RG2-VLP: a Vaccine Designed to Broadly Protect against Anogenital and Skin Human Papillomaviruses Causing Human Cancer.

The family of human papillomaviruses (HPV) includes over 400 genotypes. Genus α genotypes generally infect the anogenital mucosa, and a subset of these HPV are a necessary, but not sufficient, cause of cervical cancer. Of the 13 high-risk (HR) and 11 intermediate-risk (IR) HPV associated with cervical cancer, genotypes 16 and 18 cause 50% and 20% of cases, respectively, whereas HPV16 dominates in other anogenital and oropharyngeal cancers. A plethora of ßHPVs are associated with cutaneous squamous cell carcinoma (CSCC), especially in sun-exposed skin sites of epidermodysplasia verruciformis (EV), AIDS, and immunosuppressed patients. Licensed L1 virus-like particle (VLP) vaccines, such as Gardasil 9, target a subset of αHPV but no ßHPV. To comprehensively target both α- and ßHPVs, we developed a two-component VLP vaccine, RG2-VLP, in which L2 protective epitopes derived from a conserved αHPV epitope (amino acids 17 to 36 of HPV16 L2) and a consensus ßHPV sequence in the same region are displayed within the DE loop of HPV16 and HPV18 L1 VLP, respectively. Unlike vaccination with Gardasil 9, vaccination of wild-type and EV model mice (Tmc6Δ/Δ or Tmc8Δ/Δ) with RG2-VLP induced robust L2-specific antibody titers and protected against ß-type HPV5. RG2-VLP protected rabbits against 17 αHPV, including those not covered by Gardasil 9. HPV16- and HPV18-specific neutralizing antibody responses were similar between RG2-VLP- and Gardasil 9-vaccinated animals. However, only transfer of RG2-VLP antiserum effectively protected naive mice from challenge with all ßHPVs tested. Taken together, these observations suggest RG2-VLP's potential as a broad-spectrum vaccine to prevent αHPV-driven anogenital, oropharyngeal, and ßHPV-associated cutaneous cancers. IMPORTANCE Licensed preventive HPV vaccines are composed of VLPs derived by expression of major capsid protein L1. They confer protection generally restricted to infection by the αHPVs targeted by the up-to-9-valent vaccine, and their associated anogenital cancers and genital warts, but do not target ßHPV that are associated with CSCC in EV and immunocompromised patients. We describe the development of a two-antigen vaccine protective in animal models against known oncogenic αHPVs as well as diverse ßHPVs by incorporation into HPV16 and HPV18 L1 VLP of 20-amino-acid conserved protective epitopes derived from minor capsid protein L2.

Cutting Edge: Myosin 18A Is a Novel Checkpoint Regulator in B Cell Differentiation and Antibody-Mediated Immunity.

We investigated the function of the newly discovered myosin family protein myosin 18A (Myo18A) in Ab-mediated immunity by generating B cell-conditional Myo18A-deficient mice. Myo18A deficiency led to expansion of bone marrow progenitor B cells and mature B cells in secondary lymphoid organs. Myo18A-deficient mice displayed serum IgM hyperglobulinemia and increased splenic IgM-secreting cells, with older mice switching to IgG1 hyperglobulinemia and autoantibody development. Immunization of Myo18A-deficient mice with inactivated influenza virus led to development of more potent neutralizing Abs against the major Ag hemagglutinin, associated with persistent accumulation of Ag-specific germinal center B cells and more Ag-specific bone marrow plasma cells. In vitro stimulation with TLR7 and BCR ligands revealed a greater ability of Myo18A-deficient B cells to differentiate into Ab-secreting cells, associated with higher AID and Blimp-1 expression. Overall, our study demonstrates that Myo18A is a novel negative regulator of B cell homeostasis, differentiation, and humoral immunity.

Cutting Edge: Ezrin Regulates Inflammation by Limiting B Cell IL-10 Production.

