Molecular basis of mRNA transport by a kinesin-1-atypical tropomyosin complex.

Kinesin-1 carries cargos including proteins, RNAs, vesicles, and pathogens over long distances within cells. The mechanochemical cycle of kinesins is well described, but how they establish cargo specificity is not fully understood. Transport of oskar mRNA to the posterior pole of the Drosophila oocyte is mediated by Drosophila kinesin-1, also called kinesin heavy chain (Khc), and a putative cargo adaptor, the atypical tropomyosin, aTm1. How the proteins cooperate in mRNA transport is unknown. Here, we present the high-resolution crystal structure of a Khc-aTm1 complex. The proteins form a tripartite coiled coil comprising two in-register Khc chains and one aTm1 chain, in antiparallel orientation. We show that aTm1 binds to an evolutionarily conserved cargo binding site on Khc, and mutational analysis confirms the importance of this interaction for mRNA transport in vivo. Furthermore, we demonstrate that Khc binds RNA directly and that it does so via its alternative cargo binding domain, which forms a positively charged joint surface with aTm1, as well as through its adjacent auxiliary microtubule binding domain. Finally, we show that aTm1 plays a stabilizing role in the interaction of Khc with RNA, which distinguishes aTm1 from classical motor adaptors.

First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Selective Inhibitors with Cellular Target Engagement.

Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, especially cancer. Five HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc-dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement, e. g., in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of most other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like acetyl-putrescine or -spermidine. Hence, it is also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin-labelled acetyl-spermidine derivative to measure its PDAC activity, which is suitable for high-throughput screening. Using this assay, we identified potent inhibitors of HDAC10-mediated spermidine deacetylation in vitro. Based on the oligoamine preference of HDAC10, we also designed inhibitors with a basic moiety in appropriate distance to the zinc binding hydroxamate that showed potent inhibition of HDAC10 with high selectivity, and we solved a HDAC10-inhibitor structure using X-ray crystallography. We could demonstrate selective cellular target engagement for HDAC10 but a lysosomal phenotype in neuroblastoma cells that was previously associated with HDAC10 inhibition was not observed. Thus, we have developed new chemical probes for HDAC10 that allow further clarification of the biological role of this enzyme.

Short peptide pharmacophores developed from protein phosphatase-1 disrupting peptides (PDPs).

PP1 is a major phosphoserine/threonine-specific phosphatase that is involved in diseases such as heart insufficiency and diabetes. PP1-disrupting peptides (PDPs) are selective modulators of PP1 activity that release its catalytic subunit, which then dephosphorylates nearby substrates. Recently, PDPs enabled the creation of phosphatase-recruiting chimeras, which are bifunctional molecules that guide PP1 to a kinase to dephosphorylate and inactivate it. However, PDPs are 23mer peptides, which is not optimal for their use in therapy due to potential stability and immunogenicity issues. Therefore, we present here the sequence optimization of the 23mer PDP to a 5mer peptide, involving several attempts considering structure-based virtual screening, high throughput screening and peptide sequence optimization. We provide here a strong pharmacophore as lead structure to enable PP1 targeting in therapy or its use in phosphatase-recruiting chimeras in the future.

Structural basis for targeting the chromatin repressor Sfmbt to Polycomb response elements.

Polycomb group (PcG) protein complexes repress developmental regulator genes by modifying their chromatin. How different PcG proteins assemble into complexes and are recruited to their target genes is poorly understood. Here, we report the crystal structure of the core of the Drosophila PcG protein complex Pleiohomeotic (Pho)-repressive complex (PhoRC), which contains the Polycomb response element (PRE)-binding protein Pho and Sfmbt. The spacer region of Pho, separated from the DNA-binding domain by a long flexible linker, forms a tight complex with the four malignant brain tumor (4MBT) domain of Sfmbt. The highly conserved spacer region of the human Pho ortholog YY1 binds three of the four human 4MBT domain proteins in an analogous manner but with lower affinity. Comparison of the Drosophila Pho:Sfmbt and human YY1:MBTD1 complex structures provides a molecular explanation for the lower affinity of YY1 for human 4MBT domain proteins. Structure-guided mutations that disrupt the interaction between Pho and Sfmbt abolish formation of a ternary Sfmbt:Pho:DNA complex in vitro and repression of developmental regulator genes in Drosophila. PRE tethering of Sfmbt by Pho is therefore essential for Polycomb repression in Drosophila. Our results support a model where DNA tethering of Sfmbt by Pho and multivalent interactions of Sfmbt with histone modifications and other PcG proteins create a hub for PcG protein complex assembly at PREs.

Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2.

Fibroblast growth factor 2 (FGF2) is a potent mitogen promoting both tumor cell survival and tumor-induced angiogenesis. It is secreted by an unconventional secretory mechanism that is based upon direct translocation across the plasma membrane. Key steps of this process are (i) phosphoinositide-dependent membrane recruitment, (ii) FGF2 oligomerization and membrane pore formation, and (iii) extracellular trapping mediated by membrane-proximal heparan sulfate proteoglycans. Efficient secretion of FGF2 is supported by Tec kinase that stimulates membrane pore formation based upon tyrosine phosphorylation of FGF2. Here, we report the biochemical characterization of the direct interaction between FGF2 and Tec kinase as well as the identification of small molecules that inhibit (i) the interaction of FGF2 with Tec, (ii) tyrosine phosphorylation of FGF2 mediated by Tec in vitro and in a cellular context, and (iii) unconventional secretion of FGF2 from cells. We further demonstrate the specificity of these inhibitors for FGF2 because tyrosine phosphorylation of a different substrate of Tec is unaffected in their presence. Building on previous evidence using RNA interference, the identified compounds corroborate the role of Tec kinase in unconventional secretion of FGF2. In addition, they are valuable lead compounds with great potential for drug development aiming at the inhibition of FGF2-dependent tumor growth and metastasis.

Immunogenicity and HPV infection after one, two, and three doses of quadrivalent HPV vaccine in girls in India: a multicentre prospective cohort study.

BACKGROUND: An increase in worldwide HPV vaccination could be facilitated if fewer than three doses of vaccine are as effective as three doses. We originally aimed to compare the immunogenicity and frequency of persistent infection and cervical precancerous lesions caused by vaccine-targeted HPV after vaccination with two doses of quadrivalent vaccine on days 1 and 180 or later, with three doses on days 1, 60, and 180 or later, in a cluster-randomised trial. Suspension of the recruitment and vaccination due to events unrelated to our study meant that some enrolled girls could not be vaccinated and some vaccinated girls received fewer than the planned number of vaccinations by default. As a result, we re-analysed our data as an observational cohort study. METHODS: Our study was designed to be done in nine locations (188 clusters) in India. Participants were unmarried girls aged 10-18 years vaccinated in four cohorts: girls who received three doses of vaccine on days 1, 60, and 180 or later, two doses on days 1 and 180 or later, two doses on days 1 and 60 by default, and one dose by default. The primary outcomes were immunogenicity in terms of L1 genotype-specific binding antibody titres, neutralising antibody titres, and antibody avidity after vaccination for the vaccine-targeted HPV types 16, 18, 6, and 11 and incident and persistent infections with these HPVs. Analysis was per actual number of vaccine doses received. This study is registered with ISRCTN, number ISRCTN98283094; and with ClinicalTrials.gov, number NCT00923702. FINDINGS: Vaccination of eligible girls was initiated on Sept 1, 2009, and continued until April 8, 2010. Of 21 258 eligible girls identified at 188 clusters, 17 729 girls were recruited from 178 clusters before suspension. 4348 (25%) girls received three doses, 4979 (28%) received two doses on days 1 and 180 or later, 3452 (19%) received two doses at days 1 and 60, and 4950 (28%) received one dose. Immune response in the two-dose HPV vaccine group was non-inferior to the three-dose group (median fluorescence intensity ratio for HPV 16 1·12 [95% CI 1·02-1·23] and for HPV 18 1·04 [0·92-1·19]) at 7 months, but was inferior in the two-dose default (0·33 [0·29-0·38] for HPV 16 and 0·51 [0·43-0·59] for HPV 18) and one-dose default (0·09 [0·08-0·11] for HPV 16 and 0·12 [0·10-0·14] for HPV 18) groups at 18 months. The geometric mean avidity indices after fewer than three doses by design or default were non-inferior to those after three doses of vaccine. Fewer than three doses by design and default induced detectable concentrations of neutralising antibodies to all four vaccine-targeted HPV types, but at much lower concentration after one dose. Cervical samples from 2649 participants were tested and the frequency of incident HPV 16, 18, 6, and 11 infections was similar irrespective of the number of vaccine doses received. The testing of at least two samples from 838 participants showed that there was no persistent HPV 16 or 18 infections in any study group at a median follow-up of 4·7 years (IQR 4·2-5·1). INTERPRETATION: Despite the limitations imposed by the suspension of the HPV vaccination, our findings lend support to the WHO recommendation of two doses, at least 6 months apart, for routine vaccination of young girls. The short-term protection afforded by one dose of HPV vaccine against persistent infection with HPV 16, 18, 6, and 11 is similar to that afforded by two or three doses of vaccine and merits further assessment. FUNDING: Bill & Melinda Gates Foundation.

