The Fatty Acid Lipid Metabolism Nexus in COVID-19.

Enteric symptomology seen in early-stage severe acute respiratory syndrome (SARS)-2003 and COVID-19 is evidence of virus replication occurring in the intestine, liver and pancreas. Aberrant lipid metabolism in morbidly obese individuals adversely affects the COVID-19 immune response and increases disease severity. Such observations are in line with the importance of lipid metabolism in COVID-19, and point to the gut as a site for intervention as well as a therapeutic target in treating the disease. Formation of complex lipid membranes and palmitoylation of coronavirus proteins are essential during viral replication and assembly. Inhibition of fatty acid synthase (FASN) and restoration of lipid catabolism by activation of AMP-activated protein kinase (AMPK) impede replication of coronaviruses closely related to SARS-coronavirus-2 (CoV-2). In vitro findings and clinical data reveal that the FASN inhibitor, orlistat, and the AMPK activator, metformin, may inhibit coronavirus replication and reduce systemic inflammation to restore immune homeostasis. Such observations, along with the known mechanisms of action for these types of drugs, suggest that targeting fatty acid lipid metabolism could directly inhibit virus replication while positively impacting the patient's response to COVID-19.

Transfusion-related Epstein-Barr virus (EBV) infection: A multicenter prospective cohort study among pediatric recipients of hematopoietic stem cell transplants (TREASuRE study).

BACKGROUND: Epstein-Barr virus (EBV) is carried in the blood of most adults, and transfusion-related infections have been reported. EBV is particularly deleterious in immunosuppressed transplant patients. The aim was to determine if EBV transmission occurred through leukodepleted blood product transfusion in pediatric recipients of hematopoietic stem cell transplants (HSCT). STUDY DESIGN AND METHODS: This prospective Canadian multi-center cohort study includes 156 allogeneic HSCT pediatric recipients. The association between EBV and transfusion was analyzed using Cox regressions. EBV infection, defined by a PCR+ test in the blood of seronegative recipients of an EBV-negative graft, was monitored in order to correlate the recipient EBV strain with that of the blood donors. EBV genotypes were determined by PCR amplification followed by DNA sequencing at two loci (EBNA3b and LMP1). RESULTS: No statistically significant associations were found between transfusions and EBV. One case of post-transplant EBV infection was identified among the 21 EBV-seronegative recipients receiving an EBV-negative graft. A total of 22 blood donors were retraced to determine whether the recipient's EBV strain matched that of a donor. One donor strain showed 100% sequence homology at the EBNA3b locus, but differed by one or two point mutations and by a 132-bp deletion at the LMP1 locus. The blood donor in question was alone among the 22 donors to show amplifiable virus in plasma. Blood from this donor readily produced an immortalized lymphoblastoid cell line in culture. CONCLUSION: While considered a rare event, EBV transmission through transfusion may occur in the context of severe immunosuppression.

Construction and Characterization of a Humanized Anti-Epstein-Barr Virus gp350 Antibody with Neutralizing Activity in Cell Culture.

Acute Epstein-Barr virus (EBV) infection in immunosuppressed transplant patients can give rise to a malignant B-cell proliferation known as post-transplant lymphoproliferative disease (PTLD). The EBV major virion surface glycoprotein (gp)350 is a principal target of naturally occurring neutralizing antibodies and is viewed as the best target to prevent acute infection and PTLD in at-risk transplant recipients. We have constructed a humanized (hu) version of the murine anti-gp350 neutralizing monoclonal antibody 72a1. The hu72a1 IgG1 antibody displayed no significant anti-mouse activity, recognized both gp350 and its splice variant gp220 as well as a gp350 peptide that was shown to constitute the principal EBV gp350 neutralizing epitope when tested in immunoassays. Hu72a1 antibody blocked in vitro EBV infection of B cells at a level which equaled that of a mouse-human chimeric 72a1 antibody construct. This work provides a further structural and immunological understanding of the 72a1 antibody interaction with EBV gp350, and constitutes a launch point for future anti-EBV therapeutic antibodies designed to block EBV infection and prevent PTLD while eliminating the deleterious antigenic murine features of the original 72a1 antibody.

Genomic-Scale Interaction Involving Complementary Sequences in the Hepatitis C Virus 5'UTR Domain IIa and the RNA-Dependent RNA Polymerase Coding Region Promotes Efficient Virus Replication.

