bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct functions of IKKα and IKKβ in the induction of type I IFN and proinflammatory cytokine genes following infection with Theiler’s murine encephalomyelitis virus (TMEV). (44.29)
Authors: So Eui Young , Kim Byung S
Published: January 2023

The initial inflammatory responses and antiviral activity against virus infections are largely mediated by cellular pattern-recognition receptors (PRRs). Among these PRRs, we have recently shown that TLR3 is critical for TMEV-induced cytokine and chemokine production in mouse astrocytes (Glia, 2006). The activation of NF-κB is also involved in these TMEV-induced signaling pathways. While TMEV infection stimulates induction of proinflammatory genes in mouse astrocytes by mechanisms mainly dependent upon TLR3, the steps involved in the association of TLR engagement to NF-κB activation during TMEV infection are unknown. In this study, we showed that the upstream NF-κB activators IKKα and β are directly involved in TMEV-induced innate immune responses. MEFs deficient in IKKα and β resulted in a greatly reduced production of IFN-α/ β, IL-6, and RANTES during TMEV infection compared to normal MEFs. Interestingly, activation of IRF3 following infection with TMEV was impaired in IKKβ-KO MEFs, but not in IKKα-KO MEFs. However, TMEV-induced activation of IRF7 and NF-κB was severely impaired in both IKK-deficient MEFs. Therefore, our study strongly suggests that both IKKα and IKKβ are pivotal components of TLR3-triggered immune responses during TMEV infection. However, the roles of IKKα and IKKβ appear to be distinct in the signaling pathway for these cytokine responses.

(Supported by NS33008 and NS28752).
Sensing and recognition of bacteria by NKT cells (44.3)
Authors: Kronenberg Mitchell , Kinjo Yuki , Tupin Emanual , Nagarajan Niranjana
Published: January 2023

Natural Killer T (NKT) cells are a conserved T lymphocyte subpopulation that participates in host defense against infections. NKT cell express an invariant TCR alpha chain and they recognize glycolipids presented by CD1d. We found that NKT cells also respond to IL-12 and IL-18 produced by APC that have been activated by TLR ligands. As a result, NKT cells rapidly produce IFN gamma that stimulates the innate immune response. By responding to activated APC, NKT cells can amplify the innate immunity to many different microbes.

The invariant TCR expressed by mouse and human NKT cells recognizes glycosphingolipids with alpha linked sugars, which are unique to Sphingomonas bacteria. Mice that lack NKT cells have reduced Sphingomonas clearance. Mouse and human NKT cells with an invariant TCR also recognize glycosyl diacylglycerols from Borrelia burgdorferi, which causes Lyme disease. Interestingly, the response to these compounds was highly dependent on the nature of the aliphatic chains, with the addition of a single unsaturated bond having an enormous influence on antigenic potency. NKT cells are activated during B. burgdorferi infection. These data provide evidence that NKT cells recognize a new category of microbial glycolipids, diacylglycerols, which is more broadly distributed in pathogenic microbes, and they suggest that this TCR-mediated recognition provides protection from microbial pathogens.
CCL2 and IL-10 produced by Gr-1+CD11b+F4/80+ cells as an inhibitor of β-defensin 3 production by epidermal keratinocytes (44.35)
Authors: Jones Vickie C , Shigematsu Kenji , Kobayashi Makiko , Herndon David N , Suzuki Fujio
Published: January 2023

We have previously reported that Gr-1+CD11b+F4/80+ cells demonstrated in tissues surrounding the burn site (TSBS) are inhibitory on the production of antimicrobial peptides by epidermal keratinocytes (EK). In this study, the mechanism by which Gr-1+CD11b+F4/80+ cells inhibit the production of β-defensin 3 (MBD-3) by EK was investigated. Burn mice were BALB/c mice exposed to a gas flame (3rd degree). Twelve hrs after burn injury, Gr-1+CD11b+F4/80+ cells were isolated from their TSBS using a magnetic bead technique. To determine the effect of Gr-1+CD11b+F4/80+ cells on MBD-3 production, LPS-stimulated EK prepared from skin tissues of normal mice (1 x 106 cells/ml, lower chamber) were cultured with Gr-1+CD11b+F4/80+ cells (upper chamber) in dual-chamber transwells. Culture fluids harvested were assayed for MBD-3 by ELISA. After stimulation with LPS (0.1 μg/ml), EK produced 25 μg/ml of MBD-3, and only 0.4 μg/ml of MBD-3 was produced by EK transwell-cultured with Gr-1+CD11b+F4/80+ cells. When the same transwell cultures were performed with media supplemented with a mixture of mAbs for CCL2 and IL-10 (2 μg/ml each), 36 μg/ml of MBD-3 was detected in the culture fluids. In contrast, MBD-3 production by EK stimulated with LPS was inhibited by a mixture of recombinant CCL2 (50 ng/ml) and IL-10 (10 ng/ml). CCL2 and IL-10 were detected in the culture fluids of Gr-1+CD11b+F4/80+ cells. These results indicate that, through the production of CCL2 and IL-10, Gr-1+CD11b+F4/80+ cells appearing in association with burn injuries inhibit the production of MBD-3 from EK.

