bluuebunny/crossref_metadata_2025 huggingface dataset:
IRF5 Drives Potentiated IFN-beta and IL-12 production by dendritic cells in response to combinatorial CpG- and MPLA- delivery on microparticles
Authors: Jhita Navdeep , Toy Randall , Pradhan Pallab , Blanchard Emmeline , Narang Neha , Cortes Gabriela , Keenum Mark Cole , Santangelo Philip J , Shayakhmetov Dmitry M , Roy Krishnendu
Published: January 2023

Vaccines have enabled control of many deadly infectious diseases. However, efficacy in host protective immune responses vary greatly. Adjuvants targeting Toll like and cytosolic receptors can greatly enhance antigen-specific immunity. In this regard, pathogen-like micro-particles (PLP) co-loaded with TLR4 (MPLA) and/or TLR9 (CpG) ligands was used as a novel platform to deliver combinatorial adjuvants. We analyzed molecular signaling pathways activated in bone-marrow-derived dendritic cells (BMDCs) upon stimulation with PLPs, specifically leading to IFNb and IL-12p70 production. Stimulation of BMDCs with PLPs containing only MPLA or CpG resulted in low-level or no IFN-b and IL-12p70 release. However, stimulation of BMDCs with PLPs containing both MPLA and CpG triggered production of very high amounts of IFN-b and IL-12p70. Analysis of signaling pathways responsible for these results demonstrated IFN-b and IL12-p70 production in BMDCs harvested from WT, IRF3-KO, and IRF7-KO mice, but not from IRF5-KO animals. Exploration of IRF5 phosphorylation kinetics showed MPLA-loaded PLP transiently stimulated IRF5 (0.5 to 2 hours post stimulation) with dual adjuvant PLPs displaying a greatly prolonged p-IRF5, detectable even at 4 hours post stimulation. We are currently analyzing molecular regulators of p-IRF5 stability and kinetics of its translocation to the nucleus. Our study reveals an unexpected role for IRF5 in driving a highly potentiated production of immune-stimulatory cytokines from dendritic cells in response to dual MPLA and CpG adjuvants. These findings may help develop novel vaccines with improved efficacy when combined with PLPs loaded with MPLA and CpG adjuvants that activate IRF5-dependent cytokine production.
DNA priming vaccination to induce T cell responses to subdominant conserved HIV epitopes
Authors: Felber Barbara K. , Lu Zhongyan , Hu Xintao , Valentin Antonio , Rosati Margherita , Mullins James I. , Pavlakis George N
Published: January 2023

Background

HIV sequence diversity and epitope immunodominance exerted by the variable regions are hurdles in the development of an effective AIDS vaccine. We developed novel DNA vaccine regimens to redirect T cell responses to highly conserved regions (CE) in the HIV proteome.

Methods

Rhesus macaques were primed with DNA expressing CE immunogen and boosted with a combination of DNAs expressing CE and/or full-length (FL) immunogens. Different prime-boost protocols were compared. The CE prime/CE+FL boost vaccination co-delivered in the same anatomical site was compared to delivery of CE and FL DNA in contralateral sites.

Results

CE prime/CE+FL boost regimens induced high and broad CTL responses which cannot be achieved using FL DNAs only. Thus, priming with CE DNA profoundly alters the immunodominance exerted by the variable non-CE regions, promoting responses to subdominant CE. This vaccine regimen is currently tested in clinical trials (HVTN 119, ACTG A5369). Comparison of same site vs contralateral vaccine delivery showed that separating CE and FL DNA resulted in increased breadth of T cell responses to non-CE while maintaining CTL responses to CE. Separating the immunogens at the prime allows for induction of independent primary adaptive responses by targeting the two vaccine components to distinct lymph nodes and preventing interference.

Conclusions

HIV DNA vaccine regimens comprising CE as prime and native immunogens as boost induce responses to subdominant epitopes, maximizing breadth and focusing the immune response against vulnerable regions of the virus. Separating the immunogens results in broader epitope recognition. This has implications for many vaccine protocols promoting induction of protective responses.
Understanding the early steps of the adaptive immune response to differentially-effective vaccines during chronic schistosomiasis
Authors: Bement Zachary James , Norwood Stephanie K , Shollenberger Lisa M
Published: January 2023

Schistosomiasis is the second most burdensome parasitic disease in the world after malaria and is classified as a neglected tropical disease. Schistosomiasis is caused by an immunomodulatory helminth that can induce a Th2 biased response, which can cause Tc1 vaccine failure. Interestingly, the use of pathogenic Listeria monocytogenes vaccine vectors can overcome this failure through an unknown mechanism. Despite the promise of Listeria-vectored vaccines, there is reluctance among the vaccine community and the public to incorporate this vector into immunization programs, due to safety concerns. Here we ask what the question: Why do Listeria vaccines function when other methods, such as DNA vaccination, fail? To answer this question, we use bone-marrow derived dendritic cells (BMDCs) in vitro to model the development of vaccine immunity during the early steps of the adaptive immune response. To compare the immune environment in naïve and schistosome-infected vaccine recipients, we compare responses from both reactogenic DC1 and tolerogenic DC2 dendritic cells, respectively. The DC1 and DC2 subtypes are exposed to a DNA vaccine for HIV, a Listeria vaccine expressing the HIV-1 Gag protein, or left untouched. Through immunological and transcriptomic approaches, we examine these cells for their activation state and functionality; these results will help to elucidate the mechanism/s by which Listeria vectors can overcome schistosome-induced vaccine failure and identify unique molecular vaccine adjuvants functional in schistosome-endemic areas.
The prevention and augmentation of tetravalent recombinant measles-vectored dengue vaccine in dengue-2 infected AG129 mice
Authors: Pan Chien-Hsiung , Lin Tsung-Han , Yan Jia-Ying
Published: January 2023

