VISTA checkpoint inhibition by pH-selective antibody SNS-101 with optimized safety and pharmacokinetic profiles enhances PD-1 response.

VISTA, an inhibitory myeloid-T-cell checkpoint, holds promise as a target for cancer immunotherapy. However, its effective targeting has been impeded by issues such as rapid clearance and cytokine release syndrome observed with previous VISTA antibodies. Here we demonstrate that SNS-101, a newly developed pH-selective VISTA antibody, addresses these challenges. Structural and biochemical analyses confirmed the pH-selectivity and unique epitope targeted by SNS-101. These properties confer favorable pharmacokinetic and safety profiles on SNS-101. In syngeneic tumor models utilizing human VISTA knock-in mice, SNS-101 shows in vivo efficacy when combined with a PD-1 inhibitor, modulates cytokine and chemokine signaling, and alters the tumor microenvironment. In summary, SNS-101, currently in Phase I clinical trials, emerges as a promising therapeutic biologic for a wide range of patients whose cancer is refractory to current immunotherapy regimens.

Comparative genomics of Leishmania (Mundinia).

BACKGROUND: Trypanosomatids of the genus Leishmania are parasites of mammals or reptiles transmitted by bloodsucking dipterans. Many species of these flagellates cause important human diseases with clinical symptoms ranging from skin sores to life-threatening damage of visceral organs. The genus Leishmania contains four subgenera: Leishmania, Sauroleishmania, Viannia, and Mundinia. The last subgenus has been established recently and remains understudied, although Mundinia contains human-infecting species. In addition, it is interesting from the evolutionary viewpoint, representing the earliest branch within the genus and possibly with a different type of vector. Here we analyzed the genomes of L. (M.) martiniquensis, L. (M.) enriettii and L. (M.) macropodum to better understand the biology and evolution of these parasites. RESULTS: All three genomes analyzed were approximately of the same size (~ 30 Mb) and similar to that of L. (Sauroleishmania) tarentolae, but smaller than those of the members of subgenera Leishmania and Viannia, or the genus Endotrypanum (~ 32 Mb). This difference was explained by domination of gene losses over gains and contractions over expansions at the Mundinia node, although only a few of these genes could be identified. The analysis predicts significant changes in the Mundinia cell surface architecture, with the most important ones relating to losses of LPG-modifying side chain galactosyltransferases and arabinosyltransferases, as well as ß-amastins. Among other important changes were gene family contractions for the oxygen-sensing adenylate cyclases and FYVE zinc finger-containing proteins. CONCLUSIONS: We suggest that adaptation of Mundinia to different vectors and hosts has led to alternative host-parasite relationships and, thereby, made some proteins redundant. Thus, the evolution of genomes in the genus Leishmania and, in particular, in the subgenus Mundinia was mainly shaped by host (or vector) switches.

Molecular Characterization of Leishmania RNA virus 2 in Leishmaniamajor from Uzbekistan.

Here we report sequence and phylogenetic analysis of two new isolates of Leishmania RNA virus 2 (LRV2) found in Leishmania major isolated from human patients with cutaneous leishmaniasis in south Uzbekistan. These new virus-infected flagellates were isolated in the same region of Uzbekistan and the viral sequences differed by only nineteen SNPs, all except one being silent mutations. Therefore, we concluded that they belong to a single LRV2 species. New viruses are closely related to the LRV2-Lmj-ASKH documented in Turkmenistan in 1995, which is congruent with their shared host (L. major) and common geographical origin.

Improved panels for clinical immune phenotyping: Utilization of the violet laser.

BACKGROUND: Clinical diagnostic laboratories are subject to numerous regulations imposed by government agencies. Laboratory developed tests for flow cytometry panels are essentially restricted to the use of analyte-specific reagents (ASR) antibodies. With the advances in clinical flow cytometry systems, there is a trend toward the utilization of blue/red/violet laser flow systems and 8 to 10-color panels. Currently, the selection of commercially available ASR antibodies for the violet laser is very limited. The market is dominated by Brilliant Violet 421 (BV421) manufactured by BD Biosciences and Pacific Blue (PB) manufactured by Beckman Coulter. In this study, we compare BV421 and PB conjugated ASR antibodies. METHODS: Whole blood was stained and acquired on a Gallios flow cytometer system. For single color staining, the stain index (SI) was calculated. For the two panels, the compensation matrix was calculated and the performance of the antibody cocktails analyzed in FCS Express. RESULTS: The results show that five out of six tested BV421 conjugated antibodies have significantly higher SI than their PB counterparts. Furthermore, BV421 antibodies require less compensation for spillover than PB. Finally, BV421 conjugated antibodies give better separation between negative and positive populations in the context of an 8 and 10 color panel without affecting the intensity of the other dyes. CONCLUSIONS: Overall, using BV421 conjugated antibodies results in better separation between populations compared to PB conjugated antibodies without negatively affecting other fluorochromes in our panels. We conclude that the BV421 conjugated ASR antibodies are currently the better available option for clinical flow panels. © 2017 International Clinical Cytometry Society.

Humanized HLA-DR4 mice fed with the protozoan pathogen of oysters Perkinsus marinus (Dermo) do not develop noticeable pathology but elicit systemic immunity.

Perkinsus marinus (Phylum Perkinsozoa) is a marine protozoan parasite responsible for "Dermo" disease in oysters, which has caused extensive damage to the shellfish industry and estuarine environment. The infection prevalence has been estimated in some areas to be as high as 100%, often causing death of infected oysters within 1-2 years post-infection. Human consumption of the parasites via infected oysters is thus likely to occur, but to our knowledge the effect of oral consumption of P. marinus has not been investigated in humans or other mammals. To address the question we used humanized mice expressing HLA-DR4 molecules and lacking expression of mouse MHC-class II molecules (DR4.EA(0)) in such a way that CD4 T cell responses are solely restricted by the human HLA-DR4 molecule. The DR4.EA(0) mice did not develop diarrhea or any detectable pathology in the gastrointestinal tract or lungs following single or repeated feedings with live P. marinus parasites. Furthermore, lymphocyte populations in the gut associated lymphoid tissue and spleen were unaltered in the parasite-fed mice ruling out local or systemic inflammation. Notably, naïve DR4.EA(0) mice had antibodies (IgM and IgG) reacting against P. marinus parasites whereas parasite specific T cell responses were undetectable. Feeding with P. marinus boosted the antibody responses and stimulated specific cellular (IFNγ) immunity to the oyster parasite. Our data indicate the ability of P. marinus parasites to induce systemic immunity in DR4.EA(0) mice without causing noticeable pathology, and support rationale grounds for using genetically engineered P. marinus as a new oral vaccine platform to induce systemic immunity against infectious agents.

HLA class II (DR0401) molecules induce Foxp3+ regulatory T cell suppression of B cells in Plasmodium yoelii strain 17XNL malaria.

Unlike human malaria parasites that induce persistent infection, some rodent malaria parasites, like Plasmodium yoelii strain 17XNL (Py17XNL), induce a transient (self-curing) malaria infection. Cooperation between CD4 T cells and B cells to produce antibodies is thought to be critical for clearance of Py17XNL parasites from the blood, with major histocompatibility complex (MHC) class II molecules being required for activation of CD4 T cells. In order to better understand the correspondence between murine malaria models and human malaria, and in particular the role of MHC (HLA) class II molecules, we studied the ability of humanized mice expressing human HLA class II molecules to clear Py17XNL infection. We showed that humanized mice expressing HLA-DR4 (DR0401) molecules and lacking mouse MHC class II molecules (EA(0)) have impaired production of specific antibodies to Py17XNL and cannot cure the infection. In contrast, mice expressing HLA-DR4 (DR0402), HLA-DQ6 (DQ0601), HLA-DQ8 (DQ0302), or HLA-DR3 (DR0301) molecules in an EA(0) background were able to elicit specific antibodies and self-cure the infection. In a series of experiments, we determined that the inability of humanized DR0401.EA(0) mice to elicit specific antibodies was due to expansion and activation of regulatory CD4(+) Foxp3(+) T cells (Tregs) that suppressed B cells to secrete antibodies through cell-cell interactions. Treg depletion allowed the DR0401.EA(0) mice to elicit specific antibodies and self-cure the infection. Our results demonstrated a differential role of MHC (HLA) class II molecules in supporting antibody responses to Py17XNL malaria and revealed a new mechanism by which malaria parasites stimulate B cell-suppressogenic Tregs that prevent clearance of infection.