Autologous aldrithiol-2-inactivated HIV-1 combined with polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose as a vaccine platform for therapeutic dendritic cell immunotherapy

Abstract

Therapeutic interventions for HIV-1 that successfully augment adaptive immunity to promote killing of infected cells may be a requisite component of strategies to reduce latent cellular reservoirs. Adoptive immunotherapies utilizing autologous monocyte-derived dendritic cells (DCs) that have been activated and antigen loaded ex vivo may serve to circumvent defects in DC function that are present during HIV infection in order to enhance adaptive immune responses. Here we detail the clinical preparation of DCs loaded with autologous aldrithiol-2 (AT-2)-inactivated HIV that have been potently activated with the viral mimic, Polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (Poly-ICLC). HIV is first propagated from CD4+ T cells from HIV-infected donors and then rendered non-replicative by chemical inactivation with aldrithiol-2 (AT-2), purified, and quantified. Viral inactivation is confirmed through measurement of Tat-regulated β-galactosidase reporter gene expression following infection of TZM-bl cells. In-process testing for sterility, mycoplasma, LPS, adventitious agents, and removal of AT-2 is performed on viral preparations. Autologous DCs are generated and pulsed with autologous AT-2-inactivated virus and simultaneously stimulated with Poly-ICLC to constitute the final DC vaccine product. Phenotypic identity, maturation, and induction of HIV-specific adaptive immune responses are confirmed via flow cytometric analysis of DCs and cocultured autologous CD4+ and CD8+ T cells. Lot release criteria for the DC vaccine have been defined in accordance with Good Manufacturing Practice (GMP) guidelines. The demonstrated feasibility of this approach has resulted in approval by the FDA for investigational use in antiretroviral (ART) suppressed individuals. We discuss how this optimized DC formulation may enhance the quality of anti-HIV adaptive responses beyond what has been previously observed during DC immunotherapy trials for HIV infection.

Keywords: Dendritic cell; HIV-1; Poly-ICLC; Therapeutic vaccine.

FIGURE 1. Autologous DC Vaccine Production Schema

Following leukapheresis of viremic HIV-infected donors, virus is propagated from anti-CD3-activated CD4+ T cells in the presence of IL-2. Viral supernatants are inactivated with AT-2 and then purified. This autologous, inactivated HIV is then pulsed onto autologous monocyte-derived DCs in combination with Poly-ICLC. These mature, antigen-loaded moDCs may be injected back into the patient as a form of HIV immunotherapy.

FIGURE 2. HIV Production, Purification and Quantification

(A) p24 content of CD4+ T cell culture supernatants from HIV-infected donors (N=4) under GMP conditions was quantified by ELISA. To further establish feasibility, 14 additional donors were cultured under non-GMP conditions demonstrating similar concentration of p24 (Supplemental Figure 1). (B) Coomassie staining of purified, inactivated virus from a representative HIV-infected donor before and after treatment with AT-2. p24CA standard ranges from 500 to 15.1625 ng. This was performed on all purified virus samples cultured under GMP conditions (N=4), with an average yield of 200,071 ng p24CA [range 27,904 –489,224 ng]. Viruses from 14 additional untreated HIV-infected donors were cultured, inactivated, and purified under non-GMP conditions with similar yield of p24CA (data not shown). (C) Immunoblots for the viral proteins p24CA, gp41TM (c-terminus), and gp120 were performed on purified, inactivated virus preparations (N=4, figure displays representative donor). This was performed to confirm the presence of whole virions rather than free p24CA in the virus preparations.

FIGURE 3. Confirmation of viral inactivation and removal of AT-2 from viral preparation

(A) Evaluation for residual infectivity was performed via TZM-bl reporter assays. Infectious virus (quantified as spot forming units (SFU)) is shown before and after AT-2 treatment in donor CD4+ T cell supernatants (N=3) (left) and HIV-1 BaL/SUPT1 supernatants (right). Viral inactivation was also confirmed following purification and concentration of viral supernatants for each donor with no detectable infectivity following AT-2 treatment (data not shown). (B) HPLC analysis of 0.5M AT-2 solution (AT-2 alone control) and purified AT-2-inactivated HIV from a representative donor with and without the addition of 10 mmol r-Glutathione. HPLC was performed at a flow rate of 300 mL/min on 3 m, 2 × 100 mm Luna C18(2), (Phenomenex), using aqueous acetonitrile/trifluoroacetic/acid solvents on a Shimadzu HPLC system. Peaks were detected by UV absorption at 206 and 280 nm.

FIGURE 4. Identity, Maturation, and IL-12 Secretion

(A) To ascertain fulfillment of identity and maturation lot release criteria, thawed final DC vaccine products were stained for CD11c, CD14, and CD86. Representative donor is displayed. (B) DCs from ART-suppressed donors (N=5) were pulsed with patient-propagated AT-2 HIV and stimulated with Poly-ICLC or the standard cytokine cocktail, MCM MIMIC. Poly-ICLC resulted in upregulation of CD86 and CD40 compared with unstimulated DCs (both nonpulsed and AT-2 pulsed conditions). (C) IL-12 secretion by DCs from these same donors and conditions was evaluated. In contrast to unstimulated and MCM MIMIC DCs, Poly ICLC induced robust secretion of IL-12 by DCs.

FIGURE 5. Immunogenicity of DC Vaccine

To evaluate potency/immunogenicity, the autologous DC vaccine (Poly-ICLC matured DCs pulsed with AT-2 HIV (100ng/ml and/or 300ng/ml) or nonpulsed autologous DCs were co-cultured with autologous CD4+ (top) and CD8+ (bottom) T cells at a ratio of 1:5 (DC:T cell). Following 10 days of co-culture, expansion of HIV-specific T cell responses was measured via flow cytometry as a function of intracellular cytokine secretion in response to restimulation with corresponding autologous Poly-ICLC matured DCs ± AT-2 HIV. A) Results are displayed from representative donors for IFNγ and TNFα. Top panel shows expansion of CD4+ T cells upon coculture with autologous DCs pulsed with AT-2 HIV (100ng/ml). Bottom panel shows expansion of CD8+ T cells upon coculture with autologous DCs AT-2 HIV (300ng/ml). B) Summary of intracellular cytokines secreted by CD4+ and CD8+ T cells following expansion with autologous DCs pulsed with AT-2 HIV. Data are represented as fold change in cytokine secretion of T cells expanded and restimulated with autologous AT-HIV pulsed DCs compared with control (nonpulsed) conditions (mean, (range)).