA of Ad5 using epitope specific primers (Table 1). (B) HVR2 or HVR5 shuttle vector sites which were modified with a RGD motif. The DNA encoding for the respective RGD motifs were cloned into the HVRs of our previously modified shuttle vectors within the LGSHHHHHLGS linker, as indicated by the arrows.

Figure 2

Model epitopes incorporated in HVRs are accessible in the context of an intact virion. In the assay, varying amounts of purified viruses were immobilized in the wells of ELISA plates and incubated with anti-His₆ tag antibody. The binding was detected with an AP-conjugated secondary antibody. These results suggested that the model antigens (tagged with His₆ epitopes) and the His₆ epitopes (controls) incorporated into HVR2 or HVR5 were accessible to anti-His₆ tag antibody at the virion level, indicating that the epitopes were exposed on the virion surface. All of the Ad vectors except Ad5 present His₆or RGD-His₆within the hexon. The His₆ antigenic peptide is presented by Ad5/HVR5-His₆and Ad5/HVR5-His₆.

Figure 3

Capsid-incorporated antigens elicit an IgG immune response. C57BL/6J mice were immunized with 10¹⁰VP of Ad vectors. Post-immunization sera were collected after (A) 30 days post-injection or (B) 0–70 days post-injection for ELISA binding assays. 20 μM of synthesized antigenic peptide His₆ peptide was bound to ELISA plates. Residual unbound peptide was washed from the plates. The plates were then incubated with immunized mice sera, the binding was detected with IgG-specific HRP-conjugated anti-mouse secondary antibody. OD absorbance represents the sera levels of antibodies. Values are expressed as the mean ± standard error of three replicates. * indicates a P value of <.05., ** P < .001, *** P < .00001. Control viruses are Ad5, Ad/HVR2-His₆ and Ad/HVR5-His₆.

Figure 4

Capsid-incorporated antigens elicit a varied immune response. (A-D) C57BL/6J mice were immunized with 10¹⁰VP of Ad vectors. Post-immunization sera were collected after 50 days post-injection for ELISA binding assays. 20 μM of synthetic peptide 33RGD-His₆(RGD residue flanked by His₆/Linker) was bound to the plate. The plates were then incubated with immunized mice sera, the binding was detected with isotype-specific HRP-conjugated anti-mouse secondary antibody (A, IgG; B, IgG1; C, IgG2b; D, IgG2c). OD absorbance represents the sera levels of antibodies. Values are expressed as the mean ± standard error of three replicates. Control viruses are Ad5, Ad/HVR2-His₆ and Ad/HVR5-His₆.

Figure 5

Capsid-incorporated antigens elicit a varied T cell response. (A-B) C57BL/6J mice were immunized with 10¹⁰VP of Ad vectors. On day 40, these mice were intravenously boosted with the same dose of the same vectors. A single-cell suspension of spleen cells was prepared on day 9 after secondary virus infection. Cells were stained with a fluorescent labeled anti-CD4 antibody and then permeabilized in intracellular stain with fluorescent conjugated antibodies against IL-4 or IFN-γ. Samples were acquired on a FACSCalibur and data were analyzed with FlowJo software. Values are expressed as the mean ± standard error of three replicates.

Figure 6

Repeat administration of hexon-modified viruses results in boosting of the anti-33RGD-His₆ immune response. C57BL/6J mice were immunized with 10¹⁰VP of Ad vectors. On day 40, these mice were intravenously boosted with the same dose of the same vectors. Post-immunization sera were collected after 9 days post-injection for ELISA binding assays. 20 μM of synthetic peptide 33RGD-His₆ was bound to the plate. The plates were then incubated with immunized mice sera, the binding was detected with isotype-specific HRP-conjugated anti-mouse secondary antibody. OD absorbance represents the sera levels of antibodies. Values are expressed as the mean ± standard error of three replicates.