Human Calreticulin Research Articles

Production of active human iduronate-2-sulfatase (IDS) enzyme in Nicotiana benthamiana

Many strategies have been developed to produce high levels of biologically active recombinant proteins in plants for biopharmaceutical purposes. However, the production of an active form of human iduronate-2-sulfatase (hIDS) for the treatment of Hunter syndrome by enzyme replacement therapy (ERT) is challenging due to the requirement for cotranslational modification by a formylglycine-producing enzyme encoded by sulfatase modifying factor 1 (hSUMF1) at the Cys84 residue, which converts it to C(alpha)-formylglycine. In this study, we have shown that hIDS can be highly expressed in N. benthamiana by using different constructs. Among them, BiP-GB1-L-dCBD1-2L-8xHis-L-6xHis-3L-EK-hIDS-HDEL (GB1-CBD1-hIDS) showed a high expression level when transiently co-expressed with the turnip crinkle virus gene silencing suppressor P38 and GB1-fused human calreticulin (GB1-CRT1) as a folding enhancer. The hSUMF1 was co-expressed with hIDS for cotranslational modification. The full-length recombinant proteins were purified using Ni2+-NTA affinity resin followed by enterokinase treatment to obtain tag-free hIDS. The N-terminal fragment was removed using microcrystalline cellulose (MCC) beads. The purified active form of hIDS can successfully cleave the sulfate group from an artificial substrate, 4-nitrocatechol sulfate, at a similar level to commercial hIDS expressed in animal cells. These results suggest that plants could be a promising platform for the production of recombinant hIDS.

Human gene-engineered calreticulin mutant stem cells recapitulate MPN hallmarks and identify targetable vulnerabilities

Calreticulin (CALR) mutations present the main oncogenic drivers in JAK2 wildtype (WT) myeloproliferative neoplasms (MPN), including essential thrombocythemia and myelofibrosis, where mutant (MUT) CALR is increasingly recognized as a suitable mutation-specific drug target. However, our current understanding of its mechanism-of-action is derived from mouse models or immortalized cell lines, where cross-species differences, ectopic over-expression and lack of disease penetrance are hampering translational research. Here, we describe the first human gene-engineered model of CALR MUT MPN using a CRISPR/Cas9 and adeno-associated viral vector-mediated knock-in strategy in primary human hematopoietic stem and progenitor cells (HSPCs) to establish a reproducible and trackable phenotype in vitro and in xenografted mice. Our humanized model recapitulates many disease hallmarks: thrombopoietin-independent megakaryopoiesis, myeloid-lineage skewing, splenomegaly, bone marrow fibrosis, and expansion of megakaryocyte-primed CD41+ progenitors. Strikingly, introduction of CALR mutations enforced early reprogramming of human HSPCs and the induction of an endoplasmic reticulum stress response. The observed compensatory upregulation of chaperones revealed novel mutation-specific vulnerabilities with preferential sensitivity of CALR mutant cells to inhibition of the BiP chaperone and the proteasome. Overall, our humanized model improves purely murine models and provides a readily usable basis for testing of novel therapeutic strategies in a human setting.

Yeast Secretes High Amounts of Human Calreticulin without Cellular Stress.

The ER chaperone calreticulin (CALR) also has extracellular functions and can exit the mammalian cell in response to various factors, although the mechanism by which this takes place is unknown. The yeast Saccharomyces cerevisiae efficiently secretes human CALR, and the analysis of this process in yeast could help to clarify how it gets out of eukaryotic cells. We have achieved a secretion titer of about 140 mg/L CALR in our S. cerevisiae system. Here, we present a comparative quantitative whole proteome study in CALR-secreting yeast using non-equilibrium pH gradient electrophoresis (NEPHGE)-based two-dimensional gel electrophoresis (2DE) as well as liquid chromatography mass spectrometry in data-independent analysis mode (LC-MSE). A reconstructed carrier ampholyte (CA) composition of NEPHGE-based first-dimension separation for 2DE could be used instead of formerly commercially available gels. Using LC-MSE, we identified 1574 proteins, 20 of which exhibited differential expression. The largest group of differentially expressed proteins were structural ribosomal proteins involved in translation. Interestingly, we did not find any signs of cellular stress which is usually observed in recombinant protein-producing yeast, and we did not identify any secretory pathway proteins that exhibited changes in expression. Taken together, high-level secretion of human recombinant CALR protein in S. cerevisiae does not induce cellular stress and does not burden the cellular secretory machinery. There are only small changes in the cellular proteome of yeast secreting CALR at a high level.

