A recombinant chimeric protein composed of human and mice‐specific CD4+ and CD8+ T‐cell epitopes protects against visceral leishmaniasis

In this study, a recombinant chimeric protein (RCP), which was composed of specific CD4⁺ and CD8⁺ T‐cell epitopes to murine and human haplotypes, was evaluated as an immunogen against Leishmania infantum infection in a murine model. BALB/c mice received saline were immunized with saponin or with RCP with or without an adjuvant. The results showed that RCP/saponin‐vaccinated mice presented significantly higher levels of antileishmanial IFN‐γ, IL‐12 and GM‐CSF before and after challenge, which were associated with the reduction of IL‐4 and IL‐10 mediated responses. These animals showed significant reductions in the parasite burden in all evaluated organs, when both limiting dilution and quantitative real‐time PCR techniques were used. In addition, the protected animals presented higher levels of parasite‐specific nitrite, as well as the presence of anti‐Leishmania IgG2a isotype antibodies. In conclusion, the RCP/saponin vaccine could be considered as a prophylactic alternative to prevent against VL.     

Comparative analysis between RQ‐PCR and digital‐droplet‐PCR of immunoglobulin/T‐cell receptor gene rearrangements to monitor minimal residual disease in acute lymphoblastic leukaemia

Real‐time quantitative polymerase chain reaction (RQ‐PCR) is a standardized tool for minimal residual disease (MRD) monitoring in acute lymphoblastic leukaemia (ALL). The applicability of this technology is limited by the need of a standard curve based on diagnostic DNA. The digital droplet PCR (ddPCR) technology has been recently applied to various medical fields, but its use in MRD monitoring is under investigation. In this study, we analysed 50 ALL cases by both methods in two phases: in the first, we established analytical parameters to investigate the applicability of this new technique; in the second, we analysed MRD levels in 141 follow‐up (FU) samples to investigate the possible use of ddPCR for MRD monitoring in ALL patients. We documented that ddPCR has sensitivity and accuracy at least comparable to those of RQ‐PCR. Overall, the two methods gave concordant results in 124 of the 141 analysed MRD samples (88%, P = 0·94). Discordant results were found in 12% borderline cases. The results obtained prove that ddPCR is a reliable method for MRD monitoring in ALL, with the advantage of quantifying without the need of the calibration curves. Its application in a cohort of patients with a longer FU will conclusively define its clinical predictive value.     

Genes encoding members of the JAK‐STAT pathway or epigenetic regulators are recurrently mutated in T‐cell prolymphocytic leukaemia

T‐cell prolymphocytic leukaemia (T‐PLL) is an aggressive leukaemia. The primary genetic alteration in T‐PLL are the inv(14)(q11q32)/t(14;14)(q11;q32) leading to TRD/TRA‐TCL1A fusion, or the t(X;14)(q28;q11) associated with TRD/TRA‐MTCP1 fusion. However, additional cooperating abnormalities are necessary for emergence of the full neoplastic phenotype. Though the pattern of secondary chromosomal aberrations is remarkably conserved, targets of the changes are largely unknown. We analysed a cohort of 43 well‐characterized T‐PLL for hotspot mutations in the genes JAK3, STAT5B and RHOA. Additionally, we selected a subset of 23 T‐PLL cases for mutational screening of 54 genes known to be recurrently mutated in T‐cell and other haematological neoplasms. Activating mutations in the investigated regions of the JAK3 and STAT5B genes were detected in 30% (13/43) and 21% (8/39) of the cases, respectively, and were mutually exclusive. Further, we identified mutations in the genes encoding the epigenetic regulators EZH2 in 13% (3/23), TET2 in 17% (4/23) and BCOR in 9% (2/23) of the cases. We confirmed that the JAK‐STAT pathway is a major mutational target, and identified epigenetic regulators recurrently mutated in T‐PLL. These findings complement the mutational spectrum of secondary aberrations in T‐PLL and underscore the potential therapeutical relevance of epigenetic regulators in T‐PLL.     

Differential T‐cell responses to a chimeric Plasmodium falciparum antigen; UB05‐09, correlates with acquired immunity to malaria

The development of a sterilizing and cost‐effective vaccine against malaria remains a major problem despite recent advances. In this study, it is demonstrated that two antigens of P. falciparum UB05, UB09 and their chimera UB05‐09 can serve as protective immunity markers by eliciting higher T‐cell responses in malaria semi‐immune subjects (SIS) than in frequently sick subjects (FSS) and could be used to distinguish these two groups. UB05, UB09 and UB05‐09 were cloned, expressed in E. coli, purified and used to stimulate PBMCs isolated from 63 subjects in a malaria endemic area, for IFN‐γ production, which was measured by the ELISpot assay. The polymorphism of UB09 gene in the malaria infected population was also studied by PCR/sequencing of the gene in P. falciparum field isolates. All three antigens were preferentially recognized by PBMCs from SIS. IFN‐γ production induced by these antigens correlated with the absence of fever and parasitaemia. UB09 was shown to be relatively well‐conserved in nature. It is concluded that UB05, UB09 and the chimera UB05‐09 posses T‐cell epitopes that are associated with protection against malaria and could thus be used to distinguish SIS from FSS eventhough acute infection with malaria has been shown to reduce cytokine production in some studies. Further investigations of these antigens as potential diagnostic and/or vaccine candidates for malaria are indicated.     

