Immunology of Infectious Disease Commons^™

The Effects Of Specialized Pro-Resolving Mediator Lipoxin A4 On Pseudomonas Aeruginosa Biofilms And Interactions With Monocytes, Julianne M. Thornton

Graduate School of Biomedical Sciences Theses and Dissertations

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen known as a major cause of hospital-acquired secondary infections, commonly causing chronic respiratory infections in immunocompromised individuals, especially those with cystic fibrosis, and often found in wound infections. P. aeruginosa uses the quorum sensing pathway to readily form protective biofilms, which reduce the efficacy of antibiotics and access by host immune cells to eradicate the pathogen. Specialized pro-resolving mediators (SPMs) are lipids endogenously produced by the host immune response to infection to aid in infection resolution. One SPM, Lipoxin A4 (LxA4), has been shown to be a robust quorum sensing inhibitor.

The …

Antibody-Dependent Enhancement Activity Of A Plant-Made Vaccine Against West Nile Virus, Amber M. Paul, Haiyan Sun, Dhiraj Acharya, Huafang Lai, Junyun He, Fengwei Bai, Qiang Chen

Publications

West Nile virus (WNV) causes annual outbreaks globally and is the leading cause of mosquito-borne disease in Unite States. In the absence of licensed therapeutics, there is an urgent need to develop effective and safe human vaccines against WNV. One of the major safety concerns for WNV vaccine development is the risk of increasing infection by related flaviviruses in vaccinated subjects via antibody-dependent enhancement of infection (ADE). Herein, we report the development of a plant-based vaccine candidate that provides protective immunity against a lethal WNV challenge mice, while minimizes the risk of ADE for infection by Zika (ZIKV) and dengue …

Investigating The Pi3k/Akt/Atm Pathway, Telomeric Dna Damage, T Cell Death, And Crispr/Cas9-Mediated Gene Editing During Acute And Chronic Hiv Infection, Sushant Khanal

Electronic Theses and Dissertations

Human Immunodeficiency Virus (HIV) infection initiates major metabolic and cell- survival complications. Anti-retroviral therapy (ART) is the current approach to suppress active HIV replication to a level of undetected viral load, but it is not a curative approach. Newer and sophisticated gene editing technologies could indeed be a potent antiviral therapy to achieve a clinical sterilization/cure of HIV infection. Chronic HIV patients, even under a successful ART regimen, exhibit a low-grade inflammation, immune senescence, premature aging, telomeric DNA attrition, T cell apoptosis, and cellular homeostasis. In this dissertation, we investigated CD4 T cell homeostasis, degree of T cell apoptosis, an …

Genital Chlamydia Infection Is Influenced By The Female Sex Hormones Estrogen And Progesterone In Vivo, Amy Gail Gravitte

Electronic Theses and Dissertations

Chlamydia is the most common bacterial sexually transmitted infection in the United States and worldwide. It often goes unnoticed due to lack of symptoms and left untreated it can ascend the female genital tract to cause sequelae like pelvic inflammatory disease and irreversible tubal infertility. In reproductive-aged women, female sex hormones estrogen (E2) and progesterone (P4) concentrations fluctuate during the menstrual cycle and are influenced by hormonal contraceptives and hormone replacement therapy. E2 and P4 influence genital Chlamydia infection in women and mice, but these multifactorial interactions are not entirely mapped out. The complex interplay of E2 and P4 with …

Sodium Pyruvate Ameliorates Influenza A Virus Infection In Vitro And In Vivo, Jessica M. Reel

MSU Graduate Theses

Pyruvate is produced in duplicate at the end of glycolysis in addition to ATP and NADH. Pyruvate is the metabolite of choice in most cells, whether obtained exogenously or endogenously. Recently we found that the addition of pyruvate’s conjugate base, sodium pyruvate, to cell culture media dampened the immune response to influenza A virus (IAV) infection in cultured innate immune cells. Thus, I decided to investigate the mechanism and potential for treatment of IAV. In vitro using bone marrow derived macrophages that were infected with IAV we found that adding sodium pyruvate to the media decreased immune signaling pathways through …

B25: Generation And Characterization Of Sars-Cov-2 Spike Protein-Pseudotyped Lentivirus Particles For Use In Neutralizing Antibody Titer Testing Of Human Serum, Luis Sanchez

Annual Research Symposium

No abstract provided.

