Littérature scientifique sur le sujet « Serologic immunity »

AbstractObjective:To define measles immunity rates among employees at 2 hospitals during a community outbreak in 1990.Design:Cohort survey using enzyme-linked immunosorbant assay (ELISA) and questionnaire.Setting:Two community hospitals.Participants:Seventy-six percent of 2,060 employees.Results:Seven percent (115/1566) of participants lacked ELISA-defined measles immunity. Among employees whose ages were known, 14% (64/467) of those born after 1956 and 5% (50/1086) of those born before 1957 lacked serologic evidence of immunity. Fifty-eight percent of the susceptible persons had substantial patient contact. With ELISA results as the reference for immunity, the predictive value of an undocumented positive history of measles disease or vaccination was 95%; the predictive value of a negative history of both was 52%. Measles developed in 7 employees.Conclusions:A substantial number of hospital employees lacked ELBA-defined measles immunity, including many who had patient contact or who had been born before 1957. Undocumented disease and vaccination histories were not adequate predictors of serologic status. This study supports the recommendations and suggestions of the Immunization Practices Advisory Committee that hospitals should require documented evidence of measles immunity from employees who have patient contact.

Objective:The objective of this study was to compare the cost-effectiveness of 3 strategies of serologic enzyme-linked immunosorbent assay (ELISA) testing and post-exposure varicella zoster immune globulin (VZIG) prophylaxis for the prevention of maternal varicella pneumonia during pregnancy in patients withnegativeoruncertainhistories of varicella infection.Methods:A decision tree was constructed to compare the following strategies: 1) routine serologic testing for varicella immunity followed by targeted post-exposure VZIG prophylaxis, 2) post-exposure serologic testing followed by targeted VZIG prophylaxis, and 3) untargeted post-exposure VZIG administration. The probabilities for the model were obtained from the medical literature and supplemented by expert opinion. The costs were obtained by a review of inpatient hospitalizations for varicella pneumonia. All costs were converted to 1995 dollars.Results:Routine serologic testing followed by targeted post-exposure VZIG prophylaxis was the most costly strategy ($37.22/person), with no demonstrable increase in benefit compared with the other 2 strategies. The disutility of this strategy compared with the others was stable across a wide range of values for the probabilities and costs utilized in the sensitivity analysis. We were unable to differentiate between the cost-effectiveness of the other 2 strategies.Conclusions:Routine serologic testing for varicella immunity in patients with negative or uncertain histories of varicella infection should not be performed. The remaining options of screening and prophylaxis appear to be reasonable alternatives for dealing with varicella exposures.

Long-term persistence of immunity was assessed in 1756 healthy adults (4 - 74 years of age) with documented immunization against tick-borne encephalitis. Serologic studies indicate that the protective immunity is associated with age, number of vaccine doses and time since the last vaccine dose. 411 persons were vaccinated against tick-borne encephalitis over 10 years ago. Most of them (67,5%) had protective antibodies. In some cases, the immunity lasts up to 34 years after last vaccination.

The present study of antipertussis immunity stress and level in young and school children who was vaccinated ADTP vaccine showed that on the average 28.3% of them were seronegative. The lowest parts of seronegative children were detected in the age group of infants under 12 months of age (12.3%) and in that of 15 - 17 years old teenagers (12.1%). The maximum percent of seronegative children were detected in the age group of 6 - 8 years - 38.8%. Despite the nonsignificant increase of this indicator, compared to the previous age group, it is advisable to supplement serological monitoring of indicator group 6- 7 years. The researchers did not discover the reliable correlation between the pertussis incidence in different age groups and proportion of seronegative children in those groups. The obtained results suggest that there is an occult circulation of pertussis causing agent. They also showed that it is necessary to revise the indicator age groups for serologic monitoring of antipertussis immunity.

La terapia genica di cellule staminali ematopoietiche (HSC) ha prodotto benefici clinici in diversi pazienti affetti da una varietà di malattie genetiche. Tuttavia, l’uso di vettori che si integrano nel genoma in modo semi-casuale pone il rischio di mutagenesi inserzionale e di una espressione del transgene ectopica/non regolata. Quest’ultimo problema è particolarmente rilevante quando si trattano geni strettamente regolati attivi sulla proliferazione cellulare, come il gene CD40LG, la cui espressione sulle cellule T attivate porta all’attivazione contatto-dipendente delle cellule B, alla loro proliferazione ed allo scambio di classe delle immunoglobuline. Poichè le sue mutazioni causano l’immunodeficienza legata all’X con iper-IgM (HIGM1), il trasferimento genico in HSC è stato proposto come potenziale trattamento per questa sindrome. Anche se piccole quantità di cellule trasdotte hanno ripristinato la funzione immunitaria umorale e cellulare in un modello murino di HIGM1, l’espressione costitutiva di CD40LG in timociti o in cellule T periferiche ha portato a linfoproliferazioni, molte delle quali sono progredite a linfom. Le strategie di riparazione genica che preservano il controllo fisiologico dell’espressione del gene corretto potrebbero quindi rappresentare un approccio più promettente per il trattamento di HIGM1. In questo studio sfruttiamo il meccanismo Homology Directed Repair (HDR) per la correzione in situ della maggior parte delle mutazioni responsabili della sindrome HIGM1 presenti nel gene CD40L, con l’obiettivo di ristabilirne la funzione e il controllo dell’espressione. In particolare sfruttiamo il sistema CRISPR/Cas9 per promuovere l’integrazione sito-specifica di una copia funzionale di parte del gene CD40L a valle del suo promotore endogeno, correggendo così la maggior parte delle mutazioni responsabili della malattia. Dato che il difetto genetico non è deleterio per le cellule T, questo tipo di malattia ci offre l’opportunità unica di sviluppare una terapia genica basata sulla correzione di cellule T autologhe. Al fine di stabilire quali sono le dosi terapeutiche e le condizioni di trapianto necessarie per ottenere la ricostituzione immunitaria e il ripristino delle funzioni immunologiche con cellule corrette, abbiamo infuso diverse dosi di cellule T WT in topi HIGM1 pre-condizionati o meno con diversi regimi linfodepletanti ed eseguito trapianti competitivi di cellule staminali ematopoietiche WT e Cd40lg - / - nel modello animale. Mentre l’ analisi del sangue periferico ha dimostrato la persistenza a lungo termine di cellule T in tutte le condizioni, sono stati ottenuti livelli di attecchimento più elevati nei topi trapiantati dopo il trattamento chemioterapico con ciclofosfamide (CPA). Tutti i topi trapiantati hanno mostrato un parziale ripristino della risposta IgG specifica dopo immunizzazione con TNP-KLH, ma è stato osservato un ripristino più elevato nei topi pre-condizionati con CPA. Questi topi hanno anche mostrato la presenza di centri germinativi nella milza. Topi HIGM1 ricostituiti con dosi crescenti di HSPC WT hanno mostrato un ripristino dose-dipendente della risposta immunitaria T dipendente. In particolare, abbiamo dimostrato che il 10% di HSPC funzionali è sufficiente a ripristinare parzialmente la capacità di produrre anticorpi specifici contro diversi antigeni oltre che ad attenuare l'infezione in topi HIGM1 inoculati con il patogeno Pneumocystis murina. Il nostro obiettivo futuro è quello di dimostrare il ripristino della risposta immunitaria contro l'infezione da pneumocystis murina in topi HIGM1 trapiantati con cellule T CD4 + WT. In caso di successo, i nostri risultati saranno strumentali per stabilire il potenziale terapeutico di un approccio di correzione genica basato sulle cellule T per il trattamento della sindrome HIGM1 che potrebbe fungere da terapia ponte per una terapia definitiva basata sul trapianto di HSPC corrette.
Background The X-linked hyper-IgM syndrome type I (HIGM1) is caused by inactivating mutations in the CD40 ligand gene (CD40LG) that disrupt the T cell helper function on B cells and macrophages. This disease represents an ideal candidate for a gene correction strategy because preclinical studies of Hematopoietic Stem Cell (HSC) gene therapy have already shown i) evidence of potential efficacy even with few amounts of transduced cells; ii) critical safety issues due to unregulated transgene expression. Since in HIGM1 the genetic defect is not lethal to T cells, we aim to apply our gene editing strategy on autologous T cells that could be used to provide immediate therapeutic benefit to the patients by resolving pre-existing infections prior to a definitive HSPC transplant. Methods To establish which are the therapeutic threshold levels and transplant conditions required to achieve immune reconstitution and functional immunologic restoration with corrected cells, we infused different doses of WT T cells into HIGM1 mice pre-conditioned or not with different lymphodepleting regimens and performed competitive transplants of WT and Cd40lg-/- HSPC in the mouse model. Results While longitudinal blood analyses showed a long-term, stable T cell engraftment in all the conditions, highest engraftment rates were obtained in mice transplanted after chemotherapy treatment with cyclophosphamide (CPA). All the transplanted mice showed a partial rescue of the antigen-specific IgG response after immunization with Keyhole Limpet Hemocyanin (TNP-KLH) but a higher rescue was observed in mice pre-conditioned with CPA. These mice also showed the presence of TNP-KLH specific IgG producing B cells and germinal centers within splenic lymphoid follicles. HIGM1 mice reconstituted with increasing proportions of WT HSPC displayed a dose-dependent rescue of the T cell mediated immune response. In particular we found that 10% of WT HSPC is sufficient to partially restore serologic immunity against different antigens as well as to attenuate infection in HIGM1 mice challenged with Pneumocystis murina. Conclusions Our current efforts are aimed to demonstrate functional restoration of the immune response against Pneumocystis murina infection in HIGM1 mice that received adoptive transfer of WT CD4+ T cells. If successful, our findings will be instrumental to establish the therapeutic potential of a T cell gene correction approach for the treatment of the HIGM1 disease that could act as a bridge therapy to the HSPC-based strategy.

