INTRODUCTION:Despite the high global prevalence of Mycobacterium tuberculosis (Mtb) infection in humans, most infected individuals achieve a stable immunological equilibrium, without showing clinical signs and symptoms of tuberculosis (TB). Although the role of antibodies in TB is assumed to be relatively small compared with cell-mediated immunity, their role in TB has been documented in a few recent studies. METHODS:In this cross-sectional study, we quantitated antibody responses to Mtb antigens, lipoarabinomannan (LAM), and heparin-binding hemagglutinin adhesin (HBHA) by determining antigen-specific immunoglobulin A(IgA) and G(IgG) secretion levels using enzyme-linked immunosorbent assay in serum and saliva of pulmonary TB patients (PTB), their household contacts, and community controls (determined by QuantiFERON TB Gold assay QFT-test result). RESULTS:The HBHA-specific IgA levels were significantly higher in both saliva and serum in household contacts groups compared with PTB patients (P = 0.013, P = 0.023). Exposed contacts, who were QFT-negative, had higher serum HBHA-specific IgA responses compared with PTB patients (P = 0.04). QFT-negative household contacts and QFT-positive community controls showed higher HBHA and lipoarabinomannan-specific IgG responses (P = 0.006, P = 0.002, P = 0.0009, P = 0.006, respectively) than PTB patients. Generally, lipoarabinomannan and HBHA-specific IgA levels were significantly higher in saliva compared with serum (P < 0.0001) in all study groups. CONCLUSION:Overall, the observed higher levels of IgA and IgG in controls, and exposed but QFT-negative contacts suggest a correlation with, and perhaps a role for these antibodies in preventing the development of active TB. The findings highlighted the potential involvement of saliva IgA in the immune response to Mtb, underscoring the relevance of mucosal immunity in TB infection.

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), causes significant financial losses in the agricultural industry. Additionally, M. bovis transmission from animals to humans can result in zoonotic TB, especially in low- and middle-income countries (LMICs), highlighting the need to enhance One Health surveillance to mitigate this threat.Antibodies directed against a major mycobacterial cell wall component of M. leprae, phenolic glycolipid-I (PGL-I), have shown excellent performance in identifying M. leprae infection in humans and animals. In this study, we therefore investigated whether antibodies against M. bovis PGL similarly represent a useful biomarker for M. bovis infection in cattle.Comparing sera from naturally M. bovis-infected and the single intradermal comparative cervical tuberculin test (SICCT)-negative cattle, we assessed the potential of M. bovis PGL antibodies to identify this mycobacterial infection. Our results show that serum levels of anti-M. bovis PGL IgG and -IgM in M. bovis-infected cattle were significantly higher than in the SICCT-negative cattle. The sensitivity for anti-M. bovis PGL IgM in infected animals was, however, moderate (44.9 %) and the false-positive rate was 6.3 % in SICCT-negative cattle. Notably, vaccination with BCG- or heat-killed M. bovis did not affect serum levels of anti-M. bovis PGL IgM in cattle. Moreover, none of the 57 anti-M. bovis PGL-seropositive cattle tested positive in the anti-M. leprae PGL-I assay. This study shows for the first time that anti M. bovis PGL antibodies can be detected in infected cattle: anti-M. bovis PGL IgM is a highly specific, but moderately sensitive biomarker for M. bovis infection in cattle, showing potential for differentiate infected from vaccinated animals (DIVA). It could be a valuable component in a multi-biomarker approach for diagnosing bTB.

