Genetic Approaches for Definitive Diagnosis of Agammaglobulinemia in Consanguineous Families.

Autosomal recessive agammaglobulinemia (ARA) is a primary immunodeficiency characterized by absent peripheral B cells, severe hypogammaglobulinemia, and absent BTK gene mutations. In ARA, mutations occur in genes encoding the pre-B cell receptor (pre-BCR) or downstream signaling proteins. In this work, we used candidate gene and whole-exome sequencing to investigate the molecular basis of ARA in 6 patients from 4 consanguineous North-African families. Sanger sequencing of candidate genes encoding the pre-BCR components (ΙGΗΜ, CD79A, CD79B, IGLL1, and VPREB1) was initially performed and determined the genetic defect in five patients. Two novel mutations in IGHM (p.Val378Alafs*1 and p.Ile184Serfs*21) were identified in three patients from two unrelated kindred and a novel nonsense mutation was identified in CD79A (p.Trp66*) in two siblings from a third kindred. Whole-exome sequencing (WES) was performed on the sixth patient who harbored a homozygous stop mutation at position 407 in the RAG2 gene (p.Glu407*). We concluded that conventional gene sequencing, especially when multiple genes are involved in the defect as is the case in ARA, is costly and time-consuming, resulting in delayed diagnosis that contributes to increased morbidity and mortality. In addition, it fails to identify the involvement of novel and unsuspected gene defects when the phenotype of the patients is atypical. WES has the potential to provide a rapid and more accurate genetic diagnosis in ARA, which is crucial for the treatment of the patients.

Phlebotomus papatasi Yellow-Related and Apyrase Salivary Proteins Are Candidates for Vaccination against Human Cutaneous Leishmaniasis.

Nowadays, there is no available vaccine for human leishmaniasis. Animal experiments demonstrate that pre-exposure to sand fly saliva confers protection against leishmaniasis. Our preceding work in humans indicates that Phlebotomus papatasi saliva induces the production of IL-10 by CD8+ T lymphocytes. The neutralization of IL-10 enhanced the activation of a T-cell CD4+ population-producing IFN-γ. Herein, we used a biochemical and functional genomics approach to identify the sand fly salivary components that are responsible for the activation of the T helper type 1 immune response in humans, therefore constituting potential vaccine candidates against leishmaniasis. Fractionated P. papatasi salivary extracts were first tested on T lymphocytes of immune donors. We confirmed that the CD4+ lymphocytes proliferate and produce IFN-γ in response to stimulation with the proteins of molecular weight >30 kDa. Peripheral blood mononuclear cells from immune donors were transfected with plasmids coding for the most abundant proteins from the P. papatasi salivary gland cDNA library. Our result showed that the "yellow related proteins," PPTSP42 and PPTSP44, and "apyrase," PPTSP36, are the proteins responsible for the aforementioned cellular immune response and IFN-γ production. Strikingly, PPTSP44 triggered the highest level of lymphocyte proliferation and IFN-γ production. Multiplex cytokine analysis confirmed the T helper type 1-polarized response induced by these proteins. Importantly, recombinant PPTSP44 validated the results observed with the DNA plasmid, further supporting that PPTSP44 constitutes a promising vaccine candidate against human leishmaniasis.

Prediction of T Cell Epitopes from Leishmania major Potentially Excreted/Secreted Proteins Inducing Granzyme B Production.

Leishmania-specific cytotoxic T cell response is part of the acquired immune response developed against the parasite and contributes to resistance to reinfection. Herein, we have used an immune-informatic approach for the identification, among Leishmania major potentially excreted/secreted proteins previously described, those generating peptides that could be targeted by the cytotoxic immune response. Seventy-eight nonameric peptides that are predicted to be loaded by HLA-A*0201 molecule were generated and their binding capacity to HLA-A2 was evaluated. These peptides were grouped into 20 pools and their immunogenicity was evaluated by in vitro stimulation of peripheral blood mononuclear cells from HLA-A2+-immune individuals with a history of zoonotic cutaneous leishmaniasis. Six peptides were identified according to their ability to elicit production of granzyme B. Furthermore, among these peptides 3 showed highest affinity to HLA-A*0201, one derived from an elongation factor 1-alpha and two from an unknown protein. These proteins could constitute potential vaccine candidates against leishmaniasis.