Sequence variation in the antigenic determinants in core, NS3, NS4A/B and NS5A/B proteins, may produce genotype-specific antibodies which do not cross-react with the antigens used in current screening assays for detection of antibody to HCV. The extent of type-specific and type-common components of the humoral immune response were investigated by measuring the antibody levels in samples of each genotype with type-homologous and type-heterologous antigens. Reactivity directed against the individual genotype 1 antigens used in a commercially available anti-HCV screening assay (VK48; Murex Biotech) was compared with corresponding core, NS3 and NS4A/B antigens of genotype 3, and NS3, NS4A/B and NS5A/B antigens from genotype 4 using a panel of samples from individuals infected with different genotypes (genotype 1: n=41, 3: n=39 and 4: n=42). A combined ELISA was subsequently assembled using the core, NS3 and NS5A/B recombinant proteins and NS4A/B synthetic peptide of genotype 3a sequence, and serological reactivity in this ELISA compared quantitatively with reactivity in the (type 1-based) Murex VK48 assay, using a similar panel of samples from individuals infected with different HCV genotypes. The overall proportion of type-specific reactivity in the combined ELISAs was 46% (with the type-common component making up the other 54%). Type-specificity of reactivity to each region depended on the extent of amino acid divergence between genotypes, with the more conserved core region eliciting 26% type-specific reactivity, compared with 60% and 62-77% to the NS3 and NS5A/B regions. To investigate whether antigenic variability influenced the effectiveness of type-1 based serological assays for screening, blood donor samples collected in regions where non-genotype 1 HCV predominated, such as genotype 3 infection in Pakistan and genotype 4a infection in Egypt and the Middle East, were screened by ELISAs based on genotype 3a or 4a antigens. Additionally, samples from seroconversion panels, individuals identified with a serum positive PCR result, but a commercial assay negative result and individuals with cryptogenic hepatitis, were tested for reactivity to antigens of genotype 2, 3 or 4.  Although several samples were identified with type-specific reactivity confined to non-type 1 antigens, none were PCR positive. These results therefore indicate that while some (past, resolved) HCV infections may not be detectable using commercially available type 1-based screening assays, the lack of detectable viraemia in the samples provides no evidence at present that these serological “misses” risk the safety of blood transfusion. However, the results do indicate that a proportion of past HCV infections may remain undetected by current diagnostic methods.

Long-term antiviral antibody responses provide protection from re-infection and recurrence of persistent viruses. Using a polyomavirus (PyV) mouse model, our lab has shown that MyD88-deficient mice generate low levels of virus-specific IgG after the acute phase of infection and that these IgG responses have a skewed isotype distribution with low levels of IgG2a/c. Moreover MyD88-deficient mice have reduced numbers of long-lived plasma cells in the bone marrow. These studies suggest an important role of MyD88-mediated signaling in long-term antiviral responses. Our lab has shown that T cell-deficient mice can also maintain long-term virus-specific IgG responses following PyV infection. The goal of this thesis is to evaluate the role of innate signaling pathways in maintaining serological memory to persistent virus infection and to elaborate on how long-term antiviral responses can be maintained in an immunocompetent or partially immune compromised, T cell-deficient host. Regarding T cell-dependent B cell responses, I set out to investigate the upstream and downstream components of the MyD88-mediated pathways required for normal antibody isotype and long-term humoral responses. IgG2a is a predominant immunoglobulin isotype in most virus infections. Wild type mice, in response to PyV infection, primarily induce antiviral IgG2a with some IgG1. MyD88-deficient mice in response to PyV infection display attenuated levels of virus-specific IgG2a, but normal levels of IgG1. Using Unc93B1 mutant mice (3d mice), which are defective in TLRs 3, 7 and 9 signaling, I show that 3d mice also generated low levels of virus-specific IgG2a following PyV infection. Studies in individual TLR3-/-, TLR7-/- or TLR9-/- mice displayed PyV-specific IgG2a responses similar to wild type responses. TLR7 and TLR9 double deficient mice generated similar skewed antibody isotype responses, where virus-specific IgG2a was reduced compared to wild type mice. This shows that TLR7 and TLR9-MyD88 mediated pathways are important in regulating IgG2a responses during a PyV infection. To investigate what components downstream of MyD88 are involved in mediating IgG2a responses, I worked with IRF5-deficient mice. IRF5 is a transcription factor that is activated upon stimulation of TLR7 or TLR9-MyD88-mediated pathways. Moreover, IRF5-deficient mice cannot generate autoantibodies specifically of the IgG2a isotype in a mouse lupus model, suggesting that IRF5 plays an important function in mediating class switching to IgG2a. In vitro studies where IRF5-/- B cells were stimulated with TLR7 or TLR9 ligands also generated low levels of γ2a germ-line transcripts, suggesting a B cell-intrinsic role for IRF5 in regulating γ2a germ-line transcription. PyV infection of IRF5-deficient mice resulted in similar skewed isotypes as observed in MyD88-deficient and 3d mice. To investigate a B cell-intrinsic role for IRF5 in regulating IgG2a responses in vivo upon PyV infection, I transferred IRF5-/- B cells and WT T cells into RAG KO mice prior to infection and compared the responses of these mice with mice reconstituted with wild type B6 B and T cells. Diminished numbers of IgG2a+ B cells and reduced levels of virus-specific IgG in mice reconstituted with IRF5-/- B cells were seen compared to mice reconstituted with wild type B cells. Regarding the defect in long-term IgG production in MyD88-/- mice upon PyV infection, I conducted studies in IRF5-/-, 3d, single TLR3-/-, TLR7-/-, TLR9-/- and TLR7/9 double deficient mice. These studies reveal an important and redundant role for TLR7- and TLR9-MyD88 signaling in maintaining long-term anti-PyV IgG responses. To determine how MyD88 signaling affects the generation of long-lived plasma cells and memory B cells, I investigated germinal center (GC) responses in MyD88-deficient mice. A defect in GC B cell numbers is observed in MyD88-deficient mice after the acute phase of infection. The GC reaction is essential for the generation and maintenance of long-lived plasma cells and memory B cells. T follicular helper (TFH) cells are absolutely required to generate normal GC. l found reduced numbers of TFH cells in MyD88-deficient mice. Lower numbers of T FH cells suggests that poor T cell help may contribute to the diminished number of GC B cells. However, interaction with B cells is required for the formation of fully differentiated TFH cells. Along with B cell function, MyD88 signaling can affect T cell and dendritic cell function as well. Thus, it is not clear at this point whether the requirement for intact MyD88 signaling for the formation and maintenance of long-term B cell populations is completely B cell-intrinsic. Some viruses can induce T cell-independent B cell responses, perhaps due to their complex arrays of repetitive antigenic epitopes on virions, coupled with the induction of innate cytokines. Nevertheless, T cell help is usually necessary for generating long-term antibody responses in the form of long-lived plasma cells and memory B cells. In contrast, our lab has found that T cell-deficient mice infected with PyV develop long-lasting, protective antiviral IgG responses. I questioned whether these mice could generate TI B cell memory cells or long-lived plasma cells. I show that long-lasting anti-PyV antibody in T cell-deficient mice was not due to the presence of long-lived plasma cells or memory B cell responses. TCRβδ deficient mice, which lack both CD4 and CD8 T cells, had ~10 a times higher virus load persisting in various organs. Therefore, I hypothesized that the high level of persistent PyV antigen, in completely T cell-deficient mice, may activate naïve B cell populations continuously, thereby maintaining the long-lasting IgG responses. Prior to PyV infection, T cell-deficient mice received wild type CD8 T cells, which reduced PyV loads, and this was associated with decreased levels of antiviral serum IgG over time. As in TCRβδ deficient mice, high PyV loads were detected in the bone marrow, which is the site for B cell lymphopoiesis, I questioned how B cells develop in the presence of PyV antigen and still stay responsive to PyV, generating long-term antiviral IgG responses in the periphery. Studies have shown that self-antigens that trigger both B cell receptor signaling and TLR-MyD88 signaling pathways in the bone marrow lead to the breaking of B cell tolerance and production of autoantibody in the periphery. Thus, we hypothesized that high PyV levels in the bone marrow signal through both B cell-receptors and TLRs, allowing continuous antiviral antibody production by B cells. Using mice that are deficient in T cells and MyD88 signaling, I found that PyV-specific TI IgG levels gradually decreased, supporting this hypothesis. Thus, high PyV loads and innate signaling together can break B cell tolerance. During a persistent virus infection this can result in sustaining long-term protective T cell-independent IgG responses.

