U.S. patent application number 10/837896 was filed with the patent office on 2005-01-06 for product and method for obtaining specific immunisation with one or more antigens.
Invention is credited to Heeney, Jonathan Luke.
Application Number | 20050002967 10/837896 |
Document ID | / |
Family ID | 8239845 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050002967 |
Kind Code |
A1 |
Heeney, Jonathan Luke |
January 6, 2005 |
Product and method for obtaining specific immunisation with one or
more antigens
Abstract
The invention provides means and methods for vaccinating an
animal or a human to obtain therein an immune response against at
least one antigen, comprising different vaccine compositions for
sequential administration to said animal or said human, each
containing at least said antigen or a precursor thereof, wherein
said vaccine compositions differ from each other by the presence
therein of a different vector.
Inventors: |
Heeney, Jonathan Luke;
(Voorburg, NL) |
Correspondence
Address: |
HOFFMANN & BARON, LLP
6900 JERICHO TURNPIKE
SYOSSET
NY
11791
US
|
Family ID: |
8239845 |
Appl. No.: |
10/837896 |
Filed: |
May 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10837896 |
May 3, 2004 |
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09890379 |
Jan 9, 2002 |
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6783762 |
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Current U.S.
Class: |
424/208.1 ;
424/228.1 |
Current CPC
Class: |
A61K 2039/53 20130101;
A61K 39/21 20130101; A61K 39/12 20130101; A61K 2039/54 20130101;
C12N 2710/24143 20130101; C12N 2770/36143 20130101; C12N 2740/15034
20130101 |
Class at
Publication: |
424/208.1 ;
424/228.1 |
International
Class: |
A61K 039/21; A61K
039/29 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 28, 1999 |
EP |
99200256.8 |
Jan 28, 2000 |
WO |
PCT/NL00/00058 |
Claims
1-17. Cancel
18. A kit that induces or stimulates an immune response in an
animal or human against at least one antigen, wherein the antigen
is of a virus causing temporary, or long lasting immune impairment,
wherein the kit comprises at least three different immunogenic
compositions, wherein each of the immunogenic compositions
comprises a vector encoding the antigen; and wherein the vectors
are not the same.
19. A kit according to claim 18, wherein the antigen is a
lentivirus.
20. A kit according to claim 18, wherein at least part of the
vectors function as an adjuvant.
21. A kit according to claim 20, wherein the adjuvant function
directs the immune response toward a more T helper 1 type or T
helper 2 type of response, or both.
22. A kit according to claim 18, wherein the antigen comprises at
least an immunogenic part, derivative and/or analogue of a
lentivirus gag, pol, rev, tat, nef, or env protein or a combination
thereof.
23. A kit according to claim 18, wherein at least one of the
immunogenic compositions comprises a nucleic acid encoding at least
one proteinaceous molecule for inducing and/or boosting an immune
response against the antigen.
24. A kit according to claims 18, wherein one of the vectors
comprises a nucleic acid which encodes at least one proteinaceous
molecule capable of modulating an immune response.
25. A kit according to claim 18, wherein one of the vectors is a
nucleic acid delivery vehicle comprising said nucleic acid.
26. A kit according to claim 25, wherein said nucleic acid delivery
vehicle is selected from the group consisting of a Semliki Forest
Virus particle, a poxvirus particle, a herpes virus particle and an
adenovirus particle.
Description
FIELD OF THE INVENTION
[0001] The invention lies in the field of medicine. More
particularly the invention relates to vaccines, vaccine
compositions and vaccination strategies for obtaining improved
immune protection against infectious diseases.
BACKGROUND OF THE INVENTION
[0002] The ultimate goal of developing prophylactic and/or
therapeutic vaccines for a large number of infectious agents has
been difficult to achieve due to the inability to induce optimal
immune responses to the pathogen in a safe and effective manner.
The previously tried and proven approaches of vaccination with
whole killed or live attenuated viruses are either unsafe or
ineffective for the remaining infectious diseases of major public
health concern. To avoid possible safety problems it has been
possible to develop protein based vaccines consisting of one or
several individual viral proteins or epitopes thereof. These are
derived from individual viral genes expressed in vitro and purified
as individual subunits in the protein in the absence of genetic
material. Recombinant subunit vaccine approaches have proven
effective for certain pathogens such as Hepatitis B. However, for
many applications subunit antigens have been unsuccessful due to
expression/production difficulties, alteration of relevant
immunological epitopes or marked variability of the pathogen
requiring the continuous development, fermentation and purification
of new antigens.
