Immunological and virological analyses of rhesus macaques immunized with chimpanzee adenoviruses expressing the simian immunodeficiency virus Gag/Tat fusion protein and challenged intrarectally with repeated low doses of SIVmac

Abstract

Human adenovirus (AdHu)-based candidate AIDS vaccine can provide protection from simian immunodeficiency virus (SIV) transmission and disease progression. However, their potential use may be limited by widespread preexisting immunity to the vector. In contrast, preexisting immunity to chimpanzee adenoviruses (AdC) is relatively rare. In this study, we utilized two regimens of prime-boost immunizations with AdC serotype SAd-V23 (also called AdC6) and SAd-V24 (also called AdC7) expressing SIV Gag/Tat to test their immunogenicity and ability to protect rhesus macaques (RMs) from a repeated low-dose SIVmac239 challenge. Both AdC6 followed by AdC7 (AdC6/7) and AdC7 followed by AdC6 (AdC7/6) induced robust SIV Gag/Tat-specific T cell responses as measured by tetramer staining and functional assays. However, no significant protection from SIV transmission was observed in either AdC7/6- or AdC7/6-vaccinated RMs. Interestingly, in the RMs showing breakthrough infections, AdC7/6-SIV immunization was associated with a transient but significant (P = 0.035 at day 90 and P = 0.033 at day 120 postinfection) reduction in the setpoint viral load compared to unvaccinated controls. None of the measured immunological markers (i.e., number or functionality of SIV-specific CD8(+) and CD4(+) T cell responses and level of activated and/or CCR5(+) CD4(+) target cells) at the time of challenge correlated with protection from SIV transmission in the AdC-SIV-vaccinated RMs. The robust immunogenicity observed in all AdC-immunized RMs and the transient signal of protection from SIV replication exhibited by AdC7/6-vaccinated RMs even in the absence of any envelope immunogen suggest that AdC-based vectors may represent a promising platform for candidate AIDS vaccines.

Fig 1

Immunogenicity and protection from low-dose rectal SIV_mac239 challenge by AdC6/C7-SIVgag/tat and AdC7/C6-SIVgag/tat vectors. Shown is a schematic representation of the experimental design, which involved two immunizations with AdC6-SIVgag/tat followed after 6 months by AdC7-SIVgag/tat (group 1) or AdC7-SIVgag/tat followed after 6 months by AdC6-SIVgag/tat (group 2). Each group included six MamuA*01⁺ and 4 MamuA*01⁻ adult Indian RMs. All RMs, as well as an additional 10 unvaccinated control animals, were challenged with repeated low-dose intrarectal SIV_mac239 every 2 weeks up to 15 times. For logistical reasons, the RMs were staggered in two cohorts (A and B), with the challenge for the 1st cohort conducted 4 months after the last immunization and the challenge for the 2nd cohort conducted 5 months after the last immunization. The times of immunization, challenge, and sample collection are indicated. PB, peripheral blood.

Fig 2

Vaccination with AdC6/C7-SIVgag/tat and AdC7/C6-SIVgag/tat induces SIV-specific CD8⁺ T cell responses. (A to D) Average fractions of CD8⁺ T cells binding Gag-CM9 (A and B) or Tat-SL8 (C and D) MaMu-A*01 tetramers at various time points postimmunization in AdC6/C7-SIVgag/tat (A and C) and AdC7/C6-SIVgag/tat (B and D). (E and F) Average levels of total SIV Gag-specific CD8⁺ (E) and CD4⁺ (F) T cell responses after immunization with AdC6/C7-SIVgag/tat and AdC7/C6-SIVgag/tat. The error bars indicate the standard deviations (SD) for each vaccination group. The responses were measured by intracellular cytokine and CD107a staining after stimulation of cryopreserved PBMCs with SIV_mac239 Gag peptides. Cells showing at least one function (among CD107a, IL-2, IFN-γ, or TNF-α expression) above background were counted as positive to determine the level of total Gag-specific responses. The postvaccination time was 1 week after the last immunization, and the prechallenge time point was 2 to 4 weeks before initiation of the challenge phase.

