U.S. patent application number 09/798675 was filed with the patent office on 2002-08-08 for dna expression vectors and methods of use.
Invention is credited to Bright, Rick Arthur, Ellenberger, Dennis, Hildebrand, Donald G., Hua, Jian, Robinson, Harriet L., Ross, Ted M., Smith, James M..
Application Number | 20020106798 09/798675 |
Document ID | / |
Family ID | 26882026 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020106798 |
Kind Code |
A1 |
Robinson, Harriet L. ; et
al. |
August 8, 2002 |
DNA expression vectors and methods of use
Abstract
The present invention relates to novel plasmid constructs useful
for the delivery of DNA vaccines. The present invention provides
novel plasmids having a transcription cassette capable of directing
the expression of a vaccine nucleic acid insert encoding immunogens
derived from any pathogen, including fungi, bacteria and viruses.
The present invention, however, is particularly useful for inducing
in a patient an immune response against pathogenic viruses such as
HIV, measles or influenza. Immunodeficiency virus vaccine inserts
of the present invention express non-infectious HIV virus-like
particles (VLP) bearing multiple viral epitopes. VLPs allow
presentation of the epitopes to multiple histocompatability types,
thereby reducing the possibility of the targeted virus escaping the
immune response. Also described are methods for immunizing a
patient by delivery of a novel plasmid of the present invention to
the patient for expression of the vaccine insert therein.
Optionally, the immunization protocol may include a booster
vaccination that may be a live vector vaccine such as a recombinant
pox virus or modified vaccinia Arbora vector. The booster live
vaccine vector includes a transcription cassette expressing the
same vaccine insert as the primary immunizing vector.
Inventors: |
Robinson, Harriet L.;
(Atlanta, GA) ; Smith, James M.; (Tucker, GA)
; Ross, Ted M.; (Winterville, NC) ; Bright, Rick
Arthur; (Atlanta, GA) ; Hua, Jian; (Decatur,
GA) ; Ellenberger, Dennis; (Norcross, GA) ;
Hildebrand, Donald G.; (Athens, GA) |
Correspondence
Address: |
WONBLE CARLYLE SANDRIDGE & RICE
P.O. Box 7037
Atlanta
GA
30357-0037
US
|
Family ID: |
26882026 |
Appl. No.: |
09/798675 |
Filed: |
March 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60186364 |
Mar 2, 2000 |
|
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60251083 |
Dec 1, 2000 |
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Current U.S.
Class: |
435/456 ;
435/235.1; 435/320.1 |
Current CPC
Class: |
A61P 31/14 20180101;
C07K 2319/00 20130101; A61K 39/00 20130101; Y02A 50/30 20180101;
A61K 2039/545 20130101; C12N 2840/20 20130101; A61K 39/21 20130101;
C07K 14/472 20130101; C12N 15/67 20130101; C12N 2830/42 20130101;
C12N 2740/16334 20130101; A61K 39/12 20130101; A61P 31/16 20180101;
C12N 2740/16134 20130101; C12N 2840/107 20130101; A61K 2039/5258
20130101; C12N 2760/16134 20130101; C07K 16/10 20130101; A61K
2039/55522 20130101; C12N 15/70 20130101; C12N 2760/18434 20130101;
C07K 14/005 20130101; C12N 2740/16222 20130101; C12N 15/85
20130101; A61K 2039/5256 20130101; A61K 2039/57 20130101; Y02A
50/466 20180101; C12N 2740/16234 20130101; A61K 2039/54 20130101;
A61K 2039/53 20130101; C12N 2740/15034 20130101; C12N 2740/16122
20130101; C12N 2710/24143 20130101; C12N 15/63 20130101 |
Class at
Publication: |
435/456 ;
435/320.1; 435/235.1 |
International
Class: |
C12N 015/86; C12N
007/00 |
Goverment Interests
[0002] Work described herein may have been supported in part by
National Institutes of Health Grant 5 P01 AI43045 and National
Institutes of Health/National Institute of Allergy and Infectious
Diseases Grant R21 A144325-01. The U.S. Government may have certain
rights in this invention.
