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Ad26/MVA Therapeutic Vaccination with TLR7 Stimulation in SIV-Infected Rhesus Monkeys - Functional Cure Strategy
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"These data demonstrate the potential of therapeutic vaccination with innate immune stimulation as a strategy aimed at an HIV-1 functional cure......In summary, our data suggest the potential of combining therapeutic vaccination with innate immune stimulation as an HIV-1 cure strategy. Our findings show that these interventions can improve virology control and delay viral rebound following ART discontinuation in SIV-infected rhesus monkeys that initiated ART during acute infection. Additional preclinical and clinical studies with Ad26/MVA vaccination and TLR7 stimulation should be performed to explore this strategy in greater detail."
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Nature Published online
09 November 2016
Erica N. Borducchi, Crystal Cabral, Kathryn E. Stephenson, Jinyan Liu, Peter Abbink, David Ng'ang'a, Joseph P. Nkolola, Amanda L. Brinkman, Lauren Peter, Benjamin C. Lee, Jessica Jimenez, David Jetton, Jade Mondesir, Shanell Mojta, Abishek Chandrashekar, Katherine Molloy, Galit Alter, Jeff M. Gerold, Alison L. Hill, Mark G. Lewis, Maria G. Pau, Hanneke Schuitemaker, Joseph Hesselgesser, Romas Geleziunas, Jerome H. Kim, Merlin L. Robb, Nelson L. Michael and Dan H. Barouch
ABSTRACT
The development of immunologic interventions that can target the viral reservoir in HIV-1-infected individuals is a major goal of the HIV-1 cure field1,2. However, little evidence exists that the viral reservoir can be sufficiently targeted to improve virologic control following discontinuation of antiretroviral therapy (ART). Here we show that Ad26/MVA3,4 therapeutic vaccination with toll-like receptor 7 (TLR7) stimulation improves virologic control and delays viral rebound following ART discontinuation in SIV-infected rhesus monkeys that initiated ART during acute infection. Ad26/MVA therapeutic vaccination resulted in a dramatic increase in the magnitude and breadth of SIV-specific cellular immune responses in virologically suppressed, SIV-infected monkeys. TLR7 agonist administration led to innate immune stimulation and cellular immune activation. The combination of Ad26/MVA vaccination and TLR7 stimulation resulted in decreased levels of viral DNA in lymph nodes and peripheral blood, as well as improved virologic control and delayed viral rebound following ART discontinuation. Cellular immune breadth correlated inversely with setpoint viral loads and correlated directly with time to viral rebound. These data demonstrate the potential of therapeutic vaccination with innate immune stimulation as a strategy aimed at an HIV-1 functional cure.
The critical barrier to HIV-1 cure is the viral reservoir in latently infected CD4+ T lymphocytes5-8, which leads to viral rebound in the vast majority of HIV-1-infected individuals following discontinuation of ART9,10. Enhancing antiviral immune responses, potentially together with activation of the viral reservoir, might be able to eliminate these cells1,2,11. However, it is currently unknown whether immunologic interventions can impact the viral reservoir in vivo. In particular, it is unclear if a therapeutic vaccine will be able to induce cellular immune responses with sufficient potency and breadth to control viral rebound following ART discontinuation12. We therefore evaluated a strategy consisting of Ad26/MVA therapeutic vaccination3,4 and TLR7 agonist GS-986 administration in ART suppressed, SIV-infected rhesus monkeys.
We infected 36 Indian origin rhesus monkeys (Macaca mulatta) with SIVmac2514,13 by a single intrarectal exposure and initiated daily subcutaneous administration of a pre-formulated ART cocktail (tenofovir disoproxil fumarate, emtricitabine, dolutegravir)14 on day 7 of acute infection. Animals had median plasma SIV RNA levels of 7.10 log copies/ml (range 6.04-7.88 log copies/ml) on the day of ART initiation (Fig. 1a). SIV RNA levels were controlled in the majority of animals by day 56 and in all animals by day 224 (Fig. 1a). SIV RNA levels were comparable among the different groups, and the animals that took longer to control virus had higher starting plasma viral loads on day 7 (P = 0.04; data not shown).
