Low-dose Recipients Research Articles

Blood transcriptional correlates of BCG-induced protection against tuberculosis in rhesus macaques

Blood-based correlates of vaccine-induced protection against tuberculosis (TB) are urgently needed. Here, we analyze the blood transcriptome of rhesus macaques immunized with varying doses of intravenous (i.v.) BCG followed by Mycobacterium tuberculosis (Mtb) challenge. We use high-dose i.v. BCG recipients for "discovery" and validate our findings in low-dose recipients and in an independent cohort of macaques receiving BCG via different routes. We identify seven vaccine-induced gene modules, including an innate module (module 1) enriched for type 1 interferon and RIG-I-like receptor signaling pathways. Module 1 on day 2 post-vaccination highly correlates with lung antigen-responsive CD4 Tcells at week 8 and with Mtb and granuloma burden following challenge. Parsimonious signatures within module 1 at day 2 post-vaccination predict protection following challenge with area under the receiver operating characteristic curve (AUROC) ≥0.91. Together, these results indicate that the early innate transcriptional response to i.v. BCG in peripheral blood may provide a robust correlate of protection against TB.

2347. Blood Transcriptional Correlates of Vaccine-Induced Protection Against TB in Macaques

Abstract Background Identifying correlates of vaccine-induced protection against tuberculosis (TB) is essential for improved vaccine development and evaluation. Prior efforts have focused on antigen-specific CD4 T cell responses measured weeks following vacination, which have repeatedly failed to correlate with protection. Methods Here, we analyzed the blood transcriptional response to vaccination in rhesus macaques vaccinated intravenously (IV) with high- (N=16) or low-dose (N=18) BCG that exhibited varying protection against Mtb challenge six months later. We identified correlates of protection in high-dose recipients and validated in low-dose recipients and in an independent cohort of BCG-vaccinated macaques. We also compared the blood transcriptional response to adaptive responses in the bronchoalveolar lavage (BAL). Results We identified and independently validated seven modules of 2,499 genes induced two days, two weeks, four weeks, and/or twelve weeks post-vaccination. Adaptive modules were associated with protection against challenge in low-dose recipients but not high-dose recipients. In contrast, an innate module (Module 1) on day 2 post-vaccination was associated with protection outcomes eight months later in both high- and low-dose recipients, as well as in an independent cohort of macaques. Module 1 scores at day 2 post-vaccination were also highly correlated with BAL adaptive responses, including Mtb-specific IgA and IgG titers at week 4, log T cell (CD4+, CD8+, MAIT, Vg9) counts at week 4, and antigen-specific CD4+ T cell responses (IFNg, IL2, IL21, TNF) at week 8 (r >0.7, p< 0.0001). None of these lung adaptive markers could reliably predict protection in both high- and low-dose recipients. However, module 1 scores at day 2 post-vaccination accurately predicted protection following Mtb challenge six months later (high-dose recipients, AUC=0.8; low-dose recipients, AUC=0.99). The early innate response in peripheral blood correlates with outcomes post-challenge 8 months later. Correlation between module 1 (innate) scores on day 2 post-vaccination and total thoracic colony forming units (CFU) and number of granulomas following challenge eight months later in high- and low-dose recipients (A-B) and in an independent cohort of macaques vaccinated with BCG through different routes (C-D). AE, aerosol; HD-ID, high dose intradermal; ID, intradermal; ID/AE, intradermal and aerosol; IV, intravenous. The early innate response in peripheral blood is highly correlated with lung CD4 T cell responses 8 weeks post-vaccination. Correlation between module 1 (innate) scores at day 2 post-vaccination and cytokine expression by CD4 T cells in bronchoalveolar lavage (BAL) upon whole cell lysate restimulation. Anyg2T17, any of IFNg, IL2, TNF, or IL17. Conclusion These results suggest that adaptive responses are necessary but not sufficient for BCG-induced protection. Instead, the early innate response in peripheral blood may be a more reliable and generalizable correlate of BCG-induced protection. Disclosures Purvesh Khatri, PhD, Cepheid, Inc.: Advisor/Consultant|Inflammatix, Inc.: Advisor/Consultant|Inflammatix, Inc.: Inflammatix is in negotiations to license the 9-gene signature discussed here.|Inflammatix, Inc.: Stocks/Bonds.

