Marisol Simões

Evaluation of accuracy and reliability of the plaque reduction neutralization test (micro-PRNT) in detection of yellow fever virus antibodies.

Yellow fever is a disease caused by the prototype virus of the genus Flavivirus and remains endemic in tropical forest regions from Africa and South America, despite the availability of effective vaccines. These are capable of inducing a rapid specific immune response, with the formation of neutralizing antibodies that appear early, are protective and long lasting. The Plaque Reduction Neutralization Test is considered the most sensitive and specific test for quantification of neutralizing antibodies, and the reference method for assessing the protective immune response after vaccination. This study evaluated the reliability (repeatability and reproducibility) and accuracy (sensitivity, specificity and overall accuracy) of micro-PRNT50 and compared its performance with the micro-PRNT90. Although the micro-PRNT50 has showed satisfactory levels of reliability (ICCs ranged from 0.62 to 0.NorNormas e Manuais Técnicosas e Manuais Técnicos6 for repeatability and 0.72 for reproducibility) and accuracy (sensitivity of 91.1%, specificity of 72.9% and overall accuracy of 78%), the micro-PRNT90 showed higher performance, with ICCs for repeatability ranged from 0.78 to 0.79 and 0.81 for reproducibility, sensitivity of 100%, specificity of 94.7% and overall accuracy of 95%. Modifications in the test methodology and changes in the classification criteria in the readings of the results obtained will be important to improve the accuracy of micro-PRNT.

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.

3β-acetyl tormentic acid induces apoptosis of resistant leukemia cells independently of P-gp/ABCB1 activity or expression

SummaryChronic Myeloid Leukemia (CML) is a potentially fatal stem-cell cancer. P-glycoprotein (P-gp/ABCB1) activity has been described as a relevant factor in the chemotherapeutic failure and correlated to a poor prognosis in these malignancies. In the present study, we investigated the mechanism of the antineoplastic activity of 3β-acetyl tormentic acid (3ATA), a triterpene isolated from C. lyratiloba, on Lucena-1, an MDR leukemia cell line, that overexpressed P-gp/ABCB1. Results showing that this triterpene induced DNA-fragmentation, activation of caspase-3 and cytochrome c release indicated that its activity is mediated by the activation of the intrinsic pathway of apoptosis. Interestingly, this triterpene did not interfere with P-gp/ABCB1 expression or activity, indicating that induction of death is not mediated by any effect on this protein. Moreover, the results show that none of the others triterpenes from C. lyratiloba were able to modulate the activity of P-gp/ABCB1. Together these results suggest 3ATA and the other triterpenes as a promising material for the development of anti-neoplastic drugs for leukemia and other tumors independent of P-gp/ABCB1 activity or expression.

Seroconversion in patients with rheumatic diseases treated with immunomodulators or immunosuppressants, who were inadvertently revaccinated against yellow fever.

