Rhythmic profile of memory T and B-cells along childhood and adolescence.

Immunobiography describes the life-long effects of exogenous or endogenous stimuli on remodeling of immune cell biology, including the development of memory T and B-cells. The present study aimed at investigating the rhythms of changes in phenotypic features of memory T and B-cells along childhood and adolescence. A descriptive-observational investigation was conducted including 812 healthy volunteers, clustered into six consecutive age groups (9Mths-1Yr; 2Yrs; 3-4Yrs; 5-7Yrs; 8-10Yrs; 11-18Yrs). Immunophenotypic analysis of memory T-cell (CD4+ and CD8+) and B-cell subsets were performed by flow cytometry. The results pointed out that memory-related biomarkers of T and B-cells displayed a bimodal profile along healthy childhood and adolescence, regardless of sex. The first stage of changes occurs around 2Yrs, with predominance of naive cells, while the second and more prominent wave occurs around the age 8-10Yrs, with the prevalence of memory phenotypes. The neighborhood connectivity profile analysis demonstrated that the number of correlations reaches a peak at 11-18Yrs and lower values along the childhood. Males presented higher and conserved number of correlations when compared to females. Altogether, our results provide new insights into immunobiography and a better understanding of interactions among the cellular subsets studied here during childhood and adolescence.

Exploratory study of humoral and cellular immunity to 17DD Yellow Fever vaccination in children and adults residents of areas without circulation of Yellow Fever Virus.

The present investigation comprised two independent observational arms to evaluate the influence of pre-existing flavivirus humoral immunity and the age-impact on 17DD-YF vaccination immunity. Flavivirus (YFV; DENV; ZIKV) serology and YF-specific cellular immunity was evaluated in 288 children/9Mths-4Yrs and 288 adults/18-49Yrs residents of areas without YFV circulation. Data demonstrated that flavivirus seropositivity at baseline was higher in Adults as compared to Children (26%;87%;67% vs 6%;13%;15%, respectively). The heterologous flavivirus seropositivity (DENV; ZIKV) did not impact the YF-specific cellular immune response at baseline. However, higher levels of NCD4, EMCD8, IFN-MCD8, NCD19 and nCMCD19 were observed in subjects with pre-existing YFV seropositivity. Primary vaccination of YFV-seronegative volunteers led to higher levels of YF-neutralizing antibodies in Adults as compared to Younger Children (9Mths-2Yrs). Although similar seropositivity rates observed amongst Children and Adults at D30-45, lower rates were observed in Younger Children (9Mths-2Yrs) at D365 (94%;95%;100% vs 87%;96%;99%, respectively). A progressive decline in antibody levels were reported at D365, being more expressive in Children as compared to Adults. All age-subgroups exhibited at D30-45 increased levels of eEfCD4, EMCD4, IFN-MCD8 and nCMCD19 together with a decrease of eEfCD8 and CMCD8. While an increase of EMCD8 were observed in all subgroups at D30-45, a declined duration at D365 was reported only in Younger Children (9Mths-2Yrs). Biomarker signatures further support that only Younger Children (9Mths-2Yrs) presented a progressive decline of EMCD8 at D365. Together, these findings demonstrated that regardless the similarities observed in YF-neutralizing antibodies, the age impacts the duration of cellular immune response to primary 17DD-YF vaccination.

Corrigendum: Duration of Humoral and Cellular Immunity 8 Years After Administration of Reduced Doses of the 17DD-Yellow Fever Vaccine.

[This corrects the article DOI: 10.3389/fimmu.2019.01211.].

Short-Lived Immunity After 17DD Yellow Fever Single Dose Indicates That Booster Vaccination May Be Required to Guarantee Protective Immunity in Children.