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This thesis concerns two immunologic issues which are relevant for leprosy control activities: laboratory diagnosis of paucibacillary leprosy/PB and evaluation of the prognostic value of Mycobacterium leprae specific serology for leprosy reactions. Immunodiagnostic laboratory tests for PB leprosy (PB) depend on cellular immunity and these tests and this study evaluated a whole blood assay/WBA prototype using antigen combinations in PB and multibacillary leprosy patients (MB) and controls from two Brazilian endemic regions (Goiânia/GO and Fortaleza/CE). The M. leprae specific cellular immunity was evaluated by WBA using heparinized tubes containing different antigen combinations: 46f+LID-1 and ML0276+LID-1; controls: PBS, PHA, MLCS-M. leprae cell sonicate, PPD. After 24 hours incubation (37°C, 5% CO₂) plasma was collected and ELISA used to detect IFNγ (QuantiFERON/Qiagen, cut-off: 50pg/mL) and CXCL10 (Sigma-Aldrich®, St. Louis, Missouri, USA, cut-off: 500 pg/mL). Newly diagnosed, untreated leprosy patients (MB, n=30, PB, n=38) and controls (27 household contacts/HHC, 61 endemic controls/EC) were tested. The new platform in tube validated previous IFNγ results obtained in culture plates. Production of IFNγ to 46f+LID-1 was detected in 84% PB patients, 55% HHC; for ML0276+LID-1: 71% PB, 55% HHC. For both antigen combinations, the IFNγ response of PB leprosy patients differed from the EC (p<0.0001), however the response in PB leprosy and HHC was similar (p>0.05). Studies in tuberculosis/TB have shown that the CXCL10 response differentiate individuals with active TB from those with latent Mycobacterium tuberculosis infection. In leprosy, CXCL10 levels did not differentiate PB leprosy from HHC. Despite this limitation, our results show that WBA in tube could be a supplementary tool in assessing M. leprae infection rates and evaluating the impact of control measures. The application of leprosy serology as a predictor for the occurrence of leprosy reactions observed during clinical monitoring post treatment was evaluated by ELISA tests to detect M. leprae-specific antibodies, IgM anti PGL-I, IgG anti LID-1 and IgM and IgG anti ND-O-LID. Serum samples from 452 patients were tested and these patients participated of Clinical Trial for Uniform Multidrug Therapy Regimen for Leprosy Patients in Brazil/U-MDT/CT-B who were clinically monitored for more than six years post treatment for the development of leprosy reactions. Serological tests were performed in samples collected in untreated patients at diagnosis (baseline) and the results were analyzed taking into account the clinical outcome post treatment (reaction and reaction-free). Reactional patients (RR and ENL) had higher seropositivity rates and optical density/OD medians for PGL-I, LID-1 and ND-O-LID compared to reaction-free patients (p<0.0001). In leprosy, both the development of reactions and the serologic response are strongly influenced by the patients’ bacillary index (BI) and a positive correlation was found between both the BI and the proportion of reactional patients and the antibody levels. Negative BI group: 10% reactional patients, intermediary BI (>0<3): 50% reactional patients, high BI (>=3): 66% reactional patients. The serological results stratified according to the BI showed: in BI negative patients higher anti-PGL-I levels in RR patients compared to reaction-free ones (p=0.014); group IB> 0<3: reactional patients (RR) showed higher anti-PGL-I (p=0.014) and anti-LID-1 (p=0.035) antibody levels compared to reaction-free patients; group IB => 3: patients who developed ENL showed higher levels of anti-LID-1 antibodies (p=0.028) when compared with reaction-free ones. The accuracy of a serological test to predict the occurrence of leprosy reactions as determined by ROC curve showed that only anti-PGL-I serology provided limited value to predict RR (area-under-the-curve/AUC=0.702). The anti-PGL-I serology showed low sensitivity for RR prognosis, indicating the need to identify other plasma biomarkers for prediction of RR. In patients who developed ENL during follow-up was observed high positivity in the anti-LID-1 serology at diagnosis indicating the potential application of serology in the identification of patients at higher risk of developing ENL.
Esta tese aborda dois temas imunológicos importantes para o controle da hanseníase: diagnóstico laboratorial da hanseníase paucibacilar/ PB e valor prognóstico da sorologia específica para Mycobacterium leprae para reações hansênicas. O diagnóstico laboratorial da hanseníase PB se baseia em testes que avaliam a imunidade celular e este estudo avaliou um protótipo de ensaio de sangue total/EST com combinações antigênicas do M. leprae em pacientes multibacilares-MB e PB e controles de duas regiões endêmicas (Goiânia/GO e Fortaleza/CE). A avaliação da imunidade celular específica se baseou em EST em tubos heparinizados contendo diferentes combinações de antígenos do M. leprae: 46f+LID-1 e ML0276+LID-1; controles: PBS, PHA, MLCS-M. leprae cell sonicate, PPD. Após 24 horas de cultura (37ºC, 5% CO₂), o plasma foi coletado e ELISA utilizado para mensurar IFNγ (QuantiFERON/Qiagen, cut-off: 50pg/mL) e CXCL10 (Sigma-Aldrich®, St. Louis, Missouri, USA, cut-off: 500 pg/mL). Pacientes com hanseníase, recém diagnosticados, não tratados (30 MB, 38 PB) e controles (27 contatos domiciliares/CD, 61 controles saudáveis/CS) foram avaliados. A nova plataforma de tubos foi validada demonstrando produção de IFNγ similar a obtida em placas de cultura. A produção específica de IFNγ para a combinação 46f+LID-1 foi: 84% pacientes PB, 55% CD; para ML0276+LID-1: 71% PB, 55% CD. Para ambas as combinações antigênicas a resposta de IFNγ em pacientes PB diferiu da obtida para os CS (p<0,0001), contudo foi similar em pacientes PB e CD (p>0,05). Estudos em tuberculose/TB mostraram que a produção de CXCL10 discrimina indivíduos com TB ativa daqueles com infecção latente pelo Mycobacterium tuberculosis. Avaliamos na hanseníase se a produção de CXCL10 é capaz de diferenciar pacientes PB/infecção ativa de CD com infecção “latente”, assintomática. Nossos resultados mostraram produção de CXCL10 induzida por combinações antigênicas do M. leprae, entretanto sem diferenciar resposta de infecção ativa em PB de infecção assintomática em CD. Apesar desta limitação, nossos resultados mostram que o EST em tubo poderia ser uma ferramenta para avaliar as taxas de infecção PB pelo M. leprae e o impacto das medidas de controle. A aplicabilidade da sorologia específica para hanseníase como ferramenta preditora de reações hansênicas foi avaliada por ELISA para detectar anticorpos para específicos do M. leprae, IgM anti PGL-I, IgG anti LID-1 e IgM e IgG anti ND-O-LID Utilizamos amostras de soro de 452 pacientes que participaram do Ensaio Clínico para Avaliação da Eficácia de um Esquema Único de Multidrogaterapia em pacientes com hanseníase no Brasil (U-MDT/ CT-BR) que foram monitorados por mais de 6 anos após o tratamento quanto ao desenvolvimento de reações. Resultados da sorologia realizada em amostras coletadas ao diagnóstico, antes do início da terapia (baseline) foram analisados segundo os desfechos clínicos (ocorrência ou não de reações hansênicas). Independente do tipo de reação (reação reversa/RR ou eritema nodoso hansênico/ENH), pacientes reacionais apresentaram maior soropositividade e medianas de D.O para PGL-I, LID-1 e ND-O-LID comparados com pacientes não reacionais (p<0,0001). Na hanseníase o desenvolvimento de reações e a soropositividade são fortemente influenciadas pelo índice bacilar (IB) do paciente. Nossos resultados corroboram este paradigma mostrando associação positiva do IB tanto com a proporção de pacientes reacionais como com os níveis de anticorpos. Grupo de pacientes com IB negativo: 10% reação, IB intermediário (IB>0<3) 50% reacionais e IB >3, 66% de pacientes reacionais. Os resultados da sorologia, estratificados de acordo com o IB mostrou no grupo com IB negativo que pacientes que desenvolveram RR apresentaram maiores níveis de anticorpos anti PGL-I comparados com pacientes não reacionais (p=0,014); Grupo com IB>0<3: pacientes reacionais (RR) apresentaram maiores níveis de anticorpos para PGL-I (p=0,014) e LID-1 (p=0,035) comparados com pacientes não reacionais; Grupo IB=>3: pacientes que desenvolveram ENH apresentaram maiores níveis de anticorpos anti LID-1 comparados com pacientes não reacionais (p=0,028). De acordo com resultados da curva ROC para avaliar a acurácia de um teste sorológico para prever o desenvolvimento de reações durante o monitoramento, a sorologia anti PGL-I apresentou limitado valor para prever o desenvolvimento futuro de RR (área sob a curva/AUC=0,702). A sorologia para LID-1 mostrou capacidade de prever o desenvolvimento de ENH (AUC= 0,85). A sorologia anti-PGL-I apresentou baixa sensibilidade para prognóstico da RR, indicando a necessidade da identificação outros biomarcadores plasmáticos para predição da RR. Já em pacientes que desenvolveram ENH durante o seguimento apresentaram alta positividade na sorologia anti LID-1 ao diagnóstico, indicando a potencial aplicação da sorologia na identificação de pacientes com maior risco de desenvolver ENH.

