bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigen-presenting cells lead to severe T cell exhaustion in glioblastoma
Authors: Polania Jessica Waibl , Woroniecka Karolina , Sanchez-Perez Luis , Fecci Peter
Published: January 2023

Glioblastoma (GBM) is the most common primary brain cancer in adults and remains universally lethal. Median survival remains a bleak 15–17 months from time of diagnosis, calling for new therapeutic strategies. Current immunotherapeutic efficacy continues to be hindered by the robust immunosuppression of the GBM tumor microenvironment (TME). T cells, critical for tumor clearance, are particularly affected and many take on an exhausted phenotype within the tumor. We have previously observed significantly worse exhaustion in models of glioma relative to other solid tumor models, suggesting a TME-specific phenomenon. In an orthotopic murine model of GBM, T cell exhaustion was identified using surface expression of inhibitory ligands, PD1, TIM3, and LAG3. Functional status was examined using intracellular flow cytometry for IFNy, TNFa, and IL-2. We consistently observed T cell exhaustion among antigen-experienced tumor-specific CD8+ T cells, suggesting that tumor-imposed exhaustion is dependent upon T cells encountering their antigen. It remains undetermined, however, which cell within the GBM microenvironment provokes a uniquely severe T cell exhaustion phenotype. Using CRISPR-Cas9 gene editing, we modified tumor cells to lack antigen-presentation capacities and revealed persistence of T cell exhaustion. In contrast, when the same capacity was removed from infiltrating, hematopoietic cells, T cells in the GBM microenvironment were less exhausted and retained their function. This suggests antigen-presenting cells (APC) are critical for T cell exhaustion within GBM. Our findings uncover an important role for APCs in eliciting T cell exhaustion, which will provide novel interactions for targeted immunotherapies.
Antigen-cross presentation promotes development of terminally differentiated CD8 T cells in young individuals
Authors: Moustaki Ardiana , Crawford Jeremy Chase , Alli Shanta , Zamora Anthony E , Fan Yiping , Boi Shannon , McDonald Natalie MN , Thomas Paul Glyndwr , Pappo Alberto , Dyer Michael , Stewart Elizabeth , Federico Sara , Youngblood Benjamin A
Published: January 2023

Cancer immunotherapeutic approaches that rely on endogenous T cell responses have shown limited success in children, a pattern attributed to the low mutational burden of pediatric tumors. Here, we report that CD8 T cells isolated from a diverse set of pediatric solid tumors are enriched for an antigen-experienced phenotype. The limited ability of immune checkpoint blockade therapy (ICBT) to trigger anti-tumor responses in children, despite the presence of activated CD8 T cells, prompted us to explore alternative underlying mechanisms restricting anti-tumor responses. Using a novel mouse tumor model that expresses a well-characterized epitope coupled to an mCherry marker, we identified antigen cross-presentation by tumor infiltrating myeloid cells as a key regulator of CD8 T cell effector function in tumors. Strikingly, age-related changes in the TME resulted in a skewing of the CD8 T cell effector fate toward a terminally differentiated state in young tumor-bearing mice. Profiling of tumor infiltrating antigen presenting cells by scRNAseq revealed a proinflammatory M1 macrophage polarization in young tumors but a predominant M2 “wound healing” response in adult tumors. Consistent with our mouse findings, analysis of immune infiltrates from human pediatric solid tumors revealed a strong correlation between the expression of PDL1 on myeloid cells and enrichment of tumor-associated CD8 T cells with an exhaustion phenotype. Collectively, these data indicate that the “young” microenvironment of an actively developing tissue/individual contributes to the generation of an immune response skewed towards a terminally differentiated state with limited plasticity, thus narrowing the window for immunotherapeutic interventions.
Elucidating the Role of HDACs in T Cell Biology and Comparing Distinct HDAC Inhibitors in Augmenting Responses to Cancer Immunotherapy
Authors: Triplett Todd A , Holay Nisha , Kottapalli Srividya , VanDenBerg Carla , Capasso Anna
Published: January 2023

Histone deacetylases have canonically been known to suppress gene expression via epigenetic mechanism involving modulating histone acetylation status. While HDAC inhibitors are currently used in the clinic to treat cancer, their mechanism of action is not clear as acetylation is now known to control myriad cellular processes, such as protein stability of signaling. Importantly, it is now recognized that tumor regression during HDACi monotherapy relies on CD8+ T cells and is synergistic with immunotherapy (IMT). However, most previous studies have found HDACi to be detrimental to T cells. Thus, it is still unknown if the increase in anti-tumor immune responses is mediated through directly affecting tumor immunogenicity, T cell effector function, or both. Given the role of epigenetics in T cell differentiation, we first determined their effect on distinct populations. Rather than an expected global increase, all cytokines evaluated (IL-2, IFNy, IL-5, IL-17A) were downregulated during HDACi treatment except for TNFa, which drastically increased. Examination of the distinct subsets revealed that this effect was mostly confined to naïve T cells compared to memory. This suggests acetylation differentially affects cytokine production by distinct T cell subsets. As multiple inhibitors with varying specificities are used in the clinic, understanding their impact on T cell function could identify rational combinations. In support of this, our results demonstrate drastic differences between inhibitors on their effects on T cell cytokine production and survival. Future studies will determine the role of specific HDACs in these functions and compare inhibitors on immune infiltrates in the tumor and ability to augment immunotherapies.
CD28 and glycogen synthase kinase-3 (GSK-3) inhibitor combination therapy generates tumor infiltrating T-cells (TILs) with a reversal of markers for T-cell exhaustion
Authors: Kazanova Alexandra , Krueger Janna , Rudd Christopher E.
Published: January 2023

