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Junin virus (JUNV) is the pathogen of Argentine haemorrhagic fever which results in high lethality, while there is limited therapeutics available. Here, a series of mouse monoclonal antibodies (mAbs) were isolated through immunization with DNA and screening against glycoprotein complex (GPC). Finally, five mAbs were found to effectively neutralize JUNV. Further research indicated that they were capable of binding conformational GPC and strongly binding glycoprotein 1 which is responsible for receptor recognition. Epitope mapping revealed that they targeted loop 3 of GP1, and Tyr122 and Asp123 in loop 3 were identified as their common binding sites, which may account for their neutralizing activity. This study presents a new strategy for developing neutralizing antibodies against JUNV and provides therapeutic candidates for protection against JUNV infection. [ABSTRACT FROM AUTHOR]

To date, several new anti-CD20 monoclonal antibodies (mAbs) have been developed for potential efficacies compared with familiar mAb rituximab. Despite the recent advances in development of anti-CD20 mAbs for the treatment of B cell malignancies, the efforts should be continued to develop novel antibodies with improved properties. However, the development of mAbs against CD20 as a multi-transmembrane protein is challenging due to the difficulty of providing a lipid environment that can maintain native epitopes. To overcome this limitation, we describe a simple and efficient DNA immunization strategy for the construction of a novel anti-CD20 mAb with improved anti-tumour properties. Using a DNA immunization strategy that includes intradermal (i.d.) immunization with naked plasmid DNA encoding the CD20 gene, we generated the hybridoma cell line D4, which secretes functional mAbs against an extracellular epitope of CD20. Immunocytochemistry analysis and a cell-based enzyme-linked immunosorbent assay using a Burkitt's lymphoma cell line showed that D4 mAbs are capable of binding to native extracellular epitopes of CD20. Moreover, the binding specificity of D4 mAbs was determined by western blot analysis. Cell proliferation was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by the annexin V/propidium iodide staining and dye exclusion assay. The results showed that D4 anti-CD20 mAbs produced by DNA immunization exhibit potent growth inhibitory activity and have superior direct B-cell cytotoxicity compared to rituximab. We propose that antibody-induced apoptosis is one of the mechanisms of cell growth inhibition. Taken together, the data reported here open the path to DNA-based immunization for generating pharmacologically active monoclonal antibodies against CD20. In addition, the data support future in vivo animal testing and subsequent procedures to produce a potential therapeutic mAb. [ABSTRACT FROM AUTHOR]

The immune response has relevant physiological functions both in the male and female reproductive system, and must be tightly controlled to achieve a successful pregnancy. Several immune factors have been related to infertility, among them humoral and cellular immune responses triggered by sperm antigens. The present study was aimed at evaluating the immune profile induced by DNA immunization against the sperm protease proacrosin in CF1 male mice and its effect upon fertility. Immunized animals exhibited higher anti-proacrosin antibodies levels than controls (indirect ELISA), both in serum ( p < 0.01) and in seminal vesicle fluid (SVF; p < 0.05). IgG2a levels were higher than IgG1 in serum ( p < 0.01) and similar in SVF. IL-10 and TGF-β1 mRNA levels were lower in testis ( p < 0.05), whereas TNF-α and IFN-γ transcript levels were increased in SV tissue ( p < 0.05). Immunized mice showed a trend toward higher IFN-γ concentration in serum and SVF than controls. Male fertility rate was diminished in immunized mice ( p < 0.01) and inversely correlated with serum and SVF anti-proacrosin IgG levels ( p < 0.001). Immunized animals also had fewer pups born than controls ( p < 0.01). To our knowledge, this is the first report on DNA immunization done in CF1 mice. Injection of proacrosin DNA induces an immune response in the male reproductive tract characterized by high levels of specific antibodies and cytokine changes. These factors may alter the crucial balance of the genital tract microenvironment required for adequate fertilization and pregnancy. [ABSTRACT FROM AUTHOR]

Abstract: Molecular adjuvants were used to augment the amplitude of the immune response in many studies recently. Ubiquitin (ub), the peptide binding truncated C-terminal portion of heat shock protein 70 (hsp70c) and interleukin-2 (IL-2) are widely investigated adjuvants which have been proved to be efficient. In our study, we compared the enhancing ability of these three adjuvants based on DNA vaccination using the porcine circovirus type 2 ORF2 (capsid) gene in mice. The results of lymphocyte proliferation assay, flow cytometric analysis (FCM), antibody titer and cytokine production showed that ub conjugated plasmid induced a stronger Th1 type cellular immune response and an observably higher level of Cap-specific serum immunoglobulin G antibody compared with hsp70c or IL-2 conjugated plasmids during the period of post-immunization. Meanwhile, the ub conjugation vaccinated group elicited stronger specific immunity against PCV2 challenge than the others during most of the time of post-challenge. Thus, these data indicate that ub is a superior adjuvant for a PCV2 DNA vaccination than the hsp70c and IL-2 molecules. [Copyright &y& Elsevier]

Abstract: Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, a highly contagious enteric disease of swine. The Spike (S) protein is one of the main structural proteins of PEDV capable of inducing neutralizing antibodies in vivo. Herein, we generated three distinct DNA constructs in the eukaryotic expression plasmid pVAX1; one encoding the S protein [pVAX1-(PEDV-S)], the second encoding the N-terminal fragment (S1) [pVAX1-(PEDV-S1)] containing potent antigenic sites, and the third expressing the porcine interleukin-18 (pIL-18) [pVAX1-(IL-18)]. Immunofluorescence assays in BHK-21 cells demonstrated successful protein expression from all 3 constructs. Kunming mice were injected separately with each of these constructs or with a pVAX1-(PEDV-S1)/pVAX1-(IL-18) combination, an attenuated PEDV vaccine, or vector only control. Animals were examined for T lymphocyte proliferation, anti-PEDV antibodies, IFN-γ and IL-4 protein levels, and cytotoxic T cell function in mouse peripheral blood and spleen. In all cases, results showed that pVAX1-(PEDV-S) and the combination of pVAX1-(PEDV-S1) with pVAX1-(IL-18) induced the strongest responses; however, pIL-18 had no adjuvant effects when given in combination with pVAX1-(PEDV-S1). [Copyright &y& Elsevier]

Abstract: Antibody-based drug research involves the preparation of polyclonal and monoclonal antibodies, especially those that are reactive with native G protein-coupled receptors (GPCRs) on the cell membrane. Here, we report that DNA immunization of mice with a plasmid that encodes endothelin A receptor (ETAR) fused to Escherichia coli (E. coli) GroEL at its C-terminus (ETAR–GroEL) induced very strong and specific antibody responses to native ETAR. Co-injection of plasmids that expressed ETAR and GroEL (ETAR+GroEL) induced significantly lower antibody responses compared with the ETAR–GroEL plasmid. Monoclonal antibodies that are prepared by using GroEL as a molecular adjuvant could be used in immunoassays, such as flow cytometry, western blotting, and immunoprecipitation, to detect both exogenous and endogenous ETAR. The adjuvant activity of GroEL might involve inflammatory cytokine mediators via Toll-like receptor 4 in addition to the anticipated carrier effect. DNA immunization using GroEL might become a standard method for producing antibodies that are useful for the functional analysis of GPCRs. [Copyright &y& Elsevier]

