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Human papillomaviruses (HPV) are among the most common sexually transmitted infections and are responsible for 5% of all human cancers. HPV type 16 is the most prevalent of the high-risk HPVs (a subgroup of HPVs with potential to cause cancer), accounting for ~55% of HPV-associated cancers. HPV16 is a nonenveloped virus, composed of the major capsid protein L1, the minor capsid protein L2, and a circular double-stranded DNA genome (vDNA) condensed with human histones. HPV initially infects undifferentiated basal keratinocytes and viral replication is dependent on epithelial differentiation. Like many other DNA viruses, HPV must deliver its vDNA to the host cell nucleus to successfully replicate. Initial binding of HPV16 to host cells is through L1 interactions with cell surface heparan sulfate receptors. Shortly after virus binding, L2 is believed to undergo furin cleavage-dependent conformational changes, resulting in spanning of the protein across the local membrane and exposure of the central and C-terminal regions of L2 (which was lumenal and and inaccessible before furin cleavage) to the host cell cytosol. L2 is critical for transport of the L2/vDNA from endosomes to the trans-Golgi network (TGN). We hypothesize that furin-dependent early L2 spanning, through the direct binding and recruitment of cytosolic sorting factors, may contribute to viral endocytosis and subcellular retrograde trafficking (trafficking from endosomes to Golgi) of vDNA. We have developed a Tac receptor (CD25 or IL2 receptor, a transmembrane cell surface protein) chimera system to study L2-dependent endocytosis and trafficking. In this system the Tac ecto- and transmembrane domains are fused to the ~400 amino acid portion of L2 that is likely cytosolic upon L2 spanning. Through transient expression of Tac-L2 chimera we use anti-Tac ectodomain antibodies to label and track cell surface populations by immunofluorescence and confocal microscopy. We have also adopted this system to study endocytosis through a cell surface biotinylation approach. Both approaches suggest that L2 may enhance endocytosis and preliminary evidence suggests that the Tac-L2 chimera may recruit the cytosolic retromer complex (the host cytosolic factors help protein retrograde trafficking) to preferentially traffic to the TGN. Retromer-dependent trafficking of cargo from early endosomes to the TGN is known to involve certain members of the sorting nexin family, specifically the SNX-BAR proteins. We performed a small siRNA screen and identify SNX6 and SNX32 (aka SNX6b) as SNX-BAR proteins that may be specifically involved in retrograde trafficking of HPV16 L2/vDNA during infection. Future work will focus on the mechanisms through which L2 and SNX6 influence HPV16 entry and trafficking.

Human Papilloma Viruses (HPVs) are a large family of viruses with a capsid constituted by the L1 and L2 proteins, which bind to receptors of the basal epithelial cells, thus promoting virus entry. The majority of sexually active people become exposed to HPV, which is the most common cause of cervical cancer affecting more than 600.000 women every year. Moreover, every year more than 13.000 new cases of HPV-related cancers, including anal, penile and head and neck cancers, are diagnosed in men. Three vaccines are available based on the L1 capsid protein, which self-assembles and forms virus-like particles (VLPs) when expressed in yeast and insect cells. Although very effective, these vaccines are HPV type-restricted, and their costs limit broad vaccination campaigns, especially in low- and middle- income countries. Recently, vaccine candidates based on the conserved L2 epitope from serotypes 16, 18, 31, 33, 35, 6, 51 and 59 were shown to elicit broadly neutralizing anti-HPV antibodies, reaching a protection around 90% against all the HPV serotypes. During my research activity, we have tested whether E. coli Outer Membrane Vesicles (OMVs) could be successfully decorated with L2 polytopes and whether the engineered OMVs could induce neutralizing antibodies. OMVs represent an attractive vaccine platform for their intrinsic adjuvanticity and their low production costs. We show that strings of L2 epitopes could be efficiently expressed on the surface of the OMVs and a polypeptide constituted by the L2 epitopes from serotypes 18, 33, 35 and 59 provided broad cross-protective activity against a large panel of HPV serotypes as judged by the in vitro pseudovirus neutralization assay. In order to better characterize the vesicle and in perspective of future clinical studies of our HPV candidate vaccine, we also worked on the setting-up of a simple and reproducible production process at laboratory scale ready to be transferred at industrial level.Moreover, we focused our attention on the strategy used for the engineering of the OMVs with the L2 epitopes and in particular on the carrier used for the delivery of the fusion construct in the surface of the vesicle. More in detail, since part of the carrier is a human cancer epitope, we tested whether a similar scaffold, with less homologies to the human gene could maintain the same properties in terms of: i) expression level of the fused epitopes in the OMVs, ii) localization on the surface of the vesicle and iii) 9 immunogenicity and efficiency to stimulate the immune system in order to produce anti L2 antibodies. Considering all the results described in this work combined with the points of strength of the OMV-based vaccine platform, as the simplicity of the production process, the yields of vaccine doses and the low cost/dose, our data provide a very promising prototype of universal anti-HPV vaccine.

