The Dual-Antigen Ad5 COVID-19 Vaccine Delivered as an Intranasal Plus Subcutaneous Prime Elicits Th1 Dominant T-Cell and Humoral Responses in CD-1 Mice

In response to the need for an efficacious, thermally-stable COVID-19 vaccine that can elicit both humoral and cell-mediated T-cell responses, we have developed a dual-antigen human adenovirus serotype 5 (hAd5) COVID-19 vaccine in formulations suitable for subcutaneous (SC), intranasal (IN), or oral delivery. The vaccine expresses both the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins using an hAd5 platform with E1, E2b, and E3 sequences deleted; hAd5(E1-, E2b-, E3-); that is effective even in the presence of hAd5 immunity. In the vaccine, S is modified (S-Fusion) for enhanced cell surface display to elicit humoral responses and N is modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal pathway to increase MHC I and II presentation. Initial studies using subcutaneous (SC) prime and SC boost vaccination of CD-1 mice demonstrated that the hAd5 S-Fusion + N-ETSD vaccine elicits T-helper cell 1 (Th1) dominant T-cell and humoral responses to both S and N. We then compared SC to IN prime vaccination with either an SC or IN boost post-SC prime and an IN boost after IN prime. These studies reveal that IN prime/IN boost is as effective at generating Th1 dominant humoral responses to both S and N as the other combinations, but that the SC prime with either an IN or SC boost elicits greater T cell responses. In a third study to assess the power of the two routes of delivery when used together, we used a combined SC plus IN prime with or without a boost and found the combined prime alone to be as effective as the combined prime with either an SC or IN boost in generating both humoral and T-cell responses. The findings here in CD-1 mice demonstrate that combined SC and IN prime-only delivery has the potential to provide broad immunity, including mucosal immunity, against SARS-CoV-2 and supports further testing of this delivery approach in additional animal models and clinical trials.

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Intranasal plus subcutaneous prime vaccination with a dual antigen COVID-19 vaccine elicits T-cell and antibody responses in mice

Scientific Reports ◽

10.1038/s41598-021-94364-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Adrian Rice ◽

Mohit Verma ◽

Annie Shin ◽

Lise Zakin ◽

Peter Sieling ◽

...

Keyword(s):

T Cell ◽

T Cell Responses ◽

Human Adenovirus ◽

Wild Type ◽

T Helper ◽

Cell Responses ◽

Antigen Vaccine ◽

Serotype 5 ◽

Humoral Responses ◽

Delivery Approach

AbstractWe have developed a COVID-19 vaccine, hAd5 S-Fusion + N-ETSD, that expresses SARS-CoV-2 spike (S) and nucleocapsid (N) proteins with modifications to increase immune responses delivered using a human adenovirus serotype 5 (hAd5) platform. Here, we demonstrate subcutaneous (SC) prime and SC boost vaccination of CD-1 mice with this dual-antigen vaccine elicits T-helper cell 1 (Th1) biased T-cell and humoral responses to both S and N that are greater than those seen with hAd5 S wild type delivering only unmodified S. We then compared SC to intranasal (IN) prime vaccination with SC or IN boosts and show that an IN prime with an IN boost is as effective at generating Th1 biased humoral responses as the other combinations tested, but an SC prime with an IN or SC boost elicits greater T cell responses. Finally, we used a combined SC plus IN (SC + IN) prime with or without a boost and found the SC + IN prime alone to be as effective in generating humoral and T-cell responses as the SC + IN prime with a boost. The finding that SC + IN prime-only delivery has the potential to provide broad immunity—including mucosal immunity—against SARS-CoV-2 supports further testing of this vaccine and delivery approach in animal models of viral challenge.

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Human adenovirus-specific CD8+ T-cell responses are not inhibited by E3-19K in the presence of gamma interferon.

