Evolution of the rodent Trim5 cluster is marked by divergent paralogous expansions and independent acquisitions of TrimCyp fusions.

Evolution of cellular innate immune genes in response to viral threats represents a rich area of study for understanding complex events that shape mammalian genomes. One of these genes, TRIM5, is a retroviral restriction factor that mediates a post-entry block to infection. Previous studies on the genomic cluster that contains TRIM5 identified different patterns of gene amplification and the independent birth of CypA gene fusions in various primate species. However, the evolution of Trim5 in the largest order of mammals, Rodentia, remains poorly characterized. Here, we present an expansive phylogenetic and genomic analysis of the Trim5 cluster in rodents. Our findings reveal substantial evolutionary changes including gene amplifications, rearrangements, loss and fusion. We describe the first independent evolution of TrimCyp fusion genes in rodents. We show that the TrimCyp gene found in some Peromyscus species was acquired about 2 million years ago. When ectopically expressed, the P. maniculatus TRIMCyp shows anti-retroviral activity that is reversed by cyclosporine, but it does not activate Nf-κB or AP-1 promoters, unlike the primate TRIMCyps. These results describe a complex pattern of differential gene amplification in the Trim5 cluster of rodents and identify the first functional TrimCyp fusion gene outside of primates and tree shrews.

Immunogenicity characterization of genetically fused or chemically conjugated heat-stable toxin toxoids of enterotoxigenic Escherichia coli in mice and pigs.

Enterotoxigenic Escherichia coli (ETEC) producing type Ib heat-stable toxin (STa) are a main cause of children's diarrhea and travelers' diarrhea, thus STa needs to be targeted in ETEC vaccine development. However, because this 19-amino acid STa is poorly immunogenic, attempts to genetically fuse or chemically couple it to carrier proteins have been made to enhance STa immunogenicity. In this study, we selected one genetic fusion and one chemical conjugate to comparatively evaluate STa immunogenicity. The genetic fusion is 3xSTaN12S-mnLTR192G/L211A carrying three toxoid (STaN12S) genetically fused to a double mutant LT monomer (mnLTR192G/L211A); the chemical conjugate is BSA-STaA14T, which has toxoid STaA14T chemically coupled to bovine serum albumin (BSA). We immunized mice with the STa toxoid fusion and chemical conjugates, and examined antibody responses. Furthermore, we immunized pigs and evaluated derived antibodies for efficacy to passively provide protection against ETEC diarrhea using a piglet model. Data showed that mice subcutaneously immunized with BSA-STaA14T or 3xSTaN12S-mnLTR192G/L211A developed a strong anti-STa antibody, and the induced antibodies exhibited equivalent toxin-neutralizing activities. Pigs immunized with 3xSTaN12S-mnLTR192G/L211A or BSA-STaA14T developed similar levels of anti-STa antibodies; piglets with passively acquired antibodies induced by the genetic fusion appeared better protected against STa + ETEC. Results from the current study indicate that the fusion and conjugate approaches are viable options for facilitating STa immunogenicity and developing ETEC vaccines.

Induction of specific humoral and cellular immune responses in a mouse model following gene fusion of HSP70C and Hantaan virus Gn and S0.7 in an adenoviral vector.

Heat shock proteins (HSPs) display adjuvant functions when given as fusion proteins to enhance vaccination efficiency. To evaluate enhanced potency of Hantaan virus (HTNV) glycoprotein (GP) and nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), a recombinant adenovirus rAd-GnS0.7-pCAG-HSP70C expression vector was developed by genetically linking the HSP70 C-terminal gene (HSP70 359-610 aa, HSP70C) to the Gn and 0.7 kb fragment of the NP (aa1-274-S0.7). C57BL/6 mice were immunized with these recombinant adenoviral vectors. A series of immunological assays determined the immunogenicity of the recombinant adenoviral vectors. The results showed that rAd-GnS0.7-pCAG-HSP70C induced a stronger humoral and cellular immune response than other recombinant adenoviruses (rAd-GnS0.7-pCAG and rAd-GnS0.7) and the HFRS vaccine control. Animal protection experiments showed that rAd-GnS0.7-pCAG-HSP70C was effective at protecting C57BL/6 mice from HTNV infection. The results of the immunological experiments showed that HSP70C lead to enhanced vaccine potency, and suggested significant potential in the development of genetically engineered vaccines against HTNV.