Immune checkpoint blockade (ICB) of inhibitory receptors such as PD-1 has revolutionized the treatment of cancer. ICB is dependent on CD28 co-receptor expression introducing the question of whether the positive co-receptor could be targeted for the reversal of T-cell exhaustion in immunotherapy. We previously showed that the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) is a central regulator of programmed cell death-1 (PD-1) transcription (Taylor et al., 2016, Immunity) and that small molecule inhibitors of GSK-3 (GSK-3 SMI) are as effective as anti-PD-1 blocking antibody in the control of tumor growth (Taylor et al., 2018, Cancer Research). In this study, we now show that anti-CD28 can synergise with GSK-3α/β inhibitors to regress tumors that are resistant to anti-PD-1 immunotherapy. Combination therapy showed responses in >55% mice with an overall reduction in tumor volume of 70% in responders compared to control mice (182 mm3 vs. 611 mm3). Flow cytometry analysis of tumor infiltrating lymphocytes (TILs) shows that, while non-treated mice were enriched with TOX+TIM3+PD1+CD8+, combination therapy increased tumor infiltration of TOX-negative CD8 T cells with TIM3-PD1int CD8 phenotype. Remarkably, this reversal involved only a single injection of anti-CD28 (PV-1) and GSK-3 SMI (SB415286). These results showed that CD28/GSK-3 combination therapy greatly reduced TOX, TIM-3 and PD1markers indicative of exhaustion. Our findings offer a small molecule approach for synergy with anti-CD28 that leads to an increase tumor infiltration with TOX-negative CD8 T cells that have decrease expression of inhibitory receptors.
Effects of aging on immunosenescence and checkpoint inhibitor efficacy in a C57BL/6J syngeneic model
Authors: Yang Jiwon , Schile Andrew , Cheng Mingshan , Keck James G
Published: January 2023

Aging is a complex dynamic process that leads to increased susceptibility to various diseases including cancer. Immunosenescence is a state of aging-related gradual deterioration of the immune system in the elderly. T cells are a major part of an adaptive immune system and orchestrate the cellular immune response. However, only limited studies were conducted on how aging, immunosenescence, and cancer therapy affect each other. Our goals in this study were to establish a mouse model that shows immunosenescence and to study how the immunosenescence affects checkpoint inhibitor efficacy in a C57BL/6J syngeneic model. Young (7 weeks of age), mid-aged (54 weeks) and aged (76–78 weeks) female mice were used. The spleen and blood were collected from the naïve mice and analyzed using a flow cytometer. In both samples, we found a decrease in naïve CD4+ and CD8+ T cells in mid-aged and aged mice compared to the young counterparts, which is a sign of immunosenescence. Compared to young mice naïve aged mice showed increased regulatory T cells (Tregs) in the spleen without a significant difference in the blood. When the mice were inoculated with MC38 tumors and treated with isotype control or anti-PD1 antibody for 3 weeks, anti-PD1 treatment was found to significantly slow down tumor growth in all age groups but was the most efficacious in aged mice. Anti-PD1 treatment increased T cell infiltration into the tumor with the greatest effect observed in the mid-aged group. In conclusion, we have established a preclinical mouse model that shows immunosenescence, and immunosenescence is a possible cause of the difference in response to the checkpoint inhibitor in the elderly.
ANT2 (SLC25A5) balances the pro-apoptotic vs. effector signals of Bim in CD8 T cells
Authors: Gicobi Joanina K , Yu Xiatong , Dronca Roxana S. , Liu Xin , Barham Whitney , Wen Ti , Zhang Henan , Zhu Mojun , Harrington Susan M. , Vianzon Vianca , Hirdler Jacob , Dong Haidong
Published: January 2023

The immune response to anti-PD-1 therapy in cancer patients is not completely defined due to lack of reliable biomarkers that can determine T cell function. Bim, a member of the Bcl-2 family, was recently identified as a downstream signaling molecule in the PD-1 pathway. Within CD8 T cells, Bim appears to have a dual function, and can be pro-apoptotic or promote effector activity. Our clinical data has shown that high levels of Bim in CD8 T cells are predictive of response to anti-PD-1 therapy in metastatic melanoma whereas in colorectal cancer, a low level of Bim is associated with treatment response. Thus, we sought to determine what molecular partners might influence the role of Bim. Using co-immunoprecipitation, high resolution microscopy and FRET we identified ANT2 (Adenine Nucleotide Translocase 2), an inner membrane protein of mitochondria, as a protein that interacts with Bim. Accordingly, we found CD8 T cells with low and high mitochondrial potential demonstrated different ANT2/Bim ratios, and a low ANT2/Bim ratio was present in effector T cells with low mitochondrial potential but high cytotoxic activity. Given the pro-survival function of ANT2, our results indicate that ANT2 may balance the pro-apoptotic and effector roles of Bim in mitochondria in order to preserve effector T cells before contraction. Understanding the molecular mechanism of Bim and ANT2 interactions in CD8 T cells may provide a novel marker to predict clinical response to anti-PD-1 therapy.
Oral administration of a self-assembling nanofiber-based vaccine generates neutralizing antibodies against HIV and expands CD8+ memory T cell populations
Authors: Files Megan A , Rudra Jai S , Endsley Mark
Published: January 2023

