Assessing vaccine-mediated protection in an ultra-low dose Mycobacterium tuberculosis murine model.

Despite widespread immunization with Bacille-Calmette-Guérin (BCG), the only currently licensed tuberculosis (TB) vaccine, TB remains a leading cause of mortality globally. There are many TB vaccine candidates in the developmental pipeline, but the lack of a robust animal model to assess vaccine efficacy has hindered our ability to prioritize candidates for human clinical trials. Here we use a murine ultra-low dose (ULD) Mycobacterium tuberculosis (Mtb) challenge model to assess protection conferred by BCG vaccination. We show that BCG confers a reduction in lung bacterial burdens that is more durable than that observed after conventional dose challenge, curbs Mtb dissemination to the contralateral lung, and, in a small percentage of mice, prevents detectable infection. These findings are consistent with the ability of human BCG vaccination to mediate protection, particularly against disseminated disease, in specific human populations and clinical settings. Overall, our findings demonstrate that the ultra-low dose Mtb infection model can measure distinct parameters of immune protection that cannot be assessed in conventional dose murine infection models and could provide an improved platform for TB vaccine testing

Assessing BCG efficacy in the ultra-low dose Mtb model.

B6 mice were aerosol infected with an ULD (1–3 CFU) of H37Rv Mtb 8 weeks following either s.c. immunization with 106 BCG-Pasteur (n = 20) or no immunization (n = 20). On day 63 post-infection, CFU were enumerated from left lung, right lung, or spleen homogenates plated onto 7H10 plates. A) Combined lung CFUs or B) spleen CFUs from unimmunized and BCG-immunized mice are graphed. Counts from left lungs, right lungs and spleen are graphed separately from, C) unimmunized mice or D) BCG-immunized mice. There were 20 mice per group, and the data are graphed as mean ± SD. Single-group comparisons were analyzed using an unpaired t test and excluding mice with 0 CFU. **p<0.01, ****p<0.0001.</p

BCG-mediated reductions in Mtb lung burdens are not durable in a conventional-dose infection.

C57BL/6 (B6) mice were aerosol infected with a conventional dose (CD) (50–100 CFU) of H37Rv Mtb eight weeks following either subcutaneous (s.c.) immunization with 106 BCG-Pasteur (BCG) or no immunization (unimmunized). On day 42 or 120 post-infection, CFU were enumerated from lung homogenates plated onto 7H10 plates. These data represent 5 mice per group and are shown as mean ± SEM. Single-group comparisons were analyzed using an unpaired t test. ***p<0.001.</p

BCG immunization prevents detectable infection in some mice.

A) Table of all mice, showing the percentage with 0 CFU in the unimmunized vs BCG-immunized groups separated by timepoint post-infection. B) The proportion of unimmunized mice with 0 CFU (x-axis) vs the proportion of BCG-immunized mice with 0 CFU (y-axis) for each experiment from a compilation of 25 experiments (days 26–125) separated by timepoint post-ULD infection. Each colored symbol is an independent experiment, and the larger black circle is the compilation of all the data, which was statistically significant (mixed effects logistic regression p = 0.001). Post-hoc analyses indicated that, if each infection cohort had been analyzed separately, the 3 filled in symbols would have attained statistical significance if they had been analyzed using the same regression model (pS1 Table) In ULD-Mtb experiments in which bar-coded strains were used for infection (n = 6 for unimmunized, n = 6 for BCG-immunized), the mice are graphed according to the number of unique Mtb strains recovered from each mouse’s lungs.</p

Group sizes needed to assess vaccine-mediated prevention of detectable infection.

Group sizes needed to assess vaccine-mediated prevention of detectable infection.</p

BCG-mediated reductions in Mtb lung burden are more durable in the ULD model.

A) Combined lung CFU from a single experiment time course of ULD-infected B6 mice with or without BCG immunization. Combined lung CFU were enumerated on days 14, 42, and 115 post-infection. There were 19 or 20 mice per group, and the data are graphed as mean ± SD. Single-group comparisons were analyzed using an unpaired t test (excluding mice with 0 CFU). ***pB) Combined lung CFU from a compilation of 31 experiments, excluding mice with 0 CFU, separated by timepoint post-infection of ULD-infected B6 mice with or without BCG immunization. Error bars are 95% confidence intervals in a fixed effects negative regression model. All timepoints except days 14–15 post-infection have a p < 0.001.</p

Compilation of data from 31 experiments measuring combined lung, right lung, or left lung CFU with and without BCG immunization.

Compilation of data from 31 experiments measuring combined lung, right lung, or left lung CFU with and without BCG immunization.</p

BCG-immunization prevents Mtb dissemination to the contralateral lung.

A) Proportion of mice with bilateral lung infection (CFU in left and right lung) from a compilation of 31 experiments separated by timepoint post-infection of ULD-infected B6 mice with or without BCG immunization. Error bars are 95% confidence intervals in a mixed effects logistic regression model, with experiment as a grouping variable. All timepoints except days 14–15 post-infection have a p 0.001. B) A single ULD experiment using bar-coded Mtb strains is shown. On day 65 post-infection, right and left lung homogenates were plated onto 7H10 plates and Mtb colonies from infected lungs were scraped to make genomic DNA. DNA was sequenced, and the identity of each bar-coded Mtb strain is graphed for each lung separately. The percentage of bilateral infection for unimmunized and BCG-immunized mice from this experiment was calculated by the proportion of mice with CFUs in both lungs (C) or the proportion of mice with at least one common Mtb strain in both lungs (D). (E) The percentage of mice with bilateral Mtb strains was compiled from 5 independent experiments, excluding day 14 post-infection. Vaccine efficacy for preventing dissemination to the contralateral lung was calculated as 1-(% BCG mice with bilateral infection)/(% Unimmunized mice with bilateral infection).</p

De Novo Missense Variants in TRAF7 Cause Developmental Delay, Congenital Anomalies, and Dysmorphic Features

TRAF7 is a multi-functional protein involved in diverse signaling pathways and cellular processes. The phenotypic consequence of germ-line TRAF7 variants remains unclear. Here we report missense variants in TRAF7 in seven unrelated individuals referred for clinical exome sequencing. The seven individuals share substantial phenotypic overlap, with developmental delay, congenital heart defects, limb and digital anomalies, and dysmorphic features emerging as key unifying features. The identified variants are de novo in six individuals and comprise four distinct missense changes, including a c.1964G>A (p.Arg655Gln) variant that is recurrent in four individuals. These variants affect evolutionarily conserved amino acids and are located in key functional domains. Gene-specific mutation rate analysis showed that the occurrence of the de novo variants in TRAF7 (p = 2.6 x 10(-3)) and the recurrent de novo c.1964G>A (p.Arg655Gln) variant (p = 1.9 x 10(-8)) in our exome cohort was unlikely to have occurred by chance. In vitro analyses of the observed TRAF7 mutations showed reduced ERK1/2 phosphorylation. Our findings suggest that missense mutations in TRAF7 are associated with a multisystem disorder and provide evidence of a role for TRAF7 in human development

Interleukin-15 response signature predicts RhCMV/SIV vaccine efficacy

Simian immunodeficiency virus (SIV) challenge of rhesus macaques (RMs) vaccinated with strain 68-1 Rhesus Cytomegalovirus (RhCMV) vectors expressing SIV proteins (RhCMV/SIV) results in a binary outcome: stringent control and subsequent clearance of highly pathogenic SIV in similar to 55% of vaccinated RMs with no protection in the remaining 45%. Although previous work indicates that unconventionally restricted, SIV-specific, effector-memory (EM)-biased CD8(+) T cell responses are necessary for efficacy, the magnitude of these responses does not predict efficacy, and the basis of protection vs. non-protection in 68-1 RhCMV/SIV vector-vaccinated RMs has not been elucidated. Here, we report that 68-1 RhCMV/SIV vector administration strikingly alters the whole blood transcriptome of vaccinated RMs, with the sustained induction of specific immune-related pathways, including immune cell, toll-like receptor (TLR), inflammasome/cell death, and interleukin-15 (IL-15) signaling, significantly correlating with subsequent vaccine efficacy. Treatment of a separate RM cohort with IL-15 confirmed the central involvement of this cytokine in the protection signature, linking the major innate and adaptive immune gene expression networks that correlate with RhCMV/SIV vaccine efficacy. This change-from-baseline IL-15 response signature was also demonstrated to significantly correlate with vaccine efficacy in an independent validation cohort of vaccinated and challenged RMs. The differential IL-15 gene set response to vaccination strongly correlated with the pre-vaccination activity of this pathway, with reduced baseline expression of IL-15 response genes significantly correlating with higher vaccine-induced induction of IL-15 signaling and subsequent vaccine protection, suggesting that a robust de novo vaccine-induced IL-15 signaling response is needed to program vaccine efficacy. Thus, the RhCMV/SIV vaccine imparts a coordinated and persistent induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8(+) T cell function, that support the ability of vaccine-elicited unconventionally restricted CD8(+) T cells to mediate protection against SIV challenge