HDAC inhibitors suppressed small cell lung cancer cell growth and enhanced the suppressive effects of receptor-targeting cytotoxins via upregulating somatostatin receptor II.

Small cell lung cancer (SCLC) is a malignant human cancer and patients have very limited benefit from traditional anticancer treatments, with a poor five-year survival rate being 10% less. In present study, we observed that Notch signalling activation induced SCLC cell growth suppression via overexpressing Notch active fragments (ICN1, ICN2, ICN3 and ICN4), implying its tumor suppressive role. The histone deacetylase (HDAC) inhibitors also displayed their suppressive effects. Valproic acid (VPA) as a HDAC inhibitor was found to suppress SCLC cell growth and cell cycle arrest at phase G1, and observed to decrease HDAC4 and increase acetylation of histone H4 (AcH4) while activating Notch signalling with an increase of Notch1, Notch target gene HES1 and p21. Meanwhile, we also observed that VPA greatly stimulated the expression of somatostatin receptor type II (SSTR2) that is usually overexpressed in many cancer cells and is used as a target for anticancer drug development, providing a combination therapy with VPA and the SSTR2-targeting cytotoxins. Thus, VPA was investigated in combination with SSTR2-targeted cytotoxins captothecine-somatostatin conjugate (CPT-SST) and colchicine-somatostatin conjugate (COL-SST). Our assays showed that these combination treatments strongly led to a greater suppression as compared to each alone. In conclusion, we found that VPA suppressed SCLC cell growth and increased the expression of SSTR2. These may provide a novel clinical opportunity for enhanced anticancer therapy using the combination strategy of Notch signalling regulator and SSTR2-targeting cytotoxins in SCLC treatments.

DNA prime and virus-like particle boost from a single H5N1 strain elicits broadly neutralizing antibody responses against head region of H5 hemagglutinin.

Since 1996, highly pathogenic avian influenza (HPAI) H5N1 virus has presented a persistent threat to public health. Its high degree of genetic diversity also poses enormous challenges in developing effective vaccines. To search for vaccine regimens that could elicit broadly neutralizing antibody responses against diverse HPAI H5N1 strains, in the present study we tested H5 hemagglutinin (HA) from an A/Thailand/1(KAN)-1/2004 strain in a heterologous prime-boost vaccination. We demonstrated that priming mice with DNA and boosting with virus-like particle induced antibody responses that cross-neutralize all reported clades and subclades of HPAI H5N1 viruses and protect mice from high lethal dose HPAI H5N1 challenge in both active and passive immunizations. Unexpectedly, cross-divergent H5 neutralizing antibodies are directed to the HA head and block both attachment and postattachment of virus entry. Thus, we conclude that as a promising pan-H5 vaccine candidate this prime-boost regimen could be further developed in ferrets and in humans.

Valproic acid suppresses cervical cancer tumor progression possibly via activating Notch1 signaling and enhances receptor-targeted cancer chemotherapeutic via activating somatostatin receptor type II.

PURPOSE: We investigated the effects of the anti-epilepsy drug valproic acid (VPA) alone and in combination in treating cervical cancer. METHODS: VPA was investigated for its effects on cervical cancer Hela cell proliferation and tumor growth via in vitro and in vivo assays. RESULTS: VPA induce cell growth suppression and cell cycle arrest, with an increase of Notch1 that acts as a tumor suppressor and the change of other tumor-associated genes such as p21, p63 and PCNA. VPA was also found to induce cell morphological change, with an increase of certain cell transformation markers such as snail1, snail2 and N-cadherin. Moreover, VPA could significantly up-regulate somatostatin receptor type II (SSTR2). Our in vivo study further demonstrated that VPA via inducing SSTR2 up-regulation extremely enhanced the anti-tumor ability of the SSTR2-preferential cytotoxic COL-SST conjugate in xenografts. CONCLUSIONS: VPA could not only suppress tumor progression but also provide a novel promising therapeutic choice in combination with a receptor-targeted cytotoxic conjugate via activating the specific receptor.

A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses.

Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. In this study, by using a highly sensitive H5N1 pseudotype-based neutralization assay to screen human monoclonal antibodies produced by memory B cells from an H5N1-infected individual and molecular cloning techniques, we developed three fully human monoclonal antibodies. Among them, antibody 65C6 exhibited potent neutralization activity against all H5 clades and subclades except for subclade 7.2 and prophylactic and therapeutic efficacy against highly pathogenic avian influenza H5N1 viruses in mice. Studies on hemagglutinin (HA)-antibody complexes by electron microscopy and epitope mapping indicate that antibody 65C6 binds to a conformational epitope comprising amino acid residues at positions 118, 121, 161, 164, and 167 (according to mature H5 numbering) on the tip of the membrane-distal globular domain of HA. Thus, we conclude that antibody 65C6 recognizes a neutralization epitope in the globular head of HA that is conserved among almost all divergent H5N1 influenza stains.

Heterosubtypic antibody response elicited with seasonal influenza vaccine correlates partial protection against highly pathogenic H5N1 virus.

BACKGROUND: The spread of highly pathogenic avian influenza (HPAI) H5N1 virus in human remains a global health concern. Heterosubtypic antibody response between seasonal influenza vaccine and potential pandemic influenza virus has important implications for public health. Previous studies by Corti et al. and by Gioia et al. demonstrate that heterosubtypic neutralizing antibodies against the highly pathogenic H5N1 virus can be elicited with a seasonal influenza vaccine in humans. However, whether such response offers immune protection against highly pathogenic H5N1 virus remained to be determined. METHODOLOGY/PRINCIPAL FINDINGS: In this study, using a sensitive influenza HA (hemagglutinin) and NA (neuraminidase) pseudotype-based neutralization (PN) assay we first confirmed that low levels of heterosubtypic neutralizing antibody response against H5N1 virus were indeed elicited with seasonal influenza vaccine in humans. We then immunized mice with the seasonal influenza vaccine and challenged them with lethal doses of highly pathogenic H5N1 virus. As controls, we immunized mice with homosubtypic H5N1 virus like particles (VLP) or PBS and challenged them with the same H5N1 virus. Here we show that low levels of heterosubtypic neutralizing antibody response were elicited with seasonal influenza vaccine in mice, which were significantly higher than those in PBS control. Among them 2 out of 27 whose immune sera exhibited similar levels of neutralizing antibody response as VLP controls actually survived from highly pathogenic H5N1 virus challenge. CONCLUSIONS/SIGNIFICANCE: Therefore, we conclude that low levels of heterosubtypic neutralizing antibody response are indeed elicited with seasonal influenza vaccine in humans and mice and at certain levels such response offers immune protection against severity of H5N1 virus infection.

Superior neutralizing antibody response and protection in mice vaccinated with heterologous DNA prime and virus like particle boost against HPAI H5N1 virus.

BACKGROUND: Although DNA plasmid and virus-like particle (VLP) vaccines have been individually tested against highly pathogenic avian influenza (HPAI) H5N1 viruses, the combination of both vaccines into a heterologous prime-boost strategy against HPAI H5N1 viruses has not been reported before. METHODOLOGY/PRINCIPAL FINDINGS: We constructed DNA plasmid encoding H5HA (A/Shenzhen/406H/06, subclade 2.3.4) and generated VLP expressing the same H5HA and N1NA. We then compared neutralizing antibody responses and immune protection elicited with heterologous DNA-VLP, homologous DNA-DNA and VLP-VLP prime-boost strategies against HPAI H5N1 viruses in mice. We demonstrate that DNA-VLP elicits the highest neutralizing antibody titers among the three prime-boost strategies, whereas DNA-DNA elicits higher neutralizing antibody titers than VLP-VLP. We show that although all three prime-boost strategies protect mice from death caused by 10 MLD(50) of homologous and heterologous H5N1 challenge, only DNA-VLP and DNA-DNA protect mice from infection as manifested by no weight loss and no lung pathology. In addition, we show that although DNA-VLP and DNA-DNA protect mice from death caused by 1,000 MLD(50) of homologous H5N1 challenge, only DNA-VLP protects mice from infection. Moreover, we show that after 1,000 MLD(50) of heterologous H5N1 challenge, while all mice in PBS, VLP-VLP and DNA-DNA died, 3 of 6 mice in DNA-VLP actually survived. Finally, we show that DNA-VLP completely protects mice from infection after 1,000 MLD(50) of homologous H5N1 challenge even when the challenge was administrated at 60 days post the boost. CONCLUSIONS/SIGNIFICANCE: These results provide strong support for clinical evaluation of heterologous DNA-VLP prime-boost strategy as a public health intervention against a possible H5N1 pandemic.

