Antibodies that do not promote the destruction of pathogens in situ or prevent their internalization into target cells
through opsonization are nevertheless key factors in immunity against intracellular pathogens. An important mechanism
by which such “non-neutralizing” antibodies (nNAbs) curtail certain infections has been illuminated by the recent
discovery of antibody-dependent intracellular neutralization (ADIN). In ADIN, cytosolic nNAbs bound to pathogens are
recognized by the intracellular high-affinity Fc receptor Trim21 and the complex is degraded by the proteasome. ADIN
has been found capable of eradicating an enveloped virus. Given that enveloped viruses shed bound antibodies upon cell
membrane fusion, the means by which nNAbs access the cytosol is puzzling. That nNAbs access the cytosol in vivo but not
in vitro suggests that culture models lack a critical cell or factor for nNAb entry.
Herpes simplex viruses (HSV)-1 and -2 initially replicate in a lytic manner at mucosal surfaces but then establish life-
long residence within ganglion neurons of the peripheral nervous system. In neuronal nuclei the viral genome enters
latency, a state of quiescence in which replication is repressed and lytic proteins are produced at near-undetectable levels.
As mature neurons are non-renewable and unchecked viral replication can lead to central nervous system damage, the
control of latency is critical to the host. Ganglia are monitored by CD8+ T-cells, which deliver granzyme B at the
immunological synapse with infected neurons via perforin pores. Cytosolic granzyme B digests ICP4, an essential
transcriptional regulator. The model is therefore that without ICP4 to upregulate transcript production, latency is
perpetuated. Our data show that ICP4 antibodies exist in seropositive human trigeminal ganglia, suggestive of a check on
ICP4 expression by granzyme B and possibly by antibodies to ICP4 that mediate its ADIN.
We hypothesize that antibody-dependent intracellular neutralization of ICP4 limits HSV-1 reactivation. We will
address this in experiments of two Specific Aims: 1) assess whether antibodies gain access to the neuronal cytosol through
the close interaction between CD8+-T-cells and latently-infected neurons; and 2) determine whether non-neutralizing
antibodies direct ADIN of ICP4 produced during latent neuronal infection, stifling viral reactivation. We will use convergent
approaches to benefit from both an in vivo ¿ ex vivo mouse model of human HSV-1 latency, and an in vitro model of
latent HSV-1 infection in human induced pluripotent stem cell-derived neurons. If successful, Aim 1 will define a
heretofore undescribed route of nNAb entry into cells in the context of HSV-1 latency. Aim 2 will elucidate the molecular
basis of the complex balance between viral latency and host immunity. If successful, this R21 study will shift the focus of
HSV vaccine target design to accommodate a role for ADIN in anti-viral immunity, and indicate potential therapeutic
targets including inhibitors of reactivation, or agonists of ADIN. Advances from our studies could also inform far-reaching
approaches to antagonize human pathogens with a latency state including HIV, HCMV, MeV, and EBV.