Sheng Guo

An oligodeoxynucleotide capable of lessening acute lung inflammatory injury in mice infected by influenza virus.

Infection of influenza virus could induce acute lung inflammatory injury (ALII) that was at least partially caused by excessive innate immune responses. To study whether down-regulating Toll-like receptor (TLR)-mediated innate immune response could lessen influenza virus-induced ALII, a microsatellite DNA mimicking oligodeoxynucleotide (MS ODN), named as SAT05f capable of inhibiting TLR7/9-activation in vitro, was used to treat mice infected with FM1 virus. In parallel, two MS ODNs confirmed with less or no in vitro activities, named as MS19 and MS33, were used as controls. Unexpectedly, SAT05f failed to lessen ALII in the mice, whereas MS19 significantly inhibited the weight loss and displayed dramatic effect on lessening the ALII by reducing consolidation, hemorrhage, intra-alveolar edema and neutrophils infiltration in lungs of the mice. Meanwhile, MS19 could decrease the mortality of influenza virus infected mice and down-regulate TNF-α production in their lungs. The data suggest that MS19 might display its therapeutic role on ALII induced by influenza virus by reducing over-production of TNF-α.

Detection of the Assembly and Disassembly of PCV2b Virus-Like Particles Using Fluorescence Spectroscopy Analysis

Monitoring the assembly and disassembly of virus-like particles (VLPs) is important in developing effective VLP-based vaccines. We tried to establish a simple and rapid method to evaluate the status of VLP assembly using fluorescence spectroscopic analysis (FSA) while developing a VLP-based vaccine against porcine circovirus type 2b (PCV2b). We synthesized the gene coding for PCV2b capsid protein (CP). The CP was expressed in Escherichia coli in a soluble form, dialyzed into three different buffers, and assembled into VLPs. The immunogenicity of the VLPs was evaluated by an enzyme-linked immunosorbent assay using the sera of mice immunized with inactivated PCV2b. The VLP assembly was detected using transmission electron microscopy and FSA. The assembled VLPs showed a distinct FSA curve with a peak at 320 nm. We found that the assembly status was related to the immunogenicity, fluorescence intensity, and morphology of the VLP. The FSA assay was able to monitor the various denatured statuses of PCV2b VLPs treated with β-mercaptoethanol or β-mercaptoethanol plus urea. We have demonstrated that FSA can be used to detect the assembly of PCV2b VLPs produced in E. coli. This provides a simple solution for monitoring VLP assembly during the production of VLP-based vaccines.

Correlation of Surface Toll-Like Receptor 9 Expression with IL-17 Production in Neutrophils during Septic Peritonitis in Mice Induced by E. coli.

IL-17 is a proinflammatory cytokine produced by various immune cells. Polymorphonuclear neutrophils (PMNs) are the first line of defense in bacterial infection and express surface Toll-like receptor 9 (sTLR9). To study the relationship of sTLR9 and IL-17 in PMNs during bacterial infection, we infected mice with E. coli intraperitoneally to establish a septic peritonitis model for studying the PMNs response in peritoneal cavity. We found that PMNs and some of “giant cells” were massively accumulated in the peritoneal cavity of mice with fatal septic peritonitis induced by E. coli. Kinetically, the CD11b+ PMNs were increased from 20–40% at 18 hours to >80% at 72 hours after infection. After E. coli infection, sTLR9 expression on CD11b+ and CD11b− PMNs and macrophages in the PLCs were increased at early stage and deceased at late stage; IL-17 expression was also increased in CD11b+ PMNs, CD11b− PMNs, macrophages, and CD3+ T cells. Using experiments of in vitro blockage, qRT-PCR and cell sorting, we confirmed that PMNs in the PLCs did increase their IL-17 expression during E. coli infection. Interestingly, sTLR9−CD11b+Ly6G+ PMNs, not sTLR9+CD11b+Ly6G+ PMNs, were found to be able to increase their IL-17 expression. Together, the data may help understand novel roles of PMNs in septic peritonitis.

Single immunization with a recombinant multiple-epitope protein induced protection against FMDV type Asia 1 in cattle

To develop recombinant epitope vaccines against the foot-and-mouth disease virus (FMDV) serotype Asia 1, genes encoding six recombinant proteins (A1-A6) consisting of different combinations of B-cell and T-cell epitopes from VP1 capsid protein (VP1) of FMDV were constructed. These proteins were expressed in Escherichia coli and used to immunize animals. Our results showed that A6 elicited higher titers of neutralizing antibodies after single inoculation in guinea pigs than did the other five recombinant proteins, as determined by micro-neutralization tests. In addition, a strong lymphocyte proliferation response and Th1 type immunity were observed in splenocytes from the mice immunized with A6. Further tests carried out in cattle demonstrated that a single inoculation with A6 generated comparable levels of neutralizing antibodies as inactivated vaccine and protected 4 of 5 cattle against challenge with FMDV type Asia 1. Our results suggest that A6 might be a promising recombinant vaccine against FMDV type Asia 1 in cattle.

