bluuebunny/crossref_metadata_2025 huggingface dataset:
Adaptive development of catalytic antibodies to the gp120 superantigenic site in lupus and HIV infected patients (44.43)
Authors: Planque Stephanie , Peng Xiaochang , Karle Sangeeta , Nishiyama Yasuhiro , Mitsuda Yukie , Taguchi Hiroaki , Smith Keri C. , Salas Maria , Hanson Carl , Sheppard Haynes W. , Paul Sudhir
Published: January 2023

The B cell superantigenic site of the HIV protein gp120 (gp120SAg) is important in viral entry into host cells and gp120SAg binding IgG in the preimmune repertoire reduce the risk of infection. Like the gp120SAg binding function, the innate catalytic function of antibody (Ab) variable domains is subject to deterioration during B cell maturation, and gp120SAg binding Abs are not produced appreciably following infection. We report the selection of catalytic single chain Fv (scFv) and light chain (L chain) clones from the Ab repertoire of patients with lupus, a condition involving increased production of gp120SAg binding Abs. The Ab fragments were isolated by covalent phage selection using electrophilic gp120 analogs. Several selected fragments cleaved gp120 via a nucleophilic mechanism coordinated with noncovalent recognition of gp120(421–433), determined by electrophoretic assays. Certain clones neutralized HIV infection in cultured peripheral blood mononuclear cells (low μg/ml potency), including two scFv clones that neutralized primary clade B, C and D strains. In contrast to lupus patients, studies on polyclonal IgG and IgA from HIV infected patients did not support the presence of an amplified Ab response to gp120SAg. IgAs from a minority of infected subjects with no progression to AIDS 5.5 years following seroconversion displayed increased gp120 hydrolysis compared to rapid progressors (P<0.0001). These data suggest catalytic Abs as a protective anti-viral mechanism and open the way to considerations of immunogens that stimulate catalytic immunity as candidate vaccines.
Inhibition of Toll-like receptor and Fc receptor responses in macrophages by TREM-2 and DAP12 (44.7)
Authors: Hamerman Jessica A , Jarjoura Jessica R , Tchao Nadia K , Humphrey Mary Beth , Nakamura Mary C , Lowell Clifford A , Seaman William E , Lanier Lewis L
Published: January 2023

DAP12 is a transmembrane signaling adapter that contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain and is expressed in myeloid cells and natural killer cells. DAP12 associates with receptors that do not have intrinsic signaling ability and therefore use DAP12 to propagate signals. We examined the inflammatory response in macrophages lacking DAP12. Surprisingly, DAP12-deficient macrophages produced increased amounts of inflammatory cytokines in response to Toll-like receptor and Fc receptor stimulation, demonstrating that DAP12 inhibits inflammatory responses in wild-type macrophages. This inhibition was dependent upon the DAP12 ITAM. In vivo, DAP12-deficient mice were more susceptible to LPS/D-galactosamine-induced shock and more resistant to Listeria monocytogenes infection. TREM-2, a DAP12-associated Ig superfamily receptor, was responsible for the DAP12-mediated inhibition of TLR and FcR responses in macrophages. Additionally, a fusion protein containing the extracellular domain of TREM-2 bound to macrophages. These results suggest that the interaction of TREM-2 and its ligand on macrophages results in an inhibitory signal that can reduce the macrophage inflammatory response.
αB-crystallin protects the retina during S. aureus induced endophthalmitis (45.2)
Authors: Gregory Meredith , Whiston Emily , Sugi Norito , Sack Coralynn , Kamradt Merideth C , Heimer Susan , Gilmore Michael S , Ksander Bruce R
Published: January 2023

Purpose: Bacterial infections in the posterior of the eye (endophthalmitis) result in toxin-related retinal destruction. However, it is not known whether apoptosis contributes to infection pathology. We are investigating whether αB-crystallin protects retinal tissue during S. aureus endophthalmitis. αB-crystallin is an anti-apoptotic small heat shock protein constitutively expressed in the retina. We hypothesize that αB-crystallin protects the retina from apoptosis.

Methods: 129S6/SvEv αB-crystallin KO and wild-type mice received intravitreal injections of 500 CFU S. aureus. Clinical examinations and ERGs (electroretinalgram) were performed at 24, 48, 72 and 96 hours. Eyes were analyzed histologically for apoptosis via TUNEL staining.

Results: Infected WT mice had only low levels of apoptosis in the inner nuclear layer of the retina. By contrast, infected KO mice displayed:

decreased ERG retinal function,increased retinal destruction via histopathology, andhigher levels of apoptosis in the ganglion cell, inner nuclear and outer nuclear retinal layers at 48 hours.

Further experiments demonstrated that

S. aureus triggers up-regulation of αB-crystallin in the retina andS. aureus produces a protease that cleaves and inactivates αB-crystallin in vitro and in vivo.

Conclusions: Bacterial-induced apoptosis of the retina occurs during endophthalmitis. αB-crystallin is increased to prevent retinal apoptosis, while bacterial proteases are released in an attempt to invade the retina. NIH-EY-016145 (M Gregory)
Do immune genes influence which pigs will have persistent Porcine Reproductive and Respiratory Syndrome virus (PRRSV) infections? (45.5)
Authors: Lunney Joan K. , Rowland Raymond , Nelson Eric , Molina Ramon , Hermann Joseph , Kuhar Daniel , Christopher-Hennings Jane , Leathers Valerie , Zimmerman Jeff
Published: January 2023