Peptide-based vaccines can be used to deliver tumor-specific antigens to dendritic cells (DCs), leading to tumor-directed T cell responses. We previously developed a peptide-peptide conjugate technology enabling in vivo cross-linking of pre-existing tetanus toxin-directed antibodies, facilitating antigen delivery to, and activation of DCs. To achieve this, multiple identical tetanus toxin-derived B cell epitopes (MTTEs) are conjugated to synthetically produce target antigens of choice. Herein, we describe the generation of a prostate cancer vaccine candidate (TENDU) based on this technology. It includes long synthetic peptides harboring epitopes (CD4 and CD8) from prostate-specific antigen (PAP) and prostate-specific membrane antigen (PSMA). The preclinical efficacy of TENDU was assessed in experimental systems, and safety was evaluated in a rabbit toxicity study and a human whole blood loop assay. We also report the first clinical safety assessment of TENDU. Experimental studies showed that prostate cancer patients mounted anti-MTTE antibodies in response to tetanus vaccination with recall T cell responses detected in two patients. Transgenic humanized HLA-DR4 mice displayed T cell responses and increased anti-MTTE IgG levels after vaccination with a peptide construct including an HLA-DR4 epitope. The vaccine candidate was found safe, and a positive correlation between T cell responses and anti-MTTE antibodies was noted in the first-in-human study.

The virtually monomorphic antigen presentation molecule HLA-E can present self- and non-self peptides to the NKG2A/CD94 co-receptor inhibitory complex expressed on natural killer (NK) cells and to T cell receptors (TCRs) expressed on T cells. HLA-E presents self-peptides to NKG2A/CD94 to regulate tissue homeostasis, whereas HLA-E restricted T cells mediate regulatory and cytotoxic responses toward pathogen-infected cells. In this study, we directly compared HLA-E/peptide recognition and signaling between NKG2A/CD94 and 2 HLA-E restricted TCRs that can recognize self-peptides or identical peptide mimics from the viral UL40 protein of cytomegalovirus using position substituted peptide variants. We show that position 7 is critical for interaction with NKG2A/CD94, whereas position 8 is important for interaction with the TCRs. The Arginine at position 5 of these peptides is an essential residue for recognition by both receptors. Thus, NKG2A/CD94 and TCRs have different requirements for recognition of peptides presented in HLA-E.

Background:Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is among the leading causes of death from an infectious agent among children worldwide. Children represent a particularly vulnerable population due to the greater challenges in diagnosis and the higher risk of progression to severe forms of the disease. However, whether different pediatric outcomes relate to distinct immunologic responses remains incompletely understood. Emerging data suggest that Mtb-specific humoral immune responses represent a correlate of protection against Mtb both following natural infection and vaccination. Methods:To determine if immune profiles can distinguish children across the spectrum from Mtb infection to TB disease, as well as children with TB from non-TB lower respiratory tract infection, we mapped the humoral immune response across a panel of 4 dozen Mtb antigens across children presenting with symptoms of active TB (ATB), children with evidence of latent TB infection (LTBI) and children exhibiting non-TB lower respiratory tract infection (non-TB LRTI). Using a custom Luminex assay, Mtb-specific antibody subclass/isotype, Fc receptor (FcR) binding profiles, and functions were profiled across the pediatric groups. Findings:A robust humoral immune response was observed in children with active TB compared to non-TB LRTI, marked by a strong IgA response, that exhibited high FcαR binding. Conversely, children with LTBI uniquely elicited Mtb-specific antibodies with enhanced opsinophagocytic FcγR2A binding, as well as a higher capacity to activate NK cells and neutrophils. Interpretation:There are significant differences in humoral immune profiles across the landscape of pediatric TB, potentially contributing to differential mycobacterial control, and highlighting biomarkers that could guide both diagnostic and therapeutic approaches. Funding:US National Institutes of Health.

