bluuebunny/crossref_metadata_2025 huggingface dataset:
Ex vivo co-stimulatory 4-1BBL enhances the antitumor capacity of antigen specific CD8+ T cells after adoptive transfer in NSG mice (VAC7P.1042)
Authors: Srivastava Abhishek , Paria Biman , Chandran Smita , Kammula Udai
Published: January 2023

Adoptive T cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) can result in durable complete cancer remission in select patients with metastatic melanoma. These clinical responses have strongly correlated with the ability of the transferred cells to engraft and persist in these patients. Preclinical murine studies have further supported that T cells with superior anti-tumor efficacy have higher proliferative capacity, reduced AICD and enhanced in vivo persistence. Thus, in an effort to further improve ACT, we sought to exploit ex vivo 4-1BB co-stimulation to generate T cells with enhanced in vivo anti-tumor activity. We used an artificial APCs genetically engineered to express costimulatory 4-1BBL (eAPC-4-1BBL) to human tumor specific CD8+ T cell clones. The use of clones allowed us to precisely define the impact of the 4-1BB on the in vivo fate and function of a genetically distinct mono-specific T cell population without the ambiguity of subset outgrowth. Our results demonstrate that ex vivo eAPC-4-1BBL enhanced proliferative capacity and reduced AICD of the CD8+ T cell clones without loss of functional tumor avidity. The eAPC-4-1BBL further restored the ability of the clones to persist and survive after adoptive transfer in NSG mice and resulted in improved survival of established tumor bearing NSG mice. Similarly, eAPC-4-1BBL provided significant improvement in the in vivo persistence and antitumor capacity of human TIL after adoptive transfer in NSG mice.
Adoptive T cell therapy using rejuvenated antigen-specific T cells from induced pluripotent stem cells (VAC7P.1044)
Authors: Saito Hidehito , Ito Fumito
Published: January 2023

Current approaches to adoptive T cell therapy for cancer and chronic viral infection are limited by the difficulty of obtaining sufficient numbers of antigen-specific T cells with optimized therapeutic features. We hypothesized that this limitation of adoptive T cell therapy could be overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, and provide an unlimited source of phenotypically defined, functional, and expandable autologous T cells against targeted antigens for immunotherapy. We established iPSCs from pmel-1 T cell receptor (TCR) transgenic CD8+ T cells specific for the gp100 melanocyte differentiation antigen as a preclinical model of evaluating antitumor reactivity of rejuvenated antigen-specific T cells from iPSCs. Stably TCR retained pmel-1 T cell-derived iPSCs (pmel-1 iPSCs) were then differentiated into CD4+CD8+ double positive (DP) T cells in vitro. Stimulation of pmel-1 iPSC-derived DP T cells with the gp100 antigen generated a large number of CD8+ T cells with high antigen-specific cytokine production and cytolytic activity in vitro. Furthermore, adoptive transfer of pmel-1 iPSC-derived cytotoxic CD8+ T cells significantly delayed tumor progression and improved survival in mice with established subcutaneous B16 melanoma expressing gp100. These findings suggest that rejuvenated antigen-specific T cells from iPSCs may be useful for adoptive T cell therapy.
Targeting of a fixed bacterial immunogen to Fc receptors reverses the anti-inflammatory properties of the gram negative bacterium, Francisella tularensis, during the early stages of infection (VAC8P.1055)
Authors: Bitsaktsis Constantine
Published: January 2023

Production of pro-inflammatory cytokines by innate immune cells at the early stages of bacterial infection is important for host protection. Many intracellular bacteria, such as Francisella tularensis, utilize the anti-inflammatory cytokine IL-10 to evade the host immune response, mainly by suppressing antigen presentation. In the current study, we hypothesized that F. tularensis polarizes antigen presenting cells during the early stages of infection towards an anti-inflammatory status characterized by increased synthesis of IL-10, and decreased production of IL12p70 and TNFa, in an IFN-γ-dependent fashion. In addition, F. tularensis drives the alternative activation of alveolar macrophages at the early stages of infection, thus allowing the bacterium to avoid protective immunity. Furthermore, for the first time, we demonstrate that targeting inactivated F. tularensis (iFt) to Fcγ receptors via intranasal immunization with mAb-iFt complexes, reverses the anti-inflammatory effects of the bacterium on macrophages by down-regulating production of IL-10. More specifically, we observed that targeting of iFt to FcγRs enhances the classical activation of macrophages in the respiratory mucosa, as well as systemically, at the early stages of infection. These results provide important insight for further understanding the protective immune mechanisms generated when targeting immunogens to Fc receptors.
A single immunization with optimized DNA vaccines protects against lethal Ebola virus challenge in mice (VAC8P.1057)
Authors: Patel Ami , Scott Veronica , Wong Gary , Reuschel Emmal , Villarreal Daniel , Muthumani Karuppiah , Shedlock Devon , Yan Jian , Tierney Kevin , Sardesai Niranjan , Kobinger Gary , Weiner David
Published: January 2023

