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Broadly targeted CD8+ T cell responses restricted by major histocompatibility complex E

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Science  21 Jan 2016:
aac9475
DOI: 10.1126/science.aac9475

Abstract

Major histocompatibility complex (MHC)-E is a highly conserved, ubiquitously expressed, non-classical MHC-Ib molecule with limited polymorphism primarily involved in NK cell regulation. We found that vaccination of rhesus macaques (RM) with ΔRh157.5/.4 Rhesus Cytomegalovirus (RhCMV) vectors results in MHC-E-restricted presentation of highly varied peptide epitopes to CD8α/β+ T cells, approximately 4 distinct epitopes per 100 amino acids in all tested antigens. Computational structural analysis revealed that MHC-E provides heterogeneous chemical environments for diverse side chain interactions within a stable, open binding groove. Since MHC-E is up-regulated on cells infected with HIV/SIV and other persistent viruses to evade NK cell activity, MHC-E-restricted CD8+ T cell responses have the potential to exploit pathogen immune evasion adaptations, a capability that might endow these unconventional responses with superior efficacy.

Adaptive cellular immunity against intracellular pathogens is the primary responsibility of CD8+ T cells that recognize short pathogen-derived peptide epitopes presented by highly polymorphic MHC-Ia molecules on the surface of infected cells (1, 2). MHC-Ia allomorphs vary considerably in their peptide binding properties, and therefore the particular pathogen-derived peptides targeted by pathogen-specific CD8+ T cells are largely determined by the peptide binding specificity of the limited number of MHC-Ia allomorphs expressed by the infected individual (3). Consequently, the epitopes recognized by CD8+ T cells responding to the same pathogen are highly diverse across individuals, resulting in heterogeneity among individuals in the ability to clear or control various infections, in particular agents like human immunodeficiency virus (HIV) with a high intrinsic capacity for mutational immune escape (2, 4). Such variation can hamper efforts to develop universally effective vaccines based on CD8+ T cell responses (4, 5).

We recently reported that simian immunodeficiency virus (SIV)-targeted vaccine vectors based on strain 68-1 (Rh157.5/Rh157.4 gene-deleted) RhCMV violate the rules of MHC-Ia-restricted CD8+ T cell recognition (6), and offer a potential solution to MHC-Ia-dependent response diversity in CD8+ T cell-targeted vaccination. RhCMV/SIV vector-elicited cellular immune responses are able to stringently control and ultimately clear a highly pathogenic SIV challenge in slightly over half of vaccinated RM (7, 8). These vectors elicit SIV-specific CD8+ T cell responses that are entirely non-overlapping with conventional MHC-Ia-restricted CD8+ T cells, despite manifesting 3-fold the breadth of CD8+ T cells elicited by conventional vaccines. Part of this lack of epitope overlap was explained by the finding that many of these epitopes were restricted by MHC-II, rather than MHC-I, molecules (6). However, strain 68-1 RhCMV/SIVgag vectors also elicited CD8+ T cells that recognized MHC-I-dependent epitopes that were common to most, or even all, MHC-disparate RM, an unprecedented degree of cross-recognition for MHC-Ia-restricted CD8+ T cell responses. Indeed, two SIVgag epitopes [SIVgag276-284(69) and SIVgag482-490(120)] were targeted by 42 of 42 strain 68-1 RhCMV/SIVgag vector-immunized RM in our previous report (6), and we have since documented these “supertope” responses in 130 of 130 RM inoculated with this vector (fig. S1).