Orientador: Mary Marcondes
Banca:Wagner Luis Ferreira
Banca: Suely Regina Mogami Bomfim
Banca: Juliana Peloi Vides
Banca: Acácio Duarte Pacheco
Resumo: O presente estudo teve como objetivos investigar a presença de anticorpos anti-Leishmania spp. e anti-saliva de Lutzomyia spp., e a presença do DNA de Leishmania spp. no sangue periférico de 284 indivíduos atendidos em um hospital público de área endêmica para leishmaniose visceral (LV). Baseando-se nos resultados de sorologia e da reação em cadeia da polimerase (PCR), 21,1% dos indivíduos foram considerados expostos. A presença de anticorpos anti-Leishmania spp. e anti-saliva de Lutzomyia spp. foi observada em 20,8% e 37,7% dos indivíduos, respectivamente, observando-se uma associação significativa entre a presença dos dois anticorpos. Em 20,4% dos indivíduos verificou-se sororeatividade em ambos os testes, 17,3% apresentavam anticorpos anti-saliva de Lutzomyia spp. sem a presença de anticorpos anti-Leishmania spp. e um indivíduo apresentava somente anticorpos anti-Leishmania spp. sem a presença de anticorpos anti-saliva de flebotomíneo. Em apenas um indivíduo foi possível amplificar fragmento do DNA de Leishmania spp. no sangue periférico. Concluiu-se que a infecção por Leishmania spp. pode estar sendo subdiagnosticada em áreas endêmicas para LV e, portanto, sugere-se que indivíduos atendidos em hospitais sejam melhor avaliados quanto à possibilidade de infecção e posterior desenvolvimento da doença. Ainda, a presença de anticorpos anti-saliva de flebotomíneo pode ser um possível indicador de exposição ao vetor.
Abstract: The present study aimed to investigate the presence of anti-Leishmania spp. and anti-saliva of Lutzomyia spp. antibodies, and the presence of Leishmania spp. DNA in the peripheral blood of 284 people attended at a public hospital in an endemic area for visceral leishmaniasis (VL). Based on the results of serology and PCR, 21.1% of the people were considered exposed. The presence of antibodies anti-Leishmania spp. and anti-saliva of Lutzomyia spp. was observed in 20.8% and 37.7% of the individuals, respectively, with a significant association between the presence of both antibodies. In 20.4% of the people there was serum reactivity in both tests, 17.3% had antibodies anti-saliva of Lutzomyia spp. without the presence of anti-Leishmania spp. antibodies, while one individual had only anti-Leishmania spp. antibodies without the presence of antibodies against saliva of sand flies. In only one individual it was possible to amplify the DNA fragment of Leishmania spp. in peripheral blood. It was concluded that the infection by Leishmania spp. may be underdiagnosed in endemic areas for VL and, therefore, it is suggested that people referred to hospitals should be better evaluated for the possibility of infection and subsequent development of the disease. Furthermore, the presence of antibodies anti-sandfly saliva may be an indicator of exposure to vector.
Doutor

