Common HLA Alleles Research Articles

Safety, Maximum Tolerated Dose and Immunogenicity of CMV-MVA-Triplex in Healthy Volunteers with or without Prior Immunity to CMV and Vaccinia

In the era of preemptive antiviral therapy, cytomegalovirus (CMV) still remains the cause of major health complications, profound defects in immune reconstitution, and significant morbidity in post-transplant recovery of immune-compromised HCT recipients. Substituting antivirals with a vaccine that harnesses the native immune response to CMV may improve outcomes for HCT recipients.Modified Vaccinia Ankara (MVA), a vaccinia virus being investigated as a smallpox vaccine by the Defense Department, is a safe and robust delivery system to treat or prevent a wide range of diseases including HIV and cancer. Replication-defective MVA is safe, well tolerated and strongly immunogenic when given to HCT recipients or AIDS patients. We developed a multiple-antigen recombinant MVA with genes encoding 3 immunodominant CMV proteins: UL83 (pp65), UL123 (IE1-exon4), and UL122 (IE2-exon5) (CMV-MVA-Triplex), and established its pre-clinical safety and immunogenicity using humanized HLA transgenic mouse models and human PBMC from CMV-seropositive healthy volunteers and HCT recipients.Defining safety, persistence of the virus, maximum tolerated dose and immunogenicity of CMV-MVA-Triplex in healthy volunteers is a critical first step in its clinical development for HCT recipients as required by the FDA. In a Phase I trial (NCT01941056), these endpoints were evaluated in 24 healthy volunteers (age: 18-60), with or without prior immunity to CMV and vaccinia. Three escalating dose levels (DL) were administered intramuscularly (DL1=10xE7; DL2=5x10E7; DL3=5x10E8 pfu/dose) in 8 subjects/DL, with a booster injection 28 days later, and follow up for 1 year.As of July 2015, all 24 planned volunteers were enrolled, vaccinated and followed for at least 4 months. Vaccinations at all DL were well-tolerated, with only a few expected injection reactions and no SAE or dose limiting toxicities. Immunogenicity of the vaccine was evaluated by measuring the levels of the CD137 T-cell surface marker representing functional activation of PBL harvested from vaccinees and stimulated 24 hours with full-length pp65, IE-1 and IE2 overlapping peptide libraries, or direct measurement of CMV-specific T-cells using HLA multimers. CMV-MVA-Triplex induced robust expansion of pp65-, IE1- and IE2-specific CD8 and CD4 T-cells in vaccinated CMV-seropositives, at each DL (Cf. plot showing geometric mean with upper lower/limits of the pp65 T-cell levels for DL2 cohort). HLA multimers identified CMV-specific T-cells whose expansion closely followed CD137-activated CMV-specific T-cells in vaccinees with common HLA alleles which have a corresponding known CMV-CTL epitope (data to be presented).A statistical analysis performed using generalized estimated equations indicated that the post-vaccination levels of pp65-, IE1- or IE2-specific CD8 and CD4 T-cells were significantly increased, with p-values ranging from 3x10-5 to 0.025. For example, the pre-/post-vaccination median pp65-reactive CD4+ CD137+ T-cells rose from 1.3 to 4.4 cells/µL (p=3x10-5); and pp65-reactive CD8+ CD137+ T-cells rose from a median of 0.22 to 3.1 cells/µL (p=0.003). Importantly, robust immunity was detected in CMV-seronegatives (as shown in the plot for UPN 14 and 18), as well as in subjects who had received smallpox vaccinations. Elevated frequencies of CMV-specific CD4 and CD8 T cells for all 3 antigens plateaued after day 56, but in most cases remained elevated up to one year post-vaccination (data to be presented). Circulating MVA vector in blood was assessed by real-time PCR post-injection and showed only minimal residual vector DNA [10-30 gc/mL] in just 2 vaccinees in the DL3 cohort that disappeared within 3 months. These results provide evidence that CMV-seropositive HCT recipients, whether they receive stem cell product from CMV-seropositive or -seronegative donors could respond to CMV-MVA-Triplex by generating protective CMV-specific immunity.CMV-MVA-Triplex is the first vaccine against CMV that uses a recombinant MVA incorporating multiple CMV antigens, developed for HCT recipients, who are at risk for CMV reactivation. The safety and marked immunogenicity of CMV-MVA-Triplex in this Phase I trial, warrant testing of the vaccine in the HCT setting. A Phase 2 multicenter, placebo-controlled trial to assess CMV-MVA-Triplex in CMV seropositive recipients, receiving HCT from matched related or unrelated donors will start in Fall 2015. [Display omitted] [Display omitted] DisclosuresDiamond:Helocyte, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

OR28 Validation of 2070 common, rare, and novel HLA alleles using Illumina TruSight® HLA ultra-high-resolution sequencing

