bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential contribution of variable heavy and variable light chain domains in viral epitope recognition and neutralization function (VAC6P.950)
Authors: Pasman Yfke , Nagy Eva , Kaushik Azad 
Published: January 2023

Abstract
Antigen (Ag) may be recognized by an immunoglobulin via heavy chain variable domain (VH) alone, light chain variable domain (VL) alone and VH+VL pair. Little is known about relative contribution of VH and/or VL in Ag recognition in the context of virus neutralization function. To understand role of VH and VL, we examined Ag recognition and virus neutralization function of VH, VL and VH+VL of a neutralizing Ab against bovine herpesvirus-1 (BoHV-1), an important cattle pathogen. Monomeric scFv, VH and VL were expressed in Pichia pastoris and purified. The VH alone recognized BoHV-1 in ELISA but VL did not. This is consistent with previous studies that suggested significant role of variable heavy domain in Ag recognition. However, VH alone did not neutralize BoHV-1 in vitro but monomeric scFv, where VH and VL are linked via 18 amino acids, neutralized the virus. Such a divergence in VH and VL effector functions from Ag recognition to virus neutralization reflects fine structural complexities relevant to Ab functions. The VH alone though capable of recognizing the target neutralizing epitope is unable to neutralize BoHV-1 per se. These observations provide novel insight into non-Ag binding functional role of VL where it provides structural and configurational support to VH for accessing the neutralizing viral epitope. To conclude, such subtle differences in VH and VL contributions to Ab effector functions deserve consideration while designing Ab-based anti-viral therapeutics.
VaxCelerate: the use of MTBhsp70-avidin as an adjuvant to rapidly generate self-assembling vaccines with biotinylated, antigen-specific peptides targeting emerging pathogens. (VAC6P.941)
Authors: Fishman Jordan , Moise Leonard , LeBlanc Pierre , Brauns Timothy , Berg Eric , Richer Daniel , Boyle Christine , De Groot Anne , Martin Bill , Baker David , Zeigler Brandon , Mais Dale , Taylor William , Coleman Russell , Shaw Warren , Gelfand Jeffrey , Poznansky Mark 
Published: January 2023

Abstract
Development of effective vaccines against emerging infectious diseases can take years to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. The VaxCelerate Project’s goal is to create a platform capable of generating and pre-clinically testing a new vaccine against specific pathogen targets in less than 120 days. A self-assembling vaccine, consisting of a fusion protein M. tuberculosis MTBhsp70 and avidin (MAV), is at the core of the approach. Mixing the MAV with biotinylated pathogen specific immunogenic peptides yields a self-assembled vaccine (SAV). To minimize the time required, we used a distributed R&D model involving experts in protein engineering, bioinformatics, peptide synthesis/design and GMP/GLP manufacturing and testing. This approach was first tested using ovalbumin in C57Bl/6 mice, Flu (H1N1) specific peptides, and ultimately a Lassa fever virus (LFV) specific vaccine in transgenic HLA DR3 mice. Using a GLP validated assay we demonstrated that the MAV assembled LFV induced significantly increased class II peptide specific interferon-CD4+ T cell responses in transgenic mice compared to peptide or MAV alone controls. The use of an identical design for each vaccine may facilitate accelerated regulatory review and by developing safety assessment tools that are more relevant to human vaccine responses than current preclinical models.
Expression of microRNAs in response to a TLR dependent adjuvant PorB from Neisseria meningitidis (VAC6P.943)
Authors: Mosaheb Munir , Platt Andrew , Reiser Michael , Shaik-Dasthagirisaheb Yazdani , Toussi Deana , Liu Xiuping , Wetzler Lee 
Published: January 2023