Claims
What is claimed is:
1. A vector comprising: a termination sequence encoding for the
lambda T0 terminator; a prokaryotic origin of replication; a
selectable marker gene; and a eukaryotic transcription cassette
comprising a vaccine insert encoding one or more immunogens derived
from a pathogen.
2. The vector of claim 1, wherein the pathogen is a viral
pathogen.
3. The vector of claim 1, wherein the viral pathogen is selected
from the group consisting of HIV, measles, influenza, polio and
rubella.
4. The vector of claim 1, wherein the one or more immunogens is
selected from the group consisting of HIV Gag, HIV gp120 HIV Pol,
HIV Env, HIV VLP, measles fusion protein, measles hemagglutinin,
measles nucleoprotein, influenza hemagglutinin, mutants thereof,
and subsequences thereof.
5. The vector of claim 1, wherein the one or more immunogens are
further selected from the group consisting of HIV Gag, HIV gp120,
HIV Pol, HIV Env, and HIV VLP, mutants thereof, and subsequences
thereof.
6. The vector according to claim 1, wherein the vaccine insert
encoding one or more immunogen or immunogens further comprises at
least one C3d gene.
7. A method of immunizing or treating a patient, comprising the
step of administering a therapeutically effective amount of a
physiologically acceptable composition, comprising the vector of
claim 1.
8. The method of claim 7, further comprising the step of
subsequently administering a therapeutically effective amount of a
composition comprising a recombinant pox virus vector expressing
one or more immunogens selected from the group consisting of HIV
Gag, HIV gp120, HIV Pol, HIV Env, HIV VLP, measles fusion protein,
measles hemagglutinin, measles nucleoprotein, influenza
hemagglutinin, mutants thereof, and subsequences thereof.
9. The method of claim 7, wherein the one or more immunogens is
selected from the group consisting of HIV Gag, HIV gp120, HIV Pol,
HIV Env, HIV VLP, mutants thereof, and subsequences thereof.
10. The method of claim 7, further comprising subsequently
administering a therapeutically effective amount of a recombinant
pox virus vector expressing one or more immunogens selected from
the group consisting of HIV Gag, HIV gp120, HIV Pol, HIV Env, HIV
VLP, measles fusion protein, measles hemagglutinin, measles
nucleoprotein, influenza hemagglutinin, mutants thereof, and
subsequences thereof.
11. The method of claim 7, wherein the vector expresses one or more
immunogens selected from the group consisting of HIV Gag, HIV
gp120. HIV Pol, HIV Env, HIV VLP, mutants thereof, and subsequences
thereof.
12. A vector comprising the DNA sequence SEQ ID NO: 1.
13. The vector of claim 12, wherein the vector further comprises a
vaccine insert encoding one or more immunogens selected from the
group consisting of HIV Gag, HIV gp120, HIV Pol, HIV Env, HIV VLP,
measles fusion protein, measles hemagglutinin, measles
nucleoprotein, influenza selected hemagglutinin, influenza
transmembrane hemagglutinin mutants thereof, and subsequences
thereof; and optionally at least one C3d gene.
14. The vector of claim 12, wherein the one or more immunogens is
selected from the group consisting of HIV Gag, HIV gp12O, HIV Pol,
HIV Env, HIV VLP, mutants thereof, and subsequences thereof.
15. A vector comprising the DNA sequence SEQ ID NO: 2.
16. The vector of claim 15, wherein the vector further comprises a
vaccine insert encoding one or more immunogens selected from the
group consisting of HIV Gag, HIV gp120, HIV Pol, HIV Env, HIV VLP,
measles fusion protein, measles hemagglutinin, measles
nucleoprotein, influenza selected hemagglutinin, influenza
transmembrane hemagglutinin mutants thereof, and subsequences
thereof; and optionally at least one C3d gene.
17. The vector of claim 15, wherein the one or more immunogens is
selected from the group consisting of HIV Gag, HIV gp120, HIV Pol,
HIV Env, HIV VLP, mutants thereof, and subsequences thereof.