The Yellow Fever (YF) vaccination is recommended for people living in endemic areas and represents the most effective strategy to reduce the risk of infection. Previous studies have warned that booster regimens should be considered to guarantee the long-term persistence of 17DD-YF-specific memory components in adults living in areas with YF-virus circulation. Considering the lower seroconversion rates observed in children (9-12 months of age) as compared to adults, this study was designed in order to access the duration of immunity in single-dose vaccinated children in a 10-years cross-sectional time-span. The levels of neutralizing antibodies (PRNT) and the phenotypic/functional memory status of T and B-cells were measured at a baseline, 30-45 days, 1, 2, 4, 7, and 10 years following primary vaccination. The results revealed that a single dose induced 85% of seropositivity at 30-45 days and a progressive time-dependent decrease was observed as early as 2 years and declines toward critical values (below 60%) at time-spans of ≥4-years. Moreover, short-lived YF-specific cellular immunity, mediated by memory T and B-cells was also observed after 4-years. Predicted probability and resultant memory analysis emphasize that correlates of protection (PRNT; effector memory CD8+ T-cells; non-classical memory B-cells) wane to critical values within ≥4-years after primary vaccination. Together, these results clearly demonstrate the decline of 17DD-YF-specific memory response along time in children primarily vaccinated at 9-12 months of age and support the need of booster regimen to guarantee the long-term persistence of memory components for children living in areas with high risk of YF transmission.

Duration of Humoral and Cellular Immunity 8 Years After Administration of Reduced Doses of the 17DD-Yellow Fever Vaccine.

The present study aims to determine whether 17DD-YF-specific humoral and cellular immunological memory is maintained 8-years after primary vaccination with subdoses (10,447IU;3,013IU;587IU;158IU;31IU). For this purpose, this follow-up study was carried out in a subset of volunteers (n = 98) originally enrolled in the dose-response study in 2009 and 46 non-vaccinated controls. Our results demonstrated that vaccinees, who had seroconverted following primary vaccination and had not been revaccinated, present similar neutralizing antibodies levels and YF-specific cellular memory, particularly CMCD4 and EMCD8 as compared to the reference full dose (27,476IU). Although, PRNT seropositivity rates were similar across subgroups (94, 82, 83, 94, 80, and 91%, correspondingly), only doses above 587IU elicited similar iterative proportion of seropositivity rates, calculated as a progressive decrease on seropositivity rates along time (89, 80, 80, and 91%, respectively) as compared to 158IU and 31IU (68 and 46%, respectively). Noteworthy were the strong positive correlations ("EMCD4,EMCD8" and "TNFCD8,IFNCD8") observed in most subdoses, except for 31IU. Major similarities underscored the preserved antibody titers and the outstanding levels of EMCD8, relevant correlates of protection for YF-specific immunity. These findings provide evidences to support the regular use of dose sparing strategy for YF vaccine in adults.

17DD Yellow Fever Revaccination and Heightened Long-Term Immunity in Populations of Disease-Endemic Areas, Brazil.

We evaluated the duration of neutralizing antibodies and the status of 17DD vaccine-specific T- and B-cell memory following primary and revaccination regimens for yellow fever (YF) in Brazil. We observed progressive decline of plaque-reduction neutralization test (PRNT) seropositivity and of the levels of effector memory CD4+ and CD8+ T cells, as well as interferon-γ+CD8+ T cells, 10 years after primary vaccination. Revaccination restored PRNT seropositivity as well as the levels of effector memory CD4+, CD8+, and interferon-γ+CD8+ T cells. Moreover, secondary or multiple vaccinations guarantee long-term persistence of PRNT positivity and cell-mediated memory 10 years after booster vaccination. These findings support the relevance of booster doses to heighten the 17DD-YF-specific immune response to guarantee the long-term persistence of memory components. Secondary or multiple vaccinations improved the correlates of protection triggered by 17DD-YF primary vaccination, indicating that booster regimens are needed to achieve efficient immunity in areas with high risk for virus transmission.

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

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.

The 17D-204 and 17DD yellow fever vaccines: an overview of major similarities and subtle differences.

INTRODUCTION: The yellow fever vaccine is a live attenuated virus vaccine that is considered one of the most efficient vaccines produced to date. The original 17D strain generated the substrains 17D-204 and 17DD, which are used for the current production of vaccines against yellow fever. The 17D-204 and 17DD substrains present subtle differences in their nucleotide compositions, which can potentially lead to variations in immunogenicity and reactogenicity. We will address the main changes in the immune responses induced by the 17D-204 and 17DD yellow fever vaccines and report similarities and differences between these vaccines in cellular and humoral immunity . This is a relevant issue in view of the re-emergence of yellow fever in Uganda in 2016 and in Brazil in the beginning of 2017. AREAS COVERED: This article will be divided into 8 sections that will analyze the innate immune response, adaptive immune response, humoral response, production of cytokines, immunity in children, immunity in the elderly, gene expression and adverse reactions. EXPERT COMMENTARY: The 17D-204 and 17DD yellow fever vaccines present similar immunogenicity, with strong activation of the cellular and humoral immune responses. Additionally, both vaccines have similar adverse effects, which are mostly mild and thus are considered safe.