This study, a part of the “Big Pig” project, was aimed at highlighting differences in immune responses between control pigs and pigs with PRRSV infections, from early times post inoculation (pi), 14 dpi, to long term persistent infections (as evidenced by viral RNA in tissues). Our goal is to identify immunological or virological correlates of persistent infection. Two-week old pigs (n=109) were inoculated with PRRSV ATCC VR-2332 and 56 age-matched animals served as uninoculated controls. Sets of PRRSV infected and control pigs were euthanized at 2 week intervals through 203 dpi; blood and tissues were collected. RNA and cDNA was prepared from respiratory and regional mucosal tissues [lung, tracheobronchial lymph nodes (TBLN), tonsil] and assayed for expression of a panel of 23 immune markers, representing innate, T helper and regulatory genes. As expected, only low levels of interferon-gamma and certain innate immune genes were expressed by infected pigs early after inoculation. Our studies are aimed at determining whether there is a pattern of cytokine expression that might help predict which pigs will clear virus, and distinguish them from those that remain persistently infected. To date no pattern has been found with lung or TBLN; tests are continuing with tonsils and sera cytokine levels. We hope to identify regulatory pathways that would stimulate PRRSV immunity.

Supported by USDA ARS and NRI PRRS CAP1 funds.
Regulation of immune response by HSV-1 via inhibitory PILR (45.6)
Authors: Arase Hisashi , Kawaguchi Yasushi , Lanier Lewis L. , Satoh Takeshi
Published: January 2023

Herpes simplex virus type 1 (HSV-1) is a neurotropic human pathogen capable of infection and spread in a variety of cells. Cells infected with HSV-1 escape immune recognition by down-regulating cell surface expression of MHC class I molecules and/or by other unsolved mechanisms. In this study, we found that HSV-1-infected cells express a ligand for inhibitory paired immunoglobulin-like type 2 receptor (PILR), which is mainly expressed on macrophages, granulocytes, dendritic cells, and NK cells. When PILR ligand expressed on HSV-1-infected cells was analyzed by using mass spectrometry, the PILR ligand was revealed to be a viral product of HSV-1. Indeed, when the cDNA for the PILR ligand of HSV-1 was transfected into 293T cells, the transfectants were recognized by PILR-Ig. Furthermore, target cells transfected with PILR ligand of HSV-1 showed resistance to the cytotoxicity by NKL cells expressing inhibitory PILR. Our results suggest that HSV-1 encodes a ligand for PILR and downregulate the activation of immune cells expressing inhibitory PILR. Our findings provide a novel mechanism of immune evasion by HSV-1.
HCV does not interfere with ability of hepatocytes to express class I MHC or B7-H1 at the cell surface (45.7)
Authors: Kassel Rachel , Taylor Nicholas Alexander , Hahn Young Shin
Published: January 2023

T cell responses play a pivotal role in resolution of hepatitis C virus (HCV) infection. The high rate of HCV persistent infection (> 70% of HCV patients) is likely due to impaired T cell responses. To examine whether the impaired T cell responses result in part from ineffective antigen presenting functions of HCV-infected hepatocytes, we determined the expression of class I MHC and co-stimulatory molecules in HCV-infected hepatocytes. Modified HCV cell culture system (Huh 7.5 containing the full-length HCV genome or the non-structural region only) was used in our studies as well as parental Huh 7.5 cells. Huh 7.5 are a hepatocellular carcinoma cell line, permissive for HCV replication, which express low levels of class I MHC and the inhibitory ligand B7-H1 and lack co-stimulatory ligands CD40, CD80, or CD86. Elevated concentrations of IFN-γ characterize inflamed livers, and parental Huh 7.5 cells significantly up-regulate surface expression of class I MHC and B7-H1 following IFN-γ stimulation. Huh 7.5 cells stably transfected with HCV mRNAs encoding the complete genome are also able to up-regulate class I MHC and B7-H1 following stimulation with recombinant IFN-γ. This suggests that HCV may not interfere with the antigen processing/presentation capability.
FoxP3 Enhances HIV-1 Gene Expression by Modulating the LTR Occupancy of NFkappaB in Human T Cells (45.8)
Authors: Holmes Derek , Knudsen Geoffrey , Mackey-Cushman Stephanie , Su Lishan
Published: January 2023

FoxP3 determines the development of CD4+CD25+ regulatory T (Treg) cells and represses IL-2 expression in Treg cells. However, FoxP3+ Treg cells support higher levels of HIV-1 replication than FoxP3-CD4+ T cells. We report that FoxP3 enhances HIV-1 LTR but inhibits IL-2 promoter activity. This FoxP3 activity is mapped at the C-terminal forkhead domain and inactivated by human IPEX mutations. FoxP3 enhances HIV-1 LTR via specific NFkappaB binding sequences in an NFkappaB-dependant fashion. FoxP3 also enhances a minimal promoter containing the NFkappaB sequences derived from LTR but not from the MHC promoter in T cells, but represses both the HIV-1 LTR and MHC promoter in 293T cells. Interestingly, FoxP3 does not alter NFkappaB nuclear translocation but enhances its binding to the HIV-1 promoter in T cells. Therefore, FoxP3 distinctively modulates T cell activation and HIV-1 replication in human T cells by modulating NFAT and NFkappaB activity, respectively.
Adaptive immune cells temper initial innate responses¿ (46.1)
Authors: Fu Yang-xin , Kim kwang dong , Zhao Jie , Tang Hong
Published: January 2023

