bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation of Cross-Clade HIV-Neutralizing Human Antibodies from Memory B Cell Repertoires Using Short Term Culture and High-Throughput Primary Neutralization Screens (38.17)
Authors: Chan-hui Po-Ying , Wrin Terri , Simek Melissa , Phogat Sanjay , Olsen Ole , Hammond Phil , Poignard Pascal , Walker Laura , Mitcham Jennifer , Fling Steve , Team Protocol G , Burton Dennis , Moyle Matthew 
Published: January 2023

Abstract
Background. We have recently isolated 2 broadly neutralizing monoclonal antibodies (bNAbs), PG9 and PG16, from an HIV-infected subject in an African Protocol G cohort that are more potent than the bNAbs discovered in the past decade. The discovery highlights our human antibody (Ab) discovery approach based on short-term memory B cell culture, high-throughput primary microneutralization screening, and recombinant monoclonal antibody (mAb) reconstitution (Walker LM, et al, 2009, Science, 326:285-9). The epitope specificities of PG9 and PG16 reveal a novel target for designing vaccine immunogens that may elicit broad anti-HIV protection. To identify additional epitopes for vaccine design, we extended the bNAb discovery to more HIV-infected subjects. Method. We selected several HIV-infected subjects with broad neutralization activity in serum. Memory B cells from these elite neutralizers were activated and the IgG-containing culture media were screened for neutralizing activity against multiple pseudotyped viruses. mAbs were rescued from selected B cell culture wells by RT-PCR and subcloning. Results. From an HIV-infected elite neutralizer, we have identified in the primary screen multiple human Abs that cross-neutralize MGRM-C-26 of clade C, and 93BR020 and JR-CSF of clade B. Antibodies that neutralize either MGRM-C-26 alone or 94UG103 of clade A alone were also detected. Recombinant mAbs are currently being rescued for further characterization. (Supported by IAVI and USAID)
Pronounced T Cell Receptor-dependent Activation Drives Exhaustion but Sustains Interferon-γ Transcript Levels (38.6)
Authors: Zajac Allan , Mackerness Kathryn , Cox Maureen , Lilly Lauren , Weaver Casey , Harrington Laurie 
Published: January 2023

Abstract
The ontogeny of exhausted CD8 T cells as well as the underlying mechanisms that account for the functional inactivation of these cells remains ill-defined. We have utilized cytokine reporter mice, which mark the synthesis of interferon-γ by the expression of Thy1.1, to decipher how activation events during the early stages of a chronic infection dictate the development of exhaustion. We show that, during chronic lymphocytic choriomeningitis virus infection, the precursors of exhausted T cells all upregulate interferon-γ mRNA and become susceptible to depletion with anti-Thy1.1 antibodies. This potent phase of hyperactivation which precedes exhaustion is antigen-dependent and is dictated by viral levels and T cell precursor frequencies. Unlike acute infections, which result in massive expansion of the responding T cells, during chronic infections further expansion of the initial response becomes rapidly attenuated. The exhausted T cells which subsequently emerge in chronically infected hosts attain a distinct CD127lo, KLRG-1lo phenotype but do not silence interferon-γ transcription even though protein expression is abolished. Thus ablation of interferon-γ production by exhausted cells is not due to transcriptional silencing, implicating post-transcription regulatory mechanisms in disabling this effector module.
Virus-specific CD4+ T cells are recruited de novo during persistent polyomavirus infection (39.11)
Authors: Lin Eugene , Kemball Christopher , Hadley Annette , Wilson Jarad , Lukacher Aron 
Published: January 2023

Abstract
Virus-specific CD4+ T cells optimize anti-viral responses by providing help for anti-viral humoral responses and CD8+ T cell memory differentiation, as well as operating as direct anti-viral effectors. While CD4+ T cell responses to viral infections that undergo complete clearance have been extensively studied, comparatively less is known about virus-specific CD4+ T cell responses to viruses that persistently infect their hosts. Using a mouse polyomavirus (MPyV) infection model, we previously demonstrated that CD4+ T cells are essential for recruiting naïve MPyV-specific CD8+ T cells in persistently infected mice. In this study, we defined two dominant MPyV-specific CD4+ T cell populations, one directed toward an epitope derived from the nonstructural LT antigen and the other from VP1, the major viral capsid protein. These MPyV-specific CD4+ T cells vary in terms of their magnitude, functional competence, and phenotype during acute and persistent phases of infection. Employing a minimally myeloablative mixed bone marrow chimerism approach, we further show that naïve virus-specific CD4+ T cells, like anti-MPyV CD8+ T cells, are primed de novo during persistent virus infection. In summary, these findings reveal quantitative and qualitative differences in the CD4+ T cell response to a persistent virus infection and demonstrate that naïve antiviral CD4+ T cells are recruited during chronic polyomavirus infection.
Murine LP-BM5 viral infection induced peripheral neuropathy in C57BL/6 mice (39.2)
Authors: Cao Ling , Green Kathy , Li Wen , Tan Leonard Tan , Green William 
Published: January 2023