Immunoreactivity of Brugia malayi Calreticulin and Its Domains with Sera of Different Categories of Bancroftian Filarial Patients.

We identified calreticulin in human filaria Brugia malayi (BmCRT) that shares 97% homology with Wuchereria bancrofti calreticulin (WbCRT), but only 56% with human calreticulin. We found that BmCRT binds C1q and prevents complement-mediated parasite death; immunization with BmCRT leads to parasite death in a rodent model of the infection. BmCRT could, therefore, be a potential vaccine candidate. In the present study, we determined the levels of BmCRT-reactive IgG and its isotype in bancroftian filarial subjects. Recombinant BmCRT (rBmCRT) was prepared, and the sera of endemic normal subjects (EN), microfilaraemics (Mf+) and chronic amicrofilaraemics (ChMf-) from a bancroftian filaria-endemic area and normal subjects from filaria-non-endemic area (NEN) were probed for IgG and its isotypes reacting with rBmCRT and its domains rN, rP and rC. rBmCRT and its rN domain-reactive IgG levels were high in EN and Mf+ groups; rC domain and rP domain showed moderate and very little reactivity, respectively. NEN sera were non-reactive. Moderate levels of rBmCRT-reactive IgG1, IgG3 and IgG4 in EN and Mf+ groups and low levels of IgG2 in Mf+ were found; IgG1 and IgG3 reactivity was found for rBmCRT and its rN domain only, while IgG4 reactivity was moderate for rN domain and low for rP and rC domains. While IgG reactivity was seen in all the endemic subjects, IgG isotype reactivity was found mostly in EN and Mf+ subjects. Moderate levels of rBmCRT (and its rN domain)-reactive IgG and its isotypes are present in bancroftian subjects. Preponderance of IgG1 and IgG3 isotypes which bind and activate complement has relevance to vaccine potential of BmCRT.

Investigating Constraints Along the Plant Secretory Pathway to Improve Production of a SARS-CoV-2 Spike Vaccine Candidate.

Given the complex maturation requirements of viral glycoproteins and the challenge they often pose for expression in plants, the identification of host constraints precluding their efficient production is a priority for the molecular farming of vaccines. Building on previous work to improve viral glycoprotein production in plants, we investigated the production of a soluble SARS-CoV-2 spike comprising the ectopic portion of the glycoprotein. This was successfully transiently expressed in N. benthamiana by co-expressing the human lectin-binding chaperone calreticulin, which substantially increased the accumulation of the glycoprotein. The spike was mostly unprocessed unless the protease furin was co-expressed which resulted in highly efficient processing of the glycoprotein. Co-expression of several broad-spectrum protease inhibitors did not improve accumulation of the protein any further. The protein was successfully purified by affinity chromatography and gel filtration, although the purified product was heterogenous and the yields were low. Immunogenicity of the antigen was tested in BALB/c mice, and cellular and antibody responses were elicited after low dose inoculation with the adjuvanted protein. This work constitutes an important proof-of-concept for host plant engineering in the context of rapid vaccine development for SARS-CoV-2 and other emerging viruses.

Mapping human calreticulin regions important for structural stability

Calreticulin (CALR) is a highly conserved multifunctional chaperone protein primarily present in the endoplasmic reticulum, where it regulates Ca2+ homeostasis. Recently, CALR has gained special interest for its diverse functions outside the endoplasmic reticulum, including the cell surface and extracellular space. Although high-resolution structures of CALR exist, it has not yet been established how different regions and individual amino acid residues contribute to structural stability of the protein. In the present study, we have identified key residues determining the structural stability of CALR. We used a Saccharomyces cerevisiae expression system to express and purify 50 human CALR mutants, which were analysed for several parameters including secretion titer, melting temperature (Tm), stability and oligomeric state. Our results revealed the importance of a previously identified small patch of conserved surface residues, amino acids 166–187 (“cluster 2”) for structural stability of the human CALR protein. Two residues, Tyr172 and Asp187, were critical for maintaining the native structure of the protein. Mutant D187A revealed a severe drop in secretion titer, it was thermally unstable, prone to degradation, and oligomer formation. Tyr172 was critical for thermal stability of CALR and interacted with the third free Cys163 residue. This illustrates an unusual thermal stability of CALR dominated by Asp187, Tyr172 and Cys163, which may interact as part of a conserved structural unit.Besides structural clusters, we found a correlation of some measured parameter values in groups of CALR mutants that cause myeloproliferative neoplasms (MPN) and in mutants that may be associated with sudden unexpected death (SUD).