This study was supported by SHC NA #8610.
“Natural” antibodies against bacterial lipopolysaccharide and lipoteichoic acid in juvenile chickens (44.36)
Authors: Koppenheffer Thomas L.
Published: January 2023

Naturally occurring Abs are thought to provide protection from microbial pathogens. Chickens are susceptible to, and are potential carriers of, a number of pathogenic bacteria, yet sera of adult birds contain Abs against bacterial lipopolysaccharide (LPS) and lipoteichoic acid (LTA). This apparent anomaly led to undertaking a study of the production of these Abs during post-hatching development, the pattern of which may provide insight into understanding the factors that are involved in resistance to infection. Using Salmonella minnesota LPS and Staphylococcus aureus LTA as sentinel antigens in ELISA tests, relatively large amounts of LPS- and LTA-specific IgY were found in yolk of embryonated eggs and in serum of hatchlings. Thereafter, serum IgY levels declined rapidly over several weeks, but by 5–6 weeks of age some birds began to produce specific IgM and IgA Abs. Subsequently, the levels of LPS- and LTA-specific Abs of all isotypes gradually increased, approaching adult levels by 12 weeks of age. Thus, if resistance to infection is related to the levels of these Abs, chicks of about one month of age would be most susceptible to bacterial pathogens. Further, the concomitant appearance of specific Abs of all isotypes during the later phase of post-hatching development suggests that the birds in this study were sensitized through environmental exposure to bacterial cell wall antigens.

Supported in part by the Ralph and Louise Bricker Memorial Endowed Fund
Dissecting complement regulatory sites of the smallpox inhibitor of complement and abrogating function with a monoclonal antibody (44.37)
Authors: Liszewski M. Kathryn , Leung Marilyn K. , Bertram Paula , Atkinson John P.
Published: January 2023

Public health concerns have emerged regarding use of smallpox as a bioterrorist weapon and the possibility of natural emerging infection with monkeypox. Stockpiles of variola in rogue nations and the outbreak of monkeypox in the US in 2003 underpin these concerns. Poxviruses express virulence factors that down-modulate the host’s innate immune system. We previously constructed the smallpox inhibitor of complement enzymes, SPICE, from its homolog in vaccinia, VCP. Additionally, we cloned and functionally characterized MOPICE, the highly homologous monkeypox inhibitor. SPICE had enhanced activity over both poxviral inhibitors (PICES).

The goals of our current study were

to dissect the complement regulatory sites of SPICE andto characterize a monoclonal antibody that binds to and abrogates function of the PICES.

We created SPICE-VCP chimeras and SPICE point mutations. Generally, function increased for human C3b and C4b as more SPICE amino acids were present. We also identified SPICE residues that were critical for ligand binding and cofactor activities. Additionally, we characterized a mAb that binds SPICE, MOPICE and VCP. This mAb served as a capture reagent for ELISA to quantify PICES and as a detection mAb in Western blots. Importantly, the mAb blocked cofactor and decay accelerating activities, suggesting its possible use as a therapeutic. Thus, our studies add novel data relative to the complement inhibitory profile of SPICE. Further, a cocktail of such mAbs that inhibit virulence factors could serve in both diagnostic and therapeutic applications for poxviruses.
Proteasome activator and antigen-processing aminopeptidases are regulated by virus-induced type I interferon in the hepatitis C virus-infected liver (44.39)
Authors: Shin Eui-Cheol , Seifert Ulrike , Urban Sabrina , Truong Kim-Thuy , Feinstone Stephen M , Rice Charles M , Kloetzel Peter -M , Rehermann Barbara
Published: January 2023

Many components of the class I-antigen-processing pathway are thought to be regulated solely by IFN-γ. Recently, however, we reported type I IFN-mediated induction of immunoproteasomes in a viral infection of the liver (J Clin Invest 2006, 116:3006). In the present study, we extended this analysis to additional components of the MHC class I antigen-processing machinery. Specifically, we investigated type I IFN-mediated induction of the proteasome activator 28 (PA28) subunits α and β, the ER aminopeptidases-1 (ERAP1), -2 (ERAP2) and leucine aminopeptidase (LAP) in vitro and in vivo in a model of HCV infection. This mechanism was initiated by either synthetic or hepatitis C virus (HCV) RNA and abrogated by blocking of type I IFN. In the liver of chimpanzees with acute HCV infection, increased mRNA levels of PA28 subunits and all aminopeptidases occurred prior to IFN-γ responses and instead, coincided with type I IFN responses. Taken together, viral RNA-induced type I IFN regulates the antigen-processing machinery early during infection. Thus, viral RNA-induced type I IFN does not only exert direct antiviral functions but also enhances antigen processing in virus-infected cells to facilitate recognition by effector CD8+ T cells.
The Role of Toll-Like Receptor 7 (TLR7) in Neuroinflammatory Responses to Retrovirus Infection in Mice (44.41)
Authors: Peterson Karin E , Lewis Stephanie , Pourciau Susan , Du Min , Khaleduzzaman Mohammed
Published: January 2023

Retrovirus infection in the central nervous system of infants is often associated with strong innate immune responses including production of cytokines and chemokines, activation of astrocytes and microglia and recruitment of macrophages. Understanding how retrovirus infection leads to the induction of these innate immune responses in the CNS is important in determining mechanisms of pathogenesis. Several studies have demonstrated that single-stranded RNA viruses can stimulate macrophages and dendritic cells through toll-like receptor 7 (TLR7). In the current study, we analyzed whether TLR7 was expressed in the neonatal brain and whether TLR7 contributed to retroviral-induced innate immune response. TLR7 was expressed primarily by ependymal and endothelial cells in the brain as detected by immunohistochemistry. Deficiency in TLR7 did not alter interferon beta responses to neurovirulent murine retrovirus infection, virus replication in the brain or virus-mediated neurological disease. However, retrovirus-induced activation of astrocytes and the production of multiple chemokines were suppressed in TLR7 deficient mice compared to wildtype controls. Thus, TLR7 signaling appears to play a significant role in the activation of the innate immune response to retrovirus infection in the neonatal brain.

