Rational Design of Live-Attenuated Vaccines against Genome-Reduced Pathogens.

To develop safe and highly effective live vaccines, rational vaccine design is necessary. Here, we sought a simple approach to rationally develop a safe attenuated vaccine against the genome-reduced pathogen Erysipelothrix rhusiopathiae. We examined the mRNA expression of all conserved amino acid biosynthetic genes remaining in the genome after the reductive evolution of E. rhusiopathiae. Reverse transcription-quantitative PCR (qRT-PCR) analysis revealed that half of the 14 genes examined were upregulated during the infection of murine J774A.1 macrophages. Gene deletion was possible only for three proline biosynthesis genes, proB, proA, and proC, the last of which was upregulated 29-fold during infection. Five mutants bearing an in-frame deletion of one (ΔproB, ΔproA, or ΔproC mutant), two (ΔproBA mutant), or three (ΔproBAC mutant) genes exhibited attenuated growth during J774A.1 infection, and the attenuation and vaccine efficacy of these mutants were confirmed in mice and pigs. Thus, for the rational design of live vaccines against genome-reduced bacteria, the selective targeting of genes that escaped chromosomal deletions during evolution may be a simple approach for identifying genes which are specifically upregulated during infection. IMPORTANCE Identification of bacterial genes that are specifically upregulated during infection can lead to the rational construction of live vaccines. For this purpose, genome-based approaches, including DNA microarray analysis and IVET (in vivo expression technology), have been used so far; however, these methods can become laborious and time-consuming. In this study, we used a simple in silico approach and showed that in genome-reduced bacteria, the genes which evolutionarily remained conserved for metabolic adaptations during infection may be the best targets for the deletion and construction of live vaccines.

Two Akabane virus glycoprotein Gc domains induce neutralizing antibodies in mice.

Akabane virus (AKAV), belonging to the genus Orthobunyavirus and family Peribunyaviridae, causes reproductive and congenital abnormalities in ruminants. Its envelope glycoprotein Gc is a neutralizing antigen, on which at least five distinct antigenic regions have been identified. We attempted to identify the domains using truncated recombinant AKAV Gc proteins expressed in Escherichia coli and monoclonal antibodies (mAbs) with AKAV-neutralizing activity. Dot blot analysis revealed that amino acid positions 1-97 and 189-397 (Gc1-97 and Gc189-397) in the truncated recombinant proteins reacted with the mAbs. Additionally, AKAV was neutralized by sera from mice immunized with these recombinant proteins. The results suggested that the two domains contain neutralizing epitopes and could be potential subunit vaccines against AKAV.

Application of stabilized hypobromite for controlling membrane fouling and N-nitrosodimethylamine formation.

Chloramination is a conventional and successful pre-disinfection approach to control biological fouling for reverse osmosis (RO) treatment in water reuse. This study aimed to evaluate the possibility of using a new disinfectant-stabilized hypobromite-in controlling membrane fouling and the formation of a particular carcinogenic disinfection byproduct (DBP)-N-nitrosodimethylamine (NDMA). Our accelerated chemical exposure tests showed that the new disinfectant reduced the permeability of a polyamide RO membrane permeability from 6.7 to 4.1 L/m2hbar; however, its treatment impact was equivalent to that of chloramine. The disinfection efficacy of stabilized hypobromite was greater than that of chloramine when evaluated with intact bacterial counts, which suggests its potential for mitigating membrane biofouling. Additional pilot-scale tests using synthetic wastewater demonstrated that pre-disinfection with the use of stabilized hypobromite inhibits membrane fouling. Among 13 halogenated DBPs evaluated, the formation of bromoform by stabilized hypobromite was higher than that by chloramine at a high dose of 10 mg/L, thus suggesting the need for optimizing chemical doses for achieving sufficient biofouling mitigation. NDMA formation upon stabilized hypobromite treatment in two different types of actual treated wastewaters was found to be negligible and considerably lower than that by chloramine treatment. In addition, NDMA formation potential by stabilized hypobromite was 2-5 orders of magnitude lower than that by chloramine. Our findings suggest the potential of using stabilized hypobromite for controlling NDMA formation and biofouling, which are the keys to successful potable water reuse.

Specific Monoclonal Antibody Overcomes the Salmonella enterica Serovar Typhimurium's Adaptive Mechanisms of Intramacrophage Survival and Replication.

