Impact of CD8 Depletion and IL-15 Superagonist with anti-SIV Env RhmAbs on Latent SIV Reservoir Persistence Open Access

Abstract

It has been well established that HIV primarily infects activated CD4+ T cells and that use of antiretroviral therapy (ART) can prevent new infections by targeting various stages of the virus life cycle. However, ART is incapable of eradicating integrated provirus, thus cannot target latently infected cells. The latent reservoir is established early after infection, whereby a portion of infected CD4+ T cells enters a state of transcriptional silence. In this state, proviral transcription and expression of viral proteins are minimal, thus preventing detection by the immune system. The persistence of this latent reservoir still represents the greatest barrier to an HIV cure. The “Kick and Kill” cure approach employs a latency reversal component to reactivate latently infected CD4+ T cells and a clearance agent to eliminate the reactivated cells. It has been shown that depletion of CD8+ T cells in combination with N-803 promotes latent viral activation, thus, we hypothesized that the addition of SIV-Env specific Rhesus monoclonal antibodies (RhmAbs) would lead to infected CD4+ T cell clearance through antibody-mediated mechanisms. We used the adult SIV model of infection to investigate the reservoir-reducing capacity of the latency reversal agents MT807R1 (CD8a-lymphocyte depleting antibody) in combination with N-803 (an IL-15 superagonist) and a cocktail of four RhmAbs as clearance agents. SIVmac239 was used to infect 28 rhesus macaques (RMs), and ART was initiated 8 weeks post infection (p.i.). Plasma viral load (PVL) was monitored routinely by qPCR to measure SIV gag RNA/ml plasma. A daily ART regimen consisting of tenofovir (5.1mg kg-1), emtricitabine (40 mg kg-1), and dolutegravir (2.5 mg kg-1) was administered subcutaneously (s.c.) beginning at week 8 p.i. until analytic treatment interruption (ATI). Three study groups (ART control, RhmAbs, and MT807R1/N-803/RhmAb animals) were assigned based on criteria including peak and pre-ART PVL. Four IgG1 RhmAbs (ITS09.01-LS, ITS102.01-LS, ITS103.01-LS, ITS113.01-LS) which target the V2, CD4 binding site (BS), CD4 BS proximal, and membrane proximal external region (MPER), respectively, were co-administered twice (20 mg kg-1 s.c. each); a single dose of MT807R1 depleting antibody (50 mg kg-1 s.c.) was given; N-803 was infused a total of five times over 6 weeks (100ug kg-1 s.c.). RhmAbs reached washout threshold prior to ART interruption (ATI). Reservoir measurements were conducted on CD4+ T cells from various tissue compartments at pre- and post-intervention. We found a significant reduction of SIV-DNA within peripheral blood (PB) and lymph node (LN) of the MT807R1/N-803/RhmAb, not observed in other groups. Furthermore, we found a significant reduction in portion of intact provirus from PB and LN among MT807R1/N-803/RhmAb which was stronger than that of other groups. Time-to-viral-rebound was not impacted by MT807R1+N- 803+RhmAbs despite robust latency reversal and evidence of treatment response on the infected cell level. We did find significantly lower rebound viral setpoints within the RhmAb group and MT807R1/N-803/RhmAb group compared to the ART control group as measured by fold change of pre-ART/pre-ATI (p=0.0175 and p=0.0085, respectively). Our findings support continued use of RhmAbs in cure research and study of CD8+ T cell mechanisms which may be promoting CD4+ T cell latency. 

Table of Contents

Chapter 1: Introduction to Dissertation (p.8)

1.1 Introduction to HIV (p. 8)

1.2 Current state of therapy (p. 11)

1.3 NHP models for study of HIV (p. 12)

1.4 Review of Kick and Kill Approach (p. 15)

1.5 Brief Historical Perspective on LRA 1.0 (p. 16)

1.6 Immunomodulatory LRA 2.0 (p. 17)

1.7 PRR Agonists (p. 17)

1.8 Immune Checkpoint Inhibitors (p. 19)

1.9 Non-canonical NF-κB Stimulation as LRA 2.0 (p. 20)

1.10 The NF-κB Pathway (Canonical vs Non-Canonical) (p. 22)

1.11 SMAC Mimetics (p. 22)

1.12 Transient CD8+ Lymphocyte Depletion as LRA 2.0 (p. 23)

1.13 Promising LRA and LRA Combinations (p. 26)

1.14 LRA 2.0 in Shock and Kill Strategies (p. 27)

1.15 Remaining Questions About Latency Reversal as a Component of an HIV Cure Strategy (p. 28)

1.16 Effect Measures (p. 29)

Chapter 2: Study of CD8 Depletion and N-803 +/- RhmAbs in Rhesus Macaque Model (p.30)

2. Results (p.30)

2.1 Intervention Strategy using SIV Model of Infection (p. 30)

2.2 SIV-Env-specific Rhesus Monoclonal Antibodies as Clearance Agents (p. 31)

2.3 On-ART Reactivation during MT807R1 + N-803 + RhmAbs Treatment (p. 32)

2.4 Effect of MT807R1 + N-803 + RhmAbs on Latent Viral Reservoir (p. 32)

2.5 Analytic Treatment Interruption following RhmAb Washout (p. 34)

2.6 MT807R1 + N-803 Effect on CD4+ T Cells Reactivation (p. 35)

2.7 Effector Cell Reconstitution and Immune Responses to MT807R1 + N-803 Treatment (p. 35)

Chapter 3: Discussion (p. 38)

3.1 Observed Reservoir Reduction and Rebound Viral Setpoint (p. 38) 3.2 Therapeutic Antibodies in Other Cure Research (p. 39)

3.3 Recent Successes with Kick and Kill Models (p. 40)

3.4 Future Directions (p. 42)

3.5 Limitations (p. 42)

3.6 Clinical and Therapeutic Applications (p. 43) 3.7 Conclusions (p. 43)

Methods and Materials (p. 45)

Figures and Tables

Figure. Graphic Abstract (p. 52)

Figure 1. Experimental Design (p. 52)

Figure 2. Viral dynamics and impact on latent reservoir induced by MT807R1 + N-803 with RhmAbs (p. 56)

Figure 3. Cellular responses to intervention (p. 61)

5

Extended Data Figure 1. Gating strategies for flow cytometric analyses of T cell responses to intervention. (p. 63)

Extended Data Figure 2. Gating strategies and longitudinal assessment of CD8a expression and subsets (p. 64)

Extended Data Figure 3. Effector CD8+ T cell responses to SIVgag peptide stimulation (p. 66)

Extended Data Figure 4. ADA measurements by individual RM (p. 68)

Figure Legends (p. 69)

Abbreviations (p. 72) References (p. 73) 

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Immunology and Molecular Pathogenesis
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Virology Biology, Cell Health Sciences, Immunology
Keyword	HIV-1 Simian Immunodeficiency Virus Macaca Mulatta CD4 Lymphocyte Viral Reservoir Antibodies Latency
Committee Chair / Thesis Advisor	Ann Chahroudi, Emory University Joshy Jacob, Emory University
Committee Members	Stefan Sarafianos, Emory University Matt Parsons, U.S. Military HIV Research Program at WRAIR Deanna Kulpa, Emory University

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