Abstract
MicroRNAs (miRs) are small non-coding RNAs that are involved in post-translational modulation of gene expression. Not much is known about the role of miRs in vaccine adjuvant activity. In this study, we investigated miRs elicited by PorB, a TLR2 dependent adjuvant from N. meningitidis, in vivo and in vitro. Mice (WT) were immunized 3 times, 2 weeks apart with Ova +/- PorB. Splenic RNA was obtained 24 hours after each immunization and analyzed by microarrays. Bone marrow-derived macrophages (BMDM) from WT, MyD88 or TLR2 knock out mice were stimulated with PorB, RNA was isolated and miRs expression was determined by RT-PCR. The in vivo study revealed that miR505 and miR15a expression was increased, while miR689 and miR666 expression was decreased when PorB was included in the vaccine preparation. Interestingly the differential expression of these miRs were seen after the 2nd but not after the 1st or 3rd immunization, suggesting that the regulatory events driven by PorB occurred after the 2nd immunization. We also observed a high increase in the expression of miR155 upon stimulation with PorB in vitro, however the miR155 expression was dampened in BMDM from TLR2 knock out mice and completely abrogated in MyD88 knock out mice. PorB seems to be able to induce high level of miR155 in a TLR2 independent as well as TLR2 dependent mechanism. These data increase our understanding of the association of miR expression and PorB adjuvant activity.
A systems biology approach demonstrates that PO and ID BCG induce distinct CD4+ T cell molecular signatures. (VAC7P.970)
Authors: Xia Mei , Zhang Guang lan , Blazevic Azra , Brusic Vladimir , Hoft Daniel 
Published: January 2023

Abstract
Vaccination with Bacillus Calmette-Guérin (BCG) is and constitutes the most widely used tuberculosis (TB) control strategy in the world. Protective efficacy of BCG may be affected by the methods and routes of vaccine administration. Here we have used a systems biology approach to study innate and adaptive responses to oral (PO) and intradermal (ID) BCG vaccination in humans. 60 healthy adults vaccinated with PO and/or ID BCG was studied. Although both PO and ID BCG could induce systemic Th1 responses capable of IFN-γ production, ID BCG more strongly induced systemic Th1 responses, while significantly higher mucosal responses (TB-specific sIgA and bronchoalveolar lavage T cells) were induced by PO BCG vaccination. In order to identify early gene signatures that predict optimal mucosal and systemic immune responses, CD4+ memory T cells were isolated from both PO and ID BCG vaccinated volunteers pre- (Day 0) and post-vaccination (Day 7 and 56), and studied by Illumina BeadArray transcriptomal analysis. Notably, distinct gene expression profiles were identified both on day 7 and day 56 comparing the PO and ID BCG vaccinated groups by GSEA analysis. Correlations between specific expression patterns and mucosal vs. systemic immune responses are being studied. These data highlight the utility of systems biology approaches in studying differential mucosal and systemic immune responses.
Combined vaccination of subcutaneous BCG and intranasal HBHA with cholera toxin enhances early protective immunity against pulmonary M. tuberculosis infection (VAC7P.972)
Authors: Fukui Masayuki , Umemura Masayuki , Matsuzaki Goro 
Published: January 2023

Abstract
Effect of Mycobacterium bovis bacille de Calmette et Guérin (BCG) vaccine on preventing adult pulmonary tuberculosis has been reported to be limited. Therefore, development of a novel effective vaccination strategy against pulmonary tuberculosis has become an international research priority. We have previously reported that intranasal administration of a recombinant mycobacterial Ag, heparin-binding heamagglutinin adhesin (HBHA), combined with mucosal adjuvant cholera toxin (CT) into mice enhanced Th1-type immune response and suppressed extrapulmonary bacterial dissemination after M. tuberculosis (Mtb) infection in the lung. Here, we further report effects of the mucosal HBHA+CT vaccine on murine experimental pulmonary Mtb infection. Combination of subcutaneous BCG priming vaccine followed by the mucosal HBHA+CT vaccine as a booster significantly enhanced protective immunity against pulmonary Mtb infection on day 14. The combination of subcutaneous BCG and the mucosal HBHA+CT vaccine was more effective than the BCG vaccine alone in induction of the early protective immunity. Further, the mucosal HBHA+CT vaccine enhanced not only IFN-γ but also IL-17A production by HBHA-specific T cells in the lung after pulmonary Mtb infection. The results indicate that the combination of BCG priming vaccine and mucosal HBHA+CT boosting vaccine might be a good candidate for a new vaccine strategy against pulmonary tuberculosis.
Accelerated development of a T Cell epitope-driven biodefense vaccine for Burkholderia (VAC7P.957)
Authors: Liu Rui , Sangare Kotou , Desrosiers Joe , Martin Bill , Moise Leonard , Groot Annie 
Published: January 2023