[0003] Recombinant live viral or bacterial vaccine vectors were
developed as potential solutions to some of these problems. A
replicating live virus or bacteria which does not cause disease has
the potential to be used as a vector. Attenuated viruses such as
adenovirus, poxvirus (i.e. vaccinia, MVA, canary or fowlpox) or
bacteria such as E. coli, are being developed and evaluated as live
vectors. Due to their ability to replicate (in some cases in a
limited fashion) in a host without serious side effects, makes them
candidate to carry and express foreign genes as "vaccine" antigens.
Recombinant vaccines have the advantage that they replicate in the
host and thereby induce stronger immune responses than whole killed
viruses or bacteria or subunit proteins. An additional advantage is
that an immune response to an antigen encoded by said vector, may
be improved by the stimulation of the immune system through the
presence or the expression of additional proteins, for instance
vector specific proteins for instance through providing adjuvant
function. However, relatively few recombinant vector systems alone
have been successful enough to be widely accepted for clinical use.
Major problems other than safety have been pre-existing immunity in
the case of vectors derived from infectious agents common in
populations. Furthermore, subsequent immune responses against
vector proteins themselves have created a further immunological
barrier when more than one immunisation was required to boost
responses to the recombinant vaccine antigen(s). One problem is
that the immune system may mount an immune response against vector
or vector encoded proteins together with an immune response against
the antigen, designated the vaccination antigen, the immune
response was intended to be directed toward in order to provide the
host protection. The observation that the immune system may mount
an immune response against a vector protein or a vector encoded
protein creates a potential for competition for immune resources
such as the availability of immune cells and/or cytokines, thereby
lowering the desired response against vaccination proteins (see for
example FIG. 1A). Another problem is the potential for more
immunogenic antigens present in vector proteins or vector encoded
proteins directing the immune response away from vaccination
proteins. Additionally, immune responses against the vector
eventually limit vector replication in the host, thereby reducing
the vectors intended purpose and effectiveness. A problem that
specifically increases upon boosting of the immune response with
the same or a similar vector or vector system. For instance, the
use of different adenovirus serotypes comprising nucleic acid
encoding similar vaccination proteins as vaccines is not optimal
since the immune system will still be boosted against common
antigens present in vector proteins and/or vector encoded proteins.
A possible method to avoid this problem is to boost immune
responses induced by the recombinant vectors with subunit protein.
Several studies have shown that immune responses can be slightly
improved by this method but that there is not a substantial
improvement in the ability of the vaccine to protect from
infection.
SUMMARY OF THE INVENTION
[0004] The present invention provides novel means and methods for
obtaining a specific immune response in an individual or animal.
The invention further provides means and methods for decreasing the
negative effects of vector proteins and/or vector encoded proteins
while leaving desired effects, such as an adjuvant effect of said
proteins at least in part intact (see for a non-limiting example
the scheme depicted in FIG. 1B).
[0005] In one aspect the invention provides a product for
vaccinating an animal or a human to obtain therein an immune
response against at least one antigen, comprising at least two
different vaccine compositions for sequential administration to
said animal or said human, each containing at least said antigen or
a precursor thereof, wherein at least two of said vaccine
compositions differ from each other by the presence therein of a
different vector.
[0006] In another aspect the invention provides a method for
vaccinating an animal or human to obtain therein an immune response
against at least one antigen of a virus causing a temporary, or
long lasting immune impairment, comprising administering
sequentially to said animal, at least two different vaccine
compositions, each containing at least said antigen or a precursor
thereof and wherein at least two of said vaccine compositions
differ from each other by the presence therein of a different
vector.
[0007] In yet another aspect the invention provides a use of an
antigen, or a precursor thereof, for manufacturing a vaccine
composition for vaccinating an animal or a human to obtain therein
an immune response against said antigen, wherein said vaccine
composition is administered sequentially with at least one other
vaccine composition containing at least an immunogenic part,
derivative and/or analogue of said antigen or antigen precursor,
and a different vector.
DETAILED DESCRIPTION OF THE INVENTION.
[0008] In one aspect the invention provides a solution to
circumvent the negative effects associated with repeated exposure
of vector proteins or vector encoded proteins in a vaccination
procedure or a vaccine composition. To study problems associated
with amplification of an immune response against vector proteins
and/or vector encoded proteins a strategy was developed in which
the use of different vector systems, to consecutively deliver the
same or related antigen(s), was evaluated. The potential existed
not only to substantially boost immune responses to the recombinant
antigen, but to tailor the nature of the immune responses by
priming and then delivering subsequent boosts with different vector
combinations or by delivering the vaccine vectors to different
immunological sites and/or antigen presenting cell populations.
