bluuebunny/crossref_metadata_2025 huggingface dataset:
Neonatal vaccination primes persistent HIV Env-specific antibodies that are augmented by a single booster immunization in late infancy
Authors: Curtis Alan Dale , Dennis Maria , Eudailey Joshua , Cronin Kenneth , Pollara Justin , Ferrari Guido , Hudgens Michael , Van Rompay Koen , Permar Sallie , De Paris Kristina
Published: January 2023

Breastmilk transmission of HIV remains unacceptably high. Vaccine approaches that prime robust virus-specific immune responses in infancy may prevent acquisition during the breastfeeding period. Moreover, vaccine responses that generate robust immunological memory that persists into adolescence could prevent sexual transmission later in life.

The current study investigated the immunological benefit of late boosting following vaccination in infancy. Two groups of neonatal rhesus macaques were immunized with a clade C HIV Env protein adjuvanted with 3M-052 stable emulsion (SE) vaccine (Env Only) alone or together with a modified vaccinia Ankara (MVA) vector expressing HIV Env and SIV Gag (MVA/Env) at 0, 6, and 12 weeks of age. Env-specific B cells were present in blood yet absent from lymph nodes in the Env Only group by week 14. At week 32, 18 weeks following the last immunization, we could still detect Env-specific plasma IgG responses capable of autologous and heterologous HIV Env binding, Env-specific ADCC activity and CD4 blocking in both groups. A final booster immunization at week 32 enhanced avidity and function of Env-specific plasma IgG in both groups. These data confirmed previous work by our group that demonstrated HIV Env protein adjuvanted with the TLR7/8-based 3M-052-SE primed greater immune responses than Span85-Tween 80 Squalene.

Our data from the Env Only vaccine regimen are consistent with the induction of Env-specific antibody responses in human infants. These results support the idea that early life vaccination is an effective means to induce persistent HIV Env-specific IgG responses that can be boosted in infancy and could be exploited to drive protective immunity against HIV acquisition prior to sexual debut.
Differential protection of Mayaro virus antigen-antibody binding by various inactivation approaches: Implication in vaccine development
Authors: Sharma Anuj , Sharma Shreya , Galasso Bianca , Knollmann-Ritschel Barbara
Published: January 2023

Mayaro virus (MAYV) is an emerging alphavirus in the family Togaviridae that causes a self-limited febrile illness accompanied by maculopapular rash and arthralgia. MAYV is reported throughout South America and the Caribbean and has potential to cause widespread outbreaks. There is no FDA-approved vaccine for MAYV. The overall objectives of this study were to inactivate MAYV utilizing various inactivation approaches, and to identify a highly safe vaccine candidate with optimum immunogenicity. MAYV was inactivated by three different mechanisms: formalin; 1, 5 iodonaphthyl azide (INA); and γ-radiation. Formalin inactivation (F-iMAYV) was performed with 0.05 or 0.1% formalin at 35°C. INA-inactivation (INA-iMAYV) was performed with 10, 50, or 100 μM INA combined with UV exposure. Lastly, MAYV was inactivated with 10, 20, or 30 kGy of γ-radiation in the presence of a protein-protecting manganese-decapeptide-phosphate complex (MDP-iMAYV). MAYV was inactivated by all the three approaches. Dose- and incubation time-dependent effects were observed with formalin inactivation. 0.1% formalin with an incubation time of 48h inactivated MAYV; however, further evaluation is ongoing. MAYV was completely inactivated with >50 μM dose of INA and by all three doses of γ-radiation. Differential protection of MAYV epitopes was observed after inactivation with three agents and with different doses of the inactivating agent. The most consistent antigen (Ag)-antibody (Ab) binding was observed in INA-iMAYV, followed by MDP-iMAYV. Ag-Ab binding was almost completely lost in F-iMAYV. The data suggests that INA and MDP-inactivated MAYV can be further studied as vaccine candidates. Studies are planned to evaluate immunogenicity in-vivo.
Cervicovaginal and Alveolar Macrophage responses of Rhesus Macaques following Mucosal Adenovirus-SIV recombinant priming and systemic SIV envelope boosting
Authors: Hunegnaw Ruth , Hait Sabrina Helmold , Rahman Mohammad Arif , Enyindah-Asonye Gospel M , Hoang Tanya , Robert-Guroff Marjorie
Published: January 2023

Vaccine strategies targeting the earliest stages of HIV infection at the mucosal portal of entry may prevent acquisition. Thus, understanding the immune profiles of mucosal cells after immunization is important for vaccine design. We vaccinated rhesus macaques with replicating Ad5hr-SIVenv/gag/nef or empty Ad5hr vector orally/intranasally and intra-tracheally, followed by intramuscular gp120 boosts in alum or alum only. Macaques were repeatedly challenged intravaginally with low doses of SIVmac251. We evaluated cervicovaginal macrophage (CM) responses to Ad5hr 3d post-immunization, and alveolar macrophage (AM) responses in BAL, as initial immunization was to the upper respiratory tract. CM showed significantly increased expression of the chemokines CCL3 (p<0.01), CCL4, CCL5 and CXCL8 (p<0.0001 for all) after the first prime, which decreased post-2nd boost. CD4+ T-cell frequency in the cervical mucosa was unchanged. CD16 expression was significantly increased post 2nd Ad and positively correlated with the number of challenges needed for infection (r=0.6763; p=0.0051). Immunization also increased expression of CD16 on AMs up to the 1st boost time point. Activation of AMs was evident by increased expression of CD40 and CD80 post-2nd Ad compared to naïve macaques. APRIL expression also significantly increased post-2nd Ad compared to naive levels and correlated with B cell frequency in BAL (r=0.7329; p=0.0019) and total IgG in BAL-Fluid (r=0.525; p=0.0471). These data indicate that the vaccine regimen did not induce recruitment of susceptible cells to the vaginal mucosa but increased CM CD16 expression associated with delayed SIV acquisition, and further induced AM cytokine expression that may be associated with B cell help.
Chimeric VP1-PCV2bCap protein as antigen for construction of recombinant vaccine against porcine circovirus type 2
Authors: Vrablikova Alena , Hezova Renata , Kotoucek Jan , Kulich Pavel , Turanek-Knotigova Pavlina , Gebauer Jan , Fojtikova Martina , Psikal Ivan , Turanek Jaroslav
Published: January 2023