Journal of Virology ◽

10.1128/jvi.70.9.6314-6322.1996 ◽

1996 ◽

Vol 70 (9) ◽

pp. 6314-6322 ◽

Cited By ~ 42

Author(s):

P Flomenberg ◽

V Piaskowski ◽

R L Truitt ◽

J T Casper

Keyword(s):

T Cell ◽

T Cell Responses ◽

Human Adenovirus ◽

Cd8 T Cell ◽

Gamma Interferon ◽

Cell Responses

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418 A phase 1, dose escalation and dose expansion study of SQZ PBMC HPV as monotherapy and in combination with atezolizumab in HLA-A*02+ Patients with HPV16+ recurrent, or metastatic solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0418 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A444-A444

Author(s):

Cathy Eng ◽

Joaquina Baranda ◽

Matthew Taylor ◽

Michael Gordon ◽

Ursula Matulonis ◽

...

Keyword(s):

T Cell ◽

Solid Tumors ◽

Cancer Vaccine ◽

Phase 1 ◽

T Cell Responses ◽

Cd8 T Cell ◽

Dose Effect ◽

Statistical Hypothesis ◽

Mhc I ◽

Cell Responses

BackgroundSQZ-PBMC-HPV is a therapeutic cancer vaccine created with Cell Squeeze®, a proprietary cell-engineering system. SQZ-PBMC-HPV is a novel cancer vaccine generated from peripheral blood mononuclear cells (PBMC) squeezed with HPV16 E6 and E7 antigens, resulting in delivery into the cytosol. The resulting antigen presenting cells (APCs) provide enhanced antigen presentation on MHC-I to potentially elicit robust, antigen-specific CD8+ T cell responses. Importantly, SQZ-PBMC-HPV are neither genetically modified nor immune effector cells.Studies in MHC-I knockout mice demonstrated that activation of antigen specific CD8+ tumor infiltrating lymphocytes (TILs) was a direct effect of cytosolic antigen delivery to PBMCs. In the murine TC-1 tumor model, tumor regression correlated with an influx of HPV16-specific CD8+ TILs. In vitro studies with human volunteer PBMCs demonstrated that each subset is capable of inducing CD8+ T cell responses. The Phase 1 study includes a significant biomarker program to investigate whether pharmacodynamic effects observed in non-clinical studies correlate with potential clinical benefit. Immunogenic and pharmacodynamic endpoints include Elispot assays to measure frequency of interferon gamma secreting cells, as well as quantification and characterization of TILs and tumor microenvironment. In addition, various cytokine responses and circulating cell-free HPV16 DNA levels in plasma are measured.MethodsSQZ-PBMC-HPV-101 (NCT04084951) is open for enrollment to HLA A*02+ patients with HPV16+ recurrent, locally advanced or metastatic solid tumors and includes escalation cohorts for monotherapy and in combination with atezolizumab. After initial demonstration of safety, the study assesses dose effect by testing different cell dose levels, the effect of prolonged antigen priming in Cycle 1 [APC administration on Day 1 only compared to Days 1 and 2 (double priming)] and the impact of treatment duration to identify the optimal dose regimen. The cycle length is 3 weeks, and patients will receive SQZ-PBMC-HPV for up to 1 year or until available autologous drug product is exhausted. Atezolizumab will be administered for up to 1 year. Eligible patients including but not limited to anal, cervical and head and neck tumors will undergo a single leukapheresis at the study site. The manufacturing process includes a maturation step and takes less than 24 hours. The vein-to-vein time for the 1st administration is approximately one week. Patients must have a lesion that can be biopsied with acceptable clinical risk and agree to have a fresh biopsy at Screening and on study. A Study Safety Committee is in place. No formal statistical hypothesis testing will be performed.ResultsN/AConclusionsN/ATrial RegistrationNCT04084951Ethics ApprovalThe study is registered on clinicaltrials.gov was approved by the Ethics Board of all institution listed as recruiting.

