Evaluation of host and bacterial gene modulation during Lawsonia intracellularis infection in immunocompetent C57BL/6 mouse model

Background@#Proliferative enteritis caused by Lawsonia intracellularis undermines the economic stability of the swine industry worldwide. The development of cost-effective animal models to study the pathophysiology of the disease will help develop strategies to counter this bacterium. @*Objectives@#This study focused on establishing a model of gastrointestinal (GI) infection of L.intracellularis in C57BL/6 mice to evaluate the disease progression and lesions of proliferative enteropathy (PE) in murine GI tissue. @*Methods@#We assessed the murine mucosal and cell-mediated immune responses generated in response to inoculation with L. intracellularis. @*Results@#The mice developed characteristic lesions of the disease and shed L.intracellularis in the feces following oral inoculation with 5 × 107 bacteria. An increase in L. intracellularis 16s rRNA and groEL copies in the intestine of infected mice indicated intestinal dissemination of the bacteria. The C57BL/6 mice appeared capable of modulating humoral and cell-mediated immune responses to L. intracellularis infection. Notably, the expression of genes for the vitamin B12 receptor and for secreted and membrane-bound mucins were downregulated in L. intracellularis -infected mice. Furthermore, L. intracellularis colonization of the mouse intestine was confirmed by the immunohistochemistry and western blot analyses. @*Conclusions@#This is the first study demonstrating the contributions of bacterial chaperonin and host nutrient genes to PE using an immunocompetent mouse model. This mouse infection model may serve as a platform from which to study L. intracellularis infection and develop potential vaccination and therapeutic strategies to treat PE.

Protective efficacy of attenuated Salmonella Typhimurium strain expressing BLS, Omp19, PrpA, or SOD of Brucella abortus in goats

Background@#Attenuated Salmonella strain can be used as a vector to transport immunogens to the host antigen-binding sites. @*Objectives@#The study aimed to determine the protective efficacy of attenuated Salmonellastrain expressing highly conserved Brucella immunogens in goats. @*Methods@#Goats were vaccinated with Salmonella vector expressing individually lipoprotein outer-membrane protein 19 (Omp19), Brucella lumazine synthase (BLS), proline racemase subunit A (PrpA), Cu/Zn superoxide dismutase (SOD) at 5 × 10 9 CFU/mL and challenge of all groups was done at 6 weeks after vaccination. @*Results@#Among these vaccines inoculated at 5 × 10 9 CFU/mL in 1 mL, Omp19 or SOD showed significantly higher serum immunoglobulin G titers at (2, 4, and 6) weeks post-vaccination, compared to the vector control. Interferon-γ production in response to individual antigens was significantly higher in SOD, Omp19, PrpA, and BLS individual groups, compared to that in the vector control (all p < 0.05). Brucella colonization rate at 8 weeks post-challenge showed that most vaccine-treated groups exhibited significantly increased protection by demonstrating reduced numbers of Brucella in tissues collected from vaccinated groups. Realtime polymerase chain reaction revealed that Brucella antigen expression levels were reduced in the spleen, kidney, and parotid lymph node of vaccinated goats, compared to the nonvaccinated goats. Besides, treatment with vaccine expressing individual antigens ameliorated brucellosis-related histopathological lesions. @*Conclusions@#These results delineated that BLS, Omp19, PrpA, and SOD proteins achieved a definite level of protection, indicating that Salmonella Typhimurium successfully delivered Brucella antigens, and that individual vaccines could differentially elicit an antigen-specific immune response.

Protection of palmitic acid treatment in RAW264.7 cells and BALB/c mice during Brucella abortus 544 infection

