Vivo-induced Antigen Technology Research Articles

Development and evaluation of immunological effects of a DNA vaccine encoding phosphoketolase family protein against Nocardia seriolae in hybrid snakehead

Fish nocardiosis is a chronic disease mainly caused by Nocardia seriolae, which occurs in a variety of economically cultured freshwater and marine fish. Studies have shown that DNA vaccine is an effective treatment to protect fish from bacterial infection. In our previous experiment, an in vivo-induced gene of N. seriolae, encoding phosphoketolase (PK) family protein, was identified by in vivo-induced antigen technology. In the present study, the antigenic gene encoding PK family protein was analyzed by bioinformatics and further inserted into the eukaryotic expression vector pcDNA3.1-myc-his-A for DNA vaccine development. The immunological effects of pcDNA-PK DNA vaccine were assessed in hybrid snakehead (Channa maculata ♀ × Channa argus ♂), showing induction in several serum enzyme activity parameters (including LZM, SOD, ACP and AKP), increasing in specific-antibody IgM levels, as well as up-regulation in six immune-related genes (CD4, CD8α, TNFα, IL-1β, MHCIα and MHCIIα). Moreover, an immune-protection with a relative survival rate was provided at 53.82 % following artificial challenge with N. seriolae in vaccinated fish in comparison to the control group. In summary, these results indicate that pcDNA-PK DNA vaccine could boost strong immune responses in hybrid snakehead and show preferably protective efficacy against N. seriolae, which may be applied in aquaculture to control fish nocardiosis.

Identifying the in vivo-induced antigenic genes is a strategy to develop DNA vaccine against Nocardia seriolae in hybrid snakehead (Channa maculata ♀ × Channa argus ♂)

Nocardia seriolae has been identified as the causative agent of fish nocardiosis, resulting in serious economic losses in aquaculture. With an aim to screen potential candidates for vaccine development against N. seriolae, the in vivo-induced genes of N. seriolae in hybrid snakehead (Channa maculate ♀ × Channa argus ♂) model were profiled via in vivo-induced antigen technology (IVIAT) in the present study, and 6 in vivo-induced genes were identified as follows: IS701 family transposase (is701), membrane protein insertase YidC (yidC), ergothioneine biosynthesis glutamate-cysteine ligase (egtA), molybdopterin respectively-dependent oxidoreductase (mol), phosphoketolase family protein (Ppl), hypothetical protein 6747 (hp6747). Additionally, the yidC was inserted into eukaryotic expression vector pcDNA3.1-myc-his-A to construct a DNA vaccine named as pcDNA-YidC to evaluate immunoprotection in hybrid snakehead after artificial challenge with N. serioale. Results showed that the transcription of yidC was detected in spleen, trunk kidney, muscle and liver in vaccinated fish, suggesting that this antigenic gene can be recombinantly expressed in fish. Meanwhile, indexes of humoral immunity were evaluated in the vaccinated fish through assessing specific-antibody IgM and serum enzyme activities, including lysozyme (LZM), superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). Quantitative real-time PCR analysis indicated that pcDNA-YidC DNA vaccine could notably enhance the expression of immune-related genes (CD4、CD8α、MHCIIα、TNFα、IL-1β and MHCIα) in 4 tissues (spleen, trunk kidney, muscle and liver) of the vaccinated fish. Finally, an immuno-protection with a relative survival rate of 65.71 % was displayed in vaccinated fish in comparison to the control groups. Taken together, these results indicate that pcDNA-YidC DNA vaccine could boost strong immune responses in hybrid snakehead and show preferably protective efficacy against N. seriolae, indicating that IVIAT is a helpful strategy to screen the highly immunogenic antigens for vaccine development against fish nocardiosis.