Human immunodeficiency virus (HIV) is the world’s sixth leading cause of death. Antiretroviral therapy (ART) is the current standard of treatment, but issues of accessibility and compliance pose additional hurdles. In 2018, there were 37.9 million people living with HIV, but only 62% were on ART. The development of a successful vaccine is imperative to combat the transmission of HIV, of which one of the major goals is to generate broadly neutralizing antibodies (bnAb). The membrane-proximal external region (MPER) of gp41 in the envelope glycoprotein (Env) is a largely conserved motif, a key player in fusion, and the target of bnAb in a subset of patients with long-term HIV infections. We previously demonstrated the efficacy of a nanofiber vaccine platform as evidenced by the generation of both strong humoral and cell-mediated responses against viral targets, and enhanced protection against infection. In recent studies, we observed that serial oral dosing of nanofibers (NF) bearing the MPER epitope (NF-MPER) in mice correlated with a significant increase in serum and mucosal neutralizing antibodies against HIV. The NF-MPER vaccine also augmented CD8+ memory T cell populations in the colon, which were further characterized as effector memory T cells (TEM), using surface marker phenotypes (CD69+/− CD103+ CD44+ CD62Llo). Additionally, we observed significant expansion of CD8+ TEM pools in the spleen, a reservoir for HIV, a subset of which were similar to tissue resident memory T cells (TRM) seen in human elite controllers. These results suggest that oral immunization with a NF-based vaccine elicits the production of bnAb and enhanced CD8+ TEM populations, in non-lymphoid and lymphoid tissues, that are necessary for a successful HIV vaccine.
Bacteria-like particle bead complexes as an immunostimulatory platform for high-throughput vaccine antigen capture and delivery
Authors: Jan Sharon , Felgner Jiin , Assis Rafael , Davies Jenny , Albin Tyler , Nakajima Rie , Jasinskas Algis , Davies David Huw , Felgner Philip
Published: January 2023

Vaccines are cited as the most cost-effective public health measure in controlling infectious diseases. Scalable and high-throughput platforms can accelerate vaccine discovery, which reduces the burden on human life, morbidity and health system resources. To expedite the antigen identification and production process during the first steps of vaccine development, we have developed a highly efficient bead capture system that allows for rapid antigen capture and delivery. This technology can be used in conjunction with protein microarrays to swiftly screen and identify antigens as potential vaccine candidates.

Model antigens from Coxiella burnetii, a known biothreat and the etiological agent of Q fever, were chosen based on previous protein microarray data and expressed from plasmids containing a His-tag in a cell-free expression system. To demonstrate proof-of-concept, we used polystyrene beads with tris-NTA linkers to capture antigens from the mixture. The resulting antigen-conjugated beads were administered directly to C57BL/6 mice individually, multiplexed, and/or with an adjuvant cocktail of MPLA/CpG/squalene-in-water emulsion. Sera collected at regular intervals were screened against protein microarrays to monitor the dynamics and isotypes of the antibody response. Cytokine levels were assessed in antigen recall assays on splenocyte cell cultures through ELISPOT or by bead-based cytometric assay. Here we observed robust, durable and antigen-specific T cell and antibody responses. IgG isotypes and cytokine levels revealed that including adjuvants tuned the Th1/Th2 responses to a balanced profile. In vitro studies are currently underway to determine antibody functionality and neutralization potential.
Endogenous dendritic cell subset cDC1 mediates DC vaccine immunity against gamma herpesvirus in syngeneic BMT recipients
Authors: Zhou Xiaofeng , Chadwick Matthew M. , Chan Paul R. , Buonavolonta James J. , Wilke Carol A. , Moore Bethany B.
Published: January 2023

Dendritic cell (DC) vaccines are a promising immunotherapy for viral infections and cancers, however the mechanisms underlying their action are still not clear. The objective of this study is to understand how DC vaccines stimulate the expansion of cytotoxic T lymphocytes (CTLs) against gamma herpesvirus in bone marrow transplant (BMT) recipients. Herpesviruses commonly reactivate after transplantation of hematopoietic cells in patients and cause high mortality and morbidity. Similarly, infections with murine gamma herpesvirus 68 (MHV-68), a mouse homolog of Epstein-Barr virus, after syngeneic BMT in mice cause severe pulmonary fibrosis and pneumonitis.

We prepared DC vaccines by sorting CD11c+ cells from the lungs at 3 days post infection (dpi) with MHV-68. We administered DC vaccines i.v. to BMT mice one day before infection with MHV-68. CTLs of the lung, spleen and mediastinal draining lymph nodes were analyzed by flow cytometry at 7 dpi. Lung sections were prepared at 21 dpi for histology analysis.

Adoptive transfer of the DC vaccine stimulates the expansion of viral specific CD8 T cells in MHV-68 infected syn-BMT mice at 7 dpi, and protects them from pneumonitis and fibrosis at 21 dpi. Surprisingly, none of the transferred conventional DC (cDC) subsets, nor migration into draining lymph nodes, nor MHC class I or class II molecule expression are essential for the DC vaccine to activate CD8 T cells in BMT mice. Instead, DC vaccination of BMT mice lacking endogenous cDC1s resulted in reduced CD8 T cells and failed to suppress lytic viral infection.

