No description available.

The mortality of grouper larvae and juveniles infected by nervous necrosis virus (DGNNV) was near 100%. Vacuoles were found in photoreceptor layer, outer nuclear layer and inner nuclear layer of retina and optic tectum of mesencephalon for the dragon grouper that was infected by DGNNV, after stained with haematoxylin and eosin (H&E). Recombinantly expressed in E. coli, virus-like particles (VLPs) were used for antibody preparation. By indirect fluorescence antibody test (IFAT) with the mouse anti-VLP serum, DGNNV was detected in retina inner nuclear layer and mesencephalon optic tectum. At 96 hours post infection of DGNNV with intraocular injection, vacuoles were observed, with H&E staining, in zebrafish retina photoreceptor layer and mesencephalon optic tectum. In IFAT test, DGNNV was also detected in outer nuclear layer and optic tectum of zebrafish. This study showed antibody stimulated by the recombinant VLPs was sutible for DGNNV detection in fish nervous tissues.

Submitted by Luiz Felipe Barbosa (luiz.fbabreu2@ufpe.br) on 2015-03-13T15:00:05Z No. of bitstreams: 2 TESE_Eliane_Coimbra_ 2012.pdf: 2000368 bytes, checksum: 2ec0dc2be6907bc65cd246b36e95e551 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-13T15:00:05Z (GMT). No. of bitstreams: 2 TESE_Eliane_Coimbra_ 2012.pdf: 2000368 bytes, checksum: 2ec0dc2be6907bc65cd246b36e95e551 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012-09-04 / CAPES / O câncer cervical é considerado o segundo tipo de tumor a ocasionar maior número de mortes entre as mulheres no mundo, e a infecção pelo papilomavirus humano (HPV) é a principal causa relacionada com o desenvolvimento desta neoplasia. No Brasil, o câncer cervical produz uma média de 2,5 óbitos/100.000 indivíduos. Atualmente existem duas vacinas no mercado, baseadas em “Vírus-Like Particles” (VLPs), mas o alto custo inviabiliza seu uso pelos países em desenvolvimento. As VLPs de HPV são produzidas a partir da proteína capsidial L1, contra a qual é conferida forte imunidade humoral. Uma estratégia vacinal orientada para a prevenção do câncer cervical em humanos é muito importante para a saúde pública, no entanto, como há muitas pessoas já infectadas pelo HPV, uma abordagem terapêutica se torna necessária. Neste trabalho foi proposta a expressão do gene L1 de HPV-16 em células da levedura Pichia pastoris, como base de uma estratégia preventiva e a construção de uma vacina de DNA baseada na proteína E5 do HPV-16, identificada como potencial antígeno tumoral para terapia do câncer. O gene L1 otimizado de HPV-16 foi clonado em vetor de expressão pPGK para integração no genoma de P. pastoris, sua transcrição foi confirmada por RT-PCR e a expressão da proteína detectada por dot blot, colony blot e western blot. A vacina de DNA foi construída pela inserção do gene E5 de HPV-16 otimizado e adicionado do epítopo AU1, no vetor pCIneo. Para estudo inicial da funcionalidade da construção vacinal pCI-E5sintH16, foi realizada uma análise in vitro em células HEK-293, através de RT-PCR e western blot. O estudo do gene E5 de HPV como antígeno terapêutico, apesar de pouco explorado ainda, é bastante relevante.

