Adaptive Immunity: Focus on B cell immunology
Program leader: James Crowe
SE-RP-013: Genetic and structural determinants of neutralization by naturally-occurring human monoclonal antibodies to dengue virus
Vanderbilt University
Dengue is expanding globally with an estimated 50 million to 100 million cases of dengue world-wide annually, and more than 20,000 deaths. The virus has caused infection in Florida in the past year, and could spread widely in the Southern US in coming years. The pathogenesis of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) of course is of central importance to those interested in developing vaccines. Clearly, antibodies to dengue virus E protein can neutralize the virus in vitro, but there is also concern that cross-reactive low potency antibodies to E, and antibodies to prM protein, can enhance disease. The simple overview of current concepts of pathogenesis suggests that an initial infection with dengue virus occurs and induces antibodies, followed in some cases with a second infection with a different serotype during which pre-existing cross-reactive antibodies form non-neutralized antigen-antibody complexes that enter cells expressing Fc receptors such as monocytes, resulting in increased release of cytokines and the release of vasoactive mediator that increase vascular permeability. This process has been termed antibody-dependent enhancement (ADE) of infection, and has been demonstrated clearly to occur using cells in culture. A better understanding of the molecular, genetic, and structural basis for recognition of dengue viruses by human antibodies is sorely needed, and could lead to the rational design of vaccines that enhance the induction of neutralizing antibodies while lowering the risk of DHF. We have made significant progress in the past year, isolating large panels of novel human antibodies to dengue E, prM and capsid proteins. The studies reveal that most antibodies made by humans are low potency cross-reactive clones. We are investigating the epitope specificity of the E protein Abs, and find that most do not recognize E protein domain III, in contrast to most mouse studies. We are now isolating Abs from primary versus secondary infections, vaccinees vs. natural infection, and mild vs. severe cases to determine if certain Ab patterns are characteristic of these conditions. We are beginning to investigate the structural basis for neutralization and enhancement. In the coming year, we anticipate bringing higher throughput techniques to bear on the Ab repertoire analysis.
SE-RP-014: Regulatory B cell inhibition of immune responses to pathogens
Duke University
Immune responses are controlled, in part, by regulatory B cells, including a recently identified CD1dhiCD5+ IL-10-competent B cell subset that we call B10 cells. B10 cells expand during immune responses, inhibit inflammatory responses, regulate CD4+ T cell activation, and dampen humoral immunity. LPS, PMA, and ionomycin stimulation in vitro for 5 h induces B10 cells to express cytoplasmic IL-10. We have also identified B10 progenitor (B10pro) cells that acquire IL-10 competence during in vitro maturation. Both adaptive and innate signals regulate B10 cell development, maturation, and competence for IL-10 production.
In collaboration with Dr. Mark Heise’s group at UNC-Chapel Hill, we found that B10 cells may regulate morbidity during alphavirus infection. We utilized two strains of mice, one that lacks B10 cells (CD19-/-) and one that has an increase in B10 cells (hCD19Tg). Weight gain by young alphavirus-infected CD19-/- mice and wild-type uninfected mice is similar, indicating that these mice are protected from virus-induced pathology. By contrast, infected hCD19Tg mice closely resemble wild-type infected mice, suggesting complex interactions between B cell augmentation of immunopathology and B10 cell negative regulation of immunopathology during virus infection. To better delineate the function of B10 cells during alphaviruse infection and to expedite mechanistic studies that can also be applied to other select-agent pathogens, we have also established acute and chronic viral infection models using lymphocytic choriomeningitis virus (LCMV), and acute bacterial infections using Listeria monocytogenes.
During the past year, we have found that B10 cells modulate macrophage function and thereby inhibit pathogen clearance. As early as 5 days post-infection with L. monocytogenes or LCMV Armstrong, B10 cells expand at least two-fold in the spleen, lymph nodes, and blood. The frequency of B10pro cells declines in both infection models throughout the infection, but their numbers remain equivalent to uninfected controls. We utilized the SERCEB mAb core to produce mAbs that selectively deplete B10 cells in order to study the importance of B10 cell expansion. Selectively depleting B10 cells prior to L. monocytogenes infection enhances pathogen clearance from the spleen and liver. Adoptive transfers of CD1dhiCD5+ B cells, but not CD1dloCD5- B cells, inhibits the in vivo clearance of L. monocytogenes. Given the importance of interferon gamma (IFN-γ) in early clearance of L. monocytogenes, macrophages remarkably produce more IFN-γ and TNF-α following infections in the absence of B10 cells. The proliferation of antigen-specific CD4+ T cells is also reduced in the absence of B10 cells. This observation is most likely due to enhanced macrophage activation in the absence of B10 cells and with reduced bacterial load. Taken together, these observations support the conclusion that B10 cells inhibit pathogen clearance, which offers a likely explanation for the resistance of CD19-/- mice to alphavirus infection. Understanding how B10 cells regulate the responses to diverse pathogens and how their manipulation can be used for therapeutic benefit may identify ways to enhance immunity in response to biodefense threats.