Indeed, the ability to induce preferred type-1 or type-2 like
T-helper responses or to additionally generate specific responses
at mucosal and/or systemic sites can be foreseen with such an
approach.
[0009] In one aspect the invention provides means and methods for
vaccinating an animal or a human to obtain therein an immune
response against at least one antigen of a virus causing a
temporary, or long lasting immune impairment, comprising at least
two different vaccine compositions for sequential administration to
said animal or said human, each containing at least said antigen or
a precursor thereof, wherein at least two of said vaccine
compositions differ from each other by the presence therein of a
different vector. A much better vaccination for such viruses is
obtained with at least three different vaccine compositions wherein
at least three of said vaccine compositions differ from each other
by the presence therein of a different vector.
[0010] In another aspect the invention provides a product for
vaccinating an animal or a human to obtain therein an immune
response against an antigen comprising at least two different
vaccine compositions for sequential administration to said animal
or said human, each containing at least said antigen or a precursor
thereof, wherein at least two of said vaccine compositions differ
from each other by the presence therein of a different vector. An
improved vaccination is obtained with at least three different
vaccine compositions wherein at least three of said vaccine
compositions differ from each other by the presence therein of a
different vector. In a vaccination procedure comprising a serial
administration to said animal of at least two vaccine compositions
comprising at least said antigen or a precursor thereof and wherein
at least two of said vaccine compositions differ from each other by
the presence therein of a different vector, an amplification of an
immune response against vector antigens that may be present in one
or more of said vaccine compositions or that may be encoded by
nucleic acid present in one or more of said vaccine compositions or
both, is at least in part avoided in said animal. By at least in
part avoiding said amplification of an immune response against
vector antigens in said animal, potential masking of an immune
response against said antigen is at least in part prevented. One
method of avoiding at least in part an amplification of an immune
response against vector antigens in said animal is to avoid at
least in part the presence of vector antigens in said animal during
said vaccination procedure. This may be achieved for instance by
avoiding the presence of vector antigens in at least one of said
vaccine compositions or by avoiding at least in part, expression of
vector antigens encoded by a nucleic acid in a vaccine composition,
or both. Preferably, amplification of an immune response in said
animal or human against vector antigens is at least in part
prevented by using for said serial administration of vaccine
compositions, vaccine compositions comprising different vectors.
Another preferred method of avoiding amplification of an immune
response against vector antigens in said vaccination procedure is
to use at least one vaccine composition useful for avoiding the
presence of vector antigens in said animal and at least one vaccine
composition comprising a vector. Preferably, when more then one
vaccine composition comprising a vector is used, said vector in
said vaccine composition is essentially different.
[0011] A process for vaccinating an animal or human may be any
vaccination process provided that said process utilises serial
administration of vaccine compositions containing at least an
antigen or a precursor thereof, against which said animal or human
should at least in part be vaccinated. Vaccine compositions are
preferably administered to said animal or human in an amount
effective for eliciting an immune response in said animal or
human.
[0012] Said antigen may be a complete protein or a part of a
protein. Said antigen may also be a proteinaceous molecule, derived
from nature or synthesised chemically.
[0013] In one embodiment of the invention said animal is a human.
In one embodiment the invention provides a product for vaccinating
an animal or a human to obtain therein an immune response against
at least one antigen, comprising at least two different vaccine
compositions for sequential administration to said animal or said
human, each containing at least said antigen or a precursor
thereof, wherein at least two of said vaccine compositions differ
from each other s by the presence therein of a different vector.
Preferably said product comprises at least three of said
compositions and wherein at least three of said vaccine
compositions differ from each other by the presence therein of a
different vector.
[0014] In one embodiment at least part of, said vector or a product
thereof, functions as an adjuvant. An adjuvant in the context of
the present invention is any molecule or combination of molecules,
capable of modulating an immune response against said antigen. In
one example an adjuvant has the capability to stimulate the immune
system in said animal to elicit an immune response wherein said
stimulation also stimulates the initiation or the amplification of
an immune response against said antigen. In one example, an
adjuvant is a classical adjuvant such as complete or incomplete
freund adjuvant. In another example said adjuvant is a
proteinaceous molecule immunologically different from said antigen,
capable of eliciting an immune response in said animal or
human.
[0015] Preferably said proteinaceous molecule comprises at least a
functional part of a co-stimulatory molecule such as CD80, CD86,
CD28, CD152, CD40 or CD40 ligand; of a cell-adhesion protein; of an
immune response inhibitory protein; of an interleukin; of a major
histocompatibility complex protein or of other proteins capable of
modulating an immune response. An immune response may be modulated
through at least in part inhibiting or preventing an immune
response and/or at least in part inducing or enhancing an immune
response.
[0016] In a preferred aspect of the invention vaccination is be
performed together with a method for influencing at least in part
immune system, for example in the direction of a preferred T helper
1 type of immune response or a more T helper 2 type of immune
response. It is now widely accepted that T cell-dependent immune
responses can be classified on the basis of preferential activation
and proliferation of two distinct subsets of CD4.sup.30 T-cells
termed T.sub.H1 and T.sub.H2. These subsets can be distinguished
from each other by restricted cytokine secretion profiles. The
T.sub.N1 subset is a high producer of IFN-.gamma. with limited or
no production of IL-4, whereas the T.sub.H2 phenotype typically
shows high level production of both IL-4 and IL-5 with no
substantial production of IFN-.gamma.. Both phenotypes can develop
from naive CD4.sup.+ T cells and at present there is much evidence
indicating that IL-12 and IFN-.gamma. on the one hand and IL-4 on
the other are key stimulatory cytokines in the differentiation
process of pluripotent T.sub.H0 precursor cells into T.sub.H1 or
T.sub.H2 effector cells, respectively, in vitro and in vivo. Since
IFN-.gamma. inhibits the expansion and function of T.sub.H2
effector cells and IL-4 has the opposite effect, the preferential
expansion of either IFN-.gamma. producing cells (pc) or IL-4 pc is
indicative of whether an immune response mounts into a T.sub.H1 or
T.sub.H2 direction. The cytokine environment, however, is not the
only factor driving T.sub.H lineage differentiation. Genetic
background, antigen dose, route of antigen administration, type of
antigen presenting cell (APC) and signalling via TCR and accessory
molecules on T cells. In a preferred aspect of the invention the
immune system is directed toward a more T helper 1 or 2 type of
immune response through using vectors with the property of
modulating an immune response in one direction or the other. In a
preferred aspect of the invention at least part of said adjuvant
function comprises means for directing the immune system toward a
more T helper 1 or 2 type of immune response.
[0017] Preferably through using vectors with the property of
modulating an immune response in one direction or the other.
Examples of vectors with the capacity to stimulate either a more T
helper 1 or a more T helper 2 type of immune response or of
delivery routes such as intramuscular or epidermal delivery can be
found in Robinson 1997, Vaccine 15:785-787; Sjolander et al 1997,
Cell. Immunol. 177:69-76; Doc et al 1996, Proc. Natl. Acad. Sci.
USA 93:8578-8583; Feltquate et al 1997, J. Immunol. 158:2278-2284;
Pertmer et al 1996, J. Virol 70:6119-6125; Prayaga et al, Vaccine
15:1349-1352; Raz et al 1996, Proc. Natl. Acad. Sci. USA
93:5141-5145.
[0018] In a preferred aspect of the invention the immune system is
induced to produce innate immune responses with adjuvant potential
in the ability to induce local inflammatory responses. These
responses include interferons, -chemokines, and chemokines in
general, capable of attracting antigen processing and presenting
cells as well as certain lymphocyte populations for the production
of additional specific immune responses. These innate type
responses have different characteristics depending on the vector or
DNA used and their specific immunomodulating characteristics,
including such as encoded by CpG motifs, and as such, the site of
immunisation. By using in a specific sequence different vectors
encoding at least one common specific vaccine antigen, different
kinds of desired protective vaccine responses may be generated and
optimised for defence from a particular infectious agent. By
combining different vector systems and delivering them at different
or the same specific sites the desired vaccine effect at a
particular site of entry (i.e. oral, nasal, enteric or urogenital)
of the specific infectious agent.
[0019] In one aspect at least one of said vectors comprises antigen
presenting cells, preferably engaged in vivo but also in vitro from
said animal. Preferably said antigen presenting cells are dendritic
cells. Preferably said antigen presenting cells present said
antigen, or an immunogenic part, such as a peptide, or derivative
and/or analogue thereof, in the context of major histocompatibility
complex I or complex II.
[0020] In a preferred embodiment at least one of said compositions
comprises as an antigen precursor a nucleic acid encoding at least
one proteinaceous molecule for inducing and/or boosting an immune
response against said antigen. In a preferred embodiment said
nucleic acid is capable of replicating in a cell of the animal or
human being vaccinated. With the term boosting in this respect is
meant amplifying an immune response such, that when said animal is
s exposed to said antigen after the amplification, the immune
response to said antigen is increased in magnitude compared to
before said amplification. Said proteinaceous molecule for inducing
and/or boosting an immune response against said antigen may be said
antigen or an immunogenic part, derivative or analogue thereof.
Alternatively, antigen or an immunogenic part, derivative or
analogue thereof may be encoded by a nucleic acid present in said
vaccine composition.
[0021] In a preferred embodiment said antigen is an antigen encoded
by a nucleic acid of a pathogen, preferably of a virus. In a
particularly preferred embodiment said antigen is an antigen
encoded by a virus which causes a temporary or long lasting immune
impairment. For such viruses it has not been possible to devise a
satisfactory vaccination strategy to completely protect from
infection. The present invention is however, surprisingly suited to
provide a satisfactory vaccination for viruses causing different
degrees of immune impairment. Some vaccination is obtained using a
product comprising at least two different vaccine compositions for
sequential administration to said animal or said human, each
containing at least said antigen or a precursor thereof, wherein at
least two of said vaccine compositions differ from each other by
the presence therein of a different vector. However, vaccination is
substantially improved to provide substantial protection when at
least three different vaccine compositions are used for sequential
administration to said animal or said human, each containing at
least said antigen or a precursor thereof, wherein at least three
of said vaccine compositions differ from each other by the presence
therein of a different vector.
[0022] For effective maintenance and further boosting of the
vaccination it is preferred that the immune capacity of the
vaccinated individual is boosted at intervals with a vaccine
comprising yet another adjuvant. In a preferred embodiment said
antigen encoded by a virus causing a temporary, or preferably long
lasting immune impairement is an antigen of a lentivirus, another
retrovirus, a hepatitis C virus, another flavivirus, a measles
virus, another paramyxovirus or a Herpes Virus. In a preferred
embodiment said antigen comprises at least an immunogenic part,
derivative and/or analogue of a lentivirus gag, pol, rev, tat, nef
or env protein or a combination thereof.
[0023] In a preferred embodiment at least part of said adjuvant
function by a vector is provided by a nucleic acid which encodes at
least one proteinaceous molecule capable of modulating an immune
response. Preferably said nucleic acid is capable of replicating in
a cell of the animal of the human being vaccinated. Preferably said
proteinaceous molecule capable of modulating an immune response
comprises a functional part of a co-stimulatory molecule such as
CD80, CD86, .degree. CD28, CD152, CD40 or CD40 ligand; of a
cell-adhesion protein; of an immune response inhibitory protein; of
an interleukin; of a major histocompatibility complex protein or of
other proteins capable of modulating an immune response.
[0024] In one embodiment the invention provides vaccine
compositions wherein said vector is nucleic acid delivery vehicle
comprising said nucleic acid. In a preferred embodiment said
nucleic acid is capable of replicating in a cell of an animal or
human being vaccinated. In a preferred embodiment said replicated
nucleic acid has at least a limited capacity to spread to other
cells of the host and start a new cycle of replication and antigen
presentation and/or present adjuvant function. In a preferred
embodiment said nucleic acid comprises nucleic acid of a Semliki
Forest Virus, a poxvirus, a herpes virus and/or an adenovirus. In a
preferred embodiment said nucleic acid delivery vehicle is a
Semliki Forest Virus particle, a pox virus particle, a herpes virus
particle or an adenovirus particle.
[0025] In another embodiment the invention provides a method for
vaccinating an animal to obtain therein an immune response against
at least one antigen, comprising administering sequentially to said
animal, at least two different vaccine compositions, each
containing at least said antigen or a precursor thereof and wherein
at least two of said vaccine compositions differ from each other by
the presence therein of a different vector. Preferably said animal
is a human.
[0026] In yet another embodiment the invention provides a use of a
vaccine composition in a method or a product of the invention.
[0027] In yet another embodiment the invention provides a use of an
antigen, or a precursor thereof, for manufacturing a vaccine
composition for vaccinating an animal or a human to obtain therein
an immune response against said antigen, wherein said vaccine
composition is administered sequentially with at least one other
vaccine composition containing at least an immunogenic part,
derivative and/or analogue of said antigen or antigen precursor,
and a different vector.
[0028] As proof of principle we undertook a vaccine efficacy study
comparing one vector system alone, two different combinations of
two different vector systems, and the use of three different
vectors administered sequentially. All vectors used to immunise
animals expressed similar SIV.sub.mac antigens. Two months
following the last immunisation animals were challenged
intravenously with a highly pathogenic
SIV.sub.mac.multidot.1.times.C inoculum and followed for evidence
of protection.
EXAMPLES
Materials and Methods
[0029] Study Population
[0030] The study was carried out in outbred rhesus monkeys (Macaca
mulatta). Four groups of 4 animals and 1 group of 3 animals (19
rhesus monkeys in total) were studied. Each animal was identified
by a unique animal number tattooed on the chest. The animals were
derived from Indian genetic stock and purpose bred in captivity
either in the USA (groups A, B, C, D, E) or the Netherlands (group
F). Their age ranged from 2.5 to 3 years (groups A, B, C, D, E) or
10 to 11 years (group F). Their weights ranged between 2.7 and 3.9
kg (groups A, B, C, D, E) or 5.2 to 9.1 kg (group F). The animals
were negative for SIV, STLV, SRV and had no previous
immunosuppressive treatment. During the experiment all animals were
housed separately in individual cages.
[0031] Three different vector systems were utilised, each
containing the same genetic information for SIV gag/pol, rev, tat,
nef and env. The vectors consisted of a bacterial plasmid based DNA
expression vector, modified Vaccinia Virus Ankara (MVA) and Semliki
Forest Virus (SFV). The first group (A) consisted of four animals
immunised-with SIV-MVA chimerics alone. Secondly, the immune
responses obtained after immunisation with the DNA expression
vectors and two boosters with either MVA-SIV (group B) or SFV-SIV
(group C) vectors were compared to those obtained with a triple
vector strategy; priming by immunisation first with DNA expression
vectors, 1st booster with the MVA-SIV constructs, then 2nd booster
with the SFV-SIV constructs (group D). The virus loads (by
quantitative RNA PCR) were studied before and after virulent SIV
challenge. Animals were challenged intravenously with a
cell-associated SIV challenge stock (1.times.C).
[0032] In addition to the animals vaccinated de novo, 3 monkeys
protected from a previous SIV vaccine study served as protein
primed vector boost group (group F). They first received a boost
with MVA-SIV, followed by SFV-SIV constructs.
[0033] Experimental Design
[0034] Group A: One group of 4 animals immunised three times with
MVA vectors expressing SIV gag/pol, rev, tat, nef and env
administered intramuscularly.
[0035] Group B: One group of 4 animals immunised first
intradermally with the DNA vectors expressing SIV gag/pol, rev,
tat, nef and env, then boosted twice intramuscularly with MVA
chimerics expressing similar SIV genes.
[0036] Group C: One group of 4 animals immunised with the DNA
vectors expressing gag/pol, rev, tat, nef and env of SIV and
boosted twice intravenously with SFV-SIV recombinant vectors
expressing similar SIV genes.
[0037] Group D: One group of 4 animals vaccinated with the DNA
expression vectors, boosted first with MVA-SIV chimerics and then
with the SFV-SIV constructs.
[0038] Group E: One group of 4 control animals injected with empty
DNA, and with the empty MVA and SFV vectors as infection
controls.
[0039] Group F: One group of 3 animals which had proved to be
protected from challenge in a previous study with a protein
vaccine, then to be boosted first with the MVA-SIV chimerics, then
with the SFV-SIV constructs.
[0040] DNA Expression Vector Based Vaccines
[0041] Vectors pTH.UbgagPk, pTH.UbpolPk, pTh.UbnefPk, pTH.tat, and
pTH.rev express the gag, pol, nef, tat and rev genes of
SIV.sub.macJ5 (Rud et al., 1994) under control of the human
cytomegalovirus immediate-early (hCMV IE) enhancer/promotor (Hanke
et al., 1998a). The vector pTH and cloning sites have been
described previously (Hanke-et al., 1998a; 1998b) in which the hCMV
enhancer/promotor/intron A is cloned into the MIuI and HindIII
sites and the individual SIVmacJ5 genes tat and cloned between
HindIII and XbaI. Two vectors pTH.tat and pTH.rev contain the
respective rev genes into the BamHI site without upstream Ub-R. The
SIV.sub.macJ5 molecular clone was used as the source of these genes
as previously described (Rud et al., 1994; Rhodes, A. D. et al.,
1994; and Hanke et al., 1994). Vector pND14-G1 expresses the
SIV.sub.mac239 envelope gp120 coding sequence under control of the
hCMV IE enhancer/promotor and the simian D type retrovirus 1
(SRV-1) cis sequence was cloned between the gp120 gene and the BGH
poly A/terminator region (Rhodes, G. H. et al., 1994; Indraccolo et
al., 1998). All constructs contain the hCMV intron A sequence 5 of
the expressed genes, in order to increase expression from the hCMV
enhancer/promotor sequence, and carry the bovine growth hormone
(BGH) polyA signal/terminator sequence. Each different DNA vector
SIV construct was administered separately at a dose of 50 .mu.g of
DNA in 200 .mu.l of saline with 1/2 of the volume injected into two
separate sites intradermally.
[0042] SFV Based Vaccines
[0043] The SFV based vaccines used in this study express the
gag/pol, nef, tat, rev, and env proteins of SIV.sub.mac32HJ5. The
gag/pol, env and nef coding sequences and the tat and rev cDNAs
from the pJ5 molecular clone of the SIV.sub.mac32H proviral genome
(Rud et al., 1994; Rhodes, A. D. et al., 1994) were subcloned in
the pSFV1 vector (Liljestrom and Garoff, 1991). The gag/pol coding
sequences were obtained by PCR amplification to flank these genes
by BamHI suitable for subcloning in the pSFV1 vector (Zhang et al.,
1997). For packaging of recombinant SFV (rSFV) viral stocks a
two-helper system was used (Smerdou and Liljestrom, 1999). Virus
titres were determined by infection of BHK cells in limiting
dilutions followed by indirect immunofluorescence using antibodies
directed against relevant SIV proteins. Expression of the SIV
antigens in infected BHK cells was also demonstrated by western
blot and immunoprecipitation analysis of metabolically labelled
BHK21 cells.
[0044] MVA Based Vaccines
[0045] Modified Vaccinia Ankara (MVA) (Sutter and Moss, 1995)
recombinants in this study express the gag/pol, nef, tat, rev, and
env genes of SIV.sub.macJ5 (Rud et al., 1994; Rhodes, A. D. et al.,
1994) under transcriptional control of P7.5 vaccinia virus
early/late promotor (Sutter et al., 1994). Briefly, the gag/pol,
env and nef coding sequences and the tat and rev cDNAs from the
SIV.sub.macJ5 molecular clone (Rud et al., 1994; Rhodes et al.,
1994) were subcloned in the MVA vector plasmid pIILzP7.5 at the
SmaI site (Sutter and Moss, 1995; Sutter et al., 1994; and Seth et
al., 1998) with the exception of env which was placed under control
of a strong vaccinia vector promotor (Sutter et al., 1994). All of
these reagents are stored and accessible through the NIBSC AIDS
reagent repository, Potters Bar, U.K.
[0046] Vaccine Challenge Strain
[0047] The pathogenic, cell-associated SIV stock
(SIV.sub.mac32H.multidot.- 1.times.C) from primary, uncultured
rhesus monkey PBMC "1.times.C", described previously (Niphuis et
al., 1994), was used as the challenge virus also described in a
previous vaccine study (Heeney et al., 1994).
[0048] Administration of the Vaccines
[0049] Rhesus monkeys were sedated with ketamin (10 mg/kg, prior to
vaccine administration and bleedings. The vaccines were
administered either intradermally (DNA vectors) or intramuscularly
(MVA) or intravenously (SFV). In particular, 50 .mu.g of each DNA
expression vector in 200 .mu.l of saline was administered per
monkey, half of the volume injected into two separate sites. All
immunisations with DNA were given twice at 12 week intervals
followed by either MVA and/or SFV (see experimental design) at
additional 12 week intervals.
[0050] Virus Challenge and Follow-Up
[0051] All animals were challenged 2 months after the last
immunisation with 50 MID.sub.50 of the pathogenic cell-associated
SIV stock "1.times.C" administered by the intravenous route
(volume: 1 ml/monkey) (Niphuis et al., 1994). Post-challenge
readouts included quantification of plasma viral RNA as described
previously (Ten Haaft et al., 1998), and assessment of CD4 T-cell
numbers in peripheral blood.
[0052] Results and Discussion
[0053] To determine if protective immunity was obtained all animals
were challenged with a highly pathogenic in vivo passaged rhesus
PBMC stock of SIV.sub.mac32H.multidot.1.times.C. As observed in
FIG. 2E all of the control animals became readily infected group E)
with peak virus loads at two weeks reaching 5.times.10.sup.6 and
5.times.10.sup.7 RNA Eq/ml and remaining greater than
1.times.10.sup.4 RNA Eq/ml 12 weeks post-infection. All animals in
group A, which received MVA-SIV constructs alone, also became
infected (Table 1), although one animal had lower peak virus loads
and a load lower than 1.times.10.sup.4 RNA Eq/ml by 6 weeks
post-infection (FIG. 2A). All animals in group B which received
DNA-SIV priming and MVA-SIV boosts also became infected (Table 1),
with high virus loads persisting above pathogenic threshold levels
(>1.times.10.sup.5 viral RNA Eq/ml) after challenge. In group C
one out of four animals was protected (Table 1) from infection,
although those which became infected were not protected from virus
load (FIG. 2C).
