To further investigate whether L-4F has neurorestorative benefits in the ischemic brain after stroke in T2DM and elucidate the underlying molecular mechanisms, we subjected middle-aged, brain-ABCA1 deficient (ABCA1−B/−B), and ABCA1-floxed (ABCA1fl/fl) T2DM control mice to distal middle cerebral artery occlusion. L-4F  treatment was initiated 24 h after stroke and administered once daily for 21 days. Treatment of T2DM-stroke with L-4F improved neurological functional outcome, and decreased hemorrhage, mortality, and BBB leakage identified by decreased albumin infiltration and increased tight-junction and astrocyte end-feet densities, increased cerebral arteriole diameter and smooth muscle cell number, and increased WM density and oligodendrogenesis in the ischemic brain in both ABCA1−B/−B and ABCA1fl/fl T2DM-stroke mice compared with vehicle-control mice, respectively . The L-4F treatment reduced macrophage infiltration and neuroinflammation identified by decreases in ED-1, monocyte chemoattractant protein-1 (MCP-1), and toll-like receptor 4 (TLR4) expression, and increases in anti-inflammatory factor Insulin-like growth factor 1 (IGF-1) and its receptor IGF-1 receptor β (IGF-1Rβ) in the ischemic brain . These results suggest that post-stroke administration of L-4F may provide a restorative strategy for T2DM-stroke by promoting neurovascular and WM remodeling. Reducing neuroinflammation in the injured brain may contribute at least partially to the restorative effects of L-4F independent of the ABCA1 signaling pathway.Patients with type 2 diabetes mellitus (T2DM) exhibit a distinct and high risk of ischemic stroke with worse post-stroke neurovascular and white matter (WM) prognosis than the non-diabetic population. In the central nervous system, the ATP-binding cassette transporter member A 1 (ABCA1), a reverse cholesterol transporter that efflux cellular cholesterol, plays an important role in high-density lipoprotein (HDL) biogenesis and in maintaining neurovascular stability and WM integrity. Our previous study shows that L-4F, an economical apolipoprotein A member I (ApoA-I) mimetic peptide, has neuroprotective effects  Type-2 diabetes (T2DM) constitutes ~90% of all diabetic patients and is a major risk factor for ischemic and hemorrhagic stroke. Epidemiological investigation in both younger (15–49 years) . NeuroinAfter adjusting for all possible covariables, blood levels of low-density lipoprotein (LDL) cholesterol show a significant association with increased risk of stroke and mortality , 8. Howein situ mainly by the ATP-binding cassette transporter member A 1 (ABCA1), a reverse cholesterol transporter that efflux cellular cholesterol (−B/−B) and ABCA1-floxed (ABCA1fl/fl) control mice that were induced with T2DM and subjected to stroke, to test whether L-4F treatment initiated at 24 h after onset of ischemic stroke enhances neurological recovery. We also test if L-4F treatment improves vascular and WM rewiring in T2DM stroke and whether L-4F decreases inflammation via ABCA1 dependent signaling pathway. We demonstrate that L-4F can pass through the BBB and has neurorestorative capabilities in promoting neurovascular and WM remodeling, and oligodendrogenesis in the ischemic brain of T2DM-stroke mice. Our data also indicate that reducing neuroinflammation in the injured brain may contribute at least partially to the neurorestorative effects of L-4F independent of the ABCA1 signaling pathway.In humans, the circulating blood contains only about 40% of the total amount of HDL, and most of the HDL in the central nervous system (CNS) is synthesized lesterol , 27. ABClesterol –33. We hlesterol , 34–36. lesterol –43 and hlesterol –46. In olesterol , 46. How−B/−B and ABCA1fl/fl mice were employed. The original breeders were provided by Dr. Michael Hayden from the University of British Columbia, Canada. All mice used in this study were self-bred in the Bioresources of Henry Ford Health System, and all procedures were approved by the Institutional Animal Care and Use Committee of Henry Ford Health System . The litters were heterozygous and the ABCA1−B/−B or ABCA1fl/fl phenotype was identified with genotyping assay  were employed. These mice were induced with T2DM using a combination of a high-fat diet  and a low dose of streptozotocin  injection  or saline subcutaneously starting at 24 h after dMCAo and once daily for 21 days. The dose of L-4F employed in this study is the same as the optimal dose of D-4F which was employed in our previous study  surgery, as described previously . At 24 hus study . The treus study . Mice weTo evaluate neurological function after stroke, all animals were subject to an adhesive removal test and left foot-fault test before dMCAo (baseline) and on days 1, 3, 7, 14, and 21 after dMCAo, as described previously , 53. ExpTo test blood biochemistry, animals were fasted for 6 h, and blood was collected from a tail vein before treatment on day 1 after dMCAo as the baseline measurement and before sacrifice on day 21 after treatment. Blood glucose levels were measured using glucose test strips and a glucose analyzer . Blood levels of HDL, total cholesterol (T-CH), and triglyceride were measured using a CardioChek Plus analyzer 2,700  and test strips, following the manufacturer's instructions. Each sample was tested in triplicate and the data are presented as mg/dl.Briefly, brains were immersion fixed in 4% paraformaldehyde, paraffin-embedded, and then cut into seven (1 mm thick) coronal blocks. A series of 6 μm thick sections were prepared and stained with hematoxylin and eosin (HE), and hemorrhagic transformation was identified by blood cell infiltration in the cerebral ischemic brain and the percentage of hemorrhagic transformation volume was calculated. For lesion volume measurement, the percentage of the indirect lesion volume was calculated, in which the intact area of the ipsilateral hemisphere was subtracted from the area of the contralateral hemisphere, with a micro-computer imaging device (MCID) imaging analysis system  .For histochemical/immunostaining, a standard paraffin block was obtained from the center of the lesion (bregma −1 mm to + 1 mm). A series of 6-μm thick sections were cut from the block. Histochemical-staining for Bielshowsky silver  and Luxol fast blue , and histoimmuno-staining for antibodies against albumin , Occludin , Aquaporin-4 , von Willebrand Factor , α-smooth muscle actin , SMI31 , adenomatous polyposis coli [APC, a marker of mature oligodendrocytes-OLs , Ab-1, Ofl/fl-T2DM-stroke mice were subcutaneously administered saline or commercially available FITC-D-4F  at 24 and 48 h after dMCAo. Mice were sacrificed 4 h after last treatment and vibratome coronal sections (100 μm) were prepared for immunostaining with vWF, APC, and neuronal nuclei . Images were analyzed using laser scanning confocal microscopy.Both D-4F and L-4F have robust anti-inflammatory properties –58, impr+-vessels, αSMA+-arterioles, APC+-OLs, PDGFR+-OPCs and the perimeter of vessels or diameter of arterioles as well as the number of αSMA+-SMCs was measured in both the contralateral and the ipsilateral brain.For immunostaining measurement, each section containing 8 fields of view within the cortex and corpus callosum (CC) from the ischemic boundary zone (IBZ) were digitized using a 40× objective (Olympus BX40) using a 3-CCD color video camera (Sony DXC-970MD) interfaced with MCID. The percentage of positively stained area for albumin, Occludin, AQP-4, BS, LFB, and SMI-31 was measured using a built-in densitometry function (MCID) with a uniform threshold set above unstained for all the groups and the number of vWFn = 6 mice/group). Axonal structural changes were identified at a magnification of 14,000× in a total of 6 fields of view per animal. The analysis included measurement of the percentage of myelinated axons, thickness of myelin sheath, and G ratio (axon diameter/axon wrapped with myelin diameter ×100%) in 10 ultrathin sections, as described previously . For each sample, the cDNA was generated and used to amplify GAPDH, AQP4, MBP, ED1, MCP-1, TLR4, IGF1, and IGF-1R as described previously . All thepost hoc Test were used for analysis involving four groups, i.e., ABCA1fl/fl-T2DM and ABCA1−B/−B-T2DM stroke with or without L-4F treatment for analysis blood biochemistry, hemorrhage, mortality, lesion volume, functional outcome, immunostaining, EM measurement, WB and RT-PCR measurement. p < 0.05 was set as a significant difference, and all data are presented as mean ± standard error (SE).A total of two-way ANOVA followed by Tukey fl/fl-T2DM-stroke mice, in this study, a total of 86 T2DM-stroke mice were used.In this study, 9 survival animals in each group were employed for the measurement of hemorrhage and lesion volume, and histochemical and immunohisto-staining measurements. The number of animals employed was determined using a priori power calculation; 9 survival stroke animals per group provided 80% power at a significance level of <0.05, assuming a 20% difference in both mean and SD at the 95% CI and a two-sided test. For WB, RT-PCR, and EM analysis, 6 stroke mice per group were needed. Plus, two additional ABCA1p < 0.05, n = 21/group). ABCA1−B/−B-T2DM mice exhibit increased hemorrhage, lesion volume, and mortality compared to ABCA1fl/fl-T2DM-stroke mice . There were no changes in lesion volume at 21 days after L-4F treatment in both the ABCA1−B/−B-T2DM and the ABCA1fl/fl-T2DM-stroke mice. However, the hemorrhage volume and mortality rate was dramatically reduced in L-4F treatment groups in both ABCA1fl/fl and ABCA1−B/−B T2DM-stroke mice when compared to the vehicle control group, respectively (p < 0.05).−B/−B-T2DM stroke mice exhibited significant functional deficits as indicated by increased adhesive-removal time on 3, 7, 14, and 21 days after dMCAo compared with ABCA1fl/fl-T2DM mice . L-4F treatment significantly reduced adhesive-removal time at 14 and 21 days after stroke indicating improved sensorimotor function and decreased the left foot-fault rate at 7, 14, and 21 days after treatment indicating an improved motor function in both ABCA1fl/fl and ABCA1−B/−B T2DM-stroke mice . These data indicate that L-4F treatment increases blood HDL and reduces glucose levels, decreases cerebral hemorrhagic transformation and mortality as well as improves neurological functional outcomes in T2DM-stroke mice.ABCA1−B/−B-T2DM stroke mice, while the expression of Occludin and AQP-4 within or around blood vessels in both the contralateral and the IBZ area in the ABCA1−B/−B-T2DM stroke mice significantly decreased compared with the ABCA1fl/fl-T2DM stroke mice . In addition, WB and RT-PCR assay indicate that the level of AQP-4 in the ischemic brain is decreased in the ABCA1−B/−B-T2DM stroke mice . L-4F treatment significantly decreased albumin density indicating improved BBB integrity and increased tight junction protein in the ischemic brain in both ABCA1−B/−B and ABCA1fl/fl T2DM-stroke mice measured by immunostaining . Moreover, L-4F treatment also elevated AQP4 expression in the ischemic brain tissues measured by WB and RT-PCR  even though there is no significant difference by immunostaining analysis.