Grouper nervous necrosis viruses belong to the Betanodavirus genus in the Nodaviridae family that is a group of small, non-enveloped icosahedron viruses. More than 30 species of fish are infected by the betanodaviruses, which cause massive mortality in hatchery-reared larvae and juveniles. The infection causes great economic losses to aquaculture and sea-ranching. To study the effects of N-terminus and disulfide bonds of capsid protein on particle formation and thermal stability of grouper nervous necrosis virus, virus-like particles (VLPs) of dragon grouper nervous necrosis virus (DGNNV) were used. Deletion of 35 residues at the N-terminus completely ruined the VLP assembly. When deletions were restricted to 4, 16, or 25 N-terminal residues, the assembly of VLPs remained. Site-directed mutagenesis was used to investigate the effects of N-terminus of capsid protein on particle formation and thermal stability of grouper nervous necrosis virus. Althought all arginine mutants could produce VLPs, the relative amounts and thermal stabilities of arginine-mutated VLPs were decrease. The VLPs from £GN25-R29A and £GN25 mutants have similar structural properties on particle formation and thermal stability. Therefore, the effects of Arg29 mutations are negligible. The relative amounts and thermal stabilities of VLPs from £GN25-R30A and £GN25-R31A mutants are lower than £GN25-R29A VLP. When 25 amino acids at N-terminus of DGNNV capsid protein were removed, Arg30 and Arg31 are important for particle formation and particel stability. Although particle could form as 12 positively charged amino acids were lost (¡µN25-R293031A), the efficiency of particles assembly were decrease to 1.2 ¡Ó 0.9% as compare to wild-type VLPs (WT-VLPs). Site-directed mutagenesis and chemical reducing reagents were used to investigate the roles of disulfide bonds in particle formation and thermal stability of grouper nervous necrosis virus. The homogeneous particles from C187A, C331A and C187A/C331A mutants are indistinguishable from the native virus and WT-VLPs in their sizes and shapes. C115A and C201A mutants could not produce VLPs. The dissociated capsomers from arginine- or cysteine-mutant VLPs all can be reassembled to icosahedrons with efficiencies as high as 100%. When VLP particles are pre-fabricated, the reducing agent cannot disrupt the VLP icosahedron structure. The thiol reduction only caused effects on the disulfide linkages inside the icosahedrons. £]-mercaptoethanol-treated WT-VLPs could not tolerate the thermal effects at a temperature higher than 70¢XC. Once the disulfide linkages in dissociated capsomers were entirely disrupted by £]-mercaptoethanol treatment, the resulting capsomers could not reassemble back to icosahedron particles.These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles. In the observation of Cryo-EM, the shapes and sizes of the N-terminus truncated particle (£GN25-VLP) are indistinct from the full-length particle (WT-VLP). The maximum diameter of DGNNV is approximately 380 Å. Like that of the insect nodaviruses, the surface morphologies of £GN25-VLP and WT-VLP are consistent with a T = 3 quasi-equivalent lattice. The protrusions (~154 to 192 Å), the inner shell of the capsid (~112 to 154 Å), and the RNA (¡Õ112 Å) were observed in the DGNNV structure. The protrusion domain is consisting of three capsid subunits, and the interactions between these subunits are different. Deletion of 25 residues at the N-terminus did not affect VLPs formation and the structure of £GN25-VLP is similar to WT-VLPs. Resolutions was calculated by Fourier shell correlation, and the resolution of WT-VLPs and £GN25-VLPs is 6.5Å and 11.8Å, respectively.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-05T16:49:02Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Tese-ElianeCoimbra.pdf: 2000368 bytes, checksum: 2ec0dc2be6907bc65cd246b36e95e551 (MD5) / Made available in DSpace on 2017-04-05T16:49:02Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Tese-ElianeCoimbra.pdf: 2000368 bytes, checksum: 2ec0dc2be6907bc65cd246b36e95e551 (MD5) Previous issue date: 2012-09-04 / O câncer cervical é considerado o segundo tipo de tumor a ocasionar maior número de mortes entre as mulheres no mundo,e ainfecção pelopapilomavirus humano (HPV) éa principalcausarelacionada com o desenvolvimento desta neoplasia. No Brasil, o câncer cervicalproduz uma média de 2,5 