Identification of in vivo expressed proteins in live attenuated lipopolysaccharide mutant that mediates heterologous protection against Leptospira spp

Leptospirosis vaccines that elicit broad protection against a range of pathogenic Leptospira spp. would overcome a major drawback of currently licensed bacterin vaccines. Live attenuated vaccine produced from a lipopolysaccharide (LPS) mutant strain of L. interrogans serovar Manilae M1352 (Live M1352) stimulated better protective efficacy than heat killed M1352 (HK M1352) against a heterologous challenge with L. interrogans serovar Pomona. To identify antigens of Live M1352 potentially responsible for cross protection, in vivo-induced antigen technology (IVIAT), a powerful tool to identify in vivo-induced (ivi) genes expressed during infection, was employed in this study. Pooled sera from hamsters immunized with Live M1352 were sequentially adsorbed with various preparations of in vitro grown M1352. The pre-adsorbed sera were used to screen a genomic expression library of M1352. Nineteen strongly reactive clones were selected for DNA sequencing. These ivi genes are conserved in most Leptospira strains. Four selected genes including LIMLP_04965 (tolB), LIMLP_01535, LIMLP_06785 (fliI), and LIMLP_14930 were confirmed for their upregulated expression in kidneys of infected hamsters by RT-qPCR, suggesting their role in leptospiral infection. These ivi proteins represent potential targets for vaccine candidates that warrant further investigation for their protective efficacy.

Identification of Haemophilus parasuis genes uniquely expressed during infection using in vivo-induced antigen technology

Haemophilus parasuis is the etiological agent of Glässer’s disease which is characterized by fibrinous polyserositis, arthritis and meningitis. The pathogenesis of this bacterium remains largely unknown. Genes expressed in vivo may play an important role in the pathogenicity of H. parasuis. The development of in vivo-induced antigen technology (IVIAT) has provided a valuable tool for the identification of in vivo-induced genes during bacterial infection. In this study, IVIAT was applied to identify in vivo-induced antigens of H. parasuis. Pooled swine H. parasuis-positive sera, adsorbed against in vitro-grown cultures of H. parasuis SH0165 and Escherichia coli BL21 (DE3), were used to screen the inducible expression library of genomic proteins from whole genome sequenced H. parsuis SH0165. Finally, 24 unique genes expressed in vivo were successfully identified after secondary and tertiary screening with IVIAT. These genes were implicated in cell surface proteins, metabolism, stress response, regulation, transportation and other processes. Quantitative real-time PCR showed that the mRNA levels of 24 genes were all upregulated in vivo relative to in vitro, with 13 genes were detected significantly upregulated in H. parasuis infected pigs. Several potential virulence-associated genes were found to be uniquely expressed in vivo, including espP, lnt, hutZ, mreC, vtaA, pilB, tex, sunT and aidA. The results indicated that the proteins identified using IVIAT may play important roles in the pathogenesis of H. parasuis infection in vivo.

Identification of Actinobacillus pleuropneumoniae Genes Preferentially Expressed During Infection Using In Vivo-Induced Antigen Technology (IVIAT).

Porcine pleuropneumonia is an infectious disease caused by Actinobacillus pleuropneumoniae. The identification of A. pleuropneumoniae genes, specially expressed in vivo, is a useful tool to reveal the mechanism of infection. IVIAT was used in this work to identify antigens expressed in vivo during A. pleuropneumoniae infection, using sera from individuals with chronic porcine pleuropneumonia. Sequencing of DNA inserts from positive clones showed 11 open reading frames with high homology to A. pleuropneumoniae genes. Based on sequence analysis, proteins encoded by these genes were involved in metabolism, replication, transcription regulation, and signal transduction. Moreover, three function-unknown proteins were also indentified in this work. Expression analysis using quantitative real-time PCR showed that most of the genes tested were up-regulated in vivo relative to their expression levels in vitro. IVI (in vivoinduced) genes that were amplified by PCR in different A. pleuropneumoniae strains showed that these genes could be detected in almost all of the strains. It is demonstrated that the identified IVI antigen may have important roles in the infection of A. pleuropneumoniae.

Redefining Virulence of Bacterial Pathogens

Investigators identifying virulence genes at first did so by examining transposon mutants or individual gene mutations. Mutants of bacterial pathogens were then assessed in animals, whose symptoms mimicked human disease. Later, genome-wide screens were developed whereby genes and proteins that influence virulence could be identified, including via signature-tagged mutagenesis (STM), in vivo expression technology (IVET), and in vivo-induced antigen technology (IVIAT). Investigators then began to use RT-PCR to measure expression of individual genes, including within infected tissues. Microarray technology enables us to estimate global gene expression under defined culture conditions such as nitrogen limitation, oxygenation, and osmotic stress.

