Type I interferon responses in rhesus macaques prevent SIV infection and slow disease progression

Journal name:
Nature
Volume:
511,
Pages:
601–605
Date published:
DOI:
doi:10.1038/nature13554
Received
Accepted
Published online

Inflammation in HIV infection is predictive of non-AIDS morbidity and death1, higher set point plasma virus load2 and virus acquisition3; thus, therapeutic agents are in development to reduce its causes and consequences. However, inflammation may simultaneously confer both detrimental and beneficial effects. This dichotomy is particularly applicable to type I interferons (IFN-I) which, while contributing to innate control of infection4, 5, 6, 7, 8, 9, 10, also provide target cells for the virus during acute infection, impair CD4 T-cell recovery, and are associated with disease progression6, 7, 11, 12, 13, 14, 15, 16, 17, 18, 19. Here we manipulated IFN-I signalling in rhesus macaques (Macaca mulatta) during simian immunodeficiency virus (SIV) transmission and acute infection with two complementary in vivo interventions. We show that blockade of the IFN-I receptor caused reduced antiviral gene expression, increased SIV reservoir size and accelerated CD4 T-cell depletion with progression to AIDS despite decreased T-cell activation. In contrast, IFN-α2a administration initially upregulated expression of antiviral genes and prevented systemic infection. However, continued IFN-α2a treatment induced IFN-I desensitization and decreased antiviral gene expression, enabling infection with increased SIV reservoir size and accelerated CD4 T-cell loss. Thus, the timing of IFN-induced innate responses in acute SIV infection profoundly affects overall disease course and outweighs the detrimental consequences of increased immune activation. Yet, the clinical consequences of manipulation of IFN signalling are difficult to predict in vivo and therapeutic interventions in human studies should be approached with caution.

At a glance

Figures

  1. IFN-1ant suppresses early antiviral responses.
    Figure 1: IFN-1ant suppresses early antiviral responses.

    a, Expression of ISGs in macaques treated with IFN-1ant (n = 6) or placebo saline (n = 9) 7 days after SIV infection. FPKM (log-transformed fragments per kilobase of transcript per million fragments sequenced) reflects the relative abundance of transcripts. P values indicate differentially expressed genes at 7 d.p.i. b, Expression assessed by RNA sequencing (RNA-seq) of antiviral genes APOBEC3G, MX2 and those that code for cGAS and tetherin in PBMCs before and 7 days after SIV infection in macaques that received IFN-1ant (Ant, n = 6) or placebo (Plac, n = 9) injections. Error bars indicate range. P values were calculated by Mann–Whitney U test. c, APOBEC3G, TRIM5α and MX2 protein expression by immunohistochemistry of lymph nodes (LNs) at 4 w.p.i. in placebo (n = 6) and IFN-1ant (n = 6) macaques. Horizontal bars represent median values. P values were calculated by Mann–Whitney U test. The left panels are representative images of APOBEC3G staining from each group. d, Expression of genes involved in pattern recognition receptor signalling of IFN-1ant-treated (n = 6) macaques compared to placebo (n = 9). P values represent the differential expression between IFN-1ant and placebo macaques at 7 d.p.i. For all panels, IFN-1ant-treated macaques are represented in red, placebo-treated macaques in blue.

  2. IFN-1ant accelerates disease progression in SIV-infected rhesus macaques.
    Figure 2: IFN-1ant accelerates disease progression in SIV-infected rhesus macaques.

    a, Plasma SIV RNA levels during acute and chronic SIV infection in macaques treated with IFN-1ant (n = 6) or placebo saline (n = 9). *P < 0.05. Shading indicates treatment period. P value represents the comparison between groups of the areas under the curve (AUC) (0–4 w.p.i.). b, SIV RNA-containing cells in the lymph nodes by in situ hybridization at 4 and 12 w.p.i. in IFN-1ant (Ant, n = 6) and placebo (Plac, n = 6) macaques. Horizontal bars represent median values. P value was calculated by Mann–Whitney U test. c, Frequency of CD4 T cells in peripheral blood during acute and chronic SIV infection in macaques treated with IFN-1ant (n = 6) or placebo saline (n = 9). Error bars indicate range. Red vertical line indicates day 0 of systemic SIV infection. Shading indicates treatment period. P value represents the comparison between groups of the AUC (12-32 w.p.i.). d, Frequency of CD4 T cells in lymph nodes before SIV infection and at 4 or >12 w.p.i. for IFN-1ant (Ant, n = 6) and placebo (Plac, n = 9) macaques. Horizontal bars represent median values. P values at different time points within treatment groups were calculated by Wilcoxon matched pairs signed rank test and between groups by Mann–Whitney U test. e, Frequency of CCR5+ memory (CD28+CD95+ or CD28CD95+/−) CD4 T cells in peripheral blood in macaques treated with IFN-1ant (n = 6) or placebo saline (n = 9). Error bars indicate range. Red vertical line indicates day 0 of systemic SIV infection. Shading indicates treatment period. P values represent the comparison between groups of the AUC (0–12 w.p.i. and 4–12 w.p.i.). f, Frequency of CCR5+ memory CD4 T cells in lymph nodes in macaques treated with IFN-1ant (n = 6) or placebo saline (n = 9). Horizontal bars represent median values. P values at different time points within treatment groups were calculated by Wilcoxon matched pairs signed rank test and between groups by Mann–Whitney U test. g, Kaplan–Meier survival curve comparing macaques treated with IFN-1ant (n = 6) to macaques that received placebo (n = 9). P value indicates the significance by logrank (Mantel–Cox) test for survival by 32 w.p.i. For all panels, IFN-1ant-treated macaques are represented in red, placebo-treated macaques in blue.

