Combinations of Bacteriophage Are Efficacious against Multidrug-Resistant Pseudomonas aeruginosa and Enhance Sensitivity to Carbapenem Antibiotics.

The Gram-negative ESKAPE bacterium Pseudomonas aeruginosa has become a pathogen of serious concern due its extensive multi-drug resistance (MDR) profile, widespread incidences of hospital-acquired infections throughout the United States, and high occurrence in wound infections suffered by warfighters serving abroad. Bacteriophage (phage) therapy has received renewed attention as an alternative therapeutic option against recalcitrant bacterial infections, both as multi-phage cocktails and in combination with antibiotics as synergistic pairings. Environmental screening and phage enrichment has yielded three lytic viruses capable of infecting the MDR P. aeruginosa strain PAO1. Co-administration of each phage with the carbapenem antibiotics ertapenem, imipenem, and meropenem generated enhanced overall killing of bacteria beyond either phage or drug treatments alone. A combination cocktail of all three phages was completely inhibitory to growth, even without antibiotics. The same 3× phage cocktail also disrupted PAO1 biofilms, reducing biomass by over 75% compared to untreated biofilms. Further, the phage cocktail demonstrated broad efficacy as well, capable of infecting 33 out of 100 diverse clinical isolate strains of P. aeruginosa. Together, these results indicate a promising approach for designing layered medical countermeasures to potentiate antibiotic activity and possibly overcome resistance against recalcitrant, MDR bacteria such as P. aeruginosa. Combination therapy, either by synergistic phage-antibiotic pairings, or by phage cocktails, presents a means of controlling mutations that can allow for bacteria to gain a competitive edge.

Testable Candidate Immune Correlates of Protection for Porcine Reproductive and Respiratory Syndrome Virus Vaccination.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an on-going problem for the worldwide pig industry. Commercial and experimental vaccinations often demonstrate reduced pathology and improved growth performance; however, specific immune correlates of protection (CoP) for PRRSV vaccination have not been quantified or even definitively postulated: proposing CoP for evaluation during vaccination and challenge studies will benefit our collective efforts towards achieving protective immunity. Applying the breadth of work on human diseases and CoP to PRRSV research, we advocate four hypotheses for peer review and evaluation as appropriate testable CoP: (i) effective class-switching to systemic IgG and mucosal IgA neutralizing antibodies is required for protective immunity; (ii) vaccination should induce virus-specific peripheral blood CD4+ T-cell proliferation and IFN-γ production with central memory and effector memory phenotypes; cytotoxic T-lymphocytes (CTL) proliferation and IFN-γ production with a CCR7- phenotype that should migrate to the lung; (iii) nursery, finishing, and adult pigs will have different CoP; (iv) neutralizing antibodies provide protection and are rather strain specific; T cells confer disease prevention/reduction and possess greater heterologous recognition. We believe proposing these four CoP for PRRSV can direct future vaccine design and improve vaccine candidate evaluation.

The Local and Systemic Humoral Immune Response Against Homologous and Heterologous Strains of the Type 2 Porcine Reproductive and Respiratory Syndrome Virus.

The humoral immune response plays a crucial role in the combat and protection against many pathogens including the economically most important, highly prevalent, and diverse pig pathogen PRRSV - the Porcine Reproductive and Respiratory Syndrome Virus. In addition to viremia and viral shedding analyses, this study followed the local and systemic humoral immune response of pigs for 63 days upon inoculation with one of three types of Type-2 PRRSV (PRRSV-2) strains - one modified live virus (MLV) vaccine strain, and two lineage 1 PRRSV-2 strains, NC134 and NC174. The local response was analyzed by quantifying immunoglobulin (Ig)A in nasal swabs. The systemic response was studied by the quantification of IgG with ELISA and homo- and heterologous neutralizing antibodies (NAs) utilizing a novel method of flow cytometry. In all PRRSV-2 inoculated groups, viral nasal shedding started at 3 dpi, peaked between 3 and 7 days post inoculation, and was cleared at 28-35 dpi with sporadic rebounds thereafter. The local IgA response started 4-7 days after viral shedding occurred and showed a bi-phasic course with peaks at 14 dpi and at 28-35 dpi. Of note, the NC134 and NC174 strains induced a much stronger local IgA response. As reported earlier, main viremia lasted from 7 dpi to 28 dpi (NC174), 42 dpi (NC134) or until the end of the study (MLV). Similar to the local IgA response, the systemic IgG response started 4-7 days after viremia; but in contrast to viremia, serum IgG levels stayed high for all PRRSV-2 inoculated groups until the end of the study. A significant finding was that while the serum NA response in the MLV group was delayed by 28 days, serum NAs in pigs infected with our two NC134 and NC174 strains could be detected as early as 7 dpi (NC134) and 14 dpi (NC174). Compared to homologous NA responses, the NA responses against heterologous strains was strong but slightly delayed between our lineage 1 one strains or non-existent between the MLV and lineage 1 strains. This study improves our understanding of the relationship between local and systemic infections and the humoral immune response induced by PRRSV-2 infection or MLV vaccination. Our data also provide novel insights into the timeline of the development of homologous and heterologous NA levels - by both MLV vaccination or infection with two strains from the currently prevalent PRRSV-2 lineage 1.

Maternal Autogenous Inactivated Virus Vaccination Boosts Immunity to PRRSV in Piglets.

