bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of Potent Anti-HIV Chimeric Immune Receptors (B120)
Authors: Yang Wen , Lu Li , Junghans Richard P 
Published: January 2023

Abstract
T cells that are genetically modified to express anti-HIV chimeric immune receptors (CIR) and have the potential to eradicate the reservoirs of viral infections, may be useful to suppress or cure HIV/AIDS. CD4zeta (CD4z) can bind HIV-1gp120 antigen in an MHC nonrestricted fashion through its CD4 extracellular domain (Ec) fused to the cytoplasmic domain (Cy) of z. However, the adoptive transfer of CD4z-expressing T cells to HIV/AIDS patients failed to control the viral infection in clinical trials, with the mechanisms of failure being unknown. To date, all anti-HIV CIRs developed have been TCR Signal 1-only (e.g., via Cy of z). CIRs that also provide a costimulatory signal (Signal 2; e.g., CD28) significantly enhanced their potency in inducing T cell activation. In order to develop more potent anti-HIV CD4-based CIRs, we have constructed retroviral vectors encoding CD4-based CIR with Signal1+2 (CD4mCD28z) by fusing the Ec of CD4 to Tm and Cy of CD28 with Cy of z. This is compared with a Signal 1-CIR, CD4mz, as a control representing the former Signal 1-CIR designs. Our preliminary results showed that the two CIRs expressed equally well on Phoenix cells transfected with CIR-encoding plasmid and human Jurkat T cells retrovirally transduced individually with the two CIRs. Western blot analysis confirmed that the two CIRs were cellularly expressed intact. Human CD8 T cells will be transduced individually with the two CIRs, followed by evaluating their potencies in inducing T cell activation via the CIR as well as in inhibiting anergy of the CIR-T cells. This will demonstrate the benefits of the inclusion of Signal 2 in in vitro systems as a potential predictor of a better 2nd generation agent for subsequent human in vivo application.
Specific T lymphocyte citotoxicity induced by Cu/Zn superoxide dismutase from Brucella abortus RB51 (B124)
Authors: Moreno-Lafont Martha Cecilia , Flores-Mejia Raul , Rodriguez-Cortes Octavio , Chacon-Salinas Rommel , Cancino-Diaz Mario 
Published: January 2023

Abstract
Brucellosis is an infectious disease affecting animals and humans, caused mainly by B. abortus. Although several vaccines have been developed, B. abortus strain RB51 is the most used for cattle, but is not useful for humans since it causes severe granulomas. To develop a vaccine for humans, Cu-Zn superoxide dismutase (SOD Cu/Zn) has been identified as an antigen which induces T-cell mediated immunity in mice vaccinated with live Escherichia coli expressing B. abortus SOD Cu/Zn or a plasmid DNA carrying the gene sodC. The aim of this work was to demonstrate the induction by SOD Cu/Zn from B. abortus RB51 of specific cytotoxic T lymphocytes (CTL) recognizing in vitro macrophages infected with B. abortus strain RB51 or 2308. The protein was extracted from recombinant E. coli-pBAD-SOD and purified in a Ni-agarose column, and then by FPLC in a molecular weight exclusion column. Fractions corresponding to 20–22 KDa were collected and mice were immunized intramuscularly with three doses of 25 μg of SOD Cu/Zn each. T lymphocytes purified from spleen with an anti CD90 coupled to magnetic beads, were tested in an in vitro cytotoxicity assay, using J774A.1 cell line infected with B. abortus strain RB51 or 2308 as target. CTL activation was evident in both cases, since macrophages infected with any bacterial strain were killed as demonstrated by neutral red dye uptake or lactate dehydrogenase release assays. These results demonstrated that the protein by itself is able to prime pCTL to kill macrophages infected with B. abortus, no matter its virulence.
Grants from CONACyT 44837-Q and SIP 20060688 and 20070239.
T cell response induced by recombinant Cu/Zn superoxide dismutase from Brucella abortus RB51, in patients with brucellosis (B125)
Authors: Lopez-Santiago Ruben , Paredes-Cervantes Vladimir , Gonzalez-Gonzalez Edith , Goznalez-Peña Araceli , Cancino-Diaz Mario 
Published: January 2023

