bluuebunny/crossref_metadata_2025 huggingface dataset:
A quantitative explanation of vaccination for disease (P4315)
Authors: McAfee Megan , Johnson Philip , Antia Rustom , Blattman Joseph
Published: January 2023

Vaccination remains one of the most effective means for preventing infectious diseases. Vaccines encoding T cell epitopes have been proposed as a strategy for improving the efficacy of vaccines for chronic viral infections such as HIV. However, previous studies in mouse models of chronic viral infection have shown that vaccines eliciting narrowly focused T cell responses can result in enhanced disease. We have developed a quantitative framework for understanding how varying the precursor frequency of memory virus-specific CD8 T cells in naïve mice affects the balance between protection and pathology during subsequent infection with Lymphocytic Choriomeningitis virus. We have found that an intermediate precursor frequency of memory CD8 T cells results in maximum disease during subsequent infection. The transfer of a high number of memory CD8 T cells results in protection while low numbers undergo T cell exhaustion resulting in chronic infection. Enhancement of disease is not dependent on the breadth of the T cell response and is partially due to T cell production of TNF and IFNγ. In addition, decreased virus inoculum resulted in a lower precursor frequency of T cells needed to enhance disease. This suggests that for any given T cell response to vaccination there exists a viral dose that may result in enhanced disease. These studies highlight the importance of engaging multiple arms of the immune system during vaccination to maximize protection while minimizing pathology.
Poly gamma glutamic acid-chitosan nanogel dramatically enhances the protective efficacy and heterosubtypic cross-reactivity of H1N1 pandemic influenza vaccine in mice and ferrets (P4281)
Authors: Poo Haryoung
Published: January 2023

The global outbreak of 2009 H1N1 influenza pandemic emphasized the need for an effective vaccine adjuvant. Here, we examined the adjuvant efficacy of γ-PGA/chitosan nanogel (PC nanogel) for the 2009 H1N1 pandemic influenza vaccine antigen in mice and ferrets. Compared with alum, PC nanogel dramatically enhanced the protective efficacy of this vaccine, with animals exhibiting increased pandemic HA-specific antibody production, higher neutralization activity (HI titer), and earlier virus clearance from lung tissues after homologous (pandemic H1N1) and heterosubtypic (H3N2) viral challenges. In particular, approximately 4-fold higher influenza-specific cell-medicated immunity was observed in mice vaccinated with PC nanogel compared to alum, perhaps due to the dramatic enhancement of antigen uptake by immature dendritic cells. The results from T cell-depletion experiments indicated that the heterosubtypic cross-reactivity of PC nanogel was mediated by CD8+ T cells. Additionally, the long-lasting protection (100% at 6 months) against virus challenge after immunization and a one-twelfth dose-sparing effect were observed following vaccination with PC nanogel-adjuvanted vaccine but not with alum-adjuvanted vaccine. Together, these results strongly suggest that PC nanogel may be a promising candidate adjuvant for the influenza vaccine.
Mechanism for exploiting the complement 5a anaphylatoxin receptor on M cells by mucosal pathogen is closely related with that of mucosal immune induction (P3212)
Authors: Jang Yong-Suk , Kim Sae-Hae , Kim Ju , Choe Chung-Hyeon , Park Jisang , Lee Jeong-Chae , Kim Ji-Hae , Lee Kyung-Yeol
Published: January 2023

The mucosal epithelium is exposed to enormous amount of microorganisms containing commensal and pathogenic bacteria. Although many pathogens initiate their infection in mucosal epithelium and/or M cells of Peyer’s patch, detailed mechanism for the infection is poorly understood. We explored the possible survival mechanism of Yersinia enterocolitica in M cells in relation to crosstalk between C5aR and TLRs based on the observation that outermembrane protein H (OmpH) of Y. enterocolitica is one of the ligands for C5aR. Localization of C5aR and TLRs in human M-like cells and mouse M cells was confirmed during Y. enterocolitica infection. Also, regulation of cAMP- and/or autophagy-associated gene expression by Y. enterocolitica infection was characterized. Moreover, C5aR antagonist treatment reduced the survival of Y. enterocolitica in human M-like cells and the bacteria showed low infection efficiency in C5aR knock-out mouse. It is suggested that the OmpH of Y. enterocolitica could be used as M cell-targeting ligand in oral mucosal vaccination model. Collectively, we assume that the inhibition of cAMP expression by interaction between Y. enterocolitica and both C5aR and TLRs is closely associated with the bacterial survival in M cells and this interaction could be used as targeting of oral mucosal vaccine.
Th2-independent development of vaccine-enhanced disease associated with formalin-inactivated respiratory syncytial virus immunization (P3182)
Authors: Knudson Cory , Hartwig Stacey , Meyerholz David , Varga Steven
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants and there is currently no licensed RSV vaccine. Children immunized with a formalin-inactivated (FI) RSV vaccine during the 1960's exhibited exacerbated disease and increased mortality following a natural RSV infection. It is widely believed that a Th2-biased response directly contributes to the lung injury associated with FI-RSV vaccine-enhanced disease. This is in part due to the emphasis placed on the development of pulmonary eosinophilia noted in the autopsy reports from the FI-RSV vaccine trials. Furthermore, RSV challenge of FI-RSV-immunized mice results in extensive pulmonary inflammation including increased numbers of eosinophils and Th2 cytokine producing CD4 T cells. Depletion of CD4 T cells significantly ameliorates both airway resistance and weight loss associated with FI-RSV vaccine-enhanced disease. However, FI-RSV-immunized BALB/c mice genetically deficient in eosinophils elicit similar airway resistance, weight loss, and histopathology as compared to wild-type controls following RSV challenge. Moreover, FI-RSV-immunized STAT6-deficient mice exhibit significantly reduced Th2-associated responses, but similar airway resistance and weight loss as compared to wild-type controls following RSV challenge. Our results indicate that Th2 responses do not significantly contribute to critical disease parameters associated with FI-RSV vaccine-enhanced disease.
The effects of oral administration of silica nanoparticles on immunologic response to the food antigen (P3220)
Authors: Misato Kazuki , Yoshioka Yasuo , Uji Miyuki , Udaka Asako , Mori Takahide , Yamaguchi Manami , Hirai Toshiro , Yoshida Tokuyuki , Nabeshi Hiromi , Yoshikawa Tomoaki , Tsunoda Shin-ichi , Higashisaka Kazuma , Tsutsumi Yasuo
Published: January 2023

