Human Leukocyte Antigen Transgenic Mice Research Articles

Correlative CD4 and CD8 T-cell immunodominance in humans and mice: Implications for preclinical testing

Antigen-specific T-cell recognition is restricted by Major Histocompatibility Complex (MHC) molecules, and differences between CD4 and CD8 immunogenicity in humans and animal species used in preclinical vaccine testing are yet to be fully understood. In this study, we addressed this matter by analyzing experimentally identified epitopes based on published data curated in the Immune Epitopes DataBase (IEDB) database. We first analyzed SARS-CoV-2 spike (S) and nucleoprotein (N), which are two common targets of the immune response and well studied in both human and mouse systems. We observed a weak but statistically significant correlation between human and H-2b mouse T-cell responses (CD8 S specific (r = 0.206, p = 1.37 × 10−13); CD4 S specific (r = 0.118, p = 2.63 × 10−5) and N specific (r = 0.179, p = 2.55 × 10−4)). Due to intrinsic differences in MHC molecules across species, we also investigated the association between the immunodominance of common Human Leukocyte Antigen (HLA) alleles for which HLA transgenic mice are available, namely, A*02:01, B*07:02, DRB1*01:01, and DRB1*04:01, and found higher significant correlations for both CD8 and CD4 (maximum r = 0.702, p = 1.36 × 10−31 and r = 0.594, p = 3.04−122, respectively). Our results further indicated that some regions are commonly immunogenic between humans and mice (either H-2b or HLA transgenic) but that others are human specific. Finally, we noted a significant correlation between CD8 and CD4 S- (r = 0.258, p = 7.33 × 1021) and N-specific (r = 0.369, p = 2.43 × 1014) responses, suggesting that discrete protein subregions can be simultaneously recognized by T cells. These findings were confirmed in other viral systems, providing general guidance for the use of murine models to test T-cell immunogenicity of viral antigens destined for human use.

Acid Stripping after Infection Improves the Detection of Viral HLA Class I Natural Ligands Identified by Mass Spectrometry.

Identification of a natural human leukocyte antigen (HLA) ligandome is a key element to understand the cellular immune response. Advanced high throughput mass spectrometry analyses identify a relevant, but not complete, fraction of the many tens of thousands of self-peptides generated by antigen processing in live cells. In infected cells, in addition to this complex HLA ligandome, a minority of peptides from degradation of the few proteins encoded by the viral genome are also bound to HLA class I molecules. In this study, the standard immunopeptidomics strategy was modified to include the classical acid stripping treatment after virus infection to enrich the HLA ligandome in virus ligands. Complexes of HLA-B*27:05-bound peptide pools were isolated from vaccinia virus (VACV)-infected cells treated with acid stripping after virus infection. The HLA class I ligandome was identified using high throughput mass spectrometry analyses, yielding 37 and 51 natural peptides processed and presented untreated and after acid stripping treatment VACV-infected human cells, respectively. Most of these virus ligands were identified in both conditions, but exclusive VACV ligands detected by mass spectrometry detected on acid stripping treatment doubled the number of those identified in the untreated VACV-infected condition. Theoretical binding affinity prediction of the VACV HLA-B*27:05 ligands and acute antiviral T cell response characterization in the HLA transgenic mice model showed no differences between HLA ligands identified under the two conditions: untreated and under acid stripping condition. These findings indicated that acid stripping treatment could be useful to identify HLA class I ligands from virus-infected cells.

Evaluation of immune-mediated idiosyncratic drug toxicity using chimeric HLA transgenic mice.

Immune-mediated idiosyncratic drug toxicity (IDT) is a rare adverse drug reaction, potentially resulting in death. Although genome-wide association studies suggest that the occurrence of immune-mediated IDT is strongly associated with specific human leukocyte antigen (HLA) allotypes, these associations have not yet been prospectively demonstrated. In this study, we focused on HLA-B*57:01 and abacavir (ABC)-induced immune-mediated IDT, and constructed transgenic mice carrying chimeric HLA-B*57:01 (B*57:01-Tg) to determine if this in vivo model may be useful for evaluating immune-mediated IDT. Local lymph node assay (LLNA) results demonstrated that percentages of BrdU+, IL-2+, and IFN-γ+ in CD8+ T cells of ABC (50mg/kg/day)-applied B*57:01-Tg mice were significantly higher than those in littermates (LMs), resulting in the infiltration of inflammatory cells into the ear. These immune responses were not observed in B*57:03-Tg mice (negative control). Furthermore, oral administration of 1% (v/v) ABC significantly increased the percentage of CD44highCD62Llow CD8+ memory T cells in lymph nodes and spleen derived from B*57:01-Tg mice, but not in those from B*57:03-Tg mice and LMs. These results suggest that B*57:01-Tg mice potentially enable the reproduction and evaluation of HLA-B*57:01 and ABC-induced immune-mediated IDT.

