VXX-401, a novel anti-PCSK9 vaccine, reduces LDL-C in cynomolgus monkeys.

Atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of disease burden in the world and is highly correlated with chronic elevations of LDL-C. LDL-C-lowering drugs, such as statins or monoclonal antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9), are known to reduce the risk of cardiovascular diseases; however, statins are associated with limited efficacy and poor adherence to treatment, whereas PCSK9 inhibitors are only prescribed to a "high-risk" patient population or those who have failed other therapies. Based on the proven efficacy and safety profile of existing monoclonal antibodies, we have developed a peptide-based vaccine against PCSK9, VXX-401, as an alternative option to treat hypercholesterolemia and prevent ASCVD. VXX-401 is designed to trigger a safe humoral immune response against PCSK9, resulting in the production of endogenous antibodies and a subsequent 30-40% reduction in blood LDL-C. In this article, VXX-401 demonstrates robust immunogenicity and sustained serum LDL-C-lowering effects in nonhuman primates. In addition, antibodies induced by VXX-401 bind to human PCSK9 with high affinity and block the inhibitory effect of PCSK9 on LDL-C uptake in a hepatic cell model. A repeat-dose toxicity study conducted in nonhuman primates under good laboratory practices toxicity indicated a suitable safety and tolerability profile, with injection site reactions being the main findings. As a promising safe and effective LDL-C-lowering therapy, VXX-401 may represent a broadly accessible and convenient option to treat hypercholesterolemia and prevent ASCVD.

Maternal immunization with RSV fusion glycoprotein vaccine and substantial protection of neonatal baboons against respiratory syncytial virus pulmonary challenge.

Globally, human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children. There are no licensed vaccines despite the high worldwide disease burden. RSV fusion (F) glycoprotein vaccine is the most advanced candidate for maternal immunization. In this report, a baboon maternal immunization model was used to assess the immunogenicity and protection of infants against pulmonary challenge with human RSV/A. Vaccination in the third trimester produced high anti-RSV F IgG titers and virus-neutralizing antibodies. Infants born to immunized females had high levels of serum RSV antibodies that were comparable to maternal levels at birth and persisted for over 50 days with a half-life of 14-24 days. Furthermore, infants from immunized females and challenged with RSV/A were healthy, developed less severe disease, and had only mild pulmonary inflammatory changes whereas infants born to non-vaccinated females developed more severe disease with marked to moderate interstitial pneumonia, pulmonary edema, and bronchiolar obstruction. These results support the further development of the RSV F vaccine for maternal immunization.

Respiratory syncytial virus prefusogenic fusion (F) protein nanoparticle vaccine: Structure, antigenic profile, immunogenicity, and protection.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in the very young, elderly, and immunocompromised for which there is no vaccine. The surface exposed RSV fusion (F) glycoprotein is required for membrane fusion and infection and is a desirable vaccine candidate. RSV F glycoprotein structure is dynamic and undergoes significant rearrangements during virus assembly, fusion, and infection. We have previously described an RSV fusion-inactive prefusogenic F with a mutation of one of two furin cleavage sites resulting in the p27 region on the N-terminus of F1 with a truncated fusion peptide covalently linked to F2. A processing intermediate RSV prefusogenic F has been reported in infected cells, purified F, budded virus, and elicited a strong immune response against p27 in RSV infected young children. In this report, we demonstrate that prefusogenic F, when expressed on the cell surface of Sf9 insect and human 293T cells, binds monoclonal antibodies (mAbs) that target prefusion-specific antigenic sites Ø and VIII, and mAbs targeting epitopes common to pre- and postfusion F sites II and IV. Purified prefusogenic F bound prefusion F specific mAbs to antigenic sites Ø and VIII and mAbs targeting pre- and postfusion sites II, IV, and p27. Mice immunized with prefusogenic F antigen produced significantly higher levels of anti-F IgG and RSV neutralizing antibodies than prefusion or postfusion F antigens and induced antibodies competitive with mAbs to sites Ø, VIII, II, and IV. RSV prefusogenic F neutralization antibody responses were enhanced with aluminum phosphate adjuvant and significantly higher than prefusion F. Prefusogenic F vaccine protected cotton rats against upper and lower respiratory tract infection by RSV/A. For the first time, we present the structure, antigenic profile, immunogenicity, and protective efficacy of RSV prefusogenic F nanoparticle vaccine.

