Genetically-barcoded SIV facilitates enumeration of rebound variants and estimation of reactivation rates in nonhuman primates following interruption of suppressive antiretroviral therapy

Christine M. Fennessey, Mykola Pinkevych, Taina T. Immonen, Arnold Reynaldi, Vanessa Venturi, Priyanka Nadella, Carolyn Reid, Laura Newman, Leslie Lipkey, Kelli Oswald, William J. Bosche, Matthew T. Trivett, David E. Ott, Jacob Estes, Gregory Q. Del Prete, Jeffrey D. Lifson, Miles P. Davenport, Brandon F. Keele

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

HIV and SIV infection dynamics are commonly investigated by measuring plasma viral loads. However, this total viral load value represents the sum of many individual infection events, which are difficult to independently track using conventional sequencing approaches. To overcome this challenge, we generated a genetically tagged virus stock (SIVmac239M) with a 34-base genetic barcode inserted between the vpx and vpr accessory genes of the infectious molecular clone SIVmac239. Next-generation sequencing of the virus stock identified at least 9,336 individual barcodes, or clonotypes, with an average genetic distance of 7 bases between any two barcodes. In vitro infection of rhesus CD4+ T cells and in vivo infection of rhesus macaques revealed levels of viral replication of SIVmac239M comparable to parental SIVmac239. After intravenous inoculation of 2.2x105infectious units of SIVmac239M, an average of 1,247 barcodes were identified during acute infection in 26 infected rhesus macaques. Of the barcodes identified in the stock, at least 85.6% actively replicated in at least one animal, and on average each barcode was found in 5 monkeys. Four infected animals were treated with combination antiretroviral therapy (cART) for 82 days starting on day 6 post-infection (study 1). Plasma viremia was reduced from >106to <15 vRNA copies/mL by the time treatment was interrupted. Virus rapidly rebounded following treatment interruption and between 87 and 136 distinct clonotypes were detected in plasma at peak rebound viremia. This study confirmed that SIVmac239M viremia could be successfully curtailed with cART, and that upon cART discontinuation, rebounding viral variants could be identified and quantified. An additional 6 animals infected with SIVmac239M were treated with cART beginning on day 4 post-infection for 305, 374, or 482 days (study 2). Upon treatment interruption, between 4 and 8 distinct viral clonotypes were detected in each animal at peak rebound viremia. The relative proportions of the rebounding viral clonotypes, spanning a range of 5 logs, were largely preserved over time for each animal. The viral growth rate during recrudescence and the relative abundance of each rebounding clonotype were used to estimate the average frequency of reactivation per animal. Using these parameters, reactivation frequencies were calculated and ranged from 0.33–0.70 events per day, likely representing reactivation from long-lived latently infected cells. The use of SIVmac239M therefore provides a powerful tool to investigate SIV latency and the frequency of viral reactivation after treatment interruption.

Original languageEnglish (US)
Article numbere1006359
JournalPLoS Pathogens
Volume13
Issue number5
DOIs
StatePublished - May 1 2017
Externally publishedYes

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Primates
Viremia
Infection
Viruses
Macaca mulatta
Viral Load
Therapeutics
Virus Latency
vpr Genes
HIV Infections
Haplorhini
Clone Cells
T-Lymphocytes
Recurrence
Growth

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Molecular Biology
  • Genetics
  • Virology

Cite this

Genetically-barcoded SIV facilitates enumeration of rebound variants and estimation of reactivation rates in nonhuman primates following interruption of suppressive antiretroviral therapy. / Fennessey, Christine M.; Pinkevych, Mykola; Immonen, Taina T.; Reynaldi, Arnold; Venturi, Vanessa; Nadella, Priyanka; Reid, Carolyn; Newman, Laura; Lipkey, Leslie; Oswald, Kelli; Bosche, William J.; Trivett, Matthew T.; Ott, David E.; Estes, Jacob; Del Prete, Gregory Q.; Lifson, Jeffrey D.; Davenport, Miles P.; Keele, Brandon F.

In: PLoS Pathogens, Vol. 13, No. 5, e1006359, 01.05.2017.