  3. IFN-[agr]2a treatment transiently prevents systemic infection but results in an IFN-tolerant state.
    Figure 3: IFN-α2a treatment transiently prevents systemic infection but results in an IFN-tolerant state.

    a, Kaplan–Meier survival curve comparing the number of SIVMAC251 rectal challenges required to achieve systemic infection in macaques treated with IFN-α2a (n = 6) or placebo saline (n = 9). P value indicates the significance by logrank (Mantel–Cox) test of the number of challenges required for systemic infection, between 1 and 5 challenges. b, Correlation between the number of challenges needed to achieve systemic infection and the number of transmitted/founder (T/F) variants in IFN-α2a (green, n = 6), IFN-1ant (red, n = 6) and placebo (blue, n = 9) macaques. P value indicates the significance of the correlation between the number of challenges and the number of T/F variants in all groups. r indicates the Spearman’s rank correlation coefficient. c–f, Expression of antiviral mediators in PBMCs in IFN-α2a-treated (IFN, n = 6) macaques compared to placebo (Plac, n = 9) at 10 d.p.i. Error bars indicate range. P values represent the comparison of FPKMs between IFN-α2a and placebo at 10 d.p.i. by Mann–Whitney U test. g, Expression profile of FOXO3a, a negative regulator of type I IFN signalling. P value represents the comparison of FOXO3a FPKM between IFN-α2a (n = 6) and placebo (n = 9) macaques at 7 d.p.i. h, Gene-set enrichment analysis in IFN-α2a (n = 6) and placebo (n = 9) macaques of genes previously demonstrated to be overexpressed in FOXO3−/− macrophages25. The line plot indicates the running-sum of the enrichment score; the leading edge is indicated in magenta. The relative positions of all genes within the ranked data set are shown in the stick plot below the x axis. P value indicates statistical significance of the enrichment score, reflecting lower cumulative ranking of FOXO3a targets in IFN-α2a-treated macaques compared to placebo at 7 d.p.i. For all panels, IFN-α2a-treated macaques are represented in green, placebo-treated macaques in blue.

  4. IFN-[agr]2a accelerates disease progression.
    Figure 4: IFN-α2a accelerates disease progression.

    a, PBMC-associated SIV gag DNA at 10, 14 and 28 d.p.i. in IFN-α2a macaques (IFN, n = 6) and placebo (Plac, n = 6) macaques. LLQ indicates lower limit of quantification. Horizontal bars represent median values. P values were calculated by Mann–Whitney U test. b, Expression of differentially expressed genes involved in pattern recognition receptor signalling. P values represent the comparison between FPKMs of IFN-α2a (n = 6) and placebo (n = 9) macaques at 7 d.p.i. c, Frequency of CD4 T cells in peripheral blood during acute and early SIV infection in IFN-α2a (n = 6) and placebo (n = 9) macaques. Error bars indicate range. Shading indicates treatment period. Red vertical line indicates day 0 of systemic SIV infection. d, CD4/CD8 T-cell ratio in peripheral blood during acute and early SIV infection in IFN-α2a (n = 6) and placebo (n = 9) macaques. Error bars indicate range. Shading indicates treatment period. Red vertical line indicates day 0 of systemic SIV infection. P value represents the comparison between groups of the AUC (0-12 w.p.i.). e, Frequency of CCR5+ memory CD4 T cells in peripheral blood in IFN-α2a (n = 6) and placebo (n = 9) macaques. Error bars indicate range. Shading indicates treatment period. Red vertical line indicates day 0 of systemic SIV infection. P values represent the comparison between groups of the AUC (0-4 and 0-12 w.p.i.). f, Frequency of CCR5+ memory CD4 T cells in lymph nodes in IFN-α2a (n = 6) and placebo (n = 9) macaques. Horizontal bars represent median values. P values were calculated by Mann–Whitney U test. For all panels, IFN-α2a-treated macaques are represented in green, placebo-treated macaques in blue.

  5. Dose escalation study for IFN-1ant and experimental schema.
    Extended Data Fig. 1: Dose escalation study for IFN-1ant and experimental schema.

    ad, Effects of three times weekly IFN-1ant dosing on the frequency of CD4 T cells (a), CCR5+ CD4 T cells (b), CCR5+ CD8 T cells (c) and Ki67+ CD8 T cells (d) in 2 rhesus macaques. Dose was 50 μg in week 1, 200 μg in week 2, 500 μg in week 3 and 800 μg in week 4. Vertical dotted lines indicate the days a new dose was started. Black lines connect time points 4 days after the first dose. Grey shading indicates treatment period. e, Six macaques received 4 weeks of IFN-1ant intramuscularly starting at day 0 and were challenged intrarectally with 1 ml of a 1:25 dilution of SIVMAC251 (stock concentration 3 × 108 SIV RNA copies ml−1) at day 0 and followed until developing end-stage AIDS. Nine macaques were treated with 4 weeks of placebo saline intramuscularly starting at day 0 and challenged intrarectally with SIVMAC251 at day 0 and followed. Six macaques were injected weekly with IFN-α2a starting 1 week before the first challenge and through 4 w.p.i. Macaques required 2, 3 or 5 challenges to acquire systemic infection. Thus, macaques received 6, 7 or 9 doses of IFN-α2a. Macaques were necropsied at 12 w.p.i. per protocol.

