In silico analyses of heat shock protein 60 and calreticulin to designing a novel vaccine shifting immune response toward T helper 2 in atherosclerosis.

Recent experiments demonstrated that atherosclerosis is a Th1 dominant autoimmune condition, whereas Th2 cells are rarely detected within the atherosclerotic lesions. Several studies have indicated that Th2 type cytokines could be effective in the reduction and stabilization of atherosclerotic plaque. Therefore, the modulation of the adaptive immune response by shifting immune responses toward Th2 cells by a novel vaccine could represent a promising approach to prevent from progression and thromboembolic events in coronary artery disease. In the present study, an in silico approach was applied to design a novel multi-epitope vaccine to elicit a desirable immune response against atherosclerosis. Six novel IL-4 inducing epitopes were selected from HSP60 and calreticulin proteins. To enhance epitope presentation, IL-4 inducing epitopes were linked together by AAY and HEYGAEALERAG linkers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Moreover, cholera toxin B (CTB) was employed as an adjuvant. A multi-epitope construct was designed based on predicted epitopes which was 320 residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this chimeric protein were analyzed using bioinformatics tools and servers. Based on bioinformatics analysis, a soluble, and non-allergic protein with 35.405kDa molecular weight was designed. Expasy ProtParam classified this chimeric protein as a stable protein. In addition, predicted epitopes in the chimeric vaccine indicated strong potential to induce B-cell mediated immune response and shift immune responses toward protective Th2 immune response. Various in silico analyses indicate that this vaccine is a qualified candidate for improvement of atherosclerosis by inducing immune responses toward T helper 2.

Exogenous Calreticulin, incorporated onto non-infective Trypanosoma cruzi epimastigotes, promotes their internalization into mammal host cells.

Chagas disease is an endemic pathology in Latin America, now emerging in developed countries, caused by the intracellular protozoan Trypanosoma cruzi, whose life cycle involves three stages: amastigotes, epimastigotes, and trypomastigotes. T. cruzi Calreticulin (TcCRT), an endoplasmic reticulum resident chaperone, translocates to the external cellular membrane, where it captures complement component C1, ficolins and MBL, thus inactivating the classical and lectin pathways. Trypomastigote-bound C1 is detected as an "eat me" signal by macrophages and promotes the infective process. Unlike infective trypomastigotes, non-infective epimastigotes either do not express or express only marginal levels of TcCRT on their external membrane. We show that epimastigotes bind exogenous rTcCRT to their cellular membrane and, in the presence of C1q, this parasite form is internalized into normal fibroblasts. On the other hand, Calreticulin (CRT)-deficient fibroblasts show impaired parasite internalization. In synthesis, CRT from both parasite and host cell origin is important in the establishment of C1q-dependent first contacts between parasites and host cells.

Two endoplasmic reticulum proteins (calnexin and calreticulin) are involved in innate immunity in Chinese mitten crab (Eriocheir sinensis).

Calnexin (Cnx) and calreticulin (Crt), which are important chaperones in the endoplasmic reticulum (ER), participate in the folding and quality control of client proteins. Cnx and Crt identified from Chinese mitten crab (Eriocheir sinensis) are designated as EsCnx and EsCrt, respectively. EsCnx and EsCrt are expressed in the hemocyte, hepatopancrea, gill, and intestine at the mRNA and protein level. Immunofluorescence analysis indicated that EsCnx and EsCRT are located in the ER. Moreover, the mRNA and protein expression levels of EsCnx and EsCrt were altered by challenge with lipopolysaccharides (LPS), peptidoglycans (PGN), Staphyloccocus aureus, and Vibrio parahaemolyticus. Recombinant EsCnx and EsCrt (rEsCnx and rEsCrt, respectively) proteins can bind to various Gram-positive and Gram-negative bacteria, as well as to different polysaccharides (LPS and PGN). rEsCnx and rEsCrt assisted in the clearance of V. parahaemolyticus in vivo, and the clearance efficiency was impaired after silencing of EsCnx and EsCrt. Our results suggest that the two ER proteins are involved in anti-bacterial immunity in E. sinensis.

Calreticulin acts as an adjuvant to promote dendritic cell maturation and enhances antigen-specific cytotoxic T lymphocyte responses against non-small cell lung cancer cells.