Heparin removal by ecteola-cellulose pre-treatment enables the use of plasma samples for accurate measurement of anti-Yellow fever virus neutralizing antibodies.

Technological innovations in vaccinology have recently contributed to bring about novel insights for the vaccine-induced immune response. While the current protocols that use peripheral blood samples may provide abundant data, a range of distinct components of whole blood samples are required and the different anticoagulant systems employed may impair some properties of the biological sample and interfere with functional assays. Although the interference of heparin in functional assays for viral neutralizing antibodies such as the functional plaque-reduction neutralization test (PRNT), considered the gold-standard method to assess and monitor the protective immunity induced by the Yellow fever virus (YFV) vaccine, has been well characterized, the development of pre-analytical treatments is still required for the establishment of optimized protocols. The present study intended to optimize and evaluate the performance of pre-analytical treatment of heparin-collected blood samples with ecteola-cellulose (ECT) to provide accurate measurement of anti-YFV neutralizing antibodies, by PRNT. The study was designed in three steps, including: I. Problem statement; II. Pre-analytical steps; III. Analytical steps. Data confirmed the interference of heparin on PRNT reactivity in a dose-responsive fashion. Distinct sets of conditions for ECT pre-treatment were tested to optimize the heparin removal. The optimized protocol was pre-validated to determine the effectiveness of heparin plasma:ECT treatment to restore the PRNT titers as compared to serum samples. The validation and comparative performance was carried out by using a large range of serum vs heparin plasma:ECT 1:2 paired samples obtained from unvaccinated and 17DD-YFV primary vaccinated subjects. Altogether, the findings support the use of heparin plasma:ECT samples for accurate measurement of anti-YFV neutralizing antibodies.

Vaccination against canine leishmaniosis increases the phagocytic activity, nitric oxide production and expression of cell activation/migration molecules in neutrophils and monocytes.

Visceral leishmaniasis (VL) is transmitted by phlebotomine sandfly vectors and domestic dogs serve as a reservoir. The elimination of seropositive dogs has been a recommended strategy for managing the disease in Brazil. A protective canine vaccine would be an important tool for controlling the disease, reducing the parasites available to sandfly vectors and, consequently, reducing the number of human VL cases. Leishmune(®) is an anti-canine Leishmaniosis (VL Canine) vaccine produced by Zoetis (Pfizer, Brazil) that was commercially available in Brazil until 2014. The main goal of the present study was to investigate the protective immunological events induced by vaccination with Leishmune(®) in the time frame of one year. Healthy, non-vaccinated dogs and dogs of 1, 6 and 10 months post-vaccination were evaluated. Results showed that Leishmune(®) induced an increase in phagocytic activity of neutrophils and monocytes and also increased NO production. Immunological events were correlated with functional responses, as high levels of IgG and an increase of the receptor Fcγ were detected. Vaccination induced an increased expression of TLR (2, 4, 5, 9), integrin (CD29, CD49f), activation (MHCII) and co-stimulatory (CD80, CD81) molecules by neutrophils and monocytes. Vaccination led to decrease of IL-4 and an increase of IL-8 production by monocytes and higher IFN-γ and IL-17 production by T-cells. The results suggested that Leishmune(®) was able to induce a long-lasting change in immune response, mediated by supportive immunological events that may be participating in protective immunity against CL.

Booster dose after 10 years is recommended following 17DD-YF primary vaccination.