H1n1 Influenza Virus (Swine Flu): A Comprehensive Insight Into Escalating Catch-22 Scenarios, Muhammad Shahzaib, Ehsan Ul Haq

The University of Louisville Journal of Respiratory Infections

Introduction: Viruses have always been a major cause of various disastrous pandemics in mankind’s history. H1N1 became a threat when its original strain was first discovered back in the swine flu pandemic of 2009. It became highly catastrophic on a large scale because none of the therapeutic interventions and methodologies that were already present at the time were effective against the virus.

Methods: A vast amount of literature and research is available regarding H1N1 influenza from different reputable sources online. The data were gathered with the contrasting and relative situations of 1918 and 2009 pandemics in mind. The overall extracted …

Pseudomonas Aeruginosa Vaccine Development Using Dual-Species Whole Cell And Subunit Vaccines, Catherine Bell Blackwood

Graduate Theses, Dissertations, and Problem Reports

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen which can cause severe, recurrent, and chronic infections. The pathogen is highly adaptable, and pneumonia caused by it is associated with high morbidity and mortality, especially in individuals with the genetic disorder cystic fibrosis. Antibiotic resistance amongst clinical isolates of P. aeruginosa is steadily increasing, and multi-drug resistant strains are prevalent. There is currently no vaccine available for commercial use against P. aeruginosa. For these reasons, we sought to understand the immunity to P. aeruginosa induced by whole cell vaccination and identify antigens for development of future subunit vaccines. In this dissertation, …

Development, Expansion And Role Of Myeloid-Derived Suppressor Cells In Post-Sepsis Immune Suppression, Tuqa Alkhateeb

Electronic Theses and Dissertations

Myeloid-derived suppressor cells (MDSCs) numbers increase significantly in sepsis and are associated with high mortality rates. These myeloid cell precursors promote immunosuppression, especially in the late (post sepsis) stage. However, the mechanisms that underlie MDSC expansion and programming are not completely understood. To investigate these mechanisms, we used a cecal-ligation and puncture (CLP) mouse model of polymicrobial sepsis that progresses from an early/acute proinflammatory phase to a late/chronic immunosuppressive phase. Previous studies in our laboratory showed that microRNA (miR)-21 and miR-181b elevate levels of the transcription factor nuclear factor 1 (NFI-A) that promotes MDSC expansion. We report here that miR-21 …

Plasmodium Impairs Antibacterial Innate Immunity To Systemic Infections In Part Through Hemozoin-Bound Bioactive Molecules., Christopher Lynn Harding

Electronic Theses and Dissertations

Despite efforts to decrease the global health burden of malaria, infections with Plasmodium species continue to cause over 200 million episodes of malaria each year which resulted in 405,000 deaths in 2018 [1]. One complication of malaria is increased susceptibility to invasive bacterial infections. Plasmodium infections impair host immunity to non-Typhoid Salmonella (NTS) through activities of heme oxygenase I (HO-I) )-induced release of immature granulocytes and myeloid cell-derived IL-10. Yet, it is not known if these mechanisms are specific to NTS. We show here, that Plasmodium yoelii 17XNL (Py) infected mice had impaired clearance of systemic Listeria monocytogenes (Lm) during …

Nlrp6 In Gram-Positive Pneumonia And Sepsis, Laxman Ghimire

LSU Doctoral Dissertations

Acute lower respiratory infections (pneumonia) and pneumonia-derived sepsis are among the leading causes of death in the world causing 7.8 million deaths annually. In this regard, Methicillin-resistant Staphylococcus aureus (MRSA) is endemic in the US and implicated for causing high mortality-associated necrotizing pneumonia and aggravating viral pneumonia with superinfection. Additionally, sepsis is the 7^th leading cause of death among newborns in the US and is responsible for more than 750,000 hospitalization cases every year. Although there is a plethora of research in both pneumonia and sepsis, the detailed pathophysiology still remains elusive. Understanding the host defense mechanism will help …

Elucidating Immune Signaling Of Influenza A Virus And Aspergillus Fumigatus Co-Infections Through Pioneered Model Development, Meagan Danyelle Rippee-Brooks

