Inactivation of retroviruses with preservation of structural integrity by targeting the hydrophobic domain of the viral envelope

Abstract

We describe a new approach for the preparation of inactivated retroviruses for vaccine application. The lipid domain of the viral envelope was selectively targeted to inactivate proteins and lipids therein and block fusion of the virus with the target cell membrane. In this way, complete elimination of the infectivity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) could be achieved with preservation of antigenic determinants on the surface of the viral envelope. Inactivation was accomplished by modification of proteins and lipids in the viral envelope using the hydrophobic photoinduced alkylating probe 1,5 iodonaphthylazide (INA). Treatment of HIV and SIV isolates with INA plus light completely blocked fusion of the viral envelope and abolished infectivity. The inactivated virus remained structurally unchanged, with no detectable loss of viral proteins. Modifications to envelope and nucleocapsid proteins were detected by changes in their elution pattern on reverse-phase high-performance liquid chromatography. These modifications had no effect on primary and secondary structure epitopes as determined by monoclonal antibodies. Likewise, the inactivated HIV reacted as well as the live virus with the conformation-sensitive and broadly neutralizing anti-HIV type 1 monoclonal antibodies 2G12, b12, and 4E10. Targeting the lipid domain of biological membranes with hydrophobic alkylating compounds could be used as a general approach for inactivation of enveloped viruses and other pathogenic microorganisms for vaccine application.

FIG. 1.

The effect of INA on virus induced cell-cell fusion. AA2 cells were infected with SIV that was preinactivated by the indicated concentrations of INA, as described in Materials and Methods. At 47 h after infection, cells were tested for the presence of syncytia induced by the virus. (A) Untreated control; (B) 2 μM INA; (C) 200 μM INA; (D) 200 μM INA with no UV irradiation.

FIG.2.

Dose-dependent inactivation of SIV infectivity by INA. AA2 cells were infected with SIV and treated with INA at the concentrations indicated (0.0 μM, 2 μM, 20 μM, and 200 μM). Infectivity was tested 11 days after infection by p28 determination as described in Materials and Methods. The experiment was repeated three times with similar results.

FIG. 3.

Inactivation of HIV infectivity by INA. HIV treated with 200 μM INA and untreated control was tested for infectivity by the luciferase reporter gene assay. Serial dilutions of HIV were added to TZM-bl cells, and luminescence was plotted against the indicated amounts of virus.

FIG. 4.

INA inhibits fusion of SIV envelope with the target cell membrane. SIV Mne was treated with INA at the concentrations indicated. Photosensitized labeling was used to determine fusion activity at the plasma membrane level by measuring the extent of ¹²⁵INA incorporation into viral HLA-DR (α-chain) upon fusion with the target cell membrane. The maximal INA concentration applied for inactivation was 2 μM, because higher concentrations interfered with the incorporation of ¹²⁵INA in the assay. The experiment was repeated two times with similar results.

FIG. 5.

INA treatment does not affect the integrity of the virus. SIV isolate suspension was treated with INA as indicated, and the virus was recovered by centrifugation. The virus sample was then subjected to SDS-PAGE and Coomassie blue staining (25 μg/lane) to determine the total amount of protein recovered in each experimental group, as well as the recovery of the indicated major viral proteins. Lanes: a, control (buffer); b, 2% DMSO; c, 200 μM INA; d, 20 μM INA; e, 2 μM INA; f, 200 μM INA with no UV irradiation.

FIG. 6.

INA inactivation results in the modification of viral proteins. Samples of SIV (INA treated and nontreated control) were disrupted in 8 M guanidine hydrochloride, and proteins were subjected to separation by RP-HPLC. Individual proteins in the peaks were identified by Western blotting and sequencing. p8, p1/p2, p6, p17, and p28 are gag viral proteins, and gp120 and gp32 are viral envelope proteins. CL-II and actin are cellular proteins present in both microvesicles and virus.

FIG. 7.

INA inactivation does not affect the specific recognition of isolated viral proteins by antibodies. The indicated viral proteins from INA-treated SIV were analyzed by Western blot under reducing (R) and nonreducing (NR) conditions. Lanes: a nontreated virus; b, 1% DMSO; c, 200 μM INA; d, 20 μM INA; e, 2 μM INA; f, 200 μM INA (not photoactivated).

FIG. 8.

Inactivated HIV is recognized by conformational neutralizing antibodies, as well as the infectious virus. Antibody capture assay was carried out with increasing virus amounts with the indicated neutralizing monoclonal antibodies. 2G12 and B12 were used against epitopes on gp120 and 4E10 was used against gp41.

FIG. 9.

Electron microscopy of inactivated and control virions. Inactivated viruses and live viruses that were used in the experiments described above were fixed in glutaraldehyde, negatively stained, and visualized by electron microscopy as described in Materials and Methods. (A) HIV control; (B) inactivated HIV; (C) SIV control; (D) inactivated SIV. V, virion; Vsc, microvesicle.