This study was supported by NIH IP20RR020159-01.
TLR2/1-induced IL-15 triggers a vitamin D3-dependent antimicrobial activity against Mycobacterium tuberculosis in human macrophages (44.5)
Authors: Krutzik Stephan Richard , Robles Juan Antonio , Hewison Martin , Corcoran Kellie , Montoya Dennis , Liu Philip T , Adams John , Modlin Robert L
Published: January 2023

Toll-like receptors (TLRs) are an integral component of the innate host defense, triggering macrophage differentiation by secretion of IL-15 and antimicrobial activity against intracellular M. tuberculosis through a vitamin D-dependent pathway. Here we investigate the mechanism by which TLR2/1 activates this antimicrobial pathway. We found that the TLR2/1-mediated induction of Cyp27B1 and VDR in human monocytes was dependent on secretion of IL-15. The direct activity of IL-15 to induce this pathway was examined in comparison to IL-4, since both induce monocytes to differentiate into CD209+ macrophages but with unique FcR expression. We found that IL-15 but not IL-4 was sufficient to induce Cyp27B1 and the VDR. The IL-15-derived macrophages upregulated Cyp27B1 activity as demonstrated by their ability to convert the vitamin D prohormone 25D3 to 1,25D3. Finally, in IL-15-derived macrophages, 25D3 triggered induction of the antimicrobial peptide cathelicidin and an antimicrobial activity against intracellular M. tuberculosis. Together, our data suggest that the TLR2/1 induction of the vitamin D-dependent antimicrobial pathway is dependent on the production and direct actions of IL-15.
Trafficking of dendritic cell subsets to the lung controls viral replication and pathology in human paramyxovirus infections (44.6)
Authors: Guerrero-Plata Antonieta , Casola Antonella , Garofalo Roberto P.
Published: January 2023

Three subsets of pulmonary dendritic cells (DC) have been described in mice: conventional DC (cDC, CD11c+MHCII+), plasmacytoid DC (pDC, CD11cintB220+Ly6C+), and the recently characterized IFN-producing killer cells (IKDC, CD11cintB220+CD49b+). Here, we examined the recruitment of DC to the lung in the context of two human paramyxovirus infections: respiratory syncytial virus (RSV) and human metapneumovirus (hMPV). Recruitment of DC to the lung in infected mice was determined by flow cytometry. Both infections induced a peak of cDC recruitment (>3×106) by day 8 for RSV and by day 10 for hMPV. cDC remained elevated throughout the inflammatory and the resolution phases of infection (1×106) compared to control (1×105). RSV induced a peak of 8×105 pDC recruited by day 3, while hMPV induced similar numbers but at day 8. pDC returned to basal levels (1×105) by day 6 (RSV) and by day 12 (hMPV). IKDC mirrored the pattern of migration of pDC but overall numbers of IKDC were smaller: peak for RSV was 5×105 IKDC and <4×105 IKDC for hMPV. Furthermore, depletion of pDC with anti-mPDCA1 antibody resulted in significant increases in peak titers and pulmonary inflammation in RSV and hMPV infected mice. In conclusion, this study demonstrates that all three subsets of DC are attracted to the site of infection in the course of paramyxovirus infections and pDC play a critical role in viral replication and pathogenesis.

NIH/NIAID P01 AI062885
NADPH Oxidase but not Myeloperoxidase Protects Lymphopenic Mice from Spontaneous Multi-Organ Infections (44.9)
Authors: Ostanin Dmitry V. , Barlow Shayne , Shukla Deepti , Grisham Matthew B.
Published: January 2023

The objective of this study was to define the role that NADPH oxidase or myeloperoxidase (MPO) plays in host defense in lymphopenic mice. To do this we crossed gp91 phox deficient (gp91 ko) or MPO ko mice with mice deficient in recombinase activating gene-1 (RAG ko) to generate lymphopenic offspring deficient in either NADPH oxidase or MPO. We found that neither gp91 ko, MPO ko mice nor RAG ko mice developed spontaneous infections when raised under specific pathogen free (SPF) conditions and all animals lived normal life spans. In contrast, gp91/RAG double deficient (gp91/RAG DKO) but not MPO/RAG DKO mice developed spontaneous multi-organ bacterial and fungal infections early in life and lived only a few months. Addition of antibiotics to the drinking water attenuated some of the infections and increased modestly the survival of these mice. The enhanced mortality of the gp91/RAG DKO mice was not due to defects in inflammatory cell recruitment or NO synthase (iNOS) activity as the number of elicited PMNs and macrophages as well as PMN and macrophage-derived production of nitric oxide in these mice were similar to wt, gp91 ko or RAG ko mice. Taken together, our data suggest that that NADPH oxidase but not MPO (or iNOS) is required for host defense in lymphopenic mice and that lymphocytes and NADPH oxidase may compensate for each others deficiency in providing resistance to spontaneous infections

(Supported by DK64023).
Intrahepatic expression of HCV core impairs CD4+ T cell response in vivo (45.10)
Authors: Lassen Matthew G. , Hahn Young S.
Published: January 2023

Hepatitis C virus (HCV) is remarkably efficient in establishing a persistent infection, with >70% of acutely infected individuals developing chronic HCV. Resolution of HCV infection requires a strong, broad Th1 response, including robust IFN-γ and IL-2 production. Patients that develop chronic infection display lower proliferative capacity, a narrow clonal repertoire, and decreased IFN-γ and IL-2 production within the CD4+ T cell compartment, compared to patients who resolve infection. We sought to determine if the expression of the HCV core protein (core) within the liver compartment dysregulates intrahepatic CD4+ T cells in vivo during an inflammatory response. We preferentially targeted the expression of core protein to liver cells by injecting Balb/c mice intravenously with a replication deficient adenovirus that expresses core under the CMV promoter (Ad-core). Compared to control mice infected with β-galactosidase expressing adenovirus, intrahepatic CD4+ T cells from Ad-core infected mice expressed lower levels of CD25 on days 1 and 8 post-infection (p.i.), as well as lower IFN-γ production at day 15 p.i. In contrast, CD25 expression and IFN-γ levels on splenic CD4+ T cells were similar between Ad-core infected and control mice. These results suggest that expression of core within the liver leads to an impaired CD4+ T cell response.
The Vif Accessory Protein Alters the Cell Cycle of Human Immunodeficiency Virus Type 1 Infected Cells (45.15)
Authors: Wang Jiangfang , Shackelford Jason M. , Casella Carolyn R. , Shivers Debra K. , Rapaport Eric L. , Liu Bindong , Yu Xiao-Fang , Finkel Terri H.
Published: January 2023