Abstract: The open reading frame of the α-subunit (amino acids 1–211) of human muscle nicotinic acetylcholine receptor (hAChRα211) was inserted into eukaryotic expression vector of pPIC9K to form a recombinant plasmid. After transformation and expression, the target protein rhAChRα211 (recombinant hAChRα211) was secretively expressed in recombinant strain P. pastoris GS115/pPIC9K–hAChRα211 with a yield of 25mg/L. rhAChRα211 was purified by Q Sepharose column and gel filtration chromatography. Furthermore, the purified protein was coupled with CNBr-actived Sepharose 4B to form a special immunoadsorbent. By this immunoadsorbent, the removal rate for AChRAb in two myasthenia gravis (MG) patient sera reached 84% and 94%, respectively. The DNA fragment of hAChRα211 was cloned into shuffle vector of pcDNA3.0 to form the recombinant plasmid pcDNA–hAChRα211. Then the gene vaccine was directly injected intramuscularly into C57BL/6 mice. After immunization, the corresponding antibody, AChRAb, was detected in mice sera by ELISA. The target gene could be re-amplified by PCR in muscle, liver, spleen and kidney of immunized mice. It provides rapid and efficient methods to remove specific acetylcholine receptor antibody from the patient''s sera and establish an animal model of myasthenia gravis by recombinant hAChRα211 immunization. [Copyright &y& Elsevier]

Abstract: Introduction: Chemokines are regulated by a family of ‘atypical’ chemokine receptors, D6, DARC and CCX-CKR, each of which efficiently internalizes its cognate chemokine ligands. Development of monoclonal antibodies (MAbs) that would recognize CCX-CKR on the cell surface will be helpful to identify primary CCX-CKR-expressing cell types and analyze the fate of CCX-CKR after ligand binding to the receptor. Methods: We generated IgG MAbs recognizing the cell-surface CCX-CKR by DNA immunization using a molecular adjuvant, and analyzed the epitope recognized by the MAbs. Then, the reactivities of the MAbs with CCX-CKR-transfected cells, and also hepatocytes and hepatic tumor lines were evaluated. Finally, we also tested the ligand-like activities of the MAbs, namely, induction of internalization of CCX-CKR by the MAbs. Results: A panel of MAbs reacting with CCX-CKR expressed on the cell surface was prepared. The panel was a small one, consisting of only ten MAbs, but was rich in terms of diversity of the Ig isotypes and of the epitopes. Epitope analyses revealed that all the 10 MAbs recognized at least three different, although very close, peptide structures of the N-terminal domain. Three MAbs, namely, 2F11, 13E11 and 14F10, were selected to represent the panel. All of the MAbs were applicable for flow cytometry and immunoflurescent assays and immunoprecipitation. The reactivity of the 2F11 MAb was also confirmed by western blotting. Endogenous expression of CCX-CKR on human hepatocytes and hepatic tumor cell lines was demonstrated using the 13E11 MAb. Interestingly, binding of the 13E11 MAb with B300-19 cells expressing CCX-CKR resulted in induction of CCX-CKR internalization. Discussion: This panel of MAbs may be expected to prove valuable for further study of the functions of this silent chemokine receptor, including those related to the homeostasis of lymphoid cells, and to the growth and metastasis of hepatic cancer. [Copyright &y& Elsevier]

Abstract: High mobility group box protein 1 (HMGB1), a major non-histone protein, released from the cells induces dendritic cell (DC) maturation and Th1 polarization. While DNA immunization has become an attractive method for eliciting the production of antibodies (Abs) in animals injected with DNA encoding an antigen, the Ab responses induced by DNA immunization remain relatively weak. In this study, we investigated the release of an HMGB1-conjugated ovalbumin (HMGB1 OVA conjugate, HMGB1-OVA) from necrotic cells and the Ab responses to HMGB1-OVA following DNA immunization. HMGB1-OVA was released from 293T cells after induction of necrosis in vitro and from the muscle into the serum after DNA immunization followed by electroporation. DCs pulsed with the supernatant of necrotic 293T cells containing HMGB1-OVA induced DO11.10 CD4+ T cell proliferation and interferon-γ secretion more potently than DCs pulsed with the cell supernatant containing OVA. DNA immunization with an expression plasmid for HMGB1-OVA by intramuscular injection elicited enhanced Th1-type Ab responses to OVA. Moreover, DNA immunization with a plasmid vector for release-type HMGB1 mutant-conjugated OVA elicited an even stronger response than DNA immunization with wild-type HMGB1-OVA. HMGB1-based DNA immunization described here has the potential to enhance the immunogenicity of antigens and elicit stronger Th1-type Ab response. [ABSTRACT FROM AUTHOR]

Abstract: We identified a novel HLA-DR4-restricted CD4+ T-cell epitope on a secreted antigen of Mycobacterium tuberculosis, MPT51, in 004149-MM HLA-DR4-transgenic mice which express HLA-DRB1*0401, but not murine MHC class II molecules. The mice were immunized with plasmid DNA encoding MPT51 using gene gun and interferon (IFN)-γ production from the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, only one peptide, p191–210, stimulated the splenocytes to produce IFN-γ. Further analysis using flow cytometry and computer-assisted algorithm, ProPred, narrowed down the region of CD4+ T-cell epitope to p191–202. The CD4+ T-cell epitope would be feasible for vaccine design against tuberculosis as well as for analysis of MPT51-specific T-cells in M. tuberculosis infection. [Copyright &y& Elsevier]

Abstract: Mycobacterial DNA-binding protein 1 (MDP1) is a major protein antigen in mycobacteria and induces protective immunity against Mycobacterium tuberculosis infection in mice. In this study we determined murine T-cell epitopes on MDP1 with MDP1 DNA immunization in mice. We analyzed interferon-γ production from the MDP1 DNA-immune splenocytes in response to 20-mer overlapping peptides covering MDP1 protein. We identified several CD4+ T-cell epitopes in three inbred mouse strains and one CD8+ T-cell epitope in C57BL/6 mice. These T-cell epitopes would be feasible for analysis of the role of MDP1-specific T-cells in protective immunity and for future vaccine design against M. tuberculosis infection. [Copyright &y& Elsevier]

Abstract: The aim of this study was to evaluate the immunogenicity of NS5A protein of human hepatitis C virus (HCV) when delivered as naked DNA (NS5A DNA), or recombinant protein (rNS5A). DBA/2J mice received NS5A DNA, rNS5A, or NS5A DNA/rNS5A in different prime-boost combinations with a peptidoglycan Immunomax®. The weakest response was induced after rNS5A prime and NS5A DNA boost; rNS5A alone induced an immune response with a strong Th2-component; and NS5A DNA alone, a relatively weak secretion of IL-2 and IFN-γ. The most efficient was co-injection of NS5A DNA and rNS5A, which induced a significant increase in CD4+ and CD8+ T-cell counts, anti-NS5A antibodies, specific T-cell proliferation, and proinflammatory cytokine production in vitro against a broad spectrum of NS5A epitopes. Administration of the mixture of adjuvanted DNA and protein immunogens can be selected as the best regimen for further preclinical HCV-vaccine trials. [Copyright &y& Elsevier]

Abstract: HIV reverse transcriptase (RT) can be considered as a target and an instrument of immunotherapy aimed at limiting the emergence and spread of drug-resistant HIV. The chimeric genes coding for the wild-type and multi-drug-resistant RT (RT1.14) fused to lysosome-associated membrane protein 1 (LAMP-1) were injected intramuscularly into BALB/c mice. The immune response was assessed by ELISpot, cytokine ELISA intracellular IFN-γ staining, and antibody ELISA. The genes for RT- and RT1.14-LAMP fusions (RT-LAMP and RT1.14-LAMP) were immunogenic generating a mixed Th1/Th2-profile of immune response, while the wild-type RT gene induced only weak immune response. Specific secretion of Th1-cytokines increased with increasing level of RT modification: RT