Induced Pluripotent Stem Cells Enable Disease Modeling and Drug Screening in Calreticulin del52 and ins5 Myeloproliferative Neoplasms

Mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and primary myelofibrosis patients. To address the contribution of the human CALR mutants to the pathogenesis of myeloproliferative neoplasms (MPNs) in an endogenous context, we modeled the CALRdel52 and CALRins5 mutants by induced pluripotent stem cell (iPSC) technology using CD34+ progenitors from 4 patients. We describe here the generation of several clones of iPSC carrying heterozygous CALRdel52 or CALRins5 mutations. We showed that CALRdel52 induces a stronger increase in progenitors than CALRins5 and that both CALRdel52 and CALRins5 mutants favor an expansion of the megakaryocytic lineage. Moreover, we found that both CALRdel52 and CALRins5 mutants rendered colony forming unit–megakaryocyte (CFU-MK) independent from thrombopoietin (TPO), and promoted a mild constitutive activation level of signal transducer and activator of transcription 3 in megakaryocytes. Unexpectedly, a mild increase in the sensitivity of colony forming unit-granulocyte (CFU-G) to granulocyte-colony stimulating factor was also observed in iPSC CALRdel52 and CALRins5 compared with control iPSC. Moreover, CALRdel52-induced megakaryocytic spontaneous growth is more dependent on Janus kinase 2/phosphoinositide 3-kinase/extracellular signal-regulated kinase than TPO-mediated growth and opens a therapeutic window for treatments in CALR-mutated MPN. The iPSC models described here represent an interesting platform for testing newly developed inhibitors. Altogether, this study shows that CALR-mutated iPSC recapitulate MPN phenotypes in vitro and may be used for drug screening.

Co-expression of human calreticulin significantly improves the production of HIV gp140 and other viral glycoproteins in plants.

SummaryPlant molecular farming (PMF) is rapidly gaining traction as a viable alternative to the currently accepted paradigm of producing biologics. While the platform is potentially cheaper and more scalable than conventional manufacturing systems, expression yields and appropriate post‐translational modifications along the plant secretory pathway remain a challenge for certain proteins. Viral fusion glycoproteins in particular are often expressed at low yields in plants and, in some cases, may not be appropriately processed. Recently, however, transiently or stably engineering the host plant has shown promise as a strategy for producing heterologous proteins with more complex maturation requirements. In this study we investigated the co‐expression of a suite of human chaperones to improve the production of a human immunodeficiency virus (HIV) type 1 soluble gp140 vaccine candidate in Nicotiana benthamiana plants. The co‐expression of calreticulin (CRT) resulted in a dramatic increase in Env expression and ameliorated the endoplasmic reticulum (ER) stress response ‐ as evidenced by lower transcript abundance of representative stress‐responsive genes. The co‐expression of CRT similarly improved accumulation of glycoproteins from Epstein‐Barr virus (EBV), Rift Valley fever virus (RVFV) and chikungunya virus (CHIKV), suggesting that the endogenous chaperone machinery may impose a bottleneck for their production. We subsequently successfully combined the co‐expression of human CRT with the transient expression of human furin, to enable the production of an appropriately cleaved HIV gp140 antigen. These transient plant host engineering strategies are a promising approach for the production of high yields of appropriately processed and cleaved viral glycoproteins.

In Vitro Employment of Recombinant Taenia solium Calreticulin as a Novel Strategy Against Breast and Ovarian Cancer Stem-like Cells