Revaccination against yellow fever is contraindicated in patients receiving immunomodulators or immunosuppressants, and a mass campaign to vaccinate the population against yellow fever resulted in the inadvertent revaccination of patients with rheumatic diseases who attended the Hospital of the University of Brasilia (HUB) or a private rheumatology clinic in Brasilia, Brasil between December 2007 and May 2008. Vaccination is strongly recommended to all people living in Brasilia; thus, for the purposes of this study, we assumed that nearly 100% of the population was vaccinated. In 2007 and 2008, we collected relevant data (pertaining to rheumatic disease diagnoses, use of immunosuppressants, date of revaccination, and adverse events) within 30 days of the revaccination. Two years later, we evaluated the protective immune response in this group by analysis of neutralizing antibodies. Our study group comprised 31 patients (all women) (Table 1). A single serum analysis was conducted using a plaque reduction neutralization test. Patients with values of 794 mIU/ml were considered seropositive for protection against yellow fever. Twenty patients (64.5%) were recruited from HUB and 11 (35.5%) from the private clinic. The diagnoses among our patients were as follows: rheumatoid arthritis (RA) (n 23), systemic lupus erythematosus (SLE) (n 5), systemic sclerosis (SSc) (n 2), and ankylosing spondylitis (AS) (n 1). Seropositivity was observed in 27 individuals (87.1%). Among the 31 patients, the mean titer of neutralizing antibodies was 2,865.58 mIU/ml. (A large study by Camacho and colleagues [1] demonstrated that the geometric mean titer in revaccinated healthy individuals was equal to 14,000 mIU/ml.) Mean, geometric mean, and median titers of neutralizing antibodies were 2,535.4, 1,543.5, and 2,015.0 mIU/ ml, respectively, in the patients with RA (interquartile range [IQR] 923–3,353) and 1,934, 2,066.9, and 1,668 mIU/mL, respectively, in those with SLE (IQR 1,208.5–2,792.5). Among the RA patients, 16 were taking methotrexate (mean SD dosage 13.28 5.73 mg/week), 9 were taking leflunomide (20 mg/day), 3 were taking infliximab (3 mg/kg every 8 weeks), and 3 patients were taking rituximab (1,000 mg twice every 15 days). Four patients reported mild adverse events up to 30 days after vaccination, 24 reported no reactions, and 3 did not recall the occurrence of any adverse reaction. One patient with AS (who was taking 3.2 gm of mesalamine per day) and another patient with RA (who was taking 10 mg of prednisone per day) developed myalgia. One patient with SSc (who was receiving 1.2 gm of cyclophosphamide per month) had fever and rhinorrhea, and another patient with RA (who was taking 20 mg of methotrexate per week and 1.5 gm of sulfasalazine per day) had arthralgia. The mean SD titers of neutralizing antibodies in the patients who presented with adverse reactions and in the asymptomatic patients were 5,461 6,817 mIU/ml and 2,610.46 2,033.04 mIU/ml, respectively. No correlation was observed between the neutralizing antibodies and occurrence of adverse reactions ( 0.19) or age ( 0.246) at the time of revaccination in a sample of 28 of 31 patients. It is noteworthy that the patient with the lowest antibody titer was treated with 2 gm of rituximab 4 months before being revaccinated against yellow fever. The data presented herein are the only data currently available regarding protective immunity following yellow fever revaccination in patients with rheumatic disease. Our assessment was performed using the method that is considered to be the reference test. Although the titers of neutralizing antibodies were lower among the rheumatic disease patients than among healthy individuals, they were high enough to confer a protective response despite the use of immunosuppressive drugs. These results are compatible with the data already existing in the literature regarding revaccination in the general population (2,3). Dr. Oliveira’s work was supported by a scholarship from the National Council for Scientific and Technological Development. Dr. Mota has received speaking fees from Roche, Janssen, AbbVie, Bristol-Myers Squibb, and AstraZeneca Pharmaceuticals.

CD4/CD8 Ratio Predicts Yellow Fever Vaccine-Induced Antibody Titers in Virologically Suppressed HIV-Infected Patients.

Background:Yellow fever vaccine (YFV) induces weaker immune responses in HIV-infected individuals. However, little is known about YFV responses among antiretroviral-treated patients and potential immunological predictors of YFV response in this population. Methods:We enrolled 34 antiretroviral therapy (ART)-treated HIV-infected and 58 HIV-uninfected adults who received a single YFV dose to evaluate antibody levels and predictors of immunity, focusing on CD4+ T-cell count, CD4+/CD8+ ratio, and Human Pegivirus (GBV-C) viremia. Participants with other immunosuppressive conditions were excluded. Results:Median time since YFV was nonsignificantly shorter in HIV-infected participants than in HIV-uninfected participants (42 and 69 months, respectively, P = 0.16). Mean neutralizing antibody (NAb) titers was lower in HIV-infected participants than HIV-uninfected participants (3.3 vs. 3.6 log10mIU/mL, P = 0.044), a difference that remained significant after adjustment for age, sex, and time since vaccination (P = 0.024). In HIV-infected participants, lower NAb titers were associated with longer time since YFV (rho: −0.38, P = 0.027) and lower CD4+/CD8+ ratio (rho: 0.42, P = 0.014), but not CD4+ T-cell count (P = 0.52). None of these factors were associated with NAb titers in HIV-uninfected participant. GBV-C viremia was not associated with difference in NAb titers overall or among HIV-infected participants. Conclusions:ART-treated HIV-infected individuals seem to have impaired and/or less durable responses to YFV than HIV-uninfected individuals, which were associated with lower CD4+/CD8+ ratio, but not with CD4+ T-cell count. These results supports the notion that low CD4+/CD8+ ratio, a marker linked to persistent immune activation, is a better indicator of functional immune disturbance than CD4+ T-cell count in patients with successful ART.