Abstract
Cu/Zn superoxide dismutase (SOD), located in the periplasmic space of the cell wall of Brucella abortus, induces protective cell mediated immunity in mice challenged with virulent Brucella strains. In vitro studies have shown that the SOD induces proliferation of splenocytes, antibody production and T cell cytotoxicity. In humans, the recombinant SOD (rSOD) is recognized by serum of patients with chronic brucellosis. In order to prove the rSOD immunogenicity in humans with brucellosis, we cloned the coding sequence of the gene sodC from B. abortus RB51 into an expression system that allowed us to obtain large quantities of recombinant protein to determine the immune response in patients suffering of brucellosis. The sodC gene was cloned from B. abortus genome by PCR. A 525 bp fragment was obtained and subcloned into the pBAD-TOPO expression vector (AmpR), used to transform competent E. coli TOP-10. Ampicillin resistant clones were selected and verified for the correct 525 bp size insert. Antigenicity of rSOD purified by a Ni-column was confirmed by Western blot with a rabbit anti B. abortus RB51 serum. Patients with acute or chronic brucellosis showed a specific T cell proliferation induced by the rSOD, as assessed by BrdU incorporation assay. Furthermore, mRNA synthesis for IL-2, IL-10 and IFNγ induced by rSOD was determined by RT-PCR. It was found mRNA for IL-2 and IFNγ in acute patients, while chronically infected patients showed a tendency to express IL-10 mRNA, confirming a type 1 cytokine pattern in acute patients, whereas a type 2 in chronic patients.
Grants by CONACyT 44837-Q and SIP 20060693
IL-4 induction in RSV-infected macrophages: examining the role of NFAT family members (B128)
Authors: Levy Nyssa A , Fach Sasha J , Sacco Randy E 
Published: January 2023

Abstract
Respiratory Syncytial Virus (RSV) is the leading cause of respiratory illness in infants, causing severe bronchiolitis and pneumonia. In the lung, alveolar macrophages (AMΦ) are important antigen presenting cells responsible for pathogen clearance, antigen presentation and cytokine production. The effect of RSV infection on AMΦs is not currently well understood. The objective of our studies was to characterize the AMΦ response following bovine RSV (bRSV) infection. Previously, we have shown that bRSV infects AMΦs as detected by immunofluorescence. Active bRSV replication within AMΦs was confirmed by real time PCR analysis of viral NS2 mRNA. Interestingly, we have also found by real time PCR that bRSV infection induces AMΦs to upregulate the mRNA transcription of genes encoding immunomodulatory mediators, IL-4 and IL-10. Presently, we are examining the role of NFAT family members in the induction of IL-4 transcription in RSV-infected macrophages. Production of IL-4 by AMΦs is of particular significance because of the association of RSV and Th2-associated chronic lung disease.
Supported by NIH R01 AI062787
Changes in Cellular Composition and Induction of Cell Death in the Spleen Are Correlated to the Disease Symptoms in Response to Influenza A Virus (B130)
Authors: Sato Kayoko , Fujikura Daisuke , Takada Ayato , Kida Hiroshi , Miyazaki Tadaaki 
Published: January 2023

Abstract
The pathogenicity of influenza A viruses varies depending on the species of the host animals and the virus strains. A/PR/8 (H1N1) strain of influenza A virus causes a more severe disease than A/Aichi/2/68 (H3N2) strain in mice. However, the plausible explanation for their difference of pathogenicity is still unknown. To identify the critical determinants for the pathogenicity of these viruses, the immunological responses to these virus infection were analyzed. Mice were infected with these viruses by inhalation and the lung, spleen, and sera from the infected mice were obtained on 3, 5, and 7 days after the infection. It is noted that the weight of the spleen and the number of splenocytes in mice infected with A/PR/8 virus was decreased, whereas those in mice with A/Aichi/2/68 was increased. Furthermore, we found that A/PR/8 virus infection strongly induced cell death in the spleen and the changes of cellular profile such as the population of macrophages or T cells. Taken together, cell death induction in the spleen and the change of the composition of splenocytes could be critical characteristics for the severe pathogenicity determined by the infection with A/PR/8, compared with A/Aichi/2/68.
Cytotoxic T lymphocytes express intracellular stores of FasL via mechanisms distinct from cytolytic granule exocytosis (B16)
Authors: He Jin-Shu , Ostergaard Hanne L. 
Published: January 2023

