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Much has been learnt about the role of HLA alleles during natural infection of HIV-1, but far less is known about their role in people living with HIV (PLWH) on suppressive antiretroviral therapy (ART). In this study we used variable selection to identify predictors of HIV reservoir size, as measured by total HIV DNA in 192 participants in an acute HIV infection (AHI) cohort. Baseline clinical data including pre-ART CD4 T cell counts and plasma viral load (VL) were available from all participants along with longitudinal measurements after ART initiation during AHI. Time to VL suppression, time to CD4 reconstitution and pre-ART viremia were the strongest predictors of undetectable total HIV DNA at 24 weeks after ART initiation. We next performed HLA typing in 526 participants from the same cohort and investigated associations with the three predictors of reservoir size. HLA-B*57 and B*58 both associated significantly with time to VL suppression, which was one of the predictors of the size of the HIV reservoir. These findings are significant in PLWH and have to be considered in the context of therapeutic intervention when conducting ATI studies as participants with these alleles could impact clinical findings given the small sizes of these studies.

The recombinant, live, attenuated, tetravalent dengue vaccine CYD-TDV has shown efficacy against all four dengue serotypes. In this exploratory study (CYD59, NCT02827162), we evaluated potential associations of host human leukocyte antigen (HLA) alleles with dengue antibody responses, CYD-TDV vaccine efficacy, and virologically-confirmed dengue (VCD) cases. Children 4-11 years old, who previously completed a phase 2b efficacy study of CYD-TDV in a single center in Thailand, were included in the study. Genotyping of HLA class I and II loci was performed by next-generation sequencing from DNA obtained from 335 saliva samples. Dengue neutralizing antibody titers (NAb) were assessed as a correlate of risk and protection. Regression analyses were used to assess associations between HLA alleles and NAb responses, vaccine efficacy, and dengue outcomes. Month 13 NAb log geometric mean titers (GMTs) were associated with decreased risk of VCD. In the vaccine group, HLA-DRB1*11 was significantly associated with higher NAb log GMT levels (beta: 0.76; p = 0.002, q = 0.13). Additionally, in the absence of vaccination, HLA associations were observed between the presence of DPB1*03:01 and increased NAb log GMT levels (beta: 1.24; p = 0.005, q = 0.17), and between DPB1*05:01 and reduced NAb log GMT levels (beta: -1.1; p = 0.001, q = 0.07). This study suggests associations of HLA alleles with NAb titers in the context of dengue outcomes. This study was registered with clinicaltrials.gov: NCT02827162.

The human leukocyte antigen (HLA) genes are highly variable and are known to play an important role in disease outcomes, including infectious diseases. Prior knowledge of HLA polymorphisms in a population usually forms the basis for an effective case-control study design. As a prelude to future disease association analyses, we report HLA class I and II diversity in 334 unrelated donors from a Dengue vaccine efficacy trial conducted in Thailand. Long-range PCR amplification of six HLA loci was performed on DNA extracted from saliva samples. HLA-A, -B, -C, -DPB1, -DQB1 and -DRB1 were genotyped using a next-generation sequencing method presented at the 17th International HLA and Immunogenetics Workshop. In total, we identified 201 HLA alleles, including 35 HLA-A, 57 HLA-B, 28 HLA-C, 24 HLA-DPB1, 21 HLA-DQB1 and 36 HLA-DRB1 alleles. Very common HLA alleles with frequencies greater than 10 percent were A∗11:01:01, A∗33:03:01, A∗24:02:01, B∗46:01:01, C∗07:02:01, C∗01:02:01, C∗08:01:01, DPB1∗05:01:01, DPB1∗13:01:01, DPB1∗04:01:01, DPB1∗02:01:02, DQB1∗03:01:01, DQB1∗05:02:01, DQB1∗03:03:02, DRB1∗12:02:01, DRB1∗09:01:02, and DRB1∗15:02:01. A novel HLA allele, B∗15:450, had a non-synonymous substitution and occurred in more than one donor. Population-based full-length NGS HLA typing is more conclusive and provides a sound foundation for exploring disease association in a given population.

The RV144 HIV vaccine trial in Thailand elicited antibody responses to the envelope of HIV-1, which correlated significantly with the risk of HIV-1 acquisition. Human leukocyte antigen (HLA) class II molecules are essential in antigen presentation to CD4 T cells for activation of B cells to produce antibodies. We genotyped the classical HLA-DRB1, DQB1, and DPB1 genes in 450 individuals from the placebo arm of the RV144 study to determine the background allele and haplotype frequencies of these genes in this cohort. High-resolution 4 and 6-digit class II HLA typing data was generated using sequencing-based methods. The observed diversity for the HLA loci was 33 HLA-DRB1, 15 HLA-DQB1, and 26 HLA-DPB1 alleles. Common alleles with frequencies greater than 10% were DRB1*07:01, DRB1*09:01, DRB1*12:02, DRB1*15:02, DQB1*02:01/02, DQB1*03:01, DQB1*03:03, DQB1*05:01, DQB1*05:02, DPB1*04:01:01, DPB1*05:01:01, and DPB1*13:01:01. We identified 28 rare alleles with frequencies of less than 1% in the Thai individuals. Ambiguity for HLA-DPB1*28:01 in exon 2 was resolved to DPB1*296:01 by next-generation sequencing of all exons. Multi-locus haplotypes including HLA class I and II loci were reported in this study. This is the first comprehensive report of allele and haplotype frequencies of all three HLA class II genes from a Thai population. A high-resolution genotyping method such as next-generation sequencing avoids missing rare alleles and resolves ambiguous calls. The HLA class II genotyping data generated in this study will be beneficial not only for future disease association/vaccine efficacy studies related to the RV144 study, but also for similar studies in other diseases in the Thai population, as well as population genetics and transplantation studies.

