The aberrant expression of α defensin 5 (DEFA5) protein in colonic inflammatory bowel diseases (IBDs) underlies the distinct pathogenesis of Crohn’s colitis (CC). It can serve as a biomarker for differentiating CC from Ulcerative colitis (UC), particularly in Indeterminate colitis (IC) cases into UC and CC. We evaluated the specificity of commercially available anti-DEFA5 antibodies, emphasizing the need to further validate their appropriateness for a given application and highlighting the necessity for novel antibodies. We established two mice monoclonal DEFA5 antibody clones, 1A8 and 4F5, by immunizing mice with purified recombinant protein. We validated the specificity, sensitivity, and cross-reactivity of these antibodies in recognizing both endogenous and recombinant DEFA5 protein, especially for use in Immunohistochemistry (IHC), Western blot (WB), Immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA). Clones 1A8 and 4F5 effectively recognized the endogenous DEFA5 in active human colon tissue from patients with diverticulitis (DV), UC, CC, and IC disease samples, as well as in transiently transfected HEK293T cells expressing DEFA5 with minimal non-confounding cross reactivity. The 1A8 and 4F5 clones are useful for a wide variety of immunoassays, including WB, IHC, IP/WB, and ELISA. Their specificity enhances their potential as valuable tools for research applications in IBD colitis.

Background Robust evidence suggests that the aberrant expression of α defensin 5 protein (DEFA5) in colon inflammatory bowel diseases (IBDs) underlies the distinct pathogenesis of Crohn’s colitis, can be exploited as a reliable diagnostic biomarker to differential diagnosis of Crohn’s colitis (CC) from Ulcerative colitis (UC) in otherwise indeterminate colitis (IC). We evaluated the specificity of the commercially available anti-DEFA5 antibodies and showed further validation of their appropriateness for a given application is required. Methods We established two mouse monoclonal DEFA5 antibody clones 1A8 and 4F5 by immunizing the mice with purified recombinant protein and validated the specificity, selectivity and cross reactivity in recognizing the endogenous and recombinant DEFA5 protein, especially for Immunohistochemistry, Western blot, Immunoprecipitation, or enzyme-linked immunosorbent assay. Results Clones 1A8 and 4F5 recognized effectively the endogenous DEFA5 in active human diverticulitis (DV), UC, CC or IC disease samples, including transiently transfected HEK293T cells expressing DEFA5 with high degree of specificity and minimal non-confounding cross reactivity. Conclusions 1A8 and 4F5 clones are worth studying in larger IBD cohorts to fully address whether DEFA5 expression may be used as a diagnostic biomarker to discrimination of the diagnosis of UC from CC or IC into authentic CC or UC or a colitis with different pathological characteristics.

The recent development and mass administration of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines allowed for disease control, reducing hospitalizations and mortality. Most of these vaccines target the SARS-CoV-2 Spike (S) protein antigens, culminating with the production of neutralizing antibodies (NAbs) that disrupt the attachment of the virus to ACE2 receptors on the host cells. However, several studies demonstrated that the NAbs typically rise within a few weeks after vaccination but quickly reduce months later. Thus, multiple booster administration is recommended, leading to vaccination hesitancy in many populations. Detecting serum anti-SARS-CoV-2 NAbs can instruct patients and healthcare providers on correct booster strategies. Several in vitro diagnostics kits are available; however, their high cost impairs the mass NAbs diagnostic testing. Recently, we engineered an ACE2 mimetic that interacts with the Receptor Binding Domain (RBD) of the SARS-2 S protein. Here we present the use of this engineered mini-protein (p-deface2 mut) to develop a detection assay to measure NAbs in patient sera using a competitive ELISA assay. Serum samples from twenty-one patients were tested. Nine samples (42.8%) tested positive, and twelve (57.1%) tested negative for neutralizing sera. The data correlated with the result from the standard commercial assay that uses human ACE2 protein. This confirmed that p-deface2 mut could replace human ACE2 in ELISA assays. Using bacterially expressed p-deface2 mut protein is cost-effective and may allow mass SARS-CoV-2 NAbs detection, especially in low-income countries where economical diagnostic testing is crucial. Such information will help providers decide when a booster is required, reducing risks of reinfection and preventing the administration before it is medically necessary.

Biomedical core facilities are key resources supporting the academic research enterprise. As providers of analytical services and expertise with a commitment to rigor, reproducibility and data integrity, core facilities should be at the forefront of quality management and process improvement. Given the constantly changing research environment, an important competitive differentiator is the implementation of an operational quality management system (QMS). Additionally, cores involved in biomedical manufacturing may be subject to federal regulations, thus requiring a QMS. Linked to quality management is the adoption of quality assurance best practices to avoid unintended variation in laboratory results. This session will provide practical guidance and informative case studies for establishment of best practices and quality systems in core labs. Presentations will focus on (1) implementation of a quality management system in a genomics core facility; (2) a stepwise approach to building a quality system in a regulated academic manufacturing facility; and (3) establishment of risk-based quality assurance best practices as a strategy to reduce pre-analytical errors associated with sample quality and handling.

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Blum et al., 2015), that described how we intended to replicate selected experiments from the paper ‘Transcriptional amplification in tumor cells with elevated c-Myc’ (Lin et al., 2012). Here we report the results. We found overexpression of c-Myc increased total levels of RNA in P493-6 Burkitt’s lymphoma cells; however, while the effect was in the same direction as the original study (Figure 3E; Lin et al., 2012), statistical significance and the size of the effect varied between the original study and the two different lots of serum tested in this replication. Digital gene expression analysis for a set of genes was also performed on P493-6 cells before and after c-Myc overexpression. Transcripts from genes that were active before c-Myc induction increased in expression following c-Myc overexpression, similar to the original study (Figure 3F; Lin et al., 2012). Transcripts from genes that were silent before c-Myc induction also increased in expression following c-Myc overexpression, while the original study concluded elevated c-Myc had no effect on silent genes (Figure 3F; Lin et al., 2012). Treating the data as paired, we found a statistically significant increase in gene expression for both active and silent genes upon c-Myc induction, with the change in gene expression greater for active genes compared to silent genes. Finally, we report meta-analyses for each result.

Monkeypox (MPXV) and cowpox (CPXV) are emerging agents that cause severe human infections on an intermittent basis, and variola virus (VARV) has potential for use as an agent of bioterror. Vaccinia immune globulin (VIG) has been used therapeutically to treat severe orthopoxvirus infections but is in short supply. We generated a large panel of orthopoxvirus-specific human monoclonal antibodies (Abs) from immune subjects to investigate the molecular basis of broadly neutralizing antibody responses for diverse orthopoxviruses. Detailed analysis revealed the principal neutralizing antibody specificities that are cross-reactive for VACV, CPXV, MPXV, and VARV and that are determinants of protection in murine challenge models. Optimal protection following respiratory or systemic infection required a mixture of Abs that targeted several membrane proteins, including proteins on enveloped and mature virion forms of virus. This work reveals orthopoxvirus targets for human Abs that mediate cross-protective immunity and identifies new candidate Ab therapeutic mixtures to replace VIG.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of 50 papers in the field of cancer biology published between 2010 and 2012. This Registered report describes the proposed replication plan of key experiments from 'Transcriptional amplification in tumor cells with elevated c-Myc' by Lin et al. (2012), published in Cell in 2012. The experiments that will be replicated are those reported in Figures 3E and 3F. In these experiments, elevated levels of c-Myc in the P493-6 cell model of Burkitt's lymphoma results in an increase of the total level of RNA using UV/VIS spectrophotometry (Figure 3E; Lin et al., 2012) and on the mRNA levels/cell for a large set of genes using digital gene expression technology (Figure 3F; Lin et al., 2012). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published in eLife.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of 50 papers in the field of cancer biology published between 2010 and 2012. This Registered Report describes the proposed replication plan of key experiments from "Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion" by Straussman and colleagues, published in Nature in 2012 (Straussman et al., 2012). The key experiments being replicated in this study are from Figure 2A, C, and D (and Supplemental Figure 11) and Figure 4C (and Supplemental Figure 19) (Straussman et al., 2012). Figure 2 demonstrates resistance to drug sensitivity conferred by co-culture with some stromal cell lines and identifies the secreted factor responsible as HGF. In Figure 4, Straussman and colleagues show that blocking the HGF receptor MET abrogates HGF’s rescue of drug sensitivity. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.