A direct role for ATP1A1 in unconventional secretion of fibroblast growth factor 2.

Previous studies proposed a role for the Na/K-ATPase in unconventional secretion of fibroblast growth factor 2 (FGF2). This conclusion was based upon pharmacological inhibition of FGF2 secretion in the presence of ouabain. However, neither independent experimental evidence nor a potential mechanism was provided. Based upon an unbiased RNAi screen, we now report the identification of ATP1A1, the α1-chain of the Na/K-ATPase, as a factor required for efficient secretion of FGF2. As opposed to ATP1A1, down-regulation of the ß1- and ß3-chains (ATP1B1 and ATP1B3) of the Na/K-ATPase did not affect FGF2 secretion, suggesting that they are dispensable for this process. These findings indicate that it is not the membrane potential-generating function of the Na/K-ATPase complex but rather a so far unidentified role of potentially unassembled α1-chains that is critical for unconventional secretion of FGF2. Consistently, in the absence of ß-chains, we found a direct interaction between the cytoplasmic domain of ATP1A1 and FGF2 with submicromolar affinity. Based upon these observations, we propose that ATP1A1 is a recruitment factor for FGF2 at the inner leaflet of plasma membranes that may control phosphatidylinositol 4,5-bisphosphate-dependent membrane translocation as part of the unconventional secretory pathway of FGF2.

Comparison of the immunogenicity and reactogenicity of Cervarix and Gardasil human papillomavirus vaccines in HIV-infected adults: a randomized, double-blind clinical trial.

BACKGROUND: We compared the immunogenicity and reactogenicity of Cervarix or Gardasil human papillomavirus (HPV) vaccines in adults infected with the human immunodeficiency virus (HIV). METHODS: This was a double-blind, controlled trial randomizing HIV-positive adults to receive 3 doses of Cervarix or Gardasil at 0, 1.5, and 6 months. Immunogenicity was evaluated for up to 12 months. Neutralizing anti-HPV-16/18 antibodies were measured by pseudovirion-based neutralization assay. Laboratory tests and diary cards were used for safety assessment. The HPV-DNA status of the participants was determined before and after immunization. RESULTS: Ninety-two participants were included in the study. Anti-HPV-18 antibody titers were higher in the Cervarix group compared with the Gardasil group at 7 and 12 months. No significant differences in anti-HPV-16 antibody titers were found among vaccine groups. Among Cervarix vaccinees, women had higher anti-HPV-16/18 antibody titers compared to men. No sex-specific differences in antibody titers were found in the Gardasil group. Mild injection site reactions were more common in the Cervarix group than in the Gardasil group (91.1% vs 69.6%; P = .02). No serious adverse events occurred. CONCLUSIONS: Both vaccines were immunogenic and well tolerated. Compared with Gardasil, Cervarix induced superior vaccine responses among HIV-infected women, whereas in HIV-infected men the difference in immunogenicity was less pronounced.

Inter-epitope spacer variation within polytopic L2-based human papillomavirus antigens affects immunogenicity.

