Monoclonal Antisera Research Articles

Adenylate cyclase influences filamentous haemagglutinin-mediated attachment ofBordetella pertussisto epithelial alveolar cells

Attachment to epithelial cells in the respiratory tract is a key event in Bordetella pertussis colonization. Filamentous haemagglutinin (FHA) is an important virulence factor mediating adhesion to host cells. In this study, the relevance of the interaction between FHA and adenylate cyclase toxin (ACT) during bacterial attachment was investigated. Mutants lacking either FHA or ACT showed significantly decreased adherence to epithelial respiratory cells. The use of several ACT-specific monoclonal antibodies and antiserum showed that the decrease in attachment of strains lacking ACT expression could not be explained by the adhesin-like activity of ACT, or a change of any of the biological activities of ACT. Immunoblot analysis showed that the lack of ACT expression did not interfere with FHA localization. An heparin-inhibitable carbohydrate-binding site is crucial in the process of FHA-mediated bacterial binding to epithelial cells. In the presence of heparin attachment of wild-type B. pertussis, but not of the isogenic ACT defective mutant, to epithelial cells was significantly decreased. These results suggest that ACT enhances the adhesive functions of FHA, and modifies the performance of the FHA heparin-inhibitable carbohydrate binding site. We propose that the presence of ACT in the outer membrane of B. pertussis to play a role in the functionality of FHA.

Prohormone Convertase 1/3 Is Essential for Processing of the Glucose-dependent Insulinotropic Polypeptide Precursor

The physiology of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and their role in type 2 diabetes currently attract great interest. Recently we reported an essential role for prohormone convertase (PC) 1/3 in the cleavage of intestinal proglucagon, resulting in formation of GLP-1, as demonstrated in PC1/3-deficient mice. However, little is known about the endoproteolytic processing of the GIP precursor. This study investigates the processing of proGIP in PC1/3 and PC2 null mice and in cell lines using adenovirus-mediated overexpression. Supporting a role for PC1/3 in proGIP processing, we found co-localization of GIP and PC1/3 but not PC2 in intestinal sections by immunohistochemistry, and analysis of intestinal extracts from PC1/3-deficient animals demonstrated severely impaired processing to GIP, whereas processing to GIP was unaltered in PC2-deficient mice. Accordingly, overexpression of preproGIP in the neuroendocrine AtT-20 cell line that expresses high levels of endogenous PC1/3 and negligible levels of PC2 resulted in production of GIP. Similar results were obtained after co-expression of preproGIP and PC1/3 in GH4 cells that express no PC2 and only low levels of PC1/3. In addition, studies in GH4 cells and the alpha-TC1.9 cell line, expressing PC2 but not PC1/3, indicate that PC2 can mediate processing to GIP but also to other fragments not found in intestinal extracts. Taken together, our data indicate that PC1/3 is essential and sufficient for the production of the intestinal incretin hormone GIP, whereas PC2, although capable of cleaving proGIP, does not participate in intestinal proGIP processing and is not found in intestinal GIP-expressing cells.

Beacon immunoreactivity in the rat hypothalamus

Beacon (BC) is a peptide of 73 amino acids, whose gene expression was first reported in the hypothalamus of Psammomys obesus (or Israeli sand rat). To appreciate better the functional role of BC in normal rats and sand rats, the distribution of BC immunoreactivity (irBC) and its subcellular localization were studied in the brain of Sprague-Dawley rats. In the hypothalamus, intense staining was present in neurons of the supraoptic (SO), paraventricular (PVH), and accessory neurosecretory nuclei and in cell processes of median eminence. Double labeling of the hypothalamic sections with mouse monoclonal oxytocin (OT) antibody and rabbit polyclonal BC antiserum revealed that nearly all OT-immunoreactive cells from SO, PVH, and accessory neurosecretory nuclei were irBC. Double labeling of the sections with guinea pig vasopressin (VP) antiserum and BC antiserum showed that a population of VP-immunoreactive neurons was irBC. By immunoelectron microscopy, immunoreactive product was associated with mitochondrial membranes or appeared as electron-dense bodies in many PVH and SO neurons. Most of the neurosecretory granules were unstained for BC. Taken together, our results indicate the presence of beacon in the OT-containing neurons and a population of VP-containing neurons, mostly associated with mitochondrial membrane. Insofar as the amino acids sequence of beacon is identical to that of ubiquitin-like 5, it is possible that the distribution of BC immunoreactivity noted in our study is that of ubiquitin-like 5 peptide in the rat hypothalamus.

