Figure 1: HIV-1 sequencing and linkage results for transmission pairs. 3 Month Seroconverters were not included as study endpoints, as seroconversion occurred prior to partners’ receipt of study drug. Red circles emanating from ‘Linked Gag’ boxes show the number of linkages gained by gag sequencing. Determination of HIV transmission linkage in the Partners in Prevention Study Mary S. Campbell 1, James I. Mullins 1,2, James P. Hughes 3, Kim G. Wong 2, Dana N. Raugi 2, Stefanie Sorensen 2, Julia N. Stoddard 2, Hong Zhao 2, Wenjie Deng 2, Francine E. McCutchan 6, Jan Albert 4, Thomas Leitner 5, Connie Celum 1,7, Jairam R. Lingappa 1,7,8 Departments of Medicine 1, Microbiology 2, Global Health 7, and Pediatrics 8, University of Washington School of Medicine, Seattle, WA, USA, 3 University of Washington School of Public Health and Community Medicine and the Statistical Center for HIV/AIDS Research and Prevention, Seattle, WA, USA, 4 Karolinska Institute and Swedish Institute for Infectious Disease Control, Solna, Sweden, 5 Los Alamos National Laboratory, Los Alamos, NM, USA, 6 Bill & Melinda Gates Foundation, Seattle, WA, USA IAS 2009-LBPEC07 UNIVERSITY OF WASHINGTON INTERNATIONAL CLINICAL RESEARCH CENTER Background - Partners in Prevention Study: Randomized trial to evaluate genital herpes suppression with acyclovir in HIV-1/HSV-2 infected participants to prevent HIV- 1 transmission to their partners 1, 2 - Primary study endpoint: HIV-1 seroconversion (SC), in which the HIV-1 strain acquired by the seroconverting partner was genetically linked to his/her enrolled partner Methods - HIV-1 viral RNA extracted from plasma of index and seroconverter collected at or within 3 months of SC - cDNA synthesized and PCR amplified partial env (C2-C3-V3) and gag genes (p17/p24) - Consensus followed by single molecule (SM) dideoxy terminator sequencing or pyrosequencing, if viruses not found to be linked by consensus sequencing - Sequence quality control, viral subtyping, alignment, maximum likelihood phylogenetic tree constructed ( bin/DIVER/diver.cgi) including unrelated sequences from each study site - Reference datasets within infected individuals, between transmission pairs (mother-infant, heterosexual, MSM), between epidemiologically unlinked sequences of each subtype - Linkage assessed through monophyly, genetic distance, Bayesian posterior probability - Data adjudicated by 3 independent experts Results 155 transmission pairs evaluated (Figure 1) - Linked 108 (69.7%) - Unlinked 40 (25.8%) - Indeterminate 7 (4.5%) - Linkage by consensus env in 91 (84.3%), gag in 9 (8.3%), SM env in 8 (7.4%) Figure 2 highlights the pairs linked in gag for the subtype C/CRF10 tree. Figure 4 shows examples of linked, unlinked and indeterminate pairs. Viral Subtype - Env A 43%, C 43%, D 13% 2% intersubtype recombinants, G <1% - Gag A 44%, C 36%, D 10%, 10% intersubtype recombinants - Both genes sequenced in 128 pairs- 11.7% had discordant subtype by gene Local Control Sequences - Local controls 21.2/site (range 10-96); none split monophyly Pairwise genetic distances - Linked env 3.67% (range %), gag 1.67% (range %) - Unlinked env 18.83% (range %), gag 11.51% (range %) (Figure 3) Mean Bayesian posterior probabilities - Linked env 92.97% and gag 95.91% - Unlinked env 1.21% and gag 0.00% Single molecule env sequencing - Performed for 43 pairs, mean number of sequences 24 (range 3-62) - Linkage found in 8 (18.6%) - Linked variants accounted for 25-50% of total molecules (Figure 4B) - Amplicon pyrosequencing (Pyro) in 12 pairs, but none met criteria for linkage Adjudicator linkage determinations - 3/3 agreement initially in 96.1% of pairs - 100% agreement at end of study Transmission pairs with indeterminate linkage status - 4 pairs did not have amplifiable virus by PCR - 2 had discordant subtypes in index and seroconverter in which minority virus may have been transmitted - 1 with monophyly but large distance in env, absent linkage in gag and more closely related variants after single template sequencing of 17 env molecules Discordant env/gag results - 3 pairs linked in 1 gene but unlinked in other - 15 pairs (11.7%) with 2 genes sequenced had different subtypes in env/gag -14 linked with similar subtype pattern in both partners -1 unlinked with different mosaic subtype in each partner Figure 4: (A) shows a phylogenetic tree of consensus env sequences from linked pairs PP73 and PP82 (red circles) and an unlinked pair PP45 (black circles connected with dotted line). (B) shows sequences from a pair that was unlinked by consensus env and gag sequencing, but found to have 3 closely linked sequences during single molecule env sequencing (PP17A variant 1 and PP17B in red box). The table summarizes data and linkage decisions for pairs in (A) and (B). A B Figure 3: Distribution of pairwise genetic distances in env for reference datasets (black and grey curves) superimposed on histogram of pairwise genetic distances for linked (red bars) and unlinked (blue bars) study pairs. Curves for MACS (Multicenter AIDS Cohort Study) represent intrasubject sequence pairs. Conclusions - Our study represents the first use of viral linkage determination in an HIV-1 prevention trial. - We developed a Bayesian algorithm to account for the prior probability that enrolled partners’ HIV-1 strains were linked, in the context of genetic distances between their viral sequences and those from reference datasets, to calculate posterior probabilities of linkage. - The majority of linked transmission pairs were determined by consensus env. - Unexpectedly, more than one quarter of transmission pairs were unlinked. - A hierarchy of sequencing methods, starting with consensus env and gag, followed by env SM and pyrosequencing contributed to the efficiency of the linkage determination process. - Evaluation by independent adjudicators ensured accurate linkage assignment. - Our methods are applicable to future studies requiring assessment of HIV-1 genetic linkage Figure 2: Phylogenetic tree for subtype C and CRF10 (C/D) gag including study pairs and reference sequences. 36 linked study pairs are highlighted in red. References 1. Twice-daily acyclovir to reduce HIV-1 transmission from HIV-1 / HSV-2 co-infected persons within HIV-1 serodiscordant couples: a randomized, double-blind, placebo-controlled trial. C. Celum et al. IAS 2009, WELBC Daily acyclovir delays HIV-1 disease progression among HIV-1/HSV-2 dually-infected persons: a randomized trial. J. Lingappa et al. IAS 2009, WELBC102. PP45PP73PP82PP17 variant 1PP17 variant 2 MonophylyNoYes Distance Posterior Probability DecisionUnlinkedLinked Indeterminate