Peptide-based vaccines can be used to deliver tumor-specific antigens to dendritic cells (DCs), leading to tumor-directed T cell responses. We previously developed a peptide-peptide conjugate technology enabling in vivo cross-linking of pre-existing tetanus toxin-directed antibodies, facilitating antigen delivery to, and activation of DCs. To achieve this, multiple identical tetanus toxin-derived B cell epitopes (MTTEs) are conjugated to synthetically produce target antigens of choice. Herein, we describe the generation of a prostate cancer vaccine candidate (TENDU) based on this technology. It includes long synthetic peptides harboring epitopes (CD4 and CD8) from prostate-specific antigen (PAP) and prostate-specific membrane antigen (PSMA). The preclinical efficacy of TENDU was assessed in experimental systems, and safety was evaluated in a rabbit toxicity study and a human whole blood loop assay. We also report the first clinical safety assessment of TENDU. Experimental studies showed that prostate cancer patients mounted anti-MTTE antibodies in response to tetanus vaccination with recall T cell responses detected in two patients. Transgenic humanized HLA-DR4 mice displayed T cell responses and increased anti-MTTE IgG levels after vaccination with a peptide construct including an HLA-DR4 epitope. The vaccine candidate was found safe, and a positive correlation between T cell responses and anti-MTTE antibodies was noted in the first-in-human study.

Human leukocyte antigen E (HLA-E) communicates cellular health to natural killer (NK) cells through presentation of peptides derived from the leader sequence of classical major histocompatibility complex class I (MHC-I), inhibiting NK cell activation and lysis of healthy cells. Besides this canonical role, HLA-E can also present peptides from pathogens such as Mycobacterium tuberculosis (Mtb) to T cells and can inhibit phagocytosis by engaging with LILRB1/2. To identify additional HLA-E binding surface molecules, we utilized a CRISPR/Cas9 activation screen with HLA-E tetramers, which identified Stabilin (STAB)1 and STAB2 as novel interactors. This interaction depended on the nature of the peptide/HLA-E complex, whereby high affinity peptides did not permit the interaction while low affinity peptides did. Functionally, expression of STAB1 or STAB2 on THP-1 monocytes increased phagocytic uptake of HLA-E coated microbeads. These results provide the first evidence of an interaction between Stabilin receptors and specific HLA-E conformations.

Tuberculosis (TB) remains a significant global health challenge, latently affecting around a quarter of the global population. The sole licensed TB vaccine, Mycobacterium bovis Bacillus Calmette-Guérin (BCG), shows variable efficacy, particularly among adolescents and adults, underscoring the pressing need for more effective vaccination strategies. The administration route is crucial for vaccine efficacy, and administration via the skin, being rich in immune cells, may offer advantages over conventional subcutaneous routes, which lack direct access to abundant antigen-presenting cells.This study compared the immunogenic effects of intradermal versus subcutaneous administration of a candidate TB vaccine delivering a Ag85B-ESAT6-Rv2034 (AER) multiphase fusion recombinant protein, in lipid-poly(D,L-lactic-co-glycolic acid) (lipid-PLGA) nanoparticles in mice. In-depth evaluation of immune responses in splenocytes was performed using 27-marker spectral flow cytometry. Both routes elicited significant T-cell responses. However, intradermal administration uniquely increased polyfunctional CD4+ and CD8+ T-cells producing IL-2, IFNγ, and TNFα, associated with protection against TB. Additionally, it significantly increased CD69+ B-cell counts and induced higher AER-specific antibody titers, particularly IgG2a. These results underscore the superior immunogenic potential of intradermal vaccine administration by effectively inducing immune cells associated with TB protection, highlighting its significance in the development of new vaccine strategies.

Cytomegalovirus (CMV) is one of the most common and relevant opportunistic pathogens in people who are immunocompromised, such as kidney transplant recipients (KTRs). The exact mechanisms underlying the disability of cytotoxic T cells to provide sufficient protection against CMV in people who are immunosuppressed have not been identified yet. Here, we performed in-depth metabolic profiling of CMV-specific CD8+ T cells in patients who are immunocompromised and show the development of metabolic dysregulation at the transcriptional, protein, and functional level of CMV-specific CD8+ T cells in KTRs with noncontrolled CMV infection. These dysregulations comprise impaired glycolysis and increased mitochondrial stress, which is associated with an intensified expression of the nicotinamide adenine dinucleotide nucleotidase (NADase) CD38. Inhibiting NADase activity of CD38 reinvigorated the metabolism and improved cytokine production of CMV-specific CD8+ T cells. These findings were corroborated in a mouse model of CMV infection under conditions of immunosuppression. Thus, dysregulated metabolic states of CD8+ T cells could be targeted by inhibiting CD38 to reverse hyporesponsiveness in individuals who fail to control chronic viral infection.

