Immune Responses and Protective Efficacy Induced by 85B Antigen and Early Secreted Antigenic Target-6 kDa Antigen Fusion Protein Secreted by Recombinant Bacille Calmette-Guérin

In an attempt to improve immune responses and protective efficacy, we constructed two recombinant bacille Calmette-Guérin （rBCG） strains expressing an 85B antigen （Ag85B） and early secreted antigenic target-6 kDa antigen （ESAT6） of Mycobacterium tuberculosis （MTB） fusion protein. Both rBCG strains have the same protein insertion but in a different order （Ag85B-ESAT6 and ESAT6-Ag85B）. The cultured supernatant of rBCG strains and the sera from the mice immunized with the fusion protein Ag85B- ESAT6 or ESAT6-Ag85B formed a band with a fraction size of 37 kDa, equalivalent to the sum of Ag85B and ESAT6. Six weeks after BALB/c mice were immunized with BCG or rBCG, spleen lymphocytes showed significant proliferation in response to culture filtrate protein of MTB. Compared with the BCG group, mice vaccinated with rBCG elicited a high level increase of immunoglobulin G antibodies to culture filtrate protein in the serum. The T-interferon levels in the lymphocyte culture medium supernatants increased remarkably in the rBCG1 group, significantly higher than that of the BCG immunized group （P〈0.05）. Four weeks after vaccination, mice were infected with M. tuberculosis H37Rv and a dramatic reduction in the numbers of MTB colony forming units in the spleens and lungs was observed in the two rBCG immunization groups. Although these rBCG strains were more immunogenic, their protective effect was comparable to the classical BCG strain, and there were no significant differences between two rBCG groups （p〉0.05）.     

DNA vaccine of SARS-Cov S gene induces antibody response in mice

The spike (S) protein, a main surface antigen of SARS-coronavirus (SARS-CoV), is one of themost important antigen candidates for vaccine design. In the present study, three fragments of the truncated S protein were expressed in E. coli, and analyzed with pooled sera of convalescence phase of SARS patients. The full length S gene DNA vaccine was constructed and used to immunize BALB/c mice. The mouse serum IgG antibody against SARS-CoV was measured by ELISA with E. coli expressed truncated S protein or SARS-CoV lysate as diagnostic antigen. The results showed that all the three fragments of S protein expressed by E. coli was able to react with sera of SARS patients and the S gene DNA candidate vaccine could induce the production of specific IgG antibody against SARS-CoV efficiently in mice with seroconversion ratio of 75% after 3 times of immunization. These findings lay some foundations for further understanding the immunology of SARS-CoV and developing SARS vaccines.     

Transgenic Tobacco Expressing a Modified VP6 Gene Protects Mice Against Rotavirus Infection

Elevated expression of the rotavirus VP6 antigen in transgenic plants is a critical factor in the development of a safe and effective rotavirus vaccine. Using codon optimization, a gene that encodes the inner capsid protein VP6 of the human group A rotavirus was synthesized （sVP6）. The VP6 and sVp6 genes were transformed into tobacco （Nicotiana tabacum L.） plants using Agrobacterium tumefaciens. The expression level of the sVP6 gene in transgenic plants was 3.8-34-fold higher than that of controls containing the non-modified VP6 gene, accounting for up to 0.34% of the total soluble protein （TSP）. Then, BALB/ c female mice that had been gavaged weekly with 10 mg TSP containing 34 p.g VP6 protein, in which VP6-specific serum IgG and mucosal IgA antibodies were investigated. The severity and duration of diarrhea caused by simian rotavirus SA-11 challenge were reduced significantly in passively immunized pups, which indicates that anti-VP6 antibodies generated in orally immunized female mice can be passed onto pups and provide heterotypic protection. An edible vaccine based on the VP6 of human rotavirus group A could provide a means to protect children and young animals from severe acute diarrhea.     