Identification of in vivo-induced genes during infection of chickens with Salmonella enterica serovar Enteritidis

Chickens are an important source of food worldwide and are often infected with food-poisoning serovars of Salmonella enterica, frequently Salmonella Enteritidis (SE), without exhibiting clinical signs of disease. Ivi (in vivo induced) genes identified using in vivo-induced antigen technology (IVIAT) are expressed only during bacterial infection and have the potential value of identifying epidemic strains and antigens which can form the basis for sub-unit vaccine development. We applied IVIAT to SE strain 50041 and identified 42 ivi genes. Eight representative ivi genes were further confirmed by qRT-PCR as being expressed only in vivo within 48 h of infection compared with that of in vitro-cultured. Although our results indicated that the identified ivi genes are expressed only in vivo, further research is needed to elucidate the exact roles of these genes during infection and pathogenesis.

Identification of novel genes expressed during host infection in Streptococcus equi ssp. zooepidemicus ATCC35246

Infection with Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus, SEZ) can cause septicemia, meningitis, and mastitis in domesticated species. Identification of this organism's virulence factors is an effective way of clarifying its pathogenic mechanism. We employed in vivo-induced antigen technology (IVIAT) to find bacterial genes that were only expressed or upregulated in an infected host (IVI genes). Convalescent-phase sera from pigs infected with SEZ were pooled, adsorbed against in vitro antigens, and used to screen SEZ genomic expression libraries. This analysis identified 43 genes as IVI genes. Six of these 43 genes were verified via real-time PCR. Following the analysis, we were able to assign a putative function to 36 of the 43 proteins. These proteins included those involved in virulence and adaptation; formation of intermediary products; gene replication, transcription and expression; energy metabolism; transport and also various proteins of unknown function. The relationship between sagD gene and bacterial virulence was confirmed. This study provides new molecular data for the study of streptococcal disease in swine and is important for identifying the pathogenic mechanisms of SEZ.

Identification of in vivo-induced bacterial protein antigens during calf infection with Chlamydia psittaci

Chlamydia (C.) psittaci, the causative agent of ornithosis, is an obligate intracellular pathogen with a unique developmental cycle and a high potential for zoonotic transmission. Various mammalian hosts, such as cattle, horse, sheep and man that are in close contact with contaminated birds can get infected (referred to as psittacosis). Since little is known about long-term sequelae of chronic disease and the molecular mechanisms of chlamydial pathogenesis, a key step in understanding the in vivo situation is the identification of C. psittaci infection-associated proteins. For this, we investigated sera of infected calves. Using the immunoscreening approach In Vivo Induced Antigen Technology (IVIAT) including all relevant controls, we focused on C. psittaci proteins, which are induced in vivo during infection. Sera were pooled, extensively adsorbed against in vitro antigens to eliminate false positive results, and used to screen an inducible C. psittaci 02DC15 genomic expression library. Screening and control experiments revealed 19 immunogenic proteins, which are expressed during infection. They are involved in transport and oxidative stress response, heme and folate biosynthesis, DNA replication, recombination and repair, cell envelope, bacterial secretion systems and hypothetical proteins of so far unknown functions. Some of the proteins found may be considered as diagnostic markers or as candidates for the development of vaccines.

Use of in vivo induced antigen technology to identify genes from Aeromonas salmonicida subsp. salmonicida that are specifically expressed during infection of the rainbow trout Oncorhynchus mykiss.

BackgroundAeromonas salmonicida is a major fish pathogen associated with mass mortalities in salmonid fish. In the present study, we applied In Vivo Induced Antigen Technology (IVIAT), a technique that relies on antibodies adsorbed against in vitro cultures of the pathogen, to a clinical isolate of A. salmonicida subsp. salmonicida.ResultsThe results from IVIAT allowed identification of four proteins that were upregulated in the fish samples: A UDP-3-O-acyl-N-acetylglucosamine deacetylase, an RNA polymerase sigma factor D as well as TonB and a hypothetical protein. Subsequent investigations were performed using real-time PCR and cDNA synthesised from infected spleen, liver and anterior kidneys. These confirmed that the transcription level of each of these genes was significantly upregulated during the infection process compared to bacteria in vitro.ConclusionsThe present studied identified four genes that were upregulated during the infectious process and are likely to play a role in the virulence of A. salmonicida. Because these are antigenic they might constitute potential targets for the development of new vaccine as well as therapeutic agents.