Fig 3

Levels of CCR5⁺ and proliferating CD4⁺ T cells after AdC-SIV immunization. (A to C) Average levels of CD4⁺ CCR5⁺ T cells measured as percentages of total CD3⁺ CD4⁺ T cells in peripheral blood (A), lymph nodes (B), and rectal-biopsy specimens (C) of AdC6/7- and AdC7/6-vaccinated RMs. (D to F) Average levels of CD4⁺ Ki-67⁺ T cells measured as percentages of total CD3⁺ CD4⁺ T cells in peripheral blood (D), lymph nodes (E), and rectal-biopsy specimens (F) from the same animals. The error bars indicate SD.

Fig 4

Viral acquisition after low-dose rectal SIV_mac239 challenge. Shown are the numbers of challenges required for acquisition of SIV infection in AdC6/7- and AdC7/6-vaccinated RMs, as well as unvaccinated controls.

Fig 5

AdC-SIV-vaccinated RMs show lower virus replication after breakthrough SIV infection. The SIV plasma viral load (expressed as copies per milliliter of plasma) was measured at multiple time points after breakthrough SIV infection by real-time PCR in individual animals (A) and as geometric means for each study group (B). AdC6/7, AdC6/7-SIV-vaccinated RMs; AdC7/6, AdC7/6-SIV-vaccinated animals. The error bars represent the SD of the different study groups. p.c., postchallenge.

Fig 6

Dynamics of CD4⁺ T cells in the blood, lymph nodes, and rectal-biopsy specimens after breakthrough SIV infection. Circulating levels of CD4⁺ T cells were measured as absolute counts per cubic millimeter of blood (A to C) and percentages of CD3⁺ T cells (B to D) at multiple time points after breakthrough SIV infection in individual animals (A and B) and averaged within each study group (C and D). (E and F) Lymph node CD4⁺ T cell levels were measured as percentages of CD3⁺ T cells in individual animals (E) and averaged within each study group (F). (G and H) CD4⁺ T cell levels in rectal-biopsy specimens were also measured as percentages of CD3⁺ T cells in individual animals (G) and averaged within each study group (H). AdC6/7, AdC6/7-SIV-vaccinated RMs; AdC7/6, AdC7/6-SIV-vaccinated animals. The error bars represent the SD of the different study groups.

Fig 7

Relationship between levels and functionality of SIV-specific CD8⁺ T cells and number of challenges needed to achieve SIV infection. (A to C) The levels of SIV-specific CD8⁺ T cells in peripheral blood, lymph nodes, and rectal-biopsy specimens as measured by tetramer staining for the MamuA*01-restricted SIV Gag CM9 epitope before challenge do not predict the risk of acquiring SIV infection during the repeated intrarectal low-dose challenge. AdC6/7, AdC6/7-SIV-vaccinated RMs; AdC7/6, AdC7/6-SIV-vaccinated animals. (D) Lack of correlation between the number of functions displayed by SIV Gag-specific CD8⁺ T cells and the number of challenges needed to acquire SIV infection. HD indicates the animals that were infected with a high dose of SIV at the end of the repeated low-dose challenge phase.

Fig 8

Relationship between the levels of CD4⁺ CCR5⁺ and CD4⁺ Ki-67⁺ target T cells and the number of challenges needed to achieve SIV infection. (A and B) The levels of CD4⁺ CCR5⁺ and CD4⁺ Ki-67⁺ T cells in peripheral blood expressed as percentages of total CD3⁺ CD4⁺ T cells do not predict the risk of acquiring SIV infection during the repeated intrarectal low-dose challenge. (C and D) The levels of CD4⁺ CCR5⁺ and CD4⁺ Ki-67⁺ T cells in rectal-biopsy specimens also do not predict the risk of acquiring SIV infection during the repeated intrarectal low-dose challenge. AdC6/7, AdC6/7-SIV-vaccinated RMs; AdC7/6, AdC7/6-SIV-vaccinated animals. HD indicates the animals that were infected with a high dose of SIV at the end of the repeated low-dose challenge phase.