Dendritic cell (DC)-based immunotherapy has promising for treatment of non-small cell lung cancer (NSCLC). Melanoma-associated antigen 3 (MAGE-A3) is a tumor-specific antigen and expressed in approximately 35-40% of NSCLC tissues. Calreticulin (CALR) is a protein chaperone and can enhance DC maturation and antigen presentation. In this study, we evaluated the adjuvant activity of CALR in human DC maturation and their capacity to induce MAGE-A3-specific CD8+ cytotoxic T lymphocyte (CTL) responses to NSCLC in vitro. Infection with recombinant Ad-CALR and/or Ad-MAGE-A3, but not with control Ads, induced CALR and/or MAGE-A3 expression in DCs. Infection with Ad-CALR significantly increased the percentages of CD80+, CD83+, CD86+ and HLA-DR+ DCs and IL-12 secretion, but reduced IL-10 production in DCs. Co-culture of autologous lymphocytes with DC-Ad-CALR or DC-Ad-CM significantly increased the numbers of induced CD8+ CTLs. The percentages of IFNγ-secreting CTLs responding to SK-LU-1 and NCI-H522 NSCLC, but not to non-tumor NL-20 cells in Ad-C-CTL, Ad-M-CTL and Ad-CM-CTL were significantly higher than that of DC-CTL and Ad-null-CTL. Ad-C-CTL, Ad-M-CTL and Ad-CM-CTL, but not control DC-CTL and Ad-null-CTL, induced higher frequency of MAGE-A3+HLA-A2+ NCI-H-522 cell apoptosis, but did not affect the survival of MAGE-A3+HLA-A2- SK-LU-1 and non-tumor NL20 cells in vitro. Treatment with anti-HLA-I antibody, but not with anti-HLA-II, dramatically diminished the cytotoxicity of Ad-CM-CTLs against NCI-H522 cells. Our data indicated that CALR acted as an adjuvant to promote DC maturation, which induced CTL development and enhanced MAGE-A3-specific CTL cytotoxicity against NSCLC.

Artificial feeding of Rhipicephalus microplus female ticks with anti calreticulin serum do not influence tick and Babesia bigemina acquisition.

Ticks are obligate haematophagous ectoparasites considered the principal vectors of disease among animals. Rhipicephalus microplus and R. annulatus ticks are the most important vectors for Babesia bigemina and B. bovis, two of the most important intraerythrocytic protozoan parasites species in cattle, responsible for babesiosis which together with anaplasmosis account for substantial economic losses in the livestock industry worldwide. Anti-tick vaccines are a proved alternative to traditional tick and tick borne diseases control methods but are still limited primarily due to the lack of effective antigens. Subsequently to the identification of antigens the validation is a laborious work often expensive. Tick artificial feeding, is a low cost alternative to test antigens allowing achieving critical data. Herein, R. microplus females were successfully artificially fed using capillary tubes. Calreticulin (CRT) protein, which in a previous study has been identified as being involved in B. bigemina infection in R. annulatus ticks, was expressed as recombinant protein (rCRT) in an E. coli expression system and antibodies raised against rCRT. Anti-rCRT serum was supplemented to a blood meal, offered to partially engorged R. microplus females and their effect in feeding process as well as infection by B. bigemina was analyzed. No significant reductions in tick and egg weight were observed when ticks fed with anti-rCRT serum. Furthermore, B. bigemina infection levels did not show a statistically significant decrease when ticks fed with anti-rCRT antibodies. Results suggest that CRT is not a suitable candidate for cattle vaccination trials.

Genotoxicity induced by Taenia solium and its reduction by immunization with calreticulin in a hamster model of taeniosis.

Genotoxicity induced by neurocysticercosis has been demonstrated in vitro and in vivo in humans. The adult stage of Taenia solium lodges in the small intestine and is the main risk factor to acquire neurocysticercosis, nevertheless its carcinogenic potential has not been evaluated. In this study, we determined the genotoxic effect of T. solium infection in the hamster model of taeniosis. In addition, we assessed the effect of oral immunization with recombinant T. solium calreticulin (rTsCRT) plus cholera toxin as adjuvant on micronuclei induction, as this protein has been shown to induce 33-44% protection in the hamster model of taeniosis. Blood samples were collected from the orbital venous plexus of noninfected and infected hamsters at different days postinfection, as well as from orally immunized animals, to evaluate the frequency of micronucleated reticulocytes as a measure of genotoxicity induced by parasite exposure and rTsCRT vaccination. Our results indicate that infection with T. solium caused time-dependent DNA damage in vivo and that rTsCRT immunization reduced the genotoxic damage induced by the presence of the tapeworms.

Immunological mechanisms involved in the protection against intestinal taeniosis elicited by oral immunization with Taenia solium calreticulin.