Current understanding of the role of tyrosine kinase 2 signaling in immune responses

Immune system is a complex network that clears pathogens,toxic substrates,and cancer cells.Distinguishing self-antigens from non-self-antigens is critical for the immune cell-mediated response against foreign antigens.The innate immune system elicits an early-phase response to various stimuli,whereas the adaptive immune response is tailored to previously encountered antigens.During immune responses,B cells differentiate into antibody-secreting cells,while na?ve T cells differentiate into functionally specific effector cells[T helper 1(Th1),Th2,Th17,and regulatory T cells].However,enhanced or prolonged immune responses can result in autoimmune disorders,which are characterized by lymphocytemediated immune responses against self-antigens.Signal transduction of cytokines,which regulate the inflammatory cascades,is dependent on the members of the Janus family of protein kinases.Tyrosine kinase 2(Tyk2)is associated with receptor subunits of immune-related cytokines,such as type I interferon,interleukin(IL)-6,IL-10,IL-12,and IL-23.Clinical studies on the therapeutic effects and the underlying mechanisms of Tyk2 inhibitors in autoimmune or chronic inflammatory diseases are currently ongoing.This review summarizes the findings of studies examining the role of Tyk2 in immune and/or inflammatory responses using Tyk2-deficient cells and mice.     

Construction and characterization of a recombinant fowlpox virus containing HIV-1 multi-epitope-p24 chimeric gene in mice

The epidemic of HIV/AIDS is sweeping across the world. It is of great importance to figure out new ways to curb this disease. Epitope-based vaccine is one of these solutions. In this study, a chimeric gene was obtained by combination of a designed HIV-1 multi-epitope gene (MEG) and HIV-1 p24 gene. A re- combinant plasmid pUTA2-MEGp24 was then constructed by inserting MEGp24 gene into the down- stream of the promoter (ATI-P7.5×20) of fowlpox virus (FPV) transfer vector pUTA2. The recombinant plasmid and wild-type FPV 282E4 strain were then co-transfected into CEF cells and homologous re- combination occurred. A recombinant virus expressing HIV-1 protein MEGp24 was screened by ge- nome PCR and Western blot assay. Large scale preparation and purification of the recombinant fowl- pox virus (rFPV) were then carried out. BALB/c mice were immunized intramuscularly with the rFPV for three times on day 0, 14 and 42. Mice were executed and sampled one week after the third inoculation. Anti-HIV-1 antibody in serum and Th1 cytokines in the supernatant of cultured spleen cells were as- sayed by ELISA. The count of T lymphocyte subsets and the CTL activity of spleen lymphocytes were analyzed by flow cytometry and lactate dehydrogenase (LDH) release assay, respectively. The results showed that HIV-1 specific antibody in serum and increased T lymphocyte subsets (CD4 T, CD8 T) were detected in the immunization group. CTL target-killing activity and higher secretion of Th1 cyto- kines (IFN-γ and IL-2) of spleen lymphocytes stimulated by H-2d-restricted CTL peptide were observed in immunized mice. We concluded that the rFPV may induce HIV-1 specific immunity especially cellular immunity in mice.     

Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S, sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey     

Immunization with chlamydial plasmid protein pORF5 DNA vaccine induces protective immunity against genital chlamydial infection in mice

To validate the immune protective efficacy of pORF5 DNA vaccine and to analyze potential mechanisms related to this protection. In this study, pORF5 DNA vaccine was constructed and evaluated for its protective immunity in a mouse model of genital chlamydial infection. Groups of BALB/c mice were immunized intranasally with pORF5 DNA vaccine. Humoral and cell mediated immune responses were evaluated. The clearance ability of chlamydial challenge from the genital tract and the chlamy- dia-induced upper genital tract gross pathology and histopathological characterization were also de- tected. The results showed that the total and the IgG2a anti-pORF5 antibody levels in serum were sig- nificantly elevated after pcDNA3.1-pORF5 vaccination, as were the total antibody and IgA levels in vaginal fluids. pcDNA3.1-pORF5 induced a significantly high level of Th1 response as measured by robust gamma interferon (IFN-γ). Minimal IL-4 was produced by immune T cells in response to the re-stimulation with pORF5 protein or the inactive elementary body in vitro. pcDNA3.1-pORF5-vacci- nated mice displayed significantly reduced bacterial shedding upon a chlamydial challenge and an accelerated resolution of infection. 100% of pcDNA3.1-pORF5 vaccinated mice successfully resolved the infection by day 24. pcDNA3.1-pORF5-immunized mice also exhibited protection against patho- logical consequences of chlamydial infection. The stimulated index was significantly higher than that of mice immunized with pcDNA3.1 and PBS (P<0.05). Together, these results demonstrated that immu- nization with pORF5 DNA vaccine is a promising approach for eliciting a protective immunity against a genital chlamydial challenge.     

Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors

Protein trans-splicing based dual-vector factor VIII(FVIII) gene delivery is adversely affected by less efficiency of protein splicing.We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins,protein splicing elements,thereby facilitating protein trans-splicing.Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing,increasing the levels of spliced FVIII protein.In this study,C57BL/6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver.Forty-eight hours post-injection,plasma was collected and analyzed for FVIII antigen concentration and coagulation activity.These mice showed increased circulating FVIII heavy chain polypeptide(442±151 ng mL-1 vs.305±103 ng mL-1) and coagulation activity(1.46±0.37 IU mL-1 vs.0.85±0.23 IU mL-1) compared with control mice co-administered dual vectors expressing the heavy and light chains of wild-type FVIII.Moreover,coagulation activity was similar to that of mice receiving a single vector expressing FVIII(1.79±0.59 IU mL-1).These findings indicate that improving protein trans-splicing by inter-chain disulfide bonding is a promising approach for increasing the efficacy of dual-vector based FVIII gene transfer.     

Transcriptomic Response to Yersinia pestis:RIG-I Like Receptor Signaling Response Is Detrimental to the Host against Plague

Bacterial pathogens have evolved various mechanisms to modulate host immune responses for successful infection. In this study, RNA- sequencing technology was used to analyze the responses of human monocytes THP1 to Yersinia pestis infection. Over 6000 genes were differentially expressed over the 12 h infection. Kinetic responses of pathogen recognition receptor signaling pathways, apoptosis, antigen processing, and presentation pathway and coagulation system were analyzed in detail. Among them, RIG-I-like receptor （RLR） signaling pathway, which was established for antiviral defense, was significantly affected. Mice lacking MAVS, the adaptor of the RLR signaling pathway, were less sensitive to infection and exhibited lower IFN-13 production, higher Thl-type cytokines IFN-γ and IL-12 production, and lower Th2-type cytokines IL-4 and IL-13 production in the serum compared with wild-type mice. Moreover, infection of pathogenic bacteria other than E pestis also altered the expression of the RLR pathway, suggesting that the response of RLR pathway to bacterial infection is a universal mechanism.     

Immunogenicity of the spike glycoprotein of Bat SARS-like coronavirus

A group of SARS-like coronaviruses(SL-CoV)have been identified in horseshoe bats.Despite SL-CoVs and SARS-CoV share identical genome structure and high-level sequence similarity,SL-CoV does not bind to the same cellular receptor as for SARS-CoV and the N-terminus of the S proteins only share 64%amino acid identity,suggesting there are fundamental differences between these two groups of coronaviruses.To gain insight into the basis of this difference,we established a recombinant adenovirus system expressing the S protein from SL-CoV(rAd-Rp3-S)to investigate its immune characterization.Our results showed that immunized mice generated strong humoral immune responses against the SL-CoV S protein.Moreover,a strong cellular immune response demonstrated by elevated IFN-γand IL-6 levels was also observed in these mice.However,the induced antibody from these mice had weaker cross-reaction with the SARS-CoV S protein,and did not neutralize HIV pseudotyped with SARS-CoV S protein.These results demonstrated that the immunogenicity of the SL-CoV S protein is distinct from that of SARS-CoV,which may cause the immunological differences between human SARS-CoV and bat SL-CoV.Furthermore,the recombinant virus could serve as a potential vaccine candidate against bat SL-CoV infection.     

Cloning, expression and protective immunity evaluation of the full-length cDNA encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum

1071-bp fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3′ and 5′ ends of the incomplete expression sequence tag (EST) of succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) were amplified by the anchored PCR with 2 pairs of primers designed according to the EST of SjSDISP and the sequence of multiclone sites of the library vector. Sequence analysis indicated that the fragment was a full-length cDNA with a complete open reading frame (ORF), encoding 278 amino acid residues. The fragment was cloned into prokary- otic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coli. SDS-PAGE and Western-blot analyses showed that the recombinant protein was about 32 kD and could be recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Compared with the FCA controls, mice vaccinated with rSjSDISP (test) or rSjGST (posi- tive control) all revealed high levels of specific antibody and significant reduction in worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs. These results suggest that SjSDISP may be a novel and partially protective vaccine candidate against schistosomiasis. In contrast to the worm burden reduction rate, the higher degree of egg reduction rate in the test group also sug- gested that SjSDISP vaccine may primarily play a role in anti-embryonation or anti-fecundity immunity.     