Abstract: Plant-based oral vaccines run the risk of activating regulatory T cells (Tregs) and suppressing the antigen-specific immune response via oral tolerance. Mice humanized for two HLA alleles (HLA-A2.1 and HLA-DR1) were used to measure changes in Tregs and antigen-specific immune responses induced by the oral administration of tobacco (Nicotiana tabacum), expressing the hepatitis B surface antigen (HBsAg). Antigen-specific CD8+ T cell activation was not detected, but the plant-based oral immunization, without adjuvant, resulted in humoral responses comparable to those obtained by adjuvanted DNA immunization. Treg titers did not increase with DNA immunization. In contrast, with plant immunization, Tregs increased linearly to reach a plateau at high antigen doses. The highest humoral IgA and IgG responses correlated with the lowest plant antigen dose (0.5ng), while for DNA immunization the best antibody responses were obtained at higher antigen doses. These experiments suggest that plant-based oral vaccines could be adjusted to minimize tolerance, while still inducing an immune response. Oral tolerance and adjuvant engineering in plants are discussed. [Copyright &y& Elsevier]

Abstract: In this work, the cDNA encoding a novel P-II type metalloproteinase from Bothrops asper venom glands was cloned, sequenced and used for DNA immunization of animals with accelerated DNA-coated tungsten microparticles and the helius Gene Gun system. Specific antibodies against B. asper venom antigens were induced in mice co-immunized with the plasmid encoding the P-II metalloproteinase together with an expression plasmid encoding the murine IL-2. Similarly, specific antibodies against B. asper venom antigens were also induced in a horse co-immunized with the plasmid encoding the P-II metalloproteinase, together with a plasmid encoding the equine IL-6. The equine antibodies induced by immunization with the P-II metalloproteinase encoding plasmid cross react with several proteins of B. asper, Crotalus durissus durissus, and Lachesis stenophrys venoms in western blot, demonstrating antigenic similarity between the cloned metalloproteinase and other metalloproteinases present in these venoms. Furthermore, the equine antibodies induced by immunization with the P-II metalloproteinase encoding plasmid completely neutralized the hemorrhagic activity of the whole B. asper venom and partially the hemorrhagic activity of C. durissus durissus venom. The neutralizing ability of the produced antibodies raises, for the first time, the possibility of developing therapeutic antivenoms in horses by DNA immunization using tungsten microparticles. [Copyright &y& Elsevier]

Abstract: Little cherry, an economically important disease of cherry is caused by at least two different viruses. One of these is Little cherry virus 1 (LChV-1) for the detection of which no efficient serological tools are available, so that diagnosis is based on molecular methods. In this study, different immunization strategies for producing antibodies against the viral coat protein of LChV-1 were tried, using either purified virus preparations, or bacterially expressed protein, or a DNA vector that expressed the cloned coat protein (CP) gene in vivo. Effective induction of specific antibodies to LChV-1 CP was obtained using DNA intramuscular immunization followed by a single boost with the recombinant protein. The entire coat protein sequence was cloned in a mammalian expression vector and, after being coated by an amphiphilic non-toxic reagent was delivered into rabbit. A protein boost increased the specific immune response against the virus protein. The sensitivity of this antiserum is lower if compared with that of antisera raised conventionally against other viruses, thus it requires improvements for use for diagnostic purposes. [Copyright &y& Elsevier]

Abstract: Here we evaluated the effects of immunization with a DNA vaccine encoding a fusion protein consisting of macrophage inflammatory protein-1α (MIP-1α) and MPT51 (a major secreted protein from Mycobacterium tuberculosis) on induction of specific CD8+ T cells. The DNA vaccine encoding the fusion protein could induce significantly higher number of the antigen-specific CD8+ T cells in mice than DNA vaccine encoding MPT51 alone. Also, splenocytes from mice immunized with the fusion DNA vaccine expressed higher level of IFN-γ mRNA and protein upon stimulation with an epitope peptide derived from MPT51 than those from mice immunized with a mixture of two DNA vaccines encoding either MPT51 or MIP-1α. These results suggest that DNA vaccine encoding MIP-1α-antigen fusion protein is able to be efficiently internalized into antigen-presenting cells via the chemokine receptor and induce higher level of antigen-specific CD8+ T-cell responses. [Copyright &y& Elsevier]

Abstract: Cellular immunity is indispensable for efficient protection against intracellular bacterial infection. CD4+ and CD8+ T cells specific for a variety of antigenic peptides derived from particular bacteria are induced after the infection. T cells recognizing different antigenic peptides have been speculated to have different functions in terms of the protective immunity. We here induced individual CD4+ T cells specific for each antigenic peptide derived from Listeria monocytogenes independently with DNA vaccines using gene gun bombardment and compared the CD4+ T-cell populations for their ability on the specific protective immunity against lethal listerial challenge and analyzed their characteristics. [Copyright &y& Elsevier]

Abstract: Zinc-dependent metalloproteinases play a key role in the hemorrhage induced by viperid bite envenoming. In this work we report the cloning and sequencing of the cDNA from a novel P-III type metalloproteinase from Crotalus durissus durissus venom glands. The recombinant plasmid was used for DNA immunization in mice using accelerate DNA-coated microparticles with the Gene Gun system. The results showed that there is no significant difference in the efficiency of the immunization in mice when gold or tungsten microparticles were used. A pool of the sera from mice immunized with the metalloproteinase encoding DNA neutralized the hemorrhagic activity of C. d. durissus venom. The co-immunization with DNA encoding the metalloproteinase and a plasmid encoding the murine IL-2 increased the number of mice which show a specific antibody response towards C. d. durissus venom antigens. The neutralizing ability of the produced antibodies demonstrates that DNA immunization with tungsten microparticles may be used in strategies for antivenom production. [Copyright &y& Elsevier]

Abstract: Introduction: The generation of antibodies against G protein-coupled receptors (GPCRs) can be technically challenging. A modified DNA immunization protocol was employed in order to generate polyclonal antibodies against two herpes virus-encoded GPCRs, i.e. Epstein–Barr virus (EBV) pBILF1 and rat cytomegalovirus (RCMV) pR78. Methods: pBILF1 and pR78 expression plasmids were first injected into the tibialis anterior muscle of rats and rabbits, respectively. Subsequently, the uptake of plasmids by the muscle cells was facilitated through in vivo electroporation. Results: Potent antisera against both vGPCRs were obtained, as determined by immunoblot analysis and immunofluorescence. By using the antisera, we were able to show that the EBV BILF1 protein is expressed as a 45-kD, glycosylated protein, and that it is localized in the cytoplasmic membrane of EBV-infected cells. Interestingly, we found the R78-encoded vGPCRs to have unusual perinuclear localization in both R78-transfected and RCMV-infected cells. Discussion: The in vivo DNA electroporation method is a useful technique for generating antibodies against GPCRs. [Copyright &y& Elsevier]

Abstract: It is believed today that nucleocapsid protein (N) of severe acute respiratory syndrome (SARS)-CoV is one of the most promising antigen candidates for vaccine design. In this study, three fragments [N1 (residues: 1–422); N2 (residues: 1–109); N3 (residues: 110–422)] of N protein of SARS-CoV were expressed in Escherichia coli and analyzed by pooled sera of convalescence phase of SARS patients. Three gene fragments [N1 (1–1269 nt), N2 (1–327 nt) and N3 (328–1269 nt)—expressing the same proteins of N1, N2 and N3, respectively] of SARS-N were cloned into pVAX-1 and used to immunize BALB/c mice by electroporation. Humoral (by enzyme-linked immunosorbent assay, ELISA) and cellular (by cell proliferation and CD4+:CD8+ assay) immunity was detected by using recombinant N1 and N3 specific antigen. Results showed that N1 and N3 fragments of N protein expressed by E. coli were able to react with sera of SARS patients but N2 could not. Specific humoral and cellular immunity in mice could be induced significantly by inoculating SARS-CoV N1 and N3 DNA vaccine. In addition, the immune response levels in N3 were significantly higher for antibody responses (IgG and IgG1 but not IgG2a) and cell proliferation but not in CD4+:CD8+ assay compared to N1 vaccine. The identification of antigenic N protein fragments has implications to provide basic information for the design of DNA vaccine against SARS-CoV. The present results not only suggest that DNA immunization with pVax-N3 could be used as potential DNA vaccination approaches to induce antibody in BALB/c mice, but also illustrates that gene immunization with these SARS DNA vaccines can generate different immune responses. [Copyright &y& Elsevier]

