Xizhen Zhang

Controlled release of PEI/DNA complexes from mannose-bearing chitosan microspheres as a potent delivery system to enhance immune response to HBV DNA vaccine

Abstract A novel approach involving the preparation of mannose-bearing chitosan microspheres with entrapping complexes of HBV DNA and PEI was developed to improve the delivery of DNA into antigen-presenting cells (APCs) after intramuscular (i.m.) injection. Compared with the traditional chitosan microspheres, the microspheres could quickly release intact and penetrative PEI/DNA complexes. Whats more, chitosan was modified with mannose to target the primary APCs such as dendritic cells (DCs) owing to the high density of mannose receptors expressing on the surface of immature DCs. After i.m. immunization, the microspheres induced significantly enhanced serum antibody and cytotoxic T lymphocyte (CTL) responses in comparison to naked DNA.

Subcutaneous Administration of Modified Vaccinia Virus Ankara Expressing an Ag85B-ESAT6 Fusion Protein, but Not an Adenovirus-Based Vaccine, Protects Mice Against Intravenous Challenge with Mycobacterium tuberculosis

Recombinant virus‐based tuberculosis (TB) vaccines that are strongly immunogenic and elicit robust cellular immunity are considered ideal vaccine candidates. Here, we engineered a poxvirus‐based vaccine, MVA85B‐E6, and an adenovirus‐based vaccine, AD85B‐E6, both of which express the fusion protein Ag85B‐ESAT6. Subcutaneous vaccination of AD85B‐E6 generated strong interferon (IFN)‐γ production by both CD4 and CD8 T cells and CD8 cytotoxic T lymphocyte activity; these results indicate that strong T‐helper type 1 immune responses were elicited in mice, which is in contrast to the moderate responses induced by vaccination with MVA85B‐E6. However, MVA85B‐E6 given subcutaneously led to levels of protection comparable with that induced by the bacillus Calmette–Guérin vaccine in the lungs and spleens, whereas AD85B‐E6 given subcutaneously did not show any protective efficacy after intravenous challenge of BALB/c mice with Mycobacterium tuberculosis H37Rv. Our study emphasizes that more efficient biomarkers for vaccine efficacy and more appropriate routes of vaccine administration are necessary for the development of a successful TB vaccine.

Design and immunogenicity assessment of HIV-1 virus-like particles as a candidate vaccine

The rapid growth of the global HIV/AIDS epidemic makes it a high priority to develop an effective vaccine. Since a live attenuated or inactivated HIV vaccine is not likely to be approved for clinical application due to safety concerns, HIV virus like particles (VLPs) offer an attractive alternative because they are considered safer since they lack viral genome. We got a stable eukaryotic cell line by G418 resistance selection, engineered to express the HIV-1 structure protein Gag and Env efficiently and stably. We confirmed the presence of Gag and Env proteins in the cell culture supernatant and that they could self-assemble into VLPs. These VLPs were found to be able to elicit specific humoral and cellular immune response after immunization without any adjuvant.

Immunogenicity and protective efficacy of heterologous prime-boost regimens with mycobacterial vaccines and recombinant adenovirus- and poxvirus-vectored vaccines against murine tuberculosis

OBJECTIVES To evaluate regimens using bacillus Calmette-Guérin (BCG) or recombinant BCG (rBCG) overexpressing Ag85B for priming, followed by boosting with a modified vaccinia virus Ankara strain (MVA) and/or adenovirus vector (AD) expressing an Ag85B-ESAT6 fusion protein. METHODS Cellular and humoral immune responses were determined after subcutaneous vaccination, which was employed to trigger systemic immunity against intravenous infection in a mouse model of tuberculosis (TB). Bacterial loads and lung histology were evaluated. RESULTS The relative IgG2a and IgG1 antibody levels indicated that the viral-vectored vaccines generated a T-helper type 1 (Th1)-biased response after two doses of viral boost vaccinations. Boosting BCG-primed mice with viral vaccines induced a Th1 immune response that included both CD4 and CD8 T-cells generating antigen-specific interferon-gamma (IFN-γ) and CD8 T cytotoxic activity. Only mice vaccinated with two different viral boosters after BCG priming exhibited a significant reduction in bacterial burden in the lung after challenge. Histology examinations confirmed the attenuation of lung damage and more compact granulomas. After mycobacteria priming, boosting with AD85B-E6 followed by MVA85B-E6 afforded better protection than the reverse order of administration of the viral vectors. CONCLUSIONS This study demonstrates the potential of multiple heterologous viral booster vaccines, although the exact correlates of protection and optimal regimens should be further investigated for the rational design of future vaccine strategies.