Background: Novel vaccines targeting the world’s deadliest pathogen Mycobacterium tuberculosis (Mtb) are urgently needed as the efficacy of the Bacillus Calmette–Guérin (BCG) vaccine in its current use is limited. HLA-E is a virtually monomorphic unconventional antigen presentation molecule, and HLA-E-restricted Mtb-specific CD8+ T cells can control intracellular Mtb growth, making HLA-E a promising vaccine target for Mtb. Methods: In this study, we evaluated the frequency and phenotype of HLA-E-restricted Mtb-specific CD4+/CD8+ T cells in the circulation and bronchoalveolar lavage fluid of two independent non-human primate (NHP) studies and from humans receiving BCG either intradermally or mucosally. Results: BCG vaccination followed by Mtb challenge in NHPs did not affect the frequency of circulating and local HLA-E–Mtb CD4+ and CD8+ T cells, and we saw the same in humans receiving BCG. HLA-E–Mtb T cell frequencies were significantly increased after Mtb challenge in unvaccinated NHPs, which was correlated with higher TB pathology. Conclusions: Together, HLA-E–Mtb-restricted T cells are minimally induced by BCG in humans and rhesus macaques (RMs) but can be elicited after Mtb infection in unvaccinated RMs. These results give new insights into targeting HLA-E as a potential immune mechanism against TB.

Tuberculosis (TB) continues to pose a global threat for millennia, currently affecting over 2 billion people and causing 10.6 million new cases and 1.3 million deaths annually. The only existing vaccine, Mycobacterium Bovis Bacillus Calmette-Guérin (BCG), provides highly variable and inadequate protection in adults and adolescents. This study explores newly developed subunit tuberculosis vaccines that use a multistage protein fusion antigen Ag85b-ESAT6-Rv2034 (AER). The protection efficacy, as well as in vivo induced immune responses, were compared for five vaccines: BCG; AER-CpG/MPLA mix; poly(D,L-lactic-co-glycolic acid) (PLGA); lipid-PLGA hybrid nanoparticles (NPs); and cationic pH-sensitive liposomes (the latter three delivering AER together with CpG and MPLA). All vaccines, except the AER-adjuvant mix, induced protection in Mycobacterium tuberculosis (Mtb)-challenged C57/Bl6 mice as indicated by a significant reduction in bacterial burden in lungs and spleens of the animals. Four AER-based vaccines significantly increased the number of circulating multifunctional CD4+ and CD8+ T-cells producing IL-2, IFNγ, and TNFα, exhibiting a central memory phenotype. Furthermore, AER-based vaccines induced an increase in CD69+ B-cell counts as well as high antigen-specific antibody titers. Unexpectedly, none of the observed immune responses were associated with the bacterial burden outcome, such that the mechanism responsible for the observed vaccine-induced protection of these vaccines remains unclear. These findings suggest the existence of non-classical protective mechanisms for Mtb infection, which could, once identified, provide interesting targets for novel vaccines.

HLA-E molecules can present self- and pathogen-derived peptides to both natural killer (NK) cells and T cells. T cells that recognize HLA-E peptides via their T cell receptor (TCR) are termed donor-unrestricted T cells due to restricted allelic variation of HLA-E. The composition and repertoire of HLA-E TCRs is not known so far. We performed TCR sequencing on CD8+ T cells from 21 individuals recognizing HLA-E tetramers (TMs) folded with two Mtb-HLA-E-restricted peptides. We sorted HLA-E Mtb TM+ and TM- CD8+ T cells directly ex vivo and performed bulk RNA-sequencing and single-cell TCR sequencing. The identified TCR repertoire was diverse and showed no conservation between and within individuals. TCRs selected from our single-cell TCR sequencing data could be activated upon HLA-E/peptide stimulation, although not robust, reflecting potentially weak interactions between HLA-E peptide complexes and TCRs. Thus, HLA-E-Mtb-specific T cells have a highly diverse TCR repertoire.

