Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506
Performing Department
Diagnostic Medicne Pathobiolog
Non Technical Summary
Porcine reproductive and respiratory syndrome virus (PRRSV) is the most costly disease in the history of swine production. Vaccines and other control measures have not proved effective and a new generation of vaccines is still years away. Our previous work showed that genetically modified (GM) pigs, which lack expression of CD163 on macrophages, are completely resistant to infection with PRRSV. Since CD163 is important for homeostasis, including the regulation of inflammation and immunity, the goal of the proposed project is to construct a pig that possesses a modified CD163 that prevents PRRSV infection while retaining normal CD163 biological functions. The first objective is the use of a novel in vitro system to map the CD163 peptide sequences recognized by genotype 1 and genotype 2 PRRSV. CD163 modifications include deletions, alanine substitutions, and domain swaps with sequences from CD163-like proteins. The results will be used to develop and test the same CD163-modifications in pigs. CD163 gene modifications include the use of the latest CRISPR and homologous recombination technologies. The final objective is to take advantage of CD163 knockout pigs to understand the participation of CD163 in inflammation and immunity. Studies include gene profiling and phenotypic analysis of macrophages along with responses to infection with porcine circovirus type 2. The tools and techniques developed in the proposed project can be applied to other infectious diseases of swine.
Animal Health Component
5%
Research Effort Categories
Basic
70%
Applied
5%
Developmental
25%
Goals / Objectives
Porcine reproductive and respiratory syndrome virus (PRRSV) is the most costly disease in the history of swine production. Vaccines and other control measures have not proved effective and a new generation of vaccines is still years away. Molecular comparisons between North American and European viruses place all PRRSV isolates into one of two genotypes, Type 2 or Type 1, respectively. At the present time, both Type 1 and Type 2 viruses circulate in US herds. Even though Type 1 and Type 2 viruses possess only about 70% identity at the nucleotide level, they share a tropism for CD163-positive macrophages, establish long-term infections, and produce similar clinical signs. During the past 25 years, the economic impact of PRRSV is estimated to be between $12 and $15 billion in losses to producers. More important is the impact of the virus on animal wellbeing, as many infected pigs are "poor doers" that fail to thrive. Vaccines have been largely ineffective in controlling the disease and possess several limitations, including reversion to virulence, absence of protection against genetically diverse field isolates, continuous shedding, and transplacental infections. The next generation of vaccines designed to correct these deficiencies remain several years away. In 2016, we demonstrated that genetically modified (GM) pigs that lack CD163 expression on macrophages are completely resistant to infection with PRRSV. The results show that CD163 is required forPRRSV infection and is the first clear demonstration that CD163 modifications provide a mechanism to completely prevent the infection of pigs with PRRSV. The knockout of CD163 in pigs was accomplished using the genomic editing tool, CRISPR/Cas9. Since there is no foreign DNA involved, this genetic modification does not otherwise "adulterate" the pig genome. In fact, the CD163 knockout pigs were constructed by deleting as few as 11 bp out of the 2.7 billion bp that comprise the pig genome. In addition to its role as a PRRSV receptor, CD163 has several important functions, including the removal of excess heme and the regulation of inflammation and immunity. The main objective of the proposed research is to refine the genomic editing of CD163 to preserve these biological functions while blocking PRRSV infection. The approach is the genetic modification of pigs using CRISPR and more traditional homologous recombination techniques. A second objective is the use of CD163 knockout (KO) pigs to gain a better understanding of inflammation and immunity in the absence of CD163. The eventual deliverable is a CD163 modification that confers resistance of a pig to PRRSV, while preserving CD163 function. The same approach can be applied to other pig diseases.Objective 1. Map the CD163 peptide sequences responsible for infection with PRRSV Type 1 viruses. Both Type 1 and Type 2 viruses utilize CD163 as a PRRSV receptor; however, Type 1 and Type 2 viruses recognize different peptide sequences on the CD163 molecule. The purpose of this objective is to map regions in CD163 responsible for recognition of CD163 by Type 1viruses. Mapping the determinants for Type 2 viruses is covered by other funding. The approach is to utilize an in vitro system to test