IL-10 produced by B cells is important for controlling inflammation, thus underscoring the need to identify mechanisms regulating its production. In this study, we demonstrate that conditional deletion of ezrin in B cells increases IL-10 production induced by TLR4 ligation. The MyD88-independent Toll/IL-1R domain-containing adapter inducing IFN-ß-IFN regulatory factor 3 pathway is required for Ezrin-deficient B cells to produce higher IL-10 upon LPS stimulation. Treatment of B cells with a novel small-molecule inhibitor of ezrin induces its dephosphorylation and increases LPS-induced NF-κB and IFN regulatory factor 3 activation and IL-10 secretion, indicating a role for threonine 567 phosphorylation of ezrin in limiting IL-10. Loss of ezrin in B cells results in dampened proinflammatory response to a sublethal dose of LPS in vivo, which is dependent on increased IL-10 production. Taken together, our data yield new insights into molecular and membrane-cytoskeletal regulation of B cell IL-10 production and reveal ezrin as a potential therapeutic target in inflammatory diseases.

Ezrin tunes the magnitude of humoral immunity.

Ezrin is a member of the ezrin-radixin-moesin family of membrane-actin cytoskeleton cross-linkers that participate in a variety of cellular processes. In B cells, phosphorylation of ezrin at different sites regulates multiple processes, such as lipid raft coalescence, BCR diffusion, microclustering, and endosomal JNK activation. In this study, we generated mice with conditional deletion of ezrin in the B cell lineage to investigate the physiological significance of ezrin's function in Ag receptor-mediated B cell activation and humoral immunity. B cell development, as well as the proportion and numbers of major B cell subsets in peripheral lymphoid organs, was unaffected by the loss of ezrin. Using superresolution imaging methods, we show that, in the absence of ezrin, BCRs respond to Ag binding by accumulating into larger and more stable signaling microclusters. Loss of ezrin led to delayed BCR capping and accelerated lipid raft coalescence. Although proximal signaling proteins showed stronger activation in the absence of ezrin, components of the distal BCR signaling pathways displayed distinct effects. Ezrin deficiency resulted in increased B cell proliferation and differentiation into Ab-secreting cells ex vivo and stronger T cell-independent and -dependent responses to Ag in vivo. Overall, our data demonstrate that ezrin regulates amplification of BCR signals and tunes the strength of B cell activation and humoral immunity.

Cytokine production by bone marrow mononuclear cells in inherited bone marrow failure syndromes.

Fanconi anaemia (FA), dyskeratosis congenita (DC), Diamond-Blackfan anaemia (DBA), and Shwachman-Diamond syndrome (SDS) are characterized by the progressive development of bone marrow failure. Overproduction of tumour necrosis factor-α (TNF-α) from activated bone marrow T-cells has been proposed as a mechanism of FA-related aplasia. Whether such overproduction occurs in the other syndromes is unknown. We conducted a comparative study on bone marrow mononuclear cells to examine the cellular subset composition and cytokine production. We found lower proportions of haematopoietic stem cells in FA, DC, and SDS, and a lower proportion of monocytes in FA, DC, and DBA compared with controls. The T- and B-lymphocyte proportions were similar to controls, except for low B-cells in DC. We did not observe overproduction of TNF-α or IFN-γ by T-cells in any patients. Induction levels of TNF-α, interleukin (IL)-6, IL-1ß, IL-10, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor in monocytes stimulated with high-dose lipopolysaccharide (LPS) were similar at 4 h but lower at 24 h when compared to controls. Unexpectedly, patient samples showed a trend toward higher cytokine level in response to low-dose (0·001 µg/ml) LPS. Increased sensitivity to LPS may have clinical implications and could contribute to the development of pancytopenia by creating a chronic subclinical inflammatory micro-environment in the bone marrow.

Conformational switching in ezrin regulates morphological and cytoskeletal changes required for B cell chemotaxis.

B cell chemotaxis occurs in response to specific chemokine gradients and is critical for homeostasis and immune response. The molecular regulation of B cell membrane-actin interactions during migration is poorly understood. In this study, we report a role for ezrin, a member of the membrane-cytoskeleton cross-linking ezrin-radixin-moesin proteins, in the regulation of the earliest steps of B cell polarization and chemotaxis. We visualized chemokine-induced changes in murine B cell morphology using scanning electron microscopy and spatiotemporal dynamics of ezrin in B cells using epifluorescence and total internal reflection microscopy. Upon chemokine stimulation, ezrin is transiently dephosphorylated to assume an inactive conformation and localizes to the lamellipodia. B cells expressing a phosphomimetic conformationally active mutant of ezrin or those in which ezrin dephosphorylation was pharmacologically inhibited displayed impaired microvillar dynamics, morphological polarization, and chemotaxis. Our data suggest a 2-fold involvement of ezrin in B cell migration, whereby it first undergoes chemokine-induced dephosphorylation to facilitate membrane flexibility, followed by relocalization to the actin-rich lamellipodia for dynamic forward protrusion of the cells.

