Structures of Coxsackievirus A16 Capsids with Native Antigenicity: Implications for Particle Expansion, Receptor Binding, and Immunogenicity.

UNLABELLED: Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the primary causes of the epidemics of hand-foot-and-mouth disease (HFMD) that affect more than a million children in China each year and lead to hundreds of deaths. Although there has been progress with vaccines for EV71, the development of a CVA16 vaccine has proved more challenging, and the EV71 vaccine does not give useful cross-protection, despite the capsid proteins of the two viruses sharing about 80% sequence identity. The structural details of the expanded forms of the capsids, which possess nonnative antigenicity, are now well understood, but high resolution information for the native antigenic form of CVA16 has been missing. Here, we remedy this with high resolution X-ray structures of both mature and natural empty CVA16 particles and also of empty recombinant viruslike particles of CVA16 produced in insect cells, a potential vaccine antigen. All three structures are unexpanded native particles and antigenically identical. The recombinant particles have recruited a lipid moiety to stabilize the native antigenic state that is different from the one used in a natural virus infection. As expected, the mature CVA16 virus is similar to EV71; however, structural and immunogenic comparisons highlight differences that may have implications for vaccine production. IMPORTANCE: Hand-foot-and-mouth disease is a serious public health threat to children in Asian-Pacific countries, resulting in millions of cases. EV71 and CVA16 are the two dominant causative agents of the disease that, while usually mild, can cause severe neurological complications, leading to hundreds of deaths. EV71 vaccines do not provide protection against CVA16. A CVA16 vaccine or bivalent EV71/CVA16 vaccine is therefore urgently needed. We report atomic structures for the mature CVA16 virus, a natural empty particle, and a recombinant CVA16 virus-like particle that does not contain the viral genome. All three particles have similar structures and identical antigenicity. The recombinant particles, produced in insect cells (a system suitable for making vaccine antigen), are stabilized by recruiting from the insect cells a small molecule that is different from that used by the virus in a normal infection. We present structural and immunogenic comparisons with EV71 to facilitate structure-based drug design and vaccine development.

The ultrasound contrast imaging properties of lipid microbubbles loaded with urokinase in dog livers and their thrombolytic effects when combined with low-frequency ultrasound in vitro.

A new microbubble loaded with urokinase (uPA-MB) was explored in a previous study. However, its zeta potential and ultrasound contrast imaging properties and its thrombolytic effects when combined with low-frequency ultrasound (LFUS) were unclear. The zeta potential and ultrasound contrast imaging property of 5 uPA-MBs loading with 50,000 IU uPA was respectively detected using a Malvern laser particle analyzer and a Logiq 9 digital premium ultrasound system. Its ultrasound contrast imaging property was performed on the livers of two healthy dogs to compare with SonoVue. And the clot mass loss rate, D-dimer concentration and surface morphology of the clot residues were measured to evaluate the thrombolytic effect after treatment with three doses of 5 uPA-MBs combined with LFUS in vitro. The zeta potential of 5 uPA-MBs (-27.0 ± 2.40 mV) was higher than that of normal microbubbles (-36.95 ± 1.77 mV). Contrast-enhanced imaging of the hepatic vessels using 5 uPA-MBs was similar to SonoVue, while the imaging duration of 5 uPA-MBs (10 min) was longer than SonoVue (6 min). The thrombolytic effect of three doses of uPA-MBs combined with LFUS was significantly better than that of the control group and showed dose dependence. The 5 uPA-MBs have a negative charge on their surface and good echogenicity as ultrasound contrast agents. The 5 uPA-MBs combined with LFUS can promote thrombolysis in a dose-dependent manner.

Development of a sandwich ELISA for the quantification of enterovirus 71.

Since 2008, enterovirus 71 (EV71) has been responsible for high-mortality seasonal epidemics of hand, foot and mouth disease in China. Currently many groups in the world are in the process of developing EV71 vaccines to combat this deadly disease. We have developed three EV71-specific monoclonal antibodies, and in this study we report the establishment of a fast and cost-effective sandwich ELISA kit for measurement of virus concentration in EV71 vaccines using a pair of mouse anti-EV71 monoclonal antibodies. The system is specific for EV71 virus, with no cross-reactivity to coxsackievirus A16, H1N1, rabies, and hepatitis A. Using a reference EV71 vaccine standard, the sensitivity of the assay kit was determined to be 0.82 U/ml, with a linear range between 3.75 and 120 U/ml.

Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: A vaccine for enterovirus 71 (EV71) is needed to address the high burden of disease associated with infection. We assessed the efficacy, safety, immunogenicity, antibody persistence, and immunological correlates of an inactivated alum-adjuvant EV71 vaccine. METHODS: We did a randomised, double-blind, placebo-controlled, phase 3 trial. Healthy children aged 6-35 months from four centres in China were randomly assigned (1:1) to receive vaccine or alum-adjuvant placebo at day 0 and 28, according to a randomisation list (block size 30) generated by an independent statistician. Investigators and participants and their guardians were masked to the assignment. Primary endpoints were EV71-associated hand, foot, and mouth disease (HFMD) and EV71-associated disease during the surveillance period from day 56 to month 14, analysed in the per-protocol population. This study is registered with ClinicalTrials.gov, number NCT01508247. FINDINGS: 10,245 participants were enrolled and assigned: 5120 to vaccine versus 5125 to placebo. 4907 (with three cases of EV71-associated HFMD and eight cases of EV71-associated disease) versus 4939 (with 30 cases of EV71-associated HFMD and 41 cases of EV71-associated disease) were included in the primary efficacy analysis. Vaccine efficacy was 90·0% (95% CI 67·1-96·9) against EV71-associated HFMD (p=0·0001) and 80·4% (95% CI 58·2-90·8) against EV71-associated disease (p<0·0001). Serious adverse events were reported by 62 of 5117 (1·2%) participants in the vaccine group versus 75 of 5123 (1·5%) in the placebo group (p=0·27). Adverse events occurred in 3644 (71·2%) versus 3603 (70·3%; p=0·33). INTERPRETATION: EV71 vaccine provides high efficacy, satisfactory safety, and sustained immunogenicity. FUNDING: China's 12-5 National Major Infectious Disease Program, Beijing Vigoo Biological.

Results of a phase III clinical trial with an HBsAg-HBIG immunogenic complex therapeutic vaccine for chronic hepatitis B patients: experiences and findings.

BACKGROUND & AIMS: Even though various experimental therapeutic approaches for chronic hepatitis B infection have been reported, few of them have been verified by clinical trials. We have developed an antigen-antibody (HBsAg-HBIG) immunogenic complex therapeutic vaccine candidate with alum as adjuvant (YIC), aimed at breaking immune tolerance to HBV by modulating viral antigen processing and presentation. A double-blind, placebo-controlled, phase II B clinical trial of YIC has been reported previously, and herein we present the results of the phase III clinical trial of 450 patients. METHODS: Twelve doses of either YIC or alum alone as placebo were administered randomly to 450 CHB patients and they were followed for 24weeks after the completion of immunization. The primary end point was HBeAg seroconversion, and the secondary end points were decrease in viral load, improvement of liver function, and histology. RESULTS: In contrast to the previous phase II B trial using six doses of YIC and alum as placebo, six more injections of YIC or alum resulted in a decrease of the HBeAg seroconversion rate from 21.8% to 14.0% in the YIC group, but an increase from 9% to 21.9% in the alum group. Decrease in serum HBV DNA and normalization of liver function were similar in both groups (p>0.05). CONCLUSIONS: Overstimulation with YIC did not increase but decreased its efficacy due to immune fatigue in hosts. An appropriate immunization protocol should be explored and is crucial for therapeutic vaccination. Multiple injections of alum alone could have stimulated potent inflammatory and innate immune responses contributing to its therapeutic efficacy, and needs further investigation.

The antitumor effect of a new docetaxel-loaded microbubble combined with low-frequency ultrasound in vitro: preparation and parameter analysis.

