Analytical Treatment Interruption after Short-Term Antiretroviral Therapy in a Postnatally Simian-Human Immunodeficiency Virus-Infected Infant Rhesus Macaque Model

Abstract

To achieve long-term viral remission in human immunodeficiency virus (HIV)-infected children, novel strategies beyond early antiretroviral therapy (ART) will be necessary. Identifying clinical predictors of the time to viral rebound upon ART interruption will streamline the development of novel therapeutic strategies and accelerate their evaluation in clinical trials. However, identification of these biomarkers is logistically challenging in infants, due to sampling limitations and the potential risks of treatment interruption. To facilitate the identification of biomarkers predicting viral rebound, we have developed an infant rhesus macaque (RM) model of oral simian-human immunodeficiency virus (SHIV) SHIV.CH505.375H.dCT challenge and analytical treatment interruption (ATI) after short-term ART. We used this model to characterize SHIV replication kinetics and virus-specific immune responses during short-term ART or after ATI and demonstrated plasma viral rebound in 5 out of 6 (83%) infants. We observed a decline in humoral immune responses and partial dampening of systemic immune activation upon initiation of ART in these infants. Furthermore, we monitored SHIV replication and rebound kinetics in infant and adult RMs and found that both infants and adults demonstrated equally potent virus-specific humoral immune responses. Finally, we validated our models by confirming a well-established correlate of the time to viral rebound, namely, the pre-ART plasma viral load, as well as identified additional potential humoral immune correlates. Thus, this model of infant ART and viral rebound can be used and further optimized to define biomarkers of viral rebound following long-term ART as well as to preclinically assess novel therapies to achieve a pediatric HIV functional cure.IMPORTANCE Novel interventions that do not rely on daily adherence to ART are needed to achieve sustained viral remission for perinatally infected children, who currently rely on lifelong ART. Considering the risks and expense associated with ART interruption trials, the identification of biomarkers of viral rebound will prioritize promising therapeutic intervention strategies, including anti-HIV Env protein therapeutics. However, comprehensive studies to identify those biomarkers are logistically challenging in human infants, demanding the need for relevant nonhuman primate models of HIV rebound. In this study, we developed an infant RM model of oral infection with simian-human immunodeficiency virus expressing clade C HIV Env and short-term ART followed by ATI, longitudinally characterizing the immune responses to viral infection during ART and after ATI. Additionally, we compared this infant RM model to an analogous adult RM rebound model and identified virologic and immunologic correlates of the time to viral rebound after ATI.

Keywords: HIV reservoir; SHIV; analytical treatment interruption; pediatric HIV cure.

FIG 1

SHIV.CH505.375H.dCT replication kinetics prior to and following ATI in infant RMs. (A) Schematic representation of SHIV.CH505.375H.dCT infection (0 to 12 weeks), ART (12 to 20 weeks), and ATI (20 to 28 weeks) in infant RMs. Blood samples were collected at weekly intervals throughout the study, and peripheral lymph nodes (LNs) were collected at 12 wpi and while the RMs were on ART (20 wpi), (B) The kinetics of plasma SHIV RNA over 28 weeks were measured by qRT-PCR. (C) Peripheral lymph nodes from RMs on ART (20 wpi) were collected, and the level of naive, memory, and Tfh CD4⁺ T cell-associated SHIV DNA was estimated by qPCR. (D and E) The amounts of cell-associated SHIV DNA (CA-SHIV DNA) (D) and cell-associated SHIV RNA (CA-SHIV RNA) (E) per million CD4⁺ T cells in peripheral blood were monitored by ddPCR in the infant RMs before ART (6 wpi) and on ART (18 wpi). The sensitivity of the ddPCR assay was detection of 1 SHIV gag copy in 10,000 CD4⁺ T cells. Therefore, only those animals that had ≥10,000 CD4⁺ T cells at a particular time point were included in the analysis. Each symbol represents an individual animal. Yellow and gray boxes represent the duration of ART (weeks 12 to 20) and the duration of ATI (weeks 20 to 28), respectively. Medians are indicated as horizontal lines on the dot plots. Infants with a plasma VL of <15 copies/ml at 12 wpi are represented by open symbols.

FIG 2

Tissue-associated infectious viral loads upon ATI in mononuclear cells isolated from PBMCs and lymphoid and gastrointestinal tissues of orally infected infant RMs. (A) Tissue-associated infectious SHIV.CH505.375H.dCT titers measured through tissue mononuclear cell coculture with TZM-bl reporter cells. The reported titers represent the estimated minimum number of mononuclear cells per 10⁴ mononuclear cells required to yield detectable infection of 50% of TZM-bl cells (CID₅₀). (B and C) CD4⁺ T cell-associated proviral DNA (B) and CD4⁺ T cell-associated viral RNA (C) loads at necropsy (28 wpi), reported as the copy number per million CD4⁺ T cells in PBMCs and lymphoid and gastrointestinal tissue mononuclear cells. Each symbol represents one individual animal. Medians are indicated as horizontal lines on the dot plots. Infants with a plasma VL of <15 copies/ml at 12 wpi are represented by open symbols. The sensitivity of the ddPCR assay was detection of 1 SHIV gag copy in 10,000 CD4⁺ T cells. Therefore, only those animals that had ≥10,000 CD4⁺ T cells at a particular time point were included in the analysis. (D) Tonsil and colon sections from the SHIV.CH505.375H.dCT-infected infant RM that demonstrated the highest peak plasma VL postrebound (20,000 vRNA copies/ml plasma). Tissue sections were stained with the nuclear marker DAPI (4′,6-diamidino-2-phenylindole; dark blue) to identify cells and with antibodies specific for CD3 (green) and CD20 (red). Virus-infected cells were identified by in situ hybridization (cyan). To better visualize the virus-infected cells, we magnified a specific region (white box) in each image. Each panel consists of a larger image with the overlay of all markers and 4 smaller side panels of the same field for each individual channel. Arrow colors correspond to the color for the indicated marker. The large image has a scale bar in the lower right corner.