Our results suggest that endogenous cDC1s in BMT mice mediate DC vaccine immunity against gamma herpesvirus. We have thus revealed a previously unknown pathway of DC vaccination.
Polyanhydride-based RSV prefusion F nanoparticle vaccine establishes lasting protection mediated by humoral and cellular immune responses
Authors: Stephens Laura , Ross Kathleen A. , Hartwig Stacey M. , McLellan Jason S. , Narasimhan Balaji , Legge Kevin L. , Varga Steven M.
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children, accounting for nearly 7% of deaths in those less than one year of age. Despite the morbidity, mortality, and high economic burden caused by RSV worldwide, no licensed vaccine is available. Thus, there is a critical need for a safe, and highly efficacious vaccine to prevent RSV-induced disease. We have developed a novel vaccine utilizing a prefusion-stabilized variant of the RSV fusion (F) protein (DS-Cav1) encapsulated within polymer nanoparticles with a CpG1668 oligodeoxynucleotide adjuvant. The prefusion conformation of RSV F allows for better exposure of the major antigenic site, termed site Ø, a primary target of neutralizing antibody responses in human serum. To evaluate this vaccine candidate, BALB/c mice were vaccinated intranasally and then boosted 4 weeks later. Vaccination with the RSV nanovaccine significantly ameliorated weight loss and pulmonary dysfunction compared to unvaccinated mice in response to an RSV challenge. Additionally, vaccinated mice significantly reduced virus titers in the lungs as early as day 2 post-infection. Protection from disease and enhanced viral clearance remained until 100 days post prime vaccination. Vaccination also established an elevated number of tissue-resident memory CD4 and CD8 T cells in the lungs. The vaccine-induced protection was mediated by both humoral and cellular immune responses, as serum transfers from vaccinated mice provided only partial protection from weight loss and pulmonary dysfunction. Based on the robust immune response elicited and the high level of protection observed, our prefusion RSV F nanoparticle formulation may be a promising RSV vaccine candidate.
Vaccine-induced protection against intrarectal AIDS virus transmission in the absence of anti-Env antibodies mediated by trained innate immunity
Authors: Berzofsky Jay A , Keele Brandon F , Miller Christopher J , Sui Yongjun
Published: January 2023

Although anti-Envelope antibodies have been viewed as the primary means of protection against SIV or SHIV acquisition, we have found in two distinct vaccine studies that protection against intrarectal SHIV or SIV acquisition in repeated low-dose challenge can be achieved in the absence of anti-Env antibodies (Vaccine efficacy = 44% in one study and 81% in the other). Specific CD8+ T cells were induced but did not correlate with protection, and in one of the studies, CD8+ depletion experiments excluded any CD8-dependence of the protection. Rather, protection correlated with protective myeloid cells and inversely with suppressive myeloid-derived suppressor cells. Trained innate immunity of monocytes lasting at least 7 weeks after vaccination was confirmed by demonstrating the ability of monocytes from vaccinated animals to produce more IL-6, TNFa and/or Mip1a when re-exposed to virus than those from control animals. This trained innate response correlated with the number of exposures required to achieve infection. Trained innate immunity has been shown to be mediated by epigenetic changes in monocytes, and indeed, we have been able to detect such epigenetic changes. Thus, vaccine protection against rectal acquisition of SHIV or SIV can be achieved in the absence of anti-Env antibodies and even of CD8+ T cells, and can be mediated by trained innate monocyte immunity. These findings suggest a new mode of vaccine protection that needs to be investigated in future AIDS vaccine clinical trials.
Utilizing defined structural subunits to elucidate antibody populations elicited by a recombinant subunit Ebola virus vaccine
Authors: Ball Aquena H , Lehrer Axel T
Published: January 2023

Ebola virus (EBOV) causes lethal hemorrhagic fevers with case fatality rates of up to 90%. The recent large outbreak in West Africa and current outbreak in the DRC demonstrate an obvious need for vaccines and therapeutics. All vaccine candidates use the surface glycoprotein as the main antigen. However, the method of protection induced by the EBOV glycoprotein remains unclear. The EBOV glycoprotein is composed of GP1 and GP2 linked by a disulfide bond. GP1 contains the receptor binding domain, glycan cap, and the heavily glycosylated mucin-like domain while GP2 contains the fusion loop and transmembrane anchor. We have developed a subunit vaccine that allows for an increased safety profile as well as shows increased thermostability, which allows easier deployment in central Africa. With our studies we expect to reveal antibody patterns differentiating survivors and non-survivors from non-human primate studies as well as reveal subdomains that are more protective and/or immunodominant to guide future vaccine development as well as provide insights into correlates of protection.

Subunits of the EBOV glycoprotein have been produced in Drosophila S2 cells including full length GP, GPΔmucin-like domain, GP1, GP2, and sGP. These subdomains will be used to explore the binding patterns of antibodies elicited by a highly purified GP subunit vaccine. Ongoing analysis focuses on samples from non-human primates immunized with 3 doses of EBOV GP + adjuvant that will be analyzed for antigen binding IgG to differentiate the pre-challenge antibody binding patterns to the different EBOV subdomains.
Comparison of antibody responses in mice generated by different multivalent Filovirus vaccines based on recombinant glycoprotein subunits
Authors: Tashiro Taylor E , Wong Teri Ann , Lehrer Axel T
Published: January 2023

Filoviruses cause fulminant hemorrhagic fevers with case-fatality rates up to 90%. Recombinant glycoprotein (GP) derived from Ebola virus (EBOV), Sudan virus (SUDV) and Marburg virus (MARV) have been expressed from stably transformed Drosophila S2 cells and used to formulate recombinant subunit vaccine candidates showing efficacy in rodents and non-human primates. Immunogenicity of 11 different formulations of our trivalent subunit vaccine with adjuvant was tested.

Each group of Swiss Webster mice was administered a formulation with or without adjuvant. Mouse sera were collected two weeks after 2nd and 3rd vaccine doses and diluted 1:8,000 and 1:40,000 to determine titers of IgG binding to EBOV, SUDV and MARV GP by a microsphere immunoassay (MIA). MFI (median fluorescence intensity) was collected by Luminex xPonent software then graphed and statistically analyzed using GraphPad Prism.