Dengue is an emerging mosquito-borne disease and the use of prophylactic vaccine is still limited. We have previously developed the tetravalent dengue vaccine (rMV-TDV) by a recombinant measles virus (MV) vector expressing envelope protein domain III (ED3). In this study, we used AG129 mice to evaluate the protective and/or pathogenic immune responses induced by rMV-TDV. Immunization of rMV-TDV has induced a significant neutralizing antibody response against all serotypes of DENV, as well as a significant CD4-dependent IFN-γ response biased to DENV-3, compared to the vector controls. After DENV-2 challenge, rMV-TDV-immunized mice showed a significant lower viremia and no increase of inflammatory cytokines comparing to the vector controls, which had a ~100 times higher viremia and a significant increase of IFN-γ and TNF-α 3 days after challenge. A robust DENV-2 specific IFN-γ response detected in rMV-TDV-immunized mice suggested that pre-existing DENV-3 biased T-cell responses didn’t cross-react to DENV-2 but an anamnestic DENV-2 specific T-cell response was recalled. Prior to the challenge, only DENV-3 specific IgG demonstrated a significant higher avidity than other serotypes, whose specific IgG avidity increased after DENV-2 challenge, particular in DENV-1 and -2. However, the passive transfer of rMV-TDV immunized sera plus splenocytes resulted in the higher viremia and the significant increase of inflammatory cytokines in AG129 recipient mice after DENV-2 challenge than the vector control donors did. It suggested that while the immunity by rMV-TDV waned, the potential risks for the severe disease increased. Our data shed the lights on the role of a balanced CD4 T-cell response in the prevention of severe dengue.
Neutralizing monoclonal antibodies against Venezuelan equine encephalitis virus envelope glycoprotein E2 protect from lethal encephalitis
Authors: Kafai Natasha M. , Sukupolvi Soila , Gardner Christina L. , Klimstra William B. , Diamond Michael S.
Published: January 2023

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne New World alphavirus responsible for severe morbidity and mortality in humans and equines. The development of therapeutics against VEEV is critical due to its potential for outbreak and aerosolization as a bioterrorist agent. However, effective and widely accessible antiviral agents for VEEV do not exist. Humoral immunity is an important defense against many alphavirus infections, and we thus generated a panel of 17 highly neutralizing murine VEEV-specific monoclonal antibodies (mAbs) using the VEEV vaccine TC-83 and attenuated chimeric SINV-VEEV Trinidad donkey (TRD) subtype IAB. Nine of these mAbs demonstrate 50% effective inhibitory concentrations (EC50) values below 100 ng/ml when tested against enzootic subtype ID and epizootic VEEV subtypes IAB and IC virion envelope glycoproteins. To define specific viral epitopes important for mAb binding, mapping studies including competition assays, HDX, alanine-scanning mutagenesis, and viral escape mutants are ongoing. Preliminary data suggests the epitopes for these mAbs are found in both the A and B domains of the surface virion envelope 2 glycoprotein. Additionally, several of these mAbs confer prophylactic and therapeutic protection in mice aerosol challenged with virulent TRD VEEV. Mechanism of action studies are underway to define the steps in the virus life cycle (viral attachment, entry, fusion, and egress) at which our most potent protective antibodies act. Identification of specific epitopes targeted by these protective mAbs will better inform the development of targeted immunotherapeutics and vaccines against VEEV and related encephalitic alphaviruses.
A recombinant subunit Lassa virus vaccine elicits strong antibody responses
Authors: To Albert , Lai Chih-Yun , Wong Teri Ann , Namekar Madhuri , Lehrer Axel T
Published: January 2023

Due to the epidemic potential and high case fatality rate, the WHO has declared the development of a Lassa virus (LASV) vaccine as a high priority for preventative use in endemic regions. We used a recombinant subunit vaccine platform to generate recombinant LASV glycoprotein (GP) in an insect cell expression system and tested its immunogenicity in an outbred mouse model. Briefly, LASV GP was secreted from a stably transformed Drosophila S2 cell line and the clarified culture supernatant was purified using immunoaffinity chromatography (IAC). SDS-PAGE gel analysis showed that the purity of the antigen was sufficient for immunogenicity testing. The eluted LASV GP was composed of GP1 and GP2 subunits, the GP1–2 heterodimer, as well as trace amounts of oligomer. Swiss Webster mice immunized intramuscularly with 3 doses of purified recombinant LASV GP, formulated with various adjuvants, showed maximal GP-specific antibody response after two immunizations. We concluded that we have successfully developed a production method for highly-purified, insect-cell expressed recombinant LASV GP capable of eliciting high antibody titers when formulated with a suitable adjuvant after only two doses. These results indicate the potential for recombinant GP to be an efficacious vaccine candidate. Further work on structural enhancements of the antigen and in-depth characterization of antibody and cell-mediated responses is ongoing.
Development of a DNA vaccine for SFTSV that confers complete protection against lethal infection in ferrets
Authors: Kwak Jeong-Eun , Kim Young-Il , Park Su-Jin , Jeong Moonsup , Maslow Joel N. , Song Min-Suk , Jung Jae U. , Shin Eui-Cheol , Choi Young Ki , Park Su-Hyung
Published: January 2023

Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging tick-borne infectious disease caused by the SFTS virus (SFTSV) belonging to the Phenuiviridae family. The WHO lists SFTSV as one of the most dangerous viral pathogens, and considers it likely to cause wide epidemics in the near future. The incidence of SFTSV infection has increased from its discovery in 2012 through 2018 with a mortality rate of 10–20% and the major clinical symptoms of SFTSV infection are fever, vomiting, diarrhea, thrombocytopenia, leukopenia and multiple organ failure. However, no effective vaccines are currently available for SFTSV. Here, we describe the development of a SFTSV-specific DNA vaccine, its immunogenicity, and its protective efficacy against SFTSV lethal challenge. Vaccine candidates induced both a neutralizing antibody response and multifunctional SFTSV-specific T cell response in mice and ferrets. To investigate the vaccine efficacy in vivo, we applied a recently developed ferret model of lethal infection that recapitulates fatal clinical symptoms in SFTSV infection in humans. Vaccinated ferrets were completely protected from lethal SFTSV challenge without developing any clinical signs. Moreover, we found that anti-envelope antibodies play an important role in protective immunity and non-envelope-specific T cell responses also can contribute to protection against SFTSV infection. This study provides important insights into the development of an effective vaccine, as well as corresponding immune parameters, to control SFTSV infection.
Immunogenicity of a Purified Inactivated Zika Virus Vaccine Candidate in Humans is Diminished by Priming with a Yellow Fever or Japanese Encephalitis Virus Vaccine: Elucidating Immunologic Interference between Flavivirus Vaccines
Authors: Modjarrad Kayvon , Koren Michael , Lin Leyi , Sondergaard Erica , De La Barrera Rafael , Eckels Kenneth , Krebs Shelly , Eller Michael , Costanzo Margaret , Joyce Gordon , Dussupt Vincent , Gromowski Gregory D , Storme Casey , Amare Mihret , Jarman Richard G , Thomas Stephen , Michael Nelson
Published: January 2023

In response to the 2016 Zika virus (ZIKV) outbreak, we developed a purified, formalin-inactivated, Alum-adjuvanted ZIKV vaccine (ZPIV) candidate that was protective in animal models and advanced to human testing in four phase 1 clinical trials. We have previously reported the interim analysis of three trials. Here we present the final analysis of a phase 1, placebo controlled, double-blind trial of ZPIV. Seventy-five flavivirus-naïve adults—divided into three groups of 25—were randomly assigned to receive three 5μg doses of ZPIV or placebo at days 1, 29 and 196. Group 1 only received ZPIV, while Groups 2 and 3 first received either licensed Japanese encephalitis (IXIARO®) or yellow fever (YF-VAX®) vaccine two-months prior to receiving ZPIV. Immune responses were assessed by a live-virus microneutralization assay. ZPIV was well tolerated across groups. Group 1 exhibited neutralizing antibody (nAb) geometric mean titers (GMT) of 101 and 511 after the 2nd and 3rd doses. GMTs in Groups 2 and 3 were significantly lower at 11.8 and 6.6 after two doses, and 174 and 79 after the 3rd dose. There were no differences in binding antibody titers or in antibody affinity maturation across groups. These data suggest that flavivirus vaccine priming of B cells directed antibodies toward non-neutralizing epitopes and that decline in ZIKV nAb titers did not reflect overall decrements in B cell responses. Other studies have shown prior dengue virus infection boosts subsequent ZIKV responses. These results indicate the interplay of flavivirus immunity is contextual and likely specific to the mode and timing of antigen presentation. More studies are needed to investigate these factors, so as to optimize the rational use of vaccines against co-endemic flaviviruses.
Glycolipid-peptide vaccination induces liver-resident memory CD8+ T cells that protect against malaria
Authors: Holz Lauren , Chua Yu Cheng , Anderson Regan , Draper Sarah , Compton Benjamin , Chan Susanna , Mathew Juby , de Menezes Maria , Cozijnsen Anton , Mollard Vanessa , McFadden Geoffrey , Painter Gavin , Heath William R
Published: January 2023

Liver resident-memory CD8+ T cells (TRM cells) can kill liver-stage Plasmodium-infected cells and prevent malaria, but simple vaccines for generating this important immune population are lacking. Here, we report the development of a fully synthetic self-adjuvanting glycolipid-peptide conjugate vaccine designed to efficiently induce liver TRM cells. Upon cleavage in vivo, the glycolipid-peptide conjugate vaccine releases an MHC I-restricted peptide epitope (to stimulate Plasmodium-specific CD8+ T cells) and an adjuvant component, the NKT cell agonist a-galactosylceramide (a-GalCer). Following transfer of a transgenic CD8+ T cell population, a single dose of this vaccine induced substantial numbers of intrahepatic CD8+ T cells expressing canonical markers of liver TRM cells (CD69, CXCR6 and CD101), and these cells could be further increased in number upon vaccine boosting. We show that modifications to the peptide, such as addition of proteosomal-cleavage sequences or epitope-flanking sequences, or the use of alternative conjugation methods to link the peptide to the glycolipid, improved liver TRM cell generation and led to the development of a vaccine able to induce sterile protection in C57BL/6 mice against P. berghei sporozoite challenge after a single dose. Incorporation of a cognate malaria antigen into the vaccine resulted in the generation of large numbers of long-lived liver TRM cells derived from the endogenous T cell population that were capable of providing sterile immunity. Our findings describe an ideal synthetic vaccine platform for generating large numbers of liver TRM cells for effective control of liver-stage malaria and, potentially, a variety of other hepatotropic infections
Development of a chlamydial vaccine for koalas : Protection against infection and disease
Authors: Timms Peter
Published: January 2023