The human papillomavirus minor capsid protein L2 is being extensively explored in pre-clinical studies as an attractive vaccine antigen capable of inducing broad-spectrum prophylactic antibody responses. Recently, we have developed two HPV vaccine antigens - PANHPVAX and CUT-PANHPVAX- both based on heptameric nanoparticle antigens displaying polytopes of the L2 major cross-neutralizing epitopes of eight mucosal and twelve cutaneous HPV types, respectively. Prompted by the variable neutralizing antibody responses against some of the HPV types targeted by the antigens observed in previous studies, here we investigated the influence on immunogenicity of six distinct glycine-proline spacers inserted upstream to a specific L2 epitope. We show that spacer variants differentially influence antigen immunogenicity in a mouse model, with the antigen constructs M8merV6 and C12merV6 displaying a superior ability in the induction of neutralizing antibodies as determined by pseudovirus-based neutralization assays (PBNAs). L2-peptide enzyme-linked immunosorbent assay (ELISA) assessments determined the total anti-L2 antibody level for each antigen variant, showing for the majority of sera a correlation with their repective neutralizing antibody level. Surface Plasmon Resonance revealed that L2 epitope-specific, neutralizing monoclonal antibodies (mAbs) display distinct avidities to different antigen spacer variants. Furthermore, mAb affinity toward individual spacer variants was well correlated with their neutralizing antibody induction capacity, indicating that the mAb affinity assay predicts L2-based antigen immunogenicity. These observations provide insights on the development and optimization of L2-based HPV vaccines.

Advances on two serological assays for human papillomavirus provide insights on the reactivity of antibodies against a cross-neutralization epitope of the minor capsid protein L2.

Introduction: A second generation of prophylactic human papillomavirus (HPV) vaccines based on the minor capsid protein L2 has entered clinical trials as promising alternative to meet the gaps left out by the current vaccines concerning type-restricted protection, high costs and low penetrance in immunization programs of lowand middle-income countries. Most of the serological assays available to assess anti-HPV humoral responses are, however, not well suited for measuring vaccine-induced anti-L2 antibody responses. Methods: In this work, we have advanced our automated, purely add-on High-Throughput Pseudovirion-Based Neutralization Assay (HT-PBNA) in an L2-oriented approach for measuring antibody-mediated neutralization of HPV types 6/16/18/31/33/52/58. Results and discussion: With the optimized settings, we observed 24- to 120-fold higher sensitivity for detection of neutralizing Ab to the L2 protein of HPV6, HPV16, HPV18, and HPV31, compared to the standard HT-PBNA. Alternatively, we have also developed a highly sensitive, cell-free, colorimetric L2-peptide capture ELISA for which the results were strongly concordant with those of the advanced neutralization assay, named HT-fc-PBNA. These two high-throughput scalable assays represent attractive approaches to determine antibody-based correlates of protection for the HPV L2 vaccines that are to come.

Evaluation of immune response to single dose of quadrivalent HPV vaccine at 10-year post-vaccination.

BACKGROUND: The recent World Health Organization recommendation supporting single-dose of HPV vaccine will significantly reduce programmatic cost, mitigate the supply shortage, and simplify logistics, thus allowing more low- and middle-income countries to introduce the vaccine. From a programmatic perspective the durability of protection offered by a single-dose will be a key consideration. The primary objectives of the present study were to determine whether recipients of a single-dose of quadrivalent HPV vaccine had sustained immune response against targeted HPV types (HPV 6,11,16,18) at 10 years post-vaccination and whether this response was superior to the natural antibody titres observed in unvaccinated women. METHODS: Participants received at age 10-18 years either one, two or three doses of the quadrivalent HPV vaccine. Serology samples were obtained at different timepoints up to 10 years after vaccination from a convenience sample of vaccinated participants and from age-matched unvaccinated women at one timepoint. The evolution of the binding and neutralizing antibody response was presented by dose received. 10-year durability of immune responses induced by a single-dose was compared to that after three doses of the vaccine and in unvaccinated married women. RESULTS: The dynamics of antibody response among the single-dose recipients observed over 120 months show stabilized levels 18 months after vaccination for all four HPV types. Although the HPV type-specific (binding or neutralizing) antibody titres after a single-dose were significantly inferior to those after three doses of the vaccine (lower bounds of GMT ratios < 0.5), they were all significantly higher than those observed in unvaccinated women following natural infections (GMT ratios: 2.05 to 4.04-fold higher). The results correlate well with the high vaccine efficacy of single-dose against persistent HPV 16/18 infections reported by us earlier at 10-years post-vaccination. CONCLUSION: Our study demonstrates the high and durable immune response in single-dose recipients of HPV vaccine at 10-years post vaccination.

Impact of age at vaccination and cervical HPV infection status on binding and neutralizing antibody titers at 10 years after receiving single or higher doses of quadrivalent HPV vaccine.