Protective immunity against Rickettsia heilongjiangensis in a C3H/HeN mouse model mediated by outer membrane protein B-pulsed dendritic cells

Rickettsia heilongjiangensis is an obligate intracellular bacterium that causes Far-Eastern tick-borne spotted fever. Outer membrane protein B(Omp B) is an important surface protein antigen of rickettsiae. In the present study, the omp B gene of R. heilongjiangensis was divided into four fragments, resulting in four recombinant proteins(OmpB-p1, Omp B-p2, Omp B-p3, and Omp B-p4). Each Omp B was used in vitro to stimulate murine bone marrow-derived dendritic cells(BMDCs) of C3H/He N mice, and the Omp B-pulsed BMDCs were transferred to naive C3H/He N mice. On day 14 post-transfer of BMDCs, the mice were challenged with R. heilongjiangensis and the rickettsial loads in the mice were quantitatively determined on day 7 post-challenge. Mice receiving BMDCs pulsed with Omp B-p2, Omp B-p3, or Omp B-p4 exhibited significantly lower bacterial load compared with mice receiving Omp B-p1-pulsed BMDCs. CD4+ and CD8+ T cells isolated from the spleen of C3H/He N mice receiving BMDCs pulsed with each OmpB were co-cultured with BMDCs pulsed with the respective cognate protein. In flow cytometric analysis, the expression level of CD69 on CD4+ or CD8+ T cells from mice receiving BMDCs pulsed with Omp B-p2, OmpB-p3, or Omp B-p4 was higher than that on cells from mice receiving Omp B-p1-pulsed BMDCs, while the expression level of tumor necrosis factor(TNF)-α on CD8+ T cells and interferon(IFN)-γ on the CD4+ and CD8+ T cells from mice receiving Omp B-p2,-p3, or-p4 was significantly higher than on cells from mice receiving Omp B-p1-pulsed BMDCs. Our results suggest that the protective Omp Bs could activate CD4+ and CD8+ T cells and drive their differentiation toward CD4+ Th1 and CD8+ Tcl cells, respectively, which produce greater amounts of TNF-α and, in particular, IFN-γ, to enhance rickettsicidal activity of host cells.     

Immunogenicity and protective efficacy of recombinant M2e.Hsp70c(Hsp70_(359–610)) fusion protein against influenza virus infection in mice

New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70(mHsp70) is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte(CTL) response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2(M2e), was fused to the C-terminus of Mycobacterium tuberculosis Hsp70(Hsp70c), to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c(Hsp70359–610). And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.     

A multi-epitope vaccine based on Chlamydia trachomatis major outer membrane protein induces specific immunity in mice

We evaluated the immunogenicity and efficacy of a candidate vaccine comprising the major outer membrane protein （MOMP） multi-epitope of Chlamydia trachomatis. A short gene of muiti-epitope derived from MOMP containing multiple T- and B-cell epitopes was artificially synthesized. The recombinant plasmid pET32a（＋） containing codon optimized MOMP multi-epitope gene was constructed. Expression of the fusion protein Trx-His- MOMP multi-epitope in Escherichia coli was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Balb/c mice were inoculated with the purified fusion protein subcutaneously three times with 2-week intervals. Results showed that the MOMP multiepitope elicited not only strong humoral immune responses to C. trachomatis by generating significantly high levels of specific antibodies （lgG1 and IgG2a）, but also a cellular immune response by inducing robust cytotoxic T lymphocyte responses in mice. Furthermore, the MOMP multi- epitope substantially primed secretion of IFN-γ, revealing that this vaccine could induce a strong Thl response. Finally, the mice vaccinated with the MOMP multi-epitope displayed a reduction of C. trachomatis shedding upon a chlamydial challenge and an accelerated clearance of the infected C. trachomatis. In conclusion, the MOMP multi- epitope vaccine may have the potentiality for the development of effective prophylactic and therapeutic vaccines against the C. trachomatis infection.     

Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines

The M and NP genes of H5N1 avian influenza virus (A/chicken/Hubei/489/2004) were amplified by RT-PCR from viral RNA, and cloned into pMD18-T vector respectively. The expression plasmid containing the M gene (pHM6-m) or the NP gene (pHM6-np) was then constructed by inserting the M or NP gene into the pHM6 eukaryote expression vector; the constructed plasmid was then sequenced. 32 BALB/c mice (6-week-old) were divided into four groups at random. Three groups of BALB/c mice were inoculated one time the intramuscular route with either 30 ug of plasmid pHM6-m, 30μg of plasmid p HM6-np or the mixture of plasmid pHM6-m (15μg) and pHM6-np( 15μg) respectively. A additional group of mice were injected with 100μl PBS as controls. Two weeks later, all mice were challenged with homologous H5N1 avian influenza virus, and observed in the following 12 days. The survival rates of mice in the pHM6-m group, the pHM6-np group and mixed plasmids group were 62.5%, 25.0% and 50.0%, respectively. Results showed that effective protection could be provided by either pHM6-m or pHM6-np, but pHM6-m provided a better protective effect than pHM6-np.     

