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No description available.

Skin is an organ with a complex structure which plays a crucial role in thebody’s defence against external threats and in maintaining major homeostatic functions. The need for in vitro models that mimic the in vivo milieu is therefore high and relevant with various applications including, among others, penetration, absorption, and toxicity studies. In this context, the choice of the biomaterial that will provide a 3D scaffold to the cultured cells is defining the model’s success. The FN-4RepCT silk is here suggested as a potent biomaterial for skin tissue engineering applications. This recombinantly produced spider silk protein (FN-4RepCT), which can self-assemble into fibrils, creates a robust and elastic matrice with high bioactivity, due to its functionalization with the fibronectin derived RGD-containing peptide. Hence it overcomes the drawbacks of other available biomaterials either synthetic or based on animal derived proteins. Additionally, the FN-4RepCT silk protein can be cast in various 3D formats, two of which are utilized within this project. We herein present a bilayered skin tissue equivalent supported by the FN-4RepCT silk. This is constructed by the combination of a foam format, integrated with dermal fibroblasts and endothelial cells, and a membrane format supporting epidermal keratinocytes. As a result, a vascularized dermal layer that contains ECM components (Collagen I, Collagen III, and Elastin) is constructed and attached to an epidermal layer of differentiated keratinocytes.The protocol presented in this project offers a successful method of evenly integrating cells in the FN-4RepCT silk scaffold, while preserving their ability to resume some of their major in vivo functions like proliferation, ECM secretion, construction of vascular networks, and differentiation. The obtained results were evaluated with immunofluorescence stainings of various markers of interest and further analysed, when necessary, with image processing tools. The results that ensued from the herein presented protocol strongly suggest that the FN-4RepCT silk is a promising biomaterial for skin tissue engineering applications.

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. Since its initial detection in a Canadian commercial swine herd in 1991, PMWS has been detected in all swine producing regions of the world and is now a serious economic problem to the swine industry. The objectives of this dissertation were to biologically, genetically and experimentally characterize both PCV1 and PCV2, to identify the genetic determinant(s) for virulence and replication, and to develop an effective genetically-engineered vaccine against PCV2 infection and PMWS. The genetic heterogeneity of PCV2 and PCV1 isolates from different geographic origins were determined. We found that, although PCV1 and PCV2 genomes were very conserved, some minor genomic variation exists among PCV1 isolates and PCV2 isolates. The nonpathogenic PCV1 and pathogenic PCV2 share only about 76% nucleotide sequence identity but have similar genomic organization. The highest sequence variability among PCV isolates is found in the immunogenic ORF2 capsid gene. Based on the sequence data in this dissertation, a universal polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed that is capable of detecting all known PCV isolates and differentiating between infections by nonpathogenic PCV1 and pathogenic PCV2. In order to study the structural and functional relationship of PCV genes and to develop a genetically-engineered vaccine, we constructed infectious DNA clones of both PCV1 and PCV2. By using the PCV2 infectious clone, we showed that pigs can be infected by direct intrahepatic injection of PCV2 infectious DNA clone. The pathological lesions and clinical disease associated with PCV2 infection were more definitively characterized by using the infectious DNA clone. We found that PCV2 is the primary but not the sole causative agent of PMWS, as the full spectrum of clinical PMWS was not reproduced by the infectious PCV2 DNA clone although pathological lesions characteristic of PMWS were reproduced. A chimeric vaccine was constructed by cloning the immunogenic capsid gene of the pathogenic PCV2 into the genomic backbone of the non-pathogenic PCV1 virus. We showed that the resulting chimeric PCV1-2 vaccine virus, retained the non-pathogenic nature of PCV1 but induced a protective immune response against a wild-type PCV2 challenge. In vaccinated pigs, the chimeric PCV1-2 vaccine reduced PCV2 viremia length and serum virus loads and reduced pathological lesions such as lymphoid depletion (LD) and histiocytic replacement (HR) in lymphoid tissues, inflammation and discoloration of the lymph nodes. The amounts of PCV2 antigen and PCV2 genomic copy loads in lymph node tissues were also significantly reduced. Our results indicated that the attenuated chimeric PCV1-2 virus induces protective immunity against PCV2 infection and thus could serve as an effective vaccine against PCV2 and PMWS. To improve the safety of the vaccine, we attempted to identify the genetic determinant(s) for PCV2 virulence. An isolate of PCV2 was serially passaged for 120 times in PK-15 cells. After 120 passages, a total of two amino acid mutations were identified in the capsid protein of the passage 120 virus (VP120), P110A and R191S. Compared to other known PCV1 and PCV2 sequences, the two amino acid mutations in PCV2 VP120 are unique. The VP120 virus was biologically characterized in vitro and experimentally characterized in specific-pathogen-free (SPF) pigs. The two amino acid mutations resulted in an enhanced replication ability of PCV2 VP120 in PK-15 cells and an attenuated phenotype in infected pigs. The P110A and R191S mutations in the capsid protein either alone or collectively are likely important for PCV2 virulence and replication. In summary, we genetically characterized PCV2 isolates from different geographic regions and developed a PCR-RFLP assay. We constructed and characterized infectious DNA clones of PCV1 and PCV2, and developed a genetically engineered vaccine against PCV2 infection. We also identified the genetic determinants for PCV2 virulence and replication. The vaccine developed in this study, when it becomes available, will help the swine industry control this important pathogen. / Ph. D.