Long-term follow-up of a cohort of unmarried girls who received one, two, or three doses of quadrivalent HPV vaccine, between 10 and 18 years of age, in an Indian multi-centric study allowed us to compare antibody responses between the younger and older age cohorts at 10-years post-vaccination, and study the impact of initiation of sexual activity and cervical HPV infections on antibody levels. Among the younger (10-14 years) recipients of a single dose, 97.7% and 98.2% had detectable binding antibody titers against HPV 16 and HPV 18 respectively at ten years post-vaccination. The proportions among those receiving a single dose at age 15-18 years were 92.3% and 94.2% against HPV 16 and HPV 18 respectively. Mean HPV 16 binding antibody titers were 2.1 folds (95%CI 1.4 to 3.3) higher in those vaccinated at ages 10-14 years, and 1.9 folds (95%CI 1.2 to 3.0) higher in those vaccinated at 15-18 years compared to mean titers seen in the unvaccinated women. Compared to previous timepoints of 36 or 48 months, binding antibodies against HPV 16 and neutralizing antibodies against both HPV 16 and HPV 18 were significantly higher at 10 years. This rise was more pronounced in participants vaccinated at 15-18 years. No association of marital status or cervical HPV infections was observed with the rise in titer. Durability of antibody response in single dose recipients correlated well with the high efficacy of a single dose against persistent HPV 16/18 infections irrespective of age at vaccination, as we reported earlier.

Structural basis for tunable affinity and specificity of LxCxE-dependent protein interactions with the retinoblastoma protein family.

The retinoblastoma protein (Rb) and its homologs p107 and p130 are critical regulators of gene expression during the cell cycle and are commonly inactivated in cancer. Rb proteins use their "pocket domain" to bind an LxCxE sequence motif in other proteins, many of which function with Rb proteins to co-regulate transcription. Here, we present binding data and crystal structures of the p107 pocket domain in complex with LxCxE peptides from the transcriptional co-repressor proteins HDAC1, ARID4A, and EID1. Our results explain why Rb and p107 have weaker affinity for cellular LxCxE proteins compared with the E7 protein from human papillomavirus, which has been used as the primary model for understanding LxCxE motif interactions. Our structural and mutagenesis data also identify and explain differences in Rb and p107 affinities for some LxCxE-containing sequences. Our study provides new insights into how Rb proteins bind their cell partners with varying affinity and specificity.

Sustainability of neutralising antibodies induced by bivalent or quadrivalent HPV vaccines and correlation with efficacy: a combined follow-up analysis of data from two randomised, double-blind, multicentre, phase 3 trials.

BACKGROUND: Quadrivalent and bivalent vaccines against oncogenic human papillomavirus (HPV) are used worldwide with different reported overall efficacies against HPV infections. Although protective concentrations of vaccine-induced antibodies are still not formally defined, we evaluated the sustainability of neutralising antibodies in vaccine trial participants 2-12 years after vaccination and the correlation with reported vaccine efficacy. METHODS: We did a follow-up analysis of data from the Finnish cohorts of two international, randomised, double-blind, phase 3 trials of HPV vaccines, PATRICIA (bivalent, HPV16 and 18) and FUTURE II (quadrivalent, HPV6, 11, 16, and 18). In 2002 and 2004-05, respectively, Finnish girls aged 16-17 years participated in one of these two trials and consented to health registry follow-up with the Finnish Cancer Registry. The cohorts were also linked with the Finnish Maternity Cohort (FMC) that collects first-trimester serum samples from nearly all pregnant Finnish women, resulting in 2046 post-vaccination serum samples obtained during up to 12 years of follow-up. We obtained serum samples from the FMC-based follow-up of the FUTURE II trial (from the quadrivalent vaccine recipients) and the PATRICIA trial (from corresponding bivalent vaccine recipients who were aligned by follow-up time, and matched by the number of pregnancies). We assessed neutralising antibody concentrations (type-specific seroprevalence) to HPV6, 16, and 18, and cross-neutralising antibody responses to non-vaccine HPV types 31, 33, 45, 52, and 58 from 2 to 12 years after vaccination. FINDINGS: Up to Dec 31, 2016, we obtained and analysed 577 serum samples from the quadrivalent vaccine recipients and 568 from the bivalent vaccine recipients. In 681 first-pregnancy serum samples, neutralising antibodies to HPV6, 16, and 18 were generally found up to 12 years after vaccination. However, 51 (15%) of 339 quadrivalent vaccine recipients had no detectable HPV18 neutralising antibodies 2-12 years after vaccination, whereas all 342 corresponding bivalent vaccine recipients had HPV18 neutralising antibodies.. In seropositive quadrivalent vaccine recipients, HPV16 geometric mean titres (GMT) halved by years 5-7 (GMT 3679, 95% CI 2377 to 4708) compared with years 2-4 (6642, 2371 to 13 717). Between 5 and 12 years after vaccination, GMT of neutralising antibodies to HPV16 and 18 were 5·7 times and 12·4 times higher, respectively, in seropositive bivalent vaccine recipients than in the quadrivalent vaccine recipients. Cross-neutralising antibodies to HPV31, 33, 45, 52, and 58 were more prevalent in the bivalent vaccine recipients but, when measurable, sustainable up to 12 years after vaccination with similar GMTs in both vaccine cohorts. Seroprevalence for HPV16, 31, 33, 52, and 58 significantly correlated with vaccine efficacy against persistent HPV infections in the bivalent vaccine recipients only (rs=0·90, 95% CI 0·09 to 0·99, p=0·037, compared with rs=0·62, 95% CI -0·58 to 0·97, p=0·27 for the quadrivalent vaccine recipients). Correlation of protection with prevalence of neutralising or cross-neutralising HPV antibodies was not significant in the quadrivalent vaccine recipients. INTERPRETATION: The observed significant differences in the immunogenicity of the two vaccines are in line with the differences in their cross-protective efficacy. Protective HPV vaccine-induced antibody titres can be detected up to 12 years after vaccination. FUNDING: Academy of Finland and Finnish Cancer Foundation.

