CD8+ T cell immunogenicity induced by endogenous EVs engineered by antigens fused to a truncated Nefmut EV-anchoring protein

AbstractIntramuscular injection of DNA vectors expressing the extracellular vesicle (EV)-anchoring protein Nefmut fused at its C-terminus to viral and tumor antigens elicits a potent, effective, and anti-tolerogenic CD8+ T cell immunity against the heterologous antigen. The immune response is induced through the production of EVs incorporating Nefmut-derivatives released by muscle cells. In the perspective to a possible translation into the clinic of the Nefmut-based vaccine platform, we aimed at increasing its safety profile by identifying the minimal part of Nefmut retaining the EV-anchoring protein property. We found that a C-terminal deletion of 29-amino acids did not affect the ability of Nefmut to associate with EVs. Furthermore, the EV-anchoring function was preserved when antigens from both HPV16 (i.e., E6 and E7) and SARS-CoV-2 (i.e., S1 and S2) were fused to its C-terminus. By analyzing the immune responses induced after intramuscular injection of DNA vectors expressing fusion products based on the four viral antigens, we found that the Nefmut C-terminal deletion did not impact on the levels of antigen –specific CD8+ T lymphocytes as evaluated by IFN-γ EliSpot analysis and intracellular cytokine staining. In addition, immune responses at distal sites remained unaffected, as indicated by the similar percentages of SARS-CoV-2 S1- and S2-specific CD8+ T cells detected in spleens and lung airways of mice injected with DNA vectors expressing the viral antigens fused with either Nefmut or NefmutPL.We concluded that the C-terminal Nefmut truncation does not affect stability, EV-anchoring, and CD8+ T cell immunogenicity of the fused antigen. Hence, NefmutPL represents a safer alternative to full-length Nefmut for the design of CD8+ T cell vaccines for humans.

Download Full-text

The C-Terminal Domain of Nefmut Is Dispensable for the CD8+ T Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles

Vaccines ◽

10.3390/vaccines9040373 ◽

2021 ◽

Vol 9 (4) ◽

pp. 373

Author(s):

Chiara Chiozzini ◽

Francesco Manfredi ◽

Flavia Ferrantelli ◽

Patrizia Leone ◽

Andrea Giovannelli ◽

...

Keyword(s):

T Cell ◽

Tumor Antigens ◽

Cd8 T Cell ◽

Terminal Deletion ◽

Vaccine Platform ◽

Heterologous Antigen ◽

Cell Immunity ◽

C Terminus ◽

Anchoring Protein

Intramuscular injection of DNA vectors expressing the extracellular vesicle (EV)-anchoring protein Nefmut fused at its C-terminus to viral and tumor antigens elicit a potent, effective, and anti-tolerogenic CD8+ T cell immunity against the heterologous antigen. The immune response is induced through the production of EVs incorporating Nefmut-derivatives released by muscle cells. In the perspective of a possible translation into the clinic of the Nefmut-based vaccine platform, we aimed at increasing its safety profile by identifying the minimal part of Nefmut retaining the EV-anchoring protein property. We found that a C-terminal deletion of 29-amino acids did not affect the ability of Nefmut to associate with EVs. The EV-anchoring function was also preserved when antigens from both HPV16 (i.e., E6 and E7) and SARS-CoV-2 (i.e., S1 and S2) were fused to its C-terminus. Most important, the Nefmut C-terminal deletion did not affect levels, quality, and diffusion at distal sites of the antigen-specific CD8+ T immunity. We concluded that the C-terminal Nefmut truncation does not influence stability, EV-anchoring, and CD8+ T cell immunogenicity of the fused antigen. Hence, the C-terminal deleted Nefmut may represent a safer alternative to the full-length isoform for vaccines in humans.

