Pathogen-derived Antigens Research Articles

Temperature sensitivity of bat antibodies links metabolic state of bats with antigen-recognition diversity

The bat immune system features multiple unique properties such as dampened inflammatory responses and increased tissue protection, explaining their long lifespan and tolerance to viral infections. Here, we demonstrated that body temperature fluctuations corresponding to different physiological states in bats exert a large impact on their antibody repertoires. At elevated temperatures typical for flight, IgG from the bat species Myotis myotis and Nyctalus noctula show elevated antigen binding strength and diversity, recognizing both pathogen-derived antigens and autoantigens. The opposite is observed at temperatures reflecting inactive physiological states. IgG antibodies of human and other mammals, or antibodies of birds do not appear to behave in a similar way. Importantly, diversification of bat antibody specificities results in preferential recognition of damaged endothelial and epithelial cells, indicating an anti-inflammatory function. The temperature-sensitivity of bat antibodies is mediated by the variable regions of immunoglobulin molecules. Additionally, we uncover specific molecular features of bat IgG, such as low thermodynamic stability and implication of hydrophobic interactions in antigen binding as well as high prevalence of polyreactivity. Overall, our results extend the understanding of bat tolerance to disease and inflammation and highlight the link between metabolism and immunity.

Direct presentation of inflammation-associated self-antigens by thymic innate-like T cells induces elimination of autoreactive CD8+ thymocytes

Upregulation of diverse self-antigens that constitute components of the inflammatory response overlaps spatially and temporally with the emergence of pathogen-derived foreign antigens. Therefore, discrimination between these inflammation-associated self-antigens and pathogen-derived molecules represents a unique challenge for the adaptive immune system. Here, we demonstrate that CD8+ T cell tolerance to T cell-derived inflammation-associated self-antigens is efficiently induced in the thymus and supported by redundancy in cell types expressing these molecules. In addition to thymic epithelial cells, this included thymic eosinophils and innate-like T cells, a population that expressed molecules characteristic for all major activated T cell subsets. We show that direct T cell-to-T cell antigen presentation by minute numbers of innate-like T cells was sufficient to eliminate autoreactive CD8+ thymocytes. Tolerance to such effector molecules was of critical importance, as its breach caused by decreased thymic abundance of a single model inflammation-associated self-antigen resulted in autoimmune elimination of an entire class of effector T cells.

Functional and biological implications of clonotypic diversity among human donor-unrestricted T cells.

Tcells express a T-cell receptor (TCR) heterodimer that is the product of germline rearrangement and junctional editing resulting in immense clonotypic diversity. The generation of diverse TCR repertoires enables the recognition of pathogen-derived peptide antigens presented by polymorphic major histocompatibility complex (MHC) molecules. However, Tcells also recognize nonpeptide antigens through nearly monomorphic antigen-presenting systems, such as cluster of differentiation 1 (CD1), MHC-related protein 1 (MR1) and butyrophilins (BTNs). This potential for shared immune responses across genetically diverse populations led to their designation as donor-unrestricted Tcells (DURTs). As might be expected, some CD1-, MR1- and BTN-restricted Tcells express a TCR that is conserved across unrelated individuals. However, several recent studies have reported unexpected diversity among DURT TCRs, and increasing evidence suggests that this diversity has functional consequences. Recent reports also challenge the dogma that immune cells are either innate or adaptive and suggest that DURT TCRs may act in both capacities. Here, we review this evidence and propose an expanded view of the role for clonotypic diversity among DURTs in humans, including new perspectives on how DURT TCRs may integrate their adaptive and innate immune functions.

Lyophilized lymph nodes for improved delivery of chimeric antigen receptor T cells.

Lymph nodes are crucial organs of the adaptive immune system, orchestrating T cell priming, activation and tolerance. T cell activity and function are highly regulated by lymph nodes, which have a unique structure harbouring distinct cells that work together to detect and respond to pathogen-derived antigens. Here we show that implanted patient-derived freeze-dried lymph nodes loaded with chimeric antigen receptor T cells improve delivery to solid tumours and inhibit tumour recurrence after surgery. Chimeric antigen receptor T cells can be effectively loaded into lyophilized lymph nodes, whose unaltered meshwork and cytokine and chemokine contents promote chimeric antigen receptor T cell viability and activation. In mouse models of cell-line-derived human cervical cancer and patient-derived pancreatic cancer, delivery of chimeric antigen receptor T cells targeting mesothelin via the freeze-dried lymph nodes is more effective in preventing tumour recurrence when compared to hydrogels containing T-cell-supporting cytokines. This tissue-mediated cell delivery strategy holds promise for controlled release of various cells and therapeutics with long-term activity and augmented function.