Porcine circovirus type 2 (PCV2) is the main causative agents of post-weaning multi-systemic wasting syndrome leading to significant economic losses in swine population. Although vaccination is the most effective strategy to decrease morbidity and mortality rate, the virus persist in immunized populations and causes subclinical infections with symptoms similar to porcine circovirus diseases.

To develop new effective vaccine against PCV2 infection, we prepared chimeric complex VP1-PCV2bCap containing PCV2bCap protein fused with mouse polyomavirus (MPyV). Complex was expressed in baculovirus expression system and purified by nickel agarose. From eluate containing large aggregates of purified proteins, fraction containing pantameres of VP1-PCV2bCap protein was separated by Asymmetrical flow field-flow fractionation and fractions were analyzed by electron microscopy with nano-gold labeling of PCV2Cap portion of the fused protein.

6 weeks old BALB/c mice were subcutaneously vaccinated by 50ug of purified chimeric protein alone or in combination with incomplete Freund adjuvants (50%) or Emulsigen (10%). Immunity response was monitored by indirect ELISA. After the first immunization strong immunity response that gradually increased after the second and third immunization was detected in all mice immunized by chimeric protein. The best immunity response was detected in mice immunized by vaccine with Emulsigen (10%) as adjuvant.

Purified chimeric protein VP1-PCV2bCap has a high potential as a DIVA (differentiating infected form vaccinated animals) vaccine against PCV2b infections in pigs. The biggest benefit of this type of vaccine is possibility to distinguish between naturally infected and vaccinated animals
Exploration of roles of CD4 T cell help in recruitment of rare B cells to germinal centers
Authors: Lee Jeong Hyun , Nakao Catherine , Moyer Tyson J. , Irvine Darrell J. , Schief William R. , Crotty Shane
Published: January 2023

The elicitation of broadly neutralizing antibody (bnAb) responses is a major goal for developing an efficacious HIV vaccine. The HIV Env trimer is a weak immunogen, but it will be a necessary component in a HIV vaccine immunization schedule to drive affinity maturation to bnAbs. There are substantial biological challenges; bnAb precursor B cells are rare in humans, and B cell responses are typically directed to non-neutralizing immunodominant Env epitopes. HIV bnAb development relies heavily on germinal centers (GC) for affinity maturation. The survival of GC B cells is dependent on help from follicular helper CD4 T cells (TFH). Here, we have studied whether the modulation of TFH cells affect B cell immunodominance and competitiveness of rare B cells. We generated Env-specific TCR transgenic mice specific for a I-Ab MHCII epitope we mapped in Env. By utilizing our previously defined VRC01gHL B cell transfer model (Abbott et al., Immunity 2018), combined with co-transfer of CD4 T cells from our new Env-specific TCR transgenic mouse, we examined the competitive fitness of VRC01gHL B cells responding to Env antigens engineered to exhibit varying affinities for VRC01gHL. Increased availability of Env-specific CD4 T cells accelerated the initial expansion of GC VRC01gHL cells in response to a high affinity immunogen. However, improvements in early GC VRC01gHL cells were not observed for low affinity Env trimer immunogens. We are actively exploring whether slow delivery immunization strategies (Cirelli et al., Cell 2019) enhance the ability of Env-specific CD4 T cells to recruit rare VRC01-class B cells to GCs in mice.
Long-term sterile immunity induced by an adjuvant-containing live-attenuated AIDS virus
Authors: Yasutomi Yasuhiro
Published: January 2023

Previous studies have been performed using vaccine viruses attenuated by genetic disruption of key regulatory genes. The moderately attenuated prototypic vaccine strain SIVmac239Δnef has been used in most studies; however, it does not provide sufficient effects to prevent infection. Ag85B is one of the most dominant proteins secreted from most mycobacterial species. It has been shown to induce substantial Th1 cell proliferation and vigorous Th1 cytokine production. In the present study, we genetically constructed a live nef-deleted simian human immunodeficiency virus to express the adjuvant molecule Ag85B (SHIV-Ag85B) and assessed vaccine effects in cynomolgus macaques. SHIV-Ag85B could not be detected at 4 weeks after injection in cynomolgus macaques. When these macaques in which SHIV-Ag85B had become undetectable were challenged with pathogenic SHIV89.6P at 37 weeks after SHIV-Ag85B became undetectable, SHIV89.6P could not be detected at 36 weeks after the challenge in most of the macaques. In these macaques, SHIV antigen-specific CD8+ T cell responses with polyfunctionality were rapidly induced after SHIV89.6P injection. Eradication of pathogenic SHIV89.6P was confirmed by adoptive transfer experiments and CD8+ cell depletion study. These results suggest that SHIV-Ag85B elicited viral antigen-specific CD8+ T cell responses against pathogenic SHIV and provide the possibility of eradicating a pathogenic lentivirus from the infected cells. The results of this study provide further insights into the containment of HIV infections as well as new opportunities to develop a better therapeutic vaccines and cure.
Therapeutic vaccination with Ad48/MVA and TLR7 stimulation in SIV-infected, ART-treated infant rhesus macaques
Authors: Bricker Katherine M , Obregon-Perko Veroncia , Uddin Ferzan , Williams Brianna , Hesselgesser Joseph , Robb Merlin , Michael Nelson , Barouch Dan H , Chahroudi Ann
Published: January 2023

