scholarly journals Role for Gr-1+Cells in the Control of High-Dose Mycobacterium bovis Recombinant BCG

2014 ◽  
Vol 21 (8) ◽  
pp. 1120-1127 ◽  
Author(s):  
Michael W. Panas ◽  
Norman L. Letvin
Keyword(s):  
Immune Response ◽  
Mycobacterium Bovis ◽  
Bioluminescence Imaging ◽  
Adaptive Immune Response ◽  
Vaccine Vector ◽  
High Dose ◽  
Mycobacterial Antigen ◽  
Content Type ◽  
Inflammatory Monocytes ◽  
Recombinant Bcg

ABSTRACTMycobacterium bovisbacillus Calmette-Guérin (BCG) is an attractive target for development as a live vaccine vector delivering transgenic antigens from HIV and other pathogens. Most studies aimed at defining the clearance of BCG have been performed at doses between 102and 104CFU. Interestingly, however, recombinant BCG (rBCG) administered at doses of >106CFU effectively generates antigen-specific T-cell responses and primes for heterologous boost responses. Thus, defining clearance at high doses might aid in the optimization of rBCG as a vector. In this study, we used bioluminescence imaging to examine the kinetics of rBCG transgene expression and clearance in mice immunized with 5 × 107CFU rBCG expressing luciferase. Similar to studies using low-dose rBCG, our results demonstrate that the adaptive immune response is necessary for long-term control of rBCG beginning 9 days after immunizing mice. However, in contrast to these reports, we observed that the majority of mycobacterial antigen was eliminated prior to day 9. By examining knockout and antibody-mediated depletion mouse models, we demonstrate that the rapid clearance of rBCG occurs in the first 24 h and is mediated by Gr-1+cells. As Gr-1+granulocytes have been described as having no impact on BCG clearance at low doses, our results reveal an unappreciated role for Gr-1+neutrophils and inflammatory monocytes in the clearance of high-dose rBCG. This work demonstrates the potential of applying bioluminescence imaging to rBCG in order to gain an understanding of the immune response and increase the efficacy of rBCG as a vaccine vector.

scholarly journals Assessment of Lactobacillus gasseri as a Candidate Oral Vaccine Vector

2011 ◽  
Vol 18 (11) ◽  
pp. 1834-1844 ◽  
Author(s):  
Laura Stoeker ◽  
Shila Nordone ◽  
Sara Gunderson ◽  
Lin Zhang ◽  
Akinobu Kajikawa ◽  
...  
Keyword(s):  
Immune Response ◽  
Genetically Modified ◽  
Oral Vaccine ◽  
Adaptive Immune Response ◽  
Vaccine Vector ◽  
Human Consumption ◽  
Host Immune System ◽  
Myeloid Dendritic Cells ◽  
Content Type

ABSTRACTLactobacillusspecies are commensal bacteria that have long been recognized as probiotic microbes and are generally regarded as safe (GRAS) for human consumption. We have investigated the use ofL. gasserias a vaccine vector for oral immunization against mucosal pathogens. Recent research has shown that the immune response to different lactobacilli can vary widely depending on the species or subspecies ofLactobacillusbeing studied. While some lactobacilli seem to induce oral tolerance, others induce an adaptive immune response. This study characterized the systemic and mucosal immune response to wild-type and genetically modifiedL. gasseri. L. gasseriprimarily activates TLR2/6, with additional activation through the TLR2 homodimer. To expand the Toll-like receptor (TLR) activation profile ofL. gasseriand the immunogenicity of the vector, a plasmid containingfliC, the gene encoding bacterial flagellin, was introduced which resulted in the strong activation of TLR5. The treatment of human myeloid dendritic cells with recombinant lactobacilli expressing flagellin triggered phenotypic maturation and the release of proinflammatory cytokines. In contrast, bacterial treatment also resulted in a statistically significant increase in IL-10 production.In vivostudies established that treatment withL. gasseriled to a diversification of B-cell populations in the lamina propria of the murine colon. Furthermore, treatment with genetically modifiedL. gasseriled to a significant decrease in the percentage of FoxP3+colonic lymphocytes. Taken together, these data clarify the interaction ofL. gasseriwith the host immune system and support further investigation of thein vivoimmunogenicity ofL. gasseriexpressing both flagellin and candidate vaccine antigens.

