Lihua Hou

Safety and immunogenicity of a novel recombinant adenovirus type-5 vector-based Ebola vaccine in healthy adults in China: preliminary report of a randomised, double-blind, placebo-controlled, phase 1 trial

BACKGROUND Up to now, all tested Ebola virus vaccines have been based on the virus strain from the Zaire outbreak in 1976. We aimed to assess the safety and immunogenicity of a novel recombinant adenovirus type-5 vector-based Ebola vaccine expressing the glycoprotein of the 2014 epidemic strain. METHODS We did this randomised, double-blind, placebo-controlled, phase 1 clinical trial at one site in Taizhou County, Jiangsu Province, China. Healthy adults (aged 18-60 years) were sequentially enrolled and randomly assigned (2:1), by computer-generated block randomisation (block size of six), to receive placebo, low-dose adenovirus type-5 vector-based Ebola vaccine, or high-dose vaccine. Randomisation was pre-stratified by dose group. All participants, investigators, and laboratory staff were masked to treatment allocation. The primary safety endpoint was occurrence of solicited adverse reactions within 7 days of vaccination. The primary immunogenicity endpoints were glycoprotein-specific antibody titres and T-cell responses at day 28 after the vaccination. Analysis was by intention to treat. The study is registered with ClinicalTrials.gov, number NCT02326194. FINDINGS Between Dec 28, 2014, and Jan 9, 2015, 120 participants were enrolled and randomly assigned to receive placebo (n=40), low-dose vaccine (n=40), or high-dose vaccine. Participants were followed up for 28 days. Overall, 82 (68%) participants reported at least one solicited adverse reaction within 7 days of vaccination (n=19 in the placebo group vs n=27 in the low-dose group vs n=36 in the high-dose group; p=0·0002). The most common reaction was mild pain at the injection site, which was reported in eight (20%) participants in the placebo group, 14 (35%) participants in the low-dose group, and 29 (73%) participants in the high-dose vaccine group (p<0·0001). We recorded no statistical differences in other adverse reactions and laboratory tests across groups. Glycoprotein-specific antibody titres were significantly increased in participants in the low-dose and high-dose vaccine groups at both day 14 (geometric mean titre 421·4 [95% CI 249·7-711·3] and 820·5 [598·9-1124·0], respectively; p<0·0001) and day 28 (682·7 [424·3-1098·5] and 1305·7 [970·1-1757·2], respectively; p<0·0001). T-cell responses peaked at day 14 at a median of 465·0 spot-forming cells (IQR 180·0-1202·5) in participants in the low-dose group and 765·0 cells (400·0-1460·0) in those in the high-dose group. 21 (18%) participants had mild fever (n=9 in the placebo group, n=6 in the low-dose group, and n=6 in the high-dose group). No serious adverse events were recorded. INTERPRETATION Our findings show that the high-dose vaccine is safe and robustly immunogenic. One shot of the high-dose vaccine could mount glycoprotein-specific humoral and T-cell response against Ebola virus in 14 days. FUNDING China National Science and Technology, Beijing Institute of Biotechnology, and Tianjin CanSino Biotechnology.

Safety and immunogenicity of a recombinant adenovirus type-5 vector-based Ebola vaccine in healthy adults in Sierra Leone: a single-centre, randomised, double-blind, placebo-controlled, phase 2 trial