Construction of Antibodies for Detection and Diagnosis of Cucumber green mottle mosaic virus from Watermelon Plants

We immunized BALB/c mice with purified Cucumber green mottle mosaic virus isolate HY1 (CGMMV-HY1). Through the selection of positive clones that were grown on the HAT medium, four sensitive monoclonal clones (CG99-01, CG99-02, CG99-03, and CG99-04) were selected from 500 Hypoxanthine-guanine phosphoribosyltransferase positive hybridoma cells. Four sensitive clones of CGMMV-HYI were determined as IgM type of the subclass of mouse immunoglobulins Ig group. The titer of monoclonal antiserum against CGMMVHY1 was estimated 1:12,800 by the indirect ELISA. Although monoclonal antibodies (MAbs) from CG99-01 and from CG99-04 cross-reacted with Zucchini green mottle mosaic virus and Kyuri green mottle mosaic virus, MAb from the cell line CG99-03 was highly specific to CGMMV. No MAbs cross-reacted with Cucumber mosaic virus-Fny. Only CG99-04 reacted with Pepper mild mottle virus weakly and CG99-02 reacted with both CGMMV and KGMMV. CGMMV was detected from the rind of watermelon fruit by DAS-ELISA of CGMMV-HY1, but not from the flesh of watermelon. Average seed transmission rate of CGMMV in watermelon was from symptomatic watermelon collected from 5 regions of Gyeongnam province. CGMMV was detected by DAS-ELISA with specific MAb of CGMMVHY1 periodically from root stock, during the sequential process for nursery seedling in Haman. Necrotic spots on cotyledons of root stock seedling progressed to reveal the typical symptomatology on the primary leaves of scion upon grafting. Here, we have established MAb based ELISA system, which could accurately detect CGMMV from watermelon seeds, nursery seedlings, transplants and field samples from greenhouse or open out door field as well.

Isolation and initial identification of pathogens causing an aseptic meningitis outbreak in north area of Jiangsu province

目的 分离并初步鉴定引起2003年我国苏北地区无菌性脑膜炎暴发的病原体.方法来自70例患者的66份脑脊液和4份粪便标本,用3种细胞系(MRC-5、Vero、Hep-2)进行病毒分离,并通过电镜、逆转录聚合酶链反应(RT-PCR)和中和试验等方法,分别从形态学、分子生物学和血清学的角度对分离毒株进行鉴定.结果得到22株阳性分离株病毒,18株分离自脑脊液(分离率27.3%),4株分离自粪便标本.经电镜、肠道病毒通用引物RT-PCR以及肠道病毒组合及单价抗血清的中和试验鉴定,所有分离毒株均为Echo30型肠道病毒.结论 Echo30是本次脑膜炎暴发流行的病因病毒。

Discovery of specific tryptophan hydroxylase in the brain of the beetle Harmonia axyridis

Rabbit anti-serotonin and mouse monoclonal anti-tryptophan hydroxylase antisera were applied on the brain sections of the beetle Harmonia axyridis, butterfly Childrena zenobia, moth Antheraea pernyi and ant Camponotus japonicus, using the Streptavidin–Peroxidase immunohistochemical method and Colophony–Paraffin embedded section technique. Results revealed that all the experimental insects showed notable serotonin-like immunoreactivity in the brain. However, only the brain sections of the beetle H. axyridis were strongly labeled by mouse monoclonal anti-tryptophan hydroxylase antiserum, with the distribution pattern matching that of serotonin. These results demonstrate that specific tryptophan hydroxylase may exist in the brain of the beetle and likely reflect the diversity of serotonin synthetic mechanisms as well as the evolution of aromatic amino acid hydroxylase genes.

An open letter to our readers on the use of antibodies

An open letter to our readers on the use of antibodies

Tomato yellow leaf curl and Tomato mottle begomoviruses

<i>Tomato yellow leaf curl</i> and <i>Tomato mottle begomoviruses</i>

First Report of Narcissus mosaic virus Infecting Crocus spp. Cultivars in the Netherlands.