Aim Sanger sequencing of HLA suffers from the inability to set phase in certain heterozygous combinations. NGS overcomes these problems, but the reliability is not well described. We tested the NGS TruSight® HLA Sequencing Panel (Illumina) for its ability to provide full length, genomic, accurate, unambiguous, phase-resolved HLA genotyping in a single assay on a panel of 145 specimens. Residual clinical, PT, and IHWG DNA samples from blood, buccal swabs, cell lines of varying quality, concentration, and age, were selected to cover every known antigen, as well as rare and novel alleles. Methods The 145 samples (2070 alleles) included 26 novel variants, 7 null alleles, and 7 PT samples. We used the Illumina NGS HLA genotyping system end-to-end, from Long-Range PCR and library preparation to sequencing on the MiSeq. FASTQ files were analyzed by Conexio Assign, providing phase-resolved genotyping results for HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1, -DPB1. We calculated concordance and unambiguous allele level identity in comparison to previously typed high resolution typed results (Sanger/SSP/SSO combined). Results 87.1% of all clusters (500 cycles) had a Q30 quality score (error rate of 0.1–0.01%). Concordance with original typing was 98.5%. Subdividing concordance, we found: 65.1% identical and unambiguous in reference and NGS; 20.8% unambiguous NGS, ambiguous reference; 9% unambiguous reference, ambiguous NGS; 3.7% ambiguous in both reference and NGS; and 1.5% discordant. Unambiguous reference/ambiguous NGS was the result of primer placement. Discordance (31 alleles) was due to novel alleles, reference errors, homopolymers and microsatellites, pseudogenes, and 3 instances of contamination. Benefits of NGS included discovery and resolution of novel alleles, identification and correction of IHWG reference and clinical mistypings. Analysis of 24 samples for 11 loci took ∼3–3.5 h. Conclusions TruSight® HLA Sequencing system is a reliable, accurate, comprehensive, ultra-high-resolution HLA typing method that can be easily implemented in the laboratory. For labs needing the highest resolution typing, it reduces the number of ancillary tests that must be performed to resolve ambiguities from ∼50% down to ∼14%. For registry labs, the concordance rate equals or exceeds 98.5%. F. Yamamoto: Grant/Research Support; Company/Organization; Illumina, Conexio. A. Lindell: Grant/Research Support; Company/Organization; Illumina, Conexio. B. Baas: Grant/Research Support; Company/Organization; Illumina, Conexio. A. Crawford: Grant/Research Support; Company/Organization; Illumina, Conexio. M. Won: Grant/Research Support; Company/Organization; Illumina, Conexio. N. Baird: Grant/Research Support; Company/Organization; Illumina, Conexio. M.W. Anderson: Grant/Research Support; Company/Organization; Illumina, Conexio. J. Nytes: Grant/Research Support; Company/Organization; Illumina, Conexio. J.J. Schiller: Grant/Research Support; Company/Organization; Illumina, Conexio. D. Goodridge: Grant/Research Support; Company/Organization; Illumina, Conexio. D.B. Tyan: Grant/Research Support; Company/Organization; Illumina, Conexio.

LBP19: EXTENDED COVERAGE BY NEXT GENERATION SEQUENCING METHODS REFINES THE CHARACTERIZATION OF THE COMMON AND WELL DOCUMENTED HLA ALLELES

The accurate identification of the true allele frequency of HLA genes in various ethnic groups may be a challenging task. The current methodology to catalog the common and well-documented (CWD) HLA alleles is based on the 3rd or 4th field made from assumptions for gene segments that were not tested by Sanger Sequencing methods. We examined the allelic frequencies in all HLA loci by leveraging the power of Next Generation Sequence (NGS) and long range PCR to interrogate the entire sequence of the class I genes and most of the extent Class II genes. In a HLA typing study of 6,000 subjects we unexpectedly found several examples of alleles with common occurrence that were thought to be rare, and represent the most common allele of the 1st-2nd fields allele group. For example, we found that the alleles B ∗ 40:01:02 and DRB1 ∗ 08:01:03 represent the common alleles of their groups while the alleles with the lowest digit number (B ∗ 40:01:01, DRB1 ∗ 08:01) were absent. Extending the sequences also failed to find alleles thought to be common, while other alleles appeared to replace them; DRB3 ∗ 02:24 that associates tightly with DRB1 ∗ 14:01:01 is common while the closely related allele DRB3 ∗ 02:01 thought to be common, was not found; these alleles differ in exon-3, that had not been tested frequently. Our study also showed that several subtypes of the allele groups A ∗ 29:02, B ∗ 44:02, B ∗ 18:01, B ∗ 52:01, C ∗ 05:01, C ∗ 06:02, C ∗ 07:01, C ∗ 07:02, DQA1 ∗ 01:01, DQA1 ∗ 01:02, DQA1 ∗ 01:03, DQA1 ∗ 05:01, DQA1 ∗ 05:05, DQB1 ∗ 03:01 and DQB1 ∗ 05:01 differing at non coding regions or by silent substitutions at areas not tested frequently, are very common. Intriguingly, the alleles differing in areas not tested routinely in previously used assays, show distinctive associations with alleles of contiguous or close loci and contrary to previous predictions indicate that the evolutionary processes in HLA are more complex and modern populations show strongly conserved common haplotype blocks. Our findings show that HLA diversity may have arisen from opposing mechanisms such as allele diversification and convergent evolution.