Summary: We transfected a recombinant plasmid that co-expressed swine ubiquitin and a codon optimized GP5 encoding-gene of porcine reproductive and respiratory syndrome virus (PRRSV), designated pCA-U-optiGP5, as well as the plasmid pCA-optiGP5 encoding codon optimized GP5, and the plasmid pCA-GP5 expressing wild-type pGP5 into 293T cells. Expression of GP5 was measured by indirect immunofluorescence (IIF) assay and Western blot and found to be considerably higher in response to pCA-U-optiGP5 than the wild-type vector. GP5 protein was rapidly degraded in pCA-U-optiGP5-transfected 293T cells. The proteasome inhibitor, MG-132, however, successfully inhibited degradation. Immunogenicity of the three constructs was examined by measuring GP5-specific antibody production, lymphocyte proliferation, cytotoxic T lymphocyte (CTL) responses, and cytokine secretion in intramuscularly immunized pigs. Three weeks after the last inoculation, all animals were challenged intranasally with 2-ml 105 TCID50/ml PRRSV CH-1a. DNA immunization with pCA-optiGP5 produced a higher level of GP5-specific antibody than immunization with pCA-GP5, and the humoral response remained undetectable in the pCA-U-optiGP5 group. However, the fusion DNA had a significantly enhanced stimulation index (SI) and induced a stronger Th1 type cellular immune response than the single gene DNA, suggesting that ubiquitin conjugation improved the cellular but not the humoral immune response. Four of six pigs in the pCA-U-optiGP5 group, three of six in the pCA-optiGP5 group, and two of six in the pCA-GP5 group were devoid of visible pathological changes that were present in other vaccinated and control animals after challenge. Viral replication and distribution in the blood and tissues was lower in the pCA-U-optiGP5 vaccinated group than the other groups, suggesting that codon optimization, along with the development of rapidly processed antigen, represents a novel strategy to increase the immune efficacy of DNA vaccines against PRRSV. [Copyright &y& Elsevier]

Abstract: To identify viral proteins that induce cell-mediated cytotoxicity (CMC) against viral hemorrhagic septicemia virus (VHSV)-infected cells, rainbow trout were immunized with DNA vectors encoding the glycoprotein G or the nucleocapsid protein N of VHSV. The G protein was a more potent trigger of cytotoxic cells than the N protein. Peripheral blood leukocytes (PBL) isolated from trout immunized against the G protein killed both VHSV-infected MHC class I matched (RTG-2) and VHSV-infected xenogeneic (EPC) target cells, suggesting the involvement of both cytotoxic T lymphocytes (CTL) and NK cells, respectively. In contrast, PBL from trout that were immunized against the N protein only killed VHSV-infected RTG-2 cells, indicating that this protein only elicits a CTL response. Further, a significant killing capacity of these PBL was only observed during summer months. PBL from fish that were immunized against the VHSV G protein significantly killed VHSV-infected but not infectious hematopoietic necrosis virus (IHNV)-infected targets indicating antigen specificity. Thus, this is the first report on cytotoxic immune responses after DNA vaccination in fish. Furthermore, cells isolated from the inflamed site of DNA injection were stained and transferred to isogeneic DNA-vaccinated recipients. Most of the stained donor leukocytes accumulated at the recipients’ DNA injection site showing, for the first time, leukocyte homing in fish. Transferred donor leukocytes mainly migrated to the homologous vaccine injection site rather than to injection sites of heterologous vaccines, suggesting the antigen specificity of homing. By demonstrating CMC responses to distinct viral proteins and homing in rainbow trout, these results substantially contribute to the understanding of the teleost immune system. [Copyright &y& Elsevier]

Abstract: Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging disease in swine. The development of effective vaccines against PCV2 infection has been accepted as an important strategy in the prophylaxis of PMWS, and a DNA vaccine expressing the major immunogenic capsid (Cap) protein of PCV2 is considered to be a promising candidate. However, recent studies have revealed that interferons (IFNs), especially IFN-γ, can enhance the replication of PCV2, indicating that the high levels of IFN-γ induced by DNA vaccination seem to have potential deleterious effect on protective immunity. Strategies to improve the neutralizing antibody response and simultaneously decrease the IFN-γ response will facilitate the clinical application of DNA vaccines against PCV2. In the present study, four different DNA vaccine constructs encoding cytoplasmic (Cy-ORF2), secreted (Sc-ORF2), membrane-anchored (M-ORF2) or authentic nuclear-targeted (pc-ORF2) Cap protein were generated to evaluate the neutralizing antibody and IFN-γ responses in a mouse model. Although all four DNA constructs could elicit PCV2-specific humoral immune responses, mice inoculated with Sc-ORF2 developed a significantly higher level of neutralizing antibodies than those that received M-ORF2, pc-ORF2 or Cy-ORF2. Furthermore, mice immunized with Sc-ORF2 or M-ORF2 showed a significantly decreased or enhanced IFN-γ level, respectively, compared with those inoculated with pc-ORF2. With respect to neutralizing antibody and IFN-γ levels, Sc-ORF2 is a good candidate for DNA vaccination, and the secreted Cap protein appears to be an ideal antigen for use in development of vaccines against PCV2. [Copyright &y& Elsevier]

Abstract: Understanding the mechanisms of generation and maintenance of immunological memory is crucial for rational vaccine design. A hypothesis known as relay hypothesis was earlier proposed which explains the maintenance of immunological memory through interaction of idiotypic and anti-idiotypic lymphocytes. In the present study, we have shown that immunization with rinderpest virus hemagglutinin protein specific anti-idiotypic antibody (Ab2vβ) DNAs coding for heavy and light chains generates antigen-specific antibody and T cell responses as well as Ab1 specific T cell response. We further show that boosting with the recombinant Ab2-vβ proteins generates B and T cell memory response specific for antigen in anti-id DNA primed mice. This study provides experimental evidence for perpetuation of immunological memory through idiotypic network interactions. [Copyright &y& Elsevier]

Abstract: Human cytomegalovirus (HCMV) glycoprotein complex II (gcII) consists of two glycoproteins, gM and gN. Although gcII specific IgG purified from HCMV positive patient sera can neutralize HCMV, there has been no report describing the generation of virus-neutralizing antibodies by immunization with individual recombinant gM or gN antigens. In the current study, gM and gN antigens were expressed by the mammalian expression vector pJW4303 and used as DNA vaccines to determine the immunogenicity of these proteins. Sera from mice or rabbits immunized with individual or combinations of gM and gN DNA vaccines contained gM and gN specific antibodies as confirmed by ELISA and Western blot analyses. The combined gM and gN antigens induced the strongest antibody responses that recognized both gM and gcII complex while gM DNA vaccine alone could only elicit antibody specific for gM antigen. When given alone, the gN DNA vaccine did not induce detectable gcII specific antibody even though in vitro gN expression was confirmed by the formation of gM/gN complex in FSK cells using a gN-specific monoclonal antibody 14-16A. The neutralizing antibody titer of anti-gM/gN sera (1:128) was higher than that of anti-gM sera (1:32) against the autologous virus, HCMV AD169. Heterologous HCMV strains including Towne and Davis could also be neutralized by the anti-gM/gN antisera. Our data supported the rationale for the use of the HCMV gM/gN protein complex as protective antigens for subunit based HCMV vaccine development. DNA vaccination is an effective approach to express the gM/gN antigen complex in vivo without the need to express and purify these highly insoluble and structurally complicated antigens. [Copyright &y& Elsevier]

