In February 2008, M1 presented with a plasma viral load of 280 copies/ml, which increased over the following year. Genotypic analysis from 2008 onward revealed 25 new drug-resistance mutations to nucleoside reverse transcriptase inhibitor (NRTI) (6), non NRTI (NNRTI) (5) and protease inhibitor (14). Of note, this resistance profile shared 22 of 23 mutations (96%) found contemporaneously in M2. The shared mutations included NRTI resistance mutations 41L, 74I, 184V, and 215Y; NNRTI mutations 98G, 103N, and 108I; and protease inhibitor mutations 10V, 13V, 20R, 32I, 33I, 46I, 47V, 50V, 71I, and 77I. M1 also developed 75T (NRTI), 115/Y (NNRTI) and 82A (protease inhibitor) polymorphisms.
We obtained viral gene sequences from M1 from July 2000 [n = 1,1 ∼9 kb near full-length genomes (NFLG)], February 2008 (n = 12 gag and env genes) and May of 2008 (n = 14 gag and n = 16 env genes), and from M2 in January (n = 10 NFLG) and March of 2008 (n = 7 NFLG). All 86 viral sequences were assigned to clade B. Additionally, all sequences from M1 obtained from 2008 clustered with M2 sequences from the same year and were phylogentically unrelated to M1 sequences from 2000 (data not shown). This indicated HSI of M1 with substantial or complete replacement with virus from M2. No recombination between the M1 and M2 strains was observed.
The viral load of patient M1 increased from undetectable to above 3 logs after HSI, decreasing progressively from 2009 to 2012 without cART modification. A continuous drop of CD4+ cells of at least 10% was observed from time of HSI detection in February 2008 (30.8%) through April 2011 (18%). However, in April 2012, the CD4+ cell count for M1 recovered to 25.4% (757 cell/μl) (Fig. 1b). The follow-up for patient M2 was taken over by a general practitioner in 2009 after three consecutive undetectable viral loads following start of salvage antiretroviral treatment (Fig. 1a).
We detected superinfection and replacement by HIV-1 clade B triple-class resistant virus in a patient on long-term cART controlled infection, with the initial indicator of HSI being a detectable and increasing viral load.
Other resistance mutations not found in the superinfecting strain also emerged following HSI, though their origin is unclear. They could have been present as minority populations prior to superinfection or transmitted with the superinfecting strain but below the detection level of our assessment of M2's quasispecies (no additional specimen was available for massively parallel sequencing). Despite HSI with a triple-class resistant virus, the ART for M1 remained unchanged. During the 4 years of additional follow-up, a continuous drop of viral load occurred, followed by CD4+ recovery in the past year (Fig. 1b). The impact of HSI in CD4+ decline and disease progression following subtype B coinfection was initially suggested by Gottlieb et al. . Evidence of CD4+ T-cell decline as the initial indicator of HSI was also reported in untreated patients during primary HIV infection as well as in untreated elite controllers [14–16].
In contrast to previous studies of superinfecton among long-term known seroconcordant couples undergoing ART , this study underscores the fragile chemoprophylactic barrier exerted by ART despite excellent adherence. Apart from the complex resistance profile of the superinfecting strain present in patient M2, we cannot rule out the role of viral escape to pre-existing cytotoxic T-lymphocyte or antibody responses in the establishment of the second infection, which involves the same clade, and therefore, to some extent challenged similar immune signatures in the superinfected individual, as reported elsewhere [17,18]. Of interest, the HIV-specific T-cell responses of M1 were enriched following NYVAC-B immunizations (W0 and W4) and 21 months prior to superinfection . Briefly, two novel Env/Pol vaccine-induced responses emerged at W2 and remained present throughout the study with a clear decline by W48 , greater than 1 year prior to HSI. The protective role of these vaccine-induced responses to prevent a second infection with a resistant virus, as well as their longevity, remains unproven. Haplotypes such as human leukocyte antigen (HLA)-B3503 have been shown to be associated with HIV-1 superinfection susceptibility as a consequence of late or weak immune response priming . In this regard, patient M1 carries HLA haplotype A*30/23, B*35/44, DRB1*14/04 (performed by PCR-SSO using LabType kit on the Luminex System). He also had no CCR5-delta 32 mutations (in-house PCR modified from Wilkinson et al. ).
In summary, patient M1's superinfection onset agrees with the current knowledge on host factors and re-exposure to a resistant strain, and demonstrates the previously unexpected scenario of re-infection during well established chronic infection, despite continuously suppressed viremia with cART started during primary HIV infection.
The authors are grateful to patients M1 and M2 for their written consent to participate in this study. We also thank Brendan Larsen for submission of sequences to GenBank. This work was supported by NIH grants R37AI47734 and the Bioinformatics Core of the University of Washington Centers for AIDS Research (NIH P30AI27757).
Author contributions: E.C., J.I.M., M.C., and P.A.B. designed research; E.C. and H.Z. performed research; E.C., H.Z., J.I.M., M.C., and P.A.B. analyzed data; and E.C., G.P., J.I.M., M.C. and P.A.B. wrote the paper.
Sequence data: Nucleotide sequences were deposited in GenBank and are available under accession numbers KC797171–KC797229.
Conflicts of interest
There are no conflicts of interest.
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