Recent Scientific Advancements towards a Vaccine against Group A Streptococcus.

Group A Streptococcus (GAS), or Streptococcus pyogenes, is a gram-positive bacterium that extensively colonises within the human host. GAS is responsible for causing a range of human infections, such as pharyngitis, impetigo, scarlet fever, septicemia, and necrotising fasciitis. GAS pathogens have the potential to elicit fatal autoimmune sequelae diseases (including rheumatic fever and rheumatic heart diseases) due to recurrent GAS infections, leading to high morbidity and mortality of young children and the elderly worldwide. Antibiotic drugs are the primary method of controlling and treating the early stages of GAS infection; however, the recent identification of clinical GAS isolates with reduced sensitivity to penicillin-adjunctive antibiotics and increasing macrolide resistance is an increasing threat. Vaccination is credited as the most successful medical intervention against infectious diseases since it was discovered by Edward Jenner in 1796. Immunisation with an inactive/live-attenuated whole pathogen or selective pathogen-derived antigens induces a potent adaptive immunity and protection against infectious diseases. Although no GAS vaccines have been approved for the market following more than 100 years of GAS vaccine development, the understanding of GAS pathogenesis and transmission has significantly increased, providing detailed insight into the primary pathogenic proteins, and enhancing GAS vaccine design. This review highlights recent advances in GAS vaccine development, providing detailed data from preclinical and clinical studies across the globe for potential GAS vaccine candidates. Furthermore, the challenges and future perspectives on the development of GAS vaccines are also described.

Recombinant antigen delivery to dendritic cells as a way to improve vaccine design.

Dendritic cells are central to the development of immunity, as they are specialized in initiating antigen-specific immune responses. In this review, we briefly present the existing knowledge on dendritic cell biology and how their division in different dendritic cell subsets may impact the development of immune responses. In addition, we explore the use of chimeric monoclonal antibodies that bind to dendritic cell surface receptors, with an emphasis on the C-type lectin family of endocytic receptors, to deliver antigens directly to these cells. Promising preclinical studies have shown that it is possible to modulate the development of immune responses to different pathogens when monoclonal antibodies fused to pathogen-derived antigens are used to deliver the antigen to different subsets of dendritic cells. This approach can be used to improve the efficacy of vaccines against different pathogens.

A Novel Cell-based Luciferase Reporter Platform for the Development and Characterization of T-Cell Redirecting Therapies and Vaccine Development.

T-cell immunotherapies are promising strategies to generate T-cell responses towards tumor-derived or pathogen-derived antigens. Adoptive transfer of T cells genetically modified to express antigen receptor transgenes has shown promise for the treatment of cancer. However, the development of T-cell redirecting therapies relies on the use of primary immune cells and is hampered by the lack of easy-to-use model systems and sensitive readouts to facilitate candidate screening and development. Particularly, testing T-cell receptor (TCR)-specific responses in primary T cells and immortalized T cells is confounded by the presence of endogenous TCR expression which results in mixed alpha/beta TCR pairings and compresses assay readouts. Herein, we describe the development of a novel cell-based TCR knockout (TCR-KO) reporter assay platform for the development and characterization of T-cell redirecting therapies. CRISPR/Cas9 was used to knockout the endogenous TCR chains in Jurkat cells stably expressing a human interleukin-2 promoter-driven luciferase reporter gene to measure TCR signaling. Reintroduction of a transgenic TCR into the TCR-KO reporter cells results in robust antigen-specific reporter activation compared with parental reporter cells. The further development of CD4/CD8 double-positive and double-negative versions enabled low-avidity and high-avidity TCR screening with or without major histocompatibility complex bias. Furthermore, stable TCR-expressing reporter cells generated from TCR-KO reporter cells exhibit sufficient sensitivity to probe in vitro T-cell immunogenicity of protein and nucleic acid-based vaccines. Therefore, our data demonstrated that TCR-KO reporter cells can be a useful tool for the discovery, characterization, and deployment of T-cell immunotherapy.

Hyperoxidized Species of Heme Have a Potent Capacity to Induce Autoreactivity of Human IgG Antibodies.

