Nonprogressing HIV-infected children share fundamental immunological features of nonpathogenic SIV infection

Abstract

Disease-free infection in HIV-infected adults is associated with human leukocyte antigen-mediated suppression of viremia, whereas in the sooty mangabey and other healthy natural hosts of simian immunodeficiency virus (SIV), viral replication continues unabated. To better understand factors preventing HIV disease, we investigated pediatric infection, where AIDS typically develops more rapidly than in adults. Among 170 nonprogressing antiretroviral therapy-naïve children aged >5 years maintaining normal-for-age CD4 T cell counts, immune activation levels were low despite high viremia (median, 26,000 copies/ml). Potent, broadly neutralizing antibody responses in most of the subjects and strong virus-specific T cell activity were present but did not drive pediatric nonprogression. However, reduced CCR5 expression and low HIV infection in long-lived central memory CD4 T cells were observed in pediatric nonprogressors. These children therefore express two cardinal immunological features of nonpathogenic SIV infection in sooty mangabeys-low immune activation despite high viremia and low CCR5 expression on long-lived central memory CD4 T cells-suggesting closer similarities with nonpathogenetic mechanisms evolved over thousands of years in natural SIV hosts than those operating in HIV-infected adults.

Fig. 1.. Normal CD4 T-cell counts for age and low immune activation despite high viral loads in pediatric non-progressors, in association with evidence of low levels of microbial translocation.

a–c. Absolute CD4 T-cell count, CD4% (percentage of peripheral blood lymphocytes expressing CD4) and viral load in ART-naïve pediatric subject 517-C over the first 10 years of life. 10^th, 50^th and 90^th centile of absolute CD4 T-cell counts and CD4% are shown in panels A-B for uninfected children over the first 10 years of life^18–19. d. Longitudinal viral load data from 170 ART-naïve pediatric non-progressors. Viral load declines with age over the first 5 years (r=−0.34, p<0.0001) but then plateaus thereafter. e. Current absolute CD4 T-cell counts and viral loads in 170 pediatric non-progressors. f. Lack of correlation between CD4 T-cell count and viral load in 170 pediatric non-progressors. G–h. Immune activation (CD38/HLA-DR expression) on CD4+ T-cells (panel g) and CD8+ T-cells (panel h) is inversely correlated with absolute CD4 T-cell count in ART-naïve children aged>5yrs (n=163 HIV-infected children and n=21 HIV-uninfected children). i. Levels of soluble CD14 are significantly lower in pediatric non-progressors (absolute CD4 T-cell count >750 cells/mm³, n=14) than in progressors (absolute CD4 T-cell count <500 cells/mm³, n=16) and similar to HIV-uninfected children (n=21). j. Levels of intestinal fatty acid binding protein are lower in pediatric non-progressors (n=14) and HIV-uninfected children (n=21) compared to progressors (n=19). Comparisons between groups were calculated by Mann-Whitney tests (*p<0.05; **p<0.01). P- and r-values for bivariate associations were calculated by Spearman rank correlation tests.

Fig. 2.. CD4 T-cell differentiation and function in non-progressing pediatric infection