In This Issue

HomeCirculation ResearchVol. 120, No. 2In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published20 Jan 2017https://doi.org/10.1161/RES.0000000000000134Circulation Research. 2017;120:245SMC Reprogramming to Resident Progenitor Cells (p 296)Download figureDownload PowerPointMajesky et al discover an unexpected origin of vascular progenitor cells.The adventitia, which forms the outer layer of blood vessels, is a collagen-rich tissue that has been traditionally thought to provide only structural support to the vessel. But recent research has shown that the adventitia contains a variety of dynamic cell types, such as the vascular progenitor cells. The origin of these progenitors was unclear, but Majesky and colleagues have now discovered, quite by accident, the source of at a least a subpopulation of these cells in mice. The team was studying how, after injury, the smooth muscle cells (SMCs) within the medial layer of blood vessels dedifferentiate and migrate to the innermost intimal layer. Unexpectedly, the team observed that SMCs also migrate to the adventitia, where they lost their SMC characteristics and gained stem cell markers. Further fate-mapping experiments confirmed that SMCs do indeed give rise to a subpopulation of adventitia-residing progenitors. These SMC-derived progenitors were multipotent, giving rise to several cell types in vitro and in matrigel implants in mice. The team also found that the transition from SMC to progenitor was regulated by the transcription factor Klf4. Further studies are required to assess how these SMC-derived progenitors contribute to vascular homeostasis and pathology and how these cells could be targeted to treat vascular diseases, say the authors.CD34+ Cells for Post STEMI LV Dysfunction (p 324)Download figureDownload PowerPointQuyyumi et al report the results of a phase II stem cell trial for treating myocardial infarction.Nearly 20% of patients aged 45 and over who suffer a heart attack die within a year. Treatments to improve outcomes are desperately needed, and one possible therapeutic approach that has received much recent attention is coronary infusion of bone marrow-derived cells. Indeed, over 2600 patients have received autologous bone marrow cells in a variety of trials, but the outcomes have been inconsistent. It has been suggested that such unpredictable results may be due to variable bone marrow cell constituents, and that enriching for cells that express the stem cell marker CD34 may provide cell populations with greater clinical benefit. Indeed, a phase I trial of treatment with autologous CD34-positive bone marrow cells improved outcomes in heart attack victims. Now, Quyyumi and colleagues report that some patients enrolled in a randomized, double-blind phase II trial of CD34 cells also fared better than control subjects—with improved ejection fractions and reduced infarct sizes after 6 months. Improvement was seen in patients who were given a high-dose of cells (over 20 million), but low-dose recipients were indistinguishable from controls. The results confirm the safety of autologous CD34+ cell infusions and suggest that high doses of these cells may have some clinical benefit. Based on these results, the authors suggest that further randomized controlled trials should be performed to examine the efficacy of bone marrow-derived CD34+ cells.Mesenchymal Stem Cells for Heart Failure (p 332)Download figureDownload PowerPointButler et al present the results of an intravenous stem cell therapy trial for nonischemic cardiomyopathy.Nonischemic cardiomyopathy is a contractile dysfunction not caused by coronary artery disease (CAD) or myocardial infarction (MI). Nevertheless, as with CAD- or MI-induced heart failure, patients with nonischemic cardiomyopathy could potentially benefit from stem cell therapy. To date, cell therapies for any form of heart failure have been administered directly to the heart—because cell engraftment was considered necessary for clinical effect. However, some effects may derive from secreted factors, such as the anti-inflammatory factors produced by mesenchymal stem cells (MSCs). Butler and colleagues have, therefore, performed the first trial of intravenously administered stem cells for heart failure, giving 22 patients with nonischemic cardiomyopathy infusions of allogenic MSCs. While the team noted no difference in the occurrence of death or hospitalization between test and control subjects, patients receiving the MSCs had reduced systemic inflammation and could walk greater distances after 3 months. An inverse association between inflammation and left ventricle ejection fraction was also noted. These promising early results now pave the way for further follow up trials, say the authors. Previous Back to top Next FiguresReferencesRelatedDetails January 20, 2017Vol 120, Issue 2 Advertisement Article InformationMetrics © 2017 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000134PMID: 30520342 Originally publishedJanuary 20, 2017 PDF download Advertisement

Highly pathogenic avian influenza A virus (H5N1) can be transmitted in ferrets by transfusion

sBackgroundHighly pathogenic avian influenza A virus has been shown to infect organs other than the lung, and this is likely to be mediated by systemic spread resulting from viremia which has been detected in blood in severe cases of infection with avian H5N1 viruses. The infectivity of virus in blood and the potential for virus transmission by transfusion has not been investigated.MethodsUsing a susceptible ferret animal model, we evaluated viremia and transmission by blood transfusion. Blood was collected on day 2, 4, 6, and 10 post-infection (or before death) from donor ferrets infected with either low dose (1.0 × 102.6 EID50/ml) or high dose (1.0 × 103.6 EID50/ml) of H5N1 virus, A/VN/1203/04 and transfused to recipient animals.ResultsViremia was observed in 2/12 (16.67%) recipients that received blood from donor ferrets infected with low dose and 7/12 (58.33%) recipients who received blood from high dose infected donors. 1/12 (8.3%) low dose recipients and 6/12 (50%) high dose recipients died within 11 days after transfusion. Increased changes in body weight and temperatures were observed in high dose recipients, and high levels of viral RNA were detected in recipient ferrets after transfusion of blood from the early viremic phase, which also correlated with adverse impact on their survival.ConclusionThese data suggest that highly pathogenic avian influenza A virus, H5N1, is transmissible by blood transfusion in ferrets. Low levels of viremia were detected around the time of onset of symptoms and later in ferrets infected with highly pathogenic H5N1 virus. These findings may have implication for pathogenesis and transmissibility of H5N1.

