Mechanisms of HIV Non-progression; Robust and Sustained CD4+ T-cell Proliferative Responses to P24 Antigen Correlate With Control of Viraemia and Lack of Disease Progression After Long-term Transfusion-acquired HIV-1 Infection

Wayne B. Dyer; John J. Zaunders; Fang Fang Yuan; Bin Wang; Jennifer C. Learmont; Andrew F. Geczy; Nitin K. Saksena; Dale A. McPhee; Paul R. Gorry; John S. Sullivan

Disclosures

Retrovirology 

In This Article

Results

Status of the Non-progressor Cohort

From all reported TAHIV cases from the state of NSW, Australia, a cohort of 13 (10%) remained asymptomatic after 10 years of infection. We now report that only 5 remain non-progressors after 23 to 26 years of HIV-1 infection. Infection and treatment history for each subject is summarised in Additional file 1. Most of these individuals had a survival advantage, with 7 of 13 having at least one host genetic polymorphism associated with slow progression, and 6 of 13 were infected with the SBBC nef-defective HIV-1 strain,[12] and combined, 12 of 13 had at least one host or viral factor favouring slow progression. Acting in opposition to these survival advantages, 5 of 8 former non-progressors had the FcγRIIA polymorphism (R/R). While this genotype was absent in current LTNP, the effect of the R/R genotype in promoting disease progression was not significant in this small study of 13 individuals. On balance, these competing survival factors along with antiviral immune responses enabled a non-progressive disease course to be established early in infection.

The loss of non-progressor status was based on increasing viraemia and/or decreasing CD4 counts in 5 of 8, and initiation of ART in these individuals (Additional file 1). Patient C122 lost LTNP status due to gradually increasing viraemia, but died from unrelated causes before substantial T cell loss was observed. Another two elderly individuals (C18 and C54; both SBBC members), each with low detectable viraemia, died before losing their non-progressor status.[13,17]

Antiviral Immune Responses Associated With Non-progression

Host and viral genetic factors may have played a role in delaying disease progression into the second decade of infection in these 13 individuals, but this study also demonstrates the importance of host immune responses in sustaining this non-progressive disease course into and beyond the second decade of infection. Immune status and activity of HIV-specific CD4 T cells (proliferation) and CD8 T cells (IFN-γ response) is shown for the current non-progressors (Figure 1) compared with those that lost their non-progressor status or died (Figure 2).

Figure 1.

Immunovirological status of the surviving non-progressors, showing T cell counts; viral RNA copies/ml plasma (data generated from the Roche Amplicore standard assay, limit of detection 400, and Ultrasensitive assay, limit of detection 50, plotted separately); T cell proliferative responses to recombinant HIV-1 p24 (stimulation index; significant responses > 3, defined by the broken line); and IFNγ responses (ELISPOT) by CTL against autologous BCL expressing HIV-1 antigens after infection with recombinant vaccinia. *SBBC member.

Figure 2.

Immunovirological status of the former non-progressors (same parameters as in figure 1). Initiation of antiretroviral therapy is defined by an arrow in the viral load panels. Other reasons for loss of non-progressor status are summarised in Additional file 1.

Antiviral CTL responses were variable during the second decade of HIV infection, and did not always correlate with viremia for members of these cohorts. Strong Gag-specific CTL were detected in the Cohort 2 non-progressors (C13, C53, C122, and C105 before ART), but the predominant CTL response in the SBBC members was against Pol antigens. These CTL appeared to be equally effective in containing viral replication, whether Gag-specific as demonstrated in earlier time points in C122, or Pol-specific in C18 (Figure 2).

The main factor that differentiated LTNP from those that lost non-progressor status, was low or undetectable HIV viraemia (<100 copies/ml; p = 0.021), and low viraemia was associated with detectable p24 proliferative responses (p = 0.0047). Loss of non-progressor status was strongly associated with undetectable or declining p24 responses (p = 0.0047). The combination of detectable p24 proliferative responses and strong (>500 SFC/106 PBMC) Gag CTL responses was associated with low (<100 copies/ml) or undetectable viraemia (p = 0.032).

Illustrating the importance of these combined Gag-specific T cell responses over time, low viraemia was intermittently detected at earlier time points in C122, with sharp increases in Gag CTL temporally associated with control of transient viraemia at 17 years post infection. However, Gag CTL later failed to contain viraemia in C122 beyond approximately 20 years, coinciding with weakening proliferative responses that gradually became negative. A similar correlation between anti viral immune responses and a spike in viral replication was demonstrated in SBBC member C18, shown in more detail in Figure 3. Over the course of 12 months, in response to an increase in viraemia peaking at 3600 copies/ml, the p24 proliferative response increased, along with substantial expansions of Pol-specific CTL in both precursor[15] and effector CTL populations. The durability of immune control in this individual was not determined as he died soon after from causes unrelated to HIV disease, aged 83.

Figure 3.

Dynamics of immune responses during an episode of increased viral replication in SBBC patient C18.

