Type 3 secretion system cluster 3 is a critical virulence determinant for lung-specific melioidosis.

Burkholderia pseudomallei, the bacterial agent of melioidosis, causes disease through inhalation of infectious particles, and is classified as a Tier 1 Select Agent. Optical diagnostic imaging has demonstrated that murine respiratory disease models are subject to significant upper respiratory tract (URT) colonization. Because human melioidosis is not associated with URT colonization as a prominent presentation, we hypothesized that lung-specific delivery of B. pseudomallei may enhance our ability to study respiratory melioidosis in mice. We compared intranasal and intubation-mediated intratracheal (IMIT) instillation of bacteria and found that the absence of URT colonization correlates with an increased bacterial pneumonia and systemic disease progression. Comparison of the LD50 of luminescent B. pseudomallei strain, JW280, in intranasal and IMIT challenges of albino C57BL/6J mice identified a significant decrease in the LD50 using IMIT. We subsequently examined the LD50 of both capsular polysaccharide and Type 3 Secretion System cluster 3 (T3SS3) mutants by IMIT challenge of mice and found that the capsule mutant was attenuated 6.8 fold, while the T3SS3 mutant was attenuated 290 fold, demonstrating that T3SS3 is critical to respiratory melioidosis. Our previously reported intranasal challenge studies, which involve significant URT colonization, did not identify a dissemination defect for capsule mutants; however, we now report that capsule mutants exhibit significantly reduced dissemination from the lung following lung-specific instillation, suggesting that capsule mutants are competent to spread from the URT, but not the lung. We also report that a T3SS3 mutant is defective for dissemination following lung-specific delivery, and also exhibits in vivo growth defects in the lung. These findings highlight the T3SS3 as a critical virulence system for respiratory melioidosis, not only in the lung, but also for subsequent spread beyond the lung using a model system uniquely capable to characterize the fate of lung-delivered pathogen

Detection of dissemination of <i>B. pseudomallei</i> mutants by optical imaging.

<p>Groups of 5 female albino C57BL/6J mice were challenged by the IMIT route of infection with either wild type luminescent <i>B. pseudomallei</i> strain JW280, the Δ<i>wcb</i> capsule mutant, or the Δ<i>sctU</i>_Bp3 T3SS3 mutant. Representative images of disease endpoints are presented, with uniform image settings adjusted to a range of 2.5×10³ to 3×10⁴ p/s/cm2/sr on a logarithmic scale.</p

Detection of pulmonary growth rates of <i>B. pseudomallei</i> mutants <i>in vivo</i>.

<p>Groups of 5 female albino C57BL/6J mice were infected with wild type JW280 (10^4.2 CFU), Δ<i>wcb</i> capsule mutant (10^6.0 CFU), or Δ<i>sctU</i>Bp3 T3SS3 mutant (10^6.5 CFU), and monitored by optical diagnostic imaging once to twice daily. ROIs from the dorsally-imaged thoracic cavity were plotted as a function of infection time for each mutant. The 95% LOD was calculated for the background luminescence of uninfected mice and indicated as a dotted horizontal line. The calculated doubling rate of bioluminescent signal of each strain is indicated.</p

Host response to capsule and T3SS3 mutants of <i>B. pseudomallei</i>.

<p>Groups of 5 female albino C57BL/6J mice were challenged by the IMIT route of infection with increasing doses of either Δ<i>wcb</i> capsule mutant (A) or Δ<i>sctU</i>_Bp3 T3SS3 mutant (B) in the luminescent <i>B. pseudomallei</i> strain JW280 background. Mice were monitored for 14 days (336 hr) for disease progression and euthanized at the onset of moribund disease presentation. Survival curves are used to present the dose-dependent response of the host to increasing bacterial challenges of <i>B. pseudomallei</i> mutants. The MTTD was calculated for groups with ≥50% mortality, as indicated.</p

Host response to lung-specific delivery of <i>B. pseudomallei</i>.

<p>Groups of 5 female albino C57BL/6J mice were challenged with increasing doses of luminescent <i>B. pseudomallei</i> strain JW280 by either the intranasal (A) or IMIT (B) routes of infection. Mice were monitored for 14 days (336 hr) for disease progression and euthanized at the onset of moribund disease presentation. Survival curves are used to present the dose-dependent response of the host to increasing bacterial challenges. The MTTD was calculated for groups with ≥50% mortality, as indicated.</p

Bacterial enumeration at moribund disease for respiratory melioidosis models.

<p>Groups of 5 female albino C57BL/6J mice were infected by either the i.n. (10^5.1 CFU) or IMIT (10^4.6 CFU) routes of infection and euthanized at moribund disease endpoints. Bacteria were enumerated from tissues homogenized in 1 ml PBS, from a 1 ml PBS BAL collection, or from cardiac-drawn blood. Bacterial burden was calculated as CFU/tissue (lung, liver, and spleen) or bacteria per ml of body fluid (BAL and blood). Significant differences between log transformed data were evaluated by 2-way ANOVA with Bonferroni multiple comparisons (n.s., not significant; *, p<0.05; **, p<0.01; ****, p<0.0001).</p

Male host response to lung-specific delivery of <i>B. pseudomallei</i>.