Involvement of RP105 and toll-like receptors in the activation of mouse peritoneal macrophages by Staphylococcus aureus.

Staphylococcus aureus is the aetiological agent of many hospital‐ and community‐acquired infections. Toll‐like receptor 2 (TLR2) has been shown to play a crucial role in the host defence against S. aureus infection. The aim of this study is to investigate the roles of the heterogeneous TLR family proteins TLR2, TLR4 and RP105 during S. aureus infection. Peritoneal macrophages from mice were exposed to S. aureus. Their production of inflammatory cytokines and chemokines, their expression of cell‐surface markers and interactions between TLR2, TLR4 and RP105 were assessed in the presence or absence of inhibitory antibodies against TLR2, TLR4/MD‐2 and RP105/MD‐1 complexes. Our results demonstrate that not only TLR2 but also TLR4 and RP105 are involved in the response of macrophages to S. aureus, that TLR2, TLR4 and RP105 physically interact with each other during S. aureus infection, and that TLR2, TLR4 and RP105 both cooperate and play unique roles in the production of inflammatory cytokines (TNF‐α, IL‐12p40 and IL‐10) and chemokine (RANTES) by macrophages after S. aureus infection. This study characterizes the important roles that TLR2, TLR4 and RP105 play in host resistance against S. aureus infection.

Enhance immune response to DNA vaccine based on a novel multicomponent supramolecular assembly

Abstract DNA vaccination has tremendous potential for treating or preventing numerous diseases for which traditional vaccines are ineffective but the technique can be limited by low immunogenicity. Current synthetic DNA delivery systems are versatile and safe, but substantially less efficient than viruses. Here, a novel multicomponent supramolecular system involving the preparation of mannose-bearing chitosan oligomers microspheres with entrapping complexes of DNA vaccine and polyethylenimine was developed to mimic many of the beneficial properties of the viruses. After delivery by intramuscular immunization in BALB/c mice, the microspheres induced an enhanced serum antibody responses two orders of magnitude greater than naked DNA vaccine. Additionally, in contrast to naked DNA, the microspheres induced potent cytotoxic T lymphocyte responses at a low dose. Consequently, formulation of DNA vaccines into multicomponent vectors is a powerful means of increasing vaccine potency.

Sphere Formation Assay is Not an Effective Method for Cancer Stem Cell Derivation and Characterization from the Caco-2 Colorectal Cell Line.

Although the existence of cancer stem cells (CSCs) has been demonstrated in colorectal cancer, further investigation is hindered by controversies over their surface markers. The sphere formation assay is widely used as in vitro method for derivation and characterization of CSCs based on the intrinsic self-renewal property of these cells. Isolated cancer cells that form tumorspheres are generally recognized as CSCs with self-renewal and tumorigenic capacities. In this study, colon spheres grown from Caco-2 cells in the sphere formation assay were separated from other differentiated cells and characterized. Compared with Caco-2 cells, the derived colon spheres lost several CSC properties. The colon spheres contained decreased levels of specific colorectal CSC surface markers as well as low levels of ATP-binding cassette (ABC) transporters typically overexpressed in CSCs, resulting in the near loss of their chemoresistance ability. Furthermore, cells that developed as colon spheres with strong self-renewal ability in vitro lost their tumorigenic capacity in vivo compared with Caco-2 cells, which could establish tumors in non-obese diabetic/severe-combined immunodeficient (NOD/SCID) mice. The results indicated that the Caco-2 cell derived colon spheres did not consist of colorectal CSCs. Thus, the well-accepted sphere formation assay may not be an effective method for CSC isolation and characterization from the Caco-2 colorectal cancer cell line.