PURPOSE: To develop a novel docetaxel (DOC)-loaded lipid microbubbles (MBs) for achieving target therapy and overcoming the poor water-solubility drawback of DOC. METHODS: A novel DOC-loaded microbubble (DOC + MB) was prepared by lyophilization and the physicochemical properties including ultrasound contrast imaging of the liver were measured. The anti-tumor effect of the DOC + MBs combined with low-frequency ultrasound (LFUS; 0.8 Hz, 2.56 W/cm², 50% cycle duty) on the DLD-1 cancer cell line was examined using an MTT assay. RESULTS: The physicochemical properties of the two tested formats of DOC + MBs (1.0 mg and 1.6 mg) was shown: concentration, (6.74 ± 0.02) × 108 bubbles/mL and (8.27 ± 0.15) × 108 bubbles/mL; mean size, 3.296 ± 0.004 µm and 3.387 ± 0.005 µm; pH value, 6.67 ± 0.11 and 6.56 ± 0.05; release rate, 3.41% and 12.50%; Zeta potential, -37.95 ± 7.84 mV and -44.35 ± 8.70 mV; and encapsulation efficiency, 54.9 ± 6.21% and 46.3 ± 5.69%, respectively. Compared with SonoVue, the DOC + MBs similarly enhanced the echo signal of the liver imaging. The anti-tumor effect of the DOC + MBs/LFUS group was significantly better than that of DOC alone and that of the normal MBs/LFUS groups. CONCLUSIONS: The self-made DOC + MBs have potential as a new ultrasound contrast agent and drug-loaded microbubble, and can obviously enhance the antitumor effect of DOC under LFUS exposure.

Immunogenicity and safety of an enterovirus 71 vaccine in healthy Chinese children and infants: a randomised, double-blind, placebo-controlled phase 2 clinical trial.

BACKGROUND: Enterovirus 71 (EV71) outbreaks are a socioeconomic burden, especially in the western Pacific region. Results of phase 1 clinical trials suggest an EV71 vaccine has a clinically acceptable safety profile and immunogenicity. We aimed to assess the best possible dose and formulation, immunogenicity, and safety profile of this EV71 vaccine in healthy Chinese children. METHODS: This randomised, double-blind, placebo-controlled, phase 2 trial was undertaken at one site in Donghai County, Jiangsu Province, China. Eligible participants were healthy boys or girls aged 6­36 months. Participants were randomly assigned (1:1:1:1:1) to receive either 160 U, 320 U, or 640 U alum-adjuvant EV71 vaccine, 640 U adjuvant-free EV71 vaccine, or a placebo (containing alum adjuvant only), according to a blocked randomisation list generated by SAS 9.1. Participants and investigators were masked to the assignment. The primary endpoint was anti-EV71 neutralising antibody geometric mean titres (GMTs) at day 56, analysed according to protocol. The study is registered with ClinicalTrials.gov, number NCT01399853. FINDINGS: We randomly assigned 1200 participants, 240 (120 aged 6­11 months [infants] and 120 aged 12­36 months [children]) of whom were assigned to each dose. 1106 participants completed the study and were included in the according-to-protocol analysis. The main reasons for dropout were withdrawal of consent and refusal to donate a blood sample. Infants who received the 640 U adjuvant vaccine had the highest GMTs on day 56 (742·2 [95% CI 577·3­954·3]), followed by those who received the 320 U formulation (497·9 [383·1­647·0]). For children, those who received the 320 U formulation had the highest GMTs on day 56 (1383·2 [1037·3­1844·5]). Participants who received the vaccine had significantly higher GMTs than did who received placebo (p<0·0001). For the subgroup of participants who were seronegative at baseline, both infants and children who received the 640 U adjuvant vaccine had the highest GMTs on day 56 (522·8 [403·9­676·6] in infants and 708·4 [524·1­957·6] in children), followed by those who received the 320 U adjuvant vaccine (358·2 [280·5­457·5] in infants and 498·0 [383·4­646·9] in children). 549 (45·8%) of 1200 participants (95 CI 42·9­48·6%) reported at least one injection-site or systemic adverse reaction, but the incidence of adverse reactions did not differ significantly between groups (p=0·36). The 640 U alum-adjuvant vaccine group had a significantly higher incidence of induration than did the 640 U adjuvant-free group (p=0·001). INTERPRETATION: Taking immunogenicity, safety, and production capacity into account, the 320 U alum-adjuvant formulation of the EV71 vaccine is probably the best possible formulation for phase 3 trials. FUNDING: The National Science and Technology Major Project (2011ZX10004-902) of the Chinese Ministry of Science and Technology, China's 12­5 National Major Infectious Disease Program (2012ZX10002-001), and Beijing Vigoo Biological.

Antithrombotic effect of Z4A5 on coronary thrombosis in a canine model of acute unstable angina.

