Dayong Dong

Chimeric hepatitis B virus core particles carrying an epitope of anthrax protective antigen induce protective immunity against Bacillus anthracis

The major aim of present study is to develop and evaluate chimeric virus-like particles (VLPs) displaying a neutralizing epitope of anthrax protective antigen (PA) as a potential vaccine against anthrax. The truncated hepatitis B virus core (HBc) protein (aa 1-144) was used as a carrier, and the 2beta2-2beta3 loop of the PA domain 2 (aa 302-325) which has been shown contains a dominant neutralizing epitope was inserted into the major immunodominant region (MIR) of the HBc. The recombinant protein HBc-N144-PA-loop2 was expressed in Escherichia coli, and was able to form HBc-like particles confirmed by electron microscopy. The immunogenicity of these chimeric particles was evaluated in mice and guinea pigs. In mice the HBc-N144-PA-loop2 was able to induce PA-epitope specific antibodies; in guinea pigs it was able to induce PA-epitope specific antibodies and anthrax toxin-neutralizing antibodies regardless of whether alum adjuvant was used or not, and was able to partially protect the immunized guinea pigs against virulent anthrax spores challenge. This study suggests chimeric HBc particles carrying a neutralizing epitope of PA can induce protective immunity against Bacillus anthracis.

Hepatitis B virus core particles displaying Mycobacterium tuberculosis antigen ESAT-6 enhance ESAT-6-specific immune responses

Early secreted antigenic target-6 (ESAT-6), an important Mycobacterium tuberculosis T-cell antigen, is an attractive candidate antigen for tuberculosis subunit vaccine development. Because ESAT-6 has a low inherent immunogenicity, we used Hepatitis B virus core (HBc) protein as an immune carrier to enhance ESAT-6 immunogenicity. The ESAT-6 gene was inserted into the major immunodominant region of the HBc molecule by fusion PCR. The recombinant protein, HBc-ESAT-6 (HE6), was expressed in Escherichia coli, and electron microscopy confirmed the formation of virus-like particles. The immunogenicity of the chimeric particles was assessed in mice. Serological assays and in vitro Th1-biased cytokine assays found that immunization with HE6 particles elicited significantly higher ESAT-6-specific antibodies and CD4⁺/CD8⁺ T cell responses in mice compared to immunization with recombinant ESAT-6 protein. These data demonstrate the feasibility of HBc particles serving as an efficient immune carrier for ESAT-6 and suggest that HE6 has potential for use in a tuberculosis subunit vaccine.

Deletion modification enhances anthrax specific immunity and protective efficacy of a hepatitis B core particle-based anthrax epitope vaccine

Protective antigen (PA) is one of the major virulence factors of anthrax and is also the major constituent of the current anthrax vaccine. Previously, we found that the 2β2-2β3 loop of PA contains a dominant neutralizing epitope, the SFFD. We successfully inserted the 2β2-2β3 loop of PA into the major immunodominant region (MIR) of hepatitis B virus core (HBc) protein. The resulting fusion protein, termed HBc-N144-PA-loop2 (HBcL2), can effectively produce anthrax specific protective antibodies in an animal model. However, the protective immunity caused by HBcL2 could still be improved. In this research, we removed amino acids 79-81 from the HBc MIR of the HBcL2. This region was previously reported to be the major B cell epitope of HBc, and in keeping with this finding, we observed that the short deletion in the MIR not only diminished the intrinsic immunogenicity of HBc but also stimulated a higher titer of anthrax specific immunity. Most importantly, this deletion led to the full protection of the immunized mice against a lethal dose anthrax toxin challenge. We supposed that the conformational changes which occurred after the short deletion and foreign insertion in the MIR of HBc were the most likely reasons for the improvement in the immunogenicity of the HBc-based anthrax epitope vaccine.

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.

Secretory expression and efficient purification of recombinant anthrax toxin lethal factor with full biological activity in E. coli

Lethal factor (LF), a virulence factor of Bacillus anthracis, plays key roles in anthrax pathogenesis and host-pathogen interactions. The detailed mechanisms by which LF contributes to infection are still under investigation. While these studies require pure, homogeneous and reliable LF preparations, most methods reported for production of recombinant LF (rLF) in B. anthracis or Escherichia coli either are complicated or add extra residues to the protein. In this work, we modified our previous method by codon optimization and chromatograph workflow refinement and developed an improved strategy for efficient production of rLF from the periplasm of E. coli. We were able to obtain fully functional rLF with a purity above 95% and with a considerable yield of 5 mg/L. The preparation was characterized by SDS-PAGE, Western blot, and N-terminal sequencing, and the activity was validated by intoxication of macrophages and Fischer 344 rats. Our final product is suitable for most research involving drug development and mechanism analysis of anthrax pathogenesis.