Ebola is a hemorrhagic fever virus that is responsible for the severe, ongoing outbreak in West Africa that has infected >20000 people, exhibiting >37% mortality. Infection control has been complicated by a lack of approved vaccines and therapies and recent figures suggest that the outbreak is more severe than previously thought. We generated 3 DNA vaccines expressing Zaire ebolavirus (EBOV) glycoprotein (GP): 2 vaccines designed on consensus alignments of EBOV GPs (1976-2014) and a 3rd matched construct to a 2014 Guinea strain. BALB/c mice received 40ug of each vaccine, delivered by IM injection followed by electroporation. Strong T cell responses were detected for all 3 vaccines, including GP-specific CD4+ and CD8+ T cells. To broaden efficacy against emerging EBOV strains, we also co-administered DNA vaccines in 2 or 3 construct formulations. Groups of 10 BALB/c mice were challenged with a lethal dose of a heterologous mouse-adapted Mayinga 1976 EBOV strain. The two consensus DNA vaccines, and combinations of 2 and 3 vaccines were 100% protective in mice. The matched vaccine afforded 90% protection. Total GP-specific IgG antibody levels were high in all surviving animals and low in unprotected animals suggesting that titers may indicate therapeutic efficacy. All three DNA vaccines are immunogenic and no antigen interference was observed. These experiments in mice support further studies in non-human primates towards continued development of this potential vaccine platform.
Immunization with superantigen-derived oligopeptides protect against S. aureus bacteremia (VAC8P.1058)
Authors: Karauzum Hatice , Datta Sandip
Published: January 2023

Staphylococcal superantigens are exotoxins that play a major role in disease pathogenesis. Using a T cell epitope prediction tool from the Immune Epitope Database Analysis Resource (IEDB) site, sequences within staphylococcal enterotoxin A and B (SEA, SEB) and Toxic Shock Syndrome Toxin with high affinity towards H2-Iab were identified and oligopeptides SEA_aa116-128, SEB_aa16-28 and TSST-1_aa212-224 were generated. C57BL/6 mice were immunized with a cocktail of oligopeptides or whole toxins in Al(OH)3. Serum samples from peptide-immunized mice tested positive towards individual peptides and whole toxins, however, samples from mice immunized with whole toxins did not bind to any of the peptides. To determine peptide-specific T cell response primed CD4 T cells isolated from spleens and total LNs of immunized mice were co-cultured with peptide-pulsed BMDCs and cytokine response was evaluated. Only cells of peptide-immunized mice resulted in elevated levels of IL-4, IL-5, IL-10, IL-13, GM-CSF, IFNg and TNFa, while cells obtained from whole toxin-immunized mice did not differ from baseline levels. To evaluate protective efficacy of peptides against toxin expressing strains, immunized mice were infected intravenously with MNHOCH (SEB+) or Newman (SEA+) and monitored up to 13 days. Mice that were infected with MNHOCH showed significantly extended survival compared to non-immunized mice (P=0.03). No significant difference in survival was observed when infected with strain Newman.
The yeast quorum sensing molecule, farnesol, acts as an adjuvant to promote innate inflammatory responses (VAC8P.1060)
Authors: Brown Deborah , Hargarten Jessica , Atkin Audrey
Published: January 2023

Candida albicans is a polymorphic fungus that can cause systemic disease in immunocompromised and transplant patients, but is maintained as a commensal in healthy adults mainly through the actions of innate immunity. At low cell density, C. albicans secretes the quorum-sensing molecule, farnesol (FOH) which acts as a virulence factor. Our previous work indicates that FOH alone promotes chemotaxis of macrophages (MΦ) and synergizes with the TLR-2 agonist zymosan to promote MΦ cytokine secretion in vitro. We next determined whether FOH could act similarly to promote inflammatory responses in vivo. After intra-peritoneal injection, FOH increased the numbers of MΦ and neutrophils in the peritoneal cavity compared to untreated or thioglycollate treated mice. FOH treatment increased IL-6 and CCL2 mRNA in peritoneal exudate cells consistent with MΦ activation. In addition, cell surface expression of MHC class II and co-stimulatory molecules on dendritic cells in the peritoneum were higher in FOH injected mice compared to controls, suggesting FOH may prime antigen presenting cells for T cell activation. Currently we are investigating whether FOH can act as an adjuvant to promote antigen specific, cell mediated or antibody mediated immunity or a combination of both types. Defining the immunostimulatory properties of FOH, a potentially novel adjuvant, will improve strategies for vaccine design that promotes robust immunity against a variety of infectious diseases.
Use of heterodimeric IL-15 in immune therapeutic regimens against HIV-1 or SIV infection (VAC8P.1048)
Authors: Pavlakis George , Bergamaschi Cristina , Valentin Antonio , Kulkarni Viraj , Jalah Rashmi , Rosati Margherita , Alicea Candido , Sowder Raymond , Chertova Elena , Felber Barbara
Published: January 2023

Interleukin-15 (IL-15) promotes the growth of NK and memory CD8 T cells, which makes it an attractive candidate in immune therapeutic strategies to eradicate HIV-1 infection. We have shown that the relevant IL-15 molecule in vivo is the heterodimer (hetIL-15) composed of IL-15 chain and the so-named IL-15 Receptor alpha (IL-15Ra) chain. We developed expression vectors producing high levels of bioactive hetIL-15. We have also developed stable cell lines overproducing IL-15 heterodimers. SIV-infected and uninfected rhesus macaques were treated with these DNA plasmids by electroporation. In addition, SHIV-infected macaques received repeated subcutaneous injections of purified hetIL-15 protein or placebo. Electroporation of plasmid DNA resulted in increased levels of circulating IL-15, which typically peaked between day 4 and 5 in all the animals. Similarly, treatment with hetIL-15 protein gave sustained high cytokine plasma levels without any significant changes in plasma VL or any other adverse effects. In both studies, increased levels of IL-15 resulted in early decrease in the percentage of NK cells, probably linked to their mobilization out of the bloodstream, and preferential accumulation of CD8+ T lymphocytes with effector memory phenotype. In conclusion, the combination of CD8 effector cells expansion and lack of viral activation makes IL-15 an ideal component in immune therapeutic strategies for the treatment and potential eradication of HIV-1 infection.
Novel vaccination of supplementing live attenuated influenza vaccine with M2 ectodomain tandem repeat to confer universal protection (VAC8P.1051)
Authors: Lee Young-tae , Kim Min-Chul , Ko Eun-Ju , Lee Yu-Na , Kim Ki-Hye , Kwon Young-Man , Hwang Hye Suk , Lee Youri , Jung Yu-Jin , Kang Sang
Published: January 2023