  6. Effects of IFN-1ant on IFN-stimulated genes and virus burden.
    Extended Data Fig. 2: Effects of IFN-1ant on IFN-stimulated genes and virus burden.

    a, b, MX1 (a) and OAS2 (b) expression by qRT–PCR during acute SIV infection in IFN-1ant (red, n = 6) and placebo (blue, n = 9) macaques. P values were calculated by Mann–Whitney U test. c, ISGs in PBMCs in IFN-1ant and placebo macaques. P values represent the comparison between IFN-1ant (n = 6) and placebo (n = 9) macaque FPKMs at 7 d.p.i. d, e, SAMHD1 (d) and APOBEC3G (e) expression in the lymph nodes in IFN-1ant (n = 6) and placebo (n = 9) macaques. P values were calculated by Mann–Whitney U test. f, g, Plasma SIV RNA levels at 12 w.p.i. (f) or at peak (g) stratified by the day that MX1 or OAS2 expression peaked in PBMCs in IFN-1ant (n = 6) and placebo (n = 9) macaques. VL, viral load. P values were calculated by Mann–Whitney U test. h, SIV gag levels in PBMCs stratified by the day that MX1 or OAS2 expression peaked in PBMCs in IFN-1ant (n = 6) and placebo (n = 6) macaques. P values were calculated by Mann–Whitney U test. For all panels, IFN-1ant-treated macaques are represented in red, placebo-treated macaques in blue.

  7. Effects of IFN-1ant on CD4 T cells and on immune activation.
    Extended Data Fig. 3: Effects of IFN-1ant on CD4 T cells and on immune activation.

    a, b, CD4/CD8 T-cell ratio in peripheral blood (a) and lymph node (LN) (b) in IFN-1ant (Ant, n = 6) and placebo (Plac, n = 9) macaques. Shading indicates treatment period. Error bars indicate range. Red vertical line indicates day 0 of systemic SIV infection. For all panels, horizontal bars indicate median values, and P values at different time points within treatment groups were calculated by Wilcoxon matched pairs signed rank test and between groups by Mann–Whitney U test. c–f, T-cell activation in lymph nodes (c–f) in CD4 (c, d) and CD8 (e, f) T cells as represented by the frequency of Ki67+ (c, e) or HLA-DR+ (d, f) cells in IFN-1ant (n = 6) and placebo (n = 9) macaques. g, Frequency of circulating CD16+ or CD56+CD3CD14 NK cells in IFN-1ant (n = 6) and placebo (n = 9) macaques. h, Frequency of circulating CD16+ NK cells in IFN-1ant (n = 6) and placebo (n = 9) macaques. i, Frequency of circulating CD56+ NK cells in IFN-1ant (n = 6) and placebo (n = 9) macaques. For all panels, IFN-1ant-treated macaques are represented in red, placebo-treated macaques in blue.

  8. IFN-1ant alters innate and adaptive immune signalling.
    Extended Data Fig. 4: IFN-1ant alters innate and adaptive immune signalling.

    a, Selected pathways significantly affected by IFN-I blockade. P values were calculated by Fisher’s exact test with the Benjamini–Hochberg multiple testing correction. b, Expression of genes involved in pattern recognition receptor signalling of IFN-1ant-treated macaques (n = 6) compared to placebo (n = 9) at 7 d.p.i. Upregulation compared to pre-infection is represented by red, no change by white, downregulation by blue. P values represent the comparison between IFN-1ant and placebo macaques at 7 d.p.i. c, Selected genes in pattern recognition receptor signalling pathways. Upregulation at 7 d.p.i. is represented by red, downregulation by green.

  9. Effects of IFN-1ant on T-cell function and phenotype.
    Extended Data Fig. 5: Effects of IFN-1ant on T-cell function and phenotype.

    a–e, SIV-specific responses in peripheral blood at 4 and >12 w.p.i. in IFN-1ant (Ant, n = 6) and placebo (Plac, n = 6) macaques by frequency of IFN-γ+ (a), TNF+ (b), perforin+ (c), granzyme B+ (d) and CD107+ (e) CD8 T cells. T-cell exhaustion in peripheral blood and lymph nodes (LN) at >16 w.p.i. based on frequency of PD-1+ CD4 (f) and CD8 (h) T cells and ICOS+ (g) CD8 T cells. For all panels, P values at different time points within treatment groups were calculated by Wilcoxon matched pairs signed rank test and between groups by Mann–Whitney U test. IFN-1ant-treated macaques are represented in red, placebo-treated macaques in blue.

