Safety and immunogenicity of a novel trivalent recombinant MVA-based equine encephalitis virus vaccine: A Phase 1 clinical trial.

BACKGROUND: Three encephalitic alphaviruses-western, eastern, and Venezuelan equine encephalitis virus (WEEV, EEEV and VEEV)-can cause severe disease and have the potential to be used as biological weapons. There are no approved vaccines for human use. A novel multivalent MVA-BN-WEV vaccine encodes the envelope surface proteins of the 3 viruses and is thereby potentially able to protect against them all, as previously demonstrated in animal models. This first-in-human study assessed the safety, tolerability, and immunogenicity of MVA-BN-WEV vaccine in healthy adult participants. METHODS: Forty-five participants were enrolled into 3 dose groups (1 × 10E7 Inf.U, 1 × 10E8 Inf.U, and 2 × 10E8 Inf.U), received 2 doses 4 weeks apart, and were then monitored for 6 months. RESULTS: The safety profile of MVA-BN-WEV was acceptable at all administered doses, with incidence of local solicited AEs increased with increasing dose and no other clinically meaningful differences between dose groups. One SAE (Grade 2 pleural effusion) was reported in the lowest dose group and assessed as possibly related. No AEs resulted in death or led to withdrawal from the second vaccination or from the trial. The most common local solicited AE was injection site pain, and general solicited AEs were headache, fatigue, and myalgia. MVA-BN-WEV induced humoral immune responses; WEEV-, EEEV- and VEEV-specific neutralizing antibody responses peaked 2 weeks following the second vaccination, and the magnitude of these responses increased with dose escalation. The highest dose resulted in seroconversion of all (100 %) participants for WEEV and VEEV and 92.9 % for EEEV, 2 weeks following second vaccination, and durability was observed for 6 months. MVA-BN-WEV induced cellular immune responses to VEEV E1 and E2 (EEEV and WEEV not tested) and a dose effect for peptide pool E2. CONCLUSION: The study demonstrated that MVA-BN-WEV is well tolerated, induces immune responses, and is suitable for further development. CLINICAL TRIAL REGISTRY NUMBER: NCT04131595.

Reduced Respiratory Syncytial Virus Load, Symptoms, and Infections: A Human Challenge Trial of MVA-BN-RSV Vaccine.

BACKGROUND: Respiratory syncytial virus (RSV) causes significant disease burden in older adults. MVA-BN-RSV is a novel poxvirus-vectored vaccine encoding internal and external RSV proteins. METHODS: In a phase 2a randomized double-blind, placebo-controlled trial, healthy participants aged 18 to 50 years received MVA-BN-RSV or placebo, then were challenged 4 weeks later with RSV-A Memphis 37b. Viral load was assessed from nasal washes. RSV symptoms were collected. Antibody titers and cellular markers were assessed before and after vaccination and challenge. RESULTS: After receiving MVA-BN-RSV or placebo, 31 and 32 participants, respectively, were challenged. Viral load areas under the curve from nasal washes were lower (P = .017) for MVA-BN-RSV (median = 0.00) than placebo (median = 49.05). Total symptom scores also were lower (median = 2.50 and 27.00, respectively; P = .004). Vaccine efficacy against symptomatic, laboratory-confirmed or culture-confirmed infection was 79.3% to 88.5% (P = .022 and .013). Serum immunoglobulin A and G titers increased approximately 4-fold after MVA-BN-RSV vaccination. Interferon-γ-producing cells increased 4- to 6-fold after MVA-BN-RSV in response to stimulation with the encoded RSV internal antigens. Injection site pain occurred more frequently with MVA-BN-RSV. No serious adverse events were attributed to vaccination. CONCLUSIONS: MVA-BN-RSV vaccination resulted in lower viral load and symptom scores, fewer confirmed infections, and induced humoral and cellular responses. CLINICAL TRIALS REGISTRATION: NCT04752644.

A randomized phase 3 trial to assess the immunogenicity and safety of 3 consecutively produced lots of freeze-dried MVA-BN® vaccine in healthy adults.

