Oxford University Press Impact of Ebola Mucin-Like Domain on Antiglycoprotein Antibody Responses Induced by Ebola Virus-Like Particles
Ebola viruses (EBOVs) are enveloped, single-stranded, negative-sense RNA viruses belonging to the family
Filoviridae
that cause a hemorrhagic fever in humans, with a mortality rate of up
to 90%. EBOV attachment and entry into host cells is mediated by the
viral envelope glycoprotein (GP) [
1–
3].
The membrane-anchored GP is a trimer. GP is first produced as a
676-residue–long precursor, GP0, which is cleaved by furin into
disulfide-bonded GP1 and GP2.
These
GP1/GP2 heterodimers assemble into a
chalice-shaped trimer that is expressed on the virion surface [
4].
GP1 is distal to the membrane surface and contains the receptor-binding
domain (N-terminal residues 54–201) and a heavily glycosylated
mucin-like domain.
GP2 possesses a transmembrane domain,
the fusion
peptide, and heptad repeats required for virus–cell membrane fusion [
5,
6].
When
expressed from 293T cells, the EBOV matrix protein, virion protein 40
(VP40), induces the production of virus-like particles (VLPs) that are
biochemically and morphologically similar to EBOV. If coexpressed with
VP40, GP becomes incorporated into the VLPs and can mediate entry into
target cells [
7,
8].
GP-mediated entry likely occurs via macropinocytosis [
9,
10], although other endocytic pathways have also been implicated in entry [
9–
14].
For productive entry, GP is cleaved by cellular cathepsins B and L in
acid endosomes such that a substantial portion of the protein is removed
and the remaining approximately 19 kDa cleavage product is sufficient
to mediate membrane fusion reactions [
15–
19].
Among
the regions of GP removed by cathepsin cleavage is the highly
glycosylated mucin-like domain. The mucin-like domain is not required
for viral entry, because mucin-domain–deleted GP is able to mediate
viral attachment and entry in pseudotyped virus systems.
EBOV GP
interacts with
lectin-binding receptors present on some cell types,
including antigen-presenting cells (APCs) , thereby promoting virus attachment
and entry [
20–
24].
The mucin-like domain has specifically been shown to be
important for
interaction of GP with the human macrophage galactose-specific and
N-acetylgalactosamine-specific C-type lectin (hMGL), promoting EBOV
infection [
25].
In addition, it may serve immune-modulating functions.
For example, its
presence is correlated with
an ability to alter cellular signaling,
including mitogen-activated protein kinase (MAPK-) signaling [
26,
27] and has been shown to
enhance Ebola VLP-mediated cytokine secretion from stimulated dendritic cells [
27].
Furthermore, VLPs with wild-type but not mucin domain–deleted GP
can activate toll-like receptor 4–dependent responses [
28].
The GP mucin-like domain obstructs access to GP epitopes and epitopes expressed on other surface proteins [
29].
For example, expression of high levels of GP [
30]
blocks access to surface major histocompatibility complex class 1 molecules (MHC1) and
β-integrins [
29], resulting in loss of specific anti-MHC1 or β-integrin antibody binding, decreased CD8 T cell access to MHC1 [
31] and
induction of cell rounding as a consequence of
anchorage loss [
32].
Furthermore,
removal of the mucin-like domain with cathepsin L uncovers
epitopes for neutralizing antibodies [
16,
33].
However, a mouse model of EBOV infection has shown that immunization of
mice with a GP-expressing vaccine elicited mucin domain–specific
monoclonal antibodies, some of which were protective [
34].
The contribution of antibody responses to EBOV infection and its role in protective immunization remain unclear [
35].
Studies have demonstrated
the presence of EBOV-neutralizing antibodies in the sera of human survivors of infection [
33,
36].
However, passive transfer of neutralizing anti-EBOV antibodies has
shown mixed efficacy, failing to be protective in the more relevant
nonhuman primate models of EBOV infection but exhibiting some efficacy
in rodent models.
Furthermore, there is also
evidence of enhanced EBOV
binding to target cells in the presence of anti-EBOV antibodies [37–39].
EBOV
VLPs are immunogenic and have been used to vaccinate and protect mice
and nonhuman primates in experimental animal models of EBOV infection [
40,
41].
This immunogenicity is due, at least in part, to the mucin domain of
the GP, yet studies have shown that the mucin-like domain itself may
block access to the GP and adjacent immune molecules potentially
inhibiting immune responses, including neutralizing antibody, raising
the question of
whether vaccines would be more efficacious with or
without the GP mucin-like domain. Therefore, to test the hypothesis that
the mucin-like domain modulates antibody responses to GP, including
neutralization, mice were immunized with VLPs expressing EBOV wild-type
GP, GP lacking the mucin-like domain, and a GP expressing a heterologous
mucin-like domain; the ensuing antibody response was assessed.