As expected, groups administered equal amounts of EBOV, SUDV, and MARV GP had the most balanced antibody response as compared to groups that did not receive equal amounts of GP. Formulations with optimal antigen doses generate balanced antigen-binding antibody responses. Additional experiments are ongoing including the characterization of virus neutralization activity of the different formulations.
Characterization of immune response in mice vaccinated with highly efficacious vaccine 0ΔNLS against HSV-1
Authors: Gmyrek Grzegorz Bogdan , Carr Daniel J.J.
Published: January 2023

Using an experimental mouse model we compared the host immune response of a mutant HSV-1, termed 0ΔNLS (deficient in the nuclear location signal of ICP0), a highly efficacious prophylactic vaccine against ocular HSV-1 infection, to that of the parental HSV-1 virus, termed GFP105 (KOS background). C57BL/6 mice were infected in the footpad (FP) with 1×105 plaque forming units of virus and at times post infection (PI), organized lymphoid tissues were analyzed for changes in the immune profile. 0ΔNLS infected animals were more efficient in clearance of virus from the FP and generated a more robust chemokine responses and myeloid cell infiltration in FP at early time points (day 1–3 PI) compared to GFP105 infected mice. By day 7 PI, there were significantly more GC B cells and isotype switched B cells in the PLN of mice infected with 0ΔNLS in comparison to GFP105 infected animals. In addition, an increased number of follicular helper CD4 T cells along with enhanced IL-21 cytokine production and an increased number of antigen-specific CD8 T cells against major and sub-major HSV-1 epitopes was noted. In contrast, by day 21 PI, no difference in T cell and B cell subset was found in the PLN comparing the two groups of infected mice. Furthermore, mice which were re-exposed to 0ΔNLS 21 days PI showed an increased number of metabolically active plasma cells from the bone marrow and secretion of IgG by memory B cells after pre-activation compared to those re-infected with GFP105. In addition, an increased number of IFN-ɤ secreting, HSV-1-specific CD8 T cells from the re-exposed 0ΔNLS mice was observed as well. Collectively, these results warrant further studies to fully understand the efficacy of the 0ΔNLS virus as an effective vaccine against ocular HSV-1 infection.
Suppression of HBV replication in mice using stem cell-derived viral antigen-specific T lymphocytes
Authors: Song Jim Jianxun , Xiong Xiaofang
Published: January 2023

The viral antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) derived from pluripotent stem cells (PSCs), i.e., PSC-CTLs, have the ability to suppress the hepatitis B virus (HBV) infection. After adoptive transfer, PSC-CTLs can infiltrate into the liver to suppress HBV replication. Nevertheless, the mechanisms by which the viral Ag-specific PSC-CTLs provoke the antiviral response remain to be fully elucidated. In this study, we generated the functional HBV surface Ag-specific CTLs from the induced PSC (iPSCs), i.e., iPSC-CTLs, and investigated the underlying mechanisms of the CTL-mediated antiviral replication in a murine model. A hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver induced HBV replication in mice. We show that adoptive transfer of HBV surface Ag-specific iPSC-CTLs greatly suppressed the HBV replication in the liver and blood in the animal model and prevented the HBV surface Ag expression on hepatocytes. Furthermore, we demonstrate that the adoptive transfer significantly increased the migration of CD8+ T cells and the production of the antiviral cytokines (IFN-g, TNF-a) in the liver. These results indicate that the stem cell-derived tissue-associated CTLs can robustly accumulate in the liver and suppress the HBV replication through producing antiviral cytokine.
Lymph node and circulating TFH cells support SIV-specific antibody responses in peripheral blood and genital tract of vaccinated female rhesus macaques
Authors: Hait Sabrina Helmold , Hogge Christopher , Mushtaq Zuena , Hoang Tanya , Robert-Guroff Marjorie
Published: January 2023

TFH cells are critical for Ag-specific B cell selection in germinal centers (GCs). Circulating blood CXCR5+ CD4+ T cells (cTFH) were implicated in development of broadly neutralizing antibodies in HIV+ patients. We asked if an SIV vaccine regimen could induce GC TFH and cTFH cells that supported anti-SIV humoral immunity. Female rhesus macaques (RM) were primed twice mucosally with replicating Ad5hr-SIV recombinants (wks0&13) and boosted twice intramuscularly with SIVgp120 proteins (wks26&38). Blood, lymph nodes (LN), bone marrow (BM), vaginal and rectal swabs were collected pre-vaccination, post-2nd prime and post-2nd boost for TFH and B cell assessment. Total TFH, and Env-specific GC TFH cells and Env-specific memory (ESM) B cells were seen at wk38+3d, suggesting vaccine-mediated GC maturation. Env-specific LN TFH cells and GC B cells at wk13+3d correlated respectively with Env-specific rectal IgA and vaginal IgG titers at wk16. GC B cells and ESM B cells at wk38+3d correlated with Env-specific vaginal IgG and rectal IgA titers at wk41 respectively, indicating early GC formation contributed to SIV-specific immunity in the female genital tract (FGT). Env-specific cTFH cells peaked post-boosting and positively correlated with Env-specific IgA and IgG secreting plasmablasts and plasma cells in BM and rectal IgA titers. cTFH cells induced CD38 and CD138 upregulation in autologous B cells and supported IgA secretion in cocultures compared to B cells cultured alone or with non-cTFH cells. Our data indicate the prime/boost regimen induced GC TFH and cTFH cells, enabling robust anti-SIV T-cell-dependent mucosal and systemic B cell responses. Both LN and circulating TFH cells may play pivotal roles in FGT protective humoral immunity.
gdT cells stimulated by a novel vaccine confer protection to the naïve mice against S. aureus challenge
Authors: HUANG JIANDONG , Wang Xiaolei , Zhang Baozhong , Deng Jian , Yuen Kwok-yung
Published: January 2023