MSU Graduate Theses

Bacterial co-infections with influenza A virus (IAV) are extremely serious and life-threatening. However, there exists limited understanding about the importance of fungal infections with IAV. Clinical case reports indicate that fungal co-infections do occur and suggest the IAV pandemic of 2009 had a propensity to predispose patients to secondary fungal infections more than previous IAV strains. IAV-fungal co-infections are marked by high mortality rates of 47 to 61% in previously healthy individuals between the ages of 20 and 60. Yet, the variables involved in this co-infection remain undetermined. I achieved effective recapitulation of this co-infection using a C57Bl/6 murine (mouse) …

Enhanced Production Of Pro-Il-1Βeta Contributes To Immunopathology During The Coinfection Of Influenza A Virus And Streptococcus Pneumoniae, Angeline E. Rodriguez

MSU Graduate Theses

Viral bacterial coinfections are known to cause severe pneumonia, especially in the elderly and in pediatric patients. Antibiotics like β-Lactams kill the bacteria but fail to improve symptoms suggesting a faulty immune system may play an important role in the disease. Interleukin-1β (IL-1β) is an important immune signaling cytokine responsible for inflammation. It exists as an inactive precursor that can be activated by caspase-1 containing inflammasomes (multi-protein complex). Influenza A virus (IAV) and Streptococcus pneumoniae (S. pneumoniae) activate the inflammasome through the NOD-like receptor protein NLRP3 and/or AIM2. Previous reports in mice indicate that IL-1β levels are dramatically …

Lymphoid Hematopoiesis And The Role Of B-Cells In Transgenic Mouse Model Of Sickle Cell Disease, Christina Cotte

University Scholar Projects

Sickle cell disease (SCD) has been shown to be associated with decreased baseline immunity and thus increased susceptibility to infection. I sought to discern possible causes of this by looking into the correlations between SCD and hematopoiesis, the immune system and the neuroendocrine system, and ultimately by conducting experiments surrounding the impaired immune system of SCD. These experiments focused on the potential causes and effects of the diminution of B-1a cells in the SCD spleen. Adoptive transfers, infections with Streptococcus pneumoniae, and histologic imaging were conducted to establish if the diminution of the B-1a cells in the SCD spleen …

Tumour Inflammasome-Derived Il-1b Recruits Neutrophils And Improves Local Recurrence-Free Survival In Ebv-Induced Nasopharyngeal Carcinoma, Lih-Chyang Chen, Li-Jie Wang, Nang-Ming Tsang, David M. Ojcius, Chia-Chen Chen, Chun-Nan Ouyang, Chuen Hsueh, Ying Liang, Kai-Ping Chang, Chiu-Chin Chen, Yu-Sun Chang

David M. Ojcius

Inflammasomes sense infection and cellular damage and are critical for triggering inflammation through IL-1β production. In carcinogenesis, inflammasomes may have contradictory roles through facilitating antitumour immunity and inducing oncogenic factors. Their function in cancer remains poorly characterized. Here we show that the NLRP3, AIM2 and RIG-I inflammasomes are overexpressed in Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), and expression levels correlate with patient survival. In tumour cells, AIM2 and RIG-I are required for IL-1β induction by EBV genomic DNA and EBV-encoded small RNAs, respectively, while NLRP3 responds to extracellular ATP and reactive oxygen species. Irradiation and chemotherapy can further activate AIM2 …

The Pathological Effects Of Ccr2+ Inflammatory Monocytes Are Amplified By An Ifnar1-Triggered Chemokine Feedback Loop In Highly Pathogenic Influenza Infection, Sue-Jane Lin, Ming Lo, Rei-Lin Kuo, Shin-Ru Shih, David M. Ojcius, Jean Lu, Chien-Kuo Lee, Hui-Chen Chen, Meei Yun Lin, Chuen-Miin Leu, Chia-Ni Lin, Ching-Hwa Tsai