Both technological and experimental advancements in the field of biotechnology have allowed scientists to make leaps in areas such nucleic acid, antibody, and recombinant protein technologies. Here we focus on the use of recombinant proteins as molecular recognition motifs, wound healing biomaterials, and agents for cell cycle pathway elucidation are discussed. The author's primary project is described in chapters 2 and 3, and is focused on designed leucine-rich repeat proteins which offer increased stability, modularity, and surface area for binding interactions. These proteins bind at least two muramyl dipeptide ligands with picomolar to nanomolar affinity (Kd1 = 0.04 – 3.5 nM); as measured by fluorescence quenching experiments and ITC. The longest designed repeat, CLRR8, has a Kd app value of 1.0 nM which is comparable to full length native NOD2 protein. Molecular docking simulations revealed the locations of two potential binding sites and their respective interactions. The series of proteins represents a foundation for a high affinity and highly specific molecular recognition scaffold that has the potential to bind a variety of ligands. Previously the author contributed to the design of recombinant keratin proteins, and the work in Chapter 4 builds on the original design to allow for controlled degradation in wound healing systems. Site-directed mutagenesis was utilized to introduce these degradation sites, and modified keratin proteins were expressed with no differences to native recombinant keratin proteins. Success in engineering a variation of native keratin protein with no issues in expression lay the foundation for further engineering of native keratin or other relevant proteins for improved functionality. Chapter 5 describes steps towards producing human Aurora borealis (Bora) protein, an important substrate in cell cycle regulation, by in vitro transcription-translation with locked Ser–Pro analogues. This will allow for the elucidation of the active isomerization form to ensure proper cell division. Site-directed mutagenesis successfully introduced the amber codon to relevant Ser-Pro sites at positions 274 and 278. These mutated Bora genes along with modified ribosomes and aminoacyl tRNA will allow for the incorporation of locked dipeptide analogues. Expression of native Bora was carried out as a control, and appeared to express in dimeric form. The experiments carried out in Chapter 5 describe and outline all the molecular biology work completed and to be completed for this novel method of studying cis-trans isomerization in living cells. / Doctor of Philosophy / Sequencing of the human genome and the rapid development of gene editing and recombinant DNA technologies paved the way for a massive shift in the pharmaceutical industry. The first pharmaceutical companies in the 19th century started as fine chemicals businesses. The discovery of penicillin introduced antibiotics, and improved synthetic techniques led to the giants we know as big pharma today. Today, in the 21st century both computing and biotechnology has allowed for great leaps forward in precision medicine. Biotechnology refers to the manipulation of living organisms or their components to produce useful commercial products. In the pharmaceutical industry this refers to genetic engineering for novel pharmaceuticals. Here, we focus on the use of recombinant technology to create proteins for use in biomedical applications. Recombinant proteins are proteins formed by laboratory methods of molecular cloning. Through this technology, we are able to elucidate sequence-structure-function relationships of proteins, and determine their specific functions. Additionally, recombinant methods allow us to fine tune or modify the sequences of natural proteins to be more effective scaffolds or reagents. Chapter 3 focuses on the development of synthetic proteins for medical diagnostics. We designed a protein scaffold, based on natural innate immunity proteins, to detect bacteria cell wall components. Chapter 4 focuses on the engineering of keratin protein with applications in wound healing. We introduce controlled degradation of the biomaterial for use in potential drug delivery systems at the wound site. Chapter 5 focuses on the use of recombinant technologies aiding in the elucidation of a regulatory protein's function in cell division.

