Bacteriophage therapy: a novel method of lytic phage delivery

The widespread emergence of multi-antibiotic resistant bacteria has increased the need for alternatives to conventional antibiotic therapy. Accordingly, a significant amount of effort has been made to investigate the potential use of bacteriophages as prophylactic and therapeutic agents for bacterial infections. In this study, molecular biological techniques were applied to construct a lysogen of lytic bacteriophage lambda in an attempt to combat with multi-antibiotic resistant bacteria by a novel method of lytic phage delivery;To accomplish this goal, two plasmid-based site-specific recombination (SSR) systems for integration and recovery of DNA constructs from Escherichia coli and Salmonella typhimurium chromosomes were developed. The two systems are mediated by SSR machineries of bacteriophages lambda of E. coli and P22 of S. typhimurium. These systems utilize plasmid vectors with conditional replicating origin of replication and provide stable chromosomal integration of genes at specific bacteriophage attachment sites without disruption of any host gene or a need for antibiotic selection. E. coli contains attachment sites for both bacteriophages. When the two systems are applied consecutively, two different genes can be integrated at two specific locations. The integrated plasmids of both systems can also be completely excised and recovered from the host chromosomes to observe any genetic changes, e.g. by DNA sequencing. Both systems are also very applicable in construction of bacterial strains as well as live E. coli and S. typhimurium recombinant vaccines expressing foreign genes of interest;To construct a lysogen of lytic bacteriophage lambda, both SSR systems were applied. A lytic mutant (cI-) of bacteriophage lambda was marked with an antibiotic resistant gene cassette to facilitate a lysogen selection. The P22 SSR system helped integrate functional lambda repressor gene (cI) into a non-pathogenic E. coli strain and the marked lytic lambda phage lysogenized in the presence of the helper plasmid of the lambda SSR system. The lysogen demonstrated its efficacy in decreasing number of lambda sensitive E. coli. This lytic phage lysogen construction strategy can be applied for other bacteriophages. A pool of different lysogens infects a wider range of bacteria and could be utilized as alternatives to the use of antibiotics to control bacterial infections

Monitoring responses by use of five-color flow cytometry in subsets of peripheral T cells obtained from cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis vaccine

Objective—To monitor by use of 5-color flow cytometry the antigen-specific responses of subsets of peripheral T cells in cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis (MAP) vaccine and to compare results with those for 2 established cell-mediated immunity assays. Animals—45 female Holstein cattle with negative results for MAP in skin tests conducted at time of inoculation with MAP. Procedures—Cattle were allocated to 4 groups. Cattle of group 1 (n = 12) were 0 to 3 months old and inoculated with a killed MAP vaccine. The 10 cattle of group 2 were the same age as those in group 1 but were not inoculated with MAP vaccine. The 11 cattle of group 3 were 9 to 12 months old and inoculated with killed MAP vaccine. The 12 cattle of group 4 were the same age as those in group 3 but were not inoculated with MAP vaccine. Results—Flow cytometry identified T-cell subsets that responded specifically to the recall antigen. Results of assays for CD25 expression and wholeblood interferon-γ had the strongest correlation with results for skin tests as well as results with each other. Intracellular expression of interferon-γ was not correlated as well with results for the other tests. Conclusions and Clinical Relevance—Flow cytometry can be useful for characterizing the immune response after administration of MAP vaccine and should be evaluated with regard to its sensitivity and specificity when used in detecting cattle naturally infected with MAP

Canine Peripheral Blood Lymphocyte Phenotyping by 7-Color Multiparameter Flow Cytometry

Objective: To characterize baseline canine lymphocyte phenotypes including lymphocytes coexpressing multiple markers by novel 7-color multiparameter flow cytometry. Study Design: Fresh canine peripheral blood lymphocytes of 79 healthy 26-week-old Beagle or Beagle-mix dogs were stained and analyzed. Results: The high number of samples and acquired flow data (averaging 1.9x105 cells/sample) allowed the detection of minor lymphocyte subsets coexpressing multiple lymphocyte markers. The averaged percentages of major lymphocyte subsets of CD3+, CD4+, CD8+, CD21+ and gd TCR+ cells from this study were 74.0, 43.6, 14.3, 9.6, and 0.2, respectively, which were comparable but uniquely different from other reports as they were simultaneously detected in the same sample. We demonstrated that the commonly used CD21 and CD3 monoclonal antibody (mAb) clones, previously recommended not to be used in the same staining, could and should be used together with the proper steps of lymphocyte gating. We found a high percentage (10.3%) of unidentified CD21–CD3+CD4–CD8–gdTCR– lymphocyte subset that has never been reported. The intensive gating strategy and the mean percentages of each lymphocyte subset to their parent subsets and to the total lymphocyte population are presented and discussed. Conclusion: The canine lymphocyte phenotypes were fully characterized. This novel multiparameter flow cytometry method is a powerful approach to in-crease the accuracy of lymphocyte phenotyping in dogs

Divergent Immune Responses and Disease Outcomes in Piglets Immunized with Inactivated and Attenuated H3N2 Swine Influenza Vaccines in the Presence of Maternally-Derived Antibodies

