Monoglycosylated SARS-CoV-2 receptor binding domain fused with HAstem-scaffolded protein vaccine confers broad protective immunity against SARS-CoV-2 and influenza viruses.

The SARS-CoV-2 and influenza pandemics have posed a devastating threat to global public health. The best strategy for preventing the further spread of these respiratory viruses worldwide is to administer a vaccine capable of targeting both viruses. Here, we show that a novel monoglycosylated vaccine designed based on the influenza virus HAstem conserved domain fused with the SARS-CoV-2 spike-RBD domain (HSSRmg) can present proper antigenicity that elicits sufficient neutralization efficacy against various SARS-CoV-2 variants while simultaneously providing broad protection against H1N1 viruses in mice. Compared with the fully glycosylated HSSR (HSSRfg), HSSRmg induced higher ELISA titers targeting HAstem and spike-RBD and exhibited significantly enhanced neutralization activity against the Wuhan pseudovirus. The enhanced immune responses raised by JR300-adjuvanted HSSRmg compared to HSSRmg alone include more anti-HAstem and anti-spike-RBD antibodies that provide cross-protection against H1N1 challenges and cross-neutralization of SARS-CoV-2 pseudoviruses. Furthermore, the enhanced immune response raised by JR300-adjuvanted-HSSRmg skews toward a more balanced Th1/Th2 response than that raised by HSSRmg alone. Notably, HSSRmg elicited more plasma B cells and memory B cells, and higher IL-4 and IFN-γ cytokine immune responses than spike (S-2P) in mice with preexisting influenza-specific immunity, suggesting that B-cell activation most likely occurs through CD4+ T-cell stimulation. This study demonstrated that HSSRmg produced using a monoglycosylation process and combined with the JR300 adjuvant elicits superior cross-strain immune responses against SARS-CoV-2 and influenza viruses in mice compared with S-2P. JR300-adjuvanted HSSRmg has great potential as a coronavirus-influenza vaccine that provides dual protection against SARS-CoV-2 and influenza infections.

Increase in Akkermansiaceae in Gut Microbiota of Prostate Cancer-Bearing Mice.

Gut microbiota are reported to be associated with many diseases, including cancers. Several bacterial taxa have been shown to be associated with cancer development or response to treatment. However, longitudinal microbiota alterations during the development of cancers are relatively unexplored. To better understand how microbiota changes, we profiled the gut microbiota composition from prostate cancer-bearing mice and control mice at five different time points. Distinct gut microbiota differences were found between cancer-bearing mice and control mice. Akkermansiaceae was found to be significantly higher in the first three weeks in cancer-bearing mice, which implies its role in the early stage of cancer colonization. We also found that Bifidobacteriaceae and Enterococcaceae were more abundant in the second and last sampling week, respectively. The increments of Akkermansiaceae, Bifidobacteriaceae and Enterococcaceae were previously found to be associated with responses to immunotherapy, which suggests links between these bacteria families and cancers. Additionally, our function analysis showed that the bacterial taxa carrying steroid biosynthesis and butirosin and neomycin biosynthesis were increased, whereas those carrying naphthalene degradation decreased in cancer-bearing mice. Our work identified the bacteria taxa altered during prostate cancer progression and provided a resource of longitudinal microbiota profiles during cancer development in a mouse model.

Comparison of Chicken Immune Responses after Inoculation with H5 Avian Influenza Virus-like Particles Produced by Insect Cells or Pupae.