Salmonella-specific antibodies play an important role in host immunity; however, the mechanisms of Salmonella clearance by pathogen-specific antibodies remain to be completely elucidated since previous studies on antibody-mediated protection have yielded inconsistent results. These inconsistencies are at least partially attributable to the use of polyclonal antibodies against Salmonella antigens. Here, we developed a new monoclonal antibody (mAb)-449 and identified its related immunogen that protected BALB/c mice from infection with Salmonella enterica serovar Typhimurium. In addition, these data indicate that the mAb-449 immunogen is likely a major protective antigen. Using in vitro infection studies, we also analyzed the mechanism by which mAb-449 conferred host protection. Notably, macrophages infected with mAb-449-treated S. Typhimurium showed enhanced pathogen uptake compared to counterparts infected with control IgG-treated bacteria. Moreover, these macrophages produced elevated levels of pro-inflammatory cytokine TNFα and nitric oxide, indicating that mAb-449 enhanced macrophage activation. Finally, the number of intracellular bacteria in mAb-449-activated macrophages decreased considerably, while the opposite was found in IgG-treated controls. Based on these findings, we suggest that, although S. Typhimurium has the potential to survive and replicate within macrophages, host production of a specific antibody can effectively mediate macrophage activation for clearance of intracellular bacteria.

Mouse models for assessing the cross-protective efficacy of oral non-typhoidal Salmonella vaccine candidates harbouring in-frame deletions of the ATP-dependent protease lon and other genes.

In BALB/c mouse models of Salmonella enterica serovar Typhimurium infection, a single oral immunization with a mutant strain with an insertion of the chloramphenicol resistance gene into the ATP-dependent protease clpP or lon gene decreased the number of salmonellae in each tissue sample 5 days after oral challenge with virulent S. Typhimurium at weeks 26 and 54 post-immunization. These data suggested that an oral immunization with the ClpP- or Lon-disrupted S. Typhimurium strain could provide long-term protection against oral challenge with virulent S. Typhimurium. Accordingly, recombinant oral non-typhoidal Salmonella (NTS) vaccines were constructed by incorporating mutants of both S. Typhimurium and S. enterica serovar Enteritidis harbouring stable in-frame markerless deletions of the clpP-lon-sulA (suppressor of lon), lon-sulA or lon-msbB (acyltransferase) genes. Amongst these orally administered vaccine candidates, those with the lon-sulA gene deletion mutants of S. Typhimurium and S. Enteritidis protected BALB/c and C57BL/6J mice against oral challenge with both virulent S. Typhimurium and virulent S. Enteritidis. Therefore, the in-frame markerless lon-sulA gene deletion mutant of S. Typhimurium or S. Enteritidis could be a promising cross-protective NTS live vaccine candidate for practical use in humans.

Characterization and identification of a novel candidate vaccine protein through systematic analysis of extracellular proteins of Erysipelothrix rhusiopathiae.

Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, is a facultative intracellular Gram-positive bacterium. It has been shown that animals immunized with a filtrate from E. rhusiopathiae cultures are protected against lethal challenge. In this study, we identified and characterized the extracellular proteins of E. rhusiopathiae to search for novel vaccine antigens. A concentrated culture supernatant from the E. rhusiopathiae Fujisawa strain, which has been found to induce protection in mice, was analyzed using two-dimensional electrophoresis. From more than 40 confirmed protein spots, 16 major protein spots were selected and subjected to N-terminal amino acid sequence determination, and 14 protein spots were successfully identified. The identified proteins included housekeeping proteins and other metabolic enzymes. We searched for surface-localized proteins by analyzing the genomes of two E. rhusiopathiae strains: Fujisawa and ATCC 19414. Genome analysis revealed that the ATCC 19414 strain has three putative surface-exposed choline-binding proteins (CBPs): CbpA, CbpB, and CbpC. Each CBP contains a putative choline-binding domain. The CbpC gene is mutated in Fujisawa, becoming a nonfunctional pseudogene. Immunogold electron microscopy confirmed that CbpA and CbpB, as well as the majority of the metabolic enzymes examined, are associated with the cell surface of E. rhusiopathiae Fujisawa. Immunization with recombinant CbpB, but not with other recombinant CBPs or metabolic enzymes, protected mice against lethal challenge. A phagocytosis assay revealed that antiserum against CbpB promoted opsonin-mediated phagocytosis by murine macrophages in vitro. The protective capabilities of CbpB were confirmed in pigs, suggesting that CbpB could be used as a vaccine antigen.

Erysipelothrix rhusiopathiae exploits cytokeratin 18-positive epithelial cells of porcine tonsillar crypts as an invasion gateway.