David M. Ojcius

Background: Highly pathogenic influenza viruses cause high levels of morbidity, including excessive infiltration of leukocytes into the lungs, high viral loads and a cytokine storm. However, the details of how these pathological features unfold in severe influenza infections remain unclear. Accumulation of Gr1 + CD11b + myeloid cells has been observed in highly pathogenic influenza infections but it is not clear how and why they accumulate in the severely inflamed lung. In this study, we selected this cell population as a target to investigate the extreme inflammatory response during severe influenza infection. Results: We established H1N1 IAV-infected mouse models using …

Tolerance Of The Fetus By The Maternal Immune System: Role Of Inflammatory Mediators At The Feto-Maternal Interface, Colette Kanellopoulos-Langevin, Stephane M. Caucheteux, Philippe Verbeke, David M. Ojcius

David M. Ojcius

The adaptive immune system of placental mammals has evolved to tolerate the fetus. Rejection of the fetus by adaptive immune responses is therefore a rare event, with abortion being caused more frequently by inflammation in the placenta. This review will cover recent aspects of immune privilege and the innate immune system at the feto-maternal interface, citing examples of the role played by microbial infections in fetal demise.

Ph And Calcium Dependence Of Hemolysis Due To Rickettsia Prowazekii: Comparison With Phospholipase Activity, David M. Ojcius, M. Thibon, C. Mounier, Alice Dautry-Varsat

David M. Ojcius

Rickettsia prowazekii invades nucleated cells through phagocytosis and subsequently proliferates in the cytoplasm of the host cell. Hemolysis and a phospholipase A2 (PLA2) activity at neutral pHs have previously been reported; even though the phagosomal environment is most likely acidic. We here show that R. prowazekii and R. typhi also lyse erythrocytes at mildly acidic pHs, compatible with an early phagosomal compartment. For R. prowazekii, hemolysis at an acidic pH but not a neutral pH is enhanced by Ca2+, raising the possibility that more than one membranolytic factor may be produced by the rickettsiae. The rickettsiae alone display PLA2 activity, …

The Danger Signal Adenosine Induces Persistence Of Chlamydial Infection Through Stimulation Of A2b Receptors, Matthew A. Pettengill, Verissa W. Lam, David M. Ojcius

David M. Ojcius

Infections with intracellular bacteria such as chlamydiae affect the majority of the world population. Infected tissue inflammation and granuloma formation help contain the short-term expansion of the invading pathogen, leading also to local tissue damage and hypoxia. However, the effects of key aspects of damaged inflamed tissues and hypoxia on continued infection with intracellular bacteria remain unknown. We find that development of Chlamydia trachomatis is reversibly retarded by prolonged exposure of infected cells to extracellular adenosine, a hallmark of hypoxia and advanced inflammation. In epithelial cells, this effect was mediated by the A2b adenosine receptor, unique in the adenosine receptor …

Trail-R1 Is A Negative Regulator Of Pro-Inflammatory Responses And Modulates Long-Term Sequelae Resulting From Chlamydia Trachomatis Infections In Humans, Mufadhal Al-Kuhlani, James Rothchild, Sukumar Pal, Luis M. De La Maza, Sander Ouberg, Servaas A. Morré, Deborah Dean, David M. Ojcius

David M. Ojcius

The immune system eliminates Chlamydia trachomatis infection through inflammation. However, uncontrolled inflammation can enhance pathology. In mice, TNF-related apoptosis-inducing ligand receptor (TRAIL-R), known for its effects on apoptosis, also regulates inflammation. In humans, the four homologues of TRAIL-R had never been investigated for effects on inflammation. Here, we examined whether TRAIL-R regulates inflammation during chlamydial infection. We examined TRAIL-R1 single nucleotide polymorphisms (SNPs) in an Ecuadorian cohort with and without C. trachomatis infections. There was a highly significant association for the TRAIL+626 homozygous mutant GG for infection vs no infection in this population. To confirm the results observed in the …

Susceptibility Of Chlamydia Trachomatis To The Excipient Hydroxy-Ethylcellulose: Ph And Concentration Dependence Of Antimicrobial Activity, Ali A. Abdul-Sater, David M. Ojcius, M. P. Meyer

David M. Ojcius

Hydroxyethyl cellulose (HEC) is used as a neutral excipient in microbicides used against sexually transmitted pathogens. However, HEC inhibits the infection of cervical epithelial cells by Chlamydia trachomatis at pH 5 in a concentration-dependent manner. At pH 7, infection is inversely dependent on the concentration of HEC, possibly due to pH-dependent calcium sequestration.