An Oligodeoxynucleotide with CCT Repeats Restrains CpG ODN-Induced TLR9 Trafficking

Toll-like receptor 9 (TLR9) can sense pathogen DNA and CpG ODN or even self-DNA by trafficking assisted by Unc93B1, an endoplasmic reticulum (ER) transmembrane protein, from ER to endolysosomes or cell surface. In previous study, we found that an oligodeoxynucleotide with CCT repeats (SAT05f) could selectively inhibit TLR7/9 activation. However, the mechanism for the inhibitory activity of SAT05f is still unknown. In present research, it was found that SAT05f could inhibit CpG ODN-induced the intracellular trafficking of TLR9 and Unc93B1 with feedback the responses of decreased surface TLR9 and enhanced TLR9 mRNA expression but not influence TLR9 protein level by using human plasmacytoid dendritic cell line CAL-1 cells, suggesting that SAT05f inhibits TLR9 activation by restraining TLR9 trafficking. Since the mitochondrial DNA released from injured tissue can cause systemic inflammatory response syndrome (SIRS), this study may provide valuable data for prevention and treatment of SIRS and rescue severe trauma patients.

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.

Recombinant hepatitis B virus surface antigen formulated with B-type CpG oligodeoxynucleotide induces therapeutic immunity against hepatitis B virus surface antigen-expressing liver cancer cells in mice.

To develop a therapeutic vaccine against hepatitis B virus surface antigen (HBsAg)-expressing liver cancer, we tried to prepare a vaccine by formulating recombinant HBsAg with BW006, a B type CpG oligodeoxynucleotide (ODN) with Th1-biasing activity, and examined its potency of inducing therapeutic immunity against HBsAg-expressing liver cancer cells in mice. When applied therapeutically, BW006 could assist HBsAg to induce vigorous immune responses capable of inhibiting the growth of HBsAg-expressing liver cancer cells and prolonging the survival of mice bearing HBsAg-expressing liver cancer cells. In vivo and in vitro experiments showed that the BW006-adjuvanted HBsAg enhanced the production of IgG2a antibodies, interferon-γ, and interleukin-12 and facilitated the generation of specific cytotoxic T lymphocyte that killed the HBsAg-expressing liver cancer cells. These results suggest that the BW006-adjuvanted HBsAg might be developed into a candidate tumor vaccine for the treatment of HBsAg-expressing liver cancer.

Attribution of NKG2DL to the inhibition of early stage allogeneic tumors in mice

Allogeneic tumors are eventually rejected by adaptive immune responses, however, little is known about how allogeneic tumors are eradicated at the early stage of tumor development. In present study, we found that NKG2DL low expressing cancer cells were developed into palpable allogeneic tumors in mice within a week after the inoculation, while NKG2DL high expressing cancer cells failed to. The NKG2DL high expressing cancer cells could increase NKG2D+ NK cells in the allogeneic mice after being inoculated for 3 days. Artificially up-regulating NKG2DL on cancer cells with low level expressed NKG2DL by a CpG ODN resulted in the retardation and rejection of the allogeneic tumors at the early stage. The contribution of up-regulated NKG2DL to the early rejection was further confirmed by the results that the development of allogeneic tumors from cancer cells transfected with NKG2DL genes was significantly inhibited in mice at the early stage. Overall, hopefully, the data may provide insights for combining the allogeneic NK cell adoptive transfer with the approaches of up-regulating NKG2DL to treat cancer patients.

An immune regulatory CCT repeat containing oligodeoxynucleotide capable of causing hair loss in male mice.

An oligodeoxynucleotide with CCT repeats (CCT ODN) has been found in our previous study to selectively downregulate Toll-like receptor 7/9 (TLR7/9)-mediated immune responses both in vitro and in vivo. In this study, we unexpectedly found that CCT ODN induced severe patchy hair loss around the mouth in male F1 mice (female Balb/c × male C57BL/6) with lupus-like nephritis induced by injecting allogenic lymphocytes and also in male Balb/c mice, but not in female F1 mice and Balb/c mice and either gender of C57BL/6 mice. Increased infiltration of natural killer group 2, member D (NKG2D+) cells in hair loss skin and upregulated interferon-gamma (IFN-γ) messenger RNA expression in cultured splenocytes were observed in male Balb/c mice. The CCT ODN-conditioned supernatants of cultured mouse splenocytes caused catagen-like changes to hair follicles (HFs). We hypothesized that the CCT ODN could induce patchy hair loss in the male mice with certain genetic traits by mobilizing NKG2D+ cells to HFs and by inducing the production of IFN-γ from immune cells. Taken together these data indicated that a gender and genetic preference of immune-regulatory oligonucleotides is causing unexpected clinical situations such as hair loss.

Impact of Fighting on Antibody Response to Hepatitis B Virus Vaccine in Mice

Antibody responses to vaccines can be influenced by various behavioral and psychosocial factors. Few reports exist on the impact of fighting on antibody response to vaccines. This study unexpectedly found that fighting could significantly enhance antibody production in male mice immunized with hepatitis B virus (HBV) vaccines. To confirm the finding, a mouse-fighting model was established in which it was observed that only intense fighting, not mild fighting, enhanced the antibody response to HBV surface antigen in male mice, and that the frequency of fighting and active attacks during fighting showed no obvious relationship with the antibody levels in the male mice that experienced fighting. In addition, fighting can cause significant upregulation of CD80 in CD11c(+) cells in the spleen of male mice. These data suggest that fighting could influence the humoral immune response in individuals immunized with vaccines or infected with microbes.