WO2016135046A1 - Inhibitors of raf1, mst1, and pkl1 for use in the treatment of a retrovirus - Google Patents

Abstract

A compound selected from the group consisting of: (i) an inhibitor of Polo-like 1 (PLK1), (ii) an inhibitor of mammalian Ste20-like 1 (MST1), and (iii) an inhibitor of RAF-1, with the proviso that said compound is not an M-CSF antagonist, for use in a treatment of a subject infected with a retrovirus or for prevention of infection with a retrovirus in a subject, preferably wherein said treatment is given before seroconversion.

Description

TITLE: INHIBITORS OF RAF1, MSTl AND PLKl FOR USE IN THE TREATMENT OF A RETROVIRUS

TECHNICAL FIELD

The present invention relates to compounds, kits and pharmaceutical compositions for use in the treatment or prevention of a retrovirus. In particular, the invention relates to inhibitors of RAF1, MSTl and PLKl. More in particular, the invention relates to immune therapies, especially at an early phase of a retroviral infection.

BACKGROUND

The causative agent of AIDS is the retrovirus Human Immunodeficiency Virus (HIV). Retroviruses are distinguished by the fact that their genetic material, which is RNA, encodes the information for viral replication. Upon infection of a host cell, it acts as a template for the transcription to DNA, catalyzed by an enzyme called reverse transcriptase. The DNA so produced enters the cell nucleus where it is integrated into the host DNA as a pro virus. When properly activated, the retro viral-derived DNA is transcribed and translated to produce RNA containing virions, which are then released from the cell by a budding process.

Certain viruses, including retroviruses, may remain in a latent state for months or years before they are activated and virions are produced. Although asymptomatic, a host may nonetheless harbor the virus in a proviral form, thus being potentially at risk of disease and of infecting others.

HIV-1 is a major global health problem with over 35 million people infected and 2 million new infections every year (UNAIDS, 2013). Although antiretroviral therapy is effective, chronic infected individuals suffer from severe co-morbidities because of immune dysfunction.

Dendritic cells (DCs) are among the first immune cells to encounter HIV-1 upon sexual transmission and are crucial in host defense, linking detection of microbes to induction of pathogen-tailored adaptive immune responses. However, DCs do not mount protective immunity against HIV-1. A major determining factor for the absence of protective immunity seems to be the ability of HIV-1 to escape immune surveillance. DCs sense invading viruses through pattern recognition receptors (PRRs) that trigger innate signaling leading to intrinsic antiviral defenses during DC maturation via type I interferon (IFN-I) responses and activation of T cells.

Induction of IFN-I responses are believed to be triggered when IFNa and ΠΤΝΓβ, via autocrine signaling, which activate an antiviral program of IFN-stimulated genes (ISGs) that counteract virus replication. IFN-I also induces DC maturation, thereby facilitating (cross-)presentation of viral antigens to lymphocytes, while also shaping adaptive immunity via T helper cell polarization.

Therefore, it is believed that the absence of DC activation and IFN-I responses underlie the lack of protective immunity.

There is still a need in the art for HIV-1 immune therapies, which strengthen the immune function, prevent immune escape by the retrovirus and are more effective and safe. It is an object of the invention to overcome one or more of the above mentioned problems. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows Raf-1 activation suppresses IFN-I responses after HIV-1 infection of DCs

Raf-1 in monocyte-derived DCs was inhibited by either small molecule inhibitor GW5074 or siRNA against Raf-1, and treated DCs were infected with HIV-1. At different time points DCs were harvested and IFN production was measured by real-time PCR (A, B). To determine type I IFN response, we also measured the IFN-stimulated genes by real-time PCR after treatment of DCs with Raf inhibitor GW5074 (C). Next, primary DC subsets were isolated from different tissues and these DC subsets were treated with RAF inhibitor GW5074 and IFN production was measured as described above. DCs from different tissues all responded by a stimulated IFN production (D). Finally, we show that the induction of type I IFN responses when Raf-1 is inhibited is independent of the HIV-1 strain used since infection of monocyte-derived DCs with different HIV-1 strains (X4 and R5 tropic, as well as clinical isolates from patients) in presence of Raf inhibitor induced strong IFN responses (E,F). The IFN-stimulated genes as a measure for IFN type I responses were treated with the small molecule inhibitor against Raf or siRNA against Raf-1, and cells were subsequently infected * p < 0.05 (Student's Mest). See also Figures SI and S2. SI shows that siRNA against Raf-1 specifically downregulates/inhibits Raf-1 expression in DCs. S2 shows that Raf-1 inhibition by inhibitor GW5074 or siRNA also inhibits HIV-1 replication, since HIV-1 infection of monocyte derived DCs as well as primary DC subsets leads to tat-rev mRNA, but inhibition of Raf-1 abrogates transcription.

Figure 2 shows IFN-I responses to abortive HIV-1 RNA are mediated by DDX3-MAVS

(mitochondrial antiviral signaling protein)

In order to identify the mechanism involved in inducing type I IFN responses upon block of Raf-1, we treated DCs with different HIV-1 inhibitors and our data show that reverse transcription and integration is crucial for the induction of type I IFN responses, since both reverse transcription inhibitor AZT and integrase inhibitor raltegravir (RAL) block IFN expression when DCs are infected by HIV-1 in presence of Raf inhibitor GW5074 (A). These data suggest that transcription after integration is involved in sensing. Next we identified the RNA sensor involved. Monocyte -derived

DCs were treated with siRNA against different known or putative RNA binding proteins and our data show that DDX3 silencing by siRNA abrogates type I IFN responses (B). DDX3 signals via the known adaptor protein MAVS as shown in (C), where MAVS silencing by siRNA abrogates type I IFN responses. Mean ± SD; n = at least 2 (A-D). * p < 0.05 (Student's Mest). See also Figures SI and S2 for the silencing levels and for the effect of the different molecules on HIV-1 transcription.

Next we investigated whether MAVS and DDX3 interact and our data show that DDX3 associates with MAVS only upon treatment of monocyte-derived DCs with Raf inhibitor before HIV-1 infection (D). DCs were treated with Raf inhibitor and infected with HIV-1. After 3 h DCs were lysed and either MAV or DDX3 were immunoprecipitated (IP) with a specific antibody as denoted below immunoblot. Next IP was analysed (stained) by immunoblotting (IB) with either DDX3 or MAVS specific antibodies as shown at richt side of blot. (D) β-actin served as loading control. Representative of 2 independent experiments. (E) Cellular localization of DDX3 and mitochondria (MitoTracker) in DCs 3 h after HIV-1 infection, as determined by confocal microscopy. Right graphs show fluorescence intensity along marker (4 μιη). Representative of 3 independent experiments.

(F) Analysis of m⁷GTP cap-binding translation initiation complexes in DCs 3 h after HIV-1 infection, as determined by immunoblotting. Cap analogue m7GpppG was used as a competitor, β-actin served as loading control. Representative of 3 independent experiments.

(G) Binding of DDX3, MAVS and eIF4G to abortive HIV-1 RNA and elongated HIV-1 mRNAs in DCs 3 h after HIV-1 infection, with or without Raf-1 inhibition, as determined by RNA

immunoprecipitation (IP) and real-time PCR. IgG and no Ab indicate negative controls. Mean ± SD; n = 3. * p < 0.05 (Student's Mest).

Figure 3 shows HIV-1 infection interferes with DDX3 signaling by blocking TRAF3 recruitment to MAVS and subsequent ΤΒΚΙ/ΙΚΚε-dependent IRF3 activation

(A, C) IFN mRNA expression by DCs 4 h after infection with HIV-l_BaL, in the absence or presence of Raf inhibitor GW5074, and after silencing of TRAF3 (A), TBK1, ΙΚΚε or IRF3 (C), as determined by real-time PCR. Mean ± SD; n = 2 (A), (C). * p < 0.05 (Student's Mest). See also Figures SI and S2.

(B) Association between DDX3/MAVS and TRAF3 in DCs 3 h after HIV-1 infection, with or without Raf-1 inhibition, and after silencing of TRAF3, as determined by immunoblotting (IB) after immunoprecipitation (IP). Representative of 2 independent experiments.

(D) Serl72 phosphorylation of TBK1 and ΙΚΚε in DCs 3 h after HIV-1 infection, with or without Raf- 1 inhibition, and after silencing of DDX3 or MAVS, as determined by flow cytometry. FI, fluorescence intensity; FSC, forward scatter. Representative of 3 independent experiments.

(E) IRF3 translocation from cytoplasm (CE) to nucleus (NE) in DCs 3 h after HIV-1 infection, with or without Raf-1 inhibition, and after silencing of DDX3 or MAVS, as determined by immunoblotting. β- actin and RNA polymerase II (RNAP2) served as loading controls. Representative of 3 independent experiments. Figure 4 shows Raf-1 -dependent PLKl activation during HIV-1 infection via MST1 impedes TRAF3 recruitment to DDX3-MAVS and IFN-I responses

Here we investigated how Raf-1 activation by HIV-1 leads to suppression of MAVS signaling. PLKl was suggested in literature to be involved in regulation of MAVS signaling. Therefore, we inhibited PLKl by siRNA against PLKl and our data show that inhibition of PLKl in monocyte -derived DCs leads to strong induction of IFN expression after HIV-1 infection as determined by real time PCR (A, B). Raf-1 inhibition did not further affect type I IFN responses after PLKl silencing, suggesting that Raf-1 and PLKl are in the same pathway (A, B) IFN mRNA expression by DCs after infection (4 h in A, ) with HIV-l_BaL, in the absence or presence of Raf inhibitor GW5074, and after silencing of

PLKl (A, B) or MST1 (K), as determined by real-time PCR. Mean ± SD; n = at least 2 (A), (B), (C). * p < 0.05 (Student's Mest). See also Figures SI and S2.

Next we investigated how PLKl blocks MAVS silencing and our data show that activation of PLKl leads to binding and association of PLKl to MAVS suggesting that PLKl blocks at the level of MAVS activation, as TRAF3 recruitment, upon HIV-1 infection is blocked when PLKl associates with MAVS(C, D, J) Association between DDX3/MAVS and PLKl (C, J) or TRAF3 (D, J) in DCs 3 h after HIV-1 infection, with or without Raf-1 inhibition (C), and after silencing of MAVS (C), PLKl (D) or MST1 (J), as determined by immunoblotting (IB) after immunoprecipitation (IP).

Representative of 2 independent experiments.

Phosphorylation of Thr210 on PLKl is a measure for activation of PLKl. We investigated whether HIV-1 activates PLKl by measuring phosphorylation of Thr210 residue using specific antibodies. Both HIV-1 and gpl20 induce phosphorylation of PLKl at Thr210, which is abrogated by Raf inhibition, underscoring role for Raf-1 in activating PLKl (E, F, I). Our data suggest that PLKl is directly activated by MST1 which is a downstream kinase of Raf-1. Therefore we investigated Mstl involvement and our data show that inhibition of Mstl aborgates activation of PLKl as measured by loss of Thr210 phosphorylation (I). Moreover, in vitro kinase assay shows that recombinant PLKl is directly phosphorylated at Thr210 by recombinant MST1 but not by Raf-1 (G). We also measured Mstl kinase activity during HIV-1 infection and our data show that infection of DCs with HIV-1 leads to activation of Mstl as measured by ELISA over time (H), whereas activity was inhibited by blocking Raf-1 or DC-SIGN using a specific antibody against DC-SIGN. Representative of 3 (E), 3 (F), 3 (I) independent experiments.

Finally, we silenced Mstl in DCs and infected these DCs with HIV-1 to investigate type I IFN responses. Our data show that silencing of MST1 similar as observed for PLKl, leads to an induction of type I IFN responses that are not further affected by Raf inhibition (H). MST1 activation in DCs after HIV-1 infection, with or without Raf-1 and DC-SIGN inhibition, as determined by kinase activity assay. These data show that HIV-1 infection leads to Raf-1 activation, which triggers Mstl and subsequently PLKl. PLKl binds to MAVS and thereby prevents type I IFN responses. Abrogation of the RAF-1, Mstl, PLKl pathway leads to induction of type I IFN responses (see also fig 7) upon HIV- 1 infection. Mean ± SD; n = 4. * p < 0.05 (Student's Mest).

Figure 5 shows that IFN-I responses by DCs suppress HIV-1 replication in infected individuals PLKl inhibition prevents HIV-1 suppression and leads to IFN and type I IFN responses upon HIV-1 infection. Type I IFN responses are strongly antiviral and can inhibit viral replication. Here we investigated whether type I IFN responses induced upon PLKl inhibition affected HIV-1 infection of monocyte-derived DCs. (A).PLKl was inhibited in monocyte derived DCs by siRNA and these DCs or untreated DCs were infected with HIV-1 and infection was measured at different days by measuring HIV-1 content in the cell by detecting the viral protein p24. Strikingly, control siRNA treated DCs were efficiently infected by HIV-1, with infection levels reaching 40% but PLKl inhibition led to s strong suppression of HIV-1 replication at around 10%. Next we inhibited type I IFN responses (that are induced by IFN binding to the INF receptor) by using a specific antibody against the IFNa/β receptor. Our data show that preventing type I IFN responses by inhibiting IFNa/β receptor signaling restores HIV-1 infection of DCs in presence of PLKl inhibition to normal levels around 40%. These data show that indeed PLKl inhibition leads to type I IFN responses that suppress HIV-1 replication in DCs. (A) HIV-1 infection of DCs (% p24⁺) at 3, 5 and 7 days post-infection, with or without IFNR inhibition, and after PLKl silencing (A) or in DCs expressing either wild-type MAVS (homozygous for major alleles of rs7262903/rs7269320) or dual MAVS K198/F409 mutant (homozygous for minor alleles) (D), as determined by flow cytometry. Mean ± SD of duplicates; representative of 2 (A), (D) independent experiments.