  10. IFN-[agr]2a treatment transiently induces ISGs and subsequently induces the IFN-repressor FOXO3a but does not induce neutralizing anti-IFN antibodies.
    Extended Data Fig. 6: IFN-α2a treatment transiently induces ISGs and subsequently induces the IFN-repressor FOXO3a but does not induce neutralizing anti-IFN antibodies.

    a–d, MX1 (a, c) and OAS2 (b, d) expression during the duration of IFN-α2a treatment in the IFN-α2a group alone (a, b) and during infection in the IFN-α2a (n = 6) and placebo (n = 9) groups (c, d). P values were calculated by Wilcoxon matched pairs signed rank test. e, Percentage of in vitro IFN antiviral activity inhibited by plasma from IFN-α2a (n = 6) and placebo (n = 3) macaques. f, Expression of FOXO3a and FOXO3a-bound genes in SIV-uninfected macaques (n = 3) treated with 21 days of IFN-α2a. Large circles indicate statistically significant (P<0.05) changes from pre-IFN-α2a treatment calculated by Wilcoxon matched pairs signed rank test. Small circles indicate no statistically significant change from pre-IFN-α2a treatment. g, Expression of IFN-α-regulatory genes in IFN-α2a (n = 6) and placebo (n = 9) macaques. P values represent the comparison between FPKMs of IFN-α2a (n = 6) and placebo (n = 9) macaques at 7 d.p.i. h, Expression of FOXO3a-bound genes in IFN-α2a (green, n = 6) and placebo (blue, n = 9) macaques at 7 d.p.i.

  11. Effects of IFN-[agr]2a on IFN-stimulated and antiviral genes.
    Extended Data Fig. 7: Effects of IFN-α2a on IFN-stimulated and antiviral genes.

    a, ISGs in PBMCs in IFN-α2a (n = 6) and placebo (n = 9) macaques. Red indicates upregulation, yellow indicates no change and blue indicates downregulation relative to pre-infection. b, Expression of ISGs in macaques treated with IFN-α2a (n = 6) or placebo (n = 9). P values indicate differentially expressed genes at 10 d.p.i. c–h, Expression of TRIM22 (c, d), MX2 (e, f) and IRF7 (g, h) in SIV-uninfected macaques (n = 3) treated with weekly IFN-α2a for 3 weeks in PBMCs (c, e, g) and lymph nodes and rectum (d, f, h). Day 0 reflects baseline. Numbers indicate days since first IFN-α2a administration. Error bars indicate range. P values were calculated by Wilcoxon matched pairs signed rank test.

  12. Effects of IFN-[agr]2a on SIV control.
    Extended Data Fig. 8: Effects of IFN-α2a on SIV control.

    a, Number of transmitted/founder (T/F) variants in placebo (n = 9), IFN-1ant (n = 6) and IFN-α2a (n = 6) macaques. P value was calculated by Mann–Whitney U test. b, Antiviral protein production in lymph nodes (LN) by immunohistochemistry at 4 w.p.i. in IFN-α2a (n = 6) and placebo (n = 6) macaques. P value was calculated by Mann–Whitney U test. c, CD56+ NK-cell frequency on the day of challenge stratified by whether the macaque resisted or was susceptible to systemic infection that day. Each IFN-α2a macaque (n = 6) is indicated by a different colour. Circles indicate that the macaque was resistant to infection with the next challenge and triangles indicate that the macaque was susceptible to infection with the next challenge. P value was calculated by Mann–Whitney U test. d, Correlation between the number of challenges required to achieve systemic infection and rectal CD16+ NK-cell frequency in each macaque (n = 6) at 4 w.p.i. r indicates the Spearman’s rank correlation coefficient. P value indicates the significance of the correlation. e, Plasma SIV RNA levels in macaques treated with IFN-α2a (n = 6) or placebo (n = 9) saline. Shading reflects treatment period. Red vertical line indicates day 0 of systemic SIV infection. f–i, Frequency of IFN-γ+ (f), TNF+ (g), granzyme B+ (h) and perforin+ (i) CD8 T cells at 4 and ≥12 w.p.i. in IFN-α2a (n = 6) and placebo (n = 6) macaques. j, Frequency of circulating CD16+CD56 NK cells in IFN-α2a (n = 6) and placebo (n = 9) macaques. P values at different time points within treatment groups were calculated by Wilcoxon matched pairs signed rank test and between groups by Mann–Whitney U test.

  13. Effects of IFN-[agr]2a on T-cell activation.
    Extended Data Fig. 9: Effects of IFN-α2a on T-cell activation.

    ah, Frequency of peripheral blood (a–d) and lymph node (LN) (e–h) CD4 (a, c, e, g) and CD8 (b, d, f, h) memory T cells expressing HLA-DR (a, b, e, f) or Ki67 (c, d, g, h) in IFN-α2a (IFN, n = 6) and placebo (Plac, n = 9) macaques. Shading indicates treatment period. Error bars indicate range. ad, Red vertical line indicates day 0 of systemic SIV infection. P values represent the comparison between groups of the AUC (0-4 w.p.i.). eh, Horizontal bars indicate median values. P values were calculated by Mann–Whitney U test.

  14. Effects of IFN-[agr]2a on gene expression.
    Extended Data Fig. 10: Effects of IFN-α2a on gene expression.

    a, Selected pathways significantly affected by IFN-α2a treatment. P values were calculated by Fisher’s exact test with the Benjamini–Hochberg multiple testing correction. b, Expression of genes downstream of IL-6 signalling. Upregulation relative to before IFN-α2a or placebo treatment and SIV infection is represented by red, no change by white, downregulation by blue. P values represent the comparison between IFN-α2a (n = 6) and placebo (n = 9) macaques at 7 d.p.i. c, Selected genes in apoptosis signalling pathways. Significant upregulation at 7 d.p.i. is represented by red, downregulation by green.