Identification of Genes Expressed DuringToxoplasma gondiiInfection by in Vivo-Induced Antigen Technology (IVIAT) with Positive Porcine Sera

Infection of pigs with Toxoplasma gondii is a common source of human toxoplasmosis and causes serious economic losses. In vivo-induced antigen technology (IVIAT) is an effective immunological technique to identify the antigens that a pathogen specifically expressed during infection. To discover the genes that are important in T. gondii infection of pigs, we employed IVIAT using sera from infected pigs. Fourteen antigens were identified including microneme protein 11 (MIC11), dense granule protein 5 (GRA5), 18 kDa cyclophilin (C-18), serine proteinase inhibitor (PI), calmodulin (CaM), leucine-rich repeat protein ( LRRP), D-3-phosphoglycerate dehydrogenase (D3PD), elongation factor 1-gamma (EF1), and 6 hypothetical proteins. The increased transcription levels of 5 (MIC11, GRA5, C-18, PI, and CaM) of the 14 molecules identified by IVIAT were confirmed by real-time PCR. The full length or partial proteins encoded by these 5 genes were expressed in Escherichia coli , and their immunogenicity was confirmed by Western blot analysis with positive porcine sera. Further functional studies were conducted with CaM. Suppression of CaM expression by RNA interference decreased T. gondii tachyzoites cell attachment, invasion, and egress but did not influence their replication. The proteins identified in this study are predicted to be involved in cell invasion, ion-protein binding, protein folding, biosynthesis, and metabolism. The results of the functional analysis support the hypothesis that CaM contributes to parasite pathogenesis during infection. These results may have significant implications for the discovery of candidate molecules for the development of potential therapies and preventive measures against toxoplasmosis in pigs.

In vivo-induced argininosuccinate lyase plays a role in the replication of Brucella abortus in RAW264.7 cells

Brucellosis caused by Brucella species is a zoonotic disease with a serious impact on public health and the livestock industry. To better understand the pathogenesis of the disease, in vivo-induced antigen technology (IVIAT) was used to investigate the in vivo-induced antigens of Brucella abortus in this study. A genomic expression library of B. abortus was constructed and screened using pooled bovine B. abortus-positive sera by IVIAT. In total, 33 antigens were identified. Five antigens were further expressed and tested for their seroreactivity against 33 individual bovine B. abortus-positive sera by Western blot analysis. The results showed a highest positive rate of 32/33 for argininosuccinate lyase (ASL), indicating that ASL may be used as a candidate marker for serodiagnosis of brucellosis. Furthermore, an asl gene-deleted mutant strain S2308ΔASL was constructed, and the intracellular survival and replication of the mutant strain in RAW264.7 cells were investigated. Interestingly, the numbers of bacteria recovered from cells infected with mutant strain S2308ΔASL were similar at all time points observed from 0 h to 96 h post-infection, suggesting the asl gene plays an important role in the bacterial replication in RAW264.7 cells. Real-time quantitative PCR (qPCR) analysis showed that the mRNA levels in S2308ΔASL were decreased for BvrR, BvrS and virB5 when compared with those in S2308 (P<0.05). Our results not only expand the knowledge of Brucella intracellular replication but also expand the list of candidates for serodiagnostic markers of brucellosis.