To understand the basis of this unusual MHC-I-dependent recognition, we selected 4 strain 68-1 RhCMV/SIVgag vector-vaccinated RM for detailed MHC-I restriction analysis. We first sequenced the expressed MHC-I genes, both classical MHC-Ia and non-classical MHC-Ib (9), in each RM, and constructed a panel of MHC-I transfectants individually expressing these MHC-I molecules (fig. S2). These single MHC-I molecule transfectants were then used in a flow cytometric intra-cellular cytokine staining (ICS) assay to present 12 commonly recognized 15mer peptides to the strain 68-1 RhCMV/SIVgag vector-induced CD8+ T cells from these RM (Fig. 1, A and B, and fig. S3). Remarkably, classical MHC-Ia allomorphs were able to present only 3 of the 12 epitopic peptides to these T cells [Mamu-A1*001:01: SIVgag69-83(18) and SIVgag197-211(50); Mamu-A1*002:01: SIVgag129-143(33)], and expression of these allomorphs in RM did not track with these epitope-specific CD8+ T cell responses (fig. S4). However, all 12 peptides stimulated epitope-specific CD8+ T cells when presented by non-classical MHC-E molecules, and indeed, all peptides were presented by transfectants singly expressing three different RM MHC-E allomorphs (Mamu-E*02:04, -E*02:11, and –E*02:20), as well as by a transfectant expressing human MHC-E (HLA-E*01:03) (Fig. 1, A and B, and figs. S3 and S5).

Fig. 1 MHC restriction of strain 68-1 RhCMV/SIVgag-elicited CD8+ T cells.

(A and B) PBMC from a representative strain 68-1 RhCMV/SIVgag-vaccinated RM (Rh22034, of 4 similarly analyzed, see fig. S3) were stimulated with the indicated epitopic 15mer peptides pulsed onto the surface of parental MHC-I-negative cell lines (0.221 and K562; negative controls), autologous B lymphoblastoid cell lines (BLCL; positive controls), or the indicated MHC-I transfectants with CD8+ T cell recognition determined by detection of IFN-γ and/or TNF-α production by flow cytometric ICS assay (response frequencies of gated CD8+ T cells shown in each quadrant). The MHC-I molecules tested included both those expressed by Rh22034 (A) and additional RM and human MHC-E molecules not expressed by Rh22034 (B). (C) Mamu-E*02:04, Mamu-E*02:20 and HLA-E*01:03 transfectants were pulsed with the serially diluted concentration of the indicated optimal SIVgag 9mer epitopic peptides, and combined with PBMC from 3-4 68-1 RhCMV/SIVgag-vaccinated RM for flow cytometric ICS determination of the frequency of responding CD8+ T cells (IFN-γ+ and/or TNF-α+). Response frequencies at each peptide dose were normalized to the response observed with the transfectant pulsed with highest concentration (10μM) of peptide.

MHC-E is known to avidly bind canonical VMAPRTL(LVI)L peptides that are derived from positions 3-11 of MHC-Ia leader sequences for presentation to NKG2A/C molecules on NK cells (1014). This conserved interaction predominantly inhibits NK cell activity when cells express normal levels of MHC-Ia. However, upon interference with MHC-Ia biosynthesis by viral infection or neoplastic transformation, this inhibitory signal is reduced, facilitating NK cell responses to virally-infected or neoplastic cells (15, 16). Although a subset of CD8+ T cells can express NKG2A/C (17), phenotypic analysis of MHC-E-dependent, strain 68-1 RhCMV/SIVgag vector-elicited CD8+ T cells revealed that the vast majority of responding cells were CD8α/β+, TCRγ/δ-νεγατιϖε T cells lacking NKG2A/C expression (fig. S6). Moreover, pre-incubation of MHC-E transfectants or other antigen-presenting cells with the canonical MHC-E-binding VMAPRTLLL (VL9) peptide prior to epitopic peptide loading specifically blocked CD8+ T cell recognition of all 12 epitopic peptides (fig. S7). Indeed, the epitopic SIVgag 15mers could be truncated to optimal 9mer peptides that were common among different strain 68-1 RhCMV/SIVgag vector-vaccinated RM with MHC-E-associated CD8+ T cell responses to the parent 15mer (fig. S8) (6). These optimal 9mer peptides could trigger CD8+ T cells when pulsed on MHC-E transfectants (both Mamu-E and HLA-E) or autologous B cell lines at doses of ≤1 nM (Fig. 1C and fig. S9), functional avidities that are comparable to T cell recognition of classically MHC-Ia-restricted epitopes (18). Taken together, these data strongly suggest that the unconventional, MHC-I-dependent CD8+ T cell responses elicited by strain 68-1 RhCMV/SIVgag vectors reflect SIVgag 9mer epitope-specific, MHC-E-restricted CD8α/β+ T cells.