Abstract
Cytotoxic T lymphocytes (CTL) lyse target cells through the release of cytolytic granules and cell surface expression of Fas ligand (FasL). We found that there is prestored FasL pool in CTL, which underwent rapid TCR-regulated cell surface expression upon target cell engagement. Current models suggest that FasL is stored in cytolytic granules and that FasL cell surface expression would be subject to the same control as degranulation. The aim of the current study is to investigate if degranulation of cytolytic granules and FasL expression pathways of CTL are controlled by differential mechanisms. FasL cell surface expression and degranulation, as measured by CD107a cell surface expression in CTLs were compared by flow cytometry under different conditions. FasL underwent an immediate and transient cell surface delivery following either TCR stimulation by anti-CD3 antibody crosslinking, or phorbol ester treatment, conditions that do not permit degranulation. Furthermore, treatment with pharmacological reagents indicated that the preformed FasL cell surface transport is PI3-kinase (PI3K)-, calcineurin- and microtubule-independent, which are all indispensable for degranulation. Finally, stored FasL resides in vesicles distinct from cytolytic granules as assessed by confocal microscopy and subcellular fractionation. Our data clearly show that CTL degranulation and FasL lytic mechanisms are fully independent with respect to regulation and localization.
This work was funded by the Canadian Institutes of Health Research (CIHR).
Structural and functional features of cells from the sole (Solea senegalensis Kaup 1858) immune system. (B169)
Authors: Salinas Irene , Anchelin Monica , Esteban Maria Angeles , Meseguer Jose 
Published: January 2023

Abstract
During last years, the severe decrease of fishing resources has impulsed the growing of aquaculture with the necessary diversification of production species. The sole, Solea senegalensis Kaup 1858, is a high-value flatfish reared in intensive aquaculture production in Southern Europe, but no studies on the sole immune system cells have been conducted. The knowledge on healthy immune cells will be useful for diagnosis purposes in pathologies associated to changes in these cells. This study reports a morphological study (light and electron microscopy) of the sole mature immune cells from blood, head-kidney and spleen, the characterization of the different haemopoietic stages, and two innate immune responses of these mature cells were. Phagocytic capacity was studied by transmission electron microscopy (TEM) and the respiratory burst activity was evaluated by a chemiluminescence method. Sole blood, head-kidney and spleen leukocytes were isolated and analysed by flow cytometry showing the existence of three main cell subpopulations. Heterophilic and acidophilic granulocytes, macrophages and thrombocytes from the different studied tissues showed phagocytic capacity engulfing up to 4 yeast cells (Saccharomyces cerevisae). The head-kidney leukocytes respiratory burst response showed a slope of 4836 ± 414.
Increased T-regulatory cell number and expression of CXCR4 and mu-opioid receptors on CD4+ T-cells by methadone is associated with enhancement of HIV-1 infectivity (B17)
Authors: Rodríguez José W. , Hunter Robert , Nair Madhavan , Rios-Olivares Eddy 
Published: January 2023

Abstract
Previous in vitro, animal-model, and epidemiological studies have indicated that opiates have conditionally variable effects on HIV-1 susceptibility and disease progression. Immunovirological and pharmacological variability are two major factors that seem to dictate whether HIV-1 infection will progress more rapidly or more slowly in HIV-1 infected opiate-exposed subjects. According to previous studies, the immunovirological effect of the opioid drug methadone has not been elucidated yet. In the present study, we analyzed various immunovirological determinants to evaluate the effects of methadone. Human lymphocytes from healthy individuals were isolated and acutely infected with HIV-1 and then assayed to determine how in vitro exposure of methadone affects HIV-1 infectivity and alters the expression of HIV-1 co-receptor (CXCR4) and mu-opioid receptor (MOR) on HIV-infected CD4+ T-cells. Our results showed that in vitro exposure of methadone increases HIV-1 replication by 4-folds. This was also correlated with a significant increase in the expression of HIV-1 co-receptor CXCR4 and mu-opioid receptor on HIV-infected CD4+ T-cells. Interestingly, we also observed a significant increase of CD4+ T-regulatory cells following methadone in vitro exposure. These results provide a new insight on the immunovirological status of methadone-treated HIV-infected patients.
Supported by NIH/RCMI Grant # G12-RR03035.
Expression of human mannan-binding lectin in Pichia pastoris (B171)
Authors: Kunkalla Kranthi , Rajagopalan Rema , Salvi Veena P. , Rawal Nenoo 
Published: January 2023