modified CD163 proteins that possess mutations, including amino acid deletions and substitutions, and domain swaps with the porcine or human CD163-like protein (CD163-L1). The deliverable is to locate the smallest modification in CD163 that prevents infection of transfected HEK cells with Type 1 viruses.Objective 2. Construct CD163 pigs and evaluate for expression of CD163 and resistance to PRRSV infection. The CD163cDNA modifications developed under Objective 1 will be tested in pigs. The approach is to utilize CRSPR/Cas9 and homologous recombination (HR) to produce GM pigs that possess the same CD163 modifications described under Objective 1.Macrophages from the CD163-modified pigs will be tested for permissiveness in vitro using a panel of Type 1 and Type 2 viruses. The macrophage infection results are then confirmed by infection of CD163-modified pigs. The overall functional state of CD163 will be evaluated by measuring circulating Hp levels in the CD163-modified pigs.Objective 3. Evaluate the function of CD163 in inflammation and immunity. The macrophage M1 versus M2 dichotomy is similar to Th1-Th2 dichotomy described for T-helper responses. M1 macrophages, when activated, are associated with pro- inflammatory activities that provide the initial responses to virus infection, including antigen presentation to T cells. In contrast, M2 macrophages, are "alternatively" activated by regulatory (TH2) cytokines, such as IL-10, to mediate the anti-inflammatory activities important for the repair of damaged tissues. Macrophages possessing the CD163 surface phenotype are associated with M2 responses. We hypothesize that CD163 KO pigs will exhibit significant alterations in the overall balance between pro- and anti-inflammatory activities; i.e., favoring an environment skewed towards pro-inflammatory (M1) cytokines and responses. The purpose of Objective 3 is to assess the overall immunological state of CD163 KO pigs by performing immune gene profiling and surface phenotyping of alveolar macrophages, and by assessing the response to porcine circovirus type 2 (PCV2) infection. The approach is to compare the wild type and CD163 KO pigs. The results will be used to understand the changes that occur in the absence of CD163, especially those changes that affect immunity and growth performance.
Project Methods
Objective 1. Map the CD163 peptide sequences responsible for infection with PRRSV Type 1 viruses.CD163 cDNA fragments are cloned into a pcDNA3.1 expression vector and expressed as EGFP-labeled fusion products. Mutations in CD163 fragments are performed using standard molecular techniques, including PCR, gene synthesis and site directed mutagenesis. Fragments are cloned into the Xba, Kpn sites as described in Figure 2. Plasmid constructs are transfected into HEK cells and expression of the CD163-EGFP construct confirmed by Western blot. EGFP provides a visual tag for CD163 expression and an antigen tag for detection of CD163 in Western blots and for immuno-precipitation. The Type 1 and Type 2 recombinant viruses used for infection express a red fluorescent protein as a separate mRNA. We have in possession several recombinant viruses that possess fluorescent tags. Successful infection of the transfected HEK cells is evident by the presence of red fluorescence (see Figure 2).Modifications to the CD163 cDNA are performed at three levels or steps: 1) deletion of large and small segments containing one or more SRCR domains, 2) domain swaps between CD163 and CD163L1, and 3) alanine substitutions to identify the contribution of individual amino acids. Finer mapping will be conducted by making hybrid SRCR domains combining SRCR5 and CD163L1 SRCR8 peptide sequences.Objective 2. Construct CD163 pigs and evaluate for expression of CD163 and resistance to PRRSV infection.Guide sequences 20 nucleotides in length are used to target CRISPR deletions to specific locations and all gene modifications are confirmed by DNA sequencing. CD163 expression is confirmed using flow cytometry of macrophages or of peripheral blood mononuclear cells after stimulation with GM-CSF. Assessment of CD163 function by the measurement of serum haptoglobin (Hp) levels. provides a convenient method for determining the overall functional state of CD163 in CD163-modified pigs. The amount of Hp in serum is measured using a porcine-specific Hp ELISA kit (Genway Biotech Inc.) and steps performed according to the manufacturer's instructions.The permissiveness of CD163-modified pigs for Type 1 or Type 2 viruses is first evaluated by testing alveolar macrophages against a panel of viruses, as described above. The second step is to infect CD163-modified pigs with representative Type 1 and Type 2 viruses. The methods used to detect infection of pigs incorporate a combination of PRRSV RT-PCR and PRRSV N protein serology. The number of pigs required for each CD163 modification is based on the hypothesis to be tested. In the case where the expected