Replication-defective adenovirus serotype 5 vectors elicit durable cellular and humoral immune responses in nonhuman primates

Abstract

The magnitude and durability of immune responses induced by replication-defective adenovirus serotype 5 (ADV5) vector-based vaccines were evaluated in the simian-human immunodeficiency virus/rhesus monkey model. A single inoculation of recombinant ADV5 vector constructs induced cellular and humoral immunity, but the rapid generation of neutralizing anti-Ad5 antibodies limited the immunity induced by repeated vector administration. The magnitude and durability of the immune responses elicited by these vaccines were greater when they were delivered as boosting immunogens in plasmid DNA-primed monkeys than when they were used as single-modality immunogens. Therefore, administration of ADV5-based vectors in DNA-primed subjects may be a preferred use of this vaccine modality for generating long-term immune protection.

FIG. 1.

Mamu-A*01-restricted CD8⁺ T-lymphocyte responses following ADV5 prime/ADV5 boost or DNA prime/ADV5 boost immunizations. (A) Two Mamu-A*01⁺ rhesus monkeys received intramuscular inoculations of 10¹² particles of each of two ADV5 constructs, one expressing SIV Gag-Pol and the other expressing HIV-1 89.6P Env, on weeks 0 and 8. On weeks 0, 4, and 8, two other Mamu-A*01⁺ rhesus monkeys were inoculated with 5 mg of each of two plasmid DNA constructs, one expressing SIV Gag-Pol and the other expressing HIV-1 89.6P Env. (B) All four monkeys were boosted on week 26 with 10¹² particles of each of the two ADV5 constructs. Vaccine-elicited CD8⁺ T-cell responses specific for the immunodominant SIV Gag p11C and subdominant HIV-1 Env p41A and SIV Pol p68A epitopes were measured by tetramer staining of freshly isolated PBMC. The percentages of CD3⁺ CD8⁺ T cells that bound the Mamu-A*01/peptide-tetramer complexes are shown.

FIG. 2.

Cellular immune responses to SIV Gag and HIV-1 Env following ADV5 prime/ADV5 boost or DNA prime/ADV5 boost immunizations. (A) Two Mamu-A*01⁺ monkeys were immunized with ADV vectors (see Fig. 1A). (B) Two Mamu-A*01⁺ rhesus monkeys were inoculated with the DNA/ADV vaccine (see Fig. 1B) and analyzed by interferon gamma ELISPOT responses in freshly isolated PBMC with in vitro exposure to peptide pools spanning the SIVmac251 Gag and HIV-1 89.6P Env proteins or 9-mer peptides representing the SIV Gag p11C, HIV-1 p41A, and SIV Pol p68A epitopes. Total SFC per 10⁶ cells are shown. IFN-γ ELISPOT responses in medium control wells were consistently <15% of the response in wells containing peptide (data not shown).

FIG. 3.

Cellular immunity elicited by DNA prime and ADV5 boost immunizations is mediated by both CD4⁺ and CD8⁺ T lymphocytes. Freshly isolated PBMC from two cohorts of immunized monkeys (A and B) were depleted of CD8⁺ T lymphocytes. IFN-γ ELISPOT responses were measured in unfractionated as well as CD8⁺ T-cell-depleted PBMC of the monkeys 2 weeks following the final ADV5 immunizations.

FIG. 4.

Humoral immune responses elicited by DNA prime and ADV5 boost immunizations. (A) Neutralizing antibodies against HIV-1 89.6 were measured during the course of immunizations. The percent neutralization value for each plasma sample is based on a comparison to the corresponding preimmune plasma for each monkey. All plasma were heat inactivated and diluted 1:5. (B) ADV5-specific neutralizing antibody responses are generated following a single inoculation of an ADV5 vaccine construct. Ad5-specific neutralizing antibody responses were assessed using a luciferase-based virus neutralization assay. Ninety-percent-neutralization titers were defined as the maximum serum dilution that neutralized 90% of luciferase activity.

FIG. 4.

Humoral immune responses elicited by DNA prime and ADV5 boost immunizations. (A) Neutralizing antibodies against HIV-1 89.6 were measured during the course of immunizations. The percent neutralization value for each plasma sample is based on a comparison to the corresponding preimmune plasma for each monkey. All plasma were heat inactivated and diluted 1:5. (B) ADV5-specific neutralizing antibody responses are generated following a single inoculation of an ADV5 vaccine construct. Ad5-specific neutralizing antibody responses were assessed using a luciferase-based virus neutralization assay. Ninety-percent-neutralization titers were defined as the maximum serum dilution that neutralized 90% of luciferase activity.