Staphylococcus aureus (S. aureus) is a common pathogen found in the community and in hospitals. Most notably, Methicillin-resistant S. aureus is resistant to many antibiotics, which is a growing public health concern. The emergence of drug-resistant strains has prompted the search for alternative treatments such as immunotherapeutic approaches. Prophylactic vaccination is the best approach to combat against MRSA since it can provide protection without any concerns regarding antibiotic resistance. To date, most clinical trials of vaccines or passive immunization against S. aureus have ended in failure. In this study, we investigated multiple proteins as possible targets for a vaccine. Mice vaccinated with these purified proteins elicited high titers of specific antibodies as well as Th1- and Th17-biased immune responses in mice. Animal test indicated a protection rate over 90% in several animal models against multiple strains of S. aureus. Interestingly, gdT cells transferred from the vaccinated mice to naïve mice can confer protection to the naïve mice against S. aureus challenge in skin infection models. These findings raise the hope that the candidate antigens could be developed into multivalent and serotype-independent vaccines against S. aureus infection.
Application of a Campylobacter jejuni mouse infection model to test efficacy of a C. jejuni capsule conjugate vaccine delivered with a potent liposome adjuvant containing monophosphoryl lipid A and QS-21
Authors: Laird Renee M , Gariepy Christina L , Eggleston Heather , Shoemaker Nina M , Monteiro Mario A , Guerry Patricia , Beck Zoltan , Matyas Gary R , Poly Frédéric
Published: January 2023

Campylobacter jejuni is a major cause of infectious diarrhea worldwide. Increasing incidence of C. jejuni is attributed to new non-culture based detection methods and antibiotic resistance is unfortunately on the rise, necessitating development of interventions. Therapeutics development like vaccines have been hampered by lack of a small animal model that recapitulates campylobacteriosis symptoms. To better facilitate vaccine efficacy testing, we adapted a recently-published mouse C. jejuni infection model to adult mice fed a zinc-deficient diet and pre-treated with antibiotics prior to oral infection with C. jejuni strain 81–176. Non-vaccinated infected mice develop diarrhea, lose weight and show increased expression of fecal inflammatory markers indicating development of campylobacteriosis. We tested whether an 81–176 C. jejuni capsule conjugate vaccine delivered with a potent liposome adjuvant containing monophosphoryl lipid A and QS-21 known as ALFQ could protect mice against 81–176. Vaccinated mice developed high levels of anti-CPS IgG1 and IgG2b titers and serum bactericidal responses against 81–176. Vaccinated infected mice were protected against development of diarrhea, did not lose weight, and had significantly lower levels of fecal inflammatory marker expression. Importantly, vaccinated infected animals were protected against C. jejuni colonization indicating that parenteral vaccination with a conjugate vaccine plus the ALFQ adjuvant may provide protection against both C. jejuni disease and colonization. These promising results support further development of a multivalent C. jejuni conjugate vaccine platform delivered with potent adjuvant systems for use in human clinical studies.
Immunization with a subunit of housekeeping proteins provide partial protection against experimental necrotic enteritis in broiler chickens
Authors: Li Charles , Lillehoj Hyun , Yan Xianghe , Sun Zhifeng , Lu Mingmin , Yuan Baohong , Liu Liheng
Published: January 2023

Necrotic enteritis (NE) due to Clostridium perfringens (CP) infections is a prevalent enteric infectious disease accounting for $6 billion economical loss in global poultry industry. The increasing incidence of NE has been associated with the reduction and withdrawal of antibiotic growth promoters from animal feed during recent years. Therefore, the development of effective vaccines specific for NE assumes a priority for poultry industry. By far, no any effective vaccines are commercially available in broiler chickens. Our aim was to identify the potential CP proteins as vaccine targets for NE. We expressed 4 different recombinant CP proteins targeting 6 antigens: 3 major virulence-related toxins (alpha-toxin, NetB and TpeL), and 3 pilus subunit or enzymes (Collagen adhesion protein (Cna), Fructose-1,6-bisphosphate aldolase (FBA) and a Hypothetic protein (HP)), and their protection efficacies were evaluated with a severe Coccidiosis/NE challenge model using netB+tpeL+ CP strain. Young chicks were immunized twice subcutaneously with adjuvanted CP proteins on days 4, and 15. Prior to challenge, the chickens immunized with related antigens had much higher serum antibody titers for Cna and FBA/HP antigens than those with any of 3 toxin antigen-immunized groups. Following the challenge, the Cna-immunized group showed the least mortality and lowest lesion score among the individual antigen-vaccinated groups, while the pooled antigens-immunized group demonstrated no mortality against virulent challenges. The results indicate that the immunization with CP housekeeping protein, Cna, conferred better protection than other conventional virulence-related toxin proteins. Mucosal delivery system is being investigated next.
Intranasal vaccination induces complete protection against multidrug resistant A. baumannii pneumonia and sepsis
Authors: Shi Yun , Zeng Xi , Wang Ning , Guo Gang , Zou Quanming
Published: January 2023