Tonsils are important organs for mucosal immunity and are gateways for various pathogens, including bacteria and viruses. The purpose of the present study was to reveal how Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, invades the mucosal epithelium of the tonsils of pigs. Two germ-free piglets were orally infected with E. rhusiopathiae Koganei 65-0.15, an attenuated vaccine strain in Japan, and their tonsils of the soft palate were histologically examined four weeks after infection. Bacterial organisms were observed in dilated crypt lumens and a few epithelial cells of the crypt. Immunohistochemical examination revealed that some epithelial cells of the crypt were positive for cytokeratin (CK) 18, a specific marker for M cells in the Peyer's patches of pigs. Confocal laser scanning microscopy showed that bacterial antigens were present in the cytoplasm of CK 18-positive epithelial cells. Furthermore, an ultramicroscopic examination revealed that the bacteria-containing epithelial cells did not have microfolds or microvilli, both of which are characteristic of membranous epithelial cells (M cells), and that they were in close contact with intraepithelial phagocytes. Thus, the present observations suggest that the tonsillar crypt epithelium is a site of persistent infection for orally administered E. rhusiopathiae, and the bacteria exploit cytokeratin 18-positive epithelial cells of the crypts as portals of entry into the body.

Binding of Salmonella-specific antibody facilitates specific T cell responses via augmentation of bacterial uptake and induction of apoptosis in macrophages.

BACKGROUND: Most antigens from intracellular bacteria or vaccines induce both humoral and cell-mediated immune responses, but interactions between these responses are not fully understood. This study aims to resolve how specific antibodies participate in the activation of specific T cells in protecting hosts against Salmonella enterica serotype Typhimurium (S. typhimurium) infection. METHODS: Mice were administered anti-Salmonella immunoglobulin G (IgG) 1 day before Salmonella infection, and survival rate was observed. For in vitro assay, Salmonella bacteria were treated with anti-Salmonella IgG or control IgG before infection of the RAW264.7 or HEp2 cells. After infection, cell-associated bacteria number, induction of apoptosis, and production of nitric oxide were examined. In addition, antigen presentation assays using Salmonella-primed T cells were performed. RESULTS: Treatment of S. typhimurium with anti-Salmonella IgG enhanced the macrophages' uptake of bacteria and induced high-frequency apoptotic cell death. In vitro antigen presentation assay revealed that the extracellular vesicles isolated from apoptotic cells caused by infection with anti-Salmonella IgG-treated S. typhimurium facilitated the responses of Salmonella-specific T cells. CONCLUSION: Our findings suggest that humoral immunity cooperates with cell-mediated immunity upon induction of apoptosis in host cells to establish protective immunity against Salmonella infection, even if it does not directly eliminate intracellular microorganisms.

Expressed Salmonella antigens within macrophages enhance the proliferation of CD4+ and CD8+ T lymphocytes by means of bystander dendritic cells.

ATP-dependent Lon protease-deficient Salmonella enterica serovar Typhimurium (strain CS2022) appeared to invade successfully the mesenteric lymph nodes (MLN) and Peyer's patches (PP) of BALB/c mice and appeared to be easily eradicated by the host after oral immunization. As detected by flow cytometry, the population of major histocompatibility complex class I (MHC-I)-expressing macrophages and dendritic cells (DCs) was increased in the PP of mice immunized with CS2022 on day 6 after immunization. Thereafter, the population of splenic surface CD69(+) T lymphocytes prepared from mice immunized with CS2022 6 weeks prior to measurement increased as a result of the administration of the extracellular vesicles of RAW264.7 macrophage-like cells derived by Salmonella challenge. In addition, the proliferation of CD8(+) and even of CD4(+)T cells isolated from mouse spleens immunized with CS2022 was enhanced after cocultivation with naive DCs in the presence of the extracellular vesicles. These findings indicate that the extracellular vesicles prepared from the Salmonella-challenged macrophages carried salmonellae antigens to bystander DCs, thereby stimulating T-cell responses. Therefore, as antigen presentation after phagocytosis should be a central process in the T-cell activation that occurs in response to Salmonella infection, an oral immunization with CS2022 sufficiently induces T cell-mediated immunity in mice.

"Candidatus Helicobacter heilmannii" from a cynomolgus monkey induces gastric mucosa-associated lymphoid tissue lymphomas in C57BL/6 mice.

Both Helicobacter pylori and "Candidatus Helicobacter heilmannii" infections are associated with peptic ulcers, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphomas. However, good animal models of H. pylori clinical diseases are rare. In this study, we aimed to establish an animal model of "Candidatus Helicobacter heilmannii" gastric MALT lymphoma. We used a urease-positive gastric mucosal and mucus homogenate from a cynomolgus monkey maintained in C57BL/6 mouse stomachs. The bacterium in the homogenate was identified as "Candidatus Helicobacter heilmannii" based on a DNA sequence analysis of the 16S rRNA and urease genes. Mucosal and mucus homogenates were used to inoculate C57BL/6 mice, which were then examined for 24 months. We observed a gradual increase in the surface area of protrusive lesions in almost all infected C57BL/6 mouse fundic stomachs 6 months after infection. Light microscopic observations revealed an accumulation of B lymphocytes along with destruction of glandular elements and the presence of lymphoepithelial lesions consistent with low-grade MALT lymphomas. Electron microscopic observation revealed numerous "Candidatus Helicobacter heilmannii" bacilli in the fundic glandular lumen, the intracellular canaliculi, and the cytoplasm of intact cells, as well as damaged parietal cells. In conclusion, "Candidatus Helicobacter heilmannii" induced gastric MALT lymphomas in almost 100% of infected C57BL/6 mice after a 6-month period associated with the destruction of parietal cells.