(B) We have identified a MAVS genotype that is insensitive to PLK1 inhibition (see Figure 6). The genotype consists of linked MAVS SNPs rs7262903/rs7269320. Homozygous for these SNPs are referred to as minor genotype, heterozygous for both alleles are called Hz and major is homozygous for the normal PLK1 sensitive alleles. First we investigated in the HIV-1 cohort of Men having sex with men (MSM) from Amsterdam cohort studies on HIV-1/AIDS whether untreated HIV-1 infected individuals with the minor genotype have a different viral load than those with the major genotype. One of the important markers for viral load and how well patients are controlling HIV-1 infection is the so-called viral load at set point, which is the stabilized viral load in serum after infection, which is tat the time that the immune response and replication is balanced. A low viral load is an indication that the patient is controlling HIV-1 replication better via efficient antiviral adaptive immunity. Notably, our data show that the viral load at setpoint in patients with the minor genotype is significantly lower than those we with the major and HZ genotype 1 RNA levels (log₁₀ viral copies/ml plasma) at set point in untreated HIV-1 -infected individuals in a human MSM cohort, differentiated based on genotype of linked MAVS SNPs rs7262903/rs7269320 (Minor, homozygous for minor alleles; Hz, heterozygous for both alleles; Major, homozygous for major alleles). P values calculated using unpaired Student's Mest. (B). Next we addressed the development of the HIV-1 infection by comparing viral load levels over time as a measure for virus replication and control by immune responses in the patients. In untreated HIV-1 infected patients, the viral load increases over time because the immune system is less able to counteract HIV-1 replication, partly due to exhaustion. Our data show that the viral load of the HIV-1 patients with the minor allele remains constant over a much longer time period and progresses significantly less than those with the major or HZ genotype (C). - Kaplan-Meier survival curve comparing the time required for log₁₀ viral copies/ml plasma to reach >4.5 (VL, viral load) from seroconversion (SV), in untreated HIV-1 -infected individuals in a human MSM cohort, differentiated on genotype as in (B). P value calculated using Cox regression model. (D) Next we investigated HIV-1 infection in DCs from either individuals with the minor or major allele. Infection was measured by p24 analysis and determining amount of infected DCs. Monocyte- derived DCs from healthy individuals with the minor genotype behaved the same as PLK1 inhibited DC (See A). DCs with minor genotype were less infected by HIV-1 (around 10%) and this was dependent on type I IFN responses, as inhibition of IFNa/β receptor by antibodies enhanced infection to levels observed in DCs from healthy individuals with the major genotype. These data underscore the physiological relevance of the PLK1 pathway as individuals that are insensitive to the PLK1 pathway due to two MAVS polymorphisms are better at controlling HIV-1 replication as shown at viral set point as well as during progression of HIV-1 viral load over time. It is well accepted in literature that the viral load at set point and HIV-1 progression is controlled by the adaptive immune responses, further emphasizing the importance of preventing HIV-1 of suppressing type I IFN responses and DC maturation and cytokine production.

Figure 6 shows that PLKl-mediated suppression of DDX3 signaling after HIV-1 infection is impeded by mutation of MAVS

A) IFN mRNA expression by DCs with either major or minor genotype for MAVS SNPs

rs7262903/rs7269320 6 h after poly(I-C)-LyoVec, as determined by real-time PCR. Mean ± SD; n = 2. * p < 0.05 (Student's Mest). Here we investigated whether DCs from healthy individuals with minor genotype were indeed not affected by PLK-1 inhibition. Our data show that DCs with minor genotype induce strong type I IFN responses (both IFN as well as IFN-inducing genes) upon infection and this is independent of inhibition of Raf as expected (B-E, G) IFNp (B, D, E, G) and ISG (C) mRNA expression by DCs (primary dermal DCs in D) with either heterozygous (Hz) or minor

rs7262903/rs7269320 genotypes, after infection (4 h in D, E, G) with HIV-l_BaL, in the absence or presence of Raf inhibitor GW5074, and after silencing of DDX3, MAVS (E) or PLK1 (G), as determined by real-time PCR. Mean ± SD; n = 3 (B), (C), (D), (E), (G). * p < 0.05 (Student's Mest). See also Figures SI, S2 and S3.

(F) Association between DDX3/MAVS, TRAF3 and PLK1 in DCs with minor rs7262903/rs7269320 genotype 3 h after HIV-1 infection, with or without Raf-1 inhibition, as determined by

immunoblotting (IB) after immunoprecipitation (IP), β-actin served as loading control. Representative of 2 independent experiments. (H) Thr210 phosphorylation of PLK1 in DCs with minor rs7262903/rs7269320 genotype 3 h after HIV-1 infection, with or without Raf-1 or DC-SIG inhibition, as determined by flow cytometry. Representative of 3 independent experiments.

(I) Association between Flag-PLKl and wild-type MAVS (DC lysate major genotype) or dual MAVS K198/F409 mutant (DC lysate minor genotype), as determined by ELISA.

Figure 7 shows that HIV-1 infection attenuates DDX3/MAVS-mediated DC maturation and IL-Ιβ expression

Here we investigated how DC function after HIV-1 infection when we prevented Raf-1 -Mstl-PLKl suppression. DCs are vital to the induction of adaptive immunity to pathogens (Textbook Janeway' s Immuno Biology, 8^th Edition by Kenneth Murphy, GS Garland Science (2012)). DCs activation is a crucial step in this process and is measured by determining DC maturation as measured by upregulation of proteins CD80, CD83 and CD86. Furthermore, DCs producing type I IFN responses and cytokines are more efficient in inducing adaptive immunity (textbook). Without induction of cytokines, DCs are not able to induce adaptive immunity.

First we investigated DC activation as measured by expression of maturation markers. Monocyte- derived DCs from either major (A,B) or minor (C) genotype were infected with HIV-1 and by flow cytometry expression of the proteins CD80, CD83 and CD86 was measured. The role of type I IFN responses on DC maturation was investigated using the blocking antibody against IFNa/β receptor. Our data show that HIV-1 infection of DCs with major genotype does not lead to induction of DC maturation, since no increase in expression of CD80, CD83 and CD86 is observed (A, B). Inhibition of Rafl -Mstl-PLKl pathway by blocking Raf or blocking PLK1 (B) leads to strong induction of maturation upon HIV-1 infection and this is dependent on type I IFN responses since the antibodies against IFNa/β receptor block maturation (B). Furthermore, silencing of DDX3, MAVS also abrogate maturation as these are the signaling molecules that induce type I IFN responses. These data show that inhibition of the Raf-1 -Mstl-PLKl pathway is crucial in the induction of DC maturation upon HIV-1 infection. (C) here we investigated how DC maturation was induced in DCs with minor genotype. Similar as the PLK1 inhibited DCs, DCs with the minor genotype become activated as shown by upregulation of CD80, CD83 and CD86, upon HIV-1 infection, independent of Rafl-Mstl-PLKl pathway (C). (A-C) Expression of maturation markers CD80, CD83 and CD86 on DCs with either major (A, B) or minor (C) rs7262903/rs7269320 genotypes, 48 h after infection with HIV-l_BaL, in the absence or presence of Raf inhibitor GW5074, with or without IFNR inhibition (A, C), and after silencing of DDX3, MAVS or PLK1 (B), as determined by flow cytometry. FI, fluorescence intensity. Representative of 3 independent experiments.

Next we investigated cytokine responses and we focused on IL-Ι β since this is a crucial cytokine involved in immune activation (text book). Inhibition of the Raf-l-Mstl-PLKl pathway by GW5074 or siRNA against PLK1 lead to a strong IL-Ιβ mRNA as well as IL-β protein expression upon HIV-1 infection of DCs with major genotype. HIV-1 infection of DCs with the minor genotype also induced a strong expression of IL-1 β (H). (D-H) IL-1 β mRNA (D, F-H) and protein (E) expression by DCs with either major (D-G), heterozygous (Hz) or minor (H) rs7262903/rs7269320 genotypes, after HIV- 1 infection (48 h in E; 10 h in F, G), with or without Raf-1 inhibition, with or without IFNR inhibition (G), and after silencing of DDX3, MAVS or PLK1 (F), as determined by real-time PCR (D, F-H) and ELISA (E). Mean ± SD; n = at least 2 (D), (E), (F), (G), (H). * p < 0.05 (Student's Mest). These data underscore the importance of suppressing Raf-1 -Mstl-PLKl signaling to enhance DC maturation, type I IFN responses and cytokine responses. The natural MAVS allele that renders MAVS insensitive to Raf-1 -Mstl-PLKl inhibition is the in vivo evidence that early inhibition of Raf-1 -Mstl-PLKl leads to induction of adaptive immunity to HIV-1 and can be used to prevent or limit HIV-1 infection.

Supplementary Figure SI shows silencing of protein expression in human DCs by RNA interference (A-M). Silencing of indicated proteins using specific siRNAs and non-targeting siRNA as a control was confirmed by real-time PCR (left panels; mean ± SD, n > 7) or flow cytometry (right panels; FI, fluorescence intensity; representative of at least 3 independent experiments).

Supplementary Figure S2 shows HIV-1 replication in monocyte-derived and primary DCs, after inhibition of HIV-1 reverse transcription, HIV-1 integration, DC-SIGN and/or DDX3 signaling pathways.

(A-I) Viral Tat-Rev (A-I) and Vpu (H) mRNA expression by monocyte-derived (A, B, D-I) and primary (C) DCs after infection (4 h in C-H, Tat-Rev; 10 h in H, Vpu) with laboratory strain HIV-l_BaL (A-C, F-I), various CCR5- and CXCR4-tropic HIV-1 strains (D) or primary HIV-1 (E), in the absence or presence of Raf-1 inhibition (GW5074 in A, C-I; Raf-1 silencing (siRNA) in B), reverse transcription inhibitor AZT or integrase inhibitor raltegravir (RAL) (F), and after silencing of indicated proteins (G, H), as detemined by real-time PCR. In (I), DCs express either both wild-type and mutant MAVS (Hz, heterozygous for rs7262903/rs7269320 alleles) or only dual MAVS

K198/F409 mutants (Minor, homozygous for minor alleles). N.d., not determined. Mean ± SD; n = 6 (A, G; I, Hz), 4 (B, E), 2 (C, myeloid, vaginal, intestinal DCs), 3 (C, dermal DCs; H, Vpu; I, Minor), 6 (D), 7 (F), 8 (H, Tat-Rev). ** p < 0.01, * p < 0.05 (Student's Mest).

Supplementary Figure S3 shows that IFN-I responses are suppressed in DCs via Raf-1 after HIV-1 infection. ISG mRNA expression by DCs heterozygous (Hz) rs7262903/rs7269320 genotypes, after infection with HIV-l_BaL, in the absence or presence of Raf inhibitor GW5074, as determined by realtime PCR. Mean ± SD; n = 6. ** p < 0.01, * p < 0.05 (Student's Mest).

SUMMARY

The invention is based on the surprising finding that inhibitors are capable of overcoming the immune suppression by retroviruses.

The inventors have found that the RAF-l/Mst-l/PLKl/MAVS pathway suppresses antiviral immune responses after HIV infection of dendritic cells (DCs). It is common general knowledge that DCs are essential in the induction of strong antiviral adaptive immune responses to viral infections such as

HIV-1 (Steinman Annu Rev Immunol 2012, ;30:l-22; Textbook Janeway' s Immuno Biology, 8^th

Edition by Kenneth Murphy, GS Garland Science (2012)). It is also evident that HIV-1 infection does not lead to efficient antiviral immunity and several studies indicate that this is due to the lack of DC activation upon HIV-1 infection, since HIV-1 infection of DCs does not induce type I IFN responses

(IFN-I), cytokine responses nor DC maturation [Miller and Bhardwaj 2013; Manel et al. Nature 467,

214-217 (2010)].

The inventors have in the example of the description confirmed that suppression of antiviral IFN-I and cytokine responses as well as DC maturation play an essential role in the escape mechanism of retroviruses from effective antiviral immune responses, and this prevents the host immune system from clearing viral infection. This escape mechanism has thus far hampered the successful development of immune therapies against HIV and other retroviruses. The inventors herein provide in vitro and in vivo evidence that inhibitors of RAF-1 and/or its downstream targets Mstl and PKLl can overcome this suppression and result in a suppression of viral replication.

The inventors have shown that HIV-1 infection of monocyte-derived DCs does not lead to DC maturation nor IFN-I and cytokine induction (see Figure 1 and Figure 7A, B, D, E and G of the description), which confirms the view that the escape mechanism of HIV exerts its effect via the DCs. Surprisingly, the inventors have now found that this escape mechanism can be inhibited using specific compounds.

The inventors in the example show that this suppression of immune responses by HIV-1 can be overcome using inhibitors of RAF-1, Mstl or PLK1 and that this is sufficient to suppress or block viral replication. First, they have shown the RAF-1, Mstl and PLKl kinases are essential in the signaling pathway induced by HIV-1 that disrupts the DC maturation and activation. The inventors have shown that inhibition of any of the RAF-1 /Mstl /PLKl kinases in the signaling pathway induced by HIV-1 disrupts immune suppression and thereby leads to DC maturation and activation. The inventors have shown that RAF-1 activation by HIV-1 envelope glycoprotein gpl20 induces phosphorylation and activation of kinase Mstl (Figure 4H), and Mstl subsequently phosphorylates and thereby activates PLKl (Figure 4E, F, G, I). Inhibition of either RAF-1 by small molecule inhibitor GW5074 or small interfering RNA, or inhibition of Mstl using small interfering RNA abrogates Mstl and PLKl activation (Figure 4E, F, G, H, I). Indeed, HIV-1 infection of monocyte-derived DCs as well as primary human DCs isolated from intestine, vagina, blood and skin treated with the RAF-1 inhibitor GW5074 induces the expression of IFN-I in HIV-1 infected DCs (Figure 1), leading to a strong immune activation, similar to use of siRNA against RAF-1, PLKl and Mstl (Figure 4A, B, K). This proofs that inhibitors of RAF-1 can overcome the immune suppression by HIV. Since RAF-1 activation is required for activation of Mstl, which subsequently activates PLKl as shown by the inventors (in Figure 4), inhibitors of Mstl and PLKl also prevent the RAF-l/Mstl/PLKl pathway and therefore also overcome the immune suppression by HIV. The inventors have shown that RAF- l/Mstl/PLKl pathway activated by HIV-1 blocks MAVS function (Figure 4C, D, J). It is well known that MAVS activation leads to IFN-I and cytokine responses (Textbook Janeway's Immuno Biology, 8^th Edition by Kenneth Murphy, GS Garland Science (2012)). The inventors show that HIV-1 infection leads to MAVS -dependent DC maturation and activation (IFN-I and cytokines responses) (Figure 3). Thus, RAF-l/Mstl/PLKl pathway induces binding of PLKl to MAVS, which prevents MAVS signaling, as shown by inhibition of TRAF3 recruitment to MAVS (Figure 4C,D,J) and lack of IFN-I responses (Figure 1 and Figure 3). Inhibitors of RAF-1 /Mstl /PLKl pathway block PLKl activation and thereby prevent inhibition of MAVS activation, leading to MAVS activation and IFN-I responses (Figure 4C, D, J and Figure 3).

