DNA immunization, or the use of antigen-expressing DNAs to raise immune responses, represents a novel approach to the study and manipulation of immune responses. In this dissertation, we examine the role of antigen expression at the target site, the role of antigen presentation by bone marrow-derived cells, and the effect of secretion of antigen on DNA-raised responses in mice. Immunizations were conducted using either gene gun delivery of DNA to the epidermis or intramuscular (i.m.) saline injections. To examine the role of antigen expression at the target site, we excised target sites at different time points following immunization. We immunized with plasmid DNA expressing three different forms of antigens: influenza hemagglutinin H1, human growth hormone and influenza nucleoprotein NP (membrane-bound, secreted and intracellular, respectively). We hypothesized that antigen expression at the target site would be essential in initiating immune responses. We demonstrate here that the target site plays different roles in gene gun and i.m. immunizations. We found that the skin target site played an essential role in eliciting maximal antibody and cytotoxic T lymphocyte (CTL) responses by gene gun immunization, although low-level responses can be raised independent of the target site. In contrast, the muscle target site was not essential for eliciting maximal immune responses following i.m. immunization. We suggest that gene gun immunization results in transfection of keratinocytes and bone marrow-derived Langerhans cells at the target site, and these cells together initiate maximal responses. In i.m. immunizations, on the other hand, nonmuscle cells at distal sites, perhaps bone marrow-derived cells in lymphoid tissues, become transfected and are sufficient for initiation of maximal responses. We also examined the role of antigen presentation by bone marrow-derived cells in initiation of CTL responses to influenza NP following gene gun and i.m. immunization. We hypothesized that antigen presentation by bone marrow-derived cells would be involved in initiation of CTL responses. To test this hypothesis, irradiated F1 mice of MHC class I H-2bxd haplotype were reconstituted with bone marrow from either H-2b or H-2d donors, creating two sets of bone marrow chimeric mice (H-2b → H-2bxd and H-2d → H-2bxd, respectively). We immunized the two sets of bone marrow chimeric mice and determined the MHC haplotype restriction of the induced CTL responses using H-2b- or H-2d-restricted peptides of NP. We found that the CTL responses initiated following gene gun and i.m. immunization were restricted to the haplotype of the bone marrow donor. In H-2b→ H-2bxd chimeric mice, CTL responses were restricted to H-2b, while in H-2d→ H-2bxd chimeric mice, CTL responses were restricted to H-2d. Thus, antigen presentation by bone marrow-derived cells, and not by skin or muscle cells, initiates CTL responses following both gene gun and i.m. immunization. Finally, we examined the effect of secretion of a DNA-expressed antigen on antibody responses. We hypothesized that a secreted antigen would raise greater antibody responses than a membrane-bound antigen, due to easier access of a soluble antigen to lymphoid tissues and to uptake by professional antigen-presenting cells and by antigen-specific B cells. We immunized mice with plasmid DNA expressing either a secreted or the normal membrane-bound form of influenza hemagglutinin H1. We found that secretion of H1 (sH1) did not result in enhanced antibody responses, with sH1 appearing to be less effective than H1. We suggest that the effectiveness of DNA immunization with membrane-bound H1 in raising maximal antibody responses may be due to MHC class II presentation of H1 via an endogenous pathway, resulting from direct transfection of bone marrow-derived APCs. We also found that secretion of H1 influenced the predominant IgG subclass of antibody responses raised by i.m. immunization. Secreted H1 raised predominantly IgG1 responses and H1 raised predominantly IgG2a responses. The IgG1 response to sH1 following i.m. immunization was IL-4 dependent, suggesting that the response to sH1 had a T-helper type 2 phenotype. We propose a model for the mechanism of initiation of immune responses by DNA immunization based on our results and taking them within the context of results from other investigators in the field. We propose that DNA immunization may initiate immune responses primarily by the direct transfection of bone marrow-derived cells that then express and present the DNA vaccine-encoded antigen. However, antigen expression by nonhemopoietic cells, particularly in skin, may play a role in raising maximal responses.