Calreticulin is a chaperone and master regulator of intracellular calcium homeostasis. Several additional functions have been discovered. Human and parasite calreticulin have been shown to suppress mammary tumor growth invivo. Here, we explored the capacity of recombinant Taenia solium calreticulin (rTsCRT) to modulate cancer cell growth invitro. We used different concentrations of rTsCRT to treat cancer cell lines and analyzed viability and colony formation capacity. We also tested the combination of the IC20 or IC50 doses of rTsCRT and of the chemotherapeutic drug 5-fluorouracil on MCF7 and SKOV3 cell lines. As a control, the non-tumorigenic cell line MCF10-A was employed. The effect of the drug combinations was also assessed in cancer stem-like cells. Additionally, scavenger receptor ligands were employed to identify the role of this receptor in the rTsCRT anti-tumoral effect. rTsCRT has a dose-dependent invitro anti-tumoral effect, being SKOV3 the most sensitive cell line followed by MCF7. When rTsCRT/5-fluorouracil were used, MCF7 and SKOV3 showed a 60% reduction in cell viability; colony formation capacity was also diminished. Treatment of cancer stem-like cells from MCF7 showed a higher reduction in cell viability, while those from SKOV3 were more sensitive to colony disaggregation. Finally, pharmacological inhibition of the scavenger receptor, abrogated the reduction in viability induced by rTsCRT in both the parental and stem-like cells. Our data suggest that rTsCRT alone or in combination with 5-fluorouracil inhibits the growth of breast and ovarian cancer cell lines through its interaction with scavenger receptors.

Silencing Calreticulin Expression Inhibits Invasion Ability of SNK6 Cells in Vitro via Down-Regulating Expression of VEGF and MMP2/9

To investigate the effect of steadily down-regulating the expression of calreticulin (CALR) on the invasion of natural killer/T-cell lymphoma SNK6 cells, and explore its possible mechanism. The sequences of specific short hairpin RNA (shRNA) targeting on human CALR were designed, and were inserted into pLKO.1-puro lentivirus vector, and the reconbinant lentivirus vector was obtained; the lentivirus particles were backed by three-plasmid system and transfected into SNK6 cells, the SNK6 cells stably down-regulating the CALR expression were sercened by puromytain, the CALR-silencing effect was verified by real-time PCR and Western blot. CCK-8 assay was used to evaluate the cell viability, The transwell invasion assays was used to analyse invasion of SNK6 cells. The mRNA expression of Calreticulin, MMP2, MMP9 and VEGF was determined by real time PCR, the protein expression of Calreticulin and GAPDH was analyzed by Western blot. The recombinant lentiviral vector pLKO.1-puro-shCALR was successfully constructed, packed into the lentivirus, then the SNK6 cells stably down-regulating Calreticulin expression was obtained. When Calreticulin was down-rengulated in SNK6 cells, the proliferation rate was reduced and the invasion ability was decreased; the mRNA levels of VEGF and MMP-2/9 also were reduced. The stable down-regnlation of CALR expression in SNK6 cells can attenuate the imvasiveness of SNK6 cells, which maybe related with transcriptional decrease of MMP2, MMP9 and VEGF.

Development of a Novel Calreticulin Reporter for Determination of Immunogenic Cell Death

Immunogenic cell death (ICD) initiates a series of damage associated molecular patterns (DAMPs) that are able to activate an immune response. Of these, translocation of calreticulin (CRT) from the endoplasmic reticulum (ER) to the outer leaflet of the cell membrane is a hallmark event that leads to activation of antigen presenting cells (APC), thus priming an immune response. Induction of ICD in cancer cells may provide a manner of activating an immunological anti‐tumor response, which may be utilized in immunotherapy. Currently, a few chemotherapeutic drugs, such as doxorubicin, are known to induce ICD and have been proposed to be repurposed for this use. Thus, it is likely that other known drugs may exhibit ICD induction and may be utilized in immunotherapy. However, to evaluate such drugs, there is a need to develop a high throughput method of detecting ICD.Here, a novel ICD reporter based on translocation of CRT and the HiBit‐tag expression system is described. This system utilizes a recombinant HiBit‐tag that associates with cell impermeable LgBit, thereby forming a bioluminescent protein complex. HiBit‐tagged CRT will complex with LgBit when translocated to the outer leaflet of the cell membrane, which may be detected bioluminescently by addition of D‐luciferin. Thus, the intensity of bioluminescence may be correlated to translocation of CRT, a hallmark of ICD.To create the novel ICD reporter, the human CRT gene was amplified using polymerase chain reaction (PCR) from plasmid HsCD00322958 (Harvard Medical School, MA), and inserted onto the 3′ end of the HiBit tag (pBiT3.1‐N, Promega, WI) using restriction enzyme digestion (PspXI and SbfI) followed by ligation. The recombinant HiBiT‐CRT plasmid was transformed into competent DH5α E. coli cells using the heat‐shock method. The HiBit‐CRT cassette was validated by DNA Sanger sequencing (Genscript, Piscataway, NJ). In order to transfect human ovarian adenocarcinoma cells (SKOV‐3) with the ICD reporter, the optical concentration of geneticin was determined by performing a kill curve. In brief, SKOV‐3 cells were maintained in McCoy's 5A cell medium supplemented with 10 % fetal bovine serum (FBS) at 37°C and 5% CO2, and then treated with varying concentrations of geneticin (0.05 mg/mL, 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, 1 mg/mL, 1.25 mg/mL, 1.5 mg/mL, 1.75 mg/mL, 2 mg/mL). The cell viability was evaluated daily for 7 days using bright field microscopy. The optimal concentration was determined to be 1.25 mg/mL. Next, SKOV‐3 cells will be transfected with the ICD reporter, and used to screen the Approved Oncology Drug Set VI (kind gift from the National Cancer Institute) for compounds that induce ICD.Support or Funding InformationWestern Illinois University Research Council GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mice with Calr mutations homologous to human CALR mutations only exhibit mild thrombocytosis