Background@#We previously elucidated the protective mechanism of Korean red ginseng oil (RGO) against Brucella abortus infection, and our phytochemical analysis revealed that palmitic acid (PA) was an abundant component of RGO. Consequently, we investigated the contribution of PA against B. abortus. @*Objectives@#We aimed to investigate the efficacy of PA against B. abortus. infection using a murine cell line and a murine model. @*Methods@#Cell viability, bactericidal, internalization, and intracellular replication, western blot, nitric oxide (NO), and superoxide (O2 - ) analyses and flow cytometry were performed to determine the effects of PA on the progression of B. abortus. infection in macrophages. Flow cytometry for cytokine analysis of serum samples and bacterial counts from the spleens were performed to determine the effect of PA in a mouse model. @*Results@#PA did not affect the growth of B. abortus.. PA treatment in macrophages did not change B. abortus. uptake but it did attenuate the intracellular survivability of B. abortus.. Incubation of cells with PA resulted in a modest increase in sirtuin 1 (SIRT1) expression.Compared to control cells, reduced nitrite accumulation, augmented O2 - , and enhanced pro-inflammatory cytokine production were observed in PA-treated B. abortus.-infected cells.Mice orally treated with PA displayed a decreased serum interleukin-10 level and enhanced bacterial resistance. @*Conclusions@#Our results suggest that PA participates in the control of B. abortus. within murine macrophages, and the in vivo study results confirm its efficacy against the infection. However, further investigations are encouraged to completely characterize the mechanisms involved in the inhibition of B. abortus. infection by fatty acids.

Protective efficacy of attenuated Salmonella Typhimurium strain expressing BLS, Omp19, PrpA, or SOD of Brucella abortus in goats

Background@#Attenuated Salmonella strain can be used as a vector to transport immunogens to the host antigen-binding sites. @*Objectives@#The study aimed to determine the protective efficacy of attenuated Salmonellastrain expressing highly conserved Brucella immunogens in goats. @*Methods@#Goats were vaccinated with Salmonella vector expressing individually lipoprotein outer-membrane protein 19 (Omp19), Brucella lumazine synthase (BLS), proline racemase subunit A (PrpA), Cu/Zn superoxide dismutase (SOD) at 5 × 10 9 CFU/mL and challenge of all groups was done at 6 weeks after vaccination. @*Results@#Among these vaccines inoculated at 5 × 10 9 CFU/mL in 1 mL, Omp19 or SOD showed significantly higher serum immunoglobulin G titers at (2, 4, and 6) weeks post-vaccination, compared to the vector control. Interferon-γ production in response to individual antigens was significantly higher in SOD, Omp19, PrpA, and BLS individual groups, compared to that in the vector control (all p < 0.05). Brucella colonization rate at 8 weeks post-challenge showed that most vaccine-treated groups exhibited significantly increased protection by demonstrating reduced numbers of Brucella in tissues collected from vaccinated groups. Realtime polymerase chain reaction revealed that Brucella antigen expression levels were reduced in the spleen, kidney, and parotid lymph node of vaccinated goats, compared to the nonvaccinated goats. Besides, treatment with vaccine expressing individual antigens ameliorated brucellosis-related histopathological lesions. @*Conclusions@#These results delineated that BLS, Omp19, PrpA, and SOD proteins achieved a definite level of protection, indicating that Salmonella Typhimurium successfully delivered Brucella antigens, and that individual vaccines could differentially elicit an antigen-specific immune response.

Protection of palmitic acid treatment in RAW264.7 cells and BALB/c mice during Brucella abortus 544 infection

Background@#We previously elucidated the protective mechanism of Korean red ginseng oil (RGO) against Brucella abortus infection, and our phytochemical analysis revealed that palmitic acid (PA) was an abundant component of RGO. Consequently, we investigated the contribution of PA against B. abortus. @*Objectives@#We aimed to investigate the efficacy of PA against B. abortus. infection using a murine cell line and a murine model. @*Methods@#Cell viability, bactericidal, internalization, and intracellular replication, western blot, nitric oxide (NO), and superoxide (O2 - ) analyses and flow cytometry were performed to determine the effects of PA on the progression of B. abortus. infection in macrophages. Flow cytometry for cytokine analysis of serum samples and bacterial counts from the spleens were performed to determine the effect of PA in a mouse model. @*Results@#PA did not affect the growth of B. abortus.. PA treatment in macrophages did not change B. abortus. uptake but it did attenuate the intracellular survivability of B. abortus.. Incubation of cells with PA resulted in a modest increase in sirtuin 1 (SIRT1) expression.Compared to control cells, reduced nitrite accumulation, augmented O2 - , and enhanced pro-inflammatory cytokine production were observed in PA-treated B. abortus.-infected cells.Mice orally treated with PA displayed a decreased serum interleukin-10 level and enhanced bacterial resistance. @*Conclusions@#Our results suggest that PA participates in the control of B. abortus. within murine macrophages, and the in vivo study results confirm its efficacy against the infection. However, further investigations are encouraged to completely characterize the mechanisms involved in the inhibition of B. abortus. infection by fatty acids.

