Opportunities and challenges for T cell-based influenza vaccines.

Vaccination remains our main defence against influenza, which causes substantial annual mortality and poses a serious pandemic threat. Influenza virus evades immunity by rapidly changing its surface antigens but, even when the vaccine is well matched to the current circulating virus strains, influenza vaccines are not as effective as many other vaccines. Influenza vaccine development has traditionally focused on the induction of protective antibodies, but there is mounting evidence that T cell responses are also protective against influenza. Thus, future vaccines designed to promote both broad T cell effector functions and antibodies may provide enhanced protection. As we discuss, such vaccines present several challenges that require new strategic and economic considerations. Vaccine-induced T cells relevant to protection may reside in the lungs or lymphoid tissues, requiring more invasive assays to assess the immunogenicity of vaccine candidates. T cell functions may contain and resolve infection rather than completely prevent infection and early illness, requiring vaccine effectiveness to be assessed based on the prevention of severe disease and death rather than symptomatic infection. It can be complex and costly to measure T cell responses and infrequent clinical outcomes, and thus innovations in clinical trial design are needed for economic reasons. Nevertheless, the goal of more effective influenza vaccines justifies renewed and intensive efforts.

The antibodies 3D12 and 4D12 recognise distinct epitopes and conformations of HLA-E.

The commonly used antibodies 3D12 and 4D12 recognise the human leukocyte antigen E (HLA-E) protein. These antibodies bind distinct epitopes on HLA-E and differ in their ability to bind alleles of the major histocompatibility complex E (MHC-E) proteins of rhesus and cynomolgus macaques. We confirmed that neither antibody cross-reacts with classical HLA alleles, and used hybrids of different MHC-E alleles to map the regions that are critical for their binding. 3D12 recognises a region on the alpha 3 domain, with its specificity for HLA-E resulting from the amino acids present at three key positions (219, 223 and 224) that are unique to HLA-E, while 4D12 binds to the start of the alpha 2 domain, adjacent to the C terminus of the presented peptide. 3D12 staining is increased by incubation of cells at 27°C, and by addition of the canonical signal sequence peptide presented by HLA-E peptide (VL9, VMAPRTLVL). This suggests that 3D12 may bind peptide-free forms of HLA-E, which would be expected to accumulate at the cell surface when cells are incubated at lower temperatures, as well as HLA-E with peptide. Therefore, additional studies are required to determine exactly what forms of HLA-E can be recognised by 3D12. In contrast, while staining with 4D12 was also increased when cells were incubated at 27°C, it was decreased when the VL9 peptide was added. We conclude that 4D12 preferentially binds to peptide-free HLA-E, and, although not suitable for measuring the total cell surface levels of MHC-E, may putatively identify peptide-receptive forms.

High-throughput characterization of HLA-E-presented CD94/NKG2x ligands reveals peptides which modulate NK cell activation.

HLA-E is a non-classical class I MHC protein involved in innate and adaptive immune recognition. While recent studies have shown HLA-E can present diverse peptides to NK cells and T cells, the HLA-E repertoire recognized by CD94/NKG2x has remained poorly defined, with only a limited number of peptide ligands identified. Here we screen a yeast-displayed peptide library in the context of HLA-E to identify 500 high-confidence unique peptides that bind both HLA-E and CD94/NKG2A or CD94/NKG2C. Utilizing the sequences identified via yeast display selections, we train prediction algorithms and identify human and cytomegalovirus (CMV) proteome-derived, HLA-E-presented peptides capable of binding and signaling through both CD94/NKG2A and CD94/NKG2C. In addition, we identify peptides which selectively activate NKG2C+ NK cells. Taken together, characterization of the HLA-E-binding peptide repertoire and identification of NK activity-modulating peptides present opportunities for studies of NK cell regulation in health and disease, in addition to vaccine and therapeutic design.

HLA class I signal peptide polymorphism determines the level of CD94/NKG2-HLA-E-mediated regulation of effector cell responses.