The viral infectivity factor gene (vif) of HIV-1 increases the infectivity of viral particles by inactivation of cellular anti-viral factors, and supports productive viral replication in primary human CD4 T-cells and in certain non-permissive T cell lines. Here, we demonstrate that Vif also contributes to the arrest of HIV-1 infected cells in the G2 phase of the cell cycle. Viruses deleted in Vif or Vpr induce less cell cycle arrest than wild-type virus, while cells infected with HIV-1 deleted in both Vif and Vpr have a cell cycle profile equivalent to that of uninfected cells. Furthermore, expression of Vif alone induces accumulation of cells in the G2 phase of the cell cycle. These data demonstrate a novel role for Vif in cell cycle regulation and suggest that Vif and Vpr independently drive G2 arrest in HIV-1 infected cells. Our results may have implications for the actions and interactions of key HIV-1 accessory proteins in AIDS pathogenesis.
Loss of Antibody Neutralization Activity in Mice During Persistent γ-herpesvirus Infection (45.16)
Authors: Yager Eric J , Kim In-Jeong , Burkum Claire , Rochford Rosemary , Blackman Marcia A
Published: January 2023

Murine γ-herpesvirus-68 (γHV68), like the human γ-herpesviruses EBV and KSHV, initiates an acute, readily controlled lytic infection in mice, which is followed by the establishment of a persistent latent infection. Although T cells are important for control of both lytic and latent γHV68 infection, specific antibody alone is sufficient to prevent viral recrudescence during latency. Although chronic viral infections can affect the generation and maintenance of T cell immunity, it is unclear whether B cell immunity is also affected. Titers of γHV68-specific IgG remained relatively stable in latently infected mice up to 20 months postinfection. However, neutralization titers were found to wane in most latently infected mice, with declines observed as early as 10 months postinfection. Corresponding to a loss in neutralization activity, the transfer of immune sera taken from long-term (>18 months) γHV68-infected mice afforded naïve mice less protection against acute γHV68 infection when compared to immune sera taken from recently infected mice. No direct correlation between reduced neutralization activity and sera isotype profile, or overall affinity for viral antigen, was found. As serum levels of specific antibody are maintained by long-lived plasma cells, ongoing studies are examining whether γHV68 persistence adversely affects CD4+ T helper cell function and/or the survival of long-lived plasma cells. In summary, these observations support the novel idea that persistent viruses can adversely influence the maintenance of protective humoral immune responses.

This work was supported by NIH institutional training grant AI49823 and NIH grant AG021600.
Anti-TNF-α treatment activates Borrelia burgdorferi infection in ceftriaxone treated C3H/He mice (45.17)
Authors: Yrjänäinen Heta , Hytönen Jukka , Song Xiao-yu R. , Oksi Jarmo , Hartiala Kaija , Viljanen Matti K.
Published: January 2023

The effect of anti-TNF-α treatment in B. burgdorferi infected C3H/He mice treated with ceftriaxone was evaluated in this study. Mice were infected with B. garinii Å218 or B. burgdorferi sensu stricto N40. At two weeks of infection, a group of ten mice was treated with ceftriaxone for five days, while another group received anti-TNF-α simultaneously with ceftriaxone. A third group received anti-TNF-α four weeks after the ceftriaxone treatment and a fourth group received only anti-TNF-α. Ceftriaxone improved joint swelling and decreased B. burgdorferi specific IgG antibody responses. At 14 weeks of infection, B. burgdorferi could not be detected by cultivation or by PCR from tissue samples of any of the animals treated only with ceftriaxone. However, spirochetes grew from the tissue samples of one third of the mice treated with both anti-TNF-α and ceftriaxone, regardless of whether the treatments were received simultaneously or four weeks apart. No loss of plasmids, decreased ceftriaxone sensitivity or virulence rate was found when the activated spirochetes were compared to the bacteria used to infect the mice. All infected control mice and the mice that only received anti-TNF-α were culture positive. These results show that a number of B. burgdorferi infected mice still have live spirochetes which are activated by the anti-TNF-α treatment in their body after treatment with ceftriaxone.
Viral immune evasion protein mK3 targets MHC I proteins for rapid degradation by specifically ubiquitinating serine or lysine residues in the cytoplasmic tail of the heavy chain (45.9)
Authors: Herr Roger Alan , Lybarger Lonnie , Wiertz Emmanuel J. H. J. , Wang Xiaoli , Hansen Ted H.
Published: January 2023

The immune evasion protein mK3 is a viral RING type E3 ubiquitin ligase encoded by murine gamma herpesvirus68. mK3 specifically targets nascent MHC I heavy chain (HC) for rapid degradation in a ubiquitination-proteasome dependent manner, thus blocking detection of virally infected cells by CD8 T cells. To address the question of how HC is retrotranslocated from the ER and what role mK3 plays, we investigated ubiquitin conjugation sites in HC using mutagenesis and biochemistry approaches. Surprisingly, we found that a totally K-less HC had a similar polyubiquitination pattern and rapid degradation as wt HC in mK3 expressing cells. In addition, we found that polyubiquitinated HCs became undetectable after removal of their cytoplasmic tails by proteinase K digestion or thrombin cleavage at an engineered site. HCs with tail substitutions to remove all K,C,S & T residues abolished mK3-mediated polyubiquitination and ERAD, whereas adding backing K or S residues restored polyubiquitination and ERAD. This demonstration that S residues can function as ubiquitination sites is novel. Furthermore, the fact that mK3 specifically ubiquitinates the tail of HC suggests that it is dislocated via its C-terminus.