Production of hepatitis C virus lacking the envelope-encoding genes for single-cycle infection by providing homologous envelope proteins or vesicular stomatitis virus glycoproteins in trans.

Hepatitis C virus (HCV) infection is a major worldwide health problem. The envelope glycoproteins are the major components of viral particles. Here we developed a trans-complementation system that allows the production of infectious HCV particles in whose genome the regions encoding envelope proteins are deleted (HCVΔE). The lack of envelope proteins could be efficiently complemented by the expression of homologous envelope proteins in trans. HCVΔE production could be enhanced significantly by previously described adaptive mutations in NS3 and NS5A. Moreover, HCVΔE could be propagated and passaged in packaging cells stably expressing HCV envelope proteins, resulting in only single-round infection in wild-type cells. Interestingly, we found that vesicular stomatitis virus (VSV) glycoproteins could efficiently rescue the production of HCV lacking endogenous envelope proteins, which no longer required apolipoprotein E for virus production. VSV glycoprotein-mediated viral entry could allow for the bypass of the natural HCV entry process and the delivery of HCV replicon RNA into HCV receptor-deficient cells. Our development provides a new tool for the production of single-cycle infectious HCV particles, which should be useful for studying individual steps of the HCV life cycle and may also provide a new strategy for HCV vaccine development.

Measurement of neutralizing antibody responses against H5N1 clades in immunized mice and ferrets using pseudotypes expressing influenza hemagglutinin and neuraminidase.

Neutralizing antibody is associated with the prevention and clearance of influenza virus infection. Microneutralization (MN) and hemagglutination inhibition (HI) assays are currently used to evaluate neutralizing antibody responses against human and avian influenza viruses, including H5N1. The MN assay is somewhat labor intensive, while HI is a surrogate for neutralization. Moreover, use of replication competent viruses in these assays requires biosafety level 3 (BSL-3) containment. Therefore, a neutralization assay that does not require BSL-3 facilities would be advantageous. Toward this goal, we generated a panel of pseudotypes expressing influenza hemagglutinin (HA) and neuraminidase (NA) and developed a pseudotype-based neutralization (PN) assay. Here we demonstrate that HA/NA pseudotypes mimic release and entry of influenza virus and that the PN assay exhibits good specificity and reveals quantitative difference in neutralizing antibody titers against different H5N1 clades and subclades. Using immune ferret sera, we demonstrated excellent correlation between the PN, MN, and HI assays. Thus, we conclude that the PN assay is a sensitive and quantifiable method to measure neutralizing antibodies against diverse clades and subclades of H5N1 influenza virus.

design of multiplexed detection assays for identification of avian influenza a virus subtypes pathogenic to humans by SmartCycler real-time reverse transcription-PCR.

Influenza A virus (IAV) epidemics are the result of human-to-human or poultry-to-human transmission. Tracking seasonal outbreaks of IAV and other avian influenza virus (AIV) subtypes that can infect humans, aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts. In this study, we performed four real-time reverse transcription-PCR (rRT-PCR) assays for identification of the IAV M and hemagglutinin (HA) genes from six known AIVs infecting pigs, birds, and humans. IAV M1 gene-positive samples tested by single-step rRT-PCR and a fluorogenic Sybr green I detection system were further processed for H5 subtype identification by using two-primer-set multiplex and Sybr green I rRT-PCR assays. H5 subtype-negative samples were then tested with either a TaqMan assay for subtypes H1 and H3 or a TaqMan assay for subtypes H2, H7, and H9 and a beacon multiplex rRT-PCR identification assay. The four-tube strategy was able to detect 10 RNA copies of the HA genes of subtypes H1, H2, H3, H5, and H7 and 100 RNA copies of the HA gene of subtype H9. At least six H5 clades of H5N1 viruses isolated in Southeast Asia and China were detected by that test. Using rRT-PCR assays for the M1 and HA genes in 202 nasopharyngeal swab specimens from children with acute respiratory infections, we identified a total of 39 samples positive for the IAV M1 gene and subtypes H1 and H3. When performed with a portable SmartCycler instrument, the assays offer an efficient, flexible, and reliable platform for investigations of IAV and AIV in remote hospitals and in the field.