Human leukocyte antigen (HLA)-E binds epitopes derived from HLA-A, HLA-B, HLA-C and HLA-G signal peptides (SPs) and serves as a ligand for CD94/NKG2A and CD94/NKG2C receptors expressed on natural killer and T cell subsets. We show that among 16 common classical HLA class I SP variants, only 6 can be efficiently processed to generate epitopes that enable CD94/NKG2 engagement, which we term 'functional SPs'. The single functional HLA-B SP, known as HLA-B/-21M, induced high HLA-E expression, but conferred the lowest receptor recognition. Consequently, HLA-B/-21M SP competes with other SPs for providing epitope to HLA-E and reduces overall recognition of target cells by CD94/NKG2A, calling for reassessment of previous disease models involving HLA-B/-21M. Genetic population data indicate a positive correlation between frequencies of functional SPs in humans and corresponding cytomegalovirus mimics, suggesting a means for viral escape from host responses. The systematic, quantitative approach described herein will facilitate development of prediction algorithms for accurately measuring the impact of CD94/NKG2-HLA-E interactions in disease resistance/susceptibility.

HLA-E-restricted SARS-CoV-2-specific T cells from convalescent COVID-19 patients suppress virus replication despite HLA class Ia down-regulation.

Pathogen-specific CD8+ T cell responses restricted by the nonpolymorphic nonclassical class Ib molecule human leukocyte antigen E (HLA-E) are rarely reported in viral infections. The natural HLA-E ligand is a signal peptide derived from classical class Ia HLA molecules that interact with the NKG2/CD94 receptors to regulate natural killer cell functions, but pathogen-derived peptides can also be presented by HLA-E. Here, we describe five peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that elicited HLA-E-restricted CD8+ T cell responses in convalescent patients with coronavirus disease 2019. These T cell responses were identified in the blood at frequencies similar to those reported for classical HLA-Ia-restricted anti-SARS-CoV-2 CD8+ T cells. HLA-E peptide-specific CD8+ T cell clones, which expressed diverse T cell receptors, suppressed SARS-CoV-2 replication in Calu-3 human lung epithelial cells. SARS-CoV-2 infection markedly down-regulated classical HLA class I expression in Calu-3 cells and primary reconstituted human airway epithelial cells, whereas HLA-E expression was not affected, enabling T cell recognition. Thus, HLA-E-restricted T cells could contribute to the control of SARS-CoV-2 infection alongside classical T cells.

Intracellular trafficking of HLA-E and its regulation.

Interest in MHC-E-restricted CD8+ T cell responses has been aroused by the discovery of their efficacy in controlling simian immunodeficiency virus (SIV) infection in a vaccine model. The development of vaccines and immunotherapies utilizing human MHC-E (HLA-E)-restricted CD8+ T cell response requires an understanding of the pathway(s) of HLA-E transport and antigen presentation, which have not been clearly defined previously. We show here that, unlike classical HLA class I, which rapidly exits the endoplasmic reticulum (ER) after synthesis, HLA-E is largely retained because of a limited supply of high-affinity peptides, with further fine-tuning by its cytoplasmic tail. Once at the cell surface, HLA-E is unstable and is rapidly internalized. The cytoplasmic tail plays a crucial role in facilitating HLA-E internalization, which results in its enrichment in late and recycling endosomes. Our data reveal distinctive transport patterns and delicate regulatory mechanisms of HLA-E, which help to explain its unusual immunological functions.

Strategies for HIV-1 vaccines that induce broadly neutralizing antibodies.

After nearly four decades of research, a safe and effective HIV-1 vaccine remains elusive. There are many reasons why the development of a potent and durable HIV-1 vaccine is challenging, including the extraordinary genetic diversity of HIV-1 and its complex mechanisms of immune evasion. HIV-1 envelope glycoproteins are poorly recognized by the immune system, which means that potent broadly neutralizing antibodies (bnAbs) are only infrequently induced in the setting of HIV-1 infection or through vaccination. Thus, the biology of HIV-1-host interactions necessitates novel strategies for vaccine development to be designed to activate and expand rare bnAb-producing B cell lineages and to select for the acquisition of critical improbable bnAb mutations. Here we discuss strategies for the induction of potent and broad HIV-1 bnAbs and outline the steps that may be necessary for ultimate success.

Autoimmunity-associated T cell receptors recognize HLA-B*27-bound peptides.

Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU))1. How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8+ T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public ß-chain variable region-complementary-determining region 3ß (BV9-CDR3ß) motif2-4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide-MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9-CDR3ß TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease.

Discovery of HLA-E-Presented Epitopes: MHC-E/Peptide Binding and T-Cell Recognition.

Understanding the interactions involved during the immunological synapse between peptide, HLA-E molecules, and TCR is crucial to effectively target protective HLA-E-restricted T-cell responses in humans. Here we describe three techniques based on the generation of MHC-E/peptide complexes (MHC-E generically includes HLA-E-like molecules in human and nonhuman species, while HLA-E specifically refers to human molecules), which allow to investigate MHC-E/peptide binding at the molecular level through binding assays and by using peptide loaded HLA-E tetramers, to detect, isolate, and study peptide-specific HLA-E-restricted human T-cells.