scholarly journals Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors

2015 ◽  
Vol 22 (7) ◽  
pp. 726-741 ◽  
Author(s):  
Bryan E. Hart ◽  
Rose Asrican ◽  
So-Yon Lim ◽  
Jaimie D. Sixsmith ◽  
Regy Lukose ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Antigen Expression ◽  
Full Length ◽  
Content Type ◽  
Immunodeficiency Virus ◽  
Vaccine Stability ◽  
Hiv Gp120 ◽  
Recombinant Bcg

ABSTRACTThe well-established safety profile of the tuberculosis vaccine strain,Mycobacterium bovisbacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCDtransformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging bothin vitroandin vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCDvaccine expressing HIV gp120 that retained stable full-length expression after 1024-fold amplificationin vitroand following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >1068-fold amplificationin vitroand induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCDlots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.

scholarly journals Use of Animal Models To Support Revising Meningococcal Breakpoints of β-Lactams

2016 ◽  
Vol 60 (7) ◽  
pp. 4023-4027 ◽  
Author(s):  
Nouria Belkacem ◽  
Eva Hong ◽  
Ana Antunes ◽  
Aude Terrade ◽  
Ala-Eddine Deghmane ◽  
...  
Keyword(s):  
Human Factor ◽  
Susceptibility Testing ◽  
Bioluminescence Imaging ◽  
Factor H ◽  
Penicillin G ◽  
High Dose ◽  
Beta Lactams ◽  
Content Type ◽  
Resistance To Penicillin ◽  
Intraperitoneal Infection

ABSTRACTAntibiotic susceptibility testing (AST) inNeisseria meningitidisis an important part of the management of invasive meningococcal disease. It defines MICs of antibiotics that are used in treatment and/or prophylaxis and that mainly belong to the beta-lactams. The interpretation of the AST results requires breakpoints to classify the isolates into susceptible, intermediate, or resistant. The resistance to penicillin G is defined by a MIC of >0.25 mg/liter, and that of amoxicillin is defined by a MIC of >1 mg/liter. We provide data that may support revision of resistance breakpoints for beta-lactams in meningococci. We used experimental intraperitoneal infection in 8-week-old transgenic female mice expressing human transferrin and human factor H. Dynamic bioluminescence imaging was performed to follow the infection by bioluminescent meningococcus strains with different MICs. Three hours later, infected mice were treated intramuscularly using several doses of amoxicillin or penicillin G. Signal decreased during infection with a meningococcus strain showing a penicillin G MIC of 0.064 mg/liter at all doses. Signals decreased for the strain with a penicillin G MIC of 0.5 mg/liter only after treatment with the highest doses, corresponding to 250,000 units/kg of penicillin G or 200 mg/kg of amoxicillin, although this decrease was at a lower rate than that of the strain with a MIC of 0.064 mg/liter. The decrease in bioluminescent signals was associated with a decrease in the levels of the proinflammatory cytokine interleukin-6 (IL-6). Our data suggest that a high dose of amoxicillin or penicillin G can reduce growth during infection by isolates showing penicillin G MICs of >0.25 mg/liter and ≤1 mg/liter.

scholarly journals Promotion of Colonization and Virulence by Cholera Toxin Is Dependent on Neutrophils

2013 ◽  
Vol 81 (9) ◽  
pp. 3338-3345 ◽  
Author(s):  
Jessica Queen ◽  
Karla J. F. Satchell
Keyword(s):  
Immune Response ◽  
Cholera Toxin ◽  
Innate Immune Response ◽  
Host Response ◽  
Survival Rates ◽  
Innate Immune ◽  
Infection Model ◽  
High Dose ◽  
Content Type ◽  
Inflammatory Protein

ABSTRACTThe innate immune response toVibrio choleraeinfection is poorly understood, but this knowledge is critical for the design of safe, effective vaccines. Using an adult mouse intestinal infection model, this study examines the contribution of neutrophils to host immunity, as well as the effect of cholera toxin and other secreted factors on this response. Depletion of neutrophils from mice with anti-Ly6G IA8 monoclonal antibody led to similar survival rates of mice infected with low or moderate doses of toxigenicV. choleraeEl Tor O1. At a high dose, neutropenic mice showed increased rates of survival compared to neutrophil-replete animals. Expression of cholera toxin was found to be protective to the neutropenic host, and this phenotype can be replicated by the administration of purified toxin. Neutrophils do not effectively clear colonizing bacteria from the small intestine, nor do they alter induction of early immune-modulating signals. In both neutropenic and neutrophil-replete animals, the local response to infection is characterized by expression of interleukin 6 (IL-6), IL-10, and macrophage inflammatory protein 2 alpha (MIP-2). Overall, these data indicate that the innate immune response to toxigenicV. choleraeinfection differs dramatically from the host response to nontoxigenic infection or vaccination, where neutrophils are protective to the host. In the absence of neutrophils, cholera toxin induces immunomodulatory effects that increase host survival. In cholera toxin-producing strains, similar to nontoxigenic infection, accessory toxins are critical to virulence, indicating that cholera toxin and the other secreted toxins modulate the host response by different mechanisms, with both contributing to bacterial persistence and virulence.