Abstract
Mannan-binding lectin (MBL), a pattern-recognition molecule binds to sugars present on the cell surface of microorganisms and activates the lectin pathway of complement. Three naturally occurring point mutations that occur in exon 1 of MBL 2 gene give rise to the structural variants of MBL and are correlated with impaired immune response. Wild type human MBL A and its variant, MBL B (Gly to Asp) were expressed in the yeast, Pichia pastoris. The yeast system has several advantages such as easy to manipulate, less expensive and generally gives high expression levels. Western blot analysis under reduced conditions of rMBL A and rMBL B expressed in the yeast culture medium exhibited a molecular size similar to that of native MBL (32 kDa) purified from human plasma (hMBL). Analysis under nonreduced conditions showed rMBL A and rMBL B to form higher order oligomers (monomers to hexamers) as observed for hMBL. Functional analysis of the rMBL molecules ability to bind sugars measured by ELISA, upon extensive dialysis of the culture medium, indicated that both rMBL A and rMBL B bind to mannan. Functional studies related to complement activation are currently being investigated. The estimated expression level of rMBL A and rMBL B (~0.3mg/L) indicates that the yeast system may be a better expression system of rMBL than the insect cell system (~0.2mg/L).
Research supported by NIH grant HL073804
Proteome-wide profiling of the CD4 and humoral immune responses to vaccinia in humans reveals immunodominant antigens and limited T-B cell correlation (B184)
Authors: Jing Lichen , Davies D Huw , Chong Tiana M , Chun Sookhee , Felgner Philip L , Koelle David M 
Published: January 2023

Abstract
The aims were to identify potential immunodominant CD4 T-cell antigens, estimate within-subject CD4 diversity, and investigate CD4 T-B cell concurrence. Each of 205 predicted vaccinia ORFs was transcribed and translated in vitro. Serial PBMC and sera were obtained from vaccinated subjects. Antibody binding assays used protein-spotted arrays. CD4 proliferation assays used bulk PBMC-derived responders and autologous APC. Overall, CD4 and antibody responses were above background for 145 (70%) and 62 (30%) ORFs, respectively. The mean number of ORFs recognized per specimen by CD4 cells was 55 (range, 20 to 83). The ORFs recognized most frequently by CD4 cells were L4R, D13L, A10L, I3L, H3L, WR149, WR148, and A3L, while for antibodies, the most frequently detected responses were to A10L, B2R, H3L, WR148, I1L, D13L, D8R, and H5R. The CD4 immunodominant ORFs were mostly virion structural proteins and had late expression kinetics during virus replication. Each ORF immunodominant for antibody responses also frequently elicited CD4 responses, while antibody responses were not detected to many other CD4 antigens. This is the first proteome-wide screen of CD4 responses to a complex pathogen using full-length protein antigens. Immunodominant CD4 antigens identified using this technology may be rational subunit vaccine components for microbial pathogens requiring T-cell help for protection. First two authors contributed equally to the work.
Primary B-lymphocytes Preferentially Expand Allogeneic FoxP3+ CD4 T Cells (B19)
Authors: Chen Xinjian , Guo Xiaohua , Jensen Peter E 
Published: January 2023

Abstract
Antigen presentation by B-lymphocytes can result in immunity as well as tolerance. The potential impact of B cell-mediated tolerance and underlying mechanisms are incompletely understood. Using primary B cells as APC and allogeneic CD4 T cells as responder cells in mixed leukocyte reactions (MLR), we find that B cells, though weaker allo-stimulators than dendritic cells, preferentially expand Foxp3+ CD4 T (nTr) cells in the absence of any exogenous cytokines, such as IL-2 or TGF-beta. Partial blockade of class II MHC-TCR interaction further enhances the expansion of nTr cells partially through conversion of Foxp3- cells to Foxp3+ cells, but expansion is reduced by increasing the ratio of B cells to responder T cells or by provision of exogenous IL-2 or anti-CD28. Gamma irradiation completely abrogates the ability of B cells to expand isolated nTr cells, but exogenous IL-2 or anti-CD28 can partially restore this function. The B cell-expanded nTr cells express high levels of Foxp3 and are highly inhibitory in an antigen-specific manner. Taken together, our results suggest that
primary B cells are optimally equipped to preferentially expand nTr cells,this property is related to the modest allo-stimulatory function of B cells, anda low level of T cell stimulation favors nTr cell generation.
Single Intranasal Immunization with Recombinant Adenovirus-Based Vaccine Induces Protective Immunity against Respiratory Syncytial Virus Infection (B192)
Authors: Yu Jaerang , Kim Sol , Chang Jun 
Published: January 2023