BACKGROUND AND PURPOSE: The glycoprotein IIb/IIIa receptor is the final common pathway of platelet aggregation, regardless of the agonist, and thus represents an ideal therapeutic target for blocking coronary thrombosis. In this study, the anti-platelet and antithrombotic actions of Z4A5, a new glycoprotein IIb/IIIa receptor inhibitor, were evaluated in a canine model of acute unstable angina. EXPERIMENTAL APPROACH: Z4A5 was given i.v. as a bolus followed by 60 min of continuous infusion at doses of 30 µg·kg⁻¹ + 1 µg·kg⁻¹·min⁻¹, 30 µg·kg⁻¹ + 5 µg·kg⁻¹·min⁻¹ or 300 µg·kg⁻¹ + 5 µg·kg⁻¹·min⁻¹. Its antithrombotic effect was evaluated in a model of coronary thrombosis, the injured, stenosed left circumflex coronary artery, in which platelet-dependent cyclic flow reductions (CFRs) were induced by vascular compression and constriction to simulate clinical acute unstable angina. Platelet aggregation and coagulation parameters were determined in platelet-rich plasma and platelet poor plasma respectively. KEY RESULTS: The Z4A5 infusion induced a dose-dependent reduction in CFR frequency, which returned to baseline levels after the termination of the infusion at low doses. At medium dose that inhibited most part of platelet aggregation, it increased tongue bleeding time marginally with no dramatic changes in haemodynamic and coagulation parameters. Furthermore, the inhibition of ADP-induced platelet aggregation and prolonged bleeding time observed during Z4A5 infusion reverted to baseline levels after the termination of the infusion. CONCLUSIONS AND IMPLICATIONS: Z4A5 is an effective antithrombotic agent for coronary artery thrombosis with a rapid-on and rapid-off pharmacological profile, and could be used as an alternative treatment of coronary artery ischaemic syndromes.

Comparative analysis of the immunogenicity and protective effects of inactivated EV71 vaccines in mice.

BACKGROUND: Enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD). Three inactivated EV71 whole-virus vaccines of different strains developed by different manufacturers in mainland China have recently entered clinical trials. Although several studies on these vaccines have been published, a study directly comparing the immunogenicity and protective effects among them has not been carried out, which makes evaluating their relative effectiveness difficult. Thus, properly comparing newly developed vaccines has become a priority, especially in China. METHODS AND FINDINGS: This comparative immunogenicity study was carried out on vaccine strains (both live and inactivated), final container products (FCPs) without adjuvant, and corresponding FCPs containing adjuvant (FCP-As) produced by three manufacturers. These vaccines were evaluated by neutralizing antibody (NAb) responses induced by the same or different dosages at one or multiple time points post-immunization. The protective efficacy of the three vaccines was also determined in one-day-old ICR mice born to immunized female mice. Survival rates were observed in these suckling mice after challenge with 20 LD(50) of EV71/048M3C2. Three FCP-As, in a dose of 200 U, generated nearly 100% NAb positivity rates and similar geometric mean titers (GMTs), especially at 14-21 days post-inoculation. However, the dynamic NAb responses were different among three vaccine strains or three FCPs. The FCP-As at the lowest dose used in clinical trials (162 U) showed good protective effects in suckling mice against lethal challenge (90-100% survival), while the ED(50) of NAb responses and protective effects varied among three FCP-As. CONCLUSIONS: These studies establish a standard method for measuring the immunogenicity of EV71 vaccines in mice. The data generated from our mouse model study indicated a clear dose-response relationship, which is important for vaccine quality control and assessment, especially for predicting protective efficacy in humans when combined with future clinical trial results.

Reactogenicity and immunogenicity of an enterovirus 71 vaccine in Chinese healthy children and infants.

