Wei-an Yu

The recombinant Hc subunit of Clostridium botulinum neurotoxin serotype A is an effective botulism vaccine candidate

Vaccination with recombinant His-tagged isoforms of the Clostridium botulinum Hc domain of neurotoxin serotype A (rAHc) have effectively protected against challenge with active botulinum neurotoxin serotype A. To establish a formulation suitable for human use, rAHc was expressed in Escherichia coli without a His-tag and purified by sequential chromatography on ion-exchange and hydrophobic-interaction resins. Purified rAHc was used to vaccinate mice and survival was evaluated following challenge with active toxin. rAHc-vaccinated mice were protected against an active toxin challenge in mouse models of disease and a dose-response relationship was observed between the dose of rAHc administered and protection. Vaccination with rAHc in the presence or absence of adjuvants was also tested following intramuscular or subcutaneous vaccination to determine the optimal route of vaccination in the context of active toxin challenge. The data presented in the report suggested that rAHc administered with or without adjuvants functioned effectively over time in protecting mice against challenge with neurotoxin suggesting that this form of rAHc may be developed into a human vaccine candidate designed for the prevention of botulism.

Enhancement of the immunogenicity of DNA replicon vaccine of Clostridium botulinum neurotoxin serotype A by GM-CSF gene adjuvant

Granulocyte-macrophage clony-stimulating factor (GM-CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen-presenting cells to the site of antigen synthesis as well as stimulate the maturation of dendritic cells.This study evaluated the utility of GM-CSF as a plasmid DNA replicon vaccine adjuvants for botulinum neurotoxin serotype A (BoNT/A) in mouse model. In balb/c mice that received the plasmid DNA replicon vaccines derived from Semliki Forest virus (SFV) carrying the Hc gene of BoNT/A (AHc), both antibody and lymphoproliferative response specific to AHc were induced, the immunogenicity was enhanced by co-delivery or coexpress of the GM-CSF gene. In particular, when AHc and GM-CSF were coexpressed within the SFV based DNA vaccine, the anti-AHc antibody titers and survival rates of immunized mice after challenged with BoNT/A were significantly increased, and further enhanced by coimmunization with aluminum phosphate adjuvant.

Individual and bivalent vaccines against botulinum neurotoxin serotypes A and B using DNA-based Semliki Forest virus vectors

We evaluated individual and bivalent replicon vaccines against Clostridiumbotulinum neurotoxin serotypes A (BoNT/A) or B (BoNT/B). The DNA replicon vaccine (pSCARSBHc) encoding the Hc domain of BoNT/B (BHc) induced better responses and protection against BoNT/B mouse challenge than conventional DNA vaccine. The dual-expressing DNA vaccine (pSCARSA/BHc) protected similarly to a DNA replicon vaccine mixture (pSCARSAHc+pSCARSBHc). Additionally, recombinant SFV particles, VRP-AHc or VRP-BHc, protected mice from high-dose BoNT/A or BoNT/B challenge, respectively. Mice given either dual-expressing VRP-A/BHc or mixture of VRP-AHc and VRP-BHc were protected from challenge with serotype A/B mixtures. These data justify further testing in other animals or humans.

Neutralizing Antibodies of Botulinum Neurotoxin Serotype A Screened from a Fully Synthetic Human Antibody Phage Display Library

The botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous protein substances known. The neutralizing antibodies against botulinum neurotoxin can effectively prevent and cure the toxicosis. Using purified Hc fragments of botulinum neurotoxin serotype A (BoNT/A-Hc) as antigen, 2 specific neutralizing antibodies mapping different epitopes were selected from a fully synthetic human antibody library. The 2 antibodies can effectively inhibit the binding between BoNT/A-Hc and differentiated PC-12 cells in vitro, and the neutralization was evaluated in vivo. Although no single mAb completely protected mice from toxin, they both could prolong time to death when challenged with 20 LD 50s (50% lethal doses) of BoNT/A. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, suggesting their high neutralizing potency in vivo. The results would lead to further production of neutralizing antibody drugs against BoNT/A. It also proved that it was a quick method to obtain human therapeutic antibodies by selecting from the fully synthetic human antibody phage display library. (Journal of Biomolecular Screening 2009:991-998)