MHC-E-restricted CD8+ T cell responses have been previously identified in human CMV (HCMV), Hepatitis C virus (HCV), Mycobacterium tuberculosis (mTB), and Salmonella enterica infections, typically involving epitopes that are structurally related to the canonical MHC-Ia leader sequence peptides, but foreign to the host (13, 14, 19, 20). To determine the extent to which MHC-E restricts RM CD8+ T cell responses in different settings, we used blocking with high affinity MHC-E-binding peptide VL9 [in conjunction with blocking with anti-MHC-II CLIP peptide and anti-MHC-I monoclonal antibody (mAb) W6/32 (6)] to restriction-classify all SIVgag epitope-specific CD8+ T cell responses in RM vaccinated with strain 68-1 (ΔRh157.5/.4) RhCMV/SIVgag vectors, strain 68-1.2 (Rh157.5/.4-repaired) RhCMV/SIVgag vectors, strain 68-1.2 RhCMV/SIVgag vectors from which the Rh157.5/.4 genes were specifically re-excised (fig. S10), and Modified Vaccinia Ankara (MVA)/SIVgag vectors, as well as RM infected with SIV itself (Fig. 2A and figs. S11 and S12). This analysis revealed that essentially all SIVgag epitope-specific CD8+ T cells in strain 68-1 RhCMV/SIVgag vector- and ΔRh157.5/.4 strain 68-1.2 RhCMV/SIVgag vector-vaccinated RM were either MHC-II- or MHC-E-restricted. In contrast, 98-100% of SIVgag-specific CD8+ T cell responses in the MVA/SIVgag vector-vaccinated, strain 68-1.2 RhCMV/SIVgag vector-vaccinated and SIV-infected RM were classically MHC-Ia-restricted. In keeping with these patterns of epitope restriction, recognition of autologous SIV-infected CD4+ T cells by strain 68-1 RhCMV/SIV vector-elicited SIVgag-specific CD8+ T cells was completely blocked by a combination of the MHC-II-blocking CLIP peptide and the MHC-E-specific blocking VL9 peptide, whereas SIV-infected autologous cell recognition by SIVgag-specific CD8+ T cells elicited by MVA/SIVgag vector vaccination, strain 68-1.2 RhCMV/gag vector vaccination or SIV infection was insensitive to the CLIP + VL9 peptide combination (Fig. 2C). In addition, the VL9 peptide alone could completely block SIV-infected cell recognition by an MHC-E-restricted SIVgag482-490(120)-specific CD8+ T cell line (fig. S14). Taken together, these data confirm that strain 68-1 RhCMV vectors elicit CD8+ T cell responses that are either MHC-II- or MHC-E-restricted, and that this unusual immunobiology is a specific consequence of deletion of the RhCMV Rh157.5/.4 genes, which are orthologs of the HCMV UL128/UL130 genes and encode 2 components of the pentameric receptor complex involved in CMV infection of non-fibroblasts (21). Moreover, these data confirm that although SIV infection does not efficiently prime MHC-E-restricted CD8+ T cell responses, at least some of the SIV epitopes recognized by strain 68-1 RhCMV vector-elicited CD8+ T cells are naturally presented by SIV-infected cells, providing for their effective recognition by these heterologous responses.

Fig. 2 MHC-E restriction is limited to CD8+ T cell responses elicited by ΔRh157.5/.4 RhCMV vectors.