With the recent development of nanotechnology,nanomaterial (NM) with particle size below 100 nm have been developed and applied in various kinds of fields. For example, amorphous silica nanoparticles (nSP) have been already used in various foods as anticaking agents. Under this circumstance, there is increasing the importance of ensuring the safety of NM in foods. We previously revealed that nSP can penetrate the intestinal barrier, however,did not induce significant general toxicity. On the other hand, when we are exposed nSP, many food antigens are also exposed simultaneously.Thus, to ensure the safety of nSP, it is important to evaluate the effects of the oral exposure to nSP on immune response to food antigen. Here, we evaluated the effects of oral administration of nSP with a diameter of 30 nm on immunologic response to the food antigen. C3H/HeJ mice were orally administrated OVA or OVA plus nSP on days 0, 7, 14 and 21. Seven days after last administration, we measured OVA-specific antibody levels. Significant induction of OVA-specific IgG levels wasnot observed in OVA group compared with control. On the other hand, the mice administrated OVA plus nSP had significantly increased OVA-specific IgG levels compared with control groups. These results suggested that oral administration of nSP induced the abrogation of immune tolerance to food antigen. Currently, we are trying to evaluate the relationship between nSP and induction of food allergy.
Immunopathology in response to Vibrio parahaemolyticus infection. (P3222)
Authors: Parent Michelle , Read Kaitlin , Waters Stephanie , Guhl Shelby
Published: January 2023

Vibrio parahaemolyticus, is the most common cause of bacterial seafood-related illness in the United States. Infection results in a self-limiting gastrointestinal illness, wound infection or for those chronically ill, may lead to systemic infection and death. The CDC reports a 115% increase in laboratory confirmed cases of Vibrio species in 2012 as compared to 1996-1998 surveillance data, with an increase of 47% alone since 2011. A dearth of literature exists regarding characterization of host immunity to infection. Furthermore, a murine model of orogastric infection has not been available, prohibiting studies characterizing the host response to infection. Toward that end, employing a newly developed streptomycin-pretreatment orogastric model of infection, we have begun investigating host immunity to infection. Utilizing streptomycin pretreatment, we have established infection as confirmed by organism recovery from exposed tissue and by an influx of inflammatory cells to the site of infection as compared to control groups; therefore documenting immunopathology. Additionally, changes in mucin production and lactorferrin were also confirmatory of immunopathology. Lastly, we are investigating the pro-inflammatory response to infection by assessing IL-1β, IL-6, MIP-1α, MIP-1β. This novel orogastric murine model of infection will remove barriers, allowing for the identification of essential immune parameters required for controlling infection.
Vaginal type-II mucosa acts as an inductive site during the generation of primary CD8+ T cell mucosal immune responses (P3186)
Authors: Wang Yichuan , Sui Yongjun , Steel Jason , Morris John , Berzofsky Jay
Published: January 2023

It is widely believed that primary immune induction in type-II mucosa (vagina, mouth & cornea) occurs in the draining LNs due to a lack of mucosa-associated lymphoid tissue. In this process, naïve T cells located in the draining LNs are primed by antigen (Ag)-bearing dendritic cells migrating from the Ag-exposed mucosa. Primed T cells then travel to the mucosal site through the bloodstream. In contrast to this paradigm, we show that vaginal mucosa itself can act as an immune inductive site for generation of primary CD8+ T cell mucosal immunity. As evidence, we found that naïve CD8+ T cells routinely migrated to the female reproductive tract and that Ag-specific CD8+ T cells could be generated in the LN-deficient mice after intravaginal immunization. Further, the adoptively transferred naïve OT-1 CD8+ T cells were activated in the vaginal mucosa but not in the draining LNs at 24 hours after intravaginal immunization, even in the presence of FTY720, a drug blocking the egress of T cells from LNs. In addition, the Ag-bearing cells isolated from immunized vaginal mucosa were able to stimulate naïve TCR-transgenic RT-1 CD8+ T cells to secret IFN-γ and undergo proliferation. Finally, vaginal mucosa largely supported the expansion of Ag-specific CD8+ T cells. In conclusion, we present evidence for a new paradigm for primary CD8+ T cell immune induction in type-II mucosa of the vagina, one that occurs locally without the help of draining LNs or mucosa-associated lymphoid tissue.
HVEM is a TNF family receptor that is a crucial regulator of epithelial and lymphocyte responses in the mucosal immune system (P3192)
Authors: Kronenberg Mitchell , Shui Jr-Wen , Takahashi Daisuke , Larange Alexandre , Kim Gisen , Zahner Sonja , Vela Jose-Luis , Cheroutre Hilde
Published: January 2023

