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Distinct HIV-1 entry phenotypes are associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies.

Chikere K, Webb NE, Chou T, Borm K, Sterjovski J, Gorry PR, Lee B - Retrovirology (2014)

Bottom Line: First, we profiled a panel of reference subtype B transmitted/founder (T/F) and chronic Envs (n = 12) by analyzing the infectivity of each Env across 25 distinct combinations of CD4/CCR5 expression levels.Lastly, mutations known to confer resistance to VRC01 or PG6/PG19 BNAbs, when engineered into subtypes A-D Envs, resulted in significantly decreased CD4/CCR5 usage efficiency.GGR Affinofile profiling reveals pathophysiological phenotypes associated with varying HIV-1 entry efficiencies, and highlight the fitness costs associated with resistance to some broadly neutralizing antibodies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, Los Angeles, USA. benhur.lee@mssm.edu.

ABSTRACT

Background: The efficiency of CD4/CCR5 mediated HIV-1 entry has important implications for pathogenesis and transmission. The HIV-1 receptor affinity profiling (Affinofile) system analyzes and quantifies the infectivity of HIV-1 envelopes (Envs) across a spectrum of CD4/CCR5 expression levels and distills these data into a set of Affinofile metrics. The Affinofile system has shed light on how differential CD4/CCR5 usage efficiencies contributes to an array of Env phenotypes associated with cellular tropism, viral pathogenesis, and CCR5 inhibitor resistance. To facilitate more rapid, convenient, and robust analysis of HIV-1 entry phenotypes, we engineered a reporter Affinofile system containing a Tat- and Rev-dependent Gaussia luciferase-eGFP-Reporter (GGR) that is compatible with the use of pseudotyped or replication competent viruses with or without a virally encoded reporter gene. This GGR Affinofile system enabled a higher throughput characterization of CD4/CCR5 usage efficiencies associated with differential Env phenotypes.

Results: We first validated our GGR Affinofile system on isogenic JR-CSF Env mutants that differ in their affinity for CD4 and/or CCR5. We established that their GGR Affinofile metrics reflected their differential entry phenotypes on primary PBMCs and CD4+ T-cell subsets. We then applied GGR Affinofile profiling to reveal distinct entry phenotypes associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies (BNAbs). First, we profiled a panel of reference subtype B transmitted/founder (T/F) and chronic Envs (n = 12) by analyzing the infectivity of each Env across 25 distinct combinations of CD4/CCR5 expression levels. Affinofile metrics revealed that at low CCR5 levels, our panel of subtype B T/F Envs was more dependent on high levels of CD4 for HIV-1 entry compared to chronic Envs. Next, we analyzed a reference panel of 28 acute/early subtype A-D Envs, and noted that subtype C Envs could be distinguished from the other subtypes based on their infectivity profiles and relevant Affinofile metrics. Lastly, mutations known to confer resistance to VRC01 or PG6/PG19 BNAbs, when engineered into subtypes A-D Envs, resulted in significantly decreased CD4/CCR5 usage efficiency.

Conclusions: GGR Affinofile profiling reveals pathophysiological phenotypes associated with varying HIV-1 entry efficiencies, and highlight the fitness costs associated with resistance to some broadly neutralizing antibodies.