Use of In Vivo-Induced Antigen Technology to Identify In Vivo-Expressed Genes of Campylobacter jejuni During Human Infection

Campylobacter jejuni is a prevalent foodborne pathogen worldwide. Human infection by C. jejuni primarily arises from contaminated poultry meats. Genes expressed in vivo may play an important role in the pathogenicity of C. jejuni. We applied an immunoscreening method, in vivo-induced antigen technology (IVIAT), to identify in vivo-induced genes during human infection by C. jejuni. An inducible expression library of genomic proteins was constructed from sequenced C. jejuni NCTC 11168 and was then screened using adsorbed, pooled human sera obtained from clinical patients. We successfully identified 24 unique genes expressed in vivo. These genes were implicated in metabolism, molecular biosynthesis, genetic information processing, transport, and other processes. We selected six genes with different functions to compare their expression levels in vivo and in vitro using real-time RT-PCR. The results showed that the selected six genes were significantly upregulated in vivo but not in vitro. In short, these identified in vivo-induced genes may contribute to human infection of C. jejuni, some of which may be meaningful vaccine candidate antigens or diagnosis serologic markers for campylobacteriosis. IVIAT may present a significant and efficient method for understanding the pathogenicity mechanism of Campylobacter and for finding targets for its prevention and control.

Screening of Genes Expressed In Vivo During Interaction Between Chicken and Campylobacter jejuni

Chicken are considered as the most important source of human infection by Campylobacter jejuni, which primarily arises from contaminated poultry meats. However, the genes expressed in vivo of the interaction between chicken and C. jejuni have not been screened. In this regard, in vivo-induced antigen technology (IVIAT) was applied to identify expressed genes in vivo during interaction between chicken and C. jejuni, a prevalent foodborne pathogen worldwide. Chicken sera were obtained by inoculating C. jejuni NCTC 11168 into Leghorn chickens through oral and intramuscular administration. Pooled chicken sera, adsorbed against in vitrogrown cultures of C. jejuni, were used to screen the inducible expression library of genomic proteins from sequenced C. jejuni NCTC 11168. Finally, 28 unique genes expressed in vivo were successfully identified after secondary and tertiary screenings with IVIAT. The genes were implicated in metabolism, molecular biosynthesis, genetic information processing, transport, regulation and other processes, in addition to Cj0092, with unknown function. Several potential virulence-associated genes were found to be expressed in vivo, including chuA, flgS, cheA, rplA, and Cj0190c. We selected four genes with different functions to compare their expression levels in vivo and in vitro using real-time RT-PCR. The results indicated that these selected genes were significantly upregulated in vivo but not in vitro. In short, the expressed genes in vivo may act as potential virulence-associated genes, the protein encoded by which may be meaningful vaccine candidate antigens for campylobacteriosis. IVIAT provides an important and efficient strategy for understanding the interaction mechanisms between Campylobacter and hosts.

Identification of Immunogenic Salmonella enterica Serotype Typhi Antigens Expressed in Chronic Biliary Carriers of S. Typhi in Kathmandu, Nepal

Background Salmonella enterica serotype Typhi can colonize and persist in the biliary tract of infected individuals, resulting in a state of asymptomatic chronic carriage. Chronic carriers may act as persistent reservoirs of infection within a community and may introduce infection to susceptible individuals and new communities. Little is known about the interaction between the host and pathogen in the biliary tract of chronic carriers, and there is currently no reliable diagnostic assay to identify asymptomatic S. Typhi carriage.Methodology/Principal FindingsTo study host-pathogen interactions in the biliary tract during S. Typhi carriage, we applied an immunoscreening technique called in vivo-induced antigen technology (IVIAT), to identify potential biomarkers unique to carriers. IVIAT identifies humorally immunogenic bacterial antigens expressed uniquely in the in vivo environment, and we hypothesized that S. Typhi surviving in the biliary tract of humans may express a distinct antigenic profile. Thirteen S. Typhi antigens that were immunoreactive in carriers, but not in healthy individuals from a typhoid endemic area, were identified. The identified antigens included a number of putative membrane proteins, lipoproteins, and hemolysin-related proteins. YncE (STY1479), an uncharacterized protein with an ATP-binding motif, gave prominent responses in our screen. The response to YncE in patients whose biliary tract contained S. Typhi was compared to responses in patients whose biliary tract did not contain S. Typhi, patients with acute typhoid fever, and healthy controls residing in a typhoid endemic area. Seven of 10 (70%) chronic carriers, 0 of 8 bile culture-negative controls (0%), 0 of 8 healthy Bangladeshis (0%), and 1 of 8 (12.5%) Bangladeshis with acute typhoid fever had detectable anti-YncE IgG in blood. IgA responses were also present.Conclusions/SignificanceFurther evaluation of YncE and other antigens identified by IVIAT could lead to the development of improved diagnostic assays to identify asymptomatic S. Typhi carriers.

