Characterization of recent and minimally passaged Brazilian dengue viruses inducing robust infection in rhesus macaques

<div><p>The macaque is widely accepted as a suitable model for preclinical characterization of dengue vaccine candidates. However, the only vaccine for which both preclinical and clinical efficacy results were reported so far showed efficacy levels that were substantially different between macaques and humans. We hypothesized that this model’s predictive capacity may be improved using recent and minimally passaged dengue virus isolates, and by assessing vaccine efficacy by characterizing not only the post-dengue virus challenge viremia/RNAemia but also the associated-cytokine profile. Ten recent and minimally passaged Brazilian clinical isolates from the four dengue virus serotypes were tested for their infectivity in rhesus macaques. For the strains showing robust replication capacity, the associated-changes in soluble mediator levels, and the elicited dengue virus-neutralizing antibody responses, were also characterized. Three isolates from dengue virus serotypes 1, 2 and 4 induced viremia of high magnitude and longer duration relative to previously reported viremia kinetics in this model, and robust dengue virus-neutralizing antibody responses. Consistent with observations in humans, increased MCP-1, IFN-γ and VEGF-A levels, and transiently decreased IL-8 levels were detected after infection with the selected isolates. These results may contribute to establishing a dengue macaque model showing a higher predictability for vaccine efficacy in humans.</p></div

Characteristics of the DENV strains tested for their infectivity in rhesus macaques.

<p>Characteristics of the DENV strains tested for their infectivity in rhesus macaques.</p

Viremia and RNAemia detected after inoculation with DENV-1 0111/2011, DENV-2 0126/2010 or DENV-4 BEL 83791.

<p>Rhesus macaques were subcutaneously inoculated with ~10⁵ plaque- or focus-forming units (PFU and FFU, respectively) of DENV-1 0111/2011 (n = 5), DENV-2 0126/2010 (n = 5) or DENV-4 BEL 83791 (n = 6). Sera were collected daily during the 14 days post-inoculation and tested, in duplicate, by plaque or focus assay for their infectious virus content, <i>i</i>.<i>e</i>. viremia, expressed as PFU or FFU/mL (A) and by real-time RT-PCR for their DENV genome equivalent (ge) content, <i>i</i>.<i>e</i>. RNAemia, expressed as ge/mL (B). The individual viremia and RNAemia curves are shown. Horizontal dashed lines indicate the threshold of detection for the plaque/focus assay and the real-time RT-PCR assay, i.e. 2.5 PFU or FFU/mL and 10² ge/mL, respectively. In the absence of viremia and/or RNAemia detection, the corresponding sample was assigned an arbitrary titer corresponding to half the limit of detection.</p

Serum cytokine profiles observed after infection with DENV-1 0111/2011, DENV-2 0126/2010 or DENV-4 BEL 83791.

<p>Sera collected before (baseline) and at days 1, 4, 6, 8 and 14 after DENV inoculation were tested, in duplicate, for their concentration in the indicated cytokines. Results were expressed as pg/mL. When no signal was detected, the corresponding sample was assigned the arbitrary value of half the limit of detection for the corresponding cytokine. Shown are the mean changes from baseline and SEM from 5 (DENV-1 0111/2011 and DENV-2 0126/2010) and 6 (DENV-4 BEL 83791) animals.</p

Viremia and RNAemia as detected after DENV inoculation into rhesus macaques.

<p>Viremia and RNAemia as detected after DENV inoculation into rhesus macaques.</p

Comparative analysis of the cytokine signatures of innate and adaptive immunity triggered by the YF-17DD and YF-17D-213/77 substrains upon the <i>in vitro</i> recall of whole blood leukocytes from seroconverter children (PV-PRNT⁺) with specific YF-Ag.

<p>The diagrams highlight each leukocyte subsets with distinct tags as they display low (white rectangle) or high (black rectangle = inflammatory, gray rectangle = regulatory) cytokine producers (top panel). The ascendant frequency of volunteers with high cytokine indexes of the innate and adaptive immunity was assembled for each experimental arm and is demonstrated by bar graphs (medium panel). Comparative analysis of the overall cytokine patterns of YF-17DD (lines with black or gray triangles) and YF-17D-213/77 (lines with black or gray squares) vaccinees were further compared by overlapping the ascendant cytokine curves (bottom panel). Dotted lines highlight the 25^th and 50^th percentiles used as reference for comparative analysis. *Differences were considered relevant when the frequency for a given cytokine emerged outside the 50^th percentile as compared to the reference cytokine pattern or signature.</p

Flowchart of selection and follow-up of the Study Population from the Primary Target Sample.

