Dysregulated humoral immunity to nontyphoidal Salmonella in HIV-infected African adults

Abstract

Nontyphoidal Salmonellae are a major cause of life-threatening bacteremia among HIV-infected individuals. Although cell-mediated immunity controls intracellular infection, antibodies protect against Salmonella bacteremia. We report that high-titer antibodies specific for Salmonella lipopolysaccharide (LPS) are associated with a lack of Salmonella-killing in HIV-infected African adults. Killing was restored by genetically shortening LPS from the target Salmonella or removing LPS-specific antibodies from serum. Complement-mediated killing of Salmonella by healthy serum is shown to be induced specifically by antibodies against outer membrane proteins. This killing is lost when excess antibody against Salmonella LPS is added. Thus, our study indicates that impaired immunity against nontyphoidal Salmonella bacteremia in HIV infection results from excess inhibitory antibodies against Salmonella LPS, whereas serum killing of Salmonella is induced by antibodies against outer membrane proteins.

Fig. 1

Dysregulated humoral immunity to NTS in HIV infection. Killing of (A and C) S. Typhimurium isolate A23753, and (B and D) serum-sensitive S. Typhimurium isolate A19520 by sera at 45, 90 and 180 minutes. Negative values correspond with a decrease in viable Salmonellae compared with the initial concentration. (E and F) Serum titers of Salmonella A23753 IgG compared with killing of S. Typhimurium isolate A23753 at 180 minutes, and (G and H) C5b-9 MAC deposition on A23753. (A, B, E and G) sera from HIV-uninfected Africans (n=58). (C, D, F and H) sera from HIV-infected Africans (n=58). Each line or point represents data for serum from one individual. Note all lines are superimposed in (B). Horizontal dashed line indicates threshold for impaired killing of S. Typhimurium A23753 (−0.9 log₁₀ change in Salmonellae cfu/ml).

Fig. 2

Inhibition of HIV-uninfected control serum killing of NTS by HIV-infected sera with impaired Salmonella-killing ability. (A) Killing of S. Typhimurium isolate A23753 at 180 minutes by mixed sera consisting of different percentages of HIV-infected serum (n=12, serum from one HIV-infected subject per line) and control HIV-uninfected serum. Horizontal dashed line indicates threshold for impaired killing of S. Typhimurium A23753. (B-E) Inhibition of control serum killing of S. Typhimurium isolate D23580 (B and D), and serum-sensitive S. Typhimurium isolate D19774 (C and E) by size-fractionated (B and C) and IgG fraction (D and E) of HIV-infected serum. Data are means ± SD of 3 experiments. Inhibition of killing of both strains of Salmonella by HIV-uninfected sera with <300 kDa fraction of HIV-infected serum compared with <100 kDa fraction, and with IgG fraction of HIV-infected serum compared with IgG fraction of HIV-uninfected serum was significant by Student’s t test (P<0.0001).

Fig. 3

LPS antibodies prevent killing of NTS by HIV-infected serum. (A and B) Killing of S. Typhimurium A23753 at 180 minutes by HIV-uninfected sera (A) and HIV-infected sera (B) compared with S. Typhimurium LPS IgG titer determined by ELISA. Horizontal dashed line indicates threshold for impaired killing of S. Typhimurium A23753. r values is Spearman correlation coefficient. Median IgG titer was higher in HIV-infected sera compared with HIV-uninfected sera (Mann-Whitney P<0.002, difference in medians 20 units, 95% CI 6.0 to 39). (C) Killing of indicated S. Typhimurium D23580 strains by HIV-infected serum. (D) Effect of preabsorbing HIV-infected serum with 100 μg/ml S. Typhimurium flagellin or outer membrane proteins on killing of S. Typhimurium D23580. (E and F) Effect of preabsorbing HIV-infected serum with 1, 10 or 100μg/ml LPS on serum killing of (E) D23580 and (F) D19774. (G and H) Effect of adding LPS antibodies at one tenth concentration in inhibitory HIV-infected source serum to HIV-uninfected serum on killing of (G) D23580 and (H) D19774. Data represent means ± SD of 3 experiments. Killing of both strains of Salmonella by HIV-infected sera preabsorbed with 100 μg/ml LPS compared with unabsorbed serum, and inhibition of killing of both strains of Salmonella by HIV-uninfected serum with exogenous LPS antibody added compared with PBS added was significant by Student’s t test P<0.0001.

Fig. 4

Antibodies targeted against outer membrane proteins mediate African serum killing of NTS. (A) Effect of preabsorbing HIV-uninfected serum with 200 μg/ml of S. Typhimurium outer membrane proteins, flagellin or LPS on killing of S. Typhimurium D23580. (B) Effect of adding OMP F, C and D antibodies from four mice immunized with S. Typhimurium OMP F, C and D to antibody-deficient HIV-uninfected human serum on killing of S. Typhimurium D23580 compared with adding antibody from four unimmunized mice. Each line represents log₁₀ change of Salmonella induced by antibody from one mouse. (C) Effect of adding outer membrane protein antibodies from four HIV-uninfected sera (one per line) at one tenth concentration present in source serum, to antibody-deficient serum on killing of S. Typhimurium D23580. Note that lines are superimposed. (D and E) Effect of adding outer membrane protein antibodies and LPS antibodies extracted and purified from inhibitory HIV-infected serum to antibody-deficient serum (D) or immune HIV-uninfected serum (E) on killing of S. Typhimurium D23580. (A, E and F) Data represent means +/− SD of 3 experiments.