c mice and guinea pigs against Mycobacterium tuberculosis challenge - PubMed (original) (raw)

Listeria-vectored multi-antigenic tuberculosis vaccine protects C57BL/6 and BALB/c mice and guinea pigs against Mycobacterium tuberculosis challenge

Qingmei Jia et al. Commun Biol. 2022.

Abstract

Mycobacterium tuberculosis (Mtb) infects one-third of the world's population and is a leading cause of death from a single infectious agent. New TB vaccines are urgently needed to augment immunity conferred by the current modestly protective BCG vaccine. We have developed live attenuated recombinant Listeria monocytogenes (rLm)-vectored TB vaccines expressing five [Mpt64/23.5-EsxH/TB10.4-EsxA/ESAT6-EsxB/CFP10-Ag85B/r30] (rLmMtb5Ag) or nine (additionally EsxN-PPE68-EspA-TB8.4) immunoprotective Mtb antigens (rLmMtb9Ag) and evaluated them for safety, immunogenicity and efficacy as standalone vaccines in two mouse models and an outbred guinea pig model. In immunogenicity studies, rLmMtb5Ag administered subcutaneously induces significantly enhanced antigen-specific CD4+ and CD8+ T-cell responses in C57BL/6 and BALB/c mice, and rLmMtb9Ag induces antigen-specific CD4+ and CD8+ T-cell proliferation in guinea pigs. In efficacy studies, both rLmMtb5Ag and rLmMtb9Ag are safe and protect C57BL/6 and BALB/c mice and guinea pigs against aerosol challenge with highly virulent Mtb. Hence, multi-antigenic rLm vaccines hold promise as new vaccines against TB.

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Conflict of interest statement

The authors declare no competing interests except M.A.H. and Q.J. are inventors on a patent application and on U.S. Patent 10,010,595 filed by UCLA on Listeria monocytogenes-vectored TB vaccines described in this and previous studies.

Figures

Fig. 1. Construction and verification of rLm9Ag vaccine.

a Diagram of rLm5Ag and rLm9Ag vaccine candidates. Expression cassettes ActAN-5Ag for rLm5Ag (top panel) and ActAN-5AgII for rLm5AgII (middle panel) driven by the Lm actA promoter were integrated at the tRNA arg loci of the corresponding rLm chromosome_;_ expression cassettes ActAN-5Ag and ActAN-5AgII for rLm9Ag were integrated at the comK and tRNA arg loci of the Lm chromosome, respectively (bottom panel). b Expression of heterologous and homologous proteins by rLm vaccine candidates grown in broth medium. Stocks of rLm vaccine candidates were inoculated into Brain Heart Infusion (BHI) medium and grown overnight at 37 °C with agitation. The overnight culture was collected by centrifugation, resuspended in PBS supplemented with Halt Proteinase Inhibitor (Thermo Fisher Scientific), and lysed in SDS buffer. Equivalent amounts of bacterial lysates of each rLm were analyzed by Western blotting sequentially using rabbit polyclonal antibody to ActAN (pAb AK18) (top panel), rabbit polyclonal antibody to Mtb EsxH (middle panel); and monoclonal antibody to Lm P60 (serving as a loading control). The membrane was stripped before re-probing. c The murine macrophage-like (J774A.1) cells were uninfected (UI) or infected at a MOI of 10 with LmVector or an rLm vaccine expressing Mtb 5Ag, 5AgII, or 9Ag that had been grown to stationary phase. At 5.5 h post-infection, the infected cells were lysed and subjected to Western blotting using polyclonal antibody AK18 (upper panel); the membrane was stripped and re-probed with a monoclonal antibody to Lm P60 and a monoclonal antibody to cellular protein β-actin (lower panel), as indicated on the right border of each panel. In both panels b and c: M, protein standards; Lane 1, LmVector (LmV); Lanes 2 and 3, two clones of rLm5Ag expressing ActAN-Mpt64-EsxH-EsxA-EsxB-r30 from the tRNA arg locus; Lane 4, rLm5AgII expressing ActAN-Mpt64-EsxN-PPE68-EspA-TB8.4 from the tRNA arg locus; Lanes 5 and 6, two clones of rLm9Ag expressing a total of 9 different antigens, ActAN-5Ag from the comK locus and ActAN-5AgII from the tRNA arg locus; lane 7 (Panel c only), uninfected cells. On the left border of each panel are listed the sizes (kDa) of the molecular mass standards. Proteins of interests are indicated with arrows to the right of the protein band. Estimated Mw of ActAN-5AgII: 118-kDa; ActAN-5Ag: 94-kDa; Lm P60: 60 kDa; and β-actin, 42 kDa. The full-length images for b and c are shown in Supplementary Fig. 1.

