Escape from cytotoxic T-cell (CTL) response is a major force driving HIV-1 evolution , reviewed by , although many CTL-escape mutations revert back when transmitted to a host with a different human leucocyte antigen (HLA) type [3,4]. HLA-B alleles have a stronger effect upon viral evolution than HLA-A , probably because HIV-1 has already adapted to common HLA-A alleles encountered in the early years of its evolution . Adaptation to HLA-A alleles could be faster because fewer polymorphisms are present in the HLA-A genes as compared with HLA-B, resulting in low frequencies of specific HLA-B alleles in the human population .
Individuals with certain HLA-B alleles have a survival advantage when infected with HIV-1 , for example, HLA-B27 and HLA-B57 have been associated with long-term control of the virus, probably through targeting peptides in Gag-p24, a highly conserved viral protein (reviewed in ). Allele frequencies for HLA-B27 range from 0–3.6% in sub-Saharan Africans to 8.4–16.7% in whites (www.allelefrequencies.net). For HLA-B57, frequency ranges in both populations are more similar (3.3–8.6% in whites and 6.3–9% in sub-Saharan Africans).
The first AIDS case in The Netherlands was reported in 1983 , suggesting that HIV-1 was introduced in the country in the 1970s . By now, the virus has had over 20 years of evolution in a region where HLA-B27 is more prevalent than in sub-Saharan Africa. It could be imagined that adaptation to this host factor is ongoing. Indeed, we have detected an HIV-1 strain carrying an HLA-B27 CTL-escape mutation in the Gag-p24 KK10 epitope, R264G, together with a compensatory mutation outside this epitope, E260D. Both mutations were stable over almost 5 years of evolution in both an HLA-B27-positive and an HLA-B27-negative patient. In addition, the two mutations were also stable in two novel HIV-1 transmission cases found amongst Amsterdam men who have sex with men (MSM), dating from 2005 and 2008, respectively.
Patients and methods
Patients M28495 and M34217, participants in the Amsterdam Cohort Studies on HIV-AIDS (www.amsterdamcohortstudies.org), tested HIV-1 antibody positive in early 2003. Follow-up blood plasma and peripheral blood mononuclear cell (PBMC) samples were available from 2003 to 2007. Subsequent database analysis of pol genotyping sequences identified two additional patients, M31702 and M35843, infected with the above-described HIV-1 strain. HLA typing was done at Sanquin Diagnostiek (Amsterdam, The Netherlands). Patient characteristics are summarized in Table 1.
Amplification and sequencing of viral RNA
HIV-1 protease/reverse transcriptase gene sequences were generated with the ViroSeq HIV-1 genotyping kit version 2 (Celera Diagnostics, Alameda, California, USA).
Viral RNA was isolated from plasma with a method using silica and GuSCN . A fragment from the HIV-1 gag (nt 835–1386 of the HXB2 reference sequence) gene was amplified as described . Additional analysis of this gene was done with a second primer set, outer primers 5′GCAGAATGGGATAGATTACATCCAGT3′ and 5′TGGGTTCGCATTTTGGACCATCAT3′ and nested primers 5′ACCAAGGGGAAGTGACATAGCAGGA3′ and 5′AGTTTTATAGAACCGGTCTACATA3′.
The env V3–V4 fragment (nt 6949–7519 of HXB2) was amplified with primers ED12 and ED31 . Nested primer sequences were 5′ACAGGGCCATGYAMAAATGT3′ and 5′ATGGGAGGGGCATACATTGC3′. Amplification products were cloned with the TOPO TA cloning kit (Invitrogen, Carlsbad, California, USA), and sequenced with the BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, California, USA). Sixteen clones were analysed per sample. Electrophoresis and data collection were performed on an ABI PRISM 3100 genetic analyser (Applied Biosystems).
Chemokine (C–C motif) receptor 5 haplotype analysis
A fragment of the chemokine (C–C motif) receptor 5 (CCR5) gene was amplified from PBMC DNA to investigate the presence of the CCR5-Δ32 deletion as described .