FIG 3

SHIV.CH505.375H.dCT replication kinetics prior to and following ATI in adult RMs. (A) Schematic representation of SHIV.CH505.375H.dCT infection (0 to 12 weeks), ART (12 to 24 weeks), and ATI (24 to 32 weeks) in adult RMs. Blood samples were collected at weekly intervals throughout the study, and peripheral lymph nodes (LNs) were collected at 12 wpi and after 8 weeks of ART (20 wpi). I.V., intravenous. (B) The kinetics of plasma SHIV RNA over 32 weeks were measured by qRT-PCR. (C) Peripheral lymph nodes from macaques on ART (20 wpi) were collected, and the level of naive, memory, and Tfh CD4⁺ T cell-associated SHIV DNA was estimated by qPCR. (D and E) The amounts of cell-associated SHIV DNA (CA-SHIV DNA) (D) and cell-associated SHIV RNA (CA-SHIV RNA) (E) from CD4⁺ T cells of peripheral blood before ART (6 wpi for DNA and 12 wpi for RNA) and on ART (18 wpi) were monitored by ddPCR. The sensitivity of the ddPCR assay was detection of 1 SHIV gag copy in 10,000 CD4⁺ T cells. Therefore, only those animals that had ≥10,000 CD4⁺ T cells at a particular time point were included in the analysis. Each symbol represents an individual animal. Yellow and gray boxes represent the duration of ART (weeks 12 to 24) and the duration of ATI (weeks 24 to 32), respectively. Medians are indicated as horizontal lines on the dot plots.

FIG 4

Tissue-associated infectious virus load upon ATI in mononuclear cells isolated from PBMCs and lymphoid and gastrointestinal tissues of adult RMs intravenously infected with SHIV.CH505.375H.dCT. (A) Tissue-associated infectious SHIV.CH505.375H.dCT titers measured through tissue mononuclear cell coculture with TZM-bl reporter cells. The reported titers represent the estimated minimum number of mononuclear cells per 10⁴ mononuclear cells required to yield detectable infection of 50% of TZM-bl cells (CID₅₀). (B and C) CD4⁺ T cell-associated proviral DNA (B) and viral RNA loads (C), reported as the copy number per million CD4⁺ T cells in PBMCs and lymphoid and gastrointestinal tissue mononuclear cells. Each symbol represents one individual monkey at necropsy (week 32 postinfection). Medians are indicated as horizontal lines on the dot plots. (D) Tonsil and colon sections from the SHIV.CH505.375H.dCT-infected adult RM (animal 39950) that demonstrated the highest peak plasma VL postrebound (13,000 vRNA copies/ml plasma). Tissue sections were stained with the nuclear marker DAPI (dark blue) to identify cells and with antibodies specific for CD3 (green) and CD20 (red). Virus-infected cells were identified by in situ hybridization (cyan). Each panel consists of a larger image with the overlay of all markers and 4 smaller side panels of the same field for each individual channel. Arrow colors correspond to the color for the indicated marker. The large image has a scale bar in the lower right corner.

FIG 5

Phylogenetic tree analysis of the env gene sequences obtained pre-ART and post-ATI from the plasma of infant and adult RMs demonstrating the highest peak plasma VL postrebound. Standard SGA techniques were used to analyze the env gene from pre-ART and post-ATI samples from infants and adult RMs with the highest peak rebound plasma VL. The phylogenetic tree represents the viral env diversity in infant 46352 (A) and adult 39950 (B).

FIG 6

Magnitude and kinetics of humoral responses to acute SHIV.CH505.375H.dCT infection during ART and ATI in RMs. Plasma from infant and adult rhesus macaques was analyzed for the HIV CH505 gp120 IgG response (A), blocking of soluble CD4-gp120 interactions (B), the tier 1 neutralization response against MW965 (C), the neutralization response against the CH505 T/F virus (D), and the ADCC titer against CH505 gp120-coated target cells (E). Red symbols represent infant RMs, and blue symbols represent adult RMs. Each symbol represents an individual macaque. Yellow and gray boxes represent the duration of ART and the duration of ATI in the RMs, respectively. Infants with a plasma VL of <15 copies/ml at 12 wpi are represented with open symbols. P values were calculated using the Wilcoxon signed-rank test. eq., equivalents; Ab, antibody; ID₅₀, 50% infective dose.

FIG 7

CD4⁺ T cell activation and plasma inflammatory responses in SHIV.CH505.375H.dCT-infected infant and adult RMs. Absolute counts per milliliter of blood of activated (CD69⁺ HLA-DR⁺) CD4⁺ T cells (A) and proliferating (Ki67⁺ CD4⁺) and exhausted (PD-1⁺) CD4⁺ T cells (B). Red symbols represent infant RMs, and blue symbols represent adult RMs. Each symbol represents one animal. Yellow and gray boxes represent the duration of ART and the duration of ATI, respectively. (C) Plasma from infected infant and adult RMs was analyzed by a multiplexed Luminex assay for expression of cytokines before ART (week 12) and after 8 weeks on ART (week 20). The heat maps represent the fold change in the level of each analyte over preinfection plasma levels. Infants with a plasma VL of <15 copies/ml at 12 wpi are represented with open symbols. P values were calculated using the Wilcoxon signed-rank test.