Fig. 2. Frequency of cytokine-expressing CD4+ T cells in the lungs and spleens of C57BL/6 mice immunized three times with LmVector or rLm5Ag.

C57BL/6 mice (n = 4/group) were immunized s.q. three times with LmVector (black) or rLm5Ag (pink) at Weeks 0, 4, and 8. Six days after the last immunization, mice were euthanized; their lungs and spleens removed; single cell suspensions prepared and stimulated with individual proteins of 23.5/Mpt64 (a, i), TB10.4/EsxH (b, j), ESAT6/EsxA (c, k), CFP10/EsxB (d, l), r30/Ag85B (e, m), or pool of 5 antigens (5Ag pool) (f, n), or PPD (g, o) in the presence of anti-CD28 monoclonal antibody for 6 h; GolgiPlug (protein transport inhibitor containing Brefeldin A) diluted in T-cell medium was added to all wells; additional cells incubated with PMA and Golgiplug for 4 h served as positive control (h, p). The cells were assayed by excluding dead cells followed by staining for surface markers of CD4 and CD8 followed by CD3 and intracellular markers of IFN-γ, TNF-α, IL-2, and IL-17A, as indicated below each panel. Frequencies of live CD4+ T cells expressing any of the four cytokines were analyzed by FlowJo 10 software. Values are the mean ± standard error of the mean (SEM). *P < 0.05; and **P < 0.01 by two-way ANOVA with Sidak’s post multiple comparisons test. A similar experiment was repeated in C57BL/6 mice.

Fig. 3. Frequency of polyfunctional cytokine-expressing CD4+ T cells in the lungs and spleens of C57BL/6 mice immunized three times with LmVector or rLm5Ag.

C57BL/6 mice (n = 4/group, the same mice as shown in Fig. 2) were immunized three times s.q. with LmVector (black) or rLm5Ag (pink) at Weeks 0, 4, and 8. One week after the last immunization, animals were euthanized; their lung and spleen cells processed as described in the legend to Fig. 2. The lung and spleen cells were stimulated with r30/Ag85B (a, e), pool of 5 antigens (5Ag pool) (b, f), PPD (c, g), or PMA (positive control, (d, h), and assayed by intracellular cytokine staining for surface markers of CD4 and CD8 followed by CD3 and intracellular markers of IFN-γ, TNF-α, IL-2, and IL-17A. The frequencies of live CD4+ T cells producing any of the 15 possible combinations of the four cytokines (IFN-γ, TNF-α, IL-2, and IL-17A) were uniquely distinguished by using Boolean gates of FlowJo 10 software, as indicated below each panel. Values are the mean ± SEM. **P < 0.01; ***P < 0.001; and ****P < 0.0001 by two-way ANOVA with Sidak’s post multiple comparisons test.

Fig. 4. Frequency of cytokine-expressing CD8+ T cells in the lungs and spleens of C57BL/6 mice immunized three times with LmVector or rLm5Ag.

C57BL/6 mice (n = 4/group, the same mice as shown in Fig. 2) were immunized three times s.q. with LmVector (black) or rLm5Ag (pink) at Weeks 0, 4, and 8. One week after the last immunization, animals were euthanized and their lung and spleen cells processed as described in the legend to Fig. 2. The lung and spleen cells were stimulated with individual proteins of 23.5/Mpt64 (a, i), TB10.4/EsxH (b, j), ESAT6/EsxA (c, k), CFP10/EsxB (d, l), or r30/Ag85B (e, m), or pool of 5 antigens (5Ag pool) (f, n), PPD (g, o), or PMA (h, p), and assayed by intracellular cytokine staining for surface markers of CD4 and CD8 followed by CD3 and intracellular markers of IFN-γ, TNF-α, IL-2, and IL-17A. Frequencies of CD8+ T cells expressing any of the four cytokines are analyzed by FlowJo 10 software. Values are the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by two-way ANOVA with Sidak’s post multiple comparisons test.

Fig. 5. Frequency of polyfunctional cytokine-expressing CD8+ T cells in the lungs of C57BL/6 mice immunized three times with LmVector or rLm5Ag.

C57BL/6 mice (n = 4/group, the same mice as shown in Fig. 2) were immunized three times s.q. with LmVector (black) or rLm5Ag (pink) at Weeks 0, 4, and 8. One week after the last immunization, animals were euthanized and their lung cells processed as described in the legend to Fig. 2. The lung cells were stimulated with individual proteins of 23.5/Mpt64 (a) or TB10.4/EsxH (b), or pool of 5 antigens (5Ag pool) (c) or PPD (d), and assayed by intracellular cytokine staining for surface markers of CD4 and CD8 followed by CD3 and intracellular markers of IFN-γ, TNF-α, IL-2, and IL-17A. The frequencies of live CD8+ T cells producing any of the 15 possible combinations of the four cytokines (IFN-γ, TNF-α, IL-2, and IL-17A) were uniquely distinguished by using Boolean gates of FlowJo 10 software, as indicated below each panel. Values are the mean ± SEM. ****P < 0.0001 by two-way ANOVA with Sidak’s post multiple comparisons test.

Fig. 6. Efficacy of immunization with rLm vaccines expressing 5 Mtb antigens against Mtb aerosol challenge.

a Top Panel: C57BL/6 mice (n = 8/group) were immunized i.d. with PBS (Sham), i.d. or i.n with BCG at Week 0, or immunized i.d. or i.n. twice at Weeks 7 and 10 with rLm5Ag* (rLm30 + rLm4Ag), challenged at Week 13 with aerosolized Mtb Erdman strain (average of 24 CFU delivered to the lung of each animal, as assayed at Day 1 post challenge) and euthanized at Week 23. Middle and Bottom Panels: Lungs and spleens of mice immunized i.d. (left panels) or i.n. (right panels) were assayed for organ bacterial burden. Shown are means ± SEM. Each symbol represents one mouse. *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001 by 2-tailed non-paired t-test (Prism). b Top Panel: BALB/c mice (8/group) were immunized i.d. with PBS or i.d. with BCG at Week 0, or immunized i.m. twice (x2) at Weeks 14 and 18 or three times (x3) at Weeks 10, 14, and 18 with rLm5Ag*. The mice were then challenged with aerosolized Mtb Erdman strain (average of 30 CFU delivered to lung of each animal, as assayed at Day 1 post challenge) at Week 22 and euthanized at Week 32. Middle and Bottom Panels: Lungs and spleens were assayed for organ bacterial burden. Shown are means ± SEM. Each symbol represents one mouse. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test (Prism 9.2.0).

Fig. 7. Efficacy of immunization with rLm5Ag and rLm9Ag vaccines against Mtb aerosol challenge in C57BL/6 mice.

a C57BL/6 mice (n = 8/group) were unimmunized (UI), vaccinated i.d. with 5 × 105 CFU BCG at Week 0, or vaccinated s.q. three times with LmVector, rLm30, rLm5Ag* (rLm30 + rLm4Ag), rLm5Ag (a single vaccine expressing the same 5 Mtb antigens as rLm5Ag*), or rLm9Ag (clones #1 and #3) at Weeks 0, 3, and 6. The mice were then challenged at Week 10 with aerosolized Mtb Erdman strain (average of 10 CFU delivered to the lungs of each animal, as assayed at Day 1 post challenge) and euthanized at Week 20. b Afterwards, lungs (left) and spleens (right) were removed and assayed for bacillus burdens. Shown are means ± SEM. Each symbol represents one mouse. *P < 0.05; **P < 0.01; and ***P < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test (Prism 9.2.0). A similar experiment was repeated in BALB/c mice.

Fig. 8. Efficacy of immunization with rLm5Ag and rLm9Ag vaccines against Mtb aerosol challenge in BALB/c mice.

a BALB/c mice (n = 8/group) were unimmunized (UI), vaccinated i.d. with 5 × 105 CFU BCG at Week 0, or vaccinated s.q. three times with LmVector, rLm30, rLm5Ag* (combination of rLm30 + rLm4Ag), rLm5Ag (a single vaccine expressing the same 5 Mtb antigens as rLm5Ag*), or rLm9Ag (clones #1 and #3) at Weeks 0, 3, and 6. The mice were then challenged at Week 10 with aerosolized Mtb Erdman strain (average of 10 CFU delivered to the lungs of each animal, as assayed at Day 1 post challenge) and euthanized at Week 20. b. Afterwards, lungs (left) and spleens (right) were removed and assayed for bacillus burdens. Shown are means ± SEM. Each symbol represents one mouse. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test (Prism). A similar experiment was repeated in C57BL/6 mice.