Abstract: Plasmid pBMC2 encoding antigen Bm86 from a Colombian strain of cattle tick Boophilus microplus, was used for DNA-mediated immunization of BALB/c mice, employing doses of 10 and 50μg, delivered by intradermic and intramuscular routes. Anti-Bm86 antibody levels were significantly higher compared to control mice treated with PBS. In the evaluation of immunoglobulin isotypes, significant levels of IgG2a and IgG2b were observed in mice immunized with 50μg of pBMC2. Measurement of interleukine (IL) levels (IL-4, IL-5, IL-12(p40)) and interferon-γ (IFN-γ) in the sera of mice immunized with pBMC2 indicated high levels of IL-4 and IL-5, although there were also significant levels of IFN-γ. Mice immunized with pBMC2 showed antigen-specific stimulation of splenocytes according to the incorporation of bromodeoxyuridine and IFN-γ secretion. In all trials, mice injected intramuscularly with 50μg of pBMC2 presented the highest immune response. Moreover, cattle immunized with this DNA vaccine showed antibody production significantly different to the negative control. In conclusion, these results suggest the potential of DNA immunization with pBMC2 to induce humoral and cellular immune responses against B. microplus. [Copyright &y& Elsevier]

Abstract: Since carcinoembryonic antigen (CEA) is expressed during embryonic life, it is not immunogenic in humans. The use of anti-idiotypic (Id) antibodies as a surrogate of antigen in the immunization has been considered a promising strategy for breaking tolerance to some tumor associated antigens. We have described an anti-Id monoclonal antibody (MAb), designated 6.C4, which is able to mimic CEA functionally. The anti-Id MAb 6.C4 was shown to elicit antibodies that recognized CEA in vitro and in vivo. In the present study, we sought to verify whether a single chain (scFv) antibody obtained, the scFv 6.C4, would retain the ability to mimic CEA. Two scFv containing the variable heavy and light chain domains of 6.C4 were constructed with a 15-amino acid linker: one with and another without signal peptide. DNA immunization of mice with both forms of scFv individually elicited antibodies able to recognize CEA. [Copyright &y& Elsevier]

Abstract: To exploit Lactobacillus acidophilus as a carrier in DNA immunization against foot-and-mouth disease virus (FMDV), a recombinant eukaryotic expression plasmid (pRc/CMV2-VP1-Rep. 8014) harboring pRc/CMV2 vector, the FMDV VP1 gene, and a replication origin from Lactobacillus plantarum ATCC 8014 strain was constructed. To detect the VP1 protein, pRc/CMV2-VP1-Rep. 8014 was expressed in PK 15 cells and transfected into a L. acidophilus SW1 strain (L. acidophilus SFMD-1). To evaluate the immunization effect of L. acidophilus SFMD-1, anti-FMDV VP1 antibody, T-cell proliferation, antigen-specific delayed-type hypersensitivity (DTH), and tissue distribution were investigated using intramuscular, intraperitoneal, intranasal, and oral administration delivery routes. The results showed that L. acidophilus SFMD-1 was able to elicit a detectable antibody level on day 21. The VP1 antibody levels induced by L. acidophilus SFMD-1 and commercial inactivated FMDV vaccine rose rapidly to 0.84 and 0.70, respectively, by day 42, then sustained a high level by day 49. The route of administration had an impact on the magnitude of the systemic antigen-specific IgG responses, with intramuscularly applied L. acidophilus SFMD-1 generating the greatest FMDV VP1 antibody response, followed by intraperitoneal, intranasal, and oral administration delivery routes. Using the T-cell proliferation assay, the stimulation index of a group immunized with L. acidophilus SFMD-1 reached 2.78 versus 5.08 in a group immunized with pRc/CMV2-VP1-Rep. 8014 plasmid. Mice immunized with L. acidophilus SFMD-1 were able to induce T-cell-mediated antigen-specific DTH. In addition, the VP1 gene was detected in the muscle, kidney, spleen, and heart, but not in the liver. The results demonstrate clearly that Lactobacillus as a carrier is a promising approach of DNA vaccination, and is a potentially guard against FMDV. [Copyright &y& Elsevier]

Abstract: Rabbits were intramuscularly immunized with the plasmid pCR-X8-HBc-CETP encoding a B-cell epitope of cholesteryl ester transfer protein (CETP) C-terminal fragment (CETPC) displayed by Hepatitis B virus core (HBc) particle. This plasmid also contained immunostimulatory sequences (ISS) which included eight CpG motifs 5′-GACGTT-3′, functioning as immunomodulators. After anti-CETP antibodies were successfully produced, rabbits were fed with a high-cholesterol diet for 15 weeks, and then the antiatherogenic effects of this DNA immunization were evaluated. The results showed that the fraction of plasma cholesterol in HDL significantly increased and the fraction of plasma cholesterol in LDL decreased in the pCR-X8-HBc-CETP immunized rabbits compared with those in the saline control group and one group treated with the plasmid pCR-X8-HBc containing ISS but lacking CETP epitope. More importantly, DNA immunization with pCR-X8-HBc-CETP markedly reduced the average percentage of aortic lesions in the entire aorta area by 80.60% compared with the saline control (3.78% versus 19.48%) and the average thickness of hyperplastic coronary artery in this group was also significantly less than in the saline control group (146±11μm versus 248±18μm). Our data also showed that CpG DNA alone could be antiatherogenic in this model because the average percentage of aortic lesions in pCR-X8-HBc immunized rabbits was 16.53% lower than that of the saline control group and the average thickness of hyperplastic coronary artery was also substantially lower than saline control group (155±13μm versus 248±18μm). Thus, plasmid pCR-X8-HBc-CETP could significantly inhibit the progression of atherosclerosis and be potentially developed as a suitable DNA vaccine against atherosclerosis. [Copyright &y& Elsevier]

Abstract: We evaluated here the effect of immunization with a gene encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) inserted with a helper T cell (Th) epitope, listeriolysin O (LLO) 215–226 derived from Listeria monocytogenes on induction of a specific Th by gene gun bombardment. Immunization of C3H/He mice with pGM215m plasmid encoding murine GM-CSF inserted with LLO 215–226 Th epitope gave the epitope-specific proliferative responses of CD4+ T lymphocytes. In addition, specific interferon-γ production from the splenocytes was observed. Concomitantly, pGM215m-immunized mice showed significant protective immunity against lethal listerial challenge. These results suggest that immunization of a gene for GM-CSF inserted with a Th epitope is useful for eliciting a specific Th subset in vivo. [Copyright &y& Elsevier]

Abstract: Envelope protein E2 of human hepatitis C virus (HCV) is an attractive component of a prototype HCV vaccine. Delivered by DNA immunogens, E2 evokes specific immune response of Th1-type, failing to induce either considerable antibody production, or T-helper cell proliferation. We aimed at modulating the immunogenic performance of E2 gene by changing the mode of protein expression in eukaryotic cells. Plasmids were constructed encoding full-length E2 and nonstructural protein 1 (p7) fused to either 13 or 38 C-terminal amino acids (aa) of HCV E1 that contain second hydrophobic segment of E1 stop-transfer signal, or a complete E1 stop-transfer signal with duplicated second hydrophobic segment. Injected into BALB/c mice, E2/p7 genes induced potent antibody and T-helper cell response targeted against hypervariable region 1, aa 472–586 of E2, and a novel epitope at aa 774–796 of p7. Profile of cytokines secreted by proliferating mouse splenocytes stimulated in vitro with E2- and p7-derived peptides, indicated mixed Th1/Th2 type of immune response. Thus, the full-length E2 and p7 genes supplied in one cassette were both immunogenic. E2/p7 containing a complete E1 stop-transfer signal with prolonged membrane spanning domain was superior to the shorter E2/p7 version in terms of both antibody and cellular immunogenicity. Optimal performance of HCV E2 could thus be achieved without the aid of external/heterologous signals by easing, through modification of the E2 signal sequence, the release of E2 from the rough ER while retaining full-length E2 and p7 sequences. This finding may help to improve the Th2 performance of HCV envelope genes as prototype vaccines. [Copyright &y& Elsevier]

