Virological and immunological correlates of HIV posttreatment control after temporal antiretroviral therapy during acute HIV infection.

OBJECTIVE: People with HIV rarely control viral replication after cessation of antiretroviral therapy (ART). We present a person with HIV with extraordinary posttreatment control (PTC) for over 23 years after temporary ART during acute HIV infection (AHI) leading to a new insight in factors contributing to PTC. DESIGN/METHODS: Viral reservoir was determined by HIV qPCR, Intact Proviral DNA Assay, and quantitative viral outgrowth assay. Viral replication kinetics were determined in autologous and donor PBMC. IgG levels directed against HIV envelope and neutralizing antibodies were measured. Immune phenotyping of T cells and HIV-specific T-cell responses were analyzed by flow cytometry. RESULTS: The case presented with AHI and a plasma viral load of 2.7 million copies/ml. ART was initiated 2 weeks after diagnosis and interrupted after 26 months. Replicating virus was isolated shortly after start ART. At 18 years after treatment interruption, HIV-DNA in CD4 + T cells and low levels of HIV-RNA in plasma (<5 copies/ml) were detectable. Stable HIV envelope glycoprotein-directed IgG was present during follow-up, but lacked neutralizing activity. Strong antiviral CD8 + T-cell responses, in particular targeting HIV-gag, were detected during 25 years follow-up. Moreover, we found a P255A mutation in an HLA-B∗44 : 02 restricted gag-epitope, which was associated with decreased replication. CONCLUSION: We describe an exceptional case of PTC, which is likely associated with sustained potent gag-specific CD8 + T-cell responses in combination with a replication attenuating escape mutation in gag. Understanding the initiation and preservation of the HIV-specific T-cell responses could guide the development of strategies to induce HIV control.

Characterization of two new HLA alleles: HLA-A*33:199 and HLA-B*13:140.

Two new HLA alleles were identified during routine next generation sequencing.

Human and Rhesus Macaque KIR Haplotypes Defined by Their Transcriptomes.

The killer-cell Ig-like receptors (KIRs) play a central role in the immune recognition in infection, pregnancy, and transplantation through their interactions with MHC class I molecules. KIR genes display abundant copy number variation as well as high levels of polymorphism. As a result, it is challenging to characterize this structurally dynamic region. KIR haplotypes have been analyzed in different species using conventional characterization methods, such as Sanger sequencing and Roche/454 pyrosequencing. However, these methods are time-consuming and often failed to define complete haplotypes, or do not reach allele-level resolution. In addition, most analyses were performed on genomic DNA, and thus were lacking substantial information about transcription and its corresponding modifications. In this paper, we present a single-molecule real-time sequencing approach, using Pacific Biosciences Sequel platform to characterize the KIR transcriptomes in human and rhesus macaque (Macaca mulatta) families. This high-resolution approach allowed the identification of novel Mamu-KIR alleles, the extension of reported allele sequences, and the determination of human and macaque KIR haplotypes. In addition, multiple recombinant KIR genes were discovered, all located on contracted haplotypes, which were likely the result of chromosomal rearrangements. The relatively high number of contracted haplotypes discovered might be indicative of selection on small KIR repertoires and/or novel fusion gene products. This next-generation method provides an improved high-resolution characterization of the KIR cluster in humans and macaques, which eventually may aid in a better understanding and interpretation of KIR allele-associated diseases, as well as the immune response in transplantation and reproduction.

Extensive variation in gene copy number at the killer immunoglobulin-like receptor locus in humans.

Killer immunoglobulin-like receptors (KIRs) are involved in the regulation of natural killer cell cytotoxicity. Within the human genome seventeen KIR genes are present, which all contain a large number of allelic variants. The high level of homology among KIR genes has hampered KIR genotyping in larger cohorts, and determination of gene copy number variation (CNV) has been difficult. We have designed a multiplex ligation-dependent probe amplification (MLPA) technique for genotyping and CNV determination in one single assay and validated the results by next-generation sequencing and with a KIR gene-specific short tandem repeat assay. In this way, we demonstrate in a cohort of 120 individuals a high level of CNV for all KIR genes except for the framework genes KIR3DL3 and KIR3DL2. Application of our MLPA assay in segregation analyses of families from the Centre d'Etude du Polymorphisme Humaine, previously KIR-genotyped by classical techniques, confirmed an earlier reported duplication and resulted in the identification of a novel duplication event in one of these families. In summary, our KIR MLPA assay allows rapid and accurate KIR genotyping and CNV detection, thus rendering improved transplantation programs and oncology treatment feasible, and enables more detailed studies on the role of KIRs in human (auto)immunity and infectious disease.

Sequence-based typing of the HLA-A10/A19 group and confirmation of a pseudogene coamplified with A*3401.