How low can you go? Use of low- and standard-dose liposomal amphotericin B for treatment of invasive fungal infections

Recommended doses of liposomal amphotericin B (L-AMB) range from 3 to 6 mg/kg/day, but 1mg/kg/day may be equally effective and a lower cost alternative for many indications. The objective of this analysis was to assess indications and clinical outcomes of patients who received low-dose (1mg/kg/day rounded up in 50-mg increments) and standard-dose (≥2 mg/kg/day) L-AMB. This was a retrospective analysis of adult L-AMB recipients with suspected invasive fungal infections (IFI) at a single center from 2006 to 2011. The primary outcome was clinical response at the end of treatment. Secondary outcomes included survival and toxicity. Results were analyzed using Chi-square and descriptive statistics. Of 89 adult L-AMB recipients included, 36 had proven or probable IFIs. Nineteen (53%) received low doses and 17 (47%) received standard doses. Median doses were 1.5 and 3.0mg/kg/day. Cryptococcus was the most common fungal pathogen in the low-dose group (37%), and Candida spp. in the standard-dose group (47%). Forty-seven percent of subjects in both groups improved clinically. Sixty-eight percent of low-dose recipients and 76% of standard-dose recipients survived to discharge. Rates of nephrotoxicity and hypokalemia were comparable. Comparable rates of clinical improvement, survival to discharge, and toxicity were identified among low- and standard-dose L-AMB recipients.

Intravenous administration of replication-incompetent adenovirus to rhesus monkeys induces thrombocytopenia by increasing in vivo platelet clearance.

A replication-incompetent adenovirus vector was administered to rhesus macaques at 1, 3 and 6 x 1012 particles/kg doses to investigate its toxicity. Platelet count decrements of 28%, 82% and 90%, respectively, were observed, with corresponding platelet half-lives of 69.0, 25.2 and 22.2 h (compared with 111 h in untreated animals). The platelet decline was equivalent for all three doses for 8 h, and platelet count recovery began as early as 8 h after infusion for low-dose recipients, or as late as 24 h for the medium and high dose recipients. These observations suggest that thrombocytopenia is a saturable, reversible consumptive process.

In Vitro and In Vivo Effects of Recombinant Human Interleukin-2 in Naive Miniature Swine

Recent data in mice have shown that early administration of recombinant human interleukin-2 (rIL-2) provides significant protection from lethal graft-versus-host disease. Because of the potential clinical importance of these findings, it will be important to assess the effectiveness of this therapy in a large animal preclinical bone marrow transplantation model. We report here our initial studies of the in vitro and in vivo effects of rIL-2 in miniature swine. In vitro 4-day cultures of pig peripheral blood lymphocytes (PBL) in complete medium containing rIL-2 at 1,000 U/ml resulted in optimal proliferation and generation of lymphokine-activated killer (LAK) cells. A pig-mouse hybridoma cell line was found to be highly sensitive as a LAK cell target. Two naive pigs received 20,000 U/kg and 2 pigs received 100,000 U/kg of rIL-2 intravenously twice a day for 4 days. No clinical symptoms were seen during or after administration at the lower dose while both high dose-treated animals showed generalized erythema from days 2 to 4, and one showed mild diarrhea during this period. The disappearance of IL-2 activity from the serum showed two components: (1) an initial fast component with a half-time of approximately 10 min and (2) a slow component with a half-time of approximately 60 min. LAK cell precursors disappeared from the peripheral circulation by 6 min after rIL-2 administration and began to recover by 6 h in the low dose recipients and only after 12 h in the high dose recipients.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of etomidate on cerebral metabolism and blood flow in a canine model for hypoperfusion