Abstract A85: Passenger genes on tumor-associated amplicons function as tumor rejection antigens

Abstract Tumors frequently contain DNA copy number amplifications (amplicons) that are driven by overexpression of an oncogene but often lead to high expression of neighboring passenger genes. We hypothesized that proteins encoded on amplicons which are present at high frequency in particular types of cancer could serve as shared tumor rejection antigens. The ERBB2 (HER2) amplicon on 17q12-21 has a core region of approximately 280kB in size and is frequently present in multiple copies in 10-15% of breast and gastric cancers. Analysis of data from TCGA breast cancer project and multiple other mRNA expression studies show that ERBB2 overexpression is highly correlated with expression of some, but not all, adjacent genes. From the set of amplicon genes we selected MIEN, GRB7, PGAP3, PNMT, ORMDL3, and GSDMB as candidates for stimulating an immune response because these proteins are minimally expressed in normal tissues. Immunohistochemistry of tissue microarrays showed expression of these proteins in HER2+ breast cancer cells but not in HER2 non-amplified breast tumors. We used the epitope identification programs, NetCTLpan and NetMHCII (Center for Biological Sequence Analysis at the Technical University of Denmark) to identify several candidate peptides with high binding affinity <50 nM to common HLA alleles. In vitro sensitization of normal donor PBMC with several of these peptides led to IFN γ production. Moreover, DCs pulsed with tumor lysate from HER2 amplified tumor cells were able to sensitize T cells against epitopes derived from amplicon proteins indicating that the natural abundance of these antigens in tumors is sufficient for cross-presentation to T cells. The TM15 murine breast cancer line has amplification of a syntenic region surrounding Her2/neu and 50-fold over-expression of pnmt by qPCR. Mice vaccinated with recombinant murine PNMT rejected TM15 tumor challenge without apparent toxicity. These findings indicate that tumor genomic data can be used to identify candidate tumor antigens and epitope prediction can be used to engineer a T cell immune response to break tolerance to these normal proteins. It appears that a therapeutic window can be established to target amplicon-derived antigens due to substantially higher expression in tumor cells relative to normal tissues. Moreover, peptide sequence differences between human and murine proteins at sites of immunogenic epitopes provide an opportunity to generate high-affinity TCR in HLA transgenic mice. Although shared tumor antigens such as HER2 have served as targets for cancer immunotherapy there have been some difficulties with off-target toxicity against normal tissues due to broad expression. Selection of over-expressed passenger genes that exceed a threshold for T cell responsiveness only in tumor but not normal tissues might lead to greater efficacy. This strategy potentially could be applied to other tumor-associated amplicons such as MYCN to search for peptide epitopes from passenger genes which are predicted to have high affinity for common HLA alleles. Citation Format: Michael Berk, Arun Modi, Mona Patel, Li-Xin Wang, Gregory Plautz. Passenger genes on tumor-associated amplicons function as tumor rejection antigens. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A85.

Establishment of Alloantigen Cell Bank Allowing High Resolution Analysis of T Cell Alloimmunity.

Background: Development of multimere technologies allowed not only analysis of antigen-specific CD8 T cell but also detailed characterization of donor-specific antibodies (DSA) on a single allele level. Analysis of T cells in terms of donor-specific alloimmunity appears to be more tedious. Currently available allobanks do not allow high-resolution analysis due to expression of complete HLA-haplotypes. Artificial antigen presenting cells (aAPC) expressing single HLA-alleles is a promising approach for the high-resolution analysis of cellular allospecificity. However, current engineering of aAPC is time- and cost-intensive. Here, we developed an allele-independent HLA-haplotype amplification approach and applied a novel method for generation of aAPC expressing specific HLA-class I and II alleles. Using the developed approached we established aAPC-allobank allowing single allele resolution of T cell alloimmunity. Methods and Results: Primers allowing donor-independent amplification of HLA-class I and II alleles were designed and established. Further, using transposon-based system most common HLA-alleles were expressed in K562 cell line and several donors were screened in terms of T cell allospecificity. As a control, aAPC expressing autologous HLA-alleles were used. We found that the established aAPC induce allospecific T cells what allows high resolution analysis of T cell alloimmunity as compared with control stimulatory cells. Of note, cross-reactive HLA-class II-specific CD8 and -class I-specific CD4 T cells were also identified. Conclusions: The established approach allows fast, time- and cost-efficient expression of single HLA-alleles in a donor-independent way. The engineered aAPCs allow single allele resolution of T cell alloimmunity. The established method would allow more rigorous analysis of alloreactivity and its influence on transplant function and long-term survival. Additionally, the established approach can be applied for analysis and expansion of allospecific regulatory T cells.