Proteomics analysis of the ezrin interactome in B cells reveals a novel association with Myo18aα.

The molecular regulation of recruitment and assembly of signalosomes near the B cell receptor (BCR) is poorly understood. We have previously demonstrated a role for the ERM family protein ezrin in regulating antigen-dependent lipid raft coalescence in B cells. In this study, we addressed the possibility that ezrin may collaborate with other adaptor proteins to regulate signalosome dynamics at the membrane. Using mass spectrometry-based proteomics analysis, we identified Myo18aα as a novel binding partner of ezrin. Myo18aα is an attractive candidate as it has several protein-protein interaction domains and an intrinsic motor activity. The expression of Myo18aα varied during B cell development in the bone marrow and in mature B cell subsets suggesting functional differences. Interestingly, BCR stimulation increased the association between ezrin and Myo18aα, and induced co-segregation of Myo18aα with the BCR and phosphotyrosine-containing proteins. Our data raise an intriguing possibility that the Myo18aα/ezrin complex may facilitate BCR-mediated signaling by recruiting signaling proteins that are in close proximity of the antigen receptor. Our study is not only significant with respect to understanding the molecular regulation of BCR signaling but also provides a broader basis for understanding the mechanism of action of ezrin in other cellular systems.

Creation of a novel peptide endowing yeasts with acid tolerance using yeast cell-surface engineering.

The cell wall of Saccharomyces cerevisiae plays an essential role in the biophysical characteristics of the cell surface. The modification of the cell wall property is an important factor for cellular adaptation to a stressful environment. In this study, we randomly modified the cell wall by displaying combinatorial random peptides on the yeast cell surface, and by screening, we successfully obtained a novel peptide, Scr35, that endowed yeasts with acid tolerance. The yeast, surface-modified by Scr35, was able to grow well under acidic condition and low glucose condition and showed high glucose uptake activity. However, the growth of the modified yeast became inferior as extracellular pH became higher. This inferiority was rescued by decreasing glucose concentration in a medium. Our results suggest that the optimum pH of a medium becomes low when the newly created Scr35 affects glucose uptake activity through cell-surface modification. Therefore, such artificial modification of the cell surface has a great potential as a useful tool for breeding acid-tolerant yeasts for industrial applications of S. cerevisiae as a biocatalyst.

Enhancement of display efficiency in yeast display system by vector engineering and gene disruption.

Vector engineering and gene disruption in host cells were attempted for the enhancement of alpha-agglutinin-based display of proteins on the cell surface in yeast. To evaluate the display efficiency by flow cytometric analysis, DsRed-monomer fused with FLAG-tag was displayed and immunostained as a model protein. The use of leu2-d in the expression vector resulted in the enhanced efficiency and ratio of the accessible display of proteins. Moreover, the amount of displayed proteins in SED1-disrupted cells increased particularly during the stationary growth phase. The combination of these improvements resulted in the quantitatively enhanced accessible display of DsRed-monomer on the yeast cell surface. The improved yeast display system would be useful in a wider range of its applications in biotechnology.

Characterization of cancer omics and drug perturbations in panels of lung cancer cells.

To better understand the disruptions of transcriptional regulations and gene expression in lung cancers, we constructed a multi-omics catalogue of the responses of lung cancer cells to a series of chemical compounds. We generated and analyzed 3,240 RNA-seq and 3,393 ATAC-seq libraries obtained from 23 cell lines treated with 95 well-annotated compounds. To demonstrate the power of the created multi-omics resource, we attempted to identify drugs that could induce the designated changes alone or in combination. The basal multi-omics information was first integrated into co-expression modules. Among these modules, we identified a stress response module that may be a promising drug intervention target, as new combinations of compounds that could be used to regulate this module and the consequent phenotypic appearance of cancer cells have been identified. We believe that the multi-omics profiles generated in this study and the strategy used to stratify them will lead to more rational and efficient development of anticancer drugs.