The interaction of some human antibodies with heme results in posttranslational acquisition of binding to various self- and pathogen-derived antigens. The previous studies on this phenomenon were performed with oxidized heme (Fe3+). In the present study, we elucidated the effect of other pathologically relevant species of heme, i.e., species that were formed after contact of heme with oxidizing agents such as hydrogen peroxide, situations in which heme's iron could acquire higher oxidation states. Our data reveal that hyperoxidized species of heme have a superior capacity to heme (Fe3+) in triggering the autoreactivity of human IgG. Mechanistic studies demonstrated that oxidation status of iron was of critical importance for the heme's effect on antibodies. We also demonstrated that hyperoxidized heme species interacted at higher affinities with IgG and that this binding occurred through a different mechanism as compared to heme (Fe3+). Regardless of their profound functional impact on the antigen-binding properties of antibodies, hyperoxidized species of heme did not affect Fc-mediated functions of IgG, such as binding to the neonatal Fc receptor. The obtained data contribute to a better understanding of the pathophysiological mechanism of hemolytic diseases and of the origin of elevated antibody autoreactivity in patients with some hemolytic disorders.

An epithelial cell-derived metabolite tunes immunoglobulin A secretion by gut-resident plasma cells.

Immunoglobulin A (IgA) secretion by plasma cells, terminally differentiated B cells residing in the intestinal lamina propria, assures microbiome homeostasis and protects the host against enteric infections. Exposure to diet-derived and commensal-derived signals provides immune cells with organizing cues that instruct their effector function and dynamically shape intestinal immune responses at the mucosal barrier. Recent data have described metabolic and microbial inputs controlling T cell and innate lymphoid cell activation in the gut; however, whether IgA-secreting lamina propria plasma cells are tuned by local stimuli is completely unknown. Although antibody secretion is considered to be imprinted during B cell differentiation and therefore largely unaffected by environmental changes, a rapid modulation of IgA levels in response to intestinal fluctuations might be beneficial to the host. In the present study, we showed that dietary cholesterol absorption and commensal recognition by duodenal intestinal epithelial cells lead to the production of oxysterols, evolutionarily conserved lipids with immunomodulatory functions. Using conditional cholesterol 25-hydroxylase deleter mouse line we demonstrated that 7α,25-dihydroxycholesterol from epithelial cells is critical to restrain IgA secretion against commensal- and pathogen-derived antigens in the gut. Intestinal plasma cells sense oxysterols via the chemoattractant receptor GPR183 and couple their tissue positioning with IgA secretion. Our findings revealed a new mechanism linking dietary cholesterol and humoral immune responses centered around plasma cell localization for efficient mucosal protection.

The structure of songbird MHC class I reveals antigen binding that is flexible at the N-terminus and static at the C-terminus.

Long-distance migratory animals such as birds and bats have evolved to withstand selection imposed by pathogens across the globe, and pathogen richness is known to be particularly high in tropical regions. Immune genes, so-called Major Histocompatibility Complex (MHC) genes, are highly duplicated in songbirds compared to other vertebrates, and this high MHC diversity has been hypothesised to result in a unique adaptive immunity. To understand the rationale behind the evolution of the high MHC genetic diversity in songbirds, we determined the structural properties of an MHC class I protein, Acar3, from a long-distance migratory songbird, the great reed warbler Acrocephalus arundinaceus (in short: Acar). The structure of Acar3 was studied in complex with pathogen-derived antigens and shows an overall antigen presentation similar to human MHC class I. However, the peptides bound to Acar3 display an unusual conformation: Whereas the N-terminal ends of the peptides display enhanced flexibility, the conformation of their C-terminal halves is rather static. This uncommon peptide-binding mode in Acar3 is facilitated by a central Arg residue within the peptide-binding groove that fixes the backbone of the peptide at its central position, and potentially permits successful interactions between MHC class I and innate immune receptors. Our study highlights the importance of investigating the immune system of wild animals, such as birds and bats, to uncover unique immune mechanisms which may neither exist in humans nor in model organisms.

