Reduced protective efficacy of a blood-stage malaria vaccine by concurrent nematode infection

Abstract

Helminth infections, which are prevalent in areas where malaria is endemic, have been shown to modulate immune responses to unrelated pathogens and have been implicated in poor efficacy of malaria vaccines in humans. We established a murine coinfection model involving blood-stage Plasmodium chabaudi AS malaria and a gastrointestinal nematode, Heligmosomoides polygyrus, to investigate the impact of nematode infection on the protective efficacy of a malaria vaccine. C57BL/6 mice immunized with crude blood-stage P. chabaudi AS antigen in TiterMax adjuvant developed strong protection against malaria challenge. The same immunization protocol failed to induce strong protection in H. polygyrus-infected mice. Immunized nematode-infected mice produced significantly lower levels of malaria-specific antibody than nematode-free mice produced. In response to nematode and malarial antigens, spleen cells from immunized nematode-infected mice produced significantly lower levels of gamma interferon but more interleukin-4 (IL-4), IL-13, and IL-10 in vitro than spleen cells from immunized nematode-free mice produced. Furthermore, H. polygyrus infection also induced a strong transforming growth factor beta1 response in vivo and in vitro. Deworming treatment of H. polygyrus-infected mice before antimalarial immunization, but not deworming treatment after antimalarial immunization, restored the protective immunity to malaria challenge. These results demonstrate that concurrent nematode infection strongly modulates immune responses induced by an experimental malaria vaccine and consequently suppresses the protective efficacy of the vaccine against malaria challenge.

FIG. 1.

Course of blood-stage P. chabaudi AS challenge infection in control and H. polygyrus-infected mice with and without antimalarial vaccination. Female C57BL/6 mice were infected with 200 H. polygyrus third-stage larvae (groups C and D). Two weeks after infection, a group of nematode-infected mice (group D) and a group of naïve mice (group B) were immunized with P. chabaudi AS antigen in TiterMax and boosted as described in Materials and Methods. Two weeks after boosting, all three groups of mice and a group of naïve control mice (group A) were infected i.p. with 10⁶ P. chabaudi AS-parasitized red blood cells, and malaria parasitemia was monitored for 3 weeks. The data shown are mean parasitemia levels (n = 5); the standard errors (not shown) were less than 10% of the mean parasitemia for one of three replicate experiments which showed similar patterns of P. chabaudi AS challenge infection in all four groups of mice. Repeated-measure ANOVA was used to analyze the differences in overall parasitemia levels between groups. Mortality, indicated by a dagger, was calculated from data (n = 13) pooled from three experiments. Three asterisks indicate that the P value was <0.001 for a comparison with naïve mice in group A. Hp, H. polygyrus-infected mice; Vacc, vaccinated mice.

FIG. 2.

P. chabaudi AS-specific total immunoglobulin, IgG1, and IgG2a antibody responses in control and H. polygyrus-infected mice with and without antimalarial vaccination. Groups of mice were infected with H. polygyrus, immunized with malaria antigen in TiterMax, and challenged with blood-stage P. chabaudi AS as described in the legend to Fig. 1. Ten days after malaria challenge, mice were sacrificed, and the levels of malaria-specific total Ig and antibody isotypes in sera were determined by ELISAs. The results are expressed as means and standard errors (n = 4) for one of two replicate experiments. Two asterisks indicate that the difference was significant (P < 0.01) for a comparison with vaccinated nematode-free mice. Hp, H. polygyrus-infected mice; Vacc, vaccinated mice.

FIG. 3.

In vitro cytokine production by spleen cells from normal control and H. polygyrus-infected mice with and without antimalarial vaccination. Mice were infected with H. polygyrus and immunized with malaria antigen in TiterMax as described in the legend to Fig. 1. Two weeks after boosting, mice were sacrificed, and spleen cells were cultured in medium or in the presence of pRBC as a source of P. chabaudi AS antigen (Pc-Ag), in the presence of H. polygyrus adult worm antigen (Hp-Ag), or in the presence of both antigens (2 Ags). The levels of IFN-γ (A), IL-4 (B), and IL-13 (C) were determined by ELISAs. The results are expressed as means and standard errors (n = 4) for one of two replicate experiments. Two asterisks indicate that the difference was significant (P < 0.01) for a comparison with vaccinated nematode-free mice with the same antigen stimulation. Hp, H. polygyrus-infected mice; Vacc, vaccinated mice.

FIG. 4.

In vitro IL-10 (A) and TGF-β1 (B) production by spleen cells and levels of bioactive TGF-β1 in plasma (C) from normal control and H. polygyrus-infected mice with and without antimalarial vaccination, as described in the legend to Fig. 1. (A and B) Spleen cells were collected from the four groups of mice and cultured in vitro as described in the legend to Fig. 3. Levels of IL-10 and total TGF-β1 were determined by ELISAs. The results are expressed as means and standard errors (n = 4) for one of two replicate experiments. Two asterisks indicate that the difference was significant (P < 0.01) for a comparison with vaccinated nematode-free mice with the same antigen stimulation. (C) Plasma samples were collected from normal mice and mice 2 weeks after H. polygyrus infection (Pre-immun) or from normal control and H. polygyrus-infected mice with and without antimalarial vaccination as described above (Pre-challenge), and levels of bioactive TGF-β1 in plasma were determined by ELISAs. The results are expressed as means and standard errors (n = 4) for one of two replicate experiments. Two asterisks indicate that the difference was significant (P < 0.01) for a comparison with either normal mice or vaccinated nematode-free mice. Vacc, vaccinated mice; Hp, H. polygyrus-infected mice; Pc-Ag, cells cultured in the presence of pRBC as a source of P. chabaudi AS antigen; Hp-Ag, cells cultured in the presence of H. polygyrus adult worm antigen; 2 Ags, cells cultured in the presence of both antigens.

FIG. 5.

Effect of anthelmintic treatment on the efficacy of a malaria vaccine. (A) Four groups of mice were infected with 200 H. polygyrus third-stage larvae (Hp). Two weeks after infection, mice in group D were treated with pyrantel pamoate (100 mg/kg of body weight) (Drug). Three weeks after nematode infection, mice in groups B, C, and D were immunized with malaria vaccine (Vacc) as described in Materials and Methods. In the last week of vaccination, mice in group C were treated with the anthelmintic drug. Mice in all four groups were challenged with 10⁶ P. chabaudi AS-parasitized red blood cells (Pc). (B) Levels of parasitemia in the four groups of mice following P. chabaudi AS challenge infection. The data are mean parasitemia levels (n = 4); the standard errors (not shown) were less than 10% of the mean parasitemia levels. The results are the results of one of two replicate experiments which showed similar patterns of P. chabaudi AS challenge infection in the four groups of mice. Repeated-measure ANOVA was used to analyze the differences in overall parasitemia levels between groups. Three asterisks indicate that the P value was <0.001 for a comparison with group B.