IntroductionTuberculosis (TB) is the deadliest infectious disease worldwide and novel vaccines are urgently needed. HLA-E is a virtually monomorphic antigen presentation molecule and is not downregulated upon HIV co-infection. HLA-E restricted Mtb specific CD8+ T cells are present in the circulation of individuals with active TB (aTB) and Mtb infection (TBI) with or without HIV co-infection, making HLA-E restricted T cells interesting vaccination targets for TB.MethodsHere, we performed in-depth phenotyping of HLA-E/Mtb specific and total T cell populations in individuals with TBI and in individuals with aTB or TBI and HIV using HLA-E/Mtb tetramers.Results and DiscussionWe show that HIV co-infection is the main driver in changing the memory distribution of HLA-E/Mtb specific CD4+ and CD8+ T cell subsets. HLA-E/Mtb specific CD4+ and CD8+ T cells were found to circulate with comparable frequencies in all individuals and displayed expression of KLRG1, PD-1 and 2B4 similar to that of total T cells. The presence of HLA-E/Mtb specific T cells in individuals with aTB and TBI highlights the potential of HLA-E as a vaccine target for TB.

Tuberculosis (TB) has been and still is a global emergency for centuries. Prevention of disease through vaccination would have a major impact on disease prevalence, but the only available current vaccine, BCG, has insufficient impact. In this article, a novel subunit vaccine against TB was developed, using the Ag85B-ESAT6Rv2034 fusion antigen, two adjuvants - CpG and MPLA, and a cationic pH-sensitive liposome as a delivery system, representing a new TB vaccine delivery strategy not previously reported for TB. In vitro in human dendritic cells (DCs), the adjuvanted formulation induced a significant increase in the production of (innate) cytokines and chemokines compared to the liposome without additional adjuvants. In vivo, the new vaccine administrated subcutaneously significantly reduced Mycobacterium tuberculosis (Mtb) bacterial load in the lungs and spleens of mice, significantly outperforming results from mice vaccinated with the antigen mixed with adjuvants without liposomes. In-depth analysis underpinned the vaccine's effectiveness in terms of its capacity to induce polyfunctional CD4+ and CD8+ T-cell responses, both considered essential for controlling Mtb infection. Also noteworthy was the differential abundance of various CD69+ B-cell subpopulations, which included IL17-A-producing B-cells. The vaccine stimulated robust antigen-specific antibody titers, further extending its potential as a novel protective agent against TB.

Viral-vectored vaccines are highly amenable for respiratory mucosal delivery as a means of inducing much-needed mucosal immunity at the point of pathogen entry. Unfortunately, current monovalent viral-vectored tuberculosis (TB) vaccine candidates have failed to demonstrate satisfactory clinical protective efficacy. As such, there is a need to develop next-generation viral-vectored TB vaccine strategies which incorporate both vaccine antigen design and delivery route. In this study, we have developed a trivalent chimpanzee adenoviral-vectored vaccine to provide protective immunity against pulmonary TB through targeting antigens linked to the three different growth phases (acute/chronic/dormancy) of Mycobacterium tuberculosis (M.tb) by expressing an acute replication-associated antigen, Ag85A, a chronically expressed virulence-associated antigen, TB10.4, and a dormancy/resuscitation-associated antigen, RpfB. Single-dose respiratory mucosal immunization with our trivalent vaccine induced robust, sustained tissue-resident multifunctional CD4+ and CD8+ T-cell responses within the lung tissues and airways, which were further quantitatively and qualitatively improved following boosting of subcutaneously BCG-primed hosts. Prophylactic and therapeutic immunization with this multivalent trivalent vaccine in conventional BALB/c mice provided significant protection against not only actively replicating M.tb bacilli but also dormant, non-replicating persisters. Importantly, when used as a booster, it also provided marked protection in the highly susceptible C3HeB/FeJ mice, and a single respiratory mucosal inoculation was capable of significant protection in a humanized mouse model. Our findings indicate the great potential of this next-generation TB vaccine strategy and support its further clinical development for both prophylactic and therapeutic applications.

