Introduction
The high prevalence of recurrent malaria, tuberculosis, chronic helminth infections, and water-borne pathogens in developing countries may play an important role in the pathogenesis of HIV-1 infection in persons living in such regions [1,2]. Immune activation associated with coinfections may be associated with increases in systemic HIV-1 load [3–6], viral genotypic diversification [7,8], accelerated disease progression [9], and enhanced HIV-1 transmission risk [10]. While the great majority of cell-free HIV-1 in plasma (> 98%) is thought to be derived from short-lived lymphocytes ordinarily [11,12], HIV-1 replication within the macrophage reservoir is increased during mycobacterial and Pneumocystis carinii coinfections [13–15]. Indeed, antigen-presenting cells are important reservoirs of HIV-1 [16,17] and induction of HIV-1 replication within these cells may contribute significantly to the cofactor effect of confections on HIV-1 pathogenesis.
Plasmodium falciparum is estimated to cause 500 million episodes of symptomatic malaria annually among persons living in sub-Saharan Africa [18], and in some urban centers of this region more than 30% of sexually active adults are infected with HIV-1 [19]. P. falciparum is, therefore, likely to be a frequent copathogen in HIV-1-infected individuals living in these countries. P. falciparum antigens induce viral replication in HIV-1-infected peripheral blood mononuclear cells (PBMC) in vitro [20]. Furthermore, a cross-sectional study of HIV-1-infected adults in Malawi, southern Africa, found that plasma virus load was higher in those with symptomatic falciparum malaria compared with those who were aparasitemic [21]. Using samples from this cross-sectional study [21], we have now determined the impact of P. falciparum malaria on viral replication within macrophages and lymphocytes in HIV-1-infected persons and related this to indices of systemic immune activation.
Patients and methods
Study population
The plasma samples analyzed in this study were selected from among those collected during a previous study of the impact of P. falciparum malaria coinfection on plasma HIV-1 load [21]. As described previously in detail, the subjects (cases) were HIV-1-infected adults with confirmed symptomatic P. falciparum parasitemia with no evidence of focal infection or bacteremia and a rapid response to antimalarial therapy using either sulfadoxine/pyrimethamine or quinine sulfate [21]. Control subjects were asymptomatic adult blood donors of comparable age who were aparasitemic but who were found to be infected with HIV-1.
Informed written consent was obtained from all study participants. The study protocol conformed with the human experimentation guidelines of the US. Department of Health and Human Services and was approved by the University of Malawi College of Medicine Research Committee and the University of North Carolina Committee on the Protection of Human Subjects.
Plasma HIV-1 RNA concentrations were measured using quantitative nucleic acid sequence-based-analysis (NucliSens, Organon-Teknika, Durham, North Carolina, USA).
Determination of the cellular origin of HIV-1 particles by immunomagnetic virus capture is restricted to the analysis of samples containing ≥ 1 × 105 copies/ml virus particles [13]. Consequently, samples from patients with an enrollment virus load of ≥ 1 × 105 copies/ml were selected from lists of cases and controls consecutively recruited in the parent study [21]. Although selection of cases and controls was based on a threshold virus load, the groups were found to have no significant difference in CD4 lymphocyte count. Paired plasma samples obtained from HIV-infected patients (n = 10) at diagnosis of acute malaria and 4 weeks after antimalarial treatment were studied together with plasma samples from controls (n = 10) obtained at a single time-point.
HIV-1 replication within lymphocytes and macrophages
Since host cell-surface proteins are incorporated into the HIV-1 envelope during viral budding, HIV-1 acquires an envelope phenotype that reflects that of the host cell [reviewed in 22]. An immunomagnetic virus capture technique to detect discriminatory cell-type-specific host proteins incorporated into the envelope of HIV-1 present in plasma samples was used to define the cellular origin of HIV-1 particles [13]. Magnetic beads bearing antibody directed against CD14 selectively capture virus derived from cells of the monocytic lineage, whereas antibody directed against CD26 selectively captures virus derived from T lymphocytes [13,14]. Additionally, the extent of virus capture using antibody to HLA-DR correlates with cellular activation [23,24] and capture with antibodies to CD44 (expressed by all mononuclear cells) and CD19 (expressed by B cells) serve as the positive and negative controls, respectively [13].
Limitations in capture efficiency restrict the immunomagnetic capture technique to making semiquantitative analysis of virus production from different cell pools. It is likely that a threshold density of antigen must be present in the virion envelope to enable capture of virus using this technique [13]. Virus captured using antibody to inducible cell surface host proteins is likely to be that which is derived from the most highly activated subsets of mononuclear cells.
Measurement of immune markers
Enzyme-linked immunosorbent assays were used to measure concentrations of tumor necrosis factor-α (TNF-α) (Medgenix Biosource, Fleurus, Belgium), interleukin-6 (IL-6), interferon-γ (IFN-γ), soluble CD25 (sCD25), soluble CD14 (sCD14), β2-microglobulin (β2-m), and soluble TNF receptor type I (sTNF-RI) (R&D Systems, Minneapolis, Minnesota, USA).
