No description available.

Foamyviren (FV) weisen eine Reihe von Merkmalen auf, welche sie von Orthoretroviren unterscheidet, die sie jedoch gleichzeitig mit den Hepadnaviren teilen. Dies betrifft neben der Genomorganisation, der Proteinexpression sowie dem Replikationsverhalten auch die Partikelmorphogenese. Die zentrale Komponente in diesem Prozeß stellt das Gag-Protein dar. FV benötigen im Gegensatz zu Orthoretroviren und vergleichbar den Hepadnaviren die Koexpression des homologen Glykoproteins für den zellulären Partikelexport. Im ersten Teil dieser Arbeit wurde mittels eines chimären Konstruktes aus den Gag-Proteinen von MPMV und PFV versucht, ein Env-interagierendes Motiv sowie die für die Interaktion mit dem Glykoprotein essentiellen As in PFV Gag zu identifizieren. Dabei wiesen die chimären Gag-Proteine Gemeinsamkeiten mit PFV Gag auf, wie eine perinukleäre Akkumulation, eine Vorraussetzung für das Assembly sowohl von PFV als auch MPMV. Desweiteren waren die Gag-Chimären für einen zellulären Export auf die Koexpression des homologen Glykoproteins angewiesen. Dies deutete auf die Integrität und Funktionalität des dafür notwendigen PFV Gag N-Terminus hin. Die chimären Gag-Moleküle multimerisierten jedoch nicht zu Kapsiden oder vergleichbaren partikulären Strukturen, vermutlich aufgrund massiver sterischer Zwänge infolge der Beteiligung heterologer Proteindomänen, weswegen sie kein geeignetes System zur funktionellen Analyse der PFV Gag-Env-Interaktion darstellten. Eine weitere Besonderheit foamyviraler Gag-Proteine ist ihr äußerst geringer Lysinanteil. Im Gegensatz zu den Gag-Proteinen der Orthoretroviren wird der überwiegende Anteil basischer Aminosäuren (As) durch Arginin vertreten. Da über 60 % der Arginin-spezifizierenden Kodons über eine Einzelmutation aus Lysin-Kodons hervorgegangen sein könnten, ist es wahrscheinlich, daß im Verlauf der foamyviralen Evolution eine positive Selektionierung von Gag-Mutanten mit einer Lysin-zu-Arginin-Substitution stattfand. Im zweiten Teil dieser Arbeit wurde anhand der Beispiele von PFV sowie FFV der Frage nachgegangen, welche Funktionen Arginine in foamyviralen Gag-Proteinen während der Replikation übernehmen. Dazu wurde in infektiösen PFV- sowie FFV-Klonen eine Reihe von Argininen gegen Lysine substituiert. Zusätzlich wurde das singuläre Lysin in PFV Gag gegen Arginin substituiert. Dabei konnte gezeigt werden, daß sämtliche PFV- sowie FFV-Mutanten replikationskompetent waren. Das singuläre Lysin in PFV Gag war für dessen Replikation in immortalisierten Zellen entbehrlich, in einer primären Zellinie wies die entsprechende Mutante jedoch eine stark eingeschränkte Replikationsfähigkeit auf. Eine PFV-Substitutionsmutante (M141) induzierte in transfizierten 293T-Zellkulturen einen CPE, ein Hinweis auf eine Beteiligung dieses Gag-Abschnittes an der Interaktion mit dem PFV Glykoprotein. Nach zehnmaliger Zellkultur-Passagierung der PFV Gag-Mutanten traten weder Revertanten noch Pseudorevertanten auf, was jedoch aufgrund der kurzen Zeitspanne des Experimentes nur eine begrenzte Aussagekraft bezüglich der genetischen Stabilität der Mutanten zuließ. Mittels der Applikation von AZT, eines Inhibitors der foamyviralen reversen Transkription, entweder auf die virusproduzierenden Zellen oder die zur Infektion verwendeten Zielzellen konnte gezeigt werden, daß sich die PFV Gag-Lysinmutanten hinsichtlich ihrer Replikationsstrategie nicht von WT-Viren unterscheiden und ihre genomische RNA größtenteils noch in der Produktionszelle revers transkribieren. Desweiteren konnte mittels quantitativer real-time PCR nachgewiesen werden, daß die PFV- und FFV-Mutanten wie für FV üblich sowohl DNA als auch RNA in ihre Partikel verpacken. Die infektiöse Natur foamyviraler genomischer DNA konnte bereits in früheren Veröffentlichungen gezeigt werden. Auch in dieser Arbeit konnte nach Transfektion von Zellen mit aufgereinigter Virionen-DNA und anschließendem Überstandtransfer ein CPE in den infizierten Indikatorzellen induziert werden, was die Produktion infektiöser Viruspartikel bewies. Die -Aminogruppe von Lysin fungiert als potentieller Ubiquitinakzeptor. Für das singuläre Lysin im WT Gag-Protein von PFV konnte wie in früheren Veröffentlichungen keine Ubiquitinierung festgestellt werden, im Gegensatz dazu wurde bei vier der fünf PFV Substitutionsmutanten eine Ubiquitinierung der