Evaluation of a Recombinant Hc of Clostridium botulinum Neurotoxin Serotype F as an Effective Subunit Vaccine

ABSTRACT A new gene encoding the Hc domain of Clostridium botulinum neurotoxin serotype F (FHc) was designed and completely synthesized with oligonucleotides. A soluble recombinant Hc of C. botulinum neurotoxin serotype F was highly expressed in Escherichia coli with this synthetic FHc gene. Subsequently, the purified FHc was used to vaccinate mice and evaluate their survival against challenge with active botulinum neurotoxin serotype F (BoNT/F). After the administration of FHc protein mixed with Freund adjuvant via the subcutaneous route, a strong protective immune response was elicited in the vaccinated mice. Mice that were given two or three vaccinations with a dosage of 1 or 10 μg of FHc were completely protected against an intraperitoneal administration of 20,000 50% lethal doses (LD50) of BoNT/F. The BoNT/F neutralization assay showed that the sera from these vaccinated mice contained high titers of protective antibodies. Furthermore, mice were vaccinated once, twice, or three times at four different dosages of FHc using Alhydrogel (Sigma) adjuvant via the intramuscular route and subsequently challenged with 20,000 LD50 of neurotoxin serotype F. A dose response was observed in both the antibody titer and the protective efficacy with increasing dosage of FHc and number of vaccinations. Mice that received one injection of 5 μg or two injections of ≥0.04 μg of FHc were completely protected. These findings suggest that the recombinant FHc expressed in E. coli is efficacious in protecting mice against challenge with BoNT/F and that the recombinant FHc subunit vaccine may be useful in humans.

Potent tetravalent replicon vaccines against botulinum neurotoxins using DNA-based Semliki Forest virus replicon vectors.

Human botulism is commonly associated with botulinum neurotoxin (BoNT) serotypes A, B, E and F. This suggests that the greatest need is for a tetravalent vaccine that provides protection against all four of these serotypes. In current study, we investigated the feasibility of generating several tetravalent vaccines that protected mice against the four serotypes. Firstly, monovalent replicon vaccine against BoNT induced better antibody response and protection than that of corresponding conventional DNA vaccine. Secondly, dual-expression DNA replicon pSCARSE/FHc or replicon particle VRP-E/FHc vaccine was well resistant to the challenge of BoNT/E and BoNT/F mixture as a combination vaccine composed of two monovalent replicon vaccines. Finally, the dual-expression DNA replicon or replicon particle tetravalent vaccine could simultaneously and effectively neutralize and protect the four BoNT serotypes. Protection correlated directly with serum ELISA titers and neutralization antibody levels to BoNTs. Therefore, replicon-based DNA or particle might be effective vector to develop BoNT vaccines, which might be more desirable for use in clinical application than the conventional DNA vaccines. Our studies demonstrate the utility of combining dual-expression DNA replicon or replicon particle vaccines into multi-agent formulations as potent tetravalent vaccines for eliciting protective responses to four serotypes of BoNTs.

Co-expression of tetanus toxin fragment C in Escherichia coli with thioredoxin and its evaluation as an effective subunit vaccine candidate

The receptor-binding domain of tetanus toxin (THc), which mediates the binding of the toxin to the nerve cells, is a candidate subunit vaccine against tetanus. In this study one synthetic gene encoding the THc was constructed and highly expressed in Escherichia coli by co-expression with thioredoxin (Trx). The purified THc-vaccinated mice were completely protected against an active toxin challenge in mouse models of disease and the potency of two doses of THc was comparable to that of three doses of toxoid vaccine. And a solid-phase assay showed that the anti-THc sera inhibited the binding of THc or toxoid to the ganglioside GT1b as the anti-tetanus toxoid sera. Furthermore, mice were vaccinated once or twice at four different dosages of THc and a dose-response was observed in both the antibody titer and protective efficacy with increasing dosage of THc and number of vaccinations. The data presented in the report showed that the recombinant THc expressed in E. coli is efficacious in protecting mice against challenge with tetanus toxin suggesting that the THc protein may be developed into a human subunit vaccine candidate designed for the prevention of tetanus.

