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.