Structure basis of neutralization by a novel site II/IV antibody against respiratory syncytial virus fusion protein.

Globally, human respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in newborns, young children, and the elderly for which there is no vaccine. The RSV fusion (F) glycoprotein is a major target for vaccine development. Here, we describe a novel monoclonal antibody (designated as R4.C6) that recognizes both pre-fusion and post-fusion RSV F, and binds with nanomole affinity to a unique neutralizing site comprised of antigenic sites II and IV on the globular head. A 3.9 Å-resolution structure of RSV F-R4.C6 Fab complex was obtained by single particle cryo-electron microscopy and 3D reconstruction. The structure unraveled detailed interactions of R4.C6 with antigenic site II on one protomer and site IV on a neighboring protomer of post-fusion RSV F protein. These findings significantly further our understanding of the antigenic complexity of the F protein and provide new insights into RSV vaccine design.

Respiratory syncytial virus fusion nanoparticle vaccine immune responses target multiple neutralizing epitopes that contribute to protection against wild-type and palivizumab-resistant mutant virus challenge.

Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in newborns, young children, elderly, and immune-compromised. The RSV fusion (F) glycoprotein is a major focus of vaccine development and the target of palivizumab (Synagis®) which is licensed as an immuno-prophylactic for use in newborn children at high risk of infection. However, clinical use of a narrowly targeted monoclonal antibodies leads to the generation of escape mutant strains that are fully resistant to neutralization by the antibody. Herein, we evaluated the RSV F nanoparticle vaccine (RSV F vaccine), produced as near-full-length, pre-fusogenic F trimers that form stable protein-detergent nanoparticles. The RSV F vaccine induces polyclonal antibodies that bind to antigenic site II as well as other epitopes known to be broadly neutralizing. Cotton rats immunized with the RSV F vaccine produced antibodies that were both neutralizing and protected against wild-type RSV infection, as well as against a palivizumab-resistant mutant virus. Use of aluminum phosphate adjuvant with the RSV F vaccine increased site II antibody avidity 100 to 1000-fold, which correlated with enhanced protection against challenge. The breadth of the vaccine-induced antibody response was demonstrated using competitive binding with monoclonal antibodies targeting antigenic sites Ø, II, IV, and VIII found on pre-fusion and post-fusion conformations of RSV F. In summary, we found the RSV F vaccine induced antibodies that bind to conserved epitopes including those defined as pre-fusion F specific; that use of adjuvant increased antibody avidity that correlated with enhanced protection in the cotton rat challenge model; and the polyclonal, high-avidity antibodies neutralized and protected against both wild-type and palivizumab-resistant mutant virus. These data support the ongoing clinical development of the aluminum phosphate adjuvanted RSV F nanoparticle vaccine.

A Phase 2 randomized, observer-blind, placebo-controlled, dose-ranging trial of aluminum-adjuvanted respiratory syncytial virus F particle vaccine formulations in healthy women of childbearing age.

OBJECTIVE: Respiratory syncytial virus (RSV) causes significant morbidity and mortality in infants. We are developing an RSV fusion (F) protein nanoparticle vaccine for immunization of third trimester pregnant women to passively protect infants through transfer of RSV-specific maternal antibodies. The present trial was performed to assess the immunogenicity and safety of several formulations of RSV F vaccine in 1-dose or 2-dose schedules. METHODS: Placebo, or vaccine with 60µg or 120µg RSV F protein and 0.2, 0.4, or 0.8mg aluminum, were administered intramuscularly on Days 0 and 28 to healthy women 18-35years old. Immunogenicity was assessed from Days 0 through 91 based on anti-F IgG and palivizumab-competitive antibody (PCA) by ELISA, and RSV A and B neutralizing antibodies by microneutralization (MN) assay. Solicited adverse events were collected through Day 7 and unsolicited adverse events through Day 91. RESULTS: All formulations were well-tolerated, with no treatment-related serious adverse events. Anti-F IgG and PCA responses were correlated and increased after both doses, while MN increased significantly only after the first dose, then plateaued. The timeliest and most robust antibody responses followed one dose of 120µg RSV F protein and 0.4mg aluminum, but persistence through 91days was modestly (∼25%) superior following two doses of 60µg RSV F protein and 0.8mg aluminum. Western blot analysis showed RSV infections in active vaccinees were reduced by 52% overall (p=0.009 overall) over the Day 0 through 90 period. CONCLUSIONS: RSV F nanoparticle vaccine formulations were well tolerated and immunogenic. The optimal combination of convenience and rapid response for immunization in the third trimester occurred with 120µg RSV F and 0.4mg aluminum, which achieved peak immune responses in 14days and sufficient persistence through 91days to allow for passive transfer of IgG antibodies to the fetus. NCT01960686.