Since vaccination remains the only effective protection against orthopox virus-induced diseases such as smallpox or monkeypox, the strategic use and stockpiling of these vaccines remains of significant public health importance. The approved liquid-frozen formulation of Bavarian Nordic's Modified Vaccinia Ankara (MVA-BN) smallpox vaccine has specific cold-chain requirements, while the freeze-dried (FD) formulation of this vaccine provides more flexibility in terms of storage conditions and shelf life. In this randomized phase 3 trial, the immunogenicity and safety of 3 consecutively manufactured lots of the FD MVA-BN vaccine was evaluated. A total of 1129 healthy adults were randomized to 3 treatment groups (lots 1 to 3) and received 2 vaccinations 4 weeks apart. For both neutralizing and total antibodies, a robust increase of geometric mean titer (GMT) was observed across all lot groups 2 weeks following the second vaccination, comparable to published data. For the primary results, the ratios of the neutralizing antibody GMTs between the lot group pairs ranged from 0.936 to 1.115, with confidence ratios well within the pre-specified margin of equivalence. Results for total antibodies were similar. In addition, seroconversion rates were high across the 3 lots, ranging between 99.1 % and 99.7 %. No safety concerns were identified; particularly, no inflammatory cardiac disorders were detected. The most common local solicited adverse events (AEs) reported across lot groups were injection site pain (87.2%) and erythema (73.2%), while the most common general solicited adverse events were myalgia, fatigue, and headache in 40.6% to 45.5% of all participants, with no meaningful differences among the lot groups. No related serious AEs were reported. In conclusion, the data demonstrate consistent and robust immunogenicity and safety results with a freeze-dried formulation of MVA-BN. Clinical Trial Registry Number: NCT03699124.

One- and Two-Dose Vaccinations With Modified Vaccinia Ankara-Bavarian Nordic Induce Durable B-Cell Memory Responses Comparable to Replicating Smallpox Vaccines.

BACKGROUND: Although modified vaccinia Ankara-Bavarian Nordic (MVA-BN) vaccination is approved for smallpox and monkeypox prevention, immunological persistence and booster effects remain undescribed. METHODS: Participants naive to smallpox vaccination were randomized to 1 dose MVA-BN (1×MVA, n = 181), 2 doses MVA-BN (2×MVA, n = 183), or placebo (n = 181). Participants with previous smallpox vaccination received 1 MVA-BN booster (HSPX, n = 200). Subsets of the formerly naive groups (approximately 75 each) received an MVA-BN booster 2 years later. RESULTS: Neutralizing antibody (nAb) geometric mean titers (GMTs) increased from 1.1 (baseline, both naive groups) to 7.2 and 7.5 (week 4, 1×MVA and 2×MVA, respectively), and further to 45.6 (week 6, 2×MVA after second vaccination). In HSPX, nAb GMT rapidly increased from 21.6 (baseline) to 175.1 (week 2). At 2 years, GMTs for 1×MVA, 2×MVA, and HSPX were 1.1, 1.3, and 10.3, respectively. After boosting in the previously naive groups, nAb GMTs increased rapidly in 2 weeks to 80.7 (1×MVA) and 125.3 (2×MVA), higher than after primary vaccination and comparable to boosted HSPX subjects. Six months after boosting, GMTs were 25.6 (1×MVA) and 49.3 (2×MVA). No safety concerns were identified. CONCLUSIONS: Anamnestic responses to boosting without sustained high nAb titers support presence of durable immunological memory following primary MVA-BN immunization. Clinical Trials Registration. NCT00316524 and NCT00686582.

Broad Antibody and Cellular Immune Response From a Phase 2 Clinical Trial With a Novel Multivalent Poxvirus-Based Respiratory Syncytial Virus Vaccine.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in young children and the elderly. Protective immunity is not generated after repeated infections, but vaccination may hopefully prove effective. METHODS: This phase 2 clinical study investigated a multivalent RSV vaccine (MVA-BN-RSV) designed to induce broad antibody and cellular immune responses by encoding RSV surface proteins F, G (for both A and B subtypes), and internal antigens (M2, N). This study evaluated the immune response in adults aged ≥55 years to identify the optimal MVA-BN-RSV dose and vaccination schedule. RESULTS: A single dose increased the levels of neutralizing (plaque reduction neutralization test to RSV A and B) and total (IgG and IgA ELISA) antibodies (1.6 to 3.4-fold increase from baseline) and induced a broad Th1-biased cellular immune response (interferon-γ ELISPOT) to all 5 vaccine inserts (5.4 to 9.7-fold increases). Antibody responses remained above baseline for 6 months. A 12-month booster dose elicited a booster effect in antibody and T-cell responses (up to 2.8-fold from preboost levels). No drug-related serious adverse events were reported. CONCLUSIONS: MVA-BN-RSV induces a broad immune response that persists at least 6 months and can be boosted at 12 months, without significant safety findings. CLINICAL TRIALS REGISTRATION: NCT02873286.

