HUMORAL IMMUNOLOGY (HI)

CAN A MIXTURE OF SCFV´S TO CROTALIC AND BOTHROPIC VENOMS ENHANCES EACH ONE ABILITY OF

INHIBITING THE TOXIC EFFECTS OF THESE SPECIES ENVENOMATION?

LUCIANO COSTA E SILVA; EDUARDO CROSARA RONCOLATO; GABRIELA PESSENDA; LUCAS BENICIO

CAMPOS; JOSÉ ELPIDIO BARBOSA.

BIOCHEMISTRY AND IMMUNOLOGY DEPARTMENT - MOLECULAR IMMUNOPATOLOGY LABORATORY FMRP-

USP, RIBEIRÃO PRETO - SP - BRASIL.

In the world, the most common cause of death by animal’s poisoning is caused by snakes. In Brazil, an average of

20,000 accidents involving venomous snakes are notified annually, with an index of lethality at around 0.43%.The

administration of heterologous antibodies collected from animals immunized with crude venom of snakes is the

available treatment in cases of accidents. However, this kind of antivenom sometimes do not protect patient and may

cause hypersensitivity reactions. The construction of libraries of human antibody fragments (scFv) and selecting those

fragments by affinity to antigen of animal´s venoms are a promising alternative for the production of new and safer

immunotherapy. Based on this approach, our laboratory selected and produced by Phage Display technology, scFv

capable of inhibiting the toxic activity of various animal´s venoms, among them to Bothrops jararaca and Crotalus

durissus terrificus venoms. Considering our previous results, this paper aims to select clones with cross affinity

between those venoms, in order to check if the addition of scFv against Crotalus durissus terrificus venom to bothropic

scFv antivenom can improve the ability of this mixture to inhibit the venom´s toxic effects. Furthermore the other way

round will be also is also tested. Following this aim we selected nine clones of phage-antibodies able to cross react as

stated above. The selected clones will be tested separately and in combination in functional assays, in order to enable

the construction of a fully human anti Bothropic and anti Crotalic serum. The use of scFv, directed to the main animal

toxins, may contribute to the current treatment that uses heterologous sera for this purpose.

Financial support: INCTTox, CNPq.

CAN HUMORAL IMMUNITY ENHANCE DEVELOPMENT OF SKIN TUMORS?

ALINE ANTONIO LAVEZO1; LILIAN REGO CARVALHO

2; TATIANE CANHAMERO

3; PRISCILIA AGUILAR-

RAMIREZ4; ANDREA BORREGO

5; WAFA HANNA F. CABRERA

6; JOSE RICARDO JENSEN

7; MARCELO DE

FRANCO8; ORLANDO GARCIA RIBEIRO

9; OLGA MARIA IBAÑEZ

10; NANCY STAROBINAS

11.

1,2,3,4.UNIVERSIDADE DE SÃO PAULO, SÃO PAULO - SP - BRASIL; 5,6,7,8,9,10,11.INSTITUTO BUTANTAN,

SÃO PAULO - SP - BRASIL.

Introduction: Chronic inflammation predisposes tissue to cancer development and interactions between innate and

adaptive immune cells may potentiate malignant progression in the skin. Mice genetically selected to High (H) and

Low (L) antibody production treated with 7, 12-dimethylbenzanthracene (DMBA) was used to study the effect of

combination between humoral immunity and tumor susceptibility. Methods and Results: Skin tumor was induced in

mice by epicutaneous application of DMBA (50µg in 0,1mL acetone) for 5 consecutive days, and controls were treated

with acetone. When analyzed skin with 48 hours post treatment, males showed intense vascularization in the lesions

and both strains showed an intense superficial cutaneous inflammation around 15 days. With 30 – 60 days, L mice

healed skin lesions (74%, n=31) while H mice developed papillomas (76%, n= 33) that progressed to sarcoma with

240 days. Analyzing skin and lung, L mice were more resistant, showing few lesions and tumors. Lung tumors were

observed in all H male animals (100%, n=8). Conclusion: the genes related to high antibody production can provide

susceptibility for chemical skin tumorigenesis and predispose to lung tumors in this model. Financial support:

CAPES and FAPESP

DOES OBESITY AFFECT ANTIBODY PRODUCTION?

VINÍCIUS DANTAS MARTINS; JULIANA LAUAR GONÇALVES; MARIANA CAMILA MIRANDA; RAQUEL PINHEIRO

DAMASCENO; ROBERTA AMORIM PEREIRA; ANA MARIA CAETANO FARIA; TATIANI UCELI MAIOLI.

UFMG, BELO HORIZONTE - MG - BRASIL.

INTRODUCTION: Prominent increases in the proportion of people who suffer from obesity in modern society have

been brought about by rapid and extreme changes in lifestyle, particularly in eating habits. Obesity induces the

development of multiple metabolic and cardiovascular diseases initially triggered by insulin resistance, which is

caused by chronic, low-grade inflammation observed in obese adipose tissues. There are data showing that obesity

people and mice have altered immunoglobulin production and impaired maintenance of memory cells (Sheridan et al,

2012; Smith, et al, 2007)

So, the aim of our work was to check if diet induced obesity in C57BL/6 could affect antibody production before and

after immunization.

METHODS: Mice are treated ad libitum with a high sugar and fat diet for 12 weeks, control mice received AING93

during the same period. After that blood sample was collected to measure antibodies titles before immunization. The

immunization protocol was preceded using OVA (10μg) in AlOH3 (1 mg/ml).

After general anesthesia mice had their feces and blood collected in order to measure the immunoglobulin titles by

ELISA.

RESULTS: Not immunized obese mice showed decrease in unspecific IgM and IgG in serum, as well as, a decrease

in sIgA in feces.

Although, after immunizationju with OVA, there was no difference in anti-OVA IgM and IgG present in the serum.

CONCLUSION: Obesity, may alters antibody production in a normal condition, but after immunization with OVA in

AlOH3 the probable activation of B cells might happen at same level in obese and normal mice.

Financial support: CNPq, FAPEMIG & PRPq/UFMG.

EFFECT OF CASHEW APPLE BAGASSE FLOUR SUPPLEMENTATION IN CASHEW APPLE JUICE ON MICE

IMMUNE RESPONSE

CAMILA FREITAS BEZERRA1; ANDRESIANE SOUSA DA SILVA

2; BELISE MARIA OLIVEIRA BEZERRA

3; LUANA

OLIVEIRA LEITE4; ALBERT LAYO COSTA DE ASSIS

5; JOANA DE FREITAS ROCHA

6; BEATRIZ DE SOUSA E

LIMA7; MICHELLE APARECIDA FREITAS DE ANDRADE

8; ANA CLÁUDIA MARINHO DA SILVA

9; NEUZA FELIX

GOMES10

; ÉRIKA FREITAS MOTA11

; DIANA CÉLIA SOUSA NUNES PINHEIRO12

; DIRCE FERNANDES DE

MELO13

.

1,2,5,6,7,8,10,11,13.UNIVERSIDADE FEDERAL DO CEARÁ, FORTALEZA - CE - BRASIL; 3,4,12.UNIVERSIDADE

ESTADUAL DO CEARÁ, FORTALEZA - CE - BRASIL; 9.FACULDADE CATÓLICA DO CEARÁ, FORTALEZA - CE -

BRASIL.

Introduction: The cashew apple and cashew nut (Anacardium occidentale L.) have high nutritional value, but less

than 10% of the cashew apple from the processing of cashew nut is availed. The aim of this study was to evaluate the

phytochemical composition, total antioxidant potential and immunomodulatory activity of cashew apple juice enriched

with the mature (CAJM) and immature bagasse flour (CAJIM) of the cashew apple.