Notably, the inventors have identified a MAVS genotype consisting of linked MAVS Single

Nucleotide Polymorphism (SNP) rs7262903/rs7269320 that renders MAVS insensitive to PLKl inhibition (Figure 6). as infection of DCs from healthy donors with this MAVS polymorphism, indicated as the minor genotype, leads to strong IFN-I responses as well as cytokine responses and DC maturation even without inhibiting RAF-l/Mstl/PLKl pathway (Figure 6). This MAVS genotype therefore mimics the effects of inhibition of the RAF-l/Mstl/PLKl pathway.

The inventors have further provided in vivo and in vitro evidence which makes it plausible that treatment of patients with small molecule inhibitors or specific siRNAs against the RAF- 1/Mstl/PLKl/MAVS pathway results in suppression of viral replication in HIV-1 infected patients via induction of strong antiviral immune responses. Using blood samples from healthy donors with the wild-type MAVS (major genotype), the inventors have isolated DCs and infected said DCs with HIV- 1 in the presence of several inhibitors of the RAF-l/Mstl/PLKl/MAVS pathway. In Figure 5A, it is shown that the percentage of HIV-1 infected DCs is greatly reduced in the presence of an inhibitor of PLK1 (a specific siRNA for PLK1). A similar effect is shown in Figure 5D. In the example, the percentage of HIV-1 infected DCs is greatly reduced in isolated DCs from healthy donors with this homozygous MAVS mutant ("minor") compared to donors being heterozygous or carrying the wild type MAVS alleles ("major"). This homozygous MAVS mutant ("minor") abolishes the signaling of the RAF-l/Mstl/PLKl/MAVS pathway. Therefore, the presence of the MAVS mutant mimics the therapeutic effects of treatment using inhibitors of the RAF-l/Mstl/PLKl/MAVS pathway.

The inventors in the example also show that when the RAF- 1/Mstl/PLKl /MAVS signaling pathway is suppressed in DCs, this has a therapeutic effect in HIV infected individuals. Importantly, HIV-1 - infected individuals homozygous for the minor alleles of the "minor" genotype as discussed above, were shown to have in vivo a plasma viral load at set point which was significantly lower compared to individuals homozygous for the major alleles (major genotype) (pi = 0.044; Student's Mest), while no differences were detected between individuals with heterozygous and major genotypes (p2 = 0.215; Student's Mest) (Figure 5b). This proofs that that disruption of signaling of the RAF- 1/Mstl/PLKl/MAVS pathway has indeed a direct therapeutic effect on viral load. Moreover, they observed in these patients a significant delay in time from seroconversion to viral RNA load > 10⁴⁵ copies/ml plasma (pi = 0.025, RH = 0.106, 95% CI 0.015-0.759; Cox regression) (Figure 5c). These data signify the importance of the RAF-l/Mstl/PLKl/MAVS pathway in controlling HIV-1 replication. In the patients with the minor genotype, HIV-1 infection leads to DC maturation and activation (Figure 6) which strongly decreases HIV-1 replication at both the acute and chronic phase of the disease. . It also shows that disruption of signaling of the RAF-l/Mstl/PLKl/MAVS pathway is the key to overcome the immune escape from retroviruses.

The inventors have performed comparative experiments to show that the effect of inhibition by inhibitors of the RAF-l/Mstl/PLKl/MAVS pathway on HIV-1 infection of DCs is completely comparable to the effect of the genetic mutant (the "minor" genotype) which abolishes signaling of the RAF-l/Mstl/PLKl/MAVS pathway. Therefore, it is plausible that inhibitors of the RAF- 1/Mstl/PLKl kinases have a therapeutic effect. HIV-1 infection of monocyte-derived DCs treated with small interfering RNA (siRNA) against RAF-1, Mstl or PLKl leads to strong immune activation of DCs (Figure 1, 4 and 7). The same results are obtained when DCs derived from healthy donors carrying the homozygous MAVS mutant ("minor") alleles were infected with HIV-1. In Figure 6B, C, D, E, the inventors show that HIV-1 infection of monocyte-derived DCs as well as primary DCs isolated from donors with the minor allele leads to strong type I IFN responses (both IFN as well as IFN-inducing genes) and this is independent of inhibition of RAF-1 /Mstl /PLKl pathway. This too resulted in a strong immune activation of DCs. In Figure 7C, the inventors show that HIV-1 infection of DCs from donors with the minor genotype leads to DC maturation as shown by upregulation of CD80, CD83 and CD86. In contrast, in Figure 7A-B, the inventors show that HIV-1 infection of DCs from donors with the major genotype does not lead to DC maturation; when RAF-1 /Mstl /PLKl pathway is inhibited these DCs mature (Figure 7A,B). This supports the view that any inhibitor of RAF-1, Mstl or PLKl has a clinical effect comparable to the effect of homozygous MAVS mutant ("minor") of viral load and viral replication.

The inventors have further shown that HIV-1 infection of DCs from donors homozygous for the MAVS mutant have the same phenotype as DCs treated with the small interfering RNA against PLKl (Figure 6). Therefore, it may be concluded that is plausible that effects on viral load and viral replication in the MAVS mutant will also be achieved by inhibitors of RAF-1, Mstl and PLKl. T In conclusion, the inventors have shown the following:

A) DCs are not stimulated upon HIV-1 infection. B) DCs are stimulated, mature and secrete IFN-I and cytokines in the presence of RAF- 1/Mstl/PLKl inhibitors upon HIV-1 infection

C) DCs are stimulated upon HIV-1 infection when MAVS is insensitive to RAF- 1/Mstl /PLKl due to a mutation ("minor" genotype)

D) Patients carrying the "minor" alleles which renders MAVS insensitive to RAF- 1/Mstl /PLKl have in vivo a significant lower viral load and control virus replication better than those with the major genotype that is sensitive to RAF-l/Mstl/PLKl

E) Patients carrying the "minor" alleles which renders MAVS insensitive to RAF- 1/Mstl /PLKl have in vivo a significant delay in time from seroconversion to viral RNA load > 10^{4 5} copies/ml plasma

F) Inhibition of any of the RAF- 1/Mstl /PLKl kinases prevents HIV-1 suppression of MAVS signaling.

G) DCs treated with inhibitors of the RAF-l/Mstl/PLKl/MAVS pathway have the same

phenotype as DCs isolated from healthy donors carrying the MAVS mutant (minor genotype)

In conclusion, these cohort in vivo data together with the PLKl inhibition data strongly support the invention that inhibitors of the RAF- 1/Mstl /PLKl pathway are a novel kind of drug that allow effective control or even inhibition of retroviruses by inducing strong efficient antiviral immune responses to retroviruses. These studies also underline that the inhibitors of invention are most effective when using prior infection (prophylactic) or just after infection before seroconversion, since inhibition of the RAF- 1/Mstl /PLKl pathway is required to boost antiviral immunity induced by DCs, the first step required for any effective immune response.

US2009/0010941 Al is concerned with the treatment of HIV and described M-CSF effector kinase inhibitors for use in such treatment. The difference between the invention and US2009/0010941A1 is that inhibitors of the PLK1/MST1/RAF1 pathway are provided. PLK1/MST1/RAF1 inhibitors are therefore an alternative to M-CSF effector kinase inhibitors.

The invention therefore provides a compound selected from the group consisting of:

(i) an inhibitor of RAF-1,

(ii) an inhibitor of the mammalian Ste20-like 1 (MST1), and (iii) an inhibitor of the Polo-like 1 (PLK1)

for use in a treatment of a patient infected with a retrovirus or for prevention of infection with a retrovirus, with the proviso that said compound is not an M-CSF antagonist. Said M-CSF antagonist may be selected from: Gleevec® (Imatinib mesylate; ST1571); Nilotinib; Dasatinib; Sorafenib;

Sunitinib; GW2580, 5-{3-methoxy-4-[(4methoxybenzyl)oxy]benzyl}pyrimidine-2,4-diamine; ABT- 869; AG013736; BAY 43-9006; CHIR258; and SU11248. Preferably, said treatment is given before seroconversion.

DCs are the first cells to encounter HIV-1 and DC maturation at time of infection is paramount to induce strong adaptive antiviral immunity, inhibiting the RAF-l/Mstl/PLKl pathway at the moment of infection (before seroconversion) or even before infection (prophylaxis) allows strong induction of DC maturation and immune activation upon HIV-1 infection, which leads to lower viral replication due to effective antiviral immunity.

In a preferred embodiment, said RAF-1 inhibitor is selected from the group consisting of GDC-0879, PD-173955, PLX-4720, CHIR-265, R406, motesanib, pazopanib, AST-487, SB203580, barasertib- hQPA, cediranib, selumetinib, BI-2536, afatinib, doramapimod, BMS-345541, BMS-387032, brivanib, lestaurtinib, canertinib, CI-1040, tofacitinib, alvocidib, pictilisib, GSK-1838705A, GSK- 461364A, neratinib, ruxolitinib, JNJ-28312141, Ki-20227, KW-2449, lapatinib, enzastaurin, MLN- 120B, tandutinib, MLN-8054, PHA-665752, midostaurin, dovitinib, crizotinib, erlotinib, gefitinib, GSK690693, ruboxistaurin, A-674563, masitinib, linifanib, quizartinib, axitinib, AT-7519, tozasertib, VX-745, staurosporine, PP-242, vatalanib, R547, SGX-523, bosutinib, SU-14813, sunitinib, NVP- TAE684, TG-100-115, fedratinib, vandetanib, PI-103, foretinib, vemurafenib, CAS No. 918504-65-1, AZ628, Cas No. 878739-06-1, NVP-BHG712, CAS No. 940310-85-0, RAF265 (CAS No. 927880-90- 8), 2-Bromoaldisine (CAS No. 96562-96-8), Raf Kinase Inhibitor IV (CAS No. 303727-31-3), L- 779,450 (CAS No. 303727-31-3), PLX4032, GSK2118436, BMS-908662 (XL-281), RG-7256

(RO5212054, PLX3603), R05126766, ARQ-736, E-3810, DCC-2036, GW5074, AZ628, an anti- RAF-1 antibody, and an inhibitory RAF-1 RNA molecule.

In another preferred embodiment, said Mstl inhibitor is selected from the group consisting of staurosporine, foretinib, bosutinib, KW-2449, crizotinib, NVP-TAE684, cediranib' , AST-487, erlotinib, R406, lestaurtinib, sunitinib, Ki-20227, neratinib, tozasertib, PP-242, vandetanib, R547, doramapimod, brivanib, midostaurin, pazopanib, dovitinib, PHA-665752, ruboxistaurin, linifanib, SU- 14813, CHIR-265, fedratinib, JNJ-28312141, gefitinib, axitinib, GSK-461364A, GDC-0879, motesanib, canertinib, mxolitinib, pictilisib, BMS-387032, BMS-345541, GSK-1838705A, SGX-523, CI-1040, alvocidib, masitinib, A-674563, TG-100-115, GSK690693, VX-745, MLN-120B, tandutinib, MLN-8054, PI-103 , selumetinib, barasertib-hQPA, vatalanib, tofacitinib, enzastaurin, lapatinib, SB203580, afatinib, PD-173955, BI-2536, quizartinib, PLX-4720, AT-7519, an anti- Mammalian STE20-like kinase antibody, and an inhibitory Mammalian STE20-like kinase RNA molecule.

In another embodiment, said inhibitor of PLK1 is selected from the group consisting of Wortmannin, vatalanib, vandetanib, tozasertib, tofacitinib, tandutinib, sunitinib, staurosporine, sphingosine kinase inhibitor, semaxanib, seliciclib, purvalanol A, pazopanib, p38 MAP kinase inhibitor III, p38 MAP kinase inhibitor, mubritinib, midostaurin, masitinib, lapatinib, kenpaullone, isogranulatimidem, indirubin-3'-monoxime, indirubin derivative E804, herbimycin A, gefitinib, fasudil, fascaplysin, erlotinib, dovitinib, dorsomorphin, diacylglycerol kinase inhibitor II, compound 56 [PMID:8568816], compound 52 [PMID:9677190], chelerythrine, casein kinase II inhibitor III, casein kinase I inhibitor, bosutinib, bohemine, bisindolylmaleimide IV, aurora kinase/Cdk inhibitor, aurora kinase inhibitor III, aminopurvalanol A, alsterpaullone 2-cyanoethyl, alsterpaullone, ERK inhibitor II, PP1 analog II, p38 MAP kinase inhibitor, mubritinib, vandetanib, AG 1296, EGFR inhibitor, ERK inhibitor III, GSK- 3beta inhibitor II, H-89, KN-93, JAK3 inhibitor II, PP3, lapatinib, Syk inhibitor III, Flt-3 inhibitor, p38 MAP kinase inhibitor III, EGFR/ErbB-2/ErbB-4 inhibitor, pazopanib, herbimycin A, PKCbeta inhibitor, PKR inhibitor, CGP53353, MNK1 inhibitor, IC261, diacylglycerol kinase inhibitor II, erlotinib, Lck inhibitor, SB202190, indirubin-3'-monoxime, PI 3-Kg inhibitor, SB203580, SKF-86002, Flt-3 inhibitor III, alsterpaullone 2-cyanoethyl, SU6656, PDGF receptor tyrosine kinase inhibitor II, GF109203X, Flt-3 inhibitor II, tofacitinib, masitinib, tandutinib, IRAK-1/4 inhibitor, Rho kinase inhibitor IV, SB220025, VEGF receptor 2 kinase inhibitor IV, GSK-3 inhibitor IX, VEGF receptor tyrosine kinase inhibitor III, Akt inhibitor V, EGFR/ErbB-2 inhibitor, Ro-32-0432, AG 1295, Rho kinase inhibitor III, TGF-beta RI kinase inhibitor, GSK-3beta inhibitor VIII, ATM kinase inhibitor, VEGF receptor 2 kinase inhibitor II, PDGF receptor tyrosine kinase inhibitor IV, DMBI, SB 202474, GSK-3 inhibitor XIII, Go 6976, VEGF receptor tyrosine kinase inhibitor II, fasudil, indirubin derivative E804, SU9516, Go6983, DNA-PK inhibitor V, GSK-3beta inhibitor XII, SC-68376, semaxanib, aloisine, AG1478, TGF-beta RI inhibitor III, SU11652, GSK-3 inhibitor X, sphingosine kinase inhibitor, GTP-14564, dorsomorphin, VEGF receptor 2 kinase inhibitor I, Akt inhibitor VIII, Akt inhibitor X, ATM/ATR kinase inhibitor, an anti- PLKlantibody, and an inhibitory PLKl RNA molecule.

