Universal Non-Classical MHC I Vaccines: HLA-E-Restricted Antigenic Peptides as Universal Vaccines to Treat Allergy, Inflammation, Autoimmune and Infectious Diseases, and Cancers

Abstract

The embodiment of the invention is to enable universal non-classical MHC I peptide vaccines restricted to HLA-E, HLA-F and HLA-G. An algorithm was develop to predict HLA-E binding immunogenic or suppressorgenic peptides of the autologous origins, e.g., autoantigens, inflammatory antigens, IgE and cancer antigens, and of the microbial origins. Thus, the embodiment of the invention is to load the antigenic peptides of medical and therapeutic importance onto the non-polymorphic HLA-E, HLA-F, and HLA-G culminating in universal vaccines, bypassing highly polymorphic classical MHC I, e.g., HLA-A, HLA-B and HLA-C pathways, in order to treat autoimmune diseases, allergy, inflammatory diseases, cancers, and infectious diseases for all human population. Derlin-1 and UL40 pathways are utilized to enable antigen presentation and vaccine efficacies in the non-classical MHC I pathways.

Claims

1. A composition of the endogenous and exogenous antigenic peptides to HLA-E, -F, -G as universal vaccines, wherein the antigenic peptides are predicted by the Ige predict algorithm or overlapping 8-11-mers, processed and presented by antigen-presenting cells, and wherein the antigenic peptides are derived from auto-antigens, inflammatory molecules, IgE, cancer antigens, allergens, bacterial, fungal and viral antigens.

2. A claim according to claim 1, wherein the HLA-E-restricted IgE peptides, comprising IgE peptide sequences of SEQ ID: #40 to SEQ ID: #77, are derived from the autologous antigen, IgE (SEQ ID: #78).

3. A claim according to claim 1, wherein the HLA-E-restricted TNF-a peptides, are derived from the autologous antigen, tumor necrosis factor-1a (SEQ ID: #79, SEQ ID: #80).

4. A method of binding the endogenous and exogenous antigenic peptides to HLA-E, -F, -G as universal vaccines, wherein the antigenic peptides processed and presented by antigen-presenting cells are derived from auto-antigens, inflammatory molecules, cancer antigens, allergens, bacterial, fungal and viral antigens, and wherein antigen presenting cells are phenotypically or genetically modified for preferential loading antigenic peptides onto HLA-E, -F, -G, whereby vaccine-activated immune cells provide immune defense or orchestrate immune homeostasis to the vaccinated hosts.

5. A claim according to claim 4, wherein vaccine-activated immune cells are co-stimulated by positive enhancing second signals, whereby protective CD4+ or CD4/CD8 or CD8+ T cells are elicited for vaccine-mediated immune protection against microbial infection, inflammation and cancers.

6. A claim according to claim 4, wherein vaccine-activated immune cells are co-stimulated by negative immunoregulatory tolerance-inducing signals, whereby suppressive or immunoregulatory CD4+ or CD4+/CD8+ or CD8+ T cells are elicited for vaccine-mediated immune suppression or homeostasis against inflammation and autoimmune diseases.

7. A claim according to claim 4, wherein antigen-presenting cells are derived from monocytes, blood dendritic cells, monocyte-derived dendritic cells, tumor biopsies.

8. A claim according to claim 4, wherein the antigenic peptides are derived from immunoglobulin E, tumor necrosis factor-1.

9. A claim according to claim 4, wherein antigen-presenting cells are treated with US 11 and US2, or US11, or US6 upon loading of antigenic peptides.

10. A claim according to claim 4, wherein antigen-presenting cells are treated with siRNA/shRNA of Derlin-1, SEL-1L, HRD1, p97, Ufd1-Npl4, BiP, proteasome inhibitors, heat shock proteins, whereby uploading the antigenic peptides to HLA-E is modulated.

11. A claim according to claim 4, wherein the routes of vaccination comprise intradermal injections, tattoo gun immunization, and intramuscular injections, electroporation, gene-gun injection, subcutaneous injections, sublingual injections, targeting professional and facultative antigen-presenting cells.

12. A claim according to claim 4, wherein peptides binding to MHC Ib, e.g., HLA-E, HLA-G, and HLA-F are validated by diminishing the augmented renaturation binding via a canonical indicator peptide to conformation-specific anti-HLA-E solid phase.

13. A claim according to claim 4, wherein peptides binding to MHC Ib, e.g., HLA-E, HLA-G, and HLA-F are validated by upregulating surface MHC Ib of cell lines defective in TAP such as mutant T2 cells, and cell lines deficient of MHC Ia such as MHC Ib-transfected 0.221 cells.

14. A claim according to claim 10, wherein the length of the MHC Ib binding peptide is an 8-mer, a 9-mer, a 10-mer and from a 11- to 43-mer.

15. A claim according to claim 11, wherein the length of the MHC Ib binding peptide is an 8-mer, a 9-mer, a 10-mer and from a 11- to 43-mer.

16. A claim of using viral UL40 as a universal vaccine vehicle for inducing peptide and HLA-E, -F, -G-restricted immune immunity or immune tolerance.

17. A claim according to claim 16, further wherein the natural viral HLA-E leader peptide mimicry is substituted with leader-like peptides of autoimmune antigenic peptides or inflammation-related peptides for inducing immunoregulatory Treg/Ts for a healthy immune homeostasis.

18. A claim according to claim 16, further wherein the natural viral HLA-E leader peptide mimicry is substituted with leader-like peptides of microbial peptides or cancer antigenic peptides for inducing helper or cytotoxic CD4+, CD8+ or CD4+CD8+ T cells for immune defense against invading microbial pathogens and autochthonous cancers.

Description

EXAMPLE 1: IDENTIFICATION OF HLA-E EXPRESSION BY INCUBATING WITH HLA-E PREDICTED PEPTIDES

[0049] Surface Expression of HLA-E or HLA-G can be Induced by Retinold X Receptor (RXR) Agonist or Interferon .

[0050] HLA-E and HLA-G share the same ability to interact with NK cell receptor as well as TCRs regulating both innate and adaptive immunity. However, unlike classic MHC molecules widely expressed in tissues, non-classical MHC, HLA-E and HLA-G are characteristic of their limited polymorphism and low cell surface expression. Enhancement of expression of HLA-E and G is pivotal for non-classic MHC to regulate immunity. Bexarotene and interferon- were used as anti-cancer or anti-viral agents. (1) Thus, importantly, RXR agonist, SR11237 and bexarotene dramatically induced HLA-E expression of immature MoDC cells over a three-log intensity. However, they had little or no effect on HLA-G expression. Interferon had a similar effect, increasing intense expression of HLA-E but not that HLA-G on immature MoDCs (FIG. 1). Therefore, an embodiment of the invention is to augment HLA-E-mediated peptide presentation to achieve a curative effect for infectious diseases, inflammatory diseases, autoimmune diseases, or cancers by treating with interferon or other cytokines in a regimen. At levels of PBMCs, expression, interferon increased expression of HLA-G about two-fold in CD4.sup.+ T cells as well as in CD8.sup.+ T cells (FIG. 2A) but decrease HLA-E expression in these cells (FIG. 2B). Moreover, Interferon f increased both HLA-E and HLA-G expression in CD19.sup.+ cells. HLA-G plays an important role in downregulating immunity and inflammation. Immunoregulatory effector and target cells can mutually express HLA-G as well as ILT-1, 2, 3, 4 on cell surface, promoting a bilateral and bipartisan HLA-G/ILT-x interactive bridges amongst CD4/CD4; CD8/CD8; CD4/CD8; CD4/B cell; CD8/B cell. An embodiment of the invention is to augment the immunoregulatory efficacies of CD4 and CD8 T cells, and CD19 B cells, serving reciprocally as immunoregulatory cells and targets for manipulation for a curative effect for infectious diseases, cancers, and alleviating inflammatory diseases and autoimmune diseases.

[0051] Peptides-Pulsed MoDC Induces HLA-G Expression of CD8+ T Cells.

[0052] Nonclassical HLA-G is mainly expressed in the fetal tissues maintaining immune tolerance, and also expressed in dendritic cells, B cells and CD4 T cells and CD8 T cells, as universal targets due to limited polymorphism. In the embodiment of this invention, a cooperation between nonclassical MHC I and classical MHCI can increase the range of immunosuppression. The embodiment dictates that nonclassical HLA-G concomitantly co-expressed on nominal antigenic peptides/classical MHC I-restricted cell types, e.g., CD8 or CD4 single or CD4/CD8 double positive cells in serving the immunoregulatory T cells utilizing both TCR-mediated repertoire as well as HLA-G/ILT-x pathways, in particular, HLA-G5 can engage ILT4 on dendritic cells to stimulate IL-10, and induce Treg (Selmani et al., 2008. Stem Cell, 26: 212).