INTRODUCTION: Novel clade 2.3.4.4 H5 highly pathogenic avian influenza virus (HPAIV) outbreaks have occurred since early 2015 in Taiwan and impacted the island economically, like they have many countries. This research investigates the immunogenicity of two HPAIV-like particles to assess their promise as vaccine candidates. MATERIAL AND METHODS: The haemagglutinin (HA) gene derived from clade 2.3.4.4 H5 HPAIV and matrix protein 1 (M1) gene were cloned into the pFastBac Dual baculovirus vector. The resulting recombinant viruses were expressed in Spodoptera frugiperda moth (Sf)21 cells and silkworm pupae to generate Sf21 virus-like particles (VLP) and silkworm pupa VLP. Two-week-old specific pathogen-free chickens were immunised and their humoral and cellular immune responses were analysed. RESULTS: The silkworm pupa VLP had higher haemagglutination competence. Both VLP types elicited haemagglutination inhibition antibodies, anti-HA antibodies, splenic interferon gamma (IFN-γ) and interleukin 4 (IL-4) mRNA expression, and CD4+/CD8+ ratio elevation. However, chickens receiving silkworm pupa VLP exhibited a significantly higher anti-HA antibody titre in ELISA after vaccination. Although Sf21 VLP recipients expressed more IFN-γ and IL-4, the increase in IFN-γ did not significantly raise the CD4+/CD8+ ratio and the increase in IL-4 did not promote anti-HA antibodies. CONCLUSION: Both VLP systems possess desirable immunogenicity in vivo. However, in respect of immunogenic efficacy and the production cost, pupa VLP may be the superior vaccine candidate against clade 2.3.4.4 H5 HPAIV infection.

Sesquiterpene Lactone Deoxyelephantopin Isolated from Elephantopus scaber and Its Derivative DETD-35 Suppress BRAFV600E Mutant Melanoma Lung Metastasis in Mice.

Melanoma is a highly metastatic disease with an increasing rate of incidence worldwide. It is treatment refractory and has poor clinical prognosis; therefore, the development of new therapeutic agents for metastatic melanoma are urgently required. In this study, we created a lung-seeking A375LM5IF4g/Luc BRAFV600E mutant melanoma cell clone and investigated the bioefficacy of a plant sesquiterpene lactone deoxyelephantopin (DET) and its novel semi-synthetic derivative, DETD-35, in suppressing metastatic A375LM5IF4g/Luc melanoma growth in vitro and in a xenograft mouse model. DET and DETD-35 treatment inhibited A375LM5IF4g/Luc cell proliferation, and induced G2/M cell-cycle arrest and apoptosis. Furthermore, A375LM5IF4g/Luc exhibited clonogenic, metastatic and invasive abilities, and several A375LM5IF4g/Luc metastasis markers, N-cadherin, MMP2, vimentin and integrin α4 were significantly suppressed by treatment with either compound. Interestingly, DET- and DETD-35-induced Reactive Oxygen Species (ROS) generation and glutathione (GSH) depletion were found to be upstream events important for the in vitro activities, because exogenous GSH supplementation blunted DET and DETD-35 effects on A375LM5IF4g/Luc cells. DET and DETD-35 also induced mitochondrial DNA mutation, superoxide production, mitochondrial bioenergetics dysfunction, and mitochondrial protein deregulation. Most importantly, DET and DETD-35 inhibited lung metastasis of A375LM5IF4g/Luc in NOD/SCID mice through inhibiting pulmonary vascular permeability and melanoma cell (Mel-A+) proliferation, angiogenesis (VEGF+, CD31+) and EMT (N-cadherin) in the tumor microenvironment in the lungs. These findings indicate that DET and DETD-35 may be useful in the intervention of lung metastatic BRAFV600E mutant melanoma.

Experimental observation of flat focusing mirror based on photonic jet effect.

In this work, we experimentally demonstrate that a thin rectangle dielectric-metal structure can have a function of a flat focusing mirror based on photonic jet effect in reflection mode. Using polydimethylsiloxane (PDMS) rectangle with size length of 10 µm and wavelength-scale thickness of 1 µm on the top of a silicon wafer, we have built a flat mirror which focuses an incident beam at the focal length changing from 1.38 µm to 11.67 µm upon tuning the beam incidence angle from 30° to 75°. The focusing properties of such a mirror persist in the wavelength range of 405 nm to 671 nm. Our approach can be extended to realize other optical functionalities by properly controlling rectangle dimensions and materials. This flat focusing mirror is able to guide the incident beam in free space without perceptible diffraction at the distance equal to the photonic jet length and suitable for small-scale photonic circuits.