Abstract
Background: Burkholderia pseudomallei (BPM) and Burkholderia mallei (BM) are Category B pathogens. Burkholderia cepaciae (BC) causes chronic lung infections in cystic fibrosis patients. We are developing an epitope-driven multi-pathogen vaccine against all three Burkholderia species. Methods: Immunoinformatics tools were used to identify immunogenic consensus sequences (ICS) that are enriched for promiscuous and highly conserved CD4+ T cell epitopes in all three Burkholderia species. Top scoring 70 ICS were tested for binding affinity to 7 Class II HLA alleles in binding assays. Immunogenicity of the ICS was tested in human PBMCs, and in Balb/C (peptide immunization) and HLA DR3 transgenic mice (DNA-prime/peptide-boost immunization). Results: 100% of the ICS bound to at least two Class II HLA alleles for which they were predicted. The immunoinformatic predictions were validated in 77.3% of the HLA-peptide binding assays. Significant IFN-γ response was induced by all peptides in at least one human donor as measured by ELISpot assay. In contrast, 28.6% and 11% of the ICS peptides elicited significant IFN-γ responses in splenocytes from vaccinated Balb/C and DR3 transgenic mice as measured by ELISpot assay, respectively. Conclusions: These results illustrate the power of immunoinformatics tools for rapid selection of highly conserved epitopes for biodefense vaccines. Differences in human and murine immune responses may be due to ineffective delivery of the vaccine immunogen.
Priming protective BVDV-specific immunity using multi-component mosaic antigens (VAC7P.980)
Authors: Mwangi Waithaka , Fang Xin , Lokhandwala Shehnaz , Bray Jocelyn , Waghela Suryakant 
Published: January 2023

Abstract
Bovine viral diarrhea virus (BVDV), a Flavivirus, plays a key role in bovine respiratory disease complex, which leads to pneumonia and death of calves. Current vaccines are not very effective due, in part, to immunosuppressive traits of the virus. There are diverse BVDV strains and thus, current vaccines contain type 1 and 2 viruses (BVDV-1 & 2). Modified live virus (MLV) vaccines are superior to killed virus, but they are susceptible to antibody neutralization and complement-mediated destruction that limits their efficacy in neonates. We generated three novel mosaic polypeptide chimeras, designated NproE2123; NS231; and NS232, which represent determinants conserved among BVDV strains and these antigens are the main targets involved in protection. We confirmed that a proto-type adenovirus-vectored vaccine expressing these antigens was recognized by mAbs, sera, and T cells raised against diverse BVDV strains. In a poof-of-concept efficacy study, the vaccine induced significantly higher IFN-γ spot forming cells (p&lt;0.05), T cell proliferation (p&lt;0.05), and SN titers (p&lt;0.05) against BVDV-1 & 2 as compared to immune responses induced by a commercial MLV vaccine. Following challenge, the calves immunized with the proto-type and the MLV vaccines had mild clinical scores compared to naïve controls. These results support the hypothesis that a broadly protective subunit vaccine can be generated using mosaic polypeptides that incorporate protective motifs from diverse BVDV strains.
Modulating cathepsin cleavage sites in HIV gp120 envelope protein alters the cellular immune response. (VAC7P.989)
Authors: Frey Blake , Sui Yongjun , Wang Yichuan , Hogg Alison , Yu Bin , Berman Phillip , Berzofsky Jay 
Published: January 2023

Abstract
A major goal in HIV vaccine development is to identify antigens that can elicit long-term, protective antibody and cellular responses. Limited success with native recombinant HIV gp120 proteins suggested altering antigen processing as an approach to improve the immunogenicity of the HIV gp120 protein. Cathepsins S and D are important proteases which play critical roles in antigen processing. Here, we have utilized recombinant HIV gp120 proteins with mutations at the cathepsin S site (T322T323) and D cleavage site (L181Y182) within HIV gp120 to investigate the effects of these mutations on MHC class I/II presentation to CD8+/4+ T cells. We have found that altering the cathepsin S site in the minimal epitope, IGPGRAFYTT from T322T323 to V322V323 might improve CD8+ T cell recognition to this epitope in the V3 domain. We are investigating the mechanisms by which this mutation might enhance the CD8 response. It is possible this mutation can increase the binding affinity by changing the anchor residue with the MHC I molecule, or it can prevent degradation of the minimal epitope, or both. In a mouse model, we found that gp120 elicited antigen-specific CD4+ T cell responses against the V1V2 domain. Currently, we are investigating whether the altered cathepsin sites in gp120 affect antigen-specific CD4+T cell responses to the V1V2 regions. As antigen processing plays an important role in immunogenicity, we speculate that HIV may exploit this process to escape recognition.
Predicting T cell responses to multiple antigens in lymph nodes (VAC7P.990)
Authors: Ziraldo Cordelia , Gong Chang , Kirschner Denise , Linderman Jennifer 
Published: January 2023