óbitos/100.000 indivíduos.Atualmente existem duas vacinas no mercado, baseadas em “Vírus-Like Particles”(VLPs), mas o alto custo inviabiliza seu uso pelos países em desenvolvimento. As VLPs de HPV são produzidasa partir daproteína capsidial L1, contra aqual é conferidaforte imunidade humoral.Uma estratégia vacinalorientada para a prevenção do câncer cervical em humanosé muito importante para a saúde pública, no entanto, como hámuitas pessoas já infectadas peloHPV, uma abordagem terapêutica se torna necessária.Neste trabalho foi proposta a expressão do gene L1 de HPV-16 em células da levedura Pichia pastoris, como base de uma estratégia preventiva e a construção de uma vacina de DNA baseada na proteína E5 do HPV-16, identificadacomo potencialantígeno tumoral para terapia docâncer. O gene L1 otimizado de HPV-16 foi clonado em vetor de expressão pPGK para integração no genoma de P. pastoris, sua transcrição foi confirmada por RT-PCR e a expressão da proteína detectada por dot blot, colony blot e western blot.A vacina de DNA foi construída pela inserçãodo gene E5 de HPV-16 otimizado e adicionado doepítopo AU1, novetor pCIneo.Para estudo inicial da funcionalidadeda construção vacinalpCI-E5sintH16, foi realizadaumaanálise in vitroem célulasHEK-293, através de RT-PCRe western blot.O estudo do gene E5 de HPVcomoantígeno terapêutico,apesar depouco exploradoainda, é bastante relevante. / Cervical cancer is the second type of tumor to cause deaths among women worldwide. The infection of human papillomavirus (HPV) is the leading cause related to the development of this neoplasia.In Brazil, cervical cancer produces an average of 2.5 deaths per 100.000 individuals. There are currently two vaccines on the market, based on Virus-Like Particles, but the high cost (U.S. $ 350.00) prevents its use by developing countries. HPV VLPs are formed from the L1 capsid protein, against which it is given strong humoral immunity. A targeted vaccination strategy for the prevention of cervical cancer in humans is very important to public health;however there are many people already infected with HPV, and therefore a therapeutic approach is needed. In this study was proposed the expression of the L1 gene of HPV-16 in Pichia pastorisyeast cells as the basis for a preventive strategy and the construction of a DNA vaccine based on the HPV-16 E5 protein, identified as a potential tumor antigen for cancer therapy. The optimized L1 gene of HPV-16 was cloned into the expression vector pPGK for integration into the genome of Pichia. The transcript of this gene was confirmed by RT-PCR and L1 protein expression detected by dot blot, colony blot and western blot. The DNA vaccine was constructed by cloning the optimized E5 gene of HPV-16, with the inserted AU1 epitope, in the pCIneo vector. In vitro analysis were performed with HEK-293 cells transfected with the PCI-E5sintH16 vector, including RT-PCR and western blot for the initial evaluation of DNA vaccine functionality. The study of the cancer therapy with the E5 gene of HPV-16 although still unexplored is quite relevant.

The aim of this thesis is to reveal the potential of mouse polyomavirus (MPyV) based virus-like particles (VLPs) as possible nanocarriers for directed delivery of therapeutic or diagnostic compounds to specific cells or tissues. We have chosen mouse polyomavirus VLPs because they do not contain viral DNA and are considered safe for utilization in bio-applications. In our research, we used a chemical approach for retargeting of MPyV based VLPs from their natural receptor to cancer cells. The chemical modification of the capsid surface exposed lysines by an aldehyde-containing reagent enabled conjugation of VLPs to selected molecules: transferrin and inhibitor of glutamate carboxypeptidase II (GCPII). Transferrin, as a transporter of iron to metabolically active cells, targeted VLPs to numerous types of cancer cells overexpressing the transferrin receptor. On the other hand, GCPII serves as a transmembrane marker specific for prostate cancer cells and conjugation of its inhibitor to VLPs resulted in successful recognition of these cells. Electron microscopy was used for visualization of modified VLPs and flow cytometry together with confocal microscopy for investigation of cell specific interactions and VLP uptake. Furthermore, we explored the influence of serum proteins on VLPs. The abundance of...