Next generation sequencing (NGS) methods have been established as an efficient approach for HLA typing because unlike traditional Sanger sequencing, they provide unambiguous results at a reasonable cost. We previously developed a multi-locus index method to genotype four HLA loci (A, B, C, and DRB1) on the Illumina MiSeq platform. We have now expanded this method to include two additional loci, HLA-DPB1 and DQB1. Contiguous full-length amplicons from 5'UTR through 3'UTR regions were generated using one long-range PCR reaction per locus for each of the six loci from 96 individuals of different ethnicities. The six amplicons from each donor were pooled, enzymatically fragmented and given a donor-specific index. This approach enabled sequencing of 576 loci from 96 individuals in a single MiSeq run. Donor-specific sequence reads were demultiplexed, and allele calls were generated from FASTQ files using commercially available software. Comparison to HLA genotypes generated from Sanger sequence-based typing (SBT) identified no discordances among any of the alleles analyzed in this study. Importantly, this method was able to resolve 22 DPB1 and 20 DQB1 alleles that were ambiguous with the SBT method. Furthermore, a novel allele in each of these two loci was identified, with the DQB1*05:01:24 allele having a frequency of greater than five percent. This method was subsequently validated against a blinded panel of 22 samples from the 17th International HLA and Immunogenetics Workshop. The flexibility of the method is further highlighted by successful genotyping of eight loci comprising all classical HLA loci for a subset of the samples. We now present a high-throughput, high-resolution, scalable NGS HLA typing method to accurately and efficiently genotype all classical HLA class I and II loci.

Aim A tetravalent, live attenuated dengue vaccine demonstrated efficacy, safety and immunogenicity in several clinical trials in Asia and Latin America. Efficacy differed based on infecting serotypes, presence of pre-existing dengue neutralizing antibody (NAb) titers and age. HLA class II molecules expressed on antigen presenting cells mediate CD4+ T cell stimulation of antibody production by B cells involved in vaccine-induced responses. We hypothesized that the differences in observed vaccine efficacy could be due to variation in NAb immune responses in conjunction with host HLA class II genes. Methods Samples were available from a subset of subjects that took part in the first tetravalent dengue vaccine efficacy trial conducted in Thailand. DNA was extracted from 335 saliva samples and HLA genotyping was performed using next-generation sequencing (NGS) of full-length genes. A panel of ancestry informative markers (AIMs) was genotyped to assess population stratification. Serotype-specific NAb titers were measured by plaque-reduction neutralization test 28 days after last injection. The association of NAb titers and HLA class II on dengue infection was tested by logistic regression. Linear regression was used to test association of HLA class II alleles with NAb levels after accounting for sex, age, and serotype as covariates. A minimal false discovery rate to account for multiple comparisons, with a two-sided p-value Results NGS identified 197 HLA class I and II alleles in the Thai Dengue vaccine trial. AIMs analysis did not identify population stratification comparing cases and controls. Magnitude of NAb levels post vaccination was significantly higher in the presence of HLA-DRB1*11 (p = 0.002, q = 0.08). HLA-DPB1*03:01 and *05:01 presence correlated with pre-existing NAb titers in the placebos (p = 0.005, q = 0.09; p = 0.001, q = 0.04). We did not observe a direct effect of HLA on dengue infection in either the placebo or treatment arm. Conclusions These findings suggest that specific HLA class II alleles modulate protective NAb titers in dengue infection. This is an exploratory study to identify signals to replicate in other dengue vaccine clinical studies. Understanding this HLA class II mechanism will enable improved vaccine design.

