Although patients with primary HIV-1 infection (PHI) present with distinct clinical syndromes, only a fraction of patients are diagnosed at this early stage of infection. However, it is during this initial phase that antiviral CD8+ T cell responses first emerge and high-level HIV-1 viremia is controlled by several logs to a set point [1,2]. It is a matter of ongoing debate that therapeutic intervention initiated during PHI may alter the immune response of the host to HIV-1 and therefore allow for subsequent immune control of disease progression [3–9].
One of the strongest predictors of HIV-1 disease progression is host genetic factors [10,11], which may also play a key role in influencing the control of viral replication following short-term treatment during the acute phase of the infection. For example, patients expressing the major histocompatibility complex alleles, human leucocyte antigen (HLA)-B57, HLA-B27, HLA-B51 and HLA-A11, which dictate the specificity of the CD8+ T cell response, demonstrate an unusual ability to control viral replication regardless of early therapeutic interventions [10,12]. In addition to host factors, viral genetics have been shown to play a substantial role in clinical outcome [13–15]. Therefore, differences in both host and viral genetics can contribute substantially to disease progression in the setting of HIV-1 infections.
Unfortunately, however, the relative impact of an early therapeutic intervention during PHI in the presence of multifaceted and heterogeneous factors such as host genetics and viral diversity remains difficult to interpret. In the present study, we report on a small epidemiologically linked group, infected with virtually identical strains of HIV-1, in whom we had the rare opportunity to examine immune control and disease progression following treatment during PHI.
All participants were enrolled at the HIV clinic Jessen, Berlin, Germany (approved by the respective institutional review boards).
Autologous gag and pol was sequenced from plasma RNA using population sequencing, as described previously . A neighbor-joining phylogenetic tree was constructed using ClustalX [accession no. FJ179456-FJ179459; FJ185106-FJ185128 of the German HIV-1 seroconverterstudy (Robert Koch-Institute) and reference sequences: K03455 B.FR.83.HXB2, U63632 B.US.86.JRFL, U21135 B.US.90.WEAU 160, U88824 D.UG.94.94, U46016 C.ET.86.ET].
All participants were HLA typed using the SSP Unitray system (Invitrogen, Carlsbad, California, USA). Screening for chemokine (C-C motif) receptor 5 (CCR5), chemokine (C-C motif) receptor 2 (CCR2) and stromal cell-derived factor-1 (SDF-1) polymorphisms were performed by PCR as previously described .
HIV-1-specific cytotoxic T lymphocyte (CTL) responses were quantified by the IFN-γ–Elispot assay using previously described optimal epitopes . A response was considered positive if more than 55 spot-forming cells (SFC)/106 cells and at least three times higher than mean background activity.
Three patients with PHI (≤3 western blot bands positive) were suspected of having been serially infected within 3 months from a chronically infected index patient following unprotected sex (Fig. 1a). The status of the source person was unknown, but they did test HIV negative 5 years previously. To verify the presumed linked transmission events, the gag and pol genes from each patient were amplified and sequenced from plasma RNA. Nearly identical HIV-1 clade B sequences were identified in each patient, with a mean pairwise nucleotide sequence identity between these sequences of 99.6% (±0.2%) (Fig. 1b). These data strongly support the transmission of highly related strains of HIV-1 between these epidemiologically linked patients over a short period of time.
All patients immediately initiated highly active antiretroviral therapy (HAART) for a period of 6 months, achieving suppression of viral replication (Fig. 1a). Within 6 months of cessation of therapy, patient ‘A’, who had already established chronic infection at the time of HAART initiation, and patient ‘C’ exhibited viral load set points of 9 × 104 and 6 × 105 copies/ml, respectively, whereas patients ‘B’ and ‘D’ exhibited lower viral load set points of 7 × 103 and 3.3 × 104 copies/ml, respectively (Fig. 1a). Thus, viral loads in patients ‘A’ and ‘C’ ranged in the upper quartile, which is predictive of more rapid progression of disease, whereas patients ‘B’ and ‘D’ ranged in the second and third quartiles, associated with slower progression of disease according to the Multicenter AIDS Cohort Studies . These data indicate that patients infected with an identical viral strain, and receiving HAART during the early phase of the infection, can have a substantially different viral set point after treatment discontinuation, suggesting that additional factors may be responsible for the distinct clinical courses.