The Anti-Tumorigenic Mushroom Agaricus Blazei Murill Enhances Il-1Β Production And Activates The Nlrp3 Inflammasome In Human Macrophages, Tsung-Teng Huang, David M. Ojcius, John Ding-E. Young, Yi-Hui Wu, Yun-Fei Ko, Tsui-Yin Wong, Cheng-Yeu Wu, Chia-Chen Lu, Hsin-Chih Lai

David M. Ojcius

Agaricus blazei Murill (AbM) has been reported to possess immune activity against tumors and infections through stimulation of mononuclear phagocytes. Recently, AbM extract was shown to induce the production of the pro-inflammatory cytokine, interleukin-1β (IL-1β), in human monocytes. IL-1β is a key pro-inflammatory cytokine produced by activated macrophages and monocytes and its secretion is strictly controlled by the inflammasome. The purpose of this study is to investigate the effect of AbM water extracts on the regulation of IL-1β production and activation of the NLRP3 inflammasome in human THP-1 macrophages. The NLRP3 inflammasome consists of an NLRP3 receptor, an adaptor protein …

The Microtubule-Associated Protein, Eb1, Links Aim2 Inflammasomes With Autophagy-Dependent Secretion, Li-Jie Wang, Hsin-Yi Huang, Meng-Pin Huang, Willisa Liou, Ya-Ting Chang, Chih-Ching Wu, David M. Ojcius, Yu-Sun Chang

David M. Ojcius

Inflammasomes are multi-protein complexes that regulate chronic inflammation-associated diseases by inducing interleukin-1 β (IL-1β) secretion. Numerous components involved in inflammasome activation have been identified, but the mechanisms of inflammasome-mediated IL-1β secretion have not yet been fully explored. Here, we demonstrate that end-binding protein 1 (EB1), which is required for activation of AIM2 inflammasome complex, links the AIM2 inflammasome to autophagy-dependent secretion. Imaging studies revealed that AIM2 inflammasomes colocalize with microtubule organizing centers and autophagosomes. Biochemical analyses showed that poly(dA-dT)-activated AIM2 inflammasomes induce autophagy and IL-1β secretion in an LC3-dependent fashion. Furthermore, depletion of EB1 decreases autophagic shedding and intracellular trafficking. …

Role Of Proapoptotic Bax In Propagation Of Chlamydia Muridarum (The Mouse Pneumonitis Strain Of Chlamydia Trachomatis) And The Host Inflammatory Response, Jean-Luc Perfettini, David M. Ojcius, Charles W. Andrews Jr., Stanley J. Korsmeyer, Roger G. Rank, Toni Darville

David M. Ojcius

The BCL-2 family member BAX plays a critical role in regulating apoptosis. Surprisingly, bax-deficient mice display limited phenotypic abnormalities. Here we investigate the effect of BAX on infection by the sexually transmitted pathogen,Chlamydia muridarum (the mouse pneumonitis strain ofChlamydia trachomatis). Bax −/−cells are relatively resistant to Chlamydia-induced apoptosis, and fewer bacteria are recovered after two infection cycles from Bax −/− cells than from wild-type cells. These results suggest that BAX-dependent apoptosis may be used to initiate a new round of infection, most likely by releasingChlamydia-containing apoptotic bodies from infected cells that could be internalized by neighboring uninfected cells. Nonetheless, infected …

Separate Metabolic Pathways Leading To Dna Fragmentation And Apoptotic Nuclear Chromatin Condensation, D. Sun, Shibo Jiang, Li-Mou Zheng, David M. Ojcius, John Ding-E. Young

David M. Ojcius

Apoptosis is the predominant form of cell death observed in a variety of physiological and pathological conditions such as cancer involution, insect metamorphosis, the development of the immune and nervous systems, and embryogenesis. The typical nuclear changes taking place in apoptotic cells include extensive condensation of chromatin and internucleosomal DNA fragmentation into units of 200 base pairs. However, the mechanisms responsible for both chromatin condensation and DNA fragmentation have yet to be elucidated. In this study, micrococcal nuclease and the divalent cations, Ca2+ and Mg2+, were applied to isolated nuclei in an attempt to reconstitute in vitro the digestion of …