Download Full-text

Priming with Recombinant BCG Expressing HTI Enhances the Magnitude and Breadth of the T-Cell Immune Responses Elicited by MVA.HTI in BALB/c Mice

Vaccines ◽

10.3390/vaccines8040678 ◽

2020 ◽

Vol 8 (4) ◽

pp. 678

Author(s):

Narcís Saubi ◽

Athina Kilpeläinen ◽

Yoshiki Eto ◽

Chun-Wei Chen ◽

Àlex Olvera ◽

...

Keyword(s):

T Cell ◽

Immune Responses ◽

T Cell Responses ◽

Vaccine Platform ◽

Cell Responses ◽

Total Magnitude ◽

T Cell Immune Responses ◽

Ifn Γ ◽

Hiv 1 ◽

Recombinant Bcg

The use of Mycobacterium bovis bacillus Calmette–Guérin (BCG) as a live vaccine vehicle is a promising approach for HIV-1-specific T-cell induction. In this study, we used recombinant BCG expressing HIVACAT T-cell immunogen (HTI), BCG.HTI2auxo.int. BALB/c mice immunization with BCG.HTI2auxo.int prime and MVA.HTI boost was safe and induced HIV-1-specific T-cell responses. Two weeks after boost, T-cell responses were assessed by IFN-γ ELISpot. The highest total magnitude of IFN-γ spot-forming cells (SFC)/106 splenocytes was observed in BCG.HTI2auxo.int primed mice compared to mice receiving MVA.HTI alone or mice primed with BCGwt, although the differences between the vaccination regimens only reached trends. In order to evaluate the differences in the breadth of the T-cell immune responses, we examined the number of reactive peptide pools per mouse. Interestingly, both BCG.HTI2auxo.int and BCGwt primed mice recognized an average of four peptide pools per mouse. However, the variation was higher in BCG.HTI2auxo.int primed mice with one mouse recognizing 11 peptide pools and three mice recognizing few or no peptide pools. The recognition profile appeared to be more spread out for BCG.HTI2auxo.int primed mice and mice only receiving MVA.HTI. Here, we describe a useful vaccine platform for priming protective responses against HIV-1/TB and other prevalent infectious diseases.

Download Full-text

Intramuscular injection of plasmid DNA encoding cottontail rabbit papillomavirus E1, E2, E6 and E7 induces T cell-mediated but not humoral immune responses in rabbits

Vaccine ◽

10.1016/s0264-410x(98)00356-9 ◽

1999 ◽

Vol 17 (11-12) ◽

pp. 1558-1566 ◽

Cited By ~ 29

Author(s):

R HAN

Keyword(s):

T Cell ◽

Immune Responses ◽

Plasmid Dna ◽

Intramuscular Injection ◽

Dna Encoding ◽

Humoral Immune ◽

Humoral Immune Responses ◽

Cottontail Rabbit ◽

E6 And E7

Download Full-text

Robust Stimulation of Humoral and Cellular Immune Responses following Vaccination with Antigen-Loaded β-Glucan Particles

mBio ◽

10.1128/mbio.00164-10 ◽

2010 ◽

Vol 1 (3) ◽

Cited By ~ 87

Author(s):

Haibin Huang ◽

Gary R. Ostroff ◽

Chrono K. Lee ◽

Charles A. Specht ◽

Stuart M. Levitz

Keyword(s):