An attenuated Salmonella vaccine secreting Lawsonia intracellularis immunogenic antigens confers dual protection against porcine proliferative enteropathy and salmonellosis in a murine model

Porcine proliferative enteropathy (PPE) caused by Lawsonia intracellularis (LI) is a global cause for substantial economic losses in the swine industry. Here, we constructed live attenuated Salmonella typhimurium (ST) mutant strains expressing and secreting 4 selected immunogenic LI antigens, namely, optA, optB, Lawsonia flagellin (LfliC), and Lawsonia hemolysin (Lhly); the resultant recombinant strains were designated Sal-optA, Sal-optB, Sal-LfliC, or Sal-Lhly, respectively. Using the BALB/c mouse model, we demonstrate that mice vaccinated once orally, either with a mixture of all 4 recombinant strains or with an individual recombinant strain, show significant (p < 0.05) production of LI-specific systemic immunoglobulin (Ig) G and mucosal IgA responses compared to the Salmonella alone group. Upon restimulation of vaccinated splenocytes with the LI-specific antigens, significant (p < 0.05) and comparable production of interferon-γ responses are found in all vaccinated groups, except the Sal-Lhly group, which shows non-significant levels. Challenge studies were performed in C57BL/6 vaccinated mice. On challenge with the LI (10(6.9) 50% tissue culture infectious dose) 14 days post-vaccination, 20% (1/5) of mice in all vaccinated groups, except Sal-Lhly group, show the presence of the LI-specific genomic DNA (gDNA) in stool samples. In contrast, 40% (2/5) and 60% (3/5) of mice vaccinated with the Sal-Lhly strain and the attenuated Salmonella alone, respectively, were found positive for the LI-specific gDNA. Furthermore, 0% mortality was observed in mice vaccinated against the ST challenge compared to the 30% mortality observed in the unvaccinated control group. In conclusion, we demonstrate that the Salmonella-based LI-vaccines induce LI-specific humoral and cell-mediated immunities, and encompass the potential to offer dual protection against PPE and salmonellosis.

Vaccination of goats with a combination Salmonella vector expressing four Brucella antigens (BLS, PrpA, Omp19, and SOD) confers protection against Brucella abortus infection

Salmonella is an intracellular pathogen with a cellular infection mechanism similar to that of Brucella, making it a suitable choice for use in an anti-Brucella immune boost system. This study explores the efficacy of a Salmonella Typhimurium delivery-based combination vaccine for four heterologous Brucella antigens (Brucella lumazine synthase, proline racemase subunit A, outer-membrane protein 19, and Cu/Zn superoxide dismutase) targeting brucellosis in goats. We inoculated the attenuated Salmonella delivery-based vaccine combination subcutaneously at two different inoculation levels; 5 × 10⁹ colony-forming unit (CFU)/mL (Group B) and 5 × 10¹⁰ CFU/mL (Group C) and challenged the inoculations with virulent Brucella abortus at 6 weeks post-immunization. Serum immunoglobulin G titers against individual antigens in Salmonella immunized goats (Group C) were significantly higher than those of the non-immunized goats (Group A) at 3 and 6 weeks after vaccination. Upon antigenic stimulation, interferon-γ from peripheral blood mononuclear cells was significantly elevated in Groups B and C compared to that in Group A. The immunized goats had a significantly higher level of protection as demonstrated by the low bacterial loads in most tissues from the goats challenged with B. abortus. Relative real-time polymerase chain reaction results revealed that the expression of Brucella antigens was lower in spleen, kidney, and lung of immunized goats than of non-immunized animals. Also, treatment with our combination vaccine ameliorated histopathological lesions induced by the Brucella infection. Overall, the Salmonella Typhimurium delivery-based combination vaccine was effective in delivering immunogenic Brucella proteins, making it potentially useful in protecting livestock from brucellosis.