Immunocompromised hosts often die of acute infection, presumably, due to lack of an adaptive immune response to clear pathogens. However, we have now demonstrated that an unleashed innate immune response can also be a direct cause of death. After viral infection, nude mice produced higher levels of proinflammatory cytokines but died even though titers of virus remained comparable to wild type mice. Administration of poly I:C, a ligand for toll-like receptor 3, also led to cytokine storm in immunocompromised mice in an NK cell and TNF dependent fashion. Remarkably, addition of T cells suppressed the cytokine surge by innate cells in an antigen-independent fashion. Splenocytes of neonate also produces much higher level of cytokines in response to TLR stimulation. New pathway is required for such suppression. The concept of an unleashed innate response in the absence of adaptive modulation may lead to new diagnosis and treatment for neonate or individuals with adaptive immunodeficiencies.
IL-10 regulates movement of intestinally-derived CD4+ T cells to the liver (46.12)
Authors: Bliss Susan K. , Bliss Stuart P. , Beiting Daniel , Alcaraz Ana , Appleton Judith
Published: January 2023

Diseases that affect the intestine may have hepatic manifestations, but the mechanisms involved in establishing hepatic disease secondarily remain poorly understood. We previously reported that IL-10 knockout (KO) mice developed severe necrotizing hepatitis following oral infection with Trichinella spiralis. Here, we used this model of intestinal inflammation to further examine the role of IL-10 in regulating hepatic injury. Hepatic damage was induced by migrating newborn larvae (NBL). By delivering the parasite directly into the portal vein, we demonstrated that an ongoing intestinal immune response was necessary for the development of hepatitis. Intestinally-derived CD4+ cells accumulated in the livers of IL-10 KO mice, and depletion of CD4+ cells in wildtype (WT) mice led to a decrease in IL-10 production by hepatic leukocytes. Moreover, adoptive transfer of intestinally primed CD4+ T cells from IL-10 KO mice caused hepatitis in infected immunodeficient animals. Conversely, transfer of WT donor cells reduced the severity of hepatic inflammation in IL-10 KO recipients, demonstrating regulatory activity. Our results revealed that IL-10 prevented migration of intestinal T cells to the liver and inhibited the development of hepatitis.

This work was supported by National Institutes of Health Grants AI 14490 (J.A.A.) and DK 67290 (S.K.B.).
REGULATORY CYTOKINE ENVIRONMENT AS A PATHOGENIC MECHANISM FOR HBV PERSISTANCE (46.13)
Authors: Berrueta Lisbeth , Barboza Luisa , Salmen Siham , Goncalves Loredana , Colmenares Melisa , Peterson Darrell , Montes Henry , Cartagirone Raimondo , Gutierrez Maria del Carmen
Published: January 2023

The T cell response to HBV has been shown to be vigorous in patients with acute hepatitis who clear the virus, whereas it is weak and narrowly focused in patients with chronic disease. We hypothesized that inappropriate early activation events of T cells could contribute to viral persistence during chronic infection. Therefore, we investigated the induction of activation receptors and cytokine production in lymphocytes stimulated with HBcAg, in a group of chronically infected patients and naturally immune subjects. Chronic patients did not increase CD40L or CD69 following stimulation, compared to subjects that resolved the infection. The presence of a CD4+CD25+ cell population was observed which produced elevated levels of IL-10 and TGF-β following specific stimulation with HBcAg, cytokines that are known to be suppressors of the immune response. These results suggest that a predominant inhibitory environment may be responsible for altered T cell costimulation, representing a mechanism for viral persistence.
Is there a role for Tregs in SIV infected non-human primates? (46.15)
Authors: Pereira Lara Elizabeth , Villinger F. , Onlamoon N. , Bryan P. , Cardona A. , Pattanapanysat K. , Mori K. , Hagen S. , Picker L. , Ansari A. A.
Published: January 2023

A study was performed to identify, characterize and compare the level and function of peripheral Tregs in uninfected and SIV-infected rhesus macaques (RM) and sooty mangabeys (SM) to determine if Tregs contribute to the muted immune response that is evident in SIV-infected disease-resistant SM but readily detectable in SIV-infected disease-susceptible RM. Results show that SIV-infected SM maintain the level and function of Tregs but a substantial decline was noted in RM. A strong correlation between plasma VL and the level of Tregs was observed in SIV-infected RM but not SM. RM VL controllers exhibited lower levels of immune activation and recovered pre-infection levels of Tregs that were fully functional when tested for suppressive activity in vitro. ICC assays showed that Treg-depleted PBMC fractions from RM but not SM exhibit increased CD4+ and CD8+ T-cell responses to select SIV peptide pools suggesting that Tregs play a role in the SIV-specific immune response at least in RM. The decline in Tregs in SIV-infected RM as a general consequence of viral pathogenesis may further contribute to the increased immune activation and viral replication exhibited by disease progressors. Although the role of Tregs in SM remains unclear, the data imply that therapeutic manipulationof the frequency and function of Treg-cell in RM and perhaps HIV-infected hosts could improve immune controlof viral infection.

NIH RO1 27057, AIDS & Health Sciences Research Grants, Ministry of Health Labor & Welfare in Japan.
Rapid alteration of blood brain barrier (BBB) tight junction proteins by antigen specific CD8 T cells (46.16)
Authors: Johnson Aaron , Suidan Georgette , Chen Yi , McDole Jeremiah , Pirko Istvan
Published: January 2023

CNS vascular permeability is a feature of numerous neurological diseases as diverse as shock, multiple sclerosis, Alzheimer’s disease and viral hemorrhagic fevers. The role of inflammatory immune cells in promoting opening of the blood brain barrier (BBB) in neurological disease remains poorly understood. Our laboratory has created a novel in vivo system to study BBB disruption using the Theilers murine encephalomyelitus virus (TMEV) model of multiple sclerosis. During acute TMEV infection, 70% of CNS infiltrating CD8 T cells are specific for a TMEV peptide, VP2 121–130, presented in the context of the Db Class I molecule. Intravenous injection of the VP2 121–130 peptide during the peak of CD8 T cell expansion in the CNS results in fatal vascular permeability characterized by: 1.) Astrocyte activation as detected by GFAP upregulation, 2.) Alteration of BBB tight junction proteins Z0-2, Occludin and Claudin 5, 3.) Vascular permeability as measured by quantifiable T1 weighted MRI, and 4.) Severe motor deficit. Astrocyte activation and BBB tight junction protein alteration by CD8 T cell is rapid, occurring within four hours of peptide administration. We have also determined that perforin is critical for vascular permeability to occur. To our knowledge, this is the first demonstration of an antigen specific T cell response altering tight junction proteins of the BBB.
Novel role of NK cells in the development of adaptive immunity against mycoplasma respiratory disease (46.23)
Authors: Bodhankar Sheetal , Woolard Matthew D , Simecka Jerry W
Published: January 2023