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CXCL14 suppresses human papillomavirus-associated head and neck cancer through antigen-specific CD8+ T-cell responses by upregulating MHC-I expression

Oncogene ◽

10.1038/s41388-019-0911-6 ◽

2019 ◽

Vol 38 (46) ◽

pp. 7166-7180 ◽

Cited By ~ 4

Author(s):

Joseph A. Westrich ◽

Daniel W. Vermeer ◽

Alexa Silva ◽

Stephanie Bonney ◽

Jennifer N. Berger ◽

...

Keyword(s):

Head And Neck Cancer ◽

Human Papillomavirus ◽

T Cell ◽

Head And Neck ◽

Neck Cancer ◽

T Cell Responses ◽

Cd8 T Cell ◽

Mhc I ◽

Cell Responses

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BothCD4+andCD8+Lymphocytes Participate in the IFN-γ Response to Filamentous Hemagglutinin fromBordetella pertussisin Infants, Children, and Adults

Clinical and Developmental Immunology ◽

10.1155/2012/795958 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 17

Author(s):

Violette Dirix ◽

Virginie Verscheure ◽

Françoise Vermeulen ◽

Iris De Schutter ◽

Tessa Goetghebuer ◽

...

Keyword(s):

T Cell ◽

T Lymphocytes ◽

Bacterial Infections ◽

T Cell Responses ◽

Cell Types ◽

Production Flow ◽

Mhc I ◽

Cell Responses ◽

Filamentous Hemagglutinin ◽

Circulating Lymphocytes

Infant CD4+T-cell responses to bacterial infections or vaccines have been extensively studied, whereas studies on CD8+T-cell responses focused mainly on viral and intracellular parasite infections. Here we investigated CD8+T-cell responses uponBordetella pertussisinfection in infants, children, and adults and pertussis vaccination in infants. Filamentous hemagglutinin-specific IFN-γsecretion by circulating lymphocytes was blocked by anti-MHC-I or -MHC-II antibodies, suggesting that CD4+and CD8+T lymphocytes are involved in IFN-γproduction. Flow cytometry analyses confirmed that both cell types synthesized antigen-specific IFN-γ, although CD4+lymphocytes were the major source of this cytokine. IFN-γsynthesis by CD8+cells was CD4+T cell dependent, as evidenced by selective depletion experiments. Furthermore, IFN-γsynthesis by CD4+cells was sometimes inhibited by CD8+lymphocytes, suggesting the presence of CD8+regulatory T cells. The role of this dual IFN-γsecretion by CD4+and CD8+T lymphocytes in pertussis remains to be investigated.

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584 Powerful synergistic effects of a STING agonist and the IL-2 superkine, H9, in eliciting NK and T cell responses against MHC I- and MHC I+ tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.584 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A614-A614

Author(s):

Natalie Wolf ◽

Cristina Blaj ◽

Lora Picton ◽

Gail Snyder ◽

Li Zhang ◽

...

Keyword(s):