A Salmonella Typhi ghost induced by the E gene of phage φX174 stimulates dendritic cells and efficiently activates the adaptive immune response

Previously, we genetically engineered a Salmonella Typhi bacterial ghost (STG) as a novel inactivated vaccine candidate against typhoid fever. The underlying mechanism employed by the ghost in stimulating the adaptive immune response remains to be investigated. In this study, we aimed to evaluate the immunostimulatory effect of STG on mouse bone marrow-derived dendritic cells (BMDCs) and its activation of the adaptive immune response in vitro. Immature BMDCs were stimulated with STG, which efficiently stimulated maturation events in BMDCs, as indicated by upregulated expressions of CD40, CD80, and major histocompatibility complex class II molecules on CD11⁺ BMDCs. Immature BMDCs responded to STG stimulation by significantly increasing the expression of interleukin (IL)-6, which might indicate the induction of dendritic cell maturation in vivo (p < 0.05). In addition, ghost-stimulated murine BMDCs showed significant expressions of interferon gamma and IL-4, which can drive the development of Th1 and Th2 cells, respectively, in co-cultured CD4⁺ T cells in vitro. These results suggest that STG can effectively stimulate maturation of BMDCs and facilitate subsequent immune responses via potent immunomodulatory cytokine responses.

Effect of immunization routes and protective efficacy of Brucella antigens delivered via Salmonella vector vaccine

An anti-Brucella vaccine candidate comprised of purified Brucella lipopolysaccharide (LPS) and a cocktail of four Salmonella Typhimurium (ST)-Brucella vectors was reported previously. Each vector constitutively expressed highly conserved Brucella antigens (rB), viz., lumazine synthase (BLS), proline racemase subunit A, outer membrane protein-19 (Omp19), and Cu-Zn superoxide dismutase (SOD). The present study determined a relative level of protection conferred by each single strain. Upon virulent challenge, the challenge strain was recovered most abundantly in non-immunized control mice, with the ST-Omp19-, ST-BLS-, LPS-, and ST-SOD-immunized mice showing much less burden. Indirect enzyme-linked immunosorbent assay-based assay also confirmed the induction of antigen-specific immunoglobulin G for each antigen delivered. In a route-wise comparison of the combined vaccine candidate, intraperitoneal (IP), intramuscular (IM), and subcutaneous immunizations revealed an indication of highly efficient routes of protection. Splenocytes of mice immunized via IM and IP routes showed significant relative expression of IL-17 upon antigenic pulsing. Taken together, each of the Brucella antigens delivered by ST successfully induced an antigen-specific immune response, and it was also evident that an individual antigen strain can confer a considerable degree of protection. More effective protection was observed when the candidate was inoculated via IP and IM routes.

Development and trial of vaccines against Brucella

The search for ideal brucellosis vaccines remains active today. Currently, no licensed human or canine anti-brucellosis vaccines are available. In bovines, the most successful vaccine (S19) is only used in calves, as adult vaccination results in orchitis in male, prolonged infection, and possible abortion complications in pregnant female cattle. Another widely deployed vaccine (RB51) has a low protective efficacy. An ideal vaccine should exhibit a safe profile as well as enhance protective efficacy. However, currently available vaccines exhibit one or more major drawbacks. Smooth live attenuated vaccines suffer shortcomings such as residual virulence and serodiagnostic interference. Inactivated vaccines, in general, confer relatively low levels of protection. Recent developments to improve brucellosis vaccines include generation of knockout mutants by targeting genes involved in metabolism, virulence, and the lipopolysaccharide synthesis pathway, as well as generation of DNA vaccines, mucosal vaccines, and live vectored vaccines, have all produced varying degrees of success. Herein, we briefly review the bacteriology, pathogenesis, immunological implications, candidate vaccines, vaccinations, and models related to Brucella.