To examine whether differences in host genetics might account for these differences in viral set points between patients, we examined multiple genetic loci previously identified to contribute to the susceptibility to HIV-1 infection and the rate of progression to AIDS . In the present study, we assessed differences in HLA class I alleles , as well as polymorphisms within the chemokine receptors . Although multiple promotor mutations in CCR5, CCR2 and SDF-1 were observed in this cluster of individuals, only patient ‘B’ was found to be homozygous for the potential protective A59029G polymorphism in CCR5  (Fig. 1a). Notably, patients ‘B’ and ‘D’ also expressed the HLA alleles, HLA-B57 and HLA-A11, respectively, which are associated with control of HIV-1 replication and a more favorable disease progression . In addition, both patients immunodominantly targeted specific CTL epitopes [HLA-B57HW9 (Nef) HTQGYFPDW, HLA-B57-TW10 (Gag) TSTLQEQIGW, HLA-A11-AK11 (Gag) ACQGVGGPGHK], which have been previously associated with control of viral replication [22,23] (data not shown). The immunodominant response B51-TI8(Pol) in patient ‘C’, with a substantial higher viral load was, in contrast, not present because an escape mutation had been transmitted within this epitope to patient ‘C’. In summary, these data suggest that the expression of protective HLA alleles and the immunodominant targeting of key responses by these alleles had a strong impact on the control of viral replication following cessation of therapy.
The identification of tightly linked transmission cases of HIV-1 provides a unique opportunity to understand the different influences of host genetics and early antiretroviral therapy on disease progression. In the current study, we present data documenting the rapid transmission of a strain of HIV-1 clade B within four individuals with different genetic backgrounds, which resulted in distinctly different virologic and immunological outcomes. The high degree of similarity of sequences derived from each patient suggested that little viral diversification had taken place within the first few weeks of infection and, therefore, was not substantially contributing to differences in viral control.
After treatment discontinuation, only patients ‘B’ and ‘D’ maintained relative control of viral replication after cessation of HAART, in contrast to patients ‘A’ and ‘C’. Both patients ‘B’ and ‘D’ expressed HLA alleles – especially HLA-B57 associated with control of HIV-1 – and they mounted immune responses against immunodominantly targeted CD8+ epitopes, which have previously been associated with slower disease progression [22,23]. This observation is emphasized by revisiting the ‘Berlin patient’, the first described case of spontaneous control of viral replication following short-term treatment interruption during primary HIV-1 infection . Several subsequent studies have investigated the role of early treatment during acute HIV-1 infection with or without interruptions but have provided contradictory results [6,7,9,24,25]. A reanalysis of host genetic factors of the ‘Berlin patient’, who, to the present date, exhibits control of viral replication, has recently revealed expression of the HLA-B57 haplotype (unpublished data). Therefore, it is likely that the expression of HLA-B57 may have been at least one major contributor to the sustained control of HIV-1 replication in this patient, questioning the role of early interventional strategies for the sustained containment of HIV-1 following cessation of therapy. Taken together, these data suggest that host genetic factors should be included in trials investigating the impact of treatment initiation during acute HIV-1 infection in order to accurately discern the relative contribution of early treatment on HIV-1 clinical outcome.
We would like to thank the participants in this study. We also would like to thank Mary Carrington and Yuko Yuki from the NCI Frederick, Andreas Carganico from the HIV Clinic Dupke, Carganico, Baumgarten in Berlin, Zabrina Brumme, Chanson Brumme and Adrianne Gladden from the Partners AIDS Research Center, Boston for their outstanding help to realize this project. This study was funded by NIH grants R01-AI054178 to Todd M. Allen and U01-AI052403 to Todd M. Allen, Bruce D. Walker, and Marcus Altfeld. Hendrik Streeck is supported by the Deutscher Akademischer Austauschdiensts.
Authors' contributions: Hendrik Streeck, Todd M. Allen, Claudia Kuecherer, Marcus Altfeld and Bruce D. Walker designed the experiments/the study. Heiko Jessen and Arne B. Jessen identified and enrolled the patients of the cluster. Heiko Jessen, Arne B. Jessen, Stephan Dupke and Axel Baumgarten were treating physicians of the patients and collected clinical data. Bin Li, Todd M. Allen and Claudia Kuecherer generated viral sequence data. Hendrik Streeck generated immunological data. Hendrik Streeck, Bin Li, Claudia Kuecherer, Todd M. Allen, Jan van Lunzen and Ingrid Stahmer collected data or did experiments for the study. Hendrik Streeck and Todd M. Allen wrote the first draft of the paper. Hendrik Streeck, Heiko Jessen, Todd M. Allen, Bruce D. Walker, Claudia Kuecherer and Marcus Altfeld contributed to writing the paper.
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