The immune checkpoint NKG2A/CD94 is a promising target for cancer immunotherapy, and its ligand major histocompatibility complex E (MHC-E) is frequently upregulated in cancer. NKG2A/CD94-mediated inhibition of lymphocytes depends on the presence of specific leader peptides in MHC-E, but when and where they are presented in situ is unknown. We apply a nanobody specific for the Qdm/Qa-1b complex, the NKG2A/CD94 ligand in mouse, and find that presentation of Qdm peptide depends on every member of the endoplasmic reticulum-resident peptide loading complex. With a turnover rate of 30 min, the Qdm peptide reflects antigen processing capacity in real time. Remarkably, Qdm/Qa-1b complexes require inflammatory signals for surface expression in situ, despite the broad presence of Qa-1b molecules in homeostasis. Furthermore, we identify LILRB1 as a functional inhibition receptor for MHC-E in steady state. These data provide a molecular understanding of NKG2A blockade in immunotherapy and assign MHC-E as a convergent ligand for multiple immune checkpoints.

Immune checkpoint therapy (ICT) has the potency to eradicate cancer but the mechanisms that determine effective versus non-effective therapy-induced immune responses are not fully understood. Here, using high-dimensional single-cell profiling we examined whether T cell states in the blood circulation could predict responsiveness to a combined ICT, sequentially targeting OX40 costimulatory and PD-1 inhibitory pathways, which effectively eradicated syngeneic mouse tumors. Unbiased assessment of transcriptomic alterations by single-cell RNA sequencing and profiling of cell-surface protein expression by mass cytometry revealed unique activation states for therapy-responsive CD4 + and CD8 + T cells. Effective ICT elicited T cells with dynamic expression of distinct NK cell and chemokine receptors, and these cells were systemically present in lymphoid tissues and in the tumor. Moreover, NK cell receptor-expressing CD8 + T cells were also present in the peripheral blood of immunotherapy-responsive cancer patients. Targeting of the NK cell and chemokine receptors in tumor-bearing mice showed their functional importance for therapy-induced anti-tumor immunity. These findings provide a better understanding of ICT and highlight the use of dynamic biomarkers on effector CD4 + and CD8 + T cells to improve cancer immunotherapy.

IntroductionPlacental transfer of maternal antibodies is essential for neonatal immunity over the first months of life. In the setting of maternal HIV infection, HIV-exposed uninfected (HEU) infants are at higher risk of developing severe infections, including active tuberculosis (TB). Given our emerging appreciation for the potential role of antibodies in the control of Mycobacterium tuberculosis (Mtb), the bacteria that causes TB, here we aimed to determine whether maternal HIV status altered the quality of Mtb-specific placental antibody transfer. MethodsAntigen-specific antibody systems serology was performed to comprehensively characterize the Mtb-specific humoral immune response in maternal and umbilical cord blood from HIV infected and uninfected pregnant people in Uganda. ResultsSignificant differences were noted in overall antibody profiles in HIV positive and negative maternal plasma, resulting in heterogeneous transfer of Mtb-specific antibodies. Altered antibody transfer in HIV infected dyads was associated with impaired binding to IgG Fc-receptors, which was directly linked to HIV viral loads and CD4 counts. ConclusionsThese results highlight the importance of maternal HIV status on antibody transfer, providing clues related to alterations in transferred maternal immunity that may render HEU infants more vulnerable to TB than their HIV-unexposed peers.