HVEM is a TNF super family receptor, and previously we showed that epithelial HVEM is required for innate immune responses to pathogenic bacteria in the intestine and lung, and that it acts by enhancing epithelial Stat3 activation (Nature 488:222, 2012). Now, we show that HVEM is also an important regulator of lymphoid cells in the mucosal immune response. IL-17 and IL-22 are protective cytokines after oral infection with Citrobacter rodentium. Mice deficient for HVEM have a reduced IL-22 response by innate lymphoid cells (ILC) in the colon lamina propria at early times after infection. The adaptive immune response is also affected, with reductions in IL-17 and IL-22 by colonic lamina propria CD4+ T cells at days 9-21 after infection. The role of HVEM in mucosal lymphoid cells is more important for controlling bacterial burden and pathology at later times after infection, with the HVEM-dependent epithelial response acting earlier. Also striking, however, is the strong decrease in all intestinal epithelial lymphocyte populations (IEL) in mice deficient for HVEM, at steady state, even in the absence of infection. This reflects a decreased proliferative potential of IEL when HVEM is not expressed. Collectively, our findings establish HVEM as a crucial regulator of the mucosal immunity, affecting IEL homeostasis, innate epithelial responses and innate and adaptive responses by lamina propria lymphocytes.
Inflammatory monocytes are critical for induction of a polysaccharide-specific antibody response to an intact bacterium (P3046)
Authors: Chen Quanyi , Snapper Clifford
Published: January 2023

Although inflammatory monocytes (IM) [CD11b+Ly6Chi] play important roles in cell-mediated host protection against intracellular pathogens, their impact on humoral immune responses to extracellular bacteria are unknown. IM, localized largely in the splenic marginal zone of naïve CD11b-Diphtheria toxin receptor (DTR) bone marrow chimeric mice, were selectively depleted following treatment with DT, including no reduction of peritoneal B1a or B1b cells, which express CD11b. Depletion of IM resulted in a marked reduction in the polysaccharide (PS)-specific, T cell-independent IgM and T cell-dependent IgG responses to heat-killed Streptococcus pneumoniae (Pn) with no effect on the Pn protein-specific IgG response, or on the PS- and protein-specific IgG responses to a pneumococcal conjugate vaccine. IM acted largely within the first 48 h to induce the subsequent PS-specific IgM and IgG response. Adoptive transfer of highly purified IM from WT mice into DT-treated CD11b-DTR mice restored the PS-specific Ig response to Pn. IM were phenotypically and functionally distinct from circulating CD11b+CD11clowLy6G-/C- cells (i.e. “blood DC”) previously described to play a role in Ig responses to Pn. These data are the first to establish a critical role for IM in the induction of an Ig response to an intact extracellular bacterium.
AFLP analysis of a natural population of native Hawaiian Honeycreepers (Hemignathus virens) reveals candidate markers for tolerance to avian malaria (Plasmodium relictum) (P3066)
Authors: Farias Margaret , Bianchi Kiara , Atkinson Carter , Ohora Donna , Txakeeyang Ann , Bonnel Caroline , Jarvi Susan
Published: January 2023

A better understanding of host defenses against malaria is important, but most studies have traditionally focused on resistance and/or immunological mechanisms that limit parasite burden. Host tolerance mechanisms are thought to minimize physiological damage caused by both the parasite itself and immunopathology without directly affecting parasite burden. On Hawai`i Island, low elevation populations of amakihi (Hemignathus virens) appear to have recently evolved tolerance mechanisms against avian malaria (Plasmodium relictum). Under comparable parasite loads, low elevation amakihi have lower mortality, greater fitness, lower reticulocyte levels and lower heterophil to lymphocyte ratios than high elevation amakihi. Thus, distinct host defense mechanisms are likely operating in low vs. high elevation amakihi. To search for genes involved in host defense, AFLP analysis was completed. Group significance tests (Bionumerics v 2.0) reveal genetic distinction between groups (p = 0.000). Band frequency comparisons between low and high elevation `amakihi (Fisher’s exact test and Pearson’s chi-square test with Yate’s continuity correction, R v 2.11.1) revealed ten band classes with significantly higher (p < 0.05) frequencies in low vs. high elevation birds and 13 band classes with higher frequencies in high vs. low elevation birds. Isolation and sequencing of these candidate bands is underway and may provide a better understanding of host defense mechanisms in natural populations.
Next generation sequencing reveals major histocompatibility complex class II alleles associated with survival to avian malaria (Plasmodium relictum) in Hawaiian honeycreepers (Drepanidinae) (P3089)
Authors: Jarvi Susan , Farias Margaret , Txakeeyang Ann , McFarland Tom , Atkinson Carter
Published: January 2023

