Duration of post-vaccination humoral immunity against yellow fever in children.

INTRODUCTION: Vaccination is the most important measure for prevention and control of yellow fever. It is recommended by the World Health Organization (WHO) for residents of endemic areas and travelers to risk areas. In 2013, the WHO discontinued the recommendation of booster doses every 10 years, indicating a single dose as sufficient for lifelong protection. OBJECTIVE: Considering the lower immune response to YF vaccine in children compared to adults, this study was set out to assess the duration of immunity to YF in children vaccinated in the first two years of life. METHODS: This cross-sectional study involved children aged 9 months to 12 years with accessible vaccination records recruited in primary care units from a metropolitan area in Southeast Brazil. The serologic status (negative, indeterminate and positive), and geometric mean titers (GMT, inverse dilution) of neutralizing antibodies against YF obtained by Plaque Reduction Neutralization Test was assessed across categories of time after YF vaccination. The strength of association of seropositivity with time was assessed by the odds ratio (OR) taking recent vaccination (1-6 months) as reference. RESULTS: A total of 824 children recruited from August 2010 to July 2011were tested. The proportion of seropositivity (95% C.I.) and GMT (95% C.I.) dropped markedly across time periods: from 86.7% (80.5-91.4%), GMT 47.9 (38.3-59.9) in newly vaccinated to 59.0% (49.7-67.8%), GMT 14.8 (11.6-19.1) and 42.2% (33.8-51.0), GMT 8.6 (7.1-12.1), respectively in the subgroups vaccinated 31-72 months and 73-100 months before. CONCLUSIONS: Analogous to previous findings in adults, these data support the need for revaccination of children living in areas with yellow fever virus circulation in humans or in other primates. The data also supported the change of a booster dose to 4 years of age for those primarily vaccinated for yellow fever in the first two years of life.

Kinetic Study of Yellow Fever 17DD Viral Infection in Gallus gallus domesticus Embryos.

Yellow fever continues to be an important epidemiological problem in Africa and South America even though the disease can be controlled by vaccination. The vaccine has been produced since 1937 and is based on YFV 17DD chicken embryo infection. However, little is known about the histopathological background of virus infection and replication in this model. Here we show by morphological and molecular methods (brightfield and confocal microscopies, immunofluorescence, nested-PCR and sequencing) the kinetics of YFV 17DD infection in chicken embryos with 9 days of development, encompassing 24 to 96 hours post infection. Our principal findings indicate that the main cells involved in virus production are myoblasts with a mesenchymal shape, which also are the first cells to express virus proteins in Gallus gallus embryos at 48 hours after infection. At 72 hours post infection, we observed an increase of infected cells in embryos. Many sites are thus affected in the infection sequence, especially the skeletal muscle. We were also able to confirm an increase of nervous system infection at 96 hours post infection. Our data contribute to the comprehension of the pathogenesis of YF 17DD virus infection in Gallus gallus embryos.

Immunogenicity and safety of measles-mumps-rubella vaccine delivered by disposable-syringe jet injector in healthy Brazilian infants: a randomized non-inferiority study.

This study aimed to determine if immunogenicity to measles-mumps-rubella vaccine delivered to infants via a disposable-syringe jet injector (DSJI) was non-inferior to that administered by needle and syringe (NS). Vaccination safety was evaluated, as were the use, performance, and acceptability of each delivery method. The DSJI was the PharmaJet 2009 generation-1 device (G1) and the vaccine was measles-mumps-rubella vaccine from Bio-Manguinhos. Five hundred eighty-two healthy Brazilian infants were randomized to receive vaccine via G1 or NS. Seroconversion rates against measles and mumps viruses in the G1 treatment group did not meet non-inferiority criteria when compared with the NS group; however, responses in the G1 group to rubella virus were non-inferior to those of NS vaccinees. Most adverse events were mild or moderate. Crying after injection was more frequent in the NS group, and local skin reactions were more common in the G1 group. Five serious adverse events were judged causally unrelated to treatment and all resolved. Parents/guardians expressed a strong preference for G1 over NS for their children. Vaccinators found the G1 easy to use but noted incomplete vaccine delivery in some cases. Although the G1 has been superseded by an updated device, our results are important for the continued improvement and evaluation of DSJIs, which have the potential to overcome many of the challenges and risks associated with needle-based injections worldwide. Recommendations for future DSJI clinical studies include rigorous training of vaccinators, quantitative measurement of wetness on the skin following injection, and regular monitoring of device and vaccinator performance.