HLA alleles and hypersensitivity to carbamazepine

A considerable heterogeneity exists in the literature on the role of different HLA alleles in carbamazepine (CBZ)-induced cutaneous adverse drug reactions (cADRs) of varying severity among diverse ethnic groups. The aim of the present study was to understand and summarize this heterogeneity and evaluate the contribution of common HLA alleles to susceptibility to cADRs in patients treated with CBZ through a meta-analysis. A literature search of Embase, Medline, Web of Knowledge, and Cochrane database of systematic reviews was performed up to 28 September 2013. A total of 20 reports were identified as eligible studies, which included 720 CBZ-intolerant [Stevens-Johnson syndrome and toxic epidermal necrolysis (bullous lesions): n=277; hypersensitivity syndrome/maculopapular exanthema (nonbullous lesions): n=359; others: n=84], 1512 CBZ-tolerant, and 1113 normal controls. We observed HLA-A*3101 and HLA-B*1502 as risk markers and HLA-B*4001 as a protective marker for susceptibility to cADRs when comparing intolerant with tolerant patients. Stratification by clinical outcome showed HLA-B*1502 and HLA-B*1511 as risk and HLA-A*2402 as protective markers for bullous lesions in the Asians [HLA-B*1502: odds ratio (OR)=80.70; 95% confidence interval (CI)=45.62-142.77; P=1.8×10(-51); I(2)=33%, HLA-B*1511: OR=17.43; 95% CI=3.12-97.40; P=1.1×10(-3); I(2)=0%, HLA-A*2402: OR=0.27; 95% CI=0.11-0.64; P=2.7×10(-3); I(2)=0%]. Furthermore, HLA-A*3101 was observed to be a universal risk marker, irrespective of cADR type [OR (bullous lesions)=5.65; 95% CI =2.70-11.78; P=4.03×10(-6); I(2)=49%, OR (nonbullous lesions)=8.58; 95% CI=5.55-13.28; P=4.46×10(-22); I(2)=0%]. Sensitivity analysis showed HLA-B*4001 as a protective marker in Chinese population for showing bullous lesions (OR=0.14; 95% CI=0.06-0.32; P=3.2×10(-6); I(2)=0%). In summary, our meta-analysis showed the presence of HLA alleles contributing toward risk of as well as protection against various CBZ-induced cADRs.

Generation and Characterization of a Third Party GMP Grade Bank of CMV Specific T-Cells for Adoptive Immunotherapy of CMV Infections in Recipients of HSCT from Cord Blood or Seronegative Donors

Adoptive transfer of virus-specific T-cells (CTLs) derived from allogeneic HLA partially matched third party donors can also be effective in a proportion of patients developing EBV lymphomas, or infections due to CMV or adenovirus following transplants from seronegative donors. Such third party donor derived CTLs offer an off the shelf reagent for treatment of viral infections developing after transplant. However, the immunodominant cytotoxic activity exhibited by the CTLs is directed against specific epitopes of the viral protein and restricted by 1-2 HLA alleles. Therefore, it is critical that the T-cells administered from third party donors can recognize viral epitopes presented on shared HLA alleles. We have established a bank of 119 CMV specific T-cell lines (CMV CTLs) generated using autologous APCs loaded with a pool of overlapping peptides spanning the sequence of the dominant immunogenic protein CMVpp65. Each of these 119 CMV CTL lines has been characterized as to the epitope inducing T-cell response as well as the HLA allele restricting the epitope specific T-cell response. Epitopes were identified using an overlapping grid of peptide pools and the HLA restriction by cytotoxic activity against peptide loaded EBVBLCLs matched at a single HLA allele with the T-cell donor. The distribution of the common HLA alleles among the donors for these CTL lines was predominantly within the distribution of HLA allele frequencies represented in the caucasian and black populations, except for HLA A0201 and B0702, which were over represented ( 33% vs 25% and 21% vs 8.7% respectively). In 54% of the CTL lines, the immunodominant T-cell responses were restricted by HLA A0201 (25%), B0702 (21%) and B 3501-11(8%), and in the remaining 50%, the responses were restricted by other HLA class-I alleles, while only 16/119 lines (13%) were restricted by HLA class-II alleles. All 25 donors inheriting HLA B0702 (25/25) demonstrated HLA B0702 restricted CMV CTL responses, while 30/39 (77%) donors inheriting HLA A0201 and 9/19 (47%) donors inheriting HLA B3501-11 demonstrated HLA A0201 and B3501-11 restricted CMV CTL responses. Among all 9 donors co-inheriting HLA A0201 and B 0702, the immunodominant T-cell response was restricted by B0702. Among 12 donors co-inheriting A0201 and B 4401-04, 11/12 (91.6%) demonstrated immunodominant CMV CTL responses restricted by A0201; 1 donor also co-inherited HLA B0702 whose response was restricted by B0702. Therefore, an immunodominance hierarchy for HLA class-I alleles presenting the dominant CMVpp65 epitope was evident through this analysis among these 119 donors and was as follows: B 0702, A0201, B3501-11, A2601, B44, B40, B4201, A0101, B 1801. Strikingly, only 1 of 119 donors demonstrated T-cell responses restricted by A1101; a commonly inherited HLA class –I allele. In a series of 239 consecutive HLA matched related or unrelated transplants (MUD) and 137 HLA mismatched unrelated (MMUD) transplants, and 100 cord blood transplants conducted at our center, in 86%, 89% and 80% of the cases respectively, we could identify a CMV CTL line restricted by a shared HLA allele and matched at 2-3 alleles within this GMP grade CTL bank that would be immediately available for treatment of CMV infection. Appropriately restricted CMV CTLs would only be available in 60-70% of MMUD transplant and none of the cord blood transplants without this approach. This CMV CTL bank therefore represents a readily available clinical reagent for the treatment of resistant CMV infections developing in post transplant patients. The characterization of the CTLs has also enabled the further elucidation of immunodominant CMVpp65 epitopes and hierarchies. Since we have previously shown that CMV CTLs can be generated against subdominant epitopes presented by both common HLA alleles as well as less prevalent HLA alleles using a panel of artificial antigen presenting cells (AAPCs), expansion of this bank using T-cell sensitized against CMVpp65 presented on such AAPCs should broaden the applicability of this bank to all HSCT recipients. Disclosures: No relevant conflicts of interest to declare.