A survey to identify virus diseases affecting Crocus spp. in the Netherlands was conducted during April 2004. Crocus spp. (cvs. Flavus, Pick-wick, Remembrance, and Grand Maitre) with symptoms suggestive of virus infection (stunting, yellowing, necrosis, and flower color breaking) were collected from several fields in the Breezand and Lisse districts in northern and southern Netherlands, respectively. All samples were tested for the presence of six known crocus-infecting viruses (1,2) using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) assays. The ELISA assay was performed with the following polyclonal and monoclonal antibodies: Iris severe mosaic virus (ISMV); Tobacco rattle virus (TRV) isolates F, Y, and J obtained from the Applied Plant Research Institute, Lisse, Netherlands; Arabis mosaic virus; Cucumber mosaic virus from the Plant Research International Institute, Wageningen, Netherlands; Iris yellow spot virus (IYSV) from the Virology Department at Wageningen University, Netherlands; and the potyvirus group-specific monoclonal antiserum from the DSMZ, Braunschweig, Germany. All samples that tested positive with a potyvirus antiserum were further tested for the presence of Bean yellow mosaic virus (BYMV) using a BYMV-specific antiserum. Serological results obtained indicated that BYMV, detected with the potyvirus antiserum and BYMV-specific antiserum, and ISMV were the most commonly encountered viruses. Tobacco necrosis virus (TNV) and TRV were only found occasionally, whereas IYSV, was not detected in any of the samples tested. To study the presence of viruses not yet reported, total RNA was extracted and tested with a RT-PCR assay with carlavirus, potexvirus, necrovirus (R. Miglino, unpublished), and potyvirus (3) genus-specific oligonucleotides. In accordance with the ELISA results, PCR amplicons were obtained with the potyvirus, TNV, and TRV primer sets. Furthermore, a 280-bp amplicon corresponding to the expected size was amplified in a RT-PCR assay performed on total RNA with a potexvirus genus-specific primer set. The reverse primer (5'-AGC ATG GCG CCA TCT TGT GAC TG-3') was located upstream in the conserved viral replicaseencoding region at position 4254-4231 of Narcissus mosaic virus (NMV) RNA genome (Genbank Accession No. D13747) and the forward primer (5'-CTG AAG TCA CAA TGG GTG AAG AA-3') was located downstream at position 3969-3992. Sequence homology using BLAST analysis of the cloned and sequenced PCR product showed 98% identity with NMV. Although the virus has a very narrow host range, the results of this study may have a significant impact on the crocus industry in the Netherlands. To our knowledge, this is the first report of NMV infecting crocus.

Biliary pancreatitis-associated ascitic fluid activates the production of tumor necrosis factor-α in acinar cells*

Acute pancreatitis is associated with increased cytokine release from different cell sources. We have investigated the ability of acinar cells, in comparison with inflammatory peripheral blood cells, to produce tumor necrosis factor (TNF)-alpha in response to pancreatitis-associated ascitic fluid (PAAF). Controlled, randomized animal study. University research laboratory. Male Wistar rats. Flow cytometry using phycoerythrin-labeled monoclonal anti-TNF-alpha antiserum. PAAF (20%, v:v) obtained from rats with acute pancreatitis induced by bile-pancreatic duct obstruction significantly increased TNF-alpha production in acinar cells, as measured by flow cytometry using phycoerythrin-labeled monoclonal anti-TNF-alpha antiserum. Neither heating of PAAF nor the addition of soybean trypsin inhibitor or neutralizing amounts of anti-TNF-alpha monoclonal antiserum reduced the acinar cell TNF-alpha production. Monocytes and lymphocytes did not produce TNF-alpha in response to PAAF. Likewise, the typical monocyte and lymphocyte stimulating factors-lipopolysaccharide (10 microg/microL) and phorbol 12-myristate 13-acetate (250 ng/mL) plus ionomycin (1 microg/mL), respectively-were not able to produce TNF-alpha in acinar cells. By comparison of the two acinar cell populations differentiated by flow cytometry, R2 cells (with higher forward scatter values) showed a greater ability to produce TNF-alpha in response to PAAF than R1 cells. Acinar cell nuclear factor-kappaB was activated, but TNF-alpha production was not totally inhibited in presence of N-acetyl cysteine (30, 100 mM). The production of TNF-alpha from different cell sources is selectively activated. PAAF may be involved in the pathophysiology of acute pancreatitis by TNF-alpha production in acinar cells through mechanisms partially mediated by nuclear factor-kappaB activation. PAAF components, such as TNF-alpha or trypsin, are not responsible for acinar cell activation. TNF-alpha was induced by heat-resistant PAAF factors, displaying acinar cells with higher forward scatter (R2) a greater ability to increase the TNF-alpha production than R1 cells.