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)

Antiviral Potential of Exogenous Human Omega Interferon to Inhibit Pandemic 2009 A (H1N1) Influenza Virus

The pandemic 2009 H1N1 influenza virus broke out in North America and spread rapidly throughout the world. The type I interferon (IFN) response represents one of the first lines of defense against influenza virus infections. In this study, the protective potential of human exogenous IFN-ω against pandemic 2009 A (H1N1) influenza virus was assessed both in vitro and in guinea pigs. The viral loads of pandemic 2009 A (H1N1) influenza virus strains A/California/04/2009 and A/Beijing/501/2009 were reduced by up to 5000-fold in Caco-2 cells by the addition of human IFN-ω. With daily intranasal treatment with human IFN-ω the viral load of pandemic 2009 A (H1N1) influenza virus strain A/California/04/2009 decreased by 1000-fold in lung tissues of guinea pigs. These results provide strong support for the application of human IFN-ω pretreatment to human influenza control.

Intracellular delivery of artificial transcription factors fused to the protein transduction domain of HIV-1 Tat

Protein transduction domains (PTDs), such as the TAT peptide derived from HIV Tat protein, may transduce macromolecules into cells. In the present study, the TAT peptide-fused artificial transcription factors (ATFs) were generated by fusion of the N-terminal TAT peptide with SV40 promoter-targeted three-fingered C2H2 zinc finger proteins and the KRAB transcriptional repression domain. The fusion proteins were then expressed in an E .coli system and purified by Ni-NTA affinity chromatography. The purified fusion proteins were tested on mammalian cell lines CHO DG44 and L929. TAT-ATF-S, which contains the zinc fingers that bind to the SV40 promoter with high specificity, exhibited the desired transcriptional repression activity to the reported genes, indicating the successful cellular delivery and desired conformation of TAT-ATF-S. Our study has provided a new strategy for intracellular ATF delivery.

Recombinant HSA-CMG2 Is a Promising Anthrax Toxin Inhibitor

Anthrax toxin is the major virulence factor produced by Bacillus anthracis. Protective antigen (PA) is the key component of the toxin and has been confirmed as the main target for the development of toxin inhibitors. The inhibition of the binding of PA to its receptor, capillary morphogenesis protein-2 (CMG2), can effectively block anthrax intoxication. The recombinant, soluble von Willebrand factor type A (vWA) domain of CMG2 (sCMG2) has demonstrated potency against anthrax toxin. However, the short half-life of sCMG2 in vivo is a disadvantage for its development as a new anthrax drug. In the present study, we report that HSA-CMG2, a protein combining human serum albumin (HSA) and sCMG2, produced in the Pichia pastoris expression system prolonged the half-life of sCMG2 while maintaining PA binding ability. The IC50 of HSA-CMG2 is similar to those of sCMG2 and CMG2-Fc in in vitro toxin neutralization assays, and HSA-CMG2 completely protects rats from lethal doses of anthrax toxin challenge; these same challenge doses exceed sCMG2 at a sub-equivalent dose ratio and overwhelm CMG2-Fc. Our results suggest that HSA-CMG2 is a promising inhibitor of anthrax toxin and may contribute to the development of novel anthrax drugs.

High-level Expression of Mycobacterium tuberculosis Protein EspB in E. coli and Preparation of Novel Anti-EspB Monoclonal Antibodies

The ESX-1 secretion system plays a critical role in the virulence of Mycobacterium tuberculosis. The ESX-1 secreted protein EspB is cleaved close to its C-terminus during secretion and is necessary for inhibiting phagosome maturation. In this study, the EspB gene of M. tuberculosis H37Rv was cloned into the expression vector of pET21a(+) and was effectively expressed in Escherichia coli BL21(DE3). The expressed fusion protein existed as a soluble form in cell lysate and was first purified by a column packed with Ni-NTA resin and then a column packed with DEAE-Sepharose Fast Flow matrix. Using the purified protein to immunize BALB/c mice, five monoclonal antibodies were produced. As shown by ELISA and immunoblotting, the five respective antibodies could recognize the EspB protein. These monoclonal antibodies will provide powerful reagents for further investigation of EspB protein functions.