Die Protothekenmastitis des Rindes ist eine therapieresistente, weltweit vorkommende Infektionskrankheit. Das ätiologische Agens, die farblose Alge Prototheca (P.) zopfii, kommt ubiquitär in feuchten Habitaten vor und verursacht fakultativ akute bis chronische Entzündungen des Rindereuters. Es gibt Hinweise auf das Vorkommen eines speziellen, Mastitis-assoziierten Biotyps von P. zopfii, der sogenannten Variante II. Durch die oft zu beobachtende endemische Ausbreitung in Milchviehbeständen sowie durch die nachhaltige Therapieresistenz, welche oft zum wirtschaftlichen Totalverlust der betroffenen Milchkühe führt, stellen Protothekenmastitiden beim Rind ein großes ökonomisches Problem für den betroffenen Betrieb dar. Da bisher nur sehr beschränkte Erkenntnisse zur lokalen und systemischen Immunantwort sowie zur Erregerausscheidung im Verlaufe der Protothekenmastitis des Rindes vorlagen, wurden die verschiedenen klinischen Stadien dieser Infektion serologisch, kulturell sowie durch Bestimmung der Zahl der somatischen Zellen in der Milch charakterisiert. Zu diesem Zwecke wurden drei verschiedene ELISA-Systeme entwickelt, die anschließend auch auf ihren möglichen Einsatz bei der Diagnostik der Protothekenmastitis hin untersucht wurden. Dies geschah in einem hochgradig an Protothekenmastitis erkrankten Milchviehbestand. Darüber hinaus wurden verschiedene Isolate von P. zopfii auxanographisch, biochemisch, serologisch und genetisch untersucht, um eine Differenzierung innerhalb der Algenspezies P. zopfii vornehmen zu können. Anhand der auxanographischen, biochemischen, serologischen und genetischen Untersuchungen war eine eindeutige Differenzierung von drei verschiedenen Bio-, Sero- und Genotypen innerhalb der Algenspezies P. zopfii möglich. Alle untersuchten Mastitisisolate konnten eindeutig der Variante II von P. zopfii zugeordnet werden, womit dieser Variante eine besondere epidemiologische Bedeutung bei der Entstehung der Protothekenmastitis des Rindes zu zukommen scheint. Die Untersuchungen dieser Arbeit zeigen, dass akut infizierte Tiere sowohl die höchsten Antikörperaktivitäten an IgG im Blutserum sowie an IgA und IgG1 im Milchserum als auch die höchsten Gehalte an somatischen Zellen in der Milch aufweisen. Chronisch infizierte Milchkühe weisen zum Teil sehr hohe Antikörperaktivitäten in der Milch auf und unterschieden sich nicht signifikant von akut infizierten Tieren. Demgegenüber weisen diese chronisch infizierten Tiere signifikant höhere IgG-Aktivitäten im Blutserum sowie IgA- und IgG1-Aktivitäten in der Milch auf als nicht infizierte Tiere. Somit ist eine eindeutige Differenzierung zwischen infizierten und nichtinfizierten Kühen möglich. Die ELISAs zum Nachweis von spezifischem IgA und IgG1 im Milchserum erwiesen sich als besonders geeignet, um infizierte Kühe zu identifizieren. Beide serologischen Testsysteme wiesen Sensitivitäten von 96,3 % für IgA sowie 92,6 % für IgG1 und Spezifitäten von 94,4 % (IgA) und 96,3 % (IgG1) auf. Demgegenüber wies der ELISA zum Nachweis von spezifischem IgG im Blutserum bei einer Spezifität von 100 % nur eine Sensitivität von 81,5 % auf. Die sehr gute Reproduzierbarkeit der Tests wurde durch Intra-Assay-Variationen von 6,08 % für den Nachweis von IgA im Milchserum und 7,20 % für IgG1 sowie durch die geringe Inter-Assay-Variation von 6,32 % (IgA) und 9,74 % (IgG1) belegt. Der Einsatz dieser Testsysteme bei der Sanierung eines hochgradig mit P. zopfii infizierten Milchviehbestandes zeigte, dass die serologische Diagnostik dem bisher gebräuchlichen kulturellen Erregernachweis bei der Identifikation intermittierender Erregerausscheider überlegen ist. Es wurde deutlich, dass 70,5% der infizierten Tiere die Erreger über einen Zeitraum von 12 Monaten permanent ausschieden und mindestens weitere 4,9 %, wahrscheinlich jedoch wesentlich mehr, dieser infizierten Tiere intermittierende Erregerausscheider waren. Somit scheint der serologische Erregernachweis für die Diagnostik der Protothekenmastitis des Rindes besser geeignet zu sein als die kulturelle Diagnostik. Dabei wurde die höchste Sensitivität durch die Kombination des Nachweises von spezifischem IgA und IgG1 im Milchserum erzielt
Protothecosis is a severe, often endemic mastitis in cattle caused by colorless algae of the genus Prototheca. Only little and insufficient knowledge about the organism itself, and the host immune response to this infection existed. Therefore, the aim of this thesis was to characterize the local and systemic immune response and the possible elimination or persistence of the pathogen in the host. To gain more information on the specific immune response, different clinical stages of infection were characterized serologically, culturally, and by determination of the number of the somatic cells in milk. Three different ELISA systems were developed, which were also examined for their diagnostic application potential. For the investigations, a dairy herd highly infected with Prototheca zopfii and severe clinical manifestation of protothecal mastitis was used. The ELISA was evaluated using serum and whey from animals with different clinical stages of infection. As antibody isotypes, IgG in serum, and IgA and IgG1 in whey were used. In addition, different isolates of P. zopfii were biochemically, serologically, and genetically examined in order to allow a differentiation of individual isolates within the species P. zopfii. The biochemical, serological and genetic investigations allowed a clear differentiation of the three known Variants of P. zopfii. All examined mastitis isolates could be assigned to variant II of P. zopfii. Therefore, it can be concluded that this variant has a particular epidemiological significance in the etiology of bovine protothecal mastitis. The serological investigations showed high antibody activities during acute and chronic stage of infection. The antibody activity was low in chronically infected, but presently cultural negative animals and also in uninfected animals. A strong correlation was observed between whey IgA and whey IgG1 antibody activity and the count of somatic cells in milk. Whereas, only a weak correlation exists to the number of algae cells excreted with the milk. A sensitivity of 96 % and a specificity of 94 % were calculated for the ELISA based on IgA levels. The ELISA for detection of specific IgG1 in whey shows a sensitivity of 92,6 % and a specificity of 96,3 %. Intra-assay and interassay variations were calculated to be at 6.08 % and 6.32 %, respectively. Based on these data, these ELISAs are suitable for discrimination between infected and uninfected animals, and might therefore be used for the screening of affected herds. When used in the remediation of a high-grade infected dairy herd the serological showed clear advantages in the identification of intermittent shedders. By culturing of Prototheca from milk, it was shown that 70.5% of the infected animals were permanent shedders, whereas 4.9 % were intermittent shedders. Since intermittent shedders could be clearly identified serologically, but might not be recognized by culturing, it can be assumed that serological diagnostics is more suitable for the identification of inapparently infected, intermittent shedders

