Viable adenovirus vaccine prototypes: high-level production of a papillomavirus capsid antigen from the major late transcriptional unit

Abstract

Safe, effective, orally delivered, live adenovirus vaccines have been in use for three decades. Recombinant derivatives of the live adenovirus vaccines may prove an economical alternative to current vaccines for a variety of diseases. To explore that possibility, we constructed a series of recombinants that express the major capsid protein (L1) of canine oral papillomavirus (COPV), a model for mucosal human papillomavirus (HPV) infection. Vaccination with virus-like particles (VLPs) composed of recombinant HPV L1 completely prevents persistent HPV infection [Koutsky, L. A., Ault, K. A., Wheeler, C. M., Brown, D. R., Barr, E., Alvarez, F. B., Chiacchierini, L. M. & Jansen, K. U. (2002) N. Engl. J. Med. 347, 1645-1651], suggesting that L1 expressed from recombinant adenoviruses might provide protective immunity. In our recombinants, COPV L1 is incorporated into adenovirus late region 5 (Ad L5) and is expressed as a member of the adenoviral major late transcriptional unit (MLTU). COPV L1 production by the most prolific recombinant is comparable to that of the most abundant adenoviral protein, hexon. COPV L1 production by recombinants is influenced by Ad L5 gene order, the specific mRNA processing signals associated with COPV L1, and the state of a putative splicing inhibitor in the COPV L1 gene. Recombinant COPV L1 protein assembles into VLPs that react with an antibody specific for conformational epitopes on native COPV L1 protein that correlate with protection in vivo. The designs of these recombinants can be applied directly to the production of recombinants appropriate for assessing immunogenicity and protective efficacy in animal models and in human trials.

Fig. 1.

Structure of Ad L5 in recombinants. The organization of Ad L5 in Ad5 (A), standard-order (B), and reverse-order (C) recombinants is diagrammed. Recombination between shuttle plasmids and Ad.βgal.ΔF (D) occurs in 1-kb regions of homology that flank Ad L5 (shaded). A_n, poly(A) site. The region deleted in Ad.βgal.ΔF is indicated by a black bar (14).

Fig. 2.

Late transcription in Ad5 and recombinants. (A) The major late transcriptional region (MLTU) extends rightward from the major late promoter (MLP) and contains five late regions (Ad L1–Ad L5). Arrowheads mark polyadenylation sites. Black arrows, exons; gray lines, introns. (B) A typical multigenic adenoviral late region (Ad L4). The positions and sizes of the three Ad L4 genes are shown by black boxes; the corresponding mRNAs are indicated by arrows. The protein translated from each mRNA is indicated beside the arrow. Ad L4 mRNAs are spliced to the tripartite late leader (not shown) at their 5′ ends. L4 A_n, Ad L4 poly(A) site. (C) Recombinant Ad L5 regions and expected mRNAs. Dual SA, SAs precede each gene and the region possesses a single poly(A) site; Dual PolyA, each gene is individually associated with a SA and a poly(A) site; IRES, a poliovirus IRES separates the two genes. Single SA and poly(A) sites are located at the 5′ and 3′ ends of Ad L5, respectively.

Fig. 3.

Expression of COPV L1 by recombinant adenoviruses. Infected cell lysates were examined by immunoblotting with anti-L1 antiserum. Three recombinants not shown (FFHL, SLFF, and S*LFF) produced no detectable COPV L1. For an explanation of the nomenclature, see Table 1.

Fig. 4.

L1- and fiber-containing RNAs in recombinant-infected cells. (A) RNAs detected with a COPV L1 probe. The approximate expected positions of RNAs containing both the fiber and L1 ORFs (≈4 kb) and L1 only (≈2 kb) are indicated on the left. Superimposed on each band is the protein(s) translated from that RNA (F, fiber; L1, COPV L1). RNAs marked with an asterisk are formed by splicing to an Ad L4 SA and polyadenylation at a site in Ad L5 and do not encode L1 or fiber protein. (B) RNAs detected with a fiber probe. The ≈2-kb RNA detected by this probe encodes fiber not COPV L1. Asterisks mark bands produced by polyadenylation at a duplicated E3B site located between L1 and fiber in some recombinants.

Fig. 5.

³⁵S metabolic labeling of proteins produced by recombinants. Proteins synthesized in infected cells were labeled, fractionated on a polyacrylamide/SDS gel, and autoradiographed. The positions of adenoviral proteins II (Hexon), 100k, III, Fiber, and COPV L1 are indicated on the right. The asterisks mark bands of COPV L1 protein. Lanes labeled L1 IP and Fiber IP contain labeled, immunoprecipitated proteins.

Fig. 6.

COPV VLP production by recombinants. (A) Purified VLPs from FFSO-infected cells reacted with COPV L1 conformation-specific antibody and labeled with gold-conjugated secondary antibody. The tiny black dots are the ImmunoGold stain. (B) Adenovirus particles from the same grid are not labeled. (C) Adenovirus particles and a VLP from a grid not treated with antibody. (Scale bar, 100 nm.)