Live-attenuated influenza virus (LAIV) prime-boost vaccination previously conferred protection against heterologous H3N2 swine influenza challenge, including in piglets with maternally derived antibodies (MDA). Conversely, a whole-inactivated virus (WIV) vaccine was associated with enhanced disease. This study was aimed at identifying immune correlates of cross-protection. Piglets with and without MDA received intramuscular adjuvanted WIV or intranasal LAIV, and were challenged with heterologous H3N2. WIV induced cross-reactive IgG, inhibited by MDA, and a moderate T cell response. LAIV elicited mucosal antibodies and T cells cross-reactive to the heterologous challenge strain. The presence of MDA at LAIV vaccination blocked lung and nasal antibody production, but did not interfere with T cell priming. Even without mucosal antibodies, MDA-positive LAIV vaccinates were protected, indicating a likely role for T cells. Based on the data, one LAIV dose can induce cell-mediated immunity against antigenically divergent H3N2 influenza virus despite passive antibody interference with humoral immune responses

Comparison of Humoral and Cellular Immune Responses to Inactivated Swine Influenza Virus Vaccine in Weaned Pigs

Humoral and cellular immune responses to inactivated swine influenza virus (SIV) vaccine were evaluated and compared. Fifty 3-week-old weaned pigs were randomly divided into the non-vaccinated control group and vaccinated group containing 25 pigs each. Pigs were vaccinated intramuscularly twice with adjuvanted UV-inactivated A/SW/MN/02011/08 (MN/08) H1N2 SIV vaccine at 6 and 9 weeks of age. Whole blood samples for multi-parameter flow cytometry (MP-FCM) and serum samples for hemagglutination inhibition (HI) assay were collected at 23 and 28 days after the second vaccination, respectively. A standard HI assay and MP-FCM were performed against UV-inactivated homologous MN/08 and heterologous pandemic A/CA/04/2009 (CA/09) H1N1 viruses. While the HI assay detected humoral responses only to the MN/08 virus, the MP-FCM detected strong cellular responses against the MN/08 virus and significant heterologous responses to the CA/09 virus, especially in the CD4+CD8+ T cell subset. The cellular heterologous responses to UV-inactivated virus by MP-FCM suggested that the assay was sensitive and potentially detected a wider range of antigens than what was detected by the HI assay. Overall, the adjuvanted UV-inactivated A/SW/MN/02011/08 H1N2 SIV vaccine stimulated both humoral and cellular immune responses including the CD4−CD8+ T cell subset

Vaccination with NS1-Truncated H3N2 Swine Influenza Virus Primes T Cells and Confers Cross-Protection against an H1N1 Heterosubtypic Challenge in Pigs

The diversity of contemporary swine influenza virus (SIV) strains impedes effective immunization of swine herds. Mucosally delivered, attenuated virus vaccines are one approach with potential to provide broad cross-protection. Reverse genetics-derived H3N2 SIV virus with truncated NS1 (NS1Δ126 TX98) is attenuated and immunogenic when delivered intranasally in young pigs. We analyzed T-cell priming and cross-protective efficacy in weanling piglets after intranasal inoculation with NS1Δ126 TX98 versus wild type TX98. In vivo replication of the truncation mutant was minimal compared to the wild type virus. T-cell responses were greater in magnitude in pigs infected with the wild type virus in in vitro restimulation assays. According to the expression of activation marker CD25, peripheral T cell recall responses in NS1Δ126 TX98 infected pigs were minimal. However, intracellular IFN-γ data indicate that the attenuated virus induced virus-specific CD4+CD8–, CD4+CD8+, CD4–CD8+, and γδ T cells within 28 days. The IFN-γ response appeared to contract, as responses were reduced at later time points prior to challenge. CD4+CD8+ cells isolated 5 days after heterosubtypic H1N1 challenge (day 70 overall) showed an elevated CD25 response to virus restimulation. Pigs previously infected with wild type TX98 were protected from replication of the H1N1 challenge virus. Vaccination with NS1Δ126 TX98 was associated with significantly lower levels of Th1-associated cytokines in infected lungs but provided partial cross-protection against the H1N1 challenge. These results demonstrate that NS1Δ SIV vaccines can elicit cell-mediated cross-protection against antigenically divergent strains