A complex balance between the detrimental and beneficial effects of immunity determines the course of mycoplasma respiratory infection. NK cells were shown to dampen innate immunity in the lungs. The purpose of this study was to determine if NK cells similarly impacted the development of adaptive immunity against mycoplasma disease. Anti-asialoGM1 Ab was used to transiently deplete NK cells prior to nasal-pulmonary immunization with mycoplasma membrane Ag. Unimmunized and mice receiving only anti-asialoGM1 Ab served as controls. The treatment was repeated 7d later and the mice were challenged on day 14 with M. pulmonis. DX5+ NK cell numbers returned to normal by day 14. Colony forming units (CFUs) in lungs and nasal passages were determined 3, 7 and 14d later.

There was a significant decrease (>1 log) in CFUs in the NK cell depleted, immunized mice as compared to immunized or control mice. No such effect was seen in SCID mice, indicating adaptive immunity was affected. Further support that the protection rendered due to depletion of NK cells prior to immunization, was lymphocyte-mediated, was demonstrated when protection was shown to be adoptively transferred to naïve mice using total lung lymphocytes, purified T and/or Non T cells.

In conclusion, NK cells dampen the generation of protective adaptive immunity associated with nasal-pulmonary immunization, and this effect is on the generation of lymphoid cell mediated response. These results should provide insights into approaches to generate optimal protective immunity against mycoplasma respiratory diseases.
HIV gp120 alone may mediate lymphopenia and lymphadnopathy in HIV infected individuals (46.4)
Authors: Green Daniel Saul , Morgan Ross , Curcio-Bonner Chrystal , David Center M , Cruikshank William W
Published: January 2023

Lymphopenia is the hallmark of HIV infection and is thought to be mediated by virus infection and subsequent killing of CD4+ T cells. However, recent data suggests that decrease in peripheral CD4+ T cells may initially involve sequestration of these cells in secondary lymphoid tissues (SLT). T cell homing to SLT is facilitated by SLT-specific chemokines, CCL19 and CCL21 while egress from SLT requires expression of functional sphingosine-1-phosphate (S1P) receptors. As lymphadnopathy is observed in most HIV-1 infected individuals, we investigated the potential of HIV-1 envelope glycoprotein, gp120, to affect parameters of T cell homing to SLT. Normal human T cells were exposed to gp120 for 1 to 24h prior to migration induced by CCL19 or CCL21. We also investigated the effects of gp120 on responsiveness to S1P induced migration. We found that exposure to gp120 for 1h augmented, by 2-fold, chemoattraction induced by CCL19 and CCL21, but did not effect migration induced by the SLT independent chemokine CCL11. Conversely, these cells became completely unresponsive to S1P induced migration (2–3 fold migration blocked to 0). The gp120 mediated inhibition of migration was completely reversed by the p56lck inhibitor herbimycin A, indicating a requirement of CD4 signaling. Taken together our data indicates that gp120 alone could facilitate CD4+ T cell homing and sequestration in the SLT of HIV infected indiivduals.
In vitro and in vivo induction of B7-H1 and B7-DC expression by human rhinovirus infection of airway epithelial cells (46.5)
Authors: Kim Jean , Heinecke Lowella F , Proud David , Schleimer Robert P
Published: January 2023

We have found that human airway epithelial cells express costimulatory molecules B7-H1, B7-H2, B7-H3 and B7-DC mRNA and cell-surface protein. IFN? and TNF? selectively induce B7-H1 and B7-DC and glucocorticoid fluticasone (FP) inhibits this induction. We now report that human rhinovirus infection (HRV-16), a key trigger of exacerbations of chronic rhinosinusitis and asthma, results in selective induction of B7-H1 and B7-DC expression in airway epithelial cells both in vitro and in vivo. In vitro exposure of human primary bronchial epithelial cells (PBEC) to HRV-16 (TCID50 5 X 103.1) resulted in induction of cell surface expression of B7-H1 as measured by flow cytometry (from 42±9 to 56±8 MFI, p<0.05) and B7-DC (from 5±1 to 9±2 MFI, p<0.01) (n=6). Pretreatment with FP (10-7 M) inhibited the induction of B7-H1 by 64% (p<0.05) and B7-DC by 95% (p<0.01). Additionally, in vitro exposure of PBEC to TLR3 agonist dsRNA (25 ug/ml), a surrogate for HRV16 infection, mirrored the effect of HRV-16 infection. Nasal scrapings taken at the time of peak symptom scores 4 days after infection of 6 human subjects with HRV-16 showed selective increases in levels of mRNA for B7-H1 (8.5±2.5 fold, p<0.03) and B7-DC (3.2±1.1 fold, p<0.07). These data show that exposure to TLR3 agonist dsRNA or HRV-16 infection induces B7-H1 and B7-DC on epithelial cells and this may influence the development of adaptive immune responses in the airways.
Differential Susceptibility in a Murine Model of Melioidosis (46.7)
Authors: Tan Gladys Gek Yen , Liu Yichun , Ooi Eng Eong
Published: January 2023