O Câncer Cervical é um importante problema de saúde pública, causando aproximadamente 270 milhões de mortes anuais. Atualmente, esse câncer é o segundo mais comum em mulheres no mundo, com os maiores índices de incidência ocorrendo nos países em desenvolvimento. O Câncer cervical tem como agente etiológico o Papilomavírus Humano (HPV). O vírus possui DNA circular duplo com genes que codificam oito proteínas, E1, E2, E4, E5, E6, E7 (não estruturais), e L1 e L2 (estruturais) que formam o capsídio viral. A proteína L1 quando produzida em sistema heterólgo de expressão tem a capacidade de auto-arranjo em partículas semelhantes ao vírus chamadas de VLPs (\"Vírus like Particles\"), que são a base das vacinas profiláticas disponíveis, Gardasil® e Cervarix®. Neste trabalho, foram produzidas VLPs da proteína L1 do HPV-16 na levedura metilotrófica Pichia pastoris utilizando dois tipos de vetores de expressão para leveduras, um epissomal e outro integrativo, para os trabalhos de purificação. Inicialmente as amostras obtidas ao final da purificação apresentaram degradação e agregação da proteína L1, além de contaminantes. O protocolo de purificação foi modificado e as amostras obtidas no final do processo de purificação não apresentaram degradação e nem agregação da proteína L1, embora ainda tenha sido detectada a presença de contaminantes e, portanto, as amostras obtidas estavam semi-purificadas. Uma segunda abordagem para o desenvolvimento de uma vacina alternativa foi realizada, na qual foram produzidos candidatos vacinais baseados na proteína L2 do Papilomavírus Humano (L2 do HPV-16, L2 do HPV-18 e L2 Multimérica), e também foram expressas proteínas fusionadas ao domínio ZZ de Staphylococcus aureus, utilizado neste caso como adjuvante. Em geral as proteínas de fusão, ainda que apresentando a fusão em sua forma funcional, geraram uma fraca indução de anticorpos IgG e de anticorpos neutralizantes. Pode-se observar uma produção de anticorpos neutralizantes contra pseudovírus homólogos e heterólogos que dependeu da combinação com os adjuvantes utilizados. Numa tentativa de maximizar a eficiência imunológica dos antígenos utilizados, um novo ensaio de imunização foi realizado com uma formulação conjunta contendo três proteínas (L2 do HPV-16, L2 do HPV-18 e L2 Multimérica), sendo também avaliadas as propriedades de três adjuvantes. A combinação da formulação vacinal com os adjuvantes gerou altos níveis de anticorpos L2-específicos, destacando-se as formulações com o hidróxido de alumínio/MPLA e com a vacina celular Pertussis low. Com a utilização desses adjuvantes, os níveis de anticorpos permaneceram elevados a longo termo. Pode-se observar a imunogenicidade de cada um dos antígenos dependeu de sua formulação com os adjuvantes testados. / Cervical cancer is one of the most important problems of public health, causing nearly 270 million deaths annually. Currently, cervical cancer is the second more common type in women worldwide and the highest incidences occuring in developing countries. Human papillomavirus (HPV) are the ethiologic agents of cervical cancer. HPV have a double-stranded circular DNA genome encoding eight viral proteins, E1, E2, E4, E5, E6, E7 (non-structural), and L1, L2 (structural), the last ones forming the viral capsid. L1 protein expressed in heterologous systems self-assembles into VLPs (\"Virus like Particles\"), the base of prophylactic vaccines available commercially, Gardasil and Cervarix. In this work, we produced VLPs of HPV-16 L1 protein in the methylotrophic yeast Pichia pastoris for purification assays. Initially, samples obtained from purification presented degradation and aggregation of L1 protein and also contaminants. The purification protocol was modified and samples obtained in the new process did not present degradation nor aggregation but the presence of some contaminants could still be detected. Therefore, samples obtained were semi-purified. A second approach to the development of an alternative vaccine was performed with the production of vaccine candidates based on the HPV L2 protein (HPV-16 L2, HPV-18 L2 and Multimeric L2) and we also expressed the same proteins fused to Staphyloccocus aureus ZZ domain, using it as an adjuvant. In general, the fused proteins induced low antibodies titer and low neutralizing antibodies titer when compared to other formulations. Neutralizing antibodies elicited against homologous and heterologous pseudovirus depended on the adjuvant used. In another approach, trying to enhance immunologic antigen efficiencies, an immunization assay was performed with a formulation containing L2 HPV- 16, L2 HPV-18 and Multimeric L2 and the properties of three adjuvants were assessed. The vaccine formulations adjuvanted with alum/MPLA and cellular Pertussis low vaccine induced the highest levels of L2-specific antibodies. In addition, the usage of adjuvants maintained the antibody levels in the long term. The immunogenicity of each antigen depends on the adjuvant combination used.