Characterization of the HIV-1 strain from patients M34217 and M28495
Two patients participating in the Amsterdam Cohort Studies on HIV-AIDS were found HIV-1 antibody positive at the beginning of 2003. Patient M34217, a homosexual man, tested HIV-1 negative in September 2002, and HIV-1 positive in March 2003. By that time, he had a fully developed western blot (Fiebig stage VI ). Patient M28495, a drug user occasionally involved in sex work, tested HIV-1 negative in September 2002, and was found to be HIV-1 positive in January 2003. At that time, his western blot pattern was almost complete (Fiebig stage V ).
Genotyping showed that the virus strain infecting these patients was 100% identical at the nucleotide level in the pol gene; this was also the case for gag and env gene fragments. Analysis of the mitochondrial DNA HVR-I region (nt positions 16 046–16 479) confirmed that the samples indeed belonged to individuals harbouring divergent mitochondrial lineages that differed by 7 nt substitutions (not shown) so that sample mix-up could be excluded. Possibly, one patient infected the other during his acute HIV-1 infection or both patients were almost simultaneously infected by a third individual.
Closer inspection of the HIV-1 gag gene sequences amplified from these patients revealed that this particular HIV-1 strain is a CTL-escape mutant in the gag-p24 KRWIILGLNK263–272 or KK10 epitope targeted by HLA-B27 . Both virus isolates had an R264G mutation in this epitope combined with a compensatory mutation E260D (Table 1) that markedly increases the strongly reduced replication capacity of the R264G variant in vitro. The R264G mutation can be acquired by a relatively simple A to G transition, but the E260D mutation requires a transversion from A to C. A third mutation found to be associated with R264G, namely Q136R , was also present in all clones. Therefore, we named this HLA-B27 CTL-escape strain the Q136RE260DR264G or, in short, the RDG strain of HIV-1.
As patient M28495 carried the HLA-B27 allele, it is possible that this particular HIV-1 strain was generated de novo and subsequently transmitted. HLA-B27-escape mutations are normally found only in the later stages of infection [18,20,21], although CTL responses to the gag-p24 KK10 epitope can be detected at a very early stage [22,23]. Reanalysis of the first available sample from patient M28495 with a different primer set to circumvent eventual primer bias did not also reveal any wild-type sequences that could have pointed towards the CTL-escape strain being a de-novo variant. Also, in patient M34217, no heterogeneity was found in the sequence of the epitope.
Spread of the Q136RE260DR264G-escape strain in Amsterdam, The Netherlands
A database of current Dutch HIV-1 pol genotyping sequences is accessible from our Laboratory of Clinical Virology, encompassing sequences from patients failing antiretroviral therapy as well as from every newly diagnosed HIV-1-infected patient. Phylogenetic trees incorporating genotyping sequences retrieved two sequences clustering with a high bootstrap value (100) with those from patients M28495 and M34217 (Fig. 1[24,25]). The patients from whom these sequences were derived were MSM from Amsterdam, The Netherlands. Patient M31702 presented with an acute HIV-1 infection in September 2005. He had tested HIV antibody negative a year earlier. Patient M35843 tested HIV-1 positive in June 2008 while having been HIV antibody negative 9 months earlier. Subsequent sequencing of HIV-1 gag revealed that the CTL-escape mutations near and in the KK10 epitope were still present in these patients (Table 1). No changes occurred after 1-year follow-up of patient M31702. In the gag clones from patient M35843, a G264→R mutation was seen in a single clone, without a reversal of the compensatory mutation at position 260. So, the RDG-escape strain of HIV-1 is both stable and circulating in The Netherlands.
Fitness of the Q136RE260DR264G-escape strain
Patient M28495 showed a much better control of his HIV-1 infection than patient M34217. At their first HIV-1-positive moment, the plasma viral load in both patients was around 14 000 copies/ml; mainly increasing thereafter in patient M34217, whereas strongly decreasing in patient M28495 (not shown). CD4+ T-cell counts showed an opposite pattern; being consistently high (>740 cells/μl) in patient M28495 and being low (<480 cells/μl) in patient M34217. Patient M28495 was found to carry a CCR5 Δ32aa-deletion allele associated with an attenuated disease course .