The hepatitis C virus (HCV) genome contains structured elements thought to play important regulatory roles in viral RNA translation and replication processes. We used in vitro RNA binding assays to map interactions involving the HCV 5'UTR and distal sequences in NS5B to examine their impact on viral RNA replication. The data revealed that 5'UTR nucleotides (nt) 95⁻110 in the internal ribosome entry site (IRES) domain IIa and matching nt sequence 8528⁻8543 located in the RNA-dependent RNA polymerase coding region NS5B, form a high-affinity RNA-RNA complex in vitro. This duplex is composed of both wobble and Watson-Crick base-pairings, with the latter shown to be essential to the formation of the high-affinity duplex. HCV genomic RNA constructs containing mutations in domain IIa nt 95⁻110 or within the genomic RNA location comprising nt 8528⁻8543 displayed, on average, 5-fold less intracellular HCV RNA and 6-fold less infectious progeny virus. HCV genomic constructs containing complementary mutations for IRES domain IIa nt 95⁻110 and NS5B nt 8528⁻8543 restored intracellular HCV RNA and progeny virus titers to levels obtained for parental virus RNA. We conclude that this long-range duplex interaction between the IRES domain IIa and NS5B nt 8528⁻8543 is essential for optimal virus replication.

Adaptively introgressed Neandertal haplotype at the OAS locus functionally impacts innate immune responses in humans.

BACKGROUND: The 2'-5' oligoadenylate synthetase (OAS) locus encodes for three OAS enzymes (OAS1-3) involved in innate immune response. This region harbors high amounts of Neandertal ancestry in non-African populations; yet, strong evidence of positive selection in the OAS region is still lacking. RESULTS: Here we used a broad array of selection tests in concert with neutral coalescent simulations to demonstrate a signal of adaptive introgression at the OAS locus. Furthermore, we characterized the functional consequences of the Neandertal haplotype in the transcriptional regulation of OAS genes at baseline and infected conditions. We found that cells from people with the Neandertal-like haplotype express lower levels of OAS3 upon infection, as well as distinct isoforms of OAS1 and OAS2. CONCLUSIONS: We present evidence that a Neandertal haplotype at the OAS locus was subjected to positive selection in the human population. This haplotype is significantly associated with functional consequences at the level of transcriptional regulation of innate immune responses. Notably, we suggest that the Neandertal-introgressed haplotype likely reintroduced an ancestral splice variant of OAS1 encoding a more active protein, suggesting that adaptive introgression occurred as a means to resurrect adaptive variation that had been lost outside Africa.

Peptides designed to spatially depict the Epstein-Barr virus major virion glycoprotein gp350 neutralization epitope elicit antibodies that block virus-neutralizing antibody 72A1 interaction with the native gp350 molecule.

UNLABELLED: Epstein-Barr virus (EBV) is the etiologic agent of infectious mononucleosis and the root cause of B-cell lymphoproliferative disease in individuals with a weakened immune system, as well as a principal cofactor in nasopharyngeal carcinoma, various lymphomas, and other cancers. The EBV major virion surface glycoprotein gp350 is viewed as the best vaccine candidate to prevent infectious mononucleosis in healthy EBV-naive persons and EBV-related cancers in at-risk individuals. Previous epitope mapping of gp350 revealed only one dominant neutralizing epitope, which has been shown to be the target of the monoclonal antibody 72A1. Computer modeling of the 72A1 antibody interaction with the gp350 amino terminus was used to identify gp350 amino acids that could form strong ionic, electrostatic, or hydrogen bonds with the 72A1 antibody. Peptide DDRTTLQLAQNPVYIPETYPYIKWDN (designated peptide 2) and peptide GSAKPGNGSYFASVKTEMLGNEID (designated peptide 3) were designed to spatially represent the gp350 amino acids predicted to interact with the 72A1 antibody paratope. Peptide 2 bound to the 72A1 antibody and blocked 72A1 antibody recognition of the native gp350 molecule. Peptide 2 and peptide 3 were recognized by human IgG and shown to elicit murine antibodies that could target gp350 and block its recognition by the 72A1 antibody. This work provides a structural mapping of the interaction between the EBV-neutralizing antibody 72A1 and the major virion surface protein gp350. gp350 mimetic peptides that spatially depict the EBV-neutralizing epitope would be useful as a vaccine to focus the immune system exclusively to this important virus epitope. IMPORTANCE: The production of virus-neutralizing antibodies targeting the Epstein-Barr virus (EBV) major surface glycoprotein gp350 is important for the prevention of infectious mononucleosis and EBV-related cancers. The data presented here provide the first in silico map of the gp350 interaction with a virus-blocking monoclonal antibody. Immunization with gp350 peptides identified by in silico mapping generated antibodies that cross-react with the EBV gp350 molecule and block recognition of the gp350 molecule by a virus-neutralizing antibody. Through its ability to focus the immune system exclusively on the gp350 sequence important for viral entry, these peptides may form the basis of an EBV vaccine candidate. This strategy would sidestep the production of other irrelevant gp350 antibodies that divert the immune system from generating a protective antiviral response or that impede access to the virus-blocking epitope by protective antibodies.