Existing therapeutic options for the alleviation of nicotine addiction have been largely ineffective at stemming the tide of tobacco use. Immunopharmacotherapy, or vaccination, is a promising, alternate therapy that is currently being explored. Results from previous studies indicate that nicotine vaccines (NVs) are effective in subjects that achieve high drug-specific antibody titers, though overall efficacy has not been observed. Consequently, improvement of these vaccines is necessary before they can achieve approval for human use. In this report, three separate approaches towards NV potentiation are explored. The first approach applied physiologically-based pharmacokinetic (PBPK) modeling to better assess NV potential. Rat and human physiological and pharmacological parameters were obtained from literature and used to construct compartmentalized models for nicotine and cotinine distribution. These models were then calibrated and validated using data obtained from literature. The final models verified the therapeutic potential of the NV concept, identified four key parameters associated with vaccine success, and established correlates for success that could be used to evaluate future NVs prior to clinical trials. In the second approach, conjugate NV scaffoldings were engineered by using wild-type (WT) and chimeric human papilloma (HPV) 16 L1 protein virus-like particles (VLPs). The chimeric protein was created by removing the last 34 C-terminal residues from the WT protein and then incorporating a multi-epitope insert that could universally target major histocompatibility complex (MHC) class II molecules. The proteins were subsequently expressed in E. coli and purified using a multi-step process. Comparisons between the separation outcomes revealed that the insert was able to modulate individual process outcomes and improve overall yield without inhibiting VLP assembly. In the third approach, commonly used carrier proteins were computationally mined for their MHC class II epitope content using human leukocyte antigen (HLA) population frequency data and MHC epitope prediction software. The most immunogenic epitopes were concatenated with interspacing cathepsin cleavage sequences and the resulting protein was re-evaluated using the earlier methods. This work represents the first ever in silico design of chimeric antigens that could potentially target all of the major HLA DQ and HLA DR allotypes found in humans. / Doctor of Philosophy / Existing treatment options for addressing nicotine addiction have been largely ineffective at preventing tobacco use. Vaccination, on the other hand, is a promising, alternate treatment option that is currently being explored. Previous studies have shown that nicotine vaccines (NVs) are effective in the subjects that respond well to the vaccine. Effectiveness in the majority of vaccine recipients, however, has not been observed. Consequently, improvement of these vaccines is necessary before they can be used in humans. In this report, three separate approaches for improving NV effectiveness are explored. The first approach applied physiologically-based pharmacokinetic (PBPK) modeling to better assess NV potential. Parameters were obtained from literature and used to construct models that could predict NV effectiveness in rats and humans. These models were then calibrated and validated using data obtained from literature. The final models verified that NVs could work if certain conditions were met, identified four key parameters associated with vaccine success, and allowed for estimation of NV efficacy prior to their evaluation in humans. In the second approach, protein carriers for conjugate NVs were constructed using the human papilloma (HPV) 16 L1 protein. This protein is known for its ability to form virus-like particles (VLPs). Both a modified and an unmodified (wild-type) protein were constructed. The modified HPV 16 L1 protein was created by replacing the last 34 C-terminal amino acids with a polypeptide insert that could enhance the immunogenicity of the vaccine. The modified