Robert Mauro DiFazio

All Publications

• Boosting BCG with proteins or rAd5 does not enhance protection against tuberculosis in rhesus macaques. NPJ vaccines Darrah, P. A., DiFazio, R. M., Maiello, P., Gideon, H. P., Myers, A. J., Rodgers, M. A., Hackney, J. A., Lindenstrom, T., Evans, T., Scanga, C. A., Prikhodko, V., Andersen, P., Lin, P. L., Laddy, D., Roederer, M., Seder, R. A., Flynn, J. L. 2019; 4: 21

  Abstract

  Tuberculosis (TB) is the leading cause of death from infection worldwide. The only approved vaccine, BCG, has variable protective efficacy against pulmonary TB, the transmissible form of the disease. Therefore, improving this efficacy is an urgent priority. This study assessed whether heterologous prime-boost vaccine regimens in which BCG priming is boosted with either (i) protein and adjuvant (M72 plus AS01E or H56 plus CAF01) delivered intramuscularly (IM), or (ii) replication-defective recombinant adenovirus serotype 5 (Ad5) expressing various Mycobacterium tuberculosis (Mtb) antigens (Ad5(TB): M72, ESAT-6/Ag85b, or ESAT-6/Rv1733/Rv2626/RpfD) administered simultaneously by IM and aerosol (AE) routes, could enhance blood- and lung-localized T-cell immunity and improve protection in a nonhuman primate (NHP) model of TB infection. Ad5(TB) vaccines administered by AE/IM routes following BCG priming elicited ~10-30% antigen-specific CD4 and CD8 T-cell multifunctional cytokine responses in bronchoalveolar lavage (BAL) but did not provide additional protection compared to BCG alone. Moreover, AE administration of an Ad5(empty) control vector after BCG priming appeared to diminish protection induced by BCG. Boosting BCG by IM immunization of M72/AS01E or H56:CAF01 elicited ~0.1-0.3% antigen-specific CD4 cytokine responses in blood with only a transient increase of ~0.5-1% in BAL; these vaccine regimens also failed to enhance BCG-induced protection. Taken together, this study shows that boosting BCG with protein/adjuvant or Ad-based vaccines using these antigens, by IM or IM/AE routes, respectively, do not enhance protection against primary infection compared with BCG alone, in the highly susceptible rhesus macaque model of tuberculosis.

  View details for DOI 10.1038/s41541-019-0113-9

  View details for PubMedID 31149352

  View details for PubMedCentralID PMC6538611

• Concurrent infection with Mycobacterium tuberculosis confers robust protection against secondary infection in macaques. PLoS pathogens Cadena, A. M., Hopkins, F. F., Maiello, P., Carey, A. F., Wong, E. A., Martin, C. J., Gideon, H. P., DiFazio, R. M., Andersen, P., Lin, P. L., Fortune, S. M., Flynn, J. L. 2018; 14 (10): e1007305

  Abstract

  For many pathogens, including most targets of effective vaccines, infection elicits an immune response that confers significant protection against reinfection. There has been significant debate as to whether natural Mycobacterium tuberculosis (Mtb) infection confers protection against reinfection. Here we experimentally assessed the protection conferred by concurrent Mtb infection in macaques, a robust experimental model of human tuberculosis (TB), using a combination of serial imaging and Mtb challenge strains differentiated by DNA identifiers. Strikingly, ongoing Mtb infection provided complete protection against establishment of secondary infection in over half of the macaques and allowed near sterilizing bacterial control for those in which a secondary infection was established. By contrast, boosted BCG vaccination reduced granuloma inflammation but had no impact on early granuloma bacterial burden. These findings are evidence of highly effective concomitant mycobacterial immunity in the lung, which may inform TB vaccine design and development.

  View details for PubMedID 30312351

• Advanced model systems and tools for basic and translational human immunology. Genome medicine Wagar, L. E., DiFazio, R. M., Davis, M. M. 2018; 10 (1): 73

  Abstract

  There are fundamental differences between humans and the animals we typically use to study the immune system. We have learned much from genetically manipulated and inbred animal models, but instances in which these findings have been successfully translated to human immunity have been rare. Embracing the genetic and environmental diversity of humans can tell us about the fundamental biology of immune cell types and the elasticity of the immune system. Although people are much more immunologically diverse than conventionally housed animal models, tools and technologies are now available that permit high-throughput analysis of human samples, including both blood and tissues, which will give us deep insights into human immunity in health and disease. As we gain a more detailed picture of the human immune system, we can build more sophisticated models to better reflect this complexity, both enabling the discovery of new immunological mechanisms and facilitating translation into the clinic.