Dissertação de Mestrado Integrado em Medicina Veterinária
Bluetongue virus serotype 8 (BTV-8) was detected in Austria for the first time, in November 2008. Due to outbreaks previously occurred in German regions close to the Austrian border, an active surveillance system was in place and allowed for an early identification of BTV-8 in the country. Mass emergency vaccination was started in the western part of the country in July 2008, due to the inclusion of that area in the protection zone around German outbreaks. The main objective of this work was to study the occurrence of BTV-8 in Austria in 2008 by i) describing the outbreak in Schärding, ii) comparing the two similar districts with different preventive strategies where BTV was identified - Schärding and Bregenz, iii) evaluating the influence of population dynamics in the duration of vaccinal immunity of cattle from the region of Styria included in the emergency vaccination program, and iv) developing a transmission model for the Styria region. From the analysis of the BT cases occurred in Schärding it was concluded that the moments of infection were very likely between May and October 2008, considering the optimal temperatures for Culicoides abundance that were verified in the region between April and September. The comparison between Schärding and Bregenz, concluded that the former district gathered a higher number of risk factors for disease spread. Higher cattle density in Schärding may have contributed to a higher spread of BTV, whereas the performance of a preventive mass vaccination campaign in Bregenz, most likely contributed for the opposite. It was also found that the proportion of PCR+ results amongst c-ELISA positive sera was statistically associated to the district of origin. A much lower proportion was observed in Bregenz when compared to Schärding. The analysis of the dynamics of cattle population in Styria resulted in an estimation of 3% year variation in cattle numbers which probably has a negligible effect on the decrease of the HIT in a time-frame of one year, leading to the conclusion that the lost of population immunity to BTV in Styria will be mostly due to the lost of immunity conferred by vaccination that lasts close to one year. The results of the BT transmission model for Styria indicated that the risk of occurrence of secondary infections in the summer months is not negligible, with a maximum estimated R0 of 2.66. These studies highlight the importance of epidemiological analysis of available data, using tools like mathematical modeling and GIS in order to understand disease occurrence in animal populations.
RESUMO - Análise epidemiológica de dados de vigilância e vacinação de algumas zonas Austríacas em 2008 - O serótipo 8 do vírus da língua azul (VLA-8) foi detectado na Áustria pela primeira vez em Novembro de 2008. Devido a surtos ocorridos na Alemanha próximo da fronteira Austriaca, um sistema de vigilância activa encontrava-se em curso e identificou o VLA-8 no país. A vacinação massiva de emergência foi iniciada na zona oeste do país em Julho de 2008, devido à inclusão daquela área na zona de protecção à volta dos surtos ocorridos na Alemanha. O objectivo principal deste trabalho foi estudar a ocorrência do VLA-8 na Áustria em 2008 i) descrevendo o foco ocorrido em Schärding, ii) comparando os dois distritos semelhantes com diferentes estratégias preventivas onde o VLA foi identificado – Schärding e Bregenz, iii) avaliando a influência da dinâmica populacional na duração da imunidade vacinal dos bovinos da região da Styria, e iv) desenvolvendo um modelo de transmissão para a Styria. Da análise dos casos de LA em Schärding conclui-se que os momentos de infecção se situaram provavelmente entre Maio e Outubro de 2008, considerando as temperaturas óptimas para abundância de Culicoides que aí se verificaram entre Abril e Setembro. A comparação entre Schärding e Bregenz concluiu que Schärding reuniu um maior número de factores de risco para a disseminação da doença. A sua maior densidade de bovinos poderá ter contribuído para uma maior disseminação do VLA, ao passo que a vacinação massiva preventiva em Bregenz, muito provavelmente terá contribuído para o oposto. Foi também observado que a proporção de resultados PCR+ entre soros positivos a c-ELISA estava estatisticamente associada ao distrito de origem, sendo inferior em Bregenz relativamente a Schärding. A variação anual da população de bovinos na Styria foi de 3%, a qual terá um efeito negligenciável no decréscimo da imunidade do efectivo vacinado contra o VLA, sendo esta principalmente devida à perda da imunidade conferida pela vacinação, que dura cerca de um ano. Os resultados do modelo de transmissão de LA para a Styria indicaram que o risco de ocorrência de infecções secundárias nos meses de verão não é negligenciável, com um R0 estimado em 2.66. Estes estudos sublinham a importância da análise epidemiológica dos dados disponíveis, utilizando ferramentas como a modelação matemática e os sistemas de informação geográfica de modo a compreender a ocorrência de doença em populações animais.

碩士
國立中興大學
微生物暨公共衛生學研究所
99
Background: Trivalent inactivated influenza vaccine (TIV) is reformulated annually to contain representative strains of 2 influenza A subtypes (H1N1 and H3N2) and 1 B subtype for protection, but the impact of antigenic drift on vaccine effectiveness varies between seasons. Vaccination of children in school is one strategy to reduce the spread of influenza in households and communities. Focusing efforts for influenza vaccination on healthy children may therefore be an effective and practical method of reducing the burden of influenza in the community. Since 2008, children grades 1-4 in elementary school in Taiwan receive free TIV vaccination; moreover, during the 2009 pandemic H1N1 (pH1N1) period, anextra monovalent vaccine was used in Taiwan for protection against the 2009 pH1N1 virus. In this study, we described a serological cohort study to evaluate the dynamic change of herd immunity to different influenza viruses and component specific vaccine effectiveness (VE) during 2 consecutive influenza seasons of 2008-09 and 2009-10. Methods: Children from elementary schools in Taichung city and Nantou county were enrolled during 2008-2009 and 2009-2010 seasons. The influenza viruses used in this study included vaccine strains and wildtype component H1N1, H3N2, influenza B viruses and pH1N1 virus, which represented the majority of isolations from clinical specimens collected in the corresponding seasons in Taiwan. Serum samples were taken from three different time periods to detect the antibody titers by using hemagglutination inhibition (HI) assay. Results: Starting in the winter of 2008, age-specific 2009 pandemic H1N1 seroprevalence was calculated based on the antibodies against pH1N1 virus with an HI titer of 1:40 or more during three periods: seroprevalence in all age groups were 0% in December, 2008; seroprevalence in the age group of 5-18 years old was13.3%, 19-60 years old was 14.3%, above 60 years old was 18.8% in April–June, 2009 ; seroprevalence in the age group of 5-18 years old was 30.8%, 19-60 years old was 33.3%, above 60 years old was 25% in September– October, 2009. After the pH1N1 vaccination, seroprotecion rate among school-aged children who received 1 dose pH1N1vaccine was over 90% andthe seroconversion rate was over 50%. We also evaluated the herd immunity and component specific vaccine effectiveness (VE), it revealed that the herd immunity against one of H3N2 variants (V0) decreased from 142.17 to 55.73 and further to 17.79; in contrast, the immunity against the other H3N2 variants (V3) increased from 8.6 to 14.83 and further up to 91.1 during December, 2008, April, and December, 2009, respectively. VE for A/H3N2 components was -27% (95% CI, -106%~22%)，and 29% (95% CI,7%~46%) in the 2008/09 and 2009/10 influenza seasons, respectively. On the other hand, after receiving seasonal TIV, immunogenicity to influenza B virus was not as good as that to other vaccine components. The seroprotection rate of influenza B after vaccination were about 60% in both two influenza seasons while H3N2 were about 90%. Based on the serology data, low cross protection between two lineages (Yamagada and Victoria) was observed. VEs for influenza B subtype component were only at the range of 20% to 30% in 2008/09 and 2009/10 influenza season. Conclusion: Our serological data suggested that the herd immunity results corresponded to the numbers of virus isolation of different antigenic drifted H3N2 strains according to Taiwan-CDC. It also confirmed that the immune selection pressure is the major driving force of H3N2 evolution and there were no or little cross protection between two influenza B lineages. Therefore, keep monitoring the herd immunity in different influenza viruses among children will be crucial to understand the VE of TIV.