and unmodified proteins were then expressed in E. coli and purified. Results indicated that the insert was able to modulate individual process outcomes and improve overall process yield without preventing VLP assembly. In the third approach, commonly used carrier proteins were computationally mined for their MHC class II epitope content using human gene frequency data and MHC epitope prediction software. The epitopes that were predicted to be the most immunogenic were linked together with interspacing protease recognition sequences and the immunogenicity of the resulting protein was re-evaluated using the prediction software. This work represents the first computational design of antigens that could potentially allow a vaccine to be effective in a large portion of human population regardless of the genetic variability.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 as a highly infectious virus that spread rapidly and was declared a pandemic by the World Health Organization (WHO) in March of 2020.1 SARS-CoV-2 infection causes respiratory disease (Coronavirus Disease; COVID-19), and during the initial infection wave of the pandemic, disease severity ranged from asymptomatic to mild upper respiratory disease, to severe pneumonia resulting in hospitalization and death.1 To date, over 750 million cases of COVID-19 and almost 7 million deaths due to the disease have been confirmed (WHO COVID-19 Dashboard). Virus-like particles are nanoparticles made up of existing viral structural proteins that will assemble into a particle form. The hepatitis B virus core antigen (∆HBcAg) is a stable VLP that will maintain its ability to fold into a viral particle following incorporation of foreign epitopes into its protein sequence. In this project, we produced a vaccine against SARS-CoV-2 that was composed of three SARS-CoV-2 Spike protein epitopes inserted into the ∆HBcAg VLP. These insertions included Spike epitopes predicted to induce a humoral and/or cell-mediated immune response. The immunogenicity of the resultant vaccine was tested utilizing a K18-hACE2 transgenic C57BL/6 mouse model. Mice were challenged with live SARS-CoV-2 three weeks after the final booster dose and the vaccine was evaluated for protective efficacy. Results of these studies showed epitope-specific humoral and cell-mediated immune responses, but these responses were insufficient in protecting against SARS-CoV-2 infection. / Doctor of Philosophy / Coronaviruses have caused gastrointestinal and respiratory disease in humans and a variety of veterinary species for decades. In response to the onset of the COVID-19 (Coronavirus Disease) pandemic in late 2019, we created a vaccine against the virus that causes this disease: the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infects human cells by attaching via one of its surface proteins (the Spike protein) to the human ACE2 receptor (angiotensin converting enzyme 2). After this binding occurs, the virus can enter the cell and begin to replicate, resulting in cellular damage, organ-level dysfunction, and clinical disease. An effective vaccine against SARS-CoV-2 would induce antibodies that bind to this Spike protein, thereby blocking its ability to infect host cells and preventing the downstream effects of infection. Our vaccine consisted of a protein from the hepatitis B virus (∆HBcAg), which naturally folds into a virus-like particle (VLP). This VLP can be used as a stable backbone and foreign epitopes can be inserted into the particle for presentation to the immune system as a vaccine. We inserted three SARS-CoV-2 Spike protein epitope into the ∆HBcAg backbone and tested the vaccine's ability to elicit an immune response and protect against infection with live SARS-CoV-2. Results from these studies showed an antibody response to the vaccine and higher levels of anti-viral cytokines in vaccinated mice compared to controls, but incomplete protection against disease. Additionally, we identified areas for vaccine optimization that will inform future studies utilizing this type of vaccine platform.