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References

  1. Hunt, P. W. et al. Gut epithelial barrier dysfunction and innate immune activation predict mortality in treated HIV infection. J. Infect. Dis. http://dx.doi.org/10.1093/infdis/jiu238 (21 April 2014)
  2. Roberts, L. et al. Genital tract inflammation during early HIV-1 infection predicts higher plasma viral load set point in women. J. Infect. Dis. 205, 194203 (2012)
  3. Naranbhai, V. et al. Innate immune activation enhances HIV acquisition in women, diminishing the effectiveness of tenofovir microbicide gel. J. Infect. Dis. 206, 9931001 (2012)
  4. Schoggins, J. W. et al. A diverse range of gene products are effectors of the type I interferon antiviral response. Nature 472, 481485 (2011)
  5. Schoggins, J. W. et al. Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity. Nature 505, 691695 (2013)
  6. Gonzalez-Navajas, J. M., Lee, J., David, M. & Raz, E. Immunomodulatory functions of type I interferons. Nature Rev. Immunol. 12, 125135 (2012)
  7. Lane, H. C. et al. Anti-retroviral effects of interferon-α in AIDS-associated Kaposi’s sarcoma. Lancet 332, 12181222 (1988)
  8. Manion, M. et al. Interferon-alpha administration enhances CD8+ T cell activation in HIV infection. PLoS ONE 7, e30306 (2012)
  9. Azzoni, L. et al. Pegylated Interferon alfa-2a monotherapy results in suppression of HIV type 1 replication and decreased cell-associated HIV DNA integration. J. Infect. Dis. 207, 213222 (2013)
  10. Feld, J. J. & Hoofnagle, J. H. Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature 436, 967972 (2005)
  11. Stacey, A. R. et al. Induction of a striking systemic cytokine cascade prior to peak viremia in acute human immunodeficiency virus type 1 infection, in contrast to more modest and delayed responses in acute hepatitis B and C virus infections. J. Virol. 83, 37193733 (2009)
  12. Fraietta, J. A. et al. Type I interferon upregulates Bak and contributes to T cell loss during human immunodeficiency virus (HIV) infection. PLoS Pathog. 9, e1003658 (2013)
  13. Abel, K. et al. The relationship between simian immunodeficiency virus RNA levels and the mRNA levels of α/β interferons (IFN-alpha/beta) and IFN-α/β-inducible Mx in lymphoid tissues of rhesus macaques during acute and chronic infection. J. Virol. 76, 84338445 (2002)
  14. Teijaro, J. R. et al. Persistent LCMV infection is controlled by blockade of type I interferon signaling. Science 340, 207211 (2013)
  15. Wilson, E. B. et al. Blockade of chronic type I interferon signaling to control persistent LCMV infection. Science 340, 202207 (2013)
  16. Li, Q. et al. Glycerol monolaurate prevents mucosal SIV transmission. Nature 458, 10341038 (2009)
  17. Haas, D. W. et al. A randomized trial of interferon alpha therapy for HIV type 1 infection. AIDS Res. Hum. Retroviruses 16, 183190 (2000)
  18. Fernandez, S. et al. CD4+ T-cell deficiency in HIV patients responding to antiretroviral therapy is associated with increased expression of interferon-stimulated genes in CD4+ T cells. J. Infect. Dis. 204, 19271935 (2011)
  19. Levin, D. et al. Multifaceted activities of type I interferon are revealed by a receptor antagonist. Sci. Signal. 7, ra50 (2014)
  20. Goujon, C. et al. Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection. Nature 502, 559562 (2013)
  21. Honda, K. et al. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 434, 772777 (2005)
  22. Barouch, D. H. et al. Vaccine protection against acquisition of neutralization-resistant SIV challenges in rhesus monkeys. Nature 482, 8993 (2012)
  23. Carrington, M. & Alter, G. Innate immune control of HIV. Cold Spring Harb. Perspect. Med. 2, a007070 (2012)
  24. Asmuth, D. M. et al. Pegylated interferon-α 2a treatment of chronic SIV-infected macaques. J. Med. Primatol. 37, 2630 (2008)
  25. Litvak, V. et al. A FOXO3–IRF7 gene regulatory circuit limits inflammatory sequelae of antiviral responses. Nature 490, 421425 (2012)
  26. Schellekens, H. et al. The effect of recombinant human interferon αB/D compared to interferon α2b on SIV infection in rhesus macaques. Antiviral Res. 32, 18 (1996)
  27. Waggoner, S. N., Daniels, K. A. & Welsh, R. M. Therapeutic depletion of natural killer cells controls persistent infection. J. Virol. 88, 19531960 (2014)
  28. Parrish, N. F. et al. Phenotypic properties of transmitted founder HIV-1. Proc. Natl Acad. Sci. USA 110, 66266633 (2013)
  29. Fenton-May, A. E. et al. Relative resistance of HIV-1 founder viruses to control by interferon-alpha. Retrovirology 10, 146 (2013)
  30. McElrath, M. J. et al. Comprehensive assessment of HIV target cells in the distal human gut suggests increasing HIV susceptibility toward the anus. J. Acquir. Immune Defic. Syndr. 63, 263271 (2013)
  31. Vanderford, T. H. et al. Treatment of SIV-infected sooty mangabeys with a type-I IFN agonist results in decreased virus replication without inducing hyperimmune activation. Blood 119, 57505757 (2012)
  32. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 1521 (2013)
  33. Trapnell, C. et al. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nature Biotechnol. 31, 4653 (2013)
  34. Brenchley, J. M. et al. Differential infection patterns of CD4+ T cells and lymphoid tissue viral burden distinguish progressive and nonprogressive lentiviral infections. Blood 120, 41724181 (2012)

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Author information

Affiliations

  1. Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Netanya G. Sandler,
    • Richard T. R. Zhu,
    • Eli Boritz,
    • Sathi Wijeyesinghe,
    • Krystelle Nganou Makamdop,
    • Brenna J. Hill,
    • J. Katherina Timmer,
    • Emma Reiss,
    • Samuel Darko,
    • Eduardo Contijoch &
    • Daniel C. Douek
  2. Division of Microbiology and Immunology, Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, Georgia 30322, USA

    • Steven E. Bosinger,
    • Gregory K. Tharp &
    • Guido Silvestri
  3. Non-Human Primate Genomics Core, Yerkes National Primate Research Center, Robert W. Woodruff Health Sciences Center, Emory University, Atlanta, Georgia 30322, USA

    • Steven E. Bosinger &
    • Gregory K. Tharp
  4. AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, Frederick, Maryland 21702, USA

    • Jacob D. Estes,
    • Gregory Q. del Prete,
    • Brandon F. Keele &
    • Jeffrey D. Lifson
  5. Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel

    • Doron Levin,
    • Ganit Yarden &
    • Gideon Schreiber
  6. Laboratory of Animal Medicine, Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20892, USA

    • John Paul Todd &
    • Srinivas Rao
  7. Biostatistics Research Branch, Division of Clinical Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Martha Nason
  8. Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA

    • Robert B. Norgren Jr
  9. Department of Pharmacology, Rutgers - Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA

    • Jerome A. Langer
  10. Present address: Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch at Galveston, Galveston, Texas 77555, USA.

    • Netanya G. Sandler

Contributions

N.G.S. designed and coordinated the study, developed and performed experiments, interpreted the data and prepared the manuscript. S.E.B. analysed and interpreted the sequencing data, generated figures and contributed to manuscript preparation. J.D.E. contributed to study design and developed and performed in situ hybridization and immunohistochemistry assays. R.T.R.Z. processed samples, performed flow cytometry and analysis, performed qRT–PCR and generated the sequencing libraries. G.K.T. analysed and interpreted the sequencing data and generated figures. E.B. developed the library generation protocol and supervised library generation. D.L. and G.Y. synthesized the IFN-1ant. S.W. generated sequencing libraries and assisted in analysis of the sequencing data. K.N.M. assisted with sample processing, performed flow cytometry assays, and assessed plasma for neutralizing activity. G.Q.d.P. evaluated circulating SIV for IFN resistance. B.J.H. designed, performed and analysed qRT–PCR assays. J.K.T. processed samples and performed ELISAs. E.R. assisted with sample processing and performed flow cytometry assays. S.D. assisted with sequencing analysis. E.C. assisted with sample processing and performed flow cytometry assays. J.P.T. performed SIV inoculations and coordinated the study at Bioqual. G.Si. established the Non-Human Primate Sequencing Core and facilitated sequencing analysis and contributed to data interpretation. M.N. assisted with statistical analyses. R.B.N. generated the MuSuRCA Macaca mulatta assembly. B.F.K. sequenced the transmitted/founder variants. S.R. contributed to study design and followed the rhesus macaques clinically. J.A.L. contributed to IFN-1ant design and assisted with analysis. J.D.L. contributed to study design, assessment for IFN-resistant viruses and manuscript preparation. G.Sc. contributed to study design, IFN-1ant design and production and assisted with analysis. D.C.D. designed and supervised the study, interpreted the data and prepared the manuscript.

Competing financial interests

The type I interferon receptor antagonist used in this study and related type I interferon antagonists are covered in the Patent Application PCT/US2009/056366 held by J.A.L. and G.Sc.

Corresponding author

Correspondence to:

Gene expression data are available at the Gene Expression Omnibus under accession codes GSM1298835 through GSM1299037.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Dose escalation study for IFN-1ant and experimental schema. (274 KB)

    ad, Effects of three times weekly IFN-1ant dosing on the frequency of CD4 T cells (a), CCR5+ CD4 T cells (b), CCR5+ CD8 T cells (c) and Ki67+ CD8 T cells (d) in 2 rhesus macaques. Dose was 50 μg in week 1, 200 μg in week 2, 500 μg in week 3 and 800 μg in week 4. Vertical dotted lines indicate the days a new dose was started. Black lines connect time points 4 days after the first dose. Grey shading indicates treatment period. e, Six macaques received 4 weeks of IFN-1ant intramuscularly starting at day 0 and were challenged intrarectally with 1 ml of a 1:25 dilution of SIVMAC251 (stock concentration 3 × 108 SIV RNA copies ml−1) at day 0 and followed until developing end-stage AIDS. Nine macaques were treated with 4 weeks of placebo saline intramuscularly starting at day 0 and challenged intrarectally with SIVMAC251 at day 0 and followed. Six macaques were injected weekly with IFN-α2a starting 1 week before the first challenge and through 4 w.p.i. Macaques required 2, 3 or 5 challenges to acquire systemic infection. Thus, macaques received 6, 7 or 9 doses of IFN-α2a. Macaques were necropsied at 12 w.p.i. per protocol.