Genetic engineering has allowed the design and production of recombinant antibodies (rmAbs) in plants. Nowadays, rmAbs are used in the treatment of a wide range of pathologies such as infectious diseases, inflammatory diseases and cancer, making rmAbs an important group of biomolecules within the pharmaceutical and biotechnology industry. By the time this study was started, the immunoglobulin G (IgG) was the antibody isotype predominantly expressed in plants. In recent years Modular DNA cloning technology has facilitated antibody engineering, with the development and expression of new rmAbs formats. However, there is hardly any study where different antibody formats are produced and compared in terms of yield and neutralizing capacity. Therefore, the starting point of the first chapter of this thesis is a comparative study where five different formats of the same commercial rmAb (Infliximab) against the human cytokine Tumor Necrosis Factor (TNF-¿) were expressed and compared. The results obtained in Chapter 1 demonstrate that both the isotype and the structure of the chosen rmAb influence the yield and the neutralizing capacity of rmAb. The expression of new antibody formats not only refers to the antibody isotype or structure; the format also refers to the combination of antibody idiotypes, leading to the production of oligo or polyclonal antibodies. Therefore, the possibility of co-expressing different monoclonal antibodies simultaneously in plants (creating oligoclonal or polyclonal formats) was raised. In the second chapter of this thesis, the expression of three rmAbs against the Ebola virus glycoprotein was studied. The three rmAbs were transiently expressed in N. benthamiana individually, by establishing separated production lines; in parallel, all three rmAbs were also co-expressed simultaneously in the same production line. The results obtained in this chapter demonstrated that the individual expression of rmAbs is feasible. However, when all three rmAbs are co-expressed, a drastic decrease in the binding of the antibody to the antigen was observed due to chain shuffling, as each heavy chain (HC) can be bound to any light chain (LC) other than its cognate chain, giving rise to an antibody cocktail with lower activity. With the objective of developing a method that allows co-expression of several rmAb in a single production line, we next proposed to exploit the viral interference phenomenon (also known as superinfection exclusion, SE). The results shown in Chapter three demonstrate that the production of an oligoclonal cocktail composed of 36 rmAbs in plants was possible using a viral expression system showing SE. The data obtained in this chapter showed that the resulting oligoclonal cocktail was active and capable of neutralizing toxic activities of the venom of the snake Bothrops asper in vitro and in vivo, wich was used as a model for studying the efficacy of the oligoclonal antibodies produced. The results of this thesis confirm and support the use of plants as platforms for the expression of alternative formats of antibodies. / La ingeniería genética ha permitido el diseño y la producción de anticuerpos recombinantes (rmAbs) en plantas. Hoy en día, los rmAbs se utilizan en el tratamiento de un amplio rango de patologías como enfermedades infecciosas, enfermedades inflamatorias y cáncer, convirtiéndose en un importante grupo de biomoléculas dentro de la industria farmacéutica y biotecnológica. Hasta la fecha de este estudio, en plantas se ha producido mayoritariamente la inmunoglobulina del tipo G (IgG). Gracias al desarrollo de la ingeniería del ADN recombinante y de la ingeniería de anticuerpos, es posible diseñar y producir nuevos formatos de rmAbs. Sin embargo, apenas existen estudios comparativos donde se demuestre si el formato de anticuerpo elegido es el idóneo en términos de rendimiento y capacidad neutralizante. Por tanto, el punto de partida del primer Capítulo de esta tesis consistió en la realización de un estudio comparativo de la expresión en plantas de cinco formatos distintos de un mismo rmAb comercial (Infliximab) frente a la citoquina humana Tumor Necrosis Factor (TNF-¿). Los resultados obtenidos en el Capítulo 1 demuestran que tanto el isotipo como la estructura del rmAb elegido influye en los niveles de rendimiento y en la capacidad neutralizante del rmAb. La expresión de nuevos formatos de anticuerpos no solo afecta al isotipo o a la estructura de las regiones constantes, sino que también se puede incluir en este término la expresión conjunta de distintos idiotipos de anticuerpos recombinantes, dando lugar a anticuerpos policlonales u oligoclonales recombinantes. Por tanto en esta tesis se planteó la posibilidad de co-expresar simultáneamente distintos anticuerpos monoclonales en plantas formando un cóctel oligoclonal. En el segundo Capítulo de esta tesis se diseñaron tres rmAbs frente a la glicoproteína de la cubierta