Marcelo B. Sztein, MD

Toapanta FR, Bernal PJ, Fresnay S, Magder LS, Darton TC, Jones C, Waddington CS, Blohmke CJ, Angus B, Levine MM, Pollard AJ, Sztein MB. Oral challenge with wild-type Salmonella Typhi induces distinct changes in B cell subsets in Individuals who develop typhoid disease. PLoS Negl Trop Dis. 2016 Jun 14;10(6):e0004766.

Blohmke CJ, Darton TC, Jones C, Suarez NM, Waddington CS, Angus B, Zhou L, Hill J, Clare S, Kane L, Mukhopadhyay S, Schreiber F, Duque-Correa MA, Wright JC, Roumeliotis TI, Yu L, Choudhary JS, Mejias A, Ramilo O, Shanyinde M, Sztein MB, Kingsley RA, Lockhart S, Levine MM, Lynn DJ, Dougan G, Pollard AJ. Interferon-driven alterations of the host's amino acid metabolism in the pathogenesis of typhoid fever. J Exp Med. 2016 May 30;213(6):1061-77.

Fresnay S, McArthur MA, Magder L, Darton TC, Jones C, Waddington CS, Blohmke CJ, Angus B, Levine MM, Pollard AJ, Sztein MB. Salmonella Typhi-specific multifunctional CD8+ T cells play a dominant role in protection from typhoid fever in humans. J Transl Med. 2016 Mar 1;14:62.

Tapia MD, Sow SO, Lyke KE, Haidara FC, Diallo F, Doumbia M, Traore A, Coulibaly F, Kodio M, Onwuchekwa U, Sztein MB, Wahid R, Campbell JD, Kieny MP, Moorthy V, Imoukhuede EB, Rampling T, Roman F, De Ryck I, Bellamy AR, Dally L, Mbaya OT, Ploquin A, Zhou Y, Stanley DA, Bailer R, Koup RA, Roederer M, Ledgerwood J, Hill AV, Ballou WR, Sullivan N, Graham B, Levine MM. Use of ChAd3-EBO-Z Ebola virus vaccine in Malian and US adults, and boosting of Malian adults with MVA-BN-Filo: a phase 1, single-blind, randomised trial, a phase 1b, open-label and double-blind, dose-escalation trial, and a nested, randomised, double-blind, placebo-controlled trial. Lancet Infect Dis. 2016 Jan;16(1):31-42.

Booth JS, Salerno-Goncalves R, Blanchard TG, Patil SA, Kader HA, Safta AM, Morningstar LM, Czinn SJ, Greenwald BD, Sztein MB. Mucosal-associated invariant T cells (MAIT) in the human gastric mucosa and blood: Role in Helicobacter pylori infection. Front Immunol. 2015 Sep 17;6:466.

Toapanta FR, Bernal PJ, Fresnay S, Darton TC, Jones C, Waddington CS, Blohmke CJ, Dougan G, Angus B, Levine MM, Pollard AJ, Sztein MB. Oral wild-type Salmonella Typhi challenge induces activation of circulating monocytes and dendritic cells in individuals who develop typhoid disease. PLoS Negl Trop Dis. 2015 Jun 11;9(6):e0003837.

McArthur MA, Fresnay S, Magder LS, Darton TC, Jones C, Waddington CS, Blohmke CJ, Dougan G, Angus B, Levine MM, Pollard AJ, Sztein MB. Activation of Salmonella Typhi-specific regulatory T cells in typhoid disease in a wild-type S. Typhi challenge model. PLoS Pathog. 2015 May;11(5):e1004914.

Wahid R, Fresnay S, Levine MM, Sztein MB. Immunization with Ty21a live oral typhoid vaccine elicits cross-reactive multifunctional CD8+ T cell responses against Salmonella enterica serovar Typhi, S. Paratyphi A and S. Paratyphi B in humans. Mucosal Immunol. 2015 Nov;8(6):1349-59.

Sztein MB, Salerno-Goncalves R, McArthur MA. Complex adaptive immunity to enteric fevers in humans: Lessons learned and the path forward. Front Immunol. 2014;5:516.

Toapanta FR, Simon JK, Barry EM, Pasetti MF, Levine MM, Kotloff KL, Sztein MB. Gut-homing conventional plasmablasts and CD27- plasmablasts induced by oral immunization with an oral live attenuated Shigella vaccine in humans. Front Immunol. 2014;5:516.