Primary and secondary functions of HLA-E are determined by stability and conformation of the peptide-bound complexes.

MHC-E regulates NK cells by displaying MHC class Ia signal peptides (VL9) to NKG2A:CD94 receptors. MHC-E can also present sequence-diverse, lower-affinity, pathogen-derived peptides to T cell receptors (TCRs) on CD8+ T cells. To understand these affinity differences, human MHC-E (HLA-E)-VL9 versus pathogen-derived peptide structures are compared. Small-angle X-ray scatter (SAXS) measures biophysical parameters in solution, allowing comparison with crystal structures. For HLA-E-VL9, there is concordance between SAXS and crystal parameters. In contrast, HLA-E-bound pathogen-derived peptides produce larger SAXS dimensions that reduce to their crystallographic dimensions only when excess peptide is supplied. Further crystallographic analysis demonstrates three amino acids, exclusive to MHC-E, that not only position VL9 close to the α2 helix, but also allow non-VL9 peptide binding with re-configuration of a key TCR-interacting α2 region. Thus, non-VL9-bound peptides introduce an alternative peptide-binding motif and surface recognition landscape, providing a likely basis for VL9- and non-VL9-HLA-E immune discrimination.

GIMAP6 regulates autophagy, immune competence, and inflammation in mice and humans.

Inborn errors of immunity (IEIs) unveil regulatory pathways of human immunity. We describe a new IEI caused by mutations in the GTPase of the immune-associated protein 6 (GIMAP6) gene in patients with infections, lymphoproliferation, autoimmunity, and multiorgan vasculitis. Patients and Gimap6-/- mice show defects in autophagy, redox regulation, and polyunsaturated fatty acid (PUFA)-containing lipids. We find that GIMAP6 complexes with GABARAPL2 and GIMAP7 to regulate GTPase activity. Also, GIMAP6 is induced by IFN-γ and plays a critical role in antibacterial immunity. Finally, we observed that Gimap6-/- mice died prematurely from microangiopathic glomerulosclerosis most likely due to GIMAP6 deficiency in kidney endothelial cells.

Mouse and human antibodies bind HLA-E-leader peptide complexes and enhance NK cell cytotoxicity.

The non-classical class Ib molecule human leukocyte antigen E (HLA-E) has limited polymorphism and can bind HLA class Ia leader peptides (VL9). HLA-E-VL9 complexes interact with the natural killer (NK) cell receptors NKG2A-C/CD94 and regulate NK cell-mediated cytotoxicity. Here we report the isolation of 3H4, a murine HLA-E-VL9-specific IgM antibody that enhances killing of HLA-E-VL9-expressing cells by an NKG2A+ NK cell line. Structural analysis reveal that 3H4 acts by preventing CD94/NKG2A docking on HLA-E-VL9. Upon in vitro maturation, an affinity-optimized IgG form of 3H4 showes enhanced NK killing of HLA-E-VL9-expressing cells. HLA-E-VL9-specific IgM antibodies similar in function to 3H4 are also isolated from naïve B cells of cytomegalovirus (CMV)-negative, healthy humans. Thus, HLA-E-VL9-targeting mouse and human antibodies isolated from the naïve B cell antibody pool have the capacity to enhance NK cell cytotoxicity.

The Importance of Cellular Immune Response to HIV: Implications for Antibody Production and Vaccine Design.

Despite many years from the discovery of human immunodeficiency virus (HIV), a prophylactic vaccine against HIV is still needed. The failure of most of the vaccine clinical trials in the field has different causes, mainly due by the difficulties to identify the correct antigen able to prime the optimal B cell lineage and then make the series of somatic mutations necessary to generate broadly neutralizing antibodies (bNAbs). B cells are responsible for the bNAbs production; however, their function is strongly influenced by the presence of a population of CD4+ T lymphocytes, mainly present in the lymphoid organs, the T follicular helper cells (Tfh). In this review, the importance of the contribution of Tfh cells in HIV response is highlighted and future therapy perspectives based on these observations are described. The advanced technology available nowadays and the wide knowledge built over the past years for HIV may eventually create the best scenario for the generation of an effective vaccine.

Preexisting memory CD4+ T cells contribute to the primary response in an HIV-1 vaccine trial.