Supported by NIH grants AI019687 and T32AI007163-30
Functionally impaired T cell-dependent immunopathology in FK506-treated mice with LCMV infection (46.10)
Authors: Araki Koichi , Gangappa Shivaprakash , Rouse Barry T , Larsen Christian P , Ahmed Rafi
Published: January 2023

Lymphocytic choriomeningitis virus (LCMV) was recently shown to contaminate transplanted organs with lethal consequences in transplant recipients. The diagnosis of LCMV as a cause of death under circumstances of immunosuppression is somewhat paradoxical since LCMV is a non-cytolytic virus and is the classic example of immune-mediated disease. To understand how treatment with immunosuppressive drugs given to transplant recipients results in LCMV disease, we have initiated studies using the calcineurin inhibitor FK506. Surprisingly, FK506-treated LCMV-infected mice showed high lethality. The drug treatment resulted in a failure of infection control and interestingly induced functionally impaired virus-specific T cells. Lesions and death appeared to be the consequence of overproduction of cytokines, such as TNF-alpha. This response was dependent on the presence of T cells but these cells themselves appeared not to be the source of the disease-producing cytokines. Our results suggest that functionally impaired T cells in drug treated animals signal cells in LCMV infected tissues to overproduce the cytokines responsible for lesions.
Differential Dysfunction of Dendritic Cells and B-cells During Chronic HCV Infection (46.11)
Authors: Ayers Chris L , Averill Lynn , Pillai Vinodh , Firan Mihail , Wang CK , Lee William M , Karandikar Nitin J
Published: January 2023

Chronic hepatitis C virus (HCV) infection is characterized by attenuated antiviral T cell responses. We hypothesized that HCV-associated dysfunction of antigen presenting cells (APC) may be responsible, in part, for such attenuation. We evaluated the function and phenotype of dendritic cells (myeloid vs. plasmacytoid), B cells, and monocytes, finding distinct functional differences among the APC subsets. Myeloid DC (MDC) from chronic HCV patients showed slightly lower APC capacity with lower HLA-DR and CD86 expression. HCV-MDC also had a tendency to secrete more IL-10, when compared to healthy MDC. On the other hand, HCV-plasmacytoid DC (PDC) showed a significantly reduced ability to make IFN-α. B cells from HCV patients had a hyperactivated phenotype and a marked enhanced APC capacity. Interestingly, the enhanced B cell APC function correlated with detection of virus in these cells. Finally, increased APC functionality was paradoxically associated with the generation of a higher proportion of induced regulatory T-cells, providing a plausible explanation for T-cell attenuation. In conclusion, our studies underscore the necessity to dissect APC subsets for functional perturbations and provide evidence for their role in the immune attenuation accompanying chronic HCV infection.
Differential responses of DCs from susceptible and resistant mice to Theiler’s virus infection (46.18)
Authors: HOU Wanqiu , SO Eui Young , KIM Byung S
Published: January 2023

Although persistent viral infection-associated diseases are a major health issue, the underlying cellular mechanisms of differential susceptibility to such persistent infections among individuals are unknown. Theiler’s virus (TMEV), a natural mouse pathogen, induces a chronic demyelinating disease similar to multiple sclerosis in susceptible mice. Resistant mice completely clear the virus and do not develop chronic demyelinating disease. Here, we compared interactions between TMEV and dendritic cells (DCs) in these mice. Our results indicate that immature DCs from susceptible mice are significantly more permissive to viral infection and infection-induced DC maturation arrest than those from resistant mice, despite the production of higher levels of anti-viral cytokines, including type I/II IFNs. Further studies showed that the timing of DC exposure to type I/II IFNs is critical in controlling viral infection. Moreover, a greater level of type I/II IFNs, which are induced in DCs from susceptible mice by TMEV infection, resulted in severe inhibition of DC cytokine production in response to LPS. Thus, differential interactions between DCs and TMEV, leading to a preferential deficiency in DC maturation and activation of DCs from susceptible mice, may be a mechanism through which TMEV establishes persistent infection and chronic demyelinating disease.

(Supported by NS23349, NS28752 and NS33008)
Natural Killer cell function during foot-and-mouth disease virus infection (46.19)
Authors: Toka Felix N. , Nfon Charles K. , Endsley Janice J. , Ferman Geoffery S. , Estes D. Mark , Golde William T.
Published: January 2023

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. Immunological knowledge to assess more rapid acting vaccines against FMDV is presently limited. We examined the reactivity of swine and cattle NK cells following infection for their capability to express intracellular perforin, to kill a human tumor cell line target in vitro, and to secret IFN gamma. The cytotoxicity of NK cells from non-infected animals against the K562 cells is low with baseline levels at 5–15% in swine and 15–20% in cattle. Stimulation with rhIL-2 or rhIL-12 plus rhIL-15, increased the lytic activity against K562 cells. Infection with FMDV inhibited swine NK cell lytic activity but did not significantly increase IFN gamma secretion during the acute infection. Perforin expression increased but this did not correlate with the killing capability of the swine NK cells. Infection of cattle with FMDV initially activated the NK cells to increase target cell lysis. NK cell IFN gamma secretion and perforin expression were slightly elevated upon infection and coincided with the lytic activity in cattle. These results are a further indication of immune evasion by FMDV by inhibiting or limiting NK cell function. The potential to manipulate the innate immune response to block this evasion is discussed in the context of designing rapid acting vaccines for foot-and-mouth disease.
Increased socs-1 expression in dendritic cells may be responsible for reduced type 1 cytokine responses in HIV-1 transgenic rats (46.20)
Authors: YADAV ANJANA , REID WILLIAM
Published: January 2023

