Simultaneous immunization against tuberculosis

Abstract

Background: BCG, the only licensed vaccine against tuberculosis, provides some protection against disseminated disease in infants but has little effect on prevention of adult pulmonary disease. Newer parenteral immunization prime boost regimes may provide improved protection in experimental animal models but are unproven in man so that there remains a need for new and improved immunization strategies.

Methods and findings: Mice were immunized parenterally, intranasally or simultaneously by both routes with BCG or recombinant mycobacterial antigens plus appropriate adjuvants. They were challenged with Mycobacterium tuberculosis (Mtb) and the kinetics of Mtb growth in the lungs measured. We show that simultaneous immunization (SIM) of mice by the intranasal and parenteral routes is highly effective in increasing protection over parenteral BCG administration alone. Intranasal immunization induces local pulmonary immunity capable of inhibiting the growth of Mtb in the early phase (the first week) of infection, while parenteral immunization has a later effect on Mtb growth. Importantly, these two effects are additive and do not depend on priming and boosting the immune response. The best SIM regimes reduce lung Mtb load by up to 2 logs more than BCG given by either route alone.

Conclusions: These data establish SIM as a novel and highly effective immunization strategy for Mtb that could be carried out at a single clinic visit. The efficacy of SIM does not depend on priming and boosting an immune response, but SIM is complementary to prime boost strategies and might be combined with them.

Figure 1. Mtb cfu after SIM with BCG and subunit vaccines.

a. C57BL/6 mice were immunized once with BCG with or without simultaneous administration of 85A s.c. or 85A i.n., or b, once with BCG with or without simultaneous administration of E6 s.c. or E6 i.n. and c, once with BCG with or without simultaneous administration of 10.4 s.c. or 10.4 i.n.. Ten weeks after the last immunization they were challenged with Mtb i.n. and a further 5 weeks sacrificed later for enumeration of lung Mtb cfu. Representative data from one of two experiments with 5–7 mice/group are shown. ***p<0.001, **p<0.01, * p<0.05 between the indicated groups, one-way ANOVA with Tukey's post test. Data are means ± s.e.m.

Figure 2. Mtb cfu after SIM with subunit vaccines.

a. C57BL/6 mice were immunized 3 times at 2 weekly intervals with 85A s.c., or E6 i.n. either alone or in combination with appropriate adjuvants. Six weeks after the last immunization mice were challenged with Mtb and sacrificed for lung cfu enumeration 5 weeks later. b. Mice were immunized 3 times with 85A s.c. or E6 s.c. with MPL, separately or in combination and then challenged. c, the same antigens were administered 3 times i.n. with CT before challenge. d. Mice were immunized with 85A i.n. or s.c. in appropriate adjuvants or MPL or CT were administered 3 times s.c. or i.n separately or simultaneously and e, Rv1284 was administered s.c. or i.n. 3 times with appropriate adjuvants before Mtb challenge and enumeration as in a. Representative data from one of two experiments with 5–7 mice/group are shown. ***p<0.001, **p<0.01, * p<0.05 between the indicated groups, one-way ANOVA with Tukey's post test. Data are means ± s.e.m.

Figure 3. Mtb cfu after SIM with BCG.

a and b. In two experiments, C57BL/6 mice were immunized once with BCG s.c. or BCG i.n. or simultaneously with the same dose of BCG divided between the s.c./i.n. routes. Ten weeks later they were challenged with Mtb and lung cfu enumerated 5 weeks later. Data from the two experiments with 5–7 mice/group are shown. ***p<0.001, **p<0.01, * p<0.05 between the indicated groups, one-way ANOVA with Tukey's post test. Data are means ± s.e.m.

Figure 4. Cytokine responses of lung and spleen T cells to antigens.

a. Mice were immunized with BCG s.c., BCG s.c./85A s.c. or BCG s.c./85A i.n. as in Fig. 1a. Lung and spleen cells were isolated 10 weeks after immunization and stimulated with pooled 85A peptides for 6 hours. b. mice were immunized 3 times at 2 weekly intervals with 85A s.c., 85A i.n., E6 s.c. or E6 i.n. either alone or in combination as in Fig. 2a,b,c and cells isolated 6 weeks after immunization and stimulated with pooled 85A peptides or E6 for 6 hours. c, mice were immunized with BCG s.c., BCG i.n. or BCG s.c./i.n. as in Fig. 3b and lung cells were isolated 10 weeks after immunization and stimulated for 12 hours with PPD. After stimulation the proportion of IFNγ, IL-2 and TNF producing cells was determined by flow cytometry of CD4 gated cells (numbers of responding CD8 cells were too low for reliable analysis). Results are expressed as the means ± s.e.m. of 3 or 4 mice per group and are representative of 2 independent experiments. In a, ***p<0.001, **p<0.01 indicate significant differences between numbers of spleen IL-2 producing cells only. In b,**p<0.01 between all cytokines in the indicated groups and in c, **p<0.01 for TNF only. All other groups differ significantly from the naïve group but these comparisons are omitted for simplicity. One-way ANOVA with Tukey's post test.

Figure 5. Kinetics of Mtb growth after s.c. and i.n. immunization.

C57BL/6 mice were immunized once with BCG s.c., BCG i.n. or BCG s.c./BCG i.n. or 3 times at two weekly intervals with 85A s.c., 85A i.n., E6 s.c. or E6 i.n. Ten weeks after immunization with BCG or 4 weeks after the last immunization with 85A or E6, mice were challenged with Mtb and groups of 3–5 mice sacrificed 7, 14, 21 and 28 days later for enumeration of lung Mtb cfu. # indicates a significant difference between s.c immunized and naïve mice and * a significant difference between i.n. or SIM immunized mice and naïve controls, one-way ANOVA with Tukey's post test. Data are means ± s.e.m. Standard errors are small, so that the error bars are within the symbols when not visible.

Figure 6. Mtb growth in individual mice after s.c. and i.n. immunization.

The figure shows results for individual mice from the experiments shown in Fig. 5. C57BL/6 mice were immunized once with BCG s.c., BCG i.n. or BCG s.c./BCG i.n. or 3 times at two weekly intervals with 85A s.c., 85A i.n., E6 s.c. or E6 i.n. Ten weeks after immunization with BCG or 4 weeks after the last immunization with 85A or E6 mice were challenged with Mtb and groups of 3–5 mice sacrificed 7, 14, 21 and 28 days later for enumeration of lung Mtb cfu. Representative data from one of two experiments are shown. Horizontal lines show group means. # indicates a significant difference from naïve mice and * a significant difference between indicated groups. One-way ANOVA with Tukey's post test.