The immune checkpoint NKG2A/CD94 is a promising target for cancer immunotherapy and its ligand MHC-E is frequently upregulated in cancer. NKG2A/CD94-mediated inhibition of lymphocytes depends on the presence of specific leader peptides in MHC-E, but when and where they are presented in situ is unknown. We developed a nanobody specific for the Qdm/Qa-1b complex, the NKG2A/CD94 ligand in mouse, and found that presentation of Qdm peptide depended on every member of the ER-resident peptide loading complex. With a turnover rate of 30 minutes, the Qdm peptide reflects antigen processing capacity in real-time. Remarkably, Qdm/Qa-1b complexes required inflammatory signals for surface expression in situ, despite the broad presence of Qa-1b molecules in homeostasis. Furthermore, we identified LILRB1 as a functional inhibition receptor for MHC-E in steady state. These data provide a molecular understanding of NKG2A blockade in immunotherapy and assign MHC-E as a convergent ligand for multiple immune checkpoints.

Vaccines based on synthetic long peptides, targeting immunogenic region(s) of tumor associated proteins, are becoming increasingly used in oncology. Synthetic long peptides require endogenous processing by antigen presenting cells to be presented to T cells, thus reducing the risk of antigen presentation by non-professional antigen presenting cells lacking co-stimulation receptors. The formulation and delivery strategies require further development as peptides degrade rapidly in vivo. Herein we make use of a novel targeting and adjuvant strategy to deliver peptides to antigen presenting cells. Tumor-derived peptides are coupled to a B cell epitope element (the MTTE sequence, a tetanus toxin-derived universal B cell epitope) which induce the formation of peptide-conjugate -complex formation in vivo. This strategy, further named the TET technology, makes use of pre-existing antibodies to improve the bioavailability, stability, and immunogenicity of tumor-derived peptide therapeutics. The TET technology is here used in TENDU, a first-in-class prostate cancer vaccine candidate. TENDU incorporates selected immunogenic prostate cancer epitopes derived from PAP and PSMA. The peptides are designed to include proteasome processing elements along with a unique TAP translocation element. To assess the design, a conjugate with a known DR4 epitope embedded in a longer peptide was evaluated for T cell priming in HLA-DR4 animals and displayed T cell expansion in response to a prime/boost vaccination. To ensure that prostate cancer patients respond with antibodies to the MTTE sequence after a tetanus toxoid (TTd) vaccination, both healthy controls and patients (age and gender matched) were vaccinated using a TTd-based vaccine regime. Anti-MTTE antibody titers were evaluated. Patients and HCs responded with increased IgG1 type antibodies against MTTE after TTd vaccination. Safety with regards to hypersensitivity in response to the vaccine platform was evaluated in rabbits and a human whole blood loop system without any identified adverse events. Based on preclinical data the no-observed-adverse-effect level (NOAEL) was set to 1440µg/dose. The vaccine is currently assessed in a First-in-Human clinical study (TENDU-101, 3+3 study design) in patients with relapse after a primary radical prostatectomy. No safety concerns were noted at the lowest dose level and the 400 µg dose cohort has been initiated with the first patient dosed with no reported adverse events. The study’s primary endpoints are safety and tolerability. Secondary endpoints involve cellular immune responses, systemic PSA and PAP levels along with dose level selection. In conclusion, TENDU, a first-in-class vaccine candidate is under clinical development evaluating safety and tolerability for the TET vaccine platform technology. Citation Format: Erika Fletcher, Iliana Kerzeli, Robert Cordfunke, Aikaterini Nasi, Gunilla Törnqvist, Robert Valentijn, Frida Lindqvist, Inken Dillmann, Martin Lord, Neanke Bouwman, Jacques Neefjes, Natasja Dolezal, Kees Franken, Stephanie McArdle, Murrium Ahmad, Silvia Johansson, Ferry Ossendorp, Michael Haggman, Wolfgang Lilleby, Gustav Ullenhag, Sam Ladjevardi, Justyna Jarblad, Jan Wouter Drijfhout, Sara Mangsbo. Promoting immunogenicity of synthetic long peptide vaccines based on in vivo IgG complex formation: Preclinical evaluation and clinical entry of the TET platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4160.