A randomized phase II trial to compare safety and immunogenicity of the MVA-BN smallpox vaccine at various doses in adults with a history of AIDS.

Traditional replicating smallpox vaccines are associated with serious safety concerns in the general population and are contraindicated in immunocompromised individuals. However, this very population remains at greatest risk for severe complications following viral infections, making vaccine prevention particularly relevant. MVA-BN was developed as a non-replicating smallpox vaccine that is potentially safer for people who are immunocompromised. In this phase II trial, 3 MVA-BN dosing regimens were evaluated for safety, tolerability, and immunogenicity in persons with HIV (PWH) who had a history of AIDS. Following randomization, 87 participants who were predominately male and African American received either 2 standard doses on weeks 0 and 4 in the standard dose (SD) group (N = 27), 2 double-standard doses on the same schedule in the double dose (DD) group (N = 29), or 3 standard doses on weeks 0, 4 and 12 in the booster dose (BD) group (N = 31). No safety concerns were identified, and injection site pain was the most commonly reported solicited adverse event (AE) in all groups (66.7%), with no meaningful differences between groups. The incidence of severe (Grade 3) AEs was low across groups and no serious AEs or AEs of special interest considered related to study vaccine were reported. Doubling the standard MVA-BN dose had no significant effect on induction of neutralizing antibodies, with 100% seroconversion and comparable GMTs at week 6 in the SD and DD groups (78.9 and 100.3, respectively). A booster dose significantly increased peak neutralizing titers in the BD group (GMT: 281.1), which remained elevated at 12 months (GMT: 45.3) compared to the SD (GMT: 6.2) and DD (GMT: 10.6) groups. However, based on the immune response previously reported for healthy participants, a third dose (booster) does not appear necessary, even for immunocompromised participants. Clinical Trial Registry Number: NCT02038881.

Safety and immunogenicity of novel modified vaccinia Ankara-vectored RSV vaccine: A randomized phase I clinical trial.

Respiratory disease caused by RSV infection is recognized as a severe public health issue in infants, young children and elderly with no specific treatment option. Vaccination may be the most effective strategy to combat this highly infectious virus although no vaccine has been approved. The novel vaccine candidate MVA-BN-RSV encodes RSV surface proteins F and G (subtypes A, B) as well as internal proteins N and M2 in the MVA-BN viral vector backbone to provide broad protection against RSV. This was a first in human study to investigate safety, reactogenicity and immunogenicity of MVA-BN-RSV. Sixty-three participants were allocated to 3 groups: adult (18-49 years) low (1 × 107 TCID50) or high (1 × 108 TCID50) dose and older adult (50-65 years) high dose. Participants in each group were randomized in a 6:1 ratio to receive 2 doses of MVA-BN-RSV or placebo 4 weeks apart and were monitored for 30 weeks. All participants completed the study, receiving both doses. No serious AEs or AEs of special interest were reported. The most common AEs were injection site pain (56% in the combined high dose groups, 17% in the low dose group). MVA-BN-RSV induced robust T cell responses covering all 5 inserts with fold increases ranging from 1.8 to 3.8. Higher and broader responses were observed in the high dose groups (83% responders to at least 3 peptide pools in the combined high dose groups compared to 63% in the low dose group). Moderate but consistent humoral responses were observed against A and B RSV subtypes (up to approximately 2-fold increases in the high dose groups). No differences were observed between the adult and the older adult groups in safety, reactogenicity or immunogenicity. The study demonstrated that the well tolerated MVA-BN-RSV vaccine candidate induces broad cellular and humoral immune responses, warranting further development.

Phase 3 Efficacy Trial of Modified Vaccinia Ankara as a Vaccine against Smallpox.