O Câncer Cervical é um importante problema de saúde pública, causando aproximadamente 270 milhões de mortes anuais. Atualmente, esse câncer é o segundo mais comum em mulheres no mundo, com os maiores índices de incidência ocorrendo nos países em desenvolvimento. O Câncer cervical tem como agente etiológico o Papilomavírus Humano (HPV). O vírus possui DNA circular duplo com genes que codificam oito proteínas, E1, E2, E4, E5, E6, E7 (não estruturais), e L1 e L2 (estruturais) que formam o capsídio viral. A proteína L1 quando produzida em sistema heterólgo de expressão tem a capacidade de auto-arranjo em partículas semelhantes ao vírus chamadas de VLPs (\"Vírus like Particles\"), que são a base das vacinas profiláticas disponíveis, Gardasil® e Cervarix®. Neste trabalho, foram produzidas VLPs da proteína L1 do HPV-16 na levedura metilotrófica Pichia pastoris utilizando dois tipos de vetores de expressão para leveduras, um epissomal e outro integrativo, para os trabalhos de purificação. Inicialmente as amostras obtidas ao final da purificação apresentaram degradação e agregação da proteína L1, além de contaminantes. O protocolo de purificação foi modificado e as amostras obtidas no final do processo de purificação não apresentaram degradação e nem agregação da proteína L1, embora ainda tenha sido detectada a presença de contaminantes e, portanto, as amostras obtidas estavam semi-purificadas. Uma segunda abordagem para o desenvolvimento de uma vacina alternativa foi realizada, na qual foram produzidos candidatos vacinais baseados na proteína L2 do Papilomavírus Humano (L2 do HPV-16, L2 do HPV-18 e L2 Multimérica), e também foram expressas proteínas fusionadas ao domínio ZZ de Staphylococcus aureus, utilizado neste caso como adjuvante. Em geral as proteínas de fusão, ainda que apresentando a fusão em sua forma funcional, geraram uma fraca indução de anticorpos IgG e de anticorpos neutralizantes. Pode-se observar uma produção de anticorpos neutralizantes contra pseudovírus homólogos e heterólogos que dependeu da combinação com os adjuvantes utilizados. Numa tentativa de maximizar a eficiência imunológica dos antígenos utilizados, um novo ensaio de imunização foi realizado com uma formulação conjunta contendo três proteínas (L2 do HPV-16, L2 do HPV-18 e L2 Multimérica), sendo também avaliadas as propriedades de três adjuvantes. A combinação da formulação vacinal com os adjuvantes gerou altos níveis de anticorpos L2-específicos, destacando-se as formulações com o hidróxido de alumínio/MPLA e com a vacina celular Pertussis low. Com a utilização desses adjuvantes, os níveis de anticorpos permaneceram elevados a longo termo. Pode-se observar a imunogenicidade de cada um dos antígenos dependeu de sua formulação com os adjuvantes testados. / Cervical cancer is one of the most important problems of public health, causing nearly 270 million deaths annually. Currently, cervical cancer is the second more common type in women worldwide and the highest incidences occuring in developing countries. Human papillomavirus (HPV) are the ethiologic agents of cervical cancer. HPV have a double-stranded circular DNA genome encoding eight viral proteins, E1, E2, E4, E5, E6, E7 (non-structural), and L1, L2 (structural), the last ones forming the viral capsid. L1 protein expressed in heterologous systems self-assembles into VLPs (\"Virus like Particles\"), the base of prophylactic vaccines available commercially, Gardasil and Cervarix. In this work, we produced VLPs of HPV-16 L1 protein in the methylotrophic yeast Pichia pastoris for purification assays. Initially, samples obtained from purification presented degradation and aggregation of L1 protein and also contaminants. The purification protocol was modified and samples obtained in the new process did not present degradation nor aggregation but the presence of some contaminants could still be detected. Therefore, samples obtained were semi-purified. A second approach to the development of an alternative vaccine was performed with the production of vaccine candidates based on the HPV L2 protein (HPV-16 L2, HPV-18 L2 and Multimeric L2) and we also expressed the same proteins fused to Staphyloccocus aureus ZZ domain, using it as an adjuvant. In general, the fused proteins induced low antibodies titer and low neutralizing antibodies titer when compared to other formulations. Neutralizing antibodies elicited against homologous and heterologous pseudovirus depended on the adjuvant used. In another approach, trying to enhance immunologic antigen efficiencies, an immunization assay was performed with a formulation containing L2 HPV- 16, L2 HPV-18 and Multimeric L2 and the properties of three adjuvants were assessed. The vaccine formulations adjuvanted with alum/MPLA and cellular Pertussis low vaccine induced the highest levels of L2-specific antibodies. In addition, the usage of adjuvants maintained the antibody levels in the long term. The immunogenicity of each antigen depends on the adjuvant combination used.