Reduction in intracellular HCV RNA and virus protein expression in human hepatoma cells following treatment with 2'-O-methyl-modified anti-core deoxyribozyme.

HCV RNA is gaining greater consideration as a principal target for newer HCV antivirals because its destruction has the potential of eliminating the virus. These newer antivirals include deoxyribozymes (Dz), which are small single-stranded DNA molecules that cleave homologous RNA targets. Using a liver cell model containing functional genomic-length HCV-1b RNA we tested whether 2'-O-methyl-modified Dz, designed to recognize a highly-conserved RNA sequence located within the core-E1 coding region, could recognize and cleave its target sequence in the structural context of a functional HCV RNA molecule. Dz858-4-OMe contains four 2'-O-methyl nucleotide derivatives consecutively located on the distal ends of its two annealing arms. Intracellular HCV RNA, core protein and HCV antigen expression were reduced by 63%, 87% and 84%, respectively, when HCV RNA was challenged 6 h post-transfection with Dz858-4-OMe. The observed reduction of intracellular HCV RNA and protein by Dz858-4-OMe suggests that it may constitute an attractive HCV antiviral.

Oligonucleotide-based therapeutic options against hepatitis C virus infection.

The hepatitis C virus (HCV) is the cause of a silent pandemic that, due to the chronic nature of the disease and the absence of curative therapy, continues to claim an ever-increasing number of lives. Current antiviral regimens have proven largely unsatisfactory for patients with HCV drug-resistant genotypes. It is therefore important to explore alternative therapeutic stratagems whose mode of action allows them to bypass viral resistance. Antisense oligonucleotides, ribozymes, small interfering RNAs, aptamers and deoxyribozymes constitute classes of oligonucleotide-based compounds designed to target highly conserved or functionally crucial regions contained within the HCV genome. The therapeutic expectation for such compounds is the elimination of HCV from infected individuals. Progress in oligonucleotide-based HCV antivirals towards clinical application depends on development of nucleotide designs that bolster efficacy while minimizing toxicity, improvement in liver-targeting delivery systems, and refinement of small-animal models for preclinical testing.

Heat shock protein 90 expression in Epstein-Barr virus-infected B cells promotes gammadelta T-cell proliferation in vitro.

The aim of this study was to elucidate the in vitro response of gammadelta T cells to Epstein-Barr virus (EBV)-infected B cells and to determine whether EBV-induced heat shock proteins (HSPs) might serve as gammadelta T-cell stimulants. Cytofluorometric analysis revealed HSP90 cell surface expression in 12% of the EBV-immortalized B-cell population in all four of the B-cell lines tested. HSP27, HSP60, and HSP70 were not detected on the cell surface by cytofluorometry in these same B-cell lines. HSP90 and HSP60, but not HSP70 or HSP27, were detected on the cell surface after 125I cell surface labeling and immunoprecipitation with anti-human HSP monoclonal antibodies. In vitro kinetic studies indicated that gammadelta T cells increased at least twofold by day 11 postinfection in cultures of EBV-seronegative peripheral blood lymphocytes infected with EBV, whereas percentages of alphabeta T cells in these same cultures either decreased slightly or remained relatively unchanged in response to EBV infection. Addition of anti-human HSP90 monoclonal antibody to the EBV-infected lymphocyte cultures inhibited gammadelta T-cell expansion by 92%. The inhibition of gammadelta T-cell expansion by anti-HSP90 antibody was reversed upon treatment with exogenous HSP90. Taken together, these results indicate that HSP90 played an important role in the stimulation of gammadelta T cells during EBV infection of B cells in vitro and may serve as an important immunomodulator of gammadelta T cells during acute EBV infection.