Prognosis and immunological role of HLA-DMA in lung adenocarcinoma

<b>Background:</b> HLA-DMA presents pathogen-derived antigens to CD4+ and CD8+ T cells, respectively, and plays a significant part in initiating the immune response. So far, the impact of HLA expression on the prognosis of BC cells is controversial, because few studies have shown that the expressions of some HLA genes are related to the improvement of the survival rate. Up till now, however, the relationship between HLA-DMA and LUAD has not yet been assessed. <b>Methods:</b> We analyzed the TCGA database and assessed the prognostic value of HLA-DMA in LUAD. We conducted the Kruskal–Wallis and Wilcoxon signed-rank test and utilized logistic regression to assess the role of clinical-pathologic characteristics and HLA-DMA expression. Kaplan–Meier method and the multivariate and univariate Cox regression were also used for evaluating the prognosis-related factors of LUAD. GSEA was used to identify HLA-DMA-related key pathways. The ssGSEA of the TCGA data was used to investigate the correlations between HLA-DMA and cancer immune cell infiltration. <b>Results:</b> Low HLA-DMA expression was related to poorer disease-specific survival (DSS) and overall survival (OS) of LUAD patients. GSEA revealed that HLA-DMA was tightly interrelated with an immune response by the reactome activation of anterior hox genes in hindbrain development during the early embryogenesis signaling pathway. The expression of HLA-DMA was positively associated with cytotoxic cell infiltration and negatively related to the Th2 cell infiltration according to the ssGSEA. Western blotting and the CCK-8 assay showed that KD-HLA-DMA could significantly increase the proliferation of A549 cells and significantly reduce cell pyroptosis. <b>Conclusion:</b> All the observations implied that HLA-DMA was associated with patient prognosis and immune infiltration in LUAD.

Immunization With Lipopolysaccharide-Activated Dendritic Cells Generates a Specific CD8+ T Cell Response That Confers Partial Protection Against Infection With Trypanosoma cruzi.

Lipopolysaccharide (LPS) induces the activation of dendritic cells (DCs) throughout the engagement of toll-like receptor 4. LPS-activated DCs show increased capacity to process and present pathogen-derived antigens to activate naïve T cells. DCs-based vaccines have been successfully used to treat some cancer types, and lately transferred to the field of infectious diseases, in particular against HIV. However, there is no vaccine or DC therapy for any parasitic disease that is currently available. The immune response against Trypanosoma cruzi substantially relies on T cells, and both CD4+ and CD8+ T lymphocytes are required to control parasite growth. Here, we develop a vaccination strategy based on DCs derived from bone marrow, activated with LPS and loaded with TsKb20, an immunodominant epitope of the trans-sialidase family of proteins. We extensively characterized the CD8+ T cell response generated after immunization and compared three different readouts: a tetramer staining, ELISpot and Activation-Induced Marker (AIM) assays. To our knowledge, this work shows for the first time a proper set of T cell markers to evaluate specific CD8+ T cell responses in mice. We also show that our immunization scheme confers protection against T. cruzi, augmenting survival and reducing parasite burden in female but not male mice. We conclude that the immunization with LPS-activated DCs has the potential to prime significant CD8+ T cell responses in C57BL/6 mice independently of the sex, but this response will only be effective in female, possibly due to mice sexual dimorphisms in the response generated against T. cruzi.

Cytotoxic CD4+ T cells driven by T-cell intrinsic IL-18R/MyD88 signaling predominantly infiltrate Trypanosoma cruzi-infected hearts

Increasing attention has been directed to cytotoxic CD4+ T cells (CD4CTLs) in different pathologies, both in humans and mice. The impact of CD4CTLs in immunity and the mechanisms controlling their generation, however, remain poorly understood. Here, we show that CD4CTLs abundantly differentiate during mouse infection with the intracellular parasite Trypanosoma cruzi. CD4CTLs display parallel kinetics to Th1 cells in the spleen, mediate specific cytotoxicity against cells presenting pathogen-derived antigens and express immunoregulatory and/or exhaustion markers. We demonstrate that CD4CTL absolute numbers and activity are severely reduced in both Myd88-/- and Il18ra-/- mice. Of note, the infection of mixed-bone marrow chimeras revealed that wild-type (WT) but not Myd88-/- cells transcribe the CD4CTL gene signature and that Il18ra-/- and Myd88-/- CD4+ T cells phenocopy each other. Moreover, adoptive transfer of WT CD4+GzB+ T cells to infected Il18ra-/- mice extended their survival. Importantly, cells expressing the CD4CTL phenotype predominate among CD4+ T cells infiltrating the infected mouse cardiac tissue and are increased in the blood of Chagas patients, in which the frequency of CD4CTLs correlates with the severity of cardiomyopathy. Our findings describe CD4CTLs as a major player in immunity to a relevant human pathogen and disclose T-cell intrinsic IL-18R/MyD88 signaling as a key pathway controlling the magnitude of the CD4CTL response.

Microbial antigen in human milk: a natural vaccine?

