Lack of Therapeutic Efficacy of an Anti-α₄β₇ Antibody in SIVmac251-Infected Rhesus Macaques

Abstract

Sustained virologic control of human immunodeficiency virus type 1 (HIV-1) infection following discontinuation of antiretroviral therapy (ART) is a major goal of the HIV-1 cure field. A recent study reported that anti-α₄β₇ antibody administration induced durable virologic control following ART discontinuation in 100% of rhesus macaques infected with an attenuated strain of simian immunodeficiency virus (SIV) containing a stop codon in nef. We performed similar studies in 50 rhesus macaques infected with wildtype, pathogenic SIVmac251. In animals that initiated ART during either acute or chronic infection, anti-α₄β₇ antibody infusion had no detectable effect on the viral reservoir or viral rebound following ART discontinuation. These data demonstrate that anti-α₄β₇ antibody administration did not provide therapeutic efficacy in the model of pathogenic SIVmac251 infection of rhesus macaques.

One Sentence Summary:

An anti-α₄β₇ antibody did not result in virologic control in SIVmac251-infected rhesus macaques.

The development of therapeutic strategies that lead to sustained virologic remission in the absence of antiretroviral therapy (ART), defined as a functional cure, represents a major goal of HIV-1 cure research (1–3). Expression of the gut homing integrin α₄β₇ is upregulated on a subset of memory CD4⁺ T cells and promotes trafficking to gastrointestinal associated lymphoid tissue (GALT), where these cells are a preferred site of early viral replication (4–7). As such, α₄β₇ has been explored as a potential therapeutic target for the prevention and treatment of HIV-1 infection. It has recently been reported that administration of an anti-α₄β₇ antibody in ART-suppressed, SIV-infected rhesus macaques led to virologic suppression to undetectable levels in 100% of animals following ART withdrawal (8). Although not reported, this previous study used an attenuated strain of SIVmac239 containing a stop codon in nef to reduce viral replication during acute infection. Based on these findings, a clinical trial has been initiated to evaluate the therapeutic efficacy of anti-α₄β₇ antibody administration in HIV-1-infected humans (). To evaluate the generalizability of these preclinical findings in a more commonly used SIV model, we evaluated the therapeutic efficacy of anti-α₄β₇ antibody administration in rhesus macaques infected with wildtype, pathogenic SIVmac251.

We conducted two studies in a total of 50 SIVmac251-infected rhesus macaques that initiated ART during either early acute infection (Study 1; N=36) or late chronic infection (Study 2; N=14). In Study 1, 36 rhesus macaques were infected by the intrarectal route with 500 MID₅₀ wildtype, pathogenic SIVmac251 (Fig 1A) (9). Preformulated ART consisting of tenofovir disoproxil fumarate, emtricitabine, and dolutegravir (TDF/FTC/DTG; Gilead) (10, 11) was initiated on day 35 following infection and was continued until day 126, consistent with the previously published anti-α₄β₇ antibody therapy protocol (8). On day 63 (4 weeks after initiation of ART), animals started receiving one of three antibody infusions, which were administered every 3 weeks for a total of 8 infusions (N=12/group). Group 1 received 50 mg/kg anti-α₄β₇ antibody (8) (Clone #A4B7, primatized ACT1, MassBiologics; N=12), Group 2 received 5 mg/kg anti-α₄β₇ antibody (Clone #A4B7, MassBiologics; N=12), and Group 3 received 50 mg/kg isotype control antibody (Clone #DSPR1, MassBiologics; N=12). On day 126 (13 weeks after initiation of ART), after the fourth anti-α₄β₇ antibody infusion, ART was discontinued in all animals, as per the previously published protocol (8) (Fig. 1A).

Fig. 1. Study design and antibody pharmacokinetics.