Methods and Results Therefore, the cashew apple juice and flour antioxidant capacity (clone CCP-76) was

evaluated by two methods, ABTS and DPPH. Additionally, polyphenols, vitamin C, carotenoids, anthocyanins and

yellow flavonoids were quantified. The Ethics Committee on Animal Research (CEPA) of UFC approved in vivo tests

with male swiss mice under the protocol No. 102/2011. Toxicity tests were carried out on mice (n=10 in 8 groups)

treated for 19 days orally with CAJM and CAJIM at different doses. Physiological parameters such as body weight,

relative organ to body weight, liver lipid peroxidation and ALT and AST transaminase enzymes activities were

determined. To evaluate the humoral and cellular immune response, animals were immunized (n=10 in 7 groups) with

sheep red blood cells (SRBC) to determine the antibody titer by hemagglutination method and later challenged with

(SRBC) to evaluate the delayed type hypersensitivity (DTH) reaction. To analyze the significant differences between

the means of groups, we applied the ANOVA followed by Tukey or Newman-Keuls (p < 0.05). The immature bagasse

flour (BFIM) showed the highest antioxidant capacity with 356,52 ± 7,9 µM Trolox/g (ABTS) and 176,82 ± 22,5 g fruit/g

DPPH (DPPH). BFIM also showed highest levels of polyphenols with 4.450,62 ± 24,8 mg/100g. Physiological

parameters of toxicity tests did not showed any significant changes among groups. The cashew apple juice with no

flour enrichment and CAJIM showed the highest antibody titer with 819,2 ± 106,6 and 742,4 ± 96,9, respectively.

Moreover, DTH results showed that pure juice, CAJM and CAJIM also increased with 2,49 ± 0,04 mm; 2,49 ± 0,02

mm and 2,50 ± 0,03 mm, respectively.

Conclusion: The cashew apple juice enriched with the bagasse flour in different maturity stages can be an

alternative for improving the immune system responses. However, further studies are needed to support this

supplementation.

Financial Support: CAPES, CNPq, FUNCAP.

EFFECT OF DOSE AND ROUTE OF INOCULATION OF ANTIGEN ON PRODUCTION OF IGY ANTIBODIES

MICHELE PAGLIARI OLIVEIRA MONTINI; MIRIELE CAROLINA DA SILVA; ANA PAULA CHEIRUBIM; SANDMARY

DECHECHI CHAMBÓ; EDUARDO VIGNOTO FERNANDES; EMERSON JOSÉ VANANCIO.

UNIVERSIDADE ESTADUAL DE LONDRINA, LONDRINA - PR - BRASIL.

Introduction: The antibodies (Ab) are used in immunodiagnostic and immunotherapies (Rev. Annu. Food.

Sci.Technol. 3:163-182, 2012; Biolog. 40:313-322, 2012). IgY is an antibody class found abundantly in egg yolk of

laying hens, of which may be easily extracted by low cost processes. Therefore, IgY is an alternative to antibody

production in mammals(Comp. Bioch. And Physiol. 131:569-574, 2002; Vet . Immunol. and Immunop. 135: 173-180,

2010). The aim of this study was evaluate the effect of different doses and routes of inoculation of antigen (Ag) on the

production of IgY antibodies in laying hens.

Methods and Results: In this study, 48 laying hens (White Leghorn) were divided into 8 groups of 6 animals each. To

investigate the effect of Ag dose, the animals were inoculated with 2μg/animal, 20μg/animal or 200μg/animal of

human IgG in aluminum hydroxide by intramuscular (IM) route. The control group received only PBS in aluminum

hydroxide. To investigate the effect of route of inoculation, 20μg/animal of human IgG in aluminum hydroxide were

inoculated by IM, intradermal (ID) or subcutaneous (SC) routes. The control group was inoculated with PBS in

aluminum hydroxide. All animals were five times inoculated. Blood samples were collected before 1st inoculation and

7 days after each inoculation. The levels of IgY antibodies were analyzed by ELISA. The results were submitted to

analysis of variance (ANOVA), and differences between averages were compared by Bonferroni test at 5%. A dose of

200μg/animal resulted in high level of IgY antibodies after 1st inoculation. Doses of 20 and 200μg/animal induced

similar levels of after 2nd

inoculation and dose of 2μg/animal result in significant specific antibodies levels only after the

5th inoculation. The results show that SC route induced higher levels of specific antibodies after 1st inoculation, while

the ID and IM routes were able to induce high levels of IgY antibodies only after 2nd

and 5th inoculation, respectively.

Conclusion: In this study we show that the dose of 20μg/animal is sufficient to induce significant levels of specific

antibodies, which represents a significant reduction in the amount of antigen usually used for production of IgY

antibodies. Furthermore, the results show that the SC route is more efficient to production of specific IgY than IM

route, that is the more frequently route used to antibody production on laying hens.

Financial support: CAPES and Fundação Araucaria.

ENVELOPE SUBTYPE-CONSENSUS PEPTIDES INHIBITING NEUTRALIZATION OF HIV-1 BY PLASMA FROM

INFECTED BRAZILIAN PATIENTS

DALZIZA ALMEIDA1; MARIZA GONÇALVES MORGADO

2; FERNANDA HELOISE CORTES

3; MONICK

LINDENMEYER GUIMARÃES4; VERA BONGERTZ

5.

1,2.FIOCRUZ/ IOC, RIO DE JANEIRO - RJ - BRASIL; 3,4,5.FIOCRUZ/IOC, RIO DE JANEIRO - RJ - BRASIL.

Broadly reactive anti-Env antibodies have been detected in some HIV-1 infected individuals, mainly to the membrane

proximal external region (MPER) of the gp41 envelope glycoprotein and the third variable loop (V3) of the gp120

glycoprotein of HIV-1. However, little is known about the prevalence and subtype specificity of this antibody response

from HIV-1 infected Brazilian individuals. Four peptides corresponding to the Brazilian consensus sequences of HIV-1

subtypes B (GPGR at the top of v3), B/Bbr (GWGR), C and F1 were tested to identify epitopes of MPER (36 mer) and

V3 (15 mer) by neutralizing antibodies from plasma of HIV-1 F1 or B subtype infected Brazilian individuals. Thirty-two

plasma of HIV-1 infected (11 chronically infected individuals with typical rates of disease progression [TP], 16 Long

term non-progressor [LNTP] and 5 recent seroconvertors [RS]) were tested in the PBMC neutralization assay

against HIV-1 subtype B strain 1886 (R5 tropism). The 90% neutralizing titers were determined by competitive ELISA

using the Zeptometrix p24 antigen test. Cross-neutralizing antibodies for HIV-1B 1886 isolate were detected in

approximately 31% of the plasma (80% of the TP, 20% of the LTNP and none of the RS). The V3 and Gp41 peptides

did not inhibit neutralization in 16 (59%) plasma samples with neutralizing antibodies (NAb), indicating the presence of

other dominant NAb. The magnitude of response was mostly associated to the V3 loop peptide, especially for the

subtype C, nevertheless MPER antibodies were detected in (91%) of reactivity individuals with a low specificity.

Neutralization inhibition capacity of the peptides tested was observed in this order: V3C > gp41B > V3F > V3B/Bbr >

V3B > Gp41B/Bbr > Gp41C > Gp41F. A highly polyclonal NAb response was observed in two broadly neutralizing

plasma samples of TP individuals, binding 87.5 - 100% of peptides. This study gives a general pattern of the inhibitory

activities of the potentially immunogenic loop V3 and gp41 MPER peptides, providing insights into their potency and

breadth. A clearer understanding of immunogenic sequences that drive Nab development will further inform vaccine

design for Brazilian individuals.