Dendritic cell (DC) function appears to be compromised in HIV infected individuals, although it is unclear whether the dysregulation is a result of direct infection of the DCs or an indirect effect of exposure to HIV proteins. Since effector/memory formation depends upon clonal expansion of naïve T cells following antigenic stimulation and since cytokines play a critical role in the initiation and regulation of immune responses by DCs, events that negatively affect antigen presentation, co-stimulation and cytokine production by DCs, also negatively affect T cell immune responses. Infection with HIV-1, results in cytokine dysregulation by DCs. Suppressor of cytokine signaling-1 (SOCS-1) is an inducible negative regulator of the JAK/STAT signal pathway; SOCS-1 is expressed in DCs and negatively regulates activation and type 1 regulatory cytokine production. We have earlier reported that HIV-1 transgenic rats have defects in type 1 cytokine production, type 1 cytokine responses and generation of effector/memory CD4 T cell subsets. Here we show that B cells from the HIV-1 Tg rats are also dysregulated in KLH specific primary and secondary Th1 isotype (IgG2a) responses. Additionally, bone marrow derived DCs (BMDCs) from HIV-1 Tg rats upon stimulation with LPS and/or rIFN-gamma, exhibit reduced immunostimulatory functions depicted by reduced production of IL-12+p40, increased expression of IL-10 and SOCS-1 and reduced STAT-1 phosphorylation relative to age matched controls. These outlined immune abnormalities together with the dysregulation in memory/effector development and Th1 responses suggests a link between SOCS-1 expression and IFN-γ-induction of IL-12 by BMDCs, resulting in impaired T helper type 1 responses reported in HIV-1 Tg rat. NIH-NIAID R01-A163171
4-1BB antibody significantly enhances SIV-specific CD8+ T-cell immunity and proliferation following naked DNA vaccination in cynomolgus macaques (47.10)
Authors: Hokey David A , Hirao Lauren A. , Calarota Sandra A. , Yan Jian , Boyer Jean D. , Dai Anlan , Yoon Hanna , Jure-Kunkel Maria N. , Balimane Praveen , Weiner David B.
Published: January 2023

A major challenge in the development of an effective vaccine for HIV is to stimulate potent cell-mediated immunity that includes the induction of long term memory. Ligation of the activation-induced co-stimulatory molecule, 4-1BB, stimulates CD8+ T-cell proliferation and the generation of memory in mice. We evaluated monoclonal antibody (mAb) agonists for 4-1BB to enhance DNA vaccines. Two studies were performed in cynomolgus macaques to evaluate the ability of these mAb to augment intramuscular DNA immunizations with either plasmid SIV-Gag or with codon-optimized consensus SIV-Gag, SIV-Env, and SIV-Pol plasmid DNA. Administration of anti-4-1BB mAb doubled the CD8+ T-cell killing ability as measured by granzyme B ELISpot assays. Furthermore, the proliferative response of SIV-specific CD8+ T-cells was increased by more than 5-fold after one immunization as measured by CFSE proliferation assays. Our data demonstrate the ability of 4-1BB stimulation to enhance cellular immunity following DNA vaccination in NHPs. These findings have clinical importance in that they represent a considerable improvement in the efficacy of naked intramuscular DNA vaccines in large animals and demonstrate the ability of 4-1BB agonists to direct CD8+ T-cell responses and memory.

This work is supported in part by NIH and NIAID funds.
Induction of human primary antibody responses in vitro by ovalbumin (OVA) or anthrax (rPA) on dendritic cells (DCs) and follicular dendritic cells (FDCs) in temporally controlled germinal center reactions (GCRs) (47.19)
Authors: Shikh Mohey Eldin M El , Sayed Rania M El , Wu Yongzhong , Szakal Andras K , Tew John G
Published: January 2023

Induction of potent primary human Ab responses entirely in vitro is challenging, although such in vitro responses could be helpful in assessing vaccines and therapeutics without jeopardizing human health. Given that DCs prime T cells and that FDCs promote B cell responses, we hypothesized that primary Ab responses could be induced in temporally controlled GCRs in vitro. To test this, monocyte derived-DCs were given OVA or rPA for processing, matured with LPS, and CD4+ T cells were added for priming. Ten days later, naïve B cells and OVA or rPA bearing FDCs were added and Ab production was subsequently assayed by ELISA. The CD4+ T cells clustered with DCs through the 10 days of priming and fused with FDC-B cell clusters when GCRs were induced. IgM anti-OVA levels were 79±15 ng/ml 7 days after adding B cells and declined to 2.4±0.5 ng/ml at day 14. In contrast, IgG anti-OVA rose from 9±1.7 at day 7 to 25±6.4 ng/ml at day 14 and the IgG1:IgG2 ratio was 1:3. Similarly, IgM anti-rPA levels were 10 ng/ml by just 5 days then IgM production switched to IgG. IgG anti-rPA rose from 3 ng/ml at day 5 to 9 ng/ml by day 12. In short, Ag specific primary human Ab responses, including Ig class switching from specific IgM to IgG, can be generated in GCRs entirely in vitro. Thus, potential vaccines can be rapidly and safely assessed using the human immune system.