T Cell ◽

Combination Therapy ◽

Nk Cells ◽

Nk Cell ◽

Tumor Regression ◽

T Cell Responses ◽

Cd8 T Cell ◽

Mhc I ◽

Therapy Regimen ◽

Cell Responses

BackgroundMost current cancer immunotherapies are based on mobilizing CD8 T cell responses. However, many types of tumors evade CD8 T cell recognition by displaying few or no antigens, or losing expression of MHC I. These considerations underlie the need for complementary therapies that mobilize other antitumor effector cells, such as NK cells, which preferentially kill MHC I-deficient cells. Cyclic dinucleotides (CDNs) activate the cGAS-STING pathway of the innate immune system and are candidates as immunotherapy agents. Intratumoral CDN injections induce type I IFNs and other mediators that amplify the CD8 T cell response and induce tumor regression [1]. CDN therapy also induces long-term tumor regressions in some MHC I-deficient tumor models, mediated primarily by NK cells [2].MethodsTo extend the efficacy of CDN therapy, we combined the IL-2 superkine, H9, or half-life extended H9, with CDNs to target and activate NK cells in the tumor microenvironment and prevent or delay the onset of NK cell desensitization [3,4]. In these studies, we utilized B16-F10 and MC38 tumor cells lacking B2m to examine effects of the combination therapy on MHC I-deficient tumor growth as well as to examine the activation of NK cells by flow cytometry and cytotoxicity assays. We also utilized B16-F10 WT and the spontaneous tumor model, MCA, to assess the effect of the combination therapy on MHC I+ tumors.ResultsHere we show that H9 synergized with CDN therapy to mobilize much more powerful antitumor responses against MHC I-deficient tumors than CDN alone. The responses were mediated by NK cells and in some cases CD4 T cells, and were accompanied by increased recruitment to and sustained activation of NK cells in the tumor. This combination therapy regimen activated NK cells systemically, as shown by antitumor effects distant from the site of CDN injection and enhanced cytolytic activity of splenic NK cells against tumor cell targets ex vivo. Finally, the same combination therapy regimen synergistically mobilized powerful CD8 T cell responses in the case of MHC I+ tumor cells, suggesting the generality of the approach. The approach was effective against primary sarcomas, as well, especially when combined with checkpoint therapy, leading to tumor regressions and long-term survival of many mice with MCA-induced sarcoma.ConclusionsOverall, our work demonstrates the impact of a novel combination therapy in mobilizing powerful NK and T cell-mediated antitumor activity, providing important justification for evaluating this approach for treating cancers that are refractory to available treatment options.ReferencesCorrales, L., Glickman, L.H., McWhirter, S.M., Kanne, D.B., Sivick, K.E., Katibah, G.E., Woo, S.R., Lemmens, E., Banda, T., Leong, J.J., et al. (2015). Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity. Cell Rep 11, 1018–1030.Nicolai, C.J., Wolf, N., Chang, I.C., Kirn, G., Marcus, A., Ndubaku, C.O., McWhirter, S.M., and Raulet, D.H. (2020). NK cells mediate clearance of CD8(+) T cell-resistant tumors in response to STING agonists. Science immunology 5, eaaz2738.Levin, A.M., Bates, D.L., Ring, A.M., Krieg, C., Lin, J.T., Su, L., Moraga, I., Raeber, M.E., Bowman, G.R., Novick, P., et al. (2012). Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’. Nature 484, 529–533.Ardolino, M., Azimi, C.S., Iannello, A., Trevino, T.N., Horan, L., Zhang, L., Deng, W., Ring, A.M., Fischer, S., Garcia, K.C., and Raulet, D.H. (2014). Cytokine therapy reverses NK cell anergy in MHC-deficient tumors. J Clin Invest 124, 4781–4794.

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Transmembrane Helices Are an Over-Presented and Evolutionarily Conserved Source of Major Histocompatibility Complex Class I and II Epitopes

Frontiers in Immunology ◽

10.3389/fimmu.2021.763044 ◽

2022 ◽

Vol 12 ◽

Author(s):

Richèl J. C. Bilderbeek ◽

Maksim V. Baranov ◽

Geert van den Bogaart ◽

Frans Bianchi

Keyword(s):