BACKGROUND A recombinant adenovirus type-5 vector-based vaccine expressing the glycoprotein of Ebola Zaire Makona variant showed good safety and immunogenicity in a phase 1 trial of healthy Chinese adults. We aimed to assess the safety and immunogenicity of this vaccine in healthy adults in Sierra Leone and to determine the optimal dose. METHODS We did a single-centre, randomised, double-blind, placebo-controlled, phase 2 clinical trial at Sierra Leone-China Friendship Hospital, Freetown, Sierra Leone. We recruited healthy adults aged 18-50 years who were HIV negative, had no history of Ebola virus infection, and had no previous immunisation with other Ebola vaccine candidates. Participants were sequentially enrolled and randomly assigned (2:1:1), by computer-generated block randomisation (block size of eight), to receive the high-dose vaccine (1·6 × 1011 viral particles), low-dose vaccine (8·0 × 1010 viral particles), or placebo (containing only vaccine excipients, with no viral particles). Participants, investigators, and study staff (except two study pharmacists) were masked from treatment allocation. The primary safety outcome was occurrence of solicited adverse reactions within 7 days of vaccination, analysed by intention to treat. The primary immunogenicity outcome was glycoprotein-specific antibody responses at days 14, 28, and 168 after vaccination, analysed in all vaccinated participants who had blood samples drawn for antibody tests. The trial is registered with the Pan African Clinical Trials Registry, number PACTR201509001259869, and is completed. FINDINGS During Oct 10-28, 2015, 500 participants were enrolled and randomly assigned to receive the high-dose vaccine (n=250), low-dose vaccine (n=125), or placebo (n=125). 132 (53%) participants in the high-dose group, 60 (48%) in the low-dose group, and 54 (43%) in the placebo group reported at least one solicited adverse reaction within 7 days of vaccination. Most adverse reactions were mild and self-limiting. Solicited injection-site adverse reactions were significantly more frequent in vaccine recipients (65 [26%] in high-dose group and 31 [25%] in low-dose group) than in those receiving placebo (17 [14%]; p=0·0169). Glycoprotein-specific antibody responses were detected from day 14 onwards (geometric mean titre 1251·0 [95% CI 976·6-1602·5] in low-dose group and 1728·4 [1459·4-2047·0] in high-dose group) and peaked at day 28 (1471·8 [1151·0-1881·8] and 2043·1 [1762·4-2368·4]), but declined quickly in the following months (223·3 [148·2-336·4] and 254·2 [185·0-349·5] at day 168). Geometric mean titres in the placebo group remained around 6·0-6·8 throughout the study period. Three serious adverse events (malaria, gastroenteritis, and one fatal asthma episode) were reported in the high-dose vaccine group, but none was deemed related to the vaccine. INTERPRETATION The recombinant adenovirus type-5 vector-based Ebola vaccine was safe and highly immunogenic in healthy Sierra Leonean adults, and 8·0 × 1010 viral particles was the optimal dose. FUNDING Chinese Ministry of Science and Technology and the National Health and Family Planning Commission, Beijing Institute of Biotechnology, and Tianjin CanSino Biotechnology.

Anthrax Susceptibility: Human Genetic Polymorphisms Modulating ANTXR2 Expression.

Anthrax toxin causes anthrax pathogenesis and expression levels of ANTXR2 (anthrax toxin receptor 2) are strongly correlated with anthrax toxin susceptibility. Previous studies found that ANTXR2 transcript abundance varies considerably in individuals of different ethnic/geographical groups, but no eQTLs (expression quantitative trait loci) have been identified. By using 3C (chromatin conformation capture), CRISPR-mediated genomic deletion and dual-luciferase reporter assay, gene loci containing cis-regulatory elements of ANTXR2 were localized. Two SNPs (single nucleotide polymorphism) at the conserved CREB-binding motif, rs13140055 and rs80314910 in the promoter region of the gene, modulating ANTXR2 promoter activity were identified. Combining these two regulatory SNPs with a previously reported SNP, rs12647691, for the first time, a statistically significant correlation between human genetic variations and anthrax toxin sensitivity was observed. These findings further our understanding of human variability in ANTXR2 expression and anthrax toxin susceptibility.

Immunity duration of a recombinant adenovirus type-5 vector-based Ebola vaccine and a homologous prime-boost immunisation in healthy adults in China: final report of a randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND The 2013-15 Ebola virus disease epidemic in west Africa greatly accelerated the development of Ebola vaccine. We aimed to analyse the immune persistence induced by one shot of an adenovirus type-5 vector-based Ebola virus vaccine up to 6 months and the effect of boosting with a homologous vector in healthy adults in China. METHODS In a randomised, double-blind, placebo-controlled, phase 1 clinical trial in one site in Jiangsu Province, China, 120 healthy adults aged 18-60 years received an initial dose of intramuscular adenovirus type-5 Ebola virus vaccine of 4·0 × 1010 viral particles, 1·6 × 1011 viral particles, or placebo, and were followed up to day 168. Participants were subsequently re-recruited to receive a booster dose of the same vaccine or placebo, in the same dose, at month 6. Women who were pregnant, breastfeeding, or planned to become pregnant during the next month were excluded. Randomisation was conducted by computer-generated block randomisation. Randomisation data were unmasked for interim analysis of the data obtained between days 0-28 but not disclosed to participants or site staff. Safety and immunogenicity analysis were done on the intention-to-treat population. We aimed to assess the safety profile of the experimental vaccine and the immunity responses to a single-dose immunisation or a homologous prime-boost regimen. Primary outcomes were Ebola glycoprotein-specific ELISA antibody responses 28 days post-boost and the occurrences of adverse reactions post-boost. The original trial and the extended booster study were registered with ClinicalTrials.gov, numbers NCT02326194 and NCT02533791, respectively. FINDINGS Between Dec 28, 2014, and Jan 9, 2015, we enrolled 210 volunteers. 90 participants were not randomised due to not meeting inclusion criteria (61), meeting exclusion criteria (4), or withdrawal of consent (25). 120 people were randomly assigned to receive intramuscular Ebola vaccine at 4·0 × 1010 viral particles (low dose, n=40), Ebola vaccine at 1·6 × 1011 viral particles (high dose, n=40), or placebo (n=40, in two groups of 20). After prime vaccination, the geometric mean titer (GMT) of ELISA EC90 peaked at 682·7 (95% CI 424·3-1098·5) in the low-dose vaccine group and 1305·7 (970·1-1757·2) in the high-dose vaccine group at day 28, and then fell gradually through the next a few months to 575·5 (394·8-838·8) in the high-dose vaccine group and 197·9 (107·9-362·7) in the low-dose vaccine group at day 168. No specific response was recorded in the placebo group with a GMT of 5·0. Of the 120 participants involved in the initial trial, ten participants declined to participate, and 110 were included in the boost immunisation: 38 received the low dose, 35 received the high dose, and 37 received the placebo. At day 28 after boost vaccination, the ELISA EC90 titres rapidly rose to 6110 (95% CI 4705-7935) in the low-dose group and to 11825 (8904-15705) in the high dose group. 78 of 110 participants reported at least one solicited adverse reaction within the first 7 days after booster administration. Both of the groups who received vaccine showed significantly higher incidence of mild or moderate solicited adverse reactions than did the placebo group. INTERPRETATION The adenovirus 5-vectored Ebola vaccine of 1·6 × 1011 viral particles was highly immunogenic and safe. The lower dose of 4·0 × 1010 viral particles was also safe, but immunogenicity seemed to be more vulnerable to the pre-existing immunity of adenovirus 5. A homologous priming-boosting regimen with adenovirus type-5 Ebola vaccine at 6 months interval was able to elicit greater antibody responses with longer duration. These results support an immunisation strategy to implement a booster injection for a more durable protection against Ebola virus disease. FUNDING Chinese Ministry of Science and Technology and The National Health and Family Planning Commission, Beijing Institute of Biotechnology, and Tianjin CanSino Biotechnology.

