Immunotherapy with DNA vaccine and live attenuated rubella/SIV gag vectors plus early ART can prevent SIVmac251 viral rebound in acutely infected rhesus macaques

Abstract

Anti-retroviral therapy (ART) has been highly successful in controlling HIV replication, reducing viral burden, and preventing both progression to AIDS and viral transmission. Yet, ART alone cannot cure the infection. Even after years of successful therapy, ART withdrawal leads inevitably to viral rebound within a few weeks or months. Our hypothesis: effective therapy must control both the replicating virus pool and the reactivatable latent viral reservoir. To do this, we have combined ART and immunotherapy to attack both viral pools simultaneously. The vaccine regimen consisted of DNA vaccine expressing SIV Gag, followed by a boost with live attenuated rubella/gag vectors. The vectors grow well in rhesus macaques, and they are potent immunogens when used in a prime and boost strategy. We infected rhesus macaques by high dose mucosal challenge with virulent SIVmac251 and waited three days to allow viral dissemination and establishment of a reactivatable viral reservoir before starting ART. While on ART, the control group received control DNA and empty rubella vaccine, while the immunotherapy group received DNA/gag prime, followed by boosts with rubella vectors expressing SIV gag over 27 weeks. Both groups had a vaccine "take" to rubella, and the vaccine group developed antibodies and T cells specific for Gag. Five weeks after the last immunization, we stopped ART and monitored virus rebound. All four control animals eventually had a viral rebound, and two were euthanized for AIDS. One control macaque did not rebound until 2 years after ART release. In contrast, there was only one viral rebound in the vaccine group. Three out of four vaccinees had no viral rebound, even after CD8 depletion, and they remain in drug-free viral remission more than 2.5 years later. The strategy of early ART combined with immunotherapy can produce a sustained SIV remission in macaques and may be relevant for immunotherapy of HIV in humans.

Fig 1. Study plan.

Stage I: high-dose mucosal challenge of rhesus macaques with SIVmac251 by the intra-rectal route. ART was delayed until day 3 to allow establishment of the latent viral reservoir. Stage II: the vaccine group was immunized with DNA/gag expression plasmid followed by rubella/gag vectors, while on ART. Controls received control DNA and empty rubella vaccine. Stage III: after 27 weeks, ART was withdrawn, and the animals were followed for viral rebound. Subsequently, CD8⁺ T cells were depleted to release latent virus.

Fig 2. Immune response to rubella antigens.

The anti-rubella response indicates a vaccine “take” in all macaques, as detected by ELISA. The control group (right) received empty rubella vaccine and responded to the first dose of rubella (R1 + 6 weeks). The vaccine group (left) responded to the second dose of rubella/gag vectors (R2 + 2 and 4 weeks in red).

Fig 3. Immunogenicity of the rubella/gag vaccine insert.

(A) ELISA measurements of Gag antibody titers upon vaccination of macaques with gag DNA prime and Rubella/gag boost. (B) Antibodies to SIV Gag were detected by Western blot on Zeptometrix antigen strips one week after stopping ART. At this time (week 28), all animals were PCR negative. The vaccine group produced anti-p27^Gag antibodies, and some had anti-p17^Gag, while the controls were all negative. (C) Measurements of Gag CD4⁺ and CD8⁺ Gag T cell responses upon vaccination. (D) Cytotoxic T cells (GrzB⁺), as percentage of Gag-specific IFN-γ+-producing cells, were measured after DNA vaccination (week 11) and after rubella/gag boost in the 4 animals of the vaccine group. The blue arrows indicate DNA vaccination; green arrows indicate rubella vector vaccination; red arrows indicate time of ART withdrawal.

Fig 4. Viral rebound and T cell responses in the control group.

Each plot shows an individual animal. Red arrows indicate ART withdrawal, and red circles show the viral rebounds. T cell responses to Gag are shown as black bars. Macaques T506, T511, and T512 showed viral rebound in the expected time range, while macaque T508 had a prolonged delay before viral rebound, suggesting a correspondingly small viral reservoir. Macaques (T508, T511 and T512) were euthanized due to an AIDS-like illness (indicated by t).

Fig 5. Viral rebound and T cell response in the vaccine group.

Arrows indicate gag DNA vaccine given by electroporation (blue), last dose of rubella/gag vectors (green), and ART withdrawal (red). Viral loads (red circles) show viral rebound in just one out of four macaques (T507). The other three vaccinated macaques have remained virus-free for over 2 years after ART withdrawal. T cell responses to Gag are shown in black bars. Two animals with the most robust and durable T cell responses (T509 and T510) had complete control of viremia. Macaque T507 was euthanized before developing AIDS (indicated by t). Macaque T505 was euthanized for reasons unrelated to AIDS (indicated by *t).