Peiyin Zhang

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.

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.

A novel canine favored CpG oligodeoxynucleotide capable of enhancing the efficacy of an inactivated aluminum-adjuvanted rabies vaccine of dog use

In order to develop novel canine CpG ODNs as adjuvant for rabies vaccine of dog use, a panel of CpG ODNs containing different CpG motifs was designed and screened for their ability to induce the proliferation of canine splenocytes. Three AACGTT motif-containing CpG ODNs, designated as YW07, YW08 and YW09, respectively, were outshined with stronger ability to activate canine immune cells. The CpG ODNs were tested for their adjuvant activity for rabies vaccine in mice and dogs. It was found that YW07 could facilitate the rabies vaccine to induce more vigorous and long-lasting specific antibody response in mice and dogs, respectively. These findings suggest that YW07, a canine favored CpG ODN, could be used as a novel adjuvant for developing more efficient rabies vaccine of dog use.

Recombinant mycobacterial HSP65 in combination with incomplete Freund's adjuvant induced rat arthritis comparable with that induced by complete Freund's adjuvant.

Complete Freunds adjuvant (CFA) containing the inactivated mycobacterium has been conventionally used to induce typical arthritis in rats. In comparison, incomplete Freunds adjuvant (IFA), lacking the mycobacterium, can only induce less severe arthritis. However, the key components responsible for the arthritogenic effect of the whole mycobacterium are rarely known. Although mycobacterial heat-shock protein 65 (MHSP65) specific humoral and cellular immune responses were detected in rats with arthritis induced by CFA, MHSP65 alone cannot induce arthritis. In this study, we replaced the whole mycobacterium in CFA with recombinant MHSP65 (rMHSP65), prepared rMHSP65-IFA emulsion by mixing rMHSP65 and IFA, and investigated whether rMHSP65-IFA could induce arthritis in rats as CFA did. We found that intradermal injection of the rMHSP65-IFA emulsion induced arthritic lesions in testing animals to the same degree as those induced by CFA, manifested by severe swelling in hind paws, and synovial thickening, cartilage erosion and lymphocytes infiltration in ankle joints. Notably, the rMHSP65-IFA recipe also induced the production of anti-dsDNA and -rMHSP65 antibodies in rats. These results thus demonstrate that rMHSP65 can be used to substitute the inactivated mycobacteria in CFA to induce typical arthritis in rats.

Influence of hydrophilic amino acids and GC-content on expression of recombinant proteins used in vaccines against foot-and-mouth disease virus in Escherichia coli

Epitope-based protein expression in Escherichia coli can be improved by adjusting its amino acid composition and encoding genes. To that end, we analyzed 24 recombinant epitope proteins (rEPs) that carry multiple epitopes derived from VP1 protein of foot-and-mouth disease virus. High level expression of the rEPs was attributed to a high content of Arg, Asn, Asp and Thr, a low content of Gln, Pro and Lys, a high content of hydrophilic amino acids and a higher isoelectric point value resulting from abundant Arg. It is also attributed to the appropriate guanine and cytosine content in the encoding genes. The data provide a reference for adjusting the amino acid composition in designing epitope-based proteins used in vaccines and for adjusting the synonymous codons to improve their expressions in E. coli.

Effect of prophylactically applied CpG ODN on the development of myocarditis in mice infected with Coxsackievirus B3.

Coxsackievirus B3 was one of the major pathogens causing viral myocarditis. Toll-like receptor 9 activation contributed to the innate immune response in the process of CVB3-induced myocarditis. In order to find out how CpG oligodeoxynucleotide, known as a TLR-9 agonist, would affect the CVB3-induced myocarditis, we chose a C-type CpG oligodeoxynucleotide (YW002) injected to the mice one day before CVB3 challenge. On day 4 post CVB3 infection, 3 mice in each group were randomly sacrificed and their hearts were isolated to detect CVB3 replication. On day 10, the CVB3 neutralizing antibody and inflammatory change of the hearts were detected. The results indicated that the CVB3-induced myocarditis was aggravated with the declining body weight of mice, decreasing neutralizing antibody, and uncontrolling virus replication by injecting 20 μg YW002 per mouse. When adjusted the amount at 10 μg YW002 per mouse, there were no signs of aggravation in myocarditis. Plus, the mortality of the infected mice was reduced, the neutralizing antibody level was raised and the replication of virus was restrained. These results suggested that a proper amount of CpG oligodeoxynucleotide application could help to inhibit CVB3 infection.

Fusion Proteins Cpn10-Erns with Properties of Generating CSFV-Neutralized Antibodies1

When pigs are infected with classical swine fever virus (CSFV), the antibody primarily targets the structural gly-coprotein Erns of the virus. Previous investigations have demonstrated that Erns has low or no virus neutralizing capacity. In this study, candidate subunit marker vaccine, chaperonin 10 (Cpn10)-Erns, which possess the property of generating neutralized antibodies against lethal challenge of virulent CSFV was developed. The gene of Erns was isolated from Hog cholera lapinized virus (HCLV) -infected spleen cells of rabbits via RT-PCR method and fused to the downstream region of the cpn10 gene; the products of recombinant fusion protein (cpn10-Erns) induced expression in Escherichia coli, and the products were purified by affinity chromatography. During the course of vaccination, the candidate vaccines cpn10-Erns were used for the immunization of guinea pigs, and they induced a strong antibody response against cpn10-Erns. The antibodies can be immobilized by coating inactivated CSFV particles, indicating that these antibodies can recognize CSFV. Neutralization assay was carried out on rabbits according to National Regulations on Veterinary Drug. The results clearly indicate that the typical fever of rabbits induced by the live attenuated HCLV could be inhibited by preincubation with the antisera (dilution 1:4) induced by cpn10-Erns, but not inhibited by preincubation with the antisera induced only by Erns. Analogous results were observed for the group of the rabbits immunized with cpn10-Erns, which were protected against the typical fever induced by the challenge with HCLV. The findings of this study formed the basis of a new means for developing subunit marker vaccine against CSFV.