Abstract Immunization with vaccinia virus (VACV) confers cross-protective immunity to variola, the causative agent of smallpox, and other clinically important poxviral species, such as cowpox and monkeypox viruses. Vaccine-mediated protection has been correlated with elicitation of broad neutralizing antibody (Ab) responses, however the specificity of human neutralizing antibodies to poxviruses is poorly understood. We used a highly-optimized human hybridoma technology to generate large panels of anti-VACV monoclonal Abs (mAbs) from vaccinia-immunized subjects or from subjects who had naturally acquired monkeypox infection. Immunologic characterization and DNA sequencing of the panel revealed 16 individual mAbs that recognized the immunodominant surface protein antigen D8. Several of anti-D8 mAbs possessed neutralizing activity against VACV, cowpox and monkeypox viruses. These Abs exhibited mainly a complement-dependent pattern of neutralization and they formed at least four cross-blocking groups, suggesting the existence of several non-overlapping neutralizing epitopes for D8. The results suggest that D8 is an important target for human neutralizing Abs generated following poxviral infection or vaccination, and suggest the existence of novel epitopes targeted by anti-D8 Abs that contribute to cross-protective immunity following smallpox vaccination.

To obtain an overview of how scientists develop new antibodies and find existing ones, the Antibody and Technology Research Group (ARG) used a web-based survey to query academic antibody core facilities/resource centers and researchers. A total of four surveys were conducted using Survey Monkey. The first sought to learn the immunization strategies employed by our peer when they are trying to make antibodies against a weakly immunogenic target covering variable such as host, adjuvant and immunogen form. The second curated screening approaches the cores employ to find hybridomas secreting the desired antibodies, antibodies that bind the target in whatever their final application may be (e.g. flow cytometry, immunoflourescence and in vivo). The third survey focused on the molecular biology side of the field, specifically antibody sequencing and recombinant antibody production. As a follow up to a round table discussion entitled “The Perfect Antibody”, the fourth was conducted to understand how researchers antibodies that meet the needs of their research and if they utilize websites focused on end-user reviews and publication citations.

Respiratory syncytial virus (RSV) is a major cause of pneumonia and wheezing in infants and the elderly, but to date there is no licensed vaccine. We developed a gold nanorod construct that displayed the major protective antigen of the virus, the fusion protein (F). Nanorods conjugated to RSV F were formulated as a candidate vaccine preparation by covalent attachment of viral protein using a layer-by-layer approach. In vitro studies using ELISA, electron microscopy and circular dichroism revealed that conformation-dependent epitopes were maintained during conjugation, and transmission electron microscopy studies showed that a dispersed population of particles could be achieved. Human dendritic cells treated with the vaccine induced immune responses in primary human T cells. These results suggest that this vaccine approach may be a potent method for immunizing against viruses such as RSV with surface glycoproteins that are targets for the human immune response.

ABSTRACT The extracellular virion form (EV) of vaccinia virus (VACV) is essential for viral pathogenesis and is difficult to neutralize with antibodies. Why this is the case and how the smallpox vaccine overcomes this challenge remain incompletely understood. We previously showed that high concentrations of anti-B5 antibodies are insufficient to directly neutralize EV (M. R. Benhnia, et al., J. Virol. 83:1201–1215, 2009). This allowed for at least two possible interpretations: covering the EV surface is insufficient for neutralization, or there are insufficient copies of B5 to allow anti-B5 IgG to cover the whole surface of EV and another viral receptor protein remains active. We endeavored to test these possibilities, focusing on the antibody responses elicited by immunization against smallpox. We tested whether human monoclonal antibodies (MAbs) against the three major EV antigens, B5, A33, and A56, could individually or together neutralize EV. While anti-B5 or anti-A33 (but not anti-A56) MAbs of appropriate isotypes were capable of neutralizing EV in the presence of complement, a mixture of anti-B5, anti-A33, and anti-A56 MAbs was incapable of directly neutralizing EV, even at high concentrations. This remained true when neutralizing the IHD-J strain, which lacks a functional version of the fourth and final known EV surface protein, A34. These immunological data are consistent with the possibility that viral proteins may not be the active component of the EV surface for target cell binding and infectivity. We conclude that the protection afforded by the smallpox vaccine anti-EV response is predominantly mediated not by direct neutralization but by isotype-dependent effector functions, such as complement recruitment for antibodies targeting B5 and A33.