While most species of Hawaiian honeycreeper are highly susceptible to avian malaria, populations of the Hawaii amakihi (Hemignathus virens) are thriving at low elevations despite high infection rates of Plasmodium relictum. Due to their critical function in adaptive immunity, proteins encoded by Mhc genes may play a role in the recently evolved host defense mechanisms in these populations. A 454 Roche platform was used to generate 44,014 sequences representing 158 Mhc alleles from 46 captive amakihi involved in experimental challenges to avian malaria and 88 wild amakihi originating from Hawaii Island. Analysis of an NJ tree revealed 6 clusters of alleles occurring most often in surviving amakihi, and a significant association between survivorship and the presence of one or more alleles from these clusters was detected (p = 0.00006). Several of these alleles were also found in higher frequencies in low vs. high elevation populations. A similar analysis of 6,417 sequences representing 31 Mhc alleles from 19 experimentally infected i`iwi (Vestiaria coccinea) revealed a significant association between survivorship and the presence of one or more alleles from two clusters (p = 0.02). This is the first documentation of an Mhc association with survival to malaria in Hawaiian honeycreepers and is expected to enhance our understanding of host-parasite interactions in this natural disease system while also providing a predictive tool for conservation and management.
Macrophage migration inhibitory factor plays a role in the maturation of conventional dendritic cells in acute Toxoplasma gondii infection (P3054)
Authors: Rodriguez-Sosa Miriam , Hernández-Hernández Fidel de la Cruz , González Calixto Cecilia , Juárez-Avelar Imelda , Saavedra Rafael , Olgín Efraín , Ruiz-Rosado Juan de Dios
Published: January 2023

The role of the inflammatory cytokine MIF has been implicated on inflammatory immune response during toxoplasmosis, where an inflammatory immune response, initiated by high levels of the cytokine IL-12 produced by mature DCs, is required to control the infection. Previously we have demonstrated that MIF knockout mice are highly susceptible to T. gondii infection, due to low IL-12 levels in serum, which in turn, may be associated with a deficient maturation of DCs in the acute infection by this parasite. Therefore, the aim of this study was to determine the role of MIF on the maturation of conventional DCs during the acute infection by T. gondii. To accomplish this goal, through flow cytometry, we analyzed the maturation (IL-12 intracellular and CD40 expression) of different conventional DCs subpopulations (CD11c+ CD11b-high, CD11c+ CD11b-low and CD11c+ CD8α+) in spleens from infected and non-infected MIFKO and WT mice 24 or 48h after ip infection with T. gondii. The results demonstrated that conventional DCs subpopulations CD11c+ CD11b-high and CD11c+ CD8+α from MIF KO mice showed a lower percentage of cells producing IL-12 and reduced expression of CD40, respectively, compared to WT conventional DCs. These evidences suggest that MIF could modulate the maturation of certain subpopulations of conventional DCs during acute T. gondii.
Vaccination with GD3 synthase provides tumor protection against melanoma in mice (P4461)
Authors: Hammer Adam , Eby Jonathan , Gilbert Emily , Kahn Safia , Dilling Daniel , Le Poole I.
Published: January 2023

Expression of GD3 synthase (GD3s) in cells defines upregulated GD3 expression by melanoma tumor cells. GD3 presented in the context of CD1d is recognized by invariant NKT cells, which can generate cytokines conducive to anti-tumor responses. We hypothesized that DNA vaccination with GD3s could induce effective anti-tumor responses targeting GD3. C57BL/6 mice were subjected to weekly gene gun vaccination introducing DNA encoding either GD3s and adjuvant HSP70i, or TRP-1 ee/ng and HSP70i, versus empty vector DNA as control groups. Mice were challenged with 2.5x105 B16 mouse melanoma cells. Tumor growth was followed and any remaining tumor was resected for fluorocytometric analysis of immune infiltrates and cytokine expression by qRT-PCR. hGD3s-based vaccination provided similar protection from a B16 tumor challenge as the positive control group (60 and 62% reduced tumor sizes at 19 days, respectively). hGD3s vaccinated mice showed increased NKT abundance (2.27% ) compared to TRP-1 (1.11%) or empty vector (0.74%) among vaccinated mice. Increased CD8+ (3.3-fold), and CD4+ (1.2-fold) compared to the negative control group were also observed, combined with an increased abundance in IL-4 and IL-17 transcripts. In conclusion, GD3s based vaccination provided anti-tumor protection associated with inflammatory cytokine expression and increased (NK)T cell recruitment to tumor sites, offering an attractive concept for melanoma treatment.
Adenovirus vector-specific cellular immunity induced by engineered human dendritic cells in vitro and in vivo (P4434)
Authors: Butterfield Lisa , Naveh Hadas
Published: January 2023

Recombinant adenovirus has been used in gene transfer applications, including cancer immunotherapy and infectious disease. Immunity to the adenoviral vector is known to include a potent humoral response; however, the cellular response is poorly characterized. Here, we evaluated the virus-specific response to an AdV encoding three melanoma tumor antigens (Tyrosinase, MART-1 and MAGE-A6 (AdVTMM2)) in healthy donors and patients with melanoma. CD4+ and CD8+ T cell responses were followed weekly by multi-cytokine ELISPOT assays (IFN-gamma, IL-2, IL-10 and TNF) and flow cytometric analysis in vitro. NK cells and Treg were also assessed. We found a strong type 1-skewed response with INFg secreted by CD4+ cells beginning within 7 days of stimulation and carrying through 3 weekly stimulations. Strong type 1 CD8+ T cell immunity was also detected to the virus. Treg increased transiently and an activated, INFg-producing subset of NK cells became evident. Melanoma patient cells also show a type-1 CD8+ T cell and CD4+ T cell response to the adenovirus. While strong, these virus-specific responses did not mask responses to the melanoma antigens. Melanoma patients vaccinated with the engineered DC vaccine in a new clinical trial respond immunologically to both the viral vector and encoded melanoma antigens, and show modulation of NK cells and Treg post-vaccination. Therefore, AdV-engineered DC promote a strong type 1 virus- and melanoma antigen-specific response, in vitro and in vivo.
A novel rabies virus-based glioma immunotherapeutic strategy targeting survivin (P4440)
Authors: Bongiorno Emily , Morin-Brureau Melanie , Barkhouse Darryll , Faber Milosz , Dietzschold Bernhard , Hooper D.
Published: January 2023

