Translation Rescue by Targeting Ppp1r15a through Its Upstream Open Reading Frame in Sepsis-Induced Acute Kidney Injury in a Murine Model.

BACKGROUND: Translation shutdown is a hallmark of late-phase, sepsis-induced kidney injury. Methods for controlling protein synthesis in the kidney are limited. Reversing translation shutdown requires dephosphorylation of the eukaryotic initiation factor 2 (eIF2) subunit eIF2 α ; this is mediated by a key regulatory molecule, protein phosphatase 1 regulatory subunit 15A (Ppp1r15a), also known as GADD34. METHODS: To study protein synthesis in the kidney in a murine endotoxemia model and investigate the feasibility of translation control in vivo by boosting the protein expression of Ppp1r15a, we combined multiple tools, including ribosome profiling (Ribo-seq), proteomics, polyribosome profiling, and antisense oligonucleotides, and a newly generated Ppp1r15a knock-in mouse model and multiple mutant cell lines. RESULTS: We report that translation shutdown in established sepsis-induced kidney injury is brought about by excessive eIF2 α phosphorylation and sustained by blunted expression of the counter-regulatory phosphatase Ppp1r15a. We determined the blunted Ppp1r15a expression persists because of the presence of an upstream open reading frame (uORF). Overcoming this barrier with genetic and antisense oligonucleotide approaches enabled the overexpression of Ppp1r15a, which salvaged translation and improved kidney function in an endotoxemia model. Loss of this uORF also had broad effects on the composition and phosphorylation status of the immunopeptidome-peptides associated with the MHC-that extended beyond the eIF2 α axis. CONCLUSIONS: We found Ppp1r15a is translationally repressed during late-phase sepsis because of the existence of an uORF, which is a prime therapeutic candidate for this strategic rescue of translation in late-phase sepsis. The ability to accurately control translation dynamics during sepsis may offer new paths for the development of therapies at codon-level precision. PODCAST: This article contains a podcast at.

Filaria specific antibody response profiling in plasma from anti-retroviral naïve Loa loa microfilaraemic HIV-1 infected people.

BACKGROUND: In West and Central Africa areas of endemic Loa loa infections overlap with regions of high prevalence of human immunodeficiency virus type 1 (HIV-1) infections. Because individuals in this region are exposed to filarial parasites from birth, most HIV-1 infected individuals invariably also have a history of filarial parasite infection. Since HIV-1 infection both depletes immune system and maintains it in perpetual inflammation, this can hamper Loa loa filarial parasite mediated immune modulation, leading to enhanced loaisis. METHODS: In this study we have assessed in plasma from asymptomatic anti-retroviral (ARV) naïve Loa loa microfilaraemic HIV-1 infected people the filarial antibody responses specific to a filariasis composite antigen consisting of Wbgp29-BmR1-BmM14-WbSXP. The antibody responses specific to the filariasis composite antigen was determined by enzyme linked immunosorbent assay (ELISA) in plasma from ARV naïve Loa loa microfilaraemic HIV-1 infected participants. In addition the filarial antigen specific IgG antibody subclass profiles were also determined for both HIV-1 positive and negative people. RESULTS: Both Loa loa microfilaraemic HIV-1 positive and negative individuals showed significantly higher plasma levels of IgG1 (P < 0.0001), IgG2 (P < 0.0001) and IgM (P < 0.0001) relative to amicrofilaraemic participants. A significant increase in IgE (P < 0.0001) was observed exclusively in Loa loa microfilaraemic HIV-1 infected people. In contrast there was a significant reduction in the level of IgG4 (p < 0.0001) and IgG3 (P < 0.0001) in Loa loa microfilaraemic HIV-1 infected individuals. CONCLUSIONS: Loa loa microfilaraemia in ARV naïve HIV-1 infected people through differential reduction of plasma levels of filarial antigen specific IgG3, IgG4 and a significant increase in plasma levels of filarial antigen specific IgE could diminish Loa loa mediated immune-regulation. This in effect can result to increase loaisis mediated immunopathology in antiretroviral naive HIV-1 infected people.

Mapping immunological and host receptor binding determinants of SARS-CoV spike protein utilizing the Qubevirus platform.