Identification of Salmonella enterica Serovar Pullorum Antigenic Determinants Expressed In Vivo

Salmonella enterica serovar Pullorum affecting poultry causes pullorum disease and results in severe economic loss in the poultry industry. Currently, it remains a major threat in countries with poor poultry surveillance and no efficient control measures. As S. Pullorum could induce strong humoral immune responses, we applied an immunoscreening technique, the in vivo-induced antigen technology (IVIAT), to identify immunogenic bacterial proteins expressed or upregulated during S. Pullorum infection. Convalescent-phase sera from chickens infected with S. Pullorum were pooled, adsorbed against antigens expressed in vitro, and used to screen an S. Pullorum genomic expression library. Forty-five proteins were screened out, and their functions were implicated in molecular biosynthesis and degradation, transport, metabolism, regulation, cell wall synthesis and antibiotic resistance, environmental adaptation, or putative functions. In addition, 11 of these 45 genes were assessed for their differential expression by quantitative real-time reverse transcription-PCR (RT-PCR), revealing that 9 of 11 genes were upregulated to different degrees under in vivo conditions, especially the regulator of virulence determinants, phoQ. Then, four in vivo-induced proteins (ShdA, PhoQ, Cse3, and PbpC) were tested for their immunoreactivity in 28 clinical serum samples from chickens infected with S. Pullorum. The rate of detection of antibodies against ShdA reached 82% and was the highest among these proteins. ShdA is a host colonization factor known to be upregulated in vivo and related to the persistence of S. Typhimurium in the intestine. Furthermore, these antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis, and more potential roles, such as diagnostic, therapeutic, and preventive uses, need to be developed in future studies.

Identification ofIn Vivo-Induced Bacterial Proteins during Human Infection with Salmonella enterica Serotype Paratyphi A

Salmonella enterica serotype Paratyphi A is a human-restricted pathogen and the cause of paratyphoid A fever. Using a high-throughput immunoscreening technique, in vivo-induced antigen technology (IVIAT), we identified 20 immunogenic bacterial proteins expressed in humans who were bacteremic with S. Paratyphi A but not those expressed in S. Paratyphi A grown under standard laboratory conditions. The majority of these proteins have known or potential roles in the pathogenesis of S. enterica. These include proteins implicated in cell adhesion, fimbrial structure, adaptation to atypical conditions, oxidoreductase activity, proteolysis, antimicrobial resistance, and ion transport. Of particular interest among these in vivo-expressed proteins were S. Paratyphi A (SPA)2397, SPA2612, and SPA1604. SPA2397 and SPA2612 are prophage related, and SPA1604 is in Salmonella pathogenicity island 11 (SPI-11). Using real-time quantitative PCR (RT-qPCR), we confirmed increased levels of mRNA expressed by genes identified by IVIAT in a comparison of mRNA levels in organisms in the blood of bacteremic patients to those in in vitro cultures. Comparing convalescent- to acute-phase samples, we also detected a significant increase in the reaction of convalescent-phase antibodies with two proteins identified by IVIAT: SPA2397 and SPA0489. SPA2397 is a phage-related lysozyme, Gp19, and SPA0489 encodes a protein containing NlpC/P60 and cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains. In a previous study utilizing a different approach, we found that transcripts for 11 and 7 of the genes identified by IVIAT were detectable in organisms in the blood of humans in Bangladesh who were bacteremic with S. Paratyphi A and Salmonella enterica serovar Typhi, respectively. S. Paratyphi A antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis and might have diagnostic, therapeutic, or preventive relevance.