Abstract
Distal symmetrical polyneuropathy (DSP) is the most common form of HIV infection-associated peripheral neuropathy and is often associated with pain. C57BL/6 (B6) mice infected with LP-BM5, a murine retroviral isolate, develop a severe acquired immunodeficiency syndrome similar to humans infected with HIV, hence the term murine AIDS (MAIDS). We investigated the induction of peripheral neuropathy post- LP-BM5 infection (p.i.) in B6 mice. First, LP-BM5 infected mice displayed significant symmetrical hind paw mechanical hypersensitivity (behavioral sign of peripheral neuropathy) starting from week 6 p.i. up to the time of sacrifice at week 12. The density of PGP9.5+ (a pan-neuronal marker) intraepidermal nerve fibers (IENFs) was found to be significantly reduced at week 2 p.i. and this loss was gradually recovered with time. There were no significant differences between left and right paws. Also, significantly increased levels of viral gag RNA in all segments of spinal cord tissue were detected in infected mice via quantitative real-time PCR. Further, FACS analysis showed an early increase of lumbar spinal cord microglia p.i.. Altogether, LP-BM5 induced the development of peripheral neuropathy that occurred concurrently with the development of MAIDS and was correlated with viral infection of the spinal cord. The potential role of spinal cord microglia in LP-BM5-induced painful neuropathy will be further investigated.
Exhaustion of intra-hepatic CD8+ T cells targeting intact and escaped MHC class I epitopes in chronic phase of hepatitis C virus infection (39.20)
Authors: Callendret Benoit , Bowen David , Fuller Michael , Eccleston Heather , Rutckiewitz Jennifer , Hasselschwert Dana , Freeman Gordon , Walker Christopher 
Published: January 2023

Abstract
Hepatitis C virus (HCV)-specific CD8+ T cells persist in the chronically infected liver but provide no apparent control of virus replication. Epitopes targeted by some of these T cells contain escape mutations while others remain intact. Studies in acutely infected humans suggest that circulating CD8+ T cells targeting the intact epitopes express more PD-1, a molecule associated with loss of effector function. Whether this is true in the persistently infected liver is unknown. CD8+ T cells targeting intact or mutated epitopes were visualized using tetramers in the liver of chronically infected chimpanzees. Unexpectedly, there was no obvious relationship between expression of PD-1 on the HCV-specific CD8+ T cells and the nature of the targeted epitope. More importantly all HCV-specific CD8+ T cells we examined displayed functional incapacity regardless of the nature of the targeted epitope and anergy could not be reversed by blockade of the PD-1/PD-L1 pathway. We conclude that decreased PD-1 expression previously observed on acute phase CD8+ T cells after escape of the targeted epitope is not necessarily maintained in the liver during chronic hepatitis C. Rather, our data suggest that mechanisms other than continuous TCR triggering might be responsible for PD-1 upregulation and exhaustion of CD8+ T cells, or that escaped epitopes are still able to induce TCR engagement without T cell activation resulting in exhaustion of viral specific CD8+ T cells.
A molecular characterization of how the immunomodulatory E3-19K protein from adenovirus binds to and retains MHC class I molecules in the endoplasmic reticulum (39.23)
Authors: Bouvier Marlene , Fu Jie , Li Lenong 
Published: January 2023