Effects of 3β-Acethyl Tormentic Acid (3ATA) on ABCC Proteins Activity

Multidrug resistance (MDR) is considered the main cause of cancer chemotherapy failure and patient relapse. The active drug efflux mediated by transporter proteins of the ABC (ATP-binding cassette) family is the most investigated mechanism leading to MDR. With the aim of inhibiting this transport and circumventing MDR, a great amount of work has been dedicated to identifying pharmacological inhibitors of specific ABC transporters. We recently showed that 3β-acetyl tormentic acid (3ATA) had no effect on P-gp/ABCB1 activity. Herein, we show that 3ATA strongly inhibited the activity of MRP1/ABCC1. In the B16/F10 and Ma104 cell lines, this effect was either 20X higher or similar to that observed with MK571, respectively. Nevertheless, the low inhibitory effect of 3ATA on A549, a cell line that expresses MRP1-5, suggests that it may not inhibit other MRPs. The use of cells transfected with ABCC2, ABCC3 or ABCC4 showed that 3ATA was also able to modulate these transporters, though with an inhibition ratio lower than that observed for MRP1/ABCC1. These data point to 3ATA as a new ABCC inhibitor and call attention to its potential use as a tool to investigate the function of MRP/ABCC proteins or as a co-adjuvant in the treatment of MDR tumors.

CD4/CD8 Ratio and KT Ratio Predict Yellow Fever Vaccine Immunogenicity in HIV-Infected Patients

Background HIV-infected individuals have deficient responses to Yellow Fever vaccine (YFV) and may be at higher risk for adverse events (AE). Chronic immune activation–characterized by low CD4/CD8 ratio or high indoleamine 2,3-dioxygenase-1 (IDO) activity—may influence vaccine response in this population. Methods We prospectively assessed AE, viremia by the YFV virus and YF-specific neutralizing antibodies (NAb) in HIV-infected (CD4>350) and -uninfected adults through 1 year after vaccination. The effect of HIV status on initial antibody response to YFV was measured during the first 3 months following vaccination, while the effect on persistence of antibody response was measured one year following vaccination. We explored CD4/CD8 ratio, IDO activity (plasma kynurenine/tryptophan [KT] ratio) and viremia by Human Pegivirus as potential predictors of NAb response to YFV among HIV-infected participants with linear mixed models. Results 12 HIV-infected and 45-uninfected participants were included in the final analysis. HIV was not significantly associated with AE, YFV viremia or NAb titers through the first 3 months following vaccination. However, HIV–infected participants had 0.32 times the NAb titers observed for HIV-uninfected participants at 1 year following YFV (95% CI 0.13 to 0.83, p = 0.021), independent of sex, age and prior vaccination. In HIV-infected participants, each 10% increase in CD4/CD8 ratio predicted a mean 21% higher post-baseline YFV Nab titer (p = 0.024). Similarly, each 10% increase in KT ratio predicted a mean 21% lower post-baseline YFV Nab titer (p = 0.009). Viremia by Human Pegivirus was not significantly associated with NAb titers. Conclusions HIV infection appears to decrease the durability of NAb responses to YFV, an effect that may be predicted by lower CD4/CD8 ratio or higher KT ratio.

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.

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.