Current influenza vaccines including live attenuated influenza vaccine (LAIV) do not provide cross-protection against drift and potential pandemic strains. We hypothesized that supplementing LAIV with a highly conserved protective antigenic target M2 ectodomain (M2e) would confer broad cross-protection by inducing humoral and cellular immune responses to conserved antigenic targets (M2e, NP), overcoming strain-specific protection of current vaccines. Intranasal vaccination with LAIV (A/NL/09/H1N1) supplemented with tandem repeat M2e containing virus-like particles (M2e5x VLPs) induced M2- and H1N1 virus-specific antibodies. Upon heterosubtypic challenge with pandemic potential rgA/H5N1 virus, we found that M2e5x VLP supplemented LAIV immune mice showed significantly improved heterosubtypic protection by preventing weight loss and lowering lung viral titers compared to the LAIV alone group inducing poor heterosubtypic immunity. Further mechanistic studies on heterosubtypic immunity suggest that recall T cell responses to M2e and nucleoprotein as well as systemic and mucosal M2e antibodies were found to be major heterosubtypic immune correlates. Therefore, this study demonstrates a novel vaccination strategy to confer universal protection by supplementing current strain-specific vaccines with a highly conserved cross protective antigenic target M2e5x VLPs.
Therapeutic platform for immunotherapy of Chagas disease (VAC9P.1106)
Authors: Konduri Vanaja , Liang Dan (Vivian) , Halpert Matthew , Levitt Jonathan , Hotez Peter , Jones Kathryn , Decker William
Published: January 2023

Chagas Disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a leading cause of heart disease and sudden cardiac death in Latin America, disproportionately affecting people living in resource-poor areas. Approved treatment is limited to two drugs, benznidazole and nifurtimox, which have poor efficacy in the chronic stage of disease as well as severe side effects. Thus, there is an urgent need for alternative therapies to treat Chagas Disease. Pre-clinical models have demonstrated that antigen specific CD8+IFNγ+ responses are essential for reducing parasite burdens, increasing survival, and decreasing cardiac pathology in both the acute and chronic phases of Chagas. In the present study, we developed an adenovirus-based gene transfer cassette that was used therapeutically to prevent or delay the cardiac complications that result from infection with T. cruzi. The therapeutic cassette, containing T. cruzi Tc24 and TSA1 antigens and a dominant-negative isoform of the SHP-1 inhibitory phosphatase, mediated development of antigen-specific CD8+IFNγ+ cells when ex vivo-transduced dendritic cells were administered to Balb/c mice. Additionally, the vaccine platform reduced parasite burdens, increased survival, and reduced cardiac pathology in a Balb/c model of live T. cruzi infection. The data suggest that immunotherapeutic approaches might be successful in addressing the unmet medical need for improved Chagas treatments.
Azithromycin inhibits IL-1 secretion and non-canonical inflammasome activation (VAC9P.1110)
Authors: Gualdoni Guido , Lingscheid Tilman , Schmetterer Klaus , Hennig Annika , Steinberger Peter , Zlabinger Gerhard
Published: January 2023

Deregulation of inflammasome activation was recently identified to be involved in the pathogenesis of various inflammatory diseases. Although macrolide antibiotics have well-established immunomodulatory properties probably involved in their clinical effects, studies on their impact on this pathway are missing. Comparing the influence of macrolides on cytokine induction in human monocytes, we found that azithromycin, but not clarithromycin or roxithromycin, specifically inhibited IL-1β secretion upon LPS stimulation whereas no effects were observed after flagellin stimulation. Blockage of azithromycin’s transmembrane transport abolished the cytokine-modulatory effect, indicating intracellular azithromycin accumulation to be causative for diverging effects. Consistent with IL-1β inhibition restricted to LPS stimulation, we found impaired induction of the intracellular LPS-sensing caspase-4 whereas NF-κB signalling was unaffected. Substantiating our in vitro data, azithromycin specifically dampened IL-1β levels and enhanced survival in a murine endotoxin sepsis model. Overall, we provide first evidence of a differential impact of macrolides on the inflammasome/IL-1β axis, which might be of relevance in inflammasome driven diseases like COPD or asthma.
Fc receptor γ chain plays a critical role in lung viral clearance and reducing vaccine-enhanced disease in mice immunized with respiratory syncytial virus vaccines (VAC9P.1063)
Authors: Hwang Hye suk , Lee Young Tae , Kim Ki-Hye
Published: January 2023