BACKGROUND: Many countries have stockpiled vaccines because of concerns about the reemergence of smallpox. Traditional smallpox vaccines are based on replicating vaccinia viruses; these vaccines have considerable side effects. METHODS: To evaluate the efficacy of modified vaccinia Ankara (MVA) as a potential smallpox vaccine, we randomly assigned 440 participants to receive two doses of MVA followed by one dose of the established replicating-vaccinia vaccine ACAM2000 (the MVA group) or to receive one dose of ACAM2000 (the ACAM2000-only group). The two primary end points were noninferiority of the MVA vaccine to ACAM2000 with respect to the peak serum neutralizing antibody titers and attenuation of the ACAM2000-associated major cutaneous reaction by previous MVA vaccination, measured according to the maximum lesion area and the derived area attenuation ratio. RESULTS: A total of 220 and 213 participants were randomly assigned and vaccinated in the MVA group and ACAM2000-only group, respectively, and 208 participants received two MVA vaccinations. At peak visits, MVA vaccination induced a geometric mean titer of neutralizing antibodies of 153.5 at week 6, as compared with 79.3 at week 4 with ACAM2000 (a ratio of 1.94 [95% confidence interval {CI}, 1.56 to 2.40]). At day 14, the geometric mean titer of neutralizing antibodies induced by a single MVA vaccination (16.2) was equal to that induced by ACAM2000 (16.2), and the percentages of participants with seroconversion were similar (90.8% and 91.8%, respectively). The median lesion areas of the major cutaneous reaction were 0 mm2 in the MVA group and 76.0 mm2 in the ACAM2000-only group, resulting in an area attenuation ratio of 97.9% (95% CI, 96.6 to 98.3). There were fewer adverse events or adverse events of grade 3 or higher after both MVA vaccination periods in the MVA group than in the ACAM2000-only group (17 vs. 64 participants with adverse events of grade 3 or higher, P<0.001). CONCLUSIONS: No safety concerns associated with the MVA vaccine were identified. Immune responses and attenuation of the major cutaneous reaction suggest that this MVA vaccine protected against variola infection. (Funded by the Office of the Assistant Secretary for Preparedness and Response Biomedical Advanced Research and Development Authority of the Department of Health and Human Services and Bavarian Nordic; ClinicalTrials.gov number, NCT01913353.).

Immunogenicity and safety of three consecutive production lots of the non replicating smallpox vaccine MVA: A randomised, double blind, placebo controlled phase III trial.

BACKGROUND: Modified Vaccinia Ankara (MVA) is a live, viral vaccine under advanced development as a non-replicating smallpox vaccine. A randomised, double-blind, placebo-controlled phase III clinical trial was conducted to demonstrate the humoral immunogenic equivalence of three consecutively manufactured MVA production lots, and to confirm the safety and tolerability of MVA focusing on cardiac readouts. METHODS: The trial was conducted at 34 sites in the US. Vaccinia-naïve adults aged 18-40 years were randomly allocated to one of four groups using a 1:1:1:1 randomization scheme. Subjects received either two MVA injections from three consecutive lots (Groups 1-3), or two placebo injections (Group 4), four weeks apart. Everyone except personnel involved in vaccine handling and administration was blinded to treatment. Safety assessment focused on cardiac monitoring throughout the trial. Vaccinia-specific antibody titers were measured using a Plaque Reduction Neutralization Test (PRNT) and an Enzyme-Linked Immunosorbent Assay (ELISA). The primary immunogenicity endpoint was Geometric Mean Titers (GMTs) after two MVA vaccinations measured by PRNT at trial visit 4. This trial is registered with ClinicalTrials.gov, number NCT01144637. RESULTS: Between March 2013 and May 2014, 4005 subjects were enrolled and received at least one injection of MVA (n = 3003) or placebo (n = 1002). The three MVA lots induced equivalent antibody titers two weeks after the second vaccination, with seroconversion rates of 99·8% (PRNT) and 99·7% (ELISA). Overall, 180 (6·0%) subjects receiving MVA and 29 (2·9%) subjects in the placebo group reported at least one unsolicited Adverse Event (AE) that was considered trial-related. Vaccination was well tolerated without significant safety concerns, particularly regarding cardiac assessment. CONCLUSIONS: The neutralizing and total antibody titers induced by each of the three lots were equivalent. No significant safety concerns emerged in this healthy trial population, especially regarding cardiac safety, thus confirming the excellent safety and tolerability profile of MVA. TRIAL REGISTRATION: ClinicalTrials.gov NCT01144637.

A Randomized, Double-Blind, Placebo-Controlled Phase II Trial Investigating the Safety and Immunogenicity of Modified Vaccinia Ankara Smallpox Vaccine (MVA-BN®) in 56-80-Year-Old Subjects.