Melioidosis, an emerging infectious disease with protean clinical manifestations and caused by Burkholderia pseudomallei, is endemic to SE Asia and Northern Australia. A murine model was set up to study host-pathogen interactions associated with different acquisition modes. Aerosol, intranasal, intraperitoneal, subcutaneous routes of infection were performed on BALB/C and C57Bl/6 mice, with virulent Bp strain KHW. Based on LD50 values and disease progression, the lethality, in decreasing order, was aerosol> intranasal> intraperitoneal> subcutaneous. Regardless of the portal of entry, LD50 values of C57Bl/6 mice were at least 100-fold higher than those of Balb/c mice, suggesting C57Bl/6 mice were more resistant and Balb/c mice more susceptible to Bp infection. Comparing the pathogenesis of Bp infection with 20 CFU bacteria via inhalational routes, overwhelming bacterial burdens were found in the lungs, nasal cavity, spleen, liver and blood of Balb/c mice, against low to undetectable bacterial loads in C57Bl/6 mice post infection. High levels of proinflammatory cytokines were detected in Balb/c mice, while a transient and moderate cytokine level was observed in C57Bl/6 mice. Despite the presence of inflammation in Balb/c mice, the innate immune response was unable to control infection, leading to severe immune-mediated pathology and death.

This study was funded by D30 grant from DSTA, Singapore.
Modulation of malaria-induced immunopathology by concurrent nematode infection (46.8)
Authors: Segura Mariela , Matte Christine , Thawani Neeta , Su Zhong , Stevenson Mary M.
Published: January 2023

Gastrointestinal nematode infections, highly prevalent in malaria endemic areas, are known to modulate host immune responses to unrelated pathogens. However, the complex relationship between worms and malaria and the impact on malarial incidence vs. severity are unclear. Previously, we observed that C57BL/6 (B6) mice infected with the gastrointestinal nematode Heligosomoides polygyrus (Hp) for 2 wks before blood-stage Plasmodium chabaudi AS (Pc) infection developed significantly higher parasitemia compared to mice infected with Pc alone. Despite this, malaria-induced body weight loss, anemia and hepatosplenic damage were not exacerbated in co-infected mice. Indeed, there was less severe hypothermia and hypoglycemia and more robust reticulocytosis in co-infected mice. Co-infection with Hp suppressed type 1 immune responses induced by P. chabaudi infection with significantly reduced plasma levels of IFN-γ, TNF-α and nitric oxide, but increased IL-6 and IL-10 levels. Co-infected STAT-6−/− mice, which have disrupted Th2 responses, experienced only slightly more severe hypothermia, hypoglycemia, and anemia than co-infected WT B6 mice while in vivo administration of anti-IL-10R (1B1.2) mAb resulted in higher mortality in co-infected WT mice. Although concurrent nematode infection resulted in increased parasitemia due to impaired anti-malarial type 1 immunity, our data suggest that malaria-associated pathology is alleviated by a complex immunoregulatory network, possibly involving IL-10, and, to a lesser extent, Th2 cytokines.
Cytokine and phenotypic profiles of specific T cells induced by newborn BCG vaccination (47.1)
Authors: Hanekom Willem , Soares Andreia , Scriba Tom , Hussey Greg , Kaplan Gilla
Published: January 2023

Bacillus Calmette-Guerin (BCG), the only currently available TB vaccine, is the most widely administered vaccine in the world. However, the immune response to BCG has not been fully characterized. This knowledge is critical for studies of protective immunity induced by vaccination and for developing novel, improved TB vaccines.

We aimed to describe phenotypic and cytokine profiles of specific T cell subsets in blood of 10-week old infants following their routine vaccination with BCG at birth.

Whole blood of 30 infants was incubated with BCG for 12 hrs; Brefeldin A was added for the last 5 hrs. Red cells were lysed and white cells fixed and cryopreserved. Thawed cells were permeabilized, stained and analysed by multiparameter flow cytometry.

We observed predominant expression of presumably protective Type 1 cytokines: distinct Type 1 CD4+ and CD8+ T-cell subsets were identified, based on IL-2, TNF and IFN-gamma expression. TNF expression was virtually absent from CD8+ T cells. IL-4 could not be detected; IL-10 expression was low, but only in cells that did not express Type 1 cytokines. The distinct phenotypes of IL-2, TNF and IFN-gamma expressing cells will be described: the most common phenotypes were CD45RA-CCR7+/−CD27+.

We conclude that newborn BCG vaccination induces predominantly Type 1 cytokine responses in CD4+ and CD8+ T cells. Significant numbers of specific T-cells did not produce IFN-gamma, suggesting that assays relying on detection of this cytokine alone do not measure the entire mycobacteria-induced response. Type 1 cytokine expressing CD4+ and CD8+ T cells have predominantly effector and central memory phenotypes. We will now apply this knowledge to a large ongoing study of BCG-induced immune correlates of protection against TB in children.
Engineering filamentous phage as carriers that focus antibody responses against a peptide (47.11)
Authors: van Houten Nienke Elizabeth , Smith George P. , Scott Jamie K.
Published: January 2023

Antibody (Ab) responses to a B-cell epitope can be produced by immunization with a peptide bearing the epitope of interest. Such peptides are often poor immunogens, requiring conjugation or fusion to carrier proteins that provide T-cell help. However, carrier proteins may also divert the Ab response away from the targeted peptide via its own B-cell epitopes. Thus, an ideal peptide carrier should provide T-cell help to drive B-cell responses but contain relatively few B-cell epitopes.

