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Keywords:

  • chemokines;
  • cytokines;
  • HIV-1;
  • innate immunity;
  • primary HIV-1 infection

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Background

There are strong theoretical arguments for initiating antiretroviral therapy (ART) during primary HIV-1 infection (PHI) to preserve HIV-1-specific T-cell responses and to decrease immune activation.

Methods

We assessed the degree of immune activation during PHI and after analytical treatment interruption (ATI) in plasma samples from 22 subjects by measuring 13 cytokines/chemokines with the Luminex system. Subjects initiated quadruple ART at PHI (the QUEST cohort) and were classified as responders or nonresponders according to their HIV-1 viral load (VL) 6 months post-ATI.

Results

During PHI, nonresponders had higher levels of HIV-1 RNA, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-10 and eotaxin than responders (P≤0.05). A positive correlation was found between VL and IFN-α, TNF-α, IL-1β, macrophage inflammatory protein (MIP)-1α and MIP-1β, respectively. Post ATI, responders had higher levels of IFN-γ, MIP-1β and monocyte chemotactic protein (MCP)-1 than nonresponders, while nonresponders had higher levels of HIV-1 RNA, IL-15 and eotaxin. Cytokine/chemokine levels were higher during PHI than post-ATI.

Conclusions

High levels of immune activation during PHI are associated with a worse virological outcome post-ATI. In contrast, VL post-ATI is negatively correlated with IFN-γ and chemokines. Therefore, the degree of immune activation during PHI is associated with both the VL at PHI and the viral set-point post-ART.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The events occurring at the time of the initial encounter of HIV-1 with the immune system are understood to be critical in terms of later prognosis [1,2]. Initiation of antiretroviral therapy (ART) during primary HIV-1 infection (PHI) can decrease viral diversification and saturation of reservoirs, and preserve virus-specific CD4 T helper responses. It has also been reported to decrease rapid clinical progression [3]. All these factors may be very important in the setting of improved ARTs and use of immune modulation [4].

Measurements of cytokine and chemokine levels have been useful in the understanding of HIV-1 pathogenesis. Viral replication is regulated by a complex network of both HIV-1-suppressive [e.g. interferon (IFN)-α, interleukin (IL)-10, macrophage inflammatory protein (MIP)-1α, MIP-1β and RANTES] and inductive [e.g. tumour necrosis factor (TNF)-α, IL-1β, IL-6, IL-15 and monocyte chemotactic protein (MCP)-1] cytokines and chemokines [5]. Although there are conflicting reports regarding the role of certain cytokines during the course of the infection, their contribution to the state of generalized immune activation is well acknowledged [6–8]. The aim of this study was to characterize the pattern of immune activation using analysis of cytokines and chemokines in very early infection, in order to correlate their levels to virological outcome both at the time of PHI and during an analytical treatment interruption (ATI).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patients and blood sampling

The subjects enrolled in this substudy were participants in the QUEST PHI study, GW PROB3005 (Table 1). The trial design has been described elsewhere [9,10]. Patients were eligible for entry to the study if they had p24 antigenaemia or HIV-1 viraemia and either a negative [third-generation HIV enzyme-linked immunosorbent assay (ELISA)] or evolving (positive ELISA but ≤3 bands on HIV Western blot) antibody response [9,10]. In brief, 148 subjects started quadruple ART at PHI {Combivir® [Glaxo Smith Kline (GSK) R & D, Greenford, UK] abacavir and amprenavir twice a day} and continued this treatment for at least 72 weeks. Study subjects were then randomized into three different arms: (i) ART alone, (ii) ART plus ALVAC-HIV (vCP1452), or (iii) ART plus ALVAC-HIV (vCP1452) plus Remune (The Immune Response Corporation, Carlsbad, CA, USA). They remained on these regimens for 24 weeks, after which time all treatment was stopped. The patients were followed up for 24 weeks at regular intervals during the ATI. The outcome of the study showed that there was no statistically significant difference in terms of the primary virological endpoint [proportions of subjects with HIV-1 RNA viral load (VL)≤1000 copies/mL at 24 weeks post-ATI] between either of the two vaccine arms and the placebo arm [9,10].

Table 1.   Clinical characteristics of the subjects enrolled in the study
 Responders (n=10)Nonresponders (n=12)
  • *

    Median (range).

  • ART, antiretroviral therapy.

