Hongfei Wei

A CpG oligodeoxynucleotide acts as a potent adjuvant for inactivated rabies virus vaccine.

To develop a CpG containing oligodeoxynucleotide (CpG ODN)-enhanced rabies vaccine for stimulating an earlier production of rabies virus neutralizing antibody (RVNAb) with high titers, we designed a CpG ODN (BW006) and evaluated its adjuvant activities in enhancing the immune response to rabies vaccine with or without aluminum in mice. It was found that BW006 could facilitate the rabies vaccine to induce an earlier and more vigorous RVNAb response, resulting in more effective protection of mice from rabies virus challenge. In addition, three shots of rabies vaccine with BW006 induced compatible RVNAb level with that induced by five shots of aluminum-adjuvanted rabies vaccine. These data reveal that BW006 could be used as a promising adjuvant to replace of or combine with aluminum for developing more effective rabies vaccines.

Heat shock fusion protein induces both specific and nonspecific anti-tumor immunity

Mucin 1 (MUC1) is a tumor antigen, and the most important epitopes that can induce cytotoxic T lymphocytes (CTL) reside in the variable‐number tandem repeats (VNTR). Heat shock protein (HSP) complexes isolated from tumors have been shown to induce specific anti‐tumor immunity. HSP alone can also induce nonspecific immunity. To explore the possibility to utilize the specific anti‐tumor immunity induced by MUC1 VNTR and the nonspecific immunity induced by HSP, we constructed a recombinant protein (HSP65‐MUC1) by fusing Bacillus Calmette‐Guérin‐derived HSP65 with the MUC1 VNTR peptide and tested its ability to induce anti‐tumor activities in a tumor challenge model. The growth of MUC1‐expressing tumors was significantly inhibited in mice immunized with HSP65‐MUC1, both before and after tumor challenge. A much larger percentage of immunized mice survived the tumor challenge than non‐immunized mice. Correlating with the anti‐tumor activity, HSP65‐MUC1 was shown to induce MUC1‐specific CTL as well as nonspecific anti‐tumor immunity. In the human system, HSP65‐MUC1‐loaded human DC induced the generation of autologous MUC1‐specific CTL in vitro. These results suggest that exogenously applied HSP65‐MUC1 may be used to treat MUC1 tumors by inducing the epitope‐specific CTL as well as nonspecific anti‐tumor responses mediated by the HSP part of the fusion protein.

A recombinant truncated FMDV 3AB protein used to better distinguish between infected and vaccinated cattle

To distinguish the antibodies induced by Foot-and-mouth disease virus (FMDV) infection from those induced by vaccination, a recombinant N-terminal truncated FMDV non-structural protein (NSP) of 3AB, designated as r3aB, was constructed by deleting 80 amino acids displayed about 30% homology to transposase IS4 family protein of Escherichia coli, expressed in E. coli BL21 (DE3) and then purified. The r3aB was majorly expressed in soluble fraction and presented as homogeneous monomers after purification. Using r3aB as coating antigen, an indirect ELISA was established to specifically identify antibodies induced by FMDV infection but not those induced by vaccination. Compared with 3AB, r3aB was more specific to catch antibodies against NSP. The performance of this assay was validated by two commercial FMDV NSP ELISA kits. The result suggested that the r3aB coated ELISA could be developed into a kit to better distinguish between infected and vaccinated cattle.

MF59 formulated with CpG ODN as a potent adjuvant of recombinant HSP65-MUC1 for inducing anti-MUC1+ tumor immunity in mice

Abstract MF59 is an oil-in-water emulsion adjuvant approved for influenza vaccines for human use in Europe. Due to its Th2 inducing properties, MF59 is seldom tested for cancer vaccines. In this study, MF59 formulated with a C-type CpG oligodeoxynucleotide (YW002) was tested for its Th1 adjuvant activity to induce immune responses to HSP65-MUC1, a recombinant fusion protein incorporating a mycobacterial heat shock protein (HSP65) and mucin 1, cell surface associated (MUC1) derived peptide. Combination of YW002 with MF59 (MF59-YW002) could confer a potent Th1 biasing property to the adjuvant, which enhanced the immunogenicity of HSP65-MUC1 to induce significantly higher levels of specific IgG2c, increased IFN-γ mRNA expression in splenocytes and the generation of antigen-specific cytotoxic T lymphocytes in mice. When prophylactically applied, MF59-YW002 adjuvant containing HSP65-MUC1 inhibited the growth of MUC1+ B16 melanoma and prolonged the survival of tumor-bearing mice. In contrast, adjuvant containing MF59 with HSP65-MUC1 in the absence of YW002, promoted the growth of MUC1+ B16 melanoma in mice. These results suggest that MF59 plus CpG oligodeoxynucleotide might be developed as an efficient adjuvant for tumor vaccines against melanoma, and possibly other tumors.

Vaccination with TCL plus MHSP65 induces anti-lung cancer immunity in mice

To develop effective anti-lung cancer vaccines, we directly mixed mycobacterial heat shock protein 65 (MHSP65) and tumor cell lysate (TCL) from Lewis lung cancer cells in vitro and tested its efficacy on stimulating anti-tumor immunity. Our results showed that MHSP65–TCL immunization significantly inhibited the growth of lung cancer in mice and prolonged the survival of lung cancer bearing mice. In vivo and in vitro data suggest that MHSP65–TCL could induce specific CTL responses and non-specific immunity, both of which could contribute to the tumor inhibition. Thus, this report provides an easy approach to prepare an efficient TCL based tumor vaccine.