(Supported and cleared by DARPA on 12/20/2006 - Approved for Public Release, Distribution Unlimited)
Incorporation of the TLR5-ligand flagellin in a recombinant Influenza A hemagglutinin vaccine provides enhanced immunogenicity and efficacy in vivo (47.2)
Authors: Huleatt James William , Tang Jie , Jacobs Andrea , Jock Dallas , Price Albert , McDonald William , Song Langzhou , Nakaar Valerian , Tussey Lynda , Powell T.J.
Published: January 2023

The linkage between innate and adaptive immunity is well established. The recognition of specific pathogen associated molecular patterns (PAMPs) by members of the Toll-like receptor (TLR) family plays a prominent role in the activation of adaptive immune responses. Previously, we have demonstrated that the linkage of specific PAMPs with defined antigens provides more potent and protective antigen-specific responses in the absence of adjuvants or complex formulations. Here we utilized this approach to develop a recombinant protein based influenza vaccine. The TLR5 ligand flagellin was linked to hemagglutinin (HA) to generate a single component vaccine. The recombinant E. coli expressed protein is recognized by influenza-convalescent antisera, indicating that the HA portion of the fusion protein is properly folded and presents conformational epitopes. In mice, the recombinant vaccine induces strong humoral and cellular responses that are quantitatively and qualitatively superior to those observed when the antigen is delivered with alum or co-delivered with flagellin unlinked. Analysis of serum antibody responses reveals strong reactivity with cells infected with influenza A and the ability to inhibit viral entry in vitro. Moreover, immunization with the fusion protein fully protects BALB/c mice from a lethal intranasal challenge with influenza A. These data demonstrate that linkage of the TLR5-ligand flagellin with HA provides significant promise as a non-egg based approach in the development of novel vaccines to combat seasonal and pandemic influenza.
ANTIBODY-MEDIATED TARGETING OF “INSIDE-OUT” ANIONIC PHOSPHOLIPIDS IN VIRAL DISEASE (47.21)
Authors: Soares Melina , Syed Sameer , Barbero Gustavo , Thorpe Philip E
Published: January 2023

The anionic phospholipid phosphatidylserine (PS) is found exclusively in the inner leaflet of the plasma membrane of resting mammalian cells. We hypothesized that certain events that occur during virus replication (eg cell activation or membrane rearrangement) would trigger the exposure of anionic phospholipids on the outer surface of virus- infected cells and subsequently on the enveloped viruses that bud out of these virus- infected cells. We further hypothesized that these exposed anionic phospholipids would serve as targets for anti-viral therapy. We demonstrate here that anionic phospholipids become exposed on the enveloped Pichinde Virus (a model virus for Lassa Fever virus, a potential bioterrorism agent) and on Pichinde virus-infected cells. To detect anionic phospholipids, we used a chimeric monoclonal antibody, bavituximab, that binds anionic phospholipids in a B2-glycoprotein I dependent manner. We show that bavituximab treatment is able to cure overt disease in guinea pigs lethally infected with Pichinde virus. Bavituximab treatment reduced the amounts of virus in multiple tissues and caused direct clearance of virus from the blood. Direct clearance of free virus and antibody-dependent cellular cytotoxicity of virus-infected cells appear to be the major mechanisms that contribute to the anti-viral effect of bavituximab. Bavituximab-treated survivors were immune to reinfection. Furthermore, the murine version of bavituximab, 3G4, shows therapeutic efficacy in a lethal murine model for human cytomegalovirus. Our study demonstrates the promise of anionic phospholipids as targets for new broad-spectrum anti-viral drugs.
Creating Retroviral Vector Encoding Two Homologous Chimeric Immune Receptors with Degenerated Codons (47.28)
Authors: Yang Wen , Lu Li , Ma Qiangzhong , Junghans Richard P
Published: January 2023

T cells genetically modified to express chimeric immune receptors (CIR) have been shown to be promising for immunotherapy. T cell receptor (TCR)-based CIR can be created in that the extracellular domains (Ec) of alpha (a) and beta (b) chains are fused to signaling cytoplasmic domain (Cy) of CD3 zeta (z) (TCRa-z and TCRb-z). To reduce homologous recombination associated with high homology between two nucleotide sequences in the same vector, we applied a novel approach by substituting the nucleotide sequence of one of the two homologous genes with degenerated genetic codes without change in amino acid sequence. In this study, we created a mutated DNA sequence encoding the Cy of z with degenerated codons (muz). A retroviral vector encoding a pair of TCR based CIRs (TCRa-z and TCRb-muz; tcsv-p36z) was created, with specificity via the pair of Ec of TCR a/b to a JC virus peptide p36/HLA-A2. 293T cells were transduced with retrovirus from PG13 cells with the tcsv-p36z. The transduced cells that expressed substantial TCR Vb of the CIR were able to bind p36/A2 tetramer, indicating the expression of both a and b chains in the single cell level. Western blot confirmed that both a and b chains were cellularly expressed intact. This approach, which we employed in creating retroviral vector encoding tc-CIRs with high homology, can be universally applied to creating viral vectors encoding two or multiple homologous molecules for gene therapy.
Adjuvant properties of trout C5a in subunit protein and DNA vaccines (47.31)
Authors: Zhang Yong-An , LaPatra Scott , Li Jun , Sunyer Oriol
Published: January 2023