The poor prognosis of glioblastoma multiforme even after resection, chemotherapy and radiation has led to an interest in immunotherapy which necessitates consideration of the blood-brain barrier (BBB). Attenuated rabies viruses (RABV) induce BBB modifications that allow immune effectors to reach the CNS without immunopathology. We have shown that induction of a CNS response to an attenuated RABV can significantly prolong the survival of mice bearing intracranial GL261 tumors. This extended survival is associated with delayed tumor growth and an increase in mRNA levels of T and B cell markers and IFN-γ in CNS tissue. However it is not yet known whether the delayed tumor growth is a result of non antigen-specific effects of the antiviral response or due to BBB modifications that facilitate contact between the tumor cells and immune system. To provide a more selective target for the immune response, we have engineered a recombinant RABV to express human survivin. Survivin, an inhibitor of apoptosis protein (IAP), is preferentially expressed at high levels in certain types of cancer cells including gliomas and has been successfully used as a target in GL261 immunotherapies. The expression of survivin by RABV is expected to promote the delivery of tumor-reactive immune effectors into CNS tissues.
Enhanced immunotherapeutic effect of B cell based vaccine transduced with modified adenoviral vector (P4443)
Authors: Kim Eun Kyung , Song Bo Yeong , Chae Min Ji , Seo Hyung Seok , Park Young Jun , Yun Chae Ok , Kang Chang Yuil
Published: January 2023

For successful clinical outcome of tumor immunotherapy, proper generation of strong immune responses against tumor antigen is required. Cell based vaccine is one of the strategy to induce appropriate immune responses. In previous studies we established NKT ligand loaded, adeoviral antigen transduced-B cell based anticancer cellular vaccine that efficiently induced a wide spectrum of immune responses against tumor antigen. However the lack of the coxsakivirus-adenovirus receptor(CAR) on B cells limits the adenoviral Ag gene delivery. In this study, we established a modified adenoviral vector(Ad-k35) whose fiber structure was substituted with adenovirus serotype 35, encoding truncated breast cancer antigen Her2/neu(Ad-k35HM), to enhance tumor antigen gene delivery to B cells. Using Ad-k35HM, we observed increased expression of tumor antigen on both human and mouse B cells. And Ad-k35HM transduced B cell vaccine elicited antigen specific strong cellular and humoral immune responses and these responses was further enhanced with additional loading of derivative of NKT ligand KBC009. Ad-k35HM transduced, KBC009 loaded B cell vaccination efficiently suppressed in vivo established tumor growth in a mice model. These findings showed modified adenoviral vector Ad-k35 could be appropriate for the preclinical and clinical development of B cell based anticancer immunotherapy.
A general requirement for FcγRIIB co-engagement of agonistic anti-TNFR antibodies (P4449)
Authors: Li Fubin , Ravetch Jeffrey
Published: January 2023

The tumor necrosis factor receptor (TNFR) superfamily members are type I transmembrane proteins widely expressed on both normal and malignant tissues and control essential biological processes including cell apoptosis, activation, and proliferation1. A number of TNFR signaling pathways have been reported to be beneficial in immune and antitumor responses. For example, CD40 mediated immunostimulatory effects and DR5 mediated direct apoptotic effects display potent antitumor activity through distinct mechanisms. Agonistic antibodies targeting TNFRs have therefore been extensively investigated as a therapeutic approach to trigger TNFR signaling pathways in antitumor responses. Despite their impressive performance in animal models, clinical results of these agonistic anti-TNFR antibodies appear to be disappointing. Our studies on the role of the Fc for agonistic anti-CD40 and anti-DR5 antibodies, and the FcγR binding properties of other agonistic anti-TNFR antibodies suggest an explanation for this discrepancy between the pre-clinical animal studies and clinical application of these agonistic antibodies. It is now apparent that agonistic anti-TNFR antibodies display a general requirement for FcγRIIB co-engagement for their in vivo activity and the selection of the appropriate Fc was not taken into consideration in the advancement of therapeutic versions of these antibodies into the clinic.
Identification of a novel Th1 promoting vaccine adjuvant from natural products (P4451)
Authors: Reiman Jennifer , Zenack Dusan , Pham Ngoc , Engwerda Christian , Quinn Ronald , Good Michael
Published: January 2023

One of the great obstacles to vaccine development is the lack of adjuvants that can safely and appropriately stimulate human immune cells. There are a limited number of adjuvants that have been approved for use in human vaccines. In the USA, all but one of the adjuvanted vaccines uses aluminum salts (alum) and these promote T helper (TH) 2 but not TH1 immunity. For this screen we have utilized Eskitis's Nature Bank library as a source of compounds. This library consists of over 200,000 natural product fractions derived from a collection of over 45,000 samples of plants and marine invertebrates from Australia, China, and Papua New Guinea. Mice on a C57BL/6 background that express the IL-12p40 promoter linked to yellow fluorescent protein (YFP) have been utilized as a source of dendritic cells (DCs). Splenic DCs were cultured in the presence of Nature Bank fractions or with toll like receptor-9 (TLR9) agonist CpG 1826. Cultures were assayed for IL-12 production by measuring YFP using a Perkin Elmer Operetta high content screening system and analyzed with Harmony software. Only a single compound was identified in our screen of over 20,000 fractions and it robustly activated DCs (86% compared to 11% for CpG positive controls) to become IL-12 positive. We have preliminary structural data on the compound and are subsequently testing “compound A” for immunogenicity and adjuvant activity utilizing in vitro and in vivo assays with mice, as well as human monocyte derived DC cultures.
Neutrophil extracellular traps are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis (P4061)
Authors: Carmona-Rivera Carmelo , Khandpur Ritika , Vivekanandan-Giri Anuradha , Gizinski Alison , Yalavarthi Srilakshmi , Knight Jason , Friday Sean , Li Sam , Patel Rajiv , Thompson Paul , Chen Pojen , Fox David , Pennathur Subramaniam , Kaplan Mariana
Published: January 2023