scholarly journals Reduced Immune Response to Borrelia burgdorferi in the Absence of γδ T Cells

2011 ◽  
Vol 79 (10) ◽  
pp. 3940-3946 ◽  
Author(s):  
Cuixia Shi ◽  
Bikash Sahay ◽  
Jennifer Q. Russell ◽  
Karen A. Fortner ◽  
Nicholas Hardin ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Borrelia Burgdorferi ◽  
Adaptive Immune Response ◽  
Γδ T Cells ◽  
Content Type ◽  
Adaptive Immune ◽  
Cytokines And Chemokines

ABSTRACTLittle is known regarding the function of γδ T cells, although they accumulate at sites of inflammation in infections and autoimmune disorders. We previously observed that γδ T cellsin vitroare activated byBorrelia burgdorferiin a TLR2-dependent manner. We now observe that the activated γδ T cells can in turn stimulate dendritic cellsin vitroto produce cytokines and chemokines that are important for the adaptive immune response. This suggested thatin vivoγδ T cells may assist in activating the adaptive immune response. We examined this possibilityin vivoand observed that γδ T cells are activated and expand in number duringBorreliainfection, and this was reduced in the absence of TLR2. Furthermore, in the absence of γδ T cells, there was a significantly blunted response of adaptive immunity, as reflected in reduced expansion of T and B cells and reduced serum levels of anti-Borreliaantibodies, cytokines, and chemokines. This paralleled a greaterBorreliaburden in γδ-deficient mice as well as more cardiac inflammation. These findings are consistent with a model of γδ T cells functioning to promote the adaptive immune response during infection.

scholarly journals Virulence and Immune Response Induced by Mycobacterium avium Complex Strains in a Model of Progressive Pulmonary Tuberculosis and Subcutaneous Infection in BALB/c Mice

2013 ◽  
Vol 81 (11) ◽  
pp. 4001-4012 ◽  
Author(s):  
Mónica González-Pérez ◽  
Leonardo Mariño-Ramírez ◽  
Carlos Alberto Parra-López ◽  
Martha Isabel Murcia ◽  
Brenda Marquina ◽  
...  
Keyword(s):  
Immune Response ◽  
Pulmonary Tuberculosis ◽  
Inflammatory Cytokines ◽  
Mycobacterium Avium ◽  
High Expression ◽  
High Dose ◽  
Content Type ◽  
Subcutaneous Infection ◽  
Tnf Α ◽  
Anti Inflammatory

ABSTRACTThe genusMycobacteriumcomprises more than 150 species, including important pathogens for humans which cause major public health problems. The vast majority of efforts to understand the genus have been addressed in studies withMycobacterium tuberculosis. The biological differentiation betweenM. tuberculosisand nontuberculous mycobacteria (NTM) is important because there are distinctions in the sources of infection, treatments, and the course of disease. Likewise, the importance of studying NTM is not only due to its clinical significance but also due to the mechanisms by which some species are pathogenic while others are not.Mycobacterium aviumcomplex (MAC) is the most important group of NTM opportunistic pathogens, since it is the second largest medical complex in the genus after theM. tuberculosiscomplex. Here, we evaluated the virulence and immune response ofM. aviumsubsp.aviumandMycobacterium colombiense, using experimental models of progressive pulmonary tuberculosis and subcutaneous infection in BALB/c mice. Mice infected intratracheally with a high dose of MAC strains showed high expression of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase with rapid bacillus elimination and numerous granulomas, but without lung consolidation during late infection in coexistence with high expression of anti-inflammatory cytokines. In contrast, subcutaneous infection showed high production of the proinflammatory cytokines TNF-α and gamma interferon with relatively low production of anti-inflammatory cytokines such as interleukin-10 (IL-10) or IL-4, which efficiently eliminate the bacilli but maintain extensive inflammation and fibrosis. Thus, MAC infection evokes different immune and inflammatory responses depending on the MAC species and affected tissue.