[0053] In the embodiment, we demonstrate the feasibility to induce a centrally important HLA-A2.01 peptide induced we showed that, after pulsed with IgE epitope A2 peptide-induced CD8 T-cell bearing the immunoregulatory HLA-G positive phenotype stimulated by nuclear receptor agonist, SR11237, both immature and mature MoDC induce significant amount of HLA G expression in CD8.sup.+ T cells. But no further increase was observed after treatment of SR11237 (FIG. 3). Similarly, open conformer HLA-F can be upregulated via such types of modulators and act as a hammerhead to transmit suppressive signal via KIRx/HLA-F (open conformer) onto a NK; alternatively, CTL/Treg can recognize target via TCR/HLA-F-peptides (closed conformer). Furthermore, the state of the cells permits dual recognition in that a bifunctional CTL/Treg with HLA-F open conformer hammerhead can recruit NK cells via KIRx, as well as target closed HLA-F-peptides on targets, which maximize target lysis or suppression via CTL/Treg/NK clustering activities.

[0054] Attempt to Identify IgE Epitope HLA-E Binding Peptide by T 2 Cells.

[0055] The mutant human cell line T2, defective in antigen presentation in the context of MHC I, exhibits low levels of surface MHC I expression, but high levels of surface expression after binding to MHC I-restricted peptides (Lorente et al. 2011. J.B.C. 286:38054). HLA-E displays the same characteristic as MHC I and its surface expression in T2 cells is TAP- and tapasin-dependent and is correlated with binding of HLA-E to its peptides providing approach to screen the peptide that can bind to HLA-E. Herein, we showed that A 2 leader peptide (predicted by the IEBD and MHCPan4.0 Programs) enhanced surface expression after treatment of T2 cells with peptide overnight incubation indicating it can bind and enhance HLA-E expression. However, B3 to B5 have no effect on surface expression of HLA-E in T 2 cells (FIG. 4). In contrast, IgE peptide E33 (TMTLPATTL, SEQ ID: #68) predicted by Ige Predict, the proprietary program developed by this Inventor and explained in the Example 2 exerted a stronger upregulation of HLA-E compared to the canonical A2 leader peptide (FIG. 12); moreover, two analogous and heteroclitic peptides: SEQ ID: #71 and SEQ ID: #77 generated from the E33 exhibited a higher predicted binding scores, among others in Panel B showing comparable scores. Furthermore, a list of good HLA-E restricted IgE binders was shown in Panel C (SEQ ID: #18 to SEQ ID: #42).

[0056] Construction of Fusion Protein of HLA E and 2microglobin.

[0057] HLA-E molecules loaded with signal sequence-derive peptides can also engage the inhibitory NKG2A and protect targets from NK-mediated lysis, while peptide-processing, loading, presentation of the same leader or nominal peptide can notwithstanding induce CTL that directly recognize and lyses target cells. HLA-E forms heterodimer with 2 microglobulin in order to be stabilized on cell surface, contribute to binding to signal peptides. In this study, we have successfully used pET recombinant system to make fusion protein, HLA-E and 2 microglobulin (2m). Truncated HLA-E containing only 1, 2 and 3 domains fused with 2 microglobulin, was cloned, expressed in E coli. FIG. 5A showed that vector, pET-HLA-E-2m was digested by Bam H I and Hind III confirming fusion of HLA-E and 2 m was cloned into pET vector. FIG. 5B showed that fusion protein of HLA-E-2m was detected by western blotting with anti HLA-E or anti-2m antibody; in comparison, 2 m was detected only by anti-2 m antibody.

[0058] Biological and chemical systems are designed for characterizing and assaying HLA-E binding/-restricted peptides. In an embodiment of the invention to uncover nominal HLA-E nominal antigenic peptides, HLA-I negative K562 cells and 0.221 cells (Strong, 2003, JBC, 278:5082) are used for transfection with HLA-E*0101, and/or HLA-E*0103, and along a human .sub.2m construct. Because of the lack of endogenously processed classical MHC I processed peptides, the HLA-E transfected cells can be used for screening, purification and evaluating a repertoire of endogenous peptides loaded and bound to HLA-E. Natural HLA-E/2m with the loaded endogenous peptides, as canonical leader-like peptides or nominal, noncanonical non-leader-like peptides bound to contours varying from that to the orthodox leader peptide focal of spread, can be included in and purified via anti-HLA-E antibody-bound affinity column, or size exclusion and by ion exchange chromatography, and wherein the peptides are eluted from the HLA-E, and subjected to the tandem mass spectrometry (MS/MS) peptide sequencing at pico- to femto-mole quantity. Notably, the sequence repertoire or universe is HLA-E restricted, which can be stored in a database, feeding into, analyzed by the in-house proprietary Igepredict Program, since K562 as well as 0.221 are devoid of classical MHC I (HLA-I), e.g., HLA_A, _B, _C.

[0059] The embodiment of this invention is to use this additional knowledge base or database to train and enhance the algorithm of pythonanywhere_lgepredict, a proprietary licensing software (Example 2). In another embodiment of the invention, the endogenous and natural sequences of the endogenous protein and exogenous proteins of infectious microbial origins, can be used as a drug API as an approximation to the self-peptides relevant for treating autoimmune diseases, inflammatory diseases, infectious diseases, and cancers.

[0060] The following antibodies are used for characterization: anti-HLA-A/B/C (clone W6/32), and anti-HLA-E (clone 3D12) and anti-HLA-A2 (clone BB7.2). Normal and TAP deficient T-2 cells and TAP deficient rodent RMA-S cells (Ossevoort et al. 1993. Eur. J.I. 23: 3082), transfected with HLA_A2.01_Kb cells in the laboratory, and HLA-E01.01 or 01.03 transfected 0.221 and K562 are used for the binding studies. Upregulation of HLA-E is studied in HLA-E binding studies using leader sequence like canonical leader peptides or nominal peptides resembling binding to classical MHC I, using the in-house developed program (http://ige.pythonanywhere.com/dashboard, (Example 2), The upregulated surface HLA-E of treated cells can then be measured by a commercial FACS machines and software, and data can be analyzed with FlowJo software (Tree star, Inc). To accumulate more HLA-E via the trans-Golgi network (TGN), cells can be incubated with 10 M brefeldin A (BFA, and 200 nM bafilomycin-A1 (Streptomyces griseus) for the augmented surface expression of HLA-E.

[0061] Normal T-2 cells or reverted without TAP defects can also be used for identifying the strong HLA-E binding peptide (s). Under the normal condition, surface HLA-E is expressed at high levels, and the levels of expression can be further augmented with incubation with the leader peptides from HLA-E. Under this condition, the nonameric peptides predicted to exhibit the binding motif may not exhibit a further enhancement of the surface HLA-E expression, since endogenous HLA-A2.02.01 leader peptide, generated from proteasome pathway as well as the ER pathway can bind to HLA-E, minimally assisted by the exogenously added HLA-E binding peptides, with regards to its surface expression. Surface expressed HLA-E US11 (SEQ ID: #87, SEQ ID: #88) and US2 (SEQ ID: #89, SEQ ID: #90) are HCMV proteins that decrease classical MHC I, e.g., HLA-A2.01 and HLA-B7.02. To evaluate whether or not US11 can also diminish HLA-E. thus damaging both HLA-E-restricted viral peptide presentation and CTL induction as a viral contrivance in an HLA-E constitutively activated cell lines. T2 cells were then stably transfected with pCDNA3.1 expressing US11. FIG. 5 showed importantly that US11 impressively knock-downed expression of HLA-A2, -B7 expression, embodying the vaccine strategy to augment HLA-E nominal antigenic peptide presentation by APC (e.g., T2 cells, a fusion of B and T leukemic cells, expressing high levels of CD86 and CD40 with APC efficacies comparable to normal MoDCs observed in IGE lab facility), depleted of classical MHC I by US11/US2.

[0062] Thus, one embodiment of this invention is to employ US11-transfected TAP deficient T2 with downregulated MHC I, e.g., HLA-A, -B, -C for screening and evaluating for HLA-E binding peptides via augmented surface HLA-E expression by depriving competitor HLA-A, -B, -C pathways as well as preventing generation of HLA-A, -B, -C leader peptides, which should sabotage the loading HLA-E binding nominal peptides.

EXAMPLE 2: CONSTRUCT ALGORITHMS BASED ON PYTHON PROGRAMS IN PREDICTING THE HLA-E CANONICAL MOTIFS

[0063] A python program is written using a training set of data, which contains 8-11-meric peptides exhibiting binding to HLA-E, and exhibiting binding, weighted toward the conventional HLA-A, -B, -C leader peptide motifs, while the test sets consist of 8-11-mers amino acid peptide walks of designated protein candidates. The probability is calculated upon testing 2-mer and increased by one more residue at a given time for testing the probability of binding in order to consummate the re-iterative rounds of testing, and a score was assigned for each existing test sequence. The algorithm is presented as follows: A web site to propagate this service to the nonclassical MHC I, HLA-E, F, G community was made available by presenting the constructed website (http://ige.pythonanywhere.com) for licensable use.