18. A vector comprising the DNA sequence SEQ ID NO: 3.
19. The vector of claim 18, wherein the vector further comprises a
vaccine insert encoding one or more immunogens selected from the
group consisting of HIV Gag, HIV gp120, HIV Pol, HIV Env, HIV VLP,
measles fusion protein, measles hemagglutinin, measles
nucleoprotein, influenza selected hemagglutinin, influenza
transmembrane hemagglutinin mutants thereof, and subsequences
thereof, and optionally at least one C3d gene.
20. The vector of claim 18, wherein the one or more immunogens is
selected from the group consisting of HIV Gag, HIV gp120, HIV Pol,
HIV Env, HIV VLP, mutants thereof, and subsequences thereof.
21. A vector comprising the DNA sequence SEQ ID NO: 4.
22. The vector of claim 21, wherein the vector further comprises a
vaccine insert encoding one or more immunogens selected from the
group consisting of HIV Gag, HIV gp120, HIV Pol, HIV Env, HIV VLP,
measles fusion protein, measles hemagglutinin, measles
nucleoprotein, influenza selected hemagglutinin, influenza
transmembrane hemagglutinin mutants thereof, and subsequences
thereof, and optionally at least one C3d gene.
23. The vector of claim 21, wherein the one or more immunogens is
selected from the group consisting of HIV Gag, HIV gp120, HIV Pol,
HIV Env, HIV VLP, mutants thereof, and subsequences thereof.
24. A vector comprising the DNA sequence SEQ ID NO: 5.
25. The vector of claim 24, wherein the vector further comprises a
vaccine insert encoding one or more immunogens selected from the
group consisting of HIV Gag, HIV gp120, HIV Pol, HIV Env, HIV VLP,
measles fusion protein, measles hemagglutinin, measles
nucleoprotein, influenza selected hemagglutinin, influenza
transmembrane hemagglutinin mutants thereof, and subsequences
thereof; and optionally at least one C3d gene.
26. The vector of claim 24, wherein the one or more immunogens is
selected from the group consisting of HIV Gag, HIV gp120, HIV Pol,
HIV Env, HIV VLP, mutants thereof, and subsequences thereof.
27. A method of immunizing or treating a patient comprising
administering a therapeutically effective amount of a
physiologically acceptable composition comprising a vector wherein
the vector is a nucleic acid comprising a sequence selected from
SEQ ID NOS: 1, 2, 3, 4 and 5, and wherein administration of the
therapeutically effective amount of the composition is by an
intramuscular or intradermal route.
28. The method of claim 27, wherein the vector expresses one or
more immunogens selected from the group consisting of HIV Gag, HIV
gp120, HIV Pol, HIV Env, HIV VLP, measles fusion protein, measles
hemagglutinin, measles nucleoprotein, influenza hemagglutinin,
mutants thereof, and subsequences thereof; and optionally at least
one C3d gene.
29. The method of claim 27, wherein the vector expresses one or
more immunogens selected from the group consisting of HIV Gag, HIV
gp120. HIV Pol, HIV Env, HIV VLP, mutants thereof, and subsequences
thereof.
30. The method of claim 27, further comprising subsequently
administering a therapeutically effective amount of a recombinant
pox virus vector expressing one or more immunogens selected from
the group consisting of HIV Gag, HIV gp120, HIV Pol, HIV Env, HIV
VLP, measles fusion protein, measles hemagglutinin, measles
nucleoprotein, influenza hemagglutinin, mutants thereof, and
subsequences thereof.
31. The method of claim 27, wherein the vector expresses one or
more immunogens selected from the group consisting of HIV Gag, HIV
gp120. HIV Pol, HIV Env, HIV VLP, mutants thereof, and subsequences
thereof.
32. A method of immunizing or treating a patient in need thereof
comprising administering a therapeutically effective amount of a
composition comprising the vector of claim 29.
Description
[0001] The present application claims the benefit of priority from
U.S. provisional applications Serial No. 60/186,364, filed Mar. 2,
2000, and Serial No. 60/251,083, filed Dec. 1, 2000.
FIELD OF THE INVENTION
[0003] The present invention is directed generally to the fields of
molecular genetics and immunology. More particularly, the present
invention describes novel DNA expression vectors, novel vectors
comprising DNA encoding an immunogenic protein, and novel methods
of immunizing animals including humans by administering the novel
vectors comprising DNA encoding an immunogenic protein.
