Monitoring bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

<div><p>Both veterinarians caring for dolphins in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin (<i>Tursiops truncatus</i>) health. Quantitative PCR (qPCR) can be used to assess cytokine transcription patterns of peripheral blood mononuclear cells (PBMC). This can supplement currently available blood tests with information on immune status. Full realization of this potential requires establishment of normal ranges of cytokine gene transcription levels in bottlenose dolphins. We surveyed four dolphins over the span of seven months by serial bleeds. PBMC were stimulated with phytohaemagglutinin (1, 5, and 10 μg/mL) and concanavalin A (1 μg/mL) for 48 H in vitro. RNA from these cultures was probed by qPCR using <i>Tursiops truncatus</i>-specific primers (IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-13, IL-18, IFN-γ and TNF-α). Two blood samples from an additional bottlenose dolphin diagnosed with acute pulmonary disease add further perspective to the data. We observed that mitogen choice made a significant difference in the magnitude of gene transcription observed. On the other hand, most cytokines tested exhibited limited intra-animal variation. However, IL-6 and IL-12p40 differed between older and younger dolphins. Furthermore, the magnitude of mitogenic response clusters the tested cytokines into three groups. The data provide a reference for the selection of target cytokine mRNAs and their expected range of mitogen-stimulated cytokine gene transcription for future studies.</p></div

Higher IL-6 and IL-12p40 MSCGT in older dolphins with chronic disease.

<p>RQ values for each cytokine and stimulus were pooled into two groups. Three to four samples from dolphin A were pooled with 4 samples from dolphin D. Similarly, 5 samples from dolphin B were pooled with one to two samples from dolphin C. A, C, E, G: RQ values are plotted against sampling date. Shaded area indicates range from 0.5–2 RQ considered no different from mock. B, D, F, H: Bars indicate geometric mean with 95% confidence interval for the same data points as in A, C, E and G. Stars indicate a statistically significant difference between the groups by two-tailed Mann-Whitney test. *: <i>p</i> ≤ 00332, **: <i>p</i> ≤ 0.0021.</p

List of primers used.

<p>List of primers used.</p

Baseline MSCGT is comparable between dolphins for most cytokines tested.

<p>RQ values obtained for each cytokine are shown assorted by each dolphin and stimulus. Error bars depict geometric mean with 95% confidence interval. The error bars for the <i>n</i> = 2 data from dolphin C are often cut off due to their relative size compared with the other data sets. Stars indicate a statistically significant difference from dolphin indicated in parentheses (A: dolphin A, C: dolphin C, D, dolphin D) by Tukey’s multiple comparisons test. *: <i>p</i> ≤ 00332, ***: <i>p</i> ≤ 0.0002.</p

Hierarchical clustering of cytokine responses.

<p>MeanRQ values for each cytokine are plotted in heat maps. (A) MeanRQ values for each cytokine were averaged across the four stimulation conditions. Data were clustered by cytokine and dolphin. (B-E) MeanRQ values are plotted for each dolphin. Data were clustered by cytokine. <i>(B)</i> Dolphin A; <i>(C)</i> dolphin B; <i>(D)</i> dolphin D; <i>(E)</i> dolphin C. MeanRQ values log₂ transformed before plotting. Hierarchical clustering conducted using Genesis Software (<a href="http://genome.tugraz.at/genesisclient/genesisclient_description.shtml" target="_blank">http://genome.tugraz.at/genesisclient/genesisclient_description.shtml</a>). The Log₂ legend is to the right of (A); the scale for (A) is on the left and the scale for B-E on the right.</p

Abbreviated dolphin clinical record.

<p>Abbreviated dolphin clinical record.</p

Overlay of abnormal and normal data from each dolphin.

<p>A minimum-to-maximum bar represents the range of RQ values obtained for each cytokine from each dolphin’s normal samples. The mean of RQ values from the normal samples is represented by a horizontal line. Overlaid bullet points indicate RQ values from abnormal samples. RQ values were log₂ transformed and analyzed one-way ANOVA. Stars indicate a statistically significant difference between normal samples from cohort dolphins and the abnormal dolphin E samples by Sidak’s multiple comparisons test. *: <i>p</i> ≤ 00332, **: <i>p</i> ≤ 0.0021.</p

Safety, tolerability, and immunogenicity of the chimpanzee adenovirus type 3-vectored Marburg virus (cAd3-Marburg) vaccine in healthy adults in the USA: a first-in-human, phase 1, open-label, dose-escalation trial

BACKGROUND: WHO has identified Marburg virus as an emerging virus requiring urgent vaccine research and development, particularly due to its recent emergence in Ghana. We report results from a first-in-human clinical trial evaluating a replication-deficient recombinant chimpanzee adenovirus type 3 (cAd3)-vectored vaccine encoding a wild-type Marburg virus Angola glycoprotein (cAd3-Marburg) in healthy adults. METHODS: We did a first-in-human, phase 1, open-label, dose-escalation trial of the cAd3-Marburg vaccine at the Walter Reed Army Institute of Research Clinical Trials Center in the USA. Healthy adults aged 18-50 years were assigned to receive a single intramuscular dose of cAd3-Marburg vaccine at either 1 × 10 or 1 × 10 particle units (pu). Primary safety endpoints included reactogenicity assessed for the first 7 days and all adverse events assessed for 28 days after vaccination. Secondary immunogenicity endpoints were assessment of binding antibody responses and T-cell responses against the Marburg virus glycoprotein insert, and assessment of neutralising antibody responses against the cAd3 vector 4 weeks after vaccination. This study is registered with ClinicalTrials.gov, NCT03475056. FINDINGS: Between Oct 9, 2018, and Jan 31, 2019, 40 healthy adults were enrolled and assigned to receive a single intramuscular dose of cAd3-Marburg vaccine at either 1 × 10 pu (n=20) or 1 × 10 pu (n=20). The cAd3-Marburg vaccine was safe, well tolerated, and immunogenic. All enrolled participants received cAd3-Marburg vaccine, with 37 (93%) participants completing follow-up visits; two (5%) participants moved from the area and one (3%) was lost to follow-up. No serious adverse events related to vaccination occurred. Mild to moderate reactogenicity was observed after vaccination, with symptoms of injection site pain and tenderness (27 [68%] of 40 participants), malaise (18 [45%] of 40 participants), headache (17 [43%] of 40 participants), and myalgia (14 [35%] of 40 participants) most commonly reported. Glycoprotein-specific antibodies were induced in 38 (95%) of 40 participants 4 weeks after vaccination, with geometric mean titres of 421 [95% CI 209-846] in the 1 × 10 pu group and 545 [276-1078] in the 1 × 10 pu group, and remained significantly elevated at 48 weeks compared with baseline titres (39 [95% CI 13-119] in the 1 ×10 pu group and 27 [95-156] in the 1 ×10 pu group; both p\u3c0·0001). T-cell responses to the glycoprotein insert and neutralising responses against the cAd3 vector were also increased at 4 weeks after vaccination. INTERPRETATION: This first-in-human trial of this cAd3-Marburg vaccine showed the agent is safe and immunogenic, with a safety profile similar to previously tested cAd3-vectored filovirus vaccines. 95% of participants produced a glycoprotein-specific antibody response at 4 weeks after a single vaccination, which remained in 70% of participants at 48 weeks. These findings represent a crucial step in the development of a vaccine for emergency deployment against a re-emerging pathogen that has recently expanded its reach to new regions. FUNDING: National Institutes of Health