Abstract
Adenoviruses (Ads) cause persistent infections. The E3-19K protein from Ads retains MHC class I molecules in the endoplasmic reticulum (ER), suppressing antigen presentation to T cells. The ER-lumenal domain of E3-19K is subdivided into a variable (residues 1-81), conserved (residues 82-100), and linker (residues 101-107) regions. We show that proteolysis of serotype 2 E3-19K (residues 1-100) generates a large N-terminal 1-88 fragment and a small C-terminal 94-100 fragment that span the variable and conserved regions, respectively. Neither of these fragments associates with HLA-A*1101. However, the N-terminal 1-93 residues bind with high-affinity to HLA-A*1101; Tyr93 is critical for this interaction. The data suggest that a delicate structural interplay between the variable and conserved regions enables E3-19K to exert its MHC binding function. Interestingly, Ad37 E3-19K displays weaker binding affinities for class I molecules relative to Ad2 E3-19K but retains the same locus specificity; both proteins associate with HLA-A and -B molecules but not HLA-C molecules. We provide evidence that the class I locus specificity of E3-19K is defined by MHC residue 177. Finally, we present a model which MHC residues 56 and 177 represent “hotspots” for the variable and conserved regions, respectively, of E3-19K. In conclusion, the knowledge derived from our studies helps not only to better understand host/Ad interaction, but also to design novel antiviral therapies against Ad infections.
The activating receptor CD94-NKG2E is required for protective NK cell responses to a lethal viral infection (39.26)
Authors: Fang Min , Shamsedeen Ahamed , Orr Mark , Lanier Lewis , Sigal Luis 
Published: January 2023

Abstract
The CD94/NKG2 receptors of NK cells are C-type lectin-like heterodimers formed by CD94 and inhibitory receptor NKG2A, or activating receptor NKG2C or NKG2E. However, the physiological functions of those receptors in the NK cell responses to viral infections are not clear. Ectromelia virus (ECTV) is an orthopoxvirus (OPV) that causes mousepox, the mouse equivalent of human smallpox. Previous studies have shown that NK cell responses are essential for the natural resistance of C57BL/6 (B6) mice to mousepox. Here, we show that B6 mice deficient in CD94 (CD94 KO) and B6D2-D6 mice (a B6 congenic mouse strain harboring NK complex from the DBA2/J, also deficient in CD94) succumbed to ECTV infection due to defective NK cell cytolytic activity. In addition, [B6-CD94 KOxB6D2-D6] F1 mice remained susceptible to mousepox whereas CD94 KO or B6D2-D6 mice expressing transgenic CD94 on NK cells were resistant. Together, these data demonstrated an essential role for CD94 in resistance to mousepox. To determine the specific CD94/NKG2 receptor required for resistance, we co-incubated reporter cells expressing CD94 and different NKG2 molecules with ECTV infected cells. We found that this resulted in the activation of only the reporters co-expressing CD94 and NKG2E. These results suggest that the activating receptor CD94-NKG2E mediates the protective NK cell response to ECTV. This is the first report showing the functional involvement of CD94/NKG2 receptors in an anti-viral NK cell response.
Cytomegalovirus reactivation occurs in burn patients and correlates with increased central memory and regulatory T cell populations. (39.32)
Authors: Maile Rob , Peppercorn Amanda , Mackinnon Karen , Schmitz John , Barnes Carie , Serody Jonathon , Cairns Bruce 
Published: January 2023

Abstract
Cytomegalovirus reactivation (CMV-R) is an important cause of morbidity and mortality in immunocompromised patients. CMV-R has been recognized in critically ill patients. Severe burn injury leads to early and prolonged immune and adaptive immune dysfunction. We hypothesized that CMV-R would occur in burn patients correlating with specific changes in T cell phenotype. We prospectively evaluated incidence of CMV-R longitudinally among patients with &gt;10% total body surface area (TBSA) injury using serum PCR. We determined changes in CMV specific CD8+ T cells by utilizing CMV-specific MHC Class I tetramers, functional responsiveness to CMV peptide and the generation of Tregs using qPCR for FoxP3. 24 burn patients enrolled, with CMV-R occurring in 36.8% of patients. We detected no difference in frequency of CMV-reactive CD8+ T cells nor in vitro anti-CMV CD8+ T cell response by IFN-γ staining between R and non-R patients. CMV-R patients had a significantly decreased proportion of CMV-specific CD8+ T cells that were central-memory (CD45RO+CD62L+CD28+) and increased effector-memory phenotype (CD28-) during the first week following injury. Elevated FoxP3 expression in the 10 days prior to R correlated with R in 4/6 patients tested. CMV-R occurred following burn injury and may be related to remodeling of the CD8+ T cell population after burn. Increased T cell regulatory activity after burn may also predict for CMV-R in a subset of patients.
A Novel L-Ficolin-Mannose Binding Lectin Chimeric Molecule With Enhanced Activity Against Lethal Viral Infections (39.33)
Authors: Lear Calli , Zeitler Corinne , Karpell Marshall , Spear Gregory , Olinger Gene 
Published: January 2023