Female/male (n)2/82/10
Age (years)*35 (29–42)37 (26–52)
CD4 T-cell count at initiation of ART (cells/μL)*547 (348–1003)434 (293–519)
CD4 T-cell count at end of study (cells/μL)*834 (597–1201)644 (394–825)
HIV-1 RNA load at initiation of ART (copies/mL)*29 627 (1138–118 437)436 071 (91 327–16 193 662)
HIV-1 RNA load at end of study (copies/mL)*247 (50–619)22 509 (2526–198 548)

Twenty-two subjects were selected for the present study on the basis of virological outcome and available samples at 24 weeks after stopping ART. The median duration of ART was 31 months (range 23–51 months). Subjects included in our study were divided into two groups. Ten subjects were considered as responders as they fulfilled the predefined primary endpoint for success (VL≤1000 copies/mL at 24 weeks post-ATI), while nonresponders (n=12) had a VL of>1000 copies/mL 24 weeks post-ATI. Furthermore, there was no significant difference between responders and nonresponders in terms of symptom debut and time-point of treatment initiation.

Plasma samples were analysed during PHI on day 1, day 14, week 4 and week 28 post-initiation of ART. During the ATI phase, samples were analysed at 4 weeks prior to ART cessation, during ART cessation before VL peak, at VL peak and after VL peak.

Ethics

This study was conducted in accordance with the Declaration of Helsinki and Good Clinical Research Practice. All subjects provided signed informed consent prior to study enrolment. Independent local ethics committees and national gene therapy committees reviewed and approved the study protocol and its amendments (Karolinska Institutet: Dnr 98-015).

Plasma HIV-1 RNA quantification and CD4 and CD8 T-cell counts

Plasma HIV-1 RNA levels (VL) were quantified using the Roche Amplicor test [version 1.0; limit of detection (LOD) 400 copies/mL; Roche Molecular Systems Inc., Alameda, CA, USA] and the Roche Amplicor Monitor test (UltraSensitive version 1.5; LOD 50 copies/mL; Roche Molecular Systems Inc.). A modified version of the UltraSensitive version, with an LOD of 3 copies/mL, was used when VL was below the limit of detection of the commercial UltraSensitive assay [11]. CD4 and CD8 T-cell counts were assessed by routine flow cytometry.

Cytokine and chemokine quantification

Beta (β)-chemokines (RANTES, MIP-1α, MIP-1β, MCP-1 and eotaxin) and cytokines (IFN-α, IFN-γ, TNF-α, IL-1β, IL-6, IL-10, IL-12p70 and IL-15) were measured simultaneously using the Luminex LabMAP multiplexed bead system (Biosource International Inc., Carlsbad, CA, USA), according to the manufacturer's instructions. Results obtained from the Luminex LabMAP system were analysed automatically by the bio-plex manager software program (Bio-Rad Laboratories Inc., Hercules, CA, USA) using a standard curve derived from recombinant cytokine and chemokine standards.

Statistical methods

The area under the curve (AUC) for VL and each cytokine and chemokine was calculated for individual subjects during PHI and the ATI. The analysis was based on the four sampling time-points during PHI and post-ATI, respectively. Responders and nonresponders were then compared using a nonparametric two-tailed Mann–Whitney U-test. This test was also used to determine the statistical significance of differences between: (1) responders and the nonresponders at each time-point during the PHI and ATI phases, and (2) each time-point for the vaccinated and placebo groups during the ATI phase. The Wilcoxon signed rank test was used to compare values for VL and each cytokine and chemokine at the VL peak during the PHI phase vs. those during the ATI phase for each subject. The correlation between VL and plasma cytokine/chemokine concentrations was calculated using Spearman's rank correlation coefficient. The evaluation was performed using graphpad prism 4.0 (GraphPad Software Inc., San Diego, CA, USA). P-values ≤0.05 were considered as significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plasma HIV-1 RNA (VL) and T-cell changes during the PHI and ATI phases

The AUC analysis showed that VL during PHI was lower in responders than in nonresponders (P<0.0007; data not shown). This was confirmed by a Mann–Whitney U-test at each time-point, except for week 28 (Fig. 1a). By definition, the responders had lower VL than the nonresponders at the 24-week time-point post-ART. Additionally, the VL of the responders was lower at all other time-points during ATI than that of nonresponders. No significant difference for CD4/CD8 T-cell counts between responders and nonresponders was found during the PHI or ATI phase (data not shown).