outcome is complete resistance to virus infection by both Type 1 and Type 2 viruses, as few as eight pigs can be used: four wild type positive control pigs (two pigs per virus genotype) and four test pigs (two pigs per PRRSV genotype). Two pigs are utilized for each treatment, just in case one pig falls out of the study for a reason unrelated to infection or the CD163 modification. WT and CD163 modified pigs infected with the same virus are mingled together in a single pen to allow the continuous exposure of CD163-modified pigs to virus shed from the WT pen mates. If the expected outcome of a particular CD163 modification is less than full protection (only partial resistance to infection), the number of pigs used depends on the amount of resistance expected. For example, if the expected result is a log10 reduction in viremia for CD163-modified pigs, then 10 pigs will be used in the WT control and 10 pigs in the CD163 modified group. Again, pigs with different genetic modifications and infected with the same virus are mingled together.Objective 3. Evaluate the function of CD163 in inflammation and immunity.The data obtained from the experiments described under this objective are important for several reasons. For example, pigs that lack CD163 have an impaired ability to metabolize heme. In the absence of this mechanism for dampening inflammation, CD163 KO pigs may be skewed towards pro-inflammatory responses. An environment dominated by inflammatory cytokines, such as TNF, may not only influence immunity and disease progression, but can have a negative impact on overall growth performance of the pig. On the other hand, the absence of CD163 on macrophages can prevent the release of important cytokines normally induced through the crosslinking of CD163 by bacteria. In total, the absence of CD163 may contribute to a state of overall dysregulation, resulting in the increased susceptibility of KO pigs to other pathogens. The approach focuses on three areas of investigation: 1) gene profiling of alveolar macrophages, 2) phenotypic analysis of alveolar macrophages, and 3) the response of CD163 KO pigs to infection with a systemic pathogen, PCV2. The experimental design is to compare responses of CD163 KO with WT pigs. We already have on hand macrophages and peripheral blood mononuclear cells (PBMCs) from several pigs representing different CD163 modifications, which are stored in liquid nitrogen or Trizol (for preservation of RNA). These stored samples will used for the gene expression profiling and phenotypic analysis.Gene expression profiling of macrophages using nanostring technology (Nanostring Company) is performed as described under Preliminary Results. The gene expression profiles will be analyzed for PAMs from 10 pigs from each group. The results are presented as genes in KO pigs that are up-regulated or down-regulated differently compared to macrophages from WT pigs. Whenever possible, the elevated expression of immunologically relevant genes, such as cytokines, will be confirmed using protein assays.PAMs are naturally enriched for mature macrophages that express CD169 and CD163. The antibodies used to measure the surface marker expression on alveolar macrophages cells represent markers related to macrophage function. An example of flow cytometry for the simultaneous expression of CD169 and CD163 on alveolar macrophages is shown in Figure 4. Flow cytometry is performed using a Fortessa instrument, which can easily measure up to six colors or markers on a single cell.PCV2 infection is a convenient tool for understanding immunity against a systemic infection that affects the respiratory tract. For the model, 10 WT and 10 CD163 KO pigs, approximately four weeks of age are infected with approximately 103 TCID50 of a PCV2b isolate in 5 ml of tissue culture media. Since wild-type PCV2b (GenBank Accession #JQ692110) does not propagate to high levels in cell culture, we took advantage of the heat stability of PCV2 to make a virus preparation from a lymph node suspension enriched for PCV2. Pigs are evaluated daily for the presence of clinical signs. Blood samples are collected weekly until the end of the study, at 42 days. The lungs are scored for gross pathology and reported as the percentage of whole lung affected by pneumonia. Tissue samples are removed and immediately placed in 10% neutral buffered formalin. The fixed tissues are processed and stained with hematoxylin and eosin (H&E stain) and evaluated by a board-certified pathologist. PCV2 viremia is measured in serum. PCV2 DNA is quantified using a SYBR Green method. Standard curves and positive and negative controls are included on each plate. Plasmid DNA containing a positive control template is used to prepare standards. The PCR assay results are reported as log10 PCV2 DNA starting quantity per 20 μl reaction volume. PCV2 antibodies are using a Luminex-based. Antibody results are reported as a sample to positive (S/P) binding ratio.