Antiretroviral therapy (ART) improves outcomes for the 2.1 million children living with HIV globally but is not a cure. Therefore strategies to reduce, eliminate, or control the persistent HIV reservoir would be highly beneficial. In this study, we sought to evaluate the impact of a therapeutic vaccine and TLR7 agonist in SIV-infected, ART-treated infant rhesus macaques (RMs). Four-week-old RMs (n=16) were infected orally with SIVmac251 then after an additional 4 weeks placed on a daily ART regimen. After 22 weeks post-infection (wpi) 8 RMs received 2 doses of Ad48 vectors expressing SIVgag-pol-env 8 weeks apart and 2 doses of MVA vectors expressing SIVgag-pol-env at 38 and 50 wpi; 10 oral doses of the TLR7 agonist (GS-986) were administered every other week beginning at 40 wpi. The remaining 8 RMs served as experimental controls. The magnitude of SIV-specific cellular immune responses, measured by IFN-γ ELISPOT and multiparametric intracellular cytokine staining, was increased in vaccinated RMs following Ad48-prime and was boosted by MVA. GS-986 induced an elevation of CD14+CD169+ monocytes 24 h after doses 1, 5, and 10. Four weeks after the last GS-986 dose daily ART was stopped to assess efficacy and animals were evaluated for 8 weeks. A similar time to rebound was observed in treated and control RMs. Despite the fact that SIV-infected ART-treated infant RMs are capable of mounting a robust vaccine response and immune activation by the TLR7 agonist this regimen was not sufficient to impact viral rebound. These results are distinct from previous studies performed in acutely infected adult RMs and highlight the importance of a pediatric model to evaluate HIV cure interventions during the unique period of immune development.
Isotype-specific outcomes in Fc gamma receptor targeting of PspA using fusion proteins as a vaccination strategy against Streptococcus pneumoniae infection
Authors: Bitsaktsis Constantine , Wiedinger Kari
Published: January 2023

Streptococcus pneumoniae (Spn) remains a considerable threat to public health despite the availability of antibiotics and polysaccharide conjugate vaccines. The lack of mucosal immunity in addition to capsular polysaccharide diversity, has proved to be problematic in developing a universal vaccine against Spn. Targeting antigen to Fc receptors is an attractive way to augment both innate and adaptive immunity against mucosal pathogens. The effect of targeting Fcγ receptors (FcγR) is highly influenced by the IgG subclass, which bares differential affinities for activating and inhibitory FcγR. In the current study we demonstrate targeting activating FcγR with fusion proteins consisting of PspA and IgG2a Fc enhance PspA-specific immune responses, and effectively protect against mucosal Spn challenge. Specifically, targeting PspA to FcyR polarized alveolar macrophage to the AM1 phenotype and increased conventional dendritic cell subsets in the lung in addition to augmenting Th1 cytokines and PspA-specific IgG and IgA. In contrast, fusion proteins consisting of PspA fused to the IgG1 Fc provided minimal benefit as a result of interaction with the inhibitory FcγRIIB. Protective efficacy of the IgG1 fusion protein was significantly enhanced in animals deficient for FcγRIIB accompanied by increased B cell maturation and proliferation levels in these animals. These studies demonstrate FcγR targeting is an effective strategy for inducing potent cellular and humoral responses via mucosal immunization with Fc fusion proteins, however, careful consideration of the Fc region utilized, is required. Fc-engineering with specific attention to FcγRIIB engagement presents a valuable vaccine strategy for protecting against Spn infection.
Human B cell responses to a live attenuated cholera vaccine
Authors: Adekunle Oluwaseyi , Kauffman Robert , Cho Alice , Nyhoff Lindsay , Rouphael Nadine , Wrammert Jens
Published: January 2023

Vibrio cholerae is a bacterial pathogen which causes cholera, a severe acute diarrheal disease. A symptomatic incident of cholera can lead to long term protection against subsequent exposure; however, the generation of immunity and mechanisms of protection are undetermined. A thorough understanding of the immune response to V. cholerae is needed to identify parameters which are key for the generation and maintenance of immunity. To approach this, we utilized a live attenuated cholera vaccine to model the response to V. cholerae exposure.

In this study, we found that vaccination induced plasmablasts which were specific for the two immunodominant antigens of cholera, lipopolysaccharide (LPS) and cholera toxin (CT). These antigens induced a preferential isotype response with LPS driving IgM and IgA plasmablasts while CT drives an IgG and IgA plasmablast. Serum responses had corresponding increases in both antigen binding and functional titers that remained detectable one-year post vaccination. Additionally, LPS specific IgA memory B cells were detectable at 90 days post vaccination. These cells, along with mucosal plasma cells likely play a role in long term cholera immunity. Collectively, these findings demonstrate the use of the live attenuated cholera vaccine as an effective tool to examine the primary and long term immune response to V. cholerae exposure. Additionally, it provides insight into the phenotype and specificity of the cells which likely return to and mediate immunity at the mucosa as determined by our analysis of gut-homing receptor (CCR9) expression levels on antigen specific cells. A thorough understanding of these properties will likely inform future vaccine development to both cholera and other mucosal pathogens.
Harnessing the immunogenicity of peptide-based glycoconjugate vaccines
Authors: Stefanetti Giuseppe , Okan Nihal , Kasper Dennis L
Published: January 2023

Glycoconjugate vaccines provide enormous health benefits globally, although they have been less successful in some populations at high risk for developing disease. In most cases, conjugation of the sugar antigen to the carrier protein has been done empirically with little attention on variables critical for the immune response. Recent findings offer new insights on their mechanism of actionsuggesting that carbohydrate presentation to T cells by antigen-presenting cells may enhance the efficacy of antibody responses and suggesting the use of a peptide instead a whole protein as carrier.