Calreticulin (CALR) exon 9 frameshift mutations, commonly detected in essential thrombocythemia (ET) and primary myelofibrosis patients, activate signal transducer and activator of transcription (STAT) proteins in the presence of Myeloproliferative Leukemia Virus (MPL) and induce ET in vivo. Loss of the KDEL motif, an endoplasmic reticulum retention signal, and generation of many positively charged amino acids (AAs) in the mutated C-terminus are thought to be important for disease induction. To test this hypothesis, we generated mice harboring a Calr frameshift mutation using the CRISPR/Cas9 system. Deletion of 19-base pairs in exon 9 (c.1099-1117del), designated the del19 mutation, induced loss of the KDEL motif and generated many positively charged AAs, similar to human mutants. Calr del19 mice exhibited mild thrombocytosis, slightly increased megakaryocytes, and mild splenomegaly. In vitro experiments revealed that the murine CALR del19 mutant had a weaker ability to combine with murine MPL than the human CALR del52 mutant. Consequently, STAT5 activation was also very weak downstream of the murine mutant and murine MPL, and may be the reason for the mild disease severity. In summary, loss of the KDEL motif and positively charged AAs in the C-terminus of CALR is insufficient for MPL binding and ET development.

The Biophysical Interaction of the Danger-Associated Molecular Pattern (DAMP) Calreticulin with the Pattern-Associated Molecular Pattern (PAMP) Lipopolysaccharide.

The endoplasmic reticulum (ER) chaperone protein, calreticulin (CRT), is essential for proper glycoprotein folding and maintaining cellular calcium homeostasis. During ER stress, CRT is overexpressed as part of the unfolded protein response (UPR). In addition, CRT can be released as a damage-associated molecular pattern (DAMP) molecule that may interact with pathogen-associated molecular patterns (PAMPs) during the innate immune response. One such PAMP is lipopolysaccharide (LPS), a component of the gram-negative bacterial cell wall. In this report, we show that recombinant and native human placental CRT strongly interacts with LPS in solution, solid phase, and the surface of gram-negative and gram-positive bacteria. Furthermore, LPS induces oilgomerization of CRT with a disappearance of the monomeric form. The application of recombinant CRT (rCRT) to size exclusion and anion exchange chromatography shows an atypical heterogeneous elution profile, indicating that LPS affects the conformation and ionic charge of CRT. Interestingly, LPS bound to CRT is detected in sera of bronchiectasis patients with chronic bacterial infections. By ELISA, rCRT dose-dependently bound to solid phase LPS via the N- and C-domain globular head region of CRT and the C-domain alone. The specific interaction of CRT with LPS may be important in PAMP innate immunity.

Metal binding study of calreticulin: An immunomodulatory protein of human filarial parasite Brugia malayi

Calreticulin (CRT), a highly conserved ubiquitous eukaryotic protein with a molecular mass of 46 kDa, containing three domains (N, P, and C) is involved in promoting prolonged parasite-host relations. Brugia malayi Calreticulin (BmCRT) is involved in the establishment of parasite infection by suppression of C1q-mediated host immune response. Calcium plays important role in this immunomodulatory mechanism of BmCRT. In the present study binding of calcium with BmCRT region involved in this interaction was investigated and correlated with the accompanying changes in fluorescence, circular dichroism (CD) and UV absorption. The results obtained clearly indicated that BmCRT is a calcium binding protein and contains types two of Ca2+ binding sites, one high affinity Ca2+ binding site at P domain and another low affinity Ca2+ binding site at C domain. Zinc also binds to additional sites that do not have appreciable affinity for the calcium. These studies have provided new knowledge that allows us to describe how the structure of BmCRT responds to interactions with calcium and zinc which is different from human CRT and also discuss how this mechanism help to complex formation with host C1q.