imageimage

Figure 1.  Plasma levels of HIV-1 RNA (VL) and of cytokines and chemokines during the primary HIV-1 infection (PHI) phase. Subjects were classified in 2 groups; responders (R) and non responders (NR). The area under the cytokine-chemokine concentration curve (AUC) based on four sampling time points during PHI, was calculated for all subjects in each group and each analyte. A non-parametric two-tailed Mann-Whitney U test was applied to compare R and NR at corresponding time points during the PHI phase. This analysis was also used to reveal differences between AUC of R and NR for single cytokine and chemokine. P values *(P≤0.05), ***(P<0.001). (a) The graph shows the distribution and median levels of HIV-1 RNA. (b–f) Area under the cytokine concentration curve was calculated for R and NR and analyzed by a non-parametric two-tailed Mann-Whitney U test. Results are presented as box plots (range and median). (g–i) The graphs show the distribution and median cytokine plasma levels subsequently analyzed by a non-parametric two-tailed Mann-Whitney U test.

Nonresponders had higher levels of proinflammatory cytokines and chemokines during PHI

Throughout PHI, higher levels of cytokines and chemokines were found in the nonresponders as compared with the responders, when data were analysed using AUC (IFN-γ, P≤0.05; TNF-α, P<0.02; IL-1β, P<0.02; IL-10, P<0.04; eotaxin, P<0.04; Fig. 1b–f) with the exception of MCP-1 (data not shown). A similar pattern was found when concentrations were compared at different time-points during PHI (Fig. 1g–i). A positive correlation was seen between VL and IFN-α (r=0.3, P<0.005), TNF-α (r=0.3, P<0.02), IL-1β (r=0.3, P<0.009), MIP-1α (r=0.3, P<0.003) and MIP-1β (r=0.2, P<0.05), respectively (Table 2).

Table 2. R values for Spearman's rank correlation coefficients for the relationships between HIV-1 RNA viral load and plasma cytokine/chemokine levels in HIV-1-infected subjects during primary HIV-1 infection (PHI) and during the analytical treatment interruption (ATI) phase
 PHIATI
  1. The minus sign indicates a negative correlation with HIV-1 RNA viral load.

  2. P-values are shown in parentheses.

  3. IL, interleukin; IFN, interferon; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; TNF, tumour necrosis factor.

IFN-α0.3 (<0.005)−0.2 (<0.03)
IFN-γ0.1 (0.2)−0.4 (<0.0001)
TNF-α0.3 (<0.02)−0.06 (0.6)
IL-1β0.3 (<0.009)−0.2546 (<0.03)
IL-60.1 (0.3)−0.3 (<0.002)
IL-100.2 (0.2)0.3 (<0.01)
IL-12p700.1 (0.3)−0.3 (<0.005)
IL-150.1 (0.2)0.3 (<0.02)
MIP-1α0.3 (<0.003)−0.3 (<0.009)
MIP-1β0.2 (<0.05)−0.4 (<0.0007)
RANTES0.6 (0.05)−0.1 (0.3)
MCP-10.3 (0.1)−0.4 (<0.0002)
Eotaxin0.2 (0.1)0.3 (<0.002)

Responders had higher chemokine and IFN-γ levels after treatment cessation

During the ATI phase, AUC analysis showed higher levels of IFN-γ (P≤0.05), MIP-1β (P<0.05) and MCP-1 (P≤0.05) in responders compared with nonresponders (Fig. 2a–c), while nonresponders had higher concentrations of IL-15 (P<0.04; Fig. 2d) and eotaxin (P<0.03; data not shown). Responders had increased IFN-γ levels at baseline [median values for responders vs. nonresponders (here and below) 26.0 vs. 12.9 pg/mL, respectively; P≤0.05] and at the endpoint measurement (52.7 vs. 3.1 pg/mL; P<0.005). Increased levels of IL-6 were found during the VL peak in responders (3.8 vs. 0.1 pg/mL; P<0.03), while nonresponders had higher levels of IL-10 before the VL peak (2.1 vs. 4.1 pg/mL; P<0.03). Responders had increased MIP-1α (116.3 vs. 66.4 pg/mL; P<0.05) and MCP-1 (279.6 vs. 177.8 pg/mL; P<0.03) levels compared with nonresponders at the endpoint measurement. Nonresponders had increased eotaxin levels at baseline (64.6 vs. 121.0 pg/mL; P<0.03) and at RNA peak (69.1 vs. 125.6 pg/mL; P<0.02) compared with responders.