BACKGROUND OF THE INVENTION
[0004] Vaccines have had profound and long lasting effects on world
health. SmaIl pox has been eradicated, polio is near elimination,
and diseases such as diphtheria, measles, mumps, pertussis, and
tetanus are contained. Nonetheless, microbes remain major killers
with current vaccines addressing only a handful of the infections
of man and his domesticated animals. Common infectious diseases for
which there are no vaccines cost the United States $120 billion
dollars per year (Robinson et al., 1997). In first world countries,
emerging infections such as immunodeficiency viruses, as well as
reemerging diseases like drug resistant forms of tuberculosis, pose
new threats and challenges for vaccine development. The need for
both new and improved vaccines is even more pronounced in third
world countries where effective vaccines are often unavailable or
cost-prohibitive. Recently, direct injections of antigen-expressing
DNAs have been shown to initiate protective immune responses.
[0005] DNA-based vaccines use bacterial plasmids to express protein
immunogens in vaccinated hosts. Recombinant DNA technology is used
to clone cDNAs encoding immunogens of interest into eukaryotic
expression plasmids. Vaccine plasmids are then amplified in
bacteria, purified, and directly inoculated into the hosts being
vaccinated. DNA typically is inoculated by a needle injection of
DNA in saline, or by a gene gun device that delivers DNA-coated
gold beads into skin. The plasmid DNA is taken up by host cells,
the vaccine protein is expressed, processed and presented in the
context of self-major histocompatibility (MHC) class I and class II
molecules, and an immune response against the DNA-encoded immunogen
is generated.
[0006] The historical foundations for DNA vaccines (also known as
"genetic immunization") emerged concurrently from studies on gene
therapy and studies using retroviral vectors. Gene therapy studies
on DNA delivery into muscle revealed that pure DNA was as effective
as liposome-encapsulated DNA at mediating transfection of skeletal
muscle cells (Wolff et al., 1990). This unencapsulated DNA was
termed "naked DNA," a fanciful term that has become popular for the
description of the pure DNA used for nucleic acid vaccinations.
Gene guns, which had been developed to deliver DNA into plant
cells, were also used in gene therapy studies to deliver DNA into
skin. In a series of experiments testing the ability of
plasmid-expressed human growth hormone to alter the growth of mice,
it was realized that the plasmid inoculations, which had failed to
alter growth, had elicited antibody (Tang, De Vit, and Johnston,
1992). This was the first demonstration of the raising of an immune
response by an inoculated plasmid DNA. At the same time,
experiments using retroviral vectors, demonstrated that protective
immune responses could be raised by very few infected cells (on the
order of 10.sup.4-10.sup.5). Direct tests of the plasmid DNA that
had been used to produce infectious forms of the retroviral vector
for vaccination, performed in an influenza model in chickens,
resulted in protective immunizations (Robinson, Hunt, and Webster,
1993).
[0007] HIV-1 is projected to infect 1% of the world's population by
the year 2000, making vaccine development for this recently
emergent agent a high priority for world health. Preclinical trials
on DNA vaccines have demonstrated that DNA alone can protect
against highly attenuated HIV-1 challenges in chimpanzees (Boyer et
al., 1997), but not against more virulent SIV challenges in
macaques (Lu et al., 1997). A combination of DNA priming plus an
envelope glycoprotein boost has raised a neutralizing
antibody-associated protection against a homologous challenge with
a non-pathogenic chimera between SIV and HIV (SHIV-IIIb) (Letvin et
al., 1997). More recently, a comparative trial testing eight
different protocols for the ability to protect against a series of
challenges with SHIV-s (chimeras between simian and human
immunodeficiency viruses) revealed the best containment of
challenge infections by an immunization protocol that included
priming by intradermal inoculation of DNA and boosting with
recombinant fowl pox virus vectors (Robinson et al., 1999). This
containment of challenge infections was independent of the presence
of neutralizing antibody to the challenge virus. Protocols which
proved less effective at containing challenge infections included
immunization by both priming and boosting by intradermal or gene
gun DNA inoculations, immunization by priming with intradermal or
gene gun DNA inoculations and then boosting with a protein subunit;
immunization by priming with gene gun DNA inoculations and boosting
with recombinant fowl pox virus, immunization with protein only,
and immunization with recombinant fowl pox virus only (Robinson et
al,1999). Early clinical trials of DNA vaccines in humans have
revealed no adverse effects (MacGregor et al., 1996) and the
raising of cytolytic T-cells (Calarota et al., 1998). A number of
studies have screened for the ability of co-transfected lymphokines
and co-stimulatory molecules to increase the efficiency of
immunization (Robinson and Pertmer, in press).