Constitutive expression of the active fragment of human vasostatin Vs30 in Pichia pastoris SMD1168H

Vasostatin 30 (Vs30) is an active fragment derived from the N-terminal region (135–164 aa) of human calreticulin and has the ability to inhibit angiogenesis. In this work, the expression of Vs30 was performed using a protease-deficient strain of the methylotrophic yeast Pichia pastoris. The vs30 gene was optimized for P. pastoris preferential codon usage and inserted into constitutive expression vector pGAPZαA. In addition, a plasmid with four copies of the expression cassette was obtained and transformed into P. pastoris. The flask fermentation conditions were: culture volume of 25 mL in 250 mL baffled flasks at 28 °C, pH 6 and harvest time of 48 h. Up to 21.07 mg/L Vs30 were attained and purified by ultrafiltration with a 30-kDa cut-off membrane and the recovery was 49.7%. Bioactivity of Vs30 was confirmed by the inhibition of cell proliferation, as well as the inhibition of the capillary-like structures formation of EA.hy926 cells in vitro. This work constitutes the first report on the expression of Vs30 in Pichia pastoris using a constitutive promoter and multi-copy approach such as strategies to improve the recombinant Vs30 expression.

Calreticulin inhibits inflammation-induced osteoclastogenesis and bone resorption.

Osteoclasts play key roles in bone remodeling and pathologic osteolytic disorders such as inflammation, infection, bone implant loosening, rheumatoid arthritis, metastatic bone cancers, and pathological fractures. Osteoclasts are formed by the fusion of monocytes in response to receptor activators of NF-κB-ligand (RANKL) and macrophage colony stimulating factor 1 (M-CSF). Calreticulin (CRT), a commonly known intracellular protein as a calcium-binding chaperone, has an unexpectedly robust anti-osteoclastogenic effect when its recombinant form is applied to osteoclast precursors in vitro or at the site of bone inflammation externally in vivo. Externally applied Calreticulin was internalized inside the cells. It inhibited key pro-osteoclastogenic transcription factors such as c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1)-in osteoclast precursor cells that were treated with RANKL in vitro. Recombinant human Calreticulin (rhCRT) inhibited lipopolysaccharide (LPS)-induced inflammatory osteoclastogenesis in the mouse calvarial bone in vivo. Cathepsin K molecular imaging verified decreased Cathepsin K activity when rhCalreticulin was applied at the site of LPS application in vivo. Recombinant forms of intracellular proteins or their derivatives may act as novel extracellular therapeutic agents. We anticipate our findings to be a starting point in unraveling hidden extracellular functions of other intracellular proteins in different cell types of many organs for new therapeutic opportunities. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2658-2666, 2017.

Spontaneous and Vaccine-Induced Clearance of Mus Musculus Papillomavirus 1 Infection.

ABSTRACTMus musculus papillomavirus 1 (MmuPV1/MusPV1) induces persistent papillomas in immunodeficient mice but not in common laboratory strains. To facilitate the study of immune control, we sought an outbred and immunocompetent laboratory mouse strain in which persistent papillomas could be established. We found that challenge of SKH1 mice (Crl:SKH1-Hrhr) with MmuPV1 by scarification on their tail resulted in three clinical outcomes: (i) persistent (>2-month) papillomas (∼20%); (ii) transient papillomas that spontaneously regress, typically within 2 months (∼15%); and (iii) no visible papillomas and viral clearance (∼65%). SKH1 mice with persistent papillomas were treated by using a candidate preventive/therapeutic naked-DNA vaccine that expresses human calreticulin (hCRT) fused in frame to MmuPV1 E6 (mE6) and mE7 early proteins and residues 11 to 200 of the late protein L2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations were delivered biweekly via in vivo electroporation, and both humoral and CD8 T cell responses were mapped and measured. Previously persistent papillomas disappeared within 2 months after the final vaccination. Coincident virologic clearance was confirmed by in situ hybridization and a failure of disease to recur after CD3 T cell depletion. Vaccination induced strong mE6 and mE7 CD8+ T cell responses in all mice, although they were significantly weaker in mice that initially presented with persistent warts than in those that spontaneously cleared their infection. A human papillomavirus 16 (HPV16)-targeted version of the DNA vaccine also induced L2 antibodies and protected mice from vaginal challenge with an HPV16 pseudovirus. Thus, MmuPV1 challenge of SKH1 mice is a promising model of spontaneous and immunotherapy-directed clearances of HPV-related disease.IMPORTANCE High-risk-type human papillomaviruses (hrHPVs) cause 5% of all cancer cases worldwide, notably cervical, anogenital, and oropharyngeal cancers. Since preventative HPV vaccines have not been widely used in many countries and do not impact existing infections, there is considerable interest in the development of therapeutic vaccines to address existing disease and infections. The strict tropism of HPV requires the use of animal papillomavirus models for therapeutic vaccine development. However, MmuPV1 failed to grow in common laboratory strains of mice with an intact immune system. We show that MmuPV1 challenge of the outbred immunocompetent SKH1 strain produces both transient and persistent papillomas and that vaccination of the mice with a DNA expressing an MmuPV1 E6E7L2 fusion with calreticulin can rapidly clear persistent papillomas. Furthermore, an HPV16-targeted version of the DNA can protect against vaginal challenge with HPV16, suggesting the promise of this approach to both prevent and treat papillomavirus-related disease.