The Ab response to filamentous phage is restricted to the 12 N-terminal residues of the major coat protein (pVIII) and the external domains of the minor coat protein, pIII. Previously, we reported that the Ab response against a synthetic peptide conjugated to phage was more focused on the peptide compared to ovalbumin as the carrier. In some cases, the anti-peptide Ab titer exceeded the anti-carrier Ab titer by > 20 fold. The reduced complexity of the phage surface compared to ovalbumin’s may be responsible for this.

Here, we tested the hypothesis that Ab responses to weakly immunogenic peptides would be improved by removing immunodominant epitopes from the phage surface; immunogenic residues on pVIII were altered, and the external domains of pIII were truncated. The phage were tested alone as immunogens, and as carriers for pVIII-displayed peptides. The results and the implication for vaccine design are discussed.

Supported by NIH R01AI-49111
Complete Protective Immunity induced by Cottontail Rabbit Papillomavirus E1 Epitope DNA Vaccination in HLA-A2.1 transgenic rabbits (47.13)
Authors: Hu Jiafen , Schell Todd , Peng Xuwen , Cladel Nancy , Balogh Karla , Christensen Neil
Published: January 2023

Cottontail rabbit papillomavirus (CRPV)/HLA-A2.1 transgenic rabbit infection model has been reported previously. Because the CRPV genome can be modified extensively without loss of its function, this transgenic rabbit model can be used for testing the specificity of host immunity to specific HLA-A2.1 restricted papillomavirus protein epitopes in vivo. In this study, we incorporated online MHCI epitope prediction software, HLA-A2.1 transgenic mouse and rabbit systems together to demonstrate an efficient way to identify and test immunogenicity of HLA-A2.1 restricted epitopes from CRPVE1 (161–169 LLFRQAHSV and 303–311 MLQEKPFQL). Both epitopes were able to induce specific cytotoxic CD8 T cells in HLA-A2.1 transgenic mice immunized with peptides. Using an epitope DNA vaccination method, complete and partial protection against CRPV infection was achieved by CRPVE1/303–311 and CRPVE1/161–169 in HLA-A2.1 transgenic rabbits respectively. Our data further demonstrated the application of HLA-A2.1 transgenic rabbits as a potential in vivo model system to test papillomavirus therapeutic vaccines.
Passive immunity against infection with Trypanosoma brucei subsp. brucei in mice is conveyed by an antibody blocking the ligand-binding extracellular domain 1 (ed1) of the C2 receptor CRIT (47.20)
Authors: Inal Jameel Malhador , Rock Peter
Published: January 2023

Anti-CRIT-ed1 (recognising the first extracellular domain of CRIT) was passively transferred into mice which were subsequently challenged with 500 trypomastigotes of T. brucei subsp. brucei. At a peak of infection, eight days after challenge infection, mice that had received 0.5 mg anti-CRIT-ed1 (ascites) had significantly reduced levels of parasitaemia compared to control mice. This inhibiton was also found to be dose-dependent. As passive transfer of anti-CRIT-ed2, the antibody recognising the second extracellular domain did not reduce parasitaemia, the protection was deemed specific. The protection afforded by passive transfer of anti-CRIT-ed1 also delayed mortality. To exclude the possibility that the reduction in parasitaemia was due to intact IgG inducing complement-mediated lysis of trypomastigotes we found that whereas anti-CRIT-ed1 was unable to induce complement-mediated lysis of cultured trypomastigotes, the sera from T. brucei subsp brucei-infected mice induced lysis.
Detection of HBV Antigen-Specific Cellular Immune Responses after PrimeBoost Therapeutic Vaccination in Chronic Hepatitis B Patients (47.26)
Authors: Brown Dean , Bourne Victoria , Cripps Martin , Anderson Richard , Schneider Joerg
Published: January 2023

Hepatitis B virus (HBV)-specific T cell responses play an important role in the clearance of HBV infection. Heterologous Hi-8™ PrimeBoost vaccination using HBV surface antigen (HBsAg) is a promising approach for inducing T cell responses in chronic hepatitis B patients and results in viral suppression and liver function normalisation. Cellular immune responses against HBV antigens were determined in PBMC isolated from patients treated with Hi-8™ HBV therapy. Samples were tested by ex vivo IFN-γ ELISPOT assay and after 14 days of culture in the presence of overlapping peptides spanning the HBsAg and HBV core antigen (HBcAg) sequences. Ex vivo HBsAg-specific T cell responses were of very low frequency and transient. However, higher frequency T cell responses that could be correlated with clinical outcome were measured after culture. Notably, HBsAg but not HBcAg-specific responses increased after treatment, indicating that responses were driven by vaccination. Further experiments are being conducted to determine the relative contribution of CD4 and CD8 T cells to the immune response. In conclusion, HBsAg specific IFN-γ producing T cells were detected in PBMC from chronically infected patients and increased in those receiving Hi-8™ HBV therapy. Since the T cell immune responses could only be detected following in vitro culture, they are either of very low frequency in the peripheral blood or of a memory phenotype.
Comprehensive Analysis of the Antibody Response to Yersinia pestis by use of a Novel Whole Proteome Microarray (47.3)
Authors: Keasey Sarah L , Schmid Kara , Purcell Bret K , Morefield Garry L , Meegan James , Tikhonov Alex , Schweitzer Barry , Ulrich Robert G
Published: January 2023

A novel proteome microarray representing the majority of the 4086 Yersinia pestis KIM proteins was produced by high-throughput expression and purification. Rabbit hyper-immune sera were produced against proteomes extracted from several pathogenic gram-negative bacteria for use in validation assays. The antibody profile from each of the rabbits enabled detection of:

shared cross-reactive proteinssignature proteins common for two or more bacteria, andfingerprint proteins specific to each pathogen.