(A) CD8+ T cell responses to SIVgag were epitope-mapped using flow cytometric ICS to detect recognition of 125 consecutive 15mer gag peptides (with 11 amino acid overlap) in RM vaccinated with the indicated SIVgag-expressing viral vectors or infected with SIVmac239 (n = 4-6 per group shown; see fig. S12 for other studied RM). Peptides resulting in specific CD8+ T cell responses are indicated by a box, with the color of the box designating MHC restriction as determined by blocking with the anti-pan-MHC-I mAb W6/32, the MHC-E blocking peptide VL9 and the MHC-II blocking peptide CLIP (see Methods). The minimal number of independent epitopes in these MHC restriction categories is shown at right for each RM. (B) Analysis of SIV-infected CD4+ cell recognition by CD8β+ cells isolated from RM vaccinated with the indicated SIVgag-expressing viral vectors or infected with SIV. The flow profiles at left show IFN-γ and TNF-α production following CD8β+ T cell incubation with autologous SIVmac239-infected CD4+ T cells alone (no block), or in the presence of the pan-MHC-I-blocking mAb W6/32 plus the MHC-II-binding CLIP peptide (W6/32 + CLIP), or MHC-E-binding peptide VL9 plus CLIP (VL9 + CLIP). All plots are gated on live, CD3+, CD8+ cells. The bar graph at right shows the results from all studied RM.

Among 42 strain 68-1 RhCMV/SIVgag vector-vaccinated RM, we identified a median of 20 distinct CD8+ T cell-recognized, MHC-E-restricted, SIVgag 15mer epitopes per RM, a breadth that exceeds the median 11 and 14.5 distinct MHC-Ia-restricted SIVgag-specific epitopes identified within SIVgag-specific CD8+ T cell responses elicited by conventional vaccines or SIV infection, respectively (Fig. 3A). The ability of ΔRh157.5/.4 RhCMV vectors to elicit MHC-E- and MHC-II-restricted CD8+ T cells is not limited to SIVgag-specific responses (fig. S13), and the density of MHC-E-restricted epitopes (~4 independent MHC-E-restricted epitopes per 100 amino acids of protein length) is similar among all strain 68-1 RhCMV vector-elicited CD8+ T cell responses, regardless of nature of the antigen analyzed (Fig. 3B). Notably, among the same 42 strain 68-1 RhCMV/SIVgag vector-vaccinated RM, 109 of the 125 overlapping SIVgag 15mer peptides (87%) were recognized by MHC-E-restricted CD8+ T cells in at least one RM (Fig. 3C). Although MHC-E has been shown to bind a broader array of peptides than the canonical leader sequence peptides (14, 22), the extent of this epitope diversity and breadth is surprising, especially given the limited polymorphism of MHC-E and the observation that all tested Mamu-E and HLA-E variants present all tested MHC-E-restricted epitopes (Fig. 1, B and C, and figs. S5 and S9). In keeping with this previously unrecognized MHC-E epitope diversity, sequence analysis of 11 optimal MHC-E-restricted SIVgag 9mer epitopes showed only one epitope – the Gag273-287(69) supertope – with a canonical (M at position 2: L at position 9) MHC-E-binding motif, whereas the other 10 optimal epitopes not only lacked this motif, but manifested no statistically significant overlap with previously characterized sets of MHC-E bound peptides (22) (Fig. 3, D and E, and fig. S15). Statistical analysis of a larger group of 125 SIVgag 15mer peptides also failed to show any enrichment for previously reported MHC-E binding motifs (figs. S16 and S17). Despite this amino acid sequence diversity, 10 of the 11 immunologically-defined optimal MHC-E-restricted SIVgag 9mer epitopes were shown to physically associate with, and stabilize, MHC-E, either in an ELISA-based HLA-E plus peptide re-folding assay or in a single chain MHC-E/peptide expression assay, or both (figs. S18 and S19).

Fig. 3 Diversity of MHC-E-restricted epitopes.