scholarly journals Use of Antigen-Specific Interleukin-2 To Differentiate between Cattle Vaccinated with Mycobacterium bovis BCG and Cattle Infected with M. bovis

2013 ◽  
Vol 21 (1) ◽  
pp. 39-45 ◽  
Author(s):  
Shelley G. Rhodes ◽  
Lucy C. McKinna ◽  
Sabine Steinbach ◽  
Gilly S. Dean ◽  
Bernardo Villarreal-Ramos ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Whole Blood ◽  
Sandwich Elisa ◽  
Interleukin 2 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mycobacterial Antigen ◽  
Content Type ◽  
Purified Protein Derivative ◽  
Rapid Induction ◽  
Challenge Control

ABSTRACTWe describe here the application of a novel bovine interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) for the measurement of antigen-specific IL-2 in cattle naturally infected withMycobacterium bovisand in cattle vaccinated withMycobacterium bovisBCG and then experimentally challenged with pathogenicM. bovis. Supernatants from whole-blood cultures stimulated with mycobacterial antigen (bovine purified protein derivative [PPDB] or the peptide cocktail ESAT6-CFP10) were assessed using a sandwich ELISA consisting of a new recombinant monoclonal fragment capture antibody and a commercially available polyclonal anti-bovine-IL-2. The production of IL-2 was compared to the production of gamma interferon (IFN-γ) in the same antigen-stimulated whole-blood supernatants. The data show that cattle infected withM. bovisproduced quantifiable levels of antigen-specific IL-2, while IL-2 levels in cattle vaccinated withM. bovisBCG did not. Furthermore, cattle vaccinated withM. bovisBCG and then challenged with pathogenicM. bovisdisplayed a more rapid induction of IL-2 but ultimately had lower levels of infection-induced IL-2 than did unvaccinated challenge control cattle. These data suggest that IL-2 responses are not detectable post-BCG vaccination and that these responses may require infection with virulentM. bovisto develop. This may be useful to differentiate infected cattle from uninfected or BCG-vaccinated cattle, although the overall sensitivity is relatively low, particularly in single intradermal comparative cervical tuberculin (SICCT)-negative infected animals. Furthermore, the strength of the IL-2 response may correlate with pathology, which poses interesting questions on the immunobiology of bovine tuberculosis in contrast to human tuberculosis, which is discussed.

scholarly journals Antigen Localization Influences the Magnitude and Kinetics of Endogenous Adaptive Immune Response to Recombinant Salmonella Vaccines

2017 ◽  
Vol 85 (12) ◽  
Author(s):  
Yanina R. Sevastsyanovich ◽  
David R. Withers ◽  
Claire L. Marriott ◽  
Faye C. Morris ◽  
Timothy J. Wells ◽  
...  
Keyword(s):  
Immune Response ◽  
Adaptive Immune Response ◽  
T Cell Response ◽  
Cell Culture Supernatant ◽  
Total Serum ◽  
Igg Antibody ◽  
Content Type ◽  
Antigen Localization ◽  
Pet System ◽  
Heterologous Antigens

ABSTRACT The use of recombinant attenuated Salmonella vaccine (RASV) strains is a promising strategy for presenting heterologous antigens to the mammalian immune system to induce both cellular and humoral immune responses. However, studies on RASV development differ on where heterologous antigens are expressed and localized within the bacterium, and it is unclear how antigen localization modulates the immune response. Previously, we exploited the plasmid-encoded toxin (Pet) autotransporter system for accumulation of heterologous antigens in cell culture supernatant. In the present study, this Pet system was used to express early secretory antigen 6 (ESAT-6), an immunodominant and diagnostic antigen from Mycobacterium tuberculosis, in Salmonella enterica serovar Typhimurium strain SL3261. Three strains were generated, whereby ESAT-6 was expressed as a cytoplasmic (SL3261/cyto), surface-bound (SL3261/surf), or secreted (SL3261/sec) antigen. Using these RASVs, the relationship between antigen localization and immunogenicity in infected C57BL/6 mice was systematically examined. Using purified antigen and specific tetramers, we showed that mice infected with the SL3261/surf or SL3261/sec strain generated large numbers of Th1 CD4+ ESAT-6+ splenic T cells compared to those of mice infected with SL3261/cyto. While all mice showed ESAT-6-specific antibody responses when infected with SL3261/surf or SL3261/sec, peak total serum IgG antibody titers were reached more rapidly in mice that received SL3261/sec. Thus, how antigen is localized after production within bacteria has a more marked effect on the antibody response than on the CD4+ T cell response, which might influence the chosen strategy to localize recombinant antigen in RASVs.