image

Figure 2.  Plasma levels of cytokines and chemokines during the analytical treatment interruption (ATI) phase. The area under the cytokine-chemokine concentration curve (AUC) based on four sampling time points was calculated for all subjects in R, respective NR group, and each cytokine-chemokine. R and NR were compared by a non-parametric two tailed Mann-Whitney U-test. (a–d) Results of this analysis for IFN-γ, MIP-1β, MCP-1 and IL-15, respectively, are presented as box plots (range and median). Statistical significance is indicated as *(P≤0.05).

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During ATI most cytokines and chemokines were negatively correlated with VL

When all subjects were included in the analysis, a negative correlation was found between VL and IFN-α (r=−0.2, P<0.05), IFN-γ (r=−0.4, P<0.0001), IL-1β (r=−0.25, P<0.03), IL-6 (r=−0.34, P<0.002), IL-12p70 (r=−0.3, P<0.005), MIP-1α (r=−0.3, P<0.009), MIP-1β (r=−0.4, P<0.0007) and MCP-1 (r=−0.4, P<0.0002), respectively. In contrast, IL-10 (r=0.3, P<0.0095), eotaxin (r=0.3, P<0.002) and IL-15 (r=0.3, P<0.02) were positively correlated with VL (Table 2).

No differences in terms of cytokine/chemokine profiles between vaccinated and nonvaccinated patients after ATI

To exclude the effect of vaccination on cytokine/chemokine levels, we also divided the screened subjects into vaccinated and nonvaccinated groups. Statistical analysis showed that there were no differences in levels of cytokines and chemokines post-ATI, when vaccinated subjects (ART+ALVAC-HIV and ART+ALVAC-HIV+Remune) were compared with those who had received ART plus placebo (data not shown).

Increased levels of IFN-γ, TNF-α, IL-10 and chemokines during PHI compared with post-ATI

We compared cytokine and chemokine levels at the time of the highest VL between the two phases (PHI, median 168 784 copies/mL, range 1138–1.6 × 107 copies/mL; post-ART, median 15 050 copies/mL, range 50–1.3 × 107) using the Wilcoxon signed rank test. This analysis revealed significantly higher levels of HIV-1 RNA (P<0.004), IFN-γ (median 236.0 vs. 12.3 pg/mL for PHI vs. ATI, respectively; P<0.0005), TNF-α (7.0 vs. 4.0 pg/mL; P<0.005), IL-10 (23.5 vs. 3.5 pg/mL; P<0.0002), MIP-1α (114.0 vs. 60.1 pg/mL; P<0.002), MIP-1β (80.5 vs. 31.5 pg/mL; P<0.009), RANTES (34 806 vs. 19 509 pg/mL; P<0.04) and eotaxin (122.0 vs. 97.0 pg/mL; P<0.05) during PHI compared with the ATI phase (Fig. 3a–e).

image

Figure 3.  Concentrations of cytokines and chemokines in plasma of all subjects (responders and non-responders) at the highest HIV-1 RNA (VL) peak during the PHI and ATI. Wilcoxon signed rank test was applied to reveal variation between two phases. Statistically significant differences are indicated by P values *(P≤0.05), **(P<0.01), ***(P<0.01). (a–e) Distribution and median plasma concentrations of IFN-γ, TNF-α, IL-10, MIP-1α, and MIP-1β, respectively.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Immune activation is an important factor in the pathogenesis of chronic HIV-1 infection [12,13]. Disease stage and VL correlate with the level of immune activation [14]. However, the role of immune activation has not been determined during the very early stage of the infection which is associated with intense viral replication and major loss of some of the components of the immune system [1,15,16]. Plasma HIV-1 RNA of untreated subjects tends to reach a set-point 1–2 months after PHI [15] and is considered to be a prognostic marker of disease progression [17]. In our study, a clear correlation was seen between VL values at PHI and those after ATI. This suggests that the events immediately after HIV transmission and during the first few days after onset of PHI symptoms influence viral kinetics in later infection.