Schematic overviews of (A) Study 1 (Early ART Study) (N=36) in which ART treatment was initiated on day 35 of acute infection and (B) Study 2 (Late ART Study) (N=14) in which ART was initiated after 1 year of chronic infection. SIVmac251 infection is shown with a red arrow. Initiation and discontinuation of daily ART are indicated by blue arrows. Eight antibody infusions for each group are indicated by green arrows. Serum log anti-α₄β₇ antibody levels (µg/ml) are shown before and after each antibody infusion in (C) Study 1 and (D) Study 2. Black lines represent anti-α₄β₇ antibody levels in each individual monkey with median levels shown in red. Limit of detection is 1 µg/ml.

In Study 2, a similar treatment protocol was performed in 14 SIVmac251-infected rhesus macaques that initiated ART (TDF/FTC/DTG) during chronic infection. Rhesus macaques were infected with wildtype, pathogenic SIVmac251, and ART was initiated after 1 year of chronic infection. Animals were then virologically suppressed with ART for 6 months prior to initiation of the antibody infusions (Fig. 1B). Chronically infected rhesus macaques had setpoint viral loads of 3–5 log SIV RNA copies/ml prior to ART initiation (Fig. S1). Six months after initiation of ART, 50 mg/kg anti-α₄β₇ antibody (Group 1; Clone# A4B7, MassBiologics; N=7) or 50 mg/kg isotype control antibody (Group 2; clone #DSPR1, MassBiologics; N=7) infusions were started on day 0 and were administered every 3 weeks for a total of 8 infusions (Fig. 1B). ART was discontinued on day 63 after the fourth anti-α₄β₇ antibody infusion. Two macaques were euthanized prior to ART discontinuation, one in Group 1 due to the development of clinical AIDS shortly after ART was initiated, and one in Group 2 due to an anaphylactic reaction following administration of the control DSPR1 antibody. These two animals were therefore not included in the analysis. CD4+ T cell levels from all animals were monitored over the course of these studies and showed modest declines after ART discontinuation, particularly in the chronically treated animals in Study 2 (Fig. S2).

We measured serum anti-α₄β₇ antibody levels by ELISA prior to and 1 day after each infusion. In Study 1, anti-α₄β₇ antibody levels in animals that received the 50 mg/kg dose reached a median peak concentration of 2.86 log µg/ml (range 2.53–3.09 log µg/ml) after each infusion followed by a decline to a median of 1.72 log µg/ml three weeks after each infusion (Fig. 1C). Antibody levels were comparable after all 8 infusions without the development of suppressive anti-drug antibodies (ADA) (Fig. S3A). In Study 2, antibody levels mirrored those observed in Study 1 with a median peak antibody concentration of 2.97 log µg/ml (range 2.79–3.29 log µg/ml) after each infusion followed by a decline to a median of 1.74 log µg/ml three weeks after each infusion, and antibody levels were comparable for all 8 infusions (Fig. 1D) without development of suppressive ADA (Fig. S3B).

Plasma viral loads for all macaques were measured by RT-PCR (10). In Study 1, peak viremia for all macaques on day 14 after SIVmac251 infection was a median of 7.43 log SIV RNA copies/ml (range 6.42–8.16 log SIV RNA copies/ml) and was comparable among groups and similar to our previous experience with this challenge virus (12, 13) (Fig. 2A–C). As expected, peak viral loads in these animals were approximately 1.5 logs higher than those achieved with the attenuated SIV containing a stop codon in the nef gene (8). Initiation of ART led to virologic control in all study groups with viremia largely suppressed by day 56 and fully or nearly fully suppressed by the time of ART discontinuation on day 126. After discontinuation of ART, virologic rebound was observed in all macaques in all groups (Fig. 2A–C). Peak plasma rebound viremia was a median of 4.87 log SIV RNA copies/ml (range 4.07–6.37 log SIV RNA copies/ml) in the 50 mg/kg anti-α₄β₇ antibody Group 1 (Fig. 2A), 4.15 log SIV RNA copies/ml (range 3.41–6.67 log SIV RNA copies/ml) in the 5 mg/kg anti-α₄β₇ antibody Group 2 (Fig. 2B) and 4.91 log SIV RNA copies/ml (range 3.48–5.89 log SIV RNA copies/ml) in the sham Group 3 (Fig. 2C). Plasma setpoint viral loads did not show significant differences among the treatment groups on day 364 (Fig. 2D), indicating that the anti-α₄β₇ antibody infusions did not lead to virologic control following ART discontinuation using this protocol (8) in SIVmac251-infected rhesus macaques.