neu eingeführten Lysine detektiert. Diese kovalente Modifikation hatte jedoch keinen Einfluß auf die Env-Restriktion des PFV-Kapsidexportes aus der Zelle. Für FFV konnte sowohl für den WT als auch die Substitutionsmutanten weder eine LP- noch eine Gag-Ubiquitinierung festgestellt werden. Als wahrscheinliche Ursache dafür kommen Unterschiede in den Komponenten der Ubiquitinierungsmaschinerie zwischen humanen Zellen und Katzenzellen in Frage, weshalb die Analyse einer möglichen Ubiquitinierung der neu in FFV Gag eingeführten Lysine in Katzenzellen als Produktionszellen durchgeführt werden sollte. Die Replikationsfähigkeit mehrerer Substitutionsmutanten der Gegenwart von IFN- und - war im Vergleich zum WT stark eingeschränkt. Dies deutete darauf hin, daß IFN-vermittelte Abwehrmechanismen eine Rolle während der positiven Selektion der Lysin-zu-Arginin-Substitutionsmutanten gespielt haben könnten. Die in dieser Arbeit erzielten Resultate zeigten, daß sich die PFV- sowie FFV-Lysinmutanten in ihrem Replikationsverhalten nicht von WT-Viren unterscheiden. Im Kontext einer über Millionen von Jahren andauernden Wirts-Erreger-Koevolution stellt jedoch der durch zelluläre Restriktionsfaktoren vermittelte Selektionsdruck auf das Virus einen wichtigen Aspekt dar. Demnach könnte die Substitution von Lysin gegen Arginin beispielsweise über eine veränderte kovalente Modifikation eine Interaktion mit antiretroviralen Restriktionsfaktoren der Wirtszelle modifiziert oder inhibiert haben, wodurch diese Mutanten im Verlauf der foamyviralen Evolution positiv selektioniert wurden. / Foamyviruses (FV) harbour several features which distinguishes them from orthoretroviruses and which they share with the hepadnaviruses. Beside differences in the genomic organization, the protein expression and the replication strategy this referrs to the morphogenesis of viral particles, in which the Gag protein plays the key role. In contrast to orthoretroviruses and like hepadnaviruses, cellular egress of FV capsids depends on the presence of cognate Env protein. In the first part of this work it was tried to identify an Env-interacting motif as well as essential amino acids (aa) involved in the recognition of the glycoprotein in PFV Gag. Therefore a chimeric construct containing parts of the Gag proteins of Mason-Pfizer monkey virus (MPMV) and PFV was constructed. Like PFV Gag, the chimeric Gag proteins showed a perinuclear accumulation, a feature of PFV and MPMV assembly. Additionally, cellular export of the Gag chimerics was PFV Env restricted. This pointed to the integrity and functionality of the PFV Gag N-terminus in the chimeric construct. Nevertheless, chimeric Gag molekules didn´t multimerize to capsids or comparable particle structures. This was probably caused by massive sterical disorders, due to the involvement of heterologous protein domains. Therefore, the Gag chimerics didn´t represent an appropriate system for the functional analysis of the PFV Gag-Env interaction. Another peculiarity of foamyviral Gag proteins is their extremely low lysine content. In contrast to orthoretroviral Gag proteins, the main part of basic aa is represented by arginines. Due to the fact that more than 60 % of the arginine-specifying codons could have evolved out of lysine-specifying codons over a single point mutation, a positive selection of Gag mutants with a lysine-to-arginine substitution in the course of foamyviral evolution is likely. In the second part of this work, functions of arginines in foamyviral Gag proteins during replication were studied by using infectious molecular clones of PFV and FFV. Therefore several arginines were substituted for lysines. Additionally the single lysine in PFV Gag (K396) was mutated to arginine. All PFV as well as FFV mutants were replication-competent. Additionally, the single lysine in PFV Gag was non-essential for replication in immortalized cell culture, whereas replication was strongly inhibited in a primary cell line. One PFV substitution mutant (M141) induced an CPE in transfected 293T-cell culture, an indication of an involvement of this part of Gag in the interaction with the PFV glycoprotein. Upon ten cell-free passages of PFV Gag mutants in cell culture, neither revertants nor pseudorevertants appeared, indicating genetic stability during a short-term cell culture experiment. By application of