Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

Abstract

Introduction: Immunological damage in acute HIV infection (AHI) may predispose to detrimental clinical sequela. However, studies on the earliest HIV-induced immunological changes are limited, particularly in sub-Saharan Africa. We assessed the plasma cytokines kinetics, and their associations with virological and immunological parameters, in a well-characterized AHI cohort where participants were diagnosed before peak viremia.

Methods: Blood cytokine levels were measured using Luminex and ELISA assays pre-infection, during the hyperacute infection phase (before or at peak viremia, 1-11 days after the first detection of viremia), after peak viremia (24-32 days), and during the early chronic phase (77-263 days). Gag-protease-driven replicative capacities of the transmitted/founder viruses were determined using a green fluorescent reporter T cell assay. Complete blood counts were determined before and immediately following AHI detection before ART initiation.

Results: Untreated AHI was associated with a cytokine storm of 12 out of the 33 cytokines analyzed. Initiation of ART during Fiebig stages I-II abrogated the cytokine storm. In untreated AHI, virus replicative capacity correlated positively with IP-10 (rho = 0.84, P < 0.001) and IFN-alpha (rho = 0.59, P = 0.045) and inversely with nadir CD4⁺ T cell counts (rho = - 0.58, P = 0.048). Hyperacute HIV infection before the initiation of ART was associated with a transient increase in monocytes (P < 0.001), decreased lymphocytes (P = 0.011) and eosinophils (P = 0.003) at Fiebig stages I-II, and decreased eosinophils (P < 0.001) and basophils (P = 0.007) at Fiebig stages III-V. Levels of CXCL13 during the untreated hyperacute phase correlated inversely with blood eosinophils (rho = - 0.89, P < 0.001), basophils (rho = - 0.87, P = 0.001) and lymphocytes (rho = - 0.81, P = 0.005), suggesting their trafficking into tissues. In early treated individuals, time to viral load suppression correlated positively with plasma CXCL13 at the early chronic phase (rho = 0.83, P = 0.042).

Conclusion: While commencement of ART during Fiebig stages I-II of AHI abrogated the HIV-induced cytokine storm, significant depletions of eosinophils, basophils, and lymphocytes, as well as transient expansions of monocytes, were still observed in these individuals in the hyperacute phase before the initiation of ART, suggesting that even ART initiated during the onset of viremia does not abrogate all HIV-induced immune changes.

Keywords: Acute HIV infection; Cytokine storm; Early ART; Replication capacity.

Fig. 1

Untreated hyperacute HIV, but not ART early-treated hyperacute HIV, is associated with elevation of plasma cytokines that have distinct kinetics. a Interferon gamma-induced protein 10 (IP-10/CXCL-10). b Monokine induced by gamma interferon (MIG/CXCL-9). c Monocyte chemoattractant protein 1 (MCP-1). d Interleukin 12 (IL-12). e Soluble IL-2 receptor (IL-2R). f Interleukin 8 (IL-8). g Interferon gamma (IFN-gamma). h Interleukin-1 receptor antagonist (IL-1RA). i B cell-activating factor (BAFF/BLYS/TNFSF13B). j Chemokine (C-X-C motif) ligand 13 (CXCL13). k Soluble CD14. l Interferon alpha (IFN-alpha). N = 12 for untreated hyperacute HIV-infected participants (except CXCL13 and BAFF with N = 10). N = 8 for ART early-treated hyperacute HIV-infected individuals (except CXCL13 and BAFF with N = 6 and IFN-alpha with N = 7). Cytokine levels for one of the untreated participants were measured 434 days instead of 238–263 days after the detection of viremia. Each symbol represents an individual participant. Except for IFN-alpha, red symbols show the plasma levels in untreated participants and blue symbols show the plasma levels in ART early-treated participants. Horizontal lines and error bars in the scatter plots represent the median and interquartile range. In l (IFN-alpha), every colored line represents a participant. Statistical test used: Wilcoxon matched-pairs signed-rank test. P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001. “Pre” refers to the pre-infection time point

Fig. 2

Hyperacute HIV is associated with multicollinearity among the elevated cytokines. a Correlation analyses for all participants at each time point among the cytokines that constituted the cytokine storm (except IFN-alpha) in hyperacute HIV infection, N = 20 (12 untreated hyperacute HIV-infected participants and 8 ART early-treated hyperacute HIV-infected individuals). The colors show Spearman’s rank-order correlation coefficients (rho) for the correlations that had P < 0.05. White squares indicate associations that were not statistically significant. b The kinetics of the first and second principal components (derived from cytokines that constituted the storm except for IFN-alpha which was detected at only one time point) among untreated participants. c The kinetics of the first and second principal components (derived from cytokines that constituted the storm except for IFN-alpha) among ART early-treated participants. Cytokine levels for one of the untreated participants were measured 434 days instead of 238–263 days after the detection of viremia. In b and c, every line represents a participant (untreated hyperacute HIV-infected participants, N = 10; ART early-treated hyperacute HIV-infected individuals, N = 6). Statistical test used in b and c: Wilcoxon matched-pairs signed-rank test. P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