The early events leading to the development of rheumatoid arthritis remain unclear but formation of autoantibodies to citrullinated antigens is considered a key pathogenic phenomenon. Neutrophils isolated from patients with various autoimmune diseases display enhanced extracellular trap formation, a phenomenon that externalizes autoantigens and immunostimulatory molecules. We investigated if aberrant NETosis occurs in RA, its triggers and deleterious inflammatory consequences. Enhanced NETosis was observed in circulating and synovial fluid RA neutrophils. NETosis correlated with ACPA levels and with systemic inflammatory markers. RA sera and immunoglobulin fractions from RA patients with high levels of ACPA and/or rheumatoid factor significantly enhanced NETosis, and the NETs induced by these autoantibodies displayed distinct protein content. During NETosis, neutrophils externalized citrullinated autoantigens implicated in RA pathogenesis, while anti-citrullinated vimentin antibodies potently induced NET formation. In turn, NETs significantly augmented inflammatory responses in RA and OA synovial fibroblasts, including induction of IL-6, IL-8, chemokines and adhesion molecules. These observations implicate accelerated NETosis in RA pathogenesis, through externalization of citrullinated autoantigens and immunostimulatory molecules that may promote aberrant immune responses in the joint and in the periphery and perpetuate pathogenic mechanisms in this disease.
Nucleic acid-containing amyloid fibrils potently induce Type I interferon and stimulate systemic autoimmunity (P4075)
Authors: Cao Wei , Di Domizio Jeremy , Dorta-Estramera Stephanie , Gagea Mihai , Li Ping , Bi Liqi , Gilliet Michel
Published: January 2023

The immunopathophysiologic development of systemic autoimmunity involves numerous factors through complex mechanisms. In systemic lupus erythematosus, type I IFN (IFNI) produced by plasmacytoid dendritic cells (pDCs) critically promotes the autoimmunity through its pleiotropic effects on immune cells. However, the host-derived factors that enable abnormal IFN-I production and initial immune tolerance breakdown are largely unknown. Amyloid precursor proteins can form amyloid fibrils in the presence of nucleic acids. Here we report that nucleic acid-containing amyloid fibrils can potently activate pDCs and enable IFN-I production in response to self-DNA, self-RNA, and dead cell debris. pDCs can take up DNA-containing amyloid fibrils, which are retained in the early endosomes to activate TLR9, leading to high IFNα/β production. In mice treated with DNA-containing amyloid fibrils, a rapid IFN response correlated with pDC infiltration and activation. Immunization of nonautoimmune mice with DNA-containing amyloid fibrils induced antinuclear serology against a panel of selfantigens. The mice exhibited positive proteinuria and deposited antibodies in their kidneys. Intriguingly, pDC depletion obstructed IFN-I response and selectively abolished autoantibody generation. Our study reveals an innate immune function of nucleic acid-containing amyloid fibrils and provides a potential link between compromised protein homeostasis and autoimmunity via a pDC-IFN axis.
Regulation of pro-inflammatory responses by S100A9 (P6124)
Authors: Tsai Su-Yu , Segovia Jesus A. , Mgbemena Victoria , Chang Te-Hung , Tessier Philippe A. , Tardif Melanie , Bose Santanu
Published: January 2023

Several Damage-Associated Molecular Patterns have been known to play an important role in inflammation. However, whether DAMPs are involved in lung inflammation during infection is not clear. In order to examine the important mechanisms of DAMPs during virus infection, influenza A virus infection model was used to study the role of S100A9, one of DAMPs, during lung pathogenesis. Here, we show that S100A9 is induced and secreted from flu-infected macrophages via a “non-damage” pathway. Since DAMPs are known to activate pro-inflammatory responses, we next examined whether purified recombinant S100A9 protein triggers pro-inflammatory responses in macrophages. Indeed, treatment of macrophages with S100A9 resulted in production of pro-inflammatory cytokines. The role of S100A9 during flu infection was also assessed by studying production of S100A9 in the respiratory tract of flu-infected mice. Upon flu infection, the production of S100A9 was induced in the lung and bronchoalveolar fluid. Moreover, the pro-inflammatory activity of extracellular S100A9 in the airway was obvious from robust production of pro-inflammatory cytokines in the lung. Thus our studies have identified S100A9 as a potent activator of pro-inflammatory response in macrophages and in the respiratory tract of mice. In addition, we uncovered ability of flu to induce S100A9 expression and production in macrophages and in the lung.
4-1BBL deficiency abrogates splenic hypotrophy observed following LCMV clone 13 infection in mice (P6130)
Authors: Mbanwi Achire , Wang Chao , Watts Tania
Published: January 2023