Abstract
Ebola viruses are potentially lethal enveloped RNA viruses for which no effective therapy exists. Human mannose-binding lectin (MBL) and L-ficolin (FCN) bind to conserved epitopes on pathogens and eradicate microbes by opsonophagocytosis, complement activation, and/or regulation of adaptive immune responses. Three FCN-MBL chimeras containing the carbohydrate-recognition domains of hMBL and varying lengths of the collagen stalks of FCN were stably expressed in HEK293F cells. rhMBL (Enzon, NJ) formed octadecameric and larger oligomers vs. smaller chimera multimers. Compared with rhMBL, chimeras bound a similar range of carbohydrate ligands but FCN-MBL76 had significantly improved function as shown by: 1) greater affinity to mannan (KD, 47 vs 73 pM, p&lt;0.004); 2) greater capacity to activate complement in the range of 7-30 ng/mL, p&lt;0.001; and 3) greater binding to human placental calreticulin, a putative cellular receptor for MBL (p&lt;0.001). All chimeras and rhMBL inhibited Ebola glycoprotein-pseudotyped lentivirus and native Ebola virus infection of HepG2 cells although FCN-MBL76 was the most effective protein against native virus. Tapping-force atomic force microscopy revealed that FCN-MBL76 was less tall than the other proteins suggesting enhanced flexibility. Compared with rhMBL, a novel FCN-MBL chimeric protein exhibited superior binding to select carbohydrates, activation of complement, binding to calreticulin, and greater inhibition of Ebola virus infection in vitro.
Molecular profiling of memory CD8 T cells after repeated antigen stimulations (39.5)
Authors: Badovinac Vladimir , Wirth Thomas , Xue Hai-Hui , Rai Deepa , Harty John 
Published: January 2023

Abstract
Repetitive antigen stimulation by prime-boost vaccination or pathogen re-encounter increases memory CD8 T cell numbers, however the impact on memory T cell differentiation is unknown. Here we show that repetitive antigen stimulations induce the selective accumulation of CD8 T cells with a uniform effector memory phenotype. In contrast to this, genome-wide microarray analysis reveals that each additional antigen challenge results in the differential regulation of several hundred genes in memory CD8 T cells and therefore in stepwise diversification of T cell transcriptomes. As a consequence, repeatedly stimulated memory CD8 T cells and primary memory CD8 T cells differ significantly in their molecular signature and share expression of only a small but preserved group of memory signature genes. Our results provide new insight into the complex regulation of memory CD8 T cell differentiation and identify potential new molecular targets to dissect and manipulate the function of memory T cells in human diseases.
The role of Programmed Death-1 (PD-1) in pulmonary M. tuberculosis infection: friend or foe? (40.10)
Authors: Lazar-Molnar Eszter , Chen Bing , Almo Steven , Jacobs William , Nathenson Stanley 
Published: January 2023

Abstract
The programmed death-1 (PD-1) co-inhibitory receptor attenuates T and B cell responses and plays a crucial role in peripheral tolerance. Recently, PD-1 has been shown to inhibit T cell responses during chronic viral infections such as LCMV and HIV, and blockade of PD-1 restored anti-viral effector T cell responses. In this study, we examined the role of PD-1 in infection with Mycobacterium tuberculosis, a common co-infection with HIV. Surprisingly and paradoxically, PD-1 deficient mice showed dramatically reduced survival compared to wild-type mice. Moreover, lungs of the PD-1-/- mice showed uncontrolled bacterial proliferation, and focal necrotic areas with predominantly neutrophilic infiltrates, but a lower number of infiltrating T and B cells. Pro-inflammatory cytokines, such as TNF-α, IL-1, and especially IL-6 were significantly increased in the lung and sera of infected PD-1-/- mice, consistent with an excessive inflammation. Microarray analysis of the lungs infected with M. tuberculosis showed dramatic differences between PD-1-/- and control mice: 297 genes were differentially expressed, resulting in profoundly altered inflammatory responses, with implications in both innate and adaptive immunity. Our studies demonstrate that M. tuberculosis infection in the absence of PD-1 results in fatal inflammation with increased bacterial load; therefore we suggest that the PD-1 pathway is involved in controlling inflammatory responses after M. tuberculosis infection in the lungs.
The multiplication of invasive Streptococcus pyogenes in endothelial cell accompanies autophagy formation (40.13)
Authors: Lu Shiou-Ling , Lin Yee-Shin 
Published: January 2023