Immunogenicity and safety of a respiratory syncytial virus fusion protein (RSV F) nanoparticle vaccine in older adults.

BACKGROUND: A preventative strategy for Respiratory Syncytial Virus (RSV) infection constitutes an under-recognized unmet medical need among older adults. Four formulations of a novel recombinant RSV F nanoparticle vaccine (60 or 90 µg RSV F protein, with or without aluminum phosphate adjuvant) administered concurrently with a licensed inactivated trivalent influenza vaccine (TIV) in older adult subjects were evaluated for safety and immunogenicity in this randomized, observer-blinded study. RESULTS: A total of 220 healthy males and females ≥ 60 years of age, without symptomatic cardiopulmonary disease, were vaccinated concurrently with TIV and RSV F vaccine or placebo. All vaccine formulations produced an acceptable safety profile, with no vaccine-related serious adverse events or evidence of systemic toxicity. Vaccine-induced immune responses were rapid, rising as early as 7 days post-vaccination; and were comparable in all formulations in terms of magnitude, with maximal levels attained within 28 (unadjuvanted) or 56 (adjuvanted) days post-vaccination. Peak anti-F protein IgG antibody levels rose 3.6- to 5.6-fold, with an adjuvant effect observed at the 60 µg dose, and a dose-effect observed between the unadjuvanted 60 and 90 µg regimens. The anti-F response persisted through 12 months post-vaccination. Palivizumab-competitive antibodies were below quantifiable levels (<33 µg/mL) at day 0. The rise of antibodies with specificity for Site II peptide, and the palivizumab-competitive binding activity, denoting antibodies binding at, or in proximity to, antigenic Site II on the F protein, closely paralleled the anti-F response. However, a larger proportion of antibodies in adjuvanted vaccine recipients bound to the Site II peptide at high avidity. Day 0 neutralizing antibodies were high in all subjects and rose 1.3- to 1.7-fold in response to vaccination. Importantly, the RSV F vaccine co-administered with TIV did not impact the serum hemagglutination inhibition antibody responses to a standard-dose TIV, and TIV did not impact the immune response to the RSV F vaccine. CONCLUSIONS: RSV F protein nanoparticle vaccine induced increases in measures of functional immunity to RSV in older adults and demonstrated an acceptable safety profile. Adjuvanted formulations provided additional immunogenicity benefit as compared to increasing antigen dose alone. This trial was registered with ClinicalTrials.gov number NCT01709019.

Matrix-M adjuvant enhances antibody, cellular and protective immune responses of a Zaire Ebola/Makona virus glycoprotein (GP) nanoparticle vaccine in mice.

Ebola virus (EBOV) causes severe hemorrhagic fever for which there is no approved treatment or preventive vaccine. Immunological correlates of protective immunity against EBOV disease are not well understood. However, non-human primate studies have associated protection of experimental vaccines with binding and neutralizing antibodies to the EBOV glycoprotein (GP) as well as EBOV GP-specific CD4(+) and CD8(+) T cells. In this report a full length, unmodified Zaire EBOV GP gene from the 2014 EBOV Makona strain (EBOV/Mak) was cloned into a baculovirus vector. Recombinant EBOV/Mak GP was produced in Sf9 insect cells as glycosylated trimers and, when purified, formed spherical 30-40 nm particles. In mice, EBOV/Mak GP co-administered with the saponin adjuvant Matrix-M was significantly more immunogenic, as measured by virus neutralization titers and anti-EBOV/Mak GP IgG as compared to immunization with AlPO4 adjuvanted or non-adjuvanted EBOV/Mak GP. Similarly, antigen specific T cells secreting IFN-γ were induced most prominently by EBOV/Mak GP with Matrix-M. Matrix-M also enhanced the frequency of antigen-specific germinal center B cells and follicular helper T (TFH) cells in the spleen in a dose-dependent manner. Immunization with EBOV/Mak GP with Matrix-M was 100% protective in a lethal viral challenge murine model; whereas no protection was observed with the AlPO4 adjuvant and only 10% (1/10) mice were protected in the EBOV/Mak GP antigen alone group. Matrix-M adjuvanted vaccine induced a rapid onset of specific IgG and neutralizing antibodies, increased frequency of multifunctional CD4+ and CD8(+) T cells, specific TFH cells, germinal center B cells, and persistence of EBOV GP-specific plasma B cells in the bone marrow. Taken together, the addition of Matrix-M adjuvant to the EBOV/Mak GP nanoparticles enhanced both B and T-cell immune stimulation which may be critical for an Ebola subunit vaccine with broad and long lasting protective immunity.