Evaluation of a novel lentiviral vaccine expressing KMP11‐HASPB fusion protein against Leishmania infantum in BALB/c mice

Hydrophilic acylated surface protein B (HASPB) is an immunogenic Leishmania protein against which antibodies are produced in the sera of cutaneous and visceral Leishmaniasis (VL) patients. Kinetoplastid membrane protein 11 (KMP11) is another protein antigen of Leishmania which is reported as a promising candidate for vaccination of VL. It is a highly conserved surface protein present in all members of kinetoplastid family and is expressed in both promastigotes and amastigotes. In this study, the coding sequence of KMP11 and HASPB was cloned into a pCDH‐cGFP lentiviral vector as a fusion protein. The gene expression was confirmed using RT‐PCR and Western blot methods. After injection of the recombinant KMP11‐HASPB‐expressing lentiviruses to BALB/c mice, using ELISA technique, a significant increase in IFN‐γ and IL‐4 as well as IgG1 and IgG2a was observed compared to the control group. Furthermore, the number of parasites in the liver and spleen of vaccinated mice decreased significantly compared with the control group.     

Validation of the NCCN‐IPI for diffuse large B‐cell lymphoma (DLBCL): the addition of β2‐microglobulin yields a more accurate GELTAMO‐IPI

The study included 1848 diffuse large B‐cell lymphoma (DLBCL )patients treated with chemotherapy/rituximab. The aims were to validate the National Comprehensive Cancer Network International Prognostic Index (NCCN ‐IPI ) and explore the effect of adding high Beta‐2 microglobulin (β2M), primary extranodal presentation and intense treatment to the NCCN ‐IPI variables in order to develop an improved index. Comparing survival curves, NCCN ‐IPI discriminated better than IPI , separating four risk groups with 5‐year overall survival rates of 93%, 83%, 67% and 49%, but failing to identify a true high‐risk population. For the second aim the series was split into training and validation cohorts: in the former the multivariate model identified age, lactate dehydrogenase, Eastern Cooperative Oncology Group performance status, Stage III ‐IV , and β2M as independently significant, whereas the NCCN ‐IPI ‐selected extranodal sites, primary extranodal presentation and intense treatments were not. These results were confirmed in the validation cohort. The Grupo Español de Linfomas/Trasplante de Médula ósea (GELTAMO )‐IPI developed here, with 7 points, significantly separated four risk groups (0, 1–3, 4 or ≥5 points) with 11%, 58%, 17% and 14% of patients, and 5‐year overall survival rates of 93%, 79%, 66% and 39%, respectively. In the comparison GELTAMO IPI discriminated better than the NCCN ‐IPI . In conclusion, GELTAMO ‐IPI is more accurate than the NCCN ‐IPI and has statistical and practical advantages in that the better discrimination identifies an authentic high‐risk group and is not influenced by primary extranodal presentation or treatments of different intensity.     

Differential in vitro CD4+/CD8+ T‐cell response to live vs. killed Leishmania major

Clinical trials of killed Leishmania vaccines showed a limited efficacy compared with leishmanization (LZ). The reason for this difference in protection against cutaneous leishmaniasis (CL) is not known and in vivo studies on T‐cell function may provide valuable information. Nevertheless, there are limited studies on the nature of the stimulatory effects of live vs. killed parasites on human T cells in vitro. A total of nine Leishmanin Skin Test+ volunteers with a history of self‐healing CL (HCL) and seven healthy volunteers were included in this study. 5,6‐carboxyfluroescein diacetate succinimidyl ester‐labelled CD4⁺/CD8⁺ lymphocytes were cultured with killed Leishmania Lysate (Killed LL) or live Leishmania major (Live LM) and analysed for proliferation using flow cytometry. Culture supernatants were used for cytokine titration. In HCL volunteers, upon stimulation with killed LL, the number of proliferated CD4⁺/CD8⁺ cells was significantly more than that of unstimulated (P < 0·001) or live LM stimulated (P < 0·05) cells, or cells from controls (CD4⁺/CD8⁺: P < 0·05/P < 0·001). Stimulation of CD4⁺ cells with Live LM (P < 0·001) or Killed LL (P < 0·05) induced a significantly higher IFN‐γ production compared with that of controls, but Live LM induced significantly (P < 0·05) more IFN‐γ than Killed LL. A significantly (P < 0·05) higher IFN‐γ production was observed when CD8⁺ cells were stimulated with Live LM. Cells from HCL volunteers showed significantly more IL‐10 production to Live LM stimulation compared with that of controls (CD4⁺: P < 0·05 /CD8⁺: P < 0·001) or cells stimulated with Killed LL (CD4⁺/CD8⁺: P < 0·001/P < 0·0005). Whereas Killed LL induced more proliferation response in purified T cells, Live LM induced cytokine production without significant induction of proliferation. The results from healed CL volunteers in this study could be implicated in further studies on T‐cell response in vaccinated individuals.     

Evidence of cross‐stage CD8+ T cell epitopes in malaria pre‐erythrocytic and blood stage infections

Malaria parasites have a complex, multistage life cycle and there is a widely held view that each stage displays a distinct set of antigens presented to the immune system. Yet, molecular analysis of malaria parasites suggests that many putative antigenic targets are shared amongst the different stages. The specificities of these cross‐stage antigens and the functions of the immune responses they elicit are poorly characterized. It is well‐known that CD8+ T cells play opposing immune functions following Plasmodium berghei (Pb) infection of C57BL/6 mice. Whilst these cells play a crucial role in protective immunity against pre‐erythrocytic stages, they are implicated in the development of severe disease during blood stages. Recently, CD8+ T cell epitopes derived from proteins supposedly specific for either pre‐erythrocytic or blood stages have been described. In this brief report, we have compiled and confirmed data that the majority of the mRNAs and/or proteins from which these epitopes are derived display expression across pre‐erythrocytic and blood stages. Importantly, we provide evidence of cross‐stage immune recognition of the majority of these CD8+ T cell epitopes. Hence, our findings provide a resource to further examine the relevance of antigen‐specific cross‐stage responses during malaria infections.