The Calreticulin control of human stress erythropoiesis is impaired by JAK2V617F in polycythemia vera.

Calreticulin (CALR) is a Ca2+-binding protein that shuttles among cellular compartments with proteins bound to its N/P domains. The knowledge that activation of the human erythropoietin receptor induces Ca2+ fluxes prompted us to investigate the role of CALR in human erythropoiesis. As shown by Western blot analysis, erythroblasts generated invitro from normal sources and JAK2V617F polycythemia vera (PV) patients expressed robust levels of CALR. However, Ca2+ regulated CALR conformation only in normal cells. Normal erythroblasts expressed mostly the N-terminal domain of CALR (N-CALR) on their cell surface (as shown by flow cytometry) and C-terminal domain (C-CALR) in their cytoplasm (as shown by confocal microscopy) and expression of both epitopes decreased with maturation. In the proerythroblast (proEry) cytoplasm, C-CALR was associated with the glucocorticoid receptor (GR), which initiated the stress response. In these cells, Ca2+ deprivation and inhibition of nuclear export increased GR nuclear localization while decreasing cytoplasmic detection of C-CALR and C-CALR/GR association and proliferation in response to the GR agonist dexamethasone (Dex). C-CALR/GR association and Dex responsiveness were instead increased by Ca2+ and erythropoietin. In contrast, JAK2V617F proErys expressed normal cell-surface levels of N-CALR but barely detectable cytoplasmic levels of C-CALR. These cells contained GR mainly in the nucleus and were Dex unresponsive. Ruxolitinib rescued cytoplasmic detection of C-CALR, C-CALR/GR association, and Dex responsiveness in JAK2V617F proErys and its effects were antagonized by nuclear export and Ca2+ flux inhibitors. These results indicates that Ca2+-induced conformational changes of CALR regulate nuclear export of GR in normal erythroblasts and that JAK2V617F deregulates this function in PV.

DNA vaccine encoding human papillomavirus antigens flanked by a signal peptide and a KDEL sequence induces a potent therapeutic antitumor effect.

Cellular immune responses play a critical role in the eradication of intracellular infections and malignant cells through the recognition and subsequent removal of the infection or malignant cells. Effective antigen presentation is crucial for stimulating the immune system against malignant cells. Calreticulin (CRT) has been used to improve antigen presentation. However, CRT overexpression has been previously associated with the development of pancreatic and breast cancer. The import and retention signals of CRT in the endoplasmic reticulum (ER) can be used to overcome CRT overexpression. The present study describes the potent antitumor effect of a DNA vaccine encoding human papillomavirus type 16 E6 and E7 antigens flanked by ER import and retention signals (SP-E6E7m-KDEL). The effect of this vaccine was compared with that of E6 and E7 antigens fused to human full-length CRT (hCRT-E6E7m). In the present study, the effectiveness of SP-E6E7m-KDEL for inducing an interferon-γ antigen-specific, response and its therapeutic effect against tumors was demonstrated, which was as effective as immunization against those antigens fused to CRT. This simplified strategy, using ER import and retention signal peptides to direct antigens to this organelle, provides an efficient alternative to traditional vaccines and, more importantly, a safe and potent system to induce a therapeutic antitumor response.