Naive and memory CD4+ T cells reactive with human immunodeficiency virus type 1 (HIV-1) are detectable in unexposed, unimmunized individuals. The contribution of preexisting CD4+ T cells to a primary immune response was investigated in 20 HIV-1-seronegative volunteers vaccinated with an HIV-1 envelope (Env) plasmid DNA prime and recombinant modified vaccinia virus Ankara (MVA) boost in the HVTN 106 vaccine trial (clinicaltrials.gov NCT02296541). Prevaccination naive or memory CD4+ T cell responses directed against peptide epitopes in Env were identified in 14 individuals. After priming with DNA, 40% (8/20) of the elicited responses matched epitopes detected in the corresponding preimmunization memory repertoires, and clonotypes were shared before and after vaccination in 2 representative volunteers. In contrast, there were no shared epitope specificities between the preimmunization memory compartment and responses detected after boosting with recombinant MVA expressing a heterologous Env. Preexisting memory CD4+ T cells therefore shape the early immune response to vaccination with a previously unencountered HIV-1 antigen.

HLA-E-restricted, Gag-specific CD8+ T cells can suppress HIV-1 infection, offering vaccine opportunities.

Human leukocyte antigen-E (HLA-E) normally presents an HLA class Ia signal peptide to the NKG2A/C-CD94 regulatory receptors on natural killer (NK) cells and T cell subsets. Rhesus macaques immunized with a cytomegalovirus-vectored simian immunodeficiency virus (SIV) vaccine generated Mamu-E (HLA-E homolog)-restricted T cell responses that mediated post-challenge SIV replication arrest in >50% of animals. However, HIV-1-specific, HLA-E-restricted T cells have not been observed in HIV-1-infected individuals. Here, HLA-E-restricted, HIV-1-specific CD8 + T cells were primed in vitro. These T cell clones and allogeneic CD8 + T cells transduced with their T cell receptors suppressed HIV-1 replication in CD4 + T cells in vitro. Vaccine induction of efficacious HLA-E-restricted HIV-1-specific T cells should therefore be possible.

Interrogating the recognition landscape of a conserved HIV-specific TCR reveals distinct bacterial peptide cross-reactivity.

T cell cross-reactivity ensures that diverse pathogen-derived epitopes encountered during a lifetime are recognized by the available TCR repertoire. A feature of cross-reactivity where previous exposure to one microbe can alter immunity to subsequent, non-related pathogens has been mainly explored for viruses. Yet cross-reactivity to additional microbes is important to consider, especially in HIV infection where gut-intestinal barrier dysfunction could facilitate T cell exposure to commensal/pathogenic microbes. Here we evaluated the cross-reactivity of a 'public', HIV-specific, CD8 T cell-derived TCR (AGA1 TCR) using MHC class I yeast display technology. Via screening of MHC-restricted libraries comprising ~2×108 sequence-diverse peptides, AGA1 TCR specificity was mapped to a central peptide di-motif. Using the top TCR-enriched library peptides to probe the non-redundant protein database, bacterial peptides that elicited functional responses by AGA1-expressing T cells were identified. The possibility that in context-specific settings, MHC class I proteins presenting microbial peptides influence virus-specific T cell populations in vivo is discussed.

Detailed and atypical HLA-E peptide binding motifs revealed by a novel peptide exchange binding assay.

Diverse SIV and HIV epitopes that bind the rhesus homolog of HLA-E, Mamu-E, have recently been identified in SIVvaccine studies using a recombinant Rhesus cytomegalovirus (RhCMV 68-1) vector, where unprecedented protection against SIV challenge was achieved. Additionally, several Mycobacterial peptides identified both algorithmically and following elution from infected cells, are presented to CD8+ T cells by HLA-E in humans. Yet, a comparative and comprehensive analysis of relative HLA-E peptide binding strength via a reliable, high throughput in vitro assay is currently lacking. To address this, we developed and optimized a novel, highly sensitive peptide exchange ELISA-based assay that relatively quantitates peptide binding to HLA-E. Using this approach, we screened multiple peptides, including peptide panels derived from HIV, SIV, and Mtb predicted to bind HLA-E. Our results indicate that although HLA-E preferentially accommodates canonical MHC class I leader peptides, many non-canonical, sequence diverse, pathogen-derived peptides also bind HLA-E, albeit generally with lower relative binding strength. Additionally, our screens demonstrate that the majority of peptides tested, including some key Mtb and SIV epitopes that have been shown to elicit strong Mamu-E-restricted T cell responses, either bind HLA-E extremely weakly or give signals that are indistinguishable from the negative, peptide-free controls.