Generation and Characterization Of a Third Party GMP Grade Bank Of CMV Specific T-Cells For Adoptive Immunotherapy Of CMV Infections In Recipients Of HSCT From Cord Blood Or Seronegative Donors

Adoptive transfer of virus-specific T-cells (CTLs) derived from allogeneic HLA partially matched third party donors can also be effective in a proportion of patients developing EBV lymphomas, or infections due to CMV or adenovirus following transplants from seronegative donors. Such third party donor derived CTLs offer an off the shelf reagent for treatment of viral infections developing after transplant. However, the immunodominant cytotoxic activity exhibited by the CTLs is directed against specific epitopes of the viral protein and restricted by 1-2 HLA alleles. Therefore, it is critical that the T-cells administered from third party donors can recognize viral epitopes presented on shared HLA alleles. We have established a bank of 119 CMV specific T-cell lines (CMV CTLs) generated using autologous APCs loaded with a pool of overlapping peptides spanning the sequence of the dominant immunogenic protein CMVpp65. Each of these 119 CMV CTL lines has been characterized as to the epitope inducing T-cell response as well as the HLA allele restricting the epitope specific T-cell response. Epitopes were identified using an overlapping grid of peptide pools and the HLA restriction by cytotoxic activity against peptide loaded EBVBLCLs matched at a single HLA allele with the T-cell donor.The distribution of the common HLA alleles among the donors for these CTL lines was predominantly within the distribution of HLA allele frequencies represented in the caucasian and black populations, except for HLA A0201 and B0702, which were over represented ( 33% vs 25% and 21% vs 8.7% respectively). In 54% of the CTL lines, the immunodominant T-cell responses were restricted by HLA A0201 (25%), B0702 (21%) and B 3501-11(8%), and in the remaining 50%, the responses were restricted by other HLA class-I alleles, while only 16/119 lines (13%) were restricted by HLA class-II alleles.All 25 donors inheriting HLA B0702 (25/25) demonstrated HLA B0702 restricted CMV CTL responses, while 30/39 (77%) donors inheriting HLA A0201 and 9/19 (47%) donors inheriting HLA B3501-11 demonstrated HLA A0201 and B3501-11 restricted CMV CTL responses. Among all 9 donors co-inheriting HLA A0201 and B 0702, the immunodominant T-cell response was restricted by B0702. Among 12 donors co-inheriting A0201 and B 4401-04, 11/12 (91.6%) demonstrated immunodominant CMV CTL responses restricted by A0201; 1 donor also co-inherited HLA B0702 whose response was restricted by B0702. Therefore, an immunodominance hierarchy for HLA class-I alleles presenting the dominant CMVpp65 epitope was evident through this analysis among these 119 donors and was as follows: B 0702, A0201, B3501-11, A2601, B44, B40, B4201, A0101, B 1801. Strikingly, only 1 of 119 donors demonstrated T-cell responses restricted by A1101; a commonly inherited HLA class –I allele.In a series of 239 consecutive HLA matched related or unrelated transplants (MUD) and 137 HLA mismatched unrelated (MMUD) transplants, and 100 cord blood transplants conducted at our center, in 86%, 89% and 80% of the cases respectively, we could identify a CMV CTL line restricted by a shared HLA allele and matched at 2-3 alleles within this GMP grade CTL bank that would be immediately available for treatment of CMV infection. Appropriately restricted CMV CTLs would only be available in 60-70% of MMUD transplant and none of the cord blood transplants without this approach. This CMV CTL bank therefore represents a readily available clinical reagent for the treatment of resistant CMV infections developing in post transplant patients. The characterization of the CTLs has also enabled the further elucidation of immunodominant CMVpp65 epitopes and hierarchies. Since we have previously shown that CMV CTLs can be generated against subdominant epitopes presented by both common HLA alleles as well as less prevalent HLA alleles using a panel of artificial antigen presenting cells (AAPCs), expansion of this bank using T-cell sensitized against CMVpp65 presented on such AAPCs should broaden the applicability of this bank to all HSCT recipients. Disclosures:No relevant conflicts of interest to declare.

Filling the gaps – The generation of full genomic sequences for 15 common and well-documented HLA class I alleles using next-generation sequencing technology

Many common and well-documented (CWD) HLA alleles have only been partially characterized. The DNA sequence of these incomplete alleles, as published in the IMGT/HLA database, is most often limited to exons that code for the extracellular domains of the mature protein. Here we describe the application of next-generation sequencing technology to obtain full length genomic sequence from a single long-range PCR amplicon for 15 common and well-documented HLA Class I alleles. This technology is well suited to fill in the gaps of the current HLA allele sequence database which is largely incomplete. A more comprehensive catalog of HLA allele sequences would be beneficial in the evaluation of mismatches in transplantation, studies of population genetics, the evolution of HLAs, regulatory mechanisms and HLA expression, and issues related to the genomic organization of the MHC.