Said inhibitor is preferably an inhibitor of the mammalian Ste20-like kinase 1 (MST1), or an inhibitor of the Polo-like kinase 1 (PLKl), as these interfere more downstream in said pathway, which will have less side effects. Without wishing to be bound by theory, the inventors believe that inhibition of PLKl by silencing does not impede the signaling pathway via phosphorylation of p65 , which is important. It is believed that RAF-1 and MST1 inhibition impedes phosphorylation of p65, thereby affecting adaptive immunity, but allows induction of IFN-I responses, DC maturation and pro-inflammatory cytokine IL-lb expression. It is believed that phosphorylation of p65 as well as IFN-I responses, DC maturation and pro-inflammatory cytokines are required for the most optimal immune response to a retrovirus.

Most preferred is therefore an inhibitor of the Polo-like kinase 1 (PLKl). Preferably said inhibitor only interferes with retroviral suppression of MAVS signaling without affecting phosphorylation of p65 as observed with RAF-1 and Mstl inhibition. In a highly preferred embodiment, said inhibitor prevents the interaction of PLKl with MAVS. Without wishing to be bound by theory, the inventors believe that this interaction is required for the suppressive activation of PLKl on IFN-I responses. In a preferred embodiment, said PLKl inhibitor might prevent the interaction between PKL1 and MAVS, without altering other activities of PKL1.

The invention further provides a kit comprising a compound for use according to any of the above claims and further a compound suitable for prevention or treatment of an infection with a retrovirus for use in a treatment of a patient infected with a retrovirus or for prevention of infection with a retrovirus. Preferably, said further compound is selected from reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, fusion inhibitors, retroviral immunogens. The invention further provides a pharmaceutical composition for use in the treatment of a patient infected with a retrovirus or for prevention of infection with a retrovirus comprising a compound as defined above and a pharmaceutically acceptable carrier or excipient.

In all aspects of the invention, said treatment is preferably for stimulating and/or inducing an immune response to a retrovirus. Preferably, said treatment comprises stimulating and/or inducing I IFN responses and adaptive immunity against the retrovirus. Preferably, said treatment is given early during infection, preferably before seroconversion. In another preferred embodiment, said treatment is given during vaccination against the retrovirus.

In all aspects of the invention, said retrovirus is preferably HIV.

DETAILED DESCRIPTION

Definitions

As used herein, the term "retrovirus" refers to a virus having as its genetic material ribonucleic acid (RNA) which is transcribed into DNA which is inserted into the host genome. Examples of retroviruses include HTLV-I, HTLV-II STLV-I, and the lentivirus family including HIV, visna virus, equine infectious anemia virus, feline immunodeficiency virus and bovine immunodeficiency virus. These are described in Fauci, Science 239:617 (1988).

As used herein, the term " infection" generally encompasses infection of a host animal, particularly a human host, by a retovirus, including but not limited to the human immunodeficiency virus (HIV) family of retro viruses including, but not limited to, HIV-1 HIV-2, HIV-3 (a.k.a. HTLV- III, LAV-1, LAV -2), and the like. "HIV" can be used herein to refer to any strains, forms, subtypes, clades and variations in the HIV family. Thus, treating an infection with a retrovirus will encompass the treatment of a subject who is a carrier of any of the retroviruses mentioned above, but in particular the HIV family of retroviruses or a person who is diagnosed of active AIDS. A carrier of a retrovirus may be identified by any methods known in the art. For example, a person can be identified as HIV carrier on the basis that the person is anti-HIV antibody positive, or is HIV-positive, or has symptoms of AIDS. That is, "treating HIV infection" should be understood as treating a patient who is at any one of the several stages of HIV infection progression, which, for example, include acute primary infection syndrome (which can be asymptomatic or associated with an influenza-like illness with fevers, malaise, diarrhea and neurologic symptoms such as headache), asymptomatic infection (which is the long latent period with a gradual decline in the number of circulating CD4+ T cells), and AIDS (which is defined by more serious AIDS-defining illnesses and/or a decline in the circulating CD4 cell count to below a level that is compatible with effective immune function). In addition, "treating or preventing infection with a retrovirus" will also encompass treating suspected infection by the retrovirus after suspected past exposure to said retrovirus. In case a suspected infection by HIV, such exposure may include for example contact with HIV -contaminated blood, blood transfusion, exchange of body fluids, "unsafe" sex with an infected person, accidental needle stick, receiving a tattoo or acupuncture with contaminated instruments, or transmission of the virus from a mother to a baby during pregnancy, delivery or shortly thereafter. The term "treating or preventing an infection by a retrovirus" may also encompass a prophylactic treatment, including treating a person who has not been diagnosed as having a retrovirus infection but is believed to be at risk of infection by the retrovirus. As used herein, the term "RAF1" refers to a polypeptide or protein sequence of human origin, or homologs thereof, including allelic variants and orthologs. The protein and encoding nucleic acid sequences of RAF1 are publicly known and described including in the Genbank database. A protein sequence of RAF 1 is available under NCBI Reference Sequence XP_005265412.1.

The terms "mammalian sterile 20-like kinase-1", "Mstl" and any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to a polypeptides or protein sequences of human origin, or homologs thereof, including allelic variants and orthologs. The protein and encoding nucleic acid sequences of Mstl are publicly known. A protein sequence is available under Genbank accession AAA83254.1

As used herein, the term "PLK1" refers to a polypeptide or protein sequence of human origin, or homologs thereof, including allelic variants and orthologs. The protein and encoding nucleic acid sequences of PLK1 are publicly known and described including in the Genbank database. A protein sequence of PLK1 is available under NCBI Reference Sequence NP_005021.2.

As used herein, the term "MAVS" refers to a polypeptide or protein sequence of human origin, or homologs thereof, including allelic variants and orthologs. This gene encodes an intermediary protein necessary in the virus-triggered beta interferon signaling pathways. It is required for activation of transcription factors that regulate expression of beta interferon and contributes to antiviral immunity. Multiple transcript variants encoding different isoforms have been found for this gene. The protein and encoding nucleic acid sequences of MAVS are publicly known and described including in the Genbank database. A protein sequence of MAVS is available under for example under NCBI Reference Sequence NP_001193420.1 and UniProtKB/Swiss-Prot:Q7Z434.

The term "compound" refers generally to any entity which is normally not present or not present at the levels being administered to a cell, tissue or subject. A compound can be selected from a group comprising: polynucleotides; polypeptides; small molecules; antibodies; or functional fragments thereof.

As used herein, the term "inhibitor" refers to any suitable compound , which reduces the expression and/or activity of the gene product. The reduction in expression and/or activity can be, for example, by at least about 10%, e.g. by 10%) or more, 20% or more, 30% or more, 50% or more, 75% or more, 90% or more, 95% or more, 98%) or more, or 99% or more. The reduction of the expression and/or activity of the gene product is not intended to encompass complete inhibition or total reduction of the gene product's expression and/or activity.

In some aspects of all the embodiments, the reduction of the gene product's expression and/or activity can be a "complete reduction" or "complete inhibition", i.e. a 100%) reduction of the expression and/or activity of the gene product. In some aspects of all the inhibitor of the invention inhibits the expression level, e.g. mRNA and/or polypeptide expression product level of the gene. In preferred aspects of all the embodiments, an inhibitor can be a compound or that inhibits the activity of the gene product. In a preferred embodiment, said activity comprises the kinase activity.

Accordingly, an inhibitor may inhibit the activity of a protein that is encoded by a gene either directly or indirectly. Direct inhibition can be obtained, for instance, by binding to a protein and thereby preventing the protein from binding a target (such as a binding partner) or preventing protein activity (such as enzymatic activity). Indirect inhibition can be obtained, for instance, by binding to a protein's intended target, such as a binding partner, thereby blocking or reducing activity of the protein. The inhibitor includes compounds, which block gene expression, including transcription or translation, such as antisense nucleic acids, RNA interfering agents, siRNAs and ribozymes.

As used herein, the term "RAF1, Mstl, MAVS or PLK1 kinase inhibitor" refers to any compound, natural or synthetic, which results in a decreased kinase activity of said RAF1, Mstl, MAVS or PLK1.

The functionality of an PI K 1 inhibitor can be tested without undue burden by a skilled person using methods known in the art. High throughput methods have been described for testing PLK1 inhibitors (Wolfgang Reind et a!.: "A high-throughput assay based on fluorescence polarization for inhibitors of the polo-box domain of polo-like kinase 1", Volume 383, Issue 2, 15 December 2008, Pages 205- 209).

An "RNA interfering agent" as used herein, is defined as any agent which interferes with or inhibits expression of a target gene or genomic sequence by RNA interference (RNAi). Such RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to the target gene or genomic sequence, or a fragment thereof, short interfering RNA (siRNA), short hairpin or small hairpin RNA (shRNA), and small molecules which interfere with or inhibit expression of a target gene by RNA interference (RNAi). "RNA interference (RNAi)" is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target gene results in the sequence specific degradation or specific post- transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene (see Coburn, G. and Cullen, B. (2002) J. of Virology 76(18):9225), thereby inhibiting expression of the target gene. In one embodiment, the RNA is double stranded RNA (dsRNA). This process has been described in plants, invertebrates, and mammalian cells. In nature, RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes processive cleavage of long dsRNA into double- stranded fragments termed siRNAs. siRNAs are incorporated into a protein complex that recognizes and cleaves target mRNAs. RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silence the expression of target genes.

As used herein, a "siRNA molecule" is a duplex oligonucleotide, that is a short, double-stranded oligonucleotide, that interferes with the expression of a gene in a cell that produces RNA, after the molecule is introduced into the cell. Such molecules are constructed by techniques known to those skilled in the art. Such techniques are described in U.S. Pat. Nos. 5,898,031, 6,107,094, 6,506,559, 7,056,704 and in European Pat. Nos. 1214945 and 1230375, which are incorporated herein by reference in their entireties.

The siRNA molecule can be made of naturally occurring ribonucleotides, i.e., those found in living cells, or one or more of its nucleotides can be chemically modified by techniques known in the art. In addition to being modified at the level of one or more of its individual nucleotides, the backbone of the oligonucleotide also can be modified. Additional modifications include the use of small molecules (e.g. sugar molecules), amino acid molecules, peptides, cholesterol and other large molecules for conjugation onto the siRNA molecules.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.

The term "seroconversion" as used herein refers to the moment of appearance of antibodies to HIV-1 in a subject.

The term "subject" (alternatively referred to herein as "patient") as used herein refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.

Embodiments

In a preferred embodiment, said inhibitor of the invention is a RAFl, Mstl or PLK1 kinase inhibitor.

RAFl inhibitors

In an embodiment, said RAFl inhibitor comprises a RAFl siRNA. Methods of producing siRNAs are well known in the art. Suitable siRNAs are available, for example sc-29462 from Santa Cruz

Biotechnology, Heidelberg, Germany.

In a preferred embodiment, said RAFl inhibitor comprises a small molecule. In another preferred embodiment, said RAF1 inhibitor comprises an antibody. In a highly preferred embodiment, said RAF1 inhibitor inhibits the kinase activity of RAF1. RAF1 inhibitors are well known in the art and are commercially available.

Exemplary RAF1 inhibitors include, but ae not limited to GDC-0879, PD-173955, PLX-4720, CHIR- 265, R406, motesanib, pazopanib, AST-487, SB203580, barasertib-hQPA, cediranib, selumetinib, BI- 2536, afatinib, doramapimod, BMS-345541, BMS-387032, brivanib, lestaurtinib, canertinib, CI-1040, tofacitinib, alvocidib, pictilisib, GSK-1838705A, GSK-461364A, neratinib, ruxolitinib, JNJ- 28312141, Ki-20227, KW-2449, lapatinib, enzastaurin, MLN-120B, tandutinib, MLN-8054, PHA- 665752, midostaurin, dovitinib, crizotinib, erlotinib, gefitinib, GSK690693, ruboxistaurin, A-674563, masitinib, linifanib, quizartinib, axitinib, AT -7519, tozasertib, VX-745, staurosporine, PP-242, vatalanib, R547, SGX-523, bosutinib, SU-14813, sunitinib, NVP-TAE684, TG-100-115, fedratinib, vandetanib, PI-103, foretinib, vemurafenib (CAS No. 918504-65-1), AZ628, Cas No. 878739-06-1, NVP-BHG712, CAS No. 940310-85-0, RAF265 (CAS No. 927880-90-8), 2-Bromoaldisine (CAS No. 96562-96-8), Raf Kinase Inhibitor IV (CAS No. 303727-31-3), L-779,450 (CAS No. 303727-31-3), PLX4032, GSK2118436, BMS-908662 (XL-281), RG-7256 (RO5212054, PLX3603), R05126766, ARQ-736, E-3810, DCC-2036, GW5074, AZ628, an anti-RAF-1 antibody, and an inhibitory RAF-1 RNA molecule.