Vaccine-enhanced respiratory disease (ERD) is well-known for respiratory syncytial virus (RSV) after vaccination with formalin inactivated RSV (FI-RSV). Induction of Th2 type immune responses and deposition of immune complexes have been suggested to be possible mechanisms. We hypothesized that Fc receptor γ chain (FcRγ) would play a role in conferring protection against RSV after vaccination and that priming with Th1 inducing vaccine would prevent ERD. Virus-like particles presenting RSV fusion protein (F VLP) were found to induce Th1 type immune responses. Wild type (WT) mice and FcRγ KO mice were immunized with FI-RSV or primed with F VLP followed by FI-RSV boost (F VLP/FI-RSV). RSV specific serum IgG and isotype antibody levels were similar in both WT and FcRγ KO mice after RSV vaccination. However, F VLP/FI-RSV FcRγ KO mice showed lower levels of RSV neutralizing antibody titers and IFN-γ secreting CD8+ T cells, higher lung viral titers and more body weight loss compared to F VLP/FI-RSV WT mice. Heterologous F VLP/FI-RSV immunization induced Th1 type humoral and cellular immune responses, no overt sign of ERD in the bronchoalveolar lavage airways in both WT and FcRγ KO mice compared to homologous FI-RSV immunization. The results in this study support a critical role of FcRγ in lung viral clearance and diminishing ERD. More importantly, a strategy of priming with Th1 inducing vaccine might be a promising approach to induce protection against RSV and to prevent ERD.
Preclinical evaluation of a trivalent vaccine for treating/preventing candidal disease (VAC9P.1066)
Authors: Schmidt Clint , DeMontigny Elizabeth , Lizakowski Mary , Zacher Erica , Hoeg Jesse , Cochrane Terrence , Hennessey John
Published: January 2023

Multiple-antigen vaccines are an increasing focus of biotherapeutic development targeting infectious disease. Three recombinant antigens, based on Candida albicans proteins, have independently induced protection in animal models of candidal infections; agglutinin-like sequence 3 (rAls3), hyphally regulated protein 1 (rHyr1), and secreted aspartyl proteinase 2 (rSap2). However, a trivalent vaccine with all 3 antigens has not been previously evaluated. Here we present murine immune data for monovalent and multivalent vaccine formulations, with and without aluminum hydroxide (AlOH). IgG titers are induced against each antigen in the trivalent vaccine, and all are higher with AlOH. Interference is seen with anti-rAls3 IgG titers when equal amounts of all 3 antigens are used. This interference is abrogated by decreasing rHyr1 and rSap2 amounts relative to rAls3. Dose-dependent IL-17A splenocyte responses were detected against rAls3 and rHyr1, but not rSap2, in monovalent formulations. With trivalent formulations, Als3- and Hyr1-specific IL-17A production increased additively in magnitude and numbers of mice responding and at lower doses compared to monovalent formulations. These data support that robust immune responses can be induced by all 3 antigens in a trivalent vaccine, which may improve efficacy for some or all disease indications by targeting multiple facets of the candidal pathogenesis pathway, including adhesion, invasion and dissemination.
TCR gene expression in lymphoid and mucosal tissues of Xenopus laevis (VET1P.1122)
Authors: Geibel Gretchen , Kongprachaya Yuthana , Appasamy Pierette
Published: January 2023

γδ T cells are a small subset of T cells that are considered to function as a bridge between innate and adaptive immunity. Because these cells are relatively few in number in most laboratory animals, except during the fetal period and in mucosal tissues, it is difficult to clearly delineate the functions of γδ T cells. In order to begin to characterize the γδ T cell populations in Xenopus laevis, previous work from this lab established a semi-quantitative RT-PCR method of analysis of TCR γ and TCR β gene expression in X. laevis thymus and spleen. We have extended these studies to evaluation of expression of TCR γ, β, α and δ transcripts in the thymus and spleen as well as the skin, intestine and lungs of young adult X. laevis. Based on evaluation of TCR transcript levels by RT-PCR, γδ T cells are likely to be abundant in the mucosal tissues evaluated, and the levels of TCR γ are higher in the X. laevis thymus and spleen than expected for adult humans or mice. We suggest that X. laevis is a γδ T cell “high” organism, perhaps related to the position of frogs as relatively early in the evolution of the adaptive immune response. Evaluation of expression of other T cell-related genes including the RAG genes and other genes involved in T cell development and function is ongoing. These results point to the use of X. laevis as a uniquely powerful laboratory animal model for studies of the evolution, development and function of γδ T cells.
Investigating distinct immunization routes in zebrafish on mucosal immunity (VET2P.1133)
Authors: Criscitiello Michael , Young Matthew , Troy Savannah , Cory Hope , McCauley Naomi , Al-Kahtani Sibba , Morgan Thomas , Scholtz Sheridan , Li Ellen , Ali Hasanain , Bains Sarab , Briles Brenna , Chang Jonathan , Cruz-Vespa Diego , Diaz Navarro Jean , Eberle Brady , Gonzalez Allison , Marvin Sophia , Nelson Mary , Roubion Courtney , Watts Jiang , Chen Pat , Basler Julie , Minckler Zachary , Wu James
Published: January 2023

