Characterization of a Permissive Epitope Insertion Site in Adenovirus Hexon

Abstract

A robust immune response is generated against components of the adenovirus capsid. In particular, a potent and long-lived humoral response is elicited against the hexon protein. This is due to the efficient presentation of adenovirus capsid proteins to CD4 ؉ T cells by antigen-presenting cells, in addition to the highly repetitive structure of the adenovirus capsids, which can efficiently stimulate B-cell proliferation. In the present study, we take advantage of this immune response by inserting epitopes against which an antibody response is desired into the adenovirus hexon. We use a B-cell epitope from Bacillus anthracis protective antigen (PA) as a model antigen to characterize hypervariable region 5 (HVR5) of hexon as a site for peptide insertion. We demonstrate that HVR5 can accommodate a peptide of up to 36 amino acids without adversely affecting virus infectivity, growth, or stability. Viruses containing chimeric hexons elicited antibodies against PA in mice, with total immunoglobulin G (IgG) titers reaching approximately 1 ؋ 10 3 after two injections. The antibody response contained both IgG1 and IgG2a subtypes, suggesting that Th1 and Th2 immunity had been stimulated. Coinjection of wild-type adenovirus and a synthetic peptide from PA produced no detectable antibodies, indicating that incorporation of the epitope into the capsid was crucial for immune stimulation. Together, these results indicate that the adenovirus capsid is an efficient vehicle for presenting B-cell epitopes to the immune system, making this a useful approach for the design of epitope-based vaccines.

Protective Immunity Against a Lethal RespiratoryYersinia pestisChallenge Induced by V Antigen or the F1 Capsular Antigen Incorporated into Adenovirus Capsid

“…It has also been possible to incorporate immunogenic epitopes into permissive sites of hexon, a major capsid protein, and the resulting vaccines exhibit robust protective efficacy (Worgall et al, 2005(Worgall et al, , 2007. However, the addition of foreign immunogens into hexon is limited by size; insertions of only up to 53 amino acids in the hypervariable region 5 of hexon are tolerated for viable virus recovery (McConnell et al, 2006;Matthews et al, 2008). In contrast, studies on the minor capsid protein, pIX, have indicated that up to 1000 kDa can be fused to the C terminus of the protein, allowing surface exposure of the foreign protein without substantial impact on the formation of virions (Dmitriev et al, 2002;Campos et al, 2004;Meulenbroek et al, 2004;Le et al, 2005;Li et al, 2005;Matthews et al, 2006;Vellinga et al, 2007).…”

Vaccination with an Adenoviral Vector That Encodes and Displays a Retroviral Antigen Induces Improved Neutralizing Antibody and CD4+T-Cell Responses and Confers Enhanced Protection

“…For the presentation of the vaccine antigen we chose the pIX protein, because in contrast to hexon, fiber, and penton base, which have been used before for the display of single epitopes (5,22,25,26,43), pIX allows the incorporation of large proteins without loss of function and virion integrity (24,28). Incorporation of a whole protein is an interesting option, as the presence of multiple relevant epitopes in the vaccine allows the induction of a broader and more diverse immune response.…”

“…These were mostly inserted into external loops of the hexon protein (5,22,25,26,43), which is the main component of the adenovirus capsid, but also other components of the capsid, such as fiber, protein IX, and penton base, have been evaluated (22). These studies showed that incorporation of single epitopes into capsid proteins of adenovirus leads to induction of antibody and CD4 ϩ T-cell responses, suggesting that incorporation of epitopes into the adenovirus capsid is a useful tool for epitope-based vaccination.…”

Ternary Complex Formation on the Adenovirus Packaging Sequence by the IVa2 and L4 22-Kilodalton Proteins

“…The open reading frame encoding the 22-kDa protein was amplified using primers 22kDafor (5Ј-ACCCATGGCACCCAAAAAGAA GC-3Ј) and 22kDarev (5Ј-TTCTCGAGTCCGGTCGCCTTTGCTTC-3Ј) from the previously created cAd212 cosmid (22). The resulting PCR product was inserted into the pCR II-Blunt-TOPO vector (Invitrogen) according to the manufacturer's protocol to create pCRBT-L422kDa.…”