Reversible Inhibition Of Chlamydia Trachomatis Infection In Epithelial Cells Due To Stimulation Of P2x4 Receptors, Matthew A. Pettengill, Camila Marques-Da-Silva, Maria Luisa Avila, Verissa W. Lam, Ikechukwu Ollawa, Ali Abdul-Sater, Robson Coutinho-Silva, Georg Hacker, David M. Ojcius, Suellen D'Arc Dos Santos Oliveira

David M. Ojcius

Bacterial infections of the mucosal epithelium are a major cause of human disease. The prolonged presence of microbial pathogens stimulates inflammation of the local tissues, which leads to changes in the molecular composition of the extracellular milieu. A well-characterized molecule that is released to the extracellular milieu by stressed or infected cells is extracellular ATP and its ecto-enzymatic degradation products, which function as signaling molecules through ligation of purinergic receptors. There has been little information, however, on the effects of the extracellular metabolites on bacterial growth in inflamed tissues. Millimolar concentrations of ATP have been previously shown to inhibit irreversibly …

Role Of Bcl-2 Family Members In Caspase-Independent Apoptosis Due To Chlamydia Infection, Jean-Luc Perfettini, John C. Reed, Nicole Israel, Jean-Claude Martinou, Alice Dautry-Varsat, David M. Ojcius

David M. Ojcius

Infection with an obligate intracellular bacterium, the Chlamydia trachomatis lymphogranuloma venereum (LGV/L2) strain or the guinea pig inclusion conjunctivitis serovar of Chlamydia psittaci, leads to apoptosis of host cells. The apoptosis is not affected by a broad-spectrum caspase inhibitor, and caspase-3 is not activated in infected cells, suggesting that apoptosis mediated by these two strains of Chlamydia is independent of known caspases. Overexpression of the proapoptotic Bcl-2 family member, Bax, was previously shown to induce caspase-independent apoptosis, and we find that Bax is activated and translocates from the cytosol to the mitochondria in C. psittaci-infected cells. C. psittaci-induced apoptosis is …

Pyk2 Activates The Nlrp3 Inflammasome By Directly Phosphorylating Asc And Contributes To Inflammasome-Dependent Peritonitis, I-Che Chung, Chun-Nan Ouyang, Sheng-Ning Yuan, Hsin-Pai P. Li, Jeng-Ting Chen, Hui-Ru Shieh, Yu-Jen Chen, David M. Ojcius, Ching-Liang Chu, Jau-Song Yu, Yu-Sun Chang, Lih-Chyang Chen

David M. Ojcius

The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1β secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, …

Recruitment Of Bad By The Chlamydia Trachomatis Vacuole Correlates With Host-Cell Survival, Philippe Verbeke, Lynn Welter-Stahl, S. Ying, J. Hansen, Georg Hacker, Toni Darville, David M. Ojcius

David M. Ojcius

Chlamydiae replicate intracellularly in a vacuole called an inclusion. Chlamydial-infected host cells are protected from mitochondrion-dependent apoptosis, partly due to degradation of BH3-only proteins. The host-cell adapter protein 14-3-3β can interact with host-cell apoptotic signaling pathways in a phosphorylation-dependent manner. In Chlamydia trachomatis-infected cells, 14-3-3β co-localizes to the inclusion via direct interaction with a C. trachomatis-encoded inclusion membrane protein. We therefore explored the possibility that the phosphatidylinositol-3 kinase (PI3K) pathway may contribute to resistance of infected cells to apoptosis. We found that inhibition of PI3K renders C. trachomatis-infected cells sensitive to staurosporine-induced apoptosis, which is accompanied by mitochondrial cytochrome c …

Purification And Ligand Binding Of A Soluble Class I Mhc Molecule Consisting Of The First Three Domains Of H-2kd Fused To B2-Microglobulin Expressed In The Baculovirus/Insect Cell System, Francois Godeau, Immanuel F. Luescher, David M. Ojcius, Cecile Saucier, Estelle Mottez, Lucien Cabanie, Philippe Kourilsky

David M. Ojcius

A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative …