Pulmonary Tuberculosis in Humanized Mice Infected with HIV-1

Co-infection with HIV increases the morbidity and mortality associated with tuberculosis due to multiple factors including a poorly understood microbial synergy. We developed a novel small animal model of co-infection in the humanized mouse to investigate how HIV infection disrupts pulmonary containment of Mtb. Following dual infection, HIV-infected cells were localized to sites of Mtb-driven inflammation and mycobacterial replication in the lung. Consistent with disease in human subjects, we observed increased mycobacterial burden, loss of granuloma structure, and increased progression of TB disease, due to HIV co-infection. Importantly, we observed an HIV-dependent pro-inflammatory cytokine signature (IL-1β, IL-6, TNFα, and IL-8), neutrophil accumulation, and greater lung pathology in the Mtb-co-infected lung. These results suggest that in the early stages of acute co-infection in the humanized mouse, infection with HIV exacerbates the pro-inflammatory response to pulmonary Mtb, leading to poorly formed granulomas, more severe lung pathology, and increased mycobacterial burden and dissemination

Characterization of the <i>Burkholderia mallei tonB</i> Mutant and Its Potential as a Backbone Strain for Vaccine Development

<div><p>Background</p><p>In this study, a <i>Burkholderia mallei tonB</i> mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis.</p><p>Methodology/Principal Findings</p><p>Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following <i>B</i>. <i>mallei</i> CSM001 challenge of mice previously receiving 1.5 x 10⁴ CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of <i>B</i>. <i>mallei</i>-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with <i>B</i>. <i>pseudomallei</i>, TMM001-treated mice were significantly protected. While wild type was cleared in all <i>B</i>. <i>mallei</i> challenge studies, mice failed to clear TMM001.</p><p>Conclusions/Significance</p><p>Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis.</p></div

TMM001 (1.5x10⁴ CFU) provides 100% protection against CSM001 challenge.

<p>Mice were immunized i.n. with PBS (solid triangle), 1.5 x 10⁴ CFU (solid circle), 1.5 x 10³ CFU (solid inverted triangle), or 1.5 x 10² CFU (solid square) of TMM001. Three weeks later, mice were challenged with 1.5 x 10⁵ CFU of CSM001. The statistical significance of differences in survival times was determined by plotting Kaplan-Meier curves, followed by a log rank test. ^★★★ p ≤ 0.001, ^★★ p ≤ 0.01.</p

Treatment with TMM001 reduces the pro-inflammatory cytokine and chemokine response to <i>B</i>. <i>mallei</i> challenge.

<p>Serum cytokine/chemokine profile of PBS- or TMM001-treated mice following exposure to <i>B</i>. <i>mallei</i> CSM001 (1.5x10⁵ CFU) at 0 h (A) and 48 h (B) post challenge. Mean ± SEM plotted are representative of three animals. Statistical significance was determined by one-way ANOVA followed by the Dunnett's test. ^★ p ≤ 0.05.</p

<i>B</i>. <i>mallei-</i>specific immunoglobulin levels in TMM001- vs PBS-treated mice before challenge.

<p>Murine serum samples were taken 21 days post-immunization, diluted 1:10,000 and analyzed for <i>B</i>. <i>mallei-</i>specific IgG1 (A), IgG2a (B) and IgM (C). Mean ± SEM of three representative animals is plotted. Statistical significance was determined by the unpaired <i>t</i> test with equal SD. ^★ p ≤ 0.05 ^★★★ p ≤ 0.001, ^★★★★ p ≤ 0.0001.</p

TMM001 attenuated growth kinetics is partially rescued by iron supplementation.

<p>Overnight cultures of wild type (solid square) and TMM001 (solid circle) were diluted 1:100 in 50 mL of Luria broth with 4% glycerol (LBG). An additional overnight culture of TMM001 (solid triangle) was diluted 1:100 in 50 mL of LBG + 200 μM FeSO₄. At the indicated time points, optical densities at 600 nm of all strains were measured. The average with their SD is representative of three independent experiments. Statistical significance was determined by Dunnett’s test of multiple comparisons relative to wild-type. ^★★ p ≤ 0.001, ^★★★ p ≤ 0.0001, ^★★★★ p ≤ 0.0001.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Attenuated virulence of TMM001 is partially rescued by iron supplementation.

<p>Mice (n = 8) were challenged i.n. with 1.5 x 10⁵ CFU (solid circle/open circle), 1.5 x 10⁶ CFU (solid square/open square) or 1.5 x 10⁷ CFU (solid triangle/open triangle) of TMM001 grown in LBG with (open) or without (closed) 200 μM FeSO₄. The statistical significance of differences in survival times was determined by plotting Kaplan-Meier curves, followed by a log rank test. ^★★★★ p ≤ 0.0001.</p

Colonization of target organs by TMM001 is partially rescued by iron supplementation.

<p>Bacterial burden in the lungs (A) and spleen (B) of mice infected with CSM001 and TMM001 grown ± 200 μM FeSO₄ at 24, 48 and 72 h post infection. Bars plotted with their SD represent the mean of three independent experiments. Significant differences in colonization at 24 and 48 h were individually ascertained via one-way ANOVA followed by Tukey’s multiple comparisons test. Significant difference in colonization at 72 h was extrapolated by using an unpaired <i>t</i> test with equal SD. ^★ p ≤ 0.05, ^★★★p ≤ 0.001, ^★★★★p ≤ 0.0001, ns = no significance.</p

Histopathology of TMM001- vs PBS-treated mice 48 h post challenge.

<p>H&E-stained lung (A, B) and spleen (C, D) of CSM001 (1.5 x 10⁵ CFU) challenged mice previously immunized with PBS (A, C) or TMM001 (1.5 x 10⁴ CFU) (B, D). Scale bar = 100μM. Histological scores were assigned for the liver (E), lungs (F), and spleen (G) tissue sections after microscopic examination. Mean ± SEM, representative of three animals, is plotted. Statistical significance was determined by the Mann-Whitney test. ^★ p ≤ 0.05.</p