Fig. 2. Viral loads and viral DNA in anti-α₄β₇ antibody-treated rhesus macaques that initiated ART during acute SIVmac251 infection (Study 1).

Plasma viral loads in macaques treated with (A) 50 mg/kg anti-α₄β₇ antibody (N=12), (B) 5 mg/kg anti-α₄β₇ antibody (N=12) or (C) sham IgG (N=12). (D) Setpoint plasma viral loads on day 364. (E) Viral DNA levels in peripheral blood mononuclear cells (PBMC), lymph nodes (LN), and colorectal (CR) biopsies in macaques treated with 50 mg/kg anti-α₄β₇ antibody, 5 mg/kg anti-α₄β₇ antibody or sham IgG. (F) Viral DNA levels on day 126 in PBMC, LN and CR immediately prior to ART discontinuation. Plasma viral loads are shown as log SIV RNA copies/ml (limit of detection 100 copies/ml). Viral DNA is shown as log SIV DNA copies per 1x10⁶ cells (limit of detection 3 copies/10⁶ cells). Red lines indicate median values. Differences between groups calculated by paired Student’s t-tests.

We next assessed the potential impact of anti-α₄β₇ antibody treatment on viral DNA in peripheral blood mononuclear cells (PBMC), lymph nodes (LN), and colorectal biopsies (CR) in these animals on days 56, 126 and 210 (Fig. 2E). No significant differences in viral DNA levels were observed in these anatomical compartments in the groups that received the anti-α₄β₇ antibody as compared to the sham group on day 126 (Fig. 2F), consistent with the lack of impact on viral rebound following ART discontinuation. Discontinuation of ART led to an increase in viral DNA in all groups on day 210, as expected.

In Study 2, all animals rebounded by day 21 following ART discontinuation (study day 84) (Fig. 3A–B). Spontaneous virologic control after a low level of virus replication was observed in one monkey in the control group. Peak plasma viral loads following ART discontinuation were a median of 5.77 log SIV RNA copies/ml (range 3.18–7.12 log SIV RNA copies/ml) in the 50 mg/kg anti-α₄β₇ antibody Group 1 (Fig. 3A) and 4.90 log SIV RNA copies/ml (range 2.27–6.01 log SIV RNA copies/ml) in the sham Group 2 (Fig. 3B). No significant differences were observed in setpoint viral loads between the anti-α₄β₇ antibody group and the control group on day 364 (Fig. 3C). Viral DNA levels on day 63 and 147 in PBMC, LN, and CR compartments also showed no differences between the anti-α₄β₇ antibody group and sham group on day 63 (Fig. 3D, 3E). Discontinuation of ART led to an increase in viral DNA in both groups on day 147, as expected.

Fig. 3. Viral loads and viral DNA in anti-α₄β₇ antibody-treated rhesus macaques that initiated ART during chronic SIVmac251 infection (Study 2).

Plasma viral loads in macaques treated with (A) 50 mg/kg anti-α₄β₇ antibody (N=7) or (B) sham IgG (N=7). (C) Setpoint plasma viral loads on day 364. (D) Viral DNA levels in peripheral blood mononuclear cells (PBMC), lymph nodes (LN) and colorectal (CR) biopsies in macaques treated with 50 mg/kg anti-α₄β₇ antibody or sham IgG. (E) Viral DNA levels on day 63 in PBMC, LN, and CR immediately prior to ART discontinuation. Plasma viral loads are shown as log SIV RNA copies/ml (limit of detection 100 copies/ml). Viral DNA is shown as log SIV DNA copies per 1x10⁶ cells (limit of detection 3 copies/10⁶ cells). Red lines indicate median values. Differences between groups calculated by paired Student’s t-tests.