Herein, we have developed a platform to optimize glycopeptide vaccine design by determining optimal polysaccharide chain length, peptide composition and conjugation site. We first determined that the use of a full-length polysaccharide induce optimal immunogenicity. We have further demonstrated the role of the peptide linker and the conjugation site in enhancing both the conjugation efficiency and the immune response to vaccines. When immunizing mice, we found—using GBSIII CPS as a model—that these glycoconjugates induced full protection in the challenge model. We extended the platform to develop a prototype vaccine for Francisella tularensis, coupling the O polysaccharide to an optimized peptide and observing significant protection. Remarkably, protection with a glycoconjugate vaccine against F. tularensis has been only recently obtained by using a genetically enlarged polysaccharide.

The results show that peptide glycoconjugate vaccines provide a strong protection against bacterial challenges. We are currently applying the acquired knowledge to create more efficacious and longer lasting immunity to glycoconjugate vaccines.
Vaccination with a Live Attenuated Brucella abortus Mutant Stimulates Cytotoxic CD8+ T Cell Responses
Authors: Hoffman Carol , Goodwin Zakia I , Yang Xinghong , Pascual David W
Published: January 2023

To effect protection against mucosal and systemic tissue colonization, the B. abortus mutant strain expressing lacZ (znBAZ) was administered mucosally into BALB/c mice, and conferred nearly complete protection against pulmonary challenge with wild-type (wt) BA 2308. In fact, it was more effective than the conventional BA RB51 or S19 vaccines. Past studies with conventional brucellosis vaccines have shown the importance of stimulating CD4+ T cells for protection; however, znBAZ-vaccinated mice produced elevated levels of effector and resident memory CD8+ T cells in the lungs. In pursuit to identify cellular mechanisms of protection, we hypothesize cytolytic activity contributes to this protection. Mucosally or intravenously vaccinated mice with znBAZ induced IFN-γ-producing CD8+ T cells, both after in vitro culture or ex vivo by intracellular staining. Granzyme B+ CD8+ T cells were also induced. To assess its function, fluorescently stained RAW264.7 macrophages were infected with znBAZ at varying ratios for 2hrs, and following antibiotic treatment, were used as targets when mixed with lymph node and splenic lymphocytes for a short exposure. Cytotoxic T cell (CTL) activity was detected subsequent to granzyme B injection into znBAZ-infected macrophages, and measured by cleavage of fluorescent substrate for granzyme B. Increased CTL activity was detected with lymphocytes from vaccinated mice compared to naïve lymphocytes. Purified CD8+ T cell fractions contained this activity. Hence, these studies demonstrate that cytolytic activity is a potential effector mechanism to rid Brucella-infected cells. Work is supported by NIH AI123244 & AI125516.
Combination immunotherapy of αPD-1 and extended half-life IL-2 clears established GL261 gliomas in an MHC class I independent fashion
Authors: Tritz Zachariah Peter , Ayasoufi Kathy , Malo Courtney , Himes Benjamin , Khadka Roman , Yokanovich Lila , Goddery Emma , Fain Cori , Hansen Michael , Jin Fang , Wang Chensu , Irvine Darrell J , Wittrup K. Dane , Parney Ian F , Johnson Aaron J
Published: January 2023

Glioblastoma multiforme (GBM) is a deadly CNS malignancy with an average survival of ~12 months post diagnosis despite aggressive treatment. Immunotherapy approaches to treat GBM are being developed, but the unique immune environment of the brain and the immunosuppressive features of GBM have complicated early efforts. Even αPD-1 checkpoint blockade, highly useful in the context of melanoma, has proved ineffective in the treatment of GBM. In order to promote the efficacy of αPD-1 against the GL261 murine model of GBM, we supplemented this therapy with an engineered IL-2 cytokine. IL-2 therapy has been used clinically for metastatic melanoma and renal cancer, but its short half-life in circulation necessitated dangerously high doses to keep the drug concentration sufficiently high. Fusing IL-2 to the mouse serum albumin molecule (MSA-IL-2), extends the cytokine’s half-life. While neither αPD-1 nor MSA-IL-2 are effective monotherapies against the GL261 model tumor, we have found this combination immunotherapy to provide durable tumor clearance and to create immunologic memory capable of resisting GL261 rechallenge. Most strikingly, this combination therapy is independent of the CD8 T cell function, clearing established GL261 tumors in mice incapable of MHC class I restricted antigen presentation. Independence of MHC class I restricted antigen presentation makes this combination immunotherapy using αPD-1 checkpoint blockade both highly novel and translatable to clinical use.
CD103+ cDC1 vaccine efficacy in murine breast cancer is inhibited by STAT3
Authors: Chrisikos Taylor T , Zhou Yifan , Li Haiyan S , Babcock Rachel Lauren , Wan Xianxiu , Patel Bhakti , Newton Kathryn , Mancuso James , Watowich Stephanie S
Published: January 2023