BACKGROUND: Modified Vaccinia Ankara MVA-BN® is a live, highly attenuated, viral vaccine under advanced development as a non-replicating smallpox vaccine. In this Phase II trial, the safety and immunogenicity of Modified Vaccinia Ankara MVA-BN® (MVA) was assessed in a 56-80 years old population. METHODS: MVA with a virus titer of 1 x 108 TCID50/dose was administered via subcutaneous injection to 56-80 year old vaccinia-experienced subjects (N = 120). Subjects received either two injections of MVA (MM group) or one injection of Placebo and one injection of MVA (PM group) four weeks apart. Safety was evaluated by assessment of adverse events (AE), focused physical exams, electrocardiogram recordings and safety laboratories. Solicited AEs consisted of a set of pre-defined expected local reactions (erythema, swelling, pain, pruritus, and induration) and systemic symptoms (body temperature, headache, myalgia, nausea and fatigue) and were recorded on a memory aid for an 8-day period following each injection. The immunogenicity of the vaccine was evaluated in terms of humoral immune responses measured with a vaccinia-specific enzyme-linked immunosorbent assay (ELISA) and a plaque reduction neutralization test (PRNT) before and at different time points after vaccination. RESULTS: Vaccinations were well tolerated by all subjects. No serious adverse event related to MVA and no case of myopericarditis was reported. The overall incidence of unsolicited AEs was similar in both groups. For both groups immunogenicity responses two weeks after the final vaccination (i.e. Visit 4) were as follows: Seroconversion (SC) rates (doubling of titers from baseline) in vaccine specific antibody titers measured by ELISA were 83.3% in Group MM and 82.8% in Group PM (difference 0.6% with 95% exact CI [-13.8%, 15.0%]), and 90.0% for Group MM and 77.6% for Group PM measured by PRNT (difference 12.4% with 95% CI of [-1.1%, 27.0%]). Geometric mean titers (GMT) measured by ELISA two weeks after the final vaccination for Group MM were 804.1 and 605.8 for Group PM (with ratio of GMTs of 1.33 with 95% CI of [0.96, 1.84]). Similarly, GMTs measured by PRNT were 210.3 for Group MM and 126.7 for Group PM (with ratio 1.66 and 95% CI [0.95, 2.90]). CONCLUSIONS: One or two doses of MVA were safe and immunogenic in a 56-80 years old vaccinia-experienced population. No cases of myopericarditis were observed following vaccinations with MVA. The safety, reactogenicity and immunogenicity were similar to that seen in younger (18-55 year old) healthy populations as investigated in other MVA trials. The results suggest that a single dose of MVA in a 56-80 years old population was well tolerated and sufficient to rapidly boost the long-term B cell memory response induced by a prior vaccination with a traditional smallpox vaccine. TRIAL REGISTRATION: ClinicalTrials.gov NCT00857493.

A Multicenter, Open-Label, Controlled Phase II Study to Evaluate Safety and Immunogenicity of MVA Smallpox Vaccine (IMVAMUNE) in 18-40 Year Old Subjects with Diagnosed Atopic Dermatitis.

BACKGROUND: Replicating smallpox vaccines can cause severe complications in individuals with atopic dermatitis (AD). Prior studies evaluating Modified Vaccinia Ankara virus (MVA), a non-replicating vaccine in humans, showed a favorable safety and immunogenicity profile in healthy volunteers. OBJECTIVE: This Phase II study compared the safety and immunogenicity of MVA enrolling groups of 350 subjects with AD (SCORAD ≤ 30) and 282 healthy subjects. METHODS: Subjects were vaccinated twice with MVA, each dose given subcutaneously 4 weeks apart. Adverse events, cardiac parameters, and the development of vaccinia virus humoral immune responses were monitored. RESULTS: The overall safety of the vaccine was similar in both groups. Adverse events affecting skin were experienced significantly more often in subjects with AD, but the majority of these events were mild to moderate in intensity. Seroconversion rates and geometric mean titers for total and neutralizing vaccinia-specific antibodies in the AD group were non-inferior compared to the healthy subjects. LIMITATIONS: The size of the study population limited the detection of serious adverse events occurring at a frequency less than 1%. CONCLUSION: MVA has a favorable safety profile and the ability to elicit vaccinia-specific immune responses in subjects with AD. TRIAL REGISTRATION: ClinicalTrials.gov NCT00316602.

Sumoylation of poly(ADP-ribose) polymerase 1 inhibits its acetylation and restrains transcriptional coactivator function.

Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated nuclear protein and functions as a molecular stress sensor. At the cellular level, PARP1 has been implicated in a wide range of processes, such as maintenance of genome stability, cell death, and transcription. PARP1 functions as a transcriptional coactivator of nuclear factor kappaB (NF-kappaB) and hypoxia inducible factor 1 (HIF1). In proteomic studies, PARP1 was found to be modified by small ubiquitin-like modifiers (SUMOs). Here, we characterize PARP1 as a substrate for modification by SUMO1 and SUMO3, both in vitro and in vivo. PARP1 is sumoylated at the single lysine residue K486 within its automodification domain. Interestingly, modification of PARP1 with SUMO does not affect its ADP-ribosylation activity but completely abrogates p300-mediated acetylation of PARP1, revealing an intriguing crosstalk of sumoylation and acetylation on PARP1. Genetic complementation of PARP1-depleted cells with wild-type and sumoylation-deficient PARP1 revealed that SUMO modification of PARP1 restrains its transcriptional coactivator function and subsequently reduces gene expression of distinct PARP1-regulated target genes.

Constitutive crosspresentation of tissue antigens by dendritic cells controls CD8+ T cell tolerance in vivo.

Immature dendritic cells (DCs) sample tissue-specific antigens (TSAs) and process them for "crosspresentation" via major histocompatibility complex (MHC) class I and II molecules. Findings with adoptively transferred T cell receptor (TCR)-transgenic CD8+ T cells in transgenic mice expressing model TSA indicate that this process contributes to tolerance induction of CD8+ T cells, a phenomenon termed "crosstolerance." However, up to now it has been unknown whether "crosstolerance" can also control autoimmune T cells specific for physiological nontransgenic TSA. Here, we showed that a DC-specific deficiency in uptake of apoptotic material inhibits crosspresentation in vivo. This defect allowed the accumulation of fully functional autoreactive CD8+ T cells that could be activated for autoimmune attack in peripheral lymphoid organs. Thus, our data demonstrate the importance of crosstolerance induction by DCs as a vital instrument for controlling self-reactive T cells from the peripheral repertoire and preventing autoimmune disease.

Synergy of glucose and growth hormone signalling in islet cells through ICA512 and STAT5.

Nutrients and growth hormones promote insulin production and the proliferation of pancreatic beta-cells. An imbalance between ever-increasing metabolic demands and insulin output causes diabetes. Recent evidence indicates that beta-cells enhance insulin gene expression depending on their secretory activity. This signalling pathway involves a catalytically inactive receptor tyrosine phosphatase, ICA512, whose cytoplasmic tail is cleaved on glucose-stimulated exocytosis of insulin secretory granules and then moves into the nucleus, where it upregulates insulin transcription. Here, we show that the cleaved cytosolic fragment of ICA512 enhances the transcription of secretory granule genes (including its own gene) by binding to tyrosine phosphorylated signal transducers and activators of transcription (STAT) 5 and preventing its dephosphorylation. Sumoylation of ICA512 by the E3 SUMO ligase PIASy, in turn, may reverse this process by decreasing the binding of ICA512 to STAT5. These findings illustrate how the exocytosis of secretory granules, through a retrograde pathway that sustains STAT activity, converges with growth hormone signalling to induce adaptive changes in beta-cells in response to metabolic demands.

Down-regulation of c-Fos/c-Jun AP-1 dimer activity by sumoylation.

The inducible transcriptional complex AP-1, composed of c-Fos and c-Jun proteins, is crucial for cell adaptation to many environmental changes. While its mechanisms of activation have been extensively studied, how its activity is restrained is poorly understood. We report here that lysine 265 of c-Fos is conjugated by the peptidic posttranslational modifiers SUMO-1, SUMO-2, and SUMO-3 and that c-Jun can be sumoylated on lysine 257 as well as on the previously described lysine 229. Sumoylation of c-Fos preferentially occurs in the context of c-Jun/c-Fos heterodimers. Using nonsumoylatable mutants of c-Fos and c-Jun as well as a chimeric protein mimicking sumoylated c-Fos, we show that sumoylation entails lower AP-1 transactivation activity. Interestingly, single sumoylation at any of the three acceptor sites of the c-Fos/c-Jun dimer is sufficient to substantially reduce transcription activation. The lower activity of sumoylated c-Fos is not due to inhibition of protein entry into the nucleus, accelerated turnover, and intrinsic inability to dimerize or to bind to DNA. Instead, cell fractionation experiments suggest that decreased transcriptional activity of sumoylated c-Fos is associated with specific intranuclear distribution. Interestingly, the phosphorylation of threonine 232 observed upon expression of oncogenically activated Ha-Ras is known to superactivate c-Fos transcriptional activity. We show here that it also inhibits c-Fos sumoylation, revealing a functional antagonism between two posttranslational modifications, each occurring within a different moiety of a bipartite transactivation domain of c-Fos. Finally we report that the sumoylation of c-Fos is a dynamic process that can be reversed via multiple mechanisms. This supports the idea that this modification does not constitute a final inactivation step that necessarily precedes protein degradation.