IGE Predict:

[0064] Function of the Script.

[0065] The script for predicting binding scores consists two functions, the long sequence parsing and short sequence score prediction. Those two functions will be discussed in the following paragraph. The IgE short sequence (9-mer) prediction script takes in 1 nonamer input and breaks it down to its n-grams. Then, according to our pre-trained weight, it will add up the score for each of its local components. For example, Assume in the trained model, the following information is learned: {VM:1, LL:3, AB:-1, DD:-3} (VM,LL,AB,DD) are patterns found and the number behind them are weighted. Then, for each sequence input, the script breaks the input sequence down, and check if any of these combinations match the patter in pre-trained weight. It would add all the weight of found patter together. By doing this, a score that denotes the binding probability of these sequences would be computed. Next, the long sequence parsing tool will take in a long sequence (e.g., IgE heavy chain, CH1-CH4 sequence) and use window slides to break them down to 9-mers. Step 1: select and start with the first character, and then include 8 characters that follow in that a new sequence of 9 characters form the script; Step 2: Move forward and for each position the script would repeat what we did in step 1 and then parse the long sequence to shorter subsequences; Step 3: Fit in parsed sequence into the program for binding score for each of the parsed sequences.

[0066] SVM (Support Vector Machine) and other machine learning networks (such as convolutional neural network and deep neural network) are tested as possible candidates. However, due to the amount of data presented, significant progress in accuracy could not be made. SVM model would returns a result with low accuracy because the input data set is small and random. As a result, SVM model would make arbitrary prediction and this leads to low accuracy. In addition to that, due to the heavy imbalance presented in the data set, the prediction result is heavily biased which is also an explanation for the initial low accuracy. Next the input is fitted with some pre-trained neural network, such as CNN (convolutional neural network). Deep learning framework Keras (an open source of neural network written in Python) is tested to build a neural net with two convolutional 2D layer and two fully-connected layer. However, this approach showed an overfitted result. Although training set is associated with a very high predict accuracy, the test set showed limited accuracy. The reason is that a large training set could not be found to prevent model getting train set overfit. This kind of model would have good behavior on the training set; however, is variable upon new data feed.

[0067] Training Method.

[0068] Train set comes from the experiment results, with a nonamer peptide sequence and its binding rate. The whole train set consist of 69 peptides. Pre-trained weight: The script goes through all the sequences in a data set, separate the dataset with binding peptides and non-bind peptides, based on the binding rate. Each sequence in the training set is parsed into small pieces, and based on their binding score, a weight is assigned, which is then added to the cumulative weight for each identical sub-sequence, stored in a dictionary. For example, if a sequence is found in a non-binding peptide, a weight of (1*normalized binding probability) is given. On the other hand, if a binding peptide sub-sequence is found, a weight of (1*normalized binding probability) is assigned to itself. After the weight is assigned, it is added to the cumulative weight for this sub-sequence. Statically, this entails predicting the likelihood of a peptide sequence binding to the HLA-E. After going through the training set, a dictionary of sub-sequence weights can be computed, and they can be utilized in the prediction model. To continually improve the train set, which plays a pivotal role in precision prediction of HLA-E based vaccine candidate from a test set. Thus an embodiment of this invention is to continually explore and use this additional information of knowledge base database of HLA-E purified, nominal antigenic peptides pools from 8-25-mer, including nonamer from the HLA-E transfected 0.221 cell line by this inventor and scantily published literatures, or data mined from available resources, as well as other 0.221 cell lines, transfected with HLA-E co-transfected with HLA_A, B, _C. in order to continually train and enhance the algorithm of pythonanywhere_lgepredict, a proprietary licensing software program.

[0069] Using the pre-trained weight: For each input peptides, a scan is performed, and all the possible sub-sequences are extracted. Then, for each subsequence, the script would locate the weight in the prediction model and add them to the cumulative binding score. After performing the look up for each subsequence, the binding score will be output. The higher the binding score, the more likely it shall bind.

[0070] Test Method.

[0071] Algorithm II was established following the cumulative score assignment according to a training method or training methods in arrays. Initially, the finalized algorithm verified prediction result with the popular theory that most of the binding peptides possess a specific patter, or motif called VMAPRTLLL (SEQ ID: #51). It verified the test result with this patter and discovered that most of the results align with this patter on position 1, 3, 6, 7, 9. These are key positions to determine whether this peptide will bind. Next, the finalized algorithm also verified the results against confirmed binding peptides list. Confirmed binding peptides are read, fitted in as an aptamer with pre-trained weight and a bind score is calculated. The score is much higher than random sequence. In addition, some confirmed unbinding peptides were also fed into this algorithm, the result is a large negative number. Finally, IgE heavy chain is used as an input, and sub-sequence with high binding score from this long sequence are computed. The script successfully parses the heavy chain and assigns binding score for each nonameric sequence inside the IgE heavy chain or other sequences. The largest binding score in this chain also matches the VMAPRTLLL SEQ ID: #51) pattern. Initially, based on the information gathered, there were more than 100 sequences for input. Initially extracted data set were moderately balanced to incorporate diversity and skewness, more data base input will re-weight the predive precision and bias. The Program is under continually updated using feeder incorporating data, weighted differently in human and rhesus macaques. The Program, igepredict is reiteratively trained thereof for producing a fitting algorithm for a relative precision prediction.

[0072] Website Development.

[0073] A website is developed a website with python-based web framework Flask. Provide a front-end user interface allow users to enter their file and sequence. Put the prediction script in backend to analysis input data. Then generate result (or result file) and send back to front-end. Also provide new users register and login to track user activities. Log user input into the database, which permit only registered users to access. List of functions: 1: Registration system that allow super user to track users' prediction histories. 2: Integrated dashboard that gives user easy access to all the functionalities. 3: User Friendly interface for file upload and real time prediction result checking. 4: Downloadable format for spreadsheet of prediction results. 5: Multi-user capability that allow each user to upload their separated prediction queries without interference. 6: Single Sequence prediction interface that allows for Single Peptide Sequence prediction.

EXAMPLE 3: CONSTRUCTION OF HUMAN HLA-E AND HUMAN BETA 2 MICROGLOBULIN (2M) FOR HLA-E BASED PEPTIDE BINDING

[0074] A conformation capture of canonical/non-canonical peptide (C3N) method to HLA-E is designed to test predicted HLA-E binding peptides. HLA-E canonical and nominal peptides predicted from the python algorithm of Igepredict program and any 9 to 12-mer made collinearly with an overlapping IgE sequence can be tested by a binding assay. As illustrated herein, human HLA-E and 2m fusion construct can be made and collinearly expressed as a fusion protein as shown in FIG. 2. The assay for intact re-natured conformation of HLA-E is dependent on gaining the binding signal using anti-HLA-E coated plate to capture re-natured HLA-E in the presence of beta 2 microglobulin and leader peptides. In short, HLA-E-2m fusion product was solubilized from the inclusion bodies in the presence of 6 M urea, and then purified from the His-tag column in the continual presence of 6 M urea. The denatured material was diluted out from 6 M urea to 100-fold to 500-fold in PBS; alternatively, the three HLA-E preps were diluted in PBS at 10 g/ml final with the addition/incubation of A2 leader peptides from 0.1 g/ml to 1 g/ml for 48 h at 4 C., and captured by ELISA plate coated with anti-HLA-E. FIG. 2 showed a dramatic increment of HLA-E binding from ground zero to significant binding around 0.02 OD with HLA-E leader peptide addition at 0.1 g/ml to, and a striking increment to 0.2 OD (10 fold) and to above 0.3 OD (15 fold) with the addition of 1.0 g/ml the leader peptide. Notably, the HLA-E peptide binding capacity was enhanced with external addition of 2m (20% enhanced peptide binding compared to HLA-E heavy chain alone); in contrast, the covalently, recombinant 2m-HLA-E exhibited more than 60% peptide binding enhancement. Thus, this observation indicated that the native HLA-E structural conformation recognizable by mAb anti-HLA-E was partially restored by diluting out or dialyzing out the 6 M denaturant, and the covalently attached b2m can restore or stabilize the peptide binding capacity of HLA-E to canonical biotinylated leader peptide, and HLA-E vaccine candidate peptides can therefore compete off the biotinylated peptides to HLA-E-2m constructs.