scholarly journals Interleukin-21 Induces Short-Lived Effector CD8+ T Cells but Does Not Inhibit Their Exhaustion after Mycobacterium bovis BCG Infection in Mice

2018 ◽  
Vol 86 (8) ◽  
Author(s):  
Naoto Noguchi ◽  
Risa Nakamura ◽  
Shinya Hatano ◽  
Hisakata Yamada ◽  
Xun Sun ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Bovis ◽  
Cd8 T Cells ◽  
Absolute Number ◽  
Bacillus Calmette Guerin ◽  
Content Type ◽  
Effector Functions ◽  
Antigen 85B ◽  
Interleukin 21 ◽  
Recombinant Bcg

ABSTRACT Interleukin 21 (IL-21) is a pleiotropic common cytokine receptor γ chain cytokine that promotes the effector functions of NK cells and CD8+ T cells and inhibits CD8+ T cell exhaustion during chronic infection. We found that the absolute number of short-lived effector CD8+ T cells (SLECs) (KLRG1high CD127low) decreased significantly in IL-21 receptor-deficient (IL-21R−/−) mice during Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. Early effector CD8+ T cells (EECs) (KLRG1low CD127low) were normally generated in IL-21R−/− mice after infection. Exhausted CD8+ T cells (PD-1high KLRG1low) were also normally generated in IL-21R−/− mice after infection. Mixed bone marrow (BM) chimera and transfer experiments showed that IL-21R on CD8+ T cells was essential for the proliferation of EECs, allowing them to differentiate into SLECs after BCG infection. On the other hand, the number of SLECs increased significantly after infection with recombinant BCG (rBCG) that secreted an antigen 85B (Ag85B)–IL-21 fusion protein (rBCG–Ag85B–IL-21), but the number of exhausted CD8+ T cells did not change after rBCG–Ag85B–IL-21 infection. These results suggest that IL-21 signaling drives the differentiation of SLECs from EECs but does not inhibit the exhaustion of CD8+ T cells following BCG infection in mice.

scholarly journals Peroral Ciprofloxacin Therapy Impairs the Generation of a Protective Immune Response in a Mouse Model for Salmonella enterica Serovar Typhimurium Diarrhea, while Parenteral Ceftriaxone Therapy Does Not

2012 ◽  
Vol 56 (5) ◽  
pp. 2295-2304 ◽  
Author(s):  
Kathrin Endt ◽  
Lisa Maier ◽  
Rina Käppeli ◽  
Manja Barthel ◽  
Benjamin Misselwitz ◽  
...  
Keyword(s):  
Immune Response ◽  
Mouse Model ◽  
Antibiotic Treatment ◽  
Salmonella Enterica ◽  
Adaptive Immune Response ◽  
Content Type ◽  
Fecal Shedding ◽  
Treatment Regimens ◽  
Adaptive Immune ◽  
Serovar Typhimurium

ABSTRACTNontyphoidalSalmonella(NTS) species cause self-limiting diarrhea and sometimes severe disease. Antibiotic treatment is considered only in severe cases and immune-compromised patients. The beneficial effects of antibiotic therapy and the consequences for adaptive immune responses are not well understood. We used a mouse model forSalmonelladiarrhea to assess the effects ofper ostreatment with ciprofloxacin (15 mg/kg of body weight intragastrically 2 times/day, 5 days) or parenteral ceftriaxone (50 mg/kg intraperitoneally, 5 days), two common drugs used in human patients. The therapeutic and adverse effects were assessed with respect to generation of a protective adaptive immune response, fecal pathogen excretion, and the emergence of nonsymptomatic excreters. In the mouse model, both therapies reduced disease severity and reduced the level of fecal shedding. In line with clinical data, in most animals, a rebound of pathogen gut colonization/fecal shedding was observed 2 to 12 days after the end of the treatment. Yet, levels of pathogen shedding and frequency of appearance of nonsymptomatic excreters did not differ from those for untreated controls. Moreover, mice treated intraperitoneally with ceftriaxone developed an adaptive immunity protecting the mice from enteropathy in wild-typeSalmonella entericaserovar Typhimurium challenge infections. In contrast, the mice treated intragastrically with ciprofloxacin were not protected. Thus, antibiotic treatment regimens can disrupt the adaptive immune response, but treatment regimens may be optimized in order to preserve the generation of protective immunity. It might be of interest to determine whether this also pertains to human patients. In this case, the mouse model might be a tool for further mechanistic studies.

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