(A) Comparison of the total number of distinct MHC E- (green) vs. MHC-Ia (red)-restricted SIVgag epitopes recognized by circulating CD8+ T cells in individual RM vaccinated with strain 68-1 RhCMV/gag vs. conventional viral vectors, the latter including MVA/gag (n = 11), Ad5/gag (n = 3) and electroporated DNA/gag + IL-12 (n = 4), or in RM with controlled SIVmac239 infection (n = 12). The horizontal bars indicate median values (p values from unpaired, two-tailed Mann-Whitney test). (B) Comparison of the number of distinct MHC-E-restricted epitopes per 100 amino acids of protein length recognized by circulating CD8+ T cells in individual RM vaccinated with strain 68-1 RhCMV vectors expressing each of the indicated antigens (note: RhCMV IE1 responses were evaluated in CMV-naïve RM administered strain 68-1 RhCMV/gag). The horizontal bars indicate median values for each group. (C) Population-level analysis of the breadth of MHC-E-restricted SIVgag epitope-specific CD8+ T cell responses across 125 consecutive 15mer gag peptides (with 11 amino acid overlap) in 42 strain 68-1 RhCMV/gag vector-vaccinated RM. (D) Sequence LOGO indicating the frequency of each amino acid in a given position by the height of the letter, based on 11 optimal, MHC-E-restricted SIVgag 9mer peptide epitopes recognized by CD8+ T cells in strain 68-1 RhCMV vector-vaccinated RM. Blue indicates significant amino acid enrichment in a given position relative to their background frequency in SIVmac239 Gag (see Methods). Green highlights the 2M and L9 of the canonical MHC-E-binding motif. (E) The same LOGO as in (D) colored according to enrichment (blue or green) or underrepresentation (red) among 551 peptides eluted from HLA-E*01:03 in a TAP-deficient setting by Lampen et al. (22) (see fig. S15). Amino acids enriched in the 2nd and C-terminal anchor positions among the 551 Lampen et al. peptides were rare among our 11 optimal SIVgag peptides, while those that were significantly underrepresented were enriched, highlighted in the actual peptides on the right. The percentage of strain 68-1 RhCMV/gag-vaccinated RM (n = 42) that responded to each optimal peptide is noted as the “Recognition Frequency”.

These data suggest that the loading and binding of epitopic peptides to MHC-E must involve novel molecular mechanisms. To explore this possibility, we performed long time-scale, all-atom molecular dynamics simulations of canonical peptide bound and unbound forms of the solved structure of HLA-E*01:03 (23) in comparison to simulations of the classical HLA-Ia protein HLA-A*02:01, which overlaps with HLA-E in peptide binding specificity (22). This analysis revealed that HLA-E*01:03, but not HLA-A*02:01, preserves a relatively rigid peptide binding groove that remains open even in the absence of canonical peptide during the simulation time scales (Fig. 4, A and B). Docking analysis further suggested that the 11 optimal MHC-E-restricted SIVgag epitopes described above can bind to both HLA-E*01:03 and Mamu-E*02:04 by adopting a similar backbone configuration as the canonical peptide VL9, with diverse side chain rotamers being accommodated in the same buried positions (Fig. 4C and fig. S20). Further characterization using all-atom molecular dynamics simulations of HLA-E-peptide complexes revealed that the SIVgag epitopic peptides bind at slightly elevated positions in the binding groove than VL9, which mainly uses hydrophobic amino acid side chain interactions to stabilize its deep positioning (Fig. 4D). Consequently, in agreement with direct MHC-E/peptide binding analysis (figs. S18 and S19), computationally determined binding affinities of the 11 optimal MHC-E-restricted SIVgag epitopes are lower than that of VL9 (fig. S21). However, at these elevated positions in the binding groove, epitopic peptides encounter a much more varied binding environment in terms of available polar or charged binding sites (Fig. 4E), and indeed, charged residues appear to contribute favorably to the binding of many of these peptides to MHC-E (figs. S22 and S23). Thus, in vivo conditions such as pH, ionic strength, and/or macromolecular crowding can modulate these interactions, possibly explaining some of the differences between in vitro and intracellular MHC-E–peptide binding measurements (figs. S18 and S19). There are limits to MHC-E peptide-binding breadth, as not all peptides can be accommodated by MHC-E in the same conformation as the canonical peptide (fig. S24). These structural analyses suggest that unique conformational properties of MHC-E may drive the observed epitope diversity and breadth. The open MHC-E binding groove would provide access to binding for an unusually diverse population of peptides, the side chains of which could interact with chemically varying environments at different depths within the binding groove. At the same time, the framework of the relatively rigid MHC-E binding groove would enforce a similar “canonical” backbone conformation on the bound peptides, providing a stable MHC-E–peptide complex for T cell recognition.