Birth, decay, and reconstruction of an ancient TRIMCyp gene fusion in primate genomes.

TRIM5 is a host antiviral gene with an evolutionary history of genetic conflict with retroviruses. The TRIMCyp gene encodes a protein fusion of TRIM5 effector domains with the capsid-binding ability of a retrotransposed CyclophilinA (CypA), resulting in novel antiviral specificity against lentiviruses. Previous studies have identified two independent primate TRIMCyp fusions that evolved within the past 6 My. Here, we describe an ancient primate TRIMCyp gene (that we call TRIMCypA3), which evolved in the common ancestor of simian primates 43 Mya. Gene reconstruction shows that CypA3 encoded an intact, likely active, TRIMCyp antiviral gene, which was subject to selective constraints for at least 10 My, followed by pseudogenization or loss in all extant primates. Despite its decayed status, we found TRIMCypA3 gene fusion transcripts in several primates. We found that the reconstructed "newly born" TrimCypA3 encoded robust and broad retroviral restriction activity but that this broad activity was lost via eight amino acid changes over the course of the next 10 My. We propose that TRIMCypA3 arose in response to a viral pathogen encountered by ancestral primates but was subsequently pseudogenized or lost due to a lack of selective pressure. Much like imprints of ancient viruses, fossils of decayed genes, such as TRIMCypA3, provide unique and specific insight into paleoviral infections that plagued primates deep in their evolutionary history.

[Construction, expression and immunocompetence of Neisseria gonorrhoeae porB and Escherichia coli itB fusion gene].

OBJECTIVE: To construct prokaryotic fusion gene expression vector pET-30a/ltB-porB, express the recombinant fusion protein LTB-PorB and analyze the immunocompetence of the recombinant fusion protein in female BALB/c mice through intranasally immunization. METHODS: B subunit of Escherichia coli heat-labile enterotoxin (LTB) and Neisseria gonorrhoeae Porin B (PorB) fusion gene, LTB gene and PorB gene were cloned into prokaryotic vector pET-30a. The recombinants were identified by Polymerase Chain Reaction (PCR), enzyme digestion and DNA sequencing, and then expressed efficiently in Escherichia coli BL21 in the form of inclusion bodies. The renatured recombinant proteins had antigenicity, which was confirmed by Western blot. Female BALB/c mice were inoculated with renatured recombinant proteins without endotoxin through intranasally immunization at the days 0, 14, 28. Next, humoral immunoresponse and cellullar immunologic response were detected in female BALB/c mice by enzyme linked immunosorbent assay (ELISA) and methyl thiazolyl tetrazolium( MTT) colorimetric assay. RESULTS: The level of PorB specific sIgA in genital tract and IgG in serum shown upward trend along with the days post innoculation in LTB-PorB group, A450 of sIgA in LTB-PorB group was 0.66 at the day 42, which was significantly higher than controls (P < 0.01), and the titer was up to 1:1280. A450 of serum IgG in LTB-PorB group was 0.60 at the day 28, which was significantly higher than the LTB and the Solution Buffer controls (P < 0.01), and the titer was up to 1:2560. However, the IgG between LTB-PorB group and PorB control (A450 :0.57) had no significant difference (P > 0.05). Stimulation index of the splenic lymphocyte in LTB-PorB group was significantly higher than the LTB and the Solution Buffer controls (P < 0.05). But the level of IFN-gamma induced by splenic lymphocyte between LTB-PorB group and controls had no significant difference (P > 0.05). CONCLUSION: The recombinant fusion protein LTB-PorB could induce high level of humoral immunoresponse and slightly cellullar immunologic response in female BALB/c mice through intranasally immunization. For the first time to our knowledge, the mucosal adjuvant LTB could assist PorB to induce high level of mucosal immune response in the genital tract mucosa of mice.