Association of human leukocyte antigen DQ1 and dengue fever in a white Southern Brazilian population

Dengue is an infectious disease of viral etiology transmitted by the mosquitoes Aedes aegypti, A. albopictus, and A. scutellaris. It can develop either as a benign form or as a severe hemorrhagic form. Previous work showed an association of the hemorrhagic form with human leukocyte antigens (HLA), suggesting a role of genetic factors in disease susceptibility. Nevertheless, data on HLA association with the classical form of the disease is scarce in literature. Sixty-four patients and 667 normal individuals, living in the state of Parana, Southern Brazil, were used as test and control group, respectively. The patients developed the disease during a virus 1 dengue outbreak either in Maringa city in 1995 (47) or in Paranavai city in 1999 (17). The diagnostic was confirmed through serology and/or viral culture. HLA class I and II typing was performed by the classical microlynfocitotoxicity test using monoclonal antisera and fluorobeads. Qui-square statistical analysis confirmed a positive association with HLA-DQ1 (76.6% vs 57.7%; p = 0.005243; pc = 0.026215). HLA-DR1 also presented an increased frequency in the test group, not statistically significant after p correction though (32.8% vs 15.9%; p = 0.005729; pc = 0.080206). In conclusion, genetic factors may play a role on the susceptibility to the classical dengue, virus 1, in the Brazilian population. Further independent studies should be performed in the Brazilian population to confirm these preliminary data.

Tomato spotted wilt tospovirus, Impatiens necrotic spot tospovirus and Watermelon silver mottle tospovirus

<i>Tomato spotted wilt tospovirus</i>, <i>Impatiens necrotic spot tospovirus</i> and <i>Watermelon silver mottle tospovirus</i>

First Report of Zucchini yellow mosaic virus in Cucumber (Cucumis sativus) in India.

In August of 2002, cucumber (Cucumis sativus L. cv. Himangi) plants grown in commercial fields in Pune, western Maharashtra, India, exhibited chlorotic spots, veinal chlorosis, mosaic, blister formation and shoestring symptoms on leaves, stunted growth, and distortion of fruits. Incidence of virus infection in the fields varied from 25 to 38%. Crude sap extracted from infected cucumber leaf samples was inoculated mechanically onto cucumber and indicator host plants. The inoculated glasshouse-grown cucumber plants showed virus symptoms similar to those observed in the field. The virus produced chlorotic local lesions on Chenopodium amaranticolor and chlorotic spots followed by veinal chlorosis, mosaic, vein banding, and leaf distortion on Citrullus lanatus, C. melo, C. sativus, Cucurbita maxima, Cucurbita pepo, Luffa acutangula, and Trichosanthes anguina. The virus did not infect Nicotiana benthamiana, N. glutinosa, and N. tabacum cv. White Burley (1,2). Cucumber and indicator plants were tested using direct antigen coating enzyme-linked immunosorbent assays. A positive reaction was obtained with monoclonal antiserum to Zucchini yellow mosaic virus (ZYMV) but not with antisera to Papaya ringspot virus-P, Cucumber mosaic virus, and Watermelon mosaic virus (Agdia, Inc., Elkhart, IN). The disease was observed again in the field during July to September 2003. Natural infection of zucchini (Cucurbita pepo) by ZYMV has been reported previously from India (3). To our knowledge, this is the first report of ZYMV in cucumber in India.

Cross reactivity of polyclonal GFAP antiserum: implications for the in-vitro characterisation of brain endothelium

Following a recent claim, based on glial acidic fibrillary protein (GFAP) expression, that brain-derived astrocytes in culture are in fact endothelial cells, we immuno-labelled primary cultures of rat brain astrocytes and endothelium with various GFAP antisera. Both cell types stained positively with a polyclonal antibody, although monoclonal antiserum labelled only astrocytes. We conclude that staining of endothelial cells with the polyclonal GFAP antiserum is due to cross reactivity with another protein.