Booth JS, Toapanta FR, Salerno-Goncalves R, Patil S, Kader HA, Safta AM, Czinn SJ, Greenwald BD, Sztein MB. Characterization and functional properties of gastric tissue-resident memory T cells from children, adults and the elderly. Front Immunol. 2014 Jun 19;5:294.

Waddington CS, Darton TC, Jones C, Haworth K, Peters A, John T, Thompson BA, Kerridge SA, Kingsley RA, Zhou L, Holt KE, Yu LM, Lockhart S, Farrar JJ, Sztein MB, Dougan G, Angus B, Levine MM, Pollard AJ. An outpatient, ambulant-design, controlled human infection model using escalating doses of Salmonella Typhi challenge delivered in sodium bicarbonate solution. Clin Infect Dis. 2014 May;58(9):1230-40.

Wahid R, Zafar SJ, McArthur MA, Pasetti MF, Levine MM, Sztein MB. Live oral Salmonella enterica serovar Typhi vaccines Ty21a and CVD 909 induce opsonophagocytic functional antibodies in humans that cross-react with S. Paratyphi A and S. Paratyphi B. Clin Vaccine Immunol. 2014 Mar;21(3):427-34.

Salerno-Goncalves R, Rezwan T, Sztein MB. B cells modulate mucosal associated invariant T cell  immune responses. Front Immunol. 2014 Jan 7;4:511.

• Top viewed immunology research article in Frontiers in Immunology Jan 2014.

Fiorentino M, Levine MM, Sztein MB*, Fasano A*. *Joint senior authorship. Effect of wild-type Shigella species and attenuated Shigella vaccine candidates on small intestinal barrier function, antigen trafficking, and cytokine release. PLoS One. 2014 Jan 9;9(1):e85211.

McArthur MA, Sztein MB, Edelman R. Prospects for controlling dengue spread: Vaccines and vector control. In: Clinical Insights: Dengue: Transmission, Diagnosis & Surveillance. Future Science Group, London. e-book. 2014 Feb;103-119.

For over 25 years one of the key areas in Dr. Sztein’s laboratory has been the study of the human host responses to S. Typhi and other causative agents of enteric fevers.  Dr. Sztein’s group has been a leader in this field. Seminal findings characterizing the immunity to S. Typhi were reported by Dr. Sztein’s group in volunteers immunized with attenuated strains of S. Typhi and, more recently, challenged with wild-type S. Typhi.  He published the first reports in humans of the dominance of multifunctional Thelper 1 (Th1) and cytotoxic T cell responses, as well as the induction of Tc17 and regulatory T cell responses, the presence of classical and non-classical class-I-restricted responses, and provided a detailed characterization of S. Typhi-specific memory T cell subsets and induction of B memory (B_M) cells. In the past few years his group has also made important contributions to our understanding of the cross-reactive immune responses elicited by immunization with typhoid vaccines against S. Paratyphi A and B. Overall, these studies have contributed key information to accelerate the development of vaccines against enteric fevers. 

Another key field in Dr. Sztein’s laboratory is the identification of correlates of protective immunity to Shigella, E. coli and other gram negative enteric pathogens in humans.  This is particularly relevant since no vaccines are available to prevent these gram negative enteric infections. Thus, over the past 2 decades his laboratory has studied and made important contributions to our knowledge on human immunity to enteric gram negative pathogenic bacteria. For example, they were the first to report that volunteers immunized with attenuated Shigella vaccine candidates developed IgA and IgG B_M to S. flexneri 2a antigens. In a follow up study they made the key observation that inverse correlations between magnitude of pre-challenge IgA IpaB-B_M and post-challenge IgA LPS-B_M correlate with disease severity following challenge with wt Shigella suggesting a key role for antigen-specific B_M in protection. These findings are essential to accelerate the development of effective vaccines as the presence of B_M cells is likely to contribute to the persistence of systemic and mucosal antibodies and the ability to mount an anamnestic response when circulating antibody levels have already declined.   