EVALUATION OF IMMUNE RESPONSE IN LINES OF CHICKENS DEVELOPED BY EMBRAPA SWINE AND

POULTRY

MIRIELE CAROLINE DA SILVA1; ANA PAULA CHEIRUBIM

2; MICHELE PAGLIARI OLIVEIRA MONTINI

3; EDUARDO

VIGNOTO FERNANDES4; IVETE CONCHON COSTA

5; WAGNER LOYOLA

6; EMERSON JOSE VENANCIO

7.

1,2,3,4,5,7.UNIVERSIDADE ESTADUAL DE LONDRINA, LONDRINA - PR - BRASIL; 6.EMBRAPA AVES E SUÍNOS,

CONCÓRDIA - SC - BRASIL.

Introduction: Poultry industry is in constant evolution and depends much on the development of new lines of

chickens. Many studies have been made toward the development of chicken lines with higher disease resistance.

Within this context, the purpose of this study was to evaluate some aspects of immunity from chicken lines developed

by Embrapa Swine and Poultry.

Methods and Results: To evaluate of the humoral immune response, we analyzed three lines of laying hens (MM,

CC and CCc), and two lines of broiler (TT and LLc). Each line was represented by 9 males and 9 females. The

animals were inoculated with sheep red blood cells (SRBC) 5% on 0 and 30 days of the experiment and blood

samples were obtained at day 0 and 7 days after each inoculation. Specific antibodies to SRBC and natural antibodies

against rabbit red blood cells (RRBC) were determined by micro-hemagglutination. To evaluate phagocytosis and

nitric oxide (NO) production the animals were inoculated with 3% Sephadex G-50 by intra-abdominal route (5 males

and 5 females of each line). After two days, abdominal macrophages were collected and analyzed. The data were

submitted to the Kruskal-Wallis test followed by Dunn's test and significance level was set at p<0.05. After the first

immunization, CC line produced higher anti-SRBC antibodies titers than the MM and LLc lines, while the CCc line

exhibited higher production of anti-SRBC antibodies than MM line. After the second immunization, we observed a

significant increase in the level of anti-SRBC antibodies in CC and CCc lines than TT and LLc lines, moreover, the

CCc lines showed higher antibody production in relation to MM line. Furthermore, CC line had higher anti-RRBC

antibodies titers than LLc line. No significant differences on phagocytic activity and NO production were observed.

Conclusion: There are significant difference in humoral immune response of EMBRAPA chicken lines. CCc and CC

lines had the highest specific antibody production after antigen stimulation, while CC line had higher natural antibodies

levels than LLc line. There was no difference in the macrophages activity among lines. Further studies are needed to

identify the molecular mechanisms related to the observed differences in antibody production.

Financial support: CAPES and Fundação Araucaria.

EXPRESSION AND CHARACTERIZATION OF A RECOMBINANT MONOCLONAL ANTIBODY, SCFV, AGAINST

BAP1, A P-I METALLOPROTEINASE FROM BOTHROPS ASPER SNAKE VENOM

JUCIANE MARIA DE ANDRADE CASTRO1; CAIO RAONY FARINA SILVEIRA

2; MARIA CRISTINA CAPORRINO

3;

TAMIRES S. OLIVEIRA4; JOSÉ M. GUTIÉRREZ

5; ALEXANDRA RUCAVADO

6; GERALDO S. MAGALHÃES

7;

ELIANA L. FAQUIM-MAURO8; IRENE FERNANDES

9.

1,3.INSTITUTO BUTANTAN, SAO PAULO - SP - BRASIL; 2.INSITUTO BUTANTAN, SAO PAULO - SP - BRASIL;

4,7,8,9.INSTITUTO BUTATAN, SAO PAULO - SP - BRASIL; 5,6.INSTITUTO CLODOMIRO PICADO, SAN JOSÉ -

COSTA RICA.

Introduction: BaP1 is a P-I class snake venom metalloproteinase relevant in the local tissue damage associated with

envenomations by Bothrops asper, a medically important species in Central America and parts of South and North

America. We constructed a recombinant single chain fragment variable (scFv) monoclonal antibody against BaP1

(scFvBaP1). It contains VH and VL domains linked by a flexible (G4S)3 polypeptide. The aim was to express the

scFvBaP1 and evaluate its capacity to neutralize important actions of BaP1. Methods: ScFvBaP1 was cloned into

pMST3 vector in fusion with SUMO protein. Cytoplasmic expression of this construction was sucessfully achieved in

C43 (DE3) bacteria. The ability of monoclonal antibody (MaBaP1) and the scFv to recognize total venom from

Bothrops asper and BaP1 was assessed by ELISA. The capacity of scFvBaP1 to neutralize fibrin degradation induced

by BaP1 was evaluated using agarose gel substrate containing fibrin. The ability of scFvBaP1 to neutralize BaP1-

induced hemorrhage in skin mice and BaP1-induced gastrocnemius muscle necrosis was estimated by incubating 35

mg or 20mg of BaP1, respectively, with scFvBaP1 (10:1 molar ratio). Results: Samples of scFv and SUMO presented

bands of 38.9 and 13.6 kDa, respectively. ELISA showed that scFv was able to recognize BaP1 as well as whole

venom, while SUMO did not. BaP1-induced fibrinolysis was significantly neutralized by scFvBaP1, but not by SUMO,

in a concentration-dependent manner (ratio 20:1 and 10:1 resulted in 73.8% and 46.7% of inhibition of the fibrin

degradation, respectively). ScFvBaP1, as well as MaBaP1, completely neutralized hemorrhage and muscle necrosis

induced by BaP1. Conclusion: Our data showed that scFv specifically recognized and neutralized biological effects

of BaP1 and the whole venom of B. asper, indicating the potential use of this recombinant antibody for therapy of

poisoning caused by this snake.

Financial support: supported by FAPESP, CAPES, CNPq, INCT-TOX program of CNPq and Vicerrectoría de

Investigación (Universidad de Costa Rica).

GXM ANTIBODIES IGG RESPONSE AND COMPLEMENT SYSTEM ACTIVATION IN INDIVIDUALS NATURALLY

EXPOSED TO CRYPTOCOCCUS SP AND PATIENTS WITH NEUROCRYPTOCOCCOSIS FROM SÃO PAULO,

BRAZIL.

VIVIANA GALIMBERTI ARRUK1; KATYA CRISTINA ROCHA

2; ANETE SEVCIOVIC GRUMACH

3.

1.UNIVERSIDADE METODISTA DE SÃO PAULO, FACULDADE DE MEDICINA DO ABC, UNIVERSIDADE DE SÃO

PAULO, SÃO PAULO - SP - BRASIL; 2.FACULDADE DE MEDICINA DO ABC, UNIVERSIDADE DE SÃO PAULO,

SÃO PAULO - SP - BRASIL; 3.UNIVERSIDADE DE SÃO PAULO, SÃO PAULO - SP - BRASIL.

Cryptococcus sp is a fungal pathogen with a worldwide distribution and cryptococcal disease occurs predominantly in

immunocompromised hosts but can also occur in apparently immunocompetent individuals. The host immune

response is a major determinant of the outcome of cryptococcal infection; however, the antibodies response is poorly

understood. Complement system has soluble factors involved in defense mechanism. GXM antibodies IgG have

numerous biological activities: opsonization for phagocytosis, activation of the classical complement pathway,

suppression overall accumulation of C3; clearance facilitation of GXM from serum in vivo; protection in murine models

of cryptococcosis and facilitation of various aspects of cellular immunity to the yeast. The aim of our research was to

evaluate classical and alternative complement system pathway and to quantify MBL as well GXM antibodies IgG

response in sera of healthy individuals with high or presumed low exposure to Cryptococcus sp and patients HIV

negatives with presumed low exposure and neurocriptococcosis from São Paulo, Brazil. One hundred and four

samples were collected and classified in 3 groups: g1- 21 patients HIV negatives with neurocryptococcosis and low

exposure to the yeast; g2- 23 healthy individuals with high exposure to Cryptoccocus sp; g3- 60 healthy individuals

with presumed low exposure to the yeast. The complement system was evaluated by hemolytic assay and ELISA to

MBL and GXM antibodies IgG. Median CH 50 values were significantly different among the three groups (P<0,0001).

