Anti-Thymocyte Globulin (ATG)-Free Nonmyeloablative Haploidentical PBSCT Plus Post-Transplantation Cyclophosphamide Is a Safe and Efficient Treatment Approach for Pediatric Acquired Aplastic Anemia.

Most cases of acquired aplastic anemia (AA) arise from autoimmune destruction of hematopoietic stem and progenitor cells. Human leukocyte antigen (HLA)-haploidentical nonmyeloablative hematopoietic stem cell transplantation (HSCT) plus post-transplantation cyclophosphamide (PTCy) is increasingly applied to salvage AA using bone marrow as graft and anti-thymocyte globulin (ATG) in conditioning. Herein, we characterize a cohort of twelve AA patients clinically and molecularly, six who possessed other immunological disorders (including two also carrying germline SAMD9L mutations). Each patient with SAMD9L mutation also carried an AA-related rare BCORL1 variant or CTLA4 p.T17A GG genotype, respectively, and both presented short telomere lengths. Six of the ten patients analyzed harbored AA-risky HLA polymorphisms. All patients recovered upon non-HSCT (n = 4) or HSCT (n = 8) treatments. Six of the eight HSCT-treated patients were subjected to a modified PTCy-based regimen involving freshly prepared peripheral blood stem cells (PBSC) as graft and exclusion of ATG. All patients were engrafted between post-transplantation days +13 and +18 and quickly reverted to normal life, displaying a sustained complete hematologic response and an absence of graft-versus-host disease. These outcomes indicate most AA cases, including of the SAMD9L-inherited subtype, are immune-mediated and the modified PTCy-based regimen we present is efficient and safe for salvage.

Evolution of Graves' Disease during Immune Reconstitution following Nonmyeloablative Haploidentical Peripheral Blood Stem Cell Transplantation in a Boy Carrying Germline SAMD9L and FLT3 Variants.

Graves' disease, characterized by hyperthyroidism resulting from loss of immune tolerance to thyroid autoantigens, may be attributable to both genetic and environmental factors. Allogeneic hematopoietic stem cell transplantation (HSCT) represents a means to induce immunotolerance via an artificial immune environment. We present a male patient with severe aplastic anemia arising from a germline SAMD9L missense mutation who successfully underwent HSCT from his HLA-haploidentical SAMD9L non-mutated father together with nonmyeloablative conditioning and post-transplant cyclophosphamide at 8 years of age. He did not suffer graft-versus-host disease, but Graves' disease evolved 10 months post-transplant when cyclosporine was discontinued for one month. Reconstitution of peripheral lymphocyte subsets was found to be transiently downregulated shortly after Graves' disease onset but recovered upon antithyroid treatment. Our investigation revealed the presence of genetic factors associated with Graves' disease, including HLA-B*46:01 and HLA-DRB1*09:01 haplotypes carried by the asymptomatic donor and germline FLT3 c.2500C>T mutation carried by both the patient and the donor. Given his current euthyroid state with normal hematopoiesis, the patient has returned to normal school life. This rare event of Graves' disease in a young boy arising from special HSCT circumstances indicates that both the genetic background and the HSCT environment can prompt the evolution of Graves' disease.

Peri-treatment adverse events of primary mediastinal non-seminomatous germ cell tumors.

Primary mediastinal non-seminomatous germ cell tumors (PMNSGCT) are rare but life-threatening thoracic cancers. We report our experience from eight patients with peri-treatment adverse events. By analyzing changes in tumor extent, serum tumor markers, and pathologies between diagnosis and transfer, those events could be attributed to postbiopsy respiratory insufficiency, growing teratoma syndrome, secondary histiocytic malignancy, and PMNSGCT progression. Subjecting patients to respiratory therapy, conventional or high-dose chemotherapy, and surgery controlled the disease, with five of the eight patients surviving disease free. These outcomes indicate that integrated appropriate and timely approaches are important in tackling peri-treatment adverse events.

Analysis of site-specific glycan profiles of serum proteins in patients with multiple sclerosis or neuromyelitis optica spectrum disorder-a pilot study.