+-vessels and αSMA+-arterioles in both the contralateral hemisphere and the IBZ, and the diameter of arterioles and the number of αSMA+-SMCs in the contralateral hemispheres between the ABCA1−B/−B and ABCA1fl/fl T2DM-stroke mice, ABCA1−B/−B-T2DM stroke mice exhibit decreased vessel perimeter in both the contralateral hemisphere and the IBZ, and decreased arteriole diameter and SMC number in the IBZ compared with ABCA1fl/fl-T2DM stroke mice . L-4F treatment increased the number of SMCs in both the contralateral hemisphere and IBZ of ABCA1fl/fl-T2DM mice and increased SMC number and arteriole diameter in the IBZ of ABCA1−B/−B-T2DM stroke mice 21 days after stroke . These data indicate that T2DM-stroke induces BBB leakage and vascular injury in the ischemic brain, while brain-ABCA1 deficit exacerbates T2DM-stroke induced vascular damage. Administration of L-4F decreased T2DM-stroke induced vascular damage and BBB permeability even in brain-ABCA1-deficient mice.−B/−B-T2DM stroke mice exhibit a significant decrease in the densities of axons, phosphorylated-neurofilament, and myelin in the WM-bundles of CC , and WB/RT-PCR assay show that the protein and mRNA level of MBP was decreased in the ischemic brain tissues  at 21 days after dMCAo compared with the ABCA1fl/fl-T2DM stroke mice. The analysis from EM images indicates that both the number of myelinated-axons and the thickness of myelin-sheath decreased but G-ratio increased in the WM-bundles of CC in both the contralateral brain and the IBZ of the ischemic brain in the ABCA−B/−B-T2DM stroke mice . Moreover, the number of APC+-OLs in the CC of the IBZ and the number of PDGFRα+-OPCs in the cortex of the IBZ of ABCA−B/−B-T2DM stroke mice were also reduced . L-4F treatment significantly decreased G-ratio, increased the axon density, phosphorylated-neurofilaments, myelin density, number of myelinated-axons, myelin-sheath thickness, and the number of OLs and OPCs in the IBZ  and also the protein/mRNA levels of MBP in the ischemic brain tissues  in both ABCA1fl/fl-T2DM and ABCA−B/−B-T2DM stroke mice when compared with the vehicle-control group, respectively. These data indicate that L-4F treatment promotes WM remodeling and oligodendrogenesis in the ischemic brain after stroke in T2DM mice.The immunohisto-chemical-staining and quantification data show that the ABCA+-vessels and APC+-OLs and NeuN+-neurons in the ischemic brain.−B/−B-T2DM stroke mice exhibit an increased level of protein and mRNA of ED-1, MCP-1, and TLR-4, whereas a lower level of IGF-1/IGF-1Rβ in the ischemic brain tissues when compared that with the ABCA1fl/fl-T2DM stroke mice at 21 days after dMCAo . L-4F treatment significantly decreases ED-1, MCP-1, and TLR-4 expression and increases IGF-1 and IGF-1Rβ levels in the ischemic brain compared with the vehicle-control group, respectively . These data indicate that post-stroke administration of L-4F in T2DM-stroke mice inhibits neuroinflammation which may contribute to the L-4F treatment-induced neurorestoration after brain injury.To investigate the mechanism underlying L-4F treatment-induced neurorestoration in T2DM-stroke, the macrophage/microglial and monocyte infiltration was measured using the levels of ED-1 and MCP-1, and the inflammatory factor TLR-4 and anti-inflammatory factors IGF-1 and IGF-1Rβ were measured using WB and RT-PCR assay. fl/fl) T2DM-stroke mice. These results are consistent with our previous findings that treatment of db/db-T2DM stroke mice with L-4F reduced hemorrhage, infarct volume, mortality, BBB leakage and WM damage, and increased cerebral arteriole diameter and SMC number in the ischemic brain 4 days after stroke , pericytes, tight junction, and astrocyte end-feet and interacts directly with astrocytes and neurons in the neurovascular niche –67. The r stroke . We also glucose . D-4F tr glucose . L-4F de glucose . L-4F-trclerosis .via facilitating cholesterol and phospholipid efflux to exogenous ApoE  ABCA1 mice with nestin-cre mice, by which the ABCA1 gene is knocked out from all nestin-linage cells  in the brain including neurons, OLs, and astrocytes, and these mice exhibit reduced brain ABCA1 and ApoE and HDL content, and reduced level of ABCA1, ApoE and HDL was also found in a primary cortical neuron or OPC cultures (−B/−B-T2DM stroke mice. D-4F is capable of forming HDL-like particles and delivering cholesterol to the liver cells selectively through the scavenger receptor class B type I (SR-BI) and enhances the cholesterol delivery by native HDL , advanced glycation end-product receptor (RAGE), and plasminogen activator inhibitor-1 (PAI-1) in the ischemic brain in T2DM-stroke mice . D-4F tr−B/−B and ABCA1fl/fl T2DM stroke mice. These findings indicate that post-stroke administration of L-4F may provide a potential strategy for neurorestoration following stroke injury in the T2DM population, and reducing neuroinflammation in the injured brain may contribute to the neurorestorative effects of L-4F at least partially independent of the ABCA1 signaling pathway.In this study, we demonstrate that L-4F can pass through the BBB and has neurorestorative benefits such as promoting neurovascular and WM remodeling and oligodendrogenesis in the ischemic brain as well as improvement of neurological functional recovery in T2DM-stroke mice. L-4F-treated T2DM-stroke mice exhibit suppressed neuroinflammation in the ischemic brain of both ABCA1In this study, all brain tissue samples were collected 21 days after stroke. Tissue sampling at this late time point enables the study of long-term vascular and WM remodeling effects in the brain, however, from this delayed sampling we are unable to glean information of BBB leakage and inflammatory responses at the acute phase of stroke recovery, which warrants future investigation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institute of Animal Care and Use Committee of the Henry Ford Health System.XC and QJ conceived and designed this study. MZ, RL, AZ, YQ, JL-W, PV, and XC performed mouse experiments, acquisition of data, and statistical analysis. XC wrote and prepared this manuscript. PV and MC made critical revisions of the manuscript for intellectual content. All authors read and approved the final manuscript.This study was supported by the National Institute of Neurological Disorders and Stroke grant to QJ and XC (R01NS097747).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Interestingly, the fusion proteins were also degraded by the proteasome pathway and the resulting peptides were presented by the MHC class I system. We then asked if the aforementioned fusion proteins could improve tumor cell immunogenicity and T cell priming. The B16-F10 melanoma was chosen as the tumor cell line to express the fusion proteins. B16-F10 cells that expressed the OVA-ATG8 fused proteins stimulated OVA-specific CD8+ T cells, but demonstrated no CD4+ T cell response. In the future, these constructions may be used in vaccination trials as potential candidates to control tumor growth.Vaccine therapy is a promising method of research to promote T cell immune response and to develop novel antitumor immunotherapy protocols. Accumulating evidence has shown that autophagy is involved in antigen processing and presentation to T cells. In this work, we investigated the potential role of GABARAP and GABARAPL1, two members of the autophagic ATG8 family proteins, as surrogate tumor antigen delivery vectors to prime antitumor T cells. We showed that bone marrow-derived dendritic cells, expressing the antigen OVALBUMIN (OVA) fused with GABARAP or GABARAPL1, were able to prime OVA-specific CD4 Cancer immunotherapy clinical trials currently rely on the cytotoxic CD8+ T cell response. Accumulative evidence has shown that the CD8+ response is transient and that an efficient antitumor response also requires CD4+ T cells [+ T cell response and antitumor immunity. The effect of CD4+ T cells against tumor cells could be direct or indirect. Indeed, cytotoxic CD4+ T cells could directly contribute to the elimination of cancer cells that express MHC-II. Moreover, the helper CD4+ T cells could enhance the cell responses of both CD8+ T and natural killer cells by improving the priming and the generation of the long-lived memory CD8+ T cells [+ and CD8+ T cell responses might, therefore, become encouraging ways to improve immunotherapy [Improving T cell response by vaccinations could increase the antitumor effect induced by immunotherapy. In this context, in vitro specific targeting of antigens to the autophagosomes could enhance CD4 T cells ,2. There T cells ,4,5. Newotherapy ,7,8.+ (cytotoxic) T cells, while peptides presented by MHC-II molecules are recognized by CD4+ (helper) T cells [Classical pathways for antigen presentation involve the processing and presentation of antigen-derived peptides from intracellular and extracellular sources by the major histocompatibility complexes (MHC), class I and II (MHC-I and MHC-II). Antigen-derived peptides presented by MHC-I molecules are recognized by CD8 T cells ,10. Howe T cells ,12. Auto T cells . These c T cells , GABARAP T cells ,16. Cumu T cells ,21,22,23 T cells  presente T cells .+ T cells. This is supported by previous studies that show that the fusion of LC3, together with the antigen, ameliorated the presentation of antigen-derived peptides by MCH-II molecules, resulting in CD4+ T cell stimulation [LC3B is a ubiquitin-like protein, covalently conjugated to a phosphatidylethanolamine via its C-terminal end at a glycine in position G120 to form the LC3B- form II. LC3B-II is docked onto the inner and outer membranes of autophagosomes. Following the fusion between lysosomes and autophagosomes, LC3B-II, attached on the inner face of the autophagosomes, is rapidly degraded by lysosomal proteases. . These dmulation ,17,27.