Sequence-based in silico analysis of well studied Hepatitis C Virus epitopes and their variants in other genotypes (particularly genotype 5a) against South African human leukocyte antigen backgrounds

BackgroundHost genetics influence the outcome of HCV disease. HCV is also highly mutable and escapes host immunity. HCV genotypes are geographically distributed and HCV subtypes have been shown to have distinct repertoires of HLA-restricted viral epitopes which explains the lack of cross protection across genotypes observed in some studies. Despite this, immune databases and putative epitope vaccines concentrate almost exclusively on HCV genotype 1 class I-epitopes restricted by the HLA-A*02 allele. While both genotype and allele predominate in developed countries, we hypothesise that HCV variation and population genetics will affect the efficacy of proposed epitope vaccines in South Africa. This in silico study investigates HCV viral variability within well-studied epitopes identified in genotype 1 and uses algorithms to predict the immunogenicity of their variants from other less studied genotypes and thus rate the most promising vaccine candidates for the South African population. Six class I- and seven class II- restricted epitope sequences within the core, NS3, NS4B and NS5B regions were compared across the six HCV genotypes using local genotype 5a sequence data together with global data. Common HLA alleles in the South African population are A30:01, A02:01, B58:02, B07:02; DRB1*13:01 and DRB1*03:01. Epitope binding to 13 class I- and 8 class –II alleles were described using web-based prediction servers, Immune Epitope Database, (IEDB) and Propred. Online population coverage tools were used to assess vaccine efficacy.ResultsDespite the homogeneity of genotype 1 and genotype 5 over the epitopes, there was limited promiscuity to local HLA-alleles.Host differences will make a putative vaccine less effective in South Africa. Of the 6 well-characterized class I- epitopes, only 2 class I- epitopes were promiscuous and 3 of the 7 class-II epitopes were better conserved and promiscuous. By fine tuning the putative vaccine using an optimal cocktail of genotype 1 and 5a epitopes and local HLA data, the coverage was raised from 65.85% to 91.87% in South African Blacks.ConclusionWhile in vivo and in vitro studies are needed to confirm immunogenic epitopes, in silico HCV epitope vaccine design which takes into account HCV variation and host allele frequency will maximize population coverage in different ethnic groups.

Weak binder for MHC molecule is a potent Mycobacterium tuberculosis-specific CTL epitope in the context of HLA-A24 allele

Tuberculosis causes serious health problem for the world population. Antigenic peptides selected by pathogen-specific cytotoxic T lymphocytes (CTLs) are presented by major histocompatibility complex (MHC; or human leukocyte antigen [HLA] in humans) molecules, and HLA-A restricted responses may be of interest for vaccine development and the understanding of cellular immunity. A series of peptides derived from the 10-KDa culture filtrate protein (CFP10) and the 6 kDa early secretory antigenic target (ESAT-6) in the Mycobacterium tuberculosis (Mtb) have been screened and a CTL epitope restricted by the human leukocyte antigen HLA-A24, a common HLA allele in Asian people, has been identified. In this study, we studied a panel of CFP10 and ESAT-6-derived peptides to identify those with binding motifs for HLA-A24 molecules. The antigenicity of candidate peptides was assessed with in vitro refolding tests and an enzyme-linked immunospot (ELISPOT) assay, and by tetramer staining to determine the capacity to stimulate CTLs from peripheral blood mononuclear cells (PBMCs) of HLA-A24-positive TB Patients. We report that one novel candidate peptide at positions 5–14 of ESAT-6 of Mtb could induce peptide-specific CTLs from PBMCs of HLA-A24-positive patients, but not from HLA-A24-negative patients and HLA-A24-positive healthy controls. Identified epitope is a weak binder for HLA-A24 molecule in a mini MHC refolding assay. Since the peptide is presented by a common HLA class I molecule, it may be useful for immunotherapy against Mtb infection and vaccine development in the large population of Mtb-infected patients.

A Human CD4+T Cell Epitope in the Influenza Hemagglutinin Is Cross-Reactive to Influenza A Virus Subtypes and to Influenza B Virus

The hemagglutinin protein (HA) of the influenza virus family is a major antigen for protective immunity. Thus, it is a relevant target for developing vaccines. Here, we describe a human CD4(+) T cell epitope in the influenza virus HA that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of the HA protein of influenza A virus and the HA protein of influenza B virus. By stimulating peripheral blood mononuclear cells (PBMCs) from a healthy adult donor with peptides covering the entire HA protein based on the sequence of A/Japan/305/1957 (H2N2), we generated a T cell line specific to this epitope. This CD4(+) T cell line recognizes target cells infected with influenza A virus seasonal H1N1 and H3N2 strains, a reassortant H2N1 strain, the 2009 pandemic H1N1 strain, and influenza B virus in cytotoxicity assays and intracellular-cytokine-staining assays. It also lysed target cells infected with avian H5N1 virus. We screened healthy adult PBMCs for T cell responses specific to this epitope and found individuals who had ex vivo gamma interferon (IFN-γ) responses to the peptide epitope in enzyme-linked immunospot (ELISPOT) assays. Almost all donors who responded to the epitope had the HLA-DRB1*09 allele, a relatively common HLA allele. Although natural infection or standard vaccination may not induce strong T and B cell responses to this highly conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4(+) T cells, which are cross-reactive to both influenza A and B viruses.

