Noninfectious papilloma virus-like particles inhibit HIV-1 replication: implications for immune control of HIV-1 infection by IL-27

Abstract

Human papilloma virus (HPV)-like particles (VLPs) have been used as a vaccine to prevent HPV infection. Recent studies demonstrate that VLPs bind to dendritic cells and induce the expression of antiviral cytokines such as interferon-alpha (IFN-alpha), interleukin-10 (IL-10) and IFN-gamma. In the present study, we evaluated the effect of VLPs on HIV-1 replication in peripheral blood mononuclear cells (PBMCs), CD4+ T cells, and macrophages. Here, we show that VLPs suppress the replication of both X4 and R5 HIV-1 without affecting the expression of CD4, CXCR4, and CCR5. Soluble factor(s) released by PBMCs and macrophages on VLPs treatment inhibited HIV-1 replication. To determine the inhibitory factors, DNA microarray analysis was performed using VLP-treated PBMCs and macrophages. VLPs induced the genes associated with IFN induction, immune responses, and antiviral responses, among with the recently described cytokine IL-27. Subsequently, IL-27 was found to be a potent inhibitor of HIV-1 replication in PBMCs, CD4+ T cells, and macrophages. Taken together, our studies identify a novel role of IL-27 in restricting HIV-1 replication and suggest that further examination of the inhibitory property of IL-27 may pave the way for a novel therapy for HIV-1 infection.

Figure 1

VLPs inhibit HIV-1 replication. (A-B) PHA-stimulated PBMCs were infected with (A) X4 virus (HIV-1_NL4.3) or (B) R5 virus (HIV-1_Ad8) as described in “Materials and methods.” The infected cells were cultured for 7 days in the presence of various concentrations of VLPs. (C) PHA-stimulated PBMCs were infected with X4 virus for 2 hours and then cultured for 7 days in the absence or the presence of 1 μg/mL baculovirus extract or VLPs. (D) PHA-stimulated PBMCs were infected with X4 virus for 2 hours and then cultured for 7 days in the absence or the presence of 1 μg/mL VLPs, SV40-VLPs, or BVLPs. In all experiments, half of the culture supernatants was changed on the 4th day with fresh culture media alone. HIV-1 replication was measured by p24 antigen capture assay. Data show means ± SDs and are representative of at least 3 independent experiments.

Figure 2

Pretreatment of PBMCs with VLPs inhibit HIV-1 replication without changing in the expression of HIV receptors. (A) PHA-stimulated PBMCs were pretreated with media alone or 1 μg/mL VLPs for 2 or 24 hours and then washed with fresh media to remove unbound VLPs. The pretreated cells were infected with X4 virus for 2 hours and cultured for 7 days without addition of VLPs. Half of the culture supernatants was changed on the 4th day with fresh culture media alone. HIV-1 replication was assessed by p24 antigen capture assay. Data show means ± SDs and are representative of at least 2 independent experiments. (B) PHA-stimulated PBMCs were cultured in the absence (red) or presence (black) of VLPs (1 μg/mL) for 24 hours. The surface expression of CD4, CXCR4, and CCR5 was then assessed by flow cytometry as described in “Materials and methods.” For a positive control of down-regulated receptors, the cells were treated with 100 ng/mL of a mixture of RANTES and MCP-1 for CCR5 or SDF-1 for CXCR4, and then the expression level was analyzed. Left panels show the staining of CD3⁺ T lymphocytes; right panels show CD14⁺ monocytes. Upper, middle, and bottom panels show CD4, CXCR4, and CCR5 staining, respectively. The staining pattern of isotype control antibodies are shown in blue, and the positive control of down-regulation are shown in green. The x-axis and y-axis show florescence intensity and cell count, respectively. The data are representative of 3 independent experiments with similar outcomes.

Figure 3

VLPs inhibit HIV replication in CD8⁻ PBMCs and MDMs but not in CD4⁺ T cells. CD4⁺ T cells, CD8⁻ PBMCs, and MDMs were prepared as described in “Materials and methods.” CD4⁺ T cells and CD8⁻ PBMCs were stimulated with PHA and then infected with X4 or R5 viruses. The infected cells were cultured in the absence (□) or the presence of 1 μg/mL VLPs (■) for 7 days. MDMs were infected with R5 virus and then cultured for 10 days. Half of the culture supernatants was changed on the 4th day and the 7th day with fresh culture media alone. HIV-1 replication was measured by p24 antigen capture assay. Data show means ± SDs and represent 3 independent experiments.

Figure 4

Conditioned media derived from VLP-treated PBMCs or MDMs inhibit HIV replication in CD4⁺ T cells. Control-Sup (□) and VLP-Sup (■) from PBMCs (A) and MDMs (B) were collected as described in “Materials and methods.” PHA-stimulated CD4⁺ T cells were infected with X4 virus and then cultured in the presence of different percentages of the Sup for 7 days. Half of culture supernatants were changed on the 4th day with fresh culture media alone. HIV-1 replication was measured by p24 antigen capture assay. Data show means ± SDs. Representative results from 3 independent results are shown.

Figure 5

IL-27 suppresses HIV replication. (A) PHA-stimulated CD4⁺ T cells infected with X4 virus and PHA-stimulated PBMCs infected with R5 virus were cultured for 7 days in the absence or presence of 12.5% of VLP-Sup from PBMCs. To neutralize IFNs (IFN-α and IFN-β), MCP-2, IL-10, or CCR5 ligands, 10 μg/mL of each neutralizing antibody was added in the culture. To neutralize CCR5 ligands, an antibody mixture containing 10 μg/mL of each antibody to MIP-1α, MIP-1β, and RANTES was used. IL-27 in VLP-Sup from PBMCs was immunodepleted as described in “Materials and methods.” As a replication control, the infected cells were cultured in the absence of the VLP-Sup. On the 4th day after infection, 50% of culture supernatants were changed with fresh culture media alone. HIV-1 replication was measured by p24 antigen capture assay. The neutralization and the immunodepletion assay were performed at least 3 times, and results show the means ± SEs. (B) Relative expression of IL-27 mRNAs in PBMCs and MDMs were assessed by RT-PCR. PHA-stimulated PBMCs and MDMs were cultured in the absence or presence of 1 μg/mL VLPs for 36 and 24 hours, respectively. A total cellular RNA was isolated, and RT-PCR was performed as described in “Materials and methods.” As a control, RT-PCR was performed using RNA from SV40VLP-treated PHA-simulated PBMCs. (C-E) X4-infected CD4⁺ T cells (C) or PBMCs (D) were cultured in the presence of different concentrations of recombinant human IL-27. On the 4th day after infection, 50% of culture supernatants were changed with fresh culture media containing IL-27. HIV-1 replication was measured on 7th day by p24 antigen capture assay. MDMs were infected with R5 virus (E) and then cultured for 10 days in the presence of IL-27. Half of the culture supernatants were changed on the 4th and 7th day with fresh media with IL-27. HIV-1 replication was measured by p24 antigen capture assay. HIV-1 replication was measured by p24 antigen capture assay. Data show means ± SDs and are representative of 5 experiments.