T Cell ◽

Immune Responses ◽

Elispot Assay ◽

Cellular Response ◽

T Cell Responses ◽

Minor Role ◽

Mouse Bone Marrow ◽

Antigen Delivery ◽

Vaccine Platform ◽

Cell Responses

ABSTRACTβ-Glucan particles (GPs) are purifiedSaccharomyces cerevisiaecell walls treated so that they are primarily β1,3-d-glucans and free of mannans and proteins. GPs are phagocytosed by dendritic cells (DCs) via the Dectin-1 receptor, and this interaction stimulates proinflammatory cytokine secretion by DCs. As the hollow, porous GP structure allows for high antigen loading, we hypothesized that antigen-loaded GPs could be exploited as a receptor-targeted vaccine delivery system. Ovalbumin (OVA) was electrostatically complexed inside the hollow GP shells (GP-OVA). Incubation of C57BL/6J mouse bone marrow-derived DCs with GP-OVA resulted in phagocytosis, upregulation of maturation markers, and rapid proteolysis of OVA. Compared with free OVA, GP-OVA was >100-fold more potent at stimulating the proliferation of OVA-reactive transgenic CD8+OT-I and CD4+OT-II T cells, as measured byin vitro[3H]thymidine incorporation using DCs as antigen-presenting cells. Next, immune responses in C57BL/6J mice following subcutaneous immunizations with GP-OVA were compared with those in C57BL/6J mice following subcutaneous immunizations with OVA absorbed onto the adjuvant alum (Alum/OVA). Vaccination with GP-OVA stimulated substantially higher antigen-specific CD4+T-cell lymphoproliferative and enzyme-linked immunospot (ELISPOT) responses than that with Alum/OVA. Moreover, the T-cell responses induced by GP-OVA were Th1 biased (determined by gamma interferon [IFN-γ] ELISPOT assay) and Th17 biased (determined by interleukin-17a [IL-17a] ELISPOT assay). Finally, both the GP-OVA and Alum/OVA formulations induced strong secretions of IgG1 subclass anti-OVA antibodies, although only GP-OVA induced secretion of Th1-associated IgG2c antibodies. Thus, the GP-based vaccine platform combines adjuvanticity and antigen delivery to induce strong humoral and Th1- and Th17-biased CD4+T-cell responses.IMPORTANCEMost licensed vaccines work by promoting protective antibody responses. However, for many infectious diseases, antibody-mediated protection appears to play a relatively minor role, and vaccination has met with limited success. While live-attenuated organisms generally elicit T-cell responses, their use in vaccines is limited by the potential for causing disease. Thus, there is an urgent need for new vaccine platforms that deliver antigens in such a manner as to promote strong T-cell-mediated responses. Here we designed a novel vaccine platform consisting of yeast-derived β-glucan particles (GPs) that combines antigen delivery and adjuvant activity. GPs loaded with the model antigen ovalbumin (OVA) stimulated robust humoral and T-cell responses in mice. In addition, the cellular response was Th1 and Th17 biased. This work has implications for the design of vaccines that stimulate biased T-cell responses as well as for understanding how immunity to fungal pathogens develops.

Download Full-text

The molecular machinery for cAMP-dependent immunomodulation in T-cells

Biochemical Society Transactions ◽

10.1042/bst0340476 ◽

2006 ◽

Vol 34 (4) ◽

pp. 476-479 ◽

Cited By ~ 43

Author(s):

K. Taskén ◽

A.J. Stokka

Keyword(s):

T Cell ◽

Immune Responses ◽

Lipid Rafts ◽

T Cell Activation ◽

Negative Feedback ◽

Cell Activation ◽

Cell Receptor ◽

Membrane Microdomains ◽

Type I ◽

Anchoring Protein

cAMP inhibits Src-family kinase signalling by PKA (protein kinase A)-mediated phosphorylation and activation of Csk (C-terminal Src kinase). The PKA type I–Csk pathway is assembled and localized in membrane microdomains (lipid rafts) and regulates immune responses activated through the TCR (T-cell receptor). PKA type I is targeted to the TCR–CD3 complex during T-cell activation via an AKAP (A-kinase-anchoring protein) that serves as a scaffold for the cAMP–PKA/Csk pathway in lipid rafts of the plasma membrane during T-cell activation. Displacement of PKA by anchoring disruption peptides prevents cAMP/PKA type I-mediated inhibition of T-cell activation. These findings provide functional evidence that PKA type I regulation of T-cell responses is dependent on AKAP anchoring. Furthermore, we show that upon TCR/CD28 co-ligation, β-arrestin in complex with PDE4 (phosphodiesterase 4) is recruited to lipid rafts. The CD28-mediated recruitment of PDE4 to lipid rafts potentiates T-cell immune responses and counteracts the local, TCR-induced production of cAMP that produces negative feedback in the absence of a co-receptor stimulus. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a co-receptor stimulus.