Statistical analysis
Non-parametric tests were used to compare HIV-1 RNA concentrations and markers of immune activation. The amount of HIV-1 specifically captured from samples by each antibody type was calculated by subtraction of the background non-specific binding that was assessed by the negative control beads. To compare differences in the concentrations of HIV-1 and immune activation markers, Wilcoxon and Mann–Whitney tests were used to compare paired and unpaired data, respectively. Statistical significance was defined as P ≤ 0.05.
Results
Patient characteristics and plasma HIV-1 RNA concentrations
Among the coinfected subjects, the median malarial parasite density was 177 179 × 106/l (range, 2300–408 064). Comparing cases and controls, there was no significant difference in either median age (30.0 years and 32.5 years, respectively) or median blood CD4 lymphocyte count (206 × 106 cells/l and 200 × 106 cells/l, respectively). However, at enrollment, the median HIV-1 load was 2.1-fold greater in the subjects with malaria (5.52 log10 copies/ml; range, 5.00–5.90) compared with controls (5.20 log10 copies/ml; range, 4.99–5.62) (P = 0.023). Following antimalarial treatment, plasma virus load decreased ≥ 2.0-fold in only 10 of 27 subjects in the parent study [21]. Moreover, in this study, a decrease of this amount was seen in only 1 of the 10 selected coinfected subjects and median virus load among these patients did not decrease significantly (5.45 log10 copies/ml; range, 4.59–6.00;P = 0.4).
HIV-1 replication in lymphocytes and macrophages
A significant level of HIV-1 (≥ 3.0-fold over background) was captured from all case and control plasma samples (n = 30) using antibody directed against CD44 (positive control). The mean level of anti-CD44 antibody capture did not differ significantly between samples obtained from patients with malaria before and after antimalarial treatment or between samples from cases and controls (Fig. 1a).
;)
Fig. 1.:
Virus captured by antigen-targeted HIV-1 envelope. (a) Magnetic beads conjugated with monoclonal antibodies directed against CD44 (positive control), CD26 (lymphocyte-specific antigen), CD14 (macrophage-specific antigen) and CD19 (negative control) were used to capture HIV-1 particles from plasma samples obtained from HIV-1-infected patients with malaria (n = 10) at diagnosis and 4 weeks after antimalarial treatment and from HIV-infected controls (n = 10). Data show the percentage (group mean ± SE) of the input virus (2 × 104 virions per capture) that was captured using each of the monoclonal antibodies. Compared with aparasitaemic controls, symptomatic malaria coinfection was associated with HIV-1 derived from CD26 lymphocytes (a P = 0.028) and CD14 macrophages (b P = 0.025). The proportion of virus derived from CD14 macrophages decreased following parasite clearance (b vs b′;P = 0.010). (b) Further immunomagnetic virus analysis of these samples found that the level of virus capture using anti-HLA-DR antibody was not affected by the presence or treatment of malaria coinfection.
Using antibody directed against CD26 (T lymphocyte-specific activation marker) in the HIV-1 envelope, a significant level of HIV-1 (≥ 3.0-fold over background) was captured from a greater proportion of samples from patients with malaria both before (9 out of 10) and after (7 out of 10) antimalarial treatment compared with control samples (5 out of 10). The mean (± SE) proportion of input HIV-1 captured from plasma by anti-CD26 antibody was greater in those with malaria at diagnosis (11.3 ± 2.3%) than in controls (5.7 ± 1.4%) (P = 0.028;Fig. 1a) but did not significantly decrease in the former following antimalarial treatment (10.5 ± 3.2%). Thus, detection of lymphocyte-derived HIV-1 was greater in patients coinfected with malaria both during acute malarial infection and 4 weeks after antimalarial treatment.
Using antibody directed against CD14 (macrophage-specific marker) incorporated in the HIV-1 envelope, a significant level of virus was captured from a greater proportion of the samples obtained from the coinfected patients during acute malaria (9 out of 10) both compared with samples obtained after antimalarial treatment (5 out of 10) and compared with control samples (4 out of 10). The mean (± SE) proportion of input HIV-1 captured from plasma by anti-CD14 antibody was greater at diagnosis in those with malaria (10.0 ± 2.1%) than after treatment (4.8 ± 1.1%) (P = 0.025) and than in controls (3.5 ± 1.2%) (P = 0.010;Fig. 1a). Therefore, symptomatic malaria was associated with HIV-1 replication within CD14 macrophages that decreased following parasite clearance.
Incorporation of HLA-DR in the HIV-1 envelope
Using anti-HLA-DR antibody, a significant level of HIV-1 was captured from all plasma samples except from those of two control subjects. However, there was no difference between the sample groups in the mean level of HIV-1 captured using this antibody (Fig. 1b).
