In silico Identification and Validation of a Linear and Naturally Immunogenic B-Cell Epitope of the Plasmodium vivax Malaria Vaccine Candidate Merozoite Surface Protein-9

Synthetic peptide vaccines provide the advantages of safety, stability and low cost. The success of this approach is highly dependent on efficient epitope identification and synthetic strategies for efficacious delivery. In malaria, the Merozoite Surface Protein-9 of Plasmodium vivax (PvMSP9) has been considered a vaccine candidate based on the evidence that specific antibodies were able to inhibit merozoite invasion and recombinant proteins were highly immunogenic in mice and humans. However the identities of linear B-cell epitopes within PvMSP9 as targets of functional antibodies remain undefined. We used several publicly-available algorithms for in silico analyses and prediction of relevant B cell epitopes within PMSP9. We show that the tandem repeat sequence EAAPENAEPVHENA (PvMSP9_E795-A808) present at the C-terminal region is a promising target for antibodies, given its high combined score to be a linear epitope and located in a putative intrinsically unstructured region of the native protein. To confirm the predictive value of the computational approach, plasma samples from 545 naturally exposed individuals were screened for IgG reactivity against the recombinant PvMSP9-RIRII_729-972 and a synthetic peptide representing the predicted B cell epitope PvMSP9_E795-A808. 316 individuals (58%) were responders to the full repetitive region PvMSP9-RIRII, of which 177 (56%) also presented total IgG reactivity against the synthetic peptide, confirming it validity as a B cell epitope. The reactivity indexes of anti-PvMSP9-RIRII and anti-PvMSP9_E795-A808 antibodies were correlated. Interestingly, a potential role in the acquisition of protective immunity was associated with the linear epitope, since the IgG1 subclass against PvMSP9_E795-A808 was the prevalent subclass and this directly correlated with time elapsed since the last malaria episode; however this was not observed in the antibody responses against the full PvMSP9-RIRII. In conclusion, our findings identified and experimentally confirmed the potential of PvMSP9_E795-A808 as an immunogenic linear B cell epitope within the P. vivax malaria vaccine candidate PvMSP9 and support its inclusion in future subunit vaccines.     

MAD2γ, a novel MAD2 isoform,reduces mitotic arrest and is associated with resistance in testicular germ cell tumors

Background: Prolonged mitotic arrest in response to anti-cancer chemotherapeutics, such as DNA-damaging agents, induces apoptosis, mitotic catastrophe, and senescence. Disruptions in mitotic checkpoints contribute resistance to DNA-damaging agents in cancer. MAD2 has been associated with checkpoint failure and chemotherapy response. In this study, a novel splice variant of MAD2, designated MAD2γ, was identified, and its association with the DNA damage response was investigated.

Methods: Endogenous expression of MAD2γ and full-length MAD2 (MAD2α) was measured using RT-PCR in cancer cell lines, normal foreskin fibroblasts, and tumor samples collected from patients with testicular germ cell tumors (TGCTs). A plasmid expressing MAD2γ was transfected into HCT116 cells, and its intracellular localization and checkpoint function were evaluated according to immunofluorescence and mitotic index.

Results: MAD2γ was expressed in several cancer cell lines and non-cancerous fibroblasts. Ectopically expressed MAD2γ localized to the nucleus and reduced the mitotic index, suggesting checkpoint impairment. In patients with TGCTs, the overexpression of endogenous MAD2γ, but not MAD2α, was associated with resistance to cisplatin-based chemotherapy. Likewise, cisplatin induced the overexpression of endogenous MAD2γ, but not MAD2α, in HCT116 cells.

Conclusions: Overexpression of MAD2γ may play a role in checkpoint disruption and is associated with resistance to cisplatin-based chemotherapy in TGCTs.     