Unique proteins were recognized by convalescence sera from mice that survived plague following immunization with the experimental F1-V vaccine. Several new antigens were discovered that were recognized by antibody from rhesus that survived plague, whereas these Y. pestis proteins were not recognized by sera from animals surviving challenge with spores of the gram-positive Bacillus anthracis. Finally, analysis of sera from cynomolgus macaques acutely infected with Y. pestis or Bacillus anthracis produced antibody-binding patterns that were unique biomarkers for each disease. Our results demonstrate that the whole proteome microarray platform is very useful for establishing diagnostic biomarkers, detecting antibody responses to proteins that could potentially show promise in vaccine development, and also for determining the degree of antigen-cross reactivity between different strains or species of bacteria.
Antiviral effect of The ImmunomodulatorAS101 on West Nile Virus by AlphaVBeta3 integrin inhibition (47.33)
Authors: Sredni Benjamin , Indenbaum Victoria , Makarovsky Daria , Bin Hanna , Albeck Michael , Lustig Shlomo , Mendelson Ela
Published: January 2023

The identification of integrin αVβ3 as a receptor for West Nile virus (WNV) in recent years, created opportunities in the development of antiviral strategist against WNV infection. The immunomodulator AS101 [ammonium trichloro (dioxyethylene 0-0′)tellurate] has an antiviral effect in vivo and in vitro. Our study aimed at determining whether AS101 has an antiviral effect against WNV and its mechanism of work. Cell viability assay for Vero cells treated with AS101 before WNV infection, revealed up to 90% survival of the treated cells. More over, plaque forming unit (PFU) assay showed 83% inhibition at WNV-infected Vero cells after AS101 treatment. Significant decrease in the concentration of viral infectious particles was observed in supernatants of Vero cells infected with WNV at high MOI (5) after AS101 addition, and the viral envelope protein (E) expression decreased in Vero cells infected with WNV (MOI=5) and treated with AS101. In supernatants collected from virus infected and AS101 treated Vero cells immediately after inoculation were more virus particles, than in not treated cells. A novel AS101 effect was seen at attachment assay were AS101 inhibited Vero cells attachment to human integrin αVβ3 specific antibody in dose dependent manner. Thus, the results presented here shows that AS101 has an antiviral effect against WNV, and suggests that AS101 prevents WNV entry to the cells by inhibiting integrin αVβ3.
Directing the natural catalytic activity of antibodies to hepatitis C virus E2 protein (47.36)
Authors: Sapparapu Gopal , Planque Stephanie A , Wang Runsheng , Nishiyama Yasuhiro , Keck Zhen-Yong , Foung Steven , Paul Sudhir
Published: January 2023

The envelope protein E2 is involved in hepatitis C virus (HCV) entry into host cells. The proteolytic activity of antibodies (Abs) offers a novel neutralizing mechanism by permanent inactivation of multiple epitopes by a single Ab molecule. We studied the proteolytic cleavage of E2 linked to a glutathione S-transferase tag (E2-GST) by Abs purified from human sera. IgM but not IgG class Abs hydrolyzed recombinant E2 determined by an electrophoresis assay, consistent with previous observations that Ab catalysis is an innate immune function subject to deterioration over the course of adaptive B lymphocyte differentiation. To determine the feasibility of engineering HCV E2-specific proteolytic Abs, we cloned hybrid human IgGs composed of light chains with promiscuous proteolytic activity (clones GG63, HK13 and HK14; isolated from a lupus phage library) and the heavy chain of an HCV E2-specific Ab (clone CBH-7; isolated from an HCV infected subject). The hybrid Abs hydrolyzed E2-GST with little or no activity in the wildtype control Ab. The hybrid Abs displayed the ability to bind 125I-E2, confirming that the heavy chain subunit can imparts specificity for E2. These observations suggest the feasibility of harnessing the natural proteolytic activity of Abs to developing catalytic reagents with potential immunotherapeutic utility.
The neonatal calf tuberculosis vaccine model: immune responses to protective and non-protective vaccines after aerosol challenge with virulent Mycobacterium bovis (47.39)
Authors: Waters Ray , Palmer Mitchell , Nonnecke Brian , Thacker Tyler , Estes Mark , Jacobs William R. , Larsen Michelle
Published: January 2023

An attenuated Mycobacterium tuberculosis RD1 knockout and pantothenate auxotroph (mc26030) vaccine failed to protect neonatal calves from a low dose, aerosol M. bovis challenge. In contrast, M. bovis bacille Calmette Guerin (BCG)-vaccinates had reduced tuberculosis-associated pathology as compared to non- and mc26030-vaccinates. Mycobacterial colonization was not impacted by vaccination. Positive prognostic indicators associated with reduced pathology in the BCG-vaccinated group included: decreased antigen induced IFN-gamma, iNOS, IL-4, and MIP1-alpha responses and a diminished activation phenotype (i.e., ↓CD25+ and CD44+ cells and ↑CD62L+ cells) in mycobacterial-stimulated mononuclear cell cultures. The calf sensitization and challenge model provides an informative screen for candidate tuberculosis vaccines before their evaluation in costly non-human, primates.
Active Aβ vaccination-induced Aβ clearance enhanced by suppression of the CD40-CD40L interaction (48.11)
Authors: Bai Yun , Obregon Demian , Hou Huayan , Nikolic William , Mori Takashi , Zeng Jin , Ehrhart Jared , Giunta Brian , Fernandez Frank , Morgan Dave , Town Terrance
Published: January 2023