Construction of a live attenuated Salmonella strain expressing FanC protein to prevent bovine enterotoxigenic Escherichia coli and evaluation of its immunogenicity in mice

To construct a novel vaccine candidate against bovine enterotoxigenic Escherichia coli (ETEC), FanC, the major subunit of K99 fimbriae adhesion, was inserted into secretion plasmid pYA3560 containing a β-lactamase secretion system. This was then transformed into Δasd Δcrp Salmonella (S.) Typhimurium and designated as JOL950. Secretion of recombinant fanC fimbrial antigens was confirmed by immunoblot analysis. Groups of mice were inoculated with single or double doses of JOL950. Another group was used as a negative control. Compared to control mice, all immunized mice had significantly higher levels (p < 0.05) of serum immunoglobulin (Ig)G, and secretory IgA against FanC. The IgG2a and IgG1 titer assays revealed that immunization highly induced IgG2a compared to that of IgG1, indicating that T helper-1- related cell-mediated immune responses may be elicited by JOL950. The results show that both systemic and mucosal immunities against selected fimbrial antigens of bovine ETEC expressed by a live attenuated S. Typhimurium strain are prominently produced in mice immunized with JOL950 via an oral route.

Bacterial flagellin—a potent immunomodulatory agent

Flagellin is a subunit protein of the flagellum, a whip-like appendage that enables bacterial motility. Traditionally, flagellin was viewed as a virulence factor that contributes to the adhesion and invasion of host cells, but now it has emerged as a potent immune activator, shaping both the innate and adaptive arms of immunity during microbial infections. In this review, we summarize our understanding of bacterial flagellin and host immune system interactions and the role flagellin as an adjuvant, anti-tumor and radioprotective agent, and we address important areas of future research interests.

Protective efficacy and immune responses by homologous prime-booster immunizations of a novel inactivated Salmonella Gallinarum vaccine candidate

PURPOSE: Salmonella enterica serovar Gallinarum (SG) ghost vaccine candidate was recently constructed. In this study, we evaluated various prime-boost vaccination strategies using the candidate strain to optimize immunity and protection efficacy against fowl typhoid. MATERIALS AND METHODS: The chickens were divided into five groups designated as group A (non-immunized control), group B (orally primed and boosted), group C (primed orally and boosted intramuscularly), group D (primed and boosted intramuscularly), and group E (primed intramuscularly and boosted orally). The chickens were primed with the SG ghost at 7 days of age and were subsequently boosted at the fifth week of age. Post-immunization, the plasma IgG and intestinal secretory IgA (sIgA) levels, and the SG antigen-specific lymphocyte stimulation were monitored at weekly interval and the birds were subsequently challenged with a virulent SG strain at the third week post-second immunization. RESULTS: Chickens in group D showed an optimized protection with significantly increased plasma IgG, sIgA, and lymphocyte stimulation response compared to all groups. The presence of CD4+ and CD8+ T cells and monocyte/macrophage (M/M) in the spleen, and splenic expression of cytokines such as interferon γ (IFN-γ) and interleukin 6 (IL-6) in the immunized chickens were investigated. The prime immunization induced significantly higher splenic M/M population and mRNA levels of IFN-γ whereas the booster showed increases of splenic CD4+ and CD8+ T-cell population and IL-6 cytokine in mRNA levels. CONCLUSION: Our results indicate that the prime immunization with the SG ghost vaccine induced Th1 type immune response and the booster elicited both Th1- and Th2-related immune responses.

Hens immunized with live attenuated Salmonella strains expressing virulence-associated genes in avian pathogenic Escherichia coli passively transfer maternal antibodies to chicks

We investigated whether maternal antibodies (mAbs) elicited by dams immunized with recombinant vaccine candidates against avian pathogenic Escherichia coli (APEC) can passively confer protective immunity to chicks. In the present study, pBP244 plasmids carrying selected antigens of APEC were transformed into Salmonella Typhimurium JOL912, which was used as a vaccine candidate against APEC. The hens were immunized with the vaccine candidates using prime or booster doses. The levels of IgG and sIgA specific to the selected antigens increased significantly following prime immunization. To evaluate the persistence of passively transferred mAbs, the levels of IgY and IgA were determined in egg yolks and whites, respectively. The eggs from the immunized group showed consistently increased levels of IgY and IgA until week 16 post-laying (PL) and week 8 PL, respectively, relative to the control group. The presence of mAbs was observed in chicks that hatched from the hens, and titers of plasma IgY were consistently raised in those from the immunized hens by day 14 post-hatching. Further, chicks from the immunized hens were protected from challenge with a virulent APEC strain, whereas those from non-immunized hens showed acute mortality.