In a preferred embodiment, said compound is selected from the group consisting of vemurafenib (CAS No. 918504-65-1), AZ628, Cas No. 878739-06-1, NVP-BHG712, CAS No. 940310-85-0, RAF265

(CAS No. 927880-90-8), 2-Bromoaldisine (CAS No. 96562-96-8), Raf Kinase Inhibitor IV (CAS No. 303727-31-3), L-779,450 (CAS No. 303727-31-3), PLX4032, GSK2118436, BMS-908662 (XL-281), RG-7256 (RO5212054, PLX3603), R05126766, ARQ-736, E-3810, DCC-2036, GW5074 and AZ628.

Mammalian STE20-like kinase inhibitors

In a highly preferred embodiment, said Mstl inhibitor inhibits the kinase activity of Mstl. Such Mstl kinase inhibitors are well known in the art and described for example in US 20120225857,

WO2012121992 and in J Med Chem. 2009 Mar 26; 52(6): 1602-1611. Preferably, said Mstl kinase inhibitor is a selective Mstl kinase inhibitor. Exemplary Mammalian STE20-like kinase inhibitors include the MST1 inhibitors disclosed in US20120225857, Staurosporine, foretinib, bosutinib, KW- 2449, crizotinib, NVP-TAE684, cediranib', AST-487, erlotinib, R406, lestaurtinib, sunitinib, Ki- 20227, neratinib, tozasertib, PP-242, vandetanib, R547, doramapimod, brivanib, midostaurin, pazopanib, dovitinib, PHA-665752, ruboxistaurin, linifanib, SU-14813, CHIR-265, fedratinib, JNJ- 28312141, gefitinib, axitinib, GSK-461364A, GDC-0879, motesanib, canertinib, ruxolitinib, pictilisib, BMS-387032, BMS-345541, GSK-1838705A, SGX-523, CI-1040, alvocidib, masitinib, A-674563, TG-100-115, GSK690693, VX-745, MLN-120B, tandutinib, MLN-8054, PI- 103 , selumetinib, barasertib-hQPA, vatalanib, tofacitinib, enzastaurin, lapatinib, SB203580, afatinib, PD-173955, BI- 2536, quizartinib, PLX-4720, AT-7519, an anti- Mammalian STE20-like kinase antibody, and an inhibitory Mammalian STE20-like kinase RNA molecule.

PLK1 inhibitors

In a highly preferred embodiment, said PLK1 inhibitor inhibits the kinase activity of PLK1. Such PLK1 kinase inhibitors are well known in the art and are commercially available from for example Tocris Bioscience, UK, www.selleckchem.com, etc. Exemplary PLK1 inhibitors include volasertib (Boehringer Ingelheim GmbH), P-937 (Cielo Therapeutics, Inc.), CYC-800 (Cyclacel

Pharmaceuticals, Inc.), IPS-06 series (InnoPharmaScreen Inc), LS-008 (Le Sun Pharmaceuticals Ltd.), Small Molecules to Inhibit Polo-Like Kinase-1 for Oncology (Sareum Holdings pic), BI-2536 (Boehringer Ingelheim GmbH), CHR-4125 (Chroma Therapeutics Ltd.), CHR-4146 (Chroma Therapeutics Ltd.), DNX-1000 (Dynamix Pharmaceuticals Ltd.), GSK-461364 (GlaxoSmithKline pic), HMN-214 (Nippon Shinyaku Co., Ltd.), ICRF-193 (Zenyaku Kogyo Co., Ltd.), Mitotic

Checkpoint Inhibitor (Boehringer Ingelheim GmbH), MK-1496 (Merck & Co., Inc.), TAK-960 (Millennium Pharmaceuticals, Inc.), Wortmannin, vatalanib, vandetanib, tozasertib, tofacitinib, tandutinib, sunitinib, staurosporine, sphingosine kinase inhibitor, semaxanib, seliciclib, purvalanol A, pazopanib, p38 MAP kinase inhibitor III, p38 MAP kinase inhibitor, mubritinib, midostaurin, masitinib, lapatinib, kenpaullone, isogranulatimidem, indirubin-3'-monoxime, indirubin derivative E804, herbimycin A, gefitinib, fasudil, fascaplysin, erlotinib, dovitinib, dorsomorphin, diacylglycerol kinase inhibitor II, compound 56 [PMID:8568816], compound 52 [PMID:9677190], chelerythrine, casein kinase II inhibitor III, casein kinase I inhibitor, bosutinib, bohemine, bisindolylmaleimide IV, aurora kinase/Cdk inhibitor, aurora kinase inhibitor III, aminopurvalanol A, alsterpaullone 2- cyanoethyl, alsterpaullone, ERK inhibitor II, PP1 analog II, p38 MAP kinase inhibitor, mubritinib, vandetanib, AG 1296, EGFR inhibitor, ERK inhibitor III, GSK-3beta inhibitor II, H-89, KN-93, JAK3 inhibitor II, PP3, lapatinib, Syk inhibitor III, Flt-3 inhibitor, p38 MAP kinase inhibitor III,

EGFR/ErbB-2/ErbB-4 inhibitor, pazopanib, herbimycin A, PKCbeta inhibitor, PKR inhibitor, CGP53353, MNK1 inhibitor, IC261, diacylglycerol kinase inhibitor II, erlotinib, Lck inhibitor, SB202190, indirubin-3'-monoxime, PI 3-Kg inhibitor, SB203580, SKF-86002, Flt-3 inhibitor III, alsterpaullone 2-cyanoethyl, SU6656, PDGF receptor tyrosine kinase inhibitor II, GF109203X, Flt-3 inhibitor II, tofacitinib, masitinib, tandutinib, IRAK-1/4 inhibitor, Rho kinase inhibitor IV, SB220025, VEGF receptor 2 kinase inhibitor IV, GSK-3 inhibitor IX, VEGF receptor tyrosine kinase inhibitor III, Akt inhibitor V, EGFR/ErbB-2 inhibitor, Ro-32-0432, AG 1295, Rho kinase inhibitor III, TGF-beta RI kinase inhibitor, GSK-3beta inhibitor VIII, ATM kinase inhibitor, VEGF receptor 2 kinase inhibitor II, PDGF receptor tyrosine kinase inhibitor IV, DMBI, SB 202474, GSK-3 inhibitor XIII, Go 6976, VEGF receptor tyrosine kinase inhibitor II, fasudil, indirubin derivative E804, SU9516, G56983, DNA-PK inhibitor V, GSK-3beta inhibitor XII, SC-68376, semaxanib, aloisine, AG1478, TGF-beta RI inhibitor III, SU11652, GSK-3 inhibitor X, sphingosine kinase inhibitor, GTP-14564,

dorsomorphin, VEGF receptor 2 kinase inhibitor I, Akt inhibitor VIII, Akt inhibitor X, ATM/ATR kinase inhibitor, an anti- PLKlantibody, and an inhibitory PLK1 RNA molecule.

In a preferred embodiment, said compound is selected from the group consisting of volasertib

(Boehringer Ingelheim GmbH), P-937 (Cielo Therapeutics, Inc.), CYC-800 (Cyclacel

Pharmaceuticals, Inc.), IPS-06 series (InnoPharmaScreen Inc), LS-008 (Le Sun Pharmaceuticals Ltd.), Small Molecules to Inhibit Polo-Like Kinase-1 for Oncology (Sareum Holdings pic), BI-2536 (Boehringer Ingelheim GmbH), CHR-4125 (Chroma Therapeutics Ltd.), CHR-4146 (Chroma Therapeutics Ltd.), DNX-1000 (Dynamix Pharmaceuticals Ltd.), GSK-461364 (GlaxoSmithKline pic), HMN-214 (Nippon Shinyaku Co., Ltd.), ICRF-193 (Zenyaku Kogyo Co., Ltd.), Mitotic Checkpoint Inhibitor (Boehringer Ingelheim GmbH), MK-1496 (Merck & Co., Inc.), TAK-960 (Millennium Pharmaceuticals, Inc.).

US2004/0176380A1. US2004/0176380A1 discloses compounds of formula (I), such as exemplified volasertib (p. 53, example 110), for the treatment of diseases characterized by excessive or abnormal cell proliferation, such as HIV infection (p. 104, par. 245). To select volasertib for use in the treatment or prevention of a retrovirus, one must select volasertib from a long list of compounds according to Formula (I) and HIV infection from a list of diseases characterized by excessive or abnormal cell proliferation. Therefore, there is no specific disclosure of volasertib for the use in a treatment or prevention of HI V .

Combination therapies

In embodiments, it may be desirable to employ combination therapies to administer to a patient a compound according to the present invention in combination with one or more other anti-retroviral compounds or in particular anti-HIV compound(s) of a different class. However, it is to be understood that such other anti-retroviral or anti-HIV compounds should not interfere with or adversely affect the intended effects of the active compounds of this invention. In this combination therapy approach, the at least two different pharmaceutically active compounds can be administered separately or in the same pharmaceutical composition.

"In combination with" as used herein refers to uses where, for example, the first compound is administered during the entire course of administration of the second compound; where the first compound is administered for a period of time that is overlapping with the administration of the second compound, e.g. where administration of the first compound begins before the administration of the second compound and the administration of the first compound ends before the administration of the second compound ends; where the administration of the second compound begins before the administration of the first compound and the administration of the second compound ends before the administration of the first compound ends; where the administration of the first compound begins before administration of the second compound begins and the administration of the second compound ends before the administration of the first compound ends; where the administration of the second compound begins before administration of the first compound begins and the administration of the first compound ends before the administration of the second compound ends. As such, "in combination with" can also refer to regimen involving administration of two or more compounds. "In combination with" as used herein also refers to administration of two or more compounds which may be administered in the same or different formulations, by the same of different routes, and in the same or different dosage form type.

Compounds suitable for use in combination with the compound according to the present invention include, but are not limited to, retroviral protease inhibitors, retroviral nucleoside reverse transcriptase inhibitors, retroviral non-nucleoside reverse transcriptase inhibitors, retroviral integrase inhibitors, retroviral fusion inhibitors, immunomodulators of retroviruses, and vaccines against retroviruses. In preferred embodiments, said compound of the invention is combined with a compound selected from the group consisting of HIV protease inhibitors, nucleoside HIV reverse transcriptase inhibitors, non- nucleoside HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV fusion inhibitors, immunomodulators, and vaccines.

Nucleoside HIV reverse transcriptase inhibitors are well known in the art. Examples of nucleoside HIV reverse transcriptase inhibitors include 3 -Azido-3 - deoxythymidine (Zidovudine, also known as AZT and RETROVIR®), 2',3 -Didehydro-3 - deoxythymidine (Stavudine, also known as 2',3'- dihydro-3 -deoxythymidine, d4T, and ZERIT®), (2R-cis)-4-Amino-l-[2-(hydiOxymethyl)-l,3- oxathiolan-5-yl]-2(lH)- pyrimidinone (Lamivudine, also known as 3TC, and EPTVIR®), 2', 3'- dideoxyinosine (ddl), and 9-[(R)-2-

[[bis[[isopropoxycarbonyl)oxy]methoxy]phosphinyl]methoxy]propyl] adenine fumarate (Tenofovir disoproxil fumarate, also known as Viread).

Non-nucleoside HIV reverse transcriptase inhibitors are well known in the art and disclosed for example in WO2011120133 (Al). Further examples of non-nucleoside HIV reverse transcriptase inhibitors include (-)-6- Chloro-4-cyclopropylethynyl-4-trifluoromethyl- 1 ,4-dihydro-2H-3 , 1 - benzoxazin-2-one (efavirenz, also known as DMP-266 or SUSTIVA®) ( ee U.S. Pat. No. 5,519,021), l-[3- [( 1 -methylethyl) aminol] -2-pyridinyl] -4- [ [5- [(methylsulf onyl)amino] - 1 H -indol-2- yl]carbonyl]piperazine (Delavirdine, see PCT International Patent Application No. WO 91/09849), and (IS ,4R)-cis-4- [2-amino-6-(cycloprpoylamino)-9H-purin-9-yl] -2- cyclopentene- 1 -methanol (Abacavir).

HIV protease inhibitors are well known in the art. Several HIV protease inhibitors are presently approved for clinical use in the treatment of AIDS and HIV infection, including indinavir (see U.S. Pat. No. 5,413,999), amprenavir (U.S. Pat. No. 5,585,397), saquinavir (U.S. Pat. No. 5, 196,438), ritonavir (U.S. Pat. No. 5,484,801), nelfmavir (U.S. Pat. No. 5,484,926), and atazanavir (U.S. Pat. No. 5,849,91 1 and U.S. Pat. No. 6,087,383). Each of these protease inhibitors is a peptide -derived peptidomimetic, competitive inhibitor of the viral protease which prevents cleavage of the HIV gag- pol polyprotein precursor. Tipranavir (U.S. Pat. No. 5,852, 195) is a non-peptide peptidomimetic protease inhibitors also approved for use in treating HIV infection. The protease inhibitors are administered in combination with at least one and typically at least two other HIV antiviral agents, particularly nucleoside reverse transcriptase inhibitors such as zidovudine (AZT) and lamivudine (3TC) and/or non-nucleoside reverse transcriptase inhibitors such as efavirenz and nevirapine.

Indinavir, for example, has been found to be highly effective in reducing HIV viral loads and increasing CD4 cell counts in HIV-infected patients, when used in combination with nucleoside reverse transcriptase inhibitors. See, for example, Hammer et al., 1997, New England J. Med.

337:725-733 and Gulick et al., 1997, New England J. Med. 337:734-739. Further examples of suitable HIV protease inhibitors are disclosed in WO 2013059928 Al.

Examples of suitable HIV integrase inhibitors are disclosed in U.S. Patent Nos. 6,110,716; 6,124,327; and 6,245,806, PCT patent application publications WO05/061501, WO2010/088167 and

WO2012078844 Al, which are incorporated herein by reference. Various other antiviral agents can also be used in a combination therapy with a compound according to the present invention, including, but not limited to, 9-(2- hydroxyethoxymethyl) guanine (acyclovir), 2-amino-9-(2- hydroxyethoxymethyl)purine, suramin, ribavirin, antimoniotungstate (HPA-23), interferon, interleukin II, and phosphonoformate (Foscarnet). In addition, other medications such as levamisol or thymosin which would stimulate lymphocyte growth and/or function may also be employed. Examples of HIV fusion inhibitors include antibodies against HIV envelope proteins (e.g., gpl20, gp41) and peptides derived from the HIV envelope proteins. For example, a gp41 -derived peptide called T-20 (Trimeris Inc., Durham, NC) has been shown to be effective in treating HIV infection in a phase III clinical trial. Immunogens and vaccine compositions suitable for use in the treatment of HIV are known in the art and disclosed for example in WO2014172335 (Al), WO2014172366 (Al) and WO2013110790 (Al). A successful anti-HIV clinical studies was reported in New England Journal of Medicine 361 (23): 2209-2220.

