bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel quorum-sensing controlled Type I Effector of Pseudomonas aeruginosa, TesG, suppresses host immune response during chronic infection
Authors: Zhou Xikun , Zhao Kelei , Li Jing , Zhang Xiuyue
Published: January 2023

Pseudomonas aeruginosa is a versatile gram-negative pathogen with intricate intracellular regulatory networks that are responsible for a variety of acute and chronic diseases. The survival strategy of this microorganism during pathogenic process especially in causing chronic infections still remains largely elusive. By using in situ RNA-sequencing, here we show that P. aeruginosa undergoes a large-scale transcriptional reprogramming during lung infection. Specifically, more genes of the TCA cycle, oxidative phosphorylation, nucleotide metabolism, T3SS, and two-component system were up-regulated in acute lung infection, while more genes of lipid metabolism, amino acid metabolism, ABC transporters, the T6SS, the QS system, and biofilm formation were up-regulated in chronic lung infection. Notably, a novel virulence factor TesG (PA4141), which is dominated by the vital quorum sensing (QS) system and secreted by the adjacent type I secretion system, can suppress host inflammatory response and remarkably facilitate the development of chronic lung infection. TesG enters alveolar macrophages by clathrin-mediated endocytosis and therefore suppress subsequent adhesion and phagocytosis, as well as the secretion of cytokines and chemokines of macrophages. Mechanically, TesG can bind to and inhibit the activity of eukaryotic small GTPase RhoA by blocking the junction of RhoA with the Rho-guanine nucleotide exchange factor PDZ-RhoGEF. Overall, the characterization of TesG in our current study reveals a new functional aspect of bacterial QS system during host-pathogen interaction, and may open a new avenue for further mechanistic study of chronic respiratory disease and the development of antibacterial therapy.
Rapidly evolving PARP proteins act as both positive and negative regulators of the antiviral response
Authors: Ryan Andrew , Schwerk Johannes , Soveg Frank , Savan Ram , Daugherty Matthew
Published: January 2023

Viral manipulation of cellular post-translational modifications (PTMs) can be key to their survival and replication. One understudied PTM that has been implicated in the antiviral response is ADP-ribosylation. To manipulate this modification, several viral families encode for a macrodomain capable of binding and removing ADP-ribosylation. Importantly, disruption of this function can lead to reduction in pathogenesis or increases in sensitivity to the host interferon response. While the family of host proteins capable of catalyzing ADP-ribosylation, known as PARPs, is large, a subset show multiple signs of rapid evolution, including gene duplication and loss as well as recurrent sequence changes. Focusing in on these genes, we have found that the long isoform of PARP13 is acting directly to inhibit Sindbis virus, a member of the macrodomain-containing alphavirus family. The full antiviral activity requires the presence of the PARP domain in addition to proper localization. In contrast to this, the short isoform acts as a negative regulator of the interferon response by binding and degrading Interferon mRNAs. The balance between antiviral and proviral functions creates a novel dynamic that is potentially more resistant to viral antagonism. We are now exploring the role of ADP-ribosylation in modulating these two phenotypes.
Neutrophil maturation and their response to infectious pathogens are regulated by microbiota
Authors: Gadjeva Mihaela , Kugadas Abirami , Petenkova Anastasia , Geddes-McAlister Jennifer , Mansour Michael K , Sykes David
Published: January 2023

It has long been considered that a neutrophil’s response to various infectious challenges is innately pre-determined. Here, we provide data that demonstrates that neutrophil proteomes are modulated by the microbiota. We found that the proteomic signatures of mature neutrophils derived from germ free (GF) and specific pathogen free (SPF) mice were significantly different. In the absence of microbiota, mature neutrophils lacked GM-CSF-driven priming.

To identify molecular pathways, we set-up an in vitro system where neutrophil progenitors were transduced with lenti-guides to knock-down key microbiota-driven pathway gene targets. GF-serum exposed neutrophil progenitors did not mature efficiently and had compromised bactericidal properties when compared to progenitors matured in SPF-derived serum. To identify which of the microbiota-driven proteins directly impacted bactericidal functions of neutrophils, we knocked out 19 candidates and tested their killing efficacy. Among all tested clones, one demonstrated a superior inhibition of neutrophil’s bactericidal capacities. Excitingly, this protein had no previously identified function: the knock down of prenylcysteine oxidase-like 1 (pcyox 1l) protein reduced neutrophil’s ability to kill efficiently P. aeruginosa in vitro due to diminished ROS release. Hence, we identified a novel mechanism for microbiota-driven control of innate immunity.

Cumulatively, the data support the concept that microbiota affects neutrophil maturation by defining not only the quantity, but also the quality of mature neutrophils. We predict that neutrophil responses can be specifically tailored to pathogens.
Suppression of cGAS-STING- and RIG-I-MAVS-mediated innate immune responses by Epstein-Barr virus-encoded tegument protein BPLF1 through de-ubiquitination
Authors: Lui Wai Yin , Yuen Kit-San , Jin Dong-Yan
Published: January 2023

Epstein-Barr virus (EBV) is the first human oncogenic virus identified and it is etiologically linked with 2% of human malignancies such as Burkitt’s lymphoma, Hodgkin’s lymphoma, gastric carcinoma and nasopharyngeal carcinoma (NPC). The prevalence of EBV infection suggests that the virus may have developed effective viral countermeasures to evade host innate immunity. In this project we performed a functional screen to identify EBV deubiquitinase (DUB) BPLF1 as a potent antagonist of type I interferon production induced by DNA sensors cGAS and STING or RNA sensors RIG-I and MAVS. The large tegument protein BPLF1 exhibited a prominent suppressive effect on cGAS-STING-, and TBK1-induced interferon production. This effect disappeared when Cys61 of BPLF1 was substituted with Ala, which rendered its DUB domain catalytically inactive. This indicated the requirement of DUB activity for BPLF1’s innate immunosuppressive property. BPLF1 was an active DUB for both K63- and K48-linked ubiquitin chains on adaptor protein STING, with no ubiquitin linkage specificity. Our findings suggest that EBV large tegument protein BPLF1 mitigates innate immune responses through its DUB activity on STING and other critical components of innate immune signaling. Supported by HMRF 17160822, RGC C7027-16G and AoE/M-06/08.
Activation of human immune cells with cowpea mosaic virus
Authors: Albakri Marwah M , Beiss Veronique , Fiering Steven N , Steinmetz Nicole F , Sieg Scott F
Published: January 2023

Novel approaches to retrieve and enhance immunity against cancer antigens have been established. One promising approach involves the use of virus particles derived from the plant virus, Cowpea Mosaic Virus (CPMV). Injection of CPMV into tumor stimulates systemic antitumor immunity in tumor mouse models and canine patients with tumors. The molecular pathways that account for these effects are not well defined. In these studies, we examine the capacity of CPMV to activate human monocytes. Peripheral blood mononuclear cells were incubated overnight with CPMV and CD14+ monocytes were examined by flow cytometry for expression of various markers. Cells were also incubated with “empty” (RNA-free) CPMV (eCPMV) for comparison. Responses to CPMV were also tested with THP-1 dual reporter cells, which are activated by various surface and cytoplasm pattern recognition receptors ligands except for TLR7. CPMV, but not eCPMV, mediated activation of human monocytes as indicated by induced expression of CD86, PDL-1, HLA-DR, IL-15R, and IP-10. In contrast to positive controls, PAM3CSK4 (TLR1/2 agonist) and LPS (TLR4 agonist), neither CPMV nor eCPMV induced activation of THP-1 cells. Activation of monocytes by CPMV was markedly inhibited by a chemical inhibitor of spleen tyrosine kinase (SYK), a key signaling component of TLR activation pathways. Effects of SYK inhibition were also observed in cells stimulated by a TLR7 agonist, imiquimod, but with different outcomes than CPMV stimulation, depending on the activation index that was assessed. These data demonstrate that CPMV activates monocytes in a manner that is highly dependent on RNA and SYK signaling. Overall, these data raise the possibility that TLR7 may play a role in CPMV adjuvant-like activity.
Assessing the link between epigenetics and metabolism in acute myeloid leukemia
Authors: Greenwood Dalton L , Ramsey Haley Elizabeth , Madden Matthew Z. , Savona Michael Robert , Rathmell Jeffrey C
Published: January 2023

Hematopoietic stem cells (HSCs) undergo self-renewal or differentiation to generate the many myeloid and lymphoid cells. When dysregulated, cancers such as Acute myeloid leukemia (AML) may develop. AML is characterized by expansion and proliferation of undifferentiated myeloid cells that derive from a Leukemic Stem Cell (LSC) population. AML is the most common form of leukemia with 19,520 new cases in 2018, with an adult five-year survival rate of 24%. Here, we examine a newfound relationship between glutamine metabolism, epigenetics, and targeted differentiation of LSCs in AML to promote disease clearance. Glutamine is catalyzed by glutaminase (GLS) to supply α-ketoglutarate (αKG) to the tricarboxylic acid (TCA) cycle and as substrate to demethylase enzymes. We hypothesize that glutamine metabolism plays an integral role in balancing the fate decision of LSCs and that targeting GLS will impact epigenetic modifications and gene expression to affect cell fate. Glutaminase knockout may also be exploited to promote differentiation of AML LSCs. Preliminary data demonstrates the link between epigenetics and metabolism in AML. We profiled three different AML cell lines’ metabolic responses to INCB54329, an inhibitor of bromodomain proteins. We present a heterogeneous response to INCB54329 in oxygen consumption rate, extracellular acidification rate, reactive oxygen species generation, and mitochondrial mass and membrane potential. The precise function of glutaminolysis in hematopoiesis remains unclear. Unraveling the link between glutamine metabolism, epigenetic modifications, and stem cell function presents a novel therapeutic opportunity for various hematologic malignancies, chief among them acute myeloid leukemia.
Metabolic Reprogramming of Arginine in Tumor Stem Cells Facilitates Immune Escape
Authors: Chen KuanHui , Walker Ameae
Published: January 2023

While numerous studies have focused on metabolic reprogramming that leads to immunosuppression in the primary tumor microenvironment, it is not known whether metabolic reprogramming of metastatic tumor cells contributes to immune escape during travel in an exposed environment where they are subject to immune attack. Tumor stem cells (TSCs) are best known for their contributions to tumor metastasis. Using two models of breast cancer (mouse 4T1 and human BT474), we have determined increased arginine metabolism when TSCs encounter inactive or CD3/CD28 activated effector T cells. This manifests as increased expression of the cationic amino acid transporter 2, SLC7A2. However, other changes in arginine metabolism only occur in TSCs encountering activated T cells, including increased expression/activities of inducible nitric oxide synthase (NOS2), protein arginine deiminase and arginine decarboxylase, and a slightly increased expression/activity of Arginase. Using fluorescent Dansyl-Arginine, we have demonstrated competition between TSCs and T cells for Arginine uptake. Thus, the TSC limits activation and expansion of T cells encountered. Interestingly, a reprogramming of enzymes that favor arginine methylation (increased arginine methyltransferases and decreased arginine demethylase) occurs. As a consequence, there is an increase in secreted asymmetric dimethylarginine (ADMA). Here, we report that giving ADMA to human monocytes leads to polarization of macrophages to the M2 phenotype. In summary, our results show a novel metabolic effect in TSCs by reprogramming arginine uptake, metabolism and modification, which in turn modifies functions in approaching immune cells leading to successful immune escape.
Resident memory (TRM) cells sustain immunity to cancer at sites of frequent metastasis
Authors: Molodtsov Aleksey , Lewis Kathryn , Han Jichang , Vella Jennifer , Zhao Yanding , Malik Brian , Zhang Peisheng , Kolling Fred , Cheng Chao , Huang Yina H , Angeles Christina , Turk Mary Jo
Published: January 2023

Tissue resident memory T cells (TRM cells) have recently been recognized as durable mediators of immunity to cancer. We previously showed that regulatory T cell-depleting immunotherapy of B16 melanoma induces protective TRM cells that persist in the skin of mice that develop autoimmune vitiligo. The goal of the present study was to assess the host-wide diversity and function of memory CD8 T cells throughout peripheral tissues of these mice. Using Pmel transgenic CD8 T cells to track Ag-specific responses revealed the generation of memory T cells with a TRM-like phenotype (CD44hi CD62Llow CD103+CD69+) throughout skin, lungs and liver. Parabiotic transfer/equilibration experiments demonstrated the presence of TRM cells in each peripheral tissue and also, surprisingly, in tumor-draining lymph nodes. Tumor protection experiments in parabiotic mice, and after treatment with the S1PR antagonist FTY720, indicated roles for both TRM cells and circulating memory T cells in providing long-lived protection against melanoma re-challenge at various tissue locations. Single-cell transcriptional profiling of memory CD8 T cells isolated from the skin, lungs, liver, lymph nodes, and spleen indicated the presence of diverse populations with features of both circulating and resident memory, but with TRM cell populations dominating in peripheral tissues and tumor-draining lymph nodes. Moreover, clonally-identical TRM cells in the skin, liver, lungs, and lymph nodes each possessed unique transcriptional features exclusive to their tissue of origin. These studies highlight the tissue-specific adaptations of tumor-reactive TRM cells, and the importance of host-wide TRM responses in providing immunity to cancer.
T cell suppression by the bone microenvironment increases bone metastases from breast cancer in mice
Authors: Arellano Danna L , Verdugo-Meza Andrea , Drescher Florian , Olvera-Rodriguez Felipe , Juarez Patricia , Fournier Pierrick
Published: January 2023

Bone metastases are a frequent complication of breast cancer and cannot be cured. Immunotherapy could be used for their treatment. However, its efficiency could be limited by the ability of T cells to increase bone resorption that supports cancer cell proliferation and bone metastases.

To characterize the effect of T cells on bone metastases, we used a syngeneic mouse model with 4T1 breast cancer cells. When inoculated in normal mice, 4T1 caused more osteolysis as measured on x-rays, compared to SCID and T cell-depleted mice (253% and 61% more, respectively). Histology confirmed that normal mice have an increased tumor burden and number of osteoclasts at the tumor/bone interface compared to SCID mice.

T cells isolated from bone metastases suppressed osteoclast formation ex vivo, in contrast with in vivo data. However, this effect was due to the ex vivo activation of T cells, consistent with decreased levels of Rankl and increased expression of anti-osteoclastic Ifng and Il4 mRNA in activated T cells. In vivo, Ifng was not detected, and levels of pro-osteoclastic Rankl and Tnfa were higher in T cells from bone metastases, suggesting they are pro-osteoclastic, unlike activated T cells.

Culturing bone metastasis T cells ex vivo with ConA or anti-CD3/CD28, failed to increase the expression of the activation marker CD69, confirming the presence of suppressive factors in this microenvironment. Accordingly, there was an increase of MDSCs, including monocytic MDSCs that are PD-L1+ (87%) in 4T1 bone metastases. They could suppress the activation of T cells that are PD-1+ (71%) in bone metastases.

These results suggest that unactivated T cells increase bone metastases and their activation by immunotherapy could be used for the treatment of bone metastases.
Function of T follicular helper cells in anti-tumor immunity
Authors: Nurieva Roza I , Liu Zhongshan , Gangadharan Achintyan , Bieerkehazhi Shayahati , Zhao Yang-Zhi , Alekseev Andrei , Sahoo Anupama
Published: January 2023

Clinical relapse and metastases are the major causes of death in melanoma. Currently, adoptive T cell therapy using tumor-infiltrating lymphocytes rich in cytotoxic CD8+ T cells (CTLs) is one of the promising approaches that helps overcome this major challenge in melanoma; however CTL cell transfer alone does not improve clinical response suggesting a role of helper CD4+ T cells. Recently, it has been noted that infiltration of a CD4+ T helper subset, named as T follicular helper (Tfh) cells into tumor sites correlates with increased survival of cancer patients; however their role in tumor immunity has not been clearly elucidated till date. In our hands, both pre-clinical murine tumor studies and patient studies show correlation between increased number of intratumoral Tfh cells and reduced melanoma tumor growth and improved survival, suggesting the novel function of Tfh cells in promoting anti-tumor immunity against melanoma. Remarkably, transfer of tumor antigen-specific Tfh cells in melanoma tumor-bearing mice results in increased intratumoral CTL number and function, as well as in more efficient tumor eradication, indicating the role of Tfh cells in antitumor immunity by promoting CTL expansion and/or activity. Moreover, we determined that Tfh derived cytokine interleukin (IL)-21 contributes to intratumoral CTL activity. Our results thus for first time indicate the therapeutic potential of Tfh cells in promoting anti-tumor immunity against melanoma and provide the basis for potential usage of these cells to improve current immunotherapy approaches.
Sphingosine-1-phosphate receptor 1 (S1P1) loss mediates T cell sequestration in bone marrow in the setting of intracranial tumors: a novel mode of cancer-induced immunosuppression
Authors: Wilkinson Daniel S , Chongsathidkiet Pakawat , Dechant Cosette , Fecci Peter
Published: January 2023

Glioblastoma (GBM), the most lethal primary brain tumor, proves one of the most immunosuppressive solid tumors. Consequently, immune-based interventions, including immune checkpoint blockade, have largely failed to benefit patients with GBM. Recently, we characterized severe lymphopenia and lymphoid organ contraction in treatment-naïve patients and mice with GBM. Remarkably, bone marrow (BM) proves the lone lymphoid organ not subject to T cell disappearance, instead harboring high numbers of the previously missing T cells. Other tumors, including lung, melanoma, and breast cancers, also accumulate T cells in marrow, but only when these tumors are introduced intracranially. In this study, we sought to determine the mechanism mediating T cell sequestration in the context of intracranial tumors. We found that T cells in the BM of tumor-bearing mice had significantly lower levels of surface sphingosine-1-phosphate receptor 1 (S1P1) compared to controls. Mice that were genetically modified to stabilize S1P1 on the surface of their T cells were resistant to T cell sequestration. Moreover, liberated T cells from these mice were able to synergize with a T cell activating 4-1BB agonist to significantly prolong median survival in GBM mice. Here we describe a novel mode of cancer-induced T cell dysfunction contributed specifically by the intracranial microenvironment: S1P1-mediated BM T-cell sequestration. These findings suggest that the immunoprivileged brain harbors unique mechanisms, usurped by intracranial tumors, for preventing T cell entry into the CNS to favor immune escape. Reversal of such sequestration is anticipated as a promising immunotherapeutic adjunct for both primary brain tumors and brain metastases.
Targeting CD40 enhances antibody- and CD8-mediated protection against respiratory syncytial virus infection
Authors: Muralidharan Abenaya , Russell Marsha , Larocque Louise , Gravel Caroline , Li Changgui , Chen Wangxue , Cyr Terry , Lavoie Jessie R , Farnsworth Aaron , Rosu-Myles Michael , Wang Lisheng , Li Xuguang
Published: January 2023

Respiratory Syncytial Virus (RSV) infects almost all children under the age of one and is the leading cause of hospitalization among infants. It also causes high rates of infection in the elderly and immunocompromised patient populations. Despite several decades of research with dozens of candidate vaccines being vigorously evaluated in pre-clinical and clinical studies, there is no licensed vaccine available to date. A clinical trial conducted in the early 1960s testing a formaldehyde-inactivated RSV (FIRSV) vaccine resulted in enhanced respiratory disease, following subsequent RSV infection, leading to hospitalization of 80% of the participants and 2 deaths. Since then, other forms of RSV vaccine have also been found to induce enhanced disease in preclinical animal studies. Here, we aimed to develop a vaccine that can effectively protect mice from RSV infection and help identify facets of FIRSV-induced enhanced disease. In this study, BALB/c mice were immunized with an adenoviral vector containing the RSV fusion protein (F) fused with CD40 ligand where the CD40 ligand serves two vital functions as a molecular adjuvant and an antigen-targeting molecule. In contrast to the FIRSV vaccine, the vectored vaccine effectively protected animals against RSV without inducing enhanced respiratory disease. This protection involved a robust induction of neutralizing antibodies and memory CD8 T cells, which were not observed in the FIRSV group. Finally, the vectored vaccine was able to elicit long-lasting protection against RSV, which was mediated by increased levels of effector memory CD8 T cell 3 months post-vaccination.
Non-complexed chitosan acts as an adjuvant in an influenza A virus protein vaccine
Authors: Lampe Anna T , Farris Eric J , Ballweg Matthew D , Pannier Angela K , Brown Deborah M
Published: January 2023

During the 2017–18 season influenza A virus (IAV) induced significant morbidity and mortality with the most recent vaccine estimated to be 36% effective in preventing illness. However, IAV vaccines may be improved through the use of adjuvants that enhance or direct specific immune responses. The biomaterial chitosan (CS), a partially deacetylated derivative of chitin, has been used in nucleic acid vaccine strategies for its ability to complex with DNA to form nanoparticles. However, we and others have found that non-complexed CS induces cell death and proinflammatory cytokine responses suggesting that non-complexed CS could act as an adjuvant to enhance immunity to protein vaccination. When bone marrow derived dendritic cells (BMDC) were treated with CS in vitro, we observed increased inflammatory cytokine gene expression, including IL-6. However, at high doses of CS the cytokine response was accompanied by BMDC death. Experiments using J774-Dual™ Cells, a mouse macrophage-like cell line with a Lucia luciferase reporter and a secreted embryonic alkaline phosphatase reporter under the Interferon regulatory factors (IRF) and NF-κB respectively, suggest that CS acts as an adjuvant by activating IRF and low levels of NF-κB. In vivo, when CS was administered with an i.m. protein vaccine, enhanced IgG antibody responses were observed at 3 and 4 weeks post vaccination. As CS is a polymer, the molecular weight (MW) may play a role in adjuvanticity. We have observed that MW may impact IRF and NF-κB activation in vitro, as well as antibody production in vivo. Experiments are underway to determine the level of protection afforded by the enhanced antibody production and to investigate if T cell responses can be augmented by modifying the vaccine regimen.
The complex of poly-γ-glutamic acid and aluminum salts ameliorates the reduced efficacy of the influenza H1N1 vaccine in aged mice
Authors: Yang Jihyun , Kim Jaemoo , Kwak Chaewon , Poo Haryoung
Published: January 2023

Influenza is a highly contagious respiratory disease caused by the influenza A viruses and results in public health threat like seasonal epidemics and sporadic pandemics. Although vaccination is considered to be one of the most effective strategy for preventing the disease, the low efficacy of the influenza vaccine has been reported in the elderly. Here, we examined that the aging-related reduced efficacy of the influenza vaccine can be ameliorated by the complex of poly-γ-glutamic acid (γ-PGA) and alum (PGA/Alum), a novel adjuvant capable of inducing both humoral and cellular immune responses, using an aged mouse model. PGA/Alum induced activation of dendritic cells from aged mice, accompanied with increase of both expression of co-stimulatory molecules and production of pro-inflammatory cytokines. In addition, PGA/Alum enhanced the protective efficacy of pandemic influenza H1N1 (pH1N1) vaccine in aged mice, with earlier virus clearance after A/California/4/2009 (pH1N1) viral challenge, increased hemagglutination-inhibition titers, activity of cytotoxic T lymphocytes, and generation of multifunctional T lymphocytes. Taken together, these results suggest that PGA/Alum is a potent vaccine adjuvant that could improve the reduced vaccine efficacy of aging, thereby leading to prevention of influenza virus infection in the immune compromised populations.
The impact of pre-existing B-cell memory in the response to Influenza vaccination
Authors: Abreu Rodrigo B. , Kirchenbaum Greg A. , Clutter Emily F. , Sautto Giuseppe Andrea , Blackwell Anne-Gaelle B. , Ross Ted M.
Published: January 2023

Influenza is a highly contagious viral respiratory disease with more than 200 thousand cases reported in the US last season (2017/18). Annual vaccination is recommended by the World Health Organization with the goal to reduce influenza severity and limit transmission. However, currently available split inactivated influenza vaccines can only elicit protection up to ~60% in well-matched-strain years. Furthermore, vaccine effectiveness frequently varies between different influenza subtypes within a single influenza season for unclear reasons.

Immunological imprinting from early-life influenza infection can prominently shape the immune response to subsequent infections. Here, the impact of pre-existing B-cell memory in the response to quadrivalent influenza vaccine was assessed. Blood samples were collected from healthy subjects (18 to 85 years old) prior to vaccination and 21–28 days after vaccination with quadrivalent influenza vaccine. Samples were assessed for changes in serological antibodies to the hemagglutinin protein (HA) by ELISA and by hemagglutination-inhibition (HAI) assays against the four strains in the vaccine. The number of antigen specific memory B-cells (Bmem) were quantified by flow cytometry and the polyclonal Bmemresponse was assessed against the HA of the four vaccine strains. Influenza vaccination greatly induces the HA-specific Bmem-cell pool and, despite no differences in antigenicity between the four vaccine components, most individuals are biased towards one of the influenza A or Influenza B vaccine strains. Overall, this study unveils a new mechanism behind the differences in vaccine effectiveness against different influenza subtypes.
Neuraminidase inhibition governs protection efficacy of broadly neutralizing antiinfluenza hemagglutinin stem antibodies
Authors: Kosik Ivan , Angeletti Davide , Gibbs Jmes S. , Angel Mathew , Takeda Kazuyo , Kosikova Martina , Nair Vinod , Hickman Heather D. , Xie Hang , Brooke Christopher C. , Yewdell Jonathan W.
Published: January 2023

There is enormous interest in improving influenza A virus immunization. The most promising target for “universal” flu vaccination is the conserved stem region of the viral hemagglutinin (HA). The HA stem is the target for the vast majority of broadly neutralizing (BN) monoclonal antibodies (Abs). It is well established that BNHA Abs can prevent fusion of the viral and endosomal membranes. A discrepancy between in vitro vs. in vivo efficacy, however, suggests additional mechanisms of action. Recent studies support the role of the BNHA Abs Fc interaction with innate immune effector cells.

Here we show that BNHA mAbs potently inhibit viral neuraminidase (NA) activity against large substrates. To unravel the biological relevance of this activity, we created a panel of recombinant IAVs with shorter or longer NA stalks. Finding that neuraminidase inhibition (NI) activity is inversely proportional to NA stalk length, we use these viruses to show that the NI activity of BNHA Abs is responsible for their ability to block virus release from cells and enhanced neutralization. Critically, we show that NA stalk length governs in vivo protection mediated by BNHA Abs in mice, providing a completely novel mechanism for BNHA Abs in vivo activity. In addition, we demonstrated that the NI activity of BNHA Abs enhances the ability of virus-Ab complexes to activate Fc receptor-bearing cells, and further, that FDA approved small molecule NA inhibitors have a similar effect on Fc-mediated innate immune cell activation, providing a novel mechanism for their clinical activity.

Altogether, our findings provide surprising and clinically relevant insights into the in vivo mechanisms of protection provided by both anti-stem BN antibodies and NA inhibitors.
Development of broadly neutralizing antibodies against influenza viruses from mice immunized with monoglycosylated hemagglutinin
Authors: Ko Yi-An , Yu Yueh-Hsiang , Tseng Yung-Chieh , Ma Che , Lin Kou-I
Published: January 2023

Influenza viruses are respiratory pathogens, which cause seasonal outbreaks and result in more than 250,000 annual deaths worldwide. Vaccine inoculation is the most effective way to prevent the virus transmission. Our previous study showed that mice injected with monoglycosylated hemagglutinin (HAmg) protein vaccine designed from influenza A viruses produced cross-stain reactive antibodies and were protected better than mice injected with fully glycosylated HA (HAfg) after challenge with various influenza viruses at lethal dose. Therefore, we applied single B cell RT-PCR platform to pursue the cross-protective monoclonal antibodies (mAbs) from 2007 A (H1N1) Brisbane (Bris/07) HAmg immunized mice. A monoclonal antibody, 651, was selected, which was able to react with a broad spectrum of H1N1 viruses although it did not provide neutralizing activities. It is noted that 651 mediates antibody dependent cellular cytotoxicity (ADCC) against cells expressing HA from multiple strains of influenza viruses, including 2009 A (H1N1) swine (Cal/09), Bris/07, and H3. Alleviated weight loss and improved survival rate were observed in 651 pre-treated mice as compared with the PBS treated group after Cal/09 or Bris/07 virus infection. Interestingly, there were no protective effects against Cal/09 infection in 651 treated FcγR knockout mice. Functionally, proinflammatory cytokines accumulation in lung was ameliorated in Cal/09 infected mice pre-treated with 651. The percentage of alveolar macrophages was recovered in 651 pre-treated mice as compared with the PBS pre-treated mice after Cal/09 infection. Together, this study reveals a potent mAb that possesses broad-spectrum protective effects through the effector function of antibody.
Subunit vaccine candidate with adjuvant confers protection against clinical Mycobacterium avium in wild type and immunocompromised mouse models
Authors: Larsen Sasha E , Reese Valerie A , Pecor TIffany , Brewer Guy , Berube Bryan , Njikan Samuel , Parish Tanya , Henao-Tamayo Marcela , Baldwin Susan L , Coler Rhea N.
Published: January 2023

Nontuberculous mycobacteria (NTM) are a group of 170 species of mycobacteria that are steadily increasing in incidence and prevalence. NTM cause a significant amount of morbidity and mortality and overtake Mycobacterium tuberculosis in prevalence in certain regions of the world, including the United States. M. avium, a clinically significant NTM, is known to cause a tuberculosis-like pulmonary disease that is often difficult to treat. Due to this quietly emerging health threat, we evaluated the ability of a subunit vaccine (ID91) with the Toll-like receptor agonist glucopyranosyl lipid adjuvant formulated in an oil-in-water stable nano-emulsion (GLA-SE) to confer protection in both the C57BL/6 (wild type) and Beige (immunocompromised) mouse models. We first established a challenge model with a low dose aerosol of the virulent clinical isolate M. avium 2–151 smt and monitored bacterial burden and immune responses to infection over time. Next, cohorts of mice immunized prophylactically with ID91+GLA-SE or bacillus Calmette–Guérin (BCG) were subsequently challenged with M. avium 2–151 smt. Not surprisingly, the immunocompromised Beige mice sustained a larger bacterial burden in the lungs and spleen compared to C57BL/6 mice regardless of treatment. However, both ID91+GLA-SE and BCG significantly reduced pulmonary bacterial burden in both models. Variable innate and adaptive pulmonary immune response kinetics in C57BL/6 and Beige mice in response to M. avium 2–151 smt infection were observed. This work demonstrates the first proof-of-concept study that a subunit vaccine candidate (ID91+GLA-SE) confers prophylactic protection against NTM and suggests further vaccine development may help reduce the burden of disease.
Antigen Presenting Cell Targetted, Adjuvant free Mucosal Vaccine Induces Protection against Pneumococcal Infection
Authors: Kumar Sudeep , Sunagar Raju , Gosselin Edmund J
Published: January 2023

Protein-based subunit vaccines require adjuvants to increase their immunogenicity. However, there is lack of FDA-approved mucosal adjuvant. We have previously shown that targeting antigens to antigen presenting cells increases their immunogenicity. Specifically, Streptococcus pneumoniae (Sp) protective antigen (PspA) fused to a single-chain bivalent-anti human Fc-gamma-receptor-I (hFcgRI) antibody, when introduced into mice, generates protection against Sp. To further enhance the immunogenicity of the anti-hFcgRI-PspA fusion protein, we have incorporated an additional hFcgRI-binding moiety, with the resultant trivalent-anti-hFcgRI inducing better protection compared to the bivalent anti-hFcgRI-based vaccine. This improved vaccine requires fewer immunizations to confer over 90% protection, significantly higher than that of bivalent-anti-hFcgRI-PspA (less than 60%). The trivalent- anti-hFcgRI-PspA also induces significantly higher levels of Sp-specific antibodies. In addition, we observed elevated levels of mucosal IgA responses in trivalent-anti-hFcgRI-PspA-immunized mice compared to that of bivalent anti-hFcgRI-PspA. Both trivalent-anti-hFcgRI-PspA and bivalent anti-hFcgRI-PspA enhanced antigen presentation in FcgRI-dependent manner. We have further observed that trivalent-anti-hFcgRI-PspA induces recruitment of innate immune cells in the nasopharyngeal lymphoid tissue upon intranasal immunization, which is a hallmark of adjuvant activity. Thus, our study suggests that anti-hFcgRI-PspA compensates for the lack of adjuvant by inducing adjuvant effects, and can be utilized as a mucosal vaccine platform.
The Development of Klebsiella Pneumoniae Vaccine Strategy via Eliciting the Serotype- Independent Immunity in the Lung
Authors: Iwanaga Naoki , Sandquist Ivy , Chen Kong , Norton Elizabeth B , Moreno Javier Rangel , Kolls Jay K
Published: January 2023

Objective

Multidrug-resistant of Klebsiella pneumoniae (K. pneumoniae) has emerged as a global threat. Vaccination for at high-risk populations could be a promising strategy to prevent and control the disease.

Methods

Mice were vaccinated via intranasal with a monovalent vaccine encoding Kp serotype 2 OMPx or a quadrivalent vaccine encoding OmpC, OmpW, Omplolb, and Omp36k, adjuvanted with heat-labile enterotoxin A1 domain (LTA1). Protective immunity was assessed by performing a pulmonary challenge with a heterologous mucoid serotype K1 strain of K. pneumoniae. Lung T-cell responses were evaluated mainly by FACS, ELISPOT, and LUMINEX. We also examined inflammatory cell changes by hematoxylin-eosin stain and immunofluorescence.

Results

Flow cytometry and ELISPOT analysis revealed that CD4+ T cells were significantly increased in the lungs of the monovalent or quadrivalent vaccinated mice and that a considerable fraction of them produced IL17A. And the supernatant of these cells also showed the significant production of IL-17A. Importantly, vaccinated mice showed a significant decrease in CFU in the lung and spleen, compared with mock-vaccinated mice. Finally, we observed that only lungs of intranasally vaccinated mice had lymphocytic structures around bronchi and vessels. Characterization of these structures by immunofluorescence confirmed the presence of inducible bronchus-associated lymphoid tissue (iBALT) – a structure that can not only support local induction of protective immunity but provide a long-term niche for vaccine-induced lymphocytes.

Conclusions

These data support the therapeutic value of intranasal vaccination strategies to prevent K. pneumoniae infections.
Protective role of TH17 cells in an FcγR targeting immunization strategy against the respiratory bacterial pathogen, Francisella tularensis
Authors: Bitsaktsis Constantine , McCauley James , Devenney Kyle
Published: January 2023

Francisella tularensis is a category A intracellular bacterial pathogen with no FDA approved vaccine. Understanding the immunological mechanisms involved in host defense can improve vaccine efficacy. Previously we have shown using an F. tularensis murine infection model that targets the inactivated bacterium (iFt) to Fcγ receptors on antigen presenting cells (APCs) using anti-LPS monoclonal antibodies (mAb-iFt) provides full protection against lethal pulmonary challenge with F. tularensis live vaccine strain (LVS). The protection provided by this mucosal vaccine strategy involves both innate and adaptive immune components, with a strong inflammatory response being critical for protection. This study focuses on elucidating the role of Th 17 cells, a T helper cell subpopulation involved in pathology and host defenses against bacterial pathogens. C57BL/6 mice were immunized with iFt and mAb-iFt on day 0 and 21 and then challenged i.n. with a lethal dose of LVS on day 35. Flow cytometry data revealed a significant population of Th17 cells (characterized as CD3+/CD4+/IL- 17a+/RORγ-T+ cells) in the lungs and spleen post-infection in the mAb-iFt immunized group when compared to PBS and iFt alone. Additionally, cytokine levels of IL-6 and TGF-β1 (two known synergistic Th17 polarizing cytokines) were elevated in the lungs and spleens of mAb-iFt immunized mice on days 2 and 5 post-infection when compared to iFt alone. Lastly, cytokine levels of IL-17a, an effector cytokine secreted predominantly by active Th17 cells, was shown to be significantly increased in mAb-iFt immunized mice when compared to iFt alone. Collectively, our data suggests that Th17 cells possess a protective role in the immune response to pulmonary F. tularensis infection.
Molecular Signatures of IL-10/IFN-γ Co-Producing CD4 T Cells During Acute Dengue Virus Infection
Authors: Tian Yuan , Seumois Gregory , de la Mata Sara Herrera , Goonawardhana Suraj , de Silva Aruna D , Premawansa Sunil , Premawansa Gayani , Wijewickrama Ananda , Grifoni Alba , Vijayanand Pandurangan , Peters Bjoern , Weiskopf Daniela , Sette Alessandro
Published: January 2023

Dengue virus (DENV) is an emerging pathogen and a serious public health issue. It is estimated that ~390 million people are infected yearly. DENV infection is associated with a range of clinical manifestations, from asymptomatic to more severe presentations including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). There is currently no specific therapy available for the treatment of dengue diseases other than supportive care. Thus, a better understanding of the host’s immune response against DENV, especially during the acute phase of infection, is of great public health interests. Although previous studies suggest that increased IL-10 level in the serum and plasma is associated with severe dengue disease, whether dengue-specific CD4 T cells produce IL-10 remains unknown. Here we show evidence of DENV-specific IL-10-producing CD4 T cells during acute DENV infection. Surprisingly, an IL-10+IFN-γ+ double positive (DP) CD4 T cell population is prominent in acute hospitalized DENV cases but disappear at the convalescent stage. These cells are not conventional regulatory T (Treg) cells as they lack Foxp3 expression. In contrast, RNA-sequencing and high-dimensional CyTOF analyses reveal that these cells partially exhibit previously identified signatures associated with cytotoxic CD4 T cells and type 1 regulatory T (Tr1) cells. Notably, we identified several novel signatures of DP cells including IL-19, IL-21, and IL-22, suggesting that these cells may have complex cytokine activities and functions. Overall, this study reveals unique phenotypes of DENV-specific IL-10/IFN-γ co-producing CD4 cells and provides insights into antigen-specific T cell responses during acute DENV infection.
Increased polyfunction in CD4 T cells from persons with acute vs. remote shingles
Authors: Jin Wenjie , Fang Michael , Vangipuram Ramya , Sayin Ismail , Golden Jackelyn , Cameron Cheryl , Cameron Mark , Tyring Stephen , Canaday David H.
Published: January 2023

Little is known about CD4 T cell function in Varicella Zoster Virus (VZV) infection. Here we sampled peripheral blood of individuals (n=31) with acute shingles as a result of reactivation of VZV and persons with a remote history of shingles (n=15). Mitogen and whole VZV were added to peripheral blood mononuclear cells (PBMC) for an in vitro stimulation assay. IFN-γ, IL-2, TNF-α, CD107a, T-bet, EOMES and KLRG1 were examined by flow cytometry. Mitogen- and whole VZV-stimulated total memory CD4 T cells showed significant differences in polyfunction (p<0.0001) while the mitogen-activated groups were equal in acute vs history of shingles. There were significant differences between polyfunctional profiles in VZV-responding total and central memory CD4 T cells in acute vs remote shingles (p<0.002). Acute patients’ VZV-specific total memory cells had higher frequencies of IFN-γ (p<0.001) and IL-2 (p<0.02) producing cells, perhaps due to differences in effector memory subsets. Acute patients' VZV-specific but not mitogen-responding CD4 T cells showed significantly higher levels of T-bet+ than donors with only a history of shingles (p<=0.01). RNASeq analysis of ex vivo total memory CD4 T cells from these individuals showed differential regulation of T cell survival and differentiation pathways, including differential expression of IFN, TNF and mTOR signaling (p<0.05 for genes/pathways). These gene signatures correlated with the frequency of IFN-γ and IL-2 producing cells responding to VZV above with linear regression analysis. In summary, CD4 T cells respond differently in individuals with acute vs. remote shingles, and recognize VZV-specific epitopes upon reactivation via T-bet-mediated pathways.

Supported by NIH and VA.
Human Vγ9Vδ2-T cells exhibit potent antiviral activity against EV71 infection
Authors: Tu Wenwei
Published: January 2023

Enterovirus 71 (EV71) is a major pathogen of hand, foot and mouth disease (HFMD). It is highly contagious and usually causes mild disease. However, occasionlly HFMD caused by EV71 would develpoed into severe neurological complications or even deaths in young children. There is no effective anti-viral therapy against EV71 infection. Human Vγ9Vδ2-T cells, as the first line of human immune defense, exhibit antiviral activities against different viruses. However, whether human Vγ9Vδ2-T cells have antiviral activity against EV71 remains unkown. In this study, we used EV71-infected primary human monocyte-derived macrophages (MDMs) to examine the anti-EV71 activity of Vγ9Vδ2-T cells. We found that phosphoantigen pamidronate-expanded Vγ9Vδ2-T cells could kill EV71-infected MDMs, and thus reduce the viral load. The cytotoxicity of Vγ9Vδ2-T cells against EV71-infected MDMs required cell-cell contact and mediated by Fas/FasL pathway. Vγ9Vδ2-T cells also displayed potent capacity to produce IFN-γ and thus inhibit EV71 viral replication. CCR5 pathway was involved in the migration of Vγ9Vδ2 T cells towards EV71-infected cells. These results demonstrated that Vγ9Vδ2-T cells have both cytotoxic and non-cytolytic antiviral activities target EV71. Our data suggests a novel therapeutic approach against EV71 by boosting Vγ9Vδ2 T cell immunity.

This work is supported in part by Health and Medical Research Fund, Hong Kong SAR government (13120972) and the General Research Fund (17115015 and 17126317), Research Grants Council of the Hong Kong SAR, China.
Single-Cell Transcriptomics Identifies TOX as a Key Transcriptional Regulator of Stem- Like CD8 T Cells in Chronic Infection
Authors: Wu Tuoqi , Yao Chen , Sun Hongwei , Lacey Neal , Gattinoni Luca , McGavern Dorian B , O'Shea John J , Schwartzberg Pamela L
Published: January 2023

Stem-like CD8 T cells maintain long-term antiviral immunity during chronic infection and share regulatory pathways with memory precursor cells generated after acute infection. However, it is unclear whether stem-like CD8 T cells require distinct transcriptional and epigenetic regulation for their longevity and adaptation to the immunosuppressive environment of chronic infection. By comparing single-cell transcriptomes and epigenetic profiles of CD8 T cells responding to acute and chronic viral infections, we found that stem-like CD8 T cells became distinct from memory precursors prior to the termination of clonal expansion. We discovered a co-expression gene module containing Toxthat exhibited higher transcriptional activity associated with more active histone marks in stem-like T cells than memory precursors. Moreover, TOX promoted persistence of antiviral CD8 T cells and was required for stem-like CD8 differentiation. Thus, stem-like CD8 T cells require distinct transcriptional and epigenetic programs for their differentiation and persistence during chronic viral infection.