Robert L. Garcea, M.D.

Research Interests

Structure and assembly of small DNA viruses (polyoma and papilloma);  development of low cost vaccines for  under-resourced areas of the world.

Research Profile

My laboratory studies two fundamental cell biologic controls on DNA virus assembly:  1) how capsid assembly occurs only in the nucleus and is prevented in the cytoplasm, and 2) how the capsid proteins identify their genome for encapsidation.  We have found that cell chaperone proteins (hsc70 family) bind the polyomavirus capsid proteins immediately post translation and appear to accompany the viral proteins as they are transported into the nucleus.  We have recently recapitulated chaperone-mediated capsid assembly in vitro using recombinant capsid proteins, hsc70/hsp40, and ATP.  Genome targeting appears mediated by virus-specific DNA binding proteins (e.g., large T-antigen for polyoma and SV40), and we are now attempting to reconstitute polyomavirus assembly (including genome/minichromosome encapsidation) entirely in vitro, by linking our chaperone-mediated assembly reaction with the well-characterized in vitro SV40 DNA replication system.  In infected cells, assembly appears to take place at distinct "foci" within the nucleus termed PML-nuclear bodies.  We are investigating the role of PML-NBs in polyomavirus assembly in mouse cells, and collaborating with the cryo-EM laboratory to image these  "virus factories".  Cell chaperones may also play a role in disassembling virions at the outset of infection, suggesting a “chaperone equilibrium” during the virus life cycle.

As a consequence of having purified recombinant capsid proteins for these viruses, we have also investigated their immunology within their own host as well as in preventing virus infection.  The polyoma capsids have been studied in various immunological mouse backgrounds for induction of specific immune responses, which appear linked to the mouse innate immune system. Infection with "high-risk" human papillomaviruses is associated with the subsequent development of cervical cancer, and we are now developing new vaccine reagents to prevent and treat papillomavirus infection.  The papillomavirus L1 protein is a powerful immunogen, and we have found that the subunits (capsomeres) of the virus capsid are equivalent to the entire capsid in generating protective immunity in model animal systems. We are now developing a capsomeric vaccine as a next generation prophylactic vaccine for use in underdeveloped countries (NCI Rapid Award: Shantha Biotechnic, Hyderabad India).  The ability to induce strong immune responses without adjuvant appears related to the ability of L1 to interact avidly with dendritic cells. This immunogencity is being harnassed by fusing unrelated epitopes to L1 to enhance their immune recognition.  We are now testing fusions of the early HPV proteins, E6 and E7, to L1 in order to generate a “therapeutic” vaccine that will eradicate already infected cells (Gates Global Challenge Award:  BioSidus, Buenos Aires).

My laboratory therefore offers an environment for both basic and translational research, with opportunities for graduate students, post-doctoral fellows, and clinical fellows.  The vaccine-related projects are now being extended to such areas as inhalable delivery, expression in cow’s milk, and extension to other pathogens such as malaria.   The goal is to deliver vaccines in a form and at a cost that is reasonable for use in underdeveloped areas of the world.