[0008] Disadvantages of DNA vaccine approaches include the
limitation of immunizations to products encoded by DNA (e.g.,
proteins) and the potential for atypical processing of bacterial
and parasitic proteins by eukaryotic cells. Another significant
problem with existing approaches to DNA vaccines is the instability
of some vaccine insert sequences during the growth and
amplification of DNA vaccine plasmids in bacteria. One possible
cause of instability is exposure during plasmid growth of secondary
structures in vaccine inserts or the plasmid backbone that can be
recognized by bacterial endonucleases.
[0009] A need exists, therefore, for DNA expression vectors that
exhibit improved stability in bacterial hosts and may be safely
used in animals, including humans, for eukaryotic expression of
immunogenic proteins useful as vaccines against a variety of
infectious diseases, including HIV-1.
SUMMARY OF THE INVENTION
[0010] The present invention provides novel pGA constructs. The
novel pGA constructs are useful as vectors for the delivery of DNA
vaccines.
[0011] The present invention also provides novel pGA constructs
having vaccine inserts. The pathogen vaccine inserts can include
the DNA transcription unit of any virus, bacteria, parasite and/or
fungi.
[0012] The present invention describes novel methods of immunizing
patients by administering therapeutically effective amounts of the
novel pGA constructs comprising pathogen vaccine inserts.
[0013] The present invention describes novel methods of immunizing
patients by administering therapeutically effective amounts of the
novel pGA constructs comprising pathogen vaccine inserts followed
by booster immunizations with live vectored vaccines such as
recombinant modified vaccinia Ankara (MVA) vectors comprising the
same vaccine inserts.
[0014] The present invention also describes novel methods of
raising mult-epitope CD8 T-cell responses by administering
therapeutically effective amounts of the novel pGA constructs
comprising pathogen vaccine inserts followed by booster
immunizations with a live vectored vaccine such as recombinant
modified vaccinia Ankara (MVA) vectors comprising the same vaccine
inserts.
[0015] The present invention is described in more detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 illustrates a novel pGA1 construct of the present
invention. Designations are identities and positions of elements in
the vector. Designations in italic print are unique restriction
endonuclease sites useful for cloning vaccine inserts into the
vector.
[0017] FIG. 2 illustrates the DNA sequence SEQ ID NO: 1 of the
novel pGA1 construct shown in FIG. 1. The positions of elements in
the plasmid are indicated below the nucleotide sequence.
[0018] FIG. 3 illustrates a novel pGA2 construct of the present
invention. Designations are identities and positions of elements in
the vector. Designations in italic print are unique restriction
endonuclease sites useful for cloning vaccine inserts into the
vector.
[0019] FIG. 4 illustrates the DNA sequence SEQ ID NO: 2 of the
novel pGA2 construct shown in FIG. 3. The positions of elements in
the plasmid are indicated below the nucleotide sequence.
[0020] FIG. 5 illustrates a novel pGA3 construct of the present
invention. Designations are identities and positions of elements in
the vector. Designations in italic print are unique restriction
endonuclease sites useful for cloning vaccine inserts into the
vector.
[0021] FIG. 6 illustrates the DNA sequence SEQ ID NO: 3 of the
novel pGA3 construct shown in FIG. 5. The positions of elements in
the plasmid are indicated below the nucleotide sequence.
[0022] FIG. 7 compares the levels of anti-HA IgG raised by the
influenza HI hemagglutinin expressed in a pGA vector (pGA3/H1) and
in the pJW4303 research vector (pJW4303/H1). BALB/c mice were
immunized and boosted with a low dose (0.1 .mu.g) or a high dose (1
.mu.g), of the indicated plasmids using gene gun inoculations. A
priming immunization was followed by a booster immunization at 4
weeks.
