Photoinduced Photosensitizer-Antibody Conjugates Kill HIV Env-Expressing Cells, Also Inactivating HIV

Abstract

HIV-infected cells persist for decades in patients administered with antiretroviral therapy (ART). Meanwhile, an alarming surge in drug-resistant HIV viruses has been occurring. Addressing these issues, we propose the application of photoimmunotherapy (PIT) against not only HIV Env-expressing cells but also HIV. Previously, we showed that a human anti-gp41 antibody (7B2) conjugated to cationic or anionic photosensitizers (PSs) could specifically target and kill the HIV Env-expressing cells. Here, our photolysis studies revealed that the binding of photoimmunoconjugates (PICs) on the membrane of HIV Env-expressing cells is sufficient to induce necrotic cell death due to physical damage to the membrane by singlet oxygen, which is independent of the type of PSs. This finding persuaded us to study the virus photoinactivation of PICs using two HIV-1 strains, X4 HIV-1 NL4-3 and JR-CSF virus. We observed that the PICs could destroy the viral strains, probably via physical damage on the HIV envelope. In conclusion, we report the application of PIT as a possible dual-tool for HIV immunotherapy and ART by killing HIV-expressing cells and cell-free HIV, respectively.

Figure 1

Study of the effect of irradiation on the structure of PICs. (a and b) Microcapillary electrophoresis (a) and electropherogram (b) of 7B2 MAb and PICs of nonreduced 7B2 MAb, 7B2-porphyrin, and 7B2-IR700 with different DARs of 2, 3, and 4, before and after irradiation with 10 or 50 J/cm². (a) In a dose-dependent manner, protein bands of PICs disappeared with forming smear, while the bands of naked 7B2 antibody were preserved. Size standards are indicated on the side of each “gel.” (b) The system peaks and upper marker in the electropherogram are indicated in green and violet, respectively. (c) Histograms of PICs, before and after irradiation, monitor how the hydrodynamic radius (R_h), shape, and solubility of PICs significantly changed after irradiation with power density of 50 J/cm², while naked 7B2 antibody showed preservation. The irradiated 7B2-IR700 showed different types of aggregation in comparison to the irradiated 7B2-porphyrin. Each curve in an individual color represents the average of 10 acquisitions.

Figure 2

Study of the effect of irradiation on the binding ability of PICs. ELISA plates were coated with gp41 antigen, as a peptide representing 7B2’s epitope. PICs were kept in the dark or irradiated with 50 J/cm², before incubation with gp41. The irradiation of PICs resulted in complete loss of binding ability, while naked 7B2 antibody retained the binding ability. Mouse IgG1 Ab was used as an isotype control. Data are mean ± SEM (n = 2) with two individual experiments.

Figure 3

Study of singlet oxygen generation by PSs and PICs, in media with no applying cells, as a function of continuous 380–780 nm irradiation times. (a) Comparison of singlet oxygen production between a 4× serial concentration of porphyrin azide (156, 625, and 2500 nM) and 625 nM 7B2-porphyrin DAR4 (containing 2500 nM porphyrin in the interaction with 625 nM antibody). The graph shows that the ability of porphyrin to produce singlet oxygen has decreased after conjugation. The changes in ABDA absorption at 400 nm were measured as a function of irradiation time (0–50 min) on the generation of singlet oxygen by porphyrin azide in comparison to porphyrin–antibody (n = 3). (b) The changes in ABDA absorption at 400 nm during 50 min irradiation due to singlet oxygen generation by porphyrin-7B2 and IR700-7B2. IR700-PIC with DAR of 3 produced more singlet oxygen than the porphyrin-PIC (DAR: 4) in the aqueous solution. Singlet oxygen generation by PICs was quenched in the presence of 0.01% sodium azide. Data are mean ± SEM (n = 3) with two individual experiments. The controls include 7B2 and DD water (n = 3). (c) Photobleaching of UV–vis absorbance spectra of ABDA upon 40 min irradiation in the presence of PICs. The extra absorption bands are due to either IR700 or porphyrin, as the absorption band of porphyrin was quenched during irradiation, while the IR700 was preserved. The controls include 7B2 and DD water (n = 3).

Figure 4

(a) Schematic picture depicts three models of PIC localization on the HIV Env-transfected 293T cell membrane or internalized. The irradiation was applied in the presence or absence of sodium azide as a ¹O₂ quencher. The controls include unstained cells, the cells incubated with naked 7B2 antibody, mouse IgG1 isotype control, and porphyrin isotype. Parallel studies were performed on the cells in darkness (b) and control 293T cells (Figure S2). Red arrows indicate that the cell death was inhibited due to quenching of singlet oxygen by azide, indicating that the presence of PICs on the cell membrane is sufficient to kill the cells by singlet oxygen generation. All PIT treatments on transfected cells were in the presence of 5 μg/mL soluble CD4 (n = 3).

Figure 5

(a) Schematic picture depicts the model of study for comparing the cell internalization of porphyrin–antibody during 2P irradiation in the presence or absence of azide. (b and c) The cells were incubated with 7B2-porphyrin in PBA to block internalization. FITC anti-human IgG secondary antibody was added to detect 7B2-porphyrin. After three washes with PBS, the cells were irradiated at 800 nm. In the absence of azide, membrane damage and rapid internalization of porphyrin–antibody were observed in 10 min (b, orange curve), resulting in necrotic signs after 30 min irradiation (c). In contrast, neither membrane damage nor internalization was observed during 60 min irradiation in the presence of azide as a ¹O₂ quencher (b, green curve). The white bar indicates 10 μm.

Figure 6

Effect of PICs on X4 HIV-1 NL4-3 virus. The virus stock was incubated with 500 nM of PICs, PSs, anti-gp41 (7B2) naked MAb, mouse IgG1 isotype control, porphyrin isotype, and wells with no treatment (only virus). After irradiation, the Jurkat cells were infected with viruses. (a) HIV-1 RNA load (log₁₀ copies/mL) in each region of LTR from harvested supernatants on day 6. In the irradiated plate, the viral load was undetectable in the samples treated with PICs or PSs. In the dark plate, the samples treated with naked 7B2 MAb or PS antibodies showed a decrease in the viral load due to the non-neutralizing binding of 7B2 MAb on the gp41 of the virus. Data were subtracted from HIV stock (log₁₀ copies/mL). Data are mean ± SEM (n = 2) for the supernatant of day 6. (b) Quantification of HIV-1 proviral DNA load (HIV DNA copies/100 cells) based on the protocol of Kumar for amplification of region LTR of the virus. Total HIV DNA includes stably integrated proviruses and extrachromosomal HIV DNA forms. The results were in agreement with viral RNA load results. Data are mean ± SEM (n = 2) for the supernatant of day 3. (c) TEM images of dark and irradiated controls revealed that the near-spherical enveloped virions look intact with distinct envelope. (d) The morphology of the virions irradiated with naked 7B2 antibody showed no difference with the untreated controls. While the membrane of virions inactivated with porphyrin, porphyrin–antibody, or IR700–antibody became partially destroyed, but their membranes appeared to maintain structural integrity. PIT-treated virions with IR700 PS were mostly agglomerated.