Current projects encompass studies to investigate systemic and mucosal innate and adaptive immune responses in volunteers participating in vaccine trials being conducted at the CVD and other sites, including underdeveloped countries. These trials involve the clinical testing of genetically engineered attenuated vaccine strains developed at the CVD and other institutions, such as attenuated S. Typhi and Shigella (alone or as carriers of foreign genes).  Of particular importance, in collaboration with Dr. Pollard -Oxford, UK- and others, Dr. Sztein is performing in depth immunological studies using specimens from subjects orally challenged with wild-type S. Typhi, as well as subjects vaccinated with attenuated typhoid vaccine candidates followed by a challenge with wt S. Typhi.

Dr. Sztein is the principal investigator in an NIH Research Project Cooperative Agreement (U19) entitled “Mucosal and Systemic Immunity, Vaccines and Microbiota Interplay in Humans”, one the Cooperative Centers for Human Immunology (CCHI) NIAID network. The UMB CCHI research focuses on furthering our understanding of the protective immunological mechanisms that are elicited upon oral immunization in the gastrointestinal tract microenvironment with emphasis on S. Typhi. Moreover, the CCHI multidisciplinary team of investigators conducts pioneering studies on the interactions between the local intestinal microbiota and host immunity following oral vaccination, as well as the development of novel technologies to advance and accelerate vaccine development in humans.  Moreover, because of the importance of intestinal epithelial cells in defense and in regulating innate immunity when the enteric pathogen first encounters the gut epithelial cell layer, extensive studies are directed to investigate antigen trafficking, production of pro-inflammatory and other mediators, and other key events by using in vitro organotypic models of the human intestinal mucosa and biopsy explants.

Dr. Sztein has participated for many years in international collaborative studies and is deeply committed to the training of investigators in underdeveloped nations, including Mali, West Africa and Bangladesh.  For example, he has directed the establishment of a state-of-the-art immunology laboratory (that includes flow cytometry capabilities) at the University of Mali in Bamako and an immunology lab at the Bandiagara field site in Mali, West Africa, to study the immune responses of volunteers in malaria endemic areas.

Dr. Sztein is also an expert in flow cytometry, having been involved in this field since 1986.  For the past 26 years he has directed the CVD flow and mass cytometry facility which serves campus investigators who need access to this state-of-the-art instrumentation and technology.  

Marcelo Sztein, M.D. (PI) 
U19-AI082655
06/01/2014-05/31/2019
National Institutes of Health (NIH)
Mucosal and Systemic Immunity, Vaccines and Microbiota Interplay in Humans

Marcelo Sztein, M.D. (PI)
R01-AI036525
03/19/2013-02/28/2018
NIH
Immune Mechanisms of Protection in S. Typhi Vaccines

Myron Levine, M.D., D.T.P.H. (Center of Excellence for Translational Research (CETR) PI)
Marcelo B. Sztein, M.D. (PI Research Project 1)
U19-AI109776
03/01/2014-02/28/2019
NIH
Immunoprophylactic Strategies to Control Emerging Enteric Infections

Claire Fraser, Ph.D. (Genomic Centers for Infectious Diseases (GCID) PI)
Marcelo B. Sztein, M.D. (PI of the Immunology Core)
U19-AI110820
04/15/2014-04/14/2019
NIH
Host, Pathogen, and the Microbiome: Determinants of Infectious Disease Outcome

Karen Kotloff, M.D. (PI)
Marcelo Sztein, M.D. (Senior Immunologist)
HHSN2722013000221
09/16/2013-09/15/2023
NIH
Vaccine and Treatment Evaluation Units (VTEU)

Marcelo B. Sztein, M.D. (PI)
HHSN27200012
09/01/2015–02/28/2018

• Establishment of central facilities to analyze clinical samples for levels of circulating cytokines (including chemokines and growth factors) and activation and/or increase in subpopulations of natural immune cells, and T and B cells from multiple DMID-funded VTEU clinical studies and trials.

Marcelo B. Sztein, M.D. (Laboratory PI)
HHSN27200010

• A Phase 1 double-blind, placebo-control, dose escalating study to evaluate the safety and immunogenicity of double mutant heat labile toxin LTR192G/L211A(dmLT) from enterotoxigenic Escherichia coli (ETEC) by oral, sublingual, and intradermal vaccination in adults residing in an endemic area.  