Immune responses induced by heterologous boosting of recombinant bacillus Calmette-Guerin with Ag85B-ESAT6 fusion protein in levamisole-based adjuvant.

In the present study, we evaluated the effectiveness of a levamisole-based adjuvant (ADL) to enhance the ability of the Ag85B-ESAT6 fusion protein to boost immune responses after primary vaccination with recombinant bacillus Calmette-Guerin (rBCG) in Balb/c mice. The results were compared with that of the control adjuvant formulation of dimethyl dioctadecylammonium bromide (DDA) and monophosphoryl lipid A (MPL), which has previously been shown to induce T-helper type 1 (Th1)-biased responses. Enzyme-linked immunospot (ELISPOT) assay with Ag85B and ESAT6 derived peptides corresponding to CD4+ and CD8+ T cell restricted epitopes and cell surface immunostaining indicated that Ag85B-ESAT6/ADL predominantly triggered activation of CD4+ T cells. Functional CD8+ T cells with interferon (IFN)-γ production or cytotoxicity were undetectable all vaccinated mice. The ADL adjuvant modified T-helper (Th) subtypes by up-regulating multiple signature cytokines. Furthermore, profiles of the immunoglobulin G (IgG) subtypes indicated ADL enhanced the secretion of Th1-associated IgG2a antibodies and decreased the yield of Th2-associated IgG1 subtype. These observations suggest that the ADL adjuvant formulated with a protein booster may induce Th1-biased cellular and humoral immune responses to primary vaccination with a live attenuated bacterial TB vaccine.

Negative effects of a disulfide bond mismatch in anti-rabies G protein single-chain antibody variable fragment FV57

Rabies virus (RV) causes a fatal infectious disease requiring efficient post-exposure prophylaxis (PEP), which includes a rabies vaccine and rabies immunoglobulin (RIG). The single-chain antibody variable fragment (scFv), a small engineered antibody fragment derived from an antibody variable heavy chain and light chain, has the potential to replace the current application of RIG. In previous studies, we constructed and evaluated an anti-rabies virus G protein scFv (FV57) based on the monoclonal antibody CR57. Of the five cysteines in FV57, four are linked in intra-chain disulfide bonds (Cys-VH28/Cys-VH98 and Cys-VL16/Cys-VL84), and one is free (Cys-VL85). However, the thiol in Cys-VL85 neighboring Cys-VL84 in the CDR3 of the light chain is likely to mismatch with the thiol in Cys-VL16 during the renaturing process. In order to study effects of the mismatched disulfide bond, Cys-VL85 and Cys-VL84 of FV57 were mutated to serine to construct mutants FV57(VL85S) and FV57(VL84S). Furthermore, the disulfide bonds in the light chain of FV57, FV57(VL85S) and FV57(VL84S) were deleted by mutating Cys-VL16 to serine. All mutants were prepared and evaluated along with the original FV57. The results indicated that the mismatched disulfide bond of FV57 linking the light chain FR1 and CDR3 would confer deleterious negative effects on its activity against RV, likely due to spatial hindrance in the light chain CDR3. Moreover, avoidance of the disulfide bond mismatch provided an additional 30% protective efficacy against RV infection in the mouse RV challenge model. Thus, modifications of FV57 to eliminate the disulfide bond mismatch may provide a candidate therapeutic agent for effective PEP against rabies.

Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody

Survivin, a novel member of inhibitor of apoptosis(IAP) protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. The cancer-specific expression of survivin, coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B. The recombinant plasmid pRSET-B-Surv was expressed in E.coli BL21, and the relative molecule mass(Mr) of expressed fusion protein was approximately 21000. The recombinant protein was purified through Ni2+ affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified recombinant protein was then injected into rabbits, and antisurvivin polyclonal antibody with a high titer was obtained.