Conventional dendritic cells (cDCs) are a crucial immune population, which includes multiple subtypes that coordinate adaptive immunity and tolerance. Migratory type 1 cDC1s (CD103+ cDC1s in mice) are required to induce CD8+ T cell-mediated anti-tumor immunity, yet whether and how tumors regulate CD103+ cDC1 function is largely unknown. Many tumor-associated immunosuppressive cytokines, such as interleukin (IL)-10, require Signal Transducer and Activator of Transcription 3 (STAT3) for intracellular signaling; therefore, we sought to determine whether STAT3 inhibits CD103+ cDC1-mediated anti-tumor immunity. Indeed, we found that in vitro-derived STAT3-deficient (Stat3Δ/Δ) CD103+ cDC1sare resistant to IL-10 – mediated inhibition of Toll-like receptor 3 (TLR3)-agonist induced maturation. When used as an anti-tumor vaccine, Stat3Δ/Δ CD103+ cDC1s inhibited breast tumor growth and increased mouse survival to a greater degree than STAT3-sufficient CD103+ cDC1s. Vaccination with Stat3Δ/Δ CD103+ cDC1s increased tumor antigen-specific CD8+ T cells and IFN-g+ CD4+ T cells in tumors and tumor-draining lymph nodes compared to controls. Moreover, IL-10 receptor-deficient CD103+ cDC1s restrained tumor growth as effectively as Stat3Δ/Δ CD103+ cDC1s. Taken together, our results demonstrate STAT3 suppresses CD103+ cDC1 maturation and vaccine efficacy in breast cancer. The data also suggest IL-10 is an important factor in the murine mammary tumor environment mediating immunosuppressive STAT3 signaling in CD103+ cDC1s. Thus, STAT3 inhibition may provide an effective strategy for improving the anti-tumor efficacy of CD103+ cDC1-based vaccines.
Characterization of T cell responses induced following checkpoint inhibition therapy across multiple malignancies with a high tumor mutational burden
Authors: Moss Keith
Published: January 2023

Cancer vaccination has been idealized for the past two decades but has suffered from the lack of relevant and specific target antigens. Neoantigens, harboring neoepitopes, represent patient-specific mutated antigens which could potentially serve as immunotherapeutic targets. In the recent years, it has been observed that patients with a higher tumor-mutational burden (TMB) have better clinical responses to checkpoint inhibition therapy. TMB may become a potential criterion for the inclusion of patients in such immunotherapeutic trials. However, this observed correlation between TMB and improved clinical response has yet to be fully substantiated, especially in a pan-cancer setting.

This work presents preliminary data from a basket trial that has been undergoing at Rigshospitalet, Denmark. This trial contains patients presenting malignant tumors with a high TMB across multiple cancer types, those of which are undergoing various immunotherapies. Preliminary data indicates that neoepitope-specific CD8+ T cells can be detected in patient-derived tumor-infiltrating lymphocytes and PBMCs with the use DNA barcode labeled peptide-MHC (pMHC) multimers. Through FACS and NGS sequencing, immunogenic neopeptides may be identified from a library of personalized in silico predicted peptides. Thus, providing further support to the notion that high-throughput methods of antigen-specific T cell detection may be utilized to interrogate the contribution of TMB to clinical outcome in checkpoint inhibition therapy.
Agonistic anti-ICOS antibodies promote unexpected elimination of tumor infiltrating effector T cells in a FcγR-independent manner
Authors: Demane Dafne Sofia Franz , Joshi Kroopa , Costoya Cristóbal , Zervas Dimitrios , Solomon Isabelle , Peggs Karl S. , Quezada Sergio A.
Published: January 2023

Inducible T cell co-stimulator (ICOS) is a co-stimulatory receptor expressed on tumor infiltrating regulatory and effector T cells. ICOS potential role in cancer control is supported by the correlation between ipilimumab’s efficacy and the presence of CD4+ICOShi T cells in PBMC, as well as loss of tumor control in ICOS−/− and ICOSL−/− mice treated with anti CTLA-4.

37A10 is an anti ICOS agonistic mAb reactive to mouse and human ICOS, with a human IgG1 Fc region to promote depletion of regulatory T cells through antibody-dependent cellular cytotoxicity. We hypothesized a pure agonistic, non-depleting anti ICOS would promote co-stimulatory signals on activated T cells while avoiding depletion of activated ICOS+ T cells. Hence, we aimed to compare the efficacy in tumor control of two isotypes of 37A10. Both mIgG1 (non-depleting) and mIgG2a (depleting) isotypes failed to promote survival, as a monotherapy or in combination with anti PD-1 or anti CTLA-4. This was consistent with both isotypes promoting the elimination of activated T cells in an Fc-independent manner. Ablation of activated effector T cells with anti ICOS replicated loss of activated effector T cells upon in vivo blockade of ICOSL reflecting their ligand blocking activity. Moreover, both isotypes impaired the activity of anti CTLA-4, associated to effector T cell depletion, raising concerns regarding the use of anti ICOS mAbs as combination substrates in tumor immunotherapy.

The data suggest that agonistic antibodies that block the natural ligand binding site and have weak in vivo agonism could result in unwanted negative responses in cancer. Future work should aim to identify antibodies with robust agonistic activity in vivo while allowing signaling induced by the natural ligand.
Amplifying an abscopal effect of radiation therapy with a novel immunotherapeutic agent
Authors: Paez Patrick A , Liu Wenjie , Guo Chunqing , Mikkelsen Ross , Wang Xiang-Yang Shawn
Published: January 2023