AZT, an inhibitor of the foamyviral reverse transcription, on producer cells or target cells it was shown that the reverse transcription of the genomic RNA of the PFV Gag lysine mutants occured mainly in the producer cell and that they therefore didnt differ from wild-type (wt) virus with respect to replication strategy. By quantitative real-time PCR it was shown that PFV and FFV mutants incorporate DNA as well as RNA in particles. Like in recent publications, the infectiousness of foamyviral genomic DNA could be demonstrated in this work. By transfecting cells with extracted virion DNA and following supernatant transfer a CPE could be induced in the infected target cells, showing the production of infectious virus. The -aminogroup of lysine functions as a potential ubiquitin acceptor. Like in recent publications, no ubiquitination of the single lysine in PFV wt Gag could be detected. In contrast, four out of five PFV substitution mutants showed ubiquitination of the introduced lysines. Nevertheless, Gag mutants with this covalent modification didn´t have the ability to generate virus like particles (VLPs) or to be pseudotyped by heterologous Env. For FFV wt and mutants, neither Env leader peptide nor Gag ubiquitination could be detected. Differences in components of the ubiquitinination machinery between human cells and feline cells are a probable reason for this. Therefore the analysis of ubiquitiniation of the newly introduced lysines in FFV Gag should be carried out in feline cells as producer cells. Replication of several substitution mutants was strongly inhibited compared to wt virus in the presence of IFN- and -. These results indicated that IFN mediated defense mechanisms could have played a role in the positive selection of the lysine-to-arginine substitution mutants. These results show that the PFV and FFV mutants didn´t differ from wt viruses with respect to their replication strategy. In the context of the long-term host-pathogen coevolution the selection pressure on the virus mediated by cellular restriction factors represents an important aspect dar. The mutation from lysines to arginines implying an altered covalent modification of Gag could have lead to an modification or inhibition of the interaction with antiretroviral restriction factors of the host cell, leading to a positive selection of these mutants during foamyviral evolution.

An important step in the production of infectious HIV-1 particles is maturation of the virus core. This process is completed by cleavage of the capsid (CA) domain of Gag, from its precursor, CA-SP1, by the viral protease. Large deletions in stem-loop 1 (SL1) in the 5' untranslated region (UTR) of HIV-1 genomic RNA (gRNA) delay CA-SP1 processing. SL1 harbours the dimerisation initiation site (DIS) palindrome suggesting that efficient Gag processing may be linked to gRNA dimerization as shown in HIV-2. However, a dimerisation mutant with normal Gag processing was identified. Gag processing defects are hallmarks of late domain mutants, and SL1 mutation was found to result in reduced virus release. HIV-1 hijacks the host's endosomal complexes required for transport (ESCRT) pathway to enable budding. An ESCRT-associated protein, ALIX, is known to be capable of binding to the nucleocapsid (NC) domain of Gag using lipids or RNA as a 'bridge' in vitro. It was hypothesised that SL1 mutation disrupts an RNA-dependent interaction that occurs during virus assembly. Consistent with this, an intact SL1 was found to be required for efficient ALIX function. Increasing the abundance of gRNA in the cell by expressing it in trans accelerated CA-SP1 processing in a manner that required ALIX's binding motif in p6. Gag processing could also be accelerated by introducing previously identified compensatory mutations into the SP1 and NC domains of Gag, in a manner reminiscent of the actions of maturation inhibitor resistance mutations. The effects of the compensatory mutations were also dependent on intact late domain motifs. These data suggest that gRNA is involved in regulating virus budding and maturation through interaction with ALIX. A model is proposed whereby the packaging signal (psi) region of gRNA acts as a bridge between Gag and ALIX, acting as a checkpoint mechanism to promote Gag processing and optimise release of virions that have successfully packaged gRNA.