Microbial antigen in human milk: a natural vaccine?

Development of an Antigen Delivery System for a B Cell-Targeted Vaccine as an Alternative to Dendritic Cell-Targeted Vaccines

Vaccines have contributed to the prevention of infectious diseases for a long time. Pathogen-derived antigens and adjuvants in vaccine formulations stimulate immune cells to elicit humoral and cellular immune responses against pathogens. Achieving highly immune responses with decreased adverse effects requires the development of a system that can deliver antigens to specific immune cells. Dendritic cells (DCs) are well-known professional antigen presenting cells (APCs) that initiate acquired immune responses by presenting antigens to T cells. Accordingly, DC-targeted vaccines have been investigated and applied in clinical trials for the treatment of infectious diseases and for chronic diseases such as cancers. In addition to DCs, B lymphocytes are regarded as professional APCs despite their primary role in humoral immunity. Therefore, B cell-targeted vaccines are also expected to elicit both humoral and cellular immune responses. In this review we summarize the basic functions of DCs and B cells as APCs. We also provide information on DC and B cell targeted vaccines in preclinical and clinical settings. Finally, we introduce our novel antigen delivery system that targets splenic marginal zone B cells and the ability of this system to act as a novel vaccine that elicits both humoral and cellular immune responses.

Activation of the Innate Immune Checkpoint CLEC5A on Myeloid Cells in the Absence of Danger Signals Modulates Macrophages' Function but Does Not Trigger the Adaptive T Cell Immune Response.

C-Type lectin receptor 5A (CLEC5A) is a spleen tyrosine kinase- (Syk-) coupled pattern recognition receptor expressed on myeloid cells and involved in the innate immune response to viral and bacterial infections. Activation of the CLEC5A receptor with pathogen-derived antigens leads to a secretion of proinflammatory mediators such as TNF-α and IL-6 that may provoke a systemic cytokine storm, and CLEC5A gene polymorphisms are associated with the severity of DV infection. In addition, the CLEC5A receptor was mentioned in the context of noninfectious disorders like chronic obstructive pulmonary disease (COPD) or arthritis. Altogether, CLEC5A may be considered as an innate immune checkpoint capable to amplify proinflammatory signals, and this way contributes to infection or to aseptic inflammation. In this study, we determined CLEC5A receptor expression on different macrophage subsets (in vitro and ex vivo) and the functional consequences of its activation in aseptic conditions. The CLEC5A surface expression appeared the highest on proinflammatory M1 macrophages while intermediate on tumor-associated phenotypes (M2c or TAM). In contrast, the CLEC5A expression on ex vivo-derived alveolar macrophages from healthy donors or macrophages from ovarian cancer patients was hardly detectable. Targeting CLEC5A on noninflammatory macrophages with an agonistic α-CLEC5A antibody triggered a release of proinflammatory cytokines, resembling a response to dengue virus, and led to phenotypic changes in myeloid cells that may suggest their reprogramming towards a proinflammatory phenotype, e.g., upregulation of CD80 and downregulation of CD163. Interestingly, the CLEC5A agonist upregulated immune-regulatory molecules like CD206, PD-L1, and cytokines like IL-10, macrophage-derived chemokine (MDC/CCL22), and thymus and activation chemokine (TARC/CCL17) which are associated with an anti-inflammatory or a protumorigenic macrophage phenotype. In the absence of concomitant pathogenic or endogenous danger signals, the CLEC5A receptor activation did not amplify an autologous T cell response, which may represent a protective innate mechanism to avoid an undesirable autoimmune adaptive response.

A short HLA-DRA isoform binds the HLA-DR2 heterodimer on the outer domain of the peptide-binding site

The human leukocyte antigen (HLA) locus encodes a large group of proteins governing adaptive and innate immune responses. Among them, HLA class II proteins form α/β heterodimers on the membrane of professional antigen-presenting cells (APCs), where they display both, self and pathogen-derived exogenous antigens to CD4+ T lymphocytes. We have previously shown that a shorter HLA-DRA isoform (sHLA-DRA) lacking 25 amino acids can be presented onto the cell membrane via binding to canonical HLA-DR2 heterodimers. Here, we employed atomistic molecular dynamics simulations to decipher the binding position of sHLA-DRA and its structural impact on functional regions of the HLA-DR2 molecule. We show that a loop region exposed only in the short isoform (residues R69 to G83) is responsible for binding to the outer domain of the HLA-DR2 peptide-binding site, and experimentally validated the critical role of F76 in mediating such interaction. Additionally, sHLA-DRA allosterically modifies the peptide-binding pocket conformation. In summary, this study unravels key molecular mechanisms underlying sHLA-DRA function, providing important insights into the role of full-length proteins in structural modulation of HLA class II receptors.