Wild koala populations continue to experience serious declines as a result of several threatening factors including, (i) loss of habitat, (ii) motor vehicle trauma; (iii) dog attacks; (iv) Chlamydia. Chlamydial infections are associated with diseases ranging from ocular disease leading to blindness, as well as urinary and genital tract disease, leading to female infertility. Modeling shows that targeting chlamydial disease would have a major impact on stabilising population decline. Our studies have demonstrated that koalas can be safely immunized with a vaccine containing a mixture of chlamydial major outer membrane protein (MOMP) antigens combined with a single dose subcutaneous regime. We conducted a wild koala field trial and observed strong, specific and long-lasting immune responses in the vaccinated koalas; high titre antibody responses (as measured by ELISA and also in vitro neutralisation) as well as Chlamydia-specific cytokine responses (interferon-gamma and IL-17 in particular). For animals which were Chlamydia PCR positive at the time of vaccination, we observed a significant reduction in their infection PCR load. We also observed protection from progression to clinical disease in the vaccinated animals. We also conducted a small trial to vaccinate animals which already have clinical signs of ocular disease. Instead of the normal practice of administering antibiotics (chloramphenicol, daily for 28 days, which severely disrupts the animal’s gut microbiome) we vaccinated four animals with a single dose, 3-MOMP vaccine. For all vaccinated animals, their Chlamydia PCR load decreased, and for 6 of the 7 animals, ocular disease regressed and the animals could be released back into the wild without further treatment.
Increased immune cell infiltration contribute to enhanced protective immunity following co-delivery of VCG Chlamydia vaccine with Flt3L
Authors: Richardson Shakyra S , Lundy Stephanie , Dye Kamran , Omosun Yusuf , Eko Francis
Published: January 2023

Chlamydia trachomatis remains the commonest bacterial cause of sexually transmitted infections worldwide and can lead to pelvic inflammatory disease and infertility. Although antibiotics are effective against Chlamydia, most infections are asymptomatic suggesting the use of an effective vaccine may be the best approach for preventing these infections and the associated sequelae. We previously showed rectal (IR) immunization was a viable mucosal route for inducing female genital tract immunity and that the Fms-like tyrosine kinase 3-ligand (Flt3L; FL) enhanced the protective immunity of a Vibrio cholerae ghost (VCG)-based Chlamydia vaccine. However, the mechanism of immune enhancement was not investigated. In this study, we investigated if differences in the type and number of immune cells infiltrating mucosal and systemic tissues of mice could account for the observed immune enhancement following immunization. Mice were immunized IR with rVCG co-expressing the C. trachomatis polymorphic membrane protein D (PmpD) and porin B (PorB) proteins (rVCG-PmpD/PorB) with and without FL. Vaccine evaluation was based on measurement of IFN-γ cytokine, T cell proliferation, protection against intravaginal infection, and the differential profile of immune cells infiltrating mucosal and systemic tissues of mice using flow cytometry. The results showed co-delivery of vaccine with FL significantly enhanced IFN-γ levels and proliferation of splenic CD4+ immune T cells, as well as increased infiltration of immune cells in the spleen and genital tract tissues of mice. The results indicate the increased immune cell infiltration contribute to the enhanced protective immunity following co-delivery of VCG vaccine with FL.
Identification of toxicity and efficacy against new staphylococcal enterotoxin B subunit vaccine
Authors: Sim Euni , Kim Nayoung , Son Wonrak , Lee Minhoon , Hong Sungyoul , Shin YoungKee , Choi JunYoung
Published: January 2023

Staphylococcus aureus (S. aureus) bacteria, the gram-positive coccus and occurring irregular clusters, have the power to produce many types of enterotoxin. It is known as a foodborne poisoning agent as well as being a potential inhaled bioterrorism agent. Previously we confirmed the structure of modified-S. aureus enterotoxin B (SEB) vaccine candidate (ABN701) that can induce neutralization of antigen activity and also elicit humoral and cellular immune response. In this study, we studied the immunization efficacy and toxicity of ABN701 in various animal models. ABN701 was immunized in a dose dependent manner to BalB/C mouse and rabbit and about 20 ug ABN701 can induce an immune response in these models. Through the adjuvant tests, aluminum-composed adjuvant triggered more antibody inducement in mice and rabbit. The immunized BalB/C mouse were challenged with the SEB toxin and survived against 20LD50 SEB toxin treatment. Emesis is one of the most important evaluation factors for SEB vaccine development because emesis is related to SEB toxin symptom. House musk shrew mouse (Suncus Murinus) is widely known to be sensitive to various emetic drug evaluation and a new experimental animal model for SEB toxin related emesis. We confirmed that ABN701 did not cause emesis while SEB toxin cause emesis in this model. We also immunized ABN701 to monkey and showed no emesis symptom, but induced an effective immune response. In this study, we identified that ABN701 can induce an immune response in mouse, rabbit and monkey and ABN701 do not have toxicity related to emesis in the House musk shrew mouse model and monkey model.
An outer membrane vesicle-adjuvanted oral vaccine protects against lethal, oral Salmonella infection
Authors: Harrell Jaikin , Kurtz Jonathan R , Bauer David L , Gellings Patrick , Morici Lisa , McLachlan James B
Published: January 2023