Deregulating the CYP2C19/Epoxy-Eicosatrienoic Acid-Associated FABP4/FABP5 Signaling Network as a Therapeutic Approach for Metastatic Triple-Negative Breast Cancer.

Recurrence and metastasis are the main causes of triple-negative breast cancer (TNBC) mortality. On the basis of our clinical cohorts and integrative omics analyses, we hypothesized that understanding the interplay between fatty acid binding protein (FABP) and epoxy-eicosatrienoic acid (EET) driven metastatic progression can uncover a new opportunity for TNBC intervention. In this study, the biological relevance of increased protein expression of CYP2C19, FABP4, and FABP5 in TNBC tumors and in the TNBC cell line (MDA-MB-231), as well as its highly metastatic lung seeking variant (LM6) were delineated from publicly available datasets, shRNA-mediated knockdown, EET supplementation, cancer and stromal cell co-cultures, and an orthotopic and resection xenograft tumor mouse model. We found that the high expression levels of CYP2C19 and FABP4 and FABP5 are critical in TNBC metastatic transformation and stromal cell interactions. Furthermore, EET-associated nuclear translocation of FABP4 and FABP5 and nuclear accumulation of SREBP-2 or PPAR-γ influence TNBC cell proliferation, migratory transformation, and distal metastasis priming. Most notably, we uncovered novel bioefficacy and modes of action of the anticancer drug doxorubicin and a phytogalactolipid, 1,2-di-O-α-linolenoyl-3-O-ß-galactopyranosyl-sn-glycerol (dLGG), which effectively attenuated TNBC recurrence and lung metastasis through deregulating the FABP/EET dynamics and levels. This study, therefore, introduces a novel approach to combating TNBC by targeting the FABP/EET/CYP-associated metastatic signaling network.

O-Glycosylation-mediated signaling circuit drives metastatic castration-resistant prostate cancer.

Disseminated castration-resistant prostate cancer (CRPC) is a common disease in men that is characterized by limited survival and resistance to androgen-deprivation therapy. The increase in human epidermal growth factor receptor 2 (HER2) signaling contributes to androgen receptor activity in a subset of patients with CRPC; however, enigmatically, HER2-targeted therapies have demonstrated a lack of efficacy in patients with CRPC. Aberrant glycosylation is a hallmark of cancer and involves key processes that support cancer progression. Using transcriptomic analysis of prostate cancer data sets, histopathologic examination of clinical specimens, and in vivo experiments of xenograft models, we reveal in this study a coordinated increase in glycan-binding protein, galectin-4, specific glycosyltransferases of core 1 synthase, glycoprotein- N-acetylgalactosamine 3-ß-galactosyltransferase 1 (C1GALT1) and ST3 beta-galactoside α-2,3-sialyltransferase 1 (ST3GAL1), and resulting mucin-type O-glycans during the progression of CRPC. Furthermore, galectin-4 engaged with C1GALT1-dependent O-glycans to promote castration resistance and metastasis by activating receptor tyrosine kinase signaling and cancer cell stemness properties mediated by SRY-box 9 (SOX9). This galectin-glycan interaction up-regulated the MYC-dependent expression of C1GALT1 and ST3GAL1, which altered cellular mucin-type O-glycosylation to allow for galectin-4 binding. In clinical prostate cancer, high-level expression of C1GALT1 and galectin-4 together predict poor overall survival compared with low-level expression of C1GALT1 and galectin-4. In summary, MYC regulates abnormal O-glycosylation, thus priming cells for binding to galectin-4 and downstream signaling, which promotes castration resistance and metastasis.-Tzeng, S.-F., Tsai, C.-H., Chao, T.-K., Chou, Y.-C., Yang, Y.-C., Tsai, M.-H., Cha, T.-L., Hsiao, P.-W. O-Glycosylation-mediated signaling circuit drives metastatic castration-resistant prostate cancer.