Glycosylation is important for biological functions of proteins and greatly affected by diseases. Exploring the glycosylation profile of the protein-specific glycosylation and/or the site-specific glycosylation may help understand disease etiology, differentiate diseases and ultimately develop therapeutics. Patients with multiple sclerosis (MS) and patients with neuromyelitis optica spectrum disorder (NMOSD) are sometimes difficult to differentiate due to the similarity in their clinical symptoms. The disease-related glycosylation profiles of MS and NMOSD have not yet been well studied. Here, we analyzed site-specific glycan profiles of serum proteins of these patients by using a recently developed mass spectrometry technique. A total of 286 glycopeptides from 49 serum glycoproteins were quantified and compared between healthy controls (n = 6), remitting MS (n = 45) and remitting NMOSD (n = 23) patients. Significant differences in the levels of site-specific N-glycans on inflammation-associated components [IgM, IgG1, IgG2, complement components 8b (CO8B) and attractin], central nerve system-damage-related serum proteins [apolipoprotein D (APOD), alpha-1-antitrypsin, plasma kallikrein and ADAMTS-like protein 3] were observed among three study groups. We furthered demonstrated that site-specific N-glycans on APOD on site 98, CO8B on sites 243 and 553 are potential markers to differentiate MS from NMOSD with an area under receiver operating curve value > 0.75. All these observations indicate that remitting MS or NMOSD patients possess a unique disease-associated glyco-signature in their serum proteins. We conclude that monitoring one's serum protein glycan profile using this high-throughput analysis may provide an additional diagnostic criterion for differentiating diseases, monitoring disease status and estimating response-to-treatment effect.

Alphavirus-based hepatitis C virus therapeutic vaccines: can universal helper epitopes enhance HCV-specific cytotoxic T lymphocyte responses?

BACKGROUND: Antigen-specific T cell immune responses play a pivotal role in resolving acute and chronic hepatitis C virus (HCV) infections. Currently, no prophylactic or therapeutic vaccines against HCV are available. We previously demonstrated the preclinical potency of therapeutic HCV vaccines based on recombinant Semliki Forest virus (SFV) replicon particles. However, clinical trials do not always meet the high expectations of preclinical studies, thus, optimization of vaccine strategies is crucial. In efforts to further increase the frequency of HCV-specific immune responses in the candidate SFV-based vaccines, the authors assessed whether inclusion of three strong, so-called universal helper T cell epitopes, and an endoplasmic reticulum localization, and retention signal (collectively termed sigHELP-KDEL cassette) could enhance HCV-specific immune responses. METHODS: We included the sigHELP-KDEL cassette in two of the candidate SFV-based HCV vaccines, targeting NS3/4A and NS5A/B proteins. We characterized the new constructs in vitro for the expression and stability of the transgene-encoded proteins. Their immune efficacy with respect to HCV-specific immune responses in vivo was compared with the parental SFV vaccine expressing the corresponding HCV antigen. Further characterization of the functionality of the HCV-specific CD8+ T cells was assessed by surface and intracellular cytokine staining and flow cytometry analysis. RESULTS: Moderate, but significantly, enhanced frequencies of antigen-specific immune responses were achieved upon lower/suboptimal dosage immunization. In optimal dosage immunization, the inclusion of the cassette did not further increase the frequencies of HCV-specific CD8+ T cells when compared with the parental vaccines and the frequencies of effector and memory populations were identical. CONCLUSION: We hypothesize that the additional effect of the sigHELP-KDEL cassette in SFV-based vaccines depends on the immunogenicity, nature, and stability of the target antigen expressed by the vaccine.

Potent therapeutic efficacy of an alphavirus replicon DNA vaccine expressing human papilloma virus E6 and E7 antigens.

