Immune responses in macaques to a prototype recombinant adenovirus live oral human papillomavirus 16 vaccine

Abstract

Immunization with human papillomavirus (HPV) L1 virus-like particles (VLPs) prevents infection with HPV. However, the expense and logistical demands of current VLP vaccines will limit their widespread use in resource-limited settings, where most HPV-induced cervical cancer occurs. Live oral adenovirus vaccines have properties that are well-suited for use in such settings. We have described a live recombinant adenovirus vaccine prototype that produces abundant HPV16 L1 protein from the adenovirus major late transcriptional unit and directs the assembly of HPV16 VLPs in tissue culture. Recombinant-derived VLPs potently elicit neutralizing antibodies in mice. Here, we characterize the immune response to the recombinant after dual oral and intranasal immunization of pigtail macaques, in which the virus replicates as it would in immunized humans. The immunization of macaques induced vigorous humoral responses to adenovirus capsid and nonstructural proteins, although, surprisingly, not against HPV L1. In contrast, immunization elicited strong T-cell responses to HPV VLPs as well as adenovirus virions. T-cell responses arose immediately after the primary immunization and were boosted by a second immunization with recombinant virus. T-cell immunity contributes to protection against a wide variety of pathogens, including many viruses. The induction of a strong cellular response by the recombinant indicates that live adenovirus recombinants have potential as vaccines for those agents. These studies encourage and will inform the continued development of viable recombinant adenovirus vaccines.

FIG 1

(A) FFIL¹⁶ Genomic structure and expression cassette. Line 1, the major late transcriptional unit (MLTU) of the adenovirus genome lies between the major late promoter (MLP) and early region 4 (E4). The primary ∼27-kb transcript of the MLTU is alternatively polyadenylated and spliced to generate about 15 late mRNAs that comprise five late regions (Ad L1 to Ad L5) defined by the positions of their polyadenylation sites (arrowheads). Line 2, enlargement of the E3-fiber region of wild-type Ad5. In FFIL¹⁶ (line 3), late region 5 (Ad L5) has been expanded to include a poliovirus IRES and the HPV16 L1 gene downstream of the native fiber gene. FFIL¹⁶ also includes an E3 deletion of 2.8 kb (ΔE3) and a mutation in E2a (hr404; not shown) that confers the ability to grow in monkey cells. Both the fiber and L1 proteins are translated from a single L5 mRNA. (B) Immunization schedule. Blood samples were taken from animals to obtain PBMCs (^) and serum (*) at the times indicated. Monkeys were inoculated with 10⁹ PFU of recombinant Ad5hr-FFIL¹⁶ virus at day 0 and day 85. At day 237, the monkeys were given a protein booster consisting of 20 μg of HPV16 L1 OptiPrep-purified VLPs made from recombinant baculovirus infections.

FIG 2

Immunized macaques (numbers in key) produce anti-adenovirus antibodies. (A) Immunized macaque sera were tested for reactivity with the nonvirion late protein 100K by Western blotting. Individual filter strips containing lysates of 293 cells transfected with a 100K expression plasmid were blotted with sera taken at the indicated times. A strip probed with anti-100K rabbit serum is shown at the right of the panel for macaque 811; the positions of the two 100K species present in the transfected cells are indicated by arrows for the others. For each animal, the density and contrast of all strips, as a group, were adjusted in Photoshop. (B) Ad5-neutralizing antibodies in sera drawn at the indicated time points were measured by plaque reduction. The lowest dilution tested was 1:10; values <10 were arbitrarily assigned a value of 5.

FIG 3

Cellular responses to immunization with Ad5hr-FFIL¹⁶. Stimulation indices (SI) in response to 10 μg of HPV16 L1 VLPs (■) or an irrelevant baculovirus lysate () are plotted for PBMCs drawn from each macaque at the indicated times. The arrows indicate immunizations; the third immunization was with purified HPV16 VLPs. The error bars represent the standard errors for the triplicate wells at each time point. The asterisks indicate the time points at which postimmunization and preimmunization responses differ statistically (P < 0.05). Interruptions in the plots indicate an extended period for which SI values were not determined. A horizontal line is drawn at an SI value of 1 for reference.

FIG 4

FFIL¹⁶ does not induce CD4⁺ T-cell proliferation. PBMCs collected on day 112 from macaques 803 and 811 were labeled with CFSE and stimulated with medium (UN) or 10 μg/ml of HPV16 VLPs (VLP). Live cells were examined by flow cytometry after staining with APC-labeled anti-CD4 antibody and CFSE. The circles indicate where proliferating CD4⁺ cells are expected. The CD4⁻ proliferating populations are indicated by an ellipses.