Discovery of a modified transcription factor endowing yeasts with organic-solvent tolerance and reconstruction of an organic-solvent-tolerant Saccharomyces cerevisiae strain.

Organic-solvent tolerance in Saccharomyces cerevisiae strain KK-211, which was first isolated as an organic-solvent-tolerant strain, depends on point mutation (R821S) of the transcription factor Pdr1p. The integration of the PDR1 R821S mutation into wild-type yeast results in organic-solvent tolerance, and the PDR1 R821S mutant can reduce carbonyl compounds in organic solvents.

Stimulation of alpha2-adrenergic receptors impairs influenza virus infection.

Influenza A viruses cause seasonal epidemics and occasional pandemics. The emergence of viruses resistant to neuraminidase (NA) inhibitors and M2 ion channel inhibitors underlines the need for alternate anti-influenza drugs with novel mechanisms of action. Here, we report the discovery of a host factor as a potential target of anti-influenza drugs. By using cell-based virus replication screening of a chemical library and several additional assays, we identified clonidine as a new anti-influenza agent in vitro. We found that clonidine, which is an agonist of the alpha2-adrenergic receptor (α2-AR), has an inhibitory effect on the replication of various influenza virus strains. α2-AR is a Gi-type G protein-coupled receptor that reduces intracellular cyclic AMP (cAMP) levels. In-depth analysis showed that stimulation of α2-ARs leads to impairment of influenza virus replication and that α2-AR agonists inhibit the virus assembly step, likely via a cAMP-mediated pathway. Although clonidine administration did not reduce lung virus titers or prevent body weight loss, it did suppress lung edema and improve survival in a murine lethal infection model. Clonidine may thus protect against lung damage caused by influenza virus infection. Our results identify α2-AR-mediated signaling as a key pathway to exploit in the development of anti-influenza agents.

A comparative study of two different assay kits for the detection of secreted alkaline phosphatase in HPV antibody neutralization assays.

To assess immunogenicity and development of antibodies in the context of vaccination, it is critical to quantify titers of neutralizing antibodies. We have been employing the 293TT cell-based neutralization assay system to quantify anti-HPV neutralizing antibodies. In this system, human papillomavirus (HPV) pseudovirion (PsV) particles encapsidating secreted alkaline phosphatase (SEAP) gene are used to measure infection of 293TT cells in 72-hr cell-culture supernatants. SEAP has traditionally been measured by Great EscAPe™ SEAP Chemiluminescence Kit 2.0 (GE). To reduce the cost, and to potentially increase efficiency, we sought a cheaper kit with better detection capability. Performance characteristics of the newer chemiluminescence kit, ZiVa® Ultra SEAP Plus Assay (Ziva) and GE were compared using the 293TT system. Dose titration of HPV PsV 16 or 18 showed that signal-to-noise ratios at 48 and 72 hr post-infection were higher for ZiVa at nearly all doses. ZiVa was superior to GE as it was able to detect SEAP at 48 hr, as well as when lower numbers of 293TT cells were used. The ability of ZiVa to quantitate HPV-16 and -18 neutralizing antibody titers was tested using sera from Cervarix® immunized individuals. Spearman rank correlational analyses showed excellent correlations between the titers obtained with ZiVa and GE for anti-HPV16 (r = 0.9822, p < 0.0001) and anti-HPV18 (r = 0.9832, p < 0.0001) antibodies. We concluded that ZiVa is superior to GE in detecting SEAP, and the antibody titers in sera of vaccinated individuals were similar to those obtained with GE. Thus, Ziva is a suitable alternative to GE.

Circulating CXCR5⁺CD4⁺ T Follicular-Like Helper Cell and Memory B Cell Responses to Human Papillomavirus Vaccines.