Cervical cancer develops as a result of infection with high-risk human papillomavirus (HPV) through persistent expression of early proteins E6 and E7. Our group pioneered a recombinant viral vector system based on Semliki Forest virus (SFV) for vaccination against cervical cancer. The most striking benefit of this alphavirus vector-based vaccine platform is its high potency. DNA vaccines on the other hand, have a major advantage with respect to ease of production. In this study, the benefits associated with both SFV-based vaccines and DNA vaccines were combined with the development of a DNA-launched RNA replicon (DREP) vaccine targeting cervical cancer. Using intradermal delivery followed by electroporation, we demonstrated that DREP encoding for E6,7 (DREP-E6,7) induced effective, therapeutic antitumor immunity. While immunizations with a conventional DNA vaccine did not prevent tumor outgrowth, immunization with a 200-fold lower equimolar dose of DREP (0.05 µg of DREP) resulted in approximately 85% of tumor-free mice. To overcome the safety concern of potential malignant transformation at the vaccination site, we evaluated the anti-tumor effect of a DREP vaccine encoding a shuffled version of E7 (DREP-E7sh). DREP-E7sh delayed tumor growth yet not to the same extent as DREP-E6,7. In addition, inclusion of a helper cassette and an ER targeting signal (sigHelp) did not significantly further enhance the suppression of tumor outgrowth in the long term, albeit exhibiting better tumor control early after immunization. Collectively, this study points towards the clinical evaluation of DREP encoding HPV antigens as a potent immunotherapy for patients with HPV16 (pre)-malignancies.

Differentiation of remitting neuromyelitis optica spectrum disorders from multiple sclerosis by integrating parameters from serum proteins and lymphocyte subsets.

Differential diagnosis for neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS) is always doubtful. To differentiate these diseases, we studied the immune status in the blood of patients with MS (n = 45) or NMOSD (n = 23) at remission phase. Remitting NMOSD patients had increased levels of CXCL13 and memory B cells, while remitting MS patients had elevated levels of galectin-9 and Th1 cells. A diagnostic model with these four variables is built to distinguish remitting NMOSD from MS with a sensitivity of 91.30%. Our diagnostic model may help to improve the differentiation of remitting NMOSD from MS.

CCR6 Deficiency Impairs IgA Production and Dysregulates Antimicrobial Peptide Production, Altering the Intestinal Flora.

Intestinal immunity exists as a complex relationship among immune cells, epithelial cells, and microbiota. CCR6 and its ligand-CCL20 are highly expressed in intestinal mucosal tissues, such as Peyer's patches (PPs) and isolated lymphoid follicles (ILFs). In this study, we investigated the role of the CCR6-CCL20 axis in intestinal immunity under homeostatic conditions. CCR6 deficiency intrinsically affects germinal center reactions in PPs, leading to impairments in IgA class switching, IgA affinity, and IgA memory B cell production and positioning in PPs, suggesting an important role for CCR6 in T-cell-dependent IgA generation. CCR6 deficiency impairs the maturation of ILFs. In these follicles, group 3 innate lymphoid cells are important components and a major source of IL-22, which stimulates intestinal epithelial cells (IECs) to produce antimicrobial peptides (AMPs). We found that CCR6 deficiency reduces IL-22 production, likely due to diminished numbers of group 3 innate lymphoid cells within small-sized ILFs. The reduced IL-22 levels subsequently decrease the production of AMPs, suggesting a critical role for CCR6 in innate intestinal immunity. Finally, we found that CCR6 deficiency impairs the production of IgA and AMPs, leading to increased levels of Alcaligenes in PPs, and segmented filamentous bacteria in IECs. Thus, the CCR6-CCL20 axis plays a crucial role in maintaining intestinal symbiosis by limiting the overgrowth of mucosa-associated commensal bacteria.

Epitope Prediction Assays Combined with Validation Assays Strongly Narrows down Putative Cytotoxic T Lymphocyte Epitopes.

Tumor vaccine design requires prediction and validation of immunogenic MHC class I epitopes expressed by target cells as well as MHC class II epitopes expressed by antigen-presenting cells essential for the induction of optimal immune responses. Epitope prediction methods are based on different algorithms and are instrumental for a first screening of possible epitopes. However, their results do not reflect a one-to-one correlation with experimental data. We combined several in silico prediction methods to unravel the most promising C57BL/6 mouse-restricted Hepatitis C virus (HCV) MHC class I epitopes and validated these epitopes in vitro and in vivo. Cytotoxic T lymphocyte (CTL) epitopes within the HCV non-structural proteins were identified, and proteasomal cleavage sites and helper T cell (Th) epitopes at close proximity to these CTL epitopes were analyzed using multiple prediction algorithms. This combined in silico analysis enhances the precision of identification of functional HCV-specific CTL epitopes. This approach will be applicable to the design of human vaccines not only for HCV, but also for other antigens in which T-cell responses play a crucial role.