A Quantum Vaccinomics Approach Based on Protein-Protein Interactions.

Vaccines are the most effective preventive intervention to reduce the impact of infectious diseases worldwide. In particular, tick-borne diseases represent a growing burden for human and animal health worldwide and vaccines are the most effective and environmentally sound approach for the control of vector infestations and pathogen transmission. However, the development of effective vaccines for the control of tick-borne diseases with combined vector-derived and pathogen-derived antigens is one of the limitations for the development of effective vaccine formulations. Quantum biology arise from findings suggesting that living cells operate under non-trivial features of quantum mechanics, which has been proposed to be involved in DNA mutation biological process. Then, the electronic structure of the molecular interactions behind peptide immunogenicity led to quantum immunology and based on the definition of the photon as a quantum of light, the immune protective epitopes were proposed as the immunological quantum. Recently, a quantum vaccinomics approach was proposed based on the characterization of the immunological quantum to further advance the design of more effective and safe vaccines. In this chapter, we describe methods of the quantum vaccinomics approach based on proteins with key functions in cell interactome and regulome of vector-host-pathogen interactions for the identification by yeast two-hybrid screen and the characterization by in vitro protein-protein interactions and musical scores of protein interacting domains, and the characterization of conserved protective epitopes in protein interacting domains. These results can then be used for the design and production of chimeric protective antigens.

Mass Production of Virus-Like Particles Using Chloroplast Genetic Engineering for Highly Immunogenic Oral Vaccine Against Fish Disease.

Nervous necrosis virus (NNV) is the causative agent of viral nervous necrosis (VNN), which is one of the most serious fish diseases leading to mass mortality in a wide range of fish species worldwide. Although a few injectable inactivated vaccines are commercially available, there is a need for more labor-saving, cost-effective, and fish-friendly immunization methods. The use of transgenic plants expressing pathogen-derived recombinant antigens as edible vaccines is an ideal way to meet these requirements. In this study, chloroplast genetic engineering was successfully utilized to overexpress the red-spotted grouper NNV capsid protein (RGNNV-CP). The RGNNV-CP accumulated at high levels in all young, mature, and old senescent leaves of transplastomic tobacco plants (averaging approximately 3 mg/g leaf fresh weight). The RGNNV-CP efficiently self-assembled into virus-like particles (RGNNV-VLPs) in the chloroplast stroma of the transgenic lines, which could be readily observed by in situ transmission electron microscopy. Furthermore, intraperitoneal injection and oral administration of the crudely purified protein extract containing chloroplast-derived RGNNV-VLPs provided the sevenband grouper fish with sufficient protection against RGNNV challenge, and its immunogenicity was comparable to that of a commercial injectable vaccine. These findings indicate that chloroplast-derived VLP vaccines may play a promising role in the prevention of various diseases, not only in fish but also in other animals, including humans.

The History of Anti-Trypanosome Vaccine Development Shows That Highly Immunogenic and Exposed Pathogen-Derived Antigens Are Not Necessarily Good Target Candidates: Enolase and ISG75 as Examples.

Salivarian trypanosomes comprise a group of extracellular anthroponotic and zoonotic parasites. The only sustainable method for global control of these infection is through vaccination of livestock animals. Despite multiple reports describing promising laboratory results, no single field-applicable solution has been successful so far. Conventionally, vaccine research focusses mostly on exposed immunogenic antigens, or the structural molecular knowledge of surface exposed invariant immunogens. Unfortunately, extracellular parasites (or parasites with extracellular life stages) have devised efficient defense systems against host antibody attacks, so they can deal with the mammalian humoral immune response. In the case of trypanosomes, it appears that these mechanisms have been perfected, leading to vaccine failure in natural hosts. Here, we provide two examples of potential vaccine candidates that, despite being immunogenic and accessible to the immune system, failed to induce a functionally protective memory response. First, trypanosomal enolase was tested as a vaccine candidate, as it was recently characterized as a highly conserved enzyme that is readily recognized during infection by the host antibody response. Secondly, we re-addressed a vaccine approach towards the Invariant Surface Glycoprotein ISG75, and showed that despite being highly immunogenic, trypanosomes can avoid anti-ISG75 mediated parasitemia control.