Model organisms such as Danio rerio are used to explore physiological reactions to stress and disease. Immune response is measured by quantifying lymphocyte presence and the production of mRNA encoding different immunoglobulin proteins. Since the method through which D. rerio is exposed to an antigen affects its immune response and the degree to which immunity is developed, we will compare the immune response after visceral or mucosal introduction of dinitrophenol hapten conjugated to Keyhole Limpet hemocyanin (DNP-KLH) in D. rerio, for which there is no published data. We will either combine DNP-KLH with Freund’s incomplete adjuvant and inject the mixture into the fish (100 uL at 1 mg/mL) or combine DNP-KLH with cholera toxin and introduce the mixture to their water (35 mg/L). Tissue samples will be collected from the immunized fish and two control groups of fish at weekly points throughout the experiment and will be analyzed for lymphocyte versus granulocyte ratios and expression of IgM, IgZ-1, and IgZ-2 levels via qPCR. We expect an increase in lymphocyte percentages for both immunized groups compared to the control groups, with a higher lymphocyte ratio in the mucosal group than the visceral group. Furthermore, we expect more mucosal IgZ isotypes in the mucosal group and higher levels of the IgM, IgZ-1, and IgZ-2 antibodies in the visceral group than in the mucosal group. For future experiments, we intend to test broader data sets, including exposure to multiple antigens.
Ion efflux and influenza infection trigger NLRP3 inflammasome signaling in human dendritic cell (VIR5P.1148)
Authors: Fernandez Melissa , Miller Elizabeth , Krammer Florian , Greenbaum Benjamin , Bhardwaj Nina
Published: January 2023

The NLRP3 inflammasome is an essential intracellular mediator of anti-viral immunity. In murine dendritic cells (DCs) this complex responds to a wide array of signals, including ion efflux and influenza A virus (IAV) infection, to activate caspase-1 mediated proteolysis of interleukin (IL)-1β and IL-18 into biologically active cytokines. However, the presence and function of the NLRP3 inflammasome in human DCs in response to various triggers, including viral infection, has not been clearly defined. Here, we delineate the contribution of the NLRP3 inflammasome to the secretion of IL-1β, IL-18, and IL-1α by human DCs. Activation of the NLRP3 inflammasome in human DCs by various synthetic activators resulted in the secretion of bioactive IL-1β, IL-18, and IL-1α, and induction of pyroptotic cell death. Cellular IL-1β release depended on potassium efflux, and the activity of proteins NLRP3 and caspase-1. Similarly, IAV infection of DCs resulted in priming and activation of the NLRP3 inflammasome and secretion of IL-1β and IL-18 in an M2 and NRLP3 dependent manner. The magnitude of priming by IAV varied amongst different strains and inversely corresponded to type I IFN production. To our knowledge, this is the first report describing the existence and function of the NLRP3 inflammasome in human DCs, and the ability of IAV to prime and activate this pathway in human DCs with important implications for anti-viral immunity and pathogenesis.
LIGHT programs activated CD8 T cells to survive and become memory cells in response to a viral pathogen (VIR5P.1150)
Authors: Desai Pritesh , Tahiliani Vikas , Abboud Georges , Hutchinson Tarun , Ware Carl , Croft Michael , Salek-Ardakani Shahram
Published: January 2023

CD8 memory T cells play a critical role in protection against repeated exposure to respiratory viruses. Memory T cell development in the lung is still not fully understood in terms of the nature and source of the molecular signals that establish and maintain the memory state. LIGHT is a tumor necrosis factor family member expressed by immune cells such as activated T cells, dendritic cells, monocytes and granulocytes. It is known to interact with two receptors HVEM & LTβR, leading to differential outcomes. However, the role of LIGHT expressed specifically by CD8 T cells during respiratory viral infection is not known. Using adoptive transfer of LIGHT-deficient CD8 TCR-transgenic T cells responding to Ag in the context of respiratory vaccinia virus (VACV) infection, we found that LIGHT signaling in CD8 T cells had a small impact on the level of primary expansion, with at most 30-40% fewer CD8 T cells accumulating in the lungs at the peak of response to VACV infection. In striking contrast, very few effector CD8 T cells that lacked LIGHT survived the contraction phase to differentiate into long-lived memory cells in the lungs. Furthermore, we found that HVEM, but not LTβR, functions as a trans-activating binding partner for LIGHT expressed on CD8 T cells. These results underscore the importance of LIGHT in the memory CD8 T cell development and suggest that targeting LIGHT-HVEM interaction will help in designing CD8 T cell based vaccines against respiratory viruses.
Lymphotoxin β receptor signaling promotes influenza-induced lung damage (VIR5P.1151)
Authors: Spencer Cody , Koroleva Ekaterina , Muraoka Wayne , Blackman Marcia , Tumanov Alexei
Published: January 2023

Lymphotoxin β receptor (LTβR) has been shown to play a critical role in protection against mucosal bacterial pathogens in the gut. Surprisingly, we found that genetic or biochemical inhibition of LTβR signaling confers protection against highly pathogenic H1N1 influenza virus infection. Consistent with amelioration of disease severity, LTβR-deficient mice displayed reduced expression of pro-inflammatory cytokines, reduced inflammatory dendritic cells, and drastically reduced numbers of antigen specific CD8+ T cells in the lung. Further, LTβR-deficient mice showed attenuated viral loads during the peak of viral replication. Previous studies by our group have highlighted a potential role of LT signaling in the regulation of mucin expression in the gut. Further, increased mucin secretion in the lung is known to correlate with attenuation of influenza virus replication. Immunohistochemical and gene expression analysis revealed a marked increase in goblet cell hyperplasia and MUC5AC secretion in the lungs of influenza infected LTβR-deficient mice relative to wild type mice. Additionally, treatment of infected wild type mice with an agonistic anti-LTβR antibody resulted in almost complete inhibition of MUC5AC mRNA expression in the lung. Taken together, our data suggest that LT signaling negatively regulates mucin production in response to respiratory viral infection leading to increased viral burden, lung pathology, and death.
Monoclonal antibodies recognizing a putative immunodominant epitope in the hemagglutinin stalk do not enhance influenza A virus mediated fusion activity (VIR5P.1156)
Authors: Mullarkey Caitlin , Tan Gene , Nachbagauer Raffael , Miller Matthew , Palese Peter
Published: January 2023

