HIV-1 Protease as DNA Immunogen against Drug Resistance in HIV-1 Infection: DNA Immunization with Drug Resistant HIV-1 Protease Protects Mice from Challenge with Protease-Expressing Cells

Abstract

DNA immunization with HIV-1 protease (PR) is advanced for immunotherapy of HIV-1 infection to reduce the number of infected cells producing drug-resistant virus. A consensus PR of the HIV-1 FSU_A strain was designed, expression-optimized, inactivated (D25N), and supplemented with drug resistance (DR) mutations M46I, I54V, and V82A common for FSU_A. PR variants with D25N/M46I/I54V (PR_Ai2mut) and with D25N/M46I/I54V/V82A (PR_Ai3mut) were cloned into the DNA vaccine vector pVAX1, and PR_Ai3mut, into a lentiviral vector for the transduction of murine mammary adenocarcinoma cells expressing luciferase 4T1luc2. BALB/c mice were DNA-immunized by intradermal injections of PR_Ai, PR_Ai2mut, PR_Ai3mut, vector pVAX1, or PBS with electroporation. All PR variants induced specific CD8+ T-cell responses revealed after splenocyte stimulation with PR-derived peptides. Splenocytes of mice DNA-immunized with PR_Ai and PR_Ai2mut were not activated by peptides carrying V82A, whereas splenocytes of PR_Ai3mut-immunized mice recognized both peptides with and without V82A mutation. Mutations M46I and I54V were immunologically silent. In the challenge study, DNA immunization with PR_Ai3mut protected mice from the outgrowth of subcutaneously implanted adenocarcinoma 4T1luc2 cells expressing PR_Ai3mut; a tumor was formed only in 1/10 implantation sites and no metastases were detected. Immunizations with other PR variants were not protective; all mice formed tumors and multiple metastasis in the lungs, liver, and spleen. CD8+ cells of PR_Ai3mut DNA-immunized mice exhibited strong IFN-γ/IL-2 responses against PR peptides, while the splenocytes of mice in other groups were nonresponsive. Thus, immunization with a DNA plasmid encoding inactive HIV-1 protease with DR mutations suppressed the growth and metastatic activity of tumor cells expressing PR identical to the one encoded by the immunogen. This demonstrates the capacity of T-cell response induced by DNA immunization to recognize single DR mutations, and supports the concept of the development of immunotherapies against drug resistance in HIV-1 infection. It also suggests that HIV-1-infected patients developing drug resistance may have a reduced natural immune response against DR HIV-1 mutations causing an immune escape.

Toward a SARS-CoV-2 VLP Vaccine: HBc/G as a Carrier for SARS-CoV-2 Spike RBM and Nucleocapsid Protein-Derived Peptides

“…The mixture of mitogenic agents—phorbol myristate acetate (Sigma-Aldrich (St. Louis, MO, USA)) at 20 ng/mL and Ionomicin (Sigma-Aldrich, St. Louis, MO, USA) at 0.5 µg/mL was used as a positive control for cytokine production, and the medium alone was used as a negative control. After two hours of preincubation at +37 °C, the cell medium was supplemented with GolgyPlug reagent at a dilution of 1:1000, and the incubation was continued for another 4 h. The staining of cells for viability, T-cell surface markers (CD4, CD8), and cytokines (IFN-γ, IL-2, and TNF-α) was performed as described below and in [ 49 ]. The cytometry data were acquired using an FACSAria II cytometer (BD, Franklin Lakes, NJ, USA)with the FACSDiva™ v. 6.1.3 software (BD, Franklin Lakes, NJ, USA), exported as FCS 3.0 files, and analyzed using the FlowJo™ v. 10 software (FlowJo LLC, Ashland, OR, USA).…”

Detection of HIV-1 DNA/RNA in Peripheral Blood, Bone Marrow and Femoral Head of Patients with Osteonecrosis of the Femoral Head [Letter]

“…In addition, DNA immunization with HIV-1 protease (PR) is an advancement in immunotherapy of HIV-1 infection to reduce the number of infected cells that produce drugresistant viruses. 3 Bone marrow is a complex multicellular environment that functions in haematopoietic maintenance stem/progenitor cells (HSPC). HSPCs help regulate chemical and molecular signals as well as cell-cell interactions where HIV proteins have a direct suppressive effect on HSPC function.…”