Binding activity and immunogenic characterization of recombinant C-terminal quarter and half of the heavy chain of botulinum neurotoxin serotype A

In the present study, we explored and compared the binding activity and immunogenic characterization of the most effective part corresponding to C-terminal quarter of heavy chain of botulinum neurotoxin serotype A (AHc-C) with C-terminal half of heavy chain of botulinum neurotoxin serotype A (AHc). Firstly, the fully soluble AHc-C protein successfully expressed in Escherichia coli by co-expression with thioredoxin (Trx) was shown to bind with ganglioside as the AHc, indicating that the recombinant AHc-C protein retains a functionally active conformation. Furthermore, a solid-phase assay showed that the anti-AHc-C sera effectively inhibited the binding of AHc or AHc-C to the ganglioside GT1b, the first step in BoNT/A intoxication of neurons, as good as the anti-AHc sera. Finally, although the recombinant AHc-C protein still induced a high serum antibody titers and afforded protection level as the mice challenged with active botulinum neurotoxin serotype A, the immunization with AHc protein induced stronger protective potency than the AHc-C protein. The data presented in the report shows that there are the same ganglioside binding activity and different immunogenic characterization between the C-terminal quarter and half of heavy chain of botulinum neurotoxin serotype A. Therefore, the recombinant AHc-C protein can not only be developed into a minimal subunit candidate vaccine for prophylaxis against botulinum neurotoxin serotype A but also be used as a promising tool in the search for binding inhibitors and chimeric vaccines.

Three types of human CpG motifs differentially modulate and augment immunogenicity of nonviral and viral replicon DNA vaccines as built-in adjuvants.

NakedDNA vaccines given by intramuscular injection are efficient in mouse models, but they require improvement for human use. As the immunogenicity ofDNA vaccines depends, to a large extent, on the presence ofCpG motifs as built‐in adjuvants, we addressed this issue by inserting three types of humanCpG motifs (A‐type, B‐type, andC‐type) into the backbone of nonviralDNA and viralDNA replicon vectors with distinct immunostimulatory activities on human PBMCs. The adjuvant effects ofCpGmodifications inDNA vaccines expressing three types of antigens (β‐Gal, AHc, or PA4) were then characterized in mice and found to significantly enhance antigen‐specific humoral and cell‐mediated immune responses. The three types ofCpG motifs also differentially affected and modulated immune responses and protective potency against botulinum neurotoxin serotypeA andBacillus anthracisA16R challenge. Taken together, these results demonstrate that insertion of humanCpG motifs can differentially modulate the immunogenicity of nonviralDNA vaccines as well as viralDNA replicon vaccines. Our study provides not only a better understanding of thein vivo activities ofCpG motif adjuvants but implications for the rational design of such motifs as built‐in adjuvants forDNA vectors targeting specific antigens.

Distinct immune responses of recombinant plasmid DNA replicon vaccines expressing two types of antigens with or without signal sequences.

Here, DNA replicon vaccines encoding the Hc domain of botulinum neurotoxin serotype A (AHc) or the receptor binding domain of anthrax protective antigen (PA4) with or without signal sequences were evaluated in mice. Strong antibody and protective responses were elicited only from AHc DNA vaccines with an Ig κ signal sequence or tissue plasminogen activator signal sequence. Meanwhile, there were no differences in total antibody responses or isotypes, lymphocyte proliferative responses, cytokine profiles and protective immune responses with the PA4 DNA vaccines with or without a signal sequence. Therefore, use of targeting sequences in designing DNA replicon vaccines depends on the specific antigen.