The discovery of broadly neutralizing antibodies (Abs) which bind to the conserved hemagglutinin (HA) stalk has led to renewed interest in the development of a universal influenza virus vaccine. In swine, there have been isolated reports of vaccine-induced enhancement of disease in heterologous vaccination and challenge studies. It has been suggested that Abs recognizing the HA stalk promote virus fusion activity, contributing to pathology in pigs. Given that robust data in mouse and ferret models suggests a protective role for stalk Abs, here we examined the relationship between HA-stalk binding Abs and fusion. We generated mouse monoclonal Abs C4 and C6, which recognize a previously identified minimal epitope in the HA stalk. These Abs lack hemagglutination inhibition activity and did not inhibit or enhance virus-mediated fusion in vitro. In a mouse challenge model, we assessed the consequences of pre-exposure administration of a C4/C6 cocktail. No significant differences were observed in animals receiving C4/C6 in morbidity and mortality relative to naïve and isotype controls. We also probed the sera from a cohort of individuals who received an H5N1 vaccine. Although we detected baseline levels of Abs to the minimal stalk epitope associated with increased fusion activity in swine, titers did not increase post-vaccination. Together, our data suggests that stalk Abs do not enhance virus-mediated fusion and thus should not impede progress toward a universal influenza vaccine.
Secondary heterosubtypic influenza infections result in radically different T cell immunodominance hierarchies and clinical outcomes (VIR5P.1137)
Authors: Duan Susu , Meliopoulos Victoria , McClaren Jennifer , Guo Xizhi , Sanders Catherine , Smallwood Heather , Webby Richard , Schultz-Cherry Stacey , Doherty Peter , Thomas Paul
Published: January 2023

When human populations face a novel subtype influenza A virus (IAV) with pandemic potential, the role of CD8+ cytotoxic T lymphocyte (CTL) responses targeting epitopes conserved or cross-reactive between the novel and previously encountered IAVs can be crucial to limit disease severity. To discover immune correlates of protective efficacy, we used a model of H7N9 secondary infections in mice primed by prior H1N1or H9N2 infection. Each priming case significantly, but variably, reduced disease morbidity and mortality, virus load, and time to virus clearance after H7N9 challenge. The sizes of the respective memory CTL pools were the best predictors of protective efficacy. The secondary CTL responses were characterized by earlier, significantly greater airway infiltration of H7N9 virus-specific CTLs but with distinct epitope immunodominance hierarchies among the dominant KbPB1703, DbPA224, and DbNP366 epitopes for each priming case. The epitope conservation between the priming and challenge viruses clearly influenced but alone did not consistently predict the immunodominance. The receptor repertoire of CTLs targeting a crossreactive and non-crossreactive DbNP366 epitope variant were characterized to reveal the nature of epitope cross-reactivity. These findings will contribute to understanding human CTL responses and rational design of CTL-mediated vaccines, where protective efficacy and immunodominance hierarchies may be highly sensitive to the immunological history of hosts.
A model to non-invasively image respiratory viral infections in immunocompromised mice (VIR5P.1139)
Authors: Mostafa Heba , Srinivasan Ashok , Russell Charles
Published: January 2023

To enable real time monitoring of respiratory viral infections in living mice, we have developed a non-invasive bioluminescence imaging system for Sendai virus. Sendai virus is the murine form of human parainfluenza virus 1, a leading respiratory pathogen in children that commonly progresses to the lower respiratory tract (LRT) in immunocompromised patients with high mortality rate. Using this system, we are able to non-invasively monitor the spread of viral infection into the lower respiratory tract (LRT) based on the immune status of the host. In our ongoing studies, we demonstrate that inoculation of 7000 PFU of Sendai virus in 5ul volume mimics infection after natural contact transmission and does not penetrate well into the LRT before clearance. Immunosuppression by Dexamethasone, Cylophosphamide, or both drugs leads to a delay in clearance of infection by 1, 4, and 6 days respectively. Moreover, immunosuppression results in higher LRT viral loads. We are presently dissecting the relationship between local and systemic immune responses that result in enhanced LRT infection and clearance based on drug treatment protocols. An understanding of mechanisms by which immunosuppression leads to LRT disease may assist patients with malignancy, receiving hematopoietic stem cell transplantation, among other immunosuppressive conditions, in prevention and treatment of pneumonia that commonly develops after respiratory viral infections.
Broadly-neutralizing anti-influenza virus antibodies: Enhancement of neutralizing potency in polyclonal mixtures and IgA backbones (VIR5P.1141)
Authors: Miller Matthew , He Wenqian , Mullarkey Caitlin , Duty J. , Moran Thomas , Palese Peter
Published: January 2023

The recent discovery of broadly-neutralizing antibodies that target the hemagglutinin stalk domain has renewed hope that the development of “universal” influenza virus vaccines may be within reach. To this end, we have examined the functions of hemagglutinin stalk-binding antibodies in an endogenous setting - as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior in vitro, but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. Additionally, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk-specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context, and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during “universal” influenza virus vaccine design.
Type 1 interferon primes naïve CD8 T cells to respond to lower concentrations and affinities of cognate ligands (VIR6P.1178)
Authors: Urban Stina , Welsh Raymond
Published: January 2023