Le virus de la grippe infecte les muqueuses du tractus respiratoire. Un vaccin intranasal induit une réponse immunitaire proche de celle faisant suite à une infection naturelle en bloquant le virus directement sur le site de l'infection et permet une vaccination sans aiguille. Par ailleurs, les vaccins à base de pseudo-particules virales ou Virus-like Particles (VLP) produites sur cellules représentent une alternative intéressante au vaccin classique produit sur oeufs. Les VLP sont des particules non-réplicatives qui ressemblent au virus et qui peuvent être immunogènes même sans adjuvant, en particulier par voie intranasale. Au cours de ma thèse, une plateforme de production de VLP grippales composées d'hémagglutinine, de neuraminidase et de protéine de matrice M1 a été développée par transfection transitoire des cellules de mammifères. Des immunisations de souris BALB/c ont montré que les VLP de type A et B, purifiées et caractérisées, étaient immunogènes à de faibles doses par voie intramusculaire. L'administration par voie intranasale de VLP avec la sous-unité B de la toxine cholérique, comme adjuvant muqueux, a permis d'obtenir des taux d'anticorps sériques comparables à ceux obtenus par immunisation en intramusculaire mais également une forte réponse IgA au niveau des muqueuses. Par ailleurs, le rendement des VLP s'est révélé souche-dépendant et lié aux protéines HA et NA à la surface de la particule. Pour contourner ce problème, un vaccin quadrivalent composé de deux VLP bivalentes exprimant chacune deux HA et NA différentes à la surface a été produit montrant ainsi la flexibilité de cette plateforme / The influenza virus infects the mucous membranes of the respiratory tract. An intranasal vaccine induces an immune response close to the one induced by the natural infection by blocking the virus directly at the site of infection and allows needle-free vaccination. In addition, vaccines based on Virus-like Particles (VLP) produced in cells represent an interesting alternative to the traditional egg-based vaccine. VLPs are non-replicative particles that mimic the virus. Studies on influenza VLPs have shown protection by the intranasal route without adding an adjuvant. During my thesis, a platform for the production of influenza VLPs composed of the hemagglutinin, the neuraminidase and the M1 matrix proteins was developed by transient transfection of mammalian cells. Immunizations of BALB/c mice showed that the purified and characterized type A and B VLPs were immunogenic at low doses by the intramuscular route. The intranasal administration of VLPs with the B subunit of cholera toxin as a mucosal adjuvant resulted in serum antibody levels comparable to those obtained by intramuscular immunization but also a strong IgA response in the mucosal secretions. In addition, VLP yield was found to be strain-dependent and linked to the HA and NA proteins on the surface of the particle. To overcome this problem, a quadrivalent vaccine based on two bivalent VLPs each expressing two different HAs and NAs at the surface was produced, demonstrating the flexibility of this platform

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-04T18:15:33Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2010-Dissertação-MarceloCordeiro.pdf: 2495604 bytes, checksum: 2d51566d7f95818c5bd7b1860032afdc (MD5) / Made available in DSpace on 2017-04-04T18:15:33Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2010-Dissertação-MarceloCordeiro.pdf: 2495604 bytes, checksum: 2d51566d7f95818c5bd7b1860032afdc (MD5) Previous issue date: 2010-09-27 / Os papilomavírus são um grupo de pequenos vírus DNA dupla fita caracterizado por induzir a formação de lesões que, normalmente, são benignas e podem regredir naturalmente, mas também apresentam opotencial para se tornarem tumores malignos. O papilomavírus humano (HPV) está fortemente relacionado às doenças sexualmente transmissíveis (DST) e é uma das principais causas de câncer cervical. O papilomavírus bovino (BPV), por sua vez, representa um grave problema econômico em termos de pecuária. Em bovinos, são mais comuns as papilomatosescutânease mucosas nas regiões genital e orofaríngea. Existem bem caracterizados seis tipos diferentes de BPV (BPV 1 ao 6) e, recentemente, quatro novos tipos (BPVs 7 ao 10) foram seqüenciados. No Nordeste, especialmente no estado de Pernambuco (Zona da Mata), a incidência da papilomatose bovina é muito alta, causando grandes perdas econômicas para os criadores. Não há tratamento com eficácia comprovada. Proteção contra a infecção é conferida por anticorpos neutralizantes dirigidos contra osepítopos conformacionais de proteínas