[0075] Thus, an embodiment of the invention is to identify the leader-like peptides and nominal peptides from druggable protein targets of the proteomic database, which compete for HLA-E binding pockets by displacing the biotinylated leader peptide as an indicator. Moreover, leader-like and nominal HLA-E binders can be predicted by the Igepredict_pythonanywhere Program of Example 2, synthesized, and tested for competitive displacement of biotinylated leader peptide in the renaturation binding assay. Therefore, one embodiment of the invention resides in using the C3N restoring the native conformation of HLA-E to test and identify HLA-E peptides predicted from various sources of therapeutic proteins, according to the pythonanywhere_lgepredict Program, which is to yield API peptide drug content/sequences for rendering universal vaccines as targets for the infectious microbial diseases, inflammatory diseases, autoimmune diseases, neurological disorders, and cancers.

EXAMPLE 4: NATURAL EVASIVE UL40 CONSTRUCT HARNESSED AS A POTENT HLA-E VACCINE DELIVERY PLATFORM

[0076] Human cytomegalovirus (HCMV) is a beta-herpes virus developed a variety of mechanisms to down-regulate expression of MHC I, evading recognition of cytotoxic T lymphocytes. One way is to block the transporter associated with antigen processing (TAP), another way is via a fast degradation of MHC I via US2, US6 and US11 (Lehner et al. 1997. PNAS. 94: 6904; Lilley and Ploegh, 2004. Nature. 429: 834). Example 8 showed that fast degradation of classical MHC I in preventing CTL induction to both viral antigenic peptides bound to classical MHC I. Although more leader peptides are produced due to a fast degradation of classical MHC I, which can enhance HLA-E-mediated immunity. Although one aspect of viral immune evasion was described regarding silencing NKG2A/C pathway via the leader peptide mimicry encoded by viral UL40 protein as described, yet the embodiment of the invention focuses instead on the TCR/HLA-E peptide arm, which can be abrogated by whichever leader or leader-like or nominal peptide surrogates replacing the original viral leader peptide mimicry. Thus, the embodiment of the invention is that UL40 serves as a universal vaccine scaffold for accommodating API at will.

[0077] FIG. 11 showed that the cloned UL40 open reading frame (SEQ ID: 81; SEQ ID: 82) through synthetic gene approach designed by us. The synthetic gene sequence shown in FIG. 11 encodes a putative type I glycoprotein of 221-amino acid residues with an N-terminal 37amino acid signal sequence, containing the peptide homologous to the HLA-E binding peptide (from HLA-C), a 144 amino acid domain with 3 N-glycosylation sites, a 20-amino acid transmembrane domain, and a C-terminus of 20 amino acids (Tomasec, 2000, Science, 287: 1031). A typical leader-like peptide with the position 2 and 9 motif for stronger consensus HLA-E binding, while accompanied by position 3, 6, 7 on this nonamer as modulators for the main motif binding presentable to T cell receptors, wherein the binding of such a leader-like or nominal peptide can be blocked by the viral leader peptide mimicry co-translated in the N-terminus of UL40. Leader peptides from classical MHC I usually fill the binding site pockets of HLA-E, permitting its silencing of the NK cell counterpart. In contrast, position 5 and 8 as a main motif binder for NKG2x/CD94, which in this embodiment of invention is used as a platform to deviate NK cell recognition to the advantage of the immunized host, e.g., to augment an vaccine responses by recruiting also NK-mediated killing of the foreign targets, e.g., viral and bacterial infected cells, and tumors; or alternatively contributing to induction of NK-mediated tolerance to prevent an undesirable targeting to self.

[0078] During the fast degradation of classical MHC I, catalyzed by CMV-encoded US11/US2, the availability of classical leader peptides can be augmented if TAP is not uniformly knocked down by US6 (Matshulla et al., 2017. Sci. Rep.7:2933), and despite the competition pressure, HLA-E can be loaded with the indigenous leader peptide carried by CMV, which takes advantage of the viral mimicry leader peptides and tolerize all the leader peptides, leader-peptide-like or nominal peptides property of UL40 as an immune blindfold. The open reading frame of UL40 protein contains a 9-amino acid sequence, exactly homologous or identical to the HLA-E binding leader peptide from HLA-A2 or A2-like molecules (Tomasec et al., 2000. Science. 287: 1031). The embodiment of the invention takes advantage of this viral immune blindfold in two major ways (i) replacing a HLA-E universal autoantigenic vaccine epitope and the tolerogenic UL40 blindfold to tolerize/abort the autoimmune attack; (ii) replacing a HLA-E universal microbial or tumorous antigenic peptide vaccine epitopes together with converting tolerogenic UL40 into immunogenic UL40 signal in order to attack/neutralize the danger antigenic invasion (microbial, inflammatory and cancer).

[0079] The embodiment of the modified vector can consist of HLA-E binding motif of a nominal protein of any kind, and the site-specific mutagenesis will be prepared for make a deletion of the viral encoded HLA-A2 or -A2 like leader peptides with the leader-like nonamer (s) predicted from the igepredict program. In one version, the entirety of 221 amino acid except the swabbed out or exchanged HLA_A2 or A2-like leader peptides with a nominal leader-like peptide sequences, shall be preserved. This orthodox version should inherit in nature all possible contrivances that mediate processing out of the leader-like peptides and presentation of such peptides unto the HLA-E.

[0080] In one modified version, the transmembrane peptide of UL40 is deleted in order to have more concealed expression in the ER and peptide fragments including leader-like peptides are generated in the ER and ante-transported to the cytosol, processed by the proteasomes and retro-transported back to the ER and Golgi and permit binding and renaturation of endogenously synthesized HLA-E within the ER, and permit leader-like peptide presentation. In another version, the transmembrane sequence of UL40 is preserved to permit the natural cleavage of the leader peptide sequences from the N-terminus 37 amino acid signal peptide sequence, wherein the leader peptide-like nonameric sequences from IgE or any given nominal protein is processed and presented. In another embodiment to facilitate dimerization of the UL40, leader-like sequences swabbed in N-terminus signal sequence with the transmembrane domain deleted, is fused to an IgG Fc region, to permit dimeric leader-like peptide presentation and processing, and the fusion protein can then be employed as a leader-peptide UL 40 embedded in a protein as a tolerogenic/suppressorgenic Ying vaccine. Another main embodiment of the invention it to further expand the concept of an immunogenic carrier Yang protein for immune activation, any polypeptide chain or peptide design can be used as an immunogenic carrier protein with the appropriate built-in costimulation.

[0081] In contrast, inhibitory or modulatory position 5 and 8 of HLA-E bound (viral) peptides on targets can play a key role in interacting NK cells or bifunctional CTL with NK activities in that NK cells are activated via NKG2C/E/H, which will lyse viral infected, or cancers or inflammatory cells, autoreactive or autoimmune cells as targets versus NKG2A/B, which are inhibitory for NK or bifunctional CTL to prevent target lysis. Immune evasion by CMV consists of four parts: one evasion is by destroying the immunesurveillance of classical MHC I, HLA_A, _B, and _C mediated CTL via US2, US11, which accelerates MHC I heavy chain degradation. The second is via US6 which degrades TAP, which also abrogate CTL induction due to lack of MHC Ia-restricted viral peptides via retrograde transport from proteasomes to ER and loading to MHC I via TGN cell surface expression. The third is via the induction of inhibitory NKG2A/CD94 NK cells or bifunctional CTL, which protect or shield virus infected cells, without inducing NKG2C/CD94 lytic NK against the virus-infected cells. To tip the balance of these two activities favoring suppression, virus carries within it, the leader peptide of HLA_C, which abrogates leader like peptide immunosurveillance. An embodiment of this invention is to decipher or swap the endogenous leader peptide of UL40 with a putative vaccine epitope to render an HLA-E based universal vaccine.

[0082] The native HLA_C leader peptide must bind to HLA-E first and present the position 8 and positive 5 peptide of the mimicry HLA-E peptide to CD94, which then augments the high affinity interactions with NKG2A but not NKG2C, thus subverting the innate immunity of NK-mediated lysis via NKG2x. Furthermore, to ensure subverting adaptive immunity, UL40 will refrain from a self-destructive motif: HLA-E/VMAPRTLLL-CTL responses against itself in its own niche. The UL40L subverting immunosurveillance is to carry within it at the N-terminus human tolerogenic HLA-C leader peptide sequences as well as other tolerogenic contrivance sequences in UL40, which alone or together enable tolerance or abrogation of an induced CTL response. Thus, another embodiment of this invention is to render an immunosuppressive version of UL40 as a tolerogenic vaccine carrier for vaccine epitopes for suppressing an autoimmune attack, just as the virus deploys it in its natural history of infection to subvert/tolerize a positive protective anti-viral immune defense. Another embodiment of the invention is to turn a tolerogenic response of the tolerogenic UL40 platform into an immunogenic platform by incorporating a costimulatory signal.