Abstract: Despite foot-and-mouth disease virus (FMDV) being responsible for one of the most devastating animal diseases, little is known about the cellular immune mechanisms involved in protection against this virus. In this work we have studied the potential of DNA vaccines based on viral minigenes corresponding to three major B and T-cell FMDV epitopes (isolate C-S8c1) originally identified in natural hosts. The BTT epitopes [VP1 (133–156)-3A (11–40)-VP4 (20–34)] were cloned into the plasmid pCMV, either alone or fused to ubiquitin, the lysosomal targeting signal from LIMPII, a soluble version of CTLA4 or a signal peptide from the human prion protein, to analyze the effect of processing through different antigenic presentation pathways on the immunogenicity of the FMDV epitopes. As a first step in the analysis of modulation exerted by these target signals, a FMDV infection inhibition assay in Swiss outbred mice was developed and used to analyze the protection conferred by the different BTT-expressing plasmids. Only one of the 37 mice immunized with minigene-bearing plasmids developed specific neutralizing antibodies prior to FMDV challenge. As expected, this single mouse that had been immunized with the BTT tandem epitopes fused to a signal peptide (pCMV-spBTT) was protected against FMDV infection. Interestingly, nine more of the animals immunized with BTT-expressing plasmids did not show viremia at 48h post-infection (pi), even in the absence of anti-FMDV antibodies prior to challenge. The highest protection (50%, six out of 12 mice) was observed with the plasmid expressing BTT alone, indicating that the targeting strategies used did not result in an improvement of the protection conferred by BTT epitopes. Interestingly, peptide specific CD4+ T-cells were detected for some of the BTT-protected mice. Thus, a DNA vaccine based on single FMDV B and T cell epitopes can protect mice, in the absence of specific antibodies at the time of challenge. Further work must be done to elucidate the mechanisms involved in protection and to determine the protective potential of these vaccines in natural FMDV hosts. [Copyright &y& Elsevier]

Abstract: Nucleocapsid protein plays a critical role in SARS-CoV pathogenesis, and high-level anti-nucleocapsid antibodies are detected in the patients infected by severe acute respiratory syndrome-associated coronavirus (SARS-CoV). Several studies have shown that there exists an interaction between nucleocapsid (N) and membrane (M) protein. In this paper, we investigate whether the expression of membrane protein can affect the immune responses induced by nucleocapsid DNA immunization. Two recombinant plasmids containing M and N coding sequence were constructed. Moreover, in order to get the antigen for ELISA and in vitro stimulation assay, N protein were expressed and purified from E. coli bacteria. Injection of 20μg of the mixture of pVAX1-M and pVAX1-N into the Balb/c mice could elicit the humoral and cellular responses. The ELISA analysis using the N antigen or inactivated SARS-CoV particles as capture antigen showed that co-injection of SARS-M could enhance N-induced antibody production, especially IgG2a subclass. After lymphocytes were stimulated with 10μg/ml purified N antigen, The CD4+ and CD8+ T cells of N and M plus N group were increased compared with those of control groups, and the M protein could augment the activation of lymphocytes induced by N DNA vaccine. Cytokine ELISA analysis revealed that co-injection of M could enhance the levels of IFN-γ, IL-2 release induced by N antigen. Further experiments in field mouse also support the claim that membrane protein can augment the N-specific immune responses. Virus challenge test was conducted in BSL3 bio safety laboratory with Brandt''s vole SARS-CoV model, and the results indicated that co-immunization of M and N antigens could reduce the mortality and pathological changes in lung from the virus infection. [Copyright &y& Elsevier]

Abstract: Infections by human papillomaviruses (HPV) are the major cause of uterine cancer in women worldwide. Aiming to develop a combined prophylactic and therapeutic vaccine we have previously demonstrated immunogenicity of chimeric virus-like particles consisting of a C-terminally truncated HPV 16 L1 capsid protein fused to an E7 portion. Here we show that genetic vaccination with a corresponding DNA was inefficient in the induction of a L1-specific prophylactic immune response. DNA immunization with C-terminally truncated HPV 16 L1 genes of different lengths revealed that only short deletions (L11–498) were tolerated for eliciting a humoral immune response against viral capsids. This correlates with the observation that the C-terminal sequences are critical for nuclear localization, capsomere and capsid assembly. However, only the ability of L1 protein to form capsomeres or capsids showed a direct influence on the outcome of the immune response. C-terminal insertion of 60 amino acids of E7 was tolerated in fusion constructs, whereas insertion of full-length E71–98 or shuffled E7 (149 aa) completely abolished the humoral immune response. The L11–498/E71–60 fusion construct not only induced L1-specific antibodies but also L1- and E7-specific CTL responses after DNA vaccination. [Copyright &y& Elsevier]

Abstract: Survivin, a 16.5kDa tumor associated antigen, is the smallest member of the inhibitor of apoptosis family that is abundantly expressed during development but essentially absent in normal adult tissues. Interestingly, survivin expression is up-regulated in virtually all types of cancers studied, as well as in vascular endothelial cells during tumor associated angiogenesis. Survivin links apoptosis to cell cycle progression and plays a pivotal role in regulation of cell proliferation. These characteristics make survivin a potentially promising generic target for cancer immunotherapy. Hence, a genetic immunization strategy to induce tumor-specific immune responses against human survivin in a pre-clinical animal model was developed. In initial studies, BALB/c mice were immunized by intramuscular injection with DNA coding for human survivin (pcDNA3.1/hSurv). In addition, a construct encoding a secreted version of survivin (pSecTag2B/hSurv) was designed. A plasmid coding for murine granulocyte-macrophage colony-stimulating factor (GM-CSF) was co-injected in both cases as a molecular adjuvant. Expression of survivin following transfection in mouse cells was corroborated. Humoral responses against human survivin were detected in mice sera using two immunization protocols (injections at 2- or 3-week intervals). The humoral response was markedly improved by secretion of survivin and co-expression of GM-CSF. The predominant antibody subclass detected in responsive mice was IgG2a, suggesting that a Th1-CD4+ cellular response had been induced. Furthermore, DNA immunization with survivin encoding vectors generated an effective CD8+ T cell response measured as an increase of cytotoxic Interferon-gamma (IFN-γ) secreting CD8+ T cells. In conclusion, intramuscular genetic immunization of mice with human survivin encoding plasmids induced a survivin-specific humoral as well as cellular immune response in recipient mice. Secretion of survivin and co-injection of GM-CSF as a genetic adjuvant appear to be more important in generating an humoral than a cellular immune response. [Copyright &y& Elsevier]