Fig. 9. Frequencies of all lymphocytes and proliferating CD4+ and CD8+ T cells in the lungs and spleens of guinea pigs immunized with LmVector or rLm9Ag.

Guinea pigs (n = 4/group) were immunized three times s.q. with LmVector (black) or rLm9Ag (pink) at Weeks 0, 3, and 6. Six days after the last immunization, guinea pigs were euthanized; their spleens and lungs removed; single cell suspensions of spleen and lung cells prepared and treated with Cell Tracer Violet (CTV), followed by incubation with medium alone (negative control), medium with addition of 1 µg/ml per peptide of each of the 9 Mtb antigen peptide pools plus anti-CD28 monoclonal antibody (mAb), medium with addition of 5 µg/ml of PPD protein plus anti-CD28 mAb, or medium with addition of 5 µg/ml of ConA (positive control), as indicated beneath the horizontal axis, for 4 days. Subsequently, the cells were stained with Live/dead staining dye and surface markers of CD4-PE and CD8-FITC. Total lung lymphocytes (a) and total spleen lymphocytes (b) were gated by forward and side scatter patterns; proliferating lung CD4+ T cells (c), proliferating spleen CD4+ T cells (d), proliferating lung CD8+ T cells (e), and proliferating spleen CD8+ T cells (f) were gated by corresponding antibody staining. Values are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by two-way ANOVA with Sidak’s post multiple comparisons test (Prism 9.2.0). The experiment was repeated once with similar results.

Fig. 10. Protective efficacy of rLm9Ag and rLm5Ag vaccines against Mtb aerosol challenge in guinea pigs.

a Experimental design. Guinea pigs (n = 10/group) were immunized i.d. with 103 CFU BCG (Positive Control) at Week 0, or immunized s.q. three times with rLm9Ag at 105 (low dose, L), 106 (Medium dose, M), or 107 (High Dose, H) CFU, or with 106 rLm5Ag at Weeks 0, 3, and 6. The animals were then challenged at Week 10 with aerosolized Mtb Erdman strain. Afterwards, animals were monitored for signs of illness and weight change weekly for 10 weeks. At Week 10 post challenge, animals were euthanized and their spleen and right lungs were removed and assayed for bacillus burdens. b Percent weight change post challenge. Shown are means ± SEM. See Supplementary Fig. 8 for data points. See Supplementary Table 2 for statistical analyses comparing LmVector, rLm5Ag, rLm9Ag, and BCG groups by two-way ANOVA with Tukey’s multiple comparisons test (Prism 9.2.0). c Right lung (left) and spleen bacterial burden. Shown are means ± SEM. Each symbol represents one animal. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test.

References

1. Bloom BR. New promise for vaccines against tuberculosis. N. Engl. J. Med. 2018;379:1672–1674. doi: 10.1056/NEJMe1812483. -DOI -PubMed
1. WHO. Global Tuberculosis Report 2021, https://www.who.int/publications/i/item/9789240037021 (2021).
1. Knight GM, et al. Impact and cost-effectiveness of new tuberculosis vaccines in low- and middle-income countries. Proc. Natl. Acad. Sci. USA. 2014;111:15520–15525. doi: 10.1073/pnas.1404386111. -DOI -PMC -PubMed
1. Horwitz MA, Harth G, Dillon BJ, Maslesa-Galic S. Recombinant bacillus calmette-guerin (BCG) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein induce greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model. Proc. Natl. Acad. Sci. USA. 2000;97:13853–13858. doi: 10.1073/pnas.250480397. -DOI -PMC -PubMed
1. Horwitz MA, Harth G. A new vaccine against tuberculosis affords greater survival after challenge than the current vaccine in the guinea pig model of pulmonary tuberculosis. Infect. Immun. 2003;71:1672–1679. doi: 10.1128/IAI.71.4.1672-1679.2003. -DOI -PMC -PubMed

Listeria-vectored multi-antigenic tuberculosis vaccine protects C57BL/6 and BALB/c mice and guinea pigs against Mycobacterium tuberculosis challenge - PubMed (original) (raw)