[0083] In summary, the embodiment of this invention is to make a tolerogenic UL40 for treating autoimmune diseases or suppress inflammatory diseases. The vaccine embodied as such strengthens the tolerogenic leader peptides, leader-like peptides, or nominal autoimmune or inflammation-related antigenic peptides is conducive for inducing central tolerance of autoreactive or autoimmune cells, or induce a Treg response, which suppress autoreactive or autoimmune cells or inflammation-eliciting cells. Furthermore, the embodiment of the invention is to turn the UL40 into a vaccine with costimulations which renders an immunogenic vaccine peptides/HLA-E restricted universal CTL response due to the lack of HLA-E polymorphism; or helper CD4 T cell/antibody-mediated immune responses to defend against microbe-infected targets, and autochthonous cancers, autoreactive/autoimmune cells.

[0084] Notably, the aforementioned dual embodiment of using an HLA-E peptide related to a leader peptide, a leader-like peptide, or a non-leader nominal peptide as an immunogenic active pharmaceutical ingredient (API) vaccine versus a tolerogenic vaccine, depends on the context of an immune-stimulatory costimulation, or the lack of it thereof, or being an immunoregulatory, suppressive or downregulatory costimulation. The delivery platforms can be peptide-based, recombinant peptides in a suitable carrier protein such as UL40, or presented as a DNA vaccine, wherein costimulatory or suppressive modulators provided in an appropriate form. Thus, the following costimulatory molecules will be administered in an appropriate dosage and via an appropriate vaccination route culminating in determining a dichotomous outcome favorable for an immune response, which benefits the host in protecting against microbial infections, autochthonous tumors, and inflammatory diseases; or favorable for alleviating autoimmune attack or other inflammatory diseases.

[0085] The costimulatory and coinhibitory molecules (Vinuesa et al. 2016. ARI. 34: 335; Baumeister et al. ARI. 2016. 34: 539; Chen and Flies. 2013. Nat. Rev. Im. 13: 227; Esensten et al. 2016. Immunity. 44: 973) include and not limited to members of the immunoglobulin superfamily (IGSFs) and members of the tumor necrosis factor receptor superfamily (TNFRSFs). The invention embodies the use of two types of costimulatory and co-inhibitory molecules enabled by the use of an agonistic or antagonistic antibody or by a protein, a peptide, a DNA or RNA aptamer with adaptive binding to modulate the two classes of molecules therewith. Alternatively, a DNA vaccine packaging such a member of the two classes of molecules can be used along with the HLA-E based vaccine as a DNA vaccine adjuvant adjunct. This embodiment assists an immunogenic vaccine for the immune defense as well as a tolerogenic vaccine for immune homeostasis.

[0086] 1. TNF Receptor Superfamily (TNFSF):

[0087] TNFRSF receptors contain one or more extracellular cysteine-rich domains (CRDs), whereas the TNFSF ligands (TNFSF) contain a conserved extracellular TNF homology domain (THD), including HVEM, death receptor 3 (DR3, or TNFRSF25), CD40 (TNFRSF5) and lymphotoxin- receptor (LTBR, TNFRSF3), transmembrane activator and CAML interactor (TACI, TNFRSF13B), B cell-activating factor receptor (BAFFR, TNFRSF13C), B cell maturation protein (BCMA, TNFRSF17), interacting with APRIL, TNFSF13), B cell-activating factor (BAFF, TNFSF13B) which co-stimulates B cells; TWEAK receptor (TWEAKR, TNFRSF12A), ectodysplasin-A receptor (EDAR) and X-linked ectodysplasin-A receptor (XEDAR, TNFRSF27) as costimulatory or co-inhibitory molecules. Moreover, 4-1BB (CD137, TNFRSF9), OX40 (TNFSF4), CD27 (TNFRSF7), glucocorticoid-induced TNFR-related protein (GITR, TNFRSF18) and CD30 (TNFRSF8) as co-stimulatory molecules.

[0088] 2. Immunoglobulin Superfamily (IGSFs):

[0089] CD28 family interact primarily with members of the B7 family, albeit the co-inhibitory B and T lymphocyte attenuator (BTLA) engage the TNFRSF member herpes virus entry mediator (HVEM or TNFRSF14), and B7-H6 (NCR3LG1) engage NKp30 or NCR3 of the natural cytotoxicity receptor family in humans. Type I T cell immunoglobulin and mucin domain-containing molecules (TIM) consist of an IgV-like domain and a mucin-like domain. TIM can be co-stimulatory or co-inhibitory. CD2/SLAM: CD2 and signaling lymphocytic activation molecule (SLAM) with an IgV and an IgC domain interact with CD2 in a homophilic or heterophilic fashion. CD2 and SLAM function as co-stimulatory receptors on T cells, whereas 2B4 and LY108 function as co-inhibitory molecules. Butyrophilin (BTN) and BTN-like (BTNL) family molecules have extracellular structures are B7 family-like, while the function of a canonical b30.2-RING domain resident in the cytoplasmic tails can impart either a co-stimulatory or co-inhibitory.

[0090] In addition, CD4-like lymphocyte activation gene 3 protein (LAG3, or CD223), interacting with MHC II is co-inhibitory. Receptors CD226 (DNAM1), T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT; also known as VSIG9), cytotoxic and regulatory T cell molecule (CRTAM) and CD96 (TACTILE) as a group can interact with nectin and nectin-like ligands. CD226 and CRTAM are co-stimulatory, while TIGIT is co-inhibitory, and CD96 may be either. Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1, or CD305) of the LAIR family with a single C2-type immunoglobulin-like domain binding collagens is co-inhibitory. CD160, a glycosylphosphatidylinositol-anchored molecule with an IgV-like domain that on T cell deliver a co-inhibitory signal upon HVEM binding. Thus, one embodiment of this invention is that all the above co-stimulatory or co-inhibitory molecules of the IgSFs are employed as a vehicle of costimulation for stimulatory HLA-E-based vaccine for immune defense against tumor, microbial infections, while the co-inhibition can be harnessed to treat HLA-E mediated autoimmune diseases.

[0091] The switch between the tolerogenic and immunogenic on/off state can be manipulated at will by modulating the second stimulatory signals. The second costimulatory immunogenic signals include but are not limited to CD40, B7-1/CD80, B72 (CD86), OX-40, 41BB, TIM-1; CD28, ICOS, PD1H, TIGIT, BTLA, while the second costimulatory signals can be the high zone or low zone tolerogen by itself, PD-1, PDL-1, CTLA-4, LAG-3 (CD4 like, CD223), TGF-, IL-10, CD25, ICOS. Thus, the viral infected targets with HLA-E blocked by UL40 escape the damage NK-mediated damage. Thus, CMV can subvert classical MHC I-mediated CTL via US11/US2, as well as subverting HLA-E-mediated CTL and CD94/NKG2A/B NK-mediated lysis via UL40 evasion. And a complete optimization relies on the also the tolerance induction in the boxed in leader peptides to also evade boxed-in leader peptide, or leader-peptide like structure in HLA-E mediated CTL response. Hence the UL40 module can be engineered further into the tolerance module vs immunity modules with or without supplemented co-stimulation signals.

[0092] One main embodiment of the invention is to deploy viral UL40 (Tomasec et al., 2000, Science, 287: 1031) in a recombinant construct as a vaccine platform to deliver leader peptide-like and nominal sequences from a microbial protein, an autoimmune protein target, or IgE target respectively as a universal vaccine. Specifically, the viral mimicry leader peptide is replaced with an IgE leader peptide like sequence or nominal sequence in IgE to the N-terminal region of UL40 so that the IgE peptide(s) is juxtaposed to load onto the HLA-E binding pocket according to a similar strategy the viral delivery to arm or preempt a tolerogenic HLA-E. As an immunogenic vaccine, the vaccine-epitope inserted UL40 is further fused to an immunogenic CD4 epitope or a carrier protein or an aforementioned costimulatory signal, enabling the delivery of a second helper signal in order to provoke a vaccine epitope/HLA-E restricted CTL response, a CD4 response, a Ts/Treg response against the IgE-producing cell target for treating allergic inflammation, a viral infected cell for treating viral disease, or a target cell producing TNF- for treating rheumatoid arthritis; therefore to alleviate IgE-mediated allergy, to clear a viral infection, and to attenuate inflammatory mediators and/or alleviate cytokines-mediated inflammation.