Glycosaminoglycans (GAGs) are a group of structurally related, naturally occurring polysaccharides, found as the carbohydrate moieties of proteoglycans and sometimes as free polysaccharides. GAGs are expressed ubiquitously on animal cell surfaces and within extracellular matrices. All GAGs are sulfated to various degrees, except hyaluronan, which is always non-sulfated. In this project, GAGs and other sulfated carbohydrates were purified from different marine sources and an algae species living in soil, and their structures were characterised, mainly by NMR spectroscopy. Oversulfated dermatan sulfate (DS), fucosylated chondroitin sulfate (fCS) with interesting anti-metastatic properties, a sulfated fucosylated GlcNAc polymer, naturally-occurring cellulose sulfate and a polysaccharide with a repeating disaccharide unit of IdoA-Gal, were some of the more unusual carbohydrates identified. Common GAGs like hyaluronan, chondroitin sulfate A and C, DS and heparin/heparin sulfate-like polysaccharides were also purified from lumpsuckers and ragoworms. Different depolymerisation techniques were also investigated. The effects of Fentontype reaction and photochemical free radical depolymerisation on DS were studied. Elimination of the reducing end IdoA in the case of Fenton-type produced oligosaccharides was established. The oxidisation of reducing end GalNAc to Nacetylgalactosaminic acid, as a result of both depolymerisation techniques, was also identified. In addition, the effect of mild acid hydrolysis on fCS polysaccharides were studied and this technique was deemed unfitting for the depolymerisation of the molecule, as it causes defucosylation and partial desulfation. Finally, human beta-defensin 2 (HBD2) was expressed recombinantly, as a fusion protein with pE-Sumo, in an attempt to design a high yield expression system capable of producing correctly folded protein. The significance of NMR in the identification of the state of the protein and the need for an expression system capable of carrying out correctly the post-translational modifications was demonstrated.

Human Endogenous Retroviruses (HERV) are derived from exogenous retroviruses that infected inheritable germline tissues millions of years ago and account for 8% of the human genome. Like other retroviruses HERVs encode Gag, Pol and sometimes Env proteins. During a retroviral infection, retroviral Gag recruits the hosts Endosomal Sorting Complex Required for Transport (ESCRT) and associated proteins (ALIX and TSG101) to produce precisely sized viruses from endosomes or the plasma membrane. The ESCRT machinery is also involved in cytokinesis and control growth factor receptor signalling. HERV-K is the most recent HERV family to insert into the genome and is still able to produce mostly intact transcripts, including Gag. When expressed, Gag causes cells to release Virus-Like Particles (VLP) that lack HERV genomes. These retroviral VLP are remarkably similar to a sub-category of extracellular vesicles (EVs) called exosomes. Exosomes require ALIX, TSG101 and the ESCRT machinery for their production. It is possible that HERV-K Gag is required for exosome production or that HERV VLPs are a major contaminant of exosome preparations that account for many of the functions attributed to exosomes. Our data shows that HERV-K Gag over-expression or knockdown did not change the number of EVs released per cell in two cell lines. As well there was no difference in the amount of ALIX and TSG101 in the EVs in these conditions. The most intriguing observation made was the increase of cell number with expression of HERV-K Gag and decrease when HERV-K Gag was knocked down in HEK293T. We are currently unable to conclude the role of HERV-K Gag on EV production and content. We speculate that HERV-K Gag might affect cells through controlling cell proliferation or death, for example by competing with ESCRT machinery to impact signalling through growth factor receptors. This study begins to outline the potential effects HERV-K Gag might have on EV release and cell proliferation.

No description available.