BACKGROUND: Enterovirus 71 (EV71) is highly contagious and can cause severe complications. A safe and effective vaccine is needed. We assessed the reactogenicity and immunogenicity of an inactivated, alum-adjuvanted EV71 vaccine in this study. METHODS: A randomized, double-blind, placebo-controlled clinical trial was undertaken in 360 healthy participants who were stratified into 2 age groups (6-12 and 13-60 months), and randomly allocated to receive placebo or the investigational vaccine containing 160 U, 320 U or 640 U antigen per dose by the ratio of 1:1:1:1 at days 0 and 28. Reactogenic data within 28 days after each vaccination were recorded. Blood samples were obtained on days 0, 28 and 56 for neutralizing antibody assay. RESULTS: Overall, 193 participants reported at least 1 injection-site or systemic adverse reaction with 53.3% and 54.4% participants receiving the study vaccine and placebo, respectively. Most of the reactions were mild or moderate. Three serious adverse events were observed, but none was related to vaccination. In the participants with seronegative baseline, after 2 doses all the participants receiving EV71 vaccines were seropositive and the seroconversion rates were more than 98.1%. In the participants with seropositive baseline, 1 dose induced good seroconversion rates of more than 64.3% in participants receiving EV71 vaccines. CONCLUSIONS: This study found that the inactivated EV71 vaccine was well tolerated and had good immunogenicity in healthy children and infants. A single dose induced typical booster response in the participants with a seropositive baseline, and 2 doses were needed for the immunologically naive participants.

A novel anti-platelet peptide (Z4A5) potential for glycoprotein IIb/IIIa inhibits platelet aggregation.

INTRODUCTION: Z4A5 is a novel peptide that inhibits platelet aggregation and formation of platelet thrombi, but the mechanism of its anti-platelet effects remains unknown. This study explores the anti-platelet effect and mechanism of Z4A5. METHODS: We investigated the anti-platelet activity of Z4A5 on platelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin (TH) in human platelet-rich plasma (PRP). Fibrinogen and PAC-1 binding to glycoproteinIIb/IIIa (GPIIb/IIIa) were measured by flow cytometry. In addition, we investigated the integrin specificity of Z4A5 in attachment and detachment assays using human umbilical vein endothelial cells (HUVEC) and assessed the relative cell number using the MTT assay. RESULTS: In vitro, Z4A5 inhibited ADP-, AA- and TH-induced human platelet aggregation with IC(50) values of 0.46 ± 0.05 µM (n = 10), 0.23 ± 0.0 5 µM (n = 10) and 0.21 ± 0.02 µM (n = 10), respectively. Z4A5 inhibited fibrinogen, and PAC-1 bound to platelet GPIIb/IIIa with IC(50) values of 0.48 ± 0.07 µM (n = 8) and 0.63 ± 0.12 µM (n = 6), respectively. Z4A5 failed to inhibit α(V)ß(3) integrin-mediated HUVEC attachment to vitronectin and did not cause any significant detachment of HUVEC monolayer when compared with the controls. CONCLUSIONS: Z4A5 is a potential anti-platelet drug that inhibits fibrinogen binding to GPIIb/IIIa, but does not affect the structurally similar integrin α(V)ß(3).

A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71.

Enterovirus 71 (EV71) is a major agent of hand, foot and mouth disease in children that can cause severe central nervous system disease and death. No vaccine or antiviral therapy is available. High-resolution structural analysis of the mature virus and natural empty particles shows that the mature virus is structurally similar to other enteroviruses. In contrast, the empty particles are markedly expanded and resemble elusive enterovirus-uncoating intermediates not previously characterized in atomic detail. Hydrophobic pockets in the EV71 capsid are collapsed in this expanded particle, providing a detailed explanation of the mechanism for receptor-binding triggered virus uncoating. These structures provide a model for enterovirus uncoating in which the VP1 GH loop acts as an adaptor-sensor for cellular receptor attachment, converting heterologous inputs to a generic uncoating mechanism, highlighting new opportunities for therapeutic intervention.

Molecular determinants of enterovirus 71 viral entry: cleft around GLN-172 on VP1 protein interacts with variable region on scavenge receptor B 2.

