Introduction
Our overall goal is to understand the process of the generation of long-term humoral immunity. Long-lived plasma cells and memory B lymphocytes are the primary cellular components of long term humoral immunity, and as such are critically important for protection against many infectious diseases and are key components of the protection afforded by most vaccines.

SAP, SLAM-family receptors, and the regulation of B cell mediated immunity
Germinal centers are the critical sites for the development of long term humoral immunity in the form of antigen-specific memory B lymphocytes and long-lived plasma cells. CD4+ T cells are essential for germinal center function. Therefore it is vital to understand the role of CD4+ T  cell help to B lymphocytes to understand how to better generate long term humoral immunity to viruses. Importantly, we now have an excellent entry point for examining this process, since we have shown that SAP (SLAM-associated protein. Gene: Sh2d1a) plays a central role in CD4+ T cell help at the germinal center stage for the development of long term humoral immunity after a viral infection (Crotty et al., Nature 2003; Crotty and colleagues J. Immunology 2007). We also know that this critical function for SAP is conserved in humans. Understanding the role of SAP in greater detail may help XLP patients and may, more broadly, allow for the design of better human vaccines that take advantage of SAP’s important role in the process of generating immune memory. Our laboratory is focused on answering pivotal questions regarding T cell help to B lymphocytes and the generation of long-term humoral immunity. This knowledge will help illuminate a central process of adaptive immunity and help accelerate vaccine discovery, as a primary goal of vaccines is generation of memory B lymphocytes and long-term antibody production). Our studies focus heavily on antiviral immune responses (LCMV and vaccinia virus) because 1) SAP-deficiency results in an lethal susceptibility to infectious disease in humans, demonstrating that SAP is a crucial mediator of antiviral immune responses, and 2) the purpose of virtually all vaccine development is the prevention/control of infectious diseases.

Protective immunity generated by vaccines: roles of neutralizing antibodies. Smallpox as a gold standard model.
The smallpox vaccine is the prototypic vaccine, yet the viral targets critical for vaccine-mediated protection remain unclear in humans. We have utilized protein microarrays of a near-complete vaccinia proteome and used them to determine the major antigen specificities of the human humoral immune response to the smallpox vaccine (Dryvax®).

Vaccines are one of the most cost-effective medical treatments in modern civilization. A smallpox vaccine was the first human vaccine, and vaccinia virus (VACV) is considered the most successful human vaccine, bringing about the worldwide eradication of smallpox disease. Nevertheless, the mechanisms of adaptive immune protection elicited by the smallpox vaccine in humans generally remain unclear. There is also currently greatly renewed interest in smallpox immunity due to the possible threat of bioterrorism. Given this concern, there has been much discussion about the mechanisms of protection afforded by the smallpox vaccine.

Humoral immunity plays a major role in the protection mediated by most vaccines, including the smallpox vaccine (vaccinia virus, VACV). Memory B cells are a central component of humoral immunity, and yet little is known about their characteristics, their longevity in humans, or their protective value. In the interest of better understanding the role of human memory B cells in protection against disease, we have developed an assay to quantitate antigen-specific memory B cells in human blood (Crotty et al. JIM 2004). Using this assay we demonstrated that smallpox vaccine specific memory B cells last for greater than 50 years in immunized individuals (Crotty et al. J. Immunology 2003). Immune memory after smallpox vaccination is a valuable benchmark for understanding the longevity of B cell memory in the absence of re-exposure to antigen. These persisting human memory B cells were functional and able to mount an anamnestic antibody response upon re-vaccination. Additionally, VACV-specific CD4+ T cells and antibodies were detected decades after vaccination.

Humoral immunity plays a major role in the protection mediated by most vaccines, including the smallpox vaccine (vaccinia virus, VACV). Understanding the relevant protein targets on a pathogen is always important for understanding mechanisms and roles of humoral immunity, and VACV is no exception. These efforts are important for developing a clear understanding of the mechanisms of protection afforded by this prototypic vaccine. Hyperimmune human serum (VIG) is the licensed therapy for smallpox virus infection or disseminated vaccinia virus infection. Which envelope proteins are the major protective/neutralizing targets of the human anti-VACV antibody response? This is a complex issue, made more complex by the existence of both MV and EV virion forms. Vaccinia virions are complex macromolecular structures consisting of some 80 different proteins, of which > 20 are known envelope proteins. Given the antigenic complexity of the VACV envelope, do antibodies to multiple envelope proteins should have the potential to contribute to the overall protection afforded to an immunized person, and which viral proteins are in fact neutralizing antibody targets in humans? We and others have made substantial progress answering these questions in the past several years. A variety of immunogenic VACV antigens eliciting antibody responses have been identified previously in the literature, but the vast majority of that work was done in animal models. We have focused our efforts on understanding the human humoral immune response to VACV. These efforts have centered on identifying the antigen-specificities of the human anti-VACV antibody response and determining which antibody targets are likely to be functionally valuable in protection.
We have demonstrated that H3 is a neutralizing antibody target frequently recognized by vaccinated humans (J. Virology 2005, PNAS 2005, J. Virology 2008). Overall, we have found that diversity is a defining characteristic of the human antibody response to the smallpox vaccine. We show H3 is the most immunodominant VACV neutralizing antibody target, as determined by correlation analysis of IgG specificities to MV neutralizing antibody titers. It was determined that purified human anti-H3 IgG is sufficient for neutralization of VACV; however, depletion or blockade of anti-H3 antibodies revealed no significant reduction in neutralization activity, showing anti-H3 IgG is not required in vaccinated humans (or mice) for neutralization of MV VACV (J. Virology 2008). Comparable results were obtained for human (and mouse) anti-L1 IgG, and even anti-H3 and anti-L1 IgG in combination. In addition to H3 and L1, human antibody responses to D8, A27, D13, and A14 exhibited statistically significant correlations with virus neutralization. Altogether, these data indicate the smallpox vaccine succeeds in generating strong neutralizing antibody responses not by eliciting a stereotypic response to a single key antigen, but instead succeeds by driving neutralizing antibody development to multiple viral proteins, resulting in a “safety net” of highly redundant neutralizing antibody responses, the specificities of which can vary from individual to individual. We propose this is a fundamental attribute of the smallpox vaccine.

We have also developed fully human monoclonal antibodies against vaccinia virus, monkeypox, and smallpox as therapeutics for humans infected with either virus. The antibodies are currently undergoing rigorous pre-clinical testing, in collaborative development with Kirin Pharma USA for use in humans.