Abstract
Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses. Here, a recombinant replication-deficient adenovirus-based vaccine, rAd/3×G, expressing the soluble core domain of G glycoprotein spanning amino acids 130 to 230, was engineered by codon optimization and tandem repetition for higher level expression, and evaluated for its potential to be used as an RSV vaccine in murine model. A single intranasal but not intramuscular immunization with rAd/3xG provided potent protection against RSV challenge at an improved level over inoculation of a recombinant vaccinia virus expressing whole G protein (vvG). Strong mucosal secretory IgA responses were induced by single intranasal immunization but not by intramuscular administration or immunization of vvG, while comparable serum IgG responses were observed in each group. Interestingly, rAd/3xG immunization did not induce detectable CD4 T-cell response whereas vvG priming strongly elicited CD4 T-cell response against I-Edrestricted G epitope (amino acids 183 to 195). RSV-induced weight loss was significantly lower in rAd/3xG-immunized group than vvG-immunized group. Thus, our data demonstrate that a single intranasal administration of rAd/3xG elicits beneficial protective immunity and represents a novel vaccine regimen capable of providing mucosal protection against RSV infection.
Mining for Treasures: Systematic Profiling of 8-, 9-, 10-, and 11-mer HLA-B*0702-restricted CD8+ T cell epitopes from the Influenza A/Puerto Rico/8/34 (H1N1) Virus Hemagglutinin with Potential Application in Vaccine Development (B193)
Authors: Buchli Rico , VanGundy Rodney S. , Plompen Sonja , Rennels Aaron D. , Ede Nicholas J. , Hildebrand William H. 
Published: January 2023

Abstract
Identification of T cell epitopes is of crucial importance for the development of cancer and viral T cell eliciting vaccines. Here, we define CD8+ T cell epitopes using a novel high-throughput system for the synthesis and screening of peptide libraries that include all dominant epitope lengths (8–11mers) for MHC class I molecules. These representative peptide libraries are tested for their ability to bind various MHC class I molecules in a competitive manner. This comprehensive screening strategy begins with the synthesis of an overlapping truncated peptide library containing mixes of four peptides with a common C terminus, but having a length of 8, 9, 10, or 11 residues. Following the initial screen, positive hits are resolved into individual epitope responses. In order to identify HLA-B*0702-restricted epitopes, a peptide library was prepared for the influenza A virus Hemagglutinin (HA) protein. Screening results revealed 63 high/medium affinity epitopes at various sizes. A number of these putative influenza epitopes coincide with directly discovered influenza epitopes as well as influenza epitopes reportedly recognized by CTL. In conclusion, this study demonstrates that the rapid and methodical screening of T cell immune epitope libraries can help in detecting and monitoring infections as well as enabling immune intervention strategies with respect to treatment or prevention of the disease.
Control of viremia and preservation of intestinal CD4+ T cells in SHIVsf162P3 infected macaques intravenously challenged with pathogenic SIVmac251 (B197)
Authors: Pahar Bapi , Piatak Michael , Wang Xiaolei , Lifson Jeffrey , Montefiori David C , Ling Binhua , Lackner Andrew , Veazey Ronald 
Published: January 2023

Abstract
Attenuated vaccines in primates have provided better protection than most approaches to date. We hypothesized that SHIVsf162P3 infection might “immunize” macaques against intravenous challenge with a highly pathogenic virus. Twelve rhesus macaques of Chinese (Ch) and 2 of Indian (In) origin were intravaginally infected with SHIVsf162P3 and followed until their level of virus had subsided to undetectable or very low levels in plasma. SHIV vaccinated macaques were challenged intravenously with either 100, 10TCID50 SIVmac251 or plasma with high viral loads. Four “naïve” Ch macaques were similarly challenged with SIVmac251 as controls. Remarkably, Ch macaques “immunized” with SHIVsf162P3 were able to control viral replication of pathogenic SIVmac compared to naive (non-immunized) Ch controls. Viral loads after challenge in immunized Ch macaques ranged from undetectable to 104 in plasma compared to105 to 107 in naïve Ch controls. Indian macaques had higher levels of virus than Ch-macaques. but had better levels of protection compared to naïve challenged macaques. Control (naïve) macaques had rapid depletion of intestinal CD4+ T cells but levels remained fairly stable in immunized macaques. Control of viral load in “immunized” macaques did not correlate with the presence of neutralizing antibody and/or cell-mediated immune responses. Combined, these data suggest that effective control of SIV replication through immunization may be possible, but correlates of resistance have yet to be determined.