Alphavirus-based vaccines encoding nonstructural proteins of hepatitis C virus induce robust and protective T-cell responses.

An absolute prerequisite for a therapeutic vaccine against hepatitis C virus (HCV) infection is the potency to induce HCV-specific vigorous and broad-spectrum T-cell responses. Here, we generated three HCV vaccines based on a recombinant Semliki Forest virus (rSFV) vector expressing all- or a part of the conserved nonstructural proteins (nsPs) of HCV. We demonstrated that an rSFV vector was able to encode a transgene as large as 6.1 kb without affecting its vaccine immunogenicity. Prime-boost immunizations of mice with rSFV expressing all nsPs induced strong and long-lasting NS3-specific CD8(+) T-cell responses. The strength and functional heterogeneity of the T-cell response was similar to that induced with rSFV expressing only NS3/4A. Furthermore this leads to a significant growth delay and negative selection of HCV-expressing EL4 tumors in an in vivo mouse model. In general, as broad-spectrum T-cell responses are only seen in patients with resolved HCV infection, this rSFV-based vector, which expresses all nsPs, inducing robust T-cell activity has a potential for the treatment of HCV infections.

Therapeutic vaccination against chronic hepatitis C virus infection.

Approximately 170 million people worldwide are chronic carriers of Hepatitis C virus (HCV). To date, there is no prophylactic vaccine available against HCV. The standard-of-care therapy for HCV infection involves a combination of pegylated interferon-α and ribavirin. This therapy, which is commonly associated with side effects, has a curative rate varying from 43% (HCV genotype 1) to 80% (HCV genotype 2). In 2011, two direct-acting antiviral agents, telaprevir and boceprevir, were approved by the US Food and drug Administration and are now being used in combination with standard-of-care therapy in selected patients infected with HCV genotype 1. Although both drugs are promising, resulting in a shortening of therapy, these drugs also induce additional side effects and have reduced efficacy in patients who did not respond to standard-of-care previously. An alternative approach would be to treat HCV by stimulating the immune system with a therapeutic vaccine ideally aimed at (i) the eradication of HCV-infected cells and (ii) neutralization of infectious HCV particles. The challenge is to develop therapeutic vaccination strategies that are either at least as effective as antiviral drugs but with lower side effects, or vaccines that, when combined with antiviral drugs, can circumvent long-term use of these drugs thereby reducing their side effects. In this review, we summarize and discuss recent preclinical developments in the area of therapeutic vaccination against chronic HCV infection. Although neutralizing antibodies have been described to exert protective immunity, clinical studies on the induction of neutralizing antibodies in therapeutic settings are limited. Therefore, we will primarily discuss therapeutic vaccines which aim to induce effective cellular immune response against HCV.

Resistance to dengue virus infection in mice is potentiated by CXCL10 and is independent of CXCL10-mediated leukocyte recruitment.

CXCL10 is an IFN-inducible chemokine ligand that binds CXCR3, a receptor that is expressed on lymphocytes; CXCL10 shares the CXCR3 receptor with another two ligands, CXCL9 and CXCL11. Previously, we found that CXCL10(-/-) mice were more susceptible than wild-type (WT) mice to dengue virus (DENV) infection. In this study, we explored the mechanisms underlying this enhanced susceptibility. We found that viral loads were higher in the brains of CXCL10(-/-) mice than in WT mice. Presuming a defect in effector lymphocyte migration, we investigated whether recruitment of effector T cells and Ab-secreting cells to the infected tissues were impaired in CXCL10(-/-) mice. Unexpectedly, compared with WT, CXCL10(-/-) mice had comparable numbers of total infiltrating T cells, higher numbers of CXCR3(+) T cells, and higher numbers of Ab-secreting cells in the brain. Additionally, we found that CXCL10 was induced in neurons following DENV infection and that CXCL10 competed with DENV for binding to cell surface heparan sulfate, a coreceptor for DENV entry, thus inhibiting binding of DENV to neuronal cells. These results demonstrate that the enhanced susceptibility of CXCL10(-/-) mice to DENV infection is not due to a defect in recruitment of effector lymphocytes but rather to an antiviral activity that promotes viral clearance.