There was difference in the AP 50 values (P=0,0005) and one no activation of this pathway in group 1. There was

significant difference in MBL among the groups (P=0,0277). GXM antibodies IgG was detected in all the groups with

significant difference among them (P=0,0127). Two of the group 2 individuals had low GXM titers (1/256 and 1/32)

and no symptoms. Four patients with neurocryptococcosis died and the results showed: normal classical pathway

activation, 2/4 had low or undetectable alternative pathway values ; 3/4 had high MBL concentrations and only one

had low GXM-IgG. In conclusion, our results suggest that constant and high exposure to Cryptococcus sp can prevent

the development of cryptococcosis but not the infection. GXM antibodies IgG contribute to GXM clearence. In the

order side, genetic factors which determine MBL concentrations could influence the susceptibility to disease.

HUMORAL IMMUNE DEFICIENCY IN PATIENT WITH MUCOPOLYSACCHARIDOSES TYPE IV

MARINA C DA MATTA1; DIOGO CQ SOARES

2; MARCELO S KERSTENETZKY

3; CLAUDEIR D DA SILVA JUNIOR

4;

THOMÁS V. GOMES5; MARIA DO CARMO MB DUARTE

6; CHONG AE KIM

7; LEURIDAN C TORRES

8.

1,3,4,5,6,8.TRANSLATIONAL RESEARCH LABORATORY, INSTITUTO DE MEDICINA INTEGRAL PROF.

FERNANDO FIGUEIRA (IMIP), RECIFE - PE - BRASIL; 2,7.MEDICAL GENETICS UNIT, HOSPITAL DAS CLÍNICAS,

FACULDADE DE MEDICINA, UNIVERSIDADE DE SÃO PAULO, SÃO PAULO - SP - BRASIL.

Introduction: The mucopolysaccharidoses (MPSs) are a group of rare diseases characterized by deficiencies in

different enzymes required for degradation of complex carbohydrates. The enzymatic deficiencies lead to abnormal

accumulation of deposits of glycosaminoglycans. Once the lysosome is important for normal functioning of the

immune system, playing a key role in the expression of cellular membrane receptors, the presentation of antigens, the

secretion of cytokines and phagocytosis, we presume that these processes may be impaired in patients with MPSs

type IV. The presence of recurrent respiratory infections in these individuals may be a clinical clue of the immune

dysregulation in MPSs. Humoral immunity refers to antibody production by B cells and the accessory processes that

accompany it. Thus, the aim of this study was to evaluate the humoral immune response of a patient diagnosed with

MPSs type IV. Methods and Results: We evaluated the humoral and cellular immunity of a MPSs type IV patient with

20 years old. We performed the measurement of total serum antibodies (IgG, IgM, IgA and IgE) and

immunophenotyping of B and T cells. The patient with MPSs type IV present low levels of total IgM and IgA antibodies

with normal levels of total IgG and IgE antibodies. B cell and B- memory deficiency was observed. Relative values of

TCD4+ and TCD8+ cells were normal in this patient. Conclusion: We report here that this patient have a defect of

humoral immunity with B cells deficiency and low total IgM and IgA serum antibodies. This is the first report at

literature of a MPSs type IV patient with humoral immunodeficiency. Financial support: São Paulo Research

Foundation (FAPESP) – Grant Nº 2010/52694-8.

IGG ANTIBODIES AGAINST 10-VALENT PNEUMOCOCCAL CONJUGATE VACCINE (PCV10) IN BRAZILIAN

CHILDREN AFTER IMMUNIZATION

CAMILA SOUZA COSTA1; MILENA SOARES DOS SANTOS

2; JOICE NEVES REIS PEDREIRA

3; JACY AMARAL

FREIRE DE ANDRADE4; AJAX MARCÊS ATTA

5; MARIA LUIZA BRITO DE SOUSA ATTA

6.

1.PROGRAMA DE PÓS-GRADUAÇÃO EM IMUNOLOGIA,INSTITUTO DE CIÊNCIAS DA SAÚDE,UNIVERSIDADE

FEDERAL DA BAHIA, SALVADOR - BA - BRASIL; 2.LABORATÓRIO DE PATOLOGIA E BIOLOGIA

MOLECULAR,CENTRO DE PESQUISA GONÇALO MONIZ,FUNDAÇÃO OSWALDO CRUZ, SALVADOR - BA -

BRASIL; 3,5,6.DEPARTAMENTO DE ANÁLISES CLÍNICAS E TOXICOLÓGICAS, FACULDADE DE FARMÁCIA,

UFBA, SALVADOR - BA - BRASIL; 4.CENTRO DE REFERÊNCIA DE IMUNOBIOLÓGICOS

ESPECIAIS,UNIVERSIDADE FEDERAL DA BAHIA, SALVADOR - BA - BRASIL.

Introduction: Streptococcus pneumoniae causes different diseases in children following infection. Immunization with

10-valent pneumococcal conjugate vaccine (PCV10) is an efficient medical procedure to confer resistance to S.

pneumoniae infection, avoiding invasive and non-invasive diseases associated with this pathogenic microorganism. In

Brazil, the immunization with the PCV10 is recent, but its use has been considered safe and effective in different

countries. Nonetheless, there is no study evaluating the antibody production induced by PCV10 immunization in

brazilian children. Thus, the aim of this study was to investigate this antibody immune response against PCV10

antigens in children immunized with two consecutive doses of this vaccine in two health center in Salvador-Bahia.

Methods and Results: Were included in this study 22 children (11 boys and 11 girls, age = 2.4 ± 0.9 months) whom

were immunized twice with PCV10 during four months at the Centro de Referência de Imunobiológicos Especiais

(CRIE) and at 16th Health Center. Blood samples was collected from these children before the first and third

immunization to measure anti-pneumococcal serotype-specific IgG concentrations. To investigate anti-PCV10

antibody production, it was performed an indirect ELISA using polystyrene micro wells (Maxisorp, Nunc, Denmark)

and PCV10 as antigen. Child serum samples, positive serum (WHO 007sp) and negative reference sera were mixed

before analysis with an absorbent containing C-polysaccharide and 22F capsular polysaccharide to remove non-

specific antibodies against bacterial common antigens. The reaction was revealed using anti-human IgG antibody

horseradish peroxidase conjugate and substrate. The median levels of IgG antibodies were analyzed by the Wilcoxon

test. There was a significant increase in the concentration of anti-PVC10 IgG antibodies after the second immunization

(T0 Ab median title = 0.12 ng/mL, IQR= 0.01 - 0.34 ng/mL, range= 0.0 - 11.1 ng/mL; T2 Ab median title = 9.54 ng/mL,

IQR= 3.1 - 18.2 ng/mL, range= 0.7 - 31.5 ng/mL; P < 0.0001). Conclusion: 10-Valent pneumococcal conjugate

vaccine (PCV10) induces a vigorous antibody immune response in children following a regular schedule of two

consecutive immunization.

