Engineered deletions of HIV replicate conditionally to reduce disease in nonhuman primates

Abstract

Antiviral therapies with reduced frequencies of administration and high barriers to resistance remain a major goal. For HIV, theories have proposed that viral-deletion variants, which conditionally replicate with a basic reproductive ratio [R₀] > 1 (termed "therapeutic interfering particles" or "TIPs"), could parasitize wild-type virus to constitute single-administration, escape-resistant antiviral therapies. We report the engineering of a TIP that, in rhesus macaques, reduces viremia of a highly pathogenic model of HIV by >3log₁₀ following a single intravenous injection. Animal lifespan was significantly extended, TIPs conditionally replicated and were continually detected for >6 months, and sequencing data showed no evidence of viral escape. A single TIP injection also suppressed virus replication in humanized mice and cells from persons living with HIV. These data provide proof of concept for a potential new class of single-administration antiviral therapies.

Fig. 1.. Long-term selection of an HIV DIP.

(A) Detection of an HIV deletion variant in a CEM cell bioreactor. (Left) Cell concentration and frequency of GFP⁺ cells by flow cytometry in a long-term bioreactor infected with replication-competent HIV-GFP (26). (Inset) PCR-amplified HIV proviral DNA from day 71 cells (lane 3: red arrowhead, expected band; blue arrowhead, ~2.5-kb deletion). (Right) Putative DIP construct with the location of the 2.5-kb deletion. D2, splice donor D2; SA, splice acceptor; IRES, internal ribosome entry site; RRE, Rev response element; nef, negative factor. (B) Conditional packaging assay: DIP VLPs were assembled in the presence or absence of lentiviral packaging plasmid, and transduced MT4 cells were titered by flow cytometry relative to a lentiviral GFP control vector. (C) Interference assay: HIV outgrowth in the presence or absence of the putative DIP. Cells stably transduced with single integrations of DIP or a mock-transduced control were infected with HIV-BFP, and the frequency of BFP⁺ cells was measured by flow cytometry. (Inset) Filtered supernatants were harvested at 3 days postinfection (dpi) and titered by flow cytometry. *P = 0.004 (4 dpi); P = 0.001 (5 dpi); P = 0.001 (7 dpi); **P =5× 10⁻⁶; Student’s t test. (D) Transmission assay: The fraction of putative DIP or HIV genomic RNA in supernatant VLPs (left) compared with the fraction of proviral DNA integrated in downstream transduced cells (right) at 3 dpi.

Fig. 2.. Engineering the HIV TIP.

(A) Schematics of full-length HIV, DIP, and engineered TIP constructs; the cPPT was reintroduced in “TIP-1,” and additional coding sequences were ablated in “TIP-2” to reduce extraneous HIV protein expression (table S1). KO, knockout. (B) Transduction efficiencies of DIP, TIP-1, and TIP-2 VLPs (normalized to p24 capsid protein and assayed on MT-4 cells; mean of three biological replicates shown). *P = 0.007 and **P = 0.003 from Student’s t test. (C) TIP interference: Frequency of HIV-infected cells during long-term culture in the presence or absence of TIP-1–transduced cells. Cultures were seeded at a ratio of 1:1 of untransduced and TIP-1–transduced cells (the construct used, TIP-1/Δenv, has an additional env mutation; see table S1), infected with HIV-BFP, and monitored by flow cytometry. *P < 0.0006 from Student’s t test. (D) TIP-2 interference in donor-derived human CD4⁺ primary cells; 50% of cells were transduced with TIP-2 or lentiviral GFP control (33) infected with HIV-BFP, and new activated target cells were added. HIV-BFP⁺ cells were quantified by flow cytometry (mean ± SD of two biological replicates is shown). (E) Cells from the three-color assay were analyzed by flow cytometry 3 days post coculture to quantify mobilization of TIP (or control) from “producer” cells into mCherry⁺ “target” cells conditional on HIV (see red box for dual positives). (F) Measured R₀ values (mean ± SEM of three biological replicates). (G) Relative TIP R₀ by transmission in culture. Reactor was seeded with excess (~87%) target CEMs (mCherry⁺) and ~13% TIP-2 (GFP⁺), infected with HIV-BFP, and assayed by flow cytometry for double-positive BFP-mCherry⁺ and GFP-mCherry⁺ cells. Trendlines fit to HIV or TIP-2 viral loads [i.e., $VL\left(t\right)=C{e}^{rt}$, where r represents growth rate and C represents the initial viral load], and best-fit values are r_TIP = 0.33 and r_HIV = 0.054. (H) Quantification of interference from TIP packaging: TIP-2– or TIP-2^ΔΨ–transduced cells were infected with HIV-BFP, the supernatant was collected at 2 dpi, and levels of integration-competent HIV were titered in target cells by flow cytometry (mean ± SEM of three biological replicates; ns, nonsignificant; *P < 0.01; Student’s t test).

