Increased Frequency of Inter-Subtype HIV-1 Recombinants Identified by Near Full-Length Virus Sequencing in Rwandan Acute Transmission Cohorts

Abstract

Most studies of HIV-1 transmission have focused on subtypes B and C. In this study, we determined the genomic sequences of the transmitted founder (TF) viruses from acutely infected individuals enrolled between 2005 and 2011 into IAVI protocol C in Rwanda and have compared these isolates to viruses from more recent (2016–2019) acute/early infections in three at risk populations – MSM, high risk women (HRW), and discordant couples (DC). For the Protocol C samples, we utilized near full-length single genome (NFLG) amplification to generate 288 HIV-1 amplicons from 26 acutely infected seroconverters (SC), while for the 21 recent seroconverter samples (13 from HRW, two from DC, and six from MSM), we PCR amplified overlapping half-genomes. Using PacBio SMRT technology combined with the MDPseq workflow, we performed multiplex sequencing to obtain high accuracy sequences for each amplicon. Phylogenetic analyses indicated that the majority of recent transmitted viruses from DC and HRW clustered within those of the earlier Protocol C cohort. However, five of six sequences from the MSM cohort branched together and were greater than 97% identical. Recombination analyses revealed a high frequency (6/26; 23%) of unique inter-subtype recombination in Protocol C with 19% AC and 4% CD recombinant viruses, which contrasted with only 6.5% of recombinants defined by sequencing of the pol gene previously. The frequency of recombinants was significantly higher (12/21; 57%) in the more recent isolates, although, the five related viruses from the MSM cohort had identical recombination break points. While major drug resistance mutations were absent from Protocol C viruses, 4/21 of recent isolates exhibited transmitted nevirapine resistance. These results demonstrate the ongoing evolution and increased prevalence of recombinant and drug resistant transmitted viruses in Rwanda and highlight the importance of defining NFLG sequences to fully understand the nature of TF viruses and in particular the prevalence of unique recombinant forms (URFs) in transmission cohorts.

Assessment of a diverse panel of transmitted/founder HIV-1 infectious molecular clones in a luciferase based CD8 T-cell mediated viral inhibition assay

“…In most subjects, acquisition of HIV-1 appears to be due to transmission of a single virus or transmitted/founder (T/F) virus (37,38). The majority of IMC were TFV isolates derived from subjects with recent HIV-1 acquisition (45)(46)(47)63) with such isolates being those that vaccine elicited immune responses would potentially need to target in order to prevent HIV-1 transmission or limit subsequent HIV-1 replication. The ability of CD8 T-cells from ART naive subjects living with HIV-1 to inhibit these IMC was assessed, with subjects selected with broad coverage of HLA types present with the large protocol C cohort.…”

“…For plasma viruses from the remaining acutely infected individuals from Protocol C, near full-length single genome amplicons (NFLSGA) were sequenced within approximated 30 days from infection using PacBio ® single molecule, long-read sequencing (Pacific Biosciences, Menlo Park, CA, USA) and the Multilayer Directed Phasing and Sequencing (MDPSeq) algorithm ( 54 ). Transmitted/founder (T/F) viral sequences were defined in Geneious v9.1.8 (Biomatters, Aukland, NZ) as described previously ( 46 , 47 , 55 ). Two methods of IMC cloning were utilised; for the majority, near full-length amplicons corresponding to the T/F virus sequence and derived using the Q5 ® High-Fidelity DNA Polymerase (New England Biolabs, MA, USA) were cloned directly at Emory University, Atlanta, USA as described previously ( 55 , 56 ).…”

“…The approach for characterising these panels drew inspiration from the strategy employed in developing HIV pseudovirus panels for identifying and characterising broadly neutralising HIV-1 antibodies (26,27,29,(40)(41)(42)(43)(44). IMC-LucR representing HIV-1 T/F viruses derived from subjects with early acquisition of HIV-1 (Protocol C) (45)(46)(47) were assessed within the IMC-LucR VIA for their ability to infect and replicate within PBMCs derived from both HIV-1 uninfected volunteers and from ART naive subjects living with HIV, selected for their broad coverage of human leukocyte antigen (HLA) haplotypes present with the population and identified as having an ability for virus control (28,45,48).…”