Financial support: CNPq and CAPES

IMMUNOPROTEOMIC APPROACH USED IN THE IDENTIFICATION OF ANTIGENIC PROTEINS FROM

PROMASTIGOTE AND AMASTIGOTE-LIKE OF LEISHMANIA INFANTUM

MARIANA COSTA DUARTE1; PAULA SOUZA LAGE

2; VINICIO SILVA COELHO

3; MIGUEL CHAVEZ FUMAGALLI

4;

DIOGO GARCIA VALADARES5; MANUEL SOTO

6; CARLOS ALBERTO PEREIRA TAVARES

7; ANA PAULA

FERNANDES8; EDUARDO ANTONIO FERRAZ COELHO

9.

1,2,4,9.FACULDADE DE MEDICINA UFMG, BELO HORIZONTE - MG - BRASIL; 3,5,7.DEPARTAMENTO DE

BIOQUÍMICA E IMUNOLOGIA, ICB UFMG, BELO HORIZONTE - MG - BRASIL; 6.UNIVERSIDAD AUTÓNOMA DE

MADRID, MADRID - ESPANHA; 8.FACULDADE DE FARMÁCIA UFMG, BELO HORIZONTE - MG - BRASIL.

Introduction: The present study aims to identify antigens in protein extracts of promastigote and amastigote-like

Leishmania (Leishmania) chagasi syn. L. (L.) infantum recognized by antibodies present in the sera of dogs with

asymptomatic and symptomatic visceral leishmaniasis (VL).Methods and results: Proteins recognized by sera

samples were separated by two-dimensional electrophoresis (2DE), and identified by mass spectrometry. In the

results, a total of five hundred and fifty spots were observed in the 2DE gels, and approximately one hundred and four

proteins were identified. Several stage-specific proteins could be identified by either or both classes of sera, including,

as expected, previously known proteins identified as diagnosis, virulence factors, drug targets, or vaccine candidates.

Three, seven, and five hypothetical proteins could be identified in promastigote antigenic extracts; while two, eleven,

and three hypothetical proteins could be identified in amastigote-like antigenic extracts by asymptomatic and

symptomatic sera, as well as a combination of both, respectively. Conclusions: Therefore, the present study

represents a significant contribution not only in identifying stage-specific L. infantum molecules, but also in revealing

the expression of a large number of hypothetical proteins. Moreover, when combined, the identified proteins constitute

a significant source of information for the improvement of diagnostic tools and/or vaccine development to VL.

Financial support: INCT NanoBiofar, FAPEMIG, CNPq and PRPq/UFMG.

LATE DIAGNOSIS COMPLICATIONS OF A PATIENT WITH COMMON VARIABLE IMMUNE DEFICIENCY

MARIA DO SOCORRO VIANA DE SÁ1; LARISSA CARRERA OLIVEIRA

2; JAQUELYNE CRUZ IBIAPINA

3; BÁRBARA

ALVES DE SOUSA4; ISABEL AMORIM DE MESQUITA LIMA

5; DÉBORA BESERRA VILAR

6; HIALLY RIBEIRO

CABRAL7; HENRIQUE ALEXANDRE MIRANDA SANTOS

8; LUIZ EDUARDO GOMES NETTO

9; MARIA GABRIELA

VIANA DE SÁ10

; DIEGO JÔHN BEZERRA DO NASCIMENTO11

; FERNANDA MARIA AFONSO FERREIRA

MADEIRA12

; RAÍZA ALAPENHA BRITO13

.

1.UNIVERSIDADE FEDERAL DE CAMPINA GRANDE(UFCG)/FACULDADE DE CIÊNCIAS MEDICAS (FCM-CG),

CAMPINA GRANDE - PB - BRASIL; 2,3,4,5,6,7,8,9,10,11,12,13.FACULDADE DE CIÊNCIAS MEDICAS (FCM-CG),

CAMPINA GRANDE - PB - BRASIL.

INTRODUCTION: Common variable immunodeficiency (CVID) is an unknown etiology immunological disorder,

characterized by defective of the antibody synthesis and consequent increased susceptibility to infections, most

notably in the respiratory and gastrointestinal tracts. Its has a relatively low incidence, however, between symptomatic

primary immunodeficiencies, is the most common. It can affect any age group, diagnosed most commonly in young

adults. There is often a delay in diagnosis after onset of clinical manifestations, on average 2.5 years 5.5 years in

children and adults. Individuals with this disorder have hypogammaglobulinemia, characterized in serum

immunoglobulin levels below 300 mg / dL as well as inefficient response protocols immunization. Demonstration of the

late diagnosis complications and consequent delay in the institution of appropriate treatment for this pathology.CASE

MATERIALS AND METHODS: The methodology used was Case Report through medical and laboratory reports.

Male patient, 57 years, autonomous. At 45 began to experience recurrent and of difficult control lung infection,

progressing to bronchiectasis. After 5 years, was subjected to more detailed examinations, that showed normal B

lymphocytes, however 33 mg / dl IgA 34 mg / dl IgG, 41 mg / dL IgM. We excluded other Primary Immunodeficiency

causes with predominantly antibody defects, diagnosing by this way CVID. Thus, treatment with Human

Immunoglobulin Intravenous (HIG-IV) was instituted, however, the patient began to have complications as tremors,

limb edema, tachycardia and fever while using the HIG, precluding its use. The patient developed chronic diarrhea

and malabsorption syndrome, which caused to him extreme debilitation. CONCLUSION: The early recognition and

treatment with HIG-IV are fundamental for a better disease outcome, reducing the number of pneumonia and

progression of chronic lung disease. Adverse effects appear to use a frequency of 0.6% to 30% and may be even

larger in situations such as: infections presence, first infusion, patients with CVID and use of some specifics products.

However, in our case, the diagnosis was made only when the patient had bronchiectasis, which by itself is a poor

prognostic factor. Further treatment was not possible due to adverse events, which culminated in the worsening of his

general condition, with consequent status of extreme debilitation. That reaffirms the necessity of early diagnosis,

before the irreversible complications onset.

LGREC1, A RECOMBINANT PHOSPHOLIPASE D FROM LOXOSCELES GAUCHO SPIDER VENOM:

CHARACTERIZATION OF BIOLOGICAL PROPERTIES AND ITS IMMUNOGENICITY

KATIA CRISTINA BARBARO NOGUEIRA1; MARIA CRISTINA CAPORRINO

2; MAISA SPLENDORE DELLA-CASA

3;

LOUISE FAGGIONATO KIMURA4; JOSÉ PEDRO PREZOTTO-NETO

5; MARCELO LARAMI SANTORO

6; GERALDO

SANTANA SANTORO7.

1,2,3,4,5,7.LABORATORY OF IMMUNOPATHOLOGY- BUTANTAN INSTITUTE, SÃO PAULO - SP - BRASIL;

6.LABORATORY OF PATHOPHYSIOLOGY- BUTANTAN INSTITUTE, SÃO PAULO - SP - BRASIL.

Introduction: Loxosceles gaucho spider venom (LgV) comprises a mixture of diverse toxins that induce an intense

local inflammatory reaction, dermonecrotic injury, platelet aggregation, hemolytic anemia and acute renal failure in

patients. Among the several toxins, phospholipases D (PLD), also called dermonecrotic toxins, are responsible for the

main effects observed in loxoscelism. In this work we characterized the toxic activities of a recombinant PLD from LgV

gland (named LgRec1) and the ability of antibodies against LgRec1 to neutralize toxic activities of crude venom.