Oral immunization with functional recombinant Taenia solium calreticulin (rTsCRT) induces 37% reduction in tapeworm burden in the experimental model of intestinal taeniosis in hamsters. Furthermore, tapeworms recovered from vaccinated animals exhibit diminished length, being frequently found in more posterior parts of the small intestine. The aim of this study was to analyze the immunological mechanisms involved in protection in response to rTsCRT oral immunization. Hamsters were orally immunized with rTsCRT using cholera toxin (CT) as adjuvant, weekly for 4 weeks. Fifteen days after the last boost animals were challenged with four T. solium cysticerci. Reduction in the adult worm recovery and increased transcription of mRNA for IL-4 and IFN-γ in the mucosa of rTsCRT+CT immunized animals were observed. Immunization also induced goblet cell hyperplasia in the mucosa surrounding the implantation site of the parasite. Specific IgG and IgA antibodies in serum and fecal supernatants were detected after the second immunization, being more pronounced after challenge. Our data suggest that oral vaccination with rTsCRT+CT regulates a local expression of IL-4 and IFN-γ, stimulating secretion of IgA that, together with the increase of goblet cells and mucin production, could result in an unfavorable environment for T. solium promoting an impaired tapeworm development.

Recombinant adenovirus delivery of calreticulin-ESAT-6 produces an antigen-specific immune response but no protection against a Mycobacterium tuberculosis challenge.

Bacillus Calmette-Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT-ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT-ESAT-6-CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis.

Treatment with imiquimod enhances antitumor immunity induced by therapeutic HPV DNA vaccination.

BACKGROUND: There is an urgent need to develop new innovative therapies for the control of advanced cancer. The combination of antigen-specific immunotherapy with the employment of immunomodulatory agents has emerged as a potentially plausible approach for the control of advanced cancer. METHODS: In the current study, we explored the combination of the DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 (HPV-16) E7 antigen (CRT/E7) with the TLR7 agonist imiquimod for their ability to generate E7-specific immune responses and antitumor effects in tumor-bearing mice. RESULTS: We observed that treatment with CRT/E7 DNA in combination with imiquimod leads to an enhancement in the E7-specific CD8+ T cell immune responses and a decrease in the number of myeloid-derived suppressor cells in the tumor microenvironment of tumor-bearing mice. Furthermore, treatment with CRT/E7 DNA in combination with imiquimod leads to significantly improved antitumor effects and prolonged survival in treated mice. In addition, treatment with imiquimod led to increased number of NK1.1+ cells and F4/80+ cells in the tumor microenvironment. Macrophages and NK1.1+ cells were found to play an important role in the antitumor effects mediated by treatment with CRT/E7 DNA in combination with imiquimod. CONCLUSIONS: Thus, our data suggests that the combination of therapeutic HPV DNA vaccination with topical treatment with the TLR7 agonist imiquimod enhances the antitumor immunity induced by DNA vaccination. The current study has significant implications for future clinical translation.

Calreticulin from the intestinal nematode Heligmosomoides polygyrus is a Th2-skewing protein and interacts with murine scavenger receptor-A.

Helminth infections are commonly associated with a Th2 immune response, yet only a few parasite molecules involved in triggering such immune responses have been identified. Here, we describe the Th2-skewing property of calreticulin of Heligmosomoides polygyrus (HpCRT). HpCRT is a secreted protein most abundantly expressed by tissue invasive larvae (L4). Native HpCRT purified from adult worm extract (nHpCRT) stimulated robust IL-4 release from CD4(+) T cells of H. polygyrus infected mice. Interestingly, CD4(+) T cells also produced significant amounts of IL-10 while IFN-gamma was not detectable. Likewise, immunization with recombinant HpCRT (rHpCRT) without extrinsic adjuvant led predominantly to a specific IL-4 production implying the innate ability of HpCRT to drive Th2 responses. The triggering of a Th2-skewed immune response to rHpCRT is corroborated by the induction of HpCRT-specific IgG1 and IgE antibodies. Furthermore, rHpCRT bound to scavenger receptor type A (SR-A) on dendritic cells, and interaction of HpCRT with SR-A led to internalization of HpCRT that could be partially blocked by competition with SR-A ligands as well as with an anti-SR-A monoclonal antibody. Hence, our data imply that nematode calreticulin interacts with a mammalian scavenger receptor and at the same time induces a Th2 response.

Low-dose radiation enhances therapeutic HPV DNA vaccination in tumor-bearing hosts.