A standardized herbal extract mitigates tumor inflammation and augments chemotherapy effect of docetaxel in prostate cancer.

Activation of the NFκB pathway is often associated with advanced cancer and has thus been regarded as a rational therapeutic target. Wedelia chinensis is rich in luteolin, apigenin, and wedelolactone that act synergistically to suppress androgen receptor activity in prostate cancer. Interestingly, our evaluation of a standardized Wedelia chinensis herbal extract (WCE) concluded its efficacy on hormone-refractory prostate cancer through systemic mechanisms. Oral administration of WCE significantly attenuated tumor growth and metastasis in orthotopic PC-3 and DU145 xenografts. Genome-wide transcriptome analysis of these tumors revealed that WCE suppressed the expression of IKKα/ß phosphorylation and downstream cytokines/chemokines, e.g., IL6, CXCL1, and CXCL8. Through restraining the cytokines expression, WCE reduced tumor-elicited infiltration of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and endothelial cells into the tumors, therefore inhibiting angiogenesis, tumor growth, and metastasis. In MDSCs, WCE also reduced STAT3 activation, downregulated S100A8 expression and prevented their expansion. Use of WCE in combination with docetaxel significantly suppressed docetaxel-induced NFκB activation, boosted the therapeutic effect and reduced the systemic toxicity caused by docetaxel monotherapy. These data suggest that a standardized preparation of Wedelia chinensis extract improved prostate cancer therapy through immunomodulation and has potential application as an adjuvant agent for castration-resistant prostate cancer.

A Standardized Wedelia chinensis Extract Overcomes the Feedback Activation of HER2/3 Signaling upon Androgen-Ablation in Prostate Cancer.

Crosstalk between the androgen receptor (AR) and other signaling pathways in prostate cancer (PCa) severely affects the therapeutic outcome of hormonal therapy. Although anti-androgen therapy prolongs overall survival in PCa patients, resistance rapidly develops and is often associated with increased AR expression and upregulation of the HER2/3-AKT signaling pathway. However, single agent therapy targeting AR, HER2/3 or AKT usually fails due to the reciprocal feedback loop. Previously, we reported that wedelolactone, apigenin, and luteolin are the active compounds in Wedelia chinensis herbal extract, and act synergistically to inhibit the AR activity in PCa. Here, we further demonstrated that an herbal extract of W. chinensis (WCE) effectively disrupted the AR, HER2/3, and AKT signaling networks and therefore enhanced the therapeutic efficacy of androgen ablation in PCa. Furthermore, WCE remained effective in suppressing AR and HER2/3 signaling in an in vivo adapted castration-resistant PCa (CRPC) LNCaP cell model that was insensitive to androgen withdrawal and second-line antiandrogen, enzalutamide. This study provides preclinical evidence that the use of a defined, single plant-derived extract can augment the therapeutic efficacy of castration with significantly prolonged progression-free survival. These data also establish a solid basis for using WCE as a candidate agent in clinical studies.

A virus-like particle vaccination strategy expands its tolerance to H3N2 antigenic drift by enhancing neutralizing antibodies against hemagglutinin stalk.

Seasonal influenza viruses impact public health annually due to their continual evolution. However, the current inactivated seasonal vaccines provide poor protection against antigenically drifted viruses and require periodical reformulation through hit-and-miss predictions about which strains will circulate during the next season. To reduce the impact caused by vaccine mismatch, we investigated the drift-tolerance of virus-like particles (VLP) as an improved vaccine candidate. The cross-protective humoral immunity elicited by the H3N2-VLP vaccine constructed for the 2011-2012 season was examined against viruses isolated from 2010 to 2015 in Taiwan evolving chronologically through clades 1, 4, 5, 3B and 3C, as well as viruses that were circulating globally in 2005, 2007 and 2009. Mouse immunization results demonstrated that H3N2-VLP vaccine elicited superior immunological breadth in comparison with the cognate conventional whole-inactivated virus (WIV) vaccine. Titers of neutralizing antibodies against heterologous strains representing each epidemic period in the VLP group were significantly higher than in the WIV group, indicating the antibody repertoire induced by the H3N2-VLPs was insensitive to viral antigenic drift over a span of at least 10 years. Noticeably, H3N2-VLP elicited higher levels of anti-stalk antibodies than H3N2-WIV, which offset the ineffectiveness caused by antigenic drift. This advantageous effect was attributed to the uncleaved precursor of their HA proteins. These results suggest a mechanism through which VLP-induced humoral immunity may better tolerate the evolutionary dynamics of influenza viruses and point to the possible use of a VLP vaccine as a method by which the requirement for annual updates of seasonal influenza vaccines may be diminished.