A partir de uma biblioteca de cDNA de glândulas de veneno de Micrurus corallinus (cobra-coral), uma serpente da Família Elapidae bastante representada no Brasil e muito comum em áreas florestais tropicais, foram gerados 1.438 Expressed Sequences Tags (ESTs), agrupados em 611 clusters. O banco representa os genes mais expressos na glândula de veneno de M. corallinus. Os transcritos relacionados às toxinas apresentaram ao redor de 46% de representação nesse banco de seqüências. A composição geral das toxinas inclui: toxinas de três dígitos (3FTx) (24%), fosfolipases A2 (PLA2) (16%), lectinas do tipo C (5%), entre outros. O banco permitiu não somente a identificação de possíveis toxinas, mas também de transcritos celulares, sendo a maioria envolvida nas funções fisiológicas de células da glândula de veneno. A maior parte dessas moléculas apresenta um envolvimento na expressão gênica e protéica, o que reflete uma alta especialização do tecido para a síntese de toxinas. A análise do transcriptoma de glândulas de veneno de M. corallinus possibilitou a identificação de alguns candidatos antigênicos para um anti-soro antielapídico alternativo. Cinco candidatos antigênicos foram selecionados por meio da análise do transcriptoma obtido: Atg1 (Grupo das neurotoxinas Homolog 8), Atg2 (Grupo das neurotoxinas Homolog 7/3/1), Atg3 (Outras neurotoxinas 1), Atg4 (Outras neurotoxinas 2) e Atg5 (fosfolipase do tipo A2). Avaliamos a viabilidade de imunização com o DNA desses candidatos. Para isso, os cinco grupos de antígenos foram clonados, primeiramente em pGEM-T e, posteriormente, em pSecTag2A, que é um vetor de expressão em células de mamíferos. As clonagens foram inicialmente testadas em células do tipo COS (transfecção transiente), entretanto não ficou clara a capacidade dessas células em expressar os antígenos. Para a análise da resposta imunológica da vacina de DNA, proteínas recombinantes produzidas em E. coli foram utilizadas para o coating de ELISA para detectar anticorpos presentes no soro primário proveniente da imunização com DNA. Os resultados mostraram que o soro dos animais imunizados foi capaz de reconhecer os antígenos recombinantes. Isso indica que a imunização por DNA em camundongos poderia ser uma boa alternativa em relação à imunização do veneno puro de serpente, que é custosa e muito depende da disponibilidade do veneno. Apesar da necessidade de testes complementares, esse é um resultado promissor, já que a produção de anticorpos pode ser alcançada por via de imunização intramuscular, mais prática para objetivos de produção. / Micrurus corallinus(coral snake) is a tropical forest snake belonging to the Elapidae Family, and is very common in Brazil. From the cDNA library of its venom glands, 1.438 Expressed Sequences Tags (ESTs) were generated and grouped into 611 clusters. This database contains the most expressed genes in the M. corallinus venom glands. The transcripts related to toxins represent approximately 46% of the total genes in this database. The toxin compound consists of: three finger toxins (24%), phospholipases A2 (PLA2s) (16%), type-C lectins (5%), among others. This database allowed not only the identification of possible toxins, but also the identification of cellular transcripts, most of which seems to be involved in physiological functions of venom gland cells. The majority of these molecules are involved in gene and protein expression, revealing the high level of specialization of the tissue for toxin synthesis. The analysis of the M. corallinus venom gland transcriptome allowed the identification of some antigenic candidates for an alternative antielapidic antiserum. Five antigenic candidates were selected after analysing the transcriptome: Atg1 (Homolog group 8), Atg2 (Homolog group 7/3/1), Atg3 (Other neurotoxins 1), Atg5 (A2-type phospholipase). These five antigenic groups were used for DNA immunization. Then they were first cloned in pGEM-T and, after, in pSecTag2A, which is an expression vector in mammal cells. The cloning was tested in COS-type cells (transient transfection), without signs of expression. To analyze the immunological response, recombinant proteins were produced in E. coli and used for ELISA coating to react with the primary serum deriving from the DNA immunization. The results showed that the serum from the immunized animals was able to recognize the recombinant antigens, indicating that the DNA immunization in mice could be a feasible alternative regarding the traditional immunization with crude snake venom, which is costly and heavily dependent on the availability of the venom. Regardless the need for additional tests, this is a promising result, because the antibody production can be achieved by intramuscular immunization, a more effective method when aiming for downstream production.