a–c: IFN-γ, IL-2 and TNF-α intracellular cytokine staining (ICS) response to SEB and to Gag in pediatric non-progressors. a. Representative FACS staining in a progressor and non-progressor. b. ICS responses to SEB in non-progressors (n=14), progressors (n=32) and uninfected pediatric controls (n=19), gating on bulk CD4+ T-cells. c. ICS responses to a Gag peptide pool in non-progressors (n=26) and progressors (n=11), gating on bulk CD4+ T-cells. D–e: CD4 T-cell differentiation in pediatric progressors and non-progressors (n=161). d. Representative FACS staining in a progressor and non-progressor. e. Percentage of CD4 T-cells by absolute CD4 T-cell count defined as naïve, central memory, effector memory or terminally differentiated effector memory, by CCR7 and CD45RA expression as shown in panel d. Correlation between absolute CD4 T-cell count and % CD4 T-cells in each subset shown as Spearman rank correlation coefficient and p value. F–g: Functional differences between distinct CD4 T-cell subsets. f. Representative FACS staining in response to SEB showing IFN-γ producing cells are predominantly effector memory, and IL-2 producing cells are predominantly naïve and central memory in phenotype. g. Contribution of IL-2, IFN-γ and TNF-α to the total CD4 T-cell SEB response. The proportion of cells producing each cytokine to the total CD4 T-cell SEB response was determined; data from 11 non-progressors. H–j: PD-1 expression on CD4 T-cell subsets in pediatric progressors, non-progressors and uninfected controls h. Increased PD-1 expression on CD4+ T-cells in pediatric progressors (n=20) compared to non-progressors (n=32) and uninfected pediatric controls (n=22). i. Increased PD-1 expression on CD4+ T-cell subsets in pediatric progressors (n=20) compared to non-progressors (n=30). j. Representative FACS plots to show differential cytokine staining in relation to PD-1 expression. K–l: T-cell activation by memory subsets. Immune activation (CD38/HLA-DR expression) on CD4+ central memory T-cells (Tcm) (panel k) and other CD4+ and CD8+ T-cell memory subsets (panel l) is inversely correlated with absolute CD4 T-cell count in ART-naïve children aged>5yrs (n=97). Comparisons between groups were calculated by Mann-Whitney tests (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). P- and r-values for bivariate associations were determined by Spearman rank correlation tests.

Fig. 3.. Associations between immunological and clinical variables.

a: Selected bivariate associations between immunological and clinical measurements in HIV-infected ART-naïve children aged>5yrs (n=163). Spearman rank correlation tests. b: Correlation matrix in a subset of n=45 with available data for all regarded parameters from the same study visit. Positive correlations are indicated in blue and inverse correlations in red. Darker color shades indicate higher r-values based on Spearman rank correlation tests. Clustering of variables is based on principal component analysis using the R package ‘Corrgram’ and reveals two well-differentiated groups of parameters, one associated with disease non-progression (upper left quadrant) and the other with disease progression (lower right quadrant).

Fig. 4.. Neutralization of a panel of 16 tier-2 and −3 subtype C, B and A viruses by pediatric and adult plasma samples.

a: Neutralization breadth in pediatric non-progressors (n=66) and progressors (n=19) compared with adults (n=21). b. The frequency of bnAbs among pediatric non-progressors (median age 6.6yrs, absolute CD4 T-cell count 1,050 cells/mm³, viral load 14,000 copies/ml), pediatric progressors (median age 8.2yrs, absolute CD4 T-cell count 225 cells/mm³, viral load 71,803 copies/ml) and adults (five years after infection, median absolute CD4 T-cell count 449 cells/mm³, viral load 31,200 copies/ml). c. Correlation between viral load and % viruses neutralized among pediatric subjects. P- and r-values were calculated by Spearman rank correlation tests. D–f. Geometric means of neutralization titers for pediatric and adult samples against subtype C (n=6), B (n=6) and A (n=4) viruses. Comparisons between groups were calculated by Mann-Whitney tests.

Fig. 5.. HIV-specific CD8+ T-cell responses in pediatric infection in relation to viral load and absolute CD4 count.

a–c. Breadth of IFN-γ elispot responses correlate with viral load in a protein-specific fashion. a–b. Total breadth and Gag-specific breadth of IFN-γ elispot response (n=90). c. Env-specific breadth of IFN-γ elispot response (n=90). d–e. Total breadth and Pol-specific breadth of IFN-γ elispot response in ART-naïve pediatric subjects is inversely correlated with absolute CD4 count (n=90). f. Total breadth of IFN-γ elispot response directly correlates with CD8+ T-cell activation in pediatric infection (n=30). Spearman rank correlation tests.

Fig. 6.. CCR5 expression and HIV infection is lower in central memory CD4+ T-cells in pediatric non-progressors than in progressors.

a. Representative FACS data of CCR5 expression in pediatric progressors versus non-progressors. b. CCR5 expression on CD4+ T-cell subsets in ART-naïve children aged>5yrs by absolute CD4 T-cell count (n=59). P- and r-values were calculated by Spearman rank correlation tests. c. Absolute CD4 T-cell counts and viral loads in pediatric non-progressors and adult future progressors. d. HIV infection in Tn, Tscm, Tcm and Tem in pediatric non-progressors and adult future progressors, determined by qPCR of HIV DNA in sorted CD4 T-cell subsets. P-values were determined by Mann-Whitney tests