Metastatic Progression of Prostate Cancer Is Mediated by Autonomous Binding of Galectin-4-O-Glycan to Cancer Cells.

Metastatic prostate cancer continues to pose a difficult therapeutic challenge. Prostate cancer progression is associated with aberrant O-glycosylation of cancer cell surface receptors, but the functional impact of such events is uncertain. Here we report spontaneous metastasis of human prostate cancer xenografts that express high levels of galectin-4 along with genetic signatures of EGFR-HER2 signaling and O-glycosylation. Galectin-4 expression in clinical specimens of prostate cancer correlated with poor patient survival. Galectin-4 binding to multiple receptor tyrosine kinases stimulated their autophosphorylation, activated expression of pERK, pAkt, fibronectin, and Twist1, and lowered expression of E-cadherin, thereby facilitating epithelial-mesenchymal transition, invasion, and metastasis. In vivo investigations established that galectin-4 expression enabled prostate cancer cells to repopulate tumors in orthotopic and heterotopic tissues. Notably, these effects of galectin-4 relied upon O-glycosylation mediated by C1GALT1, a galactosyltransferase implicated in other cancers. Parallel changes in galectin-4 and O-glycosylation triggered aberrant receptor signaling and more aggressive invasive character in prostate cancer cells, which through better survival in the circulation also contributed to the bulk cell progeny of distal tumors. Our findings establish galectin-4 and C1GALT1-mediated glycosylation in a signaling axis that is activated during prostate cancer progression, with implications for therapeutic targeting of advanced metastatic disease. Cancer Res; 76(19); 5756-67. ©2016 AACR.

Development of single-chain variable fragments (scFv) against influenza virus targeting hemagglutinin subunit 2 (HA2).

Influenza A viruses (IAV) are widespread in birds and domestic poultry, occasionally causing severe epidemics in humans and posing health threats. Hence, the need to develop a strategy for prophylaxis or therapy, such as a broadly neutralizing antibody against IAV, is urgent. In this study, single-chain variable fragment (scFv) phage display technology was used to select scFv fragments recognizing influenza envelope proteins. The Tomlinson I and J scFv phage display libraries were screened against the recombinant HA2 protein (rHA2) for three rounds. Only the third-round elution sample of the Tomlinson J library showed high binding affinity to rHA2, from which three clones (3JA18, 3JA62, and 3JA78) were chosen for preparative-scale production as soluble antibody by E. coli. The clone 3JA18 was selected for further tests due to its broad affinity for influenza H1N1, H3N2 and H5N1. Simulations of the scFv 3JA18-HA trimer complex revealed that the complementarity-determining region of the variable heavy chain (VH-CDR2) bound the stem region of HA. Neutralization assays using a peptide derived from VH-CDR2 also supported the simulation model. Both the selected antibody and its derived peptide were shown to suppress infection with H5N1 and H1N1 viruses, but not H3N2 viruses. The results also suggested that the scFvs selected from rHA2 could have neutralizing activity by interfering with the function of the HA stem region during virus entry into target cells.

A novel H6N1 virus-like particle vaccine induces long-lasting cross-clade antibody immunity against human and avian H6N1 viruses.