Seroprevalence of 34 human papillomavirus types in the German general population.

The natural history of infections with many human papillomavirus (HPV) types is poorly understood. Here, we describe for the first time the age- and sex-dependent antibody prevalence for 29 cutaneous and five mucosal HPV types from 15 species within five phylogenetic genera (alpha, beta, gamma, mu, nu) in a general population. Sera from 1,797 German adults and children (758 males and 1,039 females) between 1 and 82 years (median 37 years) were analysed for antibodies to the major capsid protein L1 by Luminex-based multiplex serology. The first substantial HPV antibody reactions observed already in children and young adults are those to cutaneous types of the genera nu (HPV 41) and mu (HPV 1, 63). The antibody prevalence to mucosal high-risk types, most prominently HPV 16, was elevated after puberty in women but not in men and peaked between 25 and 34 years. Antibodies to beta and gamma papillomaviruses (PV) were rare in children and increased homogeneously with age, with prevalence peaks at 40 and 60 years in women and 50 and 70 years in men. Antibodies to cutaneous alpha PV showed a heterogeneous age distribution. In summary, these data suggest three major seroprevalence patterns for HPV of phylogenetically distinct genera: antibodies to mu and nu skin PV appear early in life, those to mucosal alpha PV in women after puberty, and antibodies to beta as well as to gamma skin PV accumulate later in life.

Peak neutralizing and cross-neutralizing antibody levels to human papillomavirus types 6/16/18/31/33/45/52/58 induced by bivalent and quadrivalent HPV vaccines.

We performed an independent comparison of neutralizing and cross-neutralizing antibody (ab) levels seven months after initiation of three-dose, six-month vaccination schedules with the bivalent and quadrivalent human papillomavirus (HPV) vaccines in adolescent Finnish and Indian females, respectively. We used a semi-automated Pseudovirion-Based Neutralization Assay and observed significantly higher HPV16/18 peak ab-levels in bivalent as compared to quadrivalent vaccine recipients. Bivalent vaccine induced cross-neutralizing HPV31/33/45/52/58 antibodies significantly more frequently and to higher levels than the quadrivalent vaccine. The correlation of bivalent vaccine-induced HPV45 ab-levels with HPV16/18 ab-levels was stronger than that of corresponding quadrivalent vaccine-induced ab-levels, suggesting a qualitatively different cross-reactive response. Our findings on the comparison of the immunogenicity of two HPV vaccine tested in two different populations indicate that further head-to-head studies are warranted.

Evaluation of serological assays to monitor antibody responses to single-dose HPV vaccines.