Non-typhoidal salmonellosis, caused by Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common disease worldwide that can be contracted from contact with contaminated food, water, or animal carriers. Salmonellosis is characterized by mild gastrointestinal distress resulting in diarrhea, chills, fever, abdominal cramps, head and body aches, nausea, and vomiting; however, increasing incidences of antibiotic resistant invasive non-typhoidal Salmonella infections makes this a global threat requiring novel treatment strategies such next-generation vaccines. The goal of the current study was to formulate a novel vaccine platform against Salmonella infection that could be delivered orally. To accomplish this, we created a vaccine consisting of Burkolderia pseudomallei outer membrane vesicles (OMVs) which have been shown to act as potent immune mediators and are currently being explored as adjuvants for the next generation of vaccines. We show here that adding OMVs to a heat-killed oral Salmonella vaccine protects against a lethal oral challenge with S. Typhimurium. We show that anti-Salmonella antibodies are induced in response to immunization and demonstrate that bacterial burdens are lessened when OMVs are included in the vaccine. We are currently exploring whether CD4 helper T cells are induced and can contribute to the observed protection as these cells are known to be essential mediators of protective anti-Salmonella immunity. This study represents a new, oral vaccine approach to combatting the increasing problem of invasive Salmonella infections.
Two for one: Identification of P. aeruginosa and B. pertussis Cross-species Antigens
Authors: Blackwood Catherine B , Kilic Emel Sen , Weaver Kelly , Boehm Dylan T , Hall Jesse , Wong Ting , Malkowski Aaron , Witt William T , Damron F. Heath , Barbier Mariette
Published: January 2023

Development of effective vaccines requires logical design and formulation of two main components: antigens and adjuvants. Some compounds, called immunogens, can assume both of these roles. Notably, Bordetella pertussis (Bp) and some of its antigenic products have been found to adjuvant immune responses to other vaccines or vaccine components. We chose to explore this ability by using B. pertussis and pertussis toxin as adjuvants for a whole cell vaccine (WCV) against Pseudomonas aeruginosa (Pa). Pa is a highly antibiotic resistant Gram-negative opportunistic pathogen for which there are no approved vaccines or immunotherapies. We formulated vaccines containing Pa with Bp or pertussis toxin to determine if these components could modulate the immune response to PaWCV, as measured by quantification of bacterial burden and immune cell response after an acute pneumonia challenge. We determined that these additions to the PaWCV were associated with increased clearance of bacteria after challenge and increased Pa-binding immunoglobulins in the blood and airway. Additionally, we determined that the BpWCV alone was sufficient to induce protection from acute Pa infection. This correlated with production of antibodies that bind Pa, indicating that there is at least one cross-reactive antigen that could be providing protection. Through utilization of immunoblotting of protein fractions of Pa and mass spectroscopy, we identified this protective antigen. It will be used to formulate a subunit vaccine and tested for efficacy in protection from Pseudomonas infection. This type of cross-protection is novel within the field and could lead to the identification of a protective antigen against P. aeruginosa in B. pertussis.
Impact of viral sensitizer-enhanced oncolytic virus immunotherapy on the anti-tumor immune response
Authors: Alluqmani Nouf N. , Bergeron Anabel , Chen Andrew , Khan Sarwat , Forbes Nicole , Arulanandam Rozanne , Souza Christiano , Crans Debbi C. , Diallo Jean-Simon
Published: January 2023

Oncolytic viruses (OVs) have emerged as promising anticancer treatment platforms, able to specifically replicate in and kill cancer cells. OVs also have immunostimulatory effects, promoting antitumor immune responses, and several strategies have been developed in combination with OVs to stimulate cancer-specific immune responses. Viral sensitizers (VSes) are molecules that alter the tumor antiviral response in order to improve the efficacy of OVs. Such compounds can improve OV spread, transgene expression, and promote long-term antitumor immunity in various tumor models. A major objective of our study is to investigate the impact of VSes compounds on T-cells and other immune cells in the context of OV therapy and cancer vaccination strategies employing OVs. Results to date, VSes in combination with OVs significantly improve the therapeutic efficacy in syngeneic murine carcinoma tumor models. Additionally, we performed peptide-MHC-class I pentameric complexes staining to evaluate the kinetics of the T cell mediated anti-tumor immune response in vivo, our data illustrated that this combination therapy induced the production of antigen specific CD8+ T cells. We screened for chemokine and cytokine secretion using multiplex assay, and we found significant increases in IFN-γ, GM-CSF, and IL-12, 24 hour and 5 days post the treatment. We currently aim to phenotype the tumor microenvironment in order to understanding the mechanism that modulates the anti-tumor immunity using flow cytometry. We anticipate that unraveling the impact of VSes on the immunological response to OV therapy will lead to better understanding of mechanism contributing to its efficacy and improve our ability to successfully translate our findings to the clinic.
N2 / Ar plasma acts as a switch from apoptosis to necroptosis by degradation of MAVS aggregates
Authors: You Young Suk , Choi Jae Hoon , Kim Chul-Ho
Published: January 2023

Inflammatory responses play an important role in cancer progression and cell survival. For this reason, cancer can be treated by targeting an inflammatory response. Previously we reported that nonthermal plasma, which is a mixture of energy particles including electrons, ions and radicals, has anti-inflammatory and anti-cancer efffects. However, the underlying mechanisms how N2/Ar plasma induces cell death is largely unknown. In this study, we investigated whether N2/Ar plasma induces apoptosis or necroptosis in HNC cells. Inflammation-related proteins and mitochondrial MAVS proteins were elevated in HNC patient samples. TCGA data also showed low survival rates for HNC patients with increased inflammatory responses. We observed that the inflammatory response and expression of mitochondrial MAVS protein decreased when N2/Ar plasma was treated in the HNC cells. In addition, N2/Ar plasma treatment reduced the inflammatory response and MAVS protein expression and induced necroptosis. Finally, it was also confirmed that the increase of RIP3 decreases the expression of mitochondrial MAVS, inhibits apoptosis and induces necroptosis. This results suggest that, in HNC cells, N2/Ar plasma may act as a molecular switch from apoptotic cell death to necrototic cell death by regulating MAVS aggregates.
Cyclooxygenase-2 dependent prostaglandin E2 signaling through the EP3 receptor is required for cell-mediated immunity to Listeria monocytogenes
Authors: McDougal Courtney E , Theisen Erin , Sauer John-Demian
Published: January 2023

