U.S. patent application number 11/187314 was filed with the patent office on 2005-12-22 for methods of use of viral vectors to deliver antigen to dendritic cells.
Invention is credited to Bender, Armin, Bhardwaj, Nina, Steinman, Ralph M..
Application Number | 20050282282 11/187314 |
Document ID | / |
Family ID | 23084035 |
Filed Date | 2005-12-22 |
United States Patent
Application |
20050282282 |
Kind Code |
A1 |
Steinman, Ralph M. ; et
al. |
December 22, 2005 |
Methods of use of viral vectors to deliver antigen to dendritic
cells
Abstract
This invention relates to methods and compositions useful for
delivering antigens to dendritic cells which are then useful for
inducing T antigen specific cytotoxic T lymphocytes. This invention
also provides assays for evaluating the activity of cytotoxic T
lymphocytes. According to the invention, antigens are provided to
dendritic cells using a viral vector such as influenza virus which
may be modified to express non-native antigens for presentation to
the dendritic cells. The dendritic cells which are infected with
the vector are then capable of presenting the antigen and inducing
cytotoxic T-lymphocyte activity or may also be used as
vaccines.
Inventors: |
Steinman, Ralph M.;
(Westport, CT) ; Bhardwaj, Nina; (West Orange,
NJ) ; Bender, Armin; (Lahntal, DE) |
Correspondence
Address: |
MORGAN & FINNEGAN, L.L.P.
3 World Financial Center
New York
NY
10281-2101
US
|
Family ID: |
23084035 |
Appl. No.: |
11/187314 |
Filed: |
July 21, 2005 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11187314 |
Jul 21, 2005 |
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10193935 |
Jul 11, 2002 |
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10193935 |
Jul 11, 2002 |
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09310560 |
May 12, 1999 |
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6455299 |
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09310560 |
May 12, 1999 |
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08693586 |
Aug 1, 1996 |
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6300090 |
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08693586 |
Aug 1, 1996 |
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08282996 |
Jul 29, 1994 |
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Current U.S.
Class: |
435/456 |
Current CPC
Class: |
A61K 2039/5252 20130101;
A61K 39/12 20130101; A61K 39/145 20130101; A61K 2039/5254 20130101;
C12N 2760/16134 20130101; A61K 39/00 20130101; A61K 2039/5154
20130101; Y02A 50/412 20180101; C12N 2760/16143 20130101; C12N
2760/16122 20130101; A61K 2039/57 20130101; Y02A 50/30 20180101;
C12N 15/86 20130101; C12N 2760/16161 20130101; C12N 7/00 20130101;
C12N 2760/16043 20130101 |
Class at
Publication: |
435/456 |
International
Class: |
C12N 015/86 |
Goverment Interests
[0001] This invention was made with United States Government
support under National Institutes of Health grants AR-39552 and
AI-24775. The United States Government has certain rights in this
invention.
Claims
1) A method of delivering antigens to human dendritic cells in situ
comprising: a) providing a non-replicating viral vector which
targets human dendritic cells, said viral vector comprising a gene
sequence encoding for said antigen; and b) exposing said human
dendritic cells in situ to said non-replicating viral vector for a
time sufficient to allow said antigen to be expressed on the
surface of said human dendritic cells.
2) (canceled)
3) The method according to claim 1, wherein targeting of human
dendritic cells is accomplished with a non-replicating influenza
viral vector.
4) The method according to claim 1, wherein said non-replicating
viral vector comprises recombinant nucleic acid.
5) The method according to claim 1, wherein said antigen is
selected from the group consisting of tumor antigens, viral
antigens, microbial antigens and autoimmune antigens.
6) The method according to claim 5, wherein said antigen comprises
amino acid sequences from at least one strain of influenza
virus.
7) The method according to claim 1, wherein said human dendritic
cells are proliferating.
8) The method according to claim 1, wherein said human dendritic
cells are non-proliferating.
9) The method according to claim 23 wherein said influenza virus is
PR8.
10-29. (canceled)
30) The method according to claim 1, wherein targeting of human
dendritic cells is accomplished by modifying the non-replicating
viral vector to encode for a protein, or part thereof, which is
recognized by a receptor on human dendritic cells.
Description
FIELD OF THE INVENTION
[0002] This invention relates to targeted antigen presentation in
the immune system. More specifically, this invention relates to the
use of viral vectors to deliver antigens to dendritic cells for
processing and presentation to the immune system. This invention
also relates to methods and compositions having preventive,
diagnostic and therapeutic applications.
BACKGROUND OF THE INVENTION
[0003] The potential role of CD8+, cytolytic T lymphocytes [CTLs]
in resistance to infectious and malignant diseases has been
emphasized by recent developments in immunology. Antigen-specific
CTLs are recognized as a possible defense mechanism in infection
with HIV-1 (1-3), cytomegalovirus (4), and in malaria (5). Antigens
that are recognized by melanoma-specific CTLs also have been
identified by Boone and colleagues (6,7). These studies document
the specificity of CTLs that recognize clinically important
targets. Less is understood about the initial generation of these
CTLs, however.
[0004] As in most T cell responses, the precursors for active CTLs
are quiescent lymphocytes that must be induced to expand clonally
and develop effector functions. For CTL activation to occur, not
only must antigens be presented as peptide fragments on MHC
products, but the antigen-MHC complexes must also be introduced on
cells with the requisite accessory functions that lead to T cell
growth and cytolytic activity. Studies of killer cells response to
transplantation antigens, provide evidence that an effective way to
induce human CTLs is to present antigens on dendritic cells (8).
Dendritic cells are specialized accessory cells for the initiation
of many T cell dependent immune responses [reviewed in (9)].
[0005] T cell receptors on CD8.sup.+ T cells recognize a complex
consisting of an antigenic peptide, .beta.-2 microglobulin and
Class I major histocompatibility complex (MHC) heavy chain (HLA-A,
B, C, in humans). Processing and presenting of peptides on
dendritic cells involves digesting of endogenously synthesized
proteins and transporting them into the endoplastic reticulum,
bound to Class I MHC heavy chain and .beta.2 microglobulin, and
finally expressing the digested peptide in the cell surface in-the
groove of the Class I MHC molecule. Therefore, T cells can detect
molecules that originate from proteins inside cells, in contrast to
antibodies that detect intact-molecules expressed on the cell
surface. Consequently, CD8+ CTL are able to kill clinically
important targets such as virus infected cells, tumors, and certain
tissues attacked during autoimmune diseases.
[0006] Dendritic cells are potent antigen presenting cells for
several immune responses. When exposed to replicating influenza
virus, mouse dendritic cells stimulate strong cytolytic responses
from CD8+ T cells. Prior work had not identified underlying
mechanisms particularly the efficiency with which influenza virus
infect dendritic cells, and whether the dendritic cells remain
viable after exposure to influenza. Accordingly, it would be
describe to identify mechanisms to deliver antigen on dendritic
cell Class I molecules for presentation to CD8+ CTLs.
[0007] The proficiency of dendritic cells as APCs has been
attributed to their ability to aggregate antigen responsive T cells
into clusters, high expression of MHC Class I and Class II
molecules, as well as adhesion and co-stimulatory molecules, and
efficient endocytic activity S for the MHC Class II pathway.
Dendritic cells can sensitize quiescent human T cells when few MHC
Class II molecules are occupied by antigen [a maximum of 0.1% of
surface MHC Class II molecules or 2000 molecules], indicating that
low levels of signal when presented on dendritic cells, are
sufficient to generate T cell responses. However, the efficiency
with which dendritic cells handle Class I restricted antigens is
not known, one difficulty being the need to identify dendritic cell
antigens which are Class I.
[0008] The development of new strategies in immunotherapy for
treatment of cancers and, pathogens is greatly needed. In
particular, improved mechanisms for prophylaxis and therapy are
needed in influenza, since control of the respiratory infection is
not readily achieved through current approaches to vaccination. For
example, presently available vaccines are not designed to induce
killer cells but instead boost antibody responses to viral antigens
that undergo antigenic drift and shift (10). It is known that
dendritic cells are a component-of the alveolar septae and airway
epithelium of the lung (11,12), and that the appearance of
influenza virus-specific CTLs is associated with a more rapid
clearance of virus from nasal washings (13).
[0009] Influenza virus is also an agent used to dissect the
different pathways for antigen presentation of dendritic cells and
analyze the specificity of CTLs. Townsend et al. reported that
viral proteins were processed in the dendritic cell cytoplasm and
presented as peptides in association with Class I MHC products of
the infected cell (14,15). Morrison et al. used influenza virus to
distinguish two pathways for antigen presentation to CTLs (16). One
emanates from acidic endocytic vesicles and leads to presentation
on MHC Class II to CD4+ CTLs; the other emanates from a nonacidic
biosynthetic S compartment for presentation on MHC Class I to CD8+
CTLs.
[0010] There is considerable evidence, primarily in mouse cell
cultures, that dendritic cells effectively present viral antigens
to T cells (26,28-30). Murine dendritic cells can be infected by
influenza virus and elicit potent CTL responses (26,29). The
responses are dependent upon the synthesis of endogenous viral
proteins (26). Efforts to extend these results to humans have been
unpredictable.
[0011] CTLs that have been the subject of investigation in humans
are usually generated from unseparated blood mononuclear cells
and/or repeated stimulation of responding lymphocytes with
exogenous IL-2 and viral antigens (4,5,13,14,17-22). For example,
Biddison et al (17) used repeatedly stimulated human blood cells in
their elegant mapping studies of influenza peptides that are
presented to CTLs. An efficient and effective system to generate
human antigen specific cyntoxic T cells in general, and in
particular influenza specific killer cells needs to be identified,
especially one that capitalizes on the efficient accessory function
of dendritic cells.
[0012] The use of dendritic cells to process and present antigens
is described in Steinman application PCT/US92/. Accordingly, it
would be useful to provide additional means of delivering various
antigens to specific populations of dendritic cells. Such delivery
systems could be useful for providing an efficient and effective
system to generate human antigen specific cytotoxic T cells in
general and, in particular, influenza specific killer cells.
[0013] Influenza A virus infection remains a major cause of
mortality and morbidity, primarily because the control of the
respiratory-illness has not been achieved through vaccination.
Current vaccines are designed to boost antibody responses to viral
antigens [HA and NA] that undergo antigenic drift and shift (10).
Consequently, the protective effects of antibodies decline with
time. Vaccines directed towards the induction of influenza
virus-specific cytotoxic T cell immune responses might be far more
effective, since CTL have been shown to express cross reactivity in
recognition of subtypes of influenza A (18). There is evidence in
humans that CTL responses play a role in recovery from infection.
McMichael et al (13) related levels of CTL immunity to clearance of
nasal virus by normal donors inoculated with live virus. A clear
association was observed between CTL responses and clearance of
virus.
[0014] An ideal vaccine would utilize cross-reactive antigens,
induce CD8+ CTL responses in most hosts, and have an efficient
means of delivery. Several approaches to induce CTLs with these
properties have been attempted in a number of systems. They include
delivery of Class I-restricted peptides with adjuvant (58-61),
conjugated to lipid (62), complexed with immune stimulating
complexes (ISCOMs) (63), or inserted into liposomes (64,65). The
injection of DNA encoding the immunizing antigen directly into
skeletal muscle (66) has also been reported to induce CTL.
[0015] Until recently, dendritic cells have not been directly
considered in strategies to design new vaccines that generate CD8+
CTLs. Targeting antigen to dendritic cells has several advantages;
one can maximize the efficiency of T cell activation (9), and avoid
anergy induction (67) or the use of adjuvants (68). For example,
dendritic cells pulsed with antigen in vitro and delivered in vivo
to mice have been highly effective for generating CD4+ immune
responses to protein antigens and microbes (68,69). Mouse dendritic
cells pulsed with Class I restricted peptides of NP (26), HIV
peptides (70), or given antigen via pH sensitive liposomes (65)
into the cytoplasm can induce CTL responses.
SUMMARY OF THE INVENTION
[0016] This invention relates to the presentation of antigens in
the immune system. More specifically, this invention relates to the
use of viral vectors to deliver antigens to dendritic cells for
processing and presentation to T cells. Delivery of antigens to
dendritic cells have preventive, diagnostic and therapeutic
applications.
[0017] In one embodiment, this invention relates to a method of
delivering antigens to dendritic cells comprising providing a viral
vector comprising a gene sequence encoding for the antigen and
exposing the dendritic cells to the viral vector for a time
sufficient to allow the antigen to be expressed on the surface of
the dendritic cells. The viral vector, in a preferred embodiment is
an influenza virus.
[0018] In another embodiment of this invention, the viral vector
comprises nucleic acid prepared by recombinant techniques so as to
encode antigens which are not encoded by the native viral vector.
Upon infection of dendritic cells, expression of the nucleic acid
results in the synthesis of protein antigens including those which
are not native to the virus. The antigens are then processed and
presented on the MHC I antigens of the dendritic cells which,
according to one embodiment of the invention, may then be used to
activate T cells, such as, for example, cytotoxic T
lymphocytes.
[0019] Accordingly, this invention also provides a method of
generating antigen specific cytotoxic T lymphocytes. This method
comprises providing a viral vector comprising a nucleic acid
sequence encoding the antigen and exposing at least one dendritic
cell to the vector for a time sufficient to allow the antigen to be
processed and expressed on the surface of the dendritic cell. The
dendritic cells are then exposed to T lymphocytes for a time
sufficient to cause their activation to antigen specific cytotoxic
T lymphocytes.
[0020] Various types of antigens are suitable for delivery by the
viral vectors. In particular, such antigens include, but are not
limited to, tumor antigens, viral antigens, bacterial antigens,
protozoans, and autoimmune antigens.
[0021] In another embodiment of the invention viral activated
dendritic cells are used to activate T cells in vitro as a method
of assaying the responsiveness of T-cells to antigens.
[0022] Methods of preventing, and treating disease are also
provided by this invention which comprise administering to an
individual in need of treatment, a therapeutically effective amount
of cytotoxic T cells which have been activated by viral activated
dendritic cells.
[0023] In addition to the methods of this invention, this invention
also provides virally activated dendritic cells, and in particular
human activated dendritic cells which are prepared according to the
methods of this invention. Cytotoxic T lymphocytes which have been
activated by the dendritic cells of this invention are another
embodiment of this invention.
[0024] It is a general object of this invention to provide a method
of using viral vectors efficiently to deliver specific antigens to
dendritic cells which are then expressed on their surface.
[0025] It is another object of this invention to provide a method
of using influenza viral vectors to deliver specific antigen to
dendritic cells which are then expressed on the surface of the
dendritic cells.
[0026] It is yet a further object of this invention to generate
antigen specific cytotoxic T lymphocytes either in vitro or in vivo
by using the dendritic cells generated by the methods described
herein.
[0027] It is yet another object of this invention to provide a
method of prophylactic or therapeutic immunization for a variety of
cancers, autoimmune diseases or pathogens using the dendritic cells
described herein.
[0028] In addition, it is another object of this invention to
provide multivalent vaccines that can be used either
prophylactically or therapeutically for immunization using the
dendritic cells generated by the methods described herein.
DESCRIPTION OF FIGURES
[0029] FIGS. 1A-H are photomicrographs which show that following
infection with influenza virus, dendritic cells express
hemagglutinin (HA) and nucleoprotein (NP) proteins. FIGS. 1A-E show
dendritic cells which were pulsed with virus for 60 min, washed
extensively and then cultured for 16 h. FIG. 1F shows dendritic
cells cultured for 1 h following the virus pulse. FIGS. 1G and 1H
show purified monocytes infected with influenza as above and
cultured for 16 h. At the end of the culture period, cells were
collected, cytospins were prepared and stained with the following
panel of monoclonal antibodies. (mAbs). B B [9.3C9,
anti-ClassII].
[0030] (1A) Control mAb [0KT8]. Black arrows depict contaminating
CD8+ T cells in the APC population. A white arrow illustrates a
negatively stained dendritic cell.
[0031] (1B) Control mAb [9.3C9. anti-Class II]. The three headed
arrow identifies typical dendritic cells.
[0032] (1C) Anti-NP [HB651. The black arrow points to the intense
nuclear location of NP in a dendritic cell.
[0033] (1D) Anti-HA [H17L2]. The arrow points to-a typical
dendritic cell.
[0034] (1E) Anti-HA [H28E23]. The arrow point to a typical
dendritic cell.
[0035] (1F) A 1 hour infection of dendritic cells.
[0036] (1G) Overnight infection of monocytes stained with anti-HA
[H28E23].
[0037] (1H) Anti-NP [HBE65]. NP is detected in several dying and
live monocytes.
[0038] In FIG. 1E, the arrows identify typical dendritic cells,
with hairy processes as expected for a plasma membrane envelope
protein. In FIG. 1F, white arrows point to the granular appearance
of HA presumably in endosomal granules, black arrows point to the
diffuse location of HA-in a few rapidly infected dendritic cells.
In FIG. 1G, dying infected monocytes are phagocytosed by
non-infected cells [black arrow].
[0039] FIG. 2 shows influenza virus-infected dendritic cells
stimulate T cell proliferative responses. Bulk T cells were tested
in a standard proliferation assay for responsiveness to influenza
virus-infected and noninfected dendritic cells, macrophages and B
cells at various T:antigen presenting cell (APC) ratio ratios.
Cultures were pulsed on day 5 for 9 h with 4 uCi/ml of .sup.3H-TdR.
Results are means of triplicates.
[0040] FIGS. 3A-B show dendritic cells are potent stimulators for
the induction of influenza specific CTL responses.
[0041] (3A) 1.times.10.sup.6 purified T cells from buffy coat
donors were stimulated with graded doses of uninfected [open
symbols] or influenza virus-infected [closed symbols] dendritic
cells or monocytes. After 7 days; CTL activity was measured using
infected syngeneic monocytes as targets at ratio of 40.1. Lysis of
uninfected target cells was <5% at all doses of stimulators
used.
[0042] (3B) Purified T cells were cultured with uninfected or
infected dendritic cells or monocytes at T:APC ratios of 30:1 for 7
days. Lytic activity was measured on syngeneic monocyte targets at
various E:T ratios, as shown. Lysis of uninfected target cells was
<5% at all E:T ratios used.
[0043] FIG. 4 shows partially enriched preparations of dendritic
cells suffice as stimulators of influenza specific CTL responses.
1.times.10.sup.6 purified T cells were cultured with graded doses
of uninfected [open symbols] or infected ER-, FcR-cells comprising
approximately 5% dendritic cells [closed symbols]. Lytic activity
was measured on syngeneic targets on day 7 of culture at E:T ratios
of 60:1 and 20:1. Lysis of uninfected target cells was 5% at all
stimulator doses and E:T ratios used.
[0044] FIG. 5 shows kinetics of CTL development. Purified T cells
obtained from a buffy coat donor were stimulated with partially
enriched populations of uninfected [open symbols] or influenza
virus-infected [closed symbols] dendritic cells at T:APC ratios of
3:1. Lytic activity was measured at day 5, 7 or 9 on infected
syngeneic monocyte targets. Lysis of uninfected monocyte targets
was <5% at all time points assayed.
[0045] FIG. 6 shows. CD4/CD8 phenotype in bulk T cell populations
responding to influenza virus antigens. Bulk T cells were cultured
with uninfected or infected APCs for 7 days, after which they were
phenotyped for CD4 or CD8 expression as described in Example 1. Dot
plots are of forward versus side scatter [left panels],
antiCD4-FITC versus forward scatter [middle panels] or anti-CD8-PE
versus forward scatter [right panels]. Note that the majority of
large cells responding to infected APCs are. CD8+.
[0046] FIG. 7 shows evidence that human influenza specific CTL are
Class I restricted. Donors A and B, who differ at Class I but share
Class II antigens [DRw52, DQwl], served as sources of T cells and
APCs. T cells were cultured with infected or uninfected syngeneic
APCs for 7 days after which the cells were harvested for CTL
activity. Lytic activity was measured on syngeneic and allogeneic
macrophage targets. Uninfected APCs failed to elicit CTL activity
[data not shown].
[0047] FIG. 8 shows purified CD4+ and CD8+ T cells proliferate to
influenza virus-infected dendritic cells. CD4+ and CD8+ T cells
were purified from a bulk T cell population to greater than 98% by
staining with specific mAbs, followed by sorting on a Facstar plus,
as described in Example 1. Bulk T cells, sorted CD4+, CD8+ and a
combination of both sorted populations [CD4:CD8; 2:11 were tested
in a standard proliferation assay for responsiveness to influenza
virus-infected ER, FcR-cells. The T:APC ratio was 5:1. Clear bars
represent the responses to uninfected APCs while shaded bars
represent the influenza virus-specific responses. Cultures were
pulsed on day s for 12 h with 4 uCi/ml of .sup.3H-TdR. Results are
means of triplicates +/-S.D. Note the log scale on the y axis.
[0048] FIG. 9 shows dendritic cells stimulate the development of
both CD4+ and CD8+ influenza virus-specific CTL. Bulk T cells,
sorted purified CD4+ or CD8+ cells were stimulated with uninfected
[open symbols] or infected [closed symbols] ER-, FcR-cells for 7
days. The T:APC ratio was 3:1. Cytolytic activity was measured on
uninfected and infected syngeneic monocytes. CD4+ and CD8+ cells
were also added together at a 2:1 ratio prior to the CTL assay
[last panel on the right]. Lysis of uninfected monocyte targets was
<5% at all E:T ratios tested.
[0049] FIGS. 10A-B. FIG. 10A shows dose response titration of live
versus heat inactivated virus. Partially enriched preparations of
dendritic cells were pulsed for 1 hr at 37.degree. with several
dilutions of live or HI virus, in serum free medium. Cells were
washed and then added to syngeneic donor T cells at 3:1 T:APC
ratio. After 7 days, CTL activity was assayed as described in Table
II. % CTL shown is on infected syngeneic monocytes targets at two
E:T ratios. Lysis of uninfected targets was <5% at all E/T
ratios used [data not shown]. FIG. 10B shows that heat inactivated
virus poorly sensitizes target cells for CTL recognition. Influenza
specific CTLs were generated from bulk cultures of T cells and
partially purified dendritic cells that had been pushed with live
influenza virus at 1000 HAU/ml. The CTLs were tested on syngeneic
macrophage targets that were infected with live, or HI virus at
dilutions of 10-1 to 103 HAU/ml. E:T ratio=60:1. Lysis of
uninfected targets was <5% at all E/T ratios used [data not
shown].
[0050] FIG. 11A-11C shows induction of influenza virus-specific CTL
with noninfectious virus. Partially purified dendritic cells pulsed
with live, HI [56.degree., 60.degree., or 100.degree. at 30-45 min]
or UV inactivated virus, were cultured with syngeneic T cells for 7
days after which CTL activity was measured on macrophage targets.
E=T ratios are 40:1 [FIG. 11A]; 50:1 [FIG. 11B]; 60:1 [FIG. 11C].
Lysis of uninfected targets was <5% at all E/T ratios used [data
not shown].
[0051] FIGS. 12A-12B-FIG. 12A shows adsorption of virus with CRBC
removes CTL inducing activity, partially enriched preparations of
dendritic cells were pulsed with live or HI influenza virus before
or after adsorption of CRBC.
[0052] We used the known hemagglutinating property of influenza
viruses to adsorb and remove virions from chicken embryo allantoic
fluids containing virus. Washed chicken red blood cells (CRBC) were
resuspended to 20% v/v in RPMI and were gently mixed with an equal
amount of allantoic fluid. After 20 min of incubation on ice, cells
and bound virus were spun down at "low speed" (1000 rpm, S min.
4.degree. C.). Supernatants were taken for 2 more cycles of
incubation with CRBC as above. The final supernatant was spun down
twice and considered to be a 1:8 dilution of the starting virus
preparation. Partially enriched preparations of dendritic cells
were pulsed with live or HI influenza virus before or after
adsorption to CRBC [CRBC-fraction] and tested for their ability to
stimulate CTL responses. % CTL activity is shown on infected
macrophage targets. Lysis of uninfected targets was <5% at all
E/T ratios used (data not shown]. FIG. 12B shows adsorption of
virus with CRBC prevents sensitization of macrophage targets. CTLs
induced with live or HI virus were tested on macrophage targets
that had been infected with live unabsorbed virus or the adsorbed
nonbound fraction [CRBC-], at 1000 HAU/ml. % CTL activity is also
shown on uninfected targets.
[0053] FIG. 13 shows influenza-virus specific CTLs generated to HI
virus are CD8+. Purified T cells were stimulated with partially
enriched populations of uninfected, live influenza virus infected
or HI influenza virus infected dendritic cells at T:APC ratios of
3:1. After 7 days of culture, T-cells stimulated with uninfected or
HI influenza-infected APCs were stained with CD4-FITC and CD8-PE
mAbs and sorted on a FACStar Plus. The CD4+ and CD8+ populations
were >98% pure. Lytic activity of each population was measured
on infected syngeneic macrophage targets. Data is representative of
three experiments. Lysis of uninfected targets was <5% at all
E/T ratios used [data not shown].
[0054] FIG. 14 shows that live and HI influenza virus stimulate the
development of both CD4+ and CD8+ CTLs. T-cells were further
purified into CD4+ or CD8+ subsets by staining with Leu2 or Leu3
mAbs, followed by panning onto plastic plates coated with goat
anti-mouse IgG. Cells depleted by panning constituted <3.5% of
the resulting subpopulation as monitored by cytofluorography. Bulk
and purified T cell subsets were stimulated with partially purified
dendritic cells that were uninfected or infected with live or HI
virus for 7 days. The T:APC ratio was 3:1. Cytolytic activity was
measured on uninfected and infected syngeneic macrophages. Lysis of
uninfected targets was <5% at all E/T ratios used [data not
shown].
DETAILED DESCRIPTION OF THE INVENTION
[0055] For the purpose of a more complete understanding of the
invention, the following definitions are described herein:
[0056] By antigen or immunogen we mean all or parts thereof of a
protein or peptide capable of causing a cellular or humoral immune
response in a mammal. Such antigens are also reactive with
antibodies from animals immunized with said protein. Examples of
antigens that can be used in this invention include, but are not
limited to, viral, bacterial, protozoan, microbial and tumor
antigens. Preferred antigens include influenza virus, malaria, HIV,
and melanoma antigens.
[0057] Mammal includes but is not limited to humans, monkeys, dogs,
cats, mice, rats, pigs, cow, horses, sheeps and goats.
[0058] For the purposes of this invention, by viral vector means a
vector that comprises all or parts thereof of a viral genome which
is capable of being introduced into dendritic cells and expressed.
Such viral vectors may include native, mutant or recombinant
viruses. Such viruses may be RNA or DNA viruses. Examples of
suitable viral vectors include adenovirus, HSV, vaccinia, vesicular
stomatitis virus (VSV) and influenza. Preferably, the virus is an
influenza virus.
[0059] Influenza viral vector means a vector comprising all or
parts thereof of an influenza viral genome.
[0060] Intended to be included in this definition are Influenza A,
B, C and strains and subtypes thereof. In a preferred embodiment
the influenza virus is PR8.
[0061] The human dendritic cells which may be used to practice this
invention preferably may be obtained as mature dendritic cells from
an appropriate tissue such as blood or bone marrow as described
herein or by methods known in the art. Proliferating cultures of
dendritic cell precursors may be obtained as described in Steinman
et al. WO 93/208185 and as described in Romani et al (1994) J. Exp.
Med. Vol. 180:83 to 93, all of which are incorporated herein by
reference. Viral vectors including influenza vectors, can be used
to deliver antigen to either proliferating or mature dendritic
cells either freshly isolated or obtained from in vitro
culture.
[0062] To deliver antigens to dendritic cells for presentation to
cells of the immune system, specifically T cells. In accordance
with this invention antigens are introduced to dendritic cells via
a viral vector comprising a nucleic acid sequence encoding for an
antigen. The viral vectors useful for practicing the method of this
invention should be capable of entering dendritic cells and
localizing to the cytoplasm. Such viral vectors are endocytosed by
the dendritic cells and pass through the endocytic membrane and are
ultimately processed by the dendritic cells for presentation of
antigens on MHC Class I receptors located on the dendritic cell
surface.
[0063] Examples of such viral vectors include those stated above,
and in particular, but are not limited to influenza virus, vascular
stomatitis virus and vaccinia virus. Preferred vectors are
influenza virus. Most preferred is influenza.
[0064] Examples of influenza viruses that can be used to construct
all or part of the vectors include, but are not limited to
Influenza A, Influenza B, Influenza C, and strains and subtypes
thereof. In a preferred embodiment, the influenza A virus, PR8,
well-known to workers in the field (Virology 2nd Edition (1990)
eds. B. A. Fields and D. M. Knipe, Raven Press, NY) is used to
deliver influenza, modified influenza or non-influenza antigens to
dendritic cells. The influenza virus may be used to infect
dendritic cells in its native or natural form without
modification.
[0065] Other suitable vectors for use with this invention may be
identified by assaying for a viral vector's ability to enter
dendritic cells to synthesize viral protein in the case of
replicating virus, and to lead to antigen presentation in the case
of replicating and non replicating virus.
[0066] In another embodiment of this invention, vectors may be
targeted to dendritic cells by modifying the viral vector to encode
for a protein or parts thereof that is recognized by a receptor on
dendritic cells, whereby occupation of the dendritic cell receptor
by the vector will initiate endocytosis of the vector allowing for
processing and presentation of antigens encoded by the nucleic
acids of the viral vector. The nucleic acid which is delivered by
the virus may be native to the virus which when expressed on the
dendritic cell encodes viral proteins which are then processed and
presented on the MHC receptor of the dendritic cell. Alternatively,
the native nucleic acid may be modified using recombinant
techniques to include nucleic acid sequences encoding amino acid
sequences which define antigens which are not native to the
infecting virus. Construction and modification of viral vectors is
performed by conventional molecular or genetic manipulation
(Sambrook et al (eds) (1989). In "Molecular Cloning--A Laboratory
Manual" Cold Spring Harbor Press, Plainview, N.Y. and Ausebei et
al. (eds) in "Current Protocols in Molecular Biology" (1987) John
Wiley an Sons New York, N.Y.).
[0067] As with other viral vectors, construction and the use of
influenza viral has been reported in the art (L1 et al. (1992))
Journal of Virology 66(1):394-404; Li et al. (1993); Proc. Natl.
Acad. Sciences (USA) 90:5214-5218) which are incorporated herein by
reference.
[0068] In one embodiment of this invention a viral vector is
comprised of the native viral genome. As discussed above, in other
embodiments mutant viral strains or recombinantly modified viral
strains are also suitable for use with this invention.
[0069] Influenza viral vectors may be used to provide influenza
antigens to the dendritic cells that may cross react with other
types, subtypes or strains of influenza virus. Alternatively, the
dendritic cells may express type, subtype, or strain specific
antigens.
[0070] In one embodiment, influenza virus may be modified to
deliver antigens for other types, subtypes or strains of influenza
thereby providing immunizing against a broad spectrum of influenza
virus. It is known on the art that cross reactive viral epitopes
are presented on Class I molecules.
[0071] Examples of noninfluenza antigens, include, but are not
limited to tumor antigens, bacterial antigens, protozoan antigens,
such as the malarial circumsporozite protein, microbial antigens,
viral antigens, autoantigens, lesteriosis and any other antigens
for which it is desired that they presented by dendritic cells.
Examples of specific antigens include, but are not limited to
MAGE-1 (Boone, et al. Ann. Rev. Immuno. 12:337-365 (1994), and the
HIV gag protein.
[0072] To prepare the influenza vectors of this invention it should
be recognized that the influenza virus is a negative strand RNA
virus (Virology, 2nd Edition (1991) eds. B. N. Fields and P. M.
Knipe et al.; Raven Press, New York). Construction and modification
of influenza viral vectors carrying foreign antigens is known to
those in the art (Luytjes; W. et al. (1989); Cell 59:1107-1113;
Enami et al. (1990); Proc. Natl. Acad. Sciences (USA) 87:3802-3805;
Enami et al. (1991); J. Virology 65:2711-2713; Muster, T. et al.
(1991); Proc. Natl. Acad. Sciences (USA) 88:5177-5181). Influenza
virus is a negative strand RNA virus, therefore modification of the
viral genome requires conversion to a cDNA form and a
ribonucleoprotein transfection methods known to workers in the
field. Restriction endonuclease sites can be introduced into the
viral cDNA by conventional methods allowing for insertion of
nucleic acid sequences encoding antigens at the restriction
endonuclease site. Preferably the endonuclease restriction site is
unique to the viral vector. Examples of how to introduce
restriction endonuclease sites include but is not limited to,
mutagenesis. Alternatively, a sequence encoding an antigen can be
inserted into the influenza viral vectors by homologous
recombination. It is preferable that the sequence encoding the
antigen be inserted into the hemagglutinins gene of the influenza
viral vector so the antigen is expressed on the surface of the
viral particle when formed. The nucleic acid sequence to be
inserted may correspond to the entire peptide in which the
antigenic region is contained or may correspond to the nucleic acid
sequence encoding the exact antigenic epitope itself. Potential
Class I antigens can be determined by one skilled in the art.
[0073] An influenza viral vector containing hemagglutinine epitopes
from different subtypes of influenza A viruses can be constructed
by using a ribonucleoprotein transfection method. (L1, S. et al.
(1992) J. of Virology, 66:399-404). In this construct, the amino
acod loop contained at antigenic site B in the A/WSN/33 (H1N1)(WSN)
influenza virus was replaced by the corresponding structures of
influenza virus A/Japan/57 (H2N2) and A/Hong Kong/868
(H.sub.3N.sub.2)(HK). This construct can be exposed to dendritic
cells and the dendritic cells may take up this construct and
thereby express epitopes of different subtypes of hemagglutins on
their surface.
[0074] Alternatively, a construct expressing non-influenza epitopes
can also be constructed. For example, a recombinant influenza virus
expressing an epitope from the pathogen causing malaria,
specifically, cytotoxic T-lymphocyte epitope can be genetically
engineered into the hemagglutin in gene of an influenza viral
construct. The influenza viral vector carrying the antigenic
epitope for the cytotoxic T-lymphocyte epitope of the malaria
parasite can then be exposed to dendritic cells allowing the
dendritic cells to take up the viral vector and express the
antigens on its surface.
[0075] Both replicating influenza viral vectors and non-replicating
influenza viral vectors can be used in these methods. Influenza
viral vectors can be made non-replicating by methods known to those
skilled in the art. Examples of how to cause the viral vector to
become non-replicating include, but are not limited to, heat
inactivation or UV exposure. Non-replicating viral vectors are
preferred for use in mammals. If a non-replicating viral vector is
used the protein antigens desired to be expressed on the surface of
the dendritic cell must be on or in the virus when it enters the
dendritic cell. If a mechanism is available for the replicating
virus to make protein in the dendritic cell then that would also be
suitable. Otherwise, the proteins which are desired to be presented
as antigens must be expressed on or by the virus prior to entry
into the dendritic cells.
[0076] The dendritic cells used in this method can be isolated from
human by conventional methods (see Example 1). Alternatively, the
dendritic cells used in this method can be cultured in vitro by
methods known to one skilled in the art and disclosed in
PCT/US93/208185.
[0077] The ratio of influenza viral vector to dendritic cells that
may be used are about 1 to 100 virus particle or vectors per cell.
Other ranges may be determined based on the methods disclosed
herein. The influenza viral vector should be exposed to the
dendritic cells for a period sufficient for the dendritic cells to
incorporate and process the viral vector or particle and express
the antigens on the surface of the dendritic cells.
[0078] In yet another embodiment of this invention T-cells isolated
from individuals can be exposed to the dendritic cells now
expressing specific or selected antigen in vitro and then
administered to a patient in need of such treatment in a
therapeutically effective amount. Sources of T-lymphocytes can
include, but are not limited to, peripheral blood, or lymph nodes.
Such lymphocytes can be isolated from the individual to be treated
or from a donor by methods known in the art and cultured in vitro.
Viability is assessed by conventional methods such as trypan blue
dye exclusion assay. Preferred ratios of dendritic cells to
lymphocytes are about 1 dendritic cell to about 10 to 100
lymphocytes.
[0079] In yet another embodiment of this invention; the antigen
specific dendritic cells or the antigen specific T-lymphocytes
generated by methods described herein may be used for either a
prophylactic or therapeutic purpose. When provided
prophylactically, the dendritic cells or antigen specific
T-lymphocytes are provided in advance of evidence or any symptom of
the condition trying to be prevented. The prophylactic
administration of the dendritic cells or T-lymphocytes serves to
prevent or attenuate the conditions in a mammal. When provided
therapeutically, the dendritic cells or T-lymphocytes are provided
at, or shortly after the onset of the condition or the onset of any
symptoms of the disease or condition trying to be prevented. The
therapeutic administration of the dendritic cells or T-lymphocytes
serves to attenuate the pathogenic condition or disease condition.
In a preferred embodiment, dendritic cells infected with an
influenzal viral vector such as PR8. Examples of how the dendritic
cells expressing the selected antigen can be administered include,
but is not limited to, intravenous, intraperitoneal or
intralesional, or intranasal.
[0080] A pivotal role for dendritic cells in human disease
prophylaxis and therapy is indicated by their ability to directly
induce strong CD8+ CTL responses, as shown here for influenza
virus. We would therefore expect that it would be possible to pulse
dendritic cells directly with peptides or with attenuated virus for
instance, and use these APCs in vivo to elicit CTL responses. By
adapting the systems described herein, dendritic cells could also
be used for generating large numbers of CD8+ CTL, for adoptive
transfer to immunosuppressed individuals who are unable to mount
normal immune responses. Immunotherapy with CD8+ CTL has been shown
to amplify the immune response. Bone marrow transplant recipients
given CMV specific CTL by adoptive transfer, do not develop disease
or viremia (4). These novel approaches for vaccine design and
prophylaxis should be applicable to several situations where CD8+
CTLs are believed to play a therapeutic role e.g. HIV infection
(1-3), malaria (5) and malignancies such as melanoma (6,7).
[0081] Examples of diseases that may be treated by the methods
disclosed herein include, but are not limited to bacterial
infections, protozoan, such as malaria, lesteriosis, microbial
infections, viral infections such as HIV or influenza, cancers or
malignancies such as melanoma, autoimmune diseases such as
psoriasis and ankolysing spondylitis.
[0082] Frequently, in clinical disease it is difficult to detect
killer cells because of inadequate presentation. This invention
also provides methods for assessing the cytoxic activity of T
lymphocytes, and in particular the ability of cytotoxic T
lymphocytes to be induced by antigen presenting dendritic cells to
express cytotoxic activity. According to this method, a sample
comprising T lymphocytes to be assayed for cytotoxic activity is
obtained. Preferably, the cells are obtained from an individual
from whom it is desirable to assess their capacity to provoke a
cytotoxic T lymphocyte response. The T lymphocytes are then exposed
to antigen presenting dendritic cells which have been caused to
present antigen. Preferably, the dendritic cells have been infected
with a viral vector, such as influenza, which has been modified to
deliver a specific antigen. After an appropriate period of time,
which may be determined by assessing the cytotoxic activity of a
control population of T lymphocytes which are known to be capable
of being induced to become cytotoxic cells, the T lymphocytes to be
assessed are tested for cytotoxic activity in a standard
cytotoxicity assay. Such assays may include the chromium release
assay described herein.
[0083] The method of assessing cytotoxic T lymphocyte activity is
particularly useful for evaluating an individual's capacity to
generate a cytotoxic response against cells expressing tumor or
viral antigens. Accordingly, this method may be useful for
evaluating an individual's ability to defend against cancers, for
example melanoma, or viruses. In addition, this method is useful to
detect autoreactive killer cells and could monitor not only the
presence of killer cells, but their response to therapy.
[0084] All books, articles, or patents reference herein are
incorporated by reference. The following examples illustrate
various aspects of the invention and in no way are intended to
limit the scope thereof.
EXAMPLE 1
Use of Replicating Influenza Viral Vector to Deliver Antigen to
Dendritic Cells
Materials & Methods
[0085] Culture medium. RPMI 1640 [Gibco Laboratories, Grand Island,
N.Y.] supplemented with gentamicin [100 ug/ml], 5% human serum and
10 mM HEPES buffer. Blood mononuclear cells. In most experiments,
buffy coats served as sources of blood mononuclear cells [PBMCs]
and were obtained from the New York Blood Center. Blood donors were
also healthy volunteers who were HLA typed by conventional methods
and selected for MHC Class I mismatch. PBMCs were separated into T
cell-enriched [ER+] (erythrocyte rosette) and T cell-depleted
fractions [ER-] as previously described (23).
[0086] T cells. ER+ cells were first depleted of monocytes by
panning on dishes coated with human gamma globulin (24). MHC Class
II+ and NK cells [CD16+ and CD11b+] were depleted by coating with
mAbs 9.3C9 [ATCC; HB180], and 3G8 [gift of Dr. Jay Unkeless and
OKM1 [ATCC; CRL 8026] respectively, followed by panning on petri
dishes coated with goat anti-mouse IgG (8). The resulting T cell
population contained fewer than 2-36 of contaminating MHC Class II+
and NK cells, as monitored by cytofluorography. In some
experiments, T cells were enriched for CD4+ or CD8+ cells by
incubation with Leu 2 or Leu 3 mAbs, respectively, followed by
panning as above. CD4+ and CD8+ cells were >95% pure when
evaluated by staining with PE-conjugated Leu 2 or Leu 3 [Becton
Dickinson and Co. Mountainview, Calif.], both before and after the
culture period that was used to generate influenza responses.
[0087] APC populations. Monocytes were obtained from ER- cells by
adhering them onto plastic dishes for 60-90 min, and dislodged by
pipetting. Nonadherent cells were subsequently used for
purification of B cells and dendritic cells, as described
previously (24,25). Residual monocytes were first removed by
panning on gamma globulin coated plates (24). The ER-, FcR-cells
proved to be adequately enriched in the dendritic cells that are
needed as APCs for strong CTL responses. However, to further enrich
the dendritic cells, and the potency of the APCs, ER-, FcR-cells
were layered onto 14% metrizamide gradients (25). After
sedimentation, dendritic cells localize to the low density
interface, while B cells and NK cells are enriched in the high
density interface. Dendritic cell purity was 50-70% with
contaminants being B cells, NK cells and a few T cells.
[0088] Virus preparation. Influenza virus strain PR8 [A/Puerto
Rico/8/1934] was kindly provided by Dr. Peter Palese [Mount Sinai
School of Medicine, New York] in the form of infectious allantoic
fluid. Virus was grown up and purified as previously described
(26). Virus stocks were replenished from seed virus by Spafas Inc.,
Storrs CT and stored in liquid nitrogen [virus stock: 20,000
HAU/ml]. Virus titers were determined using a hemagglutination
assay, as previously described (26).
[0089] Infection of cells. APCs and target cells were washed out of
medium containing serum and resuspended in RPMI at 0.5-1.times.107
cells/ml. Virus was added at a final concentration of 1000 HAU
(hemagglutininating units) PR8/ml and incubated for 60 min. at
37.degree.. This dose is saturating for the induction of
influenza-specific CTLs. To determine whether influenza infection
proceeded through an acidic compartment in APCs, the cells were
incubated in 10 mM NH.sub.4Cl for 30 min. prior to adding virus and
throughout the subsequent infection. In some cases, the NH.sub.4Cl
was added throughout the culture period, generally 24th.
[0090] Induction of influenza specific CTL. 1.times.10.sup.6
purified T cells were cultured in 24 well plates [Costar,
Cambridge] with graded doses of influenza virus-infected or
uninfected APCs, in a total volume of 1.1 ml. After 7 days of
culture, the cells were harvested and distributed in varying
numbers to 96 well microliter plates [100 ul per well]. CTL
activity was measured using a .sup.51Cr-release assay with infected
or uninfected syngeneic monocytes as targets. 1.times.10.sup.4
targets in a volume of 50 ul were added to each well to generate
Effecter:Target ratios ranging from 1:1 to 100:1.
[0091] .sup.51Cr release assay--Monocytes were obtained from ER31
cells by adhering them onto plastic dishes for 60-90 min, and
dislodged by pipetting. 1.times.10.sup.7 monocytes were cultured in
10 ml volumes in 60 ml Teflon beakers [Savillex Corp., Minnetonka,
Minn.] until use as targets in the CTL assay (27). For .sup.51Cr
labelling and infection, cells were collected on ice, washed free
of serum and brought up to 1.times.10.sup.7/ml in RPMI. 400 uCi
Na.sup.51"CrO.sup.4 [1 mCi/ml sterile stock, New England Nuclear,
Boston, Mass.] was added per 1.times.10.sup.7 monocytes. They were
simultaneously infected with 1000 HAU PR8/ml HAU PR8 for 1 h at
37.degree.. The targets were washed four times and resuspended at
2.times.10.sup.5/ml, after which 50 ul was aliquoted to each well
containing effector T cells. Spontaneous and total release samples
were prepared by adding the targets to wells-containing RPMI alone
or 0.33% SDS, respectively. The plates were centrifuged for 2 min.
at 15 g and incubated for Sh at 37.degree. C. At the termination of
the assay, the supernatant was collected with absorption cartridges
using a harvesting press [Skatron Instruments Inc., Sterling Va.]
and counted in a gamma counter. Percent specific .sup.51Cr release
was calculated from the following formula: 100.times.[[Release by
CTL-spontaneous release]/[Total release-spontaneous release]].
Spontaneous release was 15-25% of the total release.
[0092] FACS analysis of cell populations and cell sorting. In some
experiments, T cells were separated into CD4+ and CD8+ subsets by
sorting on a FACSTAR Plus [Becton Dickinson]. 1.times.10.sup.7
cells were stained with 20 ul of Simultest CD4-FITC/CD8-PE [Becton
Dickinson] for 45 min. at 4.degree. C., washed three times and
sorted. CD4+ cells were collected as FITC+ cells while CD8+ cells
were PE+.
[0093] Contamination of CD4+ cells with CD8+ cells or vice versa,
was <1%. Sorted populations were stained again following a
period of 7 days and did not demonstrate any change in their
CD4/CD8 phenotype.
[0094] Detection of influenza virus infection by
immunohistochemistry. Cytospins of various cell populations were
prepared using a Shandon Cytospin 2 Centrifuge. Slides were fixed
in acetone for 5 min at room temperature, and then incubated in
hybridoma supernatant for 45 min. The mAbs to influenza virus
proteins were kindly provided by Dr. J. Yewdell, NIH, and included
anti-NP [H16-L10-4R5; ATCC HB65], and anti-HA [H28E3, and H17L2 (A7
CC #'s)]. The cytospins were washed several times with PD/1% BSA,
and incubated with 1:200 dilution of biotinylated goat anti-mouse
Ig [Boehringer Mannheim Biochemicals] for 45 min, followed by an
HRP-Biotin-Avidin complex [Vector ABC kit, Burlingame, Calif.] for
30 min. Non-bound HRP was then washed off, and the HRP reaction
product was developed with H.sub.2O.sup.2 and DAB [diaminobenzidine
tetrahydrochloride, Polysciences, Warrington, Pa.].
[0095] Lymphocyte proliferation assays--Following infection with
influenza virus, APCs were added in graded doses to 10.sup.5 T
cells in 96 well flat bottomed plates lCostar Cambridge, MA1.
Uninfected APCs served as controls. Proliferation was determined on
days 5-6 with the addition of 4 uCi/ml of .sup.3H-TdR for 12-16 h
to triplicate microwells (mean cpm].
Results
[0096] We determined the extent to which human dendritic cells
could be infected with influenza virus. Dendritic cells were
isolated from buffy coat preparations as previously described, and
pulsed with live influenza virus for 1 hour in serum free medium.
Following multiple washes, immunohistochemistry was employed to
detect three viral proteins, NP, HA and NS1, within the cell, from
1 hour to 16 hours following infection [FIG. 1]. In addition,
dendritic cells were compared to macrophages isolated from the same
donor.
[0097] Dendritic cells failed to stain with isotype matched
antibody OKT8 [FIG. 1A], but stained intensely with mAb to Class II
[FIG. 1B]. At 16 hours following infection, NP staining was
primarily localized to the nucleus of dendritic cells although
there was clearly a cytoplasmic distribution in addition [FIG. 1C].
A diffuse distribution of HA, consistent with endogenous viral
protein synthesis, was evident [FIG. 1D and E]. Greater than 90% of
the dendritic cells were infected by these criteria, with a
viability of >90%. The NP and HA patterns of staining at 16 h
after infection indicate extensive synthesis of viral proteins in
the dendritic cells. Uninfected dendritic cells did not stain with
any mAbs for viral specific proteins [data not shown].
[0098] Following just 1 hour of infection, dendritic cells
expressed HA primarily in a granular pattern, suggesting that the
virus is first contained within endocytic vacuoles [FIG. 1F].
Evidence of viral protein synthesis was also apparent in that there
was diffuse cytoplasmic staining of a few cells, at this early time
point [FIG. 1F, black arrows]. Pretreatment of dendritic cells with
NH.sub.4Cl prior to, during and following a virus pulse, blocked
infection, i.e. few cells [<2%] stained with either anti-HA or
anti-NP mAbs [data not shown]. These findings confirm that
influenza requires an acidic compartment to deliver its genome to
the cytoplasm and engage in viral specific protein synthesis.
Macrophages were also highly susceptible to infection with
influenza. The degree of infection was generally greater than 70%
[FIG. 1 G and H]. After overnight incubation following infection,
many cells died, and appeared to be phagocytosed by viable
macrophages [FIG. 1 G, black arrow]. In contrast, dendritic cells
showed no change in viability for up to two days after
infection.
[0099] Lymphocytes also were examined by immunolabeling for their
ability to be infected by influenza virus, but none appeared to be
as infected as dendritic cells. B cells and T cells were not
susceptible to infection as assessed by staining with anti-HA and
NP mAbs. T cell blasts generated with superantigens [SEA:
staphylococcal enterotoxin], had a low level of infection [10-30%
of the total T cell preparation], while EBV transformed cells had
weak staining in 10-30% of the cells.
[0100] Relative efficacy of different influenza virus-pulsed APCs
to induce T cell proliferative responses. Enriched populations of
different APCs were pulsed with live influenza virus, and their
ability to stimulate T cell proliferation was assessed. Virus
pulsed dendritic cells were 30-50 fold mote effective than
macrophages and >200 fold more effective than B cells [FIG. 2].
Influenza specific responses were detectable even when 1 dendritic
cell was used per 300 T cells. At these stimulator:responder
ratios, influenza virus-pulsed macrophages and B cells were unable
to induce T cells to proliferate [FIG. 2]. We noted that cultures
containing significant numbers of infected macrophages e.g. at T
cell:APC ratios of 10:1, or bulk cultures of PBMCs, there was
striking toxicity and death of most cells, including T cells.
[0101] Dendritic cells are potent stimulators for the induction of
influenza specific killer cell responses. We compared dendritic
cells and macrophages for their capacity to generate human
virus-specific CTL responses. The responding T cells were
extensively depleted of APCs and added at a constant dose of
1.times.10.sup.6 [see Materials and Methods]. APCs were then added
in graded doses. In dozens of experiments, the T cells never
generated lytic activity unless APCs were added, and the APCs
themselves did not form lytic cells. Dendritic cells, if infected
with influenza virus, generated significant CTL responses even when
used at a 100:1 T cell/dendritic cell [FIG. 3A]. Significant
killing was seen in the primary effector cell populations even at
E:T ratios of 10:1 [FIG. 3B], or less [data not shown]. In
contrast, macrophages were far less stimulatory, in the order of
100 fold or less, possibly due to the significant macrophage death
observed following infection with virus. Since B cells are poor
stimulators of the proliferative response to influenza, and do not
get infected with the virus [see above], it is unlikely that
contaminating B cells in our dendritic cell population account for
the CTL that are generated. OKM1, an mAb directed towards the CD11b
antigen, known to remove NK cell precursors (8), was used to
deplete these cells from the starting T cell population. Thus the
effector cell population used in these assays is composed of T
cells. Experiments to be described below showed the killers to be
CD8+ CD4-. Most donors, >90%, could be primed with infected 2
dendritic cells, indicating that the majority of our donor pool has
been exposed to influenza. Since CTL activity was measured on
influenza A PR8-infected targets, a strain first identified in
1934, and the prevalent strains are A/Texas/36/91 and
A/Beijing/32/92, the CTLs generated appear to be crossreactive,
confirming other studies of human influenza-specific CTL (18).
[0102] Knowing that B cells do not contribute to CTL development,
we next determined whether a partially purified preparation of
dendritic cells, [i.e. omitting the metrizamide column for
enrichment] was adequate for generating CTL responses to influenza.
ER-, FcR-preparations are depleted of most T cells and monocytes
and consist of approximately 5% dendritic cells, the remaining
cells being primarily B cells. At T:APC ratios of 3:1 or 10:1,
significant CTL responses were apparent [FIG. 4]. This corresponds
to a T:dendritic cell ratio of 60:1 to about 180:1. ER-, FcR-cells
were used as stimulators for all subsequent experiments, in T:APC
ratios varying from 3:1 to 5:1. These partially enriched
populations [a]suffice to provide the cultures with dendritic cells
in the 1.5% range, [b] lack inhibitory monocytes, and [c] are
straightforward to prepare.
[0103] To ascertain when lytic activity was optimal, we measured
CTL development over the course of 9 days. Lytic activity peaked at
day 7 [FIG. 5], consistent with other studies (18) with little
variation from donor to donor. At this time, microscopic
examination routinely showed the development of large cell clusters
and released T cell blasts, as is characteristic of dendritic
cell-mediated T cell responses in vitro (9). Occasionally, assays
were done on day 8 if the clusters seemed slow to develop, and
blast release delayed.
[0104] CD8+ T cells are the principal CTLs induced with infected
dendritic cells. To establish the types of influenza-specific
effector cells in our system, we stimulated bulk T cells with
infected dendritic cells for 7 days and then separated the
populations into CD4+ and CD8+ subsets. The cultures were stained
with CD4-FITC and CD8-PE mAbs [Materials and Methods] and sorted on
a FACSTAR into >98% pure CD4+ and CD8+ populations. Unseparated
as well as sorted cells were then evaluated for lytic activity.
Table I shows the data from three individual experiments. Influenza
specific lytic activity was seen in two populations: bulk T cells
and purified CD8+ T cells. CD4+ T cells failed to demonstrate
any-lytic activity.
1TABLE I % SPECIFIC LYSIS OF MACROPHAGE TARGETS Expt. 1 Expt. 2
Expt. 3 Responding Infection M.phi. M.phi. M.phi. M.phi. M.phi.
M.phi. T cells of APCs (-) FLU (-) FLU (-) FLU Bulk (-) 0 0 0 0 2 4
FLU 0 25 0 42 9 51 CD4.sup.+ (-) 0 0 0 0 4 8 FLU 0 0 0 0 4 9
CD8.sup.+ (-) 3 7 0 0 3 9 FLU 2 37 0 57 16 54 E:T ratio* 40:1 40:1
50:1
[0105] Figure Legend for Table I. Influenza virus-specific CTL in
bulk cultures are CD8+ CD4-. Purified T cells were stimulated with
partially enriched populations of uninfected or influenza
virus-infected dendritic cells at T:APC ratios of 3:1. After 7 days
of culture, the T cells were stained with CD4-FITC and CD8-PE mAbs
and sorted on a Facstar Plus. The CD4+ and CD8+ populations were
>98% pure. Lytic activity of each population was measured on
infected syngeneic monocyte targets. Uninfected dendritic cells
failed to stimulate influenza specific CTL. Three individual
experiments are shown. * In expt. 1, the E:T ratio in the CD8+ T
cell group was 30:1.
[0106] In general the CD8+ cells were enriched for lytic activity
compared to the bulk T cells [Table 1 expt's 1 and 2]. FACs
analyses of the stimulated cultures contained ny enlarged T blasts
and most of the enlarged cells were CD8+; few CD4+ cells appeared
to enlarge in these cultures [FIG. 6].
[0107] To ascertain whether dendritic cell-induced CD8+,
influenza-specific CTLs were Class I restricted, we evaluated
responses generated in two individuals who differed at Class I loci
but shared Class II specificities [DRw52, DQwl]. Donor "A"
demonstrated significant lytic activity against syngeneic infected
macrophage targets [Fig 7], but lesser activity against Class I
mis-matched targets [donor B macrophage targets]. Likewise, donor
zB" effector cells lysed syngeneic but not allogeneic ["A"]
targets. The small degree of crossreactivity seen in the case of A
effectors vs. B targets may be due to unspecified but shared Class
I antigens, or possibly, the development of CD4+ mediated CTL
activity, which has been described in cloned human populations
(31,32). However, the latter is less likely given that no CD4+ CTLs
are generated in our system [see below].
[0108] Purified CD4+ and CD8+ T cells respond to influenza
virus-infected dendritic cells. The observation that few CD4+ T
cells seemed to be undergoing proliferation was surprising, since
their role as helper cells for influenza virus-specific CTL
responses is evident in mouse cultures (26). Also human CD4+ T cell
clones with lytic activity have been described (31,32). To
determine whether CD4+ T cells could respond to influenza
virus-infected APCs, we used cell sorting to purify CD4+ and CD8+ T
cells prior to T cell stimulation. Bulk, sorted CD4+, or CD8+ T
cells, and a combination of both sorted populations, were tested in
a standard proliferation assay for responsiveness to influenza
virus-infected ER-, FcR-cells. All four groups were able to mount
proliferative responses to these APCs [FIG. 8]. The most prominent
response was demonstrated by the CD8+ T cells [note the lower
background], compared to either bulk or CD4+ T cells. The extent of
these responses were generally similar at several time points
tested.
[0109] When CTL responses from these populations were measured, two
striking observations were made. First, sorted CD8+ T cells
developed CTL activity without a requirement for CD4+ T cells [FIG.
9]. These observations are reminiscent of human alloreactive
responses, where CD4+ helper cells are not required for the
generation of CD8+ CTL if dendritic cells are the APCs (8). Second,
CD4+ T cells also developed lytic activity, but only in the absence
of CD8+ T cells. We confirmed that the sorted populations were
>98% pure at the termination of the 7 day induction period. Thus
contamination of the CD8+ T cells with CD4+ T cells, or vice versa,
does not account for these results. It is more likely that CD4+ T
cells only exhibit the capacity to become CTLs, when few or no CD8+
T cells are present.
Discussion
[0110] Experimental conditions for the generation of human CD8+
CTLs. To generate CD8+ CTLs against infectious agents in cultures
of human T cells, one commonly uses unseparated populations of
PBMCs and/or repeated stimulation in the presence of exogenous
lymphokines like IL-2 (4,13,14,17-22). These requirements for CD8+
CTL development stand in contrast to CD4+ T cell responses, which
often are detected within 3-5 days of culture without exogenous
lymphokines. Furthermore, the primary APCs that induce CTL
responses in human T cell cultures have not been well
characterized.
[0111] Our results demonstrate that for the first time, strong
influenza virus-specific CTLs can be induced using virus-infected
dendritic cells as APCs. Partially enriched dendritic cells, which
are straightforward to isolate, suffice for the development of
CTLs. Our data which demonstrates indication of CTL's with
relatively little proliferation, is particularly surprising in view
of prior reports of human dendritic cell induction data.
[0112] Four features distinguish our system for generating CTLs
from the bulk culture systems that have been used previously.
First, only a few dendritic cells [0.5-1% suffice] are required to
generate highly potent CTL, as demonstrated by the fact that
killing is evident at E:T ratios of 1.5:1 [FIG. 5]. Such efficacy
has not been described in CTLs generated from cultures of bulk
PBMCs (13,14,18,21). Second, depletion of monocytes is necessary to
remove potential inhibitory and toxic effects on the effector
cells. In attempts to generate CTLs from unseparated PBMCs, we
often observed significant cell death that prohibited killer cell
development. This was likely secondary to cytopathic effects of
viral infection in monocytes as described [FIG. 1H]. PBMCs contain
about 1% dendritic cells (34), and an Iat cell is known to be
required for the generation of influenza virus-specific CTL in bulk
PBMC culture systems (18). We suggest that these small numbers of
dendritic cells are sufficient to permit CTL development in
circumstances where few monocytes are present or become infected.
Third, while monocytes do not induce CTLs effectively, they serve
as efficient targets in short term chromium release assays [5
hours]. During this time interval, a majority of monocytes express
viral proteins as demonstrated by immunohistochemistry [data not
shown]. In contrast, standard target cells e.g. B cell lines or
PHA-treated lymphocytes, have a low level of infection [10-30%] and
are less efficient in our hands as targets. Fourth, effector cell
populations are routinely depleted of NK cells. These cells, which
are activated by dendritic cells in vitro to lyse tumor cell
targets (8), may play a role in viral clearance. They are not
ordinarily depleted in bulk culture systems, and therefore could
potentially mask specific CTL measurement.
[0113] Antigen presenting cell requirements for the generation of
influenza virus-specific CTLs. T cell mediated immunity develops in
two stages. In the afferent phase, dendritic cells bearing antigen,
initiate T-dependent responses from resting lymphocytes. Once
activated the sensitized T lymphoblasts can interact with other
APCs in the efferent phase, to induce a number of effector
functions, e.g. B cell antibody synthesis (35), macrophage
activation and IL-1 production (36). As demonstrated here with
influenza virus, dendritic cells serve in a similar capacity to
first induce the generation of CD8+ CTLs, which then acquire the
ability to-kill infected macrophage targets. These pathways for CTL
generation are potentially important for the prevention of
cell-cell spread of virus.
[0114] Several features may account for the observed differences
between dendritic cells and monocytes in the induction of influenza
CTLs. One appears to be the manner in which influenza virus
infection is handled by these two types of APCs. Greater than 90%
of dendritic cells expressed HA, NP and NS-1 within 16 hours and
remained fully viable for >24 hours after infection. In sharp
contrast, freshly isolated monocytes or week old cultured
macrophages were infected to a lesser extent [c<70%] but died
within 16 hours of infection [FIG. 1]. In addition to influenza
virus, dendritic cells are specialized APCs for the presentation of
several other viruses to T cells, including HSV (30), Moloney
leukemia virus (28), Sendai virus (28) in the mouse and HIV in
humans (37). It is not known whether the efficacy of dendritic
cells in part reflects better developed pathways for the handling
of viral antigens e.g. efficient charging of MHC Class I and II
molecules. The findings nevertheless are consistent with other
studies showing that dendritic cells present microbial antigens [M.
tuberculosis (38), Leishmania (39), staphylococcal enterotoxins
(40)] efficiently to T cells.
[0115] Dendritic cells are also distinguished from APCs like
monocytes and B cells in the efficiency with which they deliver
signals to the TCR-CD3 complex on T cells. For example, occupancy
of only 0.1% of dendritic cell surface Class II molecules with
superantigen is sufficient to induce T cell proliferation (23).
This is due, in part, to the fact that dendritic cells express and
upregulate many accessory molecules that are critical during the
initiation of T cell immune responses [e.g. MLR (41), superantigens
(23)]. They include B7/BB1 [CD80], ICAM-1 [CD54] and LFA-3 [CD58],
ligands for CD28, CD11a and CD2, respectively. (Although we have
yet to study the role of these accessory molecules on dendritic
cells in CTL induction, there is evidence that interaction of CD28
with its ligand is a critical element in the activation of
cytotoxic CD8+ T cells (42). For example, murine Class I restricted
CTLs to alloantigens can be generated in the absence of help from
CD4+ T cells, provided a CD28-B7 interaction occurs in the
induction phase (42).)) Furthermore, B7-transfected tumor cells can
induce protective [CD8+ T cell mediated] anti-tumor responses in
vivo when CD4+ T cells are absent (43,44).
[0116] Our results reported herein are consistent with dendritic
cells being key accessories in CTL induction. Accordingly, one
would, therefore, predict that these APCs should also be effective
in the generation of CTL responses to other antigens [e.g. melanoma
antigens, alloantigens] where B7 is known to amplify the T cell
response (41,43,44). The CD28-B7/BB1 interaction provides I a
critical costimulatory mechanism for IL-2 gene expression (45-47).
This would explain the CD4 helper independence of CTL induction by
dendritic cells i.e. their ability to present antigen together with
costimulatory molecules like B7/BB1 that enhance the production of
IL-2.
[0117] Helper cells are not required for the Generation of CD8+
CTLs. Our results demonstrate resting human T cells extensively
depleted of CD4+ cells can be induced by dendritic cells to develop
influenza virus-specific cytolytic activity. As in bulk cultures of
T cells, CTL activity is generated with relatively few dendritic
cells [FIG. 9]. Dendritic cells also directly induce human and
murine CD8+ T cells to develop cytotoxic activity in the MLR
(8,48). In contrast to these findings, Nonacs et al (26) found that
mouse dendritic cells were unable to induce influenza
virus-specific CTL activity in purified CD8+ T cells, unless a
source of CD4+ T cells or helper lymphokines was available. A key
variable here may be the number of antigen-specific IL-2 producers
in the primed CD8+ population. For example, the paucity of
precursor T cells in the mouse [1:16,600 to 1:2,400 (49)] may be
insufficient to generate enough lymphokine to amplify a CTL
response. Also, far fewer murine dendritic cells [<20%, (26)]
are capable of synthesizing viral proteins than human blood
dendritic cells [>90%, FIG. 1].
[0118] There are now several examples of CTL development in the
apparent absence of CD4 help in vivo. For example, elimination of
CD4+ T cells in mice does not ablate resistance to ectromelia virus
(50,51), LCMV or vaccinia virus (52), and tumors (43,44).
[0119] Extensive proliferation of CD8+ T cells in response to
influenza virus-infected dendritic cells. The majority of T cells
that proliferate to influenza virus antigens in bulk cultures are
CD8+. We made this observation in routine Facs analyses of
stimulated cultures [FIG. 6] where most enlarged T cells stained
with antibodies to CD8. Primary populations of human CD8+ T cells,
depleted of CD4+ T cells, also proliferate extensively after
exposure to influenza virus-infected dendritic cells. The
proliferative responses were considerably greater when compared to
bulk ortCD4+ T cell responses [FIG. 81. Other than responses to MLR
antigens (8), we are not aware of other systems where such
extensive antigen-dependent, CD8 blastogenesis takes place. In
mice, following i.p. infection with LCMV, large increases in CD8+ T
cells in the spleen as well as the peritoneum occur (53).
[0120] Influenza virus-specific CD4+ CTL are also generated by
dendritic cells. Highly purified CD4+ cells can be induced by blood
dendritic cells to proliferate and develop cytolytic activity
[FIGS. 8 and 9]. However, it is necessary to remove the CD8+ T
cells to observe both the blastogenesis and CTL responses. The
reasons for this are unclear. We considered the possibility that in
bulk cultures, CD8+ CTLs might kill the CD4+ cells. Alternatively,
there might be selective inhibition of exogenous antigen
presentation via the Class II pathway, as previously described for
influenza virus infected-murine APCs (54). This seemed unlikely for
two reasons. First, CD4+ T cells can respond directly to infected
dendritic cells when separated from CD8+ T cells. Second, influenza
virus-infected APCs could present PPD to M. tuberculosis-reactive
CD4+ T cell clones as well as uninfected APCs [data not shown].
[0121] Influenza-specific CD4+ CTL have been described in both
human and mouse systems (16,31,32). In contrast to CD8+ CTLs, CD4+
Class II-restricted CTLs lysed target cells treated with
noninfectious influenza virus or purified HA preparations and Class
II presentation was sensitive to lysosomotropic agents (16). These
differences provided critical early evidence that MHC Class I and
Class II restricted CTL depended upon divergent pathways for
presentation of antigen. The role of CD4+ CTL, in viral clearance
and recovery from infection, however, remains to be determined.
Beta-2-microglobulin deficient [-/-] mice have few CD8+ T cells but
can clear vaccinia virus and nonlethal HKx31 influenza A virus, and
resist a low inoculation dose of PR8 (55), but recovery from lethal
doses of the virulent strain seems to require the presence of CD8+
T cells (56). In contrast, beta-2-microglobulin deficient (-/-]
mice infected with LCMV intracranially, develop CD4+ CTL that
mediate disease, similar to their CD8+ counterparts in infected
normal strains (57).
EXAMPLE 2
Use of Non Replicating Influenza Virus Vector to Deliver Antigen to
Dendritic Cells
[0122] The methods and materials unless otherwise specified were
the same as in Example 1
[0123] The following methods were used carry out the experiments
shown in Table II.
[0124] Poorly infectious forms of influenza virus induce human CTL
responses. 1. Influenza virus was used live, inactivated at
56.degree. for 30 minutes in a water bath or inactivated with UV
irradiation for 30 minutes by exposing it to shortwave UV radiation
[254 nml from a Mineralight UV lamp [UVGL 58; Ultraviolets
Products, San Gabrial, Calif.] for 0 mins at a distance of 4
centimeters. 2. influenza virus-specific CTLs were generated as
previously described. In brief, partially enriched preparations of
blood dendritic cells [which suffice as potent APCs] were washed
out of serum containing medium and infected with 1,000 HAU/ml of
different forms of PR8 influenza virus for 1 hr at 37.degree. C.
The APCs were washed and resuspended in RPMI containing 5% human
serum, 10 ug/ml gentamicin and 10 mM Hepes. The cells were added to
bulk cultures of purified syngeneic T cells at a 3:1 ratio
[representing a T:dendritic cell ratio of 200-60:1]. T cells were
obtained from PBMCs by sheep erythrocyte resetting. They were
depleted of contaminating monocytes by panning on gamma-globulin
coated dishes. MHC class II+ and NK cells were depleted by coating
with mAbs 9.3C9 [ATCC HB 180], and 3G8 [gift of Dr. J. Unkeless]
and OKMI [ATCC; CRL8026], respectively, followed by panning on
petri dishes coated with goat anti-mouse IgG. After 7 days of
stimulation, T cells were harvested and distributed in varying
numbers to 96 well round bottom plates. CTL activity was measured
using a standard 51Cr-release assay with infected or uninfected
syngeneic monocytes as targets. Percent specific release was
calculated from the formula: 100.times.[[Release by CTL-spontaneous
release][Total release-spontaneous release]]. Lysis of uninfected
targets was <5% at all E/T ratios used [data not shown]. 3. A
modified plaque forming assay was performed using trypsin-resistant
MDCK-II cells. These were grown in 6-well-plates to almost
monolayer density, washed once with RPMI and then inoculated with
0.33 ml of serially diluted virus preparations for 1 hr at
37.degree. C. The cells were washed again and overlaid with 3 ml of
a 0.6% agarose solution in RPMI (low melting point agarose, type L,
Behringwerke, Marburg, Germany) with freshly added trypsin final
conc. 2.5-5.0 ug/ml). Trypsin was needed for cleaving the
hemagglutinin of budding virus to facilitate infection of
neighboring cells. After 3 days, cultures were stained with 0.2%
crystal violet solution containing 4% formaldehyde. Plaque forming
units were counted from duplicate to triplicate samples and
calculated as PFU/ml. Results are averages of 4-7 experiments. 4.
Virus was titered in a standard hemagglutiantion assay.
Round-bottomed 96 well plates were used to set up serial twofold
dilutions of virus samples in 25 ul volumes. These were tested with
an equal volume of 1% CRBC suspension and incubated for >30 min
at RT. Hemagglutination titers are expressed as HAU/ml and are
representative of 3-4 expts. 5. To determine fusion activity of
virus preparations a hemolysis assay was performed according to
Huang et al with a modified protocol. 70 ul of chicken red blood
cells (CRBC, 20% v/v in PBS-d) was preincubated with 90 ul of virus
preparation at RT for 10 min. A 7.5.times. volume of 0.1M sodium
acetate buffer (pH 5.4) in saline was then added (bringing the CRBC
to a final concentration of 1%). Following 10 min. at RT, the
suspension was incubated at 37.degree. C. for 20 min to induce
fusion and subsequently hemolysis. Cell fragments were spun down
(5000 rpm for 2 min) and the supernatant was measured for
extinction of hemoglobin at 540 nm. In addition, spontaneous
release SR (in buffer) and maximum release MR (SDS-induced, final
concentration 0.015%) were determined to calculate % of hemoglobin
release as ((O.D. sample-O.D. SR).times.100):(O.D. MR-O.D. SR).
[0125] To obtain information on the nature of the antigen needed
for stimulating MHC Class I restricted responses, we first
evaluated the ability of live versus inactivated forms of influenza
virus to generate human influenza specific CTL responses. When
pulsed with heat treated. (56.degree. C., 30 min] or UV inactivated
influenza virus, dendritic cells generated CTL responses that were
equally potent to those that developed in response to live virus
[Table II].
2 TABLE II TL Resoonse.sup.2 (E:T ratio) Viral titres.sup.3
Hemagglutination.sup.4 Influenza virus.sup.1 50:1 15:1 5:1 PFU/ml
(HAU/ml) Hemolysis.sup.5 Live 31 21 8 6.4 .+-. 2.2 .times. 10.sup.8
1-2 .times. 10.sup.4 + Heat Inactivated 44 25 9 .ltoreq.3 .times.
10.sup.2 0.5-2 .times. 10.sup.4 + UV Inactivated 42 24 9 1.3 .+-.
1-3 .times. 10.sup.4 0.5-2 .times. 10.sup.4 +
[0126] Whereas the heat and UV inactivated viruses demonstrated
substantially reduced titres >10,000 fold less active virus as
determined by plaque forming assay [<3.times.10.sup.2 and
1.3.times.10.sup.4 PFU/ml, respectively], they had comparable
hemagglutinating activity to the live virus, suggesting that the
total amount of virus was not altered by inactivation treatments
[Table II]. Influenza virus attaches to sialic acid residues on
gycoconjugates on the cell surface via hemagglutinin, and is
internalized by endocytosis. Escape from the endosome into the
cytoplasm then ensues at acid pH, when the hemagglutinin undergoes
a conformational change that permits fusion of the viral envelope
with the endosomal membrane. Both heat inactivated (HI), and ultra
violet (UV) inactivated forms of influenza retained this fusion
capacity, since they could hemolyze chicken erythrocytes at acid pH
[Table II].
[0127] The ability to induce CTL responses to heat inactivated
forms of influenza virus appears to be restricted to dendritic
cells, since macrophages were ineffective in this capacity [data
not shown]. Dose response titrations demonstrate that, despite the
substantial reduction in infectious titer of the heat inactivated
virus, it retains the ability to induce CTL responses even at 10
HAU/ml, and the dose response curve is similar to that of live
virus [FIG. 10A]. Immunohistochemistry was employed to determine
the extent of infection in dendritic cells and macrophages,
following overnight culture after a 1 hr pulse with live or heat
inactivated virus. The influenza protein NP was evident in
dendritic cells infected with 1000 HAU/ml of live virus, but not
heat inactivated virus [FIG. 10B]. Similarly, HA could only be
detected on dendritic cells exposed to live but not heat
inactivated virus (data not shown]. Similar observations were made
for macrophages [data not shown]. Collectively, the data in FIG.
10A-B indicate that only small amounts of virus need be presented
on dendritic cells to elicit human cytolytic responses.
[0128] Further experiments were done to characterize the
attenuation of influenza virus with heat versus UV irradiation.
Heat treatment of influenza virus at temperature greater than
56.degree. destroyed the CTL inducing capacity of the virus [FIGS.
11A and 11B]. To determine whether the temperature affects the
ability of the virus to fuse with membranes, a fusogenic assay was
performed using chicken erythrocytes, as described in Table 1. At
60.degree., the virus can no longer lyse CRBC at an acid pH,
possibly because the HA has been altered at these higher
temperatures [data not shown]. Heat inactivated and UV virus were
equally effective at inducing CTL responses [FIG. 11C and Table
II]. Since allantoic fluid was used as the source of our virus
preparations, we ascertained whether immunogenic viral protein or
peptide fragments in the fluid might be responsible for the
observed ability of inactivated virus to elicit CTL responses. Live
and heat inactivated virus was adsorbed with CRBC, and the nonbound
fraction [CRBC-] was pulsed onto dendritic cells to test for CTL
inducing activity. The CRBC-preparation failed to generate CTL
responses [FIG. 12A]. Furthermore, the ability of the CRBC-fraction
to sensitize macrophage targets was also lost [FIG. 12B]. The
CRBC-adsorbed fraction demonstrated virus activity in a TCID 50
assay [data not shown]. Thus the CTL responses generated by
inactivated virus is due to whole virus and not contaminating viral
antigens.
[0129] To establish the types of influenza-specific effector cells
generated in response to heat inactivated virus, we stimulated bulk
T cells with heat inactivated virus-infected dendritic cells for 7
days, and then separated the populations into CD4+ and CD8+
subsets. The cultures were stained with CD4- FITC and sorted on a
Facstar into >98% pure CD4+ and CD8+ populations. Unstimulated T
cells were evaluated in the same way. Sorted subsets as well as
unseparated cells stimulated with the live virus infected dendritic
cells were then tested for lytic activity. Influenza specific CTL
activity was seen in two populations: purified CD8+ T cells and
bulk T cells [FIG. 13]. Little activity was evident in the CD4+
subset. Similar data was obtained with T cell populations obtained
after stimulation with dendritic cells pulsed with UV inactivated
virus [data not shown]. Thus, as with live virus, CD8+ CTLs are the
principal CTLs induced with dendritic cells infected with
inactivated virus.
[0130] To ascertain whether dendritic cells induced CD8+ influenza
specific CTLs were Class I restricted, blocking experiments were
performed with antibodies to Class I vs. Class II.
[0131] To determine whether purified CD8+ T cells could respond to
dendritic cells infected with attenuated virus, CD8+ and CD4+ T
cells were purified before T cell stimulation. Bulk, purified CD8+
and CD4+ cells were tested for responsiveness to dendritic cells
pulsed with live or heat inactivated virus. Both the purified
subsets developed CTL activity without a requirement for CD4+ T
cells, as shown previously for live virus [FIG. 14]. CD4+ T cells
also developed lytic activity, but only in the absence of CD8+ T
cells. Thus, as for live virus, it seems the CD4+ T cells acquire
the capacity to become CTLs when few CD8+ T cells are present.
[0132] T cells appear to recognize very small numbers of
MHC-antigen complexes in order to become activated. Little is
known, however, about the amount of antigen that is required or the
efficiency with which it is handled, in order for those MHC-peptide
complexes to be generated. Here we find that when dendritic cells
are the antigen presenting cells, only small amounts of an
attenuated virus need to be handled by the cell to elicit strong
CTL responses. The CTL responses generated are as potent as those
induced by nonattenuated virus. These results may having bearing on
the ability of dendritic cells to initiate CLTs in other contexts
like autoimmunity and tumor protection.
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