[0093] Henceforth, this embodiment is a two-fold technology innovation subverting the otherwise natural tolerogenic UL40-leader peptide in signal peptide, e.g., the original viral sin is replaced or resurrected to a protective immunity to the advantage of the host, an antigenic peptide is piggybacked together with a second helper signal in order to break tolerance to the engineered antigenic peptides or a mimetics within the viral UL40 construct. Two modalities of breaking tolerance is to deliver an IgE mimicry; (i) in conjunction with an immunogenic carrier to stimulate CD4 helper T cells, e.g., promiscuous helper T cell peptides: PADRE, tetanus toxoid peptides, diphtheria toxin peptide, measles helper peptides, and KLH, GFP and other immunogenic proteins; (ii) in conjunction with an immunogenic adjuvant, e.g., those eliciting innate immunity via TLR (Toll-like receptor), NLR, and CpG, UL40-IgE mimetics; (iii) via an HLA-E expressing APC in a cell-based immunogenic adjuvant formulation. Thus, IgE mimetics is naturally processed along with endogenous HLA-E serving as a vaccine API, while the immunogenic formulation serves to provide the second helper or suppressive/transformative signal. Thus, a main embodiment of this invention is to substitute viral leader peptide sequences with IgE or nominal protein leader peptide-like sequences that arm the HLA-E for a universal IgE peptide or nominal peptide vaccine in the presence of second signal to elicit immune responses, breaking tolerance and cause infectious tolerance.

[0094] In another embodiment of the vaccine delivery, the DNA sequence encoding HLA-E binding peptides are cloned into a mammalian expression vector with a co-expressed immunogenic cytokines such as GM-CSF, CD40L, OX40L, IL-4, IL-5, IL-12, IFN-gamma, TGF-beta, suppressive Nrf-2, IL-35, heme oxygenase 1 (HO-1) or other recombinant costimulatory factors. HLA-E biding peptides DNA vaccine is injected intradermally (id)/transcutaneously (TCI), or via intramuscularly (im) via a primed and boost schedule at appropriate intervals. Mucosal and systemic immunity of eliciting 8-11 amino acid peptides/HLA-E. In another embodiment of HLA-E vaccine delivery, immunogenic 8-11 amino acid peptides are co-administered with promiscuous helper peptides, e.g., PADRE, diphtheria toxin peptides, tetanus peptides or measles virus helper peptides, or other immunogenic carrier protein in adjuvants, e.g., KLH and others, in the presence of adjuvant such as immune-stimulatory CpG, TLR ligands for toll-like receptor1-13, NLR ligands for Nod-like receptors: NODs, NLRPs, IPAFs.

EXAMPLE 5: APPLICATION OF UNIVERSAL MHC IB, E.G., HLA-E.-F. G VACCINES AGAINST PAN-HUMAN DISEASES, INCLUDING INFECTIOUS DISEASES, INFLAMMATORY DISEASES, AUTOIMMUNE DISEASES, NEUROLOGICAL DISORDERS, AND CANCERS

[0095] The invention concept of a universal vaccine is based on the presence of the universal, non-polymorphic HLA-E protein ubiquitously expressed on all the immune cell types (CD4, CD8, B cells, dendritic cells), and all the somatic cells, including cancers. FIG. 4 showed that nearly all the CD14 monocytes, including MoDCs and cDCs in this cellular set, are intensively positive for HLA-E, and to the same extent, about 60% or more of CD4 and CD8 cells are scored positive in normal peripheral blood mononuclear cells (PBMCs) under normal non-activating conditions separated from the human plasma. Importantly, circulating CD19 B cells are negative for HLA-E, except that activated CD19+ B cells (more skewed SSC/FSC) exhibit high higher levels of HLA-E, indicating a function of surface inducible HLA-E on B cells.

[0096] Due to this ubiquitous presence of HLA-E on the CD14 containing antigen presenting cells, e.g., dendritic cells, and cells of the myeloid series, CD4 helper and immunoregulatory cells types, as well as CD8+ cytotoxic and/or immunoregulatory cells, thus as shown in FIG. 10, there exists a communication network amongst these cell types via presentation and bi-lateral recognition of the HLA-E and TCR-bearing cell and NK cell network.

[0097] Noticeably, a tri-lateral communication is built in the cellular network (FIG. 10) due to availability and feasibility of HLA-E in the Treg and Teff, which can bind to endogenous self-peptide pools generation in the ER pathway, in that CD4+, CD4+CD8+, CD8+ Treg (Ts) and/or Teff can form mutual cognitive pairs of different assortments, e.g., Treg-Teff, Teff-Teff and even Treg (Ts)-Treg (Ts), and the pairing will permit an exchange of augmenting or suppressive signals amongst the cellular interactions. For example, CD4 Teff, or Treg (e.g., CD4 bearing/nonamer-HLA-E restricted, wherein high affinity nonamer binding via TCR compensates for the lower CD4 coreceptor binding to the 3 domain of HLA-E) or CD8 Teff or Treg recognizing an HLA-E-restricted autoantigenic peptide, initiating an autoimmune attack on the nervous tissues, can itself be recognized by HLA-E-self peptide restricted Treg (Ts), which recognize self peptides processed and presented by the autoimmune CD4 Teff or Treg or CD8 Teff or Treg or autoantigenic peptides bound to itself via its own HLA-E (HLA-E ubiquitously expressed on all immune cell types, FIG. 4).

[0098] As a corollary, each T cell regardless of the functionality as an effector or a regulator bears the HLA-E decorated self-marker, which in turn can be regulated amongst HLA-E restricted T cell community in a direct cognition. Alternatively, this type of interaction can intercalate or integrate with different types of APC to form a tripartite interaction, indirectly influencing each other via an APC, by forming a constellation so that the two T cell can cement a relation via an APC chaperone. As another corollary, an HLA-E-based peptide vaccine, can initiate an HLA-E and peptide based Teff or Treg (Ts) response, while these induced T cells can also bound the vaccine peptide and becomes mutually self-regulated within a special niche community. In another variation, if two such autoimmune peptides are administered simultaneously as vaccines. The T cell pair can form heterotypic interaction of two different HLA-E specific T cell pair of different functional assortment at will.

[0099] Thus, one embodiment of this invention is to administer at least one HLA-E peptide, or two, or more peptides or such peptides synthesized or recombinantly produced in tandem in a colinear and covalent fashion in order to orchestrate a desirable and robust impact of the HLA-E vaccine.

[0100] Overall, the embodiment of the invention resides in immunizing for the protective and ameliorating effect of a positive vaccine response using the method of identifying the pertinent disease-relevant non-self, foreign, naturally processed antigenic peptides (microbial peptides) from the microbial sources of infected cells, and self-antigens from tumors (cancer antigenic peptides) and inflammatory tissues [IgE (SEQ ID: #43), rheumatoid factors, TNF- (SEQ ID: #44, SEQ ID: #45)]. These naturally processed therapeutic peptides can be used as vaccines for eliciting effector CTL mediated responses along with immune-stimulatory enhancer (ISE) adjuvants. Another embodiment of the invention resides in tolerizing an potential or ongoing autoimmune attack damaging the tissues and organs. In this embodiment, the focal point of therapeutic interest is inducing CD8+ or CD4+ Treg, which alleviate the effector cell-based immune damage. Immuno-regulatory suppressive (IRS) adjuvants will be used with the therapeutic HLA-E based universal peptide vaccines.

[0101] Since HLA-E presenting somatic antigenic, leader sequence like peptides and nominal peptides can be characterized, identified, and sequenced via affinity chromatography, HPLC and MS/MS [FAB, triple quadruple, ion trap and time of flight (TOF)]. The occurrence of these developmental driven events, e.g., somatic antigenic peptide processing, and fitting onto HLA-E is at the formation and embryogenic stages. Thus, self/non-self discrimination of the HLA-E-based system like classical MHC I and MHC II take place similarly according to clonal deletion, clonal abortion, clonal anergy, and clonal activation and clonal infectious tolerance at levels of CD8, CD4 T-cells.

[0102] One embodiment of this invention is to immunize the host with HLA-E restricted tumor antigenic peptides along with strong costimulation, resulting in HLA-E restricted CTL to the HLA-E to damage tumors expressing HLA-E bound with tumor antigenic peptides. HLA-E restricted CTL can also express CD94/NKG2C to lyse HLA-E expressing tumor cells. The appropriate costimulation provided to bifunctional CTL/NK or NK will overcome the micro-environmental suppressive effect and render the CTL/NK competent to kill the tumor cells. The embodiment of this invention resides in harnessing the nominal tumor antigens on HLA-E for inducing anti-tumor CTL responses. Thus, this new arm of sterile immunity will not only attack or lyse tumors but also cause a tumor cell to withhold its tolerizing capacity against NK via a disengagement HLA-E from NKG2A/CD94 of NK cells. Notably, in this embodiment, the CTL induced by the HLA-E/tumor antigens can be a bifunctional NK-CTL, and under strong costimulation, expressing not only TCR immunized and specific for the tumor antigenic peptides restricted or presented by nonclassical MHC I, HLA-E but also express NKG2C/CD94 and NK-mediated killing of the tumors engaging in a dual protective pathway.