Enterovirus 71 (EV71) is one of the major pathogens that cause hand, foot, and mouth disease outbreaks in young children in the Asia-Pacific region in recent years. Human scavenger receptor class B 2 (SCARB2) is the main cellular receptor for EV71 on target cells. The requirements of the EV71-SCARB2 interaction have not been fully characterized, and it has not been determined whether SCARB2 serves as an uncoating receptor for EV71. Here we compared the efficiency of the receptor from different species including human, horseshoe bat, mouse, and hamster and demonstrated that the residues between 144 and 151 are critical for SCARB2 binding to viral capsid protein VP1 of EV71 and seven residues from the human receptor could convert murine SCARB2, an otherwise inefficient receptor, to an efficient receptor for EV71 viral infection. We also identified that EV71 binds to SCARB2 via a canyon of VP1 around residue Gln-172. Soluble SCARB2 could convert the EV71 virions from 160 S to 135 S particles, indicating that SCARB2 is an uncoating receptor of the virus. The uncoating efficiency of SCARB2 significantly increased in an acidic environment (pH 5.6). These studies elucidated the viral capsid and receptor determinants of enterovirus 71 infection and revealed a possible target for antiviral interventions.

Thrombolytic effects of a combined therapy with targeted microbubbles and ultrasound in a 6 h cerebral thrombosis rabbit model.

Our previous study has shown that P1 polypeptide-loaded microbubbles (clot-targeted microbubbles, TMB) are effective for thrombolysis and recanalization in a 0.5 h cerebral thrombosis rabbit model when combined with low-frequency ultrasound (LFUS, 0.8 MHz). However, the thrombolytic effects of TMB combined with LFUS are still unclear in a 6 h cerebral thrombosis rabbit model, which closely resembles human embolic stroke. Aiming to extend the 3 h therapeutic window limitation of thrombolytic drugs, a 6 h cerebral thrombosis model of common carotid artery (CCA) occlusion was induced in rabbits, and thrombolysis using TMB by intra-arterial (IA) and intravenous (IV) application combined with LFUS was then compared to untargeted microbubbles (UTMB) and recombinant tissue plasminogen activator (rt-PA). The patency score and thrombolysis in brain ischemia (TIBI) in IA TMB combined with LFUS (IA TMB/LFUS) were significantly higher compared to the IA normal saline control with LFUS (IA SC/LFUS) (both P < 0.05) and IA UTMB plus LFUS (IA UTMB/LFUS) (both P < 0.05), respectively. The recanalization rate in the IA TMB/LFUS group (66.67%) was significantly higher compared to the IA SC/LFUS group (12.50%, P < 0.05). The patency score, TIBI and recanalization rate of IA TMB/LFUS were higher than in the IV TMB/LFUS group, but there was no significant difference between the two groups, which was similar to the infarction ratio. TMB/LFUS is an effective and safe therapy for thrombolysis in a 6 h cerebral thrombosis rabbit model, and the IA TMB/LFUS group was slightly better than the IV TMB/LFUS group.

Establishing China's national standards of antigen content and neutralizing antibody responses for evaluation of enterovirus 71 (EV71) vaccines.

Enterovirus 71 (EV71) is a highly infectious agent that causes hand-foot-mouth disease (HFMD) in humans. Effective vaccination against EV71 infection is critically important, given the recent outbreak of HFMD in the Asia-Pacific region, where it has shown significant mortality and morbidity. There is currently no approved anti-viral therapy available to treat the disease. While several vaccine manufacturers are actively developing EV71 vaccines, there are no international reference standards available to conduct quality control on EV71 vaccines or to assess the effectiveness of EV71 vaccines in immunized populations. In the current report, antigen reference standard based on the C4 subtype of the EV71 vaccine strain was developed. In addition, neutralizing antibody (NTAb) reference panels were analyzed and standards with various neutralizing titers were selected. These reference antigens were used to calibrate vaccine samples from several producers and found that five EV71 antigens and the national reference standards showed good linearity and parallelism. Moreover, mice immunized with various vaccines at doses standardized by these national references showed comparable NTAb responses. Finally, the national NTAb reference panels were found to effectively reduce assay discrepancy between different labs. Taken together, these national reference standards are highly valuable for the standardization and evaluation of EV71 vaccines.

Codon optimization enhances secretory expression of Pseudomonas aeruginosa exotoxin A in E. coli.