Mice infected with lymphocytic choriomeningitis virus (LCMV) clone 13 develop a prolonged viral infection that is used as a model to study HIV infection in humans. The tumor necrosis factor (TNF) receptor family member 4-1BB, and its ligand, 4-1BBL, have been established as powerful modulators of anti-viral immunity. Here we reveal a novel role for 4-1BBL in splenic contraction following LCMV infection. At day 8 post-infection, WT and 4-1BBL-/- mice show spleens that are 2 times the average pre-infection weight. However, by 2 months following infection, a time point at which virus is no longer detectable in the spleen, WT spleens show splenic atrophy, contracting up to 50% below baseline, whereas 4-1BBL-/- spleens contract back to baseline size. Interestingly, although 4-1BBL-/- spleens show increased cellularity, there is a deficit in the frequency of a B220-TCRβ-CD11bintCD11cintNK1.1+ natural killer (NK) cell population. The change in spleen size and NK population is observed in 4-1BBL-/-, but not 4-1BB-/- mice, suggesting a 4-1BB-independent 4-1BBL mechanism. Using bone marrow chimeras, as well as NK depleting antibodies, work is in progress to elucidate the cell type on which 4-1BBL is required for these effects and the potential role for NK cells in controlling splenic contraction following viral infection. This study suggests a novel role for 4-1BBL in splenic contraction and could have important implications for understanding the resolution of inflammatory conditions.
Understanding the role of TLR3- and MyD88-dependent signaling in the development of B cell responses to RepliVAX WN, a live attenuated single cycle flavivirus. (P6132)
Authors: Xia Jingya , Winkelmann Evandro , Gorder Summer , Milligan Gregg
Published: January 2023

TLR3- and MyD88-mediated signaling have been shown to play an important role in the development of antiviral B cell immunity. However, there has been no comparative investigation on the mechanisms by which these two pathways influence the B cell response. The role of these signaling pathways was examined by quantifying specific B cell development events during the humoral response in TLR3-/- and MyD88-/- mice following immunization with a single cycle attenuated West Nile vaccine. Expression of CD69 and CD86 early activation markers as achieved by activated B cells from wild type mice differed between TLR3-/- and MyD88-/- mice, and fewer IgM secreting B cells were detected in draining lymph nodes and spleens of MyD88-/- compared to TLR3-/- mice. Fewer germinal center B cells were detected in MyD88-/- mice compared to wild type or TLR3-/- mice. This finding was consistent with fewer IgG secreting B cells in MyD88-/- compared to TLR3-/- mice over the course of the antibody secreting cell response. Both TLR3 and MyD88 deficiency affected differentiation to long lived plasma cells whereas the absence of MyD88-dependent, but not TLR3-dependent signaling, impaired the development of memory B cells. Collectively, these results demonstrate that innate signaling through MyD88- or TLR3-dependent pathways results in differential effects on early B cell activation, germinal center cellularity, IgG-secreting cell number, memory B cell memory and long-lived plasma cell differentiation.
Toll-like receptor 3 involvement in Coxsackievirus B 4 acceleration of type 1 diabetes. (P6134)
Authors: McCall Kelly , Schwartz Frank , Thuma Jean , Benencia Fabian , James Calvin , Malgor Ramiro , Courreges Maria
Published: January 2023

Viruses are important in the pathogenesis of type 1 diabetes mellitus (T1DM). A pancreatrophic strain of coxsackievirus, CVB4, which is associated with the development of T1DM in humans, accelerates the development of T1DM in NOD mice. Toll-Like Receptor 3 (TLR3) is activated by viral dsRNA and is broadly expressed by NOD mice and human pancreatic beta cells, suggesting that TLR3 signaling may be important in CVB4 acceleration of T1DM. We used NOD mice deficient in TLR3 (TLR3 KO) to test the hypothesis that TLR3 signaling is important in CVB4 acceleration of T1DM in NOD mice. We report that TLR3 KO NOD mice are markedly protected from CVB4 acceleration of T1DM compared to wild type (WT) NOD mice. Similarly, we have observed that phenylmethimazole (C10), which has recently been shown to inhibit dsRNA-triggered TLR3 signaling, also delays CVB4 acceleration of T1DM in NOD mice. In preliminary experiments to investigate the mechanism by which TLR3 deficiency provides protection from T1DM in this model, we observed marked differences in T regulatory cell populations between TLR3 KO and WT NOD mice, and a significant enhancement of viral clearance in C10-treated NOD mice. These results indicate the distinct importance of TLR3 signaling in environmental (i.e. viral) induction of T1DM and that C10 could potentially protect β cells from virus-induced insulitis.
The effect of antibodies on endothelial cells during Dengue virus infection (P6138)
Authors: Bazzone Lindsey , Schieffelin John , Robinson James
Published: January 2023

Profound vascular leakage in conjunction with elevated viremia is the hallmark of Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS). Antibody (Ab)-dependent enhancement (ADE), in which pre-existing, cross-reactive Ab enhance virus infectivity, is thought to be responsible for increased viremia, while loss of endothelial cell (EC) barrier integrity is the precursor to plasma leakage. However, the relationship between increased viremia and vascular leak has not been established. We hypothesize that DV-Ab immune complexes enhance infection of FcγR-bearing cells causing increased virus production and release of mediators contributing to enhanced EC infection and increased vascular permeability seen in DHF/DSS. Anti-DV human monoclonal Abs (HMAb) were generated by molecular cloning to study ADE. Human dermal microvascular EC (HMEC-1) were infected directly with DV or indirectly using media from K562 cells infected with DV+/-HMAb. HMEC-1 infection and infectious virion production were quantified by qRT-PCR and plaque assay, respectively. HMEC-1 monolayers were assessed by microscopy. Indirect HMEC-1 infection under ADE conditions caused enhancement compared to infection media without Ab, and resulted in aberrant changes in HMEC-1 cell morphology, indicating that under ADE conditions, K562 cells enhance DV infection of ECs by increasing the infectious viral load and potentially releasing soluble mediators that cause EC barrier dysfunction.
The expression of mouse interferon-γ by engineered rabies viruses protects mice from lethal CNS infection through effects on innate immunity. (P6141)
Authors: Barkhouse Darryll , Faber Milosz , Dietzschold Bernhard , Hooper D.
Published: January 2023