Fig. 3.. TIP intervention in humanized mice and rhesus macaques.

(A) Human PBMC model: NSG mice were engrafted with human cells, challenged with HIV, and then administered a single IV injection of HIV TIPs (~10⁶ TU/kg) or mock at 5 days postinfection. HIV RNA plasma viral loads in NSG mice treated with TIP (n = 8) or HIV (control; n = 4) at days 5, 12, and 19 post HIV infection. **P = 0.005. LOD, limit of detection. (B) Rhesus macaque model: Kaplan-Meier survival analysis of TIP-treated animals (blue, n = 6) compared with controls (red, n = 4) [Log-rank (Mantel-Cox) test] and historical controls (gray, n = 21). (C) Quantitative PCR of SIV viral RNA (SIV gag) in peripheral blood plasma; Blue = TIP-treated animals, red = control animals (each animal represented by a unique symbol). (D) Reversetranscribed TIP DNA in peripheral blood mononuclear cells from day 1 post TIP intervention. (E) Quantitative PCR of TIP RNA copies in peripheral blood plasma.

Fig. 4.. Immune response, in situ tissue analysis, and quantitative modeling of SHIV kinetics.

(A) Seroconversion ability of TIP-treated versus control animals (ELISA for HIV Env). (B) Representative images of SHIV RNA expression within lymphoid tissue by RNAscope in situ hybridization (top scale bar, 500 μm; bottom scale bar, 100 μm). (C) Correlation between qPCR of SIV gag RNA in plasma and the quantified proportion of SHIV RNA⁺ cells within lymph nodes from RNAscope. ****P = 0.0001. (D) Best fits (nonlinear least-squares regression) of mathematical models to SHIV viral load data from a representative control animal (top) and TIP-treated animal (bottom). Data from control animals were fit to the basic ODE model (gray), whereas data from TIP-treated animals fit to both the basic model and TIP model (blue). (E) Model fitting summary: Ratio of peak to steady-state SHIV viral load data (* symbol) together with mathematical model fits from Markov chain Monte Carlo sampling (○ symbol) and the best fits from Latin-Hypercube sampling (□ symbol). Gray symbols represent the basic model of viral dynamics, whereas blue symbols represent the model with TIPs incorporated.

Fig. 5.. Effects of TIPs on HIV latency reversal, CD4 protection, and extrapolation to human intervention.

(A) Latency assay: qPCR quantification of integrated TIP DNA in Jurkat cells infected with HIV and/or transduced with TIP (optimized HIV-TIP, left). qPCR quantification of integrated HIV DNA (right) in cells infected with HIV and/or transduced with TIP during extended ART treatment (day 26) [normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)]. (B) Latency reversal upon ART cessation: fluorescence-activated cell sorting (FACS) analysis of Jurkat cells following ART cessation (+TNF-α) in the HIV control (left) versus TIP-treated samples (right). A.U., arbitrary units. (C) Percentage of HIV⁺ cells reactivated spontaneously following ART cessation (background) and in presence of TNF-α in both the HIV control versus the TIP-treated sample. *P = 0.05. (D) CD4 protection assay: FACS analysis of human donor–derived primary CD4⁺ T cells infected with HIV and transduced with either TIP (optimized HIV-TIP) or a control HIV-derived lentiviral vector, both expressing GFP. (E) HIV infection spread as assayed by CD4 depletion in the HIV-TIP– and control-treated samples on days 7 and 14. (F) qPCR quantification of integrated TIP DNA in donor-derived human primary CD4⁺ T cells from days 3 to 14. (G) Predicted reduction in HIV set-point viral load following TIP intervention at physiological HIV set point values (based on model extrapolation from rhesus macaque data).