High-avidity, high-IFNγ-producing CD8 T-cell responses following immune selection during HIV-1 infection.

HIV-1 mutations which reduce or abolish cytotoxic T lymphocyte responses against virus-infected cells are frequently selected in acute and chronic HIV-infection. Among population HIV-1 sequences, immune selection is evident as HLA allele-associated substitutions of amino acids within or near CD8 T cell epitopes. In these cases, the non-adapted epitope is susceptible to immune recognition until an escape mutation renders the epitope less immunogenic. However, several population-based studies have independently identified HLA-associated viral changes which lead to formation of a new T cell epitope, suggesting that the immune responses which these variants or “neo-epitopes” elicit provide an evolutionary advantage to the virus rather than the host. Here, we examined functional characteristics of eight CD8 T cell responses that result from viral adaptation in 125 HLA-genotyped individuals with chronic HIV-1 infection. Neo-epitopes included well-characterised immunodominant epitopes restricted by common HLA alleles and in most cases, the T cell responses against the neo-epitope exhibited significantly greater functional avidity and higher IFNγ production than T cells for non-adapted epitopes but were not more cytotoxic. Neo-epitope formation and emergence of the cognate T cell response co-incident with a rise in viral load was then observed in-vivo in an acutely infected individual. These findings demonstrate that HIV-1 adaptation not only abrogates immune recognition of early targeted epitopes, but may also increase immune recognition to other epitopes, which elicit immunodominant but non-protective T cell responses. These data have implications for immunodominance associated with polyvalent vaccines based on the diversity of chronic HIV-1 sequences.

Identification of Ara h 2 T-cell Epitopes for Use in a T cell-peptide Vaccine

RATIONALE: T cell epitope-based peptide immunotherapy has been found effective in treating certain allergic diseases. Therefore we sought to identify the most immunodominant T cell epitopes of Ara h 2, as candidates for peptide immunotherapy, and define any HLA associations with these epitopes. METHODS: T cell proliferation assays were performed on 70 peanut allergic subjects. 1x106 PBMCs were cultured for 4 days with crude peanut extract, anti-CD3/CD28 beads or over-lapping 20-mer Ara h 2 peptides and then pulsed with tritiated thymidine (1μCi). Cell supernatants from a subset of peptide-stimulated cells were evaluated for cytokine production (IL-13, IL-5, IFNγ and TNFα). DNA was isolated for HLA-DRB1* and HLA-DQB1* typing. RESULTS: Three T-cell epitope regions were identified: aa # 27-56 (cluster A), #92-126 (B) and #122-141 (C). A dominant Th2 cytokine response was induced by these epitopes [(median (pg/mL), range) A: IL-5 (58.9, 3.06-950.32), IL-13 (35.1, 2.75-1119.15); B: IL-5 (21.7, 2.98-661.96), IL-13 (13.4, 2.82-442.47); C: IL-5 (12.5, 2.79-295.11), IL-13 (11.9, 2.82-230.33)] compared to baseline response. Preliminary data show a significant correlation between SI values and cytokine production (IL-5, IL-13; Spearman r≥0.5; p≤0.0007). For all three regions, HLA-DQ6 was one of the most frequent alleles identified, accompanied by DQ7 in clusters A and C, and by DQ8 in B. HLA-DRB1*1302 showed similar distribution for all clusters, whereas *0402 was more frequent in B and *0701 in C. CONCLUSIONS: Three T-cell epitope regions recognized by common HLA alleles are responsible for Th2 cytokine responses in peanut-allergic patients. They may provide good candidates for a T-cell peptide vaccine.

91-P: Characterization of Common and Well-Documented HLA Alleles in Italy: A Multicenter Study on 24.000 Unrelated Individuals

91-P: Characterization of Common and Well-Documented HLA Alleles in Italy: A Multicenter Study on 24.000 Unrelated Individuals

Repertoire of HLA-DR1-Restricted CD4 T-Cell Responses to Capsular Caf1 Antigen ofYersinia pestisin Human Leukocyte Antigen Transgenic Mice

Yersinia pestis is the causative agent of plague, a rapidly fatal infectious disease that has not been eradicated worldwide. The capsular Caf1 protein of Y. pestis is a protective antigen under development as a recombinant vaccine. However, little is known about the specificity of human T-cell responses for Caf1. We characterized CD4 T-cell epitopes of Caf1 in "humanized" HLA-DR1 transgenic mice lacking endogenous major histocompatibility complex class II molecules. Mice were immunized with Caf1 or each of a complete set of overlapping synthetic peptides, and CD4 T-cell immunity was measured with respect to proliferative and gamma interferon T-cell responses and recognition by a panel of T-cell hybridomas, as well as direct determination of binding affinities of Caf1 peptides to purified HLA-DR molecules. Although a number of DR1-restricted epitopes were identified following Caf1 immunization, the response was biased toward a single immunodominant epitope near the C terminus of Caf1. In addition, potential promiscuous epitopes, including the immunodominant epitope, were identified by their ability to bind multiple common HLA alleles, with implications for the generation of multivalent vaccines against plague for use in humans.