An immunoinformatics-derived DNA vaccine encoding human class II T cell epitopes of Ebola virus, Sudan virus, and Venezuelan equine encephalitis virus is immunogenic in HLA transgenic mice

ABSTRACTImmunoinformatics tools were used to predict human leukocyte antigen (HLA) class II-restricted T cell epitopes within the envelope glycoproteins and nucleocapsid proteins of Ebola virus (EBOV) and Sudan virus (SUDV) and the structural proteins of Venezuelan equine encephalitis virus (VEEV). Selected epitopes were tested for binding to soluble HLA molecules representing 5 class II alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, and DRB1*1501). All but one of the 25 tested peptides bound to at least one of the DRB1 alleles, and 4 of the peptides bound at least moderately or weakly to all 5 DRB1 alleles. Additional algorithms were used to design a single “string-of-beads” expression construct with 44 selected epitopes arranged to avoid creation of spurious junctional epitopes. Seventeen of these 44 predicted epitopes were conserved between the major histocompatibility complex (MHC) of humans and mice, allowing initial testing in mice. BALB/c mice vaccinated with the multi-epitope construct developed statistically significant cellular immune responses to EBOV, SUDV, and VEEV peptides as measured by interferon (IFN)-γ ELISpot assays. Significant levels of antibodies to VEEV, but not EBOV, were also detected in vaccinated BALB/c mice. To assess immunogenicity in the context of a human MHC, HLA-DR3 transgenic mice were vaccinated with the multi-epitope construct and boosted with a mixture of the 25 peptides used in the binding assays. The vaccinated HLA-DR3 mice developed significant cellular immune responses to 4 of the 25 (16%) tested individual class II peptides as measured by IFN-γ ELISpot assays. In addition, these mice developed antibodies against EBOV and VEEV as measured by ELISA. While a low but significant level of protection was observed in vaccinated transgenic mice after aerosol exposure to VEEV, no protection was observed after intraperitoneal challenge with mouse-adapted EBOV. These studies provide proof of concept for the use of an informatics approach to design a multi-agent, multi-epitope immunogen and provide a basis for further testing aimed at focusing immune responses toward desired protective T cell epitopes.

A systematic bioinformatics approach for selection of epitope-based vaccine targets

Epitope-based vaccines provide a new strategy for prophylactic and therapeutic application of pathogen-specific immunity. A critical requirement of this strategy is the identification and selection of T-cell epitopes that act as vaccine targets. This study describes current methodologies for the selection process, with dengue virus as a model system. A combination of publicly available bioinformatics algorithms and computational tools are used to screen and select antigen sequences as potential T-cell epitopes of supertype human leukocyte antigen (HLA) alleles. The selected sequences are tested for biological function by their activation of T-cells of HLA transgenic mice and of pathogen infected subjects. This approach provides an experimental basis for the design of pathogen specific, T-cell epitope-based vaccines that are targeted to majority of the genetic variants of the pathogen, and are effective for a broad range of differences in human leukocyte antigens among the global human population.

Identification of antigenic epitopes on human allergens: studies with HLA transgenic mice.

Environmental factors play an important role in the rise and manifestation of allergic conditions in genetically predisposed subjects. Increased exposure to indoor/outdoor allergens is a significant factor in the development of allergic sensitization and asthma. Recently, strong relationships between the immune response to several highly purified allergens and specific human leukocyte antigen (HLA)-DQ and -DR haplotypes have been reported. The major antigens from clinically important allergens have been cloned and sequenced. However, whether innate structural features of major allergens or peculiar immune recognition of these molecules contribute to the overly robust immune responses is not known. We generated and used transgenic (tg) mice expressing single HLA class II transgene(s) to characterize the allergen epitopes presented by particular HLA class II molecules. Next, we generated in vivo models for asthma in the HLA tg mice by intranasal challenge with allergenic extracts. Furthermore, we used a single epitope to induce an allergic lung inflammation. Our system offers a sophisticated technique for systematically identifying the genetic (individual human class II) and antigenic (individual allergenic epitopes) basis of asthma sensitivity and has important implications for new treatment strategies.

Preclinical development of an adjuvant-free peptide vaccine with activity against CMV pp65 in HLA transgenic mice

Epitope vaccines have shown promise for inducing cellular immune responses in animal models of infectious disease. In cases where cellular immunity was augmented, peptide vaccines composed of covalently linked minimal cytotoxic T-lymphocyte (CTL) and T-helper (T(H)) epitopes generally showed the most efficacy. To address a clinical vaccine strategy for cytomegalovirus (CMV) in the context of HCT (hematopoietic cell transplantation), we observed that linking the synthetically derived pan-DR epitope peptide (PADRE) or one of several tetanus T(H) epitopes to the immunodominant human leukocyte antigen (HLA) A*0201-restricted CTL epitope from CMV-pp65 to create a fusion peptide caused robust cytotoxic cellular immune responses in HLA A*0201/K(b) transgenic mice. Significantly, the fusion peptides are immunogenic when administered in saline solution by either subcutaneous or intranasal routes. CpG-containing single-stranded DNA (ss-oligodeoxynucleotide [ODN]) added to the fusion peptides dramatically up-regulated immune recognition by either route. Notably, target cells that either expressed full-length pp65 protein from vaccinia viruses or were sensitized with the CTL epitope encoded in the vaccine were recognized by splenic effectors from immunized animals. Visualization of murine peptide-specific CTL by flow cytometry was accomplished using an HLA A*0201 tetramer complexed with the pp65(495-503) CTL epitope. T(H)-CTL epitope fusion peptides in combination with CpG ss-ODN represent a new strategy for parenteral or mucosal delivery of vaccines in a safe and effective manner that has applicability for control or prophylaxis of infectious disease, especially in situations such as vaccination of donors or recipients of HCT, where highly inflammatory adjuvants are not desired.