Radiation therapy (RT) is one of the primary treatment modalities for head and neck squamous cell carcinoma (HNSCC). At the time of diagnosis two-thirds of HNSCC patients have local-advanced disease and 50–60% of these patients will experience a local-regional or metastatic relapse within three years. Improving the immunogenic response of RT may help address this clinical problem. However, current RT regiments have failed to reliably generate robust antitumor immunity as evidenced by the rarity of clinical abscopal responses. Recently we engineered a chimeric fusion molecule called Flagrp170, a novel immunostimulatory agent highly capable of promoting antigen presentation and T-cell activation. We hypothesize that the combination of RT and Flagrp170 provides superior immunogenic signals producing effective and durable antitumor immunity against HNSCC. We report that administration of Flagrp170 to the tumor sites upon RT using a small animal radiation research platform (SARPP) results in potent activation of antigen-presenting cells, increased functionality of tumor-infiltrating T-cells, and systemic immune augmentation, when compared to either monotherapy. Additionally, the combination treatment is able to reduce the dose of RT required for tumor control and protects previously cured animals from subsequent tumor re-challenge. Finally, the combination treatment can successfully control the contralateral untreated tumors, supporting the superior activity of Flagrp170 in potentiating abscopal responses of RT. Our data suggest that the Flagrp170 may be used to enhance immunogenic cancer cell death in RT and resultant protective antitumor immunity can potentially help reduce post-RT recurrence of HNSCC.
A trifunctional bispecific antibody mediates efficient killing of B-cell lymphoma cells
Authors: Cheng Ying , Sun Rui , Tian Zhigang
Published: January 2023

Natural killer (NK) cells are a type of cytotoxic lymphocytes critical to immune surveillance. The mechanisms for NK-mediated tumor elimination including natural cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), and the secretion of interferon-γ (IFN-γ), which are essential for mediating the anti-tumor activity. CD19, a B-cell lineage-specific marker, is highly expressed in B-cell malignancies and an attractive target for therapy. Here, we generate a trifunctional bispecific antibody (TBAb) containing single chain antibody fragment (scFV) against CD16 and CD19 to create an immunologic synapse between NK cells and CD19+ lymphoma cells. X has been engineered with a modified human interleukin crosslinker for improving therapeutic efficacy. Specificity has been confirmed by investigating the ability of X to bind CD19+ tumor targets and CD16+ NK cells. We found that X TBAb mediated killing of human lymphoma B cell lines, including namalwa, daudi and raji, by human NK cells purified by microbeads or NK-cell amplification kit in vitro. X-mediated killing is accompanied by NK-cell activation, cytokine release and upregulation of TNF-related apoptosis-inducing ligand (TRAIL). Further, X TBAb demonstrated antitumor activity against lymphoma xenografts reconstituted with human PBMCs. These data provide a therapeutic candidate for the treatment of B-cell malignancies.
A new checkpoint receptor targets for cancer immunotherapy
Authors: Li yangyang , Tian Zhigang , Sun Rui
Published: January 2023

While blockade of the CTLA4 and PD1 pathways has emerged as an effective treatment of cancer, the majority of patients do not derive long term benefit. This has catalyzed enormous interest in the targeting of other immune checkpoint receptors. X protein is recently identified as a novel immune checkpoint receptor on human T cells. However, the function of murine X remains elusive. To investigate the role of murine X on immune cells, we generated anti-X antibodies and identified 11D7 with the ability to binding X and blocking X ligand binding. We found that X was not expressed on resting immune cells while it was upregulated upon cell activation. The expression of X was significantly upregulated on NK cells and CTLs in the tumor microenvironment. Furthermore, blockade of X with monoclonal antibodies inhibits tumor growth and prolong the survival of tumor-bearing mice. These data indicated that targeting X is a promising anti-cancer therapeutic strategy.
Reprogramming T Cell Specificity to Suppress Autoreactive T Cells in Type 1 Diabetes
Authors: Joglekar Alok V , Rankin Kobie , Ford Kelsey , Hicks Louise Teresa
Published: January 2023

Effector T cells (Teff) in Type 1 Diabetes (T1D) recognize antigenic targets on pancreatic β cells, thereby killing them. Insulin-derived epitopes are targeted first, followed by rapid epitope spreading, leading to progressive destruction of pancreatic islets, causing insulitis and T1D. Curbing these autoimmune responses is key to the development of immunotherapies to treat T1D. In this study, we tested the proof-of-concept of two strategies to treat T1D by reprogramming T cell specificity. We have developed cell-surface receptors called SABRs (Signaling and Antigen-presenting Bifunctional Receptors), which present a peptide-MHC complex on the extracellular domain and link it with a CD28-CD3ζ signaling domain. The intracellular signaling domains in SABRs are capable of activating T cell function. The extracellular domains can recognize TCRs specifically. We successfully demonstrated that SABRs presenting peptide epitopes on human and mouse, class I and class II MHC complexes are able to induce a functional signal through the NFAT pathway. We tested two complementary approaches to use SABRs to engineer T cells. In the first approach, we transduced human Treg cells with SABRs. SABR-armed Treg cells were able to secrete IL-10 in response to T cells bearing a diabetogenic TCR. In the second approach, we transduced human CD8+ T cells with SABRs. SABR-modified CD8+ T cells were able to specifically lyse target cells bearing a diabetogenic TCR. Ongoing studies will determine if Treg-mediated suppression or CD8+ T cell-mediated elimination of diabetogenic Teff cells will be an efficient strategy for immunotherapy for T1D. If successful, these approaches will also be applicable to other autoimmune diseases such as Multiple Sclerosis.
Optimization of CAR T cell co-stimulation reduces to MDSC suppression
Authors: Boucher Justin C , Cervantes Estelle V , Lee Sae Bom , Spitler Kristen , Reid Kayla , Davila Marco L
Published: January 2023

A major obstacle for continued development of CARs is their suppression by the tumor microenvironment. Clinical data from our lab shows patients that have increased myeloid cells have poorer outcomes to CAR T cell therapy. Based on this we examined MDSC suppression of CARs.