Following 24 weeks of suppressive ART, groups of monkeys received the following interventions: (1) Ad26/MVA vaccines alone, (2) Ad26/ MVA vaccines + TLR7 agonist GS-986, (3) TLR7 agonist GS-986 alone, or (4) sham (N = 9 animals/group). In Groups 1-2, animals were vaccinated by the intramuscular route with 3x1010 viral particles (vp) Ad26 vectors4,15 expressing SIVsmE543 Gag/Pol/Env at weeks 24 and 36, and were boosted with 108 plaque-forming units (pfu) MVA vectors4 expressing SIVsmE543 Gag/Pol/Env at weeks 48 and 60. In Groups 2-3, animals received 10 administrations of 0.3 mg/kg GS-986 (Gilead Sciences, Foster City, CA) by oral gavage every 2 weeks from weeks 50-70. In the combination intervention group, animals initiated GS-986 at peak immunity 2 weeks after the first MVA boost immunization. TLR7 triggering is known to activate dendritic cells and lymphocytes and to lead to innate immune activation, including secretion of cytokines and chemokines16,17. We observed nonspecific activation of CD8+ and CD4+ T cells as measured by CD69 expression 1-2 days following each GS-986 administration (Extended Data Figs. 1-2) as well as increased plasma levels of IFN-α (Extended Data Fig. 3), thus confirming the immunostimulatory activity of GS-986. Other pro inflammatory cytokines and chemokines were also induced by GS-986, including IL-1RA, IL-6, IL-23, CXCL9 (MIG), CXCL11 (I-TAC), CCL4 (MIP-1β ), and CCL11 (Eotaxin) (data not shown).
We next evaluated the immunogenicity of the Ad26/MVA vaccine. The groups that received the vaccine demonstrated a robust > 100-fold increase in the magnitude of Gag/Pol/Env-specific cellular immune responses as compared with pre-vaccination responses by IFN-γ ELISPOT assays (Fig. 1b). Cellular immune responses increased substantially at week 28 after Ad26 priming and further increased at week 50 after MVA boosting against both vaccine-matched SIVsmE543 peptides and virus-matched SIVmac239 peptides. We also observed induction of robust Gag/Pol/Env-specific CD8+ and CD4+ T cell responses as measured by multiparameter intracellular cytokine staining assays (Extended Data Figs. 4-5). These responses were higher magnitude than those elicited with this same vaccine in SIV-uninfected rhesus monkeys4.
Ad26/MVA vaccination also expanded cellular immune breadth by at least 9.2-fold, as measured by IFN-γ ELISPOT assays using sub pools of 10 peptides spanning Gag, Pol, and Env (Fig. 1c; Extended Data Fig. 6). Total breadth was defined as the number of Gag+ Pol+ Env positive subpools. We were unable to fine map individual epitopes due to insufficient availability of cells given the number of positive sub pools. Prior to vaccination at week 24, we observed an average of 1.9 positive subpools/animal (1.0 Gag, 0.4 Pol, 0.6 Env). Following Ad26 priming, cellular immune breadth markedly expanded to an average of 10.1 positive subpools/animal (4.4 Gag, 3.0 Pol, 3.1 Env). Following MVA boosting, cellular immune breadth further expanded to an average of 17.5 positive subpools/animal (5.4 Gag, 5.9 Pol, 6.2 Env) (Fig. 1c), which likely represents an underestimate of breadth, since some positive subpools may have contained more than one epitope. Several animals developed Gag/Pol/Env-specific T cells that targeted > 50 epitopes. The expanded cellular immune breadth involved induction of a large number of new epitopes and did not simply reflect expansion of previously established responses, since the vast majority of the epitopes following vaccination were not observed prior to vaccination in conventional IFN-γ ELISPOT assays as well as in assays using PBMC stimulated in vitro with these peptides for enhanced sensitivity (data not shown). We speculate that early initiation of ART induced preserved CD4+ T cell help (Extended Data Fig. 5), which likely contributed to vaccine immunogenicity. Consistent with this hypothesis is the observation that cellular immune breadth correlated inversely with pre-ART day 7 SIV RNA (Extended Data Fig. 7), which may be a surrogate marker for immunologic damage. In contrast with robust cellular immune responses, only modest humoral immune responses were observed following vaccination, including binding antibody
responses by ELISA as well as functional antibody-dependent cellular phagocytosis, neutrophil phagocytosis, and NK cell activation (data not shown).
