Division Photo
Row 1: M Staat, R Cardin
Row 2: M Dickey, M Steinhoff, J Jiang, E Schlaudecker
Row 3: N Hutchinson, M McNeal, S Shah, M Tan
Row 4: R Frenck, M Hostetter, D Bernstein, T Farkas
Division Highlights
Margaret K. Hostetter, MD
The Hostetter laboratory focuses on mechanisms and consequences of heparin binding by Candida albicans. Binding of heparin by C. albicans has been shown by our laboratory to be essential for:
(1) removal of candidal surface proteins that serve as targets for elimination of the organism by innate immunity
(2) exposure of a C. albicans superantigen, defined in J Inf Dis 2008; 197:981-9
(3) biofilm formation in vivo
Collaborators at the University of Cincinnati and CCHMC have helped us to define the mechanism of heparin binding. With Jason Lu, CCHMC Bioinformatics, we evaluated linear heparin binding sites in 400 C. albicans surface proteins. Fifty-six C. albicans proteins were found to have heparin-binding sites that conformed to the Cardin, Weintraub, or Sobel motifs. Of these 56, 13 were expressed at the bud neck, the site at which the yeast cell undergoes hyphal morphogenesis. These 13 proteins participated in various functions including chitin synthesis, anti-fungal resistance, GTPase activation, and phosphorylation. The protein with the most heparin binding motifs was Int1, which we had defined in our laboratory (Science 1998; 279:1355-8). In collaboration with Alexey Porollo, UC Environmental Health, we identified a cup-like structural motif in at least one of these sites that was conformationally consistent with heparin binding. A collaboration with Apryl Stallcup, UC Chemistry, provided additional information regarding the structure of heparin and possible sites of interaction with positively charged amino acids. Lastly, in a collaboration with Ken Greis, UC Proteomics laboratory, we refined the method of SILAC to examine proteins removed from the C. albicans surface after incubation with heparin. Of the top 25 proteins removed (of more than 100), 13 had identifiable heparin binding sites. The proteomics project received core grant funding from the CCTST. These collaborative efforts have provided key insights into the biochemistry of heparin binding.
David I. Bernstein, MD, MA
The pivotal trial for a genital herpes vaccine composed of herpes simplex type 2 glycoprotein D (gD) and adjuvanted with MPL and Alum was completed. Although two large trials showed efficacy in women this trial showed no efficacy for prevention of genital herpes caused by HSV type 2 (HSV-2) but surprisingly demonstrated efficacy against HSV-1 genital herpes. On a more positive note, our preclinical evaluations of vaccines that also contained gB and gH/gL combined with cationic lipid DNA complexes as an adjuvant showed protection, including protection of the dorsal route ganglia, the site of HSV latency.
The large multicenter cytomegalovirus trial that screens infants for congenital CMV has enrolled over 95,000 newborns. We have found that dried blood spots are not suitable for screening (published in JAMA) but that saliva was an excellent source for this screen (published in the NEJM).
Turning to GI viruses we completed the first human trial of a vaccine for norovirus and found it to be moderately protective (accepted for publication, NEJM) while the rotavirus vaccine we developed showed continued efficacy in preventing hospitalizations and deaths around the world.
Rebecca Brady, MD
We published three manuscripts regarding human antibody and T cell responses elicited by influenza vaccines against the hemagglutinin protein.
Rhonda Cardin, PhD
In previous studies, the Cardin lab showed that the viral chemokine receptor M33 encoded by murine cytomegalovirus (CMV) is required for long term latent infection of the bone marrow. In 2010, we identified several cell types in the bone marrow which are latently infected, and importantly, when the virus has a mutation in the M33 gene, some of these cell types do not become latently infected. This is the first identification of bone marrow cells which harbor latent CMV infection and the role that M33 plays during latent CMV infection at the cellular level. In collaboration with Dr. Helen Farrell in Australia, we have shown that the viral chemokine receptor US28 homolog encoded by human CMV rescues the latency defect of a mutant M33 virus, thus suggesting that the human CMV US28 protein plays a similar role in the establishment of CMV latency in humans. Also, in collaboration with Dr. David Bernstein and Dr. Daniel Choo, we have shown that infection of guinea pigs with guinea pig CMV induces hearing loss as measured by Auditory-Evoked Brainstem Analysis (ABR). We find that guinea pig CMV infects the cochleas of newborn guinea pigs which are experimentally infected at 48 hours after birth (neonatal CMV model) or in newborn guinea pigs which were infected in utero following infection of the pregnant guinea pig (congenital CMV model). Both models show that ~50% of the infected pups develop hearing loss. Development of the guinea pig CMV hearing loss models is significant since human CMV is the leading infectious cause of hearing loss, with approximately 0.5-2.0% of newborn infants experiencing hearing loss within the first 2-4 years of life. Studies are underway to determine the underlying mechanisms of CMV-induced hearing loss and to determine if antiviral therapy can prevent the loss.