Download Full-text

P03.01 High immunogenic VLP-based vaccines elicit new T cell specificities against melanoma neoantigens in mice

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-itoc8.24 ◽

2021 ◽

Vol 9 (Suppl 1) ◽

pp. A13.1-A13

Author(s):

C Aguilar-Gurrieri ◽

A Barajas ◽

A Pons-Grifols ◽

I Varela ◽

P Amengual-Rigo ◽

...

Keyword(s):

T Cell ◽

Immune Responses ◽

Therapeutic Vaccine ◽

T Cell Responses ◽

Advisory Board ◽

Tumor Control ◽

Vaccine Platform ◽

Anchor Residue ◽

Synonymous Mutations ◽

Cell Responses

BackgroundNeoantigens’ (neoAg) identification, which determines T-cell responses against tumors, has fostered the development of personalized vaccines with promising results. While the ranking of the most immunogenic neoAg can be addressed using predictive techniques, their formulation as vaccines needs to be improved. To maximize their therapeutic potential, optimal neoAg-based vaccines should be manufactured in a superb delivery platform that enhances robust new immune responses, able to bypass thymic tolerance and the humoral immunosuppressive microenvironment. These novel T cell responses generated at the periphery will not be exhausted, opposite to TILs. We aim to develop a highly immunogenic vaccine platform, based on engineered HIV-derived Virus-Like Particles (VLP) expressing approximately 2500 copies of each selected neoAg. We tested different neoAgs loaded VLPs (neoVLP) in a melanoma mouse model to evaluate their capability to generate new immunogenic specificities.Material and MethodsSpecific non-synonymous mutations from B16F10 cells were identified, selected and used to generate a list of prioritized peptides. NeoAgs were classified as: Tier1, acquiring a mutation that creates an anchor residue to the MHC-I, not present in the WT peptide; Tier2, acquiring a mutation in a position that largely impacts contact with the TCR respect to WT; and Tier3, acquiring a mutation in the TCR contact region but inducing a less drastic change than in Tier2. Frame shift (FS) mutations, expected to be highly immunogenic, were also included.Thirteen to fifteen selected neoAgs from each group were loaded on highly immunogenic neoVLPs. Their immunogenicity was evaluated in C57bl/6 mice by immunization with a neoVLP-coding plasmid DNA (prime) and purified neoVLPs as soluble particles (boost). Splenocytes were used to evaluate neoAg-specific T cell responses.ResultsWe have successfully generated and purified neoVLPs, exposing neoAgs from all groups by transient transfection of Expi293 cells. Protein integrity and VLP morphology were confirmed by western blot and cryo-EM. When used for immunization assays, neoVLPs, containing neoAgs from Tier2, Tier3 and FS groups, were capable of generating humoral responses against viral proteins and T cell responses against neoAgs present in the neoVLP. B16F10 inoculated animals, but not vaccinated, did not develop detectable T cell responses against neoAgs present in any tested neoVLP, suggesting that the vaccination with neoVLPs promoted new specificities against selected neoAgs that might contribute to tumor control and eradication.ConclusionOur data show that the neoVLPs promote the generation of new antitumor-specific immune responses against selected neoepitopes, suggesting that neoVLPs vaccination could be an alternative to current therapeutic vaccine approaches and a promising candidate for future personalized immunotherapy.Disclosure InformationC. Aguilar-Gurrieri: None. A. Barajas: None. A. Pons-Grifols: None. I. Varela: None. P. Amengual-Rigo: None. R. Farriol: None. M. Vazquez: None. R. Lepore: None. C. Anjos-Souza: None. J. Blanco-Heredia: None. L. de Mattos-Arruda: None. V. Guallar: None. A. Valencia: None. B. Clotet: F. Consultant/Advisory Board; Significant; Albajuna Therapeutics. J. Carrillo: Other; Significant; Albajuna Therapeutics. J. Blanco: F. Consultant/Advisory Board; Significant; Albajuna Therapeutics.