Designing antibodies for oncology.

The past five years have witnessed the emergence of monoclonal antibodies as important therapeutics for cancer treatment. Lower toxicity for antibodies versus small molecules, the potential for increased efficacy by conjugation to radioisotopes and cellular toxins, or the ability to exploit immune cell functions have led to clinical performances on par or superior to conventional drug therapies. This review outlines the various immunoglobulin design strategies currently available, techniques used to reduce Ig antigenicity and toxicity, and points to consider during the manufacture of antibodies for use in clinical oncology.

Identification, purification and characterization of a novel human blood protein with binding affinity for prostate secretory protein of 94 amino acids.

PSP94 (prostate secretory protein of 94 amino acids), an abundant protein within semen, has reported local functions within the reproductive tract and reported systemic functions. Mechanisms of action remain poorly understood, but binding to undefined molecules within the prostate, pituitary, testis and blood may initiate some of these actions. PSP94 serum measurements, especially of bound and free forms, have potential clinical utility in prostate cancer management. Identification of the binding molecules will help in the understanding of PSP94's action, and enable further development of PSP94 serum assays. PSPBP (PSP94-binding protein) was purified from human serum by ammonium sulphate fractionation, ion-exchange and affinity chromatography. The glycosylated protein ran as two bands on SDS/PAGE (70 and 95 kDa). N-terminal sequencing yielded a 30-amino-acid sequence, identical with the translated N-terminal region of a previously published cDNA (GenBank accession number AX136261). Reverse transcriptase PCR and plaque hybridization demonstrated PSPBP mRNA in peripheral blood leucocytes and in a prostate cDNA library. Northern blotting showed 2 kb mRNA species in prostate, testis, ovary and intestine. Immunohistochemistry demonstrated PSPBP in tissues, including pituitary and Leydig cells, supporting a role for PSP94 in hormonal control at the pituitary gonadal axis. ELISA demonstrated that PSPBP levels were significantly lower (P=0.0014) in the serum of a prostate cancer population (n=65) compared with a control population (n=70). PSPBP identification will help the understanding of PSP94's functions and facilitate ELISA development to address the clinical value of PSP94 serum assays.

Nucleosomes activate NF-kappaB in endothelial cells for induction of the proangiogenic cytokine IL-8.

Solid tumors often exhibit regions undergoing apoptosis and necrosis, also referred to as "aponecrosis", juxtaposed to sites of active angiogenesis. We explored whether nucleosomes resulting from aponecrosis induced the angiogenic factor IL-8 in vascular endothelial cells. Results indicate that nucleosomes induced IL-8. Nucleosomes increased IL-6 and IL-8 mRNA but not IL-10, TNF-alpha, VEGF or FGF-2 mRNA. Induction of IL-8 by nucleosomes in endothelial cells appeared to be the result of NF-kappaB/RelA transcription factor activation. The increased expression of IL-8 in vascular endothelial cells following nucleosome stimulation suggests that aponecrosis could play an important role in the promotion of tumor angiogenesis.

Nucleosomes bind fibroblast growth factor-2 for increased angiogenesis in vitro and in vivo.

Solid tumors often display sites of necrosis near regions of angiogenesis in vivo. As tumor cell necrosis would result in the release of nucleosomes into the extracellular environment, we explored the potential role of nucleosomes in the promotion of angiogenesis. Data indicate that nucleosomes acted similar to heparin and bound to several heparin-binding, proangiogenic factors [i.e., fibroblast growth factor (FGF)-1, FGF-2, vascular endothelial growth factor, and transforming growth factor-beta1]. Nucleosomes modestly enhanced FGF-2 growth of human umbilical vein endothelial cells when grown in restricted media as well as increased human umbilical vein endothelial cell migration and primitive blood vessel tube formation in vitro. On s.c. injection in mice, nucleosomes aided FGF-2 in promoting angiogenesis. These results suggest that nucleosomes released from dying tumor cells aid in the formation of blood vessels and may provide a novel means by which tumor cells increase angiogenesis.