del virus del Ébola. Los tres rmAbs se expresaron transitoriamente en N. benthamiana de manera individual mediante el establecimiento de líneas paralelas de producción y también se co-expresaron los tres rmAbs simultáneamente en una misma línea de producción. Los resultados obtenidos en este Capítulo demostraron que la expresión de los rmAbs de manera individual es factible. Sin embargo, cuando se co-expresan los tres rmAbs se observa una drástica disminución en la unión del anticuerpo al antígeno debido al barajado de cadenas, fenómeno por el cual cada cadena pesada (HC) se puede unir con cualquier cadena ligera (LC) distinta de su acompañante, dando lugar a un anticuerpo con una baja actividad. Finalmente, con el objetivo de desarrollar un método que permita co-expresar en una misma línea de producción varios rmAbs de forma reproducible se propuso explotar el fenómeno de la exclusión viral, un característica propia de los virus de plantas. Los resultados mostrados en el Capítulo 3 demuestran que es posible la producción de un cóctel oligoclonal compuesto por 36 rmAbs en N. benthamiana aprovechando el fenómeno de la exclusión viral. Los datos obtenidos en este capítulo muestran que el cóctel oligoclonal producido de esta forma mantiene intactas las actividades de los anticuerpos individuales y es capaz de neutralizar las actividades tóxicas del veneno de la serpiente Bothrops asper en ensayos in vitro e in vivo. Los resultados de esta tesis confirman y avalan el uso de las plantas como plataformas de expresión de formatos alternativos de anticuerpos. / El desenvolupament de l'enginyeria genètica ha permès el disseny i la producció d'anticossos recombinants (rmAbs) en plantes. Hui en dia, els rmAbs s'utilitzen en el tractament d'un ampli rang de patologies com malalties infeccioses, malalties inflamatòries i càncer convertint-se en un important grup de biomolècules dins de les indústries farmacèutiques i biotecnològiques. Fins a la data, s'han expressat majoritàriament la immunoglobulina del tipus G. Gràcies al desenvolupament de l'enginyeria de l'ADN recombinant i l'enginyeria dels anticossos s'han desenvolupat i expressat formats alternatius de rmAbs. Tanmateix, hi ha molts pocs estudis comparatius on es demostra si el format de l'anticòs elegit influeix en el rendiment i en la capacitat neutralitzant. Per tant, el punt de partida del primer Capítol d'esta Tesi és la realització d'un estudi comparatiu on s'expressen cinc formats diferents d'un mateix anticòs comercial (Infliximab) front a la citocina humana Tumor Necrosis Factor (TNF-¿). Els resultats obtesos demostren que tant l'isotip com l'estructura del rmAb elegit influeix en el rendiment i en la capacitat neutralitzant del rmAb. L'expressió de nous formats d'anticossos no sols afecta a l'isotip o a l'estructura del rmAb sinó que també pot incloure's dins d'aquest concepte l'expressió individual i l'expressió conjunta de diferents rmAbs. Partint d'aquesta hipòtesi, es va plantejar la possibilitat de co-expressar diferents rmAbs (còctel oligoclonal) en plantes. En el segon Capítol d'esta tesi es dissenyaren tres rmAbs front a la glicoproteïna del virus de l'Ébola. Els tres rmAbs s'expressaren transitòriament en N. benthamiana de manera individual mitjançant l'establiment de línies paral·leles de producció i també es co-expressaren els tres rmAbs en la mateixa línia de producció. Els resultats obtesos en este Capítol demostraren que l'expressió dels rmAbs de manera individual és factible. Tanmateix, quan es co-expressaren els tres rmAbs s'observà una dràstica disminució en la unió de l'anticòs a l'antigen com a conseqüència del shuffling chain, pel qual la cadena pesada (HC) s'uneix amb qualsevol cadena lleugera (LC) diferent a la seua acompanyant, formant un anticòs amb una baixa capacitat d'unió a l'antigen. Amb l'objectiu de desenvolupar un mètode que permeta co-expressar, en una mateixa línia de producció, un còctel oligoclonal es proposà explotar el fenomen de l'exclusió viral. Els resultats obtesos en el Capítol 3 demostren que l'expressió d' un còctel oligoclonal format per 36 rmAbs en plantes és possible. Els resultats mostren que el nostre còctel oligoclonal es capaç de neutralitzar activitats tòxiques del verí de la serp Bothrops asper en assaigs in vitro i in vivo. Els resultat obtesos en aquesta Tesi confirmen i avalen l'ús de les plantes com plataformes d'expressió de formats alternatius d'anticossos. / Huet Trujillo, E. (2017). Nuevas metodologías para la producción de anticuerpos recombinantes en plantas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90469