[0023] FIG. 8A presents a schematic of the parent wt BH10 provirus
from which constructs producing non-infectious virus like particles
(VLPs) were produced. Dotted regions indicate sequences that were
deleted in the VLP constructs. Positions and designations of the
various regions of the BH10 provirus are indicated in the
rectangular boxes. The U3RU5 regions which encode the long terminal
repeats contain transcriptional control elements. All other
indicated regions encode proteins. For clarity, products expressed
by pol (Prt, RT, Int) and env (SU and TM) are indicated.
[0024] FIG. 8B depicts the JS2 vaccine insert. This 6.7 kb vaccine
insert expresses the Gag, Prt, and RT sequences of the BH10 strain
of HIV-1-IIIb, Tat and Vpu proteins that are from ADA, and Rev and
Env proteins that are chimeras of ADA and BH10 sequences. The Gag
sequences include mutations of the zinc fingers to limit packaging
of viral RNA. The RT sequences encompass three point mutations to
eliminate reverse transcriptase activity. Designations are the same
as in FIG. 8A. The bracketed area indicates the region of BH10 in
which sequences from ADA have been substituted for the BH10
sequences to introduce a CCR-5 using Env. The x's indicate safety
mutations.
[0025] FIG. 8C depicts the JS5 insert. JS5 is a 6 kb vaccine insert
that expresses Gag, Prt, RT, Vpu Tat, and Rev. JS5 is comprised of
the same sequences as JS2 except that sequences in Env have been
deleted. The deleted sequences are indicated in FIG. 8B as a filled
rectangle. Designations are the same as in FIGS. 8A and 8B. The Rev
responsive element (RRE) which is in the 3' region of Env is
retained in the construct.
[0026] FIGS. 9A and 9B show Gag and Env expression, respectively,
for intermediates in the construction of the JS2 vaccine insert.
Data are from transient transfections in 293T cells. pGA1/JS1 (ADA
VLP) produced higher levels of both Gag (FIG. 9A) and Env (FIG. 9B)
than wild type HIV-1 ADA or HIV-1 IIIb proviruses, and a
VLP-producing DNA (dPol) used in previous studies.
[0027] FIG. 10 shows the expression of p24 capsid in transiently
transfected cells by vaccine vectors expressing inserts without
safety mutations (JS1 and JS4), inserts with point mutations in the
zinc fingers and in RT (JS2 and JS5), and point mutations in the
zinc fingers, RT, and protease (JS3 and JS6). Note that the safety
mutations in the zinc fingers and RT supported active VLP
expression whereas the safety mutation in Prt did not. JS2 and JS5
were chosen for continued vector development based on their high
levels of expression in the presence of safety mutations.
[0028] FIGS. 11A and 11B show Gag and Env expression, respectively,
of novel candidate vaccine constructs expressed by pGA vectors with
and without intron A. PGA1 but not pGA2 contains intron A. pGA2/JS2
and pGA1/JS5 were chosen for use in vaccines based on their
favorable levels of expression.
[0029] FIGS. 12A-12D shows Western blots of cell lysates and tissue
culture supernatants from 293T cells transfected with (1) mock, (2)
pGA2/JS2, and (3) pGA1/JS5, where the primary antibody was pooled
from anti-HIV Ig from infected patients (FIG. 12A), anti-p24 (FIG.
12B), anti-gp120 (FIG. 12C), and anti-RT (FIG. 12D)
respectively.
[0030] FIG. 13 illustrates pGA.
[0031] FIG. 14 compares Gag expression levels between pGA2/89.6,
pGA1/Gag-Pol and pGA2/JS2. Comparative studies for expression were
performed on transiently transfected 293T cells.
[0032] FIGS. 15A-15C show the temporal frequencies of Gag-specific
T cells.
[0033] FIG. 15A: Gag-specific CD8 T Cell responses raised by DNA
priming and rMVA booster immunization. The schematic presents
Gag-CM9-tetramer data generated in the high-dose i.d. DNA-immunized
animals.