Protection Against Salmonella Typhimurium, Salmonella Gallinarum, and Salmonella Enteritidis Infection in Layer Chickens Conferred by a Live Attenuated Salmonella Typhimurium Strain

In the present study, we investigated the protection conferred by a live attenuated Salmonella enterica serovar Typhimurium (ST) strain against Salmonella Typhimurium, Salmonella Gallinarum (SG), and Salmonella Enteritidis (SE) infection in layer chickens. Birds were orally primed with the attenuated ST strain at 7 days of age and then boosted at 4 weeks post prime immunization (PPI). Sequential monitoring of plasma IgG and mucosal secretory IgA (sIgA) levels revealed that inoculation with ST induced a significant antibody response to antigens against ST, SE, and SG. Moreover, significant lymphoproliferative responses to the 3 Salmonella serovars were observed in the immunized group. We also investigated protection against virulent ST, SE, and SG strain challenge. Upon virulent SG challenge, the immunized group showed significantly reduced mortality compared to the non-immunized group. The reduced persistence of the virulent ST and SE challenge strains in the liver, spleen, and cecal tissues of the immunized group suggests that immunization with the attenuated ST strain may not only protect against ST infection but can also confer cross protection against SE and SG infection.

Evaluation of systemic and mucosal immune responses in mice administered with recombinant Salmonella Typhimurium expressing IutA protein / 대한수의학회지

Avian pathogenic Escherichia coli (APEC) are known to cause extraintestinal disease in poultry, leading to substantial losses in the industry. IutA, iron-regulated aerobactin receptor is firmly associated with APEC. To assess the potential of IutA to induce protective immune responses, attenuated Salmonella Typhimurium strain expressing IutA was constructed and administered orally to BALB/c mice. The IutA-specific immune responses were measured with sera, vaginal and fecal samples by an enzyme-linked immunosorbent assay. We found that the Salmonella-IutA vaccine induced significantly higher immune responses as compared to the control inoculated with the attenuated S. Typhimurium containing the plasmid only. The IutA-specific immune responses were increased by second immunization at third week after initial immunization, whereas triple immunization induced lower immune responses than those induced by the double immunization. The Salmonella-IutA vaccine induced a nature of immunity biased to the Th1-type, as judged by the ratio of IutA-specific IgG isotypes (IgG2a/IgG1). Overall, these results suggest that the Salmonella-IutA vaccine appear to be suitable candidate for a vaccine against APEC.

Evaluation of systemic and mucosal immune responses in mice administered with novel recombinant Salmonella vaccines for avian pathogenic Esherichia coli / 대한수의학회지

Avian pathogenic Escherichia coli (APEC) is a causative agent for a number of extra intestinal diseases and account for significant losses to the poultry industry. Since protective immunity against APEC is largely directed to virulence antigens, we have individually expressed four different viulence antigens, papA, papG, IutA, and CS31A, using an attenuated Salmonella Typhimurium and a plasmid pBB244. Following oral immunization of mice with combination of two or four of these strains, serum IgG and mucosal IgA responses were elicited against each antigen represented in the mixture. The antigen-specific mucosal IgA responses were significantly higher in the group of mice immunized with the heat-labile Escherichia coli enterotoxin B subunit (LTB) strain than those in the group of mice immunized without the LTB strain. While, there was no significant difference between these two groups in antigen-specific serum IgG responses. The results showed that LTB could act as mucosal immune adjuvant. To assess the nature of immunity, the distribution of antigen-specific IgG isotypes was analyzed. All groups promoted Th1-type immunity as determined by the IgG2a/IgG1 ratio. Thus, our findings provided evidence that immunization with a combination of several vaccine strains is one of the strategies of developing effective vaccines against APEC.