Avian influenza A(H6N1) virus is one of the most common viruses isolated from migrating birds and domestic poultry in many countries. The first and only known case of human infection by H6N1 virus in the world was reported in Taiwan in 2013. This led to concern that H6N1 virus may cause a threat to public health. In this study, we engineered a recombinant H6N1 virus-like particle (VLP) and investigated its vaccine effectiveness compared to the traditional egg-based whole inactivated virus (WIV) vaccine. The H6N1-VLPs exhibited similar morphology and functional characteristics to influenza viruses. Prime-boost intramuscular immunization in mice with unadjuvanted H6N1-VLPs were highly immunogenic and induced long-lasting antibody immunity. The functional activity of the VLP-elicited IgG antibodies was proved by in vitro seroprotective hemagglutination inhibition and microneutralization titers against the homologous human H6N1 virus, as well as in vivo viral challenge analyses which showed H6N1-VLP immunization significantly reduced viral load in the lung, and protected against human H6N1 virus infection. Of particular note, the H6N1-VLPs but not the H6N1-WIVs were able to confer cross-reactive humoral immunity; antibodies induced by H6N1-VLP vaccine robustly inhibited the hemagglutination activities and in vitro replication of distantly-related heterologous avian H6N1 viruses. Furthermore, the H6N1-VLPs were found to elicit significantly greater anti-HA2 antibody responses in immunized mice than H6N1-WIVs. Collectively, we demonstrated for the first time a novel H6N1-VLP vaccine that effectively provides broadly protective immunity against both human and avian H6N1 viruses. These results, which uncover the underlying mechanisms for induction of wide-range immunity against influenza viruses, may be useful for future influenza vaccine development.

Induction of c-Cbl contributes to anti-cancer effects of HDAC inhibitor in lung cancer.

Here we found loss of c-Cbl, an E3 ligase, expression in non-small cell lung cancer (NSCLC) compared with its adjacent normal tissue in patient specimens. HDAC inhibition by WJ or knockdown of HDAC 1, HDAC2, HDAC3 or HDAC6 all induced c-Cbl. Ectopic expression of c-Cbl induced decreased EGFR, inhibited growth in NSCLC cells. Knockdown of EGFR inhibited NSCLC growth. Mutation of EGFR at Y1045 decreased WJ-induced growth inhibition as well as in vivo anti-cancer effect and EGFR degradation mediated by WJ. Time-lapse confocal analysis showed co-localization of c-Cbl and EGFR after WJ treatment. Furthermore, WJ inhibited lung tumor growth through c-Cbl induction in orthotopic and tail vein injected models. C-Cbl up-regulation induced by HDACi is a potential strategy for NSCLC treatment.

Development of a standardized and effect-optimized herbal extract of Wedelia chinensis for prostate cancer.

Herbal medicine is a popular complementary or alternative treatment for prostate cancer. Wedelia chinensis has at least three active compounds, wedelolactone, luteolin, and apigenin synergistically inhibiting prostate cancer cell growth in vitro. Here, we report a systematic study to develop a standardized and effect-optimized herbal extract, designated as W. chinensis extract (WCE) to facilitate its future scientific validation and clinical use. Ethanolic extract of dried W. chinensis plant was further condensed, acid hydrolyzed, and enriched with preparative chromatography. The chemical compositions of multiple batches of the standardized preparation WCE were quantified by LC/MS/MS, and biological activities were analyzed by in vitro and in vivo assays. Furthermore, the pharmacokinetics of the holistic WCE were compared with the combination of the equivalent principal active compounds through oral administration. The results indicated that quantitative chemical assay and PSA (prostate-specific antigen)-reporter assay together are suitable to measure the quality and efficacy of a standardized Wedelia extract on a xenograft tumor model. The presence of minor concomitant compounds in WCE prolonged the systemic exposure to the active compounds, thus augmented the anti-tumor efficacy of WCE. In conclusion, a combination of LC/MS/MS and PSA reporter assay is suitable to qualify a standardized preparation of WCE. Furthermore, the pharmacokinetics and oral bioavailability of active compounds demonstrate that holistic WCE exerted additional pharmacological synergy beyond the multi-targeted therapeutic effects caused by more than one active compound. WCE merits a higher priority to be studied for use in prostate cancer treatment.