Pharmaceutical compositions

The invention encompasses pharmaceutical compositions comprising the compound according to the invention for the prevention of a retrovirus infection or inhibition of retroviral infectivity as an active ingredient. Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically- acceptable cation or anion, as is well known in the art. Further, the compound used in the present invention may contain pharmacologically acceptable additives (e.g., carrier, excipient and diluent), stabilizers or components necessary for formulating preparations, which are generally used for pharmaceutical products, as long as it does not adversely affect the object of the present invention.

Treatments of HIV

The compound, kit and pharmaceutical composition according to the invention is highly suitable for treatment of the retrovirus at an early stage of the infection. Dendritic cells are among the first cells infected by the retrovirus and involved in dissemination of the retrovirus to T cells. Therefore, these cells are important for the induction of antiviral immune responses, and therefore play an important role in controlling virus replication in acute and chronic phases. The compound, kit and

pharmaceutical composition according to the invention may improve the induction of antiviral immune responses upon infection of dendritic cells by retroviruses, in particular by species of the HIV family of retroviruses, and help to prevent replication of the virus in dendritic cells, to block virus dissemination and/or prevent infection of the host. Without wishing to be bound by theory, the inventors believe that this may stimulate the induction of antiviral immune responses such as IFN-I, DC maturation and pro-inflammatory cytokines such as IL-lb, may lead to the induction of adaptive immunity by infected dendritic cells and thereby may help to mount efficient immune responses early during infection. The inventors believe that the induction of early antiviral immune responses upon virus infection may limit viral replication in the patient and improve the immune control over the virus. Therefore, in a preferred embodiment, the compound, kit and pharmaceutical composition according to the invention are used as early as possible, preferably before infection, to allow for the earliest induction of immune responses to the virus possible.

Selection of the therapeutically effective dose can be determined (e.g., via clinical trials) by a skilled artisan, such as a clinician or a physician, based upon the consideration of several factors which will be known to one of ordinary skill in the art. Such factors include, for example, the particular form of the inhibitor of the invention, and the compound's pharmacokinetic parameters such as bioavailability, metabolism, half -life, and the like, which is established during the development procedures typically employed in obtaining regulatory approval of a pharmaceutical compound. Further factors in considering the dose include the disease or condition to be treated, the benefit to be achieved in a subject, the subject's body mass, the subject's immune status, the route of administration, whether administration of the compound or combination therapeutic agent is acute or chronic, concomitant medications, and other factors known by the skilled artisan to affect the efficacy of administered pharmaceutical agents.

In some embodiments, the total pharmaceutically effective amount of said inhibitor administered orally per dose will be in the range of about 1 μg/kg/day to about 500 mg/kg/day, including about 10 μg/kg/day to about 200 mg/kg/day, such as, about 40 μg/kg/day to about 100 mg/kg/day, of subject body weight, although, this will be subject to a great deal of therapeutic discretion. In some embodiments, the said inhibitor therapy of the invention may be administered to the patient in the form of a single or twice daily administration of an immediate release formulation of said inhibitor. Administration of the pharmaceutical compositions of the invention includes, but is not limited to, oral, intravenous infusion, subcutaneous injection, intramuscular, topical, depo injection, implantation, time -release mode, intracavitary, intranasal, inhalation, intralesional, intraocular, immediate release, and controlled release. The pharmaceutical compositions of the invention also may be introduced parenterally, transmucosally (e.g., orally), nasally, rectally, intravaginally, sublingually, submucosally, or transdermally. In some embodiments, administration is parenteral, i.e., not through the alimentary canal but rather through some other route via, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, intraorbital, intracapsular, intraspinal, intrastemal, intra-arterial, or intradermal administration. In some embodiments, the administering of said inhibitor is by other than direct administration to the pericardial space. In other embodiments, the administering of said inhibitor is by systemic administration. In some of these embodiments, the inhibitor of the invention is systemically administered by subcutaneous bolus injection.

The present invention may be better understood by reference to the following non- limiting Examples, which are provided only as exemplary of the invention. The following examples are presented to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broader scope of the invention.

EXAMPLE

EXPERIMENTAL PROCEDURES DC isolation and treatment. Monocyte -derived DCs were generated from blood of healthy volunteer donors (Sanquin) as described (Gringhuis et al., 2014). Primary DCs were isolated using CD209 MicroBead kit (Miltenyi Biotec) from blood of healthy volunteer donors, skin and vaginal tissues obtained from healthy individuals undergoing reconstructive plastic procedure or vaginal prolapse surgery, respectively, and healthy bowel tissues from cancer or colitis ulcerosa patients undergoing colectomy; details are in Extended Experimental Procedures. Tissue harvesting procedures were approved by the AMC Medical Ethics Committee. Blood and tissues were routinely screened for SNPs rs7262903, rs7269320, and rs7267297 using TaqMan genotyping Assays (ID C_25623847,

C_25623845, and C_29168352; Applied Biosystems); only DCs with the major genotype profile for all three SNPs were used, unless otherwise stated. Details on stimuli, inhibitors, blocking antibodies and siRNAs are in Extended Experimental Procedures. Silencing of expression was verified by realtime PCR and flow cytometry (Figure SI and Table SI).

MSM cohort. Untreated HIV-1 -infected MSM participating in the Amsterdam cohort studies (ACS) on HIV-1 infection and AIDS (http://www.amsterdamcohortstudies.org) were genotyped and used for subsequent analyses. Treatment details and exclusion criteria have been described (Booiman et al., 2014, AIDS 28, 2517-2521). ACS are conducted in accordance with ethical principles set out in the declaration of Helsinki and approved by the AMC Medical Ethics Committee; all participants provided written informed consent. Viruses and infection. HIV-1 BaL, R9 (NL4.3), SF162 and LAI were produced and titers quantified as described (Gringhuis et al., 2010, Nat. Immunol. 11, 419-426; Sarrami-Forooshani et al., 2014, Retrovirology 11, 52). Clinical isolates were produced as described (Quakkelaar et al., 2007, J. Virol. 81, 8533-8542). DCs were infected at MOI 0.1-0.4; infection was determined by viral transcript (Tat- Rev, Vpu) quantification or flow cytometry (p24). niRNA isolation, cDNA synthesis and real-time PCR were performed as described (Gringhuis et al., 2010, Nat. Immunol. 11, 419-426); relative mRNA expression was obtained by setting N_t (=2^{ct(GAPDH) ct(tar}s^et)) at 1 in 4 h (Tat-Rev) or 10 h (Vpu) HIV- 1 -infected DCs, within one experiment and for each donor, except time course experiments where N_t = 1 at 8 h (Tat-Rev). Primers are listed in Table SI. Intracellular p24 was detected after 3, 5 and 7 days by anti-p24 (KC57-RD1 ; Beckman Coulter).

Immune responses. IFN , ISG and IL-Ιβ mRNA were quantified as above; N_t was set at 1 in 4 h (ΤΡΝβ), 8 h (ISGs) or 10 h (IL-Ιβ) GW5074-treated HIV- 1 -infected DCs. Primers are listed in Table SI. IL-Ιβ in supernatants was measured 48 h post-infection by ELISA (Invitrogen). DC maturation was determined 48 h post-infection by flow cytometry analysis of CD80 (557227; BD), CD83 (555658; BD) and CD86 (PN1M2218; Beckmann Coulter) cell surface expression

Gene product Forward primer Reverse primer

Raf-1 GGTGATAGTGGAGTCCCAGCA TCAGATGAGGGACTGGAGGTG

MAVS TGATTTCCTCGCAATCAGACG GAAGCCGATTTCCAGCTGTATG

PLK1 TGGTTCGAGAGACAGGTGAGG AGGCATTGACACTGTGCAGC

MST1 TGCCATGGACTCAACACTCG TCATGATGCAGGTCCGTACG

HIV-1 Tat-Rev ATGGCAGGAAGAAGCGGAG ATTCCTTCGGGCCTGTCG

HIV-1 Vpu TCTCTCGACGCAGGACTCG TCTGATGAGCTCTTCGTCGC

HIV-1 abortive RNA GGGTCTCTCTGGTTAGACCAGATC GGTTCCCTAGTTAGCCAGAGAGC

Table SI

Association of DDX3-MAVS-TRAF3 and PLK1. DDX3- or MAVS-associated proteins were immunoprecipitated from whole cell extracts of HIV-1 -infected DCs prepared 3 h post-infection usin^ RIPA Buffer (Cell Signaling), and DDX3, MAVS, TRAF3 and PLK1 were detected by

immunoblotting. Visualization of DDX3, mitochondria (MitoTracker Red CMXRos; Cell Signaling) and nucleus (Hoechst; Molecular Probes) was done with a TCS SP8 X confocal microscope (Leica). Association of MAVS with phosphorylated Flag-PLKl (Origene) was determined by capture ELISA. Details are in Extended Experimental Procedures. Activation of TBK1, ΙΚΚε, IRF3, PLK1, and MST1. Phosphorylation of TBK1, ΙΚΚε and PLK1 in HIV- 1 -infected DCs was detected 3 h post-infection by either flow cytometry or immunoblotting of whole cell extracts (see above). IRF3 activation was detected by immunoblotting of cytoplasmic and nuclear extracts of HIV- 1 -infected DCs prepared 3 h post-infection using NucBuster protein extraction kit (Novagen). MST1 activity was assayed over time in HIV- 1 -infected DCs using MST1 kinase enzyme system in combination with ADP-Glo kinase assay (both Promega).

Translation initiation complex composition. Cytoplasmic extracts of HIV- 1 -infected DCs prepared 3 h post-infection using ChIP lysis buffer (Active Motif) were treated with micrococcal nuclease (Cell Signaling). Translation initiation complexes were retained on m⁷GTP-agarose (Jena Bioscience), with cap analogue m⁷GpppG (New England Biolabs) as competitor as described (Soto-Rifo et al., 2013, Nucleic Acids Res. 41, 6286-6299), and DDX3, MAVS, eIF4G, eIF4A and PABP were detected by immunoblotting.

RNA immunoprecipitation (RIP) assay. RIP and re-RIP assays were performed using EZ- MagnaRIP kit (Millipore). Briefly, DDX3-RNA, MAVS-RNA and eIF4G-RNA complexes were immunoprecipitated from lysates of fixed cells. For re-RIP analyses, DDX3-RNA complexes underwent a second round of immunoprecipitation with anti-MAVS or anti-eIF4G. Details are in Extended Experimental Procedures. mRNA and non-mRNA fractions were separated, isolated and quantified as described (Gringhuis et al., 2010, Nat. Immunol. 11, 419-426). Primers are listed in Table SI. To normalize for RNA input, a sample for each condition was taken along which had not undergone immunoprecipitation; results are expressed as % input RNA. Statistical analysis. Used methods are Student's Mest for paired (mRNA, MST1) or unpaired (set point plasma viral load) observations, and Cox regression model for time-to-event outcomes.

Statistical significance was set at p < 0.05.

RESULTS HIV-1 innate signaling via Raf-1 blocks IFN-I responses

HIV-1 -induced activation of Raf-1 is pivotal for processive transcription of HIV-1 in DCs (Gringhuis et al., 2010, Nat. Immunol. 11, 419-426). Here we investigated whether Raf-1 is involved in suppression of IFN-I responses after HIV-1 infection of DCs. Monocyte -derived DCs did not express IFN mRNA after HIV-1 infection, however, remarkably, Raf-1 inhibition by either small molecule inhibitor GW5074 or silencing of Raf-1 expression (Figure SI) - while blocking mRNA expression of early viral genes Tat and Rev (Figure S2) - induced transient expression of IFN transcripts that peaked at 4 h post-infection (Figures la and lb), as well as expression of various ISGs, such as ISG15, Mx2, Trim5a, Trim22, and APOBEC3G (Figure lc). Similarly, HIV-1 infection of primary myeloid, dermal, intestinal and vaginal DCs, isolated from blood, skin, bowel, and vaginal tissues, induced IFN-I responses after Raf-1 inhibition (Figure Id). The observed suppression of IFN expression by Raf-1 was independent of HIV-1 tropism (Figure le). Furthermore, also primary CCR5-tropic HIV-1, isolated from infected patients, led to IFNP expression after Raf-1 inhibition (Figure If). These data show that Raf-1 activation by HIV-1 in DCs blocks the induction of antiviral IFN-I responses after HIV-1 infection.

DDX3 senses abortive HIV-1 transcripts

We next set out to identity the HIV-1 -derived ligand and its receptor involved in IFN-I responses after infection. Blocking reverse transcription and integration by azidothymidine (AZT) and raltegravir

(RAL), respectively, abrogated IFNP induction after Raf-1 inhibition (Figure 2a), excluding a role for virion components, like HIV-1 ssRNA, or reverse transcription products. These data suggest that HIV- 1 transcription products trigger IFN-I responses.

The best characterized RNA sensors are RIG-I and related RLR MDA5, however silencing of both RLRs (Figure SI) did not interfere with HIV-1 -induced IFNP expression after Raf-1 inhibition

(Figure 2c). Several DEAD box RNA helicases related to RIG-I, such as DDX1, DDX3 and DDX5, bind indirectly to HIV-1 transcripts, assisting nuclear export of unspliced HIV-1 transcripts via Rev- CRM1 interactions. DDX3 via direct binding to the HIV-1 5'UTR is also involved in translation of HIV-1 transcripts. Markedly, silencing of DDX3, but neither of DDX1 nor DDX5, completely abrogated induction of IFN-I expression after HIV-1 infection in presence of Raf-1 inhibitor (Figure 2c). DDX3 silencing shifted induction of singly spliced transcripts (Vpu) toward multiply spliced transcripts (Tat-Rev) (Figure S2), indicative of its functions in HIV-1 replication, whereby lack of viral Rev expression interferes with its function in nuclear export of singly and unspliced HIV-1 transcripts. These data indicate that DDX3 senses HIV-1 transcription products that can trigger IFN-I responses.

