Table Of ContentM E T H O D S I N M O L E C U L A R M E D I C I N ETM
HHeeppaattiittiiss BB aanndd DD
PPrroottooccoollss
VVoolluummee IIII:: IImmmmuunnoollooggyy,,
MMooddeell SSyysstteemmss,,
aanndd CClliinniiccaall SSttuuddiieess
EEddiitteedd bbyy
RRoobbeerrtt KK.. HHaammaattaakkee,,
PPhhDD
JJoohhnnssoonn YY.. NN.. LLaauu,,
MMDD
1
Studying Host Immune Responses
Against Duck Hepatitis B Virus Infection
Darren S. Miller, Edward M. Bertram, Catherine A. Scougall,
Ieva Kotlarski, and Allison R. Jilbert
1. Introduction
The duck hepatitis B virus (DHBV) is a species-specific virus that causes either tran-
sient (acute) or persistent infections,primarily in hepatocytes in the liver,with release
of high titers of infectious virions and noninfectious “empty”surface antigen particles
into the bloodstream.
Because hepadnavirus replication is noncytolytic, cell-mediated immune (CMI)
responses to viral antigens are thought to be responsible for the clearance of virus from
infected cells and for the liver damage seen in transient and persistent infections. This is
presumed to occur via a direct, cytolytic effect of viral antigen-specific cytotoxic T
lymphocytes (CTLs) on infected hepatocytes,or via the noncytopathic action of inflam-
matory cytokines. In addition, neutralizing antibodies have been shown to prevent
infection by blocking the ability of virus particles to bind to receptors on target cells.
DHBV-infected ducks and woodchuck hepatitis virus (WHV)-infected woodchucks
are the most widely accepted and frequently used animal models for the study of viral
replication,infection outcomes,and the pathogenic mechanisms related to human hep-
atitis B virus (HBV) infection. Use of the DHBV model has allowed us to study the
effects of viral dose,age,and inoculation route on the course of DHBV infection (1–4)
and the effect of immunization with various forms of vaccine on all these parameters
(5). However, until recently, studies of the immune response to DHBV infection have
been hampered by the relatively poor characterization of the duck lymphoid system and
the lack of appropriate reagents. This chapter describes a number of assays that allow
study of components of the duck immune system and the cellular and humoral immune
responses to DHBV infection.
The chapter has been divided into three sections that include:
1. Purification and characterization of duck lymphocytes and thrombocytes from peripheral
From: Methods in Molecular Medicine, vol. 96: Hepatitis B and D Protocols, volume 2
Edited by: R.K. Hamatake and J.Y.N. Lau © Humana Press Inc., Totowa, NJ
3
4 Miller et al.
blood(6)and conditions for in vitro growth and lectin stimulation of duck peripheral blood
mononuclear cells (PBMCs; 7,8).
2. Histological methods for detection of cellular and viral antigens in duck tissues including
identification of duck T lymphocytes using anti-human CD3(cid:1)antibodies(9),identification
of Kupffer cells in the liver and phagocytic cells in the spleen,and detection of DHBV anti-
gens in fixed tissues by immunoperoxidase staining.
3. Detection of viral antigens,DHBV-specific antibodies,and viral DNA in duck serum using
enzyme-linked immunosorbent assays (ELISA) for DHBV surface antigen (DHBsAg),anti-
bodies to DHBV surface antigen (anti-DHBs antibodies),antibodies to DHBV core antigen
(anti-DHBc antibodies), and polymerase chain reaction (PCR) assays for detection of
DHBV DNA.
These techniques provide the opportunity to study immune responses to DHBV but
are by no means complete. For example, we have made numerous unsuccessful
attempts to develop viral antigen-specific CTL assays but progress has been hampered
by lack of suitable major histocompatibility class (MHC)-matched target cells. The
recent cloning by Professor David Higgins and colleagues of a series of duck T-
lymphocyte and cellular markers,that includes CD3,CD4,CD8,MHC I,and MHC II
(10–13),should allow more comprehensive monitoring of immune responses to DHBV
(seeNote 1).
1.1. Purification and Characterization of Duck Lymphocytes
and Thrombocytes from Peripheral Blood
Avian blood contains lymphocytes, monocytes, thrombocytes, red blood cells, het-
erophils, and eosinophils. Duck lymphocytes are round nongranular cells with large
round nuclei and little cytoplasm and have a diameter of 4–8 (cid:2)m(6). Duck monocytes
are round cells with large, often indented, nuclei and with more cytoplasm than lym-
phocytes,although it can be difficult to distinguish one cell type from the other. Duck
thrombocytes,which are essential for blood clotting,are of similar size to lymphocytes
but are highly vacuolated, making it possible to distinguish them from lymphocytes
using flow cytometry owing to their increased side scatter (6). Duck red blood cells
(DRBCs) are nucleated and strictly ought to be considered as a subset of PBMCs. How-
ever,for the purposes of this chapter,duck PBMC preparations do not include DRBCs.
They contain the mononuclear cells that can be separated from whole blood using
Ficoll-Paque density gradients. DRBCs and heterophils pellet to the bottom of Ficoll-
Paque gradients. Further information on avian hematology and photographs of the cell
populations present in avian blood are available on the World Wide Web (14,15).
Most published reports of duck lymphocyte cultures have used PBMCs collected
from Ficoll-Paque gradients including the cells present at the plasma–Ficoll-Paque
interface and in the Ficoll-Paque above the DRBC pellet. PBMCs collected in this way
include 22–26% T lymphocytes (9) and up to 60% thrombocytes, with the remainder
not clearly identified,although most are likely to be B lymphocytes and monocytes.
Unlike the findings with mammalian and chicken lymphocytes, antibodies to duck
immunoglobulins (Ig) bind to a large proportion of duck lymphocytes from blood,
spleen,thymus,and bursa of Fabricius and therefore are not useful for identifying and
Host Immune Responses Against DHBV 5
isolating duck B lymphocytes (16). Moreover,monoclonal antibodies specific for deter-
minants on mouse,rat,human,and chicken T lymphocytes do not react with duck lym-
phocytes (D. Higgins, personal communication). However, a rabbit antiserum that
reacts with a conserved intracytoplasmic portion of the human CD3(cid:1) chain binds to
duck lymphocytes with a staining pattern similar to that of mammalian T lymphocytes
(6). These antibodies precipitate a 23-kDa protein from duck lymphoblast lysates,sug-
gesting that duck lymphoid tissues contain lymphocytes functionally equivalent to
mammalian and chicken T cells (6). Because the anti-human CD3(cid:1)antibodies are spe-
cific for an intracellular epitope, they cannot be used to identify and/or isolate viable
cells. However,they have been used to identify a subset of duck lymphocytes by FAC-
Scan analysis (seeSubheading 3.1.2.andFig. 1). The CD3(cid:1)antibodies can also be used
for immunostaining of lymphocytes in tissue sections (see Subheading 3.2.1.). Duck
thrombocytes can be distinguished from lymphocytes by both flow cytometry (Fig. 2A)
and FACScan analysis using the anti-duck thrombocyte BA3 monoclonal antibodies
(subtype IgG2a; seeSubheading 3.1.3.;Fig. 2B).
The methods described in Subheading 3.1.4.build on attempts in the 1980s to iden-
tify and separate duck lymphocytes into T and B cells (16)and to define conditions for
the in vitro culture and optimization of responses to phytohemagglutinin (PHA) and
concanavalin A (Con A) (17). We have further defined the in vitro culture conditions
that support proliferation of duck lymphocytes. These include nylon wool fractionation
of PBMCs,a technique that enriches for T lymphocytes in mammals and chickens,and
coculturing nylon wool-fractionated duck PBMCs in the presence of homologous
adherent cells (monocytes) and DRBC (8,18;Subheading 3.1.4.;Fig. 3).
Following culture of duck PBMCs large multinucleated syncytia are observed in
approx 50% of cultures from 3–7 d of incubation. The presence of these syncytia often
inhibits mitogen- and antigen-induced proliferation of the cells resulting in decreased
incorporation of [3H]thymidine. The syncytia are strikingly similar to osteoclasts that
develop on culture of human (19),mouse (20),and chicken (21–23)PBMCs. Examples
of duck syncytia are shown in Fig. 4.
Despite optimization of the in vitro proliferation assays described above,it is not yet
possible to reproducibly detect proliferation of DHBV antigen-specific T lymphocytes
from ducks immunized or infected with DHBV. Problems with reproducibility of the in
vitro assays may, in part, be due to the development of syncytia and their inhibitory
effects on lymphocyte proliferation. In any case, further efforts are required to stan-
dardize the assays before we can reliably measure CMI responses to DHBV infection.
Supernatants from PHA-stimulated duck PBMCs and spleen cells have also been
shown to contain lymphokines capable of maintaining proliferation of duck lym-
phoblasts (7; see Subheading 3.1.5.). It is possible that supernatants from DHBV
antigen-stimulated PBMCs from ducks previously infected with DHBV may contain
cytokines equivalent to those released from mammalian and chicken T cells, which
mediate CMI responses. Assays developed to detect such cytokines in culture super-
natants may also prove to be useful in measuring CMI to DHBV.