In a WHO report released in Feb 2017, Acinetobacter baumannii has been list as top one of critical antibiotic-resistant “priority pathogens” which is in an urgent need for the new treatment. A. baumannii infections have become increasingly difficult to treat because most isolates are highly resistant to a wide range of antibiotics. With the absent of effective antibiotics, an effective vaccination regimen is urgently needed. We have established a pneumonia mouse model by non-invasive intratracheal instillation with A. baumannii and sepsis model by intraperitoneal injection of bacteria. Intranasal or intramascullar Vaccination both showed 100% protection against A. baumannii infection in pneumonia and sepsis model supported by reduced bacteria burderns, pathology, and reduced proinflammatory cytokine. Intranasal immunization also showed cross protection against different type clinical A. baumannii strains. A successful vaccine against A. baumannii will help us control its infection and overcome the development of multidrug resistance.
Systemic BCG Vaccination in “Dirty Mice” induces Protective Trained Immunity against TB
Authors: Kaufmann Eva , Khan Nargis , Robbins Clinton S , Barreiro Luis B , Divangahi Maziar
Published: January 2023

After a century of discovery of Bacille Calmette-Guerin (BCG), our understanding of its protective mechanism(s) against TB or other pathogens is very limited. We have recently demonstrated that systemic BCG vaccination (intravenously, iv) in specific pathogen-free (SPF) mice imprints hematopoietic stem cells (HSCs) to generate macrophages with a unique protective program against pulmonary M. tuberculosis (Mtb) infection. While this proof of concept study was an important step to determine how we can harness the power of innate (trained) immunity in vaccination against TB, the translation of this novel approach to humans is still unknown.

As SPF lab mice incompletely recapitulate the human immune system, additional pre-clinical models are necessary to evaluate the translational potential of systemic BCG vaccination. Thus, in the current study, we hypothesize that systemic BCG vaccination will generate protective HSC-mediated trained immunity against TB in microbe-exposed “dirty” mice whose immune landscape more closely replicates adult human traits.

To test this hypothesis, we have established a bedding transfer model from pet shop mice to SPF C57BL/6 mice. Age- and sex-matched dirty or SPF mice were then iv or subcutaneously vaccinated with BCG (1×106 CFU) or PBS (control). At 4 weeks post vaccination, we generated bone marrow derived macrophages and infected them with virulent Mtb (H37Rv; MOI 1). Interestingly, the protective signature of trained immunity was completely intact in BCG-iv vaccinated dirty mice.

Collectively, our results indicate that the protective imprinting of systemic BCG vaccination for generation of trained immunity is robust and independent of previous exposure of hosts to other microbes or pathogens.
Identifying mechanistic correlates of protection against Pseudomonas aeruginosa in an acute lung pneumonia model
Authors: Kilic Emel Sen , Blackwood Catherine B , Horspool Alexander M , Huckaby Annalisa B , Weaver Kelly , Malkowski Aaron , Witt William T , Bevere Justin R , Winters Michael T , Damron F. Heath , Barbier Mariette
Published: January 2023

Pseudomonas aeruginosa (Pa) is an opportunistic Gram-negative pathogen that can lead to life-threatening pulmonary infections. Several promising vaccine candidates against Pa evaluated in clinical trials. However, there is still no commercial vaccine available against this pathogen, in part due to a lack of clarity of protective correlates of vaccine–induced immunity. To elucidate correlates of protection against Pa, we used an intranasal whole–cell Pa vaccine adjuvanted with curdlan WCV in a murine vaccination and challenge model. Immunization with WCV significantly decreased the bacterial burden and lung edema after Pa challenge. We found that WCV immunization leads to a significant increase in Th17 cellular immune response and Pa–specific serum IgG and IgA titers compared to naive animals. To identify mechanistic correlates of protection, we performed anti–CD4, – CD8, and –CD20 antibody mediated depletions prior to vaccination and challenge. Depletion of these populations did not affect the response to challenge in naive animals. Interestingly, we observed that depletion of B cells but not CD8+ or CD4+ T cells during WCV priming resulted in loss of protection against Pa. Furthermore, passive immunization of WCV sera was sufficient to protect naive mice against Pa. Taken together, vaccine-mediated humoral immune response was the critical component of immunity against Pa in acute lung pneumonia model. This study suggests that adjuvants stimulating humoral immune response may be used to enhance the efficacy of vaccine candidates against Pa.
An AAV6-based multi-antigen vaccine for treatment of murine melanoma
Authors: Krotova Karina , Aslanidi George
Published: January 2023

We recently showed that a vaccine with optimized AAV6 vector generates a superior immune response against strong antigens (Ag) such as Ovalbumin. However, tumor-associated antigens (TAA) are typically non-mutated self-proteins with low immunogenicity. To increase vaccine efficacy and to reduce tumor evasion of immune attack, we developed a multivalent AAV vaccine that targets four previously characterized melanoma TAA: tyrosinase, premelanosome protein gp100, tyrosinase-related protein 1, and dopachrome tautomerase. The vaccine represents a combination of individual AAV-based vaccines specific to unique TAA. The strength of cytotoxic T cell response against each TAA was shown via IFNγ ELISPOT analysis of re-stimulated splenocytes from immunized mice. Additionally, we showed that vaccine-induced Ag-specific antibodies directed B16F10 melanoma cells to complement-dependent lysis. The vaccine’s therapeutic potential was analyzed in prevention and treatment of B16F10 mouse melanoma. The preventative vaccination significantly reduced the number of metastatic tumor nodules in lungs. Since B16F10 melanomas are immunologically resistant tumors, we combined vaccination with aPD-1 and aPD-L1 checkpoint inhibitors for treatment of established solid tumor. The combination of both vaccine and checkpoint inhibitors significantly delayed tumor growth and extended animal survival. In summary, our preliminary data suggest that a multivalent vaccine based on modified AAV6 vectors generates strong immune responses against endogenous self-proteins, and vaccination may have a therapeutic benefit against both metastatic and solid tumor development.
The efficacy and mechanism of CD020 on the induction of specific immune responses against HPV-related cancer
Authors: Song Ying-Chyi , Yen Hung-Rong
Published: January 2023