Tibor Farkas, DVM, PhD, MBA
The Farkas lab studies enteric viral diseases of humans and animals. In 2010 we continued our work on non-human primates (NHP) enteric caliciviruses with the major goal to develop a new disease model for human norovirus (NoV) gastroenteritis. Our work demonstrated remarkable similarities between rhesus enteric caliciviruses (ReCV) and human NoVs including their epidemiology, genetic and antigenic diversity, putative receptor (histo-blood group antigen; HBGA) binding and the ability to induce gastroenteritis in challenged animals. Over15 ReCVs representing different serotypes and HBGA types were tissue culture adapted. Current work is focusing on the development and evaluation of the animal, tissue culture and surrogate model that duplicates the diversity of human NoVs.
In 2010 we described two novel picornavirus groups in turkeys and chickens, including birds with runting stunting syndrome (RSS). RSS of chickens and poult enteritis mortality syndrome (PEMS) of turkeys are the two most significant enteric diseases of poultry. The causative agent(s) of RSS and PEMS are yet unidentified. Establishing the role of picornaviruses identified in our laboratory in enteric diseases of poultry is ongoing.
Robert W. Frenck, MD
We completed the first ever human challenge model with a G2 strain of norovirus. Dr. Bernstein is the IND holder for the virus and Dr. Jiang and Dr Frenck were Principal Investigators on the study. This study has been parlayed into a project with LigoCyte to see if their G2 norovirus vaccine can protect people with subsequent challenge with our G2 strain.
Jason Jiang, PhD
Noroviruses are an important cause of acute gastroenteritis. In the past year we have made significant advancements in understanding the virus/host interaction and receptors for noroviruses. We have resolved the crystal structures of the first non-secretor binding strain (VA207) which would greatly facilitate our future study on the screening and design antivirals against noroviruses. Our preliminary study on silico screening for antivirals against noroviruses based on these crystal structures is ongoing. We have completed the first human volunteer challenge study on a GII.4 norovirus and demonstrated the association of GII.4 infection with the histo-blood types of the volunteers, which concluded one of our projects funded by the DoD. This challenge model is very valuable for future vaccine and antiviral evaluation because it is the first human volunteer challenge with a GII.4 norovirus and the GII.4 noroviruses have been found predominant in many countries around the world. In fact, following the DoD study, we have initiated another study funded by LigoCyte to determine the 50% infectious dosage of the challenge pool. We anticipate a number of vaccine trials will be initiated in the near future using this challenge model, including evaluation of our P particle vaccine licensed to LigoCyte. Our study on the evolution of noroviruses has been summarized in a review article published recently in Trend in Microbiology. Our study on the development of norovirus P particles as a candidate vaccine against noroviruses is in the second year of a NIH R01 and we have made excellent progress in the characterization of norovirus antigenic and HBGA receptor binding variations, the establishment of animal model for vaccine efficacy evaluation and scale up production of P particles for future animal and clinical evaluation. Finally, we have initiated a number of new studies in inactivation/neutralization of Tulane virus replication by silence RNA, screening antivirals against TV protease, diagnosis and passive immunization of norovirus infection with IgY developed in chickens, the study of viral host receptors of rotaviruses, and establishment of TV as a surrogate for human noroviruses in food safety research against norovirus contamination funded by the USDA.