Abstract: In the search for Onchocerca volvulus antigens possibly involved in protection against human onchocerciasis, partial amino acid sequence analysis of one of the O. volvulus antigens of the serologically identified proteins showed a close relationship to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein family. Subsequent adult worm cDNA library screening and cloning produced a clone of 1650 bp. An open reading frame spans over 1020 bp encoding for a protein of 340 amino acids with an apparent molecular weight of 38000. Comparison of the complete amino acid sequence identified this protein as a member of the GAPDH protein family. The recombinantly expressed protein shows GAPDH enzymatic activity as well as plasminogen-binding capacity. DNA sequence analysis of the corresponding gene revealed the presence of two introns. Using immunohistology Ov-GAPDH was observed in microfilariae, infective larvae, and adult male and female worms. Most striking was the labelling of the musculature of the body wall. Labelling was also observed in the pseudocoeloma cavity and in a subset of cell nuclei, suggesting additional, non-glycolytic functions of the Ov-GAPDH. Gene gun immunization with the DNA-construct in cattle led to specific humoral immune responses. Thus, the protective potential of the DNA-construct of Ov-GAPDH can be evaluated in vaccination trials using animal models such as the cattle/Onchocerca ochengi model. [Copyright &y& Elsevier]

Abstract: Immunization of domestic pigs with a DNA vaccine expressing the complete E2 protein of classical swine fever virus (CSFV) conferred total protection against a severe viral challenge. Immunization with three doses of plasmid pcDNA3.1/E2 elicited a consistent and specific, MHC class II restricted T cell response in the three domestic pigs analyzed, in the absence of detectable anti-CSFV antibodies in serum. Upon challenge specific T cell responses were boosted in the three vaccinated pigs, and a rapid rise in the titers of CSFV neutralizing antibodies was noticed in two of them, which correlated with a total protection. In these two pigs, neither disease symptoms were observed nor was virus detected at any time after CSFV infection. Neutralizing antibody titers were lower in the third vaccine, which developed a mild and transient peak of pyrexia. As expected, similar analyses in three control pigs (injected with the empty vector or PBS) did not reveal the induction of specific T cells or viral antibodies and, upon challenge, animals developed severe symptoms of the disease, including high titers of viremia, hyperthermia and virus spread to different organs. Control pigs developed, also, a marked leucopenia, resulting in SWC3+ (myelomonocytic cells) being the major PBMC population, and a drastic decrease CD3+ T cells. This T cell depletion was prevented in animals immunized with pcDNA3.1/E2. The total protection achieved, in the absence of CSFV antibodies before challenge, supports the relevance in the antiviral response observed of specific T cell responses primed by pcDNA3.1/E2 vaccine, which, upon challenge, led to a rapid induction of neutralizing antibodies. The observation that CSFV antibodies could only be detected in protected animals after viral challenge opens the possibility of exploring the potential of the DNA vaccine approach used to develop marker vaccines against CSF. [Copyright &y& Elsevier]

Abstract: An Eimeria tenella microneme recombinant gene (EtMIC2) and encoded protein were evaluated as potential vaccines against avian coccidiosis. In ovo inoculation with the EtMIC2 gene increased anti-EtMIC2 antibody titers at days 10 and 17 following E. tenella infection. In addition, vaccinated birds developed protective immunity against infection by E. tenella as assessed by significantly increased body weight gain and decreased fecal oocyst shedding compared with non-vaccinated controls. Vaccination with the EtMIC2 gene also led to protective immunity against infection by E. acervulina, but not E. maxima. Combined in ovo DNA vaccination plus post-hatch boosting with EtMIC2 DNA or protein did not improve antibody titers or protective immunity beyond that achieved with in ovo vaccination alone. These results provide evidence that in ovo immunization with a recombinant Eimeria microneme gene stimulates protective intestinal immunity against coccidiosis. [Copyright &y& Elsevier]

Abstract: Dengue fever is a growing public health concern around the world and despite vaccine development efforts, there are currently no effective dengue vaccines. In the present study we report the induction of protective antibodies against dengue virus by DNA immunization with domain III (DIII) region of the envelope protein (E) in a mouse model. The DIII region of all four dengue virus serotypes were cloned separately into pcDNA 3 plasmid. Protein expression was tested in COS-7 cells. Each plasmid, or a tetravalent combination, were used to immunize BALB/c mice by intramuscular route. Presence of specific antibodies was evaluated by ELISA, and neutralizing antibodies were tested using a cytopathogenic effect (CPE) inhibition assay in BHK-21 cells, as well as in newborn mice challenged intracranially with dengue 2 virus. Mice immunized with individual DIII constructs or the tetravalent formulation developed antibodies against each corresponding dengue serotype. Antibody titers by ELISA were similar for all serotypes and no significant differences were observed when boosters were administered, although antibody responses were dose-dependent. CPE inhibition assays using Den-2 virus showed neutralization titers of 1:10 in mice immunized with individual DIII plasmid or those immunized with the tetravalent formulations. 43% of newborn mice challenged with Den-2 in combination with sera from mice immunized with Den-2 DIII plasmid were protected, whereas sera from mice immunized with the tetravalent formulation conferred 87% protection. Our results suggest that DIII can be used as a tetravalent DNA formulation to induce neutralizing and protective antibodies against dengue virus. [Copyright &y& Elsevier]

Abstract: A DNA vaccine was tested in infant Rhesus macaques to evaluate its safety, immunogenicity and protective efficacy. Monkeys were vaccinated and challenged with a clinical isolate of human RSV. Vaccinated animals developed humoral and cellular responses following inoculation with plasmid DNA encoding the fusion (F) and nucleoprotein (N), from closely related bovine RSV. Vaccinated monkeys had decreased RSV in their lungs post-infection, and there was a qualitative difference in histopathology observed between vaccinated and unvaccinated animals. The combined result of safety and immunogenicity in a neonatal primate model is encouraging, suggesting the feasibility of DNA vaccines against RSV in infants. [Copyright &y& Elsevier]

Abstract: Although DNA vaccines have several advantages over conventional vaccines, antibody production and protection are often not adequate, particularly in single plasmid vaccine formulations. Here we assessed the potential for a combined vaccine based on plasmids encoding the membrane-anchored or secreted forms of bovine herpesvirus type 1 (BHV-1) glycoprotein B and D (gB and gD) to induce neutralizing and cell mediated immune responses in mice. Animals were injected by intramuscular, subcutaneous and intranasal routes. Mice immunized with the combined vaccine containing the secreted forms of BHV-1 glycoproteins developed higher titers of anti-BHV-1 neutralizing antibodies, compared to wild type gB/gD combined plasmids and to single plasmid injected groups. Cellular immunity was also developed in mice immunized with combined vaccines, whereas low or no response were observed in single plasmid injected animals. The data suggest the potential use of this combined vaccine in in vivo trials of calves, in order to evaluate its protective efficacy. [Copyright &y& Elsevier]

Abstract: The cytochrome P4507B1 (P4507B1) is responsible for the 7α-hydroxylation of dehydroepiandrosterone (DHEA) and other 3β-hydroxysteroids in the brain and other organs. The cDNA of human P4507B1 was used for DNA immunization of mice. The best responding mouse led to the production of monoclonal antibodies (mAbs). The clone D16-37 produced an IgM specific for P4507B1 with no cross-reaction with other human P450s. This antibody permitted the immunohistochemical detection of P4507B1 in slices of human hippocampus. P4507B1 was expressed in neurons only. This new tool will be used for the extensive examination of the P4507B1 presence and determination of its levels in slices of human normal and diseased brain and in other human tissues. [Copyright &y& Elsevier]

We have investigated the protective effect of immunization of a highly susceptible natural host of canine distemper virus (CDV) with DNA plasmids encoding the viral nucleoprotein (N) and hemagglutinin (H). The combined intradermal and intramuscular routes of immunization elicited high virus-neutralizing serum antibody titres in mink (Mustela vison). To mimic natural exposure, we also conducted challenge infection by horizontal transmission from infected contact animals. Other groups received a lethal challenge infection by administration to the mucosae of the respiratory tract and into the muscle.One of the mink vaccinated with N plasmid alone developed severe disease after challenge. In contrast, vaccination with the H plasmid together with the N plasmid conferred solid protection against disease and we were unable to detect CDV infection in PBMCs or in different tissues after challenge. Our findings show that DNA immunization by the combined intradermal and intramuscular routes can confer solid protective immunity against naturally transmitted morbillivirus infection and disease. [Copyright &y& Elsevier]