Virus infections may account for the development of several cancers, such as HPV16/18 are known to cause around 70% of cervical cancer cases. Until now, several immunotherapeutic approaches for virus-induced cancer are under development. Peptide-based vaccines have several advantages over conventional whole-protein vaccines in terms of purity, lot-to-lot consistency, production costs, and high antigenic specificity. However, the use of peptide antigens in vaccine development has been hampered by problems, such as weak immunogenicity coupled with a paucity of potent adjuvants. Specific T cell immune response is crucial for anti-tumor immunity. CD020 is a compound derived from the Chinese herbal medicine (CHM) of Hedyotis diffusa Willd. However, the efficacy and mechanism of CD020 on the induction of specific immune responses against cancer are still unclear. Therefore, we aimed to investigate whether CD020 can modulate specific T cell responses to apply for the development of a cancer vaccine. We have identified that CD020 could increase expression of maturation cytokines and activation markers of BM-DCs and induce specific T cell responses. Furthermore, in a murine TC-1 tumor-bearing model, we found that the CD020 could act as an adjuvant to induce cellular immune responses and anti-tumor effect in the peptide vaccine strategy. We suggested that immune-stimulator CD020 combined with cancer vaccine endows them with increased immunologic activity, which may be used to bypass the requirement for the conditional adjuvant. Further delineation of the mechanism may provide new clues for vaccination strategy.
Network-guided bioengineering of oncolytic immunovirotherapies for glioblastoma
Authors: Monie Dileep D. , Zhang Cheng , Correia Cristina , Ung Choong , Vile Richard G. , Li Hu
Published: January 2023

Glioblastoma is the most common and lethal primary brain tumor in adults, with a median survival less than 15 months despite current standard of care. Oncolytic immunovirotherapies (OVs) have shown promise in early clinical trials. OVs are engineered to specifically target cancer cells, trigger autophagic programmed cell death, and release tumor antigens in the context of T-cell activating cytokines. Cellular communication network factor 1 (CCN1) is an extracellular matrix protein expressed in 70% of glioblastomas that has been shown to drastically reduce OV efficacy, particularly herpes simplex virus type 1 (HSV-1). Our objectives were to decode protein-protein interaction (PPI) networks activated by CCN1, identify critical nodes in resistance networks, and use this analysis to design OVs that overcome CCN1-mediated resistance. We used NetDecoder to elucidate phenotype-specific PPI subnetworks in LN229 human glioblastoma cells with tetracycline-inducible CCN1 expression. Publicly available microarray data on CCN1-induced and control samples established differentially expressed genes, which served as sources in our PPI network to derive prioritized context-specific subnetworks. We found that 11 source genes collaborate via 39 deep nodes to confer OV resistance to CCN1-expressing glioblastomas. Of these, a router (IKBKE) and a sink (YBX1) have been previously implicated in glioblastoma pathogenesis. We conclude that autophagy regulator HSP90AA1, a critical node, may be targeted to improve OV efficacy in CCN1-expressing glioblastomas. Furthermore, WebGestalt overrepresentation analysis of subnetwork nodes suggests that oncolytic adenovirus may be more effective than HSV-1 as a synthetic biology chassis.
Immunogenicity comparison of neoantigen vaccines through different delivery platform
Authors: Wu Pinyi , Jiang Haitao , Shen Hao-chun , Tao Mi-Hua
Published: January 2023

Neoantigens are promising cancer vaccine candidates because they’re cancer-specific and can be predicted from the patient’s genomic data. A major challenge for developing potent neoantigen vaccines is the low immunogenicity of neoepitopes. DNA vaccines can deliver multiple epitopes and cytokines as an adjuvant in a single plasmid, and its immunogenicity can be further enhanced through heterologous prime-boost strategy. Here, we hypothesized utilizing DNA prime, adenovirus boost (refer to the DDA approach) strategy can improve the immunogenicity of neoantigen vaccines. To investigate whether vaccination through the DDA approach or DDA combine GM-CSF as adjuvant can improve the immunogenicity of neoantigen vaccines. Using IEDB analysis resource to predict and rank the top 21 MHC class I epitopes (short peptide) for the B16F10 tumor while 27mer peptides (long peptide) were synthesized to cover both MHC class I and class II epitopes. The 27mer neoantigen sequences of the selected 21 MHC class I epitopes were cloned into one plasmid and adenoviral vector with or without GM-CSF sequence linked by p2a. Mice were immunized through the DDA approach or peptides with polyI:C. In the peptide vaccine, the majority of the response were mediated by CD4+ T cells, only one peptide can induce CD8+ T cells response. For the DDA approach, 14% epitopes can induce CD8+ T cell response in mice immunized with DNA vaccine. Moreover, in the group with genetic encoded GM-CSF. it has significantly enhanced CD8+T cell response compare to the former, which dramatically improved the immunogenicity of neoantigen vaccines. Our preliminary data also showed that immunization of DNA vaccines significantly delayed tumor growth.