The strategy for sequencing human leukocyte antigen (HLA)-A was based on separate amplification of exons 2 and 3, followed by forward and reverse heterozygous sequencing of the alleles. Validation of the method was obtained by sequencing 11 individuals carrying alleles from all different HLA-A allele groups, except *43. All alleles could be correctly identified except A*3401. Unexpected polymorphic positions were identified in exon 3, even in individuals homozygous for A*3401. In addition, the pseudogene HLA-COQ or HLA-DEL linked to A*3401 was coamplified and sequenced. The problem was solved by using different amplification primers for exon 3 with mismatches for the two pseudogenes. A total of 252 unrelated individuals with at least one allele belonging to the A10 or A19 group were typed for HLA-A by this strategy. Ten different alleles were identified in the A10 group and 14 in the A19 group. As second allele a further 30 different subtypes from all different groups were sequenced. In 21 individuals, sequencing exon 1 was necessary to distinguish A*7401 from A*7402. The sequencing strategy, with separate amplification of the exons, has proven to be a robust method, resulting in reliable and efficient high-resolution HLA-A typing.

Elucidation of exon 1, 4, and 5 sequences of 39 infrequent HLA-B alleles.

More than 590 human leukocyte antigen (HLA)-B alleles have been identified by sequence analysis. Although the polymorphic exon 2 and 3 sequences of all HLA-B alleles are described, the sequences of the other exons of a number of infrequent B-alleles are unknown. In this study, the exon 1, 4, and 5 sequences of 39 different HLA-B alleles were elucidated by allele-specific sequencing. Overall, these exon sequences showed identity with the majority of the known sequences from the corresponding allele groups, except for four alleles B*4010, B*4415, B*4416, and B*5606. The exon 1 sequence of B*4010 had nucleotide differences with all B*40 alleles, but was identical to the B*54, *55, *56, and *59 allele groups. B*4416 differed from B*440201 at position 988, which was previously considered a conserved position. B*4415 showed exon 1, 4, and 5 sequences deviating from the other B*44 alleles, but identical to B*4501. The exon 1 and 4 sequences of B*5606 differed from other B*56 alleles, but were in complete agreement with B*7801. The deviating exon sequences of B*4415 and B*5606 confirmed the evolutionary origin of these alleles suggested by the sequences of exons 2 and 3. The polymorphism observed in exons 1, 4, and 5 merely reflects the lineage-specificity of HLA-B.

No HLA-A gene detectable on one of the haplotypes in a Caucasian family.

An unusual haplotype was detected in a family of a caucasian transplant patient. Human leukocyte antigen (HLA) analysis of the family demonstrated the absence of HLA-A on one of the haplotypes present in two family members. One was serologically typed A24, the other A2. Because they had one haplotype in common, the HLA-A allele of the shared haplotype was supposed to be a null allele. Different molecular typing methods identified only one allele in both individuals. The results suggest a deletion of the complete HLA-A gene or a major part of it. For confirmation, microsatellite analysis of the HLA-A region was performed with six microsatellite markers. Both family members were heterozygous for all markers, and a deletion of HLA-A could not be proven. Fluorescent in situ hybridization (FISH) was performed with cosmid and PAC probes encompassing the HLA-A gene. Both probes demonstrated an identical normal distribution pattern for diploid results. The absence of any serologic and molecular reaction with the results of the microsatellite and FISH analysis make a deletion of a narrow region, encompassing the HLA-A gene, the most plausible explanation.

Natural reinfection with respiratory syncytial virus does not boost virus-specific T-cell immunity.

To determine the role of respiratory syncytial virus (RSV)-specific cell-mediated immunity during natural reinfection, we investigated whether RSV-specific T-cell responses protect against reinfection and, subsequently, whether reinfection boosts virus-specific memory. In a cohort of 55 infants who were hospitalized for RSV bronchiolitis, RSV-specific lymphoproliferative responses in the peripheral blood were measured at three time-points: on admission, 4 wk after admission, and 1 y later, after the second winter season. Memory was defined as a stimulation index (SI) >2. During the second winter season, nasal secretions were collected in every case of a runny nose. Reinfection was diagnosed if immunofluorescence or PCR was positive for RSV. Virus-specific memory was found in one child on admission for primary RSV infection, whereas 4 wk later 44 infants (80%) had memory. Reinfection with RSV was found in 23 infants (43%) during the second winter season. After the second season, memory was found in 20 infants (38%). No differences in SI after the second winter season were found between infants with and without reinfection (2.3 versus 2.1). However, a highly significant correlation was found between SI measured 4 wk after primary RSV infection and SI after the second winter season (r = 0.40, p = 0.001). In conclusion, RSV-specific T-cell responses did not provide protection against reinfection. Moreover, reinfection did not boost RSV-specific T-cell proliferation. To explain both findings, it is hypothesized that RSV-specific T cells fail to expand in vivo upon reinfection.