Differential expression of sucrose transporter and polyol transporter genes during maturation of common plantain companion cells.

The cDNAs of two sorbitol transporters, common plantain (Plantago major) polyol transporter (PLT) 1 and 2 (PmPLT1 and PmPLT2), were isolated from a vascular bundle-specific cDNA library from common plantain, a dicot plant transporting Suc plus sorbitol in its phloem. Here, we describe the kinetic characterization of these sorbitol transporters by functional expression in Brewer's yeast (Saccharomyces cerevisiae) and in Xenopus sp. oocytes and for the first time the localization of plant PLTs in specific cell types of the vascular tissue. In the yeast system, both proteins were shown to be uncoupler sensitive and could be characterized as low-affinity and low-specificity polyol symporters. The Km value for the physiological substrate sorbitol is 12 mm for PmPLT1 and even higher for PmPLT2, which showed an almost linear increase in sorbitol transport rates up to 20 mm. These data were confirmed in the Xenopus sp. system, where PmPLT1 was analyzed in detail and characterized as a H+ symporter. Using peptide-specific polyclonal antisera against PmPLT1 or PmPLT2 and simultaneous labeling with the monoclonal antiserum 1A2 raised against the companion cell-specific PmSUC2 Suc transporter, both PLTs were localized to companion cells of the phloem in common plantain source leaves. These analyses revealed two different types of companion cells in the common plantain phloem: younger cells expressing PmSUC2 at higher levels and older cells expressing lower levels of PmSUC2 plus both PLT genes. The putative role of these low-affinity transporters in phloem loading is discussed.

Expression of a foot-and-mouth disease virus immunodominant epitope by a filamentous bacteriophage vector.

We described the construction of a recombinant filamentous phage displaying on its surface the immunodominant site of VP1 protein of foot-and-mouth disease virus (FMDV). The coding sequence was inserted at the amino-terminus of the major coat protein pVIII via a spacer. The hybrid phage proved to be antigenic as it was recognized by polyclonal and monoclonal anti FMDV sera. In two experiments involving immunisation of guinea-pigs with the recombinant phage, a low antibody response was generated. This suggests a possible role for phage displayed peptides in inducing anti FMDV immunity and the possibility of further development is discussed.

Increased serum levels of the specific AGE-compound methylglyoxal-derived hydroimidazolone in patients with type 2 diabetes

A time-delayed fluorescence immunoassay was developed for the determination of serum levels of methylglyoxal (MG)-derived hydroimidazolone using a monoclonal antiserum raised against Nα-acetyl-Nδ-(5-hydro-5-methyl)-4-imidazolone, Europium-labeled anti-mouse IgG antiserum as indicator, and MG modified bovine serum albumin (BSA) as standard. Serum levels of hydroimidazolone were measured in 45 patients with type 2 diabetes aged 59.4 ± 6.1 (mean ± SD) years and with duration of diabetes of 7.3 ± 3.1 years, and in 19 nondiabetic controls aged 56.3 ± 4.3 years. The serum levels of hydroimidazolone were significantly higher in patients compared to controls: median, 3.0 (5-95 percentile, 1.6 to 5.4) U/mg protein versus 1.9 (1.2 to 2.8) U/mg protein (P =.0005). Significant positive correlations were observed between the serum levels of hydroimidazolone and serum levels of advanced glycation end products (AGEs), measured with a polyclonal anti-AGE antibody: r = 0.59 for patients (P <.0001), and r = 0.65 for controls (P =.002). Similarly, significant correlations were also found between serum levels of hydroimidazolone and Nϵ-(carboxymethyl)-lysine (CML): r = 0.36 in patients and r = 0.55 for controls (both P =.02). Serum hydroimidazolone levels did not correlate with fasting plasma glucose or hemoglobin A1c (HbA1c) levels. The observed differences between patients with diabetes and nondiabetic controls seem to be comparable to differences measured for other AGE compounds. Copyright 2003, Elsevier Science (USA). All rights reserved.