Methods and Results: LgRec1 was cloned and successfully expressed in a bacterial system. LgRec1 (3 μg) evoked

an intense inflammatory reaction and dermonecrosis in rabbits, similar to those induced by LgV (3 μg), which was

used as a positive control. LgRec1 (20 μg/mL and 5 μg/mL) induced platelet aggregation in PRP, but not in washed

platelet suspensions. In addition, LgRec1 also hydrolyzed sphingomyelin and promoted mild hemolysis (~20%) and

these activities were dependent upon concentration and were slightly higher when compared with LgV. LgRec1

demonstrated to be highly immunogenic and antibodies anti-LgRec1 recognized components of LgV (titer 8,000)

evaluated by ELISA. By Western blotting, anti-LgRec1 recognized a single band in LgV (around 32-35 kDa). Anti-

LgRec1 serum partially neutralized the inflammatory reaction (~65%), and neutralized dermonecrotic activity (~100%)

induced by LgRec1 or LgV in rabbits dorsum, even when higher doses of venom were used.

Conclusion: This study shows biologic activities of a recombinant PLD that confirms the importance of PLDs to

induce toxic activities observed on LgV. Besides, the use of antibodies against LgRec1 was effective to neutralize the

local reaction and dermonecrosis induced by LgV. Therefore, the results provided here may help to improve

immunotherapy and contribute to understand the role of PLDs in the pathophysiology of Loxosceles envenomation.

Supported by FAPESP (grant #2011/23273-7), CAPES and INCTTox.

OOCYST-SPECIFIC ANTI-TOXOPLASMA GONDII ANTIBODY DETECTION IN NATURALLY INFECTED

CHICKENS IN COMPARISON WITH EXPERIMENTALLY INFECTED ANIMALS.

SILAS SILVA SANTANA1; LUIZ CARLOS GEBRIM

2; FERNANDO REIS CARVALHO

3; PATRÍCIO DA SILVA

CARDOSO BARROS4; LOURENÇO FARIA COSTA

5; FURIO SPANNO

6; TIAGO PATRIARCA MINEO

7; JOSÉ

ROBERTO MINEO8.

1,2,3,4,5,7,8.LABORATÓRIO DE IMUNOPARASITOLOGIA, UBERLÂNDIA - MG - BRASIL; 6.ISTITUTO SUPERIORI

DI SANITÀ, ROME - ITÁLIA.

Introduction: The current immunoassays applied for diagnosis or epidemiological surveys of toxoplasmosis in

humans and animals utilize whole parasite antigens. These immunoassays can determine previous exposure to the

parasite only, and the route of infection remains undertermined. Thus, it is difficult to assess which is the route of

infection, by tissue cyst or oocyst and implement measures to reduce transmission. In the present study, we used a

panel of 11 specific peptides designed from T. gondii oocyst (named as p1 to p11) and tested with serum samples

from chickens naturally infected with toxoplasma compared with chickens infected experimentally with soluble

taquizoite antigen. Methods: It was carried out indirect ELISA by using peptides and soluble Toxoplasma antigen

(STAg) against serum samples from naturally infected chickens and against samples from chickens infected with

STAg. Furthermore, Western blotting was performed to confirm the results. Results: In serum samples from naturally

infected chickens, it was observed reactivity for peptides and STAg. In contrast, in sera from experimentally infected

chickens, it was observed reactivity with STAg and peptide 3 (P3), only. These results were confirmed by Western

blotting. Conclusions: Considering that chickens are infected by oocysts ingestion only, these data suggest that

these peptides may be useful to detect exposure to sporozoites in T. gondii infection and implicates oocysts as the

agent of infection.

PEPTIDES SELECTED FROM HYPOTHETICAL PROTEINS IDENTIFIED BY AN IMMUNOPROTEOMIC

APPROACH USED IN THE SERODIAGNOSIS OF CANINE VISCERAL LEISHMANIASIS

PAULA SOUZA LAGE1; MIGUEL ANGEL CHÁVEZ-FUMAGALLI

2; VIVIAN TAMIETTI MARTINS

3; DANIELA

PAGLIARA LAGE4; LOURENA EMANUELE COSTA

5; MARIANA COSTA DUARTE

6; HENRIQUE GAMA KER

7;

ALEXANDRE BARBOSA REIS8; MANUEL SOTO

9; CARLOS ALBERTO PEREIRA TAVARES

10; ANA PAULA

FERNANDES11

; EDUARDO ANTONIO FERRAZ COELHO12

.

1,2,5,6,12.PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE: INFECTOLOGIA E MEDICINA

TROPICAL, UFMG, BELO HORIZONTE - MG - BRASIL; 3.DEPARTAMENTO DE BIOQUIMICA E IMUNOLOGIA

ICB, UFMG, BELO HORIZONTE - MG - BRASIL; 4.DEPARTAMENTO DE PATOLOGIA CLÍNICA, COLTEC, UFMG,

BELO HORIZONTE - MG - BRASIL; 7.DEPARTAMENTO DE ANÁLISES CLÍNICAS, ESCOLA DE FARMÁCIA,

UFOP, OURO PRETO - MG - BRASIL; 8.5DEPARTAMENTO DE ANÁLISES CLÍNICAS, ESCOLA DE FARMÁCIA,

UFOP, OURO PRETO - MG - BRASIL; 9.CENTRO DE BIOLOGÍA MOLECULAR SEVERO OCHOA, UNIVERSIDAD

AUTÓNOMA DE MADRID, MADRID - ESPANHA; 10.DEPARTAMENTO DE BIOQUÍMICA E IMUNOLOGIA, ICB,

UFMG, BELO HORIZONTE - MG - BRASIL; 11.DEPARTAMENTO DE ANÁLISES CLÍNICAS E TOXICOLÓGICAS,

FACULDADE DE FARMÁCIA, UFMG, BELO HORIZONTE - MG - BRASIL.

Introduction: The present work analyzed the potential of synthetic peptides mapped from hypothetical proteins

identified by an immunoproteomic approach for the serodiagnosis of canine visceral leishmaniasis (CVL). Methods

and results: From 26 identified leishmanial hypothetical proteins, eight were selected, considering that no homologies

could be identified between them and others from Trypanosoma cruzi and T. brucei species. Their sequences were

mapped to identify linear B-cell epitopes, and the peptides were synthesized. In this context, seventeen peptides were

tested in ELISA for CVL serodiagnosis. In the results, three peptides (PepLi1, PepLi2 and PepLi7) presented the

sensitivity and specificity values higher than 75% and 90%, in order to differentiate PCR(+)/sero(-) and symptomatic

animals from T. cruzi-infected and healthy animals, respectively. When peptides were combined in different mixed

formats, high sensitivity values could be observed in the diagnosis of CVL. In addition, high specificity was reached,

even when the sera from T. cruzi-infected dogs were included in the analysis. Conclusion: The study’s findings

suggest that these three top peptides, when used in isolation or in combination, can constitute a potential tool for a

more sensible and specific CVL serodiagnosis.

Financial support: INCT NanoBiofar, FAPEMIG, CNPq and INCT-NanoBiofar

PHAGE DISPLAY-BASED SUBTRACTIVE SCREENING TO IDENTIFY ANTIGENS CANDIDATES FOR THE

SERODIAGNOSIS OF CANINE VISCERAL LEISHMANIASIS

EDUARDO FERRAZ COELHO1; LOURENA EMANUELE COSTA

2; MAYARA INGRID SOUSA LIMA

3; MIGUEL

ANGEL CHÁVEZ-FUMAGALLI4; DANIELLA VIEIRA NASCIMENTO

5; MARIANA COSTA DUARTE

6; PAULA SOUZA

LAGE7; VÍVIAN TAMIETTI MARTINS

8; CARLOS ALBERTO PEREIRA TAVARES

9; LUIZ RICARDO GOULART

FILHO10

.

1,2,4,5,6,7,8,9.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL;

3,10.UNIVERSIDADE FEDERAL DE UBERLÂNDIA, UBERLÂNDIA - MG - BRASIL.

