Entry Inhibitor/Tropism Testing

Monogram has developed and provides both genotypic and phenotypic assays for assessing resistance to drugs that target the three main steps of the HIV viral entry process. We provide PhenoSense® Entry and GeneSeq® Entry to assess viral susceptibility to attachment and fusion inhibitors, and we have Trofile® technology to assess coreceptor tropism for coreceptor antagonists. We also provide clonal analysis and sequencing for in depth assessment.

Available tests and services  include:

  • PhenoSense Entry
  • GeneSeq Entry
  • Trofile
  • Trofile DNA
  • Clonal analysis
  • Next-generation sequencing

Schematic Representation for HIV Entry Into Its Host Cell


Attachment: The gp120 viral envelope protein has an affinity for the CD4 receptor, which is expressed on the cell surface of the CD4+ cell. CD4 binding inhibitors, such as the experimental drug fostemsavir, have been shown to inhibit the gp120 and CD4 receptor interaction and block HIV infection.

Coreceptor Binding

Coreceptor binding: The binding of the gp120 to the CD4 receptor causes a conformational change in the gp120 protein. This change exposes a binding site for a chemokine coreceptor (CCR5 or CXCR4). CCR5 antagonists block the binding of the gp120 protein and block HIV infection.

Virus Cell Function

Virus-cell fusion: Once gp120 binds to the coreceptor, this causes a conformational change in the gp41 protein allowing the insertion of a fusion peptide from the gp41 protein into the cell membrane. This insertion of the fusion peptide draws the virus and CD4+ cell close enough together to allow the virus envelope and cell membrane to fuse. Once this happens, the viral core can enter the CD4+ cell, and the infection begins. Enfuvirtide inhibits the conformational change and blocks the insertion of the fusion peptide.

Clonal Analysis

In Monogram Biosciences’ PhenoSense HIV and HCV assays, patient-derived sequences are tested as pools or libraries of sequences that capture the diversity of the viral quasi-species within a patient sample. In some instances, it may be beneficial to characterize these quasi-species to determine minor populations or changes in the viral population in the patient over time (longitudinal samples). This can be achieved using Monogram’s clonal testing assay. Patient pools generated in Monogram Biosciences’ PhenoSense HIV assays are used for selecting and growing individual clones (viruses). Typically, 48 or 96 clones (viruses) are prepared per patient sample, and they undergo an infectivity screen to determine clone viability. From these results, the client may select the clones (usually 12-16) that Monogram will use for subsequent testing, such as genotyping, drug susceptibility, and/or tropism.

Entry Susceptibility

Monogram has applied its PhenoSense technology to develop a phenotypic assay for the evaluation of entry inhibitor drug susceptibility. The assay has been widely used in clinical trials, including the Phase 3 studies of enfuvirtide (FUZEON®), and viral susceptibility to enfuvirtide is commercially available in Monogram’s CLIA-certified laboratory to help guide patient therapy. PhenoSense HIV Entry is complemented by Monogram’s envelope sequencing technology, GeneSeq® HIV Entry.

The successful development of new drugs in the entry inhibitor class requires unique drug resistance assay technology to assess the susceptibility of HIV, particularly multidrug-resistant HIV, to these new agents.

FUZEON is a registered trademark of Hoffmann-La Roche, Inc.

Envelope Sequencing

GeneSeq® for Entry Inhibitors (HIV Envelope Sequencing) Assay

In addition to the PhenoSense assay for entry inhibitor susceptibility, Monogram has the capability to sequence the HIV envelope (gp160) for clients. This includes V3 loop, gp41, gp120, or the entire gp160 sequence. The sequence data are provided in customized formats for convenient downloading to clinical databases. Monogram can perform envelope sequencing on virus pools or individual clones. Monogram is investigating the relationships between envelope sequence and phenotypic results and works closely with clients and collaborators to better understand the virus’ susceptibility and tropism.

Tropism Testing

What is tropism?

Coreceptor tropism is defined as the ability of a particular HIV-1 virus to infect a target cell using a specific coreceptor. HIV requires two binding events to enter into a cell. It must first bind CD4 and then, secondly, a chemokine receptor. Tropism is a label given to the virus that describes which chemokine receptor the virus is using.

The HIV-1 viruses can be characterized into four broad classifications based on their tropism status. 

RS-tripic R5-tropic: Viruses or virus populations that can use only the CCR5 chemokine coreceptor to infect CD4+ cells. X4-tropic: X4-tropic: Viruses or virus populations that can use only the CXCR4 chemokine coreceptor to infect CD4+ cells.
Dual (D)-tropicDual (D)-tropic: Viruses or virus populations that can use either the CCR5 or CXCR4 coreceptors to infect CD4+ cells. Mixed (M)-tropic: Virus populations that may contain various combinations of R5 virus, X4 virus, and/or dual-tropic viruses.

Trofile® Assays Determine Viral Tropism

Monogram currently offers two tests for tropism determination for both suppressed and nonsuppressed patients:


For patients with detectable viral loads ≥ 1000 copies/mL

trofile dna

For patients with undetectable viral loads

More than 23,000 samples have been tested using Monogram’s Trofile assays. All trials of coreceptor antagonists have used Trofile in the clinical development. Trofile has been shown to be accurate, precise, sensitive, reproducible and robust in the measurement of HIV-1 coreceptor tropism.

The current US Department of Health and Human Services (DHHS) and the Infectious Diseases Society of America (IDSA) guidelines recommend that a coreceptor tropism test should be performed whenever the use of a CCR5 inhibitor is being considered.1,2 Coreceptor tropism testing might also be considered for patients who exhibit virologic failure on a CCR5 inhibitor.1


  1. Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. Department of Health and Human Services. Available at https://aidsinfo.nih.gov/contentfiles/lvguidelines/adultandadolescentgl.pdf. Accessed June 8, 2015. Table 3.
  2. Aberg JA, Gallant JE, Ghanem KG, Emmanuel P, Zingman BS, Horberg MA. Primary care guidelines for the management of persons infected with HIV: 2013 update by the HIV Medicine Association of the Infectious Diseases Society of America. Clin Infect Dis. 2014;58(1):e1–34.