First, we examined the effect of MDSCs on CAR T cell production. We found gene transfer was lower for m1928z CAR T cells co-cultured with MDSCs compared to those that were not (24.1% vs 39.1%). There was also a reduction in total T cell counts for m1928z after MDSC co-culture (88%). We also investigated the effect of MDSCs on CAR T cells when present during antigen stimulation. Co-culture with MDSCs in vitro showed significant reductions in CAR T cell mediated killing and IFNγ secretion. Additionally, CAR T cells stimulated in the presence of MDSCs had lower expression of activation markers PD1 and LAG3. This suggests that MDSCs reduce CAR T cell gene transfer, activation, killing, and cytokine production.

To evaluate if we could create a CAR T cell resistant to MDSC suppression null mutations of YMNM and PRRP were incorporated into a CD28 CAR leaving only PYAP active (mut06). When mut06 and MDSCs were co-cultured there was a reduction in gene transfer (21% vs 38%) and T cell counts (80% vs 88%) compared to m1928z. We also found when in the presence of MDSCs mut06 was significantly better at killing compared to m1928z. To examine this in vivo we co-injected mice with CAR T cells and MDSCs. We found mice given m1928z with MDSCs had reduced CAR T cell function while mut06 was not affected by MDSCs. Overall our data shows that MDSCs can suppress CAR T cell function when present during production as well as CAR stimulation and optimizing CD28 CAR signaling creates CAR T cells that are more resistant to MDSCs.
CD4 depletion potentiates anti-tumor immunity in adoptive immunotherapy by increasing IL-18Rαhi endogenous CD8+ T cells
Authors: Kim Seon-Hee , Han Chungyong , Kwon Byoung S. , Choi Beom K.
Published: January 2023

Adoptive T cell therapy (ACT) requires lympho-depletion pre-conditioning to eliminate immune-suppressive elements to allow for the efficient engraftment of adoptively transferred tumor-reactive T cells. Because anti-CD4 monoclonal antibody depletes CD4+ immune-suppressive cells to enhance anti-tumor immunity, combinations of anti-CD4 treatment and ACT have synergistic potential in cancer therapy. We designed a post-ACT conditioning regimen that involves weekly treatment with anti-CD4 (CD4post). Using murine melanoma, cyclophosphamide and tumor-reactive CD8+ T cell infusion were included to represent an ACT model. We evaluated anti-tumor effects and immunologic changes of T cells. CD4post in ACT markedly increased tumor suppression and survival. Remarkably, CD4post worked differently on ex vivo primed CD8+ T cells versus endogenous CD8+ T cells. CD4post substantially increased the proliferation of ex vivo primed CD8+ T cells, while increasing endogenous CD8+ T cell differentiation and effector function. Endogenous CD8+ T cells upregulated activation/exhaustion markers and exhibited a skewed TCR repertoire, implying that CD4post boosted tumor-reactivity. Accordingly, CD4post-experienced endogenous CD8+ T cells showed enhanced intra-tumoral infiltration and exhibited greater anti-tumor activity against melanoma in vitro. Importantly, enrichment of the IL-18Rαhi subset was critical for boosting anti-tumor responses, as IL-18Rα+ cell-depletion CD8+ T cells resulted in diminished anti-tumor activity. This study highlights the clinical relevance of CD4post to ACT and gives insights into the characteristics of the immunological components that drive the augmented cancer–immunity cycle in ACT.
Activation of the hypoxia-inducible factor 1 alpha is necessary for type 1 interferon and IFN stimulatory gene expression during gammaherpesvirus infection
Authors: López D M , Mesri Enrique , Andreanksy Samita
Published: January 2023

Oxygen availability and the hypoxia-inducible factor 1 pathway (HIF1) influence the activity of cells involved in immune responses. Initially described as a master regulator of cellular adaptation to low oxygen concentration, activation of the hypoxia-inducible factor 1 alpha (HIF1α) is now recognized as an integral part of inflammatory cytokines production, phagocytic activity and bacterial killing by myeloid-derived cells. The goal of our studies is to determine the role of HIF1α during antiviral responses. We measure type 1 interferon gene expression in mouse embryonic fibroblasts lacking HIF1α activity (HIF1α Null MEFs) during early infection against the DNA virus, murine gammaherpesvirus 68 (MHV68). We recently reported that MHV68 activates HIF1atranscription activity and this is necessary for viral replication. In this study, analysis of mRNA levels by real-time qPCR, revealed that IFNβ1 decreased by 5-fold in HIF1α Null MEFs following 8 hours post infection (hpi), relative to wild-type, whereas levels of IFNα4, IFNα6 remained unchanged. Consistently, transcript levels of the IFN-stimulated gene OAS1β were reduced 7-fold relative to HIF1α WT MEFs. However, at 24hpi absence of HIF1α diminished expression of IFNα4, IFNα6, IFNβ1, OAS1β, IFIT18 and ISG20. In conclusion, these results suggest that HIF1α activity plays an important role during antiviral responses against dsDNA viruses.
Interferon gamma promotes long term retention of tissue resident memory T cells in the brain following alphavirus clearance in mice
Authors: Baxter Victoria Kate , Schultz Kimberly L.W. , Griffin Diane E
Published: January 2023