+ and CD4+ OT-I and OT-II T cells, respectively. Blocking autophagy in DC overexpressing GABARAP and GABARAPL1 fusion proteins drastically decreases MHC-II antigen presentation only. Our findings demonstrate, for the first time, that GABARAP and GABARAPL1 fused to OVA antigen can addressed an endogenous antigen to the autophagosomes, resulting in OVA antigen-specific CD4+ T cell responses.In this study, we investigated the ability of GABARAP and GABARAPL1 to address the antigens in MHC-I and -II presentation pathways to efficiently prime T cells. To this end, we engineered multiple GABARAP or GABARAPL1, plus OVALBUMIN (OVA) fusion proteins. We showed that dendritic cells (DC) overexpressing OVA fused with GABARAP or GABARAPL1 proteins address OVA class I and II restricted peptides to prime CD8BamHI and XhoI restriction enzyme sites. The expression vectors that encoded the fusion proteins were produced by cloning the OVA full-length sequence without the stop codon in the same open reading frame to the one coding the ATG8 protein. The primers used to amplify the OVA sequence, including the overhang sequences for the NheI and BamH1 sites, were NheI-OVA-F and BamHI-OVA-R. The cDNA encoding GABARAP was produced by PCR from a pcDNA 3.1 GFP-GABARAP vector. The primers used contained overhang sequences for the enzyme restriction sites BamHI (BamHI-GABARAP-F) and XhoI (XhoI-GABARAP-R). The cDNA encoding human GABARAPL1 was isolated from MCF7 and was amplified by PCR using the following primers, including overhang sequences for BamHI (BamHI-GABARAPL1-F) and XhoI (XhoI-GABARAPL1-R). The cDNA encoding Rattus norvegicus LC3B (GenBank ID: U05784) was amplified by PCR with primers, including overhang sequences for BamHI (BamHI-LC3B-F) and XhoI (XhoI-LC3B-R), and cloned into the ptfLC3 vector (gift from the Tamotsu Yoshimori’s Laboratory). Hybridization of specific overlapping primers (myc 1 and 2) in water at room temperature was performed to create compatible BamHI sites between the OVA and ATG8 sequences. The plasmid that encoded the OVA242–386-Myc-ATG8 (242OMATG8) proteins that expressed a deleted variant of OVA were designed by PCR using a forward primer, including overhang sequences for NheI (NheI-242OVA-F), and the precedent reverse primers, including overhang sequences for XhoI. The different fusion proteins are presented in the The pcDNA 3.1 Hygro (+)  plasmid was used as an expression vector that contains a CMV promoter. All cDNAs used in this study were cloned into this vector. The cDNA sequence of OVA was obtained from the pVAX-OVA codon optimized vector  and cloned into the pcDNA 3.1 Hygro (+) vector using the pre-existing b), derived from a murine melanoma, was obtained from the INSERM UMR 1098 Laboratory. All cell lines were cultured in Roswell Park Memorial Institute 1640 (RPM1 1640)  supplemented with antibiotics , 10 % fetal calf serum  and 0.4 mg/L amphotericin B Dutscher, L0009) in a 5 % CO2 atmosphere at 37°C.The B16-F10 cell line (H-2b) and OT-II (H-2b) mice. The total cell suspension was filtrated, erythrocytes were lysed using ammonium-chloride-potassium lysing buffer  and the cells were then rinsed with complete RPMI 1640 medium, containing FCS and penicillin-streptomycin. Splenocytes were then directly used for proliferation experiments.Splenocytes were isolated from C57BL6 OT-I  or by an incubation process with 10 µM rapamycin for 4 h  in complete growth medium. To block the fusion between autophagosomes and lysosomes, the following different autophagy inhibitors were used: bafilomycin-A1 , chloroquine  and ammonium chloride  for 2 or 4 h. Early stages of autophagy were blocked using 3-methyladenine  for 16 h. Proteasome activity was inhibited by a bortezomib treatment at 25 nM  for 16 h.The following antibodies were used for cytometry analysis: anti-CD11c Percp-Vio700 , anti-IA/IE eFluor450 , anti-CD80 APC , anti-CD86 PE , anti-CD3 PercpCy5.5 , anti-CD4 Pacific Blue  and anti-CD8 APC . The following antibodies were used for Western blotting: anti-LC3B  anti-GABARAP/GABARAPL1  anti-GABARAPL1  anti-ACTIN  and anti-OVA .Bone marrow cells were isolated from tibias and femurs of C57BL/6 mice  as described . The bon®, 114-07, 67400 Illkirch, France), according to the manufacturer’s protocol. The day after the transfection, cells were harvested and pretreated with IFN-γ  before analysis.B16-F10 cells  were plated in 6-well plates. The day after, cells were transfected using 2 µg of the different pcDNA3.1 plasmids and 4 µL of the Jetprime reagent  was used according to the standard protocol. Briefly, at day 6, the non-adherent bmDCs (4 × 105 cells) were plated in 6-well plates with complete fresh RPMI 1640 medium, containing 10 ng/mL rmGM-CSF and 10 ng/mL rmIL-4. Then, bmDCs were transfected at day 7 with 2 µg of the different pcDNA 3.1 plasmids for 16 to 24 h.For transient transfection of bmDCs, the JetPEI™-Macrophage reagent  and OVA323–339 . After 3 to 5 days, T cells were labelled with anti-CD3, anti-CD4 and anti-CD8 antibodies and proliferation was analyzed by flow cytometry using the fluorescence emission wavelength of FITC.The splenocytes were stained with CellTrace™ 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) for 10 min at 5 µM . Cells were then washed and cultured in RPMI 1640 complete medium, supplemented with β-mercaptoethanol , and plated in 96 wells (U bottom) with bmDCs at a ratio of 20,000 bmDCs for 100,000 splenocytes and B16 cells at a ratio of 100,000 B16 for 200,000 splenocytes. B16 cells and bmDCs were irradiated at 25 Gy before coculture to prevent their proliferation. OVA control peptides were OVA2HPO4, 1.8 mM KH2PO4) and lysed in Laemmli buffer . Cell lysates were sonicated for 10 s , before loading (40 µg) on a 10 or 12 % TGX Stain-Free™ FastCast™ polyacrylamide gel  and migration in a running buffer  at 100 V for 10 min, and then at 300 V for 30 min. Proteins were then transferred onto a polyvinylidene difluoride (PVDF) 0.2 µm membrane  at 25 V/2.5 mA for 7 min in a transfer buffer , using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). The membrane was blocked with 5 % non-fat milk in Tris-buffered saline, supplemented with Tween 20 (TBS-T)  for 1 h and incubated with primary antibodies in TBS-T, supplemented with 1 % non-fat milk overnight at 4°C, according to the antibody instructions. Following three washes in TBS-T, immunoreactive bands were detected using a secondary goat horseradish peroxidase (HRP)-coupled antibody and the Clarity Western ECL blotting substrate . The signals were then analyzed and quantified using the ChemiDoc XRS+ system (Bio-Rad) and the Image Lab™ 5.1 Beta software .Cells were scraped and harvested in cold phosphate-buffered saline (PBS)  were purchased from Charles River. The OT-I and OT-II mice were raised in the animal facility at the University of Franche-Comté and used at 8–12 weeks old. The T lymphocytes used in this study were isolated from OT-II and OT-I mice, which express a transgenic T cell receptor. OT-II CD4 T cells recognize the OVA323–339 epitope  in the context of I-Ab (MHC-II), while the OT-I T cells recognize the OVA257–264 peptide  in the context of H2-Kb (MHC-I).Female C57BL/6 mice ) and the experiments were performed in accordance with the approved guidelines and regulation.p-values ≤ 0.05 were considered significant.Statistical analyses were performed using GraphPad Prism version 6 and differences were analyzed using the Student’s t-test or the one-way ANOVA test. We designed different plasmids that expressed OVA fused with ATG8 autophagy proteins and used LC3B as the positive control . These dTo test whether GABARAP and GABARAPL1 fused to OVA can be addressed to autophagosomes for the MHC-II processing pathway, we performed in vitro experiments using mouse-derived bmDCs that expressed each fusion protein. Overexpressing bmDC cells were cocultured with OT-I or OT-II T cells A.4Cl) treatments. These data suggest that autophagy flux is active in bmDCs  treatment. 3-MA is an inhibitor of phosphoinositide 3-kinase that is required for autophagy initiation . We noting bmDCs D. Furtheagosomes E.+ OT-I T cells. Indeed, OT-I T cells proliferated to the same extent as peptide-pulsed bmDCs  through the autolysosomes. Melanoma cells were then transfected with OMGP, OMGL1, OMLC3B and OVA-expressing vectors B and theytometry B. As sho10 cells C,D. Next T cells B. As shoThe results suggest that a significant part of the fusion proteins was degraded by proteasomes instead of being delivered to the autophagosomes, leading to MHC-I peptide complex cell surface density that was sufficient to activate OT-I. In contrast, the inability of B16-F10 cells to activate OT-II cells suggest that targeting OVA to B16-F10 cell autophagosomes was not efficient enough to produce the high cell surface density of the OVA peptide:MCH-II complex that was necessary to prime CD4 T cells. This condition was reached only when the B16-F10 cells were directly pulsed with OVA-II peptides.+ and CD4+ T cell proliferation  led to a defect in antigen processing and presentation, in addition to impairment of the CD4+ T cell response in vivo [+ or CD4+ T cell responses.In this study, we demonstrate, for the first time, that GABARAP and GABARAPL1, as well as LC3B ATG8 proteins, can target antigens in MHC-II and I to stimulate both CD4 and CD8 T cells. These results suggest an interconnection between MHC-II-processing and autophagy pathways. Moreover, several studies have also suggested the role of autophagy in physiological MHC-II presentation of cytoplasmic and nuclear antigens ,29,30. Ferase II , the virerase II  or the terase II . Howevererase II . Indeed, in vivo . Taken t+ T cells play a critical role in cancer immunity and immunotherapy [+ T cell response.Several studies support the belief that CD4otherapy ,36. Theyotherapy ,38. In t+ T cell activation. In this work, we used GABARAP and GABARAPL1 proteins to deliver the antigen to autophagosomes to be processed, before its presentation by the MHC-II pathway. We chose to use the full-length OVA antigen, instead of limited specific epitopes, to evaluate if this strategy could be transposed to large tumor antigens for which antigenic epitopes are not clearly defined. This strategy may be particularly important to enhance the recognition of cancer cells by the immune system. This protocol would also allow for antigens to be presented by both MHC-I and II molecules. Indeed, a former study already used DCs that overexpressed Ii-OVA fusion cDNA and demonstrated that the invariant Ii chain presented OVA to the MHC-II pathway through the MIIC and elicited MHC-II-restricted T