Immunological dynamics in response to two anthrax vaccines in mice

In order to understand the variation of humoral and cellular immune responses to A16R live spore and AVA vaccine and to identify efficient immunological parameters for the early evaluation of post immu- nization in mice, we dynamically monitored the antibody production and cellular responses after the vaccination of Balb/C mice with the anthrax vaccines. The results show that both anti-AVA and anti-Spore antibodies were detectable in the A16R live spore vaccinated group while high titers of anti-AVA antibodies but not anti-Spore antibodies existed in the AVA-immunized group. IgG1 and IgG2 were the major subtypes of IgG in both of the two groups. However, the IgG2a level was significantly higher in the A16R group than in the AVA group. At the cellular level, responses of antigen-specific TH2, TH1 and plasma cells were detected. The peripheral TH2 responses could be seen on day 5 after vac- cination, and remained at a high level throughout the experiment (from day 5 post primary immuniza- tion to day 60 post the tertiary immunization); the TH1 responses to A16R vaccine appeared on day 5, while the responses to AVA could only be detected by day 7 after the secondary immunization; a low level of TH1 responses could be observed at the end of the experiment. Antigen-specific plasma cells could be found in the peripheral blood of both the immunized groups, however, the responses in the A16R group appeared earlier, lasted longer, and shown an ascending tendency until the end of the ex- periment when the plasma cell responses in the AVA group were reduced to a very low level. The re- sults suggest that the multiple antigen containing A16R live spore vaccine induces better immune re- sponses than AVA. Combined with serum antibody titers, TH2, TH1 and plasma cell responses could be used as immunological parameters for the evaluation of vaccine efficacy. These findings may afford new insight into the early evaluation of vaccination as well as being a powerful strategy for vaccine development.     

Preparation and epitope characterization of monoclonal antibodies against firefly luciferase

The 6-His tagged firefly luciferase was highly expressed in E. coli and purified to homogeneity by affinity chromatography and gel filtration. After immunizing Balb/c mice with the antigen, 6 hybridomas clones were found to secrete monoelonal antibodies (mAbs) and the mAbs were also purified separately. The competitive binding experiments show that 2 mAbs can bind heat-denatured antigen or its proteolytic fragments but not the native lueiferase, suggesting that their epitopes might be accommodated in the internal segments of the protein. On the other hand, the other 4 mAbs are capable of binding both native and denatured antigens. It infers that their epitopes locate in the segments on the protein surface. The results also suggest that the six mAbs are all sequence-specific.     

Expression of the human fast-twitch skeletal muscle troponin I cDNA in a human ovarian carcinoma sup-presses tumor growth

To explore the efficiency and mechanism of ovarian carcinoma gene therapy with the human fast-twitch skeletal muscle troponin I gene (TnI-fast), TnI-fast cDNA was transferred into human ovarian adeno-carcinoma cell-line SK-OV-3. In vitro, the cell growth and cell cycle of TnI-fast-, vector-, and mock-transfected cells were determined by MTT and flow cytometry assay, respectively. The condi-tioned media of TnI-fast-, vector-, and mock-transfected SK-OV-3 cells were collected, and the cell pro-liferation inhibiting rates of human umbilical cord venous endothelial cells (HUVECs) by the three conditioned media were assayed. All the three cell lines were implanted into node mice, and the tumor growth, cell apoptosis, angiogenesis, and expression of TnI-fast were observed or analyzed, respec-tively. In vitro, expression of TnI-fast protein had no inhibiting effect on the growth of the dominant and stable transfectant cells, but endothelium, when compared with vector-transfected cells and nontrans-fected parental SK-OV-3 cells. Implantation of stable clone expressing TnI-fast in the female BALB/c nude mice inhibits primary tumor growth by an average of 73%. The nude mice grafts expressing TnI-fast exhibit a significant decrease of microvascular density, a higher rate of tumor cells apoptosis and a comparable proliferation rate as control. Our study, to our knowledge, shows the slowed down growth of the primary ovarian carcinoma, suggested that grafts were self-inhibitory by halting angio-genesis. Our data might also provide a novel useful strategy for cancer therapy by antiangiogenic gene therapy with a specific angiogenesis inhibitor TnI-fast.