Immune strategies using single-component LipL32 and multi-component recombinant LipL32-41-OmpL1 vaccines against leptospira

Genes encoding lipoproteins LipL32, LipL41 and the outer-membrane protein OmpL1 of leptospira were recombined and cloned into a pVAX1 plasmid. BALB/c mice were immunized with LipL32 and recombined LipL32-41-OmpL1 using DNA-DNA, DNA-protein and protein-protein strategies, respectively. Prime immunization was on day 1, boost immunizations were on day 11 and day 21. Sera were collected from each mouse on day 35 for antibody, cytokine detection and microscopic agglutination test while spleen cells were collected for splenocyte proliferation assay. All experimental groups (N = 10 mice per group) showed statistically significant increases in antigen-specific antibodies, in cytokines IL-4 and IL-10, as well as in the microscopic agglutination test and splenocyte proliferation compared with the pVAX1 control group. The groups receiving the recombined LipL32-41-OmpL1 vaccine induced anti-LipL41 and anti-OmpL1 antibodies and yielded better splenocyte proliferation values than the groups receiving LipL32. DNA prime and protein boost immune strategies stimulated more antibodies than a DNA-DNA immune strategy and yielded greater cytokine and splenocyte proliferation than a protein-protein immune strategy. It is clear from these results that recombination of protective antigen genes lipL32, lipL41, and ompL1 and a DNA-protein immune strategy resulted in better immune responses against leptospira than single-component, LipL32, or single DNA or protein immunization.

Immune strategies using single-component LipL32 and multi-component recombinant LipL32-41-OmpL1 vaccines against leptospira.

Genes encoding lipoproteins LipL32, LipL41 and the outer-membrane protein OmpL1 of leptospira were recombined and cloned into a pVAX1 plasmid. BALB/c mice were immunized with LipL32 and recombined LipL32-41-OmpL1 using DNA-DNA, DNA-protein and protein-protein strategies, respectively. Prime immunization was on day 1, boost immunizations were on day 11 and day 21. Sera were collected from each mouse on day 35 for antibody, cytokine detection and microscopic agglutination test while spleen cells were collected for splenocyte proliferation assay. All experimental groups (N = 10 mice per group) showed statistically significant increases in antigen-specific antibodies, in cytokines IL-4 and IL-10, as well as in the microscopic agglutination test and splenocyte proliferation compared with the pVAX1 control group. The groups receiving the recombined LipL32-41-OmpL1 vaccine induced anti-LipL41 and anti-OmpL1 antibodies and yielded better splenocyte proliferation values than the groups receiving LipL32. DNA prime and protein boost immune strategies stimulated more antibodies than a DNA-DNA immune strategy and yielded greater cytokine and splenocyte proliferation than a protein-protein immune strategy. It is clear from these results that recombination of protective antigen genes lipL32, lipL41, and ompL1 and a DNA-protein immune strategy resulted in better immune responses against leptospira than single-component, LipL32, or single DNA or protein immunization.

[Effects of fusion gene of ubiquitin and porcine reproductive and respiratory syndrome virus M gene on the immune response in inoculated mice].

OBJECTIVE: To evaluate the effects of the fusion gene of ubiquitin (Ub) and Porcine Reproductive and Respiratory Syndrome virus (PRRSV) M gene on the immune response in inoculated mice. METHODS: Mouse Ub gene and PRRSV M gene were amplified by RT-PCR from BALB/c mice spleen cells and PRRSV Ch-1a strain, respectively, and the M and Ub gene (U-M) was fused by SOE PCR. Therefore, pVAX1-U-M and pVAX1-M recombinant plasmid were constructed for eukaryotic expression. RESULTS: The fusion U-M and M protein expressions were verified in transfected BHK-21 cells by indirect fluorescence assay. Furthermore, both pVAX1-M and pVAX1-U-M induced specific humoral and cellar immune responses against PRRSV in the recombinant plasmid injected mice. However, pVAX1-U-M was able to induce higher level of T cell response then that of pVAX1-M (P<0.05), but lower level of antibody (P<0.05). CONCLUSION: Expression of U-M fusion gene had ability to enhance specific T cell response against PRRSV, but no effect on stimulation of humoral response in inoculated mice.