Introduction: Leishmaniasis are highly endemic diseases in Brazil, and it accounts for almost 95% of reported cases

of VL in the Americas. The dog is the main reservoir in the domestic cycle of VL, and euthanasia of seropositive

animals in serologic tests has been adopted as one of the preventive measures ruled by the Ministry of Health to

control the disease. However, due to limitations of sensitivity and/or specificity of current assays, the elimination of

animals becomes difficult and sometimes questionable. Thus, the use of new techniques to identify and employ

antigens that are more refined and specific, which can be used effectively in the CVL diagnosis, is highly desirable.

Methods and Results: In this work, phage clones expressing peptides of interest were selected against IgG

antibodies purified from sera of symptomatic and asymptomatic CVL. Sera from animals without leishmaniasis

(negative) or infected with Trypanosoma cruzi were also used in the subtractive selection of clones of interest. We

have obtained six bacteriophage clones that revealed great potential in the CVL diagnosis, with all diagnostic

parameters estimated to be 100% (sensitivity, specificity, positive predictive value, negative predictive value, and

accuracy). Conclusions: In conclusion, our six novel phage clones selected by Phage Display, which mimic antigenic

epitopes of Leishmania spp., were able to detect circulating IgG of infected animals, and may be promptly used as

highly effective antigens in serological diagnosis of CVL.

Financial support: FAPEMIG, INCT Nano-Biofar, CNPq and PRPq/UFMG.

POLYCLONAL ANTIBODY ANTI-BNSP7: A PHOSPHOLIPASE A2 FROM THE SNAKE VENOM OF BOTHROPS

PAULOENSIS

LAMARTINE LEMOS DE MELO1; MIRIAN MACHADO MENDES

2; THAIS FERREIRA ISABEL

3; VERIDIANA DE

MELO RODRIGUES4; LUIZ FERNANDO MOREIRA IZIDORO

5.

1,3,4,5.UNIVERSIDADE FEDERAL DE UBERLÂNDIA, CAMPUS UBERLÂNDIA - MG - BRASIL; 2.UNIVERSIDADE

FEDERAL DE GOIAS, CAMPUS JATAÍ - GO - BRASIL.

Introduction The phospholipase A2 (PLA2) are enzymes belonging to the class of hydrolases, whose molecular

weights ranging from 13 to18 kDa and are widely distributed in the venoms of genera Bothrops (Toxicon 42:827-

840,2003). Objective This work aimed at the production of polyclonal anti-Bp (crude venom of Bothrops pauloensis)

and anti-BnSP7 (PLA2 isolated from the venom of Bp), identification of cross-reactivity between anti-BnSP7 and other

PLA2s present in crude snake venoms, and assess the potential inhibitory against activity phospholipasic and

myotoxic. Methods and Results The BnSP7-PLA2 was purified by two chromatographic steps: CM-Sepharose and

Reverse-Phase on C2C18 HPLC columns. For immunization, Bp or BnSP7 were mixed with Freund’s complete

adjuvant or Freund’s incomplete adjuvant and inoculated by i.p. route in Balb/c mice. After challenge period, the blood

obtained by cardiac puncture was centrifuged and the supernatant applied to an affinity column Protein G Sepharose

for antibodies purification. The evaluation of cross-reactivity was determined by ELISA assay, demonstrating the

ability of anti-BnSP7 to recognize the venom PLA2s present in crude Bp and Bothrops moojeni (Bm), the ratios to

1:6400 and 1:3200 (m/v) respectively. This recognition of both the venoms can be confirmed by Western blot. In

phospholipasic and myotoxic assays, anti-BnSP7 or anti-Bp were incubated for 30 min at 37°C with venoms Bp, Bm

or BpPLA2-TXI toxin, in proportions of 1:1, 1:10 and 1:20 (w/w). Compared to positive control (venom or toxin), the

results show that the catalytic activity of the phospholipases of crude venoms Bm, Bp and BpPLA2-TXI toxin was

inhibited by 41.2%, 70.0% and 72.4%, respectively, by anti-BnSP7 in the ratio 1:20 (w/w). Furthermore, it was

observed that the effect myotoxic induced by the same crude venoms (Bm and Bp) was also reduced respectively to

62.0% and 74.4% for anti-BnSP7 the same reason. Similarly, the polyclonal anti-Bp also showed high inhibitory

potential on the enzyme catalysis and the effect induced by myotoxic venom Bp with the respective percentage of

71.4% and 89.75% in the ratio 1:20 (w/w). Conclusion Our results showed the high potential of neutralizing

antibodies on activities in vitro and in vivo, highlighting the ability of recognition and inhibition that the specific antibody

anti-PLA2-BnSP7 shown to have front isoforms of phospholipases present or isolated from the venom of snakes.

Financial supported: FAPEMIG, UFU.

POLYSACCHARIDE FROM ENVIRONMENTAL FUNGUS IS ABLE TO IMMUNIZE MOUSE AND NOT CROSS-

REACT WITH OTHERS POLYSACCHARIDES

RAFAEL ANDRADE MENOLLI1; PABLO RODRIGO DA ROSA

2; CLAUDIA REJANE LIMA DE MACEDO COSTA

3;

ERICA FERNANDA OSAKU4; CLARICE AOKI OSAKU

5; PERICLES ALMEIDA DELFINO DUARTE

6; ROSIANE

GUETTER MELLO ZIBETTI7.

1,2,3,4,5,6.UNIOESTE, CASCAVEL - PR - BRASIL; 7.FACULDADES PEQUENO PRÍNCIPE, CURITIBA - PR -

BRASIL.

Introduction: Diagnostic of respiratory fungal infections is difficult and requires many approaches like: X-ray, culture

and serologic tests. Fungal polysaccharides has been used like antigens in diagnostics tests to confirm pulmonary or

systemic infections caused by environmental fungus. Characterize the immunological properties of this

exopolysaccharides (EPS) from opportunistic fungus is important to development of new tools of fungal infections

diagnostic. This work shows the ability of a polysaccharide to immunize an animal model and its relationship with

other fungal polysaccharides. Material and Methods: EPS obtained from three different environmental fungus

(Curvularia brachyspora, Aspergillus terreus and Paecilomyces variotti) were used as immunogen in this experiment.

Twenty C57BL/6 mice were separated in four groups for each immunization schedule, which group 1 and 3 received

the EPS (1 mg/ml), with in group 1 EPS plus Freund’s adjuvant, and groups 2 and 4 were controls (group 2 received

only adjuvant and group 4 only saline). Immunization was scheduled with a prime on day 0, and two boosters with 20

and 30 days. Cardiac blood puncture was performed with 40 days after the prime and with the obtained plasma was

realized an ELISA test. Indirect ELISA was determined using the EPS as antigen in a 96 plate well and revelation was

performed with an anti-mouse IgG conjugate with peroxidase and using OPD and hydrogen perxide as substrate.

Results are showed as media of OD ± SD. The mice plasma was used in pool of each group. Results and

Discussion: The sera from animals in group 1 and 3 immunized with EPS from C. brachyspora didn’t show significant

differences (p>0,05), reaching high OD in both groups in ELISA test, demonstrating a great immunogenic potential

from this polysaccharide. The groups 2 and 4 didn’t reach high OD, which was 41 times less than OD obtained with

groups 1 and 3. Sera from animals immunized with EPS from others fungi were tested against antigen from C.

brachyspora and showed weak cross reaction, with significant differences between groups 1 and 3.

Financial support: Fundação Araucária/Ministério da Saúde and Unioeste-Campus Cascavel.

PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST AVIAN INFECTIOUS

BRONCHITIS VIRUS

NEIDA LUCIA CONRAD; MARCELO MENDONÇA; CLAUDIA BALZAN; MARCELLE SILVEIRA; FABRICIO

CONCEIÇÃO; ANGELA MOREIRA.

UNIVERSIDADE FEDERAL DE PELOTAS, PELOTAS - RS - BRASIL.

Introduction: The infectious bronchitis (IB) is an acute and highly contagious disease caused by a coronavirus that

infects manly cells of the respiratory and genitourinary tract of chickens. The economic importance of this disease

results from the decrease in production and egg qualities, feed efficiency and increased mortality and condemnation of

carcasses at slaughter. The diagnosis of IB is based on virus isolation, coupled with serology. The use of monoclonal

antibodies (MAbs) has been collaborated to diagnosis of this type of disease, but their use are still limited because

mostly of MAbs used are imported, increasing the cost of such methodologies. This work reports the production and

initial characterization of MAbs against the infectious bronchitis virus (IBV). Methods and Results: For the production

of MAbs, 6-weeks old female Balb/c mice were immunized intraperitoneally with variant IBV strain mixed with

complete Freund`s adjuvant (FA) and, after two weeks, a mixture of IBV and incomplete FA was administered every

week for 8 weeks. In other to evaluate the ability of MAbs recognize the native protein, ELISA tests were carried out

using 250 ng per well of classic and variant IBV, Avian Pox virus, Newcastle virus and allantoic fluid sample (used as

negative control). Purified MAbs were diluted in PBS-T (1:200) and then goat anti-mouse conjugated with HRP was

added. The reaction was revealed using OPD chromogen solution and read at 450 nm after 15 min in the dark. All

ELISA steps were performed for 1h at 37°C. In this work, we generated two hybridomas secreting MAbs specifically to

IBV named 2G4 and 5A11, both belonging to IgM isotype. In the characterization by ELISA, both MAbs recognize

specifically the variant and classic IBV, obtaining O.D values from 0,3 to 0,9. As expected reactions with variant IBV

showed higher absorbances. Reactions against the allantoic fluid, Avian pox and Newcastle virus were three times

lower than visualized with IBV. Conclusion: Although more tests are needed to determine the specificity of the MAbs,

here we have shown that MAbs 2G4 and 5A11 have potential to use in the diagnostic tests of IBV in clinical samples.

Financial support: CNPq.

THE CONSERVATION OF IMMUNOGLOBULIN GENES: AN EVOLUTIONARY LINK BETWEEN

AUTOREACTIVITY AND INNATE RESPONSE AGAINST PATHOGENS

ANDRE M VALE1; PRATIBHA KAPOOR

2; GREG SKIBINSKI

3; ADA ELGAVISH

4; TAMER I MAHMOUD

5; COSIMA

ZEMLIN6; MICHAEL ZEMLIN

7; PETER D BURROWS

8; ALBERTO NOBREGA

9; JOHN F KEARNEY

10; DAVID E

BRILES11

; HARRY W SCHROEDER JR12

.

1,9.INSTITUTO DE MICROBIOLOGIA PROF. PAULO DE GÓES UNIVERSIDADE FEDERAL DO RIO DE JANEIRO,

RIO DE JANEIRO - RJ - BRASIL; 2,3,4,5,6,7,8,10,11,12.UNIVERSITY OF ALABAMA AT BIRMINGHAM,

BIRMINGHAM - ESTADOS UNIDOS.

Selection and physiologic production of protective natural antibodies (NAbs) have been associated with exposure to

endogenous antigens. The extent to which this association depends on germline NAb sequence is uncertain. To test

the role of natural selection of germline sequence in this cross-protection, in mice with gene-targeted alterations of DH

sequence we evaluated responses against a hazardous self antigen, oxidized low density lipoprotein (OxLDL), and

against phosphorylcholine (PC), a bacterial cell wall component. Here, we show that alterations in germline DH

sequence can sever the association between the production of self-reactive NAbs and NAbs that afford protection

against a pathogen. In unmanipulated hosts, the availability of the evolutionarily conserved DFL16.1 gene segment

sequence profoundly affected the serum levels of NAbs against bacterial phosphorylcholine (PC), but not

OxLDL. Mice with partially altered DFL16.1 sequence could use N nucleotides to recreate the amino acid sequence

associated with the classical protective T15 idiotype positive NAbs, whereas those without DFL16.1 could

not. DFL16.1 gene deficient mice proved more susceptible to challenge with live Streptococcus pneumoniae. We

present data that support the moderating concept that natural selection of conserved DH sequence, and self-antigen

driven somatic selection operate in concert to create a protective, functional NAb repertoire reactive with a bacterial

cell wall component. In remarkable contrast, NAb reactivity and function in the case of an endogenous antigen

representative of molecular debris does not show this dependence on evolutionarily conserved DH sequences. The

potential relevance of these findings for the rational design of vaccines is discussed.

USE OF RECOMBINANT MULTIEPITOPE PROTEINS FOR THE IMMUNODIAGNOSIS OF FOWL ADENOVIRUS

TYPE-I IN CHICKEN

FRANCESCA FALCONI1; DAVID REQUENA

2; LUIS SARAVIA

3; MANUEL RAMIREZ

4; HUGO VALDIVIA

5; MIRKO

ZIMIC6; MANOLO FERNANDEZ

7.

1,3,7.FARMACEUTICOS VETERINARIOS S.A.C., CHINCHA - PERU; 2,4,5,6.UNIVERSIDAD PERUANA

CAYETANO HERADIA, LIMA - PERU.

Introduction: Inclusion body hepatitis disease, caused by fowl adenovirus type I (FAdV), affects mainly broiler

chickens and is associated with a high mortality rate. There is a need for improved test methods for FAdV antibody

detection, the available commercial kits are whole virus-based and normally reported as inaccurate. In this study,

three recombinant multiepitope proteins were evaluated by an ELISA assay to determine their utility in detecting

antibodies to FAdV serotype 4 (FAdV-4). Methods and results: Thus, whole-genomes immunoinformatic analysis of

FAdV-4 was conducted employing NetMHCcons and NetMHCIIpan servers to predict in silico conserved and

immunodominant T epitopes, potentially reactive to chicken MHC-I and MHC-II. Based on these epitopes, 3 synthetic

genes were constructed which encoded multiepitope proteins comprising strong-binding class I epitopes (ISB), strong-

binding class II epitopes (IISB), and weak-binding class I epitopes (IWB). These proteins were expressed in E. coli,

purified using nickel resin and finally quantified before their evaluation as capture antigens in an indirect ELISA. Sera

from Hy-line SPF chickens: negative controls (n=25; non-infected) and positive controls (n=22; experimentally infected

with FAdV-4) were tested; additionally, sera from Charles River Laboratories positive to avian pathogens others than

FAdV were also evaluated (n=10). The results were compared with a commercial ELISA kit for FAdV detection. The

ROC (Receiver Operating Curve) curve was used to estimate the diagnostic value of each protein. The areas under

the ROC curve were 0.8571, 0.9701, 0.9325 and 0.6089, to ISB, IISB, IWB and the commercial kit, respectively. Cut-

off values of 0.58, 0.381, 0.428 y 0.4028; sensitivity and specificity estimates of 86.36% and 85.71%; 90.91% and

94.29%; 90.91% and 82.86%; and 62.50% and 62.86% were calculated respectively to ISB, IISB, IWB and the

commercial kit. Conclusion: Protein IISB showed to be the best antigen for immunodiagnostic of FAdV-4, with more

sensitivity and specificity than the commercial kit. This result is consistent with the fact that IISB is constituted with the

best predicted MHC-II epitopes and is associated to an immune response mediated by antibodies.