Although there was no correlation between treatment arms and setpoint viral loads in either study, pre-ART viral loads strongly correlated with setpoint viral loads following ART discontinuation at day 364 in both studies (Study 1: R=0.53, P=0.0006, Fig. 4A; Study 2: R=0.74, P=0.0078, Fig. 4B; Spearman rank-correlation tests). These data suggest that the viral loads prior to ART initiation were critical determinants in defining setpoint viral loads following ART discontinuation, consistent with our prior observations (10, 12).

Fig 4. Viral load correlations.

Correlations between pre-ART peak viral loads and setpoint viral loads after ART discontinuation on day 364 in (A) Study 1 and (B) Study 2 analyzed with Spearman rank-correlation tests.

We used next generation deep sequencing prior to ART initiation and after ART discontinuation in all animals in both studies to look for evidence of Env immune selection pressure (Table S1). In Study 1, we observed the emergence of V4 loop deletions and single site polymorphisms in both the anti-α₄β₇ antibody groups and the control group, but with no association with treatment arm. In Study 2, we observed multiple V4 loop deletions, insertions, and polymorphisms in both groups also with no association with treatment arm. These data suggest that V4 mutations emerged during SIVmac251 replication but did not reflect selection pressure related to anti-α₄β₇ antibody treatment.

Cellular immune responses to Env, Gag, and Pol, as measured by IFN-γ ELISPOT assays (14), were comparable among groups in Study 1 (Fig. S4A) and in Study 2 (Fig. S4B). Moreover, SIV Env-specific antibody responses, as measured by ELISA (13), were comparable among groups in Study 1 (Fig. S5A) and in Study 2 (Fig. S5B). Cellular and humoral immune responses declined during ART suppression and then increased after ART discontinuation, as expected.

The experiments described here involve two studies in SIVmac251-infected rhesus macaques (Table S2) that initiated ART during either acute or chronic infection and were designed with a comparable protocol to the published therapeutic anti-α₄β₇ antibody study, including the same source, clone, dose, and regimen for the anti-α₄β₇ antibody (8). In contrast with this prior study, we used the more common model of wildtype, pathogenic SIVmac251, instead of attenuated SIVmac239 with a stop codon in nef (15). The importance of this difference was evident in vivo by the higher peak viral loads following infection and the slower virologic control following ART initiation in the present study (Figs. 2, 3) as compared with the previous study (8). Following ART discontinuation, striking virologic control was reported in 100% of macaques in the prior study (8). In contrast, we observed rapid viral rebound in all anti-α₄β₇ antibody treated animals that was indistinguishable from controls (Figs. 2, 3). Moreover, we did not observe any impact of anti-α₄β₇ antibody administration on viral DNA in PBMC, LN, or CR biopsies. Our data demonstrate that anti-α₄β₇ antibody administration using this protocol (8) had no discernable impact on cellular and humoral immune responses, viral DNA, and viral RNA following ART discontinuation in animals infected with wildtype, pathogenic SIVmac251.

Concurrent studies from Di Mascio, Fauci et al. and Iwamoto, Roederer et al. (16, 17) also demonstrate that anti-α₄β₇ antibody infusions had no effect in the original model of attenuated SIVmac239 with a stop codon in nef (8). The reasons for these differences with the previously published study (8) remain unclear. However, we did observe that peak viral loads prior to ART initiation strongly correlated with viral rebound following ART discontinuation, as we observed in prior studies that evaluated broadly neutralizing antibodies (18). Taken together, these studies suggest that anti-α₄β₇ antibody administration is not a viable strategy to target the viral reservoir or to induce virologic remission in SIVmac251-infected rhesus macaques.