Conclusões

A expressão e a purificação das proteínas recombinantes LigANI, LigBRep, LigB7-11 e LigAFull é possível utilizando a metodologia empregada. Os Quatro lotes estão disponíveis com rendimento e pureza de >90%.

A avaliação das formulações vacinais LigANI, LigBRep e LigAFull com Hidróxido de alumínio revelou que individualmente elas protegem os hamsters do desafio letal, porém em graus variáveis de proteção (0 a 100%).

LigAFull não protege os hamsters da leptospirose letal, porém confere sobrevida aos animais imunizados.

A formulação com LigB7-11 não protege e nem confere sobrevida aos animais imunizados;

A associação entre LigANI e LigBRep confere proteção significativa (p>0,05) ao desafio letal. Dessa forma, recomenda-se que essa formulação seja utilizada em ensaios de imunogenicidade e imunoproteção heterólogo.

5 CONCLUSÃO GERAL

Os resultados obtidos em nosso estudo representam uma contribuição importante para o desenvolvimento de uma vacina contra a leptospirose devido ao grande potencial de uso das preparações testadas, as quais motivam a continuidade e futuros ensaios.

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7 ANEXOS

Anexo A – Artigo submetido para Brazilian Journal of Microbiology

First Latin American Leptospira interrogans serovar Djasiman isolate from a

human case

Amilton Clair Pinto Seixas Neto1, Samuel Rodrigues Félix2, Sandra Denize Jouglard1, Karina Colonetti1, Flávia Aleixo Vasconcellos3, Claudiomar Soares Brod2, Odir Antônio Dellagostin1, Éverton Fagonde da Silva2*

1

Centro de Desenvolvimento Tecnológico; Universidade Federal de Pelotas, Brazil. 2

Faculdade de Veterinária; Universidade Federal de Pelotas, Brazil. 3

Centro de Ciências Químicas, Farmacêuticas e de Alimentos; Universidade Federal de Pelotas, Brazil.

*

Corresponding author: Faculdade de Veterinária/UFPel, Campus Universitário Capão do Leão, prédio 1, CEP: 96010-900. Pelotas, RS, Brazil. Phone: +55 53 32757644. Fax: +55 53 32757551. E-mail: efsilva@ufpel.edu.br

Abstract

This brief report describes a human leptospirosis case, followed by the isolation of a

Leptospira interrogans serovar Djasiman previously unknown to cause human

disease in the Americas. Much more than this, the report shows how poor control measures expose the populations of underdeveloped and developing countries to the disease.

Key-words: Leptospirosis; Public Health; Veterinary Medicine; Tropical Neglected Disease

Text

Leptospirosis is a major public health issue in many countries, especially in Latin America and in South-East Asia (Picardeau, 2013). Brazil and most Latin American countries are suffering the consequences of demographic transformations due to uncontrolled growth of urban centers. Urban slums are sites of poor sanitation that favor the transmission of leptospirosis among humans (Riley et al., 2007).

We have recently reported the isolation and virulence characterization of strains obtained from humans and animals, representative of important species and serogroups for public health and veterinary medicine (Silva et al., 2009; Diniz et al., 2011). Herein, we report the isolation of Leptospira interrogans serovar Djasiman from a case of human leptospirosis. The isolate was obtained from a 24-year-old man, admitted to the Hospital Santa Casa de Misericórdia, Pelotas, RS, Brazil, with a history of fever, diarrhea, myalgia, severe headache and nausea. Prior to the onset of symptoms, he had had contact with stray dogs and rodents. A blood sample was

collected in the hospital and sent to the Zoonosis Control Center (CCZ) of the Federal University of Pelotas for the laboratory diagnosis of leptospirosis.

As part of the CCZ routine procedures, blood was inoculated aseptically in semisolid EMJH media for an isolation attempt and sera was used in the microscopic agglutination test (MAT). Clinical suspicion of leptospirosis was confirmed by high MAT titer against Leptospira interrogans Djasiman Sentot (1:1600), followed by L.

borgpetersenii Serjoe M84 (1:200) and L. biflexa Andamana CH11 (1:200). This

result was communicated to the physician, at which point the patient had already received antibiotic treatment with intravenous penicillin. After nine weeks, the presence of leptospires in the culture medium could be observed. On this occasion, the MAT with the patient´s serum was repeated and the titer for the new isolate was 1:800.

