Peptide-based vaccine targeting IL17A attenuates experimental spondyloarthritis in HLA-B27 transgenic rats.

OBJECTIVES: Spondyloarthritis (SpA) is known as series of immune-mediated inflammatory disease of the axial and peripheral joints. Human leucocyte antigen (HLA)-B27 is a genetic risk factor for SpA. Recent evidence suggests that the interleukin -17 (IL17) axis strongly contributes to SpA. This study aimed to assess the efficacy of an IL17A peptide-based vaccine on SpA manifestations in model rats. METHODS: HLA-B27/human ß2-microglobulin (hß2M) transgenic rats were immunised with heat-inactivated Mycobacterium tuberculosis (MT) to develop spondylitis and arthritis as an experimental SpA model after immunisation with a keyhole limpet hemocyanin-conjugated IL17A peptide-based vaccine with an alum adjuvant three times. The IL17A antibody titre was assessed using ELISA, and arthritis score and joint thickness were monitored two times a week. Enzyme-linked immunospot (ELISpot) assays for IL4- and interferon-γ-secreting splenocytes were conducted to evaluate IL17A-specific T cell activation. We also evaluated the effect of IL17A vaccine in SpA therapeutic model. RESULTS: The IL17A peptide-based vaccine with alum adjuvant successfully induced antibody production and suppressed the arthritis score and joint thickness. X-ray and histological analyses showed that enthesitis, bone destruction and new bone formation were inhibited by the IL17A vaccine. The ELISpot assay showed that the IL17A peptide-based vaccine did not elicit any IL17A-reactive T cell responses. IL17A vaccine tends to mitigate, but not significant, in SpA treatment model. These data showed that the peptide-based vaccine targeting IL17A alleviated the SpA phenotype in a heat-inactivated MT-induced SpA model in HLA-B27/hß2M transgenic rats. CONCLUSIONS: IL17A peptide-based vaccine may be a therapeutic option for SpA treatment.

Fv-clasp: An Artificially Designed Small Antibody Fragment with Improved Production Compatibility, Stability, and Crystallizability.

Antibody fragments are frequently used as a "crystallization chaperone" to aid structural analysis of complex macromolecules that are otherwise crystallization resistant, but conventional fragment formats have not been designed for this particular application. By fusing an anti-parallel coiled-coil structure derived from the SARAH domain of human Mst1 kinase to the variable region of an antibody, we succeeded in creating a novel chimeric antibody fragment of ∼37 kDa, termed "Fv-clasp," which exhibits excellent crystallization compatibility while maintaining the binding ability of the original IgG molecule. The "clasp" and the engineered disulfide bond at the bottom of the Fv suppressed the internal mobility of the fragment and shielded hydrophobic residues, likely contributing to the high heat stability and the crystallizability of the Fv-clasp. Finally, Fv-clasp antibodies showed superior "chaperoning" activity over conventional Fab fragments, and facilitated the structure determination of an ectodomain fragment of integrin α6ß1.

Probing conformational and functional states of human hepatocyte growth factor by a panel of monoclonal antibodies.

HGF-Met signaling contributes to various biological events by controlling cell migration. Since the abnormal activation of Met receptor causes cancer progression, inhibitors such as neutralizing antibodies are regarded as promising therapeutics. HGF is secreted as a single-chain (sc) precursor and is processed by extracellular proteases to generate disulfide-bonded two-chain (tc) HGF. Although this proteolytic processing of HGF is necessary for its biological activity, exactly how the proteolysis leads to the conversion of HGF to the active form is still unclar due to the lack of structural information. In order to gain insights about this point, we generated 6 antibodies against HGF. All antibodies recognized different epitopes on the native HGF protein and showed distinct effects when tested in a cell-based HGF-Met signaling assay. They included one antibody (t1E4) that strongly blocks Met activation by tcHGF, as well as one antibody (t8E4) exclusively recognizing the active tcHGF but not inactive scHGF. Thus, a panel of anti-HGF antibodies suitable for probing the structural mechanism of HGF activation were obtained.

Evaluation of a novel vaccine (HVJ-liposome/HSP65 DNA+IL-12 DNA) against tuberculosis using the cynomolgus monkey model of TB.

We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-liposome (HSP65+IL-12/HVJ). This vaccine provided remarkable protective efficacy in mouse and guinea pig models compared to the BCG vaccine, on the basis of an induction of the CTL activity and improvement of the histopathological tuberculosis lesions, respectively. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses. Furthermore, the combination of HSP65+IL-12/HVJ and BCG by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis for human clinical trials.

Development of vaccines and passive immunotherapy against SARS corona virus using SCID-PBL/hu mouse models.

We have investigated novel vaccine strategies against severe acute respiratory syndrome (SARS) CoV using cDNA constructs encoding the structural antigens: (S), (M), (E), or (N) protein, derived from SARS CoV. PBL from healthy human volunteers were administered i.p. into IL-2 receptor gamma-chain disrupted SCID mice, and SCID-PBL/hu mice were constructed. These mice can be used to analyze the human immune response in vivo. SARS M DNA vaccine and N DNA vaccine induced human CTL specific for SARS CoV antigens. Alternatively, SARS M DNA vaccines inducing human neutralizing antibodies and human monoclonal antibodies against SARS CoV are now being developed. These results show that these vaccines can induce virus-specific immune responses and should provide a useful tool for development of protective and therapeutic vaccines.