[0103] Many tumors are known to express high levels of HLA-E (Seliger, et al., Oncotarget, 7:67360; Huang, Oncol. Lett. 2017. 13: 3379), which bind to classical MHC I leader peptides as an immune evasive mechanism to tolerize NK-mediated defense. HLA-E expression on cancers lead to poor prognosis (Gooden, 2011. PNAS, 108:26). Lowering HLA-E levels enhanced NK-mediated cancer elimination (Enqvist et al., 2011. J. Immunol: 187:3546). Presentation of HLA-A2 leader peptide/HLA-E complexes by the tumors to NKG2A/CD94 expressing on NK cells or CTL can also render the CTL/NK or NK tolerant (Borrego et al, 1998 JEM 187:813). Typically, CD8 T cells to autologous leader peptides are well tolerized in an individual (Pietra, 2003. PNAS, 100:10896), and these anergic cells can be detected in a high percentage (10%) using autologous leader peptide-HLA-E tetramer staining method.

[0104] The principle of tolerance can be extended to leader-like self-peptides and also nominal self-peptides bound to or presented by HLA-E. In principle, a nominal autochthonous tumor antigen from cancers derived from the patients, in particular in the suppressive tumor microenvironment can be presented as a tolerogen. Since tissue proteins such as insulin, epidermal growth factor (EGF), other growth factors, cell cycle proteins etc., share identical amino acid sequences among individuals without exhibiting amino acid variation or polymorphism, of which the endogenously processed peptide presented by nonpolymorphic HLA-E renders a peptide antigenic candidate of the first in-kind universal cancer vaccine, fit for every cancer patients of the same tissue pathology, when coupled with a second costimulation.

[0105] The embodiment of this invention is to immunize the host with the HLA-E restricted antigenic peptide in the context of the second costimulatory signal for triggering or converting the tolerogenic signal to immunogenic signal, including covalent juxtaposition of the nominal peptide in the N-terminus and helper costimulation in the C terminus of the UL40 construct. Thus, this embodiment causes induction and amplification the CTL-mediated sterile immunity and NKG2C type NK-mediated lysis against this self-tissue tumor antigenic peptides restricted/presented by HLA-E on cancer cells. It follows with the peptide presentation by HLA-F and HLA-G, and recognition by T cells and NK cells. The embodiment of this invention claims a unique or universal tumor antigen presented by a nonpolymorphic HLA-E of non-classical MHCI, leading to activation of sterile CTL immunity and NK-mediated defense, wherein the tolerogenic signal is transformed by the second costimulatory signal for the product concept of a universal cancer vaccine unrestricted and unlike that of classical MHC I fit for an entire population.

[0106] An example as mentioned in Example 4 above, UL40 construct can be modified into an ISE for effector immune cells or into IRS for immunoregulation. A network of cellular communication among the three major cell types can exist in the immune system via the HLA-E+ peptides, e.g., self-peptides and foreign peptides as targets on the receptive cells, whereas HLA-E negative, or HLA-E+ peptide-specific CD4 and CD8 T cells can recognize HLA-E+ self/non-self peptides on CD4+, CD8+ T cells to form a direct network of T-T communication without the presence of APC; and the former T cell (HLA-E+ or ) is HLA-E-restricted, while the latter T cells bearing HLA-E+ self/non-self peptides can be restricted to nonclassical MHC I or classical MHC I presenting self or non-self peptides, and can like also engage in recognition of the former T cell via HLA-E restricted recognition, alternatively can recognize self peptide presented by classical MHC I plus peptides on a target. Thus, there can be bidirectional communication of these HLA-E+ self/non-self peptides CD4+ and CD8 T cells among themselves.

[0107] All the cells have HLA-E can have both a switch-on and a switch-off modality/module according to the state of the cells. Thus, this network permits an autochthonous, autologous, mono- or bidirectional mode of communications among a combinatory assembly of the immune system components in a non-classical MHC I, e.g., HLA-E and Qa1 and Qa2-based or also mixed with classical MHC I-based immunoregulatory network. In a particular case regarding controlling IgE production, naturally IgE-leader-like peptides generated in IgE lineage B cells, including IgE precursor cells, B cells and B cell blasts and plasma cells can be processed and presented onto the HLA-E (all the immune and non-immune cell types), which render itself susceptible to the cytolytic or immunoregulatory attack by the aforementioned HLA-E+ natural IgE peptides reactive CD4 and CD8 T. Hence an appropriately identified IgE natural peptide, appropriately delivered to stimulate a vaccine response can inhibit IgE production at the levels of IgE lineage cells, henceforth, ameliorate IgE-mediated allergic inflammation with the status being the HLA-E-based universal IgE allergy vaccine.

[0108] An embodiment of this invention is to combat infectious disease antigens of the viral diseases, employing microbial proteins of immune protective or therapeutic use in the presence of costimulation annulling coinhibition (Vinuesa et al. 2016. ARI. 34: 335; Baumeister et al. ARI. 2016. 34: 539; Chen and Flies. 2013. Nat. Rev. Im. 13: 227; Esensten et al. 2016. Immunity. 44: 973), e.g., those of the non-self antigens, pertaining to infectious microbes of endogenous bacterial and viral origins (Goldman's Cecil Medicine 24 ed, 2012, Elsevier) such as [Tuberculosis (Mycobacteria tuberculosis, M. bovis), Leprosy (M. leprae), Legionellosis (Legionella pneumophilia), Listeria monocytogenes infection, Salmonellosis (Salmonella enterica, S. bongori) Lyme Borreliosis (Borrelia burgdorferi), Actinomycosis (Actinomycetes, spp), Chlamydia infection (C. trachomatis, C. pneumoniae, resident in macrophages] and viral sources of infections, e.g., human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), human respiratory syncytial virus (HR,SV), influenza virus (IV), para-influenza virus (PIV), rotavirus, rhinovirus, adenovirus infection, sever acute respiratory syndrome (SRAS) virus; GI tract: norovirus, rotavirus, adenovirus, astrovirus; Exanthematous viral diseases: measles virus, rubella virus, chicken pox/shingles, roseola, smallpox, fifth virus, chikungunya virus infection; hepatitis virus: hepatitis virus A, B, C, D, E; cutaneous viral diseases: warts virus, genital warts virus, oral herpes virus, genital herpes virus, molluscum contagiosum virus; hemorrhagic diseases: Ebola virus, Lassa fever virus, dengue fever virus, yellow fever virus, Marburg hemorrhagic fever virus, Crimean-Congo hemorrhagic fever virus; neurologic viral diseases: polio virus, meningitis virus, encephalitis virus, rabies virus.

[0109] In contrast, autoimmune diseases can be initiated and maintained by effector CD4 or CD8, CD4/CD8 T cells or autoantibody-producing B cells. Numerous somatic autoantigens with exaggerated expression or neoantigenic expression in pathological conditions in a tissue and/or an organ, struck with aberrantly flared up forms of cytokines or other pathogenic proteins during inflammation, an autoimmune disease. The aberrant expression of autoantigens can be evaluated by the elevated level of mRNA via RT-PCR, and identifiable proteins via western blots. HLA-E based vaccine peptide epitopes can be evaluated and assessed via the self-peptides predicted via a given protein, or discovered by purifying HLA-E with its bound peptides, acidic elution of bound peptide, followed by MS/MS peptide sequencing (DH Hunt et al., 1992. Science 255: 1261). In the embodiment, synthetic peptide vaccines, peptide sequences contained in recombinant proteins, or recombinant DNA vectors including using UL40 can be prepared as a source of DNA vaccines. Treg or infectious tolerance can be induced by HLA-E elicited autoantigenic peptides by immunization in the absence of costimulations or provided with suppressive microenvironment conducive for Treg and infectious tolerance.