TET1 suppresses cancer invasion by activating the tissue inhibitors of metalloproteinases.

Tumor suppressor gene silencing through cytosine methylation contributes to cancer formation. Whether DNA demethylation enzymes counteract this oncogenic effect is unknown. Here, we show that TET1, a dioxygenase involved in cytosine demethylation, is downregulated in prostate and breast cancer tissues. TET1 depletion facilitates cell invasion, tumor growth, and cancer metastasis in prostate xenograft models and correlates with poor survival rates in breast cancer patients. Consistently, enforced expression of TET1 reduces cell invasion and breast xenograft tumor formation. Mechanistically, TET1 suppresses cell invasion through its dioxygenase and DNA binding activities. Furthermore, TET1 maintains the expression of tissue inhibitors of metalloproteinase (TIMP) family proteins 2 and 3 by inhibiting their DNA methylation. Concurrent low expression of TET1 and TIMP2 or TIMP3 correlates with advanced node status in clinical samples. Together, these results illustrate a mechanism by which TET1 suppresses tumor development and invasion partly through downregulation of critical gene methylation.

A VLP vaccine induces broad-spectrum cross-protective antibody immunity against H5N1 and H1N1 subtypes of influenza A virus.

The recent threats of influenza epidemics and pandemics have prioritized the development of a universal vaccine that offers protection against a wider variety of influenza infections. Here, we demonstrate a genetically modified virus-like particle (VLP) vaccine, referred to as H5M2eN1-VLP, that increased the antigenic content of NA and induced rapid recall of antibody against HA(2) after viral infection. As a result, H5M2eN1-VLP vaccination elicited a broad humoral immune response against multiple viral proteins and caused significant protection against homologous RG-14 (H5N1) and heterologous A/California/07/2009 H1N1 (CA/07) and A/PR/8/34 H1N1 (PR8) viral lethal challenges. Moreover, the N1-VLP (lacking HA) induced production of a strong NA antibody that also conferred significant cross protection against H5N1 and heterologous CA/07 but not PR8, suggesting the protection against N1-serotyped viruses can be extended from avian-origin to CA/07 strain isolated in humans, but not to evolutionally distant strains of human-derived. By comparative vaccine study of an HA-based VLP (H5N1-VLP) and NA-based VLPs, we found that H5N1-VLP vaccination induced specific and strong protective antibodies against the HA(1) subunit of H5, thus restricting the breadth of cross-protection. In summary, we present a feasible example of direction of VLP vaccine immunity toward NA and HA(2), which resulted in cross protection against both seasonal and pandemic influenza strains, that could form the basis for future design of a better universal vaccine.

Mammalian expression of virus-like particles for advanced mimicry of authentic influenza virus.

BACKGROUND: Influenza A viruses are major human and animal pathogens with huge economic and societal impact from illness, hospitalizations, and deaths. Virus-like particles (VLPs) of influenza virus have been suggested as a vaccine candidate offering improved safety and efficacy. To develop this concept further, we established a flexible platform to efficiently generate different subtypes of mammalian-expressed influenza VLPs. Here we demonstrate that these mammalian VLPs strongly resemble the authentic viruses in structure, particle size and composition of host factors, and even glycosylation of viral antigens. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a mammalian VLP system was established by stable co-expression of four influenza structural proteins (HA, NA, M1, and M2) in a Vero cell line. By replacing the surface glycoproteins of HA and NA, we converted the H3N2-VLP subtype to H5N1-VLP. After centrifugation purification of conditioned media, the particle morphologies, average sizes, and hemagglutination abilities of secreted VLPs were characterized, and the VLP constituents were identified by LC/MS/MS. Protease protection assays demonstrated that specific cellular proteins that co-purified with influenza virions were integrated into mammalian VLPs. The glycosylation profiles of mammalian VLPs as revealed by deglycosylation assays were similar to that of progeny viruses produced from Vero cells. Vaccination of mice with 2.5 microg and above of H5N1-VLP elicited H5-specific IgG1 antibodies and resulted in full protection against lethal infection with homologous virus. These results provide compelling evidence that mammalian VLPs closely emulate the exterior of authentic virus particles not only in antigen presentation but also in biological properties and should provide promising vaccine candidates. CONCLUSIONS/SIGNIFICANCE: This flexible mammalian influenza VLP system offers a superior alternative to the conventional reverse genetic vaccine platform without concerns over inadequate presentation of immune antigens or limitations imposed by the manipulation of real viruses.