Research output: Contribution to journalArticle

Fennessey, CM, Pinkevych, M, Immonen, TT, Reynaldi, A, Venturi, V, Nadella, P, Reid, C, Newman, L, Lipkey, L, Oswald, K, Bosche, WJ, Trivett, MT, Ott, DE, Estes, J, Del Prete, GQ, Lifson, JD, Davenport, MP & Keele, BF 2017, 'Genetically-barcoded SIV facilitates enumeration of rebound variants and estimation of reactivation rates in nonhuman primates following interruption of suppressive antiretroviral therapy', PLoS Pathogens, vol. 13, no. 5, e1006359. https://doi.org/10.1371/journal.ppat.1006359
Fennessey, Christine M. ; Pinkevych, Mykola ; Immonen, Taina T. ; Reynaldi, Arnold ; Venturi, Vanessa ; Nadella, Priyanka ; Reid, Carolyn ; Newman, Laura ; Lipkey, Leslie ; Oswald, Kelli ; Bosche, William J. ; Trivett, Matthew T. ; Ott, David E. ; Estes, Jacob ; Del Prete, Gregory Q. ; Lifson, Jeffrey D. ; Davenport, Miles P. ; Keele, Brandon F. / Genetically-barcoded SIV facilitates enumeration of rebound variants and estimation of reactivation rates in nonhuman primates following interruption of suppressive antiretroviral therapy. In: PLoS Pathogens. 2017 ; Vol. 13, No. 5.
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abstract = "HIV and SIV infection dynamics are commonly investigated by measuring plasma viral loads. However, this total viral load value represents the sum of many individual infection events, which are difficult to independently track using conventional sequencing approaches. To overcome this challenge, we generated a genetically tagged virus stock (SIVmac239M) with a 34-base genetic barcode inserted between the vpx and vpr accessory genes of the infectious molecular clone SIVmac239. Next-generation sequencing of the virus stock identified at least 9,336 individual barcodes, or clonotypes, with an average genetic distance of 7 bases between any two barcodes. In vitro infection of rhesus CD4+ T cells and in vivo infection of rhesus macaques revealed levels of viral replication of SIVmac239M comparable to parental SIVmac239. After intravenous inoculation of 2.2x105infectious units of SIVmac239M, an average of 1,247 barcodes were identified during acute infection in 26 infected rhesus macaques. Of the barcodes identified in the stock, at least 85.6{\%} actively replicated in at least one animal, and on average each barcode was found in 5 monkeys. Four infected animals were treated with combination antiretroviral therapy (cART) for 82 days starting on day 6 post-infection (study 1). Plasma viremia was reduced from >106to <15 vRNA copies/mL by the time treatment was interrupted. Virus rapidly rebounded following treatment interruption and between 87 and 136 distinct clonotypes were detected in plasma at peak rebound viremia. This study confirmed that SIVmac239M viremia could be successfully curtailed with cART, and that upon cART discontinuation, rebounding viral variants could be identified and quantified. An additional 6 animals infected with SIVmac239M were treated with cART beginning on day 4 post-infection for 305, 374, or 482 days (study 2). Upon treatment interruption, between 4 and 8 distinct viral clonotypes were detected in each animal at peak rebound viremia. The relative proportions of the rebounding viral clonotypes, spanning a range of 5 logs, were largely preserved over time for each animal. The viral growth rate during recrudescence and the relative abundance of each rebounding clonotype were used to estimate the average frequency of reactivation per animal. Using these parameters, reactivation frequencies were calculated and ranged from 0.33–0.70 events per day, likely representing reactivation from long-lived latently infected cells. The use of SIVmac239M therefore provides a powerful tool to investigate SIV latency and the frequency of viral reactivation after treatment interruption.",
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AU - Fennessey, Christine M.

AU - Pinkevych, Mykola

AU - Immonen, Taina T.

AU - Reynaldi, Arnold

AU - Venturi, Vanessa

AU - Nadella, Priyanka

AU - Reid, Carolyn

AU - Newman, Laura

AU - Lipkey, Leslie

AU - Oswald, Kelli

AU - Bosche, William J.

AU - Trivett, Matthew T.

AU - Ott, David E.

AU - Estes, Jacob

AU - Del Prete, Gregory Q.

AU - Lifson, Jeffrey D.

AU - Davenport, Miles P.

AU - Keele, Brandon F.

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N2 - HIV and SIV infection dynamics are commonly investigated by measuring plasma viral loads. However, this total viral load value represents the sum of many individual infection events, which are difficult to independently track using conventional sequencing approaches. To overcome this challenge, we generated a genetically tagged virus stock (SIVmac239M) with a 34-base genetic barcode inserted between the vpx and vpr accessory genes of the infectious molecular clone SIVmac239. Next-generation sequencing of the virus stock identified at least 9,336 individual barcodes, or clonotypes, with an average genetic distance of 7 bases between any two barcodes. In vitro infection of rhesus CD4+ T cells and in vivo infection of rhesus macaques revealed levels of viral replication of SIVmac239M comparable to parental SIVmac239. After intravenous inoculation of 2.2x105infectious units of SIVmac239M, an average of 1,247 barcodes were identified during acute infection in 26 infected rhesus macaques. Of the barcodes identified in the stock, at least 85.6% actively replicated in at least one animal, and on average each barcode was found in 5 monkeys. Four infected animals were treated with combination antiretroviral therapy (cART) for 82 days starting on day 6 post-infection (study 1). Plasma viremia was reduced from >106to <15 vRNA copies/mL by the time treatment was interrupted. Virus rapidly rebounded following treatment interruption and between 87 and 136 distinct clonotypes were detected in plasma at peak rebound viremia. This study confirmed that SIVmac239M viremia could be successfully curtailed with cART, and that upon cART discontinuation, rebounding viral variants could be identified and quantified. An additional 6 animals infected with SIVmac239M were treated with cART beginning on day 4 post-infection for 305, 374, or 482 days (study 2). Upon treatment interruption, between 4 and 8 distinct viral clonotypes were detected in each animal at peak rebound viremia. The relative proportions of the rebounding viral clonotypes, spanning a range of 5 logs, were largely preserved over time for each animal. The viral growth rate during recrudescence and the relative abundance of each rebounding clonotype were used to estimate the average frequency of reactivation per animal. Using these parameters, reactivation frequencies were calculated and ranged from 0.33–0.70 events per day, likely representing reactivation from long-lived latently infected cells. The use of SIVmac239M therefore provides a powerful tool to investigate SIV latency and the frequency of viral reactivation after treatment interruption.

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