Le développement des anticorps monoclonaux thérapeutiques est en plein essor notamment à cause de leur bénéfice important pour le traitement des cancers. Cependant, à l’heure actuelle, aucun anticorps monoclonal sur le marché ou en phase III ne cible de RCPGs, en dépit de l’implication grandissante de ces récepteurs dans la carcinogenèse. Parmi les RCPGs les plus pertinents pour l’oncologie, souvent cités dans la littérature et dont certains inhibiteurs chimiques sont en phase clinique avancée, on trouve les deux sous-types de récepteurs des endothélines ETAR et ETBR. Dans ce contexte, mon projet de thèse a consisté à produire des anticorps monoclonaux capables de lier spécifiquement les récepteurs des endothélines, puis à les caractériser dans le but d’évaluer leur potentiel antitumoral. Grâce à une stratégie d’immunisation génique, un ensemble de 27 anticorps monoclonaux, tous spécifiques de la forme native d’ETBR, a été obtenu. Un de ces anticorps, nommé rendomab-B1, a fait l’objet d’une caractérisation précise et s’est révélé être un puissant inhibiteur allostérique d’ETBR. De plus, cette propriété antagoniste a permis de bloquer l’action autocrine antiapoptotique de l’ET-1 sur des cellules endothéliales vasculaires, suggérant ainsi que le rendomab-B1 pourrait être utilisé comme agent thérapeutique afin d’inhiber les effets tumorigènes liés à la suractivation de l’axe ET1/ETBR au niveau de l’endothélium vasculaire tumoral. Par ailleurs, le rendomab-B1 a également été testé sur des lignées de mélanomes humains ; l’absence de fixation de l’anticorps malgré la présence de récepteurs ETB fonctionnels à la surface de ces cellules suggère l’existence d’une forme moléculaire atypique du récepteur, potentiellement spécifique aux mélanomes. A la lumière de ces résultats, le rendomab-B1 apparaît comme un outil prometteur, à la fois pour l’étude structurale et fonctionnelle d’ETBR, mais aussi pour une éventuelle thérapie anticancéreuse. Enfin, les 26 autres anticorps monoclonaux anti-ETBR, actuellement en cours de caractérisation, constituent également des molécules potentiellement intéressantes pour un usage fondamental ou thérapeutique impliquant ETBR. Pour conclure, ces travaux ont démontré l’intérêt de la méthode d’immunisation génique pour la production d’anticorps monoclonaux anti-RCPGs à visée thérapeutique. / For a decade, monoclonal antibodies have become increasingly important for the biotherapeutic management of cancer. However, none of the monoclonal antibodies currently on the market or in late stage clinical trial do target a G-protein coupled receptor in spite of the emerging role of these receptors in tumor progression. Among the therapeutically relevant GPCRs for oncology, the endothelin receptors (ETAR and ETBR) are particularly attractive considering their overexpression in a wide range of tumors and their involvement in various stages of tumorigenesis. In this context, my PhD project consisted in producing and characterizing monoclonal antibodies directed against endothelin receptors with a view to use them as anti-tumor agents. Using an original DNA immunization strategy, we produced a panel of 27 monoclonal antibodies which selectively recognized ETBR expressed at the surface of transfected cells. One of these antibodies, named rendomab-B1, was extensively characterized and proved to be a potent allosteric antagonist of ETBR. Moreover, rendomab-B1 was able to disrupt the autocrine ET1-mediated survival loop on vascular endothelial cells, suggesting that this antibody could be used to prevent the pro-tumorigenic effect due to ET-1 and ETBR upregulation in the tumor-surrounding endothelium. Furthermore, rendomab-B1 binding onto ETBR was also assessed on melanoma cell lines and revealed that a tumor-specific form of ETBR may exist, as illustrated by the poor fixation of rendomab-B1 on these cells in spite of the presence of functional ETB receptors. Together, these results present rendomab-B1 as promising agent, not only for the structural and functional study of ETBR, but also for its therapeutic modulation in the case of cancer for instance. Finally, the other 26 monoclonal antibodies, whose characterization is still ongoing, also constitute potential tools for fundamental or therapeutic applications involving ETBR. To conclude, this work has highlighted the relevance of the DNA immunization approach to generate monoclonal antibodies against the native form of GPCRs.