[0034] FIG. 15B: Gag-CM9-Mamu-A*0l tetramer-specific T cells in
Mamu-A*01 vaccinated and control macaques at various times before
challenge and at two weeks after challenge. The number at the upper
right corner of each plot represents the frequency of
tetramer-specific CD8 T cells as a % of total CD8 T cells. The
numbers above each column of plots designate individual
animals.
[0035] FIG. 15C: Gag-specific IFN-y ELISPOTs in A *01 and non-A *01
(hatched bars) vaccinated and non-vaccinated macaques at various
times before challenge and at two weeks after challenge. Three
pools of approximately 10-13 Gag peptides (22-mers overlapping by
12) were used for the analyses. The numbers above data bars
represent the arithmetic mean .+-. the standard deviation for the
ELISPOTs within each group. The numbers at the top of the graphs
designate individual animals. *, data not available; #, <20
ELISPOTs per 1.times.10.sup.6 PBMC.
[0036] FIGS. 16A-16B shows the height and breadth of
IFN-y-producing ELISPOTs against Gag and Env in the DNA/MVA memory
response.
[0037] FIG. 16A: Responses against individual Gag and Env peptide
pools. Data for animals within a group are designated by the same
symbol.
[0038] FIG. 16B: Averages of the height and breadth of ELISPOT
responses for the different groups. The heights are the mean .+-.
the standard deviation for the sums of the Gag and Env ELISPOTs for
animals in each group. The breadths are the mean .+-. the standard
deviation for the number of Gag and Env pools recognized by animals
in each group. ELISPOT responses were determined in PBMC, during
the memory phase, at 25 weeks after the rMVA booster (four weeks
prior to challenge) using 7 pools of Gag peptides (approximately
seven 22-mers overlapping by 12) representing about 70 amino acids
of Gag sequence, and 21 pools of Env peptides (approximately ten
15-mers overlapping by 11) representing about 40 amino acids of Env
sequence.
[0039] FIG. 17 shows the DNA sequence SEQ ID NO: 4 of a pGA2
construct comprising the vaccine insert, where the pathogen vaccine
insert. JS2 expresses clade B HIV-1 VLP. Both the nucleotide
sequence SEQID NO: 4 and encoded proteins are indicated.
[0040] FIG. 18 shows the DNA sequence of a pGA1 construct
comprising a pathogen vaccine insert, where the pathogen vaccine
insert. JS5 expresses lade B HIV-1 Gag-pol insert. Both the
sequence and the encoded proteins are shown.
[0041] FIGS. 19A-19E show temporal viral loads, CD4 counts and
survival after challenge of vaccinated and control animals.
[0042] FIG. 19A: Geometric mean viral loads and
[0043] FIG. 19B: geometric mean CD4 counts for vaccine and control
groups at various weeks post-challenge. The key for the groups is
in panel B.
[0044] FIG. 19C: Survival curve for vaccinated and control animals.
The dotted line represents all 24 vaccinated animals.
[0045] FIG. 19D: viral loads and
[0046] FIG. 19E: CD4 counts for individual animals in the vaccine
and control groups. The key to animal numbers is presented in FIG.
19E. Assays for the first 12 weeks post challenge had a background
of 1000 copies of RNA per ml of plasma. Animals with loads below
1000 were scored with a load of 500. For weeks 16 and 20, the
background for detection was 300 copies of RNA/ml. Animals with
levels of virus below 300 were scored at 300.
[0047] FIGS. 20A-20C show Post-challenge T-cell responses in
vaccine and control groups.
[0048] FIG. 20A: temporal tetramer+ cells and viral loads.
[0049] FIG. 20B: Intracellular cytokine assays for IFN-.gamma.
production in response to stimulation with the Gag-CM9 peptide at
two weeks post-challenge. This ex vivo assay allows evaluation of
the functional status of the peak post-challenge tetramer+ cells
displayed in FIG. 15A.
[0050] FIG. 20C: Proliferation assay at 12 weeks post-challenge.
Gag-Pol-Env (open bars) and Gag-Pol (hatched bars) produced by
transient transfections were used for stimulation. Supernatants
from mock-transfected cultures served as control antigen. Proteins
were used at approximately 1 .mu.g per ml of p27 Gag for
stimulations. Stimulation indices are the growth of cultures in the
presence of viral antigens