Viral oncoproteins E1A and E7 and cellular LxCxE proteins repress SUMO modification of the retinoblastoma tumor suppressor.

The retinoblastoma tumor suppressor protein (pRB) is a major regulator of cell-cycle progression and cellular differentiation. Central to pRB function is the pocket domain, which serves as the main binding region for cellular regulators. In tumors pRB is frequently inactivated by mutations in the pocket domain or by binding of viral oncoproteins to this region. A characteristic feature of these viral oncoproteins and many cellular pRB-binding partners is an LxCxE sequence motif, which interacts with pRB's pocket domain. Here, we show that the ubiquitin-like modifier SUMO is covalently attached to a distinct residue (K720) of pRB within the B-box of the pocket region that binds LxCxE-motif proteins. We provide evidence that SUMO preferentially targets the active, hypophosphorylated form of pRB and show that tumorigenic mutations of pRB in the pocket domain lead to a loss of SUMOylation. Notably, the level of pRB SUMOylation is controlled by the interaction of pRB with viral and cellular LxCxE-motif proteins. Inhibitors of pRB function, including the viral oncoproteins E1A and E7 and the cellular E1A-like inhibitor of differentiation EID-1, completely abolish SUMO modification of pRB. Conversely, pRB mutants deficient in binding of LxCxE-motif proteins exhibit a drastically enhanced modification by SUMO. Finally, we provide evidence that SUMOylation can influence pRB function, as the SUMO-deficient pRB(K720R) mutant exerts a slightly higher repressive potential on an E2F-responsive reporter gene than wild-type pRB. Taken together, these data identify SUMO modification as a novel post-translational modification of pRB that may control pRB activity by modulating LxCxE-pocket interactions.

SUMO: a regulator of gene expression and genome integrity.

Post-translational modification with the ubiquitin-like SUMO protein is involved in the regulation of many cellular key processes. The SUMO system modulates signal transduction pathways, including cytokine, Wnt, growth factor and steroid hormone signalling. SUMO frequently restrains the activity of downstream transcription factors in these pathways presumably by facilitating the recruitment of corepressors or mediating the assembly of repressor complexes. Additionally, evidence is accumulating that SUMO controls pathways important for the surveillance of genome integrity. SUMO regulates the PML/p53 tumour suppressor network, a key determinant in the cellular response to DNA damage. Moreover, proteins that maintain genomic stability by functioning at the interface between DNA replication, recombination and repair processes undergo SUMOylation. We will discuss some key findings that exemplify the role of SUMO in transcriptional regulation and genome surveillance.

Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity.

The activity of the p53 tumor suppressor protein and the c-Jun protooncogene is regulated by posttranslational modifications, such as phosphorylation or ubiquitination. In addition, covalent attachment of the ubiquitin-like modifier SUMO appears to modulate their transcriptional activity. Sumoylation proceeds via an enzymatic pathway that is mechanistically analogous to ubiquitination, but requires a different E1-activating enzyme and Ubc9, a SUMO-specific E2-conjugating enzyme. Here, we show that two members of the PIAS family, PIAS1 and PIASxbeta, act as specific E3-like ligases that promote sumoylation of p53 and c-Jun in vitro and in vivo. The PIAS proteins physically interact with both p53 and c-Jun. In addition, they bind to Ubc9, suggesting that they recruit the E2 enzyme to their respective substrate. The SUMO ligase activity requires the conserved zinc-finger domain, which is distantly related to the essential RING-finger motif, found in a subset of ubiquitin ligases. Furthermore, similar to RING-type ubiquitin ligases, PIASxbeta can catalyze its own modification. Hence, these data further extend the analogy between the ubiquitin and SUMO pathway. Strikingly, PIAS proteins strongly repress the transcriptional activity of p53, suggesting that the PIAS-SUMO pathway plays a crucial role in the regulation of p53 and presumably other transcription factors.