Identification and prevalence of Ehrlichia chaffeensis infection in Haemaphysalis longicornis ticks from Korea by PCR, sequencing and phylogenetic analysis based on 16S rRNA gene

Genomic DNAs extracted from 1,288 Haemaphysalis longicornis ticks collected from grass vegetation and various animals from nine provinces of Korea were subjected to screening by genus-specific (Ehrlichia spp. or Anaplasma spp.) real-time TaqMan PCR and speciesspecific (E. chaffeensis) nested-PCR based on amplification of 16S rRNA gene fragments. In all, 611 (47.4%) ticks tested positive for genus-specific amplification of 116 bp fragment of 16S rRNA of Ehrlichia spp. or Anaplasma spp. Subsequently, 396 bp E. chaffeensis-specific fragment of 16S rRNA was amplified from 4.2% (26/611) tick samples. The comparison of the nucleotide sequence of 16S rRNA gene from one tick (EC-PGHL, GeneBank accession number AY35042) with the sequences of 20 E. chaffeensis strains available in the database showed that EC-PGHL was 100% identical or similar to the Arkansas (AF416764), the Sapulpa (U60476) and the 91HE17 (U23503) strains. The phylogenetic analysis also revealed that the E. chaffeensis EC-PGHL formed a single cluster with the above strains. This is the first study to report molecular detection and phylogenetic analysis of E. chaffeensis from H. longicornis ticks in Korea. The implicit significance of E. chaffeensis infection in H. longicornis ticks in Korea is discussed.

CD8+ T Cell-mediated Immunity Induced by Heterologous Prime-boost Vaccination Based on DNA Vaccine and Recombinant Vaccinia Virus Expressing Epitope

BACKGROUND: DNA vaccination represents an anticipated approach for the control of numerous infectious diseases. Used alone, however, DNA vaccine is weak immunogen inferior to viral vectors. In recent, heterologous prime-boost vaccination leads DNA vaccines to practical reality. METHODS: We assessed prime-boost immunization strategies with a DNA vaccine (minigene, gB498-505 DNA) and recombinant vaccinia virus (vvgB498- 505) expressing epitope gB498-505 (SSIEFARL) of CD8+ T cells specific for glycoprotein B (gB) of herpes simplex virus (HSV). Animals were immunized primarily with gB498-505 epitope-expressing DNA vaccine/recombinant vaccinia virus and boosted with alternative vaccine type expressing entire Ag. RESULTS: In prime-boost protocols using vvgBw (recombinant vaccinia virus expressing entire Ag) and vvgB498-505, CD8+ T cell-mediated immunity was induced maximally at both acute and memory stages if primed with vvgBw and boosted with vvgB498-505 as evaluated by CTL activity, intracellular IFN-staining, and MHC class I tetramer staining. Similarly gB498-505 DNA prime-gBw DNA (DNA vaccine expressing entire Ag) boost immunization elicited the strongest CD8+ T cell responses in protocols based on DNA vaccine. However, the level of CD8+ T cell-mediated immunity induced with prime-boost vaccination using DNA vaccine expressing epitope or entire Ag was inferior to those based on vvgBw and vvgB498-505. Of particular interest CD8+ T cell-mediated immunity was optimally induced when vvgB498-505 was used to prime and gB DNA was used as alternative boost. Especially CD8+ T cell responses induced by such protocol was longer lasted than other protocols. CONCLUSION: These facts direct to search for the effective strategy to induce optimal CD8+ T cell-mediated immunity against cancer and viral infection.

Mucosal CD8+ T Cell-Mediated Immunity of Vaccinized Animals to Genital Infection of Herpes Simplex Virus Type 1

In the present study, we directly evaluated mucosal CD8+ T cell-mediated immunity using ex vivo cytotoxic T lymphocyte (CTL) assay and MHC class I tetramer staining method in iliac lymph node (LN) and vaginal tracts of mice immunized mucosally with several prime-boost protocols after genital infection of herpes simplex virus type 1 (HSV-1). Ex vivo CTL activity in iliac LN of infected mice was evaluated at 3-day post-infection without in vitro 5-day stimulation. Iliac LN of mice immunized with recombinant viral vaccine-priming and DNA vaccine-boosting protocol showed more potent CTL activity than those of other groups. Such ex vivo CTL activity was consistent with mucosal gB498-505 (SSIEFARL)-specific CD8+ T cell number of vaginal tract determined by MHC class I (H-2b) tetramer containing immunodominant peptide. Furthermore, the number of mucosal SSIEFARL-specific CD8+ T cells recruited into infected genital tracts appeared to decide the protective outcome against genital infection of virulent HSV-1. These results support that mucosal CD8+ T cells are principal mediators for the protection against genital infection of HSV-1.