Current therapeutic approaches to treatment of patients with bulky cervical cancer are based on conventional in situ ablative modalities including cisplatin-based chemotherapy and radiation therapy. The 5-year survival of patients with nonresectable disease is dismal. Because over 99% of squamous cervical cancer is caused by persistent infection with an oncogenic strain of human papillomavirus (HPV), particularly type 16 and viral oncoproteins E6 and E7 are functionally required for disease initiation and persistence, HPV-targeted immune strategies present a compelling opportunity in which to demonstrate proof of principle. Sublethal doses of radiation and chemotherapeutic agents have been shown to have synergistic effect in combination with either vaccination against cancer-specific antigens, or with passive transfer of tumor-specific cytotoxic T lymphocytes (CTLs). Here, we explored the combination of low-dose radiation therapy with DNA vaccination with calreticulin (CRT) linked to the mutated form of HPV-16 E7 antigen (E7(detox)), CRT/E7(detox) in the treatment of E7-expressing TC-1 tumors. We observed that TC-1 tumor-bearing mice treated with radiotherapy combined with CRT/E7(detox) DNA vaccination generated significant therapeutic antitumor effects and the highest frequency of E7-specific CD8(+) T cells in the tumors and spleens of treated mice. Furthermore, treatment with radiotherapy was shown to render the TC-1 tumor cells more susceptible to lysis by E7-specific CTLs. In addition, we observed that treatment with radiotherapy during the second DNA vaccination generated the highest frequency of E7-specific CD8(+) T cells in the tumors and spleens of TC-1 tumor-bearing mice. Finally, TC-1 tumor-bearing mice treated with the chemotherapy in combination with radiation and CRT/E7(detox) DNA vaccination generate significantly enhanced therapeutic antitumor effects. The clinical implications of the study are discussed.

Enhancement of DNA vaccine potency through coadministration of CIITA DNA with DNA vaccines via gene gun.

Administration of DNA vaccines via gene gun has emerged as an important form of Ag-specific immunotherapy. The MHC CIITA is a master regulator of MHC class II expression and also induces expression of class I molecules. We reasoned that the gene gun administration of CIITA DNA with DNA vaccines employing different strategies to improve MHC I and II processing could enhance DNA vaccine potency. We observed that DC-1 cells transfected with CIITA DNA lead to higher expression of MHC I and II molecules, leading to enhanced Ag presentation through the MHC I/II pathways. Furthermore, our data suggested that coadministration of DNA-encoding calreticulin (CRT) linked to human papillomavirus (HPV) 16 E6 Ag (CRT/E6) with CIITA DNA leads to enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. In addition, coadministration of the combination of CRT/E6 DNA with CIITA DNA and DNA encoding the invariant chain (Ii) linked to the pan HLA-DR-reactive epitope (Ii-PADRE) further enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. Treatment with the combination vaccine was also shown to enhance the antitumor effects and to prolong survival in TC-1 tumor-bearing mice. Vaccination with the combination vaccine also led to enhanced E6-specific CD8(+) memory T cells and to long-term protection against TC-1 tumors and prolonged survival in vaccinated mice. Thus, our findings suggest that the combination of CIITA DNA with CRT/E6 and Ii-PADRE DNA vaccines represents a potentially effective means to combat tumors in the clinical setting.

Cloning and characterization of a cDNA clone encoding calreticulin from Haemaphysalis qinghaiensis (Acari: Ixodidae).

The application of anti-tick vaccine has been shown to be the most promising alternative strategy compared to the current use of acaricides that suffer from a number of serious limitations. The success of this method is dependent upon identification and cloning of potential tick vaccine antigens. Previously, we have cloned 21 positive clones (named from Hq02 to Hq22) by immunoscreening complimentary DNA (cDNA) libraries of Haemaphysalis qinghaiensis; however, some of those clones did not contain open reading frames (ORF). In this study, we amplified the entire sequence of Hq07 by using rapid amplification of the cDNA ends. Hq07 contains an ORF of 1,233 bp that encodes for 410 amino acid residues with a coding capacity of 47 kDa. Search of the cloned sequences against GenBank revealed that Hq07 is a calreticulin (CRT)-similar clone and designated HqCRT. Expression analysis by reverse transcription-polymerase chain reaction showed that this gene is ubiquitously expressed at different developmental stages and in different tissues of H. qinghaiensis. The gene was expressed as glutathione S-transferase-fused proteins in a prokaryotic system. Western blot analysis revealed that native HqCRT was secreted into their hosts by ticks during blood sucking. Vaccination of sheep with rHqCRT conferred protective immunity against ticks, resulting in 54.3% mortality in adult ticks, compared to the 38.7% death rate in the control group. These results demonstrated that rHqCRT might be a useful vaccine candidate antigen for biological control of H. qinghaiensis.

Calreticulin requires an ancillary adjuvant for the induction of efficient cytotoxic T cell responses.