17DD and 17D-213/77 yellow fever substrains trigger a balanced cytokine profile in primary vaccinated children.

BACKGROUND: This study aimed to compare the cytokine-mediated immune response in children submitted to primary vaccination with the YF-17D-213/77 or YF-17DD yellow fever (YF) substrains. METHODS: A non-probabilistic sample of eighty healthy primary vaccinated (PV) children was selected on the basis of their previously known humoral immune response to the YF vaccines. The selected children were categorized according to their YF-neutralizing antibody titers (PRNT) and referred to as seroconverters (PV-PRNT(+)) or nonseroconverters (PV-PRNT(-)). Following revaccination with the YF-17DD, the PV-PRNT(-) children (YF-17D-213/77 and YF-17DD groups) seroconverted and were referred as RV-PRNT(+). The cytokine-mediated immune response was investigated after short-term in vitro cultures of whole blood samples. The results are expressed as frequency of high cytokine producers, taking the global median of the cytokine index (YF-Ag/control) as the cut-off. RESULTS: The YF-17D-213/77 and the YF-17DD substrains triggered a balanced overall inflammatory/regulatory cytokine pattern in PV-PRNT(+), with a slight predominance of IL-12 in YF-17DD vaccinees and a modest prevalence of IL-10 in YF-17D-213/77. Prominent frequency of neutrophil-derived TNF-α and neutrophils and monocyte-producing IL-12 were the major features of PV-PRNT(+) in the YF-17DD, whereas relevant inflammatory response, mediated by IL-12(+)CD8(+) T cells, was the hallmark of the YF-17D-213/77 vaccinees. Both substrains were able to elicit particular but relevant inflammatory events, regardless of the anti-YF PRNT antibody levels. PV-PRNT(-) children belonging to the YF-17DD arm presented gaps in the inflammatory cytokine signature, especially in terms of the innate immunity, whereas in the YF-17D-213/77 arm the most relevant gap was the deficiency of IL-12-producing CD8(+)T cells. Revaccination with YF-17DD prompted a balanced cytokine profile in YF-17DD nonresponders and a robust inflammatory profile in YF-17D-213/77 nonresponders. CONCLUSION: Our findings demonstrated that, just like the YF-17DD reference vaccine, the YF-17D-213/77 seed lot induced a mixed pattern of inflammatory and regulatory cytokines, supporting its universal use for immunization.

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.

Cytokine signatures of innate and adaptive immunity in 17DD yellow fever vaccinated children and its association with the level of neutralizing antibody.

BACKGROUND: The live attenuated yellow fever (YF) vaccines have been available for decades and are considered highly effective and one of the safest vaccines worldwide. METHODS: The impact of YF-17DD-antigens recall on cytokine profiles of YF-17DD-vaccinated children were characterized using short-term cultures of whole blood samples and single-cell flow cytometry. This study enrolled seroconverters and nonseroconverters after primovaccination (PV-PRNT⁺ and PV-PRNT⁻), seroconverters after revaccination (RV-PRNT⁺), and unvaccinated volunteers (UV-PRNT⁻). RESULTS: The analysis demonstrated in the PV-PRNT⁺ group a balanced involvement of pro-inflammatory/regulatory adaptive immunity with a prominent participation of innate immunity pro-inflammatory events (IL-12⁺ and TNF-α⁺ NEU and MON). Using the PV-PRNT⁺ cytokine signature as a reference profile, PV-PRNT⁻ presented a striking lack of innate immunity proinflammatory response along with an increased adaptive regulatory profile (IL-4⁺CD4⁺ T cells and IL-10⁺ and IL-5⁺CD8⁺ T cells). Conversely, the RV-PRNT⁺ shifted the overall cytokine signatures toward an innate immunity pro-inflammatory profile and restored the adaptive regulatory response. CONCLUSIONS: The data demonstrated that the overall cytokine signature was associated with the levels of PRNT antibodies with a balanced innate/adaptive immunity with proinflammatory/regulatory profile as the hallmark of PV-PRNT(MEDIUM⁺), whereas a polarized regulatory response was observed in PV-PRNT⁻ and a prominent proinflammatory signature was the characteristic of PV-PRNT(HIGH⁺).