Abstract
T cells carrying receptors specific to an epitope are primed and differentiate in lymph nodes when they encounter their peptide-MHC counterpart displayed on dendritic cells. These epitopes are subunits of proteins derived from pathogens, but very few of the epitopes from any given pathogen elicit a measureable cell-mediated immune response. For infectious diseases lacking a protective vaccine, concurrent cellular immunity to several peptides may be necessary to target multiple stages of disease. In this work, we expand upon our previously developed computational model of T cell priming and differentiation in LNs to explore simultaneous induction of memory for multiple antigens. The model incorporates key parameters describing cell interactions, including pMHC-TCR binding parameters, cognate T cell frequency, and fraction of pMHCs specific to a TCR. Our results point to the importance of binding thresholds and pMHC fractions in determining immunodominance hierarchies. These findings are relevant to design of multi-subunit vaccines, where the aim is to simultaneously induce memory to several peptides.
Therapeutic vaccination against shingles broadens the VZV-specific CD4+ T cell response (VAC7P.994)
Authors: Laing Kerry , Russell Ronnie , Dong Lichun , Haas Juergen , Stern Michael , Johnston Christine , Wald Anna , Koelle David 
Published: January 2023

Abstract
Life-long latent neuronal infection with varicella zoster virus (VZV) is a legacy of childhood chickenpox. VZV reactivation occurs in about 1/3 of persons to cause shingles. Live, attenuated VZV (vOka)—the only licensed therapeutic vaccine—moderately reduces both the incidence and the severity of shingles. The partial level of protection is poorly understood but may plausibly derive from variation in T cell boosting. Twelve subjects (&gt;50 years old) were vaccinated with vOka and blood obtained on days 0, 14, and 28. Ex vivo ICC analyses confirmed boosting of vOka-reactive T cells: the mean increase in frequency of IFNγ+IL2+ CD4+ T cells was 74% by day 14 (p=0.02). We enriched and expanded VZV-specific CD4+ T cells in an unbiased manner and defined the specificity and breadth of the resulting polyclonal CD4+ T cell lines using each of the 70 known VZV proteins. Prior to boosting, the median number of detectable responses was 9 ORFs (range 3-10). Vaccination increased the median breadth to 17 ORFs (range 9-20) at day 28. Prevalent targets before and after boosting were glycoproteins gE, gI, and gH, and the tegument proteins encoded by ORF12 and ORF44. While new responses were noted post-vaccination, baseline responses rarely disappeared. T cell breadth was well-maintained during VZV latency and further increased by therapeutic vaccination. In addition to CD4+ T cell magnitude, specificity and breadth are now accessible as candidate immune correlates of vaccine efficacy.
The αβ-double negative T cells in lymphoma patients: the predictive role and the functional attitude. (VAC8P.1003)
Authors: De Tullio Giacoma , Iacopino Pasquale , Guarini Attilio 
Published: January 2023

Abstract
Mutual interactions between the host immune system and lymphoma cells can either promote or control lymphomagenesis.A subset of CD4-CD8- double-negative T cells(DNTs) has been recently described to be involved in anti-tumor immunity acting as regulatory T cells and/or cytotoxic T cells.The aim of this study was to assess the role of DNTs on clinical outcome and progression of Lymphoma patients (pts).Peripheral blood(PB) and bone marrow(BM) samples of 46 Lymphoma pts,with non-Hodgkin’s(nHL) and classical Hodgkin Lymphoma(HL),and 16 PB of 16 healthy donors (hd),were selected and prospectively collected at diagnosis, at different time of therapy and at the time of relapse or progression.DNTs were purified and characterized for their phenotype and for tolerogenic or cytotoxic attitude with functional studies.The percentage of DNTs in BM (2.367+0.5891)of pts was lower than in PB(3.421+0.981) and decreased(p=0.006)in pts with untreated lymphoma as compared with hd,seeming to be modulated by disease relapse/progression or disease treatment.In HL αβ-DNTs were significantly increased as compared with other histotypes(p=0.0001).Interestingly,after ex vivo expansion,DNTs acquired a immunomodulatory cytokine profile.Our study demonstrated that αβ-DNTs could play an important role in both the development and the progression of lymphomas.Finally,it is likely that ex-vivo expanded DNTs exert an anti-tumor activity suggesting their possible use as a new strategy for adoptive immune-therapy.
Dynamics of the CD8+ T cell response to a model of acute viral infection (VIR4P.1017)
Authors: Emerson Ryan , DeWitt William , Lindau Paul , Desmarais Cindy , Makar Karen , Warren Edus , McElrath Juliana , Wald Anna , Robins Harlan 
Published: January 2023