Aim RNA sequencing (RNA-Seq) is a powerful next-generation sequencing method for discovering, profiling and quantifying RNA transcripts. RNA-Seq can also identify splice junctions and single nucleotide polymorphisms (SNP) in coding exons. In this study, we tested if RNA-Seq reads provide sufficient coverage and depth for high-resolution HLA genotyping. Methods RNA-Seq data was available from 24 individuals from an acutely infected HIV cohort. We used two methods to extract HLA reads from the RNA-Seq data: 1) FR-HIT recruited RNA-Seq reads to the IMGT coding sequence reference database; and 2) the SNP-tolerant aligner GSNAP aligned the reads to a constructed GMAP database consisting of cDNA from HLA genes. The resulting HLA-specific FASTQ reads that were generated were analyzed by commercial HLA typing software. Results We obtained 100% sequence coverage and a median sequence read depth of 331, which was sufficient to genotype 15 HLA loci from each of the donors. HLA genotypes for 8 classical HLA class I and II loci matched genotypes generated from the Sanger sequence-based typing method, as well as targeted next-generation sequencing of genomic DNA. We identified a novel HLA-DQB1 allele in the Thai population with a frequency greater than 5% that was identical to the 05:01:01 allele in all exons except exon 1, where it differed by one SNP at position 81. The DQB1 * 05:01:01 allele, like several other DQB1 alleles, has exon 5 spliced out. This deletion however, cannot be confirmed from the reads that are obtained by sequencing genomic DNA. RNA-Seq confirmed that the novel allele does not express exon 5, as the RNA-Seq data showed a complete absence of exon 5 reads compared to reads from targeted genomic sequences. Conclusions We have successfully genotyped 15 HLA loci and confirmed a splice variant in a novel allele using RNA-Seq. This method highlights the importance of mining RNA-Seq data for high-resolution HLA genotyping. Additional analysis methods may be able to identify allele-specific expression of HLA alleles from RNA-Seq data.

RV144 is the first phase 3 HIV vaccine clinical trial to demonstrate efficacy. This study consisted of more than 8,000 individuals in each arm of the trial, representing the four major regions of Thailand. Human leukocyte antigen (HLA) class I and killer cell immunoglobulin-like receptor (KIR) genes, as well as 96 genome-wide ancestry informative markers (AIMs) were genotyped in 450 placebo HIV-1-uninfected individuals to identify the immunogenetic diversity and population structure of this cohort. High-resolution genotyping identified the common HLA alleles as A*02:03, A*02:07, A*11:01, A*24:02, A*24:07, A*33:03, B*13:01, B*15:02, B*18:01, B*40:01, B*44:03, B*46:01, B*58:01, C*01:02, C*03:02, C*03:04, C*07:01, C*07:02, C*07:04, and C*08:01. The most frequent three-loci haplotype was B*46:01-C*01:02-A*02:07. Framework genes KIR2DL4, 3DL2, and 3DL3 were present in all samples, and KIR2DL1, 2DL3, 3DL1, 2DS4, and 2DP1 occurred at frequencies greater than 90 %. The combined HLA and KIR profile suggests admixture with neighboring Asian populations. Principal component and correspondence analyses comparing the RV144 samples to the phase 3 International HapMap Project (HapMap3) populations using AIMs corroborated these findings. Structure analyses identified a distinct profile in the Thai population that did not match the Asian or other HapMap3 samples. This shows genetic variability unique to Thais in RV144, making it essential to take into account population stratification while performing genetic association studies. The overall analyses from all three genetic markers indicate that the RV144 samples are representative of the Thai population. This will inform subsequent host genetic analyses in the RV144 cohort and provide insight for future genetic association studies in the Thai population.

Aim The Multi-locus Individual Tagging – Next Generation Sequencing (MIT-NGS) method was previously shown to efficiently genotype four HLA loci (A, B, C, and DRB1) from 96 donors while maintaining phase, and resolving ambiguities associated with traditional sequence based HLA typing (Ehrenberg et al., BMC Genomics, 2014). Here we expanded MIT-NGS to six loci to include DPB1 and DQB1, and evaluated HLA genotypes for all loci against those obtained from both the HLA sequence-based typing (SBT) method and the four loci MIT-NGS method. Methods Full-length genes of HLA-A, B, C, DPB1, DQB1 and DRB1 loci from 96 donors of Caucasian, African and Asian ethnicities were amplified using long-range PCR. Individual donor pools, each consisting of equimolar inputs corresponding to the target size of the six HLA loci amplicons were enzymatically fragmented and uniquely indexed. This 6-plex approach enabled sequencing of 576 loci from 96 individuals in a single Illumina MiSeq run. Locus specific sequence reads were demultiplexed, aligned, and genotyped using commercially available software and an in-house pipeline. Results There were no observed discordances between the 6-plex MIT-NGS method and the combined SBT or 4-plex NGS methods for all 1152 alleles analyzed in this study. Furthermore, this method was able to resolve 20 DQBI and 22 DPB1 genotypes that were ambiguous with SBT. These alleles included DPB1 ∗ 104:01,DPB1 ∗ 105:01, DPB1 ∗ 107:01, DPB1 ∗ 131:01, DPB1 ∗ 135:01, DPB1 ∗ 296:01, DPB1 ∗ 414:01, DQB1 ∗ 02:02, DQB1 ∗ 03:19 and DQB1 ∗ 06:01:03. All of these ambiguous changes affected the protein coding sequence. Conclusions We have developed a six-locus high resolution HLA typing method to accurately and efficiently genotype all classical HLA class I and II loci while maintaining phase, and resolving ambiguities associated with conventional sequence based HLA typing.