Myron Levine, M.D., D.T.P.H. (PI)
Marcelo Sztein, M.D. (Co-director of Immunology, Training Faculty and Member of the Executive Committee)
T32AI07524
07/01/2013-06/30/2018
NIH

Flow Cytometry Core Laboratory

Dr. Sztein, Director

The core facility currently houses four flow cytometer systems described below:

1. Custom Becton-Dickinson LSR-II Flow Cytometer Analyzer. This analyzer is equipped with four lasers (a 100 mW solid state 488 nm blue laser, a 100 mW solid state 640 nm red laser, a 50 mW solid state 405 nm violet laser and a solid-state 552 nm green laser). This system also includes 17 ultra-high sensitivity PMTs (e.g., is capable of collecting 15/16 colors simultaneously) plus forward and side light scatter parameters.  This analyzer is controlled by a state-of-the-art PC-based computer that includes a CD/RW drive and a 1 Tb external hard drive for data archival.

2. Beckman-Coulter MoFlo Astrios EQ Flow Cytometer/Cell Sorter System. This state-of-the art upgradable system is equipped with four lasers (a 405 nm, a 488 nm, a 640 nm and a 100 mW 355 nm JDSU UV). This system also includes 21 ultra-high sensitivity PMTs (e.g., is capable of collecting up to 19 colors simultaneously) plus side scatter and forward scatter to collect light scatter parameters.  The system can be easily upgraded to a max of 32 PMT’s if this becomes necessary due to the development of new fluorochomes, a very likely possibility in coming years. Furthermore, this system is capable of sorting simultaneously 6 distinct cells subsets into a variety of tubes and plates, including 96-well plates for single cell transcriptomics analyses, an important component of this HIPC application.  Of great importance for the proposed studies which include sorting various cell subsets in all Research Projects is that this system features some of the most advanced sorting capabilities of any instrument in the market today concerning cell yield and purity, coupled with automated features to avoid possible cell loses during the sorting process, not an uncommon occurrence.  They include Sort Rescue (auto-stops in case of a clog), IntelliSort II (a fully bead-less drop delay determination and monitoring system) and Auto QC (which automatically sets laser delay for each laser, adjusts PMT voltages, compares CV’s and PMT voltages to pre-determined values and records daily QC).  

Of importance, this system is contained in an integrated Biosafety Cabinet II (Baker) to enable the safe sorting of human cells obtained from healthy individuals participating in the vaccine and challenge studies described in this HIPC application. In addition, technical personnel routinely wear Personal Protective Equipment and use enhanced BSL-II precautions when sorting human cells.  Finally, the system also includes an integrated refrigerated waterbath (ThermoFisher Scientific) for the collection of sorted cells at defined temperatures.

3. Fluidigm CyTOF 1 Mass Cytometer. A Fluidigm CyTOF Mass Cytometer (time-of-flight atomic mass spectrometer for high-speed acquisition of highly multi-parametric single cell data) is a single-cell analyzer which uses mass spectrometry to allow the simultaneous detection of at least 35 metal-labeled antibodies and two DNA intercalators - one for cell identification and one for live/dead discrimination in a single panel. The mass spectrometer in the CyTOF has been specifically tuned to measure in the range of the Lanthanide series of elements. It has the capability to measure up to 94 independent heavy metal signals, once the reagents become available. Unlike traditional flow cytometers, there is no overlap of the emission signals, which means there is no need for compensation and there is no background above the instrument noise. The CyTOF in our facility is equipped with an Autosampler, which can handle up to three 96-well plates.

4. Fluidigm CyTOF Helios Mass Cytometer. As described above in the CytOF 1 description, mass cytometry is a remarkable novel technology by Fluidigm that enables the simultaneous detection of >35 metal-labeled antibodies and two DNA intercalators in a single panel.  Early in 2016 Fluidigm released the latest version of this instrumentation, the CyTOF Helios, which include marked improvements in performance, speed, easiness of operation (particularly in sample introduction) and the number of parameters (i.e., mass spec channels) that can be collected simultaneously, without the need for cross-beam compensation.  These improvements result in increased data quality and easiness of use. For example, compared to CyTOF 1, the Helios allows an almost doubling of the acquisition events, 135 mass spec channels, increased sensitivity ( i.e., improved signal to noise ratios, now at 600,000 counts/pg Tb, resulting in even higher detection of low abundance targets), automated calibration, real-time bivariate plots, 7.2 Tb RAID, mirrored storage, etc.      

Flow Cytometry Core Laboratory

US patent 9,200,258: Multicellular Organotypic Model of Intestinal Mucosa, 2015.

• Inventors: Rosangela Mezghanni, Alessio Fasano, and Marcelo B Sztein