A single vaccination of Yellow Fever vaccines is believed to confer life-long protection. In this study, results of vaccinees who received a single dose of 17DD-YF immunization followed over 10 y challenge this premise. YF-neutralizing antibodies, subsets of memory T and B cells as well as cytokine-producing lymphocytes were evaluated in groups of adults before (NVday0) and after (PVday30-45, PVyear1-4, PVyear5-9, PVyear10-11, PVyear12-13) 17DD-YF primary vaccination. YF-neutralizing antibodies decrease significantly from PVyear1-4 to PVyear12-13 as compared to PVday30-45, and the seropositivity rates (PRNT≥2.9Log10mIU/mL) become critical (lower than 90%) beyond PVyear5-9. YF-specific memory phenotypes (effector T-cells and classical B-cells) significantly increase at PVday30-45 as compared to naïve baseline. Moreover, these phenotypes tend to decrease at PVyear10-11 as compared to PVday30-45. Decreasing levels of TNF-α(+) and IFN-γ(+) produced by CD4(+) and CD8(+) T-cells along with increasing levels of IL-10(+)CD4(+)T-cells were characteristic of anti-YF response over time. Systems biology profiling represented by hierarchic networks revealed that while the naïve baseline is characterized by independent micro-nets, primary vaccinees displayed an imbricate network with essential role of central and effector CD8(+) memory T-cell responses. Any putative limitations of this cross-sectional study will certainly be answered by the ongoing longitudinal population-based investigation. Overall, our data support the current Brazilian national immunization policy guidelines that recommend one booster dose 10 y after primary 17DD-YF vaccination.

Cross-reactivity of commercially available anti-human monoclonal antibodies with canine cytokines: establishment of a reliable panel to detect the functional profile of peripheral blood lymphocytes by intracytoplasmic staining.

BACKGROUND: The process for obtaining monoclonal antibodies against a specific antigen is very laborious, involves sophisticated technologies and it is not available in most research laboratories. Considering that most cytokines remain partially conserved among species during evolution, the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine. In this context, the amino acid sequence from human and canine cytokines have demonstrated 49-96 % homology, suggesting high probability of cross-reactivity amongst monoclonal antibodies. For this, 17 commercially available anti-human monoclonal antibodies [IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-10, IL-12, IL-13, IL-17A, IFN-γ (#1, #2), TNF-α (#1, #2) and TGF-ß], were evaluated in vitro for intracellular cytokine detection in a stimulated canine blood culture by flow cytometry and confocal microscopy. Lymphocytes from peripheral blood of healthy and two unhealthy dogs were analyzed. RESULTS: Eleven anti-human mAbs [IL-1α, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-12, IL-17A, TNF-α (#1, #2) and TGF-ß] cross-reacted against canine intracellular cytokines. The specificity of the assays was not affected after Fc-blocking. Three anti-human cytokine mAbs [IL-4, IL-8 (#2) and TGF-ß] when evaluated by confocal microscopy also cross-reacted with intracellular canine cytokines. The identification of human mAbs that cross-reacted with canine cytokines may support their use as immunological biomarkers in veterinary medicine studies. CONCLUSION: The identification of these 11 anti-human cytokine mAbs that cross-reacted with canine cytokines will be useful immunological biomarkers for pathological conditions by flow cytometry and fluorescence microscopy in dogs.

One-year timeline kinetics of cytokine-mediated cellular immunity in dogs vaccinated against visceral leishmaniasis.

BACKGROUND: The main control strategy for visceral leishmaniasis in Brazil has been based on the elimination of seropositive dogs, although this is not widely accepted. In this context, the use of a long-lasting protective vaccine against canine visceral leishmaniasis (CVL) has been highly expected. The aim of this work was to determine the timeline kinetics of the cytokine microenvironment derived from circulating leukocytes as supportive immunological biomarkers triggered by Leishmune® vaccine. Cross-sectional kinetic analysis of cellular immunity cytokines was carried out at three times (1, 6 and 12 months) after primovaccination with Leishmune®. In vitro short-term whole blood cultures were stimulated with Leishmania infantum soluble antigen (SLAg). The secreted cytokine signatures and their major sources were determined. RESULTS: At six months after vaccination, Leishmune® induced an increase in IL-8, IFN-γ, IL-17a and TNF-α levels and a decrease in IL-10. Cytokine signature analysis revealed a shift in the microenvironment towards a pro-inflammatory profile mediated by IL-8 and IFN-γ. Both, CD4(+) (↑TNF-α(+) and ↑IFN-γ (+)) and CD8(+) (↑IL-17a and ↓IL-4) T-cells contributed to the acquired immune responses observed after stimulation with SLAg. CONCLUSIONS: The changes observed in the cytokine profile suggested that Leishmune® was able to induce an effective response at six months after primovaccination. After one year, it returned to baseline suggesting the need of additional boosting.