A partir de uma biblioteca de cDNA de glândulas de veneno de Micrurus corallinus (cobra-coral), uma serpente da Família Elapidae bastante representada no Brasil e muito comum em áreas florestais tropicais, foram gerados 1.438 Expressed Sequences Tags (ESTs), agrupados em 611 clusters. O banco representa os genes mais expressos na glândula de veneno de M. corallinus. Os transcritos relacionados às toxinas apresentaram ao redor de 46% de representação nesse banco de seqüências. A composição geral das toxinas inclui: toxinas de três dígitos (3FTx) (24%), fosfolipases A2 (PLA2) (16%), lectinas do tipo C (5%), entre outros. O banco permitiu não somente a identificação de possíveis toxinas, mas também de transcritos celulares, sendo a maioria envolvida nas funções fisiológicas de células da glândula de veneno. A maior parte dessas moléculas apresenta um envolvimento na expressão gênica e protéica, o que reflete uma alta especialização do tecido para a síntese de toxinas. A análise do transcriptoma de glândulas de veneno de M. corallinus possibilitou a identificação de alguns candidatos antigênicos para um anti-soro antielapídico alternativo. Cinco candidatos antigênicos foram selecionados por meio da análise do transcriptoma obtido: Atg1 (Grupo das neurotoxinas Homolog 8), Atg2 (Grupo das neurotoxinas Homolog 7/3/1), Atg3 (Outras neurotoxinas 1), Atg4 (Outras neurotoxinas 2) e Atg5 (fosfolipase do tipo A2). Avaliamos a viabilidade de imunização com o DNA desses candidatos. Para isso, os cinco grupos de antígenos foram clonados, primeiramente em pGEM-T e, posteriormente, em pSecTag2A, que é um vetor de expressão em células de mamíferos. As clonagens foram inicialmente testadas em células do tipo COS (transfecção transiente), entretanto não ficou clara a capacidade dessas células em expressar os antígenos. Para a análise da resposta imunológica da vacina de DNA, proteínas recombinantes produzidas em E. coli foram utilizadas para o coating de ELISA para detectar anticorpos presentes no soro primário proveniente da imunização com DNA. Os resultados mostraram que o soro dos animais imunizados foi capaz de reconhecer os antígenos recombinantes. Isso indica que a imunização por DNA em camundongos poderia ser uma boa alternativa em relação à imunização do veneno puro de serpente, que é custosa e muito depende da disponibilidade do veneno. Apesar da necessidade de testes complementares, esse é um resultado promissor, já que a produção de anticorpos pode ser alcançada por via de imunização intramuscular, mais prática para objetivos de produção. / Micrurus corallinus(coral snake) is a tropical forest snake belonging to the Elapidae Family, and is very common in Brazil. From the cDNA library of its venom glands, 1.438 Expressed Sequences Tags (ESTs) were generated and grouped into 611 clusters. This database contains the most expressed genes in the M. corallinus venom glands. The transcripts related to toxins represent approximately 46% of the total genes in this database. The toxin compound consists of: three finger toxins (24%), phospholipases A2 (PLA2s) (16%), type-C lectins (5%), among others. This database allowed not only the identification of possible toxins, but also the identification of cellular transcripts, most of which seems to be involved in physiological functions of venom gland cells. The majority of these molecules are involved in gene and protein expression, revealing the high level of specialization of the tissue for toxin synthesis. The analysis of the M. corallinus venom gland transcriptome allowed the identification of some antigenic candidates for an alternative antielapidic antiserum. Five antigenic candidates were selected after analysing the transcriptome: Atg1 (Homolog group 8), Atg2 (Homolog group 7/3/1), Atg3 (Other neurotoxins 1), Atg5 (A2-type phospholipase). These five antigenic groups were used for DNA immunization. Then they were first cloned in pGEM-T and, after, in pSecTag2A, which is an expression vector in mammal cells. The cloning was tested in COS-type cells (transient transfection), without signs of expression. To analyze the immunological response, recombinant proteins were produced in E. coli and used for ELISA coating to react with the primary serum deriving from the DNA immunization. The results showed that the serum from the immunized animals was able to recognize the recombinant antigens, indicating that the DNA immunization in mice could be a feasible alternative regarding the traditional immunization with crude snake venom, which is costly and heavily dependent on the availability of the venom. Regardless the need for additional tests, this is a promising result, because the antibody production can be achieved by intramuscular immunization, a more effective method when aiming for downstream production.