Clinical and immunological insights on severe, adverse neurotropic and viscerotropic disease following 17D yellow fever vaccination.

Yellow fever (YF) vaccines (17D-204 and 17DD) are well tolerated and cause very low rates of severe adverse events (YEL-SAE), such as serious allergic reactions, neurotropic adverse diseases (YEL-AND), and viscerotropic diseases (YEL-AVD). Viral and host factors have been postulated to explain the basis of YEL-SAE. However, the mechanisms underlying the occurrence of YEL-SAE remain unknown. The present report provides a detailed immunological analysis of a 23-year-old female patient. The patient developed a suspected case of severe YEL-AVD with encephalitis, as well as with pancreatitis and myositis, following receipt of a 17D-204 YF vaccination. The patient exhibited a decreased level of expression of Fc-gammaR in monocytes (CD16, CD32, and CD64), along with increased levels of NK T cells (an increased CD3(+) CD16(+/-) CD56(+/-)/CD3(+) ratio), activated T cells (CD4(+) and CD8(+) cells), and B lymphocytes. Enhanced levels of plasmatic cytokines (interleukin-6 [IL-6], IL-17, IL-4, IL-5, and IL-10) as well as an exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within NK cells (gamma interferon positive [IFN-gamma(+)], tumor necrosis factor alpha positive [TNF-alpha(+)], and IL-4 positive [IL-4(+)]), CD8(+) T cells (IL-4(+) and IL-5(+)), and B lymphocytes (TNF-alpha(+), IL-4(+), and IL-10(+)). The analysis of CD4(+) T cells revealed a complex profile that consisted of an increased frequency of IL-12(+) and IFN-gamma(+) cells and a decreased percentage of TNF-alpha(+), IL-4(+), and IL-5(+) cells. Depressed cytokine synthesis was observed in monocytes (TNF-alpha(+)) following the provision of antigenic stimuli in vitro. These results support the hypothesis that a strong adaptive response and abnormalities in the innate immune system may be involved in the establishment of YEL-AND and YEL-AVD.

Accuracy and repeatability of a micro plaque reduction neutralization test for vaccinia antibodies.

The detection of neutralizing antibodies against vaccinia virus is a valuable tool for the investigation of previous smallpox vaccination. Compulsory smallpox vaccination ended in Brazil during the early 1970s, although the vaccine was available until the late 1970s. The threat of smallpox as a biological weapon has called the attention of public health authorities to the need for an evaluation of the immune status of the population. Based on our previous experience with a micro plaque reduction neutralization test (PRNT) for the evaluation of yellow fever immunity, a similar test was developed for the detection and quantification of vaccinia neutralizing antibodies. A cross-sectional study to test the repeatability and validity of plaque reduction neutralization test (PRNT) for vaccinia antibodies was performed in 182 subjects divided into two categories: subjects above 31 years old and the other > or = 35 years old. Cases were subjects considered to have been vaccinated with vaccinia virus if they declared vaccination history or evidenced vaccination marks. The assay is carried out in 96-well plates, provides results within 30 h, is easily performed, has good sensitivity (92.7%) and specificity (90.8), excellent repeatability (ICC 0.89 (0.88; 0.92)) and is thus suitable for use in mass screening of a population's antibody levels.

Screening of CHO cell clones expressing histidine-tagged major S hepatitis B surface protein using a semi-quantitative PCR protocol.