  View details for PubMedID 30266097

• Integrating Non-human Primate, Human, and Mathematical Studies to Determine the Influence of BCG Timing on H56 Vaccine Outcomes FRONTIERS IN MICROBIOLOGY Joslyn, L. R., Pienaar, E., DiFazio, R. M., Suliman, S., Kagina, B. M., Flynn, J. L., Scriba, T. J., Linderman, J. J., Kirschner, D. E. 2018; 9

  View details for DOI 10.3389/fmicb.2018.01734

  View details for Web of Science ID 000441899800001

• Integrating Non-human Primate, Human, and Mathematical Studies to Determine the Influence of BCG Timing on H56 Vaccine Outcomes. Frontiers in microbiology Joslyn, L. R., Pienaar, E., DiFazio, R. M., Suliman, S., Kagina, B. M., Flynn, J. L., Scriba, T. J., Linderman, J. J., Kirschner, D. E. 2018; 9: 1734

  Abstract

  Tuberculosis (TB) is the leading cause of death by an infectious agent, and developing an effective vaccine is an important component of the WHO's EndTB Strategy. Non-human primate (NHP) models of vaccination are crucial to TB vaccine development and have informed design of subsequent human trials. However, challenges emerge when translating results from animal models to human applications, and connecting post-vaccination immunological measurements to infection outcomes. The H56:IC31 vaccine is a candidate currently in phase I/IIa trials. H56 is a subunit vaccine that is comprised of 3 mycobacterial antigens: ESAT6, Ag85B, and Rv2660, formulated in IC31 adjuvant. H56, as a boost to Bacillus Calmette-Guérin (BCG, the TB vaccine that is currently used in most countries world-wide) demonstrates improved protection (compared to BCG alone) in mouse and NHP models of TB, and the first human study of H56 reported strong antigen-specific T cell responses to the vaccine. We integrated NHP and human data with mathematical modeling approaches to improve our understanding of NHP and human response to vaccine. We use a mathematical model to describe T-cell priming, proliferation, and differentiation in lymph nodes and blood, and calibrate the model to NHP and human blood data. Using the model, we demonstrate the impact of BCG timing on H56 vaccination response and reveal a general immunogenic response to H56 following BCG prime. Further, we use uncertainty and sensitivity analyses to isolate mechanisms driving differences in vaccination response observed between NHP and human datasets. This study highlights the power of a systems biology approach: integration of multiple modalities to better understand a complex biological system.

  View details for PubMedID 30177914

  View details for PubMedCentralID PMC6109686

• Identifying mechanisms driving formation of granuloma-associated fibrosis during Mycobacterium tuberculosis infection. Journal of theoretical biology Warsinske, H. C., DiFazio, R. M., Linderman, J. J., Flynn, J. L., Kirschner, D. E. 2017; 429: 1?17

  Abstract

  Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a pulmonary pathogen of major global concern. A key feature of Mtb infection in primates is the formation of granulomas, dense cellular structures surrounding infected lung tissue. These structures serve as the main site of host-pathogen interaction in TB, and thus to effectively treat TB we must clarify mechanisms of granuloma formation and their function in disease. Fibrotic granulomas are associated with both good and bad disease outcomes. Fibrosis can serve to isolate infected tissue from healthy tissue, but it can also cause difficulty breathing as it leaves scars. Little is known about fibrosis in TB, and data from non-human primates is just beginning to clarify the picture. This work focuses on constructing a hybrid multi-scale model of fibrotic granuloma formation, in order to identify mechanisms driving development of fibrosis in Mtb infected lungs. We combine dynamics of molecular, cellular, and tissue scale models from previously published studies to characterize the formation of two common sub-types of fibrotic granulomas: peripherally fibrotic, with a cuff of collagen surrounding granulomas, and centrally fibrotic, with collagen throughout granulomas. Uncertainty and sensitivity analysis, along with large simulation sets, enable us to identify mechanisms differentiating centrally versus peripherally fibrotic granulomas. These findings suggest that heterogeneous cytokine environments exist within granulomas and may be responsible for driving tissue scale morphologies. Using this model we are primed to better understand the complex structure of granulomas, a necessity for developing successful treatments for TB.