cell response in DC/T cell co-culture in vitro [+ T cell proliferation in DC/T cell co-cultures. In addition, the inhibition of autophagy drastically reduced the proliferation of MHC-II-restricted CD4+ T cells, suggesting that the delivery of tumor antigens to the autophagy pathway could become a novel strategy to enhance the antitumoral CD4+ T cell response. Interestingly, the ability of DC to present OVA-derived peptides using MHC-I molecules to activate CD8+ T cells was conserved. Indeed, OMGP and OMGL1-fused proteins are always degraded by the proteasome pathway and processed by the classical MHC-I pathway. These data suggest that use full length antigens could be a good strategy to activate CD4+ and CD8+ T cells.Further investigations are needed to demonstrate the antitumor effect of tumor-antigens fused with GABARAP and GABARAPL1. In this context, genetically modified DCs, designed to express, process, and present a tumor antigen on MHC-II molecules, might represent an innovative strategy in immunotherapy to increase CD4in vitro . These d+ T cell activation when fused to full length OVA. Using the same strategy, a fusion protein containing the tumor testis antigen NY-ESO-1 and LC3 enhanced by up to 4-fold the presentation of NY-ESO-1 antigens on MHC-II and presentation to NY-ESO-1-specific CD4+ T cells [+ T cells [Furthermore, it appears that GABARAP and GABARAPL1 were as effective as LC3B at presenting OVA to the autophagosomes. To our knowledge, this is the first study showing that GABARAP and GABARAPL1 can be used to promote CD4 T cells . These s T cells . Similar T cells . Other s T cells ,41. More T cells ,43.+ T cell response, but not an MHC-II-restricted CD4+ T cell response. We were not able to increase the immunogenicity of cancer cells by this method. These discrepancies could be explained by the fact that B16-F10 cells are not antigen presenting cells. They only expressed MHC-II molecules when pre-treated with IFN-γ and they do not express costimulatory molecules at their surface, such as CD80 (B7-1) and CD86 (B7-2) [+ T cells. For example, the Gp100 antigen and the mutated triosephosphate isomerase antigen-derived peptides were recognized by specific CD4+ T cells in the context of MHC-II HLA-DR and the cancer testis NY-ESO-1 antigen in the context of HLA-DP4 on melanoma cell lines [In the second part of the paper, our results demonstrated that B16-F10 cells, transfected with OVA cDNA fused to LC3B, GABARAP or GABARAPL1 cDNA, elicited an efficient MHC-I-restricted CD86 B7-2) . Several-2 . Sevell lines ,46. Howe+ T cell response generation. Although CD8+ T cells are considered as the main effector of the antitumor immune response, many findings have reported that CD4+ T helper cells are important regulators of the immune response [+ T cells improve the efficiency of DC in CD8+ T-cell priming. By secretion of TNF-α, IL-2 and IFN-γ cytokines, CD4+ TH1 cells also promote NK cell and macrophage (M1) activation in vivo. Furthermore, by CD4+ T cell depletion or using CD4-knockout mice, it has been shown that the absence of CD4+ T cells abrogates the antitumor immune response [+ T cell-based cancer immunotherapy.In our study, we have validated the concept of targeting antigens to autophagosomes for MHC-II presentation and CD4response ,47. For


LDH-A gene using shRNA knock-down (KD), and compared to shRNA scrambled control (NC) cell lines. Successful LDH-A KD was confirmed by differences in the expression of LDH-A and LDH-B mRNA, protein and enzymatic activity, as well as their LDH isoenzyme profiles. mRNA expression data indicated that: (i) GL261 LDH-A KD cells may have an improved ability to metabolize lactate into the TCA cycle; and (ii) that GL261 LDH-A KD cells can upregulate lipid metabolism/fatty acid oxidation pathways, whereas the other glioma cell lines  do not have this capacity. The data suggest that GL261 LDH-A KD cells can develop/activate alternative metabolic pathways for enhanced survival in a nutrient-limited environment. Furthermore, LDH-A KD prolonged the doubling time of GL261 cells in culture and prevented the formation of subcutaneous flank tumors in immune-competent C57BL/6 mice. No differences between NC and KD cell proliferation (in vitro) or tumor growth in C57BL/6 mice (doubling time) were observed for CT2A and ALTS1C1 cells and tumors. Our results suggest that GL261 glioma cells (but not CT2A and ALTS1C1 cells) are pre-programmed with a capacity for activating different metabolic pathways with higher TCA cycle activity, and that this capacity is enhanced by LDH-A depletion, leading to a difference in growth and survival in an immune-competent environment.Three different murine glioma cell lines were modified to downregulate the expression of the murine LDH-A gene using shRNA, and compared to shRNA scrambled control (NC) cell lines. Differences in the expression of LDH-A and LDH-B mRNA, protein and enzymatic activity, as well as their LDH isoenzyme profiles, were observed in the six cell lines, and confirmed successful LDH-A KD. LDH-A KD (knock-down) resulted in metabolic changes in cells with a reduction in glycolysis (GlycoPER) and an increase in basal respiratory rate (mitoOCR). GL261 cells had a more limited ATP production capacity compared to CT2A and ALTS1C1 cells. An analysis of mRNA expression data indicated that: (i) GL261 LDH-A KD cells may have an improved ability to metabolize lactate into the TCA cycle; and (ii) that GL261 LDH-A KD cells can upregulate lipid metabolism/fatty acid oxidation pathways, whereas the other glioma cell lines do not have this capacity. These two observations suggest that GL261 LDH-A KD cells can develop/activate alternative metabolic pathways for enhanced survival in a nutrient-limited environment, and that specific nutrient limitations have a variable impact on tumor cell metabolism and proliferation. The phenotypic effects of LDH-A KD were compared to those in control (NC) cells and tumors. LDH-A KD prolonged the doubling time of GL261 cells in culture and prevented the formation of subcutaneous flank tumors in immune-competent C57BL/6 mice, whereas GL261 NC tumors had a prolonged growth delay in C57BL/6 mice. In nude mice, both LDH-A KD and NC GL261 tumors grew rapidly (more rapidly than GL261 NC tumors in C57BL/6 mice), demonstrating the impact of an intact immune system on GL261 tumor growth. No differences between NC and KD cell proliferation (in vitro) or tumor growth in C57BL/6 mice (doubling time) were observed for CT2A and ALTS1C1 cells and tumors, despite the small changes to their LDH isoenzyme profiles. These results suggest that GL261 glioma cells (but not CT2A and ALTS1C1 cells) are pre-programmed to have the capacity for activating different metabolic pathways with higher TCA cycle activity, and that this capacity is enhanced by LDH-A depletion. We observed that the combined impact of LDH-A depletion and the immune system had a significant impact on the growth of subcutaneous-located GL261 tumors.Three murine glioma cell lines  were modified to downregulate the expression of the murine  Lactate metabolism in tumors has been intensively studied recently : 1) lac lac1,2,3The role of LDH-A and LDH-B in tumor biology is complex. The relationship between tumor cell growth, the balance between LDH-A and LDH-B, the effects on tumor cell metabolism and the tumor microenvironment (TME) is variable across different types of gliomas ,24,25. IThe GL261 murine glioblastoma cell line was obtained from an NCI depository ,28. The LDH-A gene, as described previously [LDH-A knock-down function in murine cells. Although shRNA-3 LDH-A knock-down was less effective, the phenotypic changes in cells and tumors were comparable to that obtained with shRNA-2 [GL261, CT2A and ALTS1C1 cells were transfected with SureSilencing shRNA plasmids  to specifically knock-down the expression of the mouse eviously . Stably eviously ,31, we d shRNA-2 ,31. The  shRNA-2 .All immunoblotting experiments were performed as described previously ,32. CellTotal LDH enzyme activity was assessed using the Cytotoxicity Detection Kit PLUS (LDH) .Glycolytic activity and oxygen consumption of tumor cells were measured using a Seahorse XF96 Extracellular Flux Analyzer, as described previously . Data wep-values and Log2 fold changes. Genes with adjusted p-values of <0.05 and absolute log2 fold changes of >1 were called as differentially expressed genes for each comparison. Significantly differentially expressed genes were used for Gene Set Enrichment Analysis (GSEA) using the fgsea package in the R statistical software (v4.0). Gene sets (pathways) used were downloaded from the Broad Molecular Signature Database and only pathways from Gene Ontology (GO), Reactome or KEGG were used.LDH-A knock-down was verified by two approaches. First, a quantitative digital droplet PCR (ddPCR) was performed for LDH-A and LDH-B by the Genomics Core Laboratory at MSKCC. For RNA purification, cells were grown for 48 h . RNA was isolated using the RNeasy total RNA isolation kit , following the manufacturer’s protocol. Second, RNA extraction, library preparations and RNA sequencing reactions were conducted at GENEWIZ, LLC. . Total RNA was extracted from frozen cell pellet samples using Qiagen RNeasy Plus Universal mini kit, following the manufacturer’s instructions . RNA Sample QC, DNase treatment, library preparations, sequencing reactions, and read mapping and alignment were conducted at GENEWIZ, LLC.  and are described in the 5 cells were seeded in 3 mL culture media in 6-well plates, followed by counting cells at 3 different time points: 48, 72 and 96 h after seeding cells using Countess Automated Cell Counter . Each sample was triplicated, and media were changed every other day. The 2 × 106 cells in 100 µL PBS were injected into the right flank of 4–6-week-old immunocompromised Hsd: Athymic Nude Foxn/nu female  or athymic nu/nu female mice . Second, 1 × 106 cells in 100 µL PBS mixed with 100 µL matrigel were injected subcutaneously into the right flank of immunocompetent C57BL/6 male mice . The volume (V) of subcutaneous tumors was calculated from caliper measurements, where V = (π/6) × x × y × z where x, y, and z are 3 orthogonal diameters. Doubling times were calculated by the equation of trend lines using GraphPad Prism. The animal protocol was approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center . Two strains of mice were used in the animal experiments. First, 1 × 10Zymography, a common method to detect isoenzymes, was used to detect