During an acute virus infection, antigen-specific CD8 T cells undergo clonal expansion and differentiation into effector cells in order to control the infection. Studies have shown that CD8 T cells need to divide at least once before they acquire specific effector functions, including the ability to directly lyse target cells and produce certain cytokines. However, recently we have shown that pre-exposure of the host to type I interferon (IFN) sensitizes CD8 T cells to rapidly acquire effector function without the need to divide. The IFN-primed naïve CD8 T cells produced IFNγ, enhanced TNF and degranulated upon stimulation with high affinity cognate antigen. The threshold required for IFN-induced sensitized CD8 T cells to become activated and produce effector functions was reduced, as primed CD8 T cells produced cytokines in response to lower affinity ligands and lower concentrations of cognate ligands. In addition, an in vivo cytotoxicity assay showed that primed naïve CD8 T cells specifically lysed target cells. The reduced requirement of naïve CD8 T cells to produce effector functions if pre-exposed to IFN-induced environments may increase the overall number of naïve T cells able to respond to any given antigen. IFN-primed increased numbers and response of naïve T cells may help to control antigen load during an acute infection.
Mitochondrial antiviral signaling molecule-mediated mitochondrial calcium regulates CD8 T cell function during acute virus infection (VIR6P.1163)
Authors: Thomas Sunil , Huang Albert , Loo Yueh-Ming , Wilkins Courtney , McMillan Aimee , Gale Michael
Published: January 2023

MAVS is a crucial adaptor molecule through which RIG-I-like receptors (RLR)s signal to mediate downstream innate antiviral and inflammatory responses. However a role for MAVS in directly programming adaptive immunity has not been defined. Here we identified a cell-intrinsic role for MAVS in programming CD8 T cells following virus infection. We utilized a mouse model of acute Lymphocytic choriomeningitis virus (LCMV) infection to define the actions of adoptively transferred WT or MAVS deficient CD8 T cells. Lack of MAVS lead to defects in CD8 T cell expansion as well as function as measured by cytokine and cytotoxic T cell effector molecule production but these parameters were not deficient in double knockout mice lacking the RLRs MDA5 and RIG-I. We identified defects in total cytoplasmic calcium, mitochondrial calcium as well as mitochondrial membrane potential in MAVS-/- CD8 T cells in vivo. We found that the inflammatory conditions of LCMV -induced cell proliferation place a metabolic load on CD8 T cells and their mitochondria such that the absence of MAVS lead to defects in mitochondrial calcium independently of the RLRs. Importantly, the MAVS defect associated with defective nuclear factor of activated T cells c1 (NFATc1) activation and consequently decreased IL-2 production. Our study assigns a cell-intrinsic role for MAVS in regulating adaptive immunity following viral infection by means of regulating mitochondrial calcium and thereby affecting T cell fitness.
Soluble Tim-3 is shed from CD8+ T cells by the sheddase ADAM10, is increased in plasma during untreated HIV infection, and correlates with HIV disease progression (VIR6P.1165)
Authors: Clayton Kiera , Douglas-Vail Matthew , Rahman A. , Medcalf Karyn , Xie Irene , Chew Glen , Tandon Ravi , Lanteri Marion , Norris Philip , Deeks Steven , Ndhlovu Lishomwa , Ostrowski Mario
Published: January 2023

Chronic HIV infection results in a loss of HIV-specific CD8+ T cell effector function, termed “exhaustion”, mediated, in part, by the co-inhibitory receptor T cell immunoglobulin mucin domain-3 (Tim-3). Like many other receptors, a soluble form of this protein has been described in human blood plasma. However, soluble Tim-3 (sTim-3) is poorly characterized and its role in HIV disease is unknown. Here we show that Tim-3 is shed from the surface of responding CD8+ T cells by the matrix metalloproteinase, ADAM10, producing a soluble form of the co-inhibitory receptor. Despite previous reports in the mouse model, no alternatively spliced, soluble form of Tim-3 was observed in humans. Shed sTim-3 was found in human plasma, and was significantly elevated during early and chronic untreated HIV infection, but was not found differentially modulated in HAART treated HIV-infected subjects or in elite controllers, when compared to HIV-uninfected subjects. Plasma sTim-3 levels positively correlated with HIV viral load and negatively correlated with CD4 counts. Thus, plasma sTim-3 shedding correlated with HIV disease progression. Despite these correlations, we found that shedding Tim-3 did not improve the function of CD8+ T cells in terms of IFN-γ production or prevent their apoptosis through galectin-9. Further characterization studies of sTim-3 function are needed to understand the contribution of sTim-3 in HIV disease pathogenesis with implications for novel therapeutic interventions.
Peptidomic analysis of type-1 diabetes associated HLA-DQ molecules and impact of HLA-DM editing (APP5P.108)
Authors: Zhou Zemin , Reyes-Vargas Eduardo , Escobar Hernando , Chang Kuan , Rockwood Alan , Delgado Julio , He Xiao , Jensen Peter
Published: January 2023