[0054] Satisfactory protection was observed in animals which
received three different vaccine vectors (Table 1) (FIG. 2) with
which protection from SIV challenge was obtained in 50% of the
animals. Indeed, when subunit protein vaccinated animals which were
previously protected from challenge were boosted 5 years later with
a combination of two vectors (Group F, Table 1), vaccine protection
was still observed in one out of three animals. Data post-infection
revealed that immunisation did not sufficiently protect from virus
load (FIG. 2).
[0055] Improved protection against SIV infection was obtained when
three vector systems were used (groups D, and F, Table 1). In group
D, immunised with three different vector systems, protection
against infection was found in 2 out of four immunised animals
(Table 1, FIG. 2D). Clearly, the use of one vector system alone for
multiple immunisations was insufficient to protect from infection
as in the case of MVA/SIV (group A) in this study (Table 1). This
failure of protection from infection has been observed in other
studies with SFV-SIV used alone for multiple immunisations (Mossman
et al., 1996), although protection from acute symptoms (but not
chronic disease) was suggested. A vaccine strategy using DNA
priming and MVA boosting failed to protect immunised monkeys from
infection (group B, Table 1). The use of DNA plus SFV to immunise
showed limited promise in which one animal (group C, Table 1, FIG.
2C) was protected from infection.
[0056] The best result against such potent challenges as the
SIV.sub.mac32H.multidot.1.times.C used here was achieved with the
use of three different vectors (group D, Table 1, FIG. 2D). Further
proof was observed when the peripheral blood CD4.sup.+ T-cells
numbers were examined (FIG. 3). The
SIV.sub.mac32H.multidot.1.times.C used in this study causes a
marked decline in CD4.sup.+ T-cell numbers over time as observed in
the control group (E) (FIG. 3E) as well as other infected animals
in this study. Notably, the animals which were protected from
infection by the use of the triple vector strategy (animals BJV and
CTC, group D) maintained normal CD4.sup.+ T-cell levels while those
of the infected animals declined. This was also noted in the
protected animal (8645) in group F (FIG. 3F) which has received a
triple combination of a protein immunisation followed by MVA and
SFV, further supporting this concept.
[0057] Through further refinement of this strategy, using
combinations of different or divergent chimeric vectors, improved
levels of vaccine protection are likely. Furthermore, optimisation
of different combinations of vector systems delivered to different
sites and populations of antigen presenting cells will lend this
application to mucosal and/or combined mucosal/systemic vaccine
strategies. It is envisioned that in addition differential
modulation of immune responses (i.e. innate and specific such as
type 1 vs type 2 T.sub.H responses) and the induction of potent
immunological memory will be possible using combinations of
different vaccine vector systems.
1TABLE 1 Experimental group and outcome Group "prime" 1st boost 2nd
boost protected A MVA-SIV MVA-SIV MVA-SIV 0/4 B DNA-SIV MVA-SIV
MVA-SIV 0/4 C DNA-SIV SFV-SIV SFV-SIV 1/4 D DNA-SIV MVA-SIV SFV-SIV
2/4 E DNA MVA SFV 0/4 F W. Virus protein MVA-SIV SFV-SIV 1/3
"protected"
BRIEF DESCRIPTION OF THE DRAWINGS.
[0058] FIG. 1: A diagram comparing; (A) existing immunisation
strategies with one delivery (i.e. vector) system; (B) the proposed
combination of delivery (i.e., multiple vectors) systems. Immune
responses to the desired Antigen are optimised and intensified with
subsequent boosting with the combination strategy (B) as compared
to conventional single delivery systems (A).
[0059] FIG. 2 A comparison of plasma RNA virus loads in immunised
and control animals which became infected after challenge with SIV.
FIG. 2A shows the virus loads in animals which had been immunised
repeatedly with the same vector (3.times.MVA). FIG. 2E shows the
plasma virus loads in the control animals which were not immunised
with any SIV antigen. Post-challenge virus loads for each of the
other combination groups; B (DNA, 2.times.MVA), C (DNA,
2.times.SFV), D (DNA, MVA, SFV) and F (protein, MVA, SFV)
respectively.
[0060] FIG. 3 Comparison of CD4.sup.+ T-cell levels following
challenge per group. In infected animals CD4.sup.+ T-cells declined
as was especially evident in the control animals (E). The CD4.sup.+
T-cell levels can be observed to remain at normal levels in
protected animals, especially BJV and CTC in group D (D) which
received the combination immunisation protocol. Groups depicted; A
(3.times.MVA), B (DNA, 2.times.MVA), C (DNA, 2.times.SFV), D (DNA,
MVA, SFV), E (controls), F (protein, MVA, SFV), respectively.
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