The prototypic alphavirus Sindbis virus (SINV) targets neurons in mice, providing a well characterized model of alphavirus encephalomyelitis. SINV clearance from the brain requires a noncytolytic mechanism to avoid neurological damage and dysfunction and is facilitated by synergistic cooperation between anti-SINV antibody and interferon gamma (IFN-γ). While infectious virus is cleared from the brain by 7 to 10 days post infection (DPI), SINV RNA levels decrease slowly and remain detectable at low levels for at least a year following infection. Because alphaviral RNA is infectious, strict control by the immune system is required to prevent virus reactivation and relapse of disease. To characterize the long-term T cell response following clearance of infectious virus, C57BL/6 mice with intact or impaired IFN-γ signaling were infected with the nonfatal TE strain of SINV, and the frequency and functionality of different T cell populations were evaluated in the brain by flow cytometry. While highly proliferative short-lived T effector cells made up the majority of CD4+ and CD8+ T cells during active infection at 7 DPI, long-lived effector memory T cells became the predominant T cell type by 14 DPI. Following the period of virus clearance, both CD4+ and CD8+ T cell polyfunctionality increased, with more cells expressing multiple effector proteins at higher intensities, and PD-1 and TIM-3 exhaustion marker expression decreased. The percentage of CD8+ T cells characterized as CD103+ tissue resident memory (TRM) cells was decreased in mice with impaired IFN-γ signaling. This indicates IFN-γ signaling promotes the development of TRM cells in the brain during SINV infection and suggests IFN-γ supports long term control of SINV reactivation.
Maternal interferon lambda signaling limits transplacental transmission and mediates fetal pathology during congenital Zika virus infection in mice
Authors: Casazza Rebecca L , Lazear Helen M
Published: January 2023

Zika virus (ZIKV) is among the few pathogens that can surmount the physical and immune barriers of the maternal-fetal interface to cause congenital infections. Immunity at the maternal-fetal interface is carefully balanced to protect the developing fetus from maternal immune rejection as well as maternal pathogens, and includes cytokines secreted by the placenta. Interferon lambda (IFN-λ) is secreted constitutively from human placental trophoblasts, and limits ZIKV transplacental transmission in mouse congenital infection models. To determine if IFN-λ is secreted from mouse placentas, we evaluated IFN-λ activity from placentas of mid-gestation and laboring dams. IFN-λ activity was present in infected and laboring placentas, but not in uninfected mid-gestation placentas, suggesting that IFN-λ is not constitutively secreted from mouse placentas. To define the tissues that respond to IFN-λ at the maternal-fetal interface, we assessed ZIKV transplacental transmission in mouse pregnancies in which either fetal or maternal tissues lacked the IFN-λ receptor (Ifnlr1−/−). Unexpectedly, the antiviral effects of IFN-λ resulted exclusively from signaling in maternal tissues, as fetal viral loads depended only on maternal Ifnlr1 genotype, not the fetal/placental genotype. Using mice also lacking the type I IFN receptor, we studied the effect of IFN-λ signaling in pregnancies with enhanced ZIKV replication and again found an antiviral effect of maternal IFN-λ signaling. Pregnancies with maternal IFN-λ signaling also exhibited enhanced fetal pathology, suggesting that IFN-λ signaling can contribute to fetal pathology in high-viremia models.
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Immunopeptidome of Thymus, Splenic B cells, and Splenic DCs from C57BL/6 mice
Authors: Nanaware Padma Pandurang , Lu Liying , Stern Lawrence J.
Published: January 2023

Antigen presentation in the thymus is central to selection of CD4 and CD8 T cells. Despite postulated roles for differential central and peripheral antigen presentation pathways, comprehensive immunopeptidome analysis of the thymus has not been reported for either MHC I or MHC II. To map the immunopeptidome in the thymus of C57BL/6 mice presented by IAb, we used immunoaffinity coupled to mass spectrometry. We identified >1000 peptides using data dependent acquisition strategy. One challenge in thymic peptidome analysis is the possibility of co-eluting non-specifically bound peptides present as a result of the low abundance of MHC II antigen presenting cells (<10% of total thymic cells). We used several approaches to reduce non-specific background peptides, including analyzing elutions from isotype control antibody conjugated beads, considering only peptides present in nested sets and using predicted binding motif analysis to identify core epitopes. We compared the immunopeptidome of thymus to those of the more easily characterized splenic B cells and splenic DCs. Some of the core epitopes were unique to thymus, splenic B cells or splenic DCs, although most were shared. We observed a broader length distribution of thymic peptidome as compared to splenic B cell and DC peptidomes. To help understand these differences, we selected for detailed analysis 57 peptides representative of thymic derived, splenic B derived or shared peptidomes and studied their binding affinity to IAb. The results suggested that the pool of peptides presented by IAb in thymus has lower MHC affinity than does the pool presented by splenic B cells. Thus our data support the idea of differential antigen processing and presentation in central versus peripheral tissues.
Sirt1 is a requisite for the antigen presentation by B cells
Authors: Park Seok-Rae , Han Younghwan , Shin Jongdae , Kang Jaeku
Published: January 2023

Sirt1 is a NAD+-dependent deacetylase that is well known to control aging and metabolism. Many reports show that Sirt1 regulates innate and adaptive immune responses. Among them, several studies have examined the role of Sirt1 in antigen processing and presentation in dendritic cells. The aim of the present study is to determine the role of Sirt1 in antigen presentation in B cells. First, we examined the expression of major components of the MHCII antigen presentation pathway (Ii, HLA-DM, HLA-DO, and MHCII) and maturation markers (CD40, CD80, and CD86) in Sirt1-deficient B cells using qRT-PCR and flow cytometric analysis. All the components and the markers expressions were lower in Sirt1-deficient B cells than wild-type (WT). To determine whether B cell APC function is dependent on Sirt1, we performed experiments regarding CD4+ OT-II T cell responses to OVA-pulsed Sirt1-deficient B cells or WT B cells in vitro co-culture. CD4+CD69+, CD4+TNF-α+, and CD4+IL-17+ T cells were decreased in Sirt1-deficient B cells compared with that of WT. In addition, T-bet/RORγt mRNA expression and TNF-α/IL-17 secretion were diminished in Sirt1-deficient B cells in the culture. Collectively, these results indicate that Sirt1 in B cells is required for expression of components of MHCII antigen presentation pathway and APC function to activate and functionally differentiate CD4+ T cells.