  2. Extended Data Figure 2: Effects of IFN-1ant on IFN-stimulated genes and virus burden. (394 KB)

    a, b, MX1 (a) and OAS2 (b) expression by qRT–PCR during acute SIV infection in IFN-1ant (red, n = 6) and placebo (blue, n = 9) macaques. P values were calculated by Mann–Whitney U test. c, ISGs in PBMCs in IFN-1ant and placebo macaques. P values represent the comparison between IFN-1ant (n = 6) and placebo (n = 9) macaque FPKMs at 7 d.p.i. d, e, SAMHD1 (d) and APOBEC3G (e) expression in the lymph nodes in IFN-1ant (n = 6) and placebo (n = 9) macaques. P values were calculated by Mann–Whitney U test. f, g, Plasma SIV RNA levels at 12 w.p.i. (f) or at peak (g) stratified by the day that MX1 or OAS2 expression peaked in PBMCs in IFN-1ant (n = 6) and placebo (n = 9) macaques. VL, viral load. P values were calculated by Mann–Whitney U test. h, SIV gag levels in PBMCs stratified by the day that MX1 or OAS2 expression peaked in PBMCs in IFN-1ant (n = 6) and placebo (n = 6) macaques. P values were calculated by Mann–Whitney U test. For all panels, IFN-1ant-treated macaques are represented in red, placebo-treated macaques in blue.

  3. Extended Data Figure 3: Effects of IFN-1ant on CD4 T cells and on immune activation. (471 KB)

    a, b, CD4/CD8 T-cell ratio in peripheral blood (a) and lymph node (LN) (b) in IFN-1ant (Ant, n = 6) and placebo (Plac, n = 9) macaques. Shading indicates treatment period. Error bars indicate range. Red vertical line indicates day 0 of systemic SIV infection. For all panels, horizontal bars indicate median values, and P values at different time points within treatment groups were calculated by Wilcoxon matched pairs signed rank test and between groups by Mann–Whitney U test. c–f, T-cell activation in lymph nodes (c–f) in CD4 (c, d) and CD8 (e, f) T cells as represented by the frequency of Ki67+ (c, e) or HLA-DR+ (d, f) cells in IFN-1ant (n = 6) and placebo (n = 9) macaques. g, Frequency of circulating CD16+ or CD56+CD3CD14 NK cells in IFN-1ant (n = 6) and placebo (n = 9) macaques. h, Frequency of circulating CD16+ NK cells in IFN-1ant (n = 6) and placebo (n = 9) macaques. i, Frequency of circulating CD56+ NK cells in IFN-1ant (n = 6) and placebo (n = 9) macaques. For all panels, IFN-1ant-treated macaques are represented in red, placebo-treated macaques in blue.

  4. Extended Data Figure 4: IFN-1ant alters innate and adaptive immune signalling. (284 KB)

    a, Selected pathways significantly affected by IFN-I blockade. P values were calculated by Fisher’s exact test with the Benjamini–Hochberg multiple testing correction. b, Expression of genes involved in pattern recognition receptor signalling of IFN-1ant-treated macaques (n = 6) compared to placebo (n = 9) at 7 d.p.i. Upregulation compared to pre-infection is represented by red, no change by white, downregulation by blue. P values represent the comparison between IFN-1ant and placebo macaques at 7 d.p.i. c, Selected genes in pattern recognition receptor signalling pathways. Upregulation at 7 d.p.i. is represented by red, downregulation by green.

  5. Extended Data Figure 5: Effects of IFN-1ant on T-cell function and phenotype. (312 KB)

    a–e, SIV-specific responses in peripheral blood at 4 and >12 w.p.i. in IFN-1ant (Ant, n = 6) and placebo (Plac, n = 6) macaques by frequency of IFN-γ+ (a), TNF+ (b), perforin+ (c), granzyme B+ (d) and CD107+ (e) CD8 T cells. T-cell exhaustion in peripheral blood and lymph nodes (LN) at >16 w.p.i. based on frequency of PD-1+ CD4 (f) and CD8 (h) T cells and ICOS+ (g) CD8 T cells. For all panels, P values at different time points within treatment groups were calculated by Wilcoxon matched pairs signed rank test and between groups by Mann–Whitney U test. IFN-1ant-treated macaques are represented in red, placebo-treated macaques in blue.