Late toxicity for prostate cancer patients treated with hypofractionated helical tomotherapy

AimThe purpose of this study is to evaluate the long term tolerability of hypofractionated helical tomotherapy (HT) in localized prostate cancer patients.BackgroundPrevious hypofractionated schedules with conventional RT were associated with excessive toxicity, likely due to inadequate sophistication of treatment delivery. There are few data about late toxicity after HT.Materials and methodsWe evaluated 38 patients with primary adenocarcinoma of the prostate. There were 9 (24%), 15 (39%), and 14 (37%) patients with high, intermediate, and low risk, respectively. Patients were treated with hypofractionated HT from May 2008 to February 2011. Hypofractionation regimens included: 68.04 Gy at 2.52 Gy/fraction (N = 25; 66%), 70 Gy at 2.5 Gy/fraction (N = 4; 11%) and 70.2 Gy at 2.6 Gy/fraction (N = 9; 23%). Late genitourinary (GU) and gastrointestinal (GI) toxicity was scored using the Radiation Therapy Oncology Group scoring system.ResultsMedian age at diagnosis was 70 years (range 49–80) and median follow-up, 5.8 years. Late grade 1, 2 and 3 GI toxicity were 13%, 24%, and 2.6%, respectively. Late grade 1, 2, 3 GU toxicity were 29%, 21%, and 8%, respectively. Sexual toxicity was evaluated in 19 patients to be grade 1, 2 in 11% and grade 3 in 16%. Multivariate analysis showed that patients with higher values of rectum V50 associated with late GI toxicity (P = 0.025). Patients with PSA ≤8 (P = 0.048) or comorbidities (P = 0.013) at diagnosis were associated with higher late GU toxicity. Additionally, PSA ≤8 also associated with moderate (grade ≥2) late GU toxicity in the multivariate analysis (P = 0.028).ConclusionsHypofractionated HT can be delivered safely with limited rates of moderate and severe late toxicity. The proportion of the rectum that receives a moderate and high dose, having comorbidities, and PSA at diagnosis seem to associate with long term toxicity.     

Identifying molecularly defined antigens for a Histoplasma capsulatum-specific interferon gamma release assay

BackgroundIn a previous work we showed the feasibility of an interferon gamma release assay (IGRA) for detecting latent infection by Histoplasma capsulatum. While in that proof-of-concept study we used crude fungal extracts as antigens, the newest IGRAs developed for other infections are based on molecularly defined antigens, mostly on mixtures of immunogenic peptides.AimsTo identify proteins in H. capsulatum that might serve as molecularly defined antigens for an IGRA test.MethodsWe surveyed the literature looking for known H. capsulatum-immunogenic proteins and assayed two of them as antigens in an IGRA test, in a study that involved 80 volunteers. Furthermore, we used several bioinformatics tools to identify specific H. capsulatum proteins and to analyze possible strategies for the design of H. capsulatum-specific immunogenic peptides.ResultsSeven H. capsulatum-immunogenic proteins were retrieved from the literature. IGRA tests using either the heat shock protein 60 or the M antigen showed high sensitivities but low specificities, most likely due to the high sequence similarity with the corresponding orthologs in other pathogenic microorganisms. We identified around 2000 H. capsulatum-specific proteins, most of which remain unannotated. Class II T-cell epitope predictions for a small number of these proteins showed a great variability among different alleles, prompting for a “brute force” approach for peptide design.ConclusionsThe H. capsulatum genome encodes a large number of distinctive proteins, which represent a valuable source of potential specific antigens for an IGRA test. Among them, the Cfp4 protein stands out as a very attractive candidate.     

Label‐free bioanalysis of Leishmania infantum using refractive index tomography with partially coherent illumination

In this work, we report the use of refractive index (RI) tomography for quantitative analysis of unstained DH82 cell line infected with Leishmania infantum. The cell RI is reconstructed by using a modality of optical diffraction tomography technique that employs partially coherent illumination, thus enabling inherent compatibility with conventional wide‐field microscopes. The experimental results demonstrate that the cell dry mass concentration (DMC) obtained from the RI allows for reliable detection and quantitative characterization of the infection and its temporal evolution. The RI provides important insight for studying morphological changes, particularly membrane blebbing linked to an apoptosis (cell death) process induced by the disease. Moreover, the results evidence that infected DH82 cells exhibit a higher DMC than healthy samples. These findings open up promising perspectives for clinical diagnosis of Leishmania.