Interferon-γ (IFNγ) has immunostimulatory, immunomodulatory and direct anti-viral properties. Previous studies have demonstrated the importance of IFNγ signaling during the immune response to rabies virus (RABV) as well as other neurotropic viruses. Specifically, IFNγ mRNA expression in the brains of RABV-infected mice correlates with immune effector entry into the brain and virus clearance. To more directly study the effect of IFNγ on the virus host response in the CNS, we constructed three IFNγ-expressing RABV vectors with decreasingly pathogenic backbone structures. We report that intranasal infection of mice with control SPBN is highly pathogenic but similar infection with SPBNγ is not. In addition, IFNγ expression by the attenuated RABV GASγ strain renders it significantly more effective than the highly attenuated GASGAS virus in preventing the death of mice from an otherwise lethal dose of DOG4 RABV when both are simultaneously administered intracranially. Finally, post-exposure treatment (PET) with an intramuscular injection of GASγGAS prevents the generally lethal outcome of intranasal infection of mice with DOG4 RABV and significantly reduces morbidity as compared to other PET RABV strains. Analysis of a variety of parameters of immunity in vivo and in vitro indicates that the protection provided by virus-encoded IFNγ is largely due to its impact on the innate response.
Immune and neurological outcomes of maternal-transmitted cytomegalovirus infection (P6153)
Authors: Puddington Lynn , Lingenheld Elizabeth , Zhu Li , Paveglio Sara , Obar Joshua , Lefrancois Leo , Wu Carol
Published: January 2023

Human cytomegalovirus (CMV) infection is the most significant infectious cause of developmental brain disorders, likely due to the predilection of the virus to infect the central nervous system early in development and that the immune system is not fully functional to eliminate this infectious agent. The data show that mother-child transmission rates for human CMV are at least 10-fold higher when mothers have their first infection while they are pregnant than if they are immune prior to becoming pregnant (30-50% versus 3%, respectively). These numbers provide a compelling argument that to prevent congenital CMV infection, all women of child-bearing age should be immune to the virus. However, immune women harbor virus in a latent state that reactivates specifically in the mammary gland during lactation, resulting in transmission of virus together with anti-viral maternal antibodies to their breastfed infants. We have established a mouse model of murine CMV infection that closely replicates this human situation. We found that neonatal mice infected while nursed by mothers with latent murine CMV infection have widespread dissemination of infectious virus, including the brain. Interestingly, the virus-specific T cell repertoire in neonates that acquire murine CMV from breast milk orally from immune mothers is distinct from neonates that ingest virus alone. The potential to elicit protective versus detrimental immune and neurological outcomes is under investigation.
CD44 expression on insulin-secreting β cells enhances the development of type 1 diabetes (P5109)
Authors: Naor David , Assayag-Asherie1 Nathalie
Published: January 2023

CD44-knockout NOD (non-obese diabetic) females show resistance to type 1 diabetes (T1D) compared with wild type (WT) littermates. Surprisingly, resistance to T1D is effective despite the increased insulitis observed in CD44-deficient NOD females by histopathological analysis of the pancreatic islets Indeed, under inflammatory conditions, destruction or reduced function is observed in WT, but not, or to a lesser extent, in CD44-deficient NOD females. Substantial reduction of insulin content was detected in WT β cells, but not in CD44-deficient cells. Apoptotic damage to β cell after binding of hyaluronan to its CD44 receptor may enhance T1D development. The effect of CD44 expression on β cells is predominant over its expression on inflammatory cells, shifting the balance in favor of overt diabetes
Mechanical insights into the functional impacts of interactions between antigen-presenting cells and T cells (P5081)
Authors: Lim Tong Seng , Mortellaro Alessandra , Lim Chwee Teck , J. Hämmerling Günter , Ricciardi-Castagnoli Paola
Published: January 2023

Antigen recognition and discrimination by T lymphocyte are essential in initiating appropriate immune responses. The mechanisms underlying exquisite sensitivity and specificity of antigen discrimination are not fully elucidated but involved physical intercellular interactions between T cell and antigen-presenting cell (APC) including dendritic cells (DCs) and B cells. Using single cell force spectroscopy (SCFS), we have successfully probe mechanical interactions between DC and T cells in response to a panel of altered peptide ligands (APLs). In the presence of different type of APLs, DC:T conjugate displays different ranges of mechanical forces that match their corresponding T cell responsiveness. Strong cell-cell interaction forces are contributed not only by surface adhesion molecules including TCR:pMHC, CD28:B7 and ICAM-1:LFA-1 interaction pairs, but also dependent on the integrity of membrane cholesterol and cytoskeleton dynamics. As compared to B:T interactions, stronger DC:T cell-cell interactions are likely to provide a mechanically stable environment that induces potent functional T cell activation, suggesting that the measurement of mechanical forces could be a significant predictor of T cell functional activation efficacy. Our SCFS studies provide mechanical insights into the adhesive roles of cell surface proteins in regulating APC:T cell-cell interactions. Functional roles of TLR ligands in regulating APC:T interactions and T-cell responses will be discussed.