  6. Extended Data Figure 6: IFN-α2a treatment transiently induces ISGs and subsequently induces the IFN-repressor FOXO3a but does not induce neutralizing anti-IFN antibodies. (428 KB)

    a–d, MX1 (a, c) and OAS2 (b, d) expression during the duration of IFN-α2a treatment in the IFN-α2a group alone (a, b) and during infection in the IFN-α2a (n = 6) and placebo (n = 9) groups (c, d). P values were calculated by Wilcoxon matched pairs signed rank test. e, Percentage of in vitro IFN antiviral activity inhibited by plasma from IFN-α2a (n = 6) and placebo (n = 3) macaques. f, Expression of FOXO3a and FOXO3a-bound genes in SIV-uninfected macaques (n = 3) treated with 21 days of IFN-α2a. Large circles indicate statistically significant (P<0.05) changes from pre-IFN-α2a treatment calculated by Wilcoxon matched pairs signed rank test. Small circles indicate no statistically significant change from pre-IFN-α2a treatment. g, Expression of IFN-α-regulatory genes in IFN-α2a (n = 6) and placebo (n = 9) macaques. P values represent the comparison between FPKMs of IFN-α2a (n = 6) and placebo (n = 9) macaques at 7 d.p.i. h, Expression of FOXO3a-bound genes in IFN-α2a (green, n = 6) and placebo (blue, n = 9) macaques at 7 d.p.i.

  7. Extended Data Figure 7: Effects of IFN-α2a on IFN-stimulated and antiviral genes. (394 KB)

    a, ISGs in PBMCs in IFN-α2a (n = 6) and placebo (n = 9) macaques. Red indicates upregulation, yellow indicates no change and blue indicates downregulation relative to pre-infection. b, Expression of ISGs in macaques treated with IFN-α2a (n = 6) or placebo (n = 9). P values indicate differentially expressed genes at 10 d.p.i. c–h, Expression of TRIM22 (c, d), MX2 (e, f) and IRF7 (g, h) in SIV-uninfected macaques (n = 3) treated with weekly IFN-α2a for 3 weeks in PBMCs (c, e, g) and lymph nodes and rectum (d, f, h). Day 0 reflects baseline. Numbers indicate days since first IFN-α2a administration. Error bars indicate range. P values were calculated by Wilcoxon matched pairs signed rank test.

  8. Extended Data Figure 8: Effects of IFN-α2a on SIV control. (398 KB)

    a, Number of transmitted/founder (T/F) variants in placebo (n = 9), IFN-1ant (n = 6) and IFN-α2a (n = 6) macaques. P value was calculated by Mann–Whitney U test. b, Antiviral protein production in lymph nodes (LN) by immunohistochemistry at 4 w.p.i. in IFN-α2a (n = 6) and placebo (n = 6) macaques. P value was calculated by Mann–Whitney U test. c, CD56+ NK-cell frequency on the day of challenge stratified by whether the macaque resisted or was susceptible to systemic infection that day. Each IFN-α2a macaque (n = 6) is indicated by a different colour. Circles indicate that the macaque was resistant to infection with the next challenge and triangles indicate that the macaque was susceptible to infection with the next challenge. P value was calculated by Mann–Whitney U test. d, Correlation between the number of challenges required to achieve systemic infection and rectal CD16+ NK-cell frequency in each macaque (n = 6) at 4 w.p.i. r indicates the Spearman’s rank correlation coefficient. P value indicates the significance of the correlation. e, Plasma SIV RNA levels in macaques treated with IFN-α2a (n = 6) or placebo (n = 9) saline. Shading reflects treatment period. Red vertical line indicates day 0 of systemic SIV infection. f–i, Frequency of IFN-γ+ (f), TNF+ (g), granzyme B+ (h) and perforin+ (i) CD8 T cells at 4 and ≥12 w.p.i. in IFN-α2a (n = 6) and placebo (n = 6) macaques. j, Frequency of circulating CD16+CD56 NK cells in IFN-α2a (n = 6) and placebo (n = 9) macaques. P values at different time points within treatment groups were calculated by Wilcoxon matched pairs signed rank test and between groups by Mann–Whitney U test.

  9. Extended Data Figure 9: Effects of IFN-α2a on T-cell activation. (454 KB)

    ah, Frequency of peripheral blood (a–d) and lymph node (LN) (e–h) CD4 (a, c, e, g) and CD8 (b, d, f, h) memory T cells expressing HLA-DR (a, b, e, f) or Ki67 (c, d, g, h) in IFN-α2a (IFN, n = 6) and placebo (Plac, n = 9) macaques. Shading indicates treatment period. Error bars indicate range. ad, Red vertical line indicates day 0 of systemic SIV infection. P values represent the comparison between groups of the AUC (0-4 w.p.i.). eh, Horizontal bars indicate median values. P values were calculated by Mann–Whitney U test.

  10. Extended Data Figure 10: Effects of IFN-α2a on gene expression. (303 KB)

    a, Selected pathways significantly affected by IFN-α2a treatment. P values were calculated by Fisher’s exact test with the Benjamini–Hochberg multiple testing correction. b, Expression of genes downstream of IL-6 signalling. Upregulation relative to before IFN-α2a or placebo treatment and SIV infection is represented by red, no change by white, downregulation by blue. P values represent the comparison between IFN-α2a (n = 6) and placebo (n = 9) macaques at 7 d.p.i. c, Selected genes in apoptosis signalling pathways. Significant upregulation at 7 d.p.i. is represented by red, downregulation by green.

Supplementary information

PDF files

  1. Supplementary Information (879 KB)

    This file contains Supplementary Results and Supplementary Figure 1.

Additional data