This chapter cites Pneumocystis jirovecii (PJP), formerly known as Pneumocystis carinii, as an opportunistic pathogen that causes pneumonia in the immunocompromised individual. It explains how the disease caused by PJP occurs when both cellular and humoral immunity are impaired. It also looks at serologic studies that show the worldwide distributions of Pneumocystis and prevalence of antibodies to specific antigens that varies among different geographic regions. The chapter traces how PJP first came to attention when it caused interstitial pneumonia in severely malnourished and premature infants in Central and Eastern Europe during World War II. It describes PJP as one of several life-threatening opportunistic infections in patients with human immunodeficiency virus (HIV) infection worldwide and as the most common AIDS-defining illness in patients with advanced HIV infection.

Celiac disease (CD) is an immune-mediated chronic inflammatory disorder characterized by permanent gluten intolerance in genetically susceptible individuals. Exposure to gluten perpetuates an enteropathy leading to malabsorption with chronic diarrhea, weight loss, and abdominal distension. The small intestine mucosa is abnormal, and jejunal biopsy demonstrates various degrees of villous atrophy, absence of surface mucosa, and crypt hyperplasia. The diagnosis is based on the demonstration of a more or less pronounced villus atrophy in a jejunal biopsy. The villous atrophy improves after withdrawal of gluten from the diet. If undetected or neglected, CD may cause considerable late complications from malabsorption or secondary autoimmune diseases (Feigbery 1999; Maki and Collins 1997; Holmes 1996). The therapy consists of permanently excluding gluten from the diet and allows the healing of the mucosal lesion. Abnormalities of humeral and cell-mediated immunity suggest that celiac disease is an immunologic disorder (Walker-Smith 1996). It is caused by inappropriate immune response to the gliadin component in the dietary gluten (Dieterich 1997). Genetic susceptibility is present, and 90% of the patients have HLA DRG 3 DQ-2 haplotype, and some have the HLA DR4 DQ8 gene (Hadjivassiliou 1998). A close relationship exists between the biochemical properties of tissue transglutaminase and the basic molecular mechanisms responsible for CD, and possibly with the neuropsychiatric manifestations of CD (Gentile 2002). Anti–tissue transglutaminase antibody assay has been used as a serologic screening test for CD. In addition, antiendomysial, antigliadin, and antireticulin antibodies are associated with the disease. Nevertheless, the clinical symptomatology affecting the gastrointestinal (GI) system, histological abnormalities on gut biopsy, and presence of antiendomysial antibodies do not always coexist. Also, presentation with minor symptoms, such as irritable bowel syndrome, anaemia, slight weight loss, and fatigue, has become increasingly common, and in many cases the disease may be clinically silent, despite manifest small-bowel mucosal lesions. Therefore, CD is underdiagnosed (Catassi et al. 1996; Feigbery 1999; Holmes 1996; Kolho et al. 1998; Maki and Collins 1997).

An understanding of the consequences of infection and vaccination on host immunity sets the stage for interpreting pertussis epidemiology. Yet, with no known serological marker of protection, such an understanding is currently not possible. This chapter interrogates longitudinal age-stratified pertussis incidence reports from Sweden and Massachusetts, United States, with the aim of quantifying the impact of infection and immunization on protective immunity. The analysis of data from Sweden during the vaccination hiatus period (1986–1996) indicates that adults contribute little to transmission. This may either be because infection-derived immunity is very long-lasting, or that individuals whose immunity has waned are subsequently less susceptible. The analysis of data from Massachusetts (1990–2005) identifies the primary mechanism of vaccine failure—for both whole-cell and acellular pertussis vaccines—to be waning. However, the average duration of immunity is identified as many decades, though the model predicts substantial individual variability in this trait. Finally, the chapter demonstrates the estimates to be consistent with those obtained from popular measures of vaccine effectiveness, though the interpretation of these findings is quite different.

This chapter analyzes Cryptococcus neoformans, which is found worldwide as a soil organism and thought to be transmitted by inhalation and often causes disease in patients with abnormal cell-mediated immunity. It discusses the infection of Cryptococcus neoformans on patients with human immunodeficiency virus (HIV) infection, solid organ transplant recipients, immunocompetent persons. It also looks into cases of invasive cryptococcal infection that occur annually in patients with acquired immunodeficiency syndrome (AIDS) worldwide with more than 150,000 deaths each year. The chapter investigates two varieties of C. neoformans that distinguishable by serology: C. neoformans var. neoformans and C. neoformans var. gattii. It explains that C. neoformans var. gattii is endemic in Australia and recent outbreaks of its infection have occurred in the Pacific northwestern parts of North America.

Cryptococcus neoformans, which is found worldwide as a soil organism and thought to be transmitted by inhalation, most often causes disease in patients with abnormal cell-mediated immunity, notably patients with HIV infection and solid-organ transplant recipients, but the infection also occurs rarely in apparently immunocompetent people in restricted geographical areas, especially involving C. neoformans var. gattii. The most common presentation is with subacute meningoencephalitis, but other manifestations (e.g. isolated pulmonary disease or disseminated infection, are well described). Diagnosis is usually by culture or serology. Untreated cryptococcal meningitis is fatal: aside from supportive care (including monitoring for raised intracranial pressure), the therapy of choice is an initial period (at least two weeks) of amphotericin B (ideally with flucytosine), followed by at least 3 months of fluconazole. Most immunocompromised patients subsequently require maintenance suppressive therapy, usually with fluconazole.

Hepatitis B virus (HBV) is a double-stranded DNA hepadnavirus. It is an important cause of acute 5and chronic hepatitis and hepatocellular carcinoma. Worldwide about 2 billion people show serological evidence of exposure and about 400 million have active infection. High prevalence areas include sub-Saharan Africa, China, and southeast Asia. HBV was known at onset as the etiology of what is called “serum hepatitis”, this is the most common form of viral hepatitis transmitted parenterally. It is also a cause of both acute and chronic hepatitis of great significance. Hepatitis B virus has an incubation period that varies between 1 and 6 months. The clinical features of acute infection resemble those of the other viral hepatitides. Death from fulminant hepatitis occurs in about 1%. Following acute infection, there is either complete recovery (with long-term immunity) or persistent infection. The latter occurs in 5–10% infected adults, 30% infected children and 90% infants infected at birth; it is more common in the immunocompromised.

Mycobacterium ulcerans is the causative agent of the subcutaneous necrotic condition known as Buruli ulcer (BU).BU is Neglected Tropical Disease. The bacillus is the third most common mycobacteria disease-causing agent after Mycobacterium tuberculosis and Mycobacterium leprae. M. ulcerans produces the toxin-Mycolactone, which plays a key role in the pathophysiological features of the disease. Buruli ulcer has been reported in 34 countries, mainly in the tropics and subtropics. Tropical countries include Benin, Cameroon, Ghana, Democratic Republic of Congo and Nigeria. BU is also prevalent in Queensland, a subtropical region, and in Victoria, a temperate area, all within Australia. The exact mode of the transmission remains unclear. However, M. ulcerans is believed to have an aquatic niche. Initial diagnosis of BU is based on the experience of the clinician, but PCR targeting the M. ulcerans DNA, IS2404, isolation and culture of the bacillus and histopathology are used for confirmation. The current, commonly used methods for confirmatory diagnosis have logistic and resource challenges. Novel cell mediated immunity (CMI) and serology-based tests would be beneficial to provide a more accurate assessment of population exposure.

