Yongli Yu

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.

Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide.

Abstract To develop CpG oligodeoxynucleotides (CpG ODNs) based therapy for prevention and treatment of severe acute respiratory syndrome (SARS), we selected a novel CpG ODN (BW001), which displays B-type CpG ODN structure feature at the 5′ and A-type CpG ODN structure feature at the 3′, and tested for its anti-SARS-CoV activity. We found that the supernatants of human PBMCs stimulated by BW001 significantly protected Vero cells from SARS-CoV infection. BW001 could stimulate human PBMCs and pDCs to secrete high level of IFN-α and promote human PBMCs and B cells to proliferate. Furthermore, we demonstrated that BW001 could activate CD19+ B cells and CD56+ NK cells in human PBMCs. In addition, BW001 could enhance NK cytotoxicity and IFN-γ secretion in human PBMCs. Together, BW001 represents a novel type of CpG ODN and may have potential for the development of treatment and prevention for SARS as well as other viral associated diseases.

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.

A human microsatellite DNA-mimicking oligodeoxynucleotide with CCT repeats negatively regulates TLR7/9-mediated innate immune responses via selected TLR pathways

Abstract A human microsatellite DNA-mimicking ODN (MS ODN) composed of CCT repeats, designated as SAT05f, has been studied for its capacity of negatively regulating innate immunity induced by TLR7/TLR9 agonists in vitro and in mice. The result showed that SAT05f could down-regulate TLR7/9-dependent IFN-α production in cultured human PBMC stimulated by inactivated Flu virus PR8 or HSV-1 or CpG ODN or imiquimod, protect d-GalN-treated mice from lethal shock induced by TLR9 agonist, not by TLR3/4 agonist. In addition, SAT05f significantly inhibit IFN-α production from purified human plasmacytoid cells (pDCs) stimulated by CpG ODN. Interestingly, SAT05f could up-regulate CD80, CD86, and HLA-DR on the pDCs in vitro, implying that SAT05f-mediated inhibition on IFN-α production could be related to the activation of pDCs. The data suggest that SAT05f could be developed as a candidate medicament for the treatment of TLR7/9 activation-associated diseases by inhibiting TLR7/9 signaling pathways.

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.

CpG oligodeoxynucleotide and montanide ISA 206 adjuvant combination augments the immune responses of a recombinant FMDV vaccine in cattle.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. To prevent the spread of FMDV, inactivated virus vaccines are used to immunize animals in developing countries. However, there are safety concerns. In addition, it is difficult to distinguish the vaccinated animals from those naturally infected ones. In our lab, we have developed a recombinant FMDV vaccine named A7. A7 contained multiple B cell and T cell epitopes, which reside in a capsid protein (VP1) of FMDV. To enhance its immunogenicity, A7 was formulated with CpG ODN RW03 in combination with Montanide ISA 206 (ISA), and the resultant vaccine (A7+ISA+CpG ODN RW03) was used to immunize mice and cattle. It was found that CpG ODN RW03 and ISA combination could facilitate A7 to induce a vigorous and long-lasting specific antibody response in mice and cattle. After FMDV challenge, 80% (4/5) of the calves immunized with A7+ISA+CpG ODN RW03 were protected, which was superior to those immunized with A7+ISA (25%, 1/4) or inactivated FMDV vaccine (50%, 2/4). These findings suggest that CpG ODN RW03 could be used with Montanide ISA 206 as a potent adjuvant for recombinant FMDV in cattle.

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.