Fig. 4 Structural analysis of MHC-E peptide binding.

(A) Structural changes in the binding groove of canonical peptide-bound and -unbound HLA-A*02:01 and HLA-E*01:03. Calculations of changes in the volume of binding groove indicate that, unlike HLA-A*02:01, the HLA-E*01:03 binding groove doesn’t collapse when the peptide is not bound during the 0.5 microsecond all-atom molecular dynamics simulations. Error bar indicates 95% confidence interval using standard error estimates from five independent simulations for each case. Total volume is calculated from the cavity volumes from N, F and C, as shown in the three-dimensional structure and described in the Methods. (B) Root mean square fluctuations of the backbone atoms of unbound HLA-A*02:01 and HLA-E*01:03 are mapped on the X-ray structure. Consistent with (A), the binding groove of HLA-E*01:03 is less flexible compared to HLA-A*02:01. The binding groove helices partially unfold in unbound HLA-A*02:01, whereas the unbound HLA-E*01:03 binding groove remains relatively stable. Increasing flexibility is captured by the change in color gradient from blue to white to red. (C) HLA-E*01:03 binding profile obtained from a ROSETTA-based docking approach (30) of 11 optimal, MHC-E-restricted, SIVgag epitopic peptides. The backbones of these peptides adopt a similar conformation as shown with multiple colors in front of the binding groove cross-section. The bound conformations for these 11 peptides are shown in the colored insets. Residues buried in HLA-E and exposed are marked in red and white, respectively. (D) Molecular dynamics simulations of docked complexes show that the 11 epitopic peptides are bound in a slightly elevated position in the MHC-E binding groove compared to VL9 and are more solvent-exposed (inset bar graph). (E) Cross-section (view from top) of the MHC-E binding groove at two different depths show the differences in the chemical environment recognized by the buried residues of epitopic peptides and VL9. Unlike the hydrophobic environment experienced by the buried residues of VL9 peptide, epitopic peptides experience a chemically heterogeneous environment at their slightly elevated position in the binding groove (see figs. S22 and S23).

Both HCMV and RhCMV encode proteins with a strategically embedded canonical VL9 peptide within the UL40 and Rh67 genes, respectively (24, 25). The VL9 peptide of UL40 has been shown to be loaded onto nascent MHC-E chains by a transporter-associated-with-antigen-processing (TAP)-independent mechanism, and therefore functions to stabilize and up-regulate MHC-E expression in HCMV-infected cells in the face of virus-mediated TAP inhibition and profound MHC-Ia down-regulation mediated by the HCMV US2-11 gene products (15, 24). We have demonstrated a similar function for RhCMV Rh67 (fig. S25). MHC-E up-regulation is therefore thought to be a key viral strategy for evading the NK cell response to virally infected cells that lack MHC-Ia expression (15, 16). However, this evasion strategy would have the consequence of enhancing MHC-E expression in such infected cells, increasing the opportunity for loading and presentation of novel peptides to MHC-E-restricted T cells. In this regard, the canonical MHC-E-binding VL9 peptide might act as a chaperone that facilitates stable high expression of MHC-E and delivery to an endosomal compartment (26) that would facilitate peptide exchange, analogous to the invariant chain-associated CLIP peptide and MHC-II (1). The rigid, open structure of the MHC-E peptide binding groove would be expected to facilitate peptide exchange, consistent with this putative mechanism. Also, consistent with such a peptide exchange mechanism, MHC-E peptide loading has been directly demonstrated in the mTB phagolysosome (27).