Human chorionic gonadotropin has been used as an anti-fertility vaccine and as a target for cancer immunotherapy. We have explored the use of DNA immunization with the aim of improving the immunogenicity of this hormone. Stimulating the muscle with electric pulses following intramuscular injection of plasmids expressing hCGβ resulted in higher levels of human chorionic gonadotropin (hCG)-specific antibodies, which could be further enhanced following a protein boost with hCG mixed with adjuvant. DNA vaccines encoding a membrane attached or a secreted form of hCGβ produced similar—albeit relatively modest—antibody responses. Providing hCGβ with additional T cell help by vaccinating with a plasmid encoding a hCGβ-hFc fusion protein did not further increase the antibody levels in the immunized animals. However, immunization of mice with a construct encoding hCGβ fused to C3d3 produced significantly lower antibody levels relative to mice immunized with the hCGβ-alone expression plasmid, even though the hCGβ-C3d3 chimera was expected to facilitate cross-linking of the antigen-specific B-cell receptor and CR2 thereby lowering the threshold of activation. Thus the limiting factor determining the antibody levels following hCGβ immunization, at least for DNA immunization, is related to the amount of protein available rather than the form of protein produced or lack of T cell epitopes. [Copyright &y& Elsevier]

It is well known that newborns and infants respond poorly to immunization with influenza virus vaccines. The poor response of neonates may be related to restricted B cell repertoire and high susceptibility of neonates to high dose tolerance. Protective antibody response against hemagglutinin (HA) of influenza virus is a T-dependent response. While the immunization of neonates with live virus caused a long lasting unresponsiveness, the immunization with a plasmid containing influenza virus HA circumvents the neonatal unresponsiveness. Genetic immunization primes efficiently neonatal HA-specific B cells, and induces memory cell enabling the animals to develop a strong secondary response and to survive to challenge with a lethal dose. The most striking effect of neonatal immunization consists of a shift of neonatal HA-specific B cell repertoire to adult-type as assessed by analysis of reactivity pattern of HA-specific clonotypes. The diversification is associated with the induction of germinal centers, increased number of B220+GL-7+ cells and with the re-expression of RAG genes. This suggests that the receptor revision may contribute to the diversification of HA-specific neonatal B repertoire. [Copyright &y& Elsevier]

The fear of autoimmunity in DNA-vaccine recipients initiated screening for anti-DNA antibodies in rabbits immunized with genes of viral nucleic acid-binding and adapter proteins. Of 11 DNA/protein-immunized rabbits, seven had developed secondary antibodies against DNA detected at weeks 11–50 from the on-start of immunization. Two rabbits immunized with HIV-1 reverse transcriptase gene developed transient anti-double-stranded DNA antibodies of high avidity that recognized DNA in the kinetoplasts of Crithidia luciliae. Others developed antibodies reacting with DNA in ELISA and targeting nuclear-associated antigens in the immunofluoresence test. No anti-DNA antibodies were detected at these time-points in any of the controls (P=0.036). Induction of anti-DNA antibodies by epitope spreading from protein domains involved in nucleic acid-binding versus maturation of anti-protein antibodies to dual protein-DNA specificity is discussed. (126 words). [Copyright &y& Elsevier]

Patients with chronic Chagas’ heart disease (cChHD) develop a strong IgG response against the C-terminal region of the Trypanosoma cruzi ribosomal P2β protein (TcP2β). These antibodies have been shown to exert an in vitro chronotropic effect on cardiocytes through stimulation of the β1-adrenergic receptor (β1-AR). Moreover, the presence of antibodies recognizing the TcP2β C-terminus was associated with cardiac alterations in mice immunized with the corresponding recombinant protein. Here, we demonstrate that DNA immunization could be used to modulate the specificity of the anti-TcP2β humoral response in order to avoid the production of pathogenic antibodies. After DNA injection, we detected IgG antibodies that were directed only to internal epitopes of the TcP2β molecule and that did not exert anti-β1-AR functional activity, measured as an increase in intracellular cAMP levels of transfected COS-7 cells. Accordingly, DNA-immunized mice did not present electrocardiographic alterations. These data demonstrate that anti-TcP2β antibodies elicited by DNA immunization are completely different in their specificity and functional activity from those produced during T. cruzi infection. [Copyright &y& Elsevier]

Objective: To express the hCGβ-C3d3 fusion protein in a CHO cell continual expression system to investigate further the adjuvant effects of C3d on contraceptive vaccination. Method: We constructed a plasmid pcDNA3-hCGβ-C3d3 which contains three copies of murine C3d cDNA and the hCGβ gene by cloning the chimerical hCGβ-C3d3 cDNA into the eukaryotic vector pcDNA3 downstream of the CMV promoter. The plasmid was transfected into a COS-7 cell transient expression system and a CHO cell continual expression system. RIA was used to detect hCGβ in the culture supernatant. Western blot and Raji cell immunohistochemical assays were performed to evaluate the expressed protein. Then, 6–8-week-old female BALB/c mice were inoculated intramuscularly with pcDNA3-hCGβ and pcDNA3-hCGβ-C3d3, and ELISA was used to assess anti-hCGβ IgG antibody in serum. Results: In 72 h after COS-7 cells were transfected with the plasmid pcDNA3-hCGβ-C3d3, 1.0×105 cells could secrete 152 ng of the recombinant protein (calculated by hCGβ contained). The transfected CHO cells, which were then screened by G418, could continuously secrete the fusion protein at 660 ng/106 cells/48 h. The hCGβ-C3d3 protein was purified by anti-hCGβ immunoaffinity chromatography. Raji cell immunohistochemical assay demonstrated that both the hCGβ and C3d3 were successfully fused. After DNA immunization intramuscularly, the anti-hCGβ IgG antibody titer in the pcDNA3-hCGβ-C3d3 immunized group was 243-fold higher than that of the pcDNA3-hCGβ immunized group. Conclusion: We have expressed the hCGβ-C3d3 protein successfully, both in a transient expression system (COS-7 cells) and in a stable expression system (CHO cells). The C3d3 molecular adjuvant can enhance significantly the immunogenecity of hCGβ antigen in DNA immunization. [Copyright &y& Elsevier]

In this work, we have investigated the immune response in pigs to two recombinant plasmids containing immunodominant neutralizing antibody epitopes of foot-and-mouth disease virus structural protein (VP1) coexpressed with viral non-structural proteins as a source of T cell epitopes. The plasmid pcDNA3.1/3D15 contained a sequence coding for the 3D polymerase upstream of a sequence coding for peptide FMDV15, a peptide derived from VP1, previously shown to stimulate protective immunity to foot-and-mouth disease virus (FMDV), that consisted of the carboxy terminal peptide [VP1200-213] linked by ProProSer to the “loop” peptide [VP1143-160] and terminating in CysGly. The plasmid, pcDNA3.1/2B15 contained a sequence coding for the non-structural protein 2B, and the same FMDV15 peptide sequence. Pigs injected with both constructs showed antibody and T cell responses to 3D and 2B, but not to the FMDV15 peptide. Additionally, delayed type hypersensitivity responses were observed in some cases to both 3D or 2B and to FMDV virus. Finally, no protection was seen against FMDV infection in animals immunized with either of the two FMDV DNA constructs. The additional co-immunization of plasmids encoding for GMCSF did not result in any significant change in the immune responses to the plasmids encoding for FMDV. This work gives some optimism for the construction of a DNA vaccine for FMDV in the future. [Copyright &y& Elsevier]