Different mammalian cells have been used successfully for the expression of the hepatitis B surface antigen (HBsAg). The patterns of expression of the HBsAg seem to depend on the cell type and the number of gene copies integrated into cellular genome. The expression of an HBsAg fused to histidine tag (His-HBsAg), had not been reported in mammalian cells. This paper describes a semi-quantitative polymerase chain reaction (PCR) employed to investigate the patterns of expression of His-HBsAg in stably transfected Chinese hamster ovary (CHO) cell clones. pcDNA4CR20, a mammalian expressing vector, encoding His-HBsAg, was constructed. The correlation between His-HBsAg expression and the number of integrated copies of the HBsAg gene into cell clones genome was evaluated by the semi-quantitative PCR, with limit dilutions of the genomic DNA as template. The results show a positive correlation between the expression levels of His-HBsAg with the number of the HBsAg gene copies integrated into CHO cell clones. The approach of a semi-quantitative PCR proved to be a good and non-expensive alternative strategy to analyze the patterns of expression of integrated genes into CHO cells genome.

Simple immunoaffinity method to purify recombinant hepatitis B surface antigen secreted by transfected mammalian cells.

Purification of recombinant hepatitis B surface antigen (recHBsAg) produced in a stable Chinese hamster ovary (CHO) cell line was evaluated using Linx Affinity Purification System (Invitrogen, USA). To purify HBsAg secreted by this cell line, a murine monoclonal antibody (MAbAH1) raised against native HBsAg was used. The purified AH1MAb was conjugated with phenyldiboronic acid (PDBA) and immobilized on the immunoaffinity chromatographic support. Using an optimized protocol the affinity column was able to purify recHBsAg from supernatant of mammalian cells cultures with more than 80% purity. This method showed to be simple and quicker than the current ultracentrifugation methods. The method is also efficient and economical in obtaining purified recHBsAg.

Identification and characterization of avian retroviruses in chicken embryo-derived yellow fever vaccines: investigation of transmission to vaccine recipients.

All currently licensed yellow fever (YF) vaccines are propagated in chicken embryos. Recent studies of chick cell-derived measles and mumps vaccines show evidence of two types of retrovirus particles, the endogenous avian retrovirus (EAV) and the endogenous avian leukosis virus (ALV-E), which originate from the chicken embryonic fibroblast substrates. In this study, we investigated substrate-derived avian retrovirus contamination in YF vaccines currently produced by three manufacturers (YF-vax [Connaught Laboratories], Stamaril [Aventis], and YF-FIOCRUZ [FIOCRUZ-Bio-Manguinhos]). Testing for reverse transcriptase (RT) activity was not possible because of assay inhibition. However, Western blot analysis of virus pellets with anti-ALV RT antiserum detected three distinct RT proteins in all vaccines, indicating that more than one source is responsible for the RTs present in the vaccines. PCR analysis of both chicken substrate DNA and particle-associated RNA from the YF vaccines showed no evidence of the long terminal repeat sequences of exogenous ALV subgroups A to D in any of the vaccines. In contrast, both ALV-E and EAV particle-associated RNA were detected at equivalent titers in each vaccine by RT-PCR. Quantitative real-time RT-PCR revealed 61,600, 348,000, and 1,665,000 ALV-E RNA copies per dose of Stamaril, YF-FIOCRUZ, and YF-vax vaccines, respectively. ev locus-specific PCR testing of the vaccine-associated chicken substrate DNA was positive both for the nondefective ev-12 locus in two vaccines and for the defective ev-1 locus in all three vaccines. Both intact and ev-1 pol sequences were also identified in the particle-associated RNA. To investigate the risks of transmission, serum samples from 43 YF vaccine recipients were studied. None of the samples were seropositive by an ALV-E-based Western blot assay or had detectable EAV or ALV-E RNA sequences by RT-PCR. YF vaccines produced by the three manufacturers all have particles containing EAV genomes and various levels of defective or nondefective ALV-E sequences. The absence of evidence of infection with ALV-E or EAV in 43 YF vaccine recipients suggests low risks for transmission of these viruses, further supporting the safety of these vaccines.