CXCL13 is positively associated with delayed suppression of viremia in early-treated individuals. a Duration to viral suppression in days among early-treated participants. b Correlation between duration to viral suppression in days and viral load at the time of initiating ART in early-treated individuals. c Correlation between duration to viral suppression and plasma CXCL13 levels at 3 months. d Correlation between viral load at the time of initiating ART and plasma CXCL13 levels at 3 months. Each symbol represents an individual participant (N = 6). Statistical test: Spearman’s rank-order correlation. P values < 0.05 were considered significant

Fig. 4

HIV Gag-driven replicative capacity predicts the magnitude of inflammatory cytokines and CD4⁺ T cell depletion in untreated hyperacute HIV infection. a Gag-driven replicative capacity among untreated participants. b Correlation between IP-10 in the hyperacute phase and replicative capacity. c Correlation between peak IFN-alpha and replicative capacity (* indicates two overlapping data points). d Correlation between nadir CD4⁺ T cell counts and replicative capacity. e Correlation between peak viral loads and replicative capacity. Each symbol represents an individual participant (N = 12). Statistical test: Spearman’s rank-order correlation. P values < 0.05 were considered significant

Fig. 5

The magnitude of plasma cytokines predicts CD4⁺ T cell and viral load dynamics in untreated hyperacute HIV infection. a Correlation between peak IFN-alpha and peak viremia. b Correlation between hyperacute soluble IL-2 receptor and peak viremia. c Correlation between hyperacute IL-1RA and viral load set point. d Correlation between hyperacute CXCL13 and nadir CD4⁺ T cell counts. e Correlation between hyperacute soluble IL-2 receptor and nadir CD4⁺ T cell counts. f Correlation between hyperacute IL-1RA and set point CD4⁺ T cell counts. Each symbol represents an individual participant (N = 12 except CXCL13 with N = 10). Statistical test: Spearman’s rank-order correlation. P values < 0.05 were considered significant

Fig. 6

Hyperacute HIV infection is associated with dysregulation of blood lymphoid and myeloid cells. a Monocyte absolute counts. b Total lymphocyte absolute counts. c Eosinophil absolute counts. d Basophil absolute counts. e Neutrophil absolute counts. f Red cell counts. g Platelet counts. For all cellular components, measurements before HIV infection (blue symbols, N = 70), in AHI at Fiebig stages I–II (red symbols, N = 60) and in AHI at Fiebig stages III–V (purple symbols, N = 15) from the FRESH acute infection cohort are shown. Measurements from a different chronic cohort (brown symbols, N = 33) are included for comparison purposes. Each symbol represents an individual participant. Horizontal lines and error bars in the scatter plots represent the median and interquartile range. Statistical test used: Wilcoxon rank-sum test (Mann-Whitney U test). P values < 0.05 were considered significant

Fig. 7

Plasma cytokines/chemokines are associated with reduced blood counts of lymphocytes, eosinophils, and basophils in untreated acutely HIV-infected patients. a Correlation between CXCL13 and total lymphocytes. b Correlation between CXCL13 and eosinophils. c Correlation between CXCL13 and basophils. d Correlation between MIG/CXCL9 and total lymphocytes. e Correlation between MIG/CXCL9 and eosinophils. f Correlation between MIG/CXCL9 and basophils. g Correlation between soluble IL-2 receptor and total lymphocytes. h Correlation between soluble IL-2 receptor and eosinophils. i Correlation between soluble IL-2 receptor and basophils. The measurements of cytokines and blood cell counts were in the hyperacute phase of HIV infection. Each symbol represents an individual participant (N = 12 except CXCL13 (a–c) with N = 10). Statistical test: Spearman’s rank-order correlation. P values < 0.05 were considered significant

Fig. 8

Correlation network showing a summary of the relationships between cytokines, CD4⁺ T cell dynamics, viral load dynamics, Gag-driven viral replication capacity, and hematological parameters in untreated hyperacute HIV infection. Statistical test used: Spearman’s rank-order correlation. Red lines show significant positive correlations. Blue lines show significant inverse correlations. The width of the line indicates the strength of Spearman’s correlation coefficient (rho). Only correlations that have P < 0.05 are shown. Gag RC, Gag-driven viral replication capacity (N = 12 except CXCL13 and BAFF with N = 10)