Combination antiretroviral therapy (cART) suppresses viral replication but does not cure HIV infection because a reservoir of infectious (intact) HIV proviruses persists in long-lived CD4+T cells. However, a large majority (>95%) of HIV-infected cells that persist on effective cART carry defective (non-infectious) proviruses. Defective proviruses consisting of only a single LTR (solo long terminal repeat) are commonly found as endogenous retroviruses in many animal species, but the frequency of solo-LTR HIV proviruses has not been well defined. Here we show that, in five pediatric donors whose viremia was suppressed on cART for at least 5 years, the proviruses in the nine largest clones of HIV-infected cells were solo LTRs. The sizes of five of these clones were assayed longitudinally by integration site-specific quantitative PCR. Minor waxing and waning of the clones was observed, suggesting that these clones are generally stable over time. Our findings show that solo LTRs comprise a large fraction of the proviruses in infected cell clones that persist in children on long-term cART. IMPORTANCE This work highlights that severely deleted HIV-1 proviruses comprise a significant proportion of the proviral landscape and are often overlooked.

Objectives: Low-capital-layout sequencing options from Oxford Nanopore Technologies (ONT) could assist in expanding HIV drug resistance testing to resource-limited settings. HIV drug resistance mutations often occur as mixtures, but current ONT pipelines provide a consensus sequence only. Moreover, there is no integrated pipeline that provides a drug resistance report from an ONT sequence file without intervention from skilled bioinformaticists. We therefore investigated Nano-RECall, which provides seamless drug resistance interpretation while requiring low-read coverage ONT sequence data from affordable Flongle or MinION flow cells and which provides mutation mixtures similar to Sanger Sequencing. Methods: We compared Sanger sequencing to ONT sequencing of the same HIV-1 subtype C polymerase chain reaction (PCR) amplicons, respectively using RECall and the novel Nano-RECall bioinformatics pipelines. Amplicons were from separate assays: (a) Applied Biosystems HIV-1 Genotyping Kit (ThermoFisher) spanning protease (PR) to reverse transcriptase (RT) (PR-RT) (n = 46) and (b) homebrew integrase (IN) (n = 21). The agreement between Sanger sequences and ONT sequences was assessed at nucleotide level, and at codon level for Stanford HIV drug resistance database mutations at an optimal ONT read depth of 400 reads only. Results: The average sequence similarity between ONT and Sanger sequences was 99.3% (95% CI: 99.1%-99.4%) for PR-RT and 99.6% (95% CI: 99.4%-99.7%) for INT. Drug resistance mutations did not differ for 21 IN specimens; 8 mutations were detected by both ONT-and Sanger sequencing. For the 46 PR and RT specimens, 245 mutations were detected by either ONT or Sanger, of these 238 (97.1%) were detected by both. Conclusions: The Nano-RECall pipeline, freely available as a downloadable application on a Windows computer, provides Sanger-equivalent HIV drug resistance interpretation. This novel pipeline combined with a simple workflow and multiplexing samples on ONT flow-cells would contribute to making HIV drug resistance sequencing feasible for resource-limited settings. K E Y W O R D S bioinformatics pipeline, codon-aware alignment, HIV drug resistance sequencing, homopolymer error, Oxford Nanopore Technology, sanger sequencing P. Richard Harrigan and Gert Uves van Zyl contributed equally to this work.

Paediatric HIV spontaneous controllers (HSCs) are a unique and understudied population with potential to inform alternative treatment options for patients living with HIV. As HSCs are so rare and often not recognised prior to antiretroviral treatment (ART) initiation, it can be difficult for clinicians to optimally manage this group. We describe the diagnosis, history and management of three paediatric HSCs, two girls and a boy who were followed for 2, 1.25 and 10.4 years, respectively, before starting ART. All had low but detectable viral loads throughout follow-up but mostly marginally low CD4:CD8 ratios. The reason for starting ART in all was a gradual tendency to poorer virological control. This case series should assist in recognising paediatric HSCs. Clinical dilemmas arising in the management of paediatric HSCs include arriving at a correct HIV-positive diagnosis, correct diagnosis as an HSC, as well as whether to initiate ART. Decision-making for initiation of ART in paediatric HSCs should be individualised. Factors supporting ART initiation in these patients included increased frequency of viral load blips, increasing detectable viral load, CD4 percentage and CD4:CD8 ratio. Other factors included Hepatitis C serology and highly sensitive C-reactive protein. All three patients ultimately required ART, which supports universal initiation of ART in paediatric HSCs, but further research is required.

Introduction: Oxford Nanopore Technologies (ONT) offer sequencing with low-capital-layout sequencing options, which could assist in expanding HIV drug resistance testing to resource limited settings. However, sequence analysis remains time time-consuming and reliant on skilled personnel. Moreover, current ONT bioinformatic pipelines provide a single consensus sequence that is not equivalent to Sanger sequencing, as drug resistance is often detected in mixed populations. We have therefore investigated an integrated bioinformatic pipeline, Nano-RECall, for seamless drug resistance of low read coverage ONT sequence data from affordable Flongle or MinION flow cells. Methods: We compared Sanger sequencing to ONT sequencing of the same HIV-1 subtype C polymerase chain reaction (PCR) amplicons, respectively using RECall and the novel Nano-RECall bioinformatics pipelines. Amplicons were from separate assays a) Applied Biosystems HIV-1 Genotyping Kit (ThermoFisher) spanning protease (PR) to reverse transcriptase (RT) (PR-RT) (n=46) and b) homebrew integrase (IN) (n=21). We investigated optimal read-depth by assessing the coefficient of variation (CV) of nucleotide proportions for various read-depths; and between replicates of 400 reads. The agreement between Sanger sequences and ONT sequences were assessed at nucleotide level, and at codon level for Stanford HIV drug resistance database mutations. Results: The coefficient of variation of ONT minority variants plateaued after a read depth of 400-fold implying limited benefit of additional depth and replicates of 400 reads showed a CV of ~6 % for a representative position. The average sequence similarity between ONT and Sanger sequences was 99.3% (95% CI: 99.1-99.4%) for PR-RT and 99.6% (95% CI: 99.4-99.7%) for INT. Drug resistance mutations did not differ for 21 IN sequences; 16 mutations were detected by both ONT- and Sanger sequencing. For the 46 PR and RT sequences, 245 mutations were detected by either ONT or Sanger, of these 238 (97.1%) were detected by both. Conclusions: The Nano-RECall pipeline, freely available as a downloadable application on a Windows computer, provides Sanger-equivalent HIV drug resistance interpretation. This novel pipeline combined with a simple workflow and multiplexing samples on ONT flow-cells would contribute to making HIV drug resistance sequencing feasible for resource limited settings.