Lasse Eggers Pedersen

Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae

CRISPR/Cas9 is a simple and efficient tool for targeted and marker-free genome engineering. Here, we report the development and successful application of a multiplex CRISPR/Cas9 system for genome engineering of up to 5 different genomic loci in one transformation step in bakers yeast Saccharomyces cerevisiae. To assess the specificity of the tool we employed genome re-sequencing to screen for off-target sites in all single knock-out strains targeted by different gRNAs. This extensive analysis identified no more genome variants in CRISPR/Cas9 engineered strains compared to wild-type reference strains. We applied our genome engineering tool for an exploratory analysis of all possible single, double, triple, quadruple and quintuple gene disruption combinations to search for strains with high mevalonate production, a key intermediate for the industrially important isoprenoid biosynthesis pathway. Even though we did not overexpress any genes in the mevalonate pathway, this analysis identified strains with mevalonate titers greater than 41-fold compared to the wild-type strain. Our findings illustrate the applicability of this highly specific and efficient multiplex genome engineering approach to accelerate functional genomics and metabolic engineering efforts.

Immune markers and correlates of protection for vaccine induced immune responses.

Vaccines have been a major innovation in the history of mankind and still have the potential to address the challenges posed by chronic intracellular infections including tuberculosis, HIV and malaria which are leading causes of high morbidity and mortality across the world. Markers of an appropriate humoral response currently remain the best validated correlates of protective immunity after vaccination. Despite advancements in the field of immunology over the past few decades currently there are, however, no sufficiently validated immune correlates of vaccine induced protection against chronic infections in neither human nor veterinary medicine. Technological and conceptual advancements within cell-mediated immunology have led to a number of new immunological read-outs with the potential to emerge as correlates of vaccine induced protection. For T(H)1 type responses, antigen-specific production of interferon-gamma (IFN-γ) has been promoted as a quantitative marker of protective cell-mediated immune responses over the past couple of decades. More recently, however, evidence from several infections has pointed towards the quality of the immune response, measured through increased levels of antigen-specific polyfunctional T cells capable of producing a triad of relevant cytokines, as a better correlate of sustained protective immunity against this type of infections. Also the possibilities to measure antigen-specific cytotoxic T cells (CTL) during infection or in response to vaccination, through recombinant major histocompatibility complex (MHC) class I tetramers loaded with relevant peptides, has opened a new vista to include CTL responses in the evaluation of protective immune responses. Here, we review different immune markers and new candidates for correlates of a protective vaccine induced immune response against chronic infections and how successful they have been in defining the protective immunity in human and veterinary medicine.

Induction of Foot-and-Mouth Disease Virus-Specific Cytotoxic T Cell Killing by Vaccination

ABSTRACT Foot-and-mouth disease (FMD) continues to be a significant threat to the health and economic value of livestock species. This acute infection is caused by the highly contagious FMD virus (FMDV), which infects cloven-hoofed animals, including large and small ruminants and swine. Current vaccine strategies are all directed toward the induction of neutralizing antibody responses. However, the role of cytotoxic T lymphocytes (CTLs) has not received a great deal of attention, in part because of the technical difficulties associated with establishing a reliable assay of cell killing for this highly cytopathic virus. Here, we have used recombinant human adenovirus vectors as a means of delivering FMDV antigens in a T cell-directed vaccine in pigs. We tested the hypothesis that impaired processing of the FMDV capsid would enhance cytolytic activity, presumably by targeting all proteins for degradation and effectively increasing the class I major histocompatibility complex (MHC)/FMDV peptide concentration for stimulation of a CTL response. We compared such a T cell-targeting vaccine with the parental vaccine, previously shown to effectively induce a neutralizing antibody response. Our results show induction of FMDV-specific CD8+ CTL killing of MHC-matched target cells in an antigen-specific manner. Further, we confirm these results by MHC tetramer staining. This work presents the first demonstration of FMDV-specific CTL killing and confirmation by MHC tetramer staining in response to vaccination against FMDV.

Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

In all vertebrate animals, CD8+ cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC effectively individualizes the immune response of each member of the species. We have recently developed efficient methods to generate recombinant human MHC-I (also known as human leukocyte antigen class I, HLA-I) molecules, accompanying peptide-binding assays and predictors, and HLA tetramers for specific CTL staining and manipulation. This has enabled a complete mapping of all HLA-I specificities (“the Human MHC Project”). Here, we demonstrate that these approaches can be applied to other species. We systematically transferred domains of the frequently expressed swine MHC-I molecule, SLA-1*0401, onto a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules. A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data indicate that it is possible to extend the biochemical and bioinformatics tools of the Human MHC Project to other vertebrate species.

Identification of peptides from foot‐and‐mouth disease virus structural proteins bound by class I swine leukocyte antigen (SLA) alleles, SLA‐1*0401 and SLA‐2*0401

Characterization of the peptide-binding specificity of swine leukocyte antigen (SLA) class I and II molecules is critical to the understanding of adaptive immune responses of swine toward infectious pathogens. Here, we describe the complete binding motif of the SLA-2*0401 molecule based on a positional scanning combinatorial peptide library approach. By combining this binding motif with data achieved by applying the NetMHCpan peptide prediction algorithm to both SLA-1*0401 and SLA-2*0401, we identified high-affinity binding peptides. A total of 727 different 9mer and 726 different 10mer peptides within the structural proteins of foot-and-mouth disease virus (FMDV), strain A24 were analyzed as candidate T-cell epitopes. Peptides predicted by the NetMHCpan were tested in ELISA for binding to the SLA-1*0401 and SLA-2*0401 major histocompatibility complex class I proteins. Four of the 10 predicted FMDV peptides bound to SLA-2*0401, whereas five of the nine predicted FMDV peptides bound to SLA-1*0401. These methods provide the characterization of T-cell epitopes in response to pathogens in more detail. The development of such approaches to analyze vaccine performance will contribute to a more accelerated improvement of livestock vaccines by virtue of identifying and focusing analysis on bona fide T-cell epitopes.