An Improved Bicistronic CD20/tCD34 Vector for Efficient Purification and In Vivo Depletion of Gene-Modified T Cells for Adoptive Immunotherapy

T-cell-based adoptive immunotherapy is widely used to treat graft rejection and relapse after stem cell transplantation (SCT). However, this approach is hampered by a high risk of life-threatening graft-versus-host-disease (GvHD). Clinical trials have demonstrated the value of suicide genes to modify T cells for the effective control of GvHD. Herewith, we show that the combination of a codon-optimized B-cell antigen (CD20op) with a selection marker based on a cytoplasmic truncated version of the human stem cell antigen CD34 (tCD34) allows the generation of highly enriched gene-modified T cells. We demonstrate coordinate co-expression of both transgenes and high expression of CD20op resulting in an increased susceptibility to Rituximab (RTX)-induced cell death. In addition, T cells partially retained their alloreactive potential and their CD4/CD8 ratio after transduction and expansion. Long-lasting transgene expression was sustained in vivo after adoptive transfer into Rag-1(-/-) mice. Moreover, gene-modified T cells were quickly and efficiently depleted from peripheral blood (PB) and secondary lymphoid organs of transplanted animals after RTX treatment. These results warrant further steps toward a clinical application of CD20op as a suicide gene for adoptive immunotherapy.

Identification of an immunodominant region of Fel d 1 and characterization of constituent epitopes

Characterization of T cell epitopes restricted by common HLA alleles is a powerful tool in the understanding of the immune responses to allergens and for the identification of potential peptides for future peptide immunotherapy (PIT). One important requirement is the identification and use of peptides that will bind to HLA molecules covering a large proportion of the population. To identify commonly recognized CD4(+) T cell epitopes in Fel d 1, restricted through frequently expressed HLA molecules for potential future use in PIT. HLA matched antigen presenting cells, HLA blocking antibodies, and peptide truncations were used in ELISpot assays to establish HLA-restricted T cell epitopes. Cytokine responses were measured by ex vivo and cultured IFN-gamma, IL-4, and IL-10 ELISpots. Responses to an immunodominant region of chain 2 were identified in the majority of atopic individuals and epitopes restricted by HLA-DQB1(*)06 and -DPB1(*)0401 were characterized in detail. Significantly higher ex vivo IL-4 and lower IFN-gamma responses were observed to both epitopes in individuals with atopic dermatitis (AD) compared with those without disease. IL-10 responses were significantly lower in those with AD in the individuals with HLA-DPB1(*)0401. We have identified an immunodominant region of Fel d 1 which is frequently recognized by CD4(+) T cells from atopic individuals and contains epitopes that are restricted by very common HLA alleles.

A Statistical Method for Predicting Classical HLA Alleles from SNP Data

Genetic variation at classical HLA alleles is a crucial determinant of transplant success and susceptibility to a large number of infectious and autoimmune diseases. However, large-scale studies involving classical type I and type II HLA alleles might be limited by the cost of allele-typing technologies. Although recent studies have shown that some common HLA alleles can be tagged with small numbers of markers, SNP-based tagging does not offer a complete solution to predicting HLA alleles. We have developed a new statistical methodology to use SNP variation within the region to predict alleles at key class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1, HLA-DQA1, and HLA-DQB1) loci. Our results indicate that a single panel of approximately 100 SNPs typed across the region is sufficient for predicting both rare and common HLA alleles with up to 95% accuracy in both African and non-African populations. Furthermore, we show that HLA alleles can be successfully predicted by using previously genotyped SNPs that are within the MHC and that had not been chosen for their ability to predict HLA alleles, such as those included on genome-wide products. These results indicate that our methodology, combined with an extended database of reference haplotypes, will facilitate large-scale experiments, including disease-association studies and vaccine trials, in which detailed information about HLA type is valuable.

CD4 and CD8 T cell responses to tumour-associated Epstein-Barr virus antigens in nasopharyngeal carcinoma patients.

Nasopharyngeal carcinoma (NPC), an Epstein-Barr virus (EBV)-associated tumour common in Southern Chinese populations, is a potentially important target for T cell-based immunotherapy. The tumour cells are HLA class I- and II-positive and express a limited subset of EBV latent proteins, namely the nuclear antigen EBNA1 and the latent membrane proteins LMP2 and (in some cases) LMP1. To ask whether the tumour develops in the presence of a potentially protective host response or in its absence, we set out to determine the prevailing levels of CD4+ and CD8+ T cell memory to these proteins in NPC patients at tumour diagnosis. We first screened healthy Chinese donors against Chinese strain EBNA1, LMP1 and LMP2 sequences in Elispot assays of interferon-gamma release and identified the immunodominant CD4+ and CD8+ epitope peptides presented by common Chinese HLA alleles. Then, comparing 60 patients with >70 healthy controls on peptide epitope mini-panels, we found that T cell memory to CD4 epitopes in all three proteins was unimpaired in the blood of patients at diagnosis. In most cases NPC patients also showed detectable responses to CD8 epitopes relevant to their HLA type, the one consistent exception being the absence in patients of a B*4001-restricted response to LMP2. We infer that NPC arises in patients whose prevailing levels of T cell memory to tumour-associated EBV proteins is largely intact; the therapeutic goal must therefore be to re-direct the existing memory repertoire more effectively against antigen-expressing tumour cells.