DDX3 has been shown to interact with MAVS and enhance IFN-I responses by RIG-I. MAVS silencing (Figure SI) completely inhibited IFN expression in HIV-1 -infected DCs after Raf-1 inhibition (Figure 2d). HIV-1 infection induced association of DDX3 with MAVS in DCs, even without Raf-1 inhibition (Figure 2e), implying that Raf-1 interferes with signaling downstream of MAVS to block IFN-I responses. Confocal microscopy verified that HIV-1 infection invoked partial colocalization of DDX3 around mitochondria (Figure 2f). Considering its role in HIV-1 translation, we examined whether DDX3 associates with MAVS within translation initiation complexes consisting of cap-binding elongation factor eIF4E, eIF4G and eIF4A, that assemble in a highly ordered manner on the 5' m⁷GTP cap structure of endogenous transcripts. DDX3 substitutes for eIF4E on HIV-1 transcripts, and forms a trimeric complex with eIF4G and poly(A)-binding protein (PABP), allowing 43S ribosomal units to attach to the transcripts. DDX3, but not MAVS, was present together with eIF4G, eIF4A and PABP within translation initiation complexes in HIV-1 -infected but not uninfected DCs (Figure 2g), showing that DDX3 present within translation initiation complexes is not involved in induction of MAVS -dependent IFN-I responses. We next performed RNA immunoprecipation (RIP) assays to identify the HIV-1 transcripts bound by DDX3 leading to MAVS activation. Both DDX3 and eIF4G, but not MAVS, interacted with Tat-Rev mRNA as well as other HIV-1 mRNAs in HIV-1 -infected DCs (Figure 2h). Further analysis (re-RIP) of RNA-DDX3 complexes established that eIF4G, but not MAVS, was bound together with DDX3 on HIV-1 mRNAs (Figure 2h). Beside multiply, singly and unspliced mRNAs, HIV-1 transcription also generates abortive HIV-1 RNA (Kao et al., 1987), both in the absence and presence of Raf-1 inhibition. These abortive RNAs lack a poly(A) tail and therefore are unable to recruit PABP and initiate translation. We hypothesized that abortive HIV-1 RNA might be recognized by DDX3, but not eIF4G, due to the absence of PABP. Notably, we observed that DDX3-MAVS but not DDX3-eIF4G complexes were bound to abortive HIV-1 RNAs (Figure 2h). These results imply that ribosomal recruitment excludes MAVS recruitment. Thus, DDX3 binds both abortive and elongated HIV-1 transcripts, but only sensing of abortive HIV-1 RNA results in MAVS recruitment.

DDX3 induces IFN-I responses via MAVS-TRAF3-dependent IRF3 activation

We next investigated the events downstream from DDX3-MAVS. RIG-I and MDA5 induce IFN-I responses via recruitment of TRAF3 to MAVS aggregates, which activate TBKl and ΙΚΚε to phosphorylate IRF3. TRAF3 silencing (Figure SI) abrogated IFN expression after HIV-1 infection in presence of Raf-1 inhibition (Figure 3a), indicating that DDX3 signals via the MAVS-TRAF3 axis. Notably, TRAF3 immunoprecipitated together with DDX3, in a MAVS -dependent manner, from whole cell extracts of HIV-1 -infected DCs only after Raf-1 inhibition (Figure 3b). Thus, Raf-1 suppresses IFN-I responses after HIV-1 infection by impeding recruitment of TRAF3 to MAVS. DDX3-MAVS signaling downstream from TRAF3 required activation of both TBKl and ΙΚΚε, as well as IRF3 for HIV-l-induced IFN expression after Raf-1 inhibition (Figures 3c and SI). Both TBKl and ΙΚΚε were phosphorylated at Serl72, a mark of activation, in only a fraction of DCs, likely those productively infected with HIV-1, in both a DDX3- and MAVS-dependent manner (Figure 3d). We also observed that nuclear translocation of IRF3 depended on both DDX3 and MAVS (Figure 3e). These results show that sensing of abortive HIV-1 RNA by DDX3, when Raf-1 activation is blocked, leads to MAVS-TRAF3- and ΤΒΚΙ-ΙΚΚε-dependent activation of IRF3, which drives IFNB1 transcription.

HIV-l-activated PLK1 impedes TRAF3 recruitment to MAVS

We next addressed how Raf-1 blocks TRAF3 recruitment to DDX3-MAVS complexes after HIV-1 infection. Mitotic Polo-like kinase 1 (PLK1) impedes MAVS-TRAF3 interactions when it associates with MAVS via dual interactions at the N- and C-termini, thereby blocking TRAF3 binding to the C- terminus of MAVS. Notably, HIV-1 infection induced IFN expression in PLKl-silenced DCs without Raf-1 inhibition (Figures 4a and SI), suggesting that PLK1 is involved in IFN suppression by Raf-1. HIV-1 -infected PLKl -silenced DCs showed a similar transient IFN expression profile (Figure 4b), as observed after Raf-1 inhibition (Figures la and lb). PLKl silencing did not abrogate processive HIV-1 transcription (Figure S2), indicating that PLKl, unlike Raf-1, is not involved in the elongation phase of HIV-1 transcription. We detected PLKl bound to DDX3-MAVS complexes in HIV-1 -infected DCs, but not after Raf-1 inhibition (Figure 4c). Furthermore, PLKl silencing allowed TRAF3 recruitment to DDX3 complexes after HIV-1 infection (Figure 4d). These results show that HIV-1 -induced Raf-1 activation results in association of PLKl with MAVS, thereby blocking downstream DDX3-MAVS signaling to IFN expression.

PLKl resides in an inactive state due to intramolecular interactions between its kinase and polo-box (PBD) domains. PLKl phosphorylation at Thr210 abolishes this inhibitory interaction, enabling PLKl-PBD to bind substrates in a phosphodependent manner (Elia et al., 2003, Cell 115, 83-95), such as the phospho-Thr234 residue of MAVS (Vitour et al., 2009, J. Biol. Chem. 284, 21797-21809). HIV-1 infection induced DC-SIGN- and Raf-1 -dependent phosphorylation of PLKl at Thr210

(Figures 4e and 4f). Ligation of DC-SIGN by HIV-1 gpl20 also resulted in Raf-1 -mediated PLKl phosphorylation (Figure 4f), indicating that HIV-1 induces Raf-1 activation and subsequent PLK1- Thr210 phosphorylation via DC-SIGN. Notably, recombinant Raf-1, in contrast to recombinant protein kinase A as a control, was unable to phosphorylate Flag-PLKl at Thr210 (Figure 4g). Thr210 phosphorylation of PLKl has been attributed to mitotic kinase Aurora A as well as Ste20-like kinase family members, like MST1 and MST2. Raf-1 interacts with several Ste20-like kinases. Whereas MST2 activity is inhibited by Raf-1, the role of Raf-1 in MST1 activity remains largely unknown. MST1 interacts with adaptor protein CNK1 that is a component of the Raf-1 signalosome attached to DC-SIGN in resting DCs. As recombinant MST1 phosphorylated Flag-PLKl (Figure 4g), we determined whether HIV-1 infection leads to MST1 activation. MST1 activity was transiently activated in a DC-SIGN- and Raf-1 dependent manner during HIV-1 infection, peaking 1 h postinfection and declining after 2 h (Figure 4h). Indeed, MST1 silencing (Figure SI) prevented PLKl phosphorylation during HIV-1 infection (Figure 4i), abrogating PLKl association but allowing TRAF3 recruitment with DDX3 complexes (Figure 4j), leading to IFN expression in HIV-1 -infected DCs, without Raf-1 inhibition (Figure 4k). Similar to Raf-1 inhibition but unlike PLKl silencing, MST silencing blocked processive HIV-1 transcription in DCs (Figure S2), suggesting that MST1 is also involved in the elongation phase of HIV-1 transcription. Together, these data imply that HIV-1 binding to DC-SIGN triggers Raf-1 -dependent MST1 activation, which leads to PLK1

phosphorylation and its binding to MAVS, thereby blocking downstream DDX3-MAVS signaling to IFN expression.

IFN-I responses by DCs suppress HIV-1 replication in infected individuals

We next investigated the effect of IFN-I responses on HIV-1 replication in DCs. HIV-1 infection of DCs increased from 3 to 5 days, after which it remained constant or declined slightly (Figure 5a). Blocking IFNa/β receptor (IFNR) signaling did not affect productive HIV-1 infection (Figure 5a). HIV-1 infection of PLK1 -silenced DCs was consistently lower after 3 days, compared with control- silenced cells, and did not increase over time (Figure 5a). The low infection rate of PLK1 -silenced DCs was due to IFN-I responses, as blocking IFNR signaling restored DC infection to levels similar as observed in control-silenced cells (Figure 5a). Thus, HIV-1 -induced PLK1 -mediated suppression of IFN expression facilitates productive infection of DCs.

As clinical outcome ultimately gives the most comprehensive understanding of the significance of molecular mechanisms, we analyzed the effect of single nucleotide polymorphisms (SNP) for components of the DDX3-MAVS signaling pathway within a human MSM cohort of untreated HIV-1 - infected individuals. We identified three SNPs within MAVS that showed significant differences in plasma viral load, rs7262903, rs7269320 and rs7267297. rs7262903 and rs7269320 result in amino acid substitutions within MAVS (Glnl98 to Lys (Q198K), Ser409 to Phe (S409F)), whereas rs7267297 lies within flanking 3'UTR of MAVS. rs7262903 and rs7269320 are 100% linked within the MSM cohort; linkage disequilibrium in the global population is D' = 0.956, r² = 0.559 (HapMap release 23). Plasma viral load at set point was significantly lower in HIV-1 -infected individuals homozygous for the minor alleles of rs7262903/rs7269320 (minor genotype), compared to individuals homozygous for the major alleles (major genotype) (pi = 0.044; Student's Mest), while no differences were detected between individuals with heterozygous and major genotypes (p2 = 0.215; Student's t- test) (Figure 5b). Moreover, we observed a significant delay in time from seroconversion to viral RNA load > 10⁴⁵ copies/ml plasma {pi = 0.025, RH = 0.106, 95% CI 0.015-0.759; Cox regression) (Figure 5c). Minor genotype individuals showed no significant effect on disease progression (AIDS according to the CDC definition 1993), likely due to the low number of individuals with this genotype within the MSM cohort. These data signify the importance of the DDX3-MAVS pathway in controlling HIV-1 replication, not only during the acute infection stage but also the clinical latency stage.

We next examined whether MAVS K198/F409 expression affects HIV-1 replication in DCs from uninfected individuals. Notably, minor genotype DCs showed lower infection than DCs with the major genotype (Figure 5d). Remarkably, blocking IFNR signaling increased productive HIV-1 infection of minor genotype DCs to levels similar as observed in major genotype DCs (Figure 5d). These data imply that suppression of IFN-I responses by HIV-1 is defective in DCs from individuals expressing dual MAVS K198/F409 mutant.

MAVS Q198K is a functionally neutral mutant. Indeed, DCs with either major or minor genotypes for rs7262903/rs7269320 responded similarly to triggering of RIG-I/MDA5 signaling (Figure 6a).

However, minor genotype DCs, both monocyte-derived and primary dermal DCs, in contrast to those with either heterozygous or major genotypes (Figures la and lb), induced IFN-I responses after HIV- 1 infection only (Figures 6b-d), via the DDX3-MAVS axis (Figures 6e and 6f). We observed that TRAF3 instead of PLK1 was recruited by DDX3-MAVS in HIV-1 -infected DCs (Figure 6f), while PLK1 silencing did not affect IFN expression (Figure 6g). DC-SIGN/Raf-1 -mediated

phosphorylation of PLK1 was intact in minor genotype DCs after HIV-1 infection (Figure 6h), suggesting that the mutations within MAVS interfere with binding of PLK1 to MAVS. Indeed, Thr210-phosphorylated Flag-PLKl captured MAVS from lysates of major genotype but not minor genotype DCs (Figure 6i). These data show that MAVS K198/F409 is resistant to suppression by PLK1. Thus, HIV-1 -induced association between PLK1 and MAVS is essential in suppression of IFN- I responses, however is non-functional in DCs with MAVS K198/F409, therefore limiting HIV-1 replication and viral load, protecting individuals carrying minor alleles of rs7262903/rs7269320 after infection. Attenuation of DDX3-MAVS signaling by HIV-1 suppresses immune activation

We next addressed whether suppression of DDX3-MAVS signaling prevents DC activation in response to HIV-1 infection. We only observed DC maturation after HIV-1 infection in presence of Raf-1 inhibition (Figure 7a). IFNR inhibition as well as DDX3 and MAVS silencing abrogated maturation of HIV-1 -infected DCs after Raf-1 inhibition, while PLK1 silencing allowed DC maturation after HIV-1 infection only (Figures 7a and 7b). Furthermore, HIV-1 infection induced maturation of DCs from donors with minor rs7262903/rs7269320 genotype independent of Raf-1 inhibition, in an IFNR-dependent manner (Figure 7c). These data demonstrate that innate IFN-I responses during HIV-1 infection induce DC maturation, which is efficiently blocked by HIV-1. Inter leukin (IL)-l family members are at the center of adaptive responses, inducing neutrophil recruitment, lymphocyte activation and inflammatory mediators. While HIV-1 infection did not result in IL1B transcription, simultaneous Raf-1 inhibition induced transient expression of IL-1 β mRNA (Figure 7d) and protein (Figure 7e). Silencing of DDX3 and MAVS blocked IL-1 β mRNA induction, while PLK1 silencing induced IL1B transcription independent of Raf-1 inhibition (Figure 7f).

Blocking IFNR signaling did not affect IL-Ιβ mRNA expression (Figure 7g), suggesting that IL1B transcription depends on MAVS-TRAF3 -mediated NF-κΒ but not IRF3 activation. In addition, DCs expressing MAVS K198/S409 induced IL-Ιβ expression in response to HIV-1 infection (Figure 7h), as opposed to DCs with the major (Figure 7d) or heterozygous genotypes (Figure 7h). Thus, HIV-1 targets DDX3-MAVS signaling not only to block antiviral responses, but also adaptive immune responses.