  View details for PubMedID 28642013

• Rhesus macaques are more susceptible to progressive tuberculosis than cynomolgus macaques: A quantitative comparison. Infection and immunity Maiello, P., DiFazio, R. M., Cadena, A. M., Rodgers, M. A., Lin, P. L., Scanga, C. A., Flynn, J. L. 2017

  Abstract

  In the past two decades, it has become increasingly clear that non-human primates, specifically macaques, are useful models for human tuberculosis (TB). Several macaque species have been used for TB studies, and questions remain about the similarities and differences in TB pathogenesis among macaque species, which can complicate decisions about the best species for a specific experiment. Here we provide a quantitative assessment, using serial positron emission tomography and computed tomography (PET-CT) imaging and precise quantitative determination of bacterial burdens, of low-dose M. tuberculosis infection in cynomolgus macaques of Chinese origin, rhesus macaques of Chinese origin, and Mauritian cynomolgus macaques. This comprehensive study demonstrates that there is substantial variability in infection outcome within and among species. Overall, rhesus macaques have faster rates of disease progression, more lung, lymph node, and extrapulmonary involvement, and higher bacterial burdens than Chinese cynomolgus macaques. The small cohort of Mauritian cynomolgus macaques assessed here indicates that this species is more similar to rhesus than to "Chinese" cynomolgus macaques in terms of M. tuberculosis infection outcome. These data provide insights into the differences among species, providing valuable data to the field for assessing macaque studies of TB.

  View details for PubMedID 28947646

• PET CT Identifies Reactivation Risk in Cynomolgus Macaques with Latent M. tuberculosis PLOS PATHOGENS Lin, P. L., Maiello, P., Gideon, H. P., Coleman, M. T., Cadena, A. M., Rodgers, M. A., Gregg, R., O'Malley, M., Tomko, J., Fillmore, D., Frye, L. J., Rutledge, T., DiFazio, R. M., Janssen, C., Klein, E., Andersen, P. L., Fortune, S. M., Flynn, J. L. 2016; 12 (7)

  Abstract

  Mycobacterium tuberculosis infection presents across a spectrum in humans, from latent infection to active tuberculosis. Among those with latent tuberculosis, it is now recognized that there is also a spectrum of infection and this likely contributes to the variable risk of reactivation tuberculosis. Here, functional imaging with 18F-fluorodeoxygluose positron emission tomography and computed tomography (PET CT) of cynomolgus macaques with latent M. tuberculosis infection was used to characterize the features of reactivation after tumor necrosis factor (TNF) neutralization and determine which imaging characteristics before TNF neutralization distinguish reactivation risk. PET CT was performed on latently infected macaques (n = 26) before and during the course of TNF neutralization and a separate set of latently infected controls (n = 25). Reactivation occurred in 50% of the latently infected animals receiving TNF neutralizing antibody defined as development of at least one new granuloma in adjacent or distant locations including extrapulmonary sites. Increased lung inflammation measured by PET and the presence of extrapulmonary involvement before TNF neutralization predicted reactivation with 92% sensitivity and specificity. To define the biologic features associated with risk of reactivation, we used these PET CT parameters to identify latently infected animals at high risk for reactivation. High risk animals had higher cumulative lung bacterial burden and higher maximum lesional bacterial burdens, and more T cells producing IL-2, IL-10 and IL-17 in lung granulomas as compared to low risk macaques. In total, these data support that risk of reactivation is associated with lung inflammation and higher bacterial burden in macaques with latent Mtb infection.

  View details for DOI 10.1371/journal.ppat.1005739

  View details for Web of Science ID 000383366400025

  View details for PubMedID 27379816

  View details for PubMedCentralID PMC4933353

• Active transforming growth factor-ß is associated with phenotypic changes in granulomas after drug treatment in pulmonary tuberculosis. Fibrogenesis & tissue repair DiFazio, R. M., Mattila, J. T., Klein, E. C., Cirrincione, L. R., Howard, M., Wong, E. A., Flynn, J. L. 2016; 9: 6-?