De nombreuses équipes se sont intéressées aux fonctions antiangiogéniquesde PF4 (Platelet Factor 4 ou Facteur Plaquettaire 4). Ses capacités inhibitrices vis-àvisde la prolifération et de la migration des cellules endothéliales in vitro et son effetinhibiteur sur lʼangiogenèse in vivo ne sont plus à démontrer. En revanche, il existeencore de nombreuses interrogations sur les mécanismes dʼaction responsables deson activité antiangiogénique. La chimiokine PF4v1 (Platelet Factor 4 variant 1)mature ne diverge de PF4 que par trois acides aminés mais son potentielangiostatique est beaucoup plus élevé que celui de PF4. Lʼétude comparative de PF4et PF4v1 est donc susceptible de fournir des éclairages intéressants sur lesmécanismes dʼaction de lʼactivité antiangiogénique de PF4. La question se pose desavoir si la différence dʼactivité antiangiogénique entre ces deux chimiokines pourraitsʼexpliquer par des différences dʼaffinité aux GAGs (Glycoaminoglycans), à unrécepteur ou bien aux voies de transduction utilisées pour médier leurs effets ?Comme les mécanismes dʼaction de PF4v1 demeurent très largement incompris(bien que son utilisation comme agent thérapeutique antiangiogénique soit trèsprometteuse), nous avons adopté plusieurs axes de travail pour élucider lescaractéristiques spécifiques de cette chimiokine.Dans un premier temps, nous avons étudié les caractéristiques de diffusibilité etde biodisponibilité des facteurs PF4 et PF4v1. Nous avons déterminé que cesparamètres étaient liés aux affinités de PF4 et PF4v1 pour lʼhéparine et les GAGs, etnous avons identifié lʼacide aminé principalement responsable des différencesobservées.Sur le plan de lʼactivité antiangiogénique de ces deux chimiokines, nousmontrons une absence de corrélation avec lʼaffinité respective aux GAGs. Par contre,nous identifions que la liaison avec un récepteur spécifique pourrait être à lʼorigine dela différence dʼactivité antiangiogénique. Nous avons mené une étude permettant decomprendre le rôle de chaque acide aminé variant entre ces deux chimiokines dansla liaison spécifique au récepteur.Enfin, nous avons développé le premier anticorps monoclonal spécifique de laprotéine PF4v1 qui, de plus, neutralise son activité antiangiogénique. Ce nouvel outilapporte des informations sur la structure et sur lʼactivité biologique de PF4v1. Il nousa aussi permis de démontrer que la protéine PF4v1 est un nouveau biomarqueur ducancer du pancréas. Grâce à ce nouvel outil, nous avons aussi développé un dosageELISA anti-PF4v1. Dans le cadre de la recherche de nouveaux biomarqueurs pour ladétection précoce des cancers, nous pouvons envisager une utilisation de cet ELISAen collaboration avec des services cliniques. / Abstract :

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 104-125).

La protéine du VIH-1 est une protéine essentielle à la transcription et à la réplication du virus. Elle a donc un rôle crucial dans la cellule infectée. On sait qu'une partie de la Tat cellulaire peut être sécrétée, malgré l'absence de séquence signal. En effet, les 2/3 de la Tat cellulaire sont exportés par la cellule T-primaire infectée. Le mécanisme de sécrétion de Tat est non conventionnel et se produit directement à travers la membrane plasmique où Tat est recrutée grâce à sa très forte affinité pour le phosphatidylinositol(4,5)-biphosphate ou PI(4,5)P2 qui est exclusivement localisé à ce niveau. La Tat extracellulaire a un effet délétère sur de nombreux types cellulaires et agit donc comme une toxine virale. Elle constitue un facteur déterminant de l'évolution vers le SIDA. En accord avec cette efficacité de sécrétion par les cellules T primaires infectées, Tat est essentiellement localisée sur la membrane plasmique des T primaires infectées par le VIH-1. Une fraction importante de Tat est donc résidente à la membrane et nous avons recherché un mécanisme pouvant expliquer cette rétention et mis en évidence la palmitoylation. Nos travaux montrent que Tat est palmitoylée, dans des cellules T ainsi que dans les ‘cibles' comme les neurones et les macrophages. Cette palmitoylation, qui inhibe la sécrétion de Tat, est réalisée sur la cystéine 31 de Tat (qui possède 7 cystéines) par l'enzyme DHHC20 avec comme cofacteurs nécessaires les immunophilines (prolyl-isomérases), Cyclophiline A et FKBP12, qui interagissent avec Tat au niveau de la membrane. Nos résultats indiquent aussi qu'en présence de Gag, la palmitoylation de Tat est inhibée. Nous pensons que l'export de la CypA dû à son encapsidation diminuerait la quantité de CypA disponible pour Tat, inhibant la palmitoylation de Tat et permettant sa sécrétion efficace par les cellules infectées. En effet, le VIH-1 encapside 250 copies de CypA/virion, le taux de CypA régulant la virulence des virions produits. Dans les cellules cibles, Tat serait