Possible clearance of transfusion-acquired nef/LTR-deleted attenuated HIV-1 infection by an elite controller with CCR5 Δ32 heterozygous and HLA-B57 genotype

Abstract

Background: Subject C135 is one of the members of the Sydney Blood Bank Cohort, infected in 1981 through transfusion with attenuated nef/3' long terminal repeat (LTR)-deleted HIV-1, and has maintained undetectable plasma viral load and steady CD4 cell count, in the absence of therapy. Uniquely, C135 combines five factors separately associated with control of viraemia: nef/LTR-deleted HIV-1, HLA-B57, HLA-DR13, heterozygous CCR5 Δ32 genotype and vigorous p24-stimulated peripheral blood mononuclear cell (PBMC) proliferation. Therefore, we studied in detail viral burden and immunological responses in this individual.

Methods: PBMC and gut and lymph node biopsy samples were analysed for proviral HIV-1 DNA by real-time and nested PCRs, and nef/LTR alleles by nested PCR. HIV-specific antibodies were studied by Western blotting, and CD4+ and CD8+ T lymphocyte responses were measured by proliferation and cytokine production in vitro.

Results: PBMC samples from 1996, but not since, showed amplification of nef alleles with gross deletions. Infectious HIV-1 was never recovered. Proviral HIV-1 DNA was not detected in recent PBMC or gut or lymph node biopsy samples. C135 has a consistently weak antibody response and a substantial CD4+ T cell proliferative response to a previously described HLA-DR13-restricted epitope of HIV-1 p24 in vitro, which augmented a CD8+ T cell response to an immunodominant HLA-B57-restricted epitope of p24, while his T cells show reduced levels of CCR5.

Conclusions: Subject C135's early PCR and weak antibody results are consistent with limited infection with a poorly replicating nef/LTR-deleted strain of HIV-1. With his HLA-B57-restricted gag-specific CD8 and helper HLA-DR13-restricted CD4 T cell proliferative responses, C135 appears to have cleared his HIV-1 infection 37 years after transfusion.

Keywords: CCR5; CD4; HIV-1; Nef.

Figure 1.

Timeline of studies, HIV-1 serology and CD4 T cell counts for subject C135. (a) Timeline of transfusion, testing, sampling and studies for C135. See text for details. (b) Western blot analysis of longitudinal serum samples from C135, consistently showing faint bands at p18, p24 and gp160, for the following dates: 25/9/96 (lane 1), 14/4/97 (lane 2), 14/7/98 (lane 3), 23/8/99 (lane 4), 17/10/00 (lane 5), 23/8/01 (lane 6), 1/10/02 (lane 7), 23/9/03 (lane 8), 4/11/04 (lane 9), 29/6/07 (lane 10), kit negative control (lane 11), and kit positive control (lane 12). Note that the bands on the original blots are faint to the naked eye but easily visible. The level of brightness of the original digital photographic image has been reduced by 25% to make bands easier to visualise for this report, but makes the bands on the positive control (lane 12) appear less distinct than those on the original blots. (c) Longitudinal CD4 T cell counts (solid line) and CD4 : CD8 ratios (dashed line) for C135 from 1996 to 2017. Trend lines (linear regression) are shown for each data series. CTL: cytotoxic T lymphocyte; ELISpot: enzyme-linked absorbent immunospot; PBMC: peripheral blood mononuclear cell

Figure 2.

CD4 and CD8 T cell responses to HIV-1 Gag antigens for subject C135. (a) Stimulation indices for C135's PBMC proliferation in response to HIV p24, for longitudinal samples from 1996 to 2006, using the ³H-thymidine uptake assay. (b) Flow cytometric analysis showing proliferating CD4+ T lymphocytes in CFSE-labelled PBMC from C135, in response to culture with HIV-1 Gag peptide pool (middle histogram) or with peptide #63, WMTNNPPIPVGEIYK (right histogram), compared with culture in the absence of antigen (left histogram). (c) Flow cytometric analysis showing proliferation of CD8+ T lymphocyte in CFSE-labelled PBMC from C135, in response to culture with the CD8 Gag peptide #60 (AGTTSTLQEQIGWMT) in the presence (right histogram) and absence (left histogram) of the CD4 peptide #63 (WMTNNPPIPVGEIYK). (d) Flow cytometric analysis showing proliferation of CD8+ T lymphocyte in CFSE-labelled PBMC from C135, in response to culture with the CD8 Gag peptides #40 (KVVEEKAFSPEVIPM) and #41 (EKAFSPEVIPMFSAL) in the presence (right histogram) and absence (left histogram) of the CD4 peptide #63 (WMTNNPPIPVGEIYK). CFSE: carboxyfluorescein diacetate succinimidyl ester; PBMC: peripheral blood mononuclear cell

Figure 2.

CD4 and CD8 T cell responses to HIV-1 Gag antigens for subject C135. (a) Stimulation indices for C135's PBMC proliferation in response to HIV p24, for longitudinal samples from 1996 to 2006, using the ³H-thymidine uptake assay. (b) Flow cytometric analysis showing proliferating CD4+ T lymphocytes in CFSE-labelled PBMC from C135, in response to culture with HIV-1 Gag peptide pool (middle histogram) or with peptide #63, WMTNNPPIPVGEIYK (right histogram), compared with culture in the absence of antigen (left histogram). (c) Flow cytometric analysis showing proliferation of CD8+ T lymphocyte in CFSE-labelled PBMC from C135, in response to culture with the CD8 Gag peptide #60 (AGTTSTLQEQIGWMT) in the presence (right histogram) and absence (left histogram) of the CD4 peptide #63 (WMTNNPPIPVGEIYK). (d) Flow cytometric analysis showing proliferation of CD8+ T lymphocyte in CFSE-labelled PBMC from C135, in response to culture with the CD8 Gag peptides #40 (KVVEEKAFSPEVIPM) and #41 (EKAFSPEVIPMFSAL) in the presence (right histogram) and absence (left histogram) of the CD4 peptide #63 (WMTNNPPIPVGEIYK). CFSE: carboxyfluorescein diacetate succinimidyl ester; PBMC: peripheral blood mononuclear cell

Figure 3.

Intracellular cytokine assays. Antigen-specific CD4+ T cells by intracellular cytokine assay for subject C135. Intracellular flow cytometric analysis of the CD4+ T cell cytokine response in fresh whole blood from C135, cultured with a pool of overlapping HIV-1 Gag peptides, showing the production of IFN-γ and IL-2, the lack of production of the inhibitory receptor CTLA-4, in combination with the expression of the IL-7Rα chain, CD127, a marker of long-term memory T cells. Background production of IFN-γ and IL-2 in the absence of HIV-1 Gag peptides is shown in the upper left histogram. IFN: interferon; IL: interleukin

Figure 4.

Relatively low level of CCR5 expression on CD4 and CD8 T lymphocytes from subject C135. (a) Flow cytometric histogram of CCR5 expression on CD4+ T lymphocytes from C135 (solid black line) and three healthy adult controls (dashed histograms), respectively. (b) Flow cytometric histogram of CCR5 expression on CD8+ T lymphocytes from C135 (solid black line) and three healthy adult controls (dashed histograms), respectively