Dose-Dependent Differences in HIV Inhibition by Different Interferon Alpha Subtypes While Having Overall Similar Biologic Effects

Abstract

Type I interferons (IFNs) are key players in the antiviral immune response. Interferon alpha (IFN-α) belongs to this class of IFNs and comprises 12 subtypes that differ from each other in their binding affinities for a common receptor and, thus, in their signaling potencies. Recent data suggest that IFN-α6 and -α14 are the most potent IFN-α subtypes in restricting HIV replication when applied exogenously. However, in the context of antiviral therapy, IFNs are administered at high doses, which may compensate for differences in potency seen between IFN-α subtypes. In this study, we reexamined whether IFN-α subtypes induce different biological activities, with a focus on how IFN-α treatment dose affects cellular responses to HIV in primary CD4⁺ T cells, peripheral blood mononuclear cells (PBMCs), and macrophages. We found that the subtypes' antiviral activities were dose dependent, with >90% inhibition of HIV replication at a high dose of all IFN-αs except the weak IFN-α/β receptor (IFNAR) binder, IFN-α1. The quality of the responses engendered by IFN-α1, -α2, -α6, and -α14 was highly comparable, with essentially the same set of genes induced by all four subtypes. Hierarchal cluster analysis revealed that the individual donors were stronger determinants for the IFN-stimulated-gene (ISG) responses than the specific IFN-α subtype used for stimulation. Notably, IFN-α2-derived mutants with substantially reduced IFNAR2 binding still inhibited HIV replication efficiently, whereas mutants with increased IFNAR1 binding potentiated antiviral activity. Overall, our results support the idea that IFN-α subtypes do not induce different biological responses, given that each subtype is exogenously applied at bioequivalent doses.IMPORTANCE Elucidating the functional role of the IFN-α subtypes is of particular importance for the development of efficacious therapies using exogenous IFN-α. Specifically, this will help define whether IFN therapy should be based on the use of pathogen-dependent IFN subtypes or, rather, IFN mutants with optimized IFNAR binding properties.

Keywords: antiviral therapy; human immunodeficiency virus; interferons; therapeutic efficacy.

FIG 1

Signaling activities of IFN-α subtypes converge at high treatment doses, which translates into comparable ISG induction profiles between subtypes. (A) Dose-dependent induction of gene expression by all 12 IFN-α subtypes in cells carrying an ISRE-luciferase construct. Significance testing was done for each IFN-α dose using one-way ANOVAs on fold activity relative to the activity of IFN-α2. Symbols for comparisons with IFN-α2 are as follows: *, IFN-α1; #, IFN-α6. Presentation of data is described in “Statistical analysis” in Materials and Methods for all figures. (B) Heat map showing genes induced in primary CD4⁺ T cells from three independent donors stimulated with 1,000 pg/ml of IFN-α1, -α2, -α6, or -α14 compared to their induction in unstimulated controls (i.e., fold upregulation normalized to the expression in the unstimulated control; the values −3 to 115 represent the extremes of down- and upregulation as normalized to the result for the unstimulated control), and hierarchical clustering of the induced-gene-response profiles. For this assay, the interferons and receptors RT² Profile PCR array (Qiagen) was used, but only results for genes of interest (ISGs) are shown; see Fig. S2 in the supplemental material for results for the full array of genes included in the PCR array. (C) Detailed view of genes quantitatively differentially induced by IFN-α1, -α2, -α6, or -α14. Lines connect responses from each donor, and the horizontal dashed line represents 2-fold change in gene expression compared to expression in unstimulated controls. These 10 genes represent the set of genes that showed significantly different expression levels between at least one pair of IFN-α subtypes in repeated measures (RM) two-way ANOVAs of the data set shown in panel B (ANOVA results are summarized in Table S2). Note that all 10 genes were induced >2-fold.

FIG 2

Dose-dependent HIV-inhibitory activity of IFN-α subtypes as examined in primary cells infected ex vivo. (A and B) CD4⁺ T cells, PBMCs, or MDMs were pretreated with the indicated IFN-α subtype for 2 h and then inoculated with HIV YU-2 overnight. The next day, the cultures were washed and the IFN-αs added back. At days 0, 4, 8, and 12, supernatant was harvested to monitor HIV replication by quantifying p24 Ag. The area under the curve of the p24 Ag over time was calculated and normalized to that of the control (i.e., no IFN-α). (A) Data exploring anti-HIV activities of IFN-α1, -α2, -α6, and -α14 in subsets of six donors’ CD4⁺ T cells and three donors’ PBMCs at an extended dose range. (B) Data for IFN-α1, -α2, and -α6 in cells from larger numbers of donors (12, 9, and 9 donors, respectively), focusing on higher dosages of the IFN-α subtypes. Responses were compared to those measured in the IFN-α2-treated groups by significance testing with RM two-way ANOVAs, comparing all groups to the IFN-α2 group. Symbols for comparisons with IFN-α2 are as follows: *, IFN-α1; #, IFN-α6. (C) Data for fold changes in anti-HIV activities compared to that of IFN-α2 for IFN-α subtypes and donor cells as described in the legend to panel B. (D) Percentages of inhibition in donor-matched cell subsets for IFN-α1, -α2, and -α6 at 10 pg/ml. Significance testing was done for each IFN-α subtype using RM one-way ANOVAs, comparing the responses in each cell type to one another.

FIG 3

IFN subtypes converge in their anti-HIV activities at 1,000 pg/ml, with the exception of IFN-α1. PBMCs were pretreated with the IFN-α subtypes and infected with HIV YU-2 overnight. The next day, the cultures were washed and IFN-αs added back. V_res was assessed for all subtypes (n = 3 donors, in duplicates). Significance testing was done using RM one-way ANOVAs, comparing all IFN-α treatment groups to IFN-α2.

FIG 4

IFN-α2 mutants with modified IFNAR binding affinities display differential anti-HIV activities compared to that of IFN-α2. (A) Purified primary CD4⁺ T cells and PBMCs were pretreated with either IFN-α2 or the mutants for 2 h and then inoculated with HIV YU-2 overnight. The next day, the cultures were washed and IFN-αs added back. At days 0, 4, 8, and 12, supernatants were harvested for monitoring HIV replication by quantifying p24 Ag. The areas under the curve of the p24 Ag over time were calculated and normalized to that of matched controls (n = 3 donors). Significance testing was done using RM two-way ANOVAs, comparing all mutants to IFN-α2. Symbols for comparisons with IFN-α2 are as follows: *, YNS; #, YNS-α8T; §, YNS-148A; °, IFN-α2-145G; %, IFN-α2-α8T; †, IFN-α2-26A. (B) V_res values for all of the mutants (n = 3 donors, in duplicates). Significance testing was done using RM one-way ANOVAs, comparing mutants to IFN-α2.