Through the interaction of T follicular helper (Tfh) cells and B cells, efficacious vaccines can generate high-affinity, pathogen-neutralizing antibodies, and memory B cells. Using CXCR5, CXCR3, CCR6, CCR7, PD1, and ICOS as markers, Tfh-like cells can be identified in the circulation and be classified into three functionally distinct subsets that are PD1+ICOS+, PD1+ ICOS-, or PD1-ICOS-. We used these markers to identify different subsets of CXCR5+CD4+ Tfh-like cells in response to highly immunogenic and efficacious vaccines for human papillomaviruses (HPV): Cervarix and Gardasil. In this small study, we used PBMC samples from 11 Gardasil recipients, and 8 Cervarix recipients from the Vaccine Research Center 902 Study to examine the induction of circulating Tfh-like cells and IgD-CD38HiCD27+ memory B cells by flow cytometry. PD1+ICOS+ CXCR3+CCR6-CXCR5+CD4+ (Tfh1-like) cells were induced and peaked on Day (D) 7 post-first vaccination, but not as much on D7 post-third vaccination. We also observed a trend toward increase in PD1+ICOS+ CXCR3-CCR6-CXCR5+CD4+ (Tfh2-like) cells for both vaccines, and PD1+ICOS+ CXCR3-CCR6+CXCR5+CD4+ (Tfh17-like) subset was induced by Cervarix post-first vaccination. There were also minimal changes in the other cellular subsets. In addition, Cervarix recipients had more memory B cells post-first vaccination than did Gardasil recipients at D14 and D30. We found frequencies of memory B cells at D30 correlated with anti-HPV16 and 18 antibody titers from D30, and the induction levels of memory B cells at D30 and PD1+ICOS+Tfh1-like cells at D7 post-first vaccination correlated for Cervarix. Our study showed that induction of circulating CXCR5+CD4+ Tfh-like subsets can be detected following immunization with HPV vaccines, and potentially be useful as a marker of immunogenicity of vaccines. However, further investigations should be extended to different cohorts with larger sample size to better understand the functions of these T cells, as well as their relationship with B cells and antibodies.

Production of Furin-Cleaved Papillomavirus Pseudovirions and Their Use for In Vitro Neutralization Assays of L1- or L2-Specific Antibodies.

Immunization with Human Papillomavirus (HPV) L1 virus-like particles or L2 capsid protein elicits neutralizing antibodies that mediate protection. A high-throughput and sensitive in vitro neutralization assay is therefore valuable for prophylactic HPV vaccine studies. Over several hours during infection of the genital tract, virions take on a distinct intermediate conformation, including a required furin cleavage of L2 at its N-terminus. This intermediate is an important target for neutralization by L2-specific antibody, but it is very transiently exposed during in vitro infection of most cell lines resulting in insensitive measurement for L2, but not L1-specific neutralizing antibodies. To model this intermediate, we describe a protocol to generate furin-cleaved HPV pseudovirions (fc-PsV), which deliver an encapsidated reporter plasmid to facilitate infectivity measurements. We also describe a protocol for use of fc-PsV in a high-throughput in vitro neutralization assay for the sensitive measurement of both L1 and L2-specific neutralizing antibodies.

Quantitation and visualization of tumor-specific T cells in the secondary lymphoid organs during and after tumor elimination by PET.

Increased understanding in the area of trafficking behavior of adoptively transferred tumor-specific T cells could help develop better therapeutic protocols. We utilized the DUC18/CMS5 tumor model system in conjunction with a microPET scanner to study the DUC18 T cell distribution pattern in spleens and lymph nodes in live mice. Anti-Thy1.2 antibodies conjugated to 1,4,7,10-tetraazacyclododecane-N,N',N'',N''-tetraacetic acid (DOTA) and radiolabeled with (64)Cu were administered to three groups of BALB-Thy1.1 mice on days 4, 7, or 14 post-DUC18 T cell transfer. We were able to detect the transferred cells in all the major lymph nodes, spleens, and in tumors. Our findings suggest that tumor-specific T cells do not all preferentially localize to the tumors but they also home to all the major lymphoid organs; additionally the number of DUC18 T cells remains relatively constant during and after tumor elimination within each lymphoid organ.

A case of postoperative hemorrhage after a hysterectomy in which a bleeding point of the left uterine artery was identified by postmortem angiography.

We report a case of fatal postoperative hemorrhage caused by failure to ligate the left uterine artery during a hysterectomy in a 42-year-old woman, as demonstrated postmortem by conventional autopsy methods and confirmed by angiography. Angiography was confirmed to be a useful adjunctive technique for postmortem diagnosis and localization of the bleeding point. In cases of suspected fatal postoperative hemorrhage, postmortem angiography may be of value to the forensic pathologist.