The immune checkpoint blockade is a novel approach of cancer therapy, which markedly enhanced treatment efficacy of several cancer types. However, the frequency of cancer patients non-responding to this treatment is high. Establishment of predictive markers to distinguish patients suitable for the immune checkpoint blockade would enhance the number of patients receiving benefit from the therapy. This dissertation thesis focuses on the enhancement of efficacy of immune checkpoint inhibitors (ICIs) and predictive markers in experimental models of mouse tumours induced by TC-1 and TC-1/A9 cell lines and its clones with deactivation of interferon (IFN)-γ signalling (TC-1/dIfngr1 and TC-1/A9/dIfngr1), or CD80 molecule (TC-1/dCD80-1). IFN-γ is presumed to be the main inducer of programmed death ligand 1 (PD-L1) and a major histocompatibility complex I (MHC-I). Moreover, PD-L1 expression may predict sensitivity to PD-1/PD-L1 blockade. Non-functional IFN-γ signalling or downregulated MHC-I expression has been associated with resistance to ICIs in some patients. We found that IFNs type I (IFN-α and IFN-β) induced the expression of PD-L1 and MHC-I on TC-1/A9/dIfngr1 tumour cells with reversible downregulation of both molecules. We also showed that deactivation of IFN-γ signalling in TC-1/A9 cells was not a...

MHCI molecules are constitutively expressed in all nucleated cells and play a key role in antigen presentation to CD8+ T lymphocytes. One of the tumor immune evasion strategies is MHCI expression downregulation. This leads to an impaired recognition of tumor antigens by CD8+ T lymphocytes that are unable to start the immune response. Since the MHCI expression downregulation occurs in up to 90 % of some tumors it is neccesary to have a clinical relevant tumor model without a MHCI surface expression that would be used for testing of immunotherapeutic approaches. This thesis describes a production of new model cell lines of TC-1 tumor cells with irreversibly downregulated MHCI. That was achieved by an inactivation of B2m, which is a part of MHCI, by gene editing using CRISR/Cas9. The B2m inactivation was confirmed by flow cytometry, western blot and sanger sequencing of single alleles. The inactivation slowed down the cell growth for both in vitro and in vivo. The cell metastatic activity was not affected. The tumors established by cells without the B2m expression are not sensitive to DNA vaccine against HPV16 E7 oncoprotein by a pBSC/PADRE.E7GGG vaccine. The main effector function against these tumors possess the NK1.1+ cells. In a therapeutic vaccination experiment it was repeatedly achieved of...

Japanese encephalitis virus (JEV), a member of the family flaviviridae, is one of the most important pathogens of the developing countries, causing high mortality and morbidity amongst children. The present study is aimed at the development of a DNA vaccine for Japanese Encephalitis (JE). As a first step towards developing a DNA vaccine for JE, an eukaryotic expression plasmid encoding the envelope (E) glycoprotein of Japanese Encephalitis Virus (pCMXENV) was constructed. This plasmid expresses the E protein intracellularly, when transfected into Vero cells in culture. Several independent immunization and intracerebral (i.e.) JEV challenge experiments were carried out and the results indicate that 51% and 59% of the mice are protected from lethal i.e. JEV challenge, when immunized with pCMXENV via intramuscular (i.m.) and intranasal (i.n.) routes respectively. JEV-specific antibodies were not detected in pCMXENV-immunized mice either before or after challenge. JEV-specific T cells were observed in mice immunized with pCMXENV, which increased significantly after JEV challenge indicating the presence of vaccination-induced memory T cells. Enhanced production of interferon-y (EFN-y) and complete absence of interleukin-4 (IL-4) in splenocytes of pCMXENV-immunized mice on restimulation with JEV antigens in vitro indicated that the protection is likely to be mediated by T helper (Th) lymphocytes of the Thl sub type. These results demonstrated that immunization with a plasmid DNA expressing intracellular form of JEV E protein confers significant protection against i.e. JEV challenge even in the absence of detectable antiviral antibodies. We then examined the potency of JEV DNA vaccines as well as that of the inactivated mouse brain derived BIKEN vaccine in the i.e. challenge model. The results indicate that all the mice immunized with BIKEN JE vaccine were protected against i.e. JEV challenge while 50% protection was observed in case of mice immunized with pJME or pJNSl and 38% protection was observed in pCMXENV-immunized mice. Immunization with both pJME and pJNSl resulted in 66% protection. These results indicate that the BIKEN JE vaccine confers better protection against i.e. JEV challenge than DNA vaccines. The fact that the BIKEN vaccine conferred better protection against i.e. JEV challenge than DNA vaccines indicated that the i.e. JEV challenge model can be exploited further to examine the potency of different DNA vaccine constructs. Towards this goal, we constructed plasmids that encode secretory or nonsecretory forms of JEV E protein and examined their potency in the i.e. JEV challenge model. Our results indicate that i.m. immunization of mice with plasmid encoding secretory form of JEV E protein confers higher level (75%-80%) protection than those encoding nonsecretory forms. Cytokine analysis of splenocytes isolated from DNA immunized mice after stimulation in vitro with JEV revealed that immunization with plasmid encoding secretory form of JEV E protein induces both Thl and Th2 responses while those encoding nonsecretory forms induce only Thl type of response. Thus, synthesis of secretory form of JEV E protein results in an altered immune response leading better protection against i.e. JEV challenge. Based on our studies, we propose that both cellular and humoral immune responses play a key role in protective immunity against i.e. JEV challenge and DNA vaccines that can induce higher levels of neutralizing antibodies will be as efficient as the BIKEN vaccine in conferring protection against i.e. JEV challenge.

Recombinant strategies are today dominating in thedevelopment of modern subunit vaccines. This thesis describesstrategies for the production and recovery of protein subunitimmunogens, and how genetic design of the expression vectorscan be used to adapt the immunogens for incorporation intoadjuvant systems. In addition, different strategies fordelivery of subunit vaccines by RNA or DNA immunization havebeen investigated. Attempts to create general production strategies forrecombinant protein immunogens in such a way that these areadapted for association with an adjuvant formulation wereevaluated. Different hydrophobic amino acid sequences, beingeither theoretically designed or representing transmembraneregions of bacterial or viral origin, were fused on gene leveleither N-terminally or C-terminally to allow association withiscoms. In addition, affinity tags derived fromStaphylococcus aureusprotein A (SpA) or streptococcalprotein G (SpG), were incorporated to allow efficient recoveryby means of affinity chromatography. A malaria peptide, M5,derived from the central repeat region of thePlasmodium falciparumblood-stage antigen Pf155/RESA,served as model immunogen in these studies. Furthermore,strategies forin vivoorin vitrolipidation of recombinant immunogens for iscomincorporation were also investigated, with a model immunogendeltaSAG1 derived fromToxoplasma gondii. Both strategies were found to befunctional in that the produced and affinity purified fusionproteins indeed associated with iscoms. The iscoms werefurthermore capable of inducing antigen-specific antibodyresponses upon immunization of mice, and we thus believe thatthe presented strategies offer convenient methods for adjuvantassociation. Recombinant production of a respiratory syncytial virus(RSV) candidate vaccine, BBG2Na, in baby hamster kidney(BHK-21) cells was investigated. Semliki Forest virus(SFV)-based expression vectors encoding both intracellular andsecreted forms of BBG2Na were constructed and found to befunctional. Efficient recovery of BBG2Na could be achieved bycombining serum-free production with a recovery strategy usinga product-specific affinity-column based on a combinatoriallyengineered SpA domain, with specific binding to the G proteinpart of the product. Plasmid vectors encoding cytoplasmic or secreted variants ofBBG2Na, and employing the SFV replicase for self-amplification,was constructed and evaluated for DNA immunization against RSV.Both plasmid vectors were found to be functional in terms ofBBG2Na expression and localization. Upon intramuscularimmunization of mice, the plasmid vector encoding the secretedvariant of the antigen elicited significant anti-BBG2Na titersand demonstrated lung protective efficacy in mice. This studyclearly demonstrate that protective immune responses to RSV canbe elicited in mice by DNA immunization, and that differentialtargeting of the antigens expressed by nucleic acid vaccinationcould significantly influence the immunogenicity and protectiveefficacy. We further evaluated DNA and RNA constructs based on the SFVreplicon in comparison with a conventional DNA plasmid forinduction of antibody responses against theP. falciparumPf332-derived antigen EB200. In general,the antibody responses induced were relatively low, the highestresponses surprisingly obtained with the conventional DNAplasmid. Also recombinant SFV suicide particles inducedEB200-reactive antibodies. Importantly, all immunogens inducedan immunological memory, which could be efficiently activatedby a booster injection with EB200 protein. <b>Keywords</b>: Affibody, Affinity chromatography, Affinitypurification, DNA immunization, Expression plasmid, Fusionprotein, Hydrophobic tag, Iscoms, Lipid tagging, Malaria,Mammalian cell expression, Recombinant immunogen, RespiratorySyncytial Virus, Semliki Forest virus, Serum albumin,Staphylococcus aureusprotein A, Subunit vaccine,Toxoplasma gondii

<p>Recombinant strategies are today dominating in thedevelopment of modern subunit vaccines. This thesis describesstrategies for the production and recovery of protein subunitimmunogens, and how genetic design of the expression vectorscan be used to adapt the immunogens for incorporation intoadjuvant systems. In addition, different strategies fordelivery of subunit vaccines by RNA or DNA immunization havebeen investigated.</p><p>Attempts to create general production strategies forrecombinant protein immunogens in such a way that these areadapted for association with an adjuvant formulation wereevaluated. Different hydrophobic amino acid sequences, beingeither theoretically designed or representing transmembraneregions of bacterial or viral origin, were fused on gene leveleither N-terminally or C-terminally to allow association withiscoms. In addition, affinity tags derived from<i>Staphylococcus aureus</i>protein A (SpA) or streptococcalprotein G (SpG), were incorporated to allow efficient recoveryby means of affinity chromatography. A malaria peptide, M5,derived from the central repeat region of the<i>Plasmodium falciparum</i>blood-stage antigen Pf155/RESA,served as model immunogen in these studies. Furthermore,strategies for<i>in vivo</i>or<i>in vitro</i>lipidation of recombinant immunogens for iscomincorporation were also investigated, with a model immunogendeltaSAG1 derived from<i>Toxoplasma gondii</i>. Both strategies were found to befunctional in that the produced and affinity purified fusionproteins indeed associated with iscoms. The iscoms werefurthermore capable of inducing antigen-specific antibodyresponses upon immunization of mice, and we thus believe thatthe presented strategies offer convenient methods for adjuvantassociation.</p><p>Recombinant production of a respiratory syncytial virus(RSV) candidate vaccine, BBG2Na, in baby hamster kidney(BHK-21) cells was investigated. Semliki Forest virus(SFV)-based expression vectors encoding both intracellular andsecreted forms of BBG2Na were constructed and found to befunctional. Efficient recovery of BBG2Na could be achieved bycombining serum-free production with a recovery strategy usinga product-specific affinity-column based on a combinatoriallyengineered SpA domain, with specific binding to the G proteinpart of the product.</p><p>Plasmid vectors encoding cytoplasmic or secreted variants ofBBG2Na, and employing the SFV replicase for self-amplification,was constructed and evaluated for DNA immunization against RSV.Both plasmid vectors were found to be functional in terms ofBBG2Na expression and localization. Upon intramuscularimmunization of mice, the plasmid vector encoding the secretedvariant of the antigen elicited significant anti-BBG2Na titersand demonstrated lung protective efficacy in mice. This studyclearly demonstrate that protective immune responses to RSV canbe elicited in mice by DNA immunization, and that differentialtargeting of the antigens expressed by nucleic acid vaccinationcould significantly influence the immunogenicity and protectiveefficacy.</p><p>We further evaluated DNA and RNA constructs based on the SFVreplicon in comparison with a conventional DNA plasmid forinduction of antibody responses against the<i>P. falciparum</i>Pf332-derived antigen EB200. In general,the antibody responses induced were relatively low, the highestresponses surprisingly obtained with the conventional DNAplasmid. Also recombinant SFV suicide particles inducedEB200-reactive antibodies. Importantly, all immunogens inducedan immunological memory, which could be efficiently activatedby a booster injection with EB200 protein.</p><p><b>Keywords</b>: Affibody, Affinity chromatography, Affinitypurification, DNA immunization, Expression plasmid, Fusionprotein, Hydrophobic tag, Iscoms, Lipid tagging, Malaria,Mammalian cell expression, Recombinant immunogen, RespiratorySyncytial Virus, Semliki Forest virus, Serum albumin,<i>Staphylococcus aureus</i>protein A, Subunit vaccine,<i>Toxoplasma gondii</i></p>