estruturais L1. Esses anticorpos podem ser eficientemente induzidos pela imunização com virus-like particles(VLP) que se formam espontaneamente, após a expressão de L1 em sistemas heterólogos. Nos últimos anos, a levedura metilotrófica Pichia pastoris emergiu como um sistema eficaz e barato para produzir altos níveis deproteínarecombinante. O foco deste trabalho foi construir e avaliar dois plasmídeos (pPICZAαL1B2 e pPICZAL1B2), como vetores para expressão do geneL1BPV-2 em células de P. pastoris. Um deles (pPICZAαL1B2) insere na proteína recombinante um sinal de secreção (α-mating). Assim, esperou-se comparar a eficiência da expressão heteróloga através das viasintra e extracelulares. O gene L1 foi amplificado por PCR do genoma completo BPV-2, foi clonado em vetor pGEM-T(Promega ®) e, posteriormente, foi subclonado nos vetores de expressão pPICZA e pPICZAα (Invitrogen ®). PCR e análise de restrição indicarama inserção correta do gene L1 em ambos os vetores, bem como o seqüenciamento. Por eletroporação, P. pastoris (cepa de levedura X-33) foi transformada com a construção pPICZAL1B2 ou pPICZAαL1B2, cuja integração no genoma de levedura pode ser identificada por PCR da colônia. As leveduras transformadas foram selecionadas para experimentos de cultura com indução com metanol. A expressão de L1 foi indicada por RT-PCR e análise de proteínas. A proteína recombinante L1foi detectada em células de levedura, que sofreram tratamento com solução de lise (via intracelular) e em meio de cultura submetido a precipitação com acetona (via extracelular). Este estudo fornece um caminho para uma estratégia de vacina com VLP contra papilomaviroses bovina e apresenta um modelo experimental para estudos de papilomatosessimilares, além de mostrar um modelo experimental interessante para futuras aplicações em seres humanos. / Papillomaviruses are a group ofsmall double-stranded DNA viruses characterized by inducingthe formation of lesions that are usually benign and may regress naturally, but also havethe potential to become malignant tumors. The human papillomavirus (HPV) is strongly related to sexually transmitted diseases (STDs) and is a major cause of cervical cancer. The bovine papillomavirus (BPV), in turn, represents a serious economic problem in terms of livestock. In cattle,cutaneous and mucosal papillomatosis at genital and oropharyngeal regionsare the most common. There are six different types of BPV (BPV 1-6) well characterized and, recently, four new types (BPVs 7-10) havebeen sequenced.In theNortheast, especially inthe state of Pernambuco (Zona da Mata), the incidence of bovine papillomatosis isveryhigh,causing great economic losses for breeders. There isno treatment with proven efficacy. Protection against infection is conferred by neutralizing antibodies directed against structural L1proteinconformational epitopes. These antibodies can be efficiently induced by immunization with virus-like particles (VLP) that are formed spontaneously after L1 expression in heterologous systems. In recent years, the methylotrophic yeast Pichia pastorishas emerged as an efficient and inexpensive heterologous system to produce high protein levels. The focus of this paper is to construct andevaluate two plasmids (pPICZAαL1B2 and pPICZAL1B2), as vectors to gene expression of L1BPV-2 in cells of P. pastoris. One of them (pPICZAαL1B2) inserts a secretion signal (α-matting)on recombinant protein. Thus, it had been expected to compare the heterologous expression via intracellular and extracellular pathway efficiency. L1 gene has been amplified by PCR from the complete BPV-2 genome, has been cloned into pGEM-T vector (Promega®) and, subsequently, has been sub cloned into pPICZA and pPICZAαexpression vectors (Invitrogen®). PCR and restriction analysis haveindicated the correct insertion of the L1 gene in both vectors, as well as sequencing. By electroporation, P. pastoris(strain X-33 yeast) has been transformed with pPICZAL1B2 orpPICZAαL1B2 constructions, whose integration into yeast genome could be identified by colony PCR. Transformed yeast has been selected to culture experiments withmethanol induction. L1 expression has been indicated by RT-PCR and protein analysis. L1 recombinant protein has been detected in yeast cells that has undergonelysis solution treatment (intracellular pathway) and from the culture medium acetone precipitation submitted (extracellularpathway). This study provides a way for a VLP-vaccine strategyagainst bovine papillomaviroses and exhibits an experimental model for similar papillomatosis studies, whileshowingan interesting experimental model for future applications in humans.