All subjects showed an intense state of immune activation during PHI, the degree of which correlated with VL, and was higher in the nonresponder group. The only exception was for MCP-1, the levels of which were higher during PHI among responders. We found that the cytokine/chemokine pattern during the PHI phase was associated with the type of virological outcome after treatment cessation; thus, subjects who had lower levels of VL and of all soluble factors at PHI (with the exception of MCP-1) also had lower VL after ART cessation.

It has been suggested that events after ART cessation mimic those of the PHI period. Thus, in general viral kinetics follows at least partially the pattern seen during PHI, and clinical symptoms similar to those seen at PHI may occur [15,18]. However, in our study clear differences were seen in the cytokine profile after cessation of ART compared with the PHI phase. We found that in the ATI phase responders had significantly elevated levels of IFN-γ, MIP-1β and MCP-1 and significantly lower levels of IL-15 and eotaxin compared with nonresponders. This shift in the cytokine profile suggests that there may be better preservation of innate and adaptive immunity in subjects with a lower degree of immune activation during PHI. Betts et al. [19] recently suggested that the presence of polyfunctional HIV-1-specific CD8 T cells characterized by strong cytokine (IFN-γ, TNF-α and IL-2) and chemokine (MIP-1β) production is negatively correlated with disease progression. Our data for the plasma compartment are consistent with this hypothesis. The analysis of the cytokine profile at the time of the VL peak during PHI and after ART cessation also shows that levels of IFN-γ, TNF-α, IL-10, MIP-1α, MIP-1β, RANTES and eotaxin were significantly higher during PHI than after ATI. This finding was probably related to the substantially higher VL during PHI [20,21].

A beneficial effect of increased levels of β-chemokines during PHI [22,23] was not confirmed in our study. RANTES, MIP-1α and MIP-1β followed the general cytokine pattern. However, changes were observed during the ATI phase, with a tendency towards increased chemokine levels in responders and a negative correlation with VL for MCP-1, MIP-1α and MIP-1β. Thus, the chemokines studied here may have different impacts on viral replication depending on the phase of HIV-1 infection considered [24,25].

We found that plasma levels of IL-15 and eotaxin were elevated in nonresponders during ATI. Plasma IL-15 has been previously reported to be increased in subjects on ART [26] as well as in those with successful structured treatment interruption (STI) outcomes [8]. In contrast, our data do not support a beneficial effect of IL-15 after cessation of ART in terms of virological set-point.

Increased plasma levels of eotaxin have been found in several inflammatory diseases [27–29]. This protein is not only a potent chemoattractant of eosinophils but also contributes to the recruitment of immune cells following viral infection [30]. Eotaxin is a ligand for chemokine (C-C motif) receptor 3 (CCR3), which can serve as an HIV-1 co-receptor in at least in vitro settings [31,32]. Furthermore, genes for eotaxin, MCP-1 and MCP-3 are localized to the same chromosomal region, which has been linked to a role in the modulation of HIV-1 transmission, probably by activating the immune system [33]. Interestingly, Promadej-Lanier et al.[34] have shown that peak systemic levels of eotaxin coincide with initial detection of viral RNA in macaques challenged with vaginal simian/human immunodeficiency virus (SHIV) inoculate. The uniform high eotaxin levels in nonresponders during both the primary and the ATI phases strongly suggest that this chemokine does contribute to immune activation, rather than having a beneficial effect in vivo.

The cellular sources of the chemokines and cytokines studied here have not been explored. Both their active release by immune cells and their release during cell death may contribute to the elevated plasma levels observed during PHI. The initial phase of HIV-1 infection is indeed associated with a rapid decline in CD4 T cells as a result of cell death caused by necrosis and apoptosis [16,35]. Necrotic cell death (direct viral cytopathic effect) is associated with inflammation/immune activation and with the recently described involvement of the intracellular protein high mobility group box protein 1 (HMGB1) in this process [36–38].

In conclusion, our study adds important information in terms of the pattern of cytokine and chemokine production during early HIV-1 infection and after cessation of ART. We have shown that the degree of immune activation is highest during PHI and is associated with VL. Further investigations are required to clarify the role of ART in the change in cytokine/chemokine pattern observed after treatment interruption and its potential impact on the long-term virological outcome.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

For funding and support as well as members of the QUEST study group, please see references [9,10]. This study was also supported by the Swedish Physicians Against AIDS Foundation, Swedish Research Council and SIDA/SAREC. We thank all patients who participated in the study.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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