Serum and Cervicovaginal Fluid Antibody Profiling in Herpes Simplex Virus-Seronegative Recipients of the HSV529 Vaccine.

Previous herpes simplex virus type 2 (HSV-2) vaccines have not prevented genital herpes. Concerns have been raised about the choice of antigen, the type of antibody induced by the vaccine, and whether antibody is present in the genital tract where infection occurs. We reported results of a trial of an HSV-2 replication-defective vaccine, HSV529, that induced serum neutralizing antibody responses in 78% of HSV-1-/HSV-2- vaccine recipients. Here we show that HSV-1-/HSV-2- vaccine recipients developed antibodies to epitopes of several viral proteins; however, fewer antibody epitopes were detected in vaccine recipients compared with naturally infected persons. HSV529 induced antibodies that mediated HSV-2-specific natural killer (NK) cell activation. Depletion of glycoprotein D (gD)-binding antibody from sera reduced neutralizing titers by 62% and NK cell activation by 81%. HSV-2 gD antibody was detected in cervicovaginal fluid at about one-third the level of that in serum. A vaccine that induces potent serum antibodies transported to the genital tract might reduce HSV genital infection.

Serum Epitope Repertoire Analysis Enables Early Detection of Lyme Disease with Improved Sensitivity in an Expandable Multiplex Format.

Widely employed diagnostic antibody serology for Lyme disease, known as standard two-tier testing (STTT), exhibits insufficient sensitivity in early Lyme disease, yielding many thousands of false-negative test results each year. Given this problem, we applied serum antibody repertoire analysis (SERA), or next-generation sequencing (NGS)-based serology, to discover IgG and IgM antibody epitope motifs capable of detecting Lyme disease-specific antibodies with high sensitivity and specificity. Iterative motif discovery and bioinformatic analysis of epitope repertoires from subjects with Lyme disease (n = 264) and controls (n = 391) yielded a set of 28 epitope motifs representing 20 distinct IgG antibody epitopes and a set of 38 epitope motifs representing 21 distinct IgM epitopes, which performed equivalently in a large validation cohort of STTT-positive samples. In a second validation set from subjects with clinically defined early Lyme disease (n = 119) and controls (n = 257), the SERA Lyme IgG and IgM assay exhibited significantly improved sensitivity relative to STTT (77% versus 62%; Z-test; P = 0.013) and improved specificity (99% versus 97%). Early Lyme disease subjects exhibited significantly fewer reactive epitopes (Mann-Whitney U test; P < 0.0001) relative to subjects with Lyme arthritis. Thus, SERA Lyme IgG and M panels provided increased accuracy in early Lyme disease in a readily expandable multiplex assay format.

Amyloid ß peptide cleavage by kallikrein 7 attenuates fibril growth and rescues neurons from Aß-mediated toxicity in vitro.

The gradual accumulation and assembly of ß-amyloid (Aß) peptide into neuritic plaques is a major pathological hallmark of Alzheimer disease (AD). Proteolytic degradation of Aß is an important clearance mechanism under normal circumstances, and it has been found to be compromised in those with AD. Here, the extended substrate specificity and Aß-degrading capacity of kallikrein 7 (KLK7), a serine protease with a unique chymotrypsin-like specificity, was characterized. Preferred peptide substrates of KLK7 identified using a bacterial display substrate library were found to exhibit a consensus motif of RXΦ(Y/F)↓(Y/F)↓(S/A/G/T) or RXΦ(Y/F)↓(S/T/A) (Φ=hydrophobic), which is remarkably similar to the hydrophobic core motif of Aß (K16L17V18F19F20 A21) that is largely responsible for aggregation propensity. KLK7 was found to cleave after both Phe residues within the core of Aß42 in vitro, thereby inhibiting Aß fibril formation and promoting the degradation of preformed fibrils. Finally, the treatment of Aß oligomer preparations with KLK7, but not inactive pro-KLK7, significantly reduced Aß42-mediated toxicity to rat hippocampal neurons to the same extent as the known Aß-degrading protease insulin-degrading enzyme (IDE). Taken together, these results indicate that KLK7 possesses an Aß-degrading capacity that can ameliorate the toxic effects of the aggregated peptide in vitro.

Profiling antibody epitopes induced by mRNA-1273 vaccination and boosters.