Monica McNeal, MS
The Laboratory for Specialized Clinical Studies continues to supply lab support for a large number of clinical studies involving vaccine trials and vaccine development. Influenza virus vaccines and rotavirus vaccines continue to be important for overall health of children in the US and around the world. The lab is committed to help establish clinical labs in India to support rotavirus vaccine trials in that country. In addition, the lab consults with other labs around the world to provide training and support for establishing quality assays to support vaccine trials.
Nancy Sawtell, PhD
Most of the human population world-wide has been infected by herpes simplex viruses. Following the initial lytic infection, HSVs establish permanent latent infections within sensory neurons. Reactivation of latent virus not only results in viral disease (new infections, blindness and encephalitis) but also contributes to HIV infection, diabetes, cardiovascular and neurodegenerative diseases. No effective vaccine is available and no therapy eliminates latency or prevents reactivation. The long-term goal of ongoing research in the Sawtell lab is to find interventions for recurrent HSV episodes by defining mechanisms that control establishment and reactivation of HSV-1 latency.
The gene expression cascade during HSV-1 lytic infection begins with activation of immediate-early (IE) gene transcription by the virion protein VP16 with host factors Oct-1 and HCF-1. In contrast, the initial events in the reactivation from latency are still poorly defined. Ourcentral hypothesis is that regulation of both VP16 expression and activity underlie the establishment of latency and reactivation from latency. These two levels of control involve multiple positive and negative inputs to allow or inhibit viral replication in the sensory neuron in vivo.
Significant Accomplishments
Diarrheal Viruses
Jason Jiang, PhD, and Ming Tan, PhD, have extended their work on the norovirus P particle and its adaptability as a vaccine platform. The norovirus P particle is formed by 24 copies of the protruding (P) domain of the norovirus capsid, which is ideally sized for a subunit vaccine. Their studies have shown that insertion of the M2e epitope of influenza virus and the VP8 epitope of rotavirus into the three surface loops of each P domain induced high titers of neutralizing antibodies against replication of influenza virus and rotavirus and protected vaccinated mice from infection with these two viruses.
The Rotarix vaccine, invented by David Bernstein, MD, and Richard Ward, PhD, was recently found to be associated with a 42 percent decrease in deaths per 100,000 children aged 11 months or younger in Mexico. Diarrhea-related mortality was 29 percent lower for children between the ages of 12 and 23 months, although these children are typically too old to receive the vaccine. Diarrhea-related mortality among unvaccinated children between the ages 24 and 59 months was not significantly reduced. (Richardson V. et. al., New England Journal of Medicine, 2010; 362:299-305).
Herpes Viruses
Nancy Sawtell, PhD, has continued her work on the herpes simplex virus type 1 (HSV-1) virion protein VP16 as a central mediator of latency and reactivation from latency in sensory neurons. Innovative animal models allow testing of the hypothesis that the VP16 gene in the HSV-1 genome can be regulated by action of neuro-specific and stress-responsive promoter elements and corresponding transcription factors to allow the differential responses entailed in initial infection or exit from latency. Sawtell hopes to identify transcription factors or protein-modifying enzymes that regulate VP16 and its differential functions as promising targets for development of therapeutic interventions for HSV reactivation.
Rhonda Cardin, PhD, has developed a guinea pig model that accurately recapitulates transmission of cytomegalovirus (CMV) from mother to fetus and is accompanied by attendant hearing loss. Since CMV infection in humans is the major cause of nonhereditary deafness, this critically important animal model will allow dissection of each step of pathogenesis and serve as an important tool for testing or therapeutic interventions.
Expansion of Clinical Services
Effective Jan. 1, 2011, Robert Frenck, MD, has assumed the position of chief of the Clinical Service after the departure of Michael Gerber, MD. Under Frenck’s leadership, divisional revenues from inpatient consults have increased by 37 percent, and the outpatient antibiotic service has had an increase of more than 40 percent in patient encounters over the past two years. Frenck’s plans call for the opening of a travel clinic at the Cincinnati Children’s Burnet Campus and a general infectious disease clinic at Liberty Campus within the next 12 months.
In continuing to refine their model of services for internationally adopted children, Mary Staat, MD, and the International Adoption Center have added mental health services and family counseling to help internationally adopted children overcome abusive backgrounds and to facilitate families’ adjustment to the emotional and educational needs of internationally adopted children.
