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Paracoccidioidomycosis (PCM) is an infectious disease endemic to South America, caused by the thermally dimorphic fungi Paracoccidioides. Currently, there is no effective human vaccine that can be used in prophylactic or therapeutic regimes. We tested the hypothesis that the immunogenicity of the immunodominant CD4+ T-cell epitope (P10) of Paracoccidioides brasiliensis gp43 antigen might be significantly enhanced by using a hepatitis B virus-derived particle (VLP) as an antigen carrier. This chimera was administered to mice as a (His)6-purified protein (rPbT) or a replication-deficient human type 5 adenoviral vector (rAdPbT) in an immunoprophylaxis assay. The highly virulent Pb18 yeast strain was used to challenge our vaccine candidates. Fungal challenge evoked robust P10-specific memory CD4+ T cells secreting protective Th-1 cytokines in most groups of immunized mice. Furthermore, the highest level of fungal burden control was achieved when rAdPbT was inoculated in a homologous prime-boost regimen, with 10-fold less CFU recovering than in non-vaccinated mice. Systemic Pb18 spreading was only prevented when rAdPbT was previously inoculated. In summary, we present here VLP/P10 formulations as vaccine candidates against PCM, some of which have demonstrated for the first time their ability to prevent progression of this pernicious fungal disease, which represents a significant social burden in developing countries., Author summary Human paracoccidioidomycosis (PCM) represents a serious public health issue due to its disabling sequelae in working-age people and mortality rates (8th among chronic infectious parasitic diseases in endemic countries and first among mycoses in Brazil). Although antifungal drugs have been widely used and provide clinical improvement for patients, the long duration of treatments (commonly from 6 to 12 months) has contributed to non-compliance and worsening of the disease in many cases. Induction of protective immune responses (either prophylactic or immune-therapeutic) remains the most cost-effective approach against most infectious agents. Members of our group have previously reported the protective properties of P10, a peptide contained in Paracoccidioides brasiliensis gp43 antigen, against PCM. However, the magnitude of the CD4+ T-cell responses elicited lacked the capacity of completely protecting experimental animals. We demonstrate here that immunogenic virus-like particles (VLP) carrying multiple copies of P10 peptide substantially improve P10-related cellular immunity in mice. This was particularly true when an adenoviral immunization vector expressed the chimeric VLP. Moreover, VLP/P10 formulations were capable of controlling the host fungal burden and prevented fungal systemic dissemination. The efficacy of diverse VLP/P10 formulations in murine PCM showed at least one promising candidate vaccine against human PCM.

Submitted by FERNANDO CHRISTIANO GABRIEL MORELLI null (fcgmorelli@yahoo.com.br) on 2016-02-12T17:49:35Z No. of bitstreams: 1 Fernando - Tese versão final Repositório UNESP.pdf: 1900802 bytes, checksum: a8c9044a1f5a46e203d3432398fc0a07 (MD5) Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-02-15T11:32:07Z (GMT) No. of bitstreams: 1 morelli_fcg_dr_araca.pdf: 1900802 bytes, checksum: a8c9044a1f5a46e203d3432398fc0a07 (MD5) Made available in DSpace on 2016-02-15T11:32:07Z (GMT). No. of bitstreams: 1 morelli_fcg_dr_araca.pdf: 1900802 bytes, checksum: a8c9044a1f5a46e203d3432398fc0a07 (MD5) Previous issue date: 2016-02-01 Erosões e úlceras são achados comuns no abomaso e causam preocupação econômica nos mais variados sistemas de produção de gado. Muitos fatores podem predispor ao aparecimento de úlceras e acúmulo de gases no abomaso, incluindo alimentos grosseiros, estresse ambiental, deficiências de vitaminas e minerais e infecções bacterianas. Essas úlceras podem ser subclínicas, sendo descobertas nas necropsias ou após o abate do animal, ou levarem à redução da motilidade do órgão, prejudicando o fluxo do seu conteúdo e causando transtornos digestivos graves e até ao aparecimento de síndromes semelhantes à indigestão vagal. Existem informações a respeito da resposta do sistema imune na maior parte das mucosas do trato gastrintestinal de não-ruminantes e ruminantes, porém são raras a respeito do abomaso. Os objetivos desse estudo foram detectar os níveis de citocinas (IL-17A, IFN-γ, TNF-α, IL-10, IL-6, IL-4, IL-2) no conteúdo abomasal em bovinos de corte, determinar o perfil Th1 ou Th2 dessas citocinas em animais com úlceras de grau 1 e 2 na região cárdica abomasal e comparar esses valores com os níveis de citocinas de animais sem úlceras (controle), em amostras colhidas em abatedouro, para auxiliar na compreensão da fisiopatologia do processo inflamatório local. A avaliação macroscópica e a classificação das úlceras foi realizada por meio de exames visual e histológico em amostras de tecidos da parede da região cárdica abomasal ulcerada. Os níveis de citocinas produzidas do líquido abomasal dos animais com ou sem úlceras foram avaliados por citometria de fluxo (método Cytometric Bead Array). As citocinas citadas foram detectadas no líquido do abomaso dos bovinos. Não houve diferença na liberação das citocinas entre os grupos com úlceras e o grupo sem úlcera, indicando um equilíbrio entre perfis Th1 e Th2 da resposta inflamatória. Erosions and ulcers are common findings in the abomasum and cause economic concern in several livestock production systems. Many factors may predispose to ulcers and bloat in the abomasum, including roughage, environmental stress, deficiencies of vitamins and minerals and bacterial infections. These ulcers may be subclinical and are found during necropsy or after slaughter, or lead to reduction of abomasal motility, hindering the flow of your content and causing serious digestive disorders and even the appearance of syndromes similar to vagal indigestion. There are some studies evaluating the immune system response in most of the mucous membranes of the gastrointestinal tract of non-ruminants and ruminants, but rarely related to the abomasum. The aims of this study were to investigate the levels of cytokines (IL-17A, IFN-γ, TNF-α, IL-10, IL-6, IL-4, IL-2) in the abomasal fluid of beef cattle, to determine the Th1 or Th2 profile of these cytokines in animals with types 1 or 2 ulcers located in the abomasal cardic region and to compare these levels with those of animals without ulcers (controls), in samples collected in an abbatoir, to help to the understand the pathophysiology of the local inflammatory process. Ulcers from the abomasal cardic region were macroscopicaly evaluated, then classified by histology. Cytokine levels in the abomasal fluid from animals with or without ulcers were evaluated by flow cytometry (Cytometric Bead Array). Cytokines were detected in the abomasum fluid of cattle. There was no difference in the release of cytokines between groups, indicating a balance between Th1 and Th2 profiles of the inflammatory response.