A decline in Gag-specific T cell responses preceding detectable viraemia was demonstrated in C13. This decline up to year 16 was followed by a period of low detectable viraemia (50 – 100 copies/ml) between years 19 – 22. A rebound in these Gag-specific T cell responses coincided with the first detectable viraemia at 19 years. These T cell responses may have helped contain viraemia to low levels over the following two years, but the sharp increase in viral RNA at 22.7 years (Figure 1) coincided with a decline in Gag-specific CD4 and CD8 T cell responses, whereas Pol-specific CTL increased in response to rising viraemia. These examples demonstrate the influence of conserved Gag-specific responses, particularly helper T cell responses, in reduced viral replication and delayed disease progression. While the decline in these responses preceded detectable viraemia in C13, sufficient patient specimens were not available to allow this critical observation to be made in others who progressed.

Breadth of the Anti-gag CTL Response in Non-progressors

To determine why strong Gag CTL may have contained viral replication in some, but failed in others, we mapped the breadth of the Gag CTL response over time in patients with at least moderate CLT responses to whole Gag antigens. Pools of overlapping 15-mer Gag peptides were used to test sequential PBMC spanning the study period by ELISPOT. The composition of each peptide pool, and examples of responses to these are shown in Figures 4 and 5, indicating the relevant HLA-specific epitopes contained in peptides at the intersection of positive pools. Figure 4 demonstrates a broad strong response by C53's PBMC to multiple immunodominant epitopes, contrasted in Figure 5 by the restricted response from C122 to only two immunodominant epitopes. The sequential analysis revealed relatively high stability in the repertoire of Gag responses over the past 10 years in most subjects (Additional file 2). Relevant epitopes at intersecting positive peptide pools were then confirmed using individual peptides (Figure 6). This data demonstrates that retention of broadly reactive Gag CTL was associated with ongoing non-progression (C49, C64, and C53), while restriction toward a narrow CTL specificity was observed in patients that eventually lost control of viraemia (C122 and possibly C13). The SBBC non-progressors C49 and C64 had responses to several Gag epitopes, and although Gag responses were moderate to weak in C64, this needs to be viewed in the context of Pol CTL dominance in the SBBC. A strong but restricted Gag response was also seen in C18, but these Gag responses were likely to be secondary in controlling viraemia, as suggested by the kinetics of Pol CTL in response to a spike in viraemia (Figure 3). Pol CTL recognition was confirmed by subsequent analysis of responses to peptide pools derived from the full set of Pol overlapping 15-mer peptides. Moderate to strong responses to multiple pools containing epitopes in the reverse transcriptase protein were detected in SBBC members C49, C64, C18, C54, but weakly in C98 (data not shown). C18 also responded strongly to integrase peptides.

Figure 4.

Identification of responses to Gag peptide epitopes by peptide pool mapping in a stable non-progressor (C53, 21.3 years post infection). Mean INF-γ spots/106 PBMC (SFC), and representative ELISPOT images are shown. Individual peptides intersecting positive peptide pools containing HLA-relevant epitopes (Additional file 2) were then tested individually, and positive responses indicated by dark shaded cells, and dominant responses in large font.

Figure 5.

Identification of responses to Gag peptide epitopes by peptide pool mapping in an individual with increasing viraemia (C122, 20.3 years post infection). Mean INF-γ spots/106 PBMC (SFC), and representative ELISPOT images are shown. Individual peptides intersecting positive peptide pools containing HLA-relevant epitopes (Additional file 2) were then tested individually, and positive responses indicated by dark shaded cells, and dominant responses in large font.

A strong but narrow CTL response may eventually fail to control viral replication. Restricted recognition of only one A3 and two B27 Gag epitopes in C13 appeared sufficient to have contained viraemia for many years, but the most recent viral load result (Figure 1) suggested that immune escape from these B27-restricted CTL may have occurred recently. Similarly, the predominant response by C122 against an immunodominant B27 epitope (Figure 5 and 6) may have contained earlier spikes of increased viraemia, but ultimately failed to contain increasing viral replication in later years (Figure 2).

Figure 6.

Breadth of Gag CTLs, showing responses to individual peptides selected from intersecting positive peptide pools, in non-progressor C49 (A), C64 (B), C18 (C), C13 showing an early and late time point (D), C53 (E), and C122 (F). Limit of detection 50 spots/106 PBMC.

Limited Immune Escape From HLA B27-restricted CTL

To determine why immunodominant B27-restricted CTL initially contributed to reduced viral replication in C13 and C122, but not in C117, sequencing of plasma and PBMC derived virus spanning the period before and after signs of disease progression was carried out to determine if viral escape mutants had emerged in this region of Gag (Figure 7). With the exception of one sample in 1996, a well characterised escape mutant[34] was detected from the earliest time point in C117. This escape mutant was not detected in C13 or C122, and hence was not the cause for the loss of control of viraemia in C122, nor was it detected in the latest time point from C13 when viraemia first increased above 1000 copies/ml. This suggests that immune escape at this B27 Gag epitope was not a major cause of disease progression in very long term infected individuals. The sole common factor was a decline in p24-specific proliferative responses.

Figure 7.

Amino acid sequences of the HLA B27 restricted Gag epitope KRWIILGLNK).

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