Azithromycin inhibits the formation of flagellar filaments without suppressing flagellin synthesis in Salmonella enterica serovar typhimurium.

The present study shows that a sub-MIC of the macrolide antibiotic azithromycin (AZM) diminishes the virulence function of Salmonella enterica serovar Typhimurium. We first constructed an AZM-resistant strain (MS248) by introducing ermBC, an erythromycin ribosome methylase gene, into serovar Typhimurium. The MIC of AZM for MS248 exceeded 100 microg/ml. Second, we managed to determine the efficacy with which a sub-MIC of AZM reduced the virulence of MS248 in vitro. On the one hand, AZM (10 microg/ml) in the culture medium was unable to inhibit the total protein synthesis, growth rate, or survival within macrophages of MS248. On the other hand, AZM (10 microg/ml) reduced MS248's swarming and swimming motilities in addition to its invasive activity in Henle-407 cells. Electron micrographs revealed no flagellar filaments on the surface of MS248 after overnight growth in L broth supplemented with AZM (10 microg/ml). However, immunoblotting analysis showed that flagellin (FliC) was fully synthesized within the bacterial cells in the presence of AZM (10 microg/ml). In contrast, the same concentration of AZM reduced the export of FliC to the culture medium. These results indicate that a sub-MIC of AZM was able to affect the formation of flagellar filaments, specifically by reducing the amount of flagellin exported from bacterial cells, but it was not involved in suppressing the synthesis of flagellin. Unfortunately, AZM treatment was ineffective against murine salmonellosis caused by MS248.

Derepression of Salmonella pathogenicity island 1 genes within macrophages leads to rapid apoptosis via caspase-1- and caspase-3-dependent pathways.

Salmonella enterica serovar Typhimurium has been reported to induce apoptosis in infected macrophages within 14 h from the time of infection by a caspase-1-dependent mechanism. Here, we demonstrate that depletion of Lon protease in serovar Typhimurium induces rapid and massive apoptosis in macrophages by a mechanism involving both caspases-1 and -3. This excessive induction of apoptosis was abrogated by disruption of invF, which is required for the expression of the Salmonella pathogenicity island 1 (SPI1) genes. Expression of hilA, a central regulator of SPI1 transcription, was repressed in the macrophages after phagocytosis, but this gene was continuously expressed when the DeltaLon mutant grew within the macrophages, so the SPI1 proteins accumulated. Thus, the increase in macrophage apoptosis induced by the DeltaLon mutant could result from continued expression of SPI1 genes under conditions where they are normally repressed. Once Salmonella has established a systemic infection, excess apoptosis of macrophages cells upon which the organism is reliant would be detrimental to the pathogen. Therefore, the Lon protease may be required to suppress apoptosis sufficiently to allow time for the bacterium to replicate, escape and invade new macrophages.

Oral immunization with ATP-dependent protease-deficient mutants protects mice against subsequent oral challenge with virulent Salmonella enterica serovar typhimurium.

We evaluated the efficacy of mutants with a deletion of the stress response protease gene as candidates for live oral vaccine strains against Salmonella infection through infection studies with mice by using a Salmonella enterica serovar Typhimurium mutant with a disruption of the ClpXP or Lon protease. In vitro, the ClpXP protease regulates flagellum synthesis and the ClpXP-deficient mutant strain exhibits hyperflagellated bacterial cells (T. Tomoyasu et al., J. Bacteriol. 184:645-653, 2002). On the other hand, the Lon protease negatively regulates the efficacy of invading epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). When 5-week-old BALB/c mice were orally administered 5 x 10(8) CFU of the ClpXP- or Lon-deficient strain, bacteria were detected with 10(3) to 10(4) CFU in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum 1 week after inoculation and the bacteria then decreased gradually in each tissue. Significant increases of lipopolysaccharide-specific immunoglobulin G (IgG) and secretory IgA were detected at week 4 and maintained until at least week 12 after inoculation in serum and bile, respectively. Immunization with the ClpXP- or Lon-deficient strain protected mice against oral challenge with the serovar Typhimurium virulent strain. Both the challenged virulent and immunized avirulent salmonellae were completely cleared from the spleen, mesenteric lymph nodes, Peyer's patches, and even cecum 5 days after the challenge. These data indicate that Salmonella with a disruption of the ATP-dependent protease ClpXP or Lon can be useful in developing a live vaccine strain.