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Sensitivity vector metrics reflect biologically relevant differences in T cell subset tropism. (A) Total PBMCs were infected with luciferase reporter pseudotypes bearing wt, S142N, or K421D JR-CSF envelopes. VSV-G pseudotypes were used as positive controls. All infections (except for VSV-G) could be inhibited by maraviroc (>95%). Error bars represent ranges between two experiments. (B) Scheme for using CCR7 (PE-Cy7) and CD45RO (FITC) to identify the following T-cell subsets: Naïve (CCR7+ CD45RO-), Central Memory (TCM, CCR7+ CD45RO+), Effector Memory (TEM, CCR7- CD45RO+), and Effector Memory RA (TEMRA, CCR7- CD45RO-). (C) and (D) CD8-depleted PBMCs were infected with the indicated pseudotyped viruses at an MOI of 20 (as titered on Ghost-R5 cells). Three days post-infection, cells were analyzed by multi-color flow cytometry. (C) Infected cells were identified by intracellular p24 staining using PE-conjugated KC57 Mab. (D) Uninfected T-cell subset distribution is shown in grey density plot, while infected p24+ cells are overlaid as the red dots. The percent of total p24+ cells are indicated in each quadrant. All infections could be inhibited by maraviroc (>90%). Data shown here is a representative of two independent donors.
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Figure 4: Sensitivity vector metrics reflect biologically relevant differences in T cell subset tropism. (A) Total PBMCs were infected with luciferase reporter pseudotypes bearing wt, S142N, or K421D JR-CSF envelopes. VSV-G pseudotypes were used as positive controls. All infections (except for VSV-G) could be inhibited by maraviroc (>95%). Error bars represent ranges between two experiments. (B) Scheme for using CCR7 (PE-Cy7) and CD45RO (FITC) to identify the following T-cell subsets: Naïve (CCR7+ CD45RO-), Central Memory (TCM, CCR7+ CD45RO+), Effector Memory (TEM, CCR7- CD45RO+), and Effector Memory RA (TEMRA, CCR7- CD45RO-). (C) and (D) CD8-depleted PBMCs were infected with the indicated pseudotyped viruses at an MOI of 20 (as titered on Ghost-R5 cells). Three days post-infection, cells were analyzed by multi-color flow cytometry. (C) Infected cells were identified by intracellular p24 staining using PE-conjugated KC57 Mab. (D) Uninfected T-cell subset distribution is shown in grey density plot, while infected p24+ cells are overlaid as the red dots. The percent of total p24+ cells are indicated in each quadrant. All infections could be inhibited by maraviroc (>90%). Data shown here is a representative of two independent donors.

Mentions: To determine how these Affinofile metrics reflect the ability of a viral Env to infect primary CD4+ T-cells, we infected total PBMCs with pseudotyped luciferase reporter viruses bearing wt JR-CSF, S142N or the K421D Env mutants. Figure 4A shows that the S142N virus infected PBMCs better than wt JR-CSF while the K421D virus exhibited the lowest level of infection. This pattern reflected the θ and M metrics of the respective viruses, as the limiting parameter on primary CD4+ T-cells are the levels of CCR5 (low), not CD4 (high).


Distinct HIV-1 entry phenotypes are associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies.

Chikere K, Webb NE, Chou T, Borm K, Sterjovski J, Gorry PR, Lee B - Retrovirology (2014)

Sensitivity vector metrics reflect biologically relevant differences in T cell subset tropism. (A) Total PBMCs were infected with luciferase reporter pseudotypes bearing wt, S142N, or K421D JR-CSF envelopes. VSV-G pseudotypes were used as positive controls. All infections (except for VSV-G) could be inhibited by maraviroc (>95%). Error bars represent ranges between two experiments. (B) Scheme for using CCR7 (PE-Cy7) and CD45RO (FITC) to identify the following T-cell subsets: Naïve (CCR7+ CD45RO-), Central Memory (TCM, CCR7+ CD45RO+), Effector Memory (TEM, CCR7- CD45RO+), and Effector Memory RA (TEMRA, CCR7- CD45RO-). (C) and (D) CD8-depleted PBMCs were infected with the indicated pseudotyped viruses at an MOI of 20 (as titered on Ghost-R5 cells). Three days post-infection, cells were analyzed by multi-color flow cytometry. (C) Infected cells were identified by intracellular p24 staining using PE-conjugated KC57 Mab. (D) Uninfected T-cell subset distribution is shown in grey density plot, while infected p24+ cells are overlaid as the red dots. The percent of total p24+ cells are indicated in each quadrant. All infections could be inhibited by maraviroc (>90%). Data shown here is a representative of two independent donors.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230403&req=5