A Randomized, Blinded, Controlled, Dose-Ranging Study of a Respiratory Syncytial Virus Recombinant Fusion (F) Nanoparticle Vaccine in Healthy Women of Childbearing Age.

BACKGROUND: Respiratory syncytial virus (RSV) is a leading cause of infant morbidity and mortality. A recombinant RSV fusion protein nanoparticle vaccine (RSV F vaccine) candidate for maternal immunization was tested for safety and immunogenicity in women of childbearing age. METHODS: Three hundred thirty women (18-35 years) were randomized to receive 1 or 2 doses of RSV F vaccine (60 or 90 µg) with or without aluminum phosphate adjuvant, or placebo at days 0 and 28. Safety was evaluated over 180 days; immunogenicity and RSV infection rates were evaluated over 112 days. RESULTS: All vaccine formulations were well tolerated, without vaccine-related serious adverse events. Anti-F immunoglobulin G antibodies rose 6.5-15.6-fold, with significantly higher levels in 2-dose, adjuvanted regimens at day 56. Palivizumab-competitive antibody levels were undetectable at day 0 but increased up to 325 µg/mL at day 56. A 2.7- and 3.5-fold rise in RSV/A and RSV/B microneutralization antibodies were noted at day 56. Between days 56 and 112, 21% (12/56) of placebo recipients and 11% of vaccinees (26/244) showed evidence of a recent RSV infection (P = .04). CONCLUSIONS: The vaccine appeared safe, immunogenic, and reduced RSV infections. Further development as a vaccine for use in maternal immunization is warranted. CLINICAL TRIALS REGISTRATION: NCT01704365.

Modeling maternal fetal RSV F vaccine induced antibody transfer in guinea pigs.

BACKGROUND: Protection of newborns and young infants against RSV disease via maternal immunization mediated by transplacental transfer of antibodies is under evaluation in third-trimester pregnant women with the RSV recombinant F nanoparticle vaccine (RSV F vaccine). Since the hemichorial placental architecture in guinea pigs and humans is similar, the guinea pig model was employed to assess RSV F vaccine immunogenicity in pregnant sows and to compare RSV-specific maternal antibody levels in their pups. METHODS: Thirty (30) presumptive pregnant guinea pigs were immunized on gestational day 25 and 46 with placebo (PBS), 30µg RSV F, or 30µg RSV F+400µg aluminum phosphate. Sera at delivery/birth (sows/pups) and 15 and 30 days post-partum (pups) were analyzed for the presence of anti-F IgG, palivizumab-competitive antibody (PCA) and RSV/A microneutralization (MN). RESULTS: The rates of pregnancy and stillbirth were similar between controls and vaccinees. The vaccine induced high levels of anti-F IgG, PCA and MN in sows, with the highest levels observed in adjuvanted vaccinees. Placental transfer to pups was proportional to the maternal antibody levels, with concentration effects observed for all immune measures. CONCLUSIONS: The RSV F vaccine was safe and immunogenic in pregnant guinea pigs and supported robust transplacental antibody transfer to their pups. Relative concentration of antibodies in the pups was observed even in the presence of high levels of maternal antibody. Guinea pigs may be an important safety and immunogenicity model for preclinical assessment of candidate vaccines for maternal immunization.

An insect cell derived respiratory syncytial virus (RSV) F nanoparticle vaccine induces antigenic site II antibodies and protects against RSV challenge in cotton rats by active and passive immunization.