The effects of etomidate, a nonbarbiturate cerebral metabolic depressant, on cerebral metabolism and blood flow were studied in 29 dogs during cerebral hypoperfusion. Three groups of animals were studied during a 45-minute normotensive and a 30-minute hypotensive period: 10 control animals without etomidate, 11 animals receiving a 0.1-mg/kg etomidate bolus followed by an infusion of 0.05 mg/kg/min etomidate (low-dose group), and eight animals receiving doses of etomidate sufficient to suppress electroencephalographic bursts (high-dose group). The mean arterial pressure fell to similar levels (p less than 0.05) during hypotension in all three groups (40 +/- 5, 38 +/- 3, and 27 +/- 6 mm Hg, respectively). The mean cerebral oxygen extraction fraction rose (p less than 0.05) from 0.23 +/- 0.02 to 0.55 +/- 0.08 in the five control animals tested and from 0.33 +/- 0.02 to 0.53 +/- 0.02 in the seven animals tested in the low-dose group, but did not increase (p greater than 0.05) in the four animals tested in the high-dose group (0.24 +/- 0.03 to 0.23 +/- 0.05). Mean cerebral blood flow levels decreased in all groups during hypotension (p less than 0.05): 42 +/- 3 to 21 +/- 4 ml/100 gm/min (52% +/- 12% decrease) in the five animals tested in the control group, 60 +/- 8 to 24 +/- 6 ml/100 gm/min (56% +/- 13% decrease) in the four animals tested in the low-dose group, and 55 +/- 8 to 22 +/- 3 ml/100 gm/min (60% +/- 4% decrease) in the four animals tested in the high-dose group. In summary, the cerebral oxygen extraction fraction increased in the control animals and low-dose recipients during hypotension, suggesting the presence of threatened cerebral tissue. In contrast, the cerebral oxygen extraction did not change during hypotension when high-dose etomidate was administered. It is concluded that high-dose etomidate may preserve the cerebral metabolic state during hypotension in the present model.

T-cell effector function and unresponsiveness in the murine lymphocytic choriomeningitis virus infection. I. On the mechanism of a selective suppression of the virus-specific delayed-type hypersensitivity response.

When the virus dose is increased from 10(2) (low dose) to 10(4) LD50 (high dose) a fatal lymphocytic choriomeningitis virus (LCMV) infection is changed into a subclinical one, and a selective virus-specific delayed-type hypersensitivity (DTH) unresponsiveness is induced, while the cytotoxic T-cell response remains essentially unchanged. When low-dose spleen effectors were transferred intravenously into intracerebrally infected high-dose mice, fatal LCM disease occurred, which means that infected central nervous system target structures in these animals are sensitive to virus-specific T cells. When low-dose cells were transferred to intravenously infected high-dose mice, these animals regained their TD function (the effect of T cells mediating DTH). Since this indicates that the survival of intracerebrally infected high-dose mice is intimately linked with the absence of virus-specific DTH reactivity, a search for T suppressor (TS) activity in these animals was performed by transferring high-dose spleen cells to lethally (intracerebrally) infected low-dose recipients. In this way we obtained an afferent suppression, which was not H-2 restricted, but was abrogated when the spleen cells were pretreated with neutralizing anti-LCMV serum, indicating a suppressive effect of virus transferred with the infected cells. When tolerance induction was attempted with virus alone, a potentially fatal immune reaction could be altered to unresponsiveness (i.e. survival) as late as 4 days after an otherwise lethal infection with LCMV. The results indicate that the maturation of the virus-specific TD response is sensitive to large amounts of virus antigen. We conclude that this impairment and the resulting DTH unresponsiveness is due to a clonal deletion or anergy rather than to the effect of TS cells, and that the TD effector function is critical to the development of fatal LCM disease.

Dengue-2 vaccine: viremia and immune responses in rhesus monkeys.

Studies were undertaken in Indian rhesus monkeys (Macaca mulatta) to determine the safety, potency, immunogenicity, and mosquito infectivity of a small-plaque, temperature-sensitive variant of dengue type 2 (DEN-2) virus, a vaccine candidate. Fifteen monkeys were inoculated subcutaneously with the vaccine virus, ten receiving 10(3.1) plaque-forming units (PFU) and five receiving 10(4.5) PFU. After primary immunization, viremia was detected in only one monkey, a recipient of the higher dose of vaccine. The recovered virus had the same growth characteristics as the vaccine strain. Aedes aegypti mosquitoes did not become infected when they were allowed to feed on monkeys that received the lower dose of vaccine. As expected, the immunization produced no evidence of illness in any of the animals. A dose response to vaccine was detected; all five of the high-dose recipients developed neutralizing antibodies, whereas only five of ten low-dose recipients did so. In both groups, neutralizing antibody was often transient. Its presence at 30 days did not always correlate with protection from viremia in those animals challenged 4 to 6 months after vaccination with wild-type DEN-2 virus. However, immunized animals developed anamnestic antibody responses after challenge, and none demonstrated adverse effects to infection. Reimmunization of monkeys 4 months after primary immunization led to the production of low-titered but persistent neutralizing antibody which protected the animals from a wild-type virus challenge.