HLA-DM is critical in editing peptide repertoires presented by APC, thus influencing the selection/activation of CD4+ T cells. We previously reported that type-1 diabetes (T1D) associated HLA-DQ2, 8 & DQ2/8 molecules (DQs) are relatively resistant to DM editing of the CLIP peptides. In this study, we further studied DM sensitivity of T1D-associated DQs by peptidomic analysis. Compared to a T1D-protective DQ6, the percentages of shared peptides among T1D-associated DQs were significantly higher. There were greater numbers of nested peptide sets and long peptides (>20 aa) eluted from T1D-associated DQs. Predicted peptide binding motifs of DQ2, 8 & 2/8 shared charged anchor residues, while hydrophobic anchors were present in DQ6 peptides. Competition binding assays comparing the eluted peptides from DM sensitive, dependent, or resistant groups showed that the binding affinities of the DM sensitive peptides were significantly lower than that of CLIP1 peptide. The increased sDQ8-CLIP1 complex stability, with the intrinsic half-life of 58 h measured by fluorescence polarization assay, was 8 times longer than that of sDQ6-CLIP1 (7 h), further supporting the DM resistance of T1D associated DQs. Collectively, T1D associated DQs share distinct characteristics, including binding of peptides from known T1D auto-antigens. Our new data further support the hypothesis that DM resistance in T1D associated DQs affects the peptide repertoires, thus contributing to the pathogenesis of T1D.
Varying structural environments of an ectopic epitope influence CD4+ T cell immunogenicity (APP5P.117)
Authors: Rust Blake
Published: January 2023

Preliminary studies have indicated that epitopes adjacent to large flexible loops of antigens are often specialized for the production of interferon gamma (IFNγ). Variants of the HIV envelope glycoprotein gp120 protein were created that feature the ectopic CD4+ T-cell epitope 2W1S swapped in at two points in a variable loop (V4) of gp120. The variants (T38 and T39) were designed to place the epitope in different environments of conformational “depth” within gp120. Both antigen sequence and antigen structure contribute to CD4+ T cell immunodominance through their effects on MHCII binding and antigen processing, respectively. In order to isolate one of these two factors, antigen variants with the same ectopic CD4+ T cell epitope at different structural locations within the protein were created to eliminate the variation in MHCII binding affinity. We hypothesize that 2W1S-specific CD4+ T cells that arise from T38 immunization will display increased IFNγ expression as compared to mice immunized with T39 due to the conformationally “shallow” context of the epitope. However, any interpretation of epitope immunogenicity must take into account the global stability of the protein, and not just the local context of epitope in the native gp120. Preliminary studies indicate that T39 adopts a conformation that is more similar to that of native gp120 as compared to T38. Current studies are investigating immunogenicity of both ectopic and endogenous epitopes by multiple assays.
Processing of native or citrullinated myelin oligodendrocyte glycoprotein peptides in B cells is affected by EBV infection: implications for multiple sclerosis (APP5P.118)
Authors: Morandi Elena , Jagessar Anwar , Dijkman Karin , t` Hart Bert , Gran Bruno
Published: January 2023

Multiple Sclerosis is a neurodegenerative disease with an immune-mediated pathogenesis in which Epstein Barr Virus (EBV) is a risk factor. EBV infects human B cells, in which it becomes latent. Our hypothesis is that infection renders B cells potent APC for autoreactive T cells, through influences on intracellular proteolysis and post-translational modifications of self antigens. The aim of this study is to assess the effect of EBV infection of B cells on processing of myelin oligodendrocyte glycoprotein (MOG). We investigated the processing of recombinant extracellular MOG and of the immunodominant peptides MOG1-20, MOG35-55 and through SDS PAGE gel analysis in the presence or absence of Cathepsin inhibitors. We also studied the effect of MOG35-55 citrullination in position 41 and 46. In addition, we determined the proteolytic activity of Cathepsin G and H by activity assay. We found that EBV infection increases the activity of Cathepsin G and H, leading to increased degradation of MOG35-55 by B cells. However, infection also rescues rhMOG from total degradation. By contrast, inhibition of Cathepsin G or citrullination of Arg 46 abrogated the degradation of MOG35-55. These results indicate that citrullination of an immunodominant peptide of MOG protects it from destructive processing in EBV infected B cells. This could facilitate presentation of a disease-relevant myelin autoantigen that may be involved in disease induction and progression.
Peptide sequence determinants in HLA-DM susceptibility (APP5P.105)
Authors: Reyes-Vargas Eduardo , Barker Adam , Zhou Zemin , He Xiao , Jensen Peter
Published: January 2023

HLA-DM catalyzes peptide exchange reactions in MHCII molecules. DM functions by accelerating the peptide loading process and editing the epitopes that are presented to CD4 T cells. The catalytic potency of DM differs by MHCII-peptide complex such that some are variably edited while others are immutable. To date, the catalytic mechanism and peptide sequence determinants of susceptibility to DM editing remain unresolved. In this study, we use an FP assay to measure the effect of DM on the binding kinetics of HLA-DR1 to peptides with anchor residues of neutral, disfavored, or favored amino acids. We provide evidence for a model in which the peptide sequence determines the “effective affinity” of an MHCII-peptide complex for DM and specifies DM catalytic potency. Our results support a mechanism whereby interactions between peptide and MHC regions far from the DM contact interface can alter the effective binding affinity of the complex. We show that the peptide P1 anchor impacts kcat, which implies that the peptide N-terminus may occupy the binding groove in the “functional” catalytic complex. We show that association rates are not sensitive to peptide sequence, indicating that DM has little effect on the association of peptide to empty class II molecules. Together, these findings provide insight into the DM catalytic mechanism and suggest that stable interactions with DM at the contact surface are linked to unstable interactions in the peptide-binding groove and vice versa.