Download Full-text

Wilms’ tumour antigen 1 Immunity via DNA fusion gene vaccination in haematological malignancies by intramuscular injection followed by intramuscular electroporation: a Phase II non-randomised clinical trial (WIN)

Efficacy and Mechanism Evaluation ◽

10.3310/eme03030 ◽

2016 ◽

Vol 3 (3) ◽

pp. 1-80 ◽

Cited By ~ 2

Author(s):

Christian Ottensmeier ◽

Megan Bowers ◽

Debbie Hamid ◽

Tom Maishman ◽

Scott Regan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

Phase Ii ◽

Intramuscular Injection ◽

Fusion Gene ◽

Control Group ◽

Cell Responses ◽

Gene Vaccination ◽

Epitope Sequence

BackgroundIn the UK almost 7000 people are diagnosed with leukaemia each year, but despite continuing advances in diagnosis and treatment with new drugs, such as the tyrosine kinase inhibitors, the majority of these patients will eventually die from their disease. Until quite recently, the only treatment to offer the possibility of long-term disease-free survival was allogeneic stem cell transplantation. However, this carries a substantial risk of mortality and is available to only a minority of patients.ObjectivesThe aim of the study was to test the hypothesis that molecular and clinical responses, induced by T lymphocytes (T cells), can be predicted by increases in the number of CD8+ (cluster of differentiation 8-positive) T cells specific for the vaccine-encoded T-cell epitopes. This project also aimed to build on the established programme of deoxyribonucleic acid (DNA) fusion-gene vaccination delivered by intramuscular injection, exploiting a unique experience with electroporation, to induce durable immune responses with the aim of controlling disease by precision attack of the tumour by CD8+ T cells.MethodA non-randomised, open-label, single-dose-level Phase II clinical trial in two patient groups [chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML)] on stable doses of imatinib. Human leucocyte antigen A2-positive (HLA A2+) patients were vaccinated with two DNA vaccines: (1) p.DOM–WT1-37 (epitope sequence: VLDFAPPGA); and (2) p.DOM–WT1-126 (epitope sequence: RMFPNAPYL). The HLA A2-negative patients formed an unvaccinated control group. The sample size for the HLA A2+ group was originally determined following Simon’s optimal Phase II trial design (Simon R. Optimal two-stage designs for phase II clinical trials.Control Clin Trials1989;10:1–10). This was changed to A’Hern’s single-stage design during the course of the trial (A’Hern RP. Sample size tables for single-stage phase II designs.Stat Med2001;20:859–66), which was endorsed by the trial’s independent oversight committees.ResultsThe study included 12 patients with CML who were vaccinated and nine patients with CML who were unvaccinated as the control group. Both the vaccines and the electroporation were safe, with no new or unexpected toxicities. The evaluation adverse events of special interest (heart, bone marrow, renal) did not reveal safety concerns. TwoBCR–ABL(breakpoint cluster region–Abelson murine leukaemia viral oncogene homolog 1) responses were observed, both of which were defined as a major response, with one in each group. Two Wilms’ tumour antigen 1 (WT1) molecular responses were observed in the vaccinated group and one was observed in the control group. At an immunological level, the vaccine performed as expected.ConclusionsThe study met its primary decision-making target with one major molecular response inBCR–ABLtranscript levels. Overall, the data showed, in this clinical setting, the immunogenicity and safety of the vaccine.LimitationsThe study did not complete recruitment and there were multiple hurdles that contributed to this failure. This is disappointing given the robust induction immune responses againstWT1T-cell responses in 7 out of 10 evaluable patients.Future workEvaluation of the p.DOM–WT1 vaccines in AML remains attractive clinically, but it is unlikely to be feasible at this time. Combination of the DNA vaccine approach with strategies to expand T-cell responses with immunomodulatory antibodies is in development.Funding detailsThis project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council (MRC) and National Institute for Health Research (NIHR) partnership, and Bloodwise.