Edwardsiella tarda Eta1, an In Vivo -Induced Antigen That Is Involved in Host Infection

Edwardsiella tarda, a Gram-negative bacterium, is a severe fish pathogen that can also infect humans. In this study, we identified, via in vivo-induced antigen technology, an E. tarda antigen, Eta1, and analyzed its function in a Japanese flounder (Paralichthys olivaceus) model. Eta1 is composed of 226 residues and shares homology with putative bacterial adhesins. Quantitative real-time reverse transcriptase (RT)-PCR analysis indicated that when cultured in vitro, eta1 expression was growth phase dependent and reached maximum at mid-logarithmic phase. During infection of flounder lymphocytes, eta1 expression was drastically increased at the early stage of infection. Compared to the wild type, the eta1-defective mutant, TXeta1, was unaffected in growth but exhibited attenuated overall virulence, reduced tissue dissemination and colonization capacity, and impaired ability to invade flounder lymphocytes and to block the immune response of host cells. The lost virulence of TXeta1 was restored when a functional eta1 gene was reintroduced into the strain. Western blot and immunodetection analyses showed that Eta1 is localized to the outer membrane and exposed on the surface of E. tarda and that recombinant Eta1 (rEta1) was able to interact with flounder lymphocytes. Consistent with these observations, antibody blocking of Eta1 inhibited E. tarda infection at the cellular level. Furthermore, when used as a subunit vaccine, rEta1 induced strong protective immunity in flounder against lethal E. tarda challenge. Taken together, these results indicate that Eta1 is an in vivo-induced antigen that mediates pathogen-host interaction and, as a result, is required for optimal bacterial infection.

Identification of In Vivo -Induced Antigens Including an RTX Family Exoprotein Required for Uropathogenic Escherichia coli Virulence

Uncomplicated urinary tract infections (UTI) are caused most commonly by uropathogenic Escherichia coli (UPEC). Whole-genome screening approaches, including transcriptomic, proteomic, and signature-tagged mutagenesis, have shown that UPEC highly expresses or requires genes for translational machinery, capsule, lipopolysaccharide, type 1 fimbriae, and iron acquisition systems during UTI. To identify additional genes expressed by UPEC during UTI, an immunoscreening approach termed in vivo-induced antigen technology (IVIAT) was employed to identify antigens produced during experimental infection that are not produced during in vitro culture. An inducible protein expression library, constructed from genomic DNA isolated from UPEC strain CFT073, was screened using exhaustively adsorbed pooled sera from 20 chronically infected female CBA/J mice. Using this approach, we identified 93 antigens induced by UPEC in vivo. A representative subset of these genes was tested by quantitative PCR for expression by CFT073 in vivo and during growth in human urine or LB medium in vitro; proWX, narJI, lolA, lolD, tosA (upxA), c2432, katG, ydhX, kpsS, and yddQ were poorly expressed in vitro but highly expressed in vivo. Of these, tosA, a gene encoding a predicted repeat-in-toxin family member, was expressed exclusively during UTI. Deletion of tosA in UPEC strain CFT073 resulted in significant attenuation in bladder and kidney infections during ascending UTI. By screening for in vivo-induced antigens, we identified a novel UPEC virulence factor and additional proteins that could be useful as potential vaccine targets.

Vaccination with Brucella abortus Recombinant In Vivo-Induced Antigens Reduces Bacterial Load and Promotes Clearance in a Mouse Model for Infection

Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT) on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA) were selected for further characterization and compared with three additional antigens with virulence potential (Hia, PrpA, MltA). All eight genes were PCR-amplified from B. abortus and cloned into E. coli. The recombinant products were then expressed, purified, adjuvanted, and delivered subcutaneously to BALB/c mice. After primary immunization and two boosts, mice were challenged i.p. with 5×104 CFU of B. abortus strain 19. Spleens from challenged animals were harvested and bacterial loads determined by colony count at various time points. While vaccination with four of the eight individual proteins appeared to have some effect on clearance kinetics, mice vaccinated with recombinant Mdh displayed the most significant reduction in bacterial colonization. Furthermore, mice immunized with Mdh maintained higher levels of IFN-γ in spleens compared to other treatment groups. Collectively, our in vivo data gathered from the S19 murine colonization model suggest that vaccination with at least three of the IVIAT antigens conferred an enhanced ability of the host to respond to infection, reinforcing the utility of this methodology for the identification of potential vaccine candidates against brucellosis. Mechanisms for immunity to one protein, Mdh, require further in vitro exploration and evaluation against wild-type B. abortus challenge in mice, as well as other hosts. Additional studies are being undertaken to clarify the role of Mdh and other IVI antigens in B. abortus virulence and induction of protective immunity.