Pseudomonas aeruginosa exotoxin A (PEA) is a number of family of bacterial ADP-ribosylating toxins and possesses strong immunogenicity. The detoxified exotoxin A, as a potent vaccine adjuvant and vaccine carrier protein, has been extensively used in human and animal vaccinations. However, the expression level of PEA gene in Escherichia coli is relative low which is likely due to the presence of rare codon and high levels of GC content. In order to enhance PEA gene expression, we optimized PEA gene using E. coli preferred codons and expressed it in E. coli BL21 (DE3) by using pET-20b(+) secretory expression vector. Our results showed that codon optimization significantly reduced GC content and enhanced PEA gene expression (70% increase compared with that of the wild-type). Moreover, the codon-optimized PEA possessed biological activity and had the similar toxic effects on mouse L292 cells compared with the wild-type PEA gene. Codon optimization will not only improve PEA gene expression but also benefit further modification of PEA gene using nucleotide-mediated site-directed mutagenesis. A large number of purified PEA proteins will provide the necessary conditions for further PEA functional research and application.

[Preparation and identification of the monoclonal antibodies against VP1 capsid protein of Enterovius 71].

OBJECTIVE: To prepare monoclonal antibodies (McAbs) against VP1 capsid protein of Enterovirus 71. METHODS: Two peptides, SP55 and SP70, containing amino acid 163-177 and 208-222 of VP1, were synthesized respectively. Immunized BALB/c mice with the synthetic peptides to establish the hybridoma cell strains secreting specific McAb to VP1. After the specific McAbs were prepared, identified and analyzed the titer by indirect ELISA assay. The positive clones were selected and their neutralization titer were determined by neutralization test. RESULTS: Two high titered anti-VP1 antibodies secreted by the hybridoma cells showed good neutralization reaction with enterovirus 71 on RD cells, and the neutralization titer were 1:8 and 1:16 respectively. CONCLUSION: Two high titered anti-VP1 antibodies, with good neutralization activity, secreted by the hybridoma cells, which lays the foundation for further study.

Generation of neutralizing monoclonal antibodies against Enterovirus 71 using synthetic peptides.

Enterovirus 71 (EV71) has led to recent outbreaks of hand, foot and mouth disease (HFMD) in China, resulting in high mortality. In this study, several monoclonal antibodies were generated by immunizing mice with two synthetic peptides, SP55 and SP70, containing amino acids 163-177 and 208-222 of VP1. The specificities of the anti-EV71 peptide monoclonal antibodies were confirmed by Western blot analysis and immunocytochemistry against EV71 virus. Most importantly, we have identified a monoclonal antibody, clone 22A12, which shows strong neutralizing activity against EV71 in an in vitro neutralization assay. Because there is no vaccine available and treatment is very limited, mouse anti-EV71 monoclonal antibody, clone 22A12, could be a promising candidate to be humanized and used for treatment of EV71 infection.

Association between mannose-binding lectin and HIV infection and progression in a Chinese population.

Mannose-binding lectin (MBL) is a circulating pattern recognition molecule involved in the innate immune system that mediates phagocytosis and activates complement by binding to a carbohydrate extremity. Some MBL genetic polymorphisms result in deficient protein levels and increased susceptibility to infection. The objective of this study was to investigate the association between MBL2 exon 1 polymorphisms, serum levels of normal MBL, and HIV-1 infection and progression in a Chinese population. A total of 1075 adult patients infected with HIV-1 (532 male and 543 female) were recruited. The genotype of 145 patients was determined and the genotype frequencies compared with healthy population controls. The disease status of patients was evaluated for different MBL2 genotypes and normal MBL serum levels. MBL2 exon 1 polymorphisms (A/O or O/O) were significantly more common in HIV-1-infected patients than in the healthy controls. Patients in clinical categories B/C with severe diseases were significantly more likely to have one variant allele. There was a statistical relationship between MBL2 genotypes and MBL serum levels. In addition, higher CD4(+) T cell counts and ratios of CD4(+) T cells:CD8(+) T cells were observed in patients with medium MBL levels than with low or high MBL levels. Patients with mild diseases were also more likely to have medium MBL levels than high levels. Analysis of MBL levels with respect to sex yielded differences. Median MBL levels were substantially higher in males than in females in HIV-1-infected patients. Lower CD4(+) T cell counts were detected in males with low MBL levels, but the opposite was observed in females. Our results suggest that genetic polymorphisms resulting in MBL deficiency are associated with increased susceptibility to HIV-1 infection and disease progression in the studied population. Moreover, serum circulating levels of normal MBL in HIV-1-infected patients could be an important auxiliary biological marker in association with CD4(+) T cell counts in the evaluation of HIV-1 disease progression. The sex differences in the regulation of MBL serum levels during infection merit further exploration.