An essential role for the stalk region of CD8 beta in the coreceptor function of CD8.

The CD8alphabeta heterodimer is integral to the selection of the class I-restricted lineage in the thymus; however, the contribution of the CD8beta chain to coreceptor function is poorly understood. To understand whether the CD8beta membrane proximal stalk region played a role in coreceptor function, we substituted it with the corresponding sequence from the CD8alpha polypeptide and expressed the hybrid molecule in transgenic mice in place of endogenous CD8beta. Although the stalk-swapped CD8beta was expressed on the cell surface as a disulfide-bonded heterodimer at equivalent levels of expression to an endogenous CD8beta molecule, it failed to restore selection of CD8(+) class I MHC-restricted T cells and it altered the response of peripheral T cells. Thus, the stalk region of the CD8beta polypeptide has an essential role in ensuring functionality of the CD8alphabeta heterodimer and its replacement compromises the interaction of CD8 with peptide-MHC complexes.

Expression of the human hepatitis B virus large surface antigen gene in transgenic tomato plants.

The original hepatitis B virus (HBV) large surface antigen gene was synthesized. In order to optimize the expression of this gene in tomato plants, the tobacco pathogenesis-related protein S signal peptide was fused to the 5' end of the modified gene and the sequence encoding amino acids S, E, K, D, E, and L was placed at the 3' end. The gene encoding the modified HBV large surface antigen under the control of a fruit-specific promoter was constructed and expressed in transgenic tomato plants. The expression of the antigen from transgenic plants was confirmed by PCR and reverse transcriptase PCR. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed that the maximal level of HBsAg was about 0.02% of the soluble protein in transgenic tomato fruit. The amount of HBsAg in mature fruits was found to be 65- to 171-fold larger than in small or medium fruits and leaf tissues. Examination of transgenic plant samples by transmission electron microscopy proved that HBsAg had been expressed and had accumulated. The HBsAg protein was capable of assembling into capsomers and virus-like particles. To our knowledge, this is the first time the HBV large surface antigen has been expressed in plants. This work suggests the possibility of producing a new alternative vaccine for human HBV.

DNA fusion vaccines induce epitope-specific cytotoxic CD8(+) T cells against human leukemia-associated minor histocompatibility antigens.

The graft-versus-leukemia effect of allogeneic stem-cell transplantation is believed to be mediated by T-cell recognition of minor histocompatibility antigens on recipient cells. For minor histocompatibility antigens HA-1 and HA-2, normal cell expression is restricted to hemopoietic cells, and boosting the immune response to these antigens may potentiate graft-versus-leukemia effect without accompanying graft-versus-host disease. To increase efficacy, expansion of HA-1- or HA-2-specific CTL before transplantation is desirable. However, primary HA-1- or HA-2-specific CTL expanded in vitro are often of low avidity. An alternative approach is to prime specific CTL responses in vivo by vaccination. Clearly, donor vaccination must be safe and specific. We have developed DNA fusion vaccines able to induce high levels of epitope-specific CTL using linked CD4(+) T-cell help. The vaccines incorporate a domain of tetanus toxin (DOM) fused to a sequence encoding a candidate MHC class I binding peptide. This design generates antitumor CD8(+) T-cell responses and protective immunity in preclinical models. For clinical application, we constructed vaccines encoding HLA-A*0201-restricted peptides from human HA-1 and HA-2, which were fused to DOM, and tested their performance in HLA-A*0201-transgenic mice. Priming induced epitope-specific, IFNgamma-producing CD8(+) T cells with cytotoxic function boosted to high levels with electroporation. Strikingly, these mouse T cells efficiently killed human lymphoblastoid cell lines expressing endogenous HA-1 or HA-2. High avidity is indicated by the independence of cytolysis from CD8/MHC class I interaction. These safe epitope-specific vaccines offer a potential strategy to prime HA-1- or HA-2-specific CTL in transplant donors before adoptive transfer.