Maternal-derived immunity is a critical component for the survival and success of offspring in pigs to protect from circulating pathogens such as Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2). The purpose of this study is to investigate the transfer of anti-PRRSV immunity to piglets from gilts that received modified-live virus (MLV) alone (treatment (TRT) 0), or in combination with one of two autogenous inactivated vaccines (AIVs, TRT 1+2). Piglets from these gilts were challenged with the autogenous PRRSV-2 strain at two weeks of age and their adaptive immune response (IR) was evaluated until 4 weeks post inoculation (wpi). The systemic humoral and cellular IR was analyzed in the pre-farrow gilts, and in piglets, pre-inoculation, and at 2 and 4 wpi. Both AIVs partially protected the piglets with reduced lung pathology and increased weight gain; TRT 1 also lowered piglet viremia, best explained by the AIV-induced production of neutralizing antibodies in gilts and their transfer to the piglets. In piglets, pre-inoculation, the main systemic IFN-γ producers were CD21α+ B cells. From 0 to 4 wpi, the role of these B cells declined and CD4 T cells became the primary systemic IFN-γ producers. In the lungs, CD8 T cells were the primary and CD4 T cells were the secondary IFN-γ producers, including a novel subset of porcine CD8α-CCR7- CD4 T cells, potentially terminally differentiated CD4 TEMRA cells. In summary, this study demonstrates that maternal AIV vaccination can improve protection of pre-weaning piglets against PRRSV-2; it shows the importance of transferring neutralizing antibodies to piglets, and it introduces two novel immune cell subsets in pigs-IFN-γ producing CD21α+ B cells and CD8α-CCR7- CD4 T cells.

Mucosal Vaccination with UV-Inactivated Chlamydia suis in Pre-Exposed Outbred Pigs Decreases Pathogen Load and Induces CD4 T-Cell Maturation into IFN-γ+ Effector Memory Cells.

Chlamydia trachomatis (Ct) infections are the most frequent bacterial sexually transmitted disease, and they can lead to ectopic pregnancy and infertility. Despite these detrimental long-term sequelae, a vaccine is not available. Success in preclinical animal studies is essential for vaccines to move to human clinical trials. Pigs are the natural host to Chlamydia suis (Cs)-a chlamydia species closely related to Ct, and are susceptible to Ct, making them a valuable animal model for Ct vaccine development. Before making it onto market, Ct vaccine candidates must show efficacy in a high-risk human population. The high prevalence of human Ct infection combined with the fact that natural infection does not result in sterilizing immunity, results in people at risk likely having been pre-exposed, and thus having some level of underlying non-protective immunity. Like human Ct, Cs is highly prevalent in outbred pigs. Therefore, the goal of this study was to model a trial in pre-exposed humans, and to determine the immunogenicity and efficacy of intranasal Cs vaccination in pre-exposed outbred pigs. The vaccine candidates consisted of UV-inactivated Cs particles in the presence or absence of an adjuvant (TriAdj). In this study, both groups of vaccinated pigs had a lower Cs burden compared to the non-vaccinated group; especially the TriAdj group induced the differentiation of CD4+ cells into tissue-trafficking CCR7- IFN-γ-producing effector memory T cells. These results indicate that Cs vaccination of pre-exposed pigs effectively boosts a non-protective immune response induced by natural infection; moreover, they suggest that a similar approach could be applied to human vaccine trials.

The T-Cell Response to Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause severe reproductive and respiratory pathologies resulting in immense monetary and welfare costs for the swine industry. The vaccines against PRRSV are available; but they struggle with providing protection against the plethora of heterologous PRRSV strains. To improve PRRSV vaccine development, the aim of this study was to provide an in-depth analysis of the crucial heterologous T-cell response to type-2 PRRSV. Following PRRSV modified live virus (MLV) vaccination or infection using one high- or one low-pathogenic PRRSV-strain, this nine-week study evaluated the T-cell response to different PRRSV strains. Our results demonstrate an important role for T cells in this homo- and heterologous response. Specifically, the T-helper cells were the main responders during viremia. Their peak response at 28 dpi correlated with a reduction in viremia, and their homing receptor expression indicated the additional importance for the anti-PRRSV response in the lymphatic and lung tissue. The cytocoxic T lymphocyte (CTL) response was the strongest at the site of infection-the lung and bronchoalveolar lavage. The TCR-γδ T cells were the main responders post viremia and PRRSV induced their expression of the lymph node homing the chemokine receptor, CCR7: This indicates a crucial role for TCR-γδ T cells in the anti-PRRSV response in the lymphatic system.

Evaluation of peripheral lymphocytes after weaning and vaccination for Mycoplasma hyopneumoniae.

This study evaluated immune cell populations in pigs following weaning and vaccination for Mycoplasma hyopneumoniae. Piglets (n=24) were weaned (day 0) at 16 (±1) days of age, and randomly assigned to the vaccination group (n=16) or control group (n=8). Complete blood cell counts, flow cytometry and serology were completed for blood samples collected on days 0 (within hours of weaning), 3, 7, 14, 30 and 60. The M. hyopneumoniae S:P ratios (sample optical density: positive control optical density) were negative in the vaccination group until days 30 and 60, when the S:P ratios were 1.3 and 1.0, respectively. Control animals remained serologically negative. The percentage of CD4(+) T cells was less (P<0.01) in control pigs than vaccinated pigs at day 3. In contrast, numbers of CD8(+) and CD4(+)CD8(+) T cells were greater (P<0.01) in control pigs than in vaccinated pigs at days 3 and 7. After day 7, few differences in immune cell types were evident between the groups. Differences in lymphocyte populations could not be solely attributed to vaccination, due at least in part, to the confounding influence of weaning. It was difficult to distinguish the influence of vaccination from the impact of weaning on peripheral immune cell populations.