INTRODUCTION: Whether existing serological assays are sufficiently robust to measure the lower antibody levels expected following single-dose HPV vaccination is unknown. METHODS: We evaluated seven assays measuring HPV-16/18 immunological responses overall and by number of doses in 530 serum samples from participants receiving varying doses of Cervarix or Gardasil up to 36-months post-vaccination. Serum was evaluated by simplex (HPV-16 ELISA, HPV-18 ELISA), multiplex (LIA-4, VLP-MIA, M9ELISA, GST-L1), and high-throughput pseudovirion-based neutralization assays (HT-PBNA), and results were compared to the gold standard HPV-16/18 secreted alkaline phosphatase neutralization assay (SEAP-NA). Reproducibility was assessed by the coefficient of variation (CV) and intraclass correlation coefficient (ICC). Percent agreement, Pearson correlation, and weighted-kappa were used to assess validity. Determinants of seronegativity were evaluated by chi-squared test. RESULTS: HPV-16: Seropositivity range was 97.1-99.5% for single dose and 98.8-99.8% overall. CV range was 4.0-18.0% for single dose and 2.9-19.5% overall. ICC range was 0.77-0.99 for single dose and 0.74-0.99 overall. Correlation with SEAP-NA range was 0.43-0.85 for single dose and 0.51-0.90 overall. Weighted-kappa range was 0.34-0.82 for single dose and 0.45-0.84 overall. HPV-18: Seropositivity range was 63.9-94.7% for single dose and 86.2-97.9% overall. CV range was 8.1-18.2% for single dose and 4.6-18.6% overall. ICC range was 0.75-0.99 for single dose and 0.83-0.99 overall. Correlation with SEAP-NA range was 0.31-0.99 for single dose and 0.27-0.96 overall. Weighted-kappa range was 0.35-0.83 for single dose and 0.45-0.84 overall. HPV-16 seronegativity was <5% for all assays. HPV-18 seronegativity range was 5.5-17.3%. For LIA-4 and GST-L1 where the proportion of seronegativity was >10%, the strongest correlates of seronegativity were receiving a single vaccine dose and receiving Gardasil. CONCLUSIONS: These results support the utility of existing serological assays to monitor antibody responses following single-dose HPV vaccination.

Design and Synthesis of Dihydroxamic Acids as HDAC6/8/10 Inhibitors.

We report the synthesis and evaluation of a class of selective multitarget agents for the inhibition of HDAC6, HDAC8, and HDAC10. The concept for this study grew out of a structural analysis of the two selective inhibitors Tubastatin A (HDAC6/10) and PCI-34051 (HDAC8), which we recognized share the same N-benzylindole core. Hybridization of the two inhibitor structures resulted in dihydroxamic acids with benzyl-indole and -indazole core motifs. These substances exhibit potent activity against HDAC6, HDAC8, and HDAC10, while retaining selectivity over HDAC1, HDAC2, and HDAC3. The best substance inhibited the viability of the SK-N-BE(2)C neuroblastoma cell line with an IC50 value similar to a combination treatment with Tubastatin A and PCI-34051. This compound class establishes a proof of concept for such hybrid molecules and could serve as a starting point for the further development of enhanced HDAC6/8/10 inhibitors.

Loss of N-Glycanase 1 Alters Transcriptional and Translational Regulation in K562 Cell Lines.

N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.

Selective Inhibition of Histone Deacetylase 10: Hydrogen Bonding to the Gatekeeper Residue is Implicated.

The discovery of isozyme-selective histone deacetylase (HDAC) inhibitors is critical for understanding the biological functions of individual HDACs and for validating HDACs as drug targets. The isozyme HDAC10 contributes to chemotherapy resistance and has recently been described to be a polyamine deacetylase, but no studies toward selective HDAC10 inhibitors have been published. Using two complementary assays, we found Tubastatin A, an HDAC6 inhibitor, to potently bind HDAC10. We synthesized Tubastatin A derivatives and found that a basic amine in the cap group was required for strong HDAC10 binding. HDAC10 inhibitors mimicked knockdown by causing dose-dependent accumulation of acidic vesicles in a neuroblastoma cell line. Furthermore, docking into human HDAC10 homology models indicated that a hydrogen bond between a cap group nitrogen and the gatekeeper residue Glu272 was responsible for potent HDAC10 binding. Taken together, our data provide an optimal platform for the development of HDAC10-selective inhibitors, as exemplified with the Tubastatin A scaffold.