The activation of the complement system results in the generation of C3a, C4a and C5a anaphylotoxins. C5a is the most potent and pro-inflammatory of all three anaphylatoxins. In mammals, C5a has been shown to influence the generation ofantigen-specific antibody responses, thereby acting as a molecular adjuvant. We have previously reported that rainbow trout C5a has potent chemotactic properties. In addition, we have also characterized a trout C5a receptor that is expressed at high levels both in granulocytes and B cells. In this study, we tested the possible role of trout C5a as a molecular adjuvant. To this end, we generated recombinant infectious hematopoietic necrosis virus glycoprotein (G), and a G-C5a fusion protein in E.coli. We showed that in contrast to G alone, G-C5a displayed a potent chemoattractant activity, indicating that the C5a portion of the fusion protein was functional. G-C5a was injected in incomplete freunds adjuvant (IFA) to one group of trout, while two other groups of trout were inoculated with the same dose of recombinant G in incomplete or complete freunds adjuvant (CFA) respectively. At six week postinjection, the serum antibody titers of the group injected with recombinant G-C5a in IFA were significantly higher than those of the group injected with G protein in IFA. Thus, these results suggest for the first time a role of C5a as molecular adjuvant in teleost fish. Experiments in progress will evaluate the effect of a boost injection on enhancing secondary and memory immune responses in the same fish. In addition, the presence of neutralizing antibodies against IHNV in the immunized fish will be reported.
Are Broadly Neutralizing Antibodies against HIV-1 Unusual? (47.32)
Authors: Scott Jamie Kathleen , Wang X. , Wu S. , Lepik C. , Singh H. , Richman D. D. , Utz P. J. , Breden F.
Published: January 2023

To date, AIDS vaccines cannot induce broadly(b)-neutralizing (Nt) antibodies (Abs) against HIV-1. It has been suggested that HIV’s Nt epitopes mimic “self” epitopes, and that bNt Abs are rare because they develop from autoreactive B cell clones that are normally deleted or made anergic by self antigens (Agns); such Abs are thought to have long CDR-H3 loops (H3s). Thus, self-tolerance must be broken to accumulate self-reactive naïve B cells whose Abs recognize Nt, HIV-1 epitopes. It has been reported that most of the bNt monoclonal (M)Abs have very long H3s, and that most react with self molecules (e.g., cardiolipin, CL). Alternatively, bNt Abs may arise out of “normal” Ab responses, with HIV Agns selecting bNt Abs with long H3s, and/or long H3s may develop via processes driven by persistent Agn.

We have begun to test these models by several approaches. Using autoAgn ELISAs and microarrays, bNt MAbs were shown not to be unusually self-reactive, compared to genuine autoreactive MAbs or autoimmune sera; also, bNt sera did not react significantly with CL. Finally, sequence analysis of ~700 human MAbs indicated that the bNt MAbs are no different than non-Nt MAbs against HIV-1 or other chronic viruses, or autoimmune anti-protein MAbs; but they are very different from autoimmune MAbs against non-protein autoAgns. Taken together, our results support the conclusion that HIV-bNt Abs are not unusual in their reactivity or structures.
IL-21 augments Monocyte Derived Dendritic Cell (mDC) maturation and upregulates PD-L1 gene expression (47.37)
Authors: Desai Seema , Liu Huanliang , Pahwa Savita
Published: January 2023

IL-21 and IL-4 bind overlapping epitopes on the common γ-chain, and IL-21 is reported to inhibit DC maturation and function in a murine model. To investigate the role of IL-21 on different stages of DC, monocyte derived immature dendritic cells (iDC) were generated in GMCSF and IL-4 in the presence and absence of IL-21, 20ng/mL for 5 days, termed as IL-21 iDC and control (c) iDC. IL-21iDC had significantly higher CD83 expression than ciDC on Day 5 (n=5, P<0.004) with equivalent CD86 and HLADR expression. IL21iDC had 30–50 times greater PD-L1 mRNA expression compared to ciDC as quantified by real-time PCR, but with similar expression of SOCS-1 mRNA at three doses of IL21 (20,50,100ng/ml). Day 5 iDC were further matured by the addition of maturation cocktail (TNF-α, IL1β and PGE2) for 2 days. Day7 cDC and IL-21DC expressed high PD-L1 and did not differ in their expression of phenotypic markers. Autologous T cell response to flu antigen presenting cell (APC) function of IL-21DC was 2.6 fold greater (IFN-γ Elispot response) than of cDC, whereas allogeneic T cells had a similar MLR proliferative response to IL21DC as to cDC. We conclude that IL-21 enhances iDC differentiation with mild augmentation of mDC APC function. DC maturation under optimal culture conditions is unaffected by IL-21.

Source of Research Support: Institutional Support
Antibody-mediated protection against lethal respiratory tularemia (47.5)
Authors: Kirimanjeswara Girish Soorappa , Golden Jacqueline M , Metzger Dennis W
Published: January 2023

It has been previously shown that B cells are necessary and LPS-specific antibodies are sufficient for vaccine-induced immunity to Francisella tularensis, suggesting that antibodies play a major role in protection against tularemia. However, due to the intracellular nature of the bacterium, it is widely believed that T cells, but not antibodies, are critical for protection. We assessed the relative contribution of serum antibodies in protection against respiratory tularemia by investigating the ability of serum antibodies to clear bacteria from the lungs of intranasally infected mice and prevent systemic infection. Passive administration of immune serum collected from immunized mice was found to confer protection to naïve mice against lethal F. tularensis (LVS) challenge. Adoptively transferred antibodies rapidly cleared bacteria from the lungs and prevented spread of bacteria to liver and spleen. This protection was dependent on FcγRs and phagocytes such as macrophages and neutrophils, but not complement. Interestingly, phagocytosis and killing of opsonized bacteria was dependent on the presence of IFN-γ. Consistent with this finding, in vivo antibody-mediated protection required IFN-γ and T cells. Together, these data suggest that a close and critical interaction of humoral and cellular immune responses is necessary for providing sterilizing immunity to F. tularensis. Furthermore, serum antibodies conferred protection against lethal respiratory tularemia when given 24 to 48 hr post-exposure and thus this study provides evidence for the use of antibodies for both prophylactic and therapeutic purposes.

(This work was supported by NIH Grant PO1 AI056320 (DWM) and UO1 AI057158 (GSK))