Proinflammatory cytokines and cell activation markers
Median plasma concentrations of TNF-α, IL-6, IFN-γ, sTNF-RI, sCD25, sCD14 and β2-m were all elevated in those coinfected with malaria compared with controls (Fig. 2); following antimalarial treatment all, except β2-m, decreased significantly in those with malaria to levels comparable to those of the control group (Fig. 2).
Fig. 2.:
Plasma concentrations of proinflammatory cytokines and immune activation markers in patients with HIV-1 infection and symptomatic P. falciparum malaria coinfection (n = 10) at diagnosis (0), 4 weeks after successful antimalarial treatment (4), and in aparasitaemic HIV-1-infected controls (n = 10). Box and whisker plots indicate the median, 25th, and 75th percentiles and the range of values. Plasma concentrations of (a) tumor necrosis factor-α (TNF-α), (b) interleukin-6 (IL-6), (c) soluble TNF receptor type I (sTNF-RI), (d) interferon-γ (IFN-γ), (e) soluble CD25 (sCD25), (f) soluble CD14 (sCD14) and (g) β2-microglobulin (β2-m) were all significantly greater in patients at the time of malaria diagnosis compared with in controls. With the exception of β2-m, plasma concentrations of all these immune markers significantly decreased in those with malaria following successful antimalarial treatment. However, despite this decrease, concentrations of sCD25 remained significantly elevated in the coinfected patients after treatment compared with the controls.
Discussion
In this study, immunological and virological analyses of plasma samples from HIV-1-infected individuals and controls revealed that symptomatic P. falciparum malaria coinfection impacts HIV-1-host dynamics in vivo at the cellular level. Mathematical modelling has previously suggested that the macrophage cell pool ordinarily contributes minimally (< 2%) to the total cell-free HIV-1 pool [11] as was found in control subjects in this study (Fig. 1a). However, during acute malaria, approximately 10% of the HIV-1 pool was derived from CD14 macrophages, and this is likely to be an underestimate in view of the limitations in efficiency inherent in immunomagnetic capture. This effect of malaria coinfection is similar to the effect of tuberculosis on HIV-1 replication [14] and is supported by the finding in an HIV transgenic mouse model that murine malaria markedly upregulated proviral transcription, predominantly within antigen-presenting cells [25]. Macrophages are long-lived, migratory reservoirs of HIV-1 infection that disseminate virus to lymphocytes during cell–cell interactions [1,16]. These cells may, therefore, have a sustained effect on viral propagation and dissemination, thereby promoting disease progression. HIV-1 replicating within macrophages may also be less accessible to antiretroviral drugs [26].
Increased detection of HIV-1 bearing the lymphocyte activation marker CD26 [27] during acute malaria coinfection indicates HIV-1 replication within activated lymphocytes. Ongoing detection of CD26-bearing HIV-1 in the majority of the malaria patients 4 weeks after treatment may also suggest persistence of HIV-1 replication within a subset of malaria-activated lymphocytes at this time-point. Indeed, although plasma concentrations of sCD25 decreased to some extent following antimalarial treatment, the median concentration nevertheless still remained significantly higher at 4 weeks of follow up in the malaria-infected patients compared with the controls (P = 0.01) (Fig. 2). More prolonged follow up after antimalarial treatment is clearly desirable in future such studies. The finding that, in contrast to coinfection with tuberculosis [14,24], acute malaria was not associated with increased viral incorporation of HLA-DR may be attributable to minimal upregulation of HLA-DR expression on PBMC of individuals with acute malaria living in malaria endemic areas [28]. Furthermore, compared with HIV-infected individuals in industrialized countries [24], the control population showed an unusually high basal level of viral incorporation of HLA-DR.
Induction of proinflammatory cytokine secretion by P. falciparum pigments [29] may have been responsible for the 6.7-fold greater median plasma HIV-1 load seen in those coinfected with malaria compared with controls in the parent study [21]. Since IFN-γ [30] and sCD25 [31] are produced predominantly by activated lymphocytes and sCD14 is shed exclusively by cells of the monocytic lineage [32], increased concentrations of these markers indicate activation of both lymphocyte and macrophage cell pools, corroborating the finding of increased virus production from both cellular reservoirs. The striking absence of a correlation between changes in immune activation and plasma HIV-1 load in these individuals is consistent with findings from other studies conducted in sub-Saharan Africa [33,34] and contrasts with those conducted in industrialized countries [3,5,6]. This suggests there are significant differences in virus–host dynamics among HIV-infected persons living in different parts of the world, perhaps resulting from variations in host immune regulation [35] or in transcriptional regulation of diverse HIV-1 subtypes [36].
In summary, acute P. falciparum malaria coinfection impacts virus–host dynamics in HIV-1-infected persons at the cellular level, notably showing a reversible induction of HIV-1 replication in CD14 macrophages that is associated with changes in immune activation. Increased viral replication within the macrophage cell pool may have an impact on the natural history of HIV-1 in populations frequently exposed to malaria.
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