T Cell ◽

Membrane Proteins ◽

T Cell Responses ◽

Class I ◽

Complex Class ◽

Transmembrane Helices ◽

Class I Mhc ◽

Mhc I ◽

Cell Responses ◽

Histocompatibility Complex

Cytolytic T cell responses are predicted to be biased towards membrane proteins. The peptide-binding grooves of most alleles of histocompatibility complex class I (MHC-I) are relatively hydrophobic, therefore peptide fragments derived from human transmembrane helices (TMHs) are predicted to be presented more often as would be expected based on their abundance in the proteome. However, the physiological reason of why membrane proteins might be over-presented is unclear. In this study, we show that the predicted over-presentation of TMH-derived peptides is general, as it is predicted for bacteria and viruses and for both MHC-I and MHC-II, and confirmed by re-analysis of epitope databases. Moreover, we show that TMHs are evolutionarily more conserved, because single nucleotide polymorphisms (SNPs) are present relatively less frequently in TMH-coding chromosomal regions compared to regions coding for extracellular and cytoplasmic protein regions. Thus, our findings suggest that both cytolytic and helper T cells are more tuned to respond to membrane proteins, because these are evolutionary more conserved. We speculate that TMHs are less prone to mutations that enable pathogens to evade T cell responses.

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Transmembrane helices are an overlooked and evolutionarily conserved source of major histocompatibility complex class II epitopes

10.1101/2021.05.02.441235 ◽

2021 ◽

Author(s):

Richel Bilderbeek ◽

Maksim Baranov ◽

Frans Bianchi ◽

Geert van den Bogaart

Keyword(s):

T Cell ◽

Membrane Proteins ◽

T Cell Responses ◽

Nucleotide Polymorphisms ◽

Complex Class ◽

Transmembrane Helices ◽

Class I Mhc ◽

Mhc I ◽

Cell Responses ◽

Histocompatibility Complex

Cytolytic T cell responses are predicted to be biased towards membrane proteins. The peptide-binding grooves of most haplotypes of histocompatibility complex class I (MHC-I) are relatively hydrophobic, therefor peptide fragments derived from human transmembrane helices (TMHs) are predicted to be presented more often as would be expected based on their abundance in the proteome. However, the physiological reason of why membrane proteins might be over-presented is unclear. In this study, we show that the over-presentation of TMH-derived peptides is general, as it is predicted for bacteria and viruses and for both MHC-I and MHC-II. Moreover, we show that TMHs are evolutionarily more conserved, because single nucleotide polymorphisms (SNPs) are present relatively less frequently in TMH-coding chromosomal regions compared to regions coding for extracellular and cytoplasmic protein regions. Thus, our findings suggest that both cytolytic and helper T cells respond more to membrane proteins, because these are evolutionary more conserved. We speculate that TMHs therefor are less prone to escape mutations that enable pathogens to evade T cell responses.

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MHC Class I Presented T Cell Epitopes as Potential Antigens for Therapeutic Vaccine against HBV Chronic Infection

Hepatitis Research and Treatment ◽

10.1155/2014/860562 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11 ◽

Cited By ~ 8

Author(s):

Joseph D. Comber ◽

Aykan Karabudak ◽

Vivekananda Shetty ◽

James S. Testa ◽

Xiaofang Huang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Infection ◽

Acute Infection ◽

Therapeutic Vaccine ◽

T Cell Responses ◽

Hbv Infection ◽

Mhc I ◽

Cell Responses ◽

Cell Immunity

Approximately 370 million people worldwide are chronically infected with hepatitis B virus (HBV). Despite the success of the prophylactic HBV vaccine, no therapeutic vaccine or other immunotherapy modality is available for treatment of chronically infected individuals. Clearance of HBV depends on robust, sustained CD8+ T activity; however, the limited numbers of therapeutic vaccines tested have not induced such a response. Most of these vaccines have relied on peptide prediction algorithms to identify MHC-I epitopes or characterization of T cell responses during acute infection. Here, we took an immunoproteomic approach to characterize MHC-I restricted epitopes from cells chronically infected with HBV and therefore more likely to represent the true targets of CD8+ T cells during chronic infection. In this study, we identified eight novel MHC-I restricted epitopes derived from a broad range of HBV proteins that were capable of activating CD8+ T cells. Furthermore, five of the eight epitopes were able to bind HLA-A2 and A24 alleles and activated HBV specific T cell responses. These epitopes also have potential as new tools to characterize T cell immunity in chronic HBV infection and may serve as candidate antigens for a therapeutic vaccine against HBV infection.

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