Genome editing technologies have advanced from methods that rely on nucleases to catalyze programmed double-strand breaks (DSBs), which are known to cause deleterious side effects, to next-generation reagents that perform more controlled chemistry using DSB-independent approaches. Base editing and prime editing are ideally suited for small-scale modifications, but methods to achieve large-payload gene insertion have been lacking. Recent technology development efforts have advanced strategies that employ eukaryotic transposases, bacterial recombinases/transposases, and retroelements, yet these enzymes broadly suffer from either inflexibility of target site requirements or non-specific, genome-wide insertion profiles. The ability to precisely and safely insert kilobase-scale DNA cargos at user-defined loci remained challenging due to a dearth of programmable transposase tools. CRISPR-associated transposon (CAST) systems represent a unique opportunity to solve this longstanding challenge. CASTs are diverse (types I-B, I-D, I-F, and V-K) heteromeric, macromolecular machineries that require multivalent protein-protein interactions, each of which represents a potential kinetic bottleneck, inefficiency, or failure point when porting from bacteria to mammalian cells. Through meticulous, step-by-step engineering involving functional assays, bioinformatic mining, homolog screening, structure-guided engineering, and directed evolution, we have iteratively improved overall editing efficiencies of type I-F CASTs, identified an essential bacterial co-factor, and reached editing efficiencies that approach therapeutic relevance. Together, this work represents a critical advancement towards a broad platform for targeted genomic integration of large DNA payloads.

The ability of a genetically-engineered <i>Erwirzia carotovora</i> subsp. <i>carotovora</I> (Ecc) strain to transfer recombinant chromosomal DNA or plasmids to wildtype Ecc or <i>Pseudomonas fluorescens</i> was tested on filters, within soil microcosms, and <i>in planta</i>. Ecc was engineered by chromosomal insertion of a disarmed <i>endo</i>-pectate lyase gene marked with a 1.4kb DNA fragment conferring kanamycin resistance. Plasmids RPI and pBR322 were introduced separately into engineered Ecc clones. These strains served as donors in genetic transfer experiments. No transfer of the inserted kan marker or of pBR322 was observed under any experimental condition. In filter matings, RPI was transferred to wildtype Ecc at a frequency of 3.6 X 10⁻² transconjugants per donor (TPD) and to P. <i>fluorescens</i> at a frequency of 2.4 X 10⁻⁵ TPD. In matings conducted in potato tubers inoculated using sewing needles, the respective frequencies were 4.0 X 10⁻³ and 2.0 X 10⁻³, while matings on potato slices yielded frequencies of 4.7 X 10⁻² and 2.3 X 10⁻². In soil microcosms, the maximum transfer frequencies observed were 2.3 X 10³ and 8.4 X 10⁻⁵ TPD. / Master of Science

The unfolding of recombinant DNA, from research technique to political issue, is described. As a research technique, recombinant DNA (abbreviated rDNA) has opened up new vistas in biological and other fields of research. But its potential yet unproven hazard has created uneasy feelings toward the technique. The controversial nature of the issue finally launched rDNA into the political sphere, involving scientists, the public at large, and Congress in efforts to control the development of the field. The first group to regulate rDNA was the scientists. The scientific community called for a voluntary moratorium on experiments perceived as potentially dangerous at the time. It was an unprecedented act. The National Institutes of Health subsequently issued guidelines for a safe execution of rDNA experiments to minimize potential dangers to public health and well-being. Efforts of the scientific community to control rDNA was seen, however, as a politics of expertise. Challenges to this "technocratic" approach soon emerged. Vocal members of the public suspected expert decision makers as being biased toward scientific interests, reducing rDNA to a technical issue. They rejected the experts’ tunnel vision and demanded a say in decisions. Public participation in the decision-making process precipitated community debates at locations where rDNA research was ongoing. A democratic approach to decision-making proved to be a viable policy-making mode. The ensuing local and state laws, however, seemed inadequate to cover global consequences of rDNA. In an effort to unify regulations of the field, Congress attempted to legislate on the subject. Resistance from the scientific community, which regard legislative control as rigid and unnecessary, was one of the causes of diminishing congressional interest in the matter. None of the introduced bills was enacted. For complex policy areas with uncertain yet far-reaching scientific and societal consequences -- like rDNA -- this dissertation recommends a policy-making process where scientists, interested lay persons, politicians, public administrators, and other relevant parties participate in structured communications prior to an emerging controversy. To facilitate the process, establishment of National Science Fora is recommended. / Ph. D.