  Abstract

  Tuberculosis (TB) chemotherapy clears bacterial burden in the lungs of patients and allows the tuberculous lesions to heal through a fibrotic process. The healing process leaves pulmonary scar tissue that can impair lung function. The goal of this study was to identify fibrotic mediators as a stepping-stone to begin exploring mechanisms of tissue repair in TB.Hematoxylin and eosin staining and Masson's trichrome stain were utilized to determine levels of collagenization in tuberculous granulomas from non-human primates. Immunohistochemistry was then employed to further interrogate these granulomas for markers associated with fibrogenesis, including transforming growth factor-? (TGF?), ?-smooth muscle actin (?SMA), phosphorylated SMAD-2/3, and CD163. These markers were compared across states of drug treatment using one-way ANOVA, and Pearson's test was used to determine the association of these markers with one another.TGF? and ?SMA were present in granulomas from primates with active TB disease. These molecules were reduced in abundance after TB chemotherapy. Phosphorylated SMAD-2/3, a signaling intermediate of TGF?, was observed in greater amounts after 1 month of drug treatment than in active disease, suggesting that this particular pathway is blocked in active disease. Collagen production during tissue repair is strongly associated with TGF? in this model, but not with CD163+ macrophages.Tissue repair and fibrosis in TB that occurs during drug treatment is associated with active TGF? that is produced during active disease. Further work will identify mechanisms of fibrosis and work towards mitigating lung impairment with treatments that target those mechanisms.

  View details for DOI 10.1186/s13069-016-0043-3

  View details for PubMedID 27148404

  View details for PubMedCentralID PMC4855369

• IFN-gamma from CD4 T Cells Is Essential for Host Survival and Enhances CD8 T Cell Function during Mycobacterium tuberculosis Infection JOURNAL OF IMMUNOLOGY Green, A. M., Difazio, R., Flynn, J. L. 2013; 190 (1): 270-277

  Abstract

  IFN-? is necessary in both humans and mice for control of Mycobacterium tuberculosis. CD4 T cells are a significant source of IFN-? during acute infection in mice and are required for control of bacterial growth and host survival. However, several other types of cells can and do produce IFN-? during the course of the infection. We sought to determine whether IFN-? from sources other than CD4 T cells was sufficient to control M. tuberculosis infection and whether CD4 T cells had a role in addition to IFN-? production. To investigate the role of IFN-? from CD4 T cells, a murine adoptive transfer model was developed in which all cells were capable of producing IFN-?, with the exception of CD4 T cells. Our data in this system support that CD4 T cells are essential for control of infection, but also that IFN-? from CD4 T cells is necessary for host survival and optimal long-term control of bacterial burden. In addition, IFN-? from CD4 T cells was required for a robust CD8 T cell response. IFN-? from T cells inhibited intracellular replication of M. tuberculosis in macrophages, suggesting IFN-? may be necessary for intracellular bactericidal activity. Thus, although CD4 T cells play additional roles in the control of M. tuberculosis infection, IFN-? is a major function by which these cells participate in resistance to tuberculosis.

  View details for DOI 10.4049/jimmunol.1200061

  View details for Web of Science ID 000312832500030

  View details for PubMedID 23233724

  View details for PubMedCentralID PMC3683563

• Toxin-antitoxin (TA) systems are prevalent and transcribed in clinical isolates of Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus FEMS MICROBIOLOGY LETTERS Williams, J. J., Halvorsen, E. M., Dwyer, E. M., DiFazio, R. M., Hergenrother, P. J. 2011; 322 (1): 41-50

  Abstract

  The percentage of bacterial infections refractory to standard antibiotic treatments is steadily increasing. Among the most problematic hospital and community-acquired pathogens are methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA). One novel strategy proposed for treating infections of multidrug-resistant bacteria is the activation of latent toxins of toxin-antitoxin (TA) protein complexes residing within bacteria; however, the prevalence and identity of TA systems in clinical isolates of MRSA and PA has not been defined. We isolated DNA from 78 MRSA and 42 PA clinical isolates and used PCR to probe for the presence of various TA loci. Our results showed that the genes for homologs of the mazEF TA system in MRSA and the relBE and higBA TA systems in PA were present in 100% of the respective strains. Additionally, reverse transcriptase PCR analysis revealed that these transcripts are produced in the clinical isolates. These results indicate that TA genes are prevalent and transcribed within MRSA and PA and suggest that activation of the toxin proteins could be an effective antibacterial strategy for these pathogens.

  View details for DOI 10.1111/j.1574-6968.2011.02330.x

  View details for Web of Science ID 000293028000006

  View details for PubMedID 21658105

  View details for PubMedCentralID PMC3184004