CMV is not the only intracellular pathogen to up-regulate MHC-E expression. HCV also up-regulates MHC-E expression (28), and both HIV and SIV up-regulate MHC-E in concert with MHC-Ia down-regulation (29) (fig. S26). This common adaptation suggests that for these and likely other intracellular pathogens, the evolutionary pressure to up-regulate MHC-E to counter NK cell responses outweighs the potential risk of increased susceptibility to MHC-E-restricted CD8+ T cells, perhaps because MHC-E-restricted CD8+ T cells are poorly primed during infection with these agents. The reason why MHC-E-restricted CD8+ T cell responses are such a minor component of the modern mammalian immune system is unclear, especially given our finding that such responses can be quite diverse and broad (although, arguably, less diverse and broad on a population level than polymorphic MHC-Ia; fig. S27). However, ΔRh157.5/.4 RhCMV vectors are able to bypass the intrinsic constraint of MHC-E-restricted CD8+ T cell priming, perhaps by modulation of vector cell tropism and the priming microenvironment (6). The ability of these vectors to strongly elicit broad, diverse MHC-E-restricted CD8+ T cell responses therefore offers the potential opportunity to develop vaccines that exploit MHC-E up-regulation, an intrinsic vulnerability in the immune-evasion strategies of many highly adapted, persistent pathogens. Moreover, because of limited MHC-E polymorphism, an MHC-E-restricted CD8+ T cell response-targeted vaccine would elicit largely similar responses in all or most vaccinees, potentially providing for efficacy in all individuals regardless of MHC-I genotype. Evolution may have disfavored MHC-E as a primary restricting molecule for CD8+ T cells in modern mammals, but if HCMV vectors are able to recapitulate in humans the biology of Rh157.5/.4 gene-deleted RhCMV vectors in RM (or, alternatively, if non-CMV-based strategies to elicit broadly targeted MHC-E-restricted CD8+ T cell responses can be developed), vaccinologists may be able to resurrect this largely dormant MHC-E-based adaptive immune system to attack pathogens with novel immune responses that they are not adapted to effectively evade.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/cgi/content/full/science.aac9475/DC1

Materials and Methods

Figs. S1 to S27

Tables S1 and S2

References (3154)

REFERENCES AND NOTES

  1. ACKNOWLEDGMENTSWe are grateful to T. van Hall for the HLA-E*01:03 transfectant; D. Geraghty for the 4D12 mAb; D. O’Connor and R. Wiseman for 454-based MHC typing; and Y. Guo, D. Laddy, and M. Stone (Aeras) for provision of the strain 68-1 RhCMV construct expressing the M. tuberculosis RpFA protein. We thank A. Townsend for help with the graphics, E. McDonald, A. Klug, A. Bhusari, G. Xu, J. Bae, C. Kahl, S. Hagen, A. Sylwester, and L. Boshears for technical or administrative assistance, J. Theiler for coding assistance, and P. Borrow for helpful discussions on HLA-E biology and peptide binding. This research was supported by National Institutes of Health Grants P01-AI094417, R37-AI054292, R01-DE021291, R01-AI095113, R01-AI117802, R01-AI059457, U24-OD010850, P51-OD011092 and P50-GM065794 and Contract HHSN272201100013C; the Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID; UM1-AI100645-01) of the National Institute of Allergy and Infectious Diseases; the Bill and Melinda Gates Foundation (Global Health Grants OPP1108533 and OPP1133649); Aeras Global TB Vaccine Foundation; and Center for Nonlinear Studies at the Los Alamos National Labs (LANL). LANL institutional computing was used for carrying out all-atom molecular dynamics simulations. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or other funders. OHSU has submitted a provisional patent entitled “Methods and compositions useful in generating non-canonical CD8+ T cell responses” (inventors: L.J.P., K.F., J.B.S, D.M, and S.G.H.). OHSU and Drs. Picker, Hansen, Früh, and Nelson have a significant financial interest in TomegaVax, Inc., a company that may have a commercial interest in the results of this research and technology. Drs. Picker, Früh, and Nelson serve on the board of TomegaVax and Dr. Früh is also an uncompensated officer of the company. The potential individual and institutional conflicts of interest have been reviewed and managed by OHSU. Additional data used in this report are available on Science online in the supplementary materials.
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