For species identification, the 16S rRNA gene was amplified and sequenced (Cerqueira et al., 2010), and in agreement with BLAST results, strain was closely related to L. interrogans strains. Culture samples were submitted to serogrouping (Silva et al., 2008), at which point the new isolate was classified as serogroup Djasiman. To determine the serovar, a previously described VNTR assay was used (Salaün et al., 2006). Results showed the new isolate to be of the Djasiman serovar. Virulence tests were also undertaken (Silva et al., 2008) however, the isolate was not capable of causing death in the hamster model for leptopirosis. The lack of virulence of strain might be explained by the successive in vitro propagation (31 passages), and it has already been reported that high-passage strains can present impaired virulence (Cullen et al., 2003). The strain was deposited in the Fiocruz/BA collection under the responsibility of Albert Ko, and it is available to the scientific community.

To the best of our knowledge, this is the first isolate of serovar Djasiman from humans in Latin America. How these new strains are introduced is important information for control measures, and appropriate studies should be undertaken by molecular epidemiologists. The findings described in this report are worrisome. It is not new that underdeveloped and developing countries are at a greater risk of leptospirosis outbreaks. However, the measures to control the disease in these countries are failing miserably. Furthermore, the diversity of species and serovars in Pelotas seems to be unique. Unlike other well studied settings, such as Salvador city, Northeast of Brazil, where few strains are responsible for most cases (Felzemburgh

et al., 2014), in Pelotas we have a great diversity of strains causing disease of

human and veterinary importance (Silva et al., 2008; Diniz et al., 2011; Forster et al., 2013). This diversity is not only a challenge regarding diagnosis, but also control through epidemiological measures or animal vaccination, since most vaccines are serovar specific.

Acknowledgements

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Foundation (CAPES, Brazilian Government) and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Processes n° 482180/2011-0).

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Picardeau M (2013) Diagnosis and epidemiology of leptospirosis. Med Mal Infect 43:1-9.

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Salaün L, Mérien F, Gurianova S, Baranton G, Picardeau M (2006) Application of multilocus variable-number tandem-repeat analysis for molecular typing of the agent of leptospirosis. J Clin Microbiol 44:3954-3962.

Silva ÉF, Cerqueira GM, Seyffert N, Seixas FK, Hartwig DD, Athanazio DA, Pinto LS, Queiroz A, Ko AI, Brod CS, Dellagostin AO (2009) Leptospira noguchii and human and animal leptospirosis, Southern Brazil. Emerg Infect Dis 15:621-622.

Silva ÉF, Santos CS, Athanazio DA, Seyffert N, Seixas FK, Cerqueira GM, Fagundes MQ, Brod CS, Reis MG, Dellagostin OA, Ko AI (2008) Characterization of virulence of Leptospira isolates in a hamster model. Vaccine 26:3892-3896.

Anexo B – Manuscrito a ser submetido ao periódico PLOS ONE

TESTING GREEN PROPOLIS TO BOOST PROTECTION OF LEPTOSPIROSIS

VACCINES

Michel Quevedo Fagundes 1,2*, Amilton Clair Pinto Seixas Neto 1,2*, Karina Colonetti 1,2, Najara Carneiro Bittencourt 1,2, Leonardo Marins 1, Flávia Aleixo Vasconcellos 3, Samuel Rodrigues Félix 1,2, Geferson Fischer 1, Odir Antônio Dellagostin 2, Marco Alberto Medeiros4, Éverton Fagonde da Silva1**

1. Faculdade de Veterinária, Universidade Federal de Pelotas, RS, Brazil.

2. Centro de desenvolvimento tecnológico, Universidade Federal de Pelotas, RS, Brazil. 3. Programa de Pós-graduação em Bioquímica e Bioprospecção, UFPel

4. Bio-Manguinhos, Oswaldo Cruz Foundation, Av. Brasil, 4.365, CEP 21040-360, Rio de Janeiro, Rio de Janeiro, Brazil; bGonçalo Moniz Research Center, Oswaldo Cruz