Evolution of the gag-p24 KK10 epitope of the Q136RE260DR264G-escape strain of HIV-1
Serial plasma and PBMC samples were available from patient M34217 (2003–2006, eight time-points) and for patient M28495 (2003–2007, seven time-points). HIV-1 gag fragments were amplified from blood plasma and analysed. No reversal of the KK10 CTL-escape or compensatory mutation was seen in any clone over the years (Table 1).
We have identified, amongst Dutch HIV-1-infected patients, an HIV-1 strain that is adapted to escape CTL recognition by HLA-B27 of a conserved epitope in the gag-p24 protein. The HIV-1 RDG-escape strain contains at least one, E260D, and possibly a second, Q136R, compensatory mutation that restores the replicative defect produced by the R264G mutation in gag-p24. The Q136R has not been definitely proven to be a compensatory mutation to R264G, but was strongly associated with it in a database analysis , and was consistently present in HIV-1 sequences from our patients. Although in-vitro experiments suggest that viruses with E260D and R264G mutations in gag-p24 still do not replicate as well as wild-type HIV-1, the subtype B RDG-escape strain is stable and circulating in Amsterdam, The Netherlands amongst the high-risk group of MSM. Spread of this virus strain is recent, the first infection was detected in 2003 and the last in 2008. The RDG strain can infect heterozygous CCR5+/Δ32 individuals, who generally have a lower CCR5 cell surface density  and a lower number of cells expressing CCR5  than CCR5+/+ individuals, suggesting that it is not less fit than other strains. In a patient carrying wild-type CCR5 alleles, the disease course is typical and not attenuated, again suggesting that the virus strain has no in-vivo fitness defect.
Detection of an HLA-B27-escape strain of HIV-1 that is both stable and circulating shows that HIV-1 is in the process of adapting to HLA-B27 alleles. It also shows that HIV-1 can circumvent the severe replication defects associated with mutations in its most conserved protein-coding regions. Reversal of these mutations is probably rare as associated compensatory mutations are needed and a significant fitness loss accompanies single position changes. Indeed, no reversal of E260D and R264G was seen over time in both an HLA-B27-positive individual and two HLA-B27-negative individuals and only a single clone out of eight from the first sample of a fourth patient showed a reversal at position 264. The adaptation of HIV-1 to HLA-B27 is also evident from another, a recent study  on transmitted mutations in the gag-p24 KK10 epitope in 211 HLA-B27-negative individuals with acute/early HIV-1 subtype B infection in North America, Europe and Australia. In 23 (11%) of these patients, a R264 mutation was found, in most cases with compensatory mutations outside the epitope. Two of the patients displayed the R264G mutation in combination with E260D; the other 21 individuals had the more common R264K mutation, mostly together with compensatory mutation S173A and with L268M. The two patients with the E260DR264G mutations were from Berlin, Germany. Berlin is only 600 km from Amsterdam, suggesting there could be an epidemiological link. So, it is not unlikely that in the near future protection from disease progression by HLA-B27 in HIV-1-infected individuals will be lost.
The Amsterdam Cohort Studies on HIV infection and AIDS, a collaboration between the Amsterdam Health Service, the Academic Medical Center of the University of Amsterdam, Sanquin Blood Supply Foundation and the University Medical Center Utrecht, are part of the Netherlands HIV Monitoring Foundation and financially supported by the Netherlands National Institute for Public Health and the Environment. The authors thank Debbie van Baarle for arranging the HLA typing of the first two patients.
M.C. and A.C.vdK. designed the experiments and the study. S.J. and N.K.T.B. performed the pol genotyping, HIV serology and viral load assays, and identified the first two patients together with M.P. M.B. kept the genotyping database and identified the next two patients. K.B. was the treating physician and collected the clinical data. F.M.H. and F.Z. performed the PCR amplifications, cloning and sequencing. A.C.vdK. analysed the data and drafted the paper.
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