Download Full-text

Dose Effects of Recombinant Adenovirus Immunization in Rodents

Vaccines ◽

10.3390/vaccines7040144 ◽

2019 ◽

Vol 7 (4) ◽

pp. 144 ◽

Cited By ~ 1

Author(s):

Eric A. Weaver

Keyword(s):

T Cell ◽

Immune Responses ◽

Recombinant Adenovirus ◽

T Cell Responses ◽

Antibody Responses ◽

Vaccine Platform ◽

Cell Responses ◽

Immune Correlates ◽

Cell Immunity ◽

Dose Dependent

Recombinant adenovirus type 5 (rAd) has been used as a vaccine platform against many infectious diseases and has been shown to be an effective vaccine vector. The dose of the vaccine varies significantly from study to study, making it very difficult to compare immune responses and vaccine efficacy. This study determined the immune correlates induced by serial dilutions of rAd vaccines delivered intramuscularly (IM) and intranasally (IN) to mice and rats. When immunized IM, mice had substantially higher antibody responses at the higher vaccine doses, whereas, the IN immunized mice showed a lower response to the higher rAd vaccine doses. Rats did not show dose-dependent antibody responses to increasing vaccine doses. The IM immunized mice and rats also showed significant dose-dependent T cell responses to the rAd vaccine. However, the T cell immunity plateaued in both mice and rats at 109 and 1010 vp/animal, respectively. Additionally, the highest dose of vaccine in mice and rats did not improve the T cell responses. A final vaccine analysis using a lethal influenza virus challenge showed that despite the differences in the immune responses observed in the mice, the mice had very similar patterns of protection. This indicates that rAd vaccines induced dose-dependent immune responses, especially in IM immunized animals, and that immune correlates are not as predictive of protection as initially thought.

Download Full-text

Acute myeloid leukemia cell membrane-coated nanoparticles for cancer vaccination immunotherapy

Leukemia ◽

10.1038/s41375-021-01432-w ◽

2021 ◽

Author(s):

Daniel T. Johnson ◽

Jiarong Zhou ◽

Ashley V. Kroll ◽

Ronnie H. Fang ◽

Ming Yan ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

T Cell ◽

Cell Membrane ◽

Immune Responses ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Antigen Presenting Cells ◽

Vaccine Platform ◽

Antigen Presenting ◽

Acute Myeloid

AbstractCancer vaccines are promising treatments to prevent relapse after chemotherapy in acute myeloid leukemia (AML) patients, particularly for those who cannot tolerate intensive consolidation therapies. Here, we report the development of an AML cell membrane-coated nanoparticle (AMCNP) vaccine platform, in which immune-stimulatory adjuvant-loaded nanoparticles are coated with leukemic cell membrane material. This AMCNP vaccination strategy stimulates leukemia-specific immune responses by co-delivering membrane-associated antigens along with adjuvants to antigen-presenting cells. To demonstrate that this AMCNP vaccine enhances leukemia-specific antigen presentation and T cell responses, we modified a murine AML cell line to express membrane-bound chicken ovalbumin as a model antigen. AMCNPs were efficiently acquired by antigen-presenting cells in vitro and in vivo and stimulated antigen cross-presentation. Vaccination with AMCNPs significantly enhanced antigen-specific T cell expansion and effector function compared with control vaccines. Prophylactic vaccination with AMCNPs enhanced cellular immunity and protected against AML challenge. Moreover, in an AML post-remission vaccination model, AMCNP vaccination significantly enhanced survival in comparison to vaccination with whole leukemia cell lysates. Collectively, AMCNPs retained AML-specific antigens, elicited enhanced antigen-specific immune responses, and provided therapeutic benefit against AML challenge.

Download Full-text