Abstract
Streptococcus pyogenes, also known as group A streptococcus (GAS), is one of most common human pathogens. Severe life-threatening disease, such as sepsis, results from the breakdown of blood vessel barrier. Although GAS has been identified as an extracellular bacterium, internalization of GAS by many eukaryotic cells may provide a mechanism of clinical severe damage due to evading from immune surveillance or antibiotic treatment. Previous report showed that GAS can be efficiently killed by autophagy (an intracellular innate immune system) in some nonphagocytes including epithelial cells. In this study, we showed that invasive GAS, but not noninvasive GAS strains, multiplied inside endothelial cells and bacterial growth reached plateau at 6 ~ 8 h. The intracellular sites of multiplication of GAS were first in vesicles and then in cytoplasm where most of the GAS was liberated from vesicle by streptolysin before endosomal degradation. GAS further induced autophagy formation but only a small portion of GAS was completely enveloped by GAS-containing autophagosome-like vacuoles at 6 h. Furthermore, endothelial cells underwent a caspase-indepandent cell death caused by persistent multiplication of GAS. The interrelationship between autophagy induction, cell death, and GAS multiplication remains further investigation. Our studies demonstrate that the insufficient autophagy induction during invasive GAS infection, particularly in endothelial cells, results in multiplication of GAS.
T lymphocytes are essential for initiation of fungal keratitis in mice (40.14)
Authors: Wang Yiqiang , Zhang Hongbo , Jia Changkai , Xi Haijie , Li Siyuan 
Published: January 2023

Abstract
In preliminary study, we found that experimental fungal keratitis (FK) induced an adaptive immune response that involved T lymphocytes infiltration and antibody accumulation in corneas. Here we explored the role of T lymphocytes in the progess of FK. Immunocompetent Balb/c mice and nude mice on Balb/c background were utilized. For adoptive transfer of lymphocytes, B cells and T cells were isolated from Balb/c mice spleens and transferred into nude mice via intravenous injection respectively. Experimental FK was induced by intrastromal injection of Candida albicans or Aspergillus fumigatus spores into the corneas one day after reconstitution. Development of corneal disease was measured by scoring the lesions in the corneas. We found that while immunocompetent Balb/c mice developed typical FK after pathogen challenge, T-lymphocytes deficient nude mice did not show any sign of microbial infection after challenge with same amount of fungal spores. Pre-infection reconstitution of nude mice with 2x10^7 T lymphocytes, and not with same number of B lymphocytes nor with 0.66x10^7 T lymphocytes, recovered the susceptibility of the nude mice to FK, and the disease in T cells-reconstituted nude mice were much more severe than in similarly infected Balb/c mice. We conclude that T lymphocytes above a threshold amount are essential in the initiation of keratitis when corneas are insulted by fungi, and T lymphocytes play a dual role in the pathogenesis and prognosis of fungal keratitis.
Elevated Bcl2 expression in mice decreases CD4 lymphocyte apoptosis and facilitates clearance of Pneumocystis in mice (40.6)
Authors: Opata Michael , Hollifield Melissa , Garvy Beth 
Published: January 2023

Abstract
Lymphocyte and TNF deficiency leads to susceptibility to opportunistic infections such as Pneumocystis carinii (PC). This strongly indicates that protection against the fungal pathogen PC is dependent upon B cells, T cells and TNF. Although it has been documented that macrophage derived TNF is essential for PC clearance, the role played by TNF produced by lymphocytes has not been addressed. Furthermore, the functional role of B cells in the T cell-mediated protection against PC Pneumonia (PCP) has not been determined. In this study we report that B cells produce TNF in response to PC predominantly in the TBLN. B cells enhanced expansion of CD4 T cells and secretion of TNF in the lungs of adoptive transfer recipients and hence clearance of the organisms. Importantly, the presence of B cells resulted in upregulation of Bcl2 in CD4 T cells and down regulation of Fas and Fas Ligand consistent with a decrease in apoptotic cells as determined by annexin V staining. Lastly, we observed that T cell-derived TNF is critical for PC clearance. Taken together, our data suggest that B cells are essential for CD4 T cell survival and increased expansion leading to protection against PCP in mouse models.