Acute subdural hematoma due to arteriovenous malformation primarily in dura mater: a case report.

We report a 42-year-old female with alcohol addiction who suddenly died of subdural hematoma (SDH) caused by dural arteriovenous malformation (AVM). In autopsy, there was seen a massive SDH with a total weight of 181 g that covered an entire part of the left cerebral hemisphere, although either serious external injuries of the head or any visible internal injuries of the brain were observed. SDH subsequently resulted in the tonsillar, transtentorial and subfalcial herniations with a right-sided shift of the left-lateral and third ventricles, and the left thalamus as well. Histopathological examination on the serial sections cut from the falx cerebri revealed abnormal distribution of arteries and veins with various sizes, which were comprehensively highlighted by immunohistochemical stainings with alpha-SMA and CD31. Although a very point of bleeding was not identified even by careful histological observation, we concluded that dural AVM could be critical for acute SDH in the present case. The value of ethanol concentration examined in the samples from SDH supported that the lesion could be not chronic, but acute.

ABC transporters and cell wall proteins involved in organic solvent tolerance in Saccharomyces cerevisiae.

Pleiotropic drug resistance 1 (Pdr1p) protein is a key transcription factor in multidrug resistance. Specifically, R821S point mutation in the PDR1 gene in Saccharomyces cerevisiae diploid KK-211 strain plays an important role in tolerance to hydrophobic organic solvents, though the molecular mechanisms underlying organic solvent tolerance are unclear. In KK-211, several ABC transporters and cell wall proteins were upregulated. PDR1 R821S mutant derived from the laboratory haploid strain MT8-1 confirmatively tolerated hydrophobic organic solvents, and this study is the first to reveal its tolerance of the hydrophilic organic solvent, dimethyl sulfoxide (DMSO). To identify the genes involved in the organic solvent tolerance, we focused on the upregulated 4 ABC transporters and 6 cell wall proteins in KK-211, and demonstrated 2 ABC transporters, Pdr10p and Snq2p, and the 4 cell wall proteins, Wsc3p, Pry3p, Pir1p, and Ynl190wp were responsible for hydrophobic organic solvent (n-decane and n-undecane) tolerance. Tolerance to the hydrophilic organic solvent, DMSO, was facilitated by the overproduction of 2 ABC transporters, Snq2p and Yor1p and, 3 cell wall proteins, Wsc3p, Pry3p, and Ynl190wp. Our results suggest that overexpression of genes encoding proteins effective for tolerance to specific organic solvents would enable enhanced tolerances for practical use.

Portal blood supply to locally progressed hepatocellular carcinoma after transcatheter arterial chemoembolization: Observation on CT during arterial portography.

AIM: To analyze the clinical features of locally progressed hepatocellular carcinoma (HCC) supplied by portal blood (PB) after transcatheter arterial chemoembolization (TACE). METHODS: This cohort included 12 tumors (mean diameter ± SD, 1.8 ± 0.8 cm) in 10 patients. PB supply to tumors was judged by CT during arterial portography (CTAP). Imaging data and the clinical course were retrospectively evaluated. RESULTS: Six tumors initially had a small tumor portion supplied by PB. In four tumors, TACE was incomplete because of technical problems. PB supply to recurrent tumors was demonstrated 7.3 ± 3.7 months after TACE. On follow-up arteriography, all embolized branches were occluded or severely attenuated. Four tumors showing a partial stain were treated by additional TACE (n = 3) or TACE plus radiofrequency (RF) ablation (n = 1), one without staining was treated by RF ablation, and seven were followed-up. All tumors progressed except for one treated by RF ablation. On serial CTAP images, relatively large-diameter portal veins directly entered 11 tumors (91.7%) and connected with intratumoral vessels in nine (75%). During follow-up, partial arterial supply was demonstrated in two tumors and additional TACE was performed. Nine patients died after 31.4 ± 16.2 months due to tumor progression (n = 8), or hepatic failure (n = 1). One patient has survived for 53 months despite multiple tumors. CONCLUSIONS: PB supply to locally progressed tumor after TACE became apparent on CTAP. Arterial damage by TACE, incomplete TACE, and preexisting tumor tissues supplied by PB may be the main causes.