Industrial glucoamylase fed‐batch benefits from oxygen limitation and high osmolarity

The market for glucoamylase is large and very competitive and the production process has been optimized through several decades. So far a thorough characterization of the process has not been published, but previous academic reports suggest that the process suffers from severe byproduct formation. In this study we have carried out a thorough characterization of a process as close as possible to the industrial reality. The results show that the oxygen‐limited phases of the process have the highest glucoamylase yields on carbon and that the byproducts are efficiently reused in late phases of the process. An alternative process with low glucose concentration show that high osmolarity is beneficial for the process, and we conclude that oxygen limitation, high osmolarity, and the associated byproduct metabolism are important for the efficiency of the process. Biotechnol. Bioeng. 2012;109: 116–124.

Swine Leukocyte Antigen (SLA) class I allele typing of Danish swine herds and identification of commonly occurring haplotypes using sequence specific low and high resolution primers

The swine major histocompatibility complex (MHC) genomic region (SLA) is extremely polymorphic comprising high numbers of different alleles, many encoding a distinct MHC class I molecule, which binds and presents endogenous peptides to circulating T cells of the immune system. Upon recognition of such peptide-MHC complexes (pMHC) naïve T cells can become activated and respond to a given pathogen leading to its elimination and the generation of memory cells. Hence SLA plays a crucial role in maintaining overall adaptive immunologic resistance to pathogens. Knowing which SLA alleles that are commonly occurring can be of great importance in regard to future vaccine development and the establishment of immune protection in swine through broad coverage, highly specific, subunit based vaccination against viruses such as swine influenza, porcine reproductive and respiratory syndrome virus, vesicular stomatitis virus, foot-and-mouth-disease virus and others. Here we present the use of low- and high-resolution PCR-based typing methods to identify individual and commonly occurring SLA class I alleles in Danish swine. A total of 101 animals from seven different herds were tested, and by low resolution typing the top four most frequent SLA class I alleles were those of the allele groups SLA-3*04XX, SLA-1*08XX, SLA-2*02XX, and SLA-1*07XX, respectively. Customised high resolution primers were used to identify specific alleles within the above mentioned allele groups as well as within the SLA-2*05XX allele group. Our studies also suggest the most common haplotype in Danish pigs to be Lr-4.0 expressing the SLA-1*04XX, SLA-2*04XX, and SLA-3*04XX allele combination.

Identification of swine influenza virus epitopes and analysis of multiple specificities expressed by cytotoxic T cell subsets

BackgroundMajor histocompatibility complex (MHC) class I peptide binding and presentation are essential for antigen-specific activation of cytotoxic T lymphocytes (CTLs) and swine MHC class I molecules, also termed swine leukocyte antigens (SLA), thus play a crucial role in the process that leads to elimination of viruses such as swine influenza virus (SwIV). This study describes the identification of SLA-presented peptide epitopes that are targets for a swine CTL response, and further analyses multiple specificities expressed by SwIV activated CTL subsets.FindingsFour SwIV derived peptides were identified as T cell epitopes using fluorescent influenza:SLA tetramers. In addition, multiple CTL specificities were analyzed using peptide sequence substitutions in two of the four epitope candidates analyzed. Interestingly both conserved and substituted peptides were found to stain the CD4-CD8+ T cell subsets indicating multiple specificities.ConclusionsThis study describes a timely and cost-effective approach for viral epitope identification in livestock animals. Analysis of T cell subsets showed multiple specificities suggesting SLA-bound epitope recognition of different conformations.

Increased B and T Cell Responses in M. bovis Bacille Calmette-Guérin Vaccinated Pigs Co-Immunized with Plasmid DNA Encoding a Prototype Tuberculosis Antigen.

The only tuberculosis vaccine currently available, bacille Calmette-Guérin (BCG) is a poor inducer of CD8+ T cells, which are particularly important for the control of latent tuberculosis and protection against reactivation. As the induction of strong CD8+ T cell responses is a hallmark of DNA vaccines, a combination of BCG with plasmid DNA encoding a prototype TB antigen (Ag85A) was tested. As an alternative animal model, pigs were primed with BCG mixed with empty vector or codon-optimized pAg85A by the intradermal route and boosted with plasmid delivered by intramuscular electroporation. Control pigs received unformulated BCG. The BCG-pAg85A combination stimulated robust and sustained Ag85A specific antibody, lymphoproliferative, IL-6, IL-10 and IFN-γ responses. IgG1/IgG2 antibody isotype ratio reflected the Th1 helper type biased response. T lymphocyte responses against purified protein derivative of tuberculin (PPD) were induced in all (BCG) vaccinated animals, but responses were much stronger in BCG-pAg85A vaccinated pigs. Finally, Ag85A-specific IFN-γ producing CD8+ T cells were detected by intracellular cytokine staining and a synthetic peptide, spanning Ag85A131-150 and encompassing two regions with strong predicted SLA-1*0401/SLA-1*0801 binding affinity, was promiscuously recognized by 6/6 animals vaccinated with the BCG-pAg85A combination. Our study provides a proof of concept in a large mammalian species, for a new Th1 and CD8+ targeting tuberculosis vaccine, based on BCG-plasmid DNA co-administration.