Physiological Expression of Calr Mutant Increases Cell Growth and Cytokine Independency in Human Cell Lines Expressing Mpl, and Develops Essential Thrombocythemia in Mice

Calreticulin (CALR) exon 9 mutations were reported in about two-thirds of JAK2 or MPL mutation negative ET and PMF patients. The mutations cause frameshifts that result in proteins with novel C-terminus.Retrovirus-mediated gene transfer into cell lines and mouse bone marrow (BM) cells is a common technique, but the expression level is very high compared to the physiological expression.We investigated the effects of physiological expression of mutant CALR using CRISPR/Cas9 gene editing techniques for cell lines, and as for the mouse model, we generated a transgenic mice (TG) expressing human CALR del52 mutant.We used two human cell lines expressing MPL: human acute megakaryoblastic leukemia cell line CMK11-5 which expressed endogenous MPL, and F-36P-MPL cell line which was generated by introducing MPL to GM-CSF-dependent erythroleukemia cell line F-36P. Plasmids coexpressing hCas9 and single-guide RNA were prepared by ligating oligonucleotides (5'-CACCGACAAGAAACGCAAAGAGGAGG-3', 5'-AAACCCTCCTCTTTGCGTTTCTTGTC-3') for the target sequence of human CALR exon 9 into pX330. The plasmids were introduced with a electroporator to each of the cell lines. After limiting dilution cloning, we identified cell lines which have indel mutation at the target site.We produced two types of CMK11-5 subline knocked in a CALR mutation, namely CALR del25 CMK cells and CALR del25/del17 CMK cells, respectively. The former lacks 25 bases in one CALR allele, causing a frameshift that results in a protein resembling human CALR mutant, while the latter lacks an additional 17 bases in another allele in CALR exon 9 and induces a frameshift that causes a deletion in CALR exon 9. Both kinds of CALR mutant CMK11-5 cells showed increased cell proliferation compared to WT cells. We also produced one type of F-36P-MPL subline, CALR del1/ins1 F-36P-MPL cells which had 1 base deletion in one CALR allele resembling human mutation and 1 base insertion in another allele. Though the growth of this subline in the presence of GM-CSF was comparable to WT cells, it showed GM-CSF independent autonomous cell growth.We generated TG mice expressing human CALR del52 mutant driven by the murine H2Kb promoter. We compared the expression level of human CALR mRNA in TG BM cells with the expression of endogenous WT CALR in human cell lines (CMK11-5, F-36P-MPL, CHRF288) using Rn18s as an endogenous control. The expression of human CALR in TG BM was approximately 0.6 times that of endogenous WT CALR in human cell lines, and the physiological expression level was obtained.They exhibited thrombocytosis, with platelet (PLT) counts as high as 2,000 x 109/L. Leukocyte number and the proportion of granulocytes and T and B lymphocytes, were comparable to WT mice. CALR mutation had no impact on Hb level or spleen weight. There was a striking difference in the number of megakaryocytes (Mgks), which was 2-fold higher in BM from TG mice than in WT mice. The TG Mgks were also more mature, with larger diameter, and contained higher number of alpha-granules compared to WT cells. In one year of observation, there is no fibrosis in BM. These observations showed that TG developed human ET-like disease. The survival of TG mice was comparable to that of WT mice. The disease phenotype was transplantable into WT recipient mice. To characterize in detail the impact of MPNs induced by the CALR del52 mutant, we evaluated the frequencies of HSCs and progenitors in BM. The frequency of both LT-HSC and ST-HSC in BM was higher inTG mice compared to WT mice. The frequencies of progenitors (CMP, MEP, and MKP) were also greater in BM from TG mice than from WT mice. However, BM cells did not have enhanced replating capacity. We next examined whether or not ruxolitinib (RUX) treatment ameliorated thrombocytosis in TG mice. Either 90 mg/kg bid of RUX or vehicle was administrated to TG mice for 4 weeks.TG mice treated with vehicle showed a mean 16% increase in PLT count during the treatment period, probably due to the disease progression. RUX treatment attenuated the increase in the number of PLTs in TG mice by a mean of 22%, but the overall count was still higher than that in WT mice. BM sections showed that RUX reduced the Mgks number in TG.In summary, physiological expression of CALR mutant increases cell growth and cytokine independency in human cell lines expressing MPL, and develops ET in mice. RUX therapy attenuated the increased numbers of peripheral blood PLTs and BM Mgks, and ameliorated CALR mutation-induced ET. DisclosuresNo relevant conflicts of interest to declare.