<p>Groups of 3 male albino C57BL/6J mice were challenged with increasing doses of luminescent <i>B. pseudomallei</i> strain JW280 by IMIT infection. Mice were monitored for 14 days (336 hr) for disease progression and euthanized at the onset of moribund disease presentation. Survival curves are used to present the dose-dependent response of the host to increasing bacterial challenges. The MTTD was calculated for groups with ≥50% mortality, as indicated.</p

Bacterial enumeration of <i>B. pseudomallei</i> mutants in moribund respiratory disease.

<p>Groups of 5 female albino C57BL/6J mice were infected with wild type JW280 (10^4.2 CFU), Δ<i>wcb</i> capsule mutant (10^6.0 CFU), or Δ<i>sctU</i>Bp3 T3SS3 mutant (10^6.5 CFU), and euthanized at moribund disease endpoints. Bacteria were enumerated from tissues <i>ex vivo</i> by optical imaging (left Y-axis: cps/tissue) with presentation of the estimated tissue CFU burdens based on calculated tissue-specific cps:CFU correlation (right Y-axis: CFU/tissue est.) for lung (A), liver (B) and spleen (C). The 95% LOD was calculated as a technical background luminescence and indicated as a dotted horizontal line. Data points below the 95% LOD were set to the 95% LOD value. Significant differences (1-way ANOVA with Tukey posttest) between log transformed data sets are indicated with an adjoining line (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Lassa Virus Vaccine Candidate ML29 Generates Truncated Viral RNAs Which Contribute to Interfering Activity and Attenuation

Defective interfering particles (DIPs) are naturally occurring products during virus replication in infected cells. DIPs contain defective viral genomes (DVGs) and interfere with replication and propagation of their corresponding standard viral genomes by competing for viral and cellular resources, as well as promoting innate immune antiviral responses. Consequently, for many different viruses, including mammarenaviruses, DIPs play key roles in the outcome of infection. Due to their ability to broadly interfere with viral replication, DIPs are attractive tools for the development of a new generation of biologics to target genetically diverse and rapidly evolving viruses. Here, we provide evidence that in cells infected with the Lassa fever (LF) vaccine candidate ML29, a reassortant that carries the nucleoprotein (NP) and glycoprotein (GP) dominant antigens of the pathogenic Lassa virus (LASV) together with the L polymerase and Z matrix protein of the non-pathogenic genetically related Mopeia virus (MOPV), L-derived truncated RNA species are readily detected following infection at low multiplicity of infection (MOI) or in persistently-infected cells originally infected at high MOI. In the present study, we show that expression of green fluorescent protein (GFP) driven by a tri-segmented form of the mammarenavirus lymphocytic choriomeningitis virus (r3LCMV-GFP/GFP) was strongly inhibited in ML29-persistently infected cells, and that the magnitude of GFP suppression was dependent on the passage history of the ML29-persistently infected cells. In addition, we found that DIP-enriched ML29 was highly attenuated in immunocompetent CBA/J mice and in Hartley guinea pigs. Likewise, STAT-1-/- mice, a validated small animal model for human LF associated hearing loss sequelae, infected with DIP-enriched ML29 did not exhibit any hearing abnormalities throughout the observation period (62 days)

Lassa Virus Vaccine Candidate ML29 Generates Truncated Viral RNAs Which Contribute to Interfering Activity and Attenuation

Defective interfering particles (DIPs) are naturally occurring products during virus replication in infected cells. DIPs contain defective viral genomes (DVGs) and interfere with replication and propagation of their corresponding standard viral genomes by competing for viral and cellular resources, as well as promoting innate immune antiviral responses. Consequently, for many different viruses, including mammarenaviruses, DIPs play key roles in the outcome of infection. Due to their ability to broadly interfere with viral replication, DIPs are attractive tools for the development of a new generation of biologics to target genetically diverse and rapidly evolving viruses. Here, we provide evidence that in cells infected with the Lassa fever (LF) vaccine candidate ML29, a reassortant that carries the nucleoprotein (NP) and glycoprotein (GP) dominant antigens of the pathogenic Lassa virus (LASV) together with the L polymerase and Z matrix protein of the non-pathogenic genetically related Mopeia virus (MOPV), L-derived truncated RNA species are readily detected following infection at low multiplicity of infection (MOI) or in persistently-infected cells originally infected at high MOI. In the present study, we show that expression of green fluorescent protein (GFP) driven by a tri-segmented form of the mammarenavirus lymphocytic choriomeningitis virus (r3LCMV-GFP/GFP) was strongly inhibited in ML29-persistently infected cells, and that the magnitude of GFP suppression was dependent on the passage history of the ML29-persistently infected cells. In addition, we found that DIP-enriched ML29 was highly attenuated in immunocompetent CBA/J mice and in Hartley guinea pigs. Likewise, STAT-1-/- mice, a validated small animal model for human LF associated hearing loss sequelae, infected with DIP-enriched ML29 did not exhibit any hearing abnormalities throughout the observation period (62 days)