The hereditary periodic fever syndromes or hereditary autoinflammatory diseases are disorders of innate immunity that mostly present in childhood and are characterized by recurrent, self-limiting, seemingly unprovoked episodes of fever and systemic inflammation that occur in the absence of autoantibody production or identifiable infection. Disorders include (1) familial Mediterranean fever (FMF), due to mutations in the gene encoding pyrin; (2) tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), due to mutations in a gene for a TNF receptor; (3) mevalonate kinase deficiency and period fever (MKD), caused by mutations in the mevalonate kinase gene; and (4) the cryopyrin-associated periodic syndromes (CAPS), which include (a) familial cold urticarial syndrome, (b) Muckle–Wells syndrome, and (c) chronic infantile neurological, cutaneous, and articular syndrome. With advances in genetics, further syndromes are continually being recognized. These are all extremely rare and in the majority are only known to affect a handful of kindred or individuals. Diagnosis relies on recognition of suggestive clinical features that are almost always accompanied by a substantial acute phase response, and is supported by genetic testing. With the exception of FMF, which is a common disease in certain geographic areas, hereditary periodic fever syndromes are rare and easily overlooked in the differential diagnosis of recurrent fevers. Clinical features and management—attacks can be mild to debilitating and short to prolonged, while their most feared complication is AA amyloidosis. Effective therapies are available for some syndromes, for example: (1) FMF—daily prophylactic colchicine prevents clinical attacks and susceptibility to AA amyloidosis, (2) CAPS—treatment with anti-IL-1 agents produces rapid and often complete clinical and serological remission, and (3) TRAPS—anti-IL therapies are extremely effective.

 Evidence suggests that the majority of adults develop detectable levels of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies following infection with SARS-CoV-2 (moderate strength of evidence* [SoE]).  IgM levels peak approximately 20 days after symptom onset or RT-PCR diagnosis and subsequently decline. IgG levels peak approximately 25 days after symptom onset or RT-PCR diagnosis and may remain detectable for at least 120 days (moderate SoE*).  Almost all adults develop neutralizing antibodies in response to SARS-CoV-2 infection, and these antibodies may remain detectable for at least 152 days (low SoE*).  A small percentage of people do not develop antibodies in response to SARS-CoV-2 infection for reasons that are largely unclear but may be related to less severe disease or absence of symptoms.  Antibody prevalence does not appear to vary by age or sex, but older age may be associated with higher antibody levels (low SoE*). Non-White race may be associated with higher antibody prevalence and levels (low SoE*). COVID-19 severity and presence of symptoms may also be associated with higher antibody prevalence or levels (low SoE*). More evidence is needed to draw stronger conclusions regarding how the antibody response varies by patient characteristics and disease factors.  Studies to date have not established the relationship between the development of antibodies after RT-PCR-diagnosed SARS-CoV-2 infection and the risk of reinfection. Studies based on index serologic testing suggest that the presence of antibodies is associated with a lower risk of a subsequent positive SARS-CoV-2 RT-PCR test.

The BARD project was a continuation of a previous BARD funded research project. It was aimed at characterization of the 12kDa immunodominant protein and subsequently the cloning and expression of the gene in E. coli. Additional immunodominant proteins were sought among genomic B. abortus expression library clones using T-lymphocyte proliferation assay as a screening method. The 12kDa protein was identified as the L7/L12 ribosomal protein demonstrating in the first time the role a structural protein may play in the development of the host's immunity against the organism. The gene was cloned from B. abortus (USA) and B. melitensis (Israel) showing identity of the oligonucleotide sequence between the two species. Further subcloning allowed expression of the protein in E. coli. While the native protein was shown to have DTH antigenicity its recombinant analog lacked this activity. In contrast the two proteins elicited lymphocyte proliferation in experimental murine brucellosis. CD4+ cells of the Th1 subset predominantly responded to this protein demonstrating the development of protective immunity (g-IFN, and IL-2) in the host. Similar results were obtained with bovine Brucella primed lymphocytes. UvrA, GroE1 and GroEs were additional Brucella immunodominant proteins that demonstrated MHC class II antigenicity. The role cytotoxic cells are playing in the clearance of brucella cells was shown using knock out mice defective either in their CD4+ or CD8+ cells. CD4+ defective mice were able to clear brucella as fast as did normal mice. In contrast mice which were defective in their CD8+ cells could not clear the organisms effectively proving the importance of this subtype cell line in development of protective immunity. The understanding of the host's immune response and the expansion of the panel of Brucella immunodominant proteins opened new avenues in vaccine design. It is now feasible to selectively use immunodominant proteins either as subunit vaccine to fortify immunity of older animals or as diagnostic reagents for the serological survaillance.

The goal of the one-year feasibility study was to identify specific cytotoxic T-lymphocyte (CTL) epitopes to Neosporacaninum in the natural bovine host in order to make progress toward developing an effective peptide-based vaccine against bovine neosporosis. We tested the hypothesis that: N. caninum SRS2 peptides contain immunogenicCTLepitope clusters cross-presented by multiple bovine MHC-I and MHC-IIhaplotypes. The specific objectives were: (1) Map bovine CTLepitopes of N. caninum NcSRS-2 and identify consensus MHC-I and class-II binding motifs; and (2) Determine if subunit immunization with peptides containing N. caninum-specificCTLepitopes cross-reactive to multiple bovine MHChaplotypes induces a CTL response in cattle with disparate MHChaplotypes. Neosporosis is a major cause of infectious abortion and congenital disease in cattle, persisting in cattle herds via vertical transmission.5 N. caninum abortions are reported in Israel; a serological survey of 52 Israeli dairy herds with reported abortions indicated a 31% infection rate in cows and 16% infection rate in aborted fetuses.9,14 Broad economic loss due to bovine neosporosis is estimated at $35,000,000 per year in California, USA, and $100,000,000 (Australian) per year in Australia and New Zealand.13 Per herd losses in a Canadian herd of 50 cattle are estimated more conservatively at $2,305 (Canadian) annually.4 Up to date practical measures to reduce losses from neosporosis in cattle have not been achieved. There is no chemotherapy available and, although progress has been made toward understanding immunity to Neospora infections, no efficacious vaccine is available to limit outbreaks or prevent abortions. Vaccine development to prevent N. caninum abortion and congenital infection remains a high research priority. To this end, our research group has over the past decade: 1) Identified the importance of T-lymphocyte-mediated immunity, particularly IFN-γ responses, as necessary for immune protection to congenital neosporosis in mice,1,2,10,11 and 2) Identified MHC class II restricted CD4+ CTL in Neosporainfected Holstein cattle,16 and 3) Identified NcSRS2 as a highly conserved surface protein associated with immunity to Neospora infections in mice and cattle.7,8,15 In this BARD-funded 12 month feasibility study, we continued our study of Neospora immunity in cattle and successfully completed T-lymphocyte epitope mapping of NcSRS2 surface protein with peptides and bovine immune cells,15 fulfilling objective 1. We also documented the importance of immune responses NcSRS2 by showing that immunization with native NcSRS2 reduces congenital Neospora transmission in mice,7 and that antibodies to NcSRS2 specifically inhibition invasion of placental trophoblasts.8 Most importantly we showed that T-lymphocyte responses similar to parasite infection, namely induction of activated IFN-γ secreting Tlymphocytes, could be induced by subunit immunization with NcSRS2 peptides containing the Neospora-specificCTLepitopes (Baszler et al, In preparation) fulfilling objective 2. Both DNA and peptide-based subunit approaches were tested. Only lipopeptide-based NcSRS2 subunits, modified with N-terminal linked palmitic acid to enhance Toll-like receptors 2 and 1 (TLR2-TLR1), stimulated robust antigen-specific T-lymphocyte proliferation, IFN-γ secretion, and serum antibody production across different MHC-IIhaplotypes. The discovery of MHC-II cross-reactive T-cellinducing parasite peptides capable of inducing a potentially protective immune response following subunit immunization in cattle is of significant practical importance to vaccine development to bovine neosporosis. In addition, our findings are more widely applicable in future investigations of protective T-cell, subunit-based immunity against other infectious diseases in outbred cattle populations.