Abstract
During an acute viral infection T cells specific to viral antigens, as determined by the T-cell receptor (TCR), activate and expand. To determine the breadth of immune response to infection, and to understand recruitment to immunological memory, characterizing the TCR repertoire before, during, and after a model of acute viral infection is necessary. Here we use the yellow fever vaccine (YFV) to model acute viral infection. Blood was collected from 9 subjects at 0, 14, and 90 days after YFV administration. In addition to isolating PBMC for all time points, we sorted for YFV-specific effector T-cells at day 14 and for central and effector memory T-cells in 6 of 9 subjects at day 90. For each sample, gDNA was extracted and the TCR repertoire was assayed by high-throughput TCRβ sequencing. Expanded T cell clones can specifically bind a pathogen-derived epitope (specific), or receive proliferation signals without antigen-specificity (bystander). YFV-specific clones were identified by enrichment in the YFV-specific day 14 sort vs. day 14 PBMC; in each subject, we estimate that the antigen-specific immune response comprises approximately 103 T cell clones. Bystanders were identified as clones expanding from day 0 to day 14 PBMC that are not YFV-specific. Many more YFV-specific clones were recruited to memory at day 90 than bystander clones (40% vs. 3%), indicating that antigen specificity, rather than just proliferation, is a major determinant of memory recruitment.
The transcription factor ZBTB32 negatively regulates CD8+ T cell responses during acute and chronic viral infection (VIR4P.1010)
Authors: Shin Hyun Mu , Kapoor Varun , Welsh Raymond , Berg Leslie 
Published: January 2023

Abstract
The transcription factor, ZBTB32, is not expressed in resting naïve CD8+ T cells, but is upregulated following TCR stimulation in the presence of cytokines IFNγ, IL-12 or IL-2. To investigate the role of ZBTB32 in the development of anti-viral CD8+ T cells, Zbtb32-/- mice were infected with LCMV-Armstrong or LCMV-Clone 13. Following infection with LCMV-Armstrong, Zbtb32-/- mice exhibited normal viral clearance, but generated increased numbers of virus-specific CD8+ T cells, relative to wild-type mice, resulting in an increased memory cell population. The Zbtb32-/- CD8+ memory T cell population showed rapid proliferation and enhanced protective memory potential to secondary challenge. In contrast, upon infection with LCMV-Clone 13, 70% of Zbtb32-/- mice succumbed to a fatal disease starting at day 9 post-infection, and exhibited severe immune pathology in the lung compared to WT mice. Examination of the surviving mice indicated reduced LCMV-clone 13 viral titers in Zbtb32-/- mice. Gene expression analysis showed up-regulation of Eomes, Cd27, Pvr and Cd7 in virus-specific Zbtb32-/- CD8+ T cells. ChIP assays confirmed that ZBTB32 bound to the regulatory regions of these genes and recruited HDAC1 and HDAC2 to promote repressive histone modifications. These data indicate that ZBTB32 acts an epigenetic regulator and negatively regulates T cell responses and memory generation during acute and chronic LCMV infection.
An ultrafast refolding protocol for producing cell surface immunoreceptors and viral receptors (VIR5P.1029)
Authors: Zhai Lu , Wu Ling , Li Feng , Xu Bin 
Published: January 2023

Abstract
Functional cell surface immunoreceptors and viral receptors are often a cumbersome and time-consuming process and a bottleneck step that limits detailed biochemical and functional characterization of this important class of molecules. Here we introduce an ultrafast procedure that can be used to refold inclusion-body-based immunoreceptor and viral receptors within one day from the point of harvesting culture, a speed equivalent to soluble protein production. The protocol utilizes modular on-column Ni-NTA purification followed by efficient on-column refolding. We demonstrated the utility of this method for multiple immunoreceptors and viral surface receptors, including host immunoreceptors MHC class I chain A (MICA) and NKp46 and viral receptors UL144 and UL146. The method can be easily adapted into high throughput format for structure biology, chemical biology, and proteomics screenings.