Therapeutic injection of C-class CpG ODN in draining lymph node area induces potent activation of immune cells and rejection of established breast cancer in mice

In order to develop novel CpG ODNs for the treatment of breast cancer, we have designed a series of CpG ODNs and evaluated their anti-tumor activity in a breast cancer mouse model. Interestingly, a C-class CpG ODN, designated as YW002, showed a vigorous activity on the inhibition of tumor growth in mice and completely cured some of the tumor-bearing mice through injection at tumor draining lymph node (TDLN) area. The expansion of immune cells in the TDLN and tumor and the generation of tumor specific immune memory were found associated with YW002-induced anti-tumor activity in mice. These results indicate that C-class CpG ODN could be developed into a medicament in a monotherapeutic regimen for the treatment of breast cancer through injection at TDLN area in clinic.

Recombinant heat shock protein 65 carrying PADRE and HBV epitopes activates dendritic cells and elicits HBV-specific CTL responses

BCG Hsp65 and PADRE have been shown to be potent to enhance antigen specific immunity. In order to explore the possibility to utilize them for the development of HBV therapeutic vaccine, a chimeric protein, Hsp65-HBV, was created by fusing PADRE and epitopes from HBV to the carboxyl-terminus of BCG Hsp65 and expressed in E. coli. We evaluated its effects on human dendritic cell maturation and specific CTL induction in vitro. Results showed that Hsp65-HBV could activate human dendritic cells by up-regulating the expressions of HLA-A2, HLA-DR and CD86, companioning with high level of IL-12 secretion. Furthermore, Hsp65-HBV matured DCs could significantly stimulate human autologous CD8(+) T cell proliferation and induce HBV-specific CTLs. Hsp65-HBV was also shown to generate HBsAg-specific CTLs in vivo in mice. These results indicated that Hsp65-HBV might be a candidate for the treatment of chronic HBV infection.

Vaccination with B16 Tumor Cell Lysate Plus Recombinant Mycobacterium tuberculosis Hsp70 Induces Antimelanoma Effect in Mice

Tumor cell lysate (TCL) has an advantage of containing an extensive repertoire of tumor antigens but requires proper adjuvants to enhance its immunogenicity when used as an efficient tumor vaccine. Mycobacterium tuberculosis-derived heat shock protein 70 (TBHsp70) has been shown to assist crosspresentation of exogenously applied tumor antigens and activate innate immunity against tumor cells. In this study, TBHsp70-B16TCL, a preparation generated by mixing recombinant TBHsp70 and TCL of B16 melanoma cells directly, was tested for its immunogenicity as a tumor vaccine. The TBHsp70-B16TCL induced a significant inhibition of the growth and metastasis of B16 melanoma in mice and prolonged the survival of B16 melanoma-bearing mice. The inhibition was correlated with the specific immune responses induced by TBHsp70-B16TCL. The data suggest that recombinant TBHsp70-adjuvanted TCL might be developed into effective tumor vaccines for melanomas and possibly for other tumors.

Replacing the decoy epitope of PCV2b capsid protein with a protective epitope enhances efficacy of PCV2b vaccine

Microbial pathogens may evolve decoy epitopes to evade host immune responses. In recent years, a decoy epitope has been identified in the capsid protein (CP) of porcine circovirus type 2b (PCV2b) to divert the immune response away from protective epitopes in pigs. To avoid the decoy effect, we designed and produced a recombinant PCV2b CP (ΔCP) by replacing the decoy epitope with a neutralizing B cell epitope derived from CP and tested the ability of ΔCP to induce protective antibody responses in mice and pigs. As expected, the ΔCP, unlike inactivated PCV2b vaccines, recombinant PCV2b CP, and natural PCV2b infection, did not induce anti-decoy epitope antibodies. Although unable to form typical virus-like particles (VLPs), the ΔCP could increase the production of the anti-PCV2b antibodies among which no antibody against the decoy epitopes, and therefore induces improved protective immune responses in pigs challenged with PCV2b. These results provide an alternative strategy for development of recombinant subunit vaccines against PCV2b, and possibly other viruses, by replacing the decoy epitope with a protective epitope.

Detection of the Assembly and Disassembly of PCV2b Virus-Like Particles Using Fluorescence Spectroscopy Analysis

Monitoring the assembly and disassembly of virus-like particles (VLPs) is important in developing effective VLP-based vaccines. We tried to establish a simple and rapid method to evaluate the status of VLP assembly using fluorescence spectroscopic analysis (FSA) while developing a VLP-based vaccine against porcine circovirus type 2b (PCV2b). We synthesized the gene coding for PCV2b capsid protein (CP). The CP was expressed in Escherichia coli in a soluble form, dialyzed into three different buffers, and assembled into VLPs. The immunogenicity of the VLPs was evaluated by an enzyme-linked immunosorbent assay using the sera of mice immunized with inactivated PCV2b. The VLP assembly was detected using transmission electron microscopy and FSA. The assembled VLPs showed a distinct FSA curve with a peak at 320 nm. We found that the assembly status was related to the immunogenicity, fluorescence intensity, and morphology of the VLP. The FSA assay was able to monitor the various denatured statuses of PCV2b VLPs treated with β-mercaptoethanol or β-mercaptoethanol plus urea. We have demonstrated that FSA can be used to detect the assembly of PCV2b VLPs produced in E. coli. This provides a simple solution for monitoring VLP assembly during the production of VLP-based vaccines.