Background: Characterizing the antibody epitope profiles of messenger RNA (mRNA)-based vaccines against SARS-CoV-2 can aid in elucidating the mechanisms underlying the antibody-mediated immune responses elicited by these vaccines. Methods: This study investigated the distinct antibody epitopes toward the SARS-CoV-2 spike (S) protein targeted after a two-dose primary series of mRNA-1273 followed by a booster dose of mRNA-1273 or a variant-updated vaccine among serum samples from clinical trial adult participants. Results: Multiple S-specific epitopes were targeted after primary vaccination; while signal decreased over time, a booster dose after >6 months largely revived waning antibody signals. Epitope identity also changed after booster vaccination in some subjects, with four new S-specific epitopes detected with stronger signals after boosting than with primary vaccination. Notably, the strength of antibody responses after booster vaccination differed by the exact vaccine formulation, with variant-updated mRNA-1273.211 and mRNA-1273.617.2 booster formulations inducing significantly stronger S-specific signals than a mRNA-1273 booster. Conclusion: Overall, these results identify key S-specific epitopes targeted by antibodies induced by mRNA-1273 primary and variant-updated booster vaccination.

Amyloid beta-mediated cell death of cultured hippocampal neurons reveals extensive Tau fragmentation without increased full-length tau phosphorylation.

A variety of genetic and biochemical evidence suggests that amyloid ß (Aß) oligomers promote downstream errors in Tau action, in turn inducing neuronal dysfunction and cell death in Alzheimer and related dementias. To better understand molecular mechanisms involved in Aß-mediated neuronal cell death, we have treated primary rat hippocampal cultures with Aß oligomers and examined the resulting cellular changes occurring before and during the induction of cell death with a focus on altered Tau biochemistry. The most rapid neuronal responses upon Aß administration are activation of caspase 3/7 and calpain proteases. Aß also appears to reduce Akt and Erk1/2 kinase activities while increasing GSK3ß and Cdk5 activities. Shortly thereafter, substantial Tau degradation begins, generating relatively stable Tau fragments. Only a very small fraction of full-length Tau remains intact after 4 h of Aß treatment. In conflict with expectations based on suggested increases of GSK3ß and Cdk5 activities, Aß does not cause any major increases in phosphorylation of full-length Tau as assayed by immunoblotting one-dimensional gels with 11 independent site- and phospho-specific anti-Tau antibodies as well as by immunoblotting two-dimensional gels probed with a pan-Tau antibody. There are, however, subtle and transient increases in Tau phosphorylation at 3-4 specific sites before its degradation. Taken together, these data are consistent with the notion that Aß-mediated neuronal cell death involves the loss of full-length Tau and/or the generation of toxic fragments but does not involve or require hyperphosphorylation of full-length Tau.

High-resolution epitope mapping and characterization of SARS-CoV-2 antibodies in large cohorts of subjects with COVID-19.

As Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to spread, characterization of its antibody epitopes, emerging strains, related coronaviruses, and even the human proteome in naturally infected patients can guide the development of effective vaccines and therapies. Since traditional epitope identification tools are dependent upon pre-defined peptide sequences, they are not readily adaptable to diverse viral proteomes. The Serum Epitope Repertoire Analysis (SERA) platform leverages a high diversity random bacterial display library to identify proteome-independent epitope binding specificities which are then analyzed in the context of organisms of interest. When evaluating immune response in the context of SARS-CoV-2, we identify dominant epitope regions and motifs which demonstrate potential to classify mild from severe disease and relate to neutralization activity. We highlight SARS-CoV-2 epitopes that are cross-reactive with other coronaviruses and demonstrate decreased epitope signal for mutant SARS-CoV-2 strains. Collectively, the evolution of SARS-CoV-2 mutants towards reduced antibody response highlight the importance of data-driven development of the vaccines and therapies to treat COVID-19.

Antibody epitope repertoire analysis enables rapid antigen discovery and multiplex serology.