The bacterial pathogen Listeria monocytogenes (Lm) is being developed as a cancer immunotherapeutic platform due to its ability to elicit robust CD8+ T cell responses. Though the role of cytokines in Lm-stimulated immunity is well studied, the role of eicosanoids, lipid modulators of inflammation, is unclear. We previously demonstrated that cyclooxygenase-2 (COX-2) dependent prostaglandin E2 (PGE2) is essential for optimal T-cell priming. Antigen specific T cells and protective immunity were decreased following either treatment with the COX-2 inhibitor celecoxib or immunization of mice deficient in microsomal PGE2 synthase-1. Analysis of splenic eicosanoid levels revealed an upregulation of PGE2 twelve hours post immunization and administration of exogenous PGE2 at this time restored immunity during COX-2 inhibition. To understand how PGE2 impacts T-cell priming we assessed the role of the receptors EP1–4 in mediating this response using small molecule agonists and antagonists. EP3 blockade impaired immune responses while the use of an EP3 specific agonist restored immunity during COX-2 inhibition, suggesting that PGE2 signaling through EP3 is critical for T-cell priming. Conversely, consistent with its predicted inverse role in cAMP signaling, EP4 blockade enhanced responses, providing a mechanism by which to improve Listeria-based immunotherapeutics. Ongoing studies with floxed COX-2, EP3 and EP4 mice will illuminate the cell types producing and responding to PGE2. In conclusion, PGE2 signaling through EP3 is necessary Lm-stimulated immunity while EP4 signaling is detrimental. Further understanding these signaling pathways and where they function will be key to optimizing Lm as therapeutic platform.
Exploring γδ T cells for use in non-MHC restricted immunotherapy
Authors: Schriefer Kalyn , Chang Dan , Garett-Engele Phillip , Li Lixia , Spooner Ed , Monslow Morgan , Killough Jason , Lieberman Linda , Mohammadi Sina , Sana Theodore , Wyche Thomas , Guo Amanda , Howell Bonnie , Peterson Vanessa , Martinelli Roberta
Published: January 2023

γδ T cells are a unique subset of T cells found in both blood (Vδ2) and tissues (Vδ1). Unlike canonical αβ T cells that recognize peptide antigens through MHC class I/II molecules, γδ T cells recognize lipids and metabolites presented by non-MHC molecules. γδ T cells are highly cytotoxic and can rapidly kill both infected and cancer cells. Their cytotoxicity and lack of MHC restriction makes γδ T cells a promising target for allogeneic cell transfer therapy. In collaboration with MRL Oncology and ID/Vax we are investigating γδ T cells as a potential “off-the-shelf” cell transfer therapy. The ESC is currently optimizing ex vivo expansion protocols for both Vδ1 and Vδ2 T cells. Vδ2 cells rapidly expand from PBMCs after exposure to Zometa/IL-2, and are efficient cancer killers in vitro. To assess activation state, targeted metabolomics analysis was performed during Vδ2 expansion, and displayed significant differences in glutamate utilization, lactic acid, myo-inositol, and several essential and non-essential amino acids. We have also isolated and expanded Vδ1 T cells from PBMCs and human tissues (colon). These cells express high levels of NKG2D, which is suggestive of cytotoxic potential and we are currently assessing their cancer cell killing capacity in vitro. In collaboration with GpGx, we are identifying the dominant T-cell receptor (TCR) repertoires present in these populations by performing sc-TCRseq, sc-REAPseq, and comprehensive immunophenotyping. The results of these assays will be used to identify the optimum expansion protocol as well as to inform on the mechanisms underlying γδ T cell cancer targeting.
Characterization of a novel type NK cell line KHYG-1 carrying EGFRvIII-specific CAR in glioblastoma cells
Authors: Morimoto Takayuki , Nakazawa Tsutomu , Natsume Atsushi , NIshimura Fumihiko , Matsuda Ryosuke , Murakami Toshiharu , Nakamura Mitsutoshi , Nakagawa Ichiro , Motoyama Yasushi , Park Young-Soo , Tsujimura Takahiro , Wakabayashi Toshihiko , Nakase Hiroyuki
Published: January 2023

Background

Natural killer (NK) cells are considered potential antitumor effector cells. The aim of this study was to establish a novel type of a chimeric antigen receptor (CAR)-NK cell line (CAR-KHYG-1) specific for epidermal growth factor receptor variant III (EGFRvIII)-expressing glioblastoma (GBM) cells and investigate the antitumor effects of EGFRvIII-specific-CAR-expressing KHYG-1 (EvCAR-KHYG-1) in vitro and in vivo.

Materials and Methods

EvCAR-KHYG-1 was established by lentiviral-based transduction of the EvCAR gene and magnetic bead-based purification of EvCAR-expressing NK cells. The antitumor effects of EvCAR-KHYG-1 were evaluated using growth inhibition and apoptosis detection assays on GBM cell lines (EGFRvIII-expressing and non-expressing U87MG glioma cells). Cytokine production was determined by cytometric beads array. In vivo xenograft assays were performed in NOG mice subcutaneously implanted with EGFRvIII-expressing U87MG (Ev-U87MG).