The motifs involved in tropism and immunological interactions of SARS-CoV spike (S) protein were investigated utilizing the Qubevirus platform. We showed that separately, 14 overlapping peptide fragments representing the S protein (F1-14 of 100 residues each) could be inserted into the C-terminus of A1 on recombinant Qubevirus without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F6 from S 425-525 , was found to contain the binding determinant of the recombinant human angiotensin converting enzyme 2 (rhACE2), with the shortest active binding motif situated between residues S 437-492 . Upstream, another fragment, F7, containing an overlapping portion of F6 would not bind to rhACE2, confirming not just only that residues were linear but equally also the appropriate structural orientation of F6 upon the Qubevirus. The F6 (S 441-460 ) and other inserts, including F7/F8 (S 601-620 ) and F10 (S 781-800 ), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes, improved substantially the recognition and binding to their cognate antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of subunit vaccines or other biologics against SARS-CoV. Significance: Mapping epitopes within the receptor binding domains of viruses which are essential for viral tropism is critical for developing antiviral agents and subunit vaccines. In this study we have engineered the surface of Qubevirus to display a peptide library derived from the SARS-CoV S protein. In biopanning with S protein antibodies, we have identified three peptide fragments (EP1, EP2 and EP3) which reacted selectively with antibodies specific to the S protein. We demonstrated that all recombinant phage displayed peptide fragments both individually and as chimera exposed important immunological epitopes to their cognate antibodies. A peptide fragment F6 situated at S 425-525 , was found containing the binding determinant of the recombinant human angiotensin converting enzyme 2 (rhACE2), with the shortest active binding motif situated between residues S 437-492 . The platform is rapidly to identify epitopes and receptor binding sites within viral receptors found in target host cell. Thus, this platform holds great significance.

In vivo targeting of protein antigens to dendritic cells using anti-DEC-205 single chain antibody improves HIV Gag specific CD4+ T cell responses protecting from airway challenge with recombinant vaccinia-gag virus.

INTRODUCTION: Targeting antigens to dendritic cells (DCs) in vivo via a DC-restricted endocytic receptor, DEC205, has been validated to enhance immunity in several vaccine platforms. Particularly atttractive is selected delivery of proteins to DCs in vivo because it enables proteins to be more immunogenic and provides a cheaper and effective way for repeated immunizations. METHODS: In this study, we tested the efficacy of a single chain antibody to DEC205 (scDEC) to deliver protein antigens selectively to DCs in vivo and to induce protective immunity. RESULTS: In comparison to soluble Ovalbumin (OVA) antigen, when recombinant scDEC:OVA protein was injected subcutaneously (s.c.) into mice, the OVA protein was selectively presented by DCs to both TCR transgenic CD8+ and CD4+ T cells approximately 500 and 100 times more efficient than soluble OVA, respectively, and could persist for seven days following s.c. injection of the scDEC205:OVA. Similarly selective targeting of HIV Gag P24 to DCs in vivo using scDEC-Gag protein plus polyICLC vaccine resulted in strong, long lasting, polyfuntional CD4+ T cells in mice which were protective against airway challenge by a recombinant vaccinia-gag virus. CONCLUSION: Thus targeting protein antigens to DCs using scDEC can be used either alone or in combination with other strategies for effective immunization.

Function of the RNA Coliphage Qß Proteins in Medical In Vitro Evolution.

Qß is a positive (+) single-stranded RNA bacteriophage covered by a 25 nm icosahedral shell. Qß belongs to the family of Leviviridae and is found throughout the world (bacterial isolates and sewage). The genome of Qß is about 4.2 kb, coding for four proteins. This genome is surrounded by 180 copies of coat proteins (capsomers) each comprised of 132 residues of amino acids. The other proteins, the subunit II (ß) of a replicase, the maturation protein (A2) and the read-through or minor coat protein (A1), play a key role in phage infection. With the replicase protein, which lacks proofreading activity, as well as its short replication time, and high population size, Qß phage has attractive features for in vitro evolution. The A1 protein gene shares the same initiation codon with the coat protein gene and is produced during translation when the coat protein's UGA stop codon triplet (about 400 nucleotides from the initiation) is suppressed by a low level of ribosome misincorporation of tryptophan. Thus, A1 is termed the read-through protein. This RNA phage platform technology not only serves to display foreign peptides but is also exceptionally suited to address questions about in vitro evolution. The C-terminus of A1 protein confers to this RNA phage platform an exceptional feature of not only a linker for foreign peptide to be displayed also a model for evolution. This platform was used to present a peptide library of the G-H loop of the capsid region P1 of the foot-and-mouth disease virus (FMDV) called VP1 protein. The library was exposed on the exterior surface of Qß phages, evolved and selected with the monoclonal antibodies (mAbs) SD6 of the FMDV. These hybrid phages could principally be good candidates for FMDV vaccine development. Separately, the membrane proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) epitopes was fused with the A1 proteins and exposed on the Qß phage exterior surface. The engineered phages with MPER epitopes were recognized by anti-MPER specific antibodies. This system could be used to overcome the challenge of effective presentation of MPER to the immune system. A key portion of this linear epitope could be randomized and evolved with the Qß system. Overall, antigens and epitopes of RNA viruses relevant to public health can be randomized, evolved and selected in pools using the proposed Qß model to overcome their plasticity and the challenge of vaccine development. Major epitopes of a particular virus can be engineered or displayed on the Qß phage surface and used for vaccine efficacy evaluation, thus avoiding the use of live viruses.