[show abstract][hide abstract] ABSTRACT: Foot-and-mouth disease virus (FMDV) causes a severe livestock disease, and the virus is an interesting target for virology and vaccine studies.
Here we evaluated comparatively three different viral antigen-encoding DNA sequences, delivered via two physical means (i.e., gene gun delivery into skin and electroporation delivery into muscle), for naked DNA-mediated vaccination in a mouse system.
Both methods gave similar results, demonstrating commonality of the observed DNA vaccine effects. Immunization with a cDNA vector expressing the major viral antigen (VP1) alone routinely failed to induce the production of anti-VP1 or neutralizing antibodies in test mice. As a second approach, the plasmid L-VP1 that produces a transgenic membrane-anchored VP1 protein elicited a strong antibody response, but all test mice failed in the FMDV challenge experiment. In contrast, for mice immunized with the viral capsid precursor protein (P1) cDNA expression vector, both neutralizing antibodies and 80-100% protection in test mice were detected.
This strategy of using the whole capsid precursor protein P1 cDNA for vaccination, intentionally without the use of virus-specific protease or other encoding genes for safety reasons, may thus be employed as a relevant experimental system for induction or upgrading of effective neutralizing antibody response, and as a convenient surrogate test system for DNA vaccination studies of FMDV and presumably other viral diseases.
The Journal of Gene Medicine 07/2005; 7(6):708-17. · 2.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Toll-like receptors (TLRs) are known to play an important role in binding of various pathogen associated molecular patterns (PAMPs) or non-PAMPs and signalizing the perception of stimuli from pathogens and that results in subsequent immune responses. Many reports have shown that plant polysaccharides can stimulate the immune system, but so far there is no clear evidence of molecular mechanism involved. We hypothesized that specific plant polysaccharides might mimic the functions of exogenous danger signals to mammalian immune cells and confer multiple effects in stimulating the immune cell systems via the TLR4-mediated NF-κB signaling pathway. The main objective of our study is to find the potential immune modulators from plant extracts which act as Toll-like receptors (TLRs) ligands. In a primary screening, we identified few extracts enriched with polysaccharides significantly enhanced the transgenic NF-kB driven reporter gene activity (pNiFty-SEAP) in B-16 cell line. These extracts also showed increased IL-8 secretion in a monocytic cell line, THP-1. Further studies with DxI, a polysaccharide rich fraction from Dioscorea tuber, showed involvement of TLR4 in the secretion of pro-inflammatory cytokine (IL-8) and uric acid in primary monocytes. Treatment of THP-1 cells with polysaccharide rich fractions in combination with IL-4 resulted in up-regulation of DC-SIGN. Similarly, the treatment of human monocyte-derived immature DCs with DxI up-regulated many surface markers related to antigen-presentation. This suggested that polysaccharides may enhance the antigen-presenting activity of immune cells. These results thus reveal an apparently unrecognized strong interaction between the plant polysaccharides and the mammalian defense system.
[show abstract][hide abstract] ABSTRACT: Adjuvant can be used to enhance the immunogenicity of antigen and improve the efficacy of vaccine. Potent adjuvant action is often correlated with NF-kB activation, as exemplified by the case of MPL or LPS. However, NF-kB activation is thought to play an important role in the development and/or maintenance of many types of cancer. The potential of phyto-compounds to modulate NF-kB offers a fruitful field of research into exploiting this clinically relevant transcription factor. We have therefore set to study the possible adjuvant role of polysaccharide and phyto-compounds in the development and progression of murine B16 melanoma by using DNA vaccine model. The human melanoma-associated antigen, hgp100, expression vectors were transfected into mouse skin via gene gun delivery, and followed by subcutaneous application of various phyto-compound formulations onto testing mouse skin tissues. We hypothesize that Dioscorea polysaccharide (Dx-I) might mimic the functions of exogenous danger signals to mammalian immune cells. We observed that co-administration of low dose Dx-I or LPS with the test DNA vaccine could significantly protect from tumor cell challenge with gp100-B16 melanoma cells, as compared to high dose and no treatment groups. On the other strategy, using anti-inflammatory compounds can partially filtrate the inflammation-associated tumorigenesis which elicited by Dx-I or LPS. Our data indicated that use of adjuvant containing LPS combination with emodin (an anti-inflammatory phyto-compound) has lowest tumor size and highest survival percentage than other adjuvant treatment and control in melanoma DNA vaccine model. These results suggest that balance the expression level of NF-kB is important in tumorigenesis and vaccination.
[show abstract][hide abstract] ABSTRACT: VP1, a capsid protein of foot-and-mouth disease virus (FMDV), contains neutralizing epitopes of the virus. Due to its poor water solubility, recombinant Escherichia coli derived VP1 (rVP1) has previously been used mainly in a denatured form and is not well characterized. Here, using SDS to assist protein refolding and then removing SDS with a detergent removing column, we have successfully purified rVP1 in two aqueous-soluble forms, i.e. monomer and dimer. Studies showed that dimerization occurs by an inter-molecular disulfide bond between two cysteine residues at position 187 of each monomer. Heat treatment revealed that rVP1 dimer exhibited a more thermal-stable conformation than the monomeric form. Both monomeric and dimeric rVP1 reacted with anti-FMDV antibodies. Immunization studies demonstrated that vaccination of swine with either forms of rVP1 was effective in generating immune responses and protecting them from viral challenge.