Enterovirus 71 inhibits cellular type I interferon signaling by downregulating JAK1 protein expression.

Enterovirus 71 (EV71) infection can cause severe disease and lead to death in children. Recurring outbreaks of EV71 have been reported in several countries. Interferons (IFNs) have been used for decades to treat several types of viral infection, but have a limited ability to inhibit EV71 replication. Herein, we intend to investigate the mechanisms by which EV71 inhibits the cellular type I IFN response. In this study, MRC-5 (human embryonic lung fibroblast) or RD (human rhabdomyosarcoma) cells were infected with EV71, and then treated with or without IFN-α2b. Cells were harvested and analyzed by flow cytometry to determine the level of IFNAR1. Cell lysis were prepared to detect the levels of STAT1, STAT2, phosphorylated STAT1, phosphorylated STAT2, IFNAR1, JAK1, and TYK2 by Western blotting. The phosphorylation of STAT1 and STAT2 induced by IFN were inhibited without significant downregulation of IFNAR1 in EV71-infected cells. The EV71-induced suppression of STAT1 and STAT2 phosphorylation was not rescued by the protein tyrosine phosphatases inhibitor, and was independent of suppressor of cytokine signaling protein 1/3 levels. The phosphorylation of JAK1 and TYK2 were inhibited accompanied by EV71-induced downregulation of JAK1, which occurred at a post-transcriptional level and was proteasome independent. JAK1 expression did not decrease, and IFN-α-stimulated STAT1 and STAT2 phosphorylation were not blocked in HEK293T cells overexpressing the EV71 viral protein 2A or 3C. This study demonstrates that EV71 inhibits the cellular type I IFN antiviral pathway by downregulating JAK1, while the expression of IFNAR1 does not significantly alter in EV71-infected cells. Additionally, the EV71 viral proteins 2A and 3C do not act as antagonists of cellular type I IFN signaling.

Prediction and identification of mouse cytotoxic T lymphocyte epitopes in Ebola virus glycoproteins

BackgroundEbola viruses (EBOVs) cause severe hemorrhagic fever with a high mortality rate. At present, there are no licensed vaccines or efficient therapies to combat EBOV infection. Previous studies have shown that both humoral and cellular immune responses are crucial for controlling Ebola infection. CD8+ T cells play an important role in mediating vaccine-induced protective immunity. The objective of this study was to identify H-2d-specific T cell epitopes in EBOV glycoproteins (GPs).ResultsComputer-assisted algorithms were used to predict H-2d-specific T cell epitopes in two species of EBOV (Sudan and Zaire) GP. The predicted peptides were synthesized and identified in BALB/c mice immunized with replication-deficient adenovirus vectors expressing the EBOV GP. Enzyme-linked immunospot assays and intracellular cytokine staining showed that the peptides RPHTPQFLF (Sudan EBOV), GPCAGDFAF and LYDRLASTV (Zaire EBOV) could stimulate splenoctyes in immunized mice to produce large amounts of interferon-gamma.ConclusionThree peptides within the GPs of two EBOV strains were identified as T cell epitopes. The identification of these epitopes should facilitate the evaluation of vaccines based on the Ebola virus glycoprotein in a BALB/c mouse model.