DISCUSSION

Adaptive immunity to HIV-1 requires DC activation to elicit antiviral IFN-I responses as well as CD4 and CD8 T cell activation. However, HIV-1 avoids immune surveillance, preventing mounting of such protective responses. Here we identified DDX3 as an intracellular sensor that couples recognition of abortive HIV-1 RNA to induction of IFN-I responses via MAVS. However, HIV-1 suppressed DDX3 signaling in primary and monocyte-derived DCs by triggering innate DC-SIGN signaling during HIV- 1 infection, which prevented IFN-I responses, DC maturation and proinflammatory cytokine responses. We identified MAVS as the Achilles' heel in the induction of protective host defenses; HIV-1 recognition by DC-SIGN activates PLK1, which targets MAVS, thereby impeding TRAF3 recruitment and IRF3 activation to drive IFN expression. Suppression of viral replication in HIV-1 - infected individuals was associated with homozygosity for a rare MAVS allele that encodes a dual mutant, which renders MAVS insensitive to PLK1 inhibition, thereby orchestrating effective antiviral defense via induction of DC maturation, IFN-I and proinflammatory cytokine responses. Thus, therapeutic targeting of PLK1 might boost early antiviral responses to HIV-1 in DCs, which will not only limit HIV-1 replication but also improve adaptive immunity in infected humans, strengthening immune defenses and limiting immune dysfunction.

RNA helicase DDX3 has been acknowledged for years as a host factor essential for HIV-1 replication. It facilitates translation of HIV-1 transcripts by replacing cap-binding protein eIF4E when translation initiation complexes assemble on the 5'UTR of HIV-1 transcripts to unwind the highly structured

TAR hairpin and make it accessible to ribosomes. Moreover, DDX3 assists CRMl and viral Rev in the nuclear export of singly or unspliced HIV-1 transcripts. Paradoxically, its function in HIV-1 translation and RNA transport makes it ideally suited as a RNA sensor to restrict HIV-1 infection. All HIV-1 transcripts, whether they are spliced or not, abortive or elongated, share the same highly structured -60 nt of the 5'UTR, and interact with DDX3. Notably, DDX3 bound to abortive HIV-1 RN As is not assembled in translation complexes, likely due to the absence of a poly(A) tail and concomitant PABP recruitment, but associated with MAVS at the mitochondrial membrane, thereby inducing MAVS-dependent IFN-I and IL-Ι β expression. The roles of DDX3 in HIV-1 translation and antiviral defense are thus mutually exclusive, which might prevent triggering of antiviral immune responses to endogenous transcripts that also interact with DDX3. These data indicate that DDX3 only elicits antiviral defenses in DCs after productive HIV-1 infection, when abortive HIV-1 RNAs are present.

DDX3 signaling after HIV-1 infection is blocked by DC-SIGN signaling at the level of TRAF3 recruitment to MAVS, hence facilitating HIV-1 propagation. Our data underline the importance of host innate receptors for HIV-1 to suppress antiviral host defense.

DC-SIGN-induced Raf-1 activates MST1 -mediated phosphorylation of PLK1, inducing a

conformational change, leading to association of PLK1 with MAVS, hence blocking signaling events downstream of MAVS. PLK1 plays a critical role in cell division, however its role in nondividing, differentiated cells is poorly defined. Our data identify PLK1 as an inhibitor of antiviral defense against HIV-1 in DCs. Although PLK1 association with MAVS blocks recruitment of TRAF3 to MAVS, the exact underlying mechanism remains to be elucidated. Our data indicate that PLK1 inhibitors constitute a novel class of antiretroviral drugs that enhances endogenous antiviral immunity in potentially HIV-1 -infected individuals.

The importance of IFN-I responses in HIV-1 infection became fully evident with the identification of two linked SNPs within the MAVS gene that encode a dual K198/F409 mutant that is unable to interact with PLK1. The dual MAVS mutant renders DCs resistant to PLK1 -mediated suppression and in a human MSM HIV-1 infection cohort, patients homozygous for the minor genotype of the two SNPs have a significantly prolonged suppression of plasma viral load, compared to those either homozygous or heterozygous for the major genotype. Strikingly, HIV-1 infection of DCs expressing the dual MAVS mutant induced efficient IFN-I responses that inhibited HIV-1 replication. IFN-I expression was also involved in DC maturation, which is crucial in the induction of adaptive T cell responses. DDX3-mediated but IFNR-independent IL-Ι β induction further ignites these inflammatory responses. IFN-I responses especially are important to limit viral spread, however, during HIV-1 infection, might also create more T cell activation and therefore more HIV-1 target cells. Our data in untreated HIV-1 patients who express the dual MAVS mutant corroborate that early antiviral responses during infection are beneficial in host control of viral replication. Overall, this study underscores the importance of antiviral IFN-I responses in acute retroviral exposure, when DCs are a prominent target for HIV-1. Thus, DDX3 acts a cytosolic sensor for abortive HIV-1 RNA that directs innate antiviral responses upon HIV-1 infection. However, HIV-1 evades these responses by impeding MAVS signaling via innate activation of PLKl. The importance of unimpeded MAVS activation early during infection in HIV-1 -infected individuals expressing the dual MAVS mutant reveals the identified pathways as important novel targets for early therapeutic intervention to boost endogenous antiviral immunity in acute exposure or even as a prophylactic measure.

Claims

1. A compound selected from the group consisting of:

(i) an inhibitor of Polo-like 1 (PLK1),

(ii) an inhibitor of mammalian Ste20-like 1 (MST1), and

(iii) an inhibitor of RAF-1,

with the proviso that said compound is not an M-CSF antagonist,

for use in a treatment of a subject infected with a retrovirus or for prevention of infection with a retrovirus in a subject, preferably wherein said treatment is given before seroconversion.

2. Compound for use according to claim 1, wherein said inhibitor of PLK1 is selected from the group consisting of volasertib, P-937, CYC-800, IPS-06, LS-008, BI-2536, CHR-4125, CHR-4146, DNX-1000, GSK-461364, HMN-214, ICRF-193, Mitotic Checkpoint Inhibitor, MK-1496, TAK-960, wortmannin, vatalanib, vandetanib, tozasertib, tofacitinib, tandutinib, sunitinib, staurosporine, sphingosine kinase inhibitor, semaxanib, seliciclib, purvalanol A, pazopanib, p38 MAP kinase inhibitor III, p38 MAP kinase inhibitor, mubritinib, midostaurin, masitinib, lapatinib, kenpaullone, isogranulatimidem, indirubin-3'-monoxime, indirubin derivative E804, herbimycin A, gefitinib, fasudil, fascaplysin, erlotinib, dovitinib, dorsomorphin, diacylglycerol kinase inhibitor II, compound 56 [PMID:8568816], compound 52 [PMID:9677190], chelerythrine, casein kinase II inhibitor III, casein kinase I inhibitor, bosutinib, bohemine, bisindolylmaleimide IV, aurora kinase/Cdk inhibitor, aurora kinase inhibitor III, aminopurvalanol A, alsterpaullone 2-cyanoethyl, alsterpaullone, ERK inhibitor II, PP1 analog II, p38 MAP kinase inhibitor, mubritinib, vandetanib, AG 1296, EGFR inhibitor, ERK inhibitor III, GSK-3beta inhibitor II, H-89, KN-93, JAK3 inhibitor II, PP3, lapatinib, Syk inhibitor III, Flt-3 inhibitor, p38 MAP kinase inhibitor III, EGFR/ErbB -2/ErbB -4 inhibitor, pazopanib, herbimycin A, PKCbeta inhibitor, PKR inhibitor, CGP53353, MNK1 inhibitor, IC261, diacylglycerol kinase inhibitor II, erlotinib, Lck inhibitor, SB202190, indirubin-3'-monoxime, PI 3-Kg inhibitor, SB203580, SKF-86002, Flt-3 inhibitor III, alsterpaullone 2-cyanoethyl, SU6656, PDGF receptor tyrosine kinase inhibitor II, GF109203X, Flt-3 inhibitor II, tofacitinib, masitinib, tandutinib, IRAK-1/4 inhibitor, Rho kinase inhibitor IV, SB220025, VEGF receptor 2 kinase inhibitor IV, GSK-3 inhibitor IX, VEGF receptor tyrosine kinase inhibitor III, Akt inhibitor V, EGFR/ErbB -2 inhibitor, Ro-32-0432, AG 1295, Rho kinase inhibitor III, TGF-beta RI kinase inhibitor, GSK-3beta inhibitor VIII, ATM kinase inhibitor, VEGF receptor 2 kinase inhibitor II, PDGF receptor tyrosine kinase inhibitor IV, DMBI, SB 202474, GSK-3 inhibitor XIII, Go 6976, VEGF receptor tyrosine kinase inhibitor II, fasudil, indirubin derivative E804, SU9516, Go6983, DNA-PK inhibitor V, GSK-3beta inhibitor XII, SC-68376, semaxanib, aloisine, AG1478, TGF-beta RI inhibitor III, SU11652, GSK-3 inhibitor X, sphingosine kinase inhibitor, GTP-14564, dorsomorphin, VEGF receptor 2 kinase inhibitor I, Akt inhibitor VIII, Akt inhibitor X, ATM/ATR kinase inhibitor, an anti- PLK1 antibody, and an inhibitory PLK1 RNA molecule.

3. Compound for use according to claim 1, wherein said inhibitor of Mstl is selected from the group consisting of staurosporine, foretinib, bosutinib, KW-2449, crizotinib, NVP-TAE684, cediranib', AST-487, erlotinib, R406, lestaurtinib, sunitinib, Ki-20227, neratinib, tozasertib, PP-242, vandetanib, R547, doramapimod, brivanib, midostaurin, pazopanib, dovitinib, PHA-665752, ruboxistaurin, linifanib, SU-14813, CHIR-265, fedratinib, JNJ-28312141, gefitinib, axitinib, GSK- 461364A, GDC-0879, motesanib, canertinib, ruxolitinib, pictilisib, BMS-387032, BMS-345541, GSK- 1838705A, SGX-523, CI-1040, alvocidib, masitinib, A-674563, TG-100-115, GSK690693, VX-745, MLN-120B, tandutinib, MLN-8054, PI-103, selumetinib, barasertib-hQPA, vatalanib, tofacitinib, enzastaurin, lapatinib, SB203580, afatinib, PD-173955, BI-2536, quizartinib, PLX-4720, AT-7519, an anti- Mammalian STE20-like kinase antibody, and an inhibitory Mammalian STE20-like kinase RNA molecule.

4. Compound for use according to claim 1, wherein said RAF-1 inhibitor is selected from the group consisting of GDC-0879, PD-173955, PLX-4720, CHIR-265, R406, motesanib, pazopanib, AST-487, SB203580, barasertib-hQPA, cediranib, selumetinib, BI-2536, afatinib, doramapimod, BMS-345541, BMS-387032, brivanib, lestaurtinib, canertinib, CI-1040, tofacitinib, alvocidib, pictilisib, GSK-1838705A, GSK-461364A, neratinib, ruxolitinib, JNJ-28312141, Ki-20227, KW- 2449, lapatinib, enzastaurin, MLN-120B, tandutinib, MLN-8054, PHA-665752, midostaurin, dovitinib, crizotinib, erlotinib, gefitinib, GSK690693, ruboxistaurin, A-674563, masitinib, linifanib, quizartinib, axitinib, AT-7519, tozasertib, VX-745, staurosporine, PP-242, vatalanib, R547, SGX-523, bosutinib, SU-14813, sunitinib, NVP-TAE684, TG-100-115, fedratinib, vandetanib, PI-103, foretinib, vemurafenib, CAS No. 918504-65-1, AZ628, CAS No. 878739-06-1, NVP-BHG712, CAS No.

940310-85-0, RAF265 (CAS No. 927880-90-8), 2-Bromoaldisine (CAS No. 96562-96-8), Raf Kinase Inhibitor IV (CAS No. 303727-31-3), L-779,450 (CAS No. 303727-31-3), PLX4032, GSK2118436, BMS-908662 (XL-281), RG-7256 (RO5212054, PLX3603), R05126766, ARQ-736, E-3810, DCC- 2036, GW5074, AZ628, an anti-RAF-1 antibody, and an inhibitory RAF-1 RNA molecule.

5. Kit comprising a compound as defined according to any of the above claims and further a compound suitable for prevention or treatment of a retrovirus infection for use in a treatment of a patient infected with a retrovirus or for prevention of infection with a retrovirus.

6 Kit for use according to claim 5, wherein said further compound is selected from reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, fusion inhibitors, retroviral immunogens.

7. A pharmaceutical composition comprising a compound as defined in any of the above claims or a kit as defined according to claim 5 or 6 and a pharmaceutically acceptable carrier or excipient for use as a medicament.

8 Pharmaceutical composition for use according to claim 7, wherein said pharmaceutical composition is for use in the treatment of a patient infected with a retrovirus or for prevention of infection with a retrovirus.

9. Compound for use according to anyone of claims 1-4, kit for use according to claim 5 or 6, or pharmaceutical composition for use according to claim 7 or 8, wherein said treatment is for stimulating and/or inducing an immune response to a retrovirus.

10. Compound for use according to anyone of claims 1-4 or 9, kit for use according to claim 5, 6, or 9, or pharmaceutical composition for use according to anyone of claims 7-9, wherein said treatment comprises stimulating and/or inducing I IFN responses and adaptive immunity against the retrovirus.

11. Compound for use according to anyone of claims 1-4, and 9-10, kit for use according to anyone of claims 5, 6, and 9-10, or pharmaceutical composition for use according to anyone of claims 7-10, wherein said treatment is given early during infection, preferably before seroconversion.

12. Compound for use according to anyone of claims 1-4, or 9-11, kit for use according to anyone of claims 5, 6, and 9-11, or pharmaceutical composition for use according to claim 7-11, wherein said treatment is given during vaccination against the retrovirus.

13. Compound for use according to anyone of claims 1-4, and 9-12, kit for use according to claim 5, 6, and 9-12, or pharmaceutical composition for use according to anyone of claims 7-12, wherein said retrovirus is HIV.