Post-infectious immunity to respiratory syncytial virus (RSV) infection results in limited protection as evidenced by the high rate of infant hospitalization in the face of high titer, maternally derived RSV-specific antibodies. By contrast, RSV fusion (F) glycoprotein antigenic site II humanized monoclonal antibodies, palivizumab and motavizumab, have been shown to reduce RSV-related hospitalization in infants. Immunogenicity and efficacy studies were conducted in cotton rats comparing a recombinant RSV F nanoparticle vaccine with palivizumab and controlled with live RSV virus intranasal immunization and, formalin inactivated RSV vaccine. Active immunization with RSV F nanoparticle vaccine containing an alum adjuvant induced serum levels of palivizumab competing antibody (PCA) greater than passive administration of 15 mg/kg palivizumab (human prophylactic dose) in cotton rats and neutralized RSV-A and RSV-B viruses. Immunization prevented detectable RSV replication in the lungs and, unlike passive administration of palivizumab, in the nasal passage of challenged cotton rats. Histology of lung tissues following RSV challenge showed no enhanced disease in the vaccinated groups in contrast to formalin inactivated 'Lot 100' vaccine. Passive intramuscular administration of RSV F vaccine-induced immune sera one day prior to challenge of cotton rats reduced viral titers by 2 or more log10 virus per gram of lung and nasal tissue and at doses less than palivizumab. A recombinant RSV F nanoparticle vaccine protected lower and upper respiratory tract against both RSV A and B strain infection and induced polyclonal palivizumab competing antibodies similar to but potentially more broadly protective against RSV than palivizumab.

Safety and immunogenicity of a Sf9 insect cell-derived respiratory syncytial virus fusion protein nanoparticle vaccine.

OBJECTIVE: We performed a Phase 1 randomized, observer-blinded, placebo-controlled trial to evaluate the safety and immunogenicity of a recombinant respiratory syncytial virus (RSV) fusion (F) protein nanoparticle vaccine. METHODS: Six formulations with (5, 15, 30 and 60 µg) and without (30 and 60 µg) aluminum phosphate (AdjuPhos) were administered intramuscularly on day 0 and 30 in a dose escalating fashion to healthy adults 18-49 years of age. Solicited and unsolicited events were collected through day 210. Immunogenicity measures taken at day 0, 30 and 60 included RSV A and B microneutralization, anti-F IgG, antigenic site II peptide and palivizumab competitive antibodies. RESULTS: The vaccine was well-tolerated, with no evident dose-related toxicity or attributable SAEs. At day 60 both RSV A and B microneutralization was significantly increased in vaccinees versus placebo. Across all vaccinees there was a 7- to 19-fold increase in the anti-F IgG and a 7- to 24-fold increase in the antigenic site II binding and palivizumab competitive antibodies. CONCLUSIONS: The RSV F nanoparticle vaccine candidate was well tolerated without dose-related increases in adverse events. Measures of immunity indicate that neutralization, anti-RSV F IgG titers and palivizumab competing antibodies were induced at levels that have been associated with decreased risk of hospitalization. NCT01290419.

Respiratory syncytial virus fusion glycoprotein expressed in insect cells form protein nanoparticles that induce protective immunity in cotton rats.

Respiratory Syncytial Virus (RSV) is an important viral agent causing severe respiratory tract disease in infants and children as well as in the elderly and immunocompromised individuals. The lack of a safe and effective RSV vaccine represents a major unmet medical need. RSV fusion (F) surface glycoprotein was modified and cloned into a baculovirus vector for efficient expression in Sf9 insect cells. Recombinant RSV F was glycosylated and cleaved into covalently linked F2 and F1 polypeptides that formed homotrimers. RSV F extracted and purified from insect cell membranes assembled into 40 nm protein nanoparticles composed of multiple RSV F oligomers arranged in the form of rosettes. The immunogenicity and protective efficacy of purified RSV F nanoparticles was compared to live and formalin inactivated RSV in cotton rats. Immunized animals induced neutralizing serum antibodies, inhibited virus replication in the lungs, and had no signs of disease enhancement in the respiratory track of challenged animals. RSV F nanoparticles also induced IgG competitive for binding of palivizumab neutralizing monoclonal antibody to RSV F antigenic site II. Antibodies to this epitope are known to protect against RSV when passively administered in high risk infants. Together these data provide a rational for continued development a recombinant RSV F nanoparticle vaccine candidate.

A rapid Flp-In system for expression of secreted H5N1 influenza hemagglutinin vaccine immunogen in mammalian cells.