Molecular chaperones stimulate the immune system to induce both protective immune responses and therapeutic tumor rejection. However, the underlying basis for this immunogenic activity is not well understood. A variety of chaperones, including calreticulin, hsp70 and grp94, function as vehicles to efficiently traffic associated peptides into professional antigen presenting cells. Importantly, these chaperones have also been proposed to function as adjuvants by stimulating the dendritic cell activation and co-stimulatory responses required to elicit peptide-specific CD8(+) T cell cytolytic activity. The efficacy of chaperone-mediated tumor rejection has been attributed to the ability of chaperones to function in both of these capacities. However, purified calreticulin has not previously been assessed for its ability to elicit DC maturation and, moreover, recent data indicates that it is not efficient at inducing Nf-kappaB activity which often accompanies or stimulates DC maturation. Here we use two complementary methods to produce endotoxin-free calreticulin and demonstrate that it does not measurably mature or activate dendritic cells both in vitro and in vivo. Additionally, a calreticulin/peptide complex required the addition of an exogenous adjuvant to elicit in vivo cytotoxic CD8(+) T cell responses. These data are discussed with respect to current models for chaperone-derived immune responses and in regard to rational vaccine design.

Breakthrough of immune self-tolerance to calreticulin induced by CpG-oligodeoxynucleotides as adjuvant.

Reportedly, bacterial DNA containing unmethylated cytosine-guanosine dinucleotide motif-containing oligodeoxynucleotides (CpG-ODNs) can induce Th1-type adjuvant effects. We produced autoantibodies and induced hepatitis in mice using extracted proteins from human hepatocytes with CpG-ODNs as adjuvant. Western blot analysis was performed of sera from immunized mice and two patients with autoimmune hepatitis (AIH). When a common band was detected, N-terminal amino acid sequencing was performed to determine its site. For detection of antibodies against the identified protein (calreticulin), ELISA was performed of sera of 50 patients with AIH: 45 with primary biliary cirrhosis (PBC), 24 with chronic hepatitis C (CH), and 24 healthy controls. Mice were immunized with calreticulin protein with CpG-ODNs as adjuvant. Several reacted bands were detected in their sera; in addition, a common band to the sera of patients with AIH was detected at 60 kDa. Subsequent N-terminal amino acid sequencing revealed that the protein was human calreticulin. ELISA showed that, of patients with AIH, PBC, and CH, 30.0% (15/50), 17.8% (8/45), and 12.5% (3/24), respectively, were positive for anti calreticulin antibodies. Splenocytes from immunized mice produced IFN-gamma after they were pulsed with calreticulin protein. Histological analyses of liver specimens taken from mice immunized with calreticulin protein together with CpG-ODNs showed spotty and focal necrosis. Immunofluorescence analysis showed increased expression of calreticulin in the liver treated with CpG-ODNs. These results suggest that a breakthrough of immune self tolerance to calreticulin is induced with CpG-ODNs as adjuvant and that calreticulin protein might be a target antigen in this model.

The assessment of hookworm calreticulin as a potential vaccine for necatoriasis.

A vaccine against the human hookworm Necator americanus is urgently required to reduce hookworm-induced morbidity in endemic areas. In the present study, recombinant hookworm calreticulin, a nominated vaccine candidate, has been tested in mice. Mice given calreticulin had 43-49% fewer worms in their lungs, compared to non-vaccinated controls, following challenge infection with infective hookworm larvae. These levels of protection were achieved in the absence of adjuvant following intraperitoneal administration of three doses of 15 microg antigen. Antigen was also encapsulated in PLG microparticles. Encapsulated calreticulin elicited higher levels of anti-calreticulin IgG1 than free antigen but failed to induce protective immunity. The protection induced by free calreticulin was associated with low levels of serum IgE and moderate lung eosinophilia whilst administration of calreticulin-loaded microparticles was associated with high levels of serum IgE and higher lung eosinophil activity, suggesting that the classical Th2 phenotype may not always be associated with protective immunity, albeit in experimental necatoriasis.

Cloning, characterization, and functional expression of Taenia solium calreticulin.

Calreticulin is an endoplasmic reticulum protein involved in the homeostasis of intracellular Ca++ and other physiological processes. A complementary DNA clone containing the complete coding sequence of Taenia solium calreticulin (TsCRT) was isolated and characterized. Recombinant TsCRT was expressed in bacteria as a 50-kDa protein that specifically bound calcium when tested in a radioassay. The deduced amino acid sequence has 47-50% identity with other reported calreticulins. Poor recognition of TsCRT by human and pig sera with confirmed cysticercosis discourages its use for diagnosis of the disease. However, further characterization and localization studies could provide insights into the role of TsCRT in T. solium physiology and host-parasite interactions.