Herbal extract of Wedelia chinensis attenuates androgen receptor activity and orthotopic growth of prostate cancer in nude mice.

PURPOSE: Wedelia chinensis is a common ingredient of anti-inflammatory herbal medicines in Taiwan and southern China. Inflammation is involved in promoting tumor growth, invasion, and metastasis. This study aims to test the biological effects in vivo of W. chinensis extract on prostate cancer. EXPERIMENTAL DESIGN: The in vivo efficacy and mechanisms of action of oral administration of a standardized extract of W. chinensis were analyzed in animals bearing a subcutaneous or orthotopic prostate cancer xenograft. RESULTS: Exposure of prostate cancer cells to W. chinensis extract induced apoptosis selectively in androgen receptor (AR)-positive prostate cancer cells and shifted the proportion in each phase of cell cycle toward G(2)-M phase in AR-negative prostate cancer cells. Oral herbal extract (4 or 40 mg/kg/d for 24-28 days) attenuated the growth of prostate tumors in nude mice implanted at both subcutaneous (31% and 44%, respectively) and orthotopic (49% and 49%, respectively) sites. The tumor suppression effects were associated with increased apoptosis and lower proliferation in tumor cells as well as reduced tumor angiogenesis. The antitumor effect of W. chinensis extract was correlated with accumulation of the principle active compounds wedelolactone, luteolin, and apigenin in vivo. CONCLUSION: Anticancer action of W. chinensis extract was due to three active compounds that inhibit the AR signaling pathway. Oral administration of W. chinensis extract impeded prostate cancer tumorigenesis. Future studies of W. chinensis for chemoprevention or complementary medicine against prostate cancer in humans are thus warranted.

A novel diterpene suppresses CWR22Rv1 tumor growth in vivo through antiproliferation and proapoptosis.

Androgen receptor (AR) is the main therapeutic target for treatment of metastatic prostate cancers (PCa). As recurrent tumors restore AR activity independent of hormones, new therapies that abolish AR activity have been sought to prevent or delay the emergence of ablation-resistant disease. Here, we report that a novel abietane diterpene, 6-hydroxy-5,6-dehydrosugiol (HDHS), isolated from the stem bark of Cryptomeria japonica, was a potent AR antagonist in PCa cells. HDHS treatment of androgen-dependent LNCaP and androgen-responsive 22Rv1 cells induced apoptosis as shown by nucleosome release, activation of caspase-3 and caspase-7, and cleavage of poly(ADP-ribose) polymerase accompanied with concomitant up-regulation of tumor suppressor p53. HDHS also decreased the protein expression of cyclins (D1 and E), cyclin-dependent kinases (CDK2, CDK4, and CDK6), and retinoblastoma phosphorylation in PCa cells, which suggest cell cycle arrest in the G(1) phase. Oral administration of HDHS at 0.5 and 2.5 mg/kg once daily for 24 days to 22Rv1 PCa xenografted mice suppressed tumor growth by 22% and 39%, respectively, in association with decreased proliferation and increased apoptosis in tumor cells, which further correlated with increased levels of HDHS in plasma and tumors. Overall, our data suggest that HDHS has potential for use in chemoprevention and chemotherapy of PCa.