BACKGROUND: Continuing transmissions of highly pathogenic H5N1 viruses in poultry and humans underscores the need for a rapid response to potential pandemic in the form of vaccine. Recombinant technologies for production of immunogenic hemagglutinin (HA) could provide an advantage over the traditional inactivated vaccine manufacturing process. Generation of stably transfected mammalian cells secreting properly folded HA proteins is important for scalable controlled manufacturing. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a Flp-In based 293 stable cell lines through targeted site-specific recombination for expression of secreted hemagglutinin (HA) proteins and evaluated their immunogenicity. H5N1 globular domain HA1(1-330) and HA0(1-500) proteins were purified from the supernatants of 293 Flp-In stable cell lines. Both proteins were properly folded as confirmed by binding to H5N1-neutralizing conformation-dependent human monoclonal antibodies. The HA0 (with unmodified cleavage site) was monomeric, while the HA1 contained oligomeric forms. Upon rabbit immunization, both HA proteins elicited neutralizing antibodies against the homologous virus (A/Vietnam/1203/2004, clade 1) as well as cross-neutralizing antibodies against heterologous H5N1 clade 2 strains, including A/Indonesia/5/2005. These results exceeded the human antibody responses against the inactivated sub-virion H5N1 vaccine. CONCLUSIONS/SIGNIFICANCE: Our data suggest that the 293 Flp-In system could serve as a platform for rapid expression of HA immunogens in mammalian cells from emerging influenza strains.

Novel functions for TAF7, a regulator of TAF1-independent transcription.

The transcription factor TFIID components TAF7 and TAF1 regulate eukaryotic transcription initiation. TAF7 regulates transcription initiation of TAF1-dependent genes by binding to the acetyltransferase (AT) domain of TAF1 and inhibiting the enzymatic activity that is essential for transcription. TAF7 is released from the TAF1-TFIID complex upon completion of preinitiation complex assembly, allowing transcription to initiate. However, not all transcription is TAF1-dependent, and the role of TAF7 in regulating TAF1-independent transcription has not been defined. The IFNγ-induced transcriptional co-activator CIITA activates MHC class I and II genes, which are vital for immune responses, in a TAF1-independent manner. Activation by CIITA depends on its intrinsic AT activity. We now show that TAF7 binds to CIITA and inhibits its AT activity, thereby repressing activated transcription. Consistent with this TAF7 function, siRNA-mediated depletion of TAF7 resulted in increased CIITA-dependent transcription. A more global role for TAF7 as a regulator of transcription was revealed by expression profiling analysis: expression of 30-40% of genes affected by TAF7 depletion was independent of either TAF1 or CIITA. Surprisingly, although TAF1-dependent transcripts were largely down-regulated by TAF7 depletion, TAF1-independent transcripts were predominantly up-regulated. We conclude that TAF7, until now considered only a TFIID component and regulator of TAF1-dependent transcription, also regulates TAF1-independent transcription.

TFIID component TAF7 functionally interacts with both TFIIH and P-TEFb.

Transcription consists of a series of highly regulated steps: assembly of the preinitiation complex (PIC) at the promoter, initiation, elongation, and termination. PIC assembly is nucleated by TFIID, a complex composed of the TATA-binding protein (TBP) and a series of TBP-associated factors (TAFs). One component, TAF7, is incorporated in the PIC through its interaction with TFIID but is released from TFIID upon transcription initiation. We now report that TAF7 interacts with the transcription factors, TFIIH and P-TEFb, resulting in the inhibition of their Pol II CTD kinase activities. Importantly, in in vitro transcription reactions, TAF7 inhibits steps after PIC assembly and formation of the first phosphodiester bonds. Further, in vivo TAF7 coelongates with P-TEFb and Pol II downstream of the promoter. We propose a model in which TAF7 contributes to the regulation of the transition from PIC assembly to initiation and elongation.

Coactivator-associated arginine methyltransferase 1 enhances transcriptional activity of the human T-cell lymphotropic virus type 1 long terminal repeat through direct interaction with Tax.

In this study, we demonstrate that the coactivator-associated arginine methyltransferase 1 (CARM1), which methylates histone H3 and other proteins such as p300/CBP, is positively involved in the regulation of Tax transactivation. First, transfection studies demonstrated that overexpression of CARM1 wild-type protein resulted in increased Tax transactivation of the human T-cell lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR). In contrast, transfection of a catalytically inactive CARM1 methyltransferase mutant did not enhance Tax transactivation. CARM1 facilitated Tax transactivation of the CREB-dependent cellular GEM promoter. A direct physical interaction between HTLV-1 Tax and CARM1 was demonstrated using in vitro glutathione S-transferase-Tax binding assays, in vivo coimmunoprecipitation, and confocal microscopy experiments. Finally, chromatin immunoprecipitation analysis of the activated HTLV-1 LTR promoter showed the association of CARM1 and methylated histone H3 with the template DNA. In vitro, Tax facilitates the binding of CARM1 to the transcription complex. Together, our data provide evidence that CARM1 enhances Tax transactivation of the HTLV-1 LTR through a direct interaction between CARM1 and Tax and this binding promotes methylation of histone H3 (R2, R17, and R26).