We next assessed viral DNA in lymph nodes and PBMC using an RT-PCR assay14 with a sensitivity of 3 DNA copies/106 CD4+ T cells (Fig. 2a-b). In sham controls, viral DNA declined slightly between weeks 20 and 48, presumably reflecting the impact of suppressive ART, but no further decline was observed at week 70. In contrast, the two groups that received the Ad26/MVA vaccine demonstrated marked reductions of viral DNA to undetectable levels in the majority of animals by week 70 in both lymph nodes (Fig. 2a) and PBMC (Fig. 2b), suggesting that vaccination led to substantial reductions in SIV-infected CD4+ T cells in these tissue compartments. It is possible that a larger fraction of proviruses might be transcriptionally active following early ART initiation as compared with ART initiation during chronic infection, although this remains to be determined8. Viral outgrowth assays using 20 million PBMC were negative in all animals including controls at week 70 (data not shown), presumably as a result of early initiation of ART.
To evaluate the therapeutic efficacy of the interventions, we discontinued ART at week 72. Viral rebound was observed in all animals (Fig. 3a). All sham controls rebounded by day 10-14 following ART discontinuation in a stereotypical fashion and exhibited median set point plasma SIV RNA levels of 4.89 log copies/ml (range 4.27-5.57 log copies/ml) on day 168 following ART discontinuation. The monkeys that received GS-986 alone did not demonstrate any discernible delay or control of viral rebound, indicating that TLR7 stimulation alone exerted no detectable antiviral effect in this study. Animals that received the Ad26/MVA vaccine alone exhibited a 0.66 log reduction of median setpoint plasma SIV RNA levels to 4.23 log copies/ml (range 2.70-4.91 log copies/ml) (P = 0.002, Wilcoxon rank-sum test) but only a marginal delay of viral rebound (P = 0.01, Wilcoxon rank-sum test) as compared with controls (Fig. 3b-c). In contrast, monkeys that received both the Ad26/MVA vaccine and GS-986 showed a striking 1.74 log reduction of median setpoint plasma SIV RNA levels to 3.15 log copies/ml (range < 2.30-4.09 log copies/ml) (P < 0.0001) and a 2.5-fold delay of viral rebound from a median of 10 to 25 days as compared with controls (P = 0.003) (Fig. 3b-c). Moreover, 33% (3 of 9) of the monkeys in the combination intervention group showed effective virologic control to undetectable setpoint viral loads (< 2.30 log copies/ml) following ART discontinuation. These data demonstrate that the combination of Ad26/ MVA vaccination and TLR7 stimulation improved virology control and delayed viral rebound following ART discontinuation.
We next evaluated the immunologic and virologic correlates of virologic control. Cellular immune breadth immediately prior to ART discontinuation (Fig. 4a) as well as at peak immunity (Extended Data Fig. 8) correlated inversely with setpoint viral loads following ART discontinuation, particularly the breadth of Gag, Env, and total responses (P < 0.0001, Spearman rank-correlation tests). Gag, Env, and total cellular immune breadth also correlated directly with the time to viral rebound (Fig. 4b, Extended Data Fig. 9; P = 0.0001 to P = 0.001). Consistent with the correlates analyses, mathematical modeling of viral dynamics further suggested that the combination of changes in the reservoir exit rate and the early viral growth rate accounted for the differences in the time to viral rebound, whereas the virus-specific immune proliferation rate was likely responsible for virology control (Extended Data Fig. 10).