[0110] One main embodiment herein is to induce HLA-E/autoantigenic peptide specific Treg to suppress effector CD4, CD8 T cell-mediated, or antibody-producing B cells-mediated tissue damage in the following types of autoimmune diseases (Sinmaz et al., 2016: 13:219; Riedhammer and Werssert. 2015. Front. Im. 2015. 6: 322; Tong et al. J Inf. Dis. 2017. 10: 97; Pianta et al. 2017. J.C.I. 127: 2946; Meched et al. 2016. FASEB J. 30:2123), including and not limited to organ specific (Type I diabetes mellitus, thyroiditis, mysasthenia gravis, primary biliary cirrhosis, Goodpasture's syndrome), or systemic (rheumatoid arthritis, progressive systemic sclerosis, systemic lupus erythematosus). The neurological autoantigens involved in autoimmune neurological lesions are as follows. Autoantigens found in CNS antibody-mediated disorders include N-methyl-D-aspartate receptor (NMDAR), -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), glycine receptor (GlyR), components of the voltage-gated potassium channel (VGKC) complex, including leucine-rich glioma-inactivated protein 1 (Lgi1) and contactin-associated protein-like 2 (Caspr2), -aminobutyric acid receptor-B (GABA.sub.BR), -aminobutyric acid receptor-A (GABA.sub.AR), metabotropic glutamate receptor 5 (mGluR5), dipeptidyl-peptidase-like protein-6 (DPPX), dopamine-2 receptor (D2R), myelin oligodendrocyte glycoprotein (MOG), aquaporin-4 (AQP4), 65 kDa glutamic acid decarboxylase (GAD65), neurofascin (NF), and contactin. Furthermore, a number of different autoantigens have been discovered in neuromuscular junction antibody-mediated disorders. These include acetylcholine receptor (AChR), muscle-specific kinase (MuSK), lipoprotein receptor-related protein 4 (Lrp4), all associated with myasthenia gravis (MG), and voltage-gated calcium channel (VGCC) associated with PNS Lambert-Eaton myasthenic syndrome (LEMS); collagen type II, human cartilage gp39 (HCgp39), gp130-RAPS associated with rheumatoid arthritis; fibrillarin, small nucleolar protein (snoRNP) associated with scleroderma; thyroid stimulating factor receptor (TSH-R) associated with Graves' disease; nuclear antigens, DNA, histone, glycoprotein gp70, ribosomes associated with systemic lupus erythematosus; PDC-E2 (mitochondrial enzyme, pyruvate dehydrogenase dehydrolipoamide acetyltransferase) associated with primary billiary cirrhosis; Hair follicle antigens associated with Alopecia areata; Pancreatic -cell antigens, insulin, GAD and its isoforms associated with IDDM; MBP, PLP, MOG associated with multiple sclerosis; Human tropomyosin isoform 5 (hTM5) associated with Ulcerative colitis.

[0111] Highly reactive lipid intermediates (Sinmaz et al., 2016: 13:219; Riedhammer and Werssert. 2015. Front. Im. 2015. 6: 322; Tong et al. J Inf. Dis. 2017. 10: 97; Pianta et al. 2017. J.C.I. 127: 2946; Meched et al. 2016. FASEB J. 30:2123): MDA and HNE modified low-density lipoproteins (LDL), oxidized LDL (oxLDL), and 8-oxodeoxyguanine modified LDL in atherosclerotic plaques; oxLDL in Behcet's disease; oxidation-specific surface antigens on apoptotic cells; oxidized liver antigens in alcoholic liver disease; HNE-modified 60-kDa Ro in animal model of SLE; oxidized carbohydrates; IgG modified with advanced glycation end (AGE) product; pentosidine modified IgG in rheumatoid arthritis; Oxidized glutamic acid hydroxylase in IDDM; Type I diabetes: Carboxypeptidase H, Chromogranin A, Glutamate decarboxylase, Imogen-38, insulin, Insulinoma antigen-2 and 2, Islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP), proinsulin; MS: -enolase, Aquaporin-4, -arrestin, Myelin basic protein, Myelin oligodendrocytic glycoprotein, Proteolipid protein, S100-; Rheumatoid arthritis: Citrullinated protein, Collagen II, Heat shock proteins; Systemic lupus erythematosus: Double-stranded DNA, La antigen, Nucleosomal histones and ribonucleoproteins (snRNP), Phospholipid--2 glycoprotein I complex, Poly(ADP-ribose) polymerase, Sm antigens of U-1 small ribonucleoprotein complex. The embodiment of the invention is to utilize the above autoantigenic peptides to elicit an HLA-E mediated immune protective responses to dampen autoimmunity attack.

Derlin-1 and US11 as Vaccine Adjuvant

[0112] MHC I destined for the surface membrane expression bound to endogenous or antigenic peptides survive in the ER lumen, whereas misfolded MHC I in a quality-control process is transferred from the ER to the cytosol in a ubiquitin-dependent pathway in exported via AAA ATPase (Cdc48/p97), Derlin-1 forming complexes with co-factors, Ufd1p and Npl4p, into cytosol for degradation by the 26S proteasome. Normally the leader peptides from HLA-A, -B, -C are constitutively available and loaded onto HLA-E; and this constitutive level of endogenous leader peptide loading can compromise the vaccine peptide loading onto HLA-E.

[0113] Thus, one embodiment of vaccine peptide loading unto the HLA-E compartment resides in clearing house of endogenously already occupied house-keeping HLA-A, -B leader peptides. Protein quality control process entails a mechanism whereby misfolded HLA-A, -B, -C proteins were loaded onto an internal antegraded ER-associated degradation (ERAD) assembly exporting into cytosol. Breaking down of leader peptide sequences and HLA-A, -B, -C subsequently take place in cytosolic proteasomes, whereby leader peptides of HLA-A, -B, -C are retrograded transported via the TAP transporter, to be imported and reloaded onto HLA-E. The quality control process can be controlled by two human cytomegalovirus proteins, US11 (SEQ ID: #87, SEQ ID: #88), and US2 (SEQ ID: #89, SEQ ID: #90) causes a rapid transfer of MHC I heavy chain from the ER to the cytosol, and Derlin-1 as an ER membrane protein essential for US11-mediated antegrade translocation of the class I HC. where N-glycanase, ubiquitin-conjugating enzymes and finally the proteasomes act on them. A human homologue of yeast Der1p, Derlin-1 (SEQ ID: #85, SEQ ID: #86), a protein essentially required for the degradation of a misfolded ER luminal proteins, including MHC I (Lilley and Ploegh, 2004. Nature, 429:834) as the partner essential for US11 to perform its function. US11 is a 215 amino acid glycoprotein, whereby the luminal domain interacts with MHC I, while the transmembrane domain interacts with Derlin-1, which is in a large complex tight in with the antegrade pathway exporting to cytosol for degradation. The US11 enormously accelerate the association of misfolded MHC I with Derlin-1, the degradation process within minutes of its synthesis. It is possible that Derlin-1 acts to degrade cellular ER proteins independently of the US11 and US2.

[0114] Derlin-1 forms an integral part of the antegraded machinery required for transporting classical MHCI, e.g., HLA_A, _B, _C for proteasome degradation. Table I and FIG. 9 showed that we have uncovered three anti-Derlin-1 siRNA, which knockdown Derlin-1 expression up to 65% in three types of antigen presenting cells by sensitive RT-PCR, those of MoDCs, T2 (B and T cell hybrid lymphoid tumor), and 0.221 B cells (HLA-A, -B, -C, -E not expressed). In contrast, the protein band of Derlin-1 is entirely missing in Derlin-1 siRNA-treated MoDCs. Transfection of the primary cells were performed using PEI reagents (Viromer Blue vs. Viromer Red). The embodiment of the invention is three-fold. One aspect of the embodiment resides in treating APC with Derlin-1 RNA interference to prevent the generation of endogenous leader peptide as well as to cause dissociation of the HLA-E bound endogenous leader peptides so that Derlin-1-treated APC can be fully loaded with the HLA-E vaccines in the presence of the second costimulatory signal for immune induction.

[0115] Alternatively in the absence of costimulation to cause immunoregulation of autoimmune or inflammatory diseases. Notably, although US11 and US2 accelerate the fast degradation of classical MHCI: HLA-A, -B, and -C it does not affect the integrity of nonclassical MHC I (Schust, 1998. JEM, 188:497): HLA-G, and HLA-E, HLA-F by extension. Thus, another aspect of embodiment resides in treating APC with US11 plasmid constructed from the CMV (FIG. 11), and the intact, unaffected HLA-E compartment without a competing classic MHC I antigenic peptide presentation from the microenvironmental milieu, can further augment vaccine peptide presentation. In. this embodiment, a fast degradation of MHC I by US11 can ease up antigen presenting energy space/time to facilitate nonclassical MHCI-based vaccine peptide presentation. In this freed-up space/time, a modification of this embodiment resides in a co-transfection of the vaccine leader-peptide like or nominal vaccine peptide constructed within the UL-40 plasmid mimics natural infection, while co-administered with a second signal, mimicking a reversal phase of the natural infection.

[0116] The embodiment of this invention residing in slowing down the export of HLA-A, -B, -C by downregulating the Derlin-1 based exporter complexes, hence permitting dissociation of already bound endogenous leader peptide, and the freeing up the binding pocket and availability of HLA-E for exchanging the tolerogenic leader peptides with the vaccine epitopes, which can be imported via the TAP exporter pathway, or inside the ER via UL-40 which can directly load the IgE leader peptide-like sequence. An aspect of this embodiment resides in using MoDCs or PBMC, blood dendritic cells, B cells or monocytes, treated with Derlin-1 inhibitors or RNA interference, thus facilitating HLA-E vaccine peptide pickups and loading into the empty binding pocket of HLA-E.