Active and passive Aβ immunization efficiently reduces amyloid plaque load and memory impairment in transgenic mouse models of Alzheimer’s disease. Immunization has also yielded favorable results for many patients; but a significant percentage also developed severe meningioencephalitis. Prior studies by our group suggest CD40 receptor-CD40 ligand interaction between T-cells and microglia initiates release of proinflammatory factors, which drive phenotypic switching from a phagocytic to an antigen presenting cell phenotype thus impairing the clearance of Aβ. We therefore tested whether a CD40 blockade could enhance Aβ vaccination-mediated Aβ clearance in vivo. Importantly in our present study, Aβ vaccinated CD40 deficient heterozygous transgenic mice over-expressing mutant amyloid precursor protein and presenilin-1 demonstrated enhanced Aβ clearance; compared with vaccinated PSAPP mice. Similar results were obtained with Aβ vaccinated PSAPP mice passively immunized with CD40L antibody injections. Taken together with our previous studies these data suggest that CD40 blockade, in the context of active Aβ vaccination, acts to enhance CNS Aβ clearance by maintaining microglia in a phagocytic phenotype. These data also may provide the basis for a method whereby enhancing Aβ vaccination-mediated Aβ clearance strategies.
4-1BB is Superior to CD28 Costimulation for Generating Effector CD8+ T Cells for Adoptive Immunotherapy (48.12)
Authors: zhang hua , Snyder Kristen , Suhoski Megan , Maus Marcela , Kapoor Veena , June Carl H , Mackall Crystal L
Published: January 2023

Artificial APCs (aAPCs) provide a new approach to expand cells for adoptive cell therapy, but optimal requirements for CD8+ expansion have not been defined. Beads expressing αCD3/αCD28 efficiently expand CD4+ cells, but not cytolytic CD8+ T cells. 4-1BB, a costimulatory molecule expressed by a minority of resting CD8+ T cells, is transiently upregulated by all CD8+ T cells following activation. We compared the capacity for aAPCs providing costimulation via 4-1BB vs. CD28 to expand cytolytic CD8+ T cell populations. Whereas αCD3/αCD28 aAPCs preferentially expand naive cells, αCD3/4-1BBL aAPCs preferentially expand memory cells, resulting in cultures with enhanced reactivity to previously primed viral antigens and leading to efficient expansion of electronically sorted CD8+ populations reactive toward viral or tumor-associated antigens. Moreover, using HLA-A2-Fc fusion proteins, 4-1BBL aAPCs selectively induced a 3 log expansion of antigen-specific CTL from polyclonal CD8+ PBL over 14 days; similar expansions were not induced using HLA-A2-Fc/αCD28 aAPCs. Compared to αCD3/αCD28 expanded CD8+ CTL, αCD3/4-1BBL expanded CD8+ T cells also mediate enhanced cytolytic capacity as a result of 4-1BB signaling itself and NKG2D upregulation. These results demonstrate that 4-1BB costimulation is superior to CD28 costimulation for expanding antigen specific CD8+ CTL for use in adoptive immunotherapy.
Genetic immunizations in the presence of a specific neurokinin 1 receptor agonist triggers Th1-Tc1 biased immune responses that protect against B16 melanomas (48.13)
Authors: Mathers Alicia R , Tkacheva Olga A , Janelsins Brian , Schuffeski William , Morelli Adrian E , Larregina Adriana T
Published: January 2023

Antigen (Ag) specific Th1-Tc1 cellular immunity induced by genetic immunizations is critical to prevent / eradicate tumors. The high number of resident dendritic cells (DCs) makes the skin an ideal microenvironment for genetic immunizations against tumors. Skin DCs become potent stimulators of Ag specific T cells by signals triggered during acute inflammation in peripheral tissues. The secretion of the pro-inflammatory neuropeptide substance P signaling via the neurokinin 1 receptor (NK1R) represents an early danger signal that stimulates APC functions of DCs. Here we tested whether signaling via the NK1R during skin genetic immunizations favors Th1-Tc1 immunity in C57BL/6 mice injected with B16 melanoma cells. We utilized the gene gun to deliver transgenic (tg) OVA or TRP-2 Ag to mouse skin and the NK1R agonist Sar9Met (O2)11-SP as a potential Th1 adjuvant. Our strategy expressed Ag exclusively in the epidermis and induced substantial migration of activated Langerhans cells (LCs) transporting tg Ag to skin draining lymph nodes, which resulted in generation of Th1-Tc1 immunity. Importantly delivery of tg TRP-2 plus NK1R agonist was able to protect mice against B16 melanoma tumor cell growth. Our data demonstrate that NK1R signaling provides the Th1 adjuvant effect that positively modulates the immunostimulatory functions of LCs during skin genetic immunizations leading to productive immune responses.
Form of antigen dictates immunity: Irradiated cell vs. whole cell lysate vaccination (48.16)
Authors: Buckwalter Matthew , Srivastava Pramod
Published: January 2023

The context in which antigen is perceived by the immune system dictates the quality of the ensuing immune response, which can range from tolerance to lasting immunity. In the current report we investigated the effects of vaccinating mice with antigen in two separate contexts, namely: Irradiated cells, or whole cell lysates. Using the MethA tumor model we observed that although a single vaccination with irradiated MethA cells leads to immunity in BALB/c mice, vaccination with the same cell equivalents of whole cell lysate does not. These results were surprising in light of the substantial body of literature demonstrating the immunostimulatory effects of cell lysates. We hypothesize that although each vaccination contains the same potential antigens, due to the different context in which they are delivered, the immune system will react to them differently. To test this we analyzed multiple characteristics of the immune response following vaccination with irradiated cells or whole cell lysates including: Tumor protection, the quantity and quality of an antigen specific T cell response, stability and persistence of antigen, and the effects of multiple vaccinations. We report dramatic differences between the two immunogens at each of the aspects tested. Our data suggests that although whole cell lysates contain antigen and the same potentially immunostimulatory components as irradiated cells, vaccinating mice with irradiated tumor cells leads to a much greater T cell response and tumor protection than does vaccinating with whole cell lysates.