Results

EvCAR-KHYG-1 inhibited GBM cell-growth via apoptosis in an EGFRvIII-expression specific manner in vitro. In addition, EvCAR-KHYG-1 produced IL-2, INF-γ and TNF-α on Ev-U87MG cells. In vivo xenograft assays showed that EvCAR-KHYG-1 did not decrease the volume of subcutaneous tumors but inhibited the occupancy of cancer cells in the subcutaneous tumor.

Discussion

EvCAR-KHYG-1 enhanced antitumor effects and produced Th1-type cytokines on EGFRvIII-expressing GBM cells, but did not inhibit progression of subcutaneous GBM cell-derived tumors to induce a pseudo progression pathological phenotype.

Conclusion

A novel type CAR-NK cell line was established. In vivo experiments are required to determine ways it can be used to inhibit tumor progression.
Phosphatidylserine targeting antibody enhances anti-tumor activity of adoptive cell therapies in a mouse melanoma model
Authors: Schad Sara , Hirschhorn-Cymerman Daniel , Budhu Sadna , Zhong Hong , Yang Xia , Merghoub Taha , Wolchok Jedd D
Published: January 2023

Adoptive cell therapy has emerged as a viable strategy to treat cancer. T cells that recognize tumor antigens can be reinvigorated ex-vivo or autologous T cells can be genetically modified to express anti-tumor T cell receptors (TCRs) or chimeric antigen receptors (CARs). However, once re-infused into patients, these tumor specific T cells are subjected to immunosuppressive signals within the tumor. A critical immune checkpoint within tumors is phosphatidylserine (PS), a phospholipid that is exposed on apoptotic cells and tumor cells. Innate cells exposed to PS secrete suppressive cytokines that can significantly impair the function of tumor specific T cells. Antibodies that target PS can reactivate anti-tumor immunity by reducing the number of MDSCs in tumors and promoting the maturation of functional APCs. Our lab has shown that the mouse chimeric version of PS Targeting monoclonal antibody Bavituximab (1N11), in combination with transgenic CD4+ T cells that recognize melanoma antigen Trp1, can regress advanced melanoma tumors in mice. Here, we demonstrate a 2nd generation CAR T cell, that binds Trp1 on the surface of B16 melanoma, in combination with 1N11 can improve anti-tumor activity and survival in B16 tumor bearing mice. Flow cytometry analysis of immune responses in the tumor of mice treated with tumor specific T cells and 1N11 shows a decrease in M2 macrophages and FoxP3+ regulatory T cells. These findings highlight that diminishing suppressive mechanisms locally with PS targeting can enhance the efficacy of transgenic TCR and CAR T cells to improve the outcome in patients with advanced-stage melanoma. Our studies may inform the design of clinical trials combining PS Targeting antibodies with CAR T cell therapy in solid tumors.
T-cells modified with CD70 as an alternative cellular vaccine for antitumor immunity
Authors: Cho Hyun-Il , Lee Sang-Eun , Shin A-Ri , Sohn Hyun-Jung , Kim Tai-Gyu
Published: January 2023

Successful tumor eradication primarily depends on generation and maintenance of a large population of tumor-reactive CD8 T-cells. Dendritic cells (DCs) have applied to clinics as potent antitumor therapeutic agents. However, high cost and difficulty in obtaining sufficient amounts for clinical use are the crucial drawbacks of DC-based vaccines. Here, antigenic peptide-loaded T-cells transfected with retrovirus encoding costimulatory ligands CD70, CD80, OX40L, or 4-1BBL were assessed for antigen-specific CD8 T-cell responses, and evaluated antitumor effects along with immunization of a mixture of synthetic peptides, poly-IC and anti-CD40 antibodies (TriVax). T-cells expressing CD70 (CD70-T) exhibited similar level of stimulatory functionality and therapeutic efficacy as DCs. Moreover, CD70-T prime followed by TriVax booster heterologous vaccination elicited therapeutic antitumor effect against B16 melanoma where mediated by CD8 T-cells but not CD4 T-cells or NK cells. The combination with PD-L1 blockade led to potent therapeutic efficacy which exhibited increased tumor infiltrating CD8 T-cells. CD70-T pulsed with multi-antigenic peptide generated multiple antigen-specific polyvalent CD8 T-cells that were capable of inhibiting tumor growth effectively. Moreover, CD70-T vaccination resulted in higher expansion and migration of adoptively transferred T-cells into tumor sites, and elicits enhanced therapeutic effects with peptide-based booster immunization. These results imply that T-cells endowed with CD70 enable the design of effective vaccination strategies against solid cancer, which may overcome current limitations of DC-based vaccines.
Lysosomal Protease Creation of Chimeric Epitopes for Diabetogenic CD4 T cells via Transpeptidation
Authors: Reed Brendan Kearney , Crawford Frances , Hill Ryan , Jin Niyun , Krovi Sai Harsha , White Janice , Marrack Philippa , Hansen Kirk , Kappler John
Published: January 2023

The identification of the peptide epitopes presented by major histocompatibility complex class II molecules (MHCII) that drive the CD4 T cell component of autoimmune diseases has presented a formidable challenge over several decades. In type-1 diabetes (T1D) recent insight into this problem has come from the realization that several of the important epitopes are not directly processed from a protein source, but rather pieced together by fusion of different peptide fragments to create new chimeric epitopes. We have proposed that this fusion is performed by a reverse proteolysis reaction called transpeptidation, occurring during the catabolic turnover of pancreatic proteins when secretory granules fuse with lysosomes. Here we demonstrate that highly antigenic chimeric epitopes for diabetogenic CD4 T cells are produced by digestion of the appropriate fragments of the diabetogenic granule proteins with the lysosomal protease, cathepsin L (CatL). This pathway has implications for how self-tolerance can be broken in T1D and other autoimmune diseases.
Lipid dysregulation and antigen presentation during intestinal inflammation
Authors: Russo Matthew V , Tordoff Jesse , Chiang Shian-Huey , Berasi Stephen , Buhlmann Janet
Published: January 2023