*

Autores contribuíram igualmente na execução do artigo

ABSTRACT

Vaccination would be the preferred to leptospirosis control measure. However, current vaccines are heat killed whole-cell bacterins that generate serovar specific protection and several side effects. Modern molecular assays have revealed antigens that may replace traditional bacterins. Foremost among these are LipL32 and LigB, present in all pathogenic and absent in saprophytic species. Some unsuccessful attempts at using these antigens as vaccines have occurred, and we believe immune modulation through alternative adjuvants and co-adjuvants may overcome previous setbacks. In this light, our study assessed the protective immune response in hamsters vaccinated with the aforementioned antigens using oil adjuvant, with or without green propolis (GP) as a co-adjuvant. Doses were prepared with 40 µg of recombinant proteins in oil adjuvant with 5 mg of green propolis. and inoculated with the different preparations on day 0 and 14. All hamsters suffered lethal challenge with 1000 live leptospires on day 28. Groups immunized with LigBNI/oil adjuvant and LigBNI/oil adjuvant/green propolis were capable of generating statistically significant (p<0.05) protection. Our preliminary results show that rLigB may protect hamsters against homologous lethal challenge. The green propolis, though unable to produce statistically different results, seems to have a positive effect on protection. Further assays with a greater number of animals per group and optimal propolis doses may reveal these subtle differences.

INTRODUCTION

In the last decades, the World Health Organization (WHO) has recognized leptospirosis as an emergent global public health issue [1]. The disease is mostly an occupational hazard in developed countries. However, in underdeveloped or developing countries, lack of sanitation, favorable climate conditions, and the presence of reservoir hosts, especially Rattus

norvegicus, makes tropical regions ideal for the development of leptospirosis [2]. An

estimated 873,000 human cases of severe leptospirosis occur annually, with 49,000 deaths [3]. Leptospirosis is caused by a Gram negative like bacteria of the Leptospira genus which comprises 21 species and more than 200 serovars [4]. In humans the initial infection occurs when leptospires in the environment (soil or water) penetrate abraded skin or mucosal membranes. The bacteria then migrates through tissues barriers until it reaches the blood stream, quickly establishing a systematic infection and compromising several organs, especially kidney, liver and lungs . Immune response can also be jeopardous, secreting pro- inflammatory cytokines, leading to inflammation and tissue damage, a reaction known as Jarish-Herxheimer [5].

Vaccines represent a cost-effective approach to prevent neglected tropical diseases, however, leptospirosis vaccines are mostly used in veterinary medicine due to side effects generated by the traditional bacterins (killed, whole-cell suspensions) [6]. Attempts to circumvent these setbacks include vector-delivered vaccines, subunit vaccines, and DNA vaccines, using recombinant outer-membrane proteins (OMPs) as antigens [7, 8, 9]. The subsurface lipoprotein LipL32 is a candidate of interest, and several research groups have assessed it for this end [10,11]. However, none of the approaches using this antigen produced significant protection against lethal challenge so far [12]. Other proteins that have been extensively studied are the leptospiral immunoglobulin-like proteins (Lig), LigA, LigB and LigC. Ligs have biological characteristics that also make them attractive candidates for vaccine

development, especially LigB, with some preparations affording partial, but not full protection [13,14].

Efforts are being made to circumvent this apparent lack of protection. Propolis has been shown as an adjuvant substance in serositis [15], swine herpesvirus type 1 (SuHV-1) [16] and bovine herpesvirus type 5 (BoHV-5) [17] inactivated vaccines. This natural product is a resinous material produced by bees that displays a variety of biological activities against viruses, bacteria, fungi, pathogenic protozoa, and tumor cells [18]. Thus, the aim of this study was to assess the adjuvant potential of a green propolis extract when coadministered with the most promissing leptospirosis vaccinal antigens, through humoral immune response. Furthermore, these preparations were preliminary assessed with a lethal challenge assay in the hamster model.

MATERIAL AND METHODS

Leptospira culture. L. interrogans serovar Copenhageni strain Fiocruz L1- 130 was cultivated in Ellinghausen-McCullough-Johnson-Harris (EMJH) liquid medium (Difco Laboratories) at 30°C. The procedures for the maintenance of the culture and challenge experiments were conducted as previously described [9].

Cloning, expression, and purification of recombinant proteins. The recombinant vectors

used in this work had been previously constructed. The vector pET/LigBNI, containing the non-identical portion of LigB (1873-3773 nt), coding a 633-amino-acid peptide, and