fortement palmitoylée ce qui induirait sa fixation quasi irréversible au PI(4,5)P2, l'empêchant de ressortir de la cellule et permet ainsi un effet cumulatif des doses reçues par la cellules. Cette accumulation de Tat perturbe des processus membranaires dépendant du PI(4,5)P2 tels que la phagocytose et la neurosécrétion. La palmitoylation de Tat est nécessaire pour ces effets. Ces actions de la Tat extracellulaire pourraient participer au développement des infections opportunistes et des troubles neurologiques observé lors du SIDA. / HIV-1 Tat is a small protein that is required for viral transcription and multiplication. It thus has a crucial role in the infected cell. It was known that Tat can be secreted despite its lack of signal sequence. In fact 2/3 of cellular Tat are exported by infected primary T-cells. The unconventional secretion of Tat relies on its interaction with phosphatidylinositol(4,5)-biphosphate or PI(4,5)P2, a lipid that is concentrated within the inner leaflet of the plasma membrane and is strictly required for Tat secretion. Exogenous Tat has deleterious effects on several cell types, indicating that extracellular Tat is involved in evolution to AIDS. Consistent with this secretion efficiency, Tat is mainly localized at the plasma membrane of primary T-cells infected by HIV-1. A large fraction of Tat is resident at the membrane and we looked for a mechanism that could explain this retention and discovered that Tat is palmitoylated. Our studies show that Tat is palmitoylated, both in T-cells and also in ‘target' cells such as neurons and macrophages. Tat palmitoylation inhibits its secretion and is performed on Tat cysteine 31 (Tat has seven cyteines) by the enzyme DHHC20 using immunophilins (prolyl ismerases), Cyclophilin A (CypA) and FKPB12, as cofactors. Our results also indicate that the presence of Gag inhibits Tat palmitoylation. We believe that the export of CypA due to its encapsidation will make less CypA available for Tat, thereby inhibiting Tat palmitoylation. Indeed, HIV-1 encapsidates 250 copies of CypA/virion and the amount of CypA regulates the virulence of produced virions. In target cells, Tat is strongly palmitoylated and this modification induces its almost irreversible binding to PI(4,5)P2, preventing its secretion and allowing cumulative effect of minute Tat doses.Tat palmitoylation enables Tat to severely inhibit various PI(4,5)P2-dependent processes such as phagocytosis and neurosecretion. These effects of extracellular Tat likely contribute to the development of opportunistic infections and neurological disorders observed during AIDS.

Esporos de Bacillus subtilis apresentam propriedades adjuvantes, sendo capazes de aumentar a resposta humoral após a sua coadministração com antígenos misturados ou adsorvidos à sua superfície. Mas, para isso, é necessária a produção de esporos altamente purificados e com rendimentos elevados. Neste trabalho, realizamos com sucesso uma análise quantitativa das condições de esporulação e dos métodos de purificação, o que melhorou a reprodutibilidade do processo e a obtenção de amostras com elevado grau de pureza e rendimento. Avaliamos também as propriedades imunomodulatórias destes esporos, utilizando como antígeno modelo a proteína recombinante Gag-p24 do HIV-1. A coadministração, mas não a adsorção à superfície do esporo, aumentou a imunogenicidade do antígeno sem induzir efeitos deletérios após a administração parenteral em camundongos BALB/c e C57BL/6. Além de promoveram a ativação das APCs, os esporos interagem com receptores relacionados à imunidade inata, devido à ausência do efeito adjuvante em camundongos nocautes para TLR2. Esses resultados abrem perspectivas interessantes para a utilização de esporos como adjuvantes vacinais. / Bacillus subtilis spores have been shown to behave as vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. Nonetheless, such specialized application requires highly purified spore preparations at high yields. In this work, we successfully performed a systematic quantitative analysis of sporulation conditions and spore purification methods, which improved the reproducibility of the process and the obtainment of samples with high purity and yield. Afterwards, we further evaluated the immune modulatory properties of these spores using a recombinant HIV-1 Gag-p24 protein as a model antigen. The co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen, without inducing deleterious effects, after subcutaneous administration to BALB/c and C57BL/6 mice. Besides promoting activation of antigen presenting cells, spores interact with receptors related to innate immunity, due to the absence of the adjuvant effect on TLR2 knockout mice. These results open interesting perspectives for the use of B. subtilis spores as vaccine adjuvants.