Inflammatory bowel diseases are associated with a loss of epithelial barrier integrity that can lead to an imbalance in gut homeostasis and severe inflammation. Studies in mice have shown that CD1d, a lipid antigen-presenting molecule, and natural killer T (NKT) cells play important roles in forming and maintaining homeostasis in the intestines. However, it is currently unknown what specific lipids are presented to NKT cells during homeostasis or inflammation in the gut. We hypothesize that during inflammation, CD1d ligands are replaced with pathogenic lipids that alter the activation of NKT cells and contribute to inflammation. Mice were administered dextran sodium sulfate (DSS) in their drinking water for 7 days to induce colitis symptoms, then switched to normal water for an additional 7 days to recover. Lipid species were measured in colons from day 7 inflamed mice and day 14 recovered mice. Compared to control mice, the lipid composition of inflamed colon was significantly altered. Day 7 enriched ceramide and hexosylceramide species remained elevated at the recovery time-point. These lipids stimulated inflammatory cytokine production in lamina propria cultures in a CD1d-dependent manner. To determine if these lipids were being generated endogenously, we measured the gene expression of several key enzymes in the hexosylceramide synthesis pathway. Expression of Degs1 and Ugcg were up-regulated in lamina propria cells during colitis, suggesting that immune cells could be responsible for the altered lipid composition we observed in DSS treated mice. Overall, we have demonstrated that colitis-induced lipid alterations in lamina propria cells of the gut can contribute to intestinal inflammation through CD1d antigen presentation.
The Outer Face of the IgV Domain of Butyrophilin 3A1 is Critical for the Sensing of Prenyl Pyrophosphates by Human Vγ2Vδ2 T cells: A New Paradigm for γδ T Cell Recognition
Authors: Morita Craig T , Rhodes David A. , Al-Janabi Mohanad Hameed Nada , Tanaka Yoshimasa , Sakuraba Shun , Wang Hong
Published: January 2023

γδ T cells expressing Vγ2Vδ2 TCR play critical roles in human immunity to microbes and tumors. These cells are uniquely able to use their TCRs for pattern recognition to monitor phosphorylated metabolites in isoprenoid biosynthesis in a process requiring butyrophilin (BTN) 3A1. Metabolites are sensed by binding to the intracellular B30.2 domain and requires the juxtamembrane coiled coil region. The role played by the extracellular domains (ECD) is unclear. To assess the ECD, surface IgV and IgC residues were mutagenized to alanine. Mutant BTN3A1 cDNAs were expressed in 293 T embryonic kidney cells lacking BTN3A1, -A2, and -A3, and the transfected cells pulsed with zoledronic acid and used to stimulate Vγ2Vδ2 T cells. Mutation of 13 residues scattered on the IgC domain had no effect on stimulation. Mutation of 22 residues scattered on the inner, side, and outer faces of the IgV domain also had no effect. However, 4 residues localized to a region on the outer face of the IgV domain either abrogated or diminished stimulation. Others have identified 2 additional residues in this region and mapped recognition to the Vγ2 HV4 loop paralleling BTNL3 and Btnl6 recognition by other γδ TCR. BTN3A2 and BTN3A3 can complement mutant BTN3A1 proteins suggesting that they form heterodimers or can alter their conformation. Modeling of the potential binding interface identifies BTN3 residues that can form ionic or hydrogen bonds to HV4. Given that our previous studies have shown the requirement for all CDRs of Vγ2Vδ2 TCR, a putative second ligand for the conventional binding side is also required. Taken together, we propose that Vγ2Vδ2 T cell stimulation is dependent on the simultaneous binding of BTN3A1 to HV4 and a second ligand to the conventional Vγ2Vδ2 binding site.
TLR7/8 agonist accelerates lupus development with heart disease in lupus-prone mice
Authors: Elshikha Ahmed Samir , Teng Xiangyu , Li Wei , Brown Josephine Michelle , Abboud Georges , Choi Seung-Chul , Kanda Nathalie , Zou Xueyang , Morel Laurence
Published: January 2023

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by loss of self-tolerance to nuclear antigens leading to the production of pathogenic autoantibodies, immune complex deposition and multi-organ damage. Although the prevalence of heart disease is higher than 50% in SLE patients, its etiology remains unclear but both genetic and environment factors are likely involved. Here, we compared the effect of TLR7/8 agonist Resiquimod (R848) on lupus and cardiac lesion development in lupus-prone B6.Sle1.Sle2.Sle3 (TC) and congenic normal mice B6 mice. Two weeks of R848 treatment increased the production of antinuclear antibodies and induced antiphospholipid autoantibodies lined to vascular pathology in TC mice. Correspondently, R848 expanded the frequency of Tfh cells and plasma cells in TC mice as compared to R848 treated B6 mice. More importantly, unlike the near normal appearance in hearts of R848-treated B6 mice, R848 induced severe cardiac pathology in TC mice with infiltration of CD45+ cells, edema, hemorrhage, enlarged endothelial cells and myocyte damage. Our results indicate that heart disease develops by combining genetic predisposition to lupus together with TLR7/8 activation. This novel model may lead to the identification of autoimmune mechanisms responsible for lupus-associated heart disease.