tissue-specific differences in LDH isoenzymes. This approach can directly observe 5 isozyme bands in the active state . Based oDissected tumors were placed into 4% paraformaldehyde for further immunohistochemistry (IHC). The immunofluorescent (IF) staining was performed at the Molecular Cytology Core Facility of MSKCC using Discovery XT processor . The 5 µm thick, paraffin-embedded sections were stained for H&E and LDH-A and LDH-B staining. The sections of tumors from nude or immunocompetent mice were stained by anti-LDH-A and anti-LDH-B. The small 5-day tumors from GL261 NC and KD were stained with immune markers: anti-CD68 antibody , anti-CD4  and anti-CD3 antibody . Quantification of morphological characteristics was performed using trainable Weka Segmentation (Image J segmentation plugin) to assess the fraction of viable tumor cells, stroma, hemorrhage and necrosis in the H&E sections. The same approach was used to quantify LDH-A and LDH-B staining ,37.p-value of <0.05 was considered significant. All data presented for T cells assessment using IF staining were analyzed using GraphPad Prism  and are presented as mean +/− SD. Results were analyzed using the unpaired Student’s t-test, and statistical significance was defined as p < 0.05. Results are presented as mean ± standard error unless otherwise specified. Statistical significance was determined by a two-tailed Student t-test. A We chose three murine brain tumor models to understand the impact of LDH-A downregulation on tumor phenotype and growth potential: GL261 , CT2A 3 and ALTSAs expected, following LDH-A knock-down with shRNA, LDH-A mRNA levels, protein expression and enzyme activity were all significantly reduced compared to the control (NC) cell lines. The most notable difference was a significantly higher LDH-B/LDH-A ratio for mRNA, protein expression and LDH enzyme activity in GL261 KD cells F–H. To better understand the effect of LDH-A KD on the metabolism of the glioma cell lines and the effect of differences in the LDH-A/LDH-B ratio on the metabolic and phenotypic changes in glioma cells, we performed metabolic assays. The extracellular acidification rate (ECAR), the glycolytic proton efflux rate (glycoPER) and the oxygen consumption rate (OCR) were obtained, as described previously ,31,38. TScl16A3). Given the increased OCR rates observed in GL261 and CT2A LDH-A KD cells (We performed RNA sequencing (RNASeq) to analyze the cellular transcriptome in all six cell lines  to investigate changes in their transcriptomes. We confirmed LDH-A depletion in our LDH-A KD cell samples, and also observed a significantly increased expression of LDH-B in the GL261 KD cells compared to all other cells A. In addKD cells , we perfKD cells B. This aKD cells C,D. A siPDHA1 , which catalyzes the conversion of pyruvate into acetyl-CoA, which may further be used in the TCA cycle or for fatty acid synthesis  had the highest levels of ynthesis E. The effect of LDH-A knock-down on cell proliferation (in vitro growth profile) was compared to that of control NC cells A–C. The 3) tumors, whereas most GL261 NC tumors grew after a long 40-day delay period  tumors were grown in both immune-competent (C57BL/6)  and incoy period E. Once Gy period F–H. Wildp = 0.002)  E. The diLDH zymography is a common method to detect LDH isoenzymes and provides for the direct observation of five isozyme bands in the active state . The LDHAll NC tumors have an isoenzyme pattern similar to skeletal muscle (LDH5- and LDH-A-dominant), with some formation of LDH 4, 3 and 2. CT2A and ALTS1C1 LDH-A KD tumors have a similar LDH isoenzyme pattern as the NC tumors. Only GL261 LDH-A KD tumors were strikingly different, with a LDH isoenzyme pattern similar to the heart, where LDH1, 2, 3 and 4 isoenzymes are most highly expressed . The LDHLDH-A and LDH-B levels between control and knock-down ALTS1C1 cells  of subcutaneous GL261 NC and KD tumors growing in nude mice (GL261 KD tumors did not grow in immune-competent C57BL/6 mice) and that of CT2A NC and KD tumors growing in C57BL/6 mice . We chosC1 cells A. There C1 cells , consistC1 cells C,E,F. CoC1 cells , but no C1 cells , also coC1 cells D,E,H.In many, but not all, GL261 tumor regions, there was an inverse relationship between LDH-A and LDH-B expression intensity . This innu/nu) mice. Our objective was to better understand the impact of LDH-A downregulation (KD) on glioma tumor phenotype and growth potential in a subcutaneous location. In an accompanying manuscript [It has been shown that that LDH-A-associated lactic acid accumulation in melanoma inhibits tumor surveillance by T and NK cells, and that elevated levels of lactic acid prevent the upregulation of the nuclear factor of activated T cells (NFAT) in T and NK cells, resulting in diminished IFN-γ production . These rnuscript , we comp3) tumors, whereas most GL261 NC tumors grew after a long 40-day delay period. No significant difference was observed between the s.c. growth profiles of the NC and KD groups of CT2A and ALTS1C1 tumors in C57BL/6 mice. The differences and similarities in the growth profiles between the six cell lines are consistent with the differences and similarities observed in their mRNA, LDH zymogram, enzymatic and Western blot profiles. Our observations in GL261 LDH-A KD and control NC tumors are more similar to the growth delay and inhibition we observed in 4T1 orthotopic murine breast tumors in immune-competent and immune-suppressed host animals, following LDH-A KD [Here, we found that the growth of s.c. GL261 LDH-A KD tumors regressed after developing small (<50 mmLDH-A KD . HoweverThe major distinguishing characteristics of the GL261 LDH-A knock-down cells and tumors (compared to ALTS1C1 or CT2A) include a high level of LDH-B and the ability of GL261 LDH-A KD cells (and most likely tumors) to accumulate intracellular lactate, due in part to the downregulation of the MCT 4 transporter and the ability to convert lactate to pyruvate and further processing due to the up-regulation of the oxidative phosphorylation pathway. Only the GL261 LDH-A KD cell line showed a higher expression of LDH-B mRNA, and higher LDH-B/LDH-A mRNA and protein ratios compared to the other five cell lines. Additionally, control GL261 NC cells had the lowest levels of LDH-A and LDH-B, and LDH enzyme activity of the three control cell lines. The metabolic profile of these cells, using Seahorse XF technology, showed a limited energetic phenotype of GL261 glioma (with the lowest ATP production) compared to CT2A and ALTS1C1 cells, thereby linking the LDH activity in GL261 cells with its observed metabolic phenotype.LDH is the enzyme catalyzing the final step of glycolysis and contains two subunits, A and B, encoded by two genes . LDH-A iThe LDH-A and LDH-B immunohistochemistry and Weka analysis confirmed the isoenzyme patterns observed with LDH zymography. GL261 and CT2A KD tumors showed significantly less LDH-A staining than their control NC tumors. Additionally, the significantly greater LDH-B staining, only in GL261 LDH-A KD, was consistent (compared to NC tumors); no significant differences in LDH-B staining was observed between CT2A LDH-A KD and NC tumors. We also noted an “inverse” LDH-A/LDH-B staining relationship (high vs. low) in many, but not in all GL261 tumor regions. In contrast, CT2A tumors showed a more “direct” LDH-A/LDH-B staining relationship (high vs. low) in many tumor regions. These results are consistent with the known spatial heterogeneity of gliomas ,50. The The association of LDH-A and lactate with cancer metabolism and tumor growth has been studied extensively, including that in human gliomas ,47,48,49It has been suggested that glioma cells express one of at least two different metabolic phenotypes ,58, as rSCL16A3). We also observed that GL261 cells  had the highest levels of PDHA1 , an enzyme which catalyzes the conversion of pyruvate into acetyl-CoA in the mitochondria, and feeds acetyl-CoA into the TCA cycle. This suggests that GL261 glioma cells are pre-programed to have the capacity for high TCA cycle activity and that this capacity is enhanced by LDH-A depletion with the predominance of LDH-B expression. The data suggest that GL261 KD cells also have the ability to reprogram their transcriptome to support alternative (non-glycolytic) metabolic pathways in LDH-A-depleted cells. Furthermore, it suggests that GL261 LDH-A-depleted cells, through their unique upregulation of LDH-B, may be able to metabolize lactate into pyruvate and through the TCA cycle.Our RNA-seq bioinformatics analysis provides additional evidence that GL261 LDH-A KD cells may have an improved ability to convert lactate into pyruvate and entry into the TCA cycle. A Gene Set Enrichment Analysis (GSEA) showed that GL261 KD cells had a significant enrichment of the oxidative phosphorylation pathway, but little or no change in the expression of glycolytic genes (with the exception of PGK1). Neither CT2A or ALTS1C1 LDH-A-depleted cells showed any consistent enrichment of either glycolytic or oxidative pathway genes when compared to their respective NC controls. In addition to decreased LDH-A and increased LDH-B, GL261 KD cells also showed a significant downregulation of the lactate exporter MCT4 (The high maximum respiration capacity detected in GL261 LDH-A KD cells (following the addition of the uncoupler FCCP), demonstrates the magnitude of physiological “energy demand” which can be achieved by rapid oxidation of substrates  to meet this metabolic challenge. We and others ,48 suggeSix genetically altered murine glioma cell lines and tumors (with LDH-A shRNA knock-down (KD) vs. scrambled shRNA controls—NC) were compared for differences in their levels of LDH-A and LDH-B mRNA, protein and enzymatic activity, as well as their glycolytic and oxidative metabolic pathway activity. An RNA sequencing (RNASeq) analysis and the above studies showed that GL261 glioma cells (but not CT2A and ALTS1C1 cells) are pre-programmed to have the capacity for high TCA cycle activity and that this capacity is enhanced by LDH-A depletion. The expression of glycolytic pathway genes was not significantly affected by LDH-A KD. These results show the combined impact of LDH-A depletion (LDH-A KD vs. NC control) and the immune system (C57BL/6 vs. nude mice) on the growth of s.c. located tumors and suggest that GL261 glioma cells (but not CT2A and ALTS1C1 cells) can adopt different metabolic pathways with higher TCA cycle activity under environmental stress, and that this capacity is enhanced by LDH-A depletion.


Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly. Although the RSV matrix (M) protein has key roles in the nucleus early in infection, and in the cytoplasm later, the molecular basis of switching between the nuclear and cytoplasmic compartments is not known. Here, we show that protein kinase CK2 can regulate M nucleocytoplasmic distribution, whereby inhibition of CK2 using the specific inhibitor 4,5,6,7-tetrabromobenzo-triazole (TBB) increases M nuclear accumulation in infected cells as well as when ectopically expressed in transfected cells. We use truncation/mutagenic analysis for the first time to show that serine (S) 95 and threonine (T) 205 are key CK2 sites that regulate M nuclear localization. Dual alanine (A)-substitution to prevent phosphorylation abolished TBB- enhancement of nuclear accumulation, while aspartic acid (D) substitution to mimic phosphorylation at S95 increased nuclear accumulation. D95 also induced cytoplasmic aggregate formation, implying that a negative charge at S95 may modulate M oligomerization. A95/205 substitution in recombinant RSV resulted in reduced virus production compared with wild type, with D95/205 substitution resulting in an even greater level of attenuation. Our data support a model where unphosphorylated M is imported into the nucleus, followed by phosphorylation of T205 and S95 later in infection to facilitate nuclear export and cytoplasmic retention of M, respectively, as well as oligomerization/virus budding. In the absence of widely available, efficacious treatments to protect against RSV, the results raise the possibility of antiviral strategies targeted at CK2. Pneumoviridae family. Its nonsegmented negative-sense RNA genome [Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract disease in infants , as wellA genome  encodes A genome ,10,11,12Previously, we showed that M protein localization in the nucleus at the early stages of infection correlates with decreased host cell transcription, correlating strongly with M association with the host chromatin ,14,15. IWe previously reported that RSV M’s contribution to virus assembly/release is strongly dependent on the CK2 consensus site threonine 205 (T205), which may play a role in regulating M oligomerization . Since Tp < 0.0001) increased M nuclear accumulation (Fn/c of 3.5) compared with untreated cells, with this significantly (p < 0.001) higher accumulation compared with untreated cells still evident at 30 h p.i.Building on previous observations ,17,24, rp < 0.0001) reduced nuclear accumulation in the presence compared with the absence of TBB (p < 0.0003) in the nuclear levels of GFP-M in both Vero and A549 cells  by live cell confocal laser scanning microscopy, CLSM c,d. As ee of TBB d. Striki95LDE, single letter code) as a high-confidence CK2 site within RSV M, additionally to the previously reported threonine at position 205 (T205VTD). To begin to assess the functionality of this site, and its potential relationship to T205, truncation constructs (p < 0.01) increased nuclear accumulation  in similar fashion to full-length GFP-M (95LDE and T205VTD), within aa1-110 and 183-256, that modulate nucleocytoplasmic trafficking.Use of the ExPaSy public domain software enabled us to identify the serine at position 95 , and the mutant derivatives were expressed as full-length GFP-fusions in transfected cells (95A and T205A derivatives) had no significant effect on nuclear accumulation of M and did not appear to impair sensitivity to TBB, with both derivatives showing significantly increased (p < 0.0001) nuclear accumulation in the presence of TBB compared with its absence  resulted in increased nuclear accumulation (Fn/c of c. 1.2) in the absence of TBB compared with wild type GFP-M (Fn/c of c. 0.5) and loss of responsiveness to TBB  also resulted in increased nuclear accumulation compared with GFP-M:T205A, but the levels of nuclear accumulation were not significantly higher than wild type M; it also had reduced sensitivity to TBB .The substitution of Ss to TBB b. GFP-M:ells see ; these cp < 0.0001) increased nuclear accumulation of wild type GFP-M , with GFP-M:S 95A, -M:T 205A and -M:T 205D all showing similar responses. In stark contrast, GFP-M:S 95D was not sensitive to LMB treatment , implying that negative charge at S95, normally supplied by phosphorylation, inhibits M nuclear export via CRM1.The effect of the CK2 site mutations on M localization in cells were also assessed in the presence of the specific CRM1 inhibitor leptomycin B (LMB) a,c. As p95 or T205 (through A substitution) or negative charge at T 205 is not sufficient to alter the sensitivity to TBB of M subcellular localization. Negative charge at S 95, however, increases nuclear accumulation which is not sensitive to TBB or LMB treatment. This increased nuclear accumulation would appear to be in part due to the inhibition of M nuclear export and/or enhanced nuclear retention.Overall, the lack of phosphorylation at either S95 and T205 site substitutions; in all cases, combined mutation of both S95 and T205 eliminated TBB sensitivity , AD (GFP-M:AD) or DD (GFP-M:DD) substitution had little effect on M localization compared with wild type M in the absence of TBB/LMB, DA substitution increased nuclear accumulation in similar fashion to substitution of D95 alone (p < 0.05) increased nuclear accumulation, implying that nuclear export is completely comparable to that of wild type M  abrogated responsiveness to LMB, indicating impaired CRM1-dependent nuclear export. M with negative charge at position 95 with a nonphosphorylatable mutant at position 205 (GFP-M:DA) retained LMB sensitivity.Analysis was extended to cells expressing GFP-M with dual S95 alone d,e; cytod type M d,f. In c95 and T205 CK2 sites coregulate M nucleocytoplasmic trafficking, where negative charge mimicking phosphorylation at either S95 or T205 results in reduced M nuclear export, leading to increased nuclear accumulation.Results overall are consistent with the idea that the S95A and S95D mutations, with or without T205A, were introduced into the full-length genome of RSV rA2 in a reverse genetics system and multiple attempts were made to recover recombinant viruses. As for previous studies that were unable to recover rA2 carrying M:T205D [95D was not recovered, but the rA2-M:S95A and rA2-M:S95A/T205A) viruses were able to be recovered. Replication fitness was assessed in a multicycle replication assay in three different cell lines  reduced fitness compared with rA2 in all three cell lines (Since a role for Tll lines a. The in95A at 24 h p.i. (1−2 × 106 pfu/mL in both cases), rA2-M:S95A/T205A, in contrast, had markedly reduced titer (0.6 × 106 pfu/mL), while rA2-M:S95D/T205D was below the level of detection in the assay (<0.7 pfu/mL). N, P, and M were present in all samples of whole-cell lysates; N was also present in all samples of pelleted virus. P and M, in contrast, were detectable in all pelleted virus samples except that from cells infected with rA2-M:S95D/T205D. That there are equivalent levels of RSV structural proteins  in cells infected with rA2 and mutant viruses .Human alveolar epithelial A549 cells, human epithelial Hep2 cells, and African green monkey kidney Vero cells were obtained from European tissue culture collection (ECCAC) and used for rescue of recombinant virus, virus culture, and all infections. Cells were cultured in a humidified 5% COCells were transfected using Lipofectamine  24 h before analysis by CLSM; see below. Where relevant, LMB (2.8 ng/mL) was added at 18 h post-transfection for 6 h, or TBB (25 μm) at 6 h post-transfection for 18 h, prior to CLSM and image analysis.RSV titers in cell lysates (cell-associated virus) or supernatant (released virus) were determined as described in ,39. BrieThe M coding sequence was generated by PCR from full-length rA2 cDNA  and intr95 and/or T205 mutations were cloned into the shuttle vector pGEM-HX. The shuttle vectors so obtained were digested with SpeI and XhoI to subclone the coding sequences for the mutated M genes into vector D51 [Recombinant RSV strain rA2 has been described in . The Hinctor D51 , digestector D51 ,44.205A, rA2-M:S95A, rA2-M:S95A/T205A, rA2-M:S95D/T205D) were used to infect cells at an MOI of 0.1. Virus was harvested at the times indicated, and titer was determined in immunoplaque assays [Recombinant wild type (rA2) or the mutants  in PBS, followed by incubation with anti-RSV antibody overnight at 4°C with rocking. Blots were incubated with species-specific secondary antibodies conjugated to horseradish peroxidase, followed by washing and detection of bound antibodies with Enhanced Chemiluminescence . Bound protein images were captured on the Licor Odyssey Fc, and digital images were analyzed using ImageStudio software (Licor). Infectious virus titers were determined in the culture supernatant as above.Cells were grown to 80% confluence on glass coverslips before being infected at an MOI of 3 with rA2 ; certainFixed and live (transfected) cell samples were imaged as previously in , and ImaGraphpad Prism 6 was used for the determination of statistically significant differences using the Mann–Whitney test or two-way ANOVA as appropriate.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic which has brought a great challenge to public health. After the first emergence of novel coronavirus SARS-CoV-2 in the city of Wuhan, China, in December 2019. As of March 2020, SARS-CoV-2 was first reported in Bangladesh and since then the country has experienced a steady rise in infections, resulting in 13,355,191 cases and 29,024 deaths as of 27 February 2022. Bioinformatics techniques are used to predict B cell and T cell epitopes from the new SARS-CoV-2 spike glycoprotein in order to build a unique multiple epitope vaccine. The immunogenicity, antigenicity scores, and toxicity of these epitopes were evaluated and chosen based on their capacity to elicit an immune response.The best multi-epitope of the possible immunogenic property was created by combining epitopes. EAAAK, AAY, and GPGPG linkers were used to connect the epitopes. In several computer-based immune response analyses, this vaccine design was found to be efficient, as well as having high population coverage.in silico findings would represent a major step forward in the development of a vaccine that might eradicate SARS-CoV-2 in Bangladeshi patients.This research is entirely reliant on the development of epitope-based vaccines, and these  As of March 2022, more than 140 million doses of currently approved vaccines had been administered in Bangladesh. Bangladesh observed the second wave of COVID-19 in March 2021, employing the UK variant as well as the Beta, V2 variant from South Africa, during a vaccination program. Furthermore, the third wave of COVID-19, caused by Delta variant, arrived in mid-May 2021 and in Bangladesh, it turned out to be the most lethal, resulting in a huge increase in COVID-19 cases and deaths domestically. The Omicron (B.1.1.529) a new variant was identified in November from South Africa and quickly spread to over 57 countries throughout the world. According to recent research, the development of complicated mutations in the spike glycoprotein may enable this variant to elude the protective immune response induced by vaccination.Globally, the World Health Organization recorded 430,257564 confirmed cases and 5,922049 fatalities, prompting major health concerns [On January 12, 2020, Chinese scientists disclosed and reported the SARS-CoV-2 genome sequence, which was then uploaded to the GenBank database . As the Researchers have proposed a number of ways for developing SARS-CoV-2 vaccines . The staNasopharyngeal specimens were collected from suspected COVID-19 patients in Bangladesh. Specimens were kept at 4 °C, before and during transport when sampled and processed on the day of collection or stored at – 20 °C. The specimens were transported on ice and processed within 24 h of collection.ORF1ab and N genes of SARS-CoV-2.Cycle threshold (Ct) values were used to select nasopharyngeal swab samples for RNA extraction. SARS-CoV-2 has predominantly been detected using the SANSURE qRT-PCR kit  according to the manufacturer’s instructions. The qRT-PCR was performed for TM Viral Total Nucleic Acid Purification Kit was used ; Illumina RNA Prep with Enrichment, (L) Tagmentation protocol were followed with step-by-step instructions to prepare and enrich libraries . A paired-end (2 × 74 bp) read was generated from each sample after tagmentation and amplification. Based on default parameters, Basespace Dragen RNA Pathogen Detection programme (version 3.5.14) was used to construct SARS-CoV-2 genome sequence reads.Whole-genome sequencing of SARS-CoV-2 isolates were conducted by the Genomic Research Laboratory, BCSIR, Dhaka, Bangladesh using NextSeq 550. In this case, ReliaPrephttps://www.ezbiocloud.net/tools/ sc2) of EZBioCloud, the samples are subjected to SARS-CoV-2 consensus generation. SARS-CoV-2 genome coverage averaged 2×.SARS-CoV-2 genome readings were found in qRT-PCR positive samples after sequencing. The sequencing method generates 1.2 million reads per sample. With EzCOVID19 (https://swissmodel.expasy.org/interactive). Protein structure identification and analyses were reported through Expasy (https://web.expasy.org/protparam/) [http://bioinf.cs.ucl.ac.uk/psipred/) [http://clavius.bc.edu/~clotelab/DiANNA/) [Whole-genome sequence of 15 isolates of SARS-CoV-2 were done using NextSeq 550. SARS-CoV-2 spike glycoprotein sequence was extracted from the FASTA file after alignment of 15 SARS-CoV-2 isolates with a reference sequence (Wuhan) using Mega-X. From all the data the spike glycoprotein of BCSIR-NILMRC_355 was selected for vaccine design as the data quality of this isolates was good. The PDB file of the particular protein was built using Swiss-model (tparam/) . Secondasipred/) . Disulfihttp://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) with the threshold 4.0 [https; /www.ddg.pharmfac.net/AllerTop). The trRosetta [https://services.healthtech.dtu.dk/service.php?TMHMM-2.0).To determine the antigenic behaviors of the vaccine protein, the sequence was first subjected to online server VaxiJen (hold 4.0 . while arRosetta  and I-TAhttp://tool.iedb.org/main/) [50 scores were assigned by the online server. A threshold score of more than 0.5 was taken, good epitope candidate. IC50 and binding affinity are inversely proportional to each other. Mean if IC50 value is small, epitope binding affinity to MHC-II will be high. IC50 values < 10 nM < 100 nM < 1000 nM means high, intermediate and low binding affinity with MHC-II. MHC-II and binding affinity between epitope are inversely proportional to percentile rank [Selecting effective B cell epitopes requires surface accessibility. Thus, the Emini surface accessibility tool was used to analyses. IEDB Resource (g/main/)  was usedile rank .To check the antigenic and allergic behavior of B cells and T cells epitopes, online servers VaxiJen v2.0  and Alletools.iedb.org/population/). The recovery score of more than 60 was considered as a good population coverage [Using online IEDB Analysis Resource, MHC-I and MHC-II epitopes were used to predict population coverage  and TrRosetta web server were used to generate the secondary and tertiary structures of the vaccine construct. The 3D structure of the vaccine construct was generated after analysis of the domain with 3D structure. Galaxy Refine  was used to validate the vaccine. For further verification by ProSA-Web Ramachandran Plot, and PROCHECK (https://saves.mbi.ucla.edu/) [PSIPRED 3.3 online server (la.edu/)  online shttps://cluspro.bu.edu) most extensively used docking server, which is based on six energy functions and depends on the type of protein. Cluspro predicts 10 docking results in about 5 h based on densely packed low-energy clusters for each docking parameter. This procedure illustrates how to use various choices such as creating extra restrictions files, selecting various energy parameters, and analyzing the outcomes. PDBsum were used to validate the vaccine and TLR-4 graphical representations.The three-dimensional structure of TLR-4 with (PDB ID: 4G8A) was downloaded from PDB. The structure was first check for missing residues and breaks. The water molecules were removed. The MOE software were used for the correct protonation. Then the structure was save in TLR-4.pdb format. The vaccine sequence was docked with the human Toll-like receptor 4 through Cluspro Surface exposed residues were detected at position 1–14, 69–114, 222–230, and 305–318, whereas residues from 38 to 48, 138–198, 254–281, and 342–380 were found inside the transmembrane region. The core area of the SARS CoV-2 spike glycoprotein was determined to have residues from positions 15–37, 49–68, 115–137,199–221, and 319–341.B cell epitopes are crucial for viral infection resistance. Potential B cell epitopes have changed characteristics that instruct B cells to recognize and activate a wide range of immune responses directed to specific viral infections. A total of ten linear epitopes were calculated with a threshold score of 0.500 through IEBD analysis tool. For linear epitope maximum score was 0.698 and the minimum score was 0.191. However, average prediction score was 0.476. Allergenic and poisonous epitopes were eliminated, and only antigenic and non-allergic epitopes were chosen. On average, 10 out of 25 B cell epitopes were considered effective. (RSYLTPGDSSSGFTVEYQTSNFRVQPSNKKFLPFQTLVNNSYECDIPIQLGQSKRVDFCEDR) that can evoke B-lymphocytes. After compensating of data, sequences of peptides from 350 to 440 residues boost the immune responses Table Selecting effective B cell epitopes requires surface accessibility. Thus, the Emini surface accessibility tool was used to analyses. After detecting the default threshold value of 1.000, 13 epitopes were selected. The area with the most accessibility was 89–95 residues, with an average observed value of 1.00. Although the least value was 0.078 and the greatest value was 5.199, it was discovered that the minimum value was 0.078, maximum value was 5.199. Table Homo sapiens as the MHC source. This tool can compute epitope HLA binding affinity using the IC50 nM unit. A lesser IC50 suggests greater affinity for MHC-I molecules. In order to maximize affinity for MHC-1 alleles, the total number of epitopes was designed to be fewer than 100. On the basis of MHC-1 allele interactions, 60 epitopes were chosen among 950. The antigenicity, allergenicity, and toxicity of 60 epitopes were assessed. The antigenic scores of toxic and allergic epitopes were ruled out. The MHC-1 epitopes were finalized. They bind to alleles HLA-B*58:01, HLA-B*57:01, HLA-A*23:01, and HLA-A*24:02. YSLEFQSFGW has an antigenic score of 1.713   for the vaccination chimaeras. The vaccine included 50S ribosomal protein L7/L12 as an adjuvant. The adjuvant and the B cell epitope was linked by EAAAK linker. B cell and MHC-I epitopes were linked by GPGPG linkers and MHC-I and MHC-II epitopes was linked through AAY linkers. The vaccine was made smaller by combining B cell, CTL, and HLT epitopes. The vaccine sequence has a 6x His tag for protein identification and purification. The resulting vaccine construct sequence has a molecular weight of 50689.18 kDa and 309 a.a sequence. The Vaccine build is shown in the diagram below.>vaccine proteinEAAAKYSLEFQSFGWYMSLGPMVLYLVPCYTCLWWLLPVIVEVWTAGAAAYYWLLPVIVEVSIFGCLLPQKFQLKYLSQKVTNRFWWFFFSTNGSKAEKKVSSEFILVRYCCYSLISITLKLEVIPFHLMMVFILLITRFYRLRYGMAIELLEATCPGPGYYKLVPFWSFYWQNSRLTFYWQNSRLTFFYSLEFQSFGWYMSLGPMVLYLVPCYTCLWWLLPVIVEVWTAGAAAYYWLLPVIVEVSIFGCLLPQKAAYLQSYGFQPTLQYGSFCTQVLSFELLHAVVVLSFELLAAYYVGYLQAVLYQDVNCAYSNNSIACVADYSVLYCVIAWNSNNCVLGQSKRVFAMQMAYRFDVVNQNAQAAYRFNGIGVHHHHHH.https://web.expasy.org/protparam/) was used to calculate the physical and chemical parameters of the vaccine, which consisted of 380 residues, and a molecular weight of 50689.18 kDa. The theoretical isoelectric point (PI) value is 8.96 which indicates that the protein is positively charged, as is the case with isoelectric points over 7.0. An instability index (II) of 42.46 determined by Protparam classified our protein to be stable. The aliphatic index is 73.10 which is thermo-stable across a wide temperature range. The aliphatic index is 73.10, indicating that it is thermo-stable over a wide temperature range. At 0.007, the grand average of hydropathcity (GRAVY) was computed using the chemical formula C2283H3452N620O664S15.Protparam (Expasy) , Northeast Asia (91.85%), South Asia (95.13%), Europe (95.13%), and North America (88.65%). The lowest population was found in Central America (7.76%). South Asia has the highest percentage of MHC-I alleles in the population (91%), followed by Europe. Central Africa was found to have the lowest population density (51.46 %) .Four epitopes  are crucial for the majority of interactions in MHC-II alleles describe a considerable coverage in contrast to the whole world population. For LQSYGFQPT, the proportion of concentrated population coverage in the world was anticipated at 69.75%. In the instance of MHC-I, five epitopes  were predicted to interact with common MH Class-I alleles, resulting in a massive worldwide coverage. The population coverage data for the abundant binders to MHC-I and MHC-II alleles reveal 97.55 and 98.65% coverage, respectively .Z-score was calculated to see if the input structure was within the range of similar-sized of natural proteins. Figure Z-score for the input structure was − 8.77, indicating that it was outside the normal range for natural proteins of the same size. For Ramachandran analysis, the PROCHECK server computed 87.5% of the residues in the most favored areas, 12.5% in extra permitted regions, 0.0% in generously allowed regions, and 0.0% in forbidden region, with 0.0% residues in disallowed regions.The PROSA 3D server was used to predict the 3D structure of the multi-epitope vaccine sequence, affording ten predicted structures for a given query sequence. The model five was taken for further investigation in Fig. TLR-4 and the vaccine construct was docked using the Clustpro docking server to understand the interaction and to predict the final 3D model. A totals of ten models were created. Using the Pymol program, all ten docking models were visually examined and analyzed, Among the 10 model docking complex model 5 proved good docking result with total 17 H-bond interaction with the binding score − 290.5. For a graphical illustration of the residual interaction between the vaccine construct and TLR-4, the PDBsum online databases were used. To show hydrogen bonding between the vaccine construct complex and TLR-4, a graphical picture was generated. There were 17 hydrogen bond interactions between TLR-4 and the vaccine construct. Analysis of the vaccine-TLR complex involved the direct interaction of the residues which revealed that hydrogen bonds were formed between ARG355-GLU42, ARG227-PHE64, GLN547-PRO157, SER545-LYS163, GLU474-ARG185, THR499-ARG185, ARG496-TRP218, GLU494-TRP219, THR493-GLU226, GLN436-ASP324, GLN436-ASP324, SER415-LEU328, ASN417-LEU328, ARG447-ILE332, GLU422-ARG346, GLU422-ARG346, ASN448-ARG346 with distances of 2.82 Å, 2.81 Å, 2.85 Å, 2.70 Å, 2.70 Å, 2.73 Å, 2.67 Å, 2.65 Å, 2.89 Å, 2.86 Å, 2.96 Å, 3.13 Å, 2.72 Å, 2.86 Å, 2.63Å, 2.73 Å, 2.79 Å, and 2.93 Å, respectively Fig. iMODS performs an expository study of the structure by altering force field of the complex with regard to various time periods. The final model shows less deformation at each residue capacity level. The complex has an eigen value of 9.798369e−06. In heat maps, low RMSD and highly co-related regions indicated improved interactions between individual residues  of 0.95, with a GC content of 63.42%. Because the GC content ranged between average of 35 and 60%, these figures imply steady vector expression in li. Fig. .Fig. 7CoWe evaluated the vaccine's ability to trigger an effective immune response in practical use using the C-ImmSim webserver. After the primary reaction, the secondary and tertiary immune responses rose steadily. Antibodies  levels have risen dramatically. Figure , by using the immunoinformatics technique. The protein sequence was reported as the near-complete genome sequence of a SARS-CoV-2 Delta variant strain in Bangladesh, after which the antigenicity, Non-Allergenicity, and non-toxic properties of the protein were investigated. Mapping of epitopes was performed for further analysis. MHC-1 and MHC-2 were predicated. The selected epitopes are combined by the linker . The stability and efficiency of the vaccine constructs were checked via an online server. Furthermore, clinical research on MHC targeting with human leukocyte antigen (HLA) is emerging [The disease is causing global issues, and now is the moment to take preventative measures. Computational methods have become increasingly important in the design of multi-epitope-based vaccines. Multiple epitopes are highly recommended because of their cost-effectiveness, and higher efficiency. The study aims to design a vaccine against SARS-CoV-2emerging . Antigenemerging . For immemerging . This premerging . Accordiemerging . Populatemerging . Analysiemerging . To mimiemerging –40. Throemerging , 40.Using immunoinformatics methods a convenient method of vaccination was done against SARS-CoV-2 in Bangladeshi patients. This scientific endeavor starts with the NGS sequencing. Various immunoinformatics methods were utilized to create the vaccine protein. Online tools were utilized to build B cell and T cell epitope vaccines. CTL and HTL epitope were joined using adjuvant and linker. Then antigenicity and allergenicity were determined to ensure the vaccination is safe and stable. Molecular docking and MD modelling were used to test human-TLR-4 interaction and stability. Using immunoinformatics technologies ensures that the vaccination design is safe and stable.


The V179I substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is selected in humans or mouse models treated with certain nonnucleoside reverse transcriptase inhibitors (NNRTIs). While it is often observed together with other NNRTI resistance mutations, V179I does not confer drug resistance. To understand how V179I arises during NNRTI treatment, we characterized it in HIV-1 molecular clones with or without the NNRTI resistance mutations Y181C or Y181V. While V179I alone did not confer resistance to any NNRTIs tested, when present with Y181C/V it enhanced drug resistance to some NNRTIs by 3- to 8-fold. In replication competition experiments in the presence of the NNRTI rilpivirine (RPV), V179I modestly enhanced Y181C HIV-1 or Y181V HIV-1 replication compared to viruses without V179I. As V179I arises from a G to A mutation, we evaluated whether it could arise due to host APOBEC3 deaminase activity and be maintained in the presence of a NNRTI to provide a selective advantage for the virus. V179I was detected in some humanized mice treated with RPV and was associated with G to A mutations characteristic of APOBEC3 activity. In RPV selection experiments, the frequency of V179I in HIV-1 was accelerated in CD4+ T cells expressing higher APOBEC3F and APOBEC3G levels. Our results provide evidence that V179I in HIV-1 RT can arise due to APOBEC-mediated G to A hypermutation and can confer a selective advantage to drug-resistant HIV-1 isolates in the presence of some NNRTIs. The goals of antiretroviral drugs are to suppress replication of HIV-1 in infected individuals  and to prevent infection of HIV-1 in uninfected individuals . Nonnucleoside reverse transcriptase inhibitors (NNRTIs) comprise a class of antiretroviral drugs that target the HIV-1 enzyme reverse transcriptase (RT) that are used for both ART and PrEP. They inhibit reverse transcription by binding to a hydrophobic pocket near the polymerase active site, which prevents DNA polymerization . In 2021Due to high frequencies of circulating NNRTI-resistant isolates, NNRTIs are no longer recommended as first line ART in the U.S. . The morV179 is a polymorphic residue in HIV-1 RT, particularly subtypes A and C, which is detected in treatment-naive individuals –20. AminThe apolipoprotein B messenger RNA-editing enzyme catalytic polypeptide 3 (APOBEC3) family of mammalian proteins are cytidine deaminases that convert cytidine residues to uridines. APOBEC3G and APOBEC3F are packaged into HIV-1 particles and deaminate negative-sense viral DNA, resulting in G to A mutations in the viral positive-sense strand that can restrict infection . The antIn a recent humanized mouse study, we reported low frequencies of V179I in plasma HIV-1 RNA from mice infected with WT or Y181V HIV-1 after treatment with RPV-LA PrEP . In the NL4–3 and molecular clones containing V179I, Y181C, and Y181V alone or in combination with multiple concentrations of EFV, DAP, ETR, RPV, and DOR. The specific infectivity of each virus was determined, which was similar for all viruses (50) and fold-changes compared to WT for the NNRTIs were calculated for the WT and mutant viruses  of RPV. Additionally, V179I was selected in infectedCEM and CEM-SS cells in the presence of high levels of RPV, suggesting that it provides a selective advantage. Y181C HIV-1 can be rapidly selected by NNRTIs and has relatively high prevalence  supplemented with 10% fetal bovine serum , 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.292 mg/ml of L-glutamine . GHOST-R3/X4/R5 cells  were maiEFV, ETR, FTC, and RPV were obtained from the AIDS Reagent Program, Division of AIDS, National Institutes of Health. DAP and DOR were obtained from Dr. Nicolas Sluis-Cremer. Drug stocks were made in dimethyl sulfoxide (DMSO) and diluted to final concentrations in cell culture medium.pol gene of plasmids encoding the HIV-1NL4–3 with a deletion in env and the luciferase gene in place of nef (NLdE-luc) (NL4–3 or HIV-1NL4-BAL proviruses were amplified by PCR and were subcloned into pCR2.1-TOPO (Life Technologies). RT mutations E138K, V179I, Y181C, Y181V, E138K/V179I, V179I/Y181C, and V179I/Y181V were introduced into these plasmids using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies). After verification by Sanger sequencing, the mutated fragment was cloned into the plasmids.The RT coding region of the LdE-luc)  or full-WT and mutant viruses were produced by transfection of 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific). NLdE-luc viruses were pseudotyped with VSV-G, using the pLVSV-G plasmid. Virus-containing supernatants were harvested 48h after transfection.via ELISA (XpressBio).Viruses were titered on GHOST R3/X4/R5 cells using an Accuri C6 flow cytometer (BD Biosciences), as previously described . NLdE-lu4 cells/well of a 96-well plate) were incubated with or without dilutions of drugs and infected with each virus at a multiplicity of infection (MOI) of0.02 in phenol red-free medium. After 48h, luciferase was measured using the Britelite Plus Reporter Gene Assay System (PerkinElmer). Infectivity was measured by luciferase expression in the cells, with uninfected cells set as background and infected cells in the absence of inhibitor set as 100%. Experiments were performed in triplicate in 3 or 4 independent experiments. The 50% effective concentration (EC50) was calculated in Prism (GraphPad) using nonlinear regression for each experiment.Susceptibility assays for WT and mutant HIV-1 (NLdE-luc) with NNRTIs DAP, DOR, EFV, ETR, and RPV as well as the NRTI FTC were performed in TZM-bl cells as previously described . BrieflyNL4–3 or Y181V and V179I/Y181V HIV-1NL4–3 in two independent experiments, as previously described (50 for Y181C HIV-1 or Y181V HIV-1 (with or without V179I): 1.4 nM and 13 nM, respectively. Cultures were split every 2 days for 8 days. HIV-1 RNA was isolated from cell supernatants at each time point using the RNeasy kit (Qiagen). PrimerID MiSeq was performed to sequence part of the RT coding region as described previously (Competition experiments were performed comparing Y181C and V179I/Y181C HIV-1escribed . BrieflyNL4–3 were performed in CEM cells and CEM-SS cells. CEM and CEM-SS cells were infected with Y181C HIV-1 at a MOI of 0.1 in the absence or presence of 0.5 nM RPV (Experiment 1) or 1 nM RPV (Experiment 2). Cultures were split every 2–3 days. When significant cytopathic effects were observed, cells were pelleted and supernatant was collected from each flask. Each culture was replenished with new cells and fresh medium with or without 2- to 3-fold higher RPV concentrations. The selection process was repeated for 4 rounds. Due to the COVID-19 pandemic, supernatants from cultures after round 1 in Experiment 1 were frozen at −80 C and were used to infect new cells in round 2. HIV-1 RNA was isolated from supernatant collected at the last time point from each round of selection using the RNeasy kit. PrimerID MiSeq for the RT coding region was performed as previously described (Selections of Y181C HIV-1escribed  to deterCg-PrkdcscidIl2rgtm1Wjl/SzJ) mice from Jackson Laboratory were implanted with fetal thymus, liver, and CD34 + cells obtain from the University of Pittsburgh Biospecimen Core with an Institutional Review Board-approved Honest Broker System. Mice (n = 30) were injected intramuscularly with 150 mg/kg RPV LA and challenged 1 or 7 days later with WT or mutant HIV-1NL4-BAL and compared to untreated and challenged mice (n = 26). Of the RPV LA-treated mice, sixteen became infected as determined by detection of HIV-1 RNA in the plasma, as described previously (pol. A total of 715 sequences were obtained from these mice, of which 6 had stop premature stop codons. Two-tailed unpaired t tests were performed in Prism to compare viremia and mutations between groups.We previously evaluated the ability of RPV LA to prevent vaginal transmission of WT, Y181C, or Y181V HIV-1 in humanized mice . Female eviously . HIV-1 seviously  to ident