Viral DNA in lymph nodes and PBMC correlated poorly with virology control following ART discontinuation and time to viral rebound (P = 0.03, data not shown), presumably because all animals with undetectable viral DNA still rebounded. These findings suggest that these viral DNA assays are not sufficiently sensitive to predict functional cure, consistent with recent clinical observations18,19. Moreover, there was no correlation between day 7 pre-ART SIV RNA or time to initial virology suppression on ART and virologic control following ART discontinuation (P = NS).
In this study, we demonstrate that Ad26/MVA therapeutic vaccination robustly augmented cellular immune magnitude and breadth in ART-suppressed, SIV-infected rhesus monkeys and that the TLR7 agonist GS-986 led to innate immune stimulation and cellular activation. The combination of Ad26/MVA vaccination and GS-986 resulted in a significant 1.74 log reduction in median setpoint viral loads and a 2.5-fold delay in the time to viral rebound following ART discontinuation as compared with sham controls. Moreover, 3 of 9 animals in this group demonstrated virologic control to undetectable levels in the absence of ART. These three animals were characterized by high cellular immune magnitude and breadth and negative viral DNA prior to ART discontinuation.
Taken together, these data demonstrate the proof-of-concept that the combination of therapeutic vaccination and innate immune stimulation can impact viral rebound following ART discontinuation.
The Ad26/MVA vaccine induced remarkably potent IFN-γ ELISPOT responses in the ART-suppressed, SIV-infected monkeys in the present study, consistent with a previous prophylactic vaccine study in uninfected monkeys4. We speculate that preserved CD4+ T cell help, primed by short-term viral replication following SIV infection prior to ART initiation on day 7, likely enhanced vaccine immunogenicity. The Ad26/MVA vaccine also expanded cellular immune breadth by nearly 10-fold, including induction of responses to a large number of epitopes that were not detectable following SIV infection. This may be critical for a therapeutic vaccine, because the viral reservoir typically contains viruses with T cell epitope escape mutations12. We did not detect evidence for viral "blipping" on day 1 or day 2 following TLR7 agonist administration (J. Whitney, unpublished data), which may reflect the early initiation of ART on day 7 of infection and thus the limited size of the viral reservoirs in this study14. We were therefore unable to determine whether the beneficial effect of the TLR7 agonist reflected its potential role as a vaccine adjuvant, a latency reversing agent, or both. Future studies should be performed in SIV-infected monkeys that initiate ART during chronic infection, which would be more representative of the majority of HIV-1-infected individuals. Moreover, future studies could explore longer periods of ART suppression, potentially to reduce residual viral replication, although the majority of animals appear to have a stable reservoir after 24-72 weeks of ART14. The capacity of immunologic interventions to target follicular helper CD4+ T cells in lymph nodes should also be explored20.
Previous studies of poxvirus-, adenovirus-, and DNA-based therapeutic vaccines have typically shown only a modest impact on viral rebound following ART discontinuation in both rhesus monkeys21 and humans22,23. The present study extends these prior observations by combining therapeutic vaccination with innate immune stimulation. Of note, the combination of Ad26/MVA vaccination and TLR7 stimulation proved more potent than either component alone. This finding is consistent with a prior in vitro study that showed that robust CD8+ T cells may be able to facilitate elimination of the viral reservoir following reactivation11. The present study also demonstrates that the breadth of Gag/Pol/Env-specific T cell responses correlated inversely with setpoint viral loads following ART discontinuation, suggesting
that the mechanism underlying the therapeutic efficacy of the vaccine involved expansion of cellular immune breadth and immunologic control of virus rebounding from the reservoir.
In summary, our data suggest the potential of combining therapeutic vaccination with innate immune stimulation as an HIV-1 cure strategy. Our findings show that these interventions can improve virology control and delay viral rebound following ART discontinuation in SIV-infected rhesus monkeys that initiated ART during acute infection. Additional preclinical and clinical studies with Ad26/MVA vaccination and TLR7 stimulation should be performed to explore this strategy in greater detail.
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