Bacteriophage therapy: a novel method of lytic phage delivery

The widespread emergence of multi-antibiotic resistant bacteria has increased the need for alternatives to conventional antibiotic therapy. Accordingly, a significant amount of effort has been made to investigate the potential use of bacteriophages as prophylactic and therapeutic agents for bacterial infections. In this study, molecular biological techniques were applied to construct a lysogen of lytic bacteriophage lambda in an attempt to combat with multi-antibiotic resistant bacteria by a novel method of lytic phage delivery;To accomplish this goal, two plasmid-based site-specific recombination (SSR) systems for integration and recovery of DNA constructs from Escherichia coli and Salmonella typhimurium chromosomes were developed. The two systems are mediated by SSR machineries of bacteriophages lambda of E. coli and P22 of S. typhimurium. These systems utilize plasmid vectors with conditional replicating origin of replication and provide stable chromosomal integration of genes at specific bacteriophage attachment sites without disruption of any host gene or a need for antibiotic selection. E. coli contains attachment sites for both bacteriophages. When the two systems are applied consecutively, two different genes can be integrated at two specific locations. The integrated plasmids of both systems can also be completely excised and recovered from the host chromosomes to observe any genetic changes, e.g. by DNA sequencing. Both systems are also very applicable in construction of bacterial strains as well as live E. coli and S. typhimurium recombinant vaccines expressing foreign genes of interest;To construct a lysogen of lytic bacteriophage lambda, both SSR systems were applied. A lytic mutant (cI-) of bacteriophage lambda was marked with an antibiotic resistant gene cassette to facilitate a lysogen selection. The P22 SSR system helped integrate functional lambda repressor gene (cI) into a non-pathogenic E. coli strain and the marked lytic lambda phage lysogenized in the presence of the helper plasmid of the lambda SSR system. The lysogen demonstrated its efficacy in decreasing number of lambda sensitive E. coli. This lytic phage lysogen construction strategy can be applied for other bacteriophages. A pool of different lysogens infects a wider range of bacteria and could be utilized as alternatives to the use of antibiotics to control bacterial infections.</p

Monitoring responses by use of five-color flow cytometry in subsets of peripheral T cells obtained from cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis vaccine

Objective—To monitor by use of 5-color flow cytometry the antigen-specific responses of subsets of peripheral T cells in cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis (MAP) vaccine and to compare results with those for 2 established cell-mediated immunity assays. Animals—45 female Holstein cattle with negative results for MAP in skin tests conducted at time of inoculation with MAP. Procedures—Cattle were allocated to 4 groups. Cattle of group 1 (n = 12) were 0 to 3 months old and inoculated with a killed MAP vaccine. The 10 cattle of group 2 were the same age as those in group 1 but were not inoculated with MAP vaccine. The 11 cattle of group 3 were 9 to 12 months old and inoculated with killed MAP vaccine. The 12 cattle of group 4 were the same age as those in group 3 but were not inoculated with MAP vaccine. Results—Flow cytometry identified T-cell subsets that responded specifically to the recall antigen. Results of assays for CD25 expression and wholeblood interferon-γ had the strongest correlation with results for skin tests as well as results with each other. Intracellular expression of interferon-γ was not correlated as well with results for the other tests. Conclusions and Clinical Relevance—Flow cytometry can be useful for characterizing the immune response after administration of MAP vaccine and should be evaluated with regard to its sensitivity and specificity when used in detecting cattle naturally infected with MAP.This article is published as Platt, Ratree, James A. Roth, Ryan L. Royer, and Charles O. Thoen. "Monitoring responses by use of five-color flow cytometry in subsets of peripheral T cells obtained from cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis vaccine." American journal of veterinary research 67, no. 12 (2006): 2050-2058. doi: 10.2460/ajvr.67.12.2050. Posted with permission.</p

Comparison of humoral and T-cell-mediated immune responses to a single dose of Bovela® live double deleted BVDV vaccine or to a field BVDV strain

The objective of this study was to determine and compare the humoral and cellular immune responses of calves exposed to a single dose of Bovela® bovine viral diarrhea virus (BVDV) live double deleted vaccine or a field strain virus (FSV) of BVDV type 2 (strain 890). Thirty seronegative, colostrum-deprived 5 month-old Holstein steer calves that tested negative for persistent BVDV by ear notch immunohistochemistry and seronegative to BVDV types 1 and 2 were used. Calves were screened by multi-parameter flow cytometry (MP-FCM) 1 week before vaccination to ensure that they were negative for T cell responses to the BVDV types 1 and 2 viruses in the Bovela® vaccine. Calves were assigned to 3 treatment groups: control (PBS), FSV inoculated, and Bovela® vaccinated. The humoral response was tested by standard serum virus neutralization (SVN) test to BVDV types 1 (Singer strain) and 2 (strain 125). The response by CD4, CD8, and gamma delta (γδ TCR) T cells was evaluated by MP-FCM using individual BVDV types 1 and 2 from Bovela® vaccine as recall antigens at 5, 6, and 7 weeks after vaccination. Activation markers used were upregulation of surface CD25 (IL-2R), intracellular interferon gamma (IFNγ) and intracellular interleukin 4 (IL-4). Each T cell subset was evaluated for increased expression of each activation marker compared to non-antigen stimulated cells of the same animal. All Bovela® vaccinated and FSV inoculated calves produced SVN antibodies to both BVDV types 1 and 2 while control animals remained seronegative throughout the study. The mean (weeks 5, 6, and 7) T cell recall responses to Bovela® BVDV type 1 and type 2 recall antigens were numerically higher in all three T cell subsets (CD4, CD8, and γδ TCR) for all three activation markers (CD25, IFNγ, and IL-4) when compared to either the control animals or to the FSV inoculated animals. These differences were often, but not always, statistically significant (P This article is from Veterinary Immunology and Immunopathology 187 (2017): 20, doi: 10.1016/j.vetimm.2017.03.003. Posted with permission.</p