The serologic response of horses to equine arteritis virus as determined by competitive enzyme-linked immunosorbent assays (c-ELISAs) to structural and non-structural viral proteins

In an effort to further characterize the humoral immune response of horses to equine arteritis virus (EAV), direct and competitive enzyme-linked immunosorbent assays (c-ELISAs) were developed using monoclonal and polyclonal anti-sera to structural (G L, N and M) and non-structural (nsp1) viral proteins. A nsp1-specific monoclonal antibody was produced to facilitate development of a c-ELISA to this protein. Data obtained using the various c-ELISAs confirm that the M protein is a major target of the antibody response of horses to EAV. However, none of the c-ELISAs that were developed were as sensitive in detecting EAV-specific antibodies in horse sera as the existing serum neutralization test.

P53 in nonneoplastic central nervous system lesions: an immunohistochemical and genetic sequencing study.

Immunostaining for p53 commonly is considered a marker of neoplasia. Previous studies of nonneoplastic processes have yielded conflicting results. To test the assumption that p53 immunoreactivity indicates neoplasia, we examined 60 formalin-fixed, paraffin-embedded biopsies of nonneoplastic central nervous system lesions, including gliosis (n = 12), infarction (n = 9), demyelinating disease (n = 23), progressive multifocal leukoencephalopathy (n = 11), and herpes simplex virus encephalitis (n = 5). Diffuse astrocytomas (n = 50) of World Health Organization Grades 2 to 4 also were studied, as were six control autopsy brains. The avidin-biotin-peroxidase complex method was used with commercially available monoclonal antisera to both p53 (clone DO7; Dako, Carpinteria, CA) and mdm2 (Dako), a protein known to stabilize p53. Two samples of each nonneoplastic lesion also were subjected to deoxyribonucleic acid isolation, amplification, and sequencing of exons 5 to 8 of TP53. Although it was low level in most instances, p53 immunoreactivity was noted in all but normal control samples. In reactive lesions, staining was largely observed in astrocytes and histiocytes. Scant oligodendroglia also were labeled in demyelinating disease. The progressive multifocal leukoencephalopathy samples revealed exceptionally strong staining in astrocytes and infected oligodendrocytes. Staining also was noted in occasional endothelial cells and neurons, and in rare lymphocytes. Immunoreactivity for mdm2, studied only in nonneoplastic lesions, was moderate to strong in all cases and limited to reactive astrocytes and histiocytes. No TP53 mutations were noted in the nonneoplastic lesions studied. To some extent, all astrocytomas exhibited p53 immunopositivity, particularly high-grade lesions. p53 immunoreactivity is not limited to astrocytomas, but it can be observed in lesions that often are mistaken for glioma. No TP53 mutations accompany p53 expression in nonneoplastic lesions, and mdm2 may be responsible for persistence of p53 expression in these processes.

Construction of multivalent DNA vaccines forMycobacterium tuberculosis and its immunogenicity

The coding regions of Ag85B MPT-64, and ESAT-6 secreted proteins were cloned initially into the eukaryotic expression vector pJW4303, then transformed toE coli Top 10 strain for plasmid DNA extraction and further analysis. Plasmids containing the right insertion were sequenced to confirm their identity. COS7 cells were transfected with a mixture containing serially diluted plasmid DNA encoding three secreted proteins and Lipofectin (Gibco). The supernatants and pellets prepared from various cell lines were run on SDS-PAGE gel and the expression of these proteins in COS7 cells were demonstrated by immunoblot using polyclonal or monoclonal antiserum of M.TBH37Rv. 21 days after first vaccination of C57BL-6 mice by all three recombinant eukaryotic expressing vectors, antibody titer for Ag85B reached 1: 3200. 21 days after second vaccination, the antibody titer reached 1: 102400. The highest antibody levels induced by multivalent vaccines after the second injection were equal to or even greater than the highest antibody levels of single DNA vaccine reported in literature after third injections. Antibody titer of MPT-64 was 1: 50 after the first injection and it reached 1: 200 after the second injection. No antigen-specific antibody against ESAT-6 was detected in sera harvested from immunized mice 21 days after both injections. Antigen-specific IFN-γ level of Ag85B was 110 pg/mL while no antigen-specific IFN-γ level of ESAT-6 and MPT-64 was detected even after third injections. To our knowledge, it is the first time that studies of polyvalent recombinant DNA vaccines against TB were carried out in C57BL-6 mice. Our results indicated that multiple DNA vaccines could be used to enhance protective responses against MTB.