HDAC1 acetylation is linked to progressive modulation of steroid receptor-induced gene transcription.

Although histone deacetylases (HDACs) are generally viewed as corepressors, we show that HDAC1 serves as a coactivator for the glucocorticoid receptor (GR). Furthermore, a subfraction of cellular HDAC1 is acetylated after association with the GR, and this acetylation event correlates with a decrease in promoter activity. HDAC1 in repressed chromatin is highly acetylated, while the deacetylase found on transcriptionally active chromatin manifests a low level of acetylation. Acetylation of purified HDAC1 inactivates its deacetylase activity, and mutation of the critical acetylation sites abrogates HDAC1 function in vivo. We propose that hormone activation of the receptor leads to progressive acetylation of HDAC1 in vivo, which in turn inhibits the deacetylase activity of the enzyme and prevents a deacetylation event that is required for promoter activation. These findings indicate that HDAC1 is required for the induction of some genes by the GR, and this activator function is dynamically modulated by acetylation.

Tax interacts with P-TEFb in a novel manner to stimulate human T-lymphotropic virus type 1 transcription.

Human T-lymphotropic virus type 1 (HTLV-1) encodes a transcriptional activator, Tax, whose function is essential for viral transcription and replication. Tax transactivates the viral long-terminal repeat through a series of protein-protein interactions which facilitate CREB and CBP/p300 binding. In addition, Tax dissociates transcription repressor histone deacetylase 1 interaction with the CREB response element. The subsequent events through which Tax interacts and communicates with RNA polymerase II and cyclin-dependent kinases (CDKs) are not clearly understood. Here we present evidence that Tax recruits positive transcription elongation factor b (P-TEFb) (CDK9/cyclin T1) to the viral promoter. This recruitment likely involves protein-protein interactions since Tax associates with P-TEFb in vitro as demonstrated by glutathione S-transferase fusion protein pull-down assays and in vivo as shown by co-immunoprecipitation assays. Functionally, small interfering RNA directed toward CDK9 inhibited Tax transactivation in transient assays. Consistent with these findings, the depletion of CDK9 from nuclear extracts inhibited Tax transactivation in vitro. Reconstitution of the reaction with wild-type P-TEFb, but not a kinase-dead mutant, recovered HTLV-1 transcription. Moreover, the addition of the CDK9 inhibitor flavopiridol blocked Tax transactivation in vitro and in vivo. Interestingly, we found that Tax regulates CDK9 kinase activity through a novel autophosphorylation pathway.

Tax relieves transcriptional repression by promoting histone deacetylase 1 release from the human T-cell leukemia virus type 1 long terminal repeat.

Expression of human T-cell leukemia virus type 1 (HTLV-1) is regulated by the viral transcriptional activator Tax. Tax activates viral transcription through interaction with the cellular transcription factor CREB and the coactivators CBP/p300. In this study, we have analyzed the role of histone deacetylase 1 (HDAC1) on HTLV-1 gene expression from an integrated template. First we show that trichostatin A, an HDAC inhibitor, enhances Tax expression in HTLV-1-transformed cells. Second, using a cell line containing a single-copy HTLV-1 long terminal repeat, we demonstrate that overexpression of HDAC1 represses Tax transactivation. Furthermore, a chromatin immunoprecipitation assay allowed us to analyze the interaction of transcription factors, coactivators, and HDACs with the basal and activated HTLV-1 promoter. We demonstrate that HDAC1 is associated with the inactive, but not the Tax-transactivated, HTLV-1 promoter. In vitro and in vivo glutathione S-transferase-Tax pull-down and coimmunoprecipitation experiments demonstrated that there is a direct physical association between Tax and HDAC1. Importantly, biotinylated chromatin pull-down assays demonstrated that Tax inhibits and/or dissociates the binding of HDAC1 to the HTLV-1 promoter. Our results provide evidence that Tax interacts directly with HDAC1 and regulates binding of the repressor to the HTLV-1 promoter.