Figure 4: Sensitivity vector metrics reflect biologically relevant differences in T cell subset tropism. (A) Total PBMCs were infected with luciferase reporter pseudotypes bearing wt, S142N, or K421D JR-CSF envelopes. VSV-G pseudotypes were used as positive controls. All infections (except for VSV-G) could be inhibited by maraviroc (>95%). Error bars represent ranges between two experiments. (B) Scheme for using CCR7 (PE-Cy7) and CD45RO (FITC) to identify the following T-cell subsets: Naïve (CCR7+ CD45RO-), Central Memory (TCM, CCR7+ CD45RO+), Effector Memory (TEM, CCR7- CD45RO+), and Effector Memory RA (TEMRA, CCR7- CD45RO-). (C) and (D) CD8-depleted PBMCs were infected with the indicated pseudotyped viruses at an MOI of 20 (as titered on Ghost-R5 cells). Three days post-infection, cells were analyzed by multi-color flow cytometry. (C) Infected cells were identified by intracellular p24 staining using PE-conjugated KC57 Mab. (D) Uninfected T-cell subset distribution is shown in grey density plot, while infected p24+ cells are overlaid as the red dots. The percent of total p24+ cells are indicated in each quadrant. All infections could be inhibited by maraviroc (>90%). Data shown here is a representative of two independent donors.
Mentions: To determine how these Affinofile metrics reflect the ability of a viral Env to infect primary CD4+ T-cells, we infected total PBMCs with pseudotyped luciferase reporter viruses bearing wt JR-CSF, S142N or the K421D Env mutants. Figure 4A shows that the S142N virus infected PBMCs better than wt JR-CSF while the K421D virus exhibited the lowest level of infection. This pattern reflected the θ and M metrics of the respective viruses, as the limiting parameter on primary CD4+ T-cells are the levels of CCR5 (low), not CD4 (high).

Bottom Line: First, we profiled a panel of reference subtype B transmitted/founder (T/F) and chronic Envs (n = 12) by analyzing the infectivity of each Env across 25 distinct combinations of CD4/CCR5 expression levels.Lastly, mutations known to confer resistance to VRC01 or PG6/PG19 BNAbs, when engineered into subtypes A-D Envs, resulted in significantly decreased CD4/CCR5 usage efficiency.GGR Affinofile profiling reveals pathophysiological phenotypes associated with varying HIV-1 entry efficiencies, and highlight the fitness costs associated with resistance to some broadly neutralizing antibodies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, Los Angeles, USA. benhur.lee@mssm.edu.

ABSTRACT

Background: The efficiency of CD4/CCR5 mediated HIV-1 entry has important implications for pathogenesis and transmission. The HIV-1 receptor affinity profiling (Affinofile) system analyzes and quantifies the infectivity of HIV-1 envelopes (Envs) across a spectrum of CD4/CCR5 expression levels and distills these data into a set of Affinofile metrics. The Affinofile system has shed light on how differential CD4/CCR5 usage efficiencies contributes to an array of Env phenotypes associated with cellular tropism, viral pathogenesis, and CCR5 inhibitor resistance. To facilitate more rapid, convenient, and robust analysis of HIV-1 entry phenotypes, we engineered a reporter Affinofile system containing a Tat- and Rev-dependent Gaussia luciferase-eGFP-Reporter (GGR) that is compatible with the use of pseudotyped or replication competent viruses with or without a virally encoded reporter gene. This GGR Affinofile system enabled a higher throughput characterization of CD4/CCR5 usage efficiencies associated with differential Env phenotypes.

Results: We first validated our GGR Affinofile system on isogenic JR-CSF Env mutants that differ in their affinity for CD4 and/or CCR5. We established that their GGR Affinofile metrics reflected their differential entry phenotypes on primary PBMCs and CD4+ T-cell subsets. We then applied GGR Affinofile profiling to reveal distinct entry phenotypes associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies (BNAbs). First, we profiled a panel of reference subtype B transmitted/founder (T/F) and chronic Envs (n = 12) by analyzing the infectivity of each Env across 25 distinct combinations of CD4/CCR5 expression levels. Affinofile metrics revealed that at low CCR5 levels, our panel of subtype B T/F Envs was more dependent on high levels of CD4 for HIV-1 entry compared to chronic Envs. Next, we analyzed a reference panel of 28 acute/early subtype A-D Envs, and noted that subtype C Envs could be distinguished from the other subtypes based on their infectivity profiles and relevant Affinofile metrics. Lastly, mutations known to confer resistance to VRC01 or PG6/PG19 BNAbs, when engineered into subtypes A-D Envs, resulted in significantly decreased CD4/CCR5 usage efficiency.

Conclusions: GGR Affinofile profiling reveals pathophysiological phenotypes associated with varying HIV-1 entry efficiencies, and highlight the fitness costs associated with resistance to some broadly neutralizing antibodies.

Show MeSH
Related in: MedlinePlus