The detection of pathogen-specific antibodies remains a cornerstone of clinical diagnostics. Yet, many test exhibit undesirable performance or are completely lacking. Given this, we developed serum epitope repertoire analysis (SERA), a method to rapidly discover conserved, pathogen-specific antigens and their epitopes, and applied it to develop an assay for Chagas disease caused by the protozoan parasite Trypanosoma cruzi. Antibody binding peptide motifs were identified from 28 Chagas repertoires using a bacterial display random 12-mer peptide library and next-generation sequencing (NGS). Thirty-three motifs were selected and mapped to candidate Chagas antigens. In a blinded validation set (n = 72), 30/30 Chagas were positive, 30/30 non-Chagas were negative, and 1/12 Leishmania sp. was positive. After unblinding, a Leishmania cross-reactive epitope was identified and removed from the panel. The Chagas assay exhibited 100% sensitivity (30/30) and specificity (90/90) in a second blinded validation set including individuals with other parasitic infections. Amongst additional epitope repertoires with unknown Chagas serostatus, assay specificity was 99.8% (998/1000). Thus, the Chagas assay achieved a combined sensitivity and specificity equivalent or superior to diagnostic algorithms that rely on three separate tests to achieve high specificity. NGS-based serology via SERA provides an effective approach to discover antigenic epitopes and develop high performance multiplex serological assays.

Bendamustine (Treanda) displays a distinct pattern of cytotoxicity and unique mechanistic features compared with other alkylating agents.

PURPOSE: Bendamustine has shown clinical activity in patients with disease refractory to conventional alkylator chemotherapy. The purpose of this study was to characterize the mechanisms of action of bendamustine and to compare it with structurally related compounds. EXPERIMENTAL DESIGN: Bendamustine was profiled in the National Cancer Institute in vitro antitumor screen. Microarray-based gene expression profiling, real-time PCR, immunoblot, cell cycle, and functional DNA damage repair analyses were used to characterize response to bendamustine and compare it with chlorambucil and phosphoramide mustard. RESULTS: Bendamustine displays a distinct pattern of activity unrelated to other DNA-alkylating agents. Its mechanisms of action include activation of DNA-damage stress response and apoptosis, inhibition of mitotic checkpoints, and induction of mitotic catastrophe. In addition, unlike other alkylators, bendamustine activates a base excision DNA repair pathway rather than an alkyltransferase DNA repair mechanism. CONCLUSION: These results suggest that bendamustine possesses mechanistic features that differentiate it from other alkylating agents and may contribute to its distinct clinical efficacy profile.

Antibody Biomarker Discovery Via In Vitro Evolution of Peptides.

Antigen discovery and mapping strategies that enable the precise identification of serum antibody-binding epitopes in human diseases will be invaluable for translational diagnostics and therapeutic development. Protein and peptide library display screening techniques have shown utility for the discovery of antigens associated with disease onset and progression. Here, we describe a screening methodology using bacterial peptide library display to identify consensus families of disease-specific binding motifs to multiple pools of human serum antibodies. The sensitivity and specificity of identified disease-specific peptide motifs are then optimized using in vitro evolution techniques.

Identification of disease-specific motifs in the antibody specificity repertoire via next-generation sequencing.

Disease-specific antibodies can serve as highly effective biomarkers but have been identified for only a relatively small number of autoimmune diseases. A method was developed to identify disease-specific binding motifs through integration of bacterial display peptide library screening, next-generation sequencing (NGS) and computational analysis. Antibody specificity repertoires were determined by identifying bound peptide library members for each specimen using cell sorting and performing NGS. A computational algorithm, termed Identifying Motifs Using Next- generation sequencing Experiments (IMUNE), was developed and applied to discover disease- and healthy control-specific motifs. IMUNE performs comprehensive pattern searches, identifies patterns statistically enriched in the disease or control groups and clusters the patterns to generate motifs. Using celiac disease sera as a discovery set, IMUNE identified a consensus motif (QPEQPF[PS]E) with high diagnostic sensitivity and specificity in a validation sera set, in addition to novel motifs. Peptide display and sequencing (Display-Seq) coupled with IMUNE analysis may thus be useful to characterize antibody repertoires and identify disease-specific antibody epitopes and biomarkers.

Effects of Paclitaxel and Eribulin in Mouse Sciatic Nerve: A Microtubule-Based Rationale for the Differential Induction of Chemotherapy-Induced Peripheral Neuropathy.

Microtubule targeting agents (MTAs) often lead to treatment limiting and life threatening side effects, including chemotherapy-induced peripheral neuropathy (CIPN). The frequency of severe CIPN varies among different MTAs. Since the microtubule binding interactions and mechanisms of action also vary among MTAs, we hypothesized that these distinct mechanisms may underlie the variability in frequency of severe CIPN. Using a two-week, maximum tolerated dose model, we morphologically and biochemically analyzed sciatic nerves from mice treated with either paclitaxel or eribulin. These drugs differ in their manner of microtubule binding and mechanisms of action and reports indicate paclitaxel also induces a higher frequency of severe CIPN than does eribulin. Morphologically, paclitaxel increased the frequency of observed signs of axon degeneration more significantly than did eribulin. Alternatively, eribulin but not paclitaxel induced occasional myelin "halo" structures. Biochemically, paclitaxel, and eribulin both induced α-tubulin expression (~1.9- and ~2.5-fold, respectively) and tubulin acetylation, a marker for microtubule stability, (~5- and ~11.7-fold, respectively). Eribulin but not paclitaxel-induced EB1 expression ~2.2-fold while paclitaxel but not eribulin mildly suppressed EB3 expression. Both EB proteins are associated with microtubule growth. Eribulin's combination of relatively mild deleterious morphological effects coupled with more potent biochemical changes promoting microtubule stability and growth in mice correlate with lower frequencies of severe CIPN in humans. We suggest that these eribulin-induced effects create a relatively stable microtubule network that compensates, in part, for the toxic anti-cancer effects of the drug, leading to fewer reported incidences of CIPN than for paclitaxel.

Halogenated DOPA in a Marine Adhesive Protein.

The sandcastle worm Phragmatopoma californica, a marine polychaete, constructs a tube-like shelter by cementing together sand grains using a glue secreted from the building organ in its thorax. The glue is a mixture of post-translationally modified proteins, notably the cement proteins Pc-1 and Pc-2 with the amino acid, 3,4-dihydroxyphenyl-L-alanine (DOPA). Significant amounts of a halogenated derivative of DOPA were isolated from the worm cement following partial acid hydrolysis and capture of catecholic amino acids by phenylboronate affinity chromatography. Analysis by tandem mass spectrometry and (1)H NMR indicates the DOPA derivative to be 2-chloro-4, 5-dihydroxyphenyl-L-alanine. The potential roles of 2-chloro-DOPA in chemical defense and underwater adhesion are considered.

Regulation of fibronectin and metalloproteinase expression by Wnt signaling in rheumatoid arthritis synoviocytes.

OBJECTIVE: The enhanced release of extracellular matrix proteins by fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients is suggestive of joint remodeling. Because Wnt proteins play a critical role in joint development, we investigated whether up-regulated Wnt signaling plays a role in the enhanced synthesis of extracellular matrix proteins. The purpose of the present experiments was to determine the role of Wnt-1-like molecules in the expression of matrix proteins by RA FLS and to ascertain the effects of Wnt antagonists on RA FLS function and survival. METHODS: Transfection with a reporter plasmid (TOPflash) was performed to assess whether Wnt signaling is active in RA FLS. Wnt signaling was up-regulated in normal FLS by transfection with a Wnt-1 expression plasmid and was down-regulated in RA FLS by transfection with dominant-negative lymphoid enhancer factor 1 (LEF-1)/T cell factor 4 (TCF-4) and secreted Frizzled receptor protein 1 (sFRP-1) expression plasmids. Recombinant sFRP-1 and anti-Wnt-1 antibody were also administered to RA FLS to block Wnt signaling. RESULTS: RA FLS had constitutive activation of the canonical Wnt signaling pathway. Transfection of normal FLS with a Wnt-1 expression vector enhanced not only fibronectin, but also pro-matrix metalloproteinase 3 (proMMP-3) expression. In a complementary manner, interference with Wnt signaling using anti-Wnt-1 antibody, the Wnt antagonist sFRP-1, or dominant-negative vectors that inhibited the transcription factors TCF-4/LEF-1 blocked the expression of fibronectin by RA FLS and reduced cell survival. Anti-Wnt-1 antibody and sFRP-1 also blocked the expression of proMMP-3. CONCLUSION: Our results indicate that the canonical Wnt pathway regulates fibronectin and metalloproteinase expression in RA FLS.