<p>A total of 3,060 children, 9–12 months-old were elected for an epidemiological studies reported elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049828#pone.0049828-Collaborative1" target="_blank">[14]</a> and referred as “Primary Target Sample”. The Clinical Trial design is highlighted by dashed format. The study population enrolled in the present investigation was selected from the “Primary Target Sample” according to the PRNT results and comprise 30 PV-PRNT⁺ and 10 PV-PRNT⁻ individuals on each experimental arm (17DD and 17D-213/77), reaching a total of 80 volunteers. The current Immunological Study design is highlighted by solid format.</p

Impact of serum titers of anti-YF neutralizing antibodies on the cytokine-mediated immune response triggered by the YF-17DD and YF-17D-213/77 substrains upon <i>in vitro</i> recall of whole blood leukocytes from seroconverter children (PV-PRNT⁺) with specific YF-Ag.

<p>The PRNT⁺ groups from each experimental arm were first categorized into two subgroups referred to as PV-PRNT^MEDIUM+ (2.5≤ serum titers ≤3.5 log₁₀ mIU/mL) and PV-PRNT^HIGH+ (serum titers >3.5 log₁₀ mIU/mL). The cytokine profile of the PV-PRNT^MEDIUM+ and PV-PRNT^HIGH+ subgroups were evaluated, considering relevant the percentages of a given inflammatory cytokine that emerged higher than the 50^th percentile, as indicate by an upward arrow (↑).</p

Comparative inflammatory and regulatory cytokine signatures triggered by the YF-17DD and YF-17D-213/77 substrains upon the <i>in vitro</i> recall of whole blood leukocytes from (A) seroconverter (PV-PRNT⁺) and nonseroconverter primary vaccinees (PV-PRNT⁻) as well as (B) seroconverter revaccinees (RV-PRNT⁺) with specific YF-Ag.

<p>The ascendant frequency of volunteers with high inflammatory and regulatory cytokine indexes was assembled for each experimental arm and demonstrated by bar graphs. Comparative analysis between PV-PRNT⁺, PV-PRNT⁻, and RV-PRNT⁺ within the same experimental arm was performed taking the ascendant cytokine curve of the YF-17DD (lines with black or gray triangles) or YF-17D-213/77 (lines with black or gray squares) groups as reference. #Differences were considered relevant when the percentage of a given cytokine emerged below the quartile of the reference cytokine signatures.</p

Multi-parameter approach to evaluate the timing of memory status after 17DD-YF primary vaccination

<div><p>In this investigation, machine-enhanced techniques were applied to bring about scientific insights to identify a minimum set of phenotypic/functional memory-related biomarkers for post-vaccination follow-up upon yellow fever (YF) vaccination. For this purpose, memory status of circulating T-cells (Naïve/early-effector/Central-Memory/Effector-Memory) and B-cells (Naïve/non-Classical-Memory/Classical-Memory) along with the cytokine profile (IFN/TNF/IL-5/IL-10) were monitored before-NV(day0) and at distinct time-points after 17DD-YF primary vaccination—PV(day30-45); PV(year1-9) and PV(year10-11). A set of biomarkers (eEfCD4; EMCD4; CMCD19; EMCD8; IFNCD4; IL-5CD8; TNFCD4; IFNCD8; TNFCD8; IL-5CD19; IL-5CD4) were observed in PV(day30-45), but not in NV(day0), with most of them still observed in PV(year1-9). Deficiencies of phenotypic/functional biomarkers were observed in NV(day0), while total lack of memory-related attributes was observed in PV(year10-11), regardless of the age at primary vaccination. Venn-diagram analysis pre-selected 10 attributes (eEfCD4, EMCD4, CMCD19, EMCD8, IFNCD4, IL-5CD8, TNFCD4, IFNCD8, TNFCD8 and IL-5CD4), of which the overall mean presented moderate accuracy to discriminate PV(day30-45)&PV(year1-9) from NV(day0)&PV(year10-11). Multi-parameter approaches and decision-tree algorithms defined the EMCD8 and IL-5CD4 attributes as the top-two predictors with moderated performance. Together with the PRNT titers, the top-two biomarkers led to a resultant memory status observed in 80% and 51% of volunteers in PV(day30-45) and PV(year1-9), contrasting with 0% and 29% found in NV(day0) and PV(year10-11), respectively. The deficiency of memory-related attributes observed at PV(year10-11) underscores the conspicuous time-dependent decrease of resultant memory following17DD-YF primary vaccination that could be useful to monitor potential correlates of protection in areas under risk of YF transmission.</p></div