Enhanced expression of soluble recombinant tetanus neurotoxin Hc in Escherichia coli as a tetanus vaccine candidate.

The expression of the carboxyl fragment of the heavy chain of tetanus neurotoxin (TeNT-Hc) in Escherichia coli has been hampered by the unusually high AT content and the presence of rarely used codons by Clostridium. The gene encoding TeNT-Hc was optimized for E. coli by replacing rare codons and decreasing the AT pairs from 72.57% to 52.47%. The reconstructed gene was expressed in E. coli BL21(DE3) and resulted in a soluble product which was about 46% of the total bacterial protein. TeNT-Hc produced in a 42 L fermentor was purified to >95% at 87 g/kg of cell paste (approximately 333 mg/L). BALB/c mice vaccinated with three bi-weekly doses of TeNT-Hc with Freunds adjuvant were fully protected against an intraperitoneally challenge of 2 × 10(3) 50% lethal doses (LD(50)s) of tetanus neurotoxin. NIH mice vaccinated with TeNT-Hc adsorbed to aluminum hydroxide gel adjuvant demonstrated a potency of 168 IU/mL, which was 2 times higher than the national standard for tetanus vaccines. These results suggest that TeNT-Hc may be a promising second-generation vaccine candidate for clinical use against tetanus neurotoxin.

Protection against anthrax and plague by a combined vaccine in mice and rabbits

The protective antigen (PA) of Bacillus anthracis and the Fraction 1 Capsular Antigen (F1 antigen), V antigen of Yersinia pestis have been demonstrated to be potential immunogens and candidate vaccine sub-units against anthrax and plague respectively. In this study, the authors have investigated the antibody responses and the protective efficacy when the antigens were administered separately or in combination intramuscularly formulation adsorbed to an aluminum hydroxide adjuvant. Results show that immunized rF1 + rV and rPA antigen together was as effective as separately for induction of serological antibody response, and these titers were maintained for over 1 year in mice. An isotype analysis of the serum indicates that the co-administration of these antigens did not influence the antigen-specific IgG1/IgG2a ratio which was consistent with a Th2 bias. Furthermore, the combined vaccine comprising the protein antigens rF1 + rV + rPA has been demonstrated to protect mice from subcutaneous challenge with 10(7) colony-forming units (CFU) virulent Y. pestis strain, and to fully protect rabbit against subcutaneous challenge with 1.2x10(5) colony-forming units (CFU) virulent B. anthracis spores. These data show that the protective efficacy was unaffected when the antigens were administered in combination.

Fusion protein of Δ27LFn and EFn has the potential as a novel anthrax toxin inhibitor

MINT‐7014735, MINT‐7014747, MINT‐7014761: PA63 (uniprotkb:P13423) and LF (uniprotkb:P15917) bind (MI:0407) by surface plasmon resonance (MI:0107)

Creation of a Six-fingered Artificial Transcription Factor That Represses the Hepatitis B Virus HBx Gene Integrated into a Human Hepatocellular Carcinoma Cell Line

Chronic hepatitis B virus (HBV) infection is an independent risk factor for the development of hepatocellular carcinoma (HCC). The HBV HBx gene is frequently identified as an integrant in the chromosomal DNA of patients with HCC. HBx encodes the X protein (HBx), a putative viral oncoprotein that affects transcriptional regulation of several cellular genes. Therefore, HBx may be an ideal target to impede the progression of HBV infection–related HCC. In this study, integrated HBx was transcriptionally downregulated using an artificial transcription factor (ATF). Two three-fingered Cys2-His2 zinc finger (ZF) motifs that specifically recognized two 9-bp DNA sequences regulating HBx expression were identified from a phage-display library. The ZF domains were linked into a six-fingered protein that specified an 18-bp DNA target in the Enhancer I region upstream of HBx. This DNA-binding domain was fused with a Krüppel-associated box (KRAB) transcriptional repression domain to produce an ATF designed to downregulate HBx integrated into the Hep3B HCC cell line. The ATF significantly repressed HBx in a luciferase reporter assay. Stably expressing the ATF in Hep3B cells resulted in significant growth arrest, whereas stably expressing the ATF in an HCC cell line lacking integrated HBx (HepG2) had virtually no effect. The targeted downregulation of integrated HBx is a promising novel approach to inhibiting the progression of HBV infection–related HCC.