6 Miller et al.
Fig. 1. FACScan analysis of single-cell suspensions of duck lymphoid organs. Cells were pre-
treated with acetone–paraformaldehyde and labeled with either rabbit anti-human CD3(cid:1) anti-
serum (black line) or the negative control rabbit anti-bovine myoglobin antiserum (gray line)
before the addition of FITC-conjugated sheep anti-rabbit IgG as described in the text.
1.2. Histological Methods for Detection of Cellular and
Viral Antigens in Duck Tissues
Histological and immunostaining techniques have been developed for the identifica-
tion of duck T lymphocytes, Kupffer cells, and phagocytic cells in a range of tissues,
and for the detection of DHBV antigens in liver, pancreas, kidney, and spleen. Using
these techniques it is possible to monitor infected tissues for changes in cellular infiltra-
Host Immune Responses Against DHBV 7
Fig. 2. FACScan analysis of duck PBMCs. Dot plot of duck PBMC (A). The gated region was
analyzed further using the anti-duck thrombocyte BA3 monoclonal antibodies (black line)or a
negative control monoclonal antibodies before the addition of FITC-conjugated sheep anti-mouse
IgG (B). The cell populations in the gated region of A were also separated on a FACStar cell
sorter(data not shown)and were morphologically identified as thrombocytes (with increased side
scatter) and lymphocytes (with decreased side scatter).
Fig. 3. Comparison of duck in vitro T-cell responses to PHA. Eight different ducks were bled
and stimulation of their T lymphocytes by PHA (5 (cid:2)g/mL) was measured following the method
described in the text.
8 Miller et al.
Fig. 4. Demonstration of giant cells (syncytia) in cultures of duck PBMCs (A)and adherent
cells alone (B)following 5 d of culture as described in Subheading 3.1.4.andNote 8.In addition
to the very large syncytia,DRBC and T lymphocytes can also be seen in A.Bar = 100 (cid:2)m. Final
magnification = ×90.5.
tion and viral expression,and relate these to the development of viraemia and antibody
responses in the bloodstream (3).
Duck T lymphocytes can be detected in sections of formalin-fixed tissues using
anti-human CD3(cid:1)antibodies (seeSubheading 3.2.2.). Phagocytic cells can be identi-
fied in duck liver and spleen by intravenous inoculation of ducks with colloidal car-
bon followed by histological identification of carbon containing cells (see
Subheading 3.2.3.). In the liver the phagocytic Kupffer cells are located within the
hepatic sinusoids (Fig. 5A),while the phagocytic cells present in the spleen are pres-
ent around the periellipsoid sheath in a similar location to the ellipsoid-associated
cells described in chicken spleen (24,25). Phagocytic cells in duck liver and spleen
can also be identified in sections of ethanol-fixed tissues using mouse monoclonal
antibodies, 2E.12, raised against duck liver and kindly supplied to us by Dr. John
Pugh. This reagent identifies both Kupffer cells in the liver (Fig. 5B) and ellipsoid-
associated cells in the spleen. Similar reagents that detect Kupffer and ellipsoid-
associated cells have been described for the chicken (26,27). DHBV-infected cells
can be identified in ethanol–acetic acid fixed tissues using polyclonal rabbit anti-
recombinant DHBV core antigen (rDHBcAg; 1) and anti-DHBV pre-S/S monoclonal
antibodies (1H.1; 28).
The primary cell type in the liver supporting DHBV replication is the hepatocyte,
and high levels of viral antigens and viral DNA can readily be detected in the cytoplasm
of infected cells within the liver lobule (Fig. 5C). We have found no evidence that Kupf-
fer or endothelial cells support DHBV replication (1–3); DHBV antigens and DHBV
DNA have been detected within Kupffer cells only during the clearance phase of acute,
Host Immune Responses Against DHBV 9
Fig. 5. (A)A section of formalin-fixed duck liver collected at autopsy 24 h after intravenous
inoculation with 165 mg/kg body wt of colloidal carbon. Phagocytic (Kupffer) cells located
within the hepatic sinusoids have taken up carbon. Counterstained with hematoxylin and eosin.
(B). Section of ethanol-fixed duck liver after immunostaining with the 2E.12 monoclonal anti-
bodies specific for duck Kupffer and phagocytic cells. Stained cells are located within the hepatic
sinusoids. Counterstained with hematoxylin. (C)A section of ethanol–acetic acid fixed duck liver
collected from an adult duck (B47) 5 d following intravenous inoculation with a high dose of
DHBV. Detection of DHBV pre-S/S antigen in the cytoplasm of hepatocytes using anti-DHBV
pre-S/S monoclonal antibodies (1H.1). Counterstained with hematoxylin. Bar = 100 (cid:2)m. Final
magnificationA–C=×163.
transient DHBV infections or following challenge of immune ducks with high doses of
DHBV (3; A. Jilbert,unpublished data).
1.3. Detection of Antigens, Antibodies, and Viral DNA in Duck Serum
Antibody responses to the HBV surface,core,and e antigens have been detected in
the sera of humans following transient HBV infection. Anti-surface (anti-HBs) antibod-
ies are a marker of resolution of transient HBV infection. In chronic HBV infection,
antibodies to the viral surface proteins are generally not detected in serum,although it
is possible their presence is masked by the formation of immune complexes with sur-
face antigen particles. Antibodies to the HBV core protein (anti-HBc antibodies) can be
readily detected in the sera of patients with chronic HBV infection as can antibodies to
e antigen (anti-HBe antibodies) that develop following seroconversion from e antigene-
mia. Anti-HBe antibodies are unable to neutralize viral infectivity.
ELISAs have been developed for quantitation of DHBsAg (Fig. 6)and detection of
anti-DHBs(Fig. 7A)and anti-DHBc (Fig. 7B)antibodies. In the DHBsAg ELISA rab-
bit anti-DHBs antibodies (seeSubheading 3.3.1.) are used to coat the plates and cap-
ture DHBsAg from duck serum samples. Bound DHBsAg is then detected using
anti-DHBV pre-S/S monoclonal antibodies (1H.1; 28). In the anti-DHBs ELISA the
10 Miller et al.
Fig. 6. (A)Diagrammatic representation of the quantitative ELISA used to detect DHBsAg in
duck sera. (B)A typical standard curve for the quantitative DHBsAg ELISA generated using high
titer DHBV-positive duck serum and NDS (negative control). The levels of DHBsAg in test sam-
ples are calculated using the standard curves. The cutoff for negative/positive results is set at three
times the standard deviation from the mean value obtained with NDS.
plates are coated with 1H.1,followed by sucrose gradient purified DHBsAg to capture
the antibodies,and bound antibodies are detected using rabbit anti-duck IgY. In the anti-
DHBc ELISA plates are coated with rDHBcAg (1), and bound antibodies are again
detected using rabbit anti-duck IgY antibodies. Rabbit anti-duck IgY antibodies are pre-
pared by immunization of rabbits with duck IgY from egg yolk (18;seeSubheadings
3.3.2. and 3.3.3.). The ELISAs for detection of anti-DHBs and anti-DHBc antibodies
thus detect total bound Ig and allow investigation of the overall humoral responses to
DHBV infection (2–5) but do not distinguish between IgM, IgY, and IgY ((cid:3)Fc) (29)
subtype antibodies.
In congenitally DHBV-infected ducks, anti-DHBc antibodies can be detected in
the serum from approx 80 d post-hatch (4), while in experimentally DHBV-infected
ducks anti-DHBc antibodies are detected from as early as 7–10 d post-inoculation and
Host Immune Responses Against DHBV 11
Fig. 7. (A)Diagrammatic representation of the ELISA used to detect anti-DHBs antibodies.
Levels of anti-DHBs antibodies are expressed as the reciprocal of the log serum dilution that
gives an OD of 0.4 at an absorbance of 490 nm. (B)Diagrammatic representation of the ELISA
used to detect anti-DHBc antibodies. Levels of anti-DHBc antibodies are expressed as the recip-
rocal of the log serum dilution that gave an OD of 0.5 at an absorbance of 490 nm.
persist throughout the course of infection (4). Anti-DHBs antibodies are detected at
high levels only in the sera of ducks that have resolved their DHBV infection,but can
also be detected at low levels in the sera of congenitally and experimentally DHBV-
infected ducks with persistent DHBV infection (Wendy Foster, personal communica-
tion). In this case anti-DHBs antibodies may be masked by the formation of immune
complexes between the anti-DHBs antibodies and circulating DHBsAg.
Detailed analyses of humoral immune responses to DHBV infection have been per-
formed in 4-mo-old ducks inoculated with 1 ×103,1 ×106,1 ×109or 2 ×1011DHBV
genomes(3). In these studies increasing the dose of inoculated virus shortened the time
to appearance and increased the levels of detectable antibodies. An increase in the
inoculum from 1 ×109to 2 ×1011DHBV genomes resulted in (cid:4)1 log increases in anti-