Bei bhPIV3 handelt es sich um ein bovines Parainfluenzavirus Typ 3 (bPIV3), dessen Ober-flächenproteingene gegen jene des humanen Parainfluenzavirus Typ 3 (hPIV3) ausgetauscht wurden. Dieses ursprünglich als experimenteller Impfstoff gegen hPIV3 entwickelte Virus wurde darüber hinaus als Impfvektor zur Expression anderer viraler Antigene verwendet. Im Rahmen der hier vorgestellten Arbeit wurden die ersten bhPIV3-basierten Vektoren für nicht-virale Antigene hergestellt und in einem ersten Versuch evaluiert. Dazu wurden ein reverses Genetiksystem zur Herstellung rekombinanter bhPIV3 in einem neuen Labor aufgebaut und fünf neue rekombinante Viren erhalten, welche zusätzlich Antigene des Mycobacterium tuberculosis (M. tb.) exprimieren. Balb/c-Mäuse wurden intranasal mit den bhPIV3-Vektoren infiziert, so dass sowohl deren Replikation als auch der induzierte protektive Effekt gegenüber M. tb.-Neuinfektionen getestet werden konnte. In einem ersten Versuch zeigte sich, dass eine Immunisierung mit den rekombinanten Viren allein keine Schutzwirkung entfaltet. Als Boost-Impfung nach Gabe des Bacille Calmette Guérin (BCG) zeigten einige Vektoren jedoch einen signifikanten protektiven Effekt. In einem Folgeversuch konnten diese Beobachtungen jedoch bislang nicht bestätigt werden, so dass weitere Versuche durchzuführen sind, bevor eine endgültige Aussage bezüglich des hervorgerufenen Schutzeffektes getroffen werden kann. In einem weiteren Tierversuch wurde gezeigt, dass die Baumwollratte ein Tiermodell darstellt, in dem bhPIV3 erheblich schlechter repliziert als hPIV3. Trotz der eingeschränkten Replikation induzierte bhPIV3 neutralisierende Antikörpertiter gegen hPIV3, die mit durch hPIV3 induzierten Titern vergleichbar waren. Mit Hilfe eines neu generierten rekombinanten Virus, welches das grün fluoreszierende Protein EGFP exprimiert, konnte ein Weg aufgewiesen werden, die Bestimmung neutralisierender Antikörpertiter deutlich zu vereinfachen. / The initial objective of this project was to establish a reverse genetic system for generation of recombinant bovine/human parainfluenza virus type 3 (bhPIV3), a bovine PIV3 (bPIV3) in which the bhPIV3 glycoprotein genes are replaced by their counterparts of human PIV3 (hPIV3). In addition, methods needed to characterise virus infectivity, genetic integrity and relevant in vitro phenotypes were established. The reverse genetics system was used to add individual mycobacterium tuberculosis (M. tb.) open reading frames (ORFs) as supernumerary gene units to the bhPIV3 genome and to rescue bhPIV3 vectors that expressed M. tb. antigens. In addition, a similar vector expressing the enhanced green fluorescent protein (EGFP) was constructed. Following the in vitro characterization of the derived viral vectors, the M. tb. vectors were evaluated for their efficacy to protect against M. tb. aerosole challenge in the Balb/c mouse model for tuberculosis. Although, in a single experiment, vaccination with bhPIV3 vectors alone did not confer any protection against M. tb. challenge, a boost with selected bhPIV3 vectors after Bacille Calmette Guérin (BCG) priming was successful in conferring protective efficacy against M. tb. challenge. A repeat of this challenge study could not confirm the initial observation, and further experiments are needed to determine whether the observed protection can be reliably reproduced. Evaluation of the bhPIV3 vectors in the cotton rat model showed that this small animal model is suitable to evaluate the attenuation phenotype of bhPIV3 compared to human parainfluenza virus type 3 (hPIV3). Although replication of bhPIV3 was highly restricted compared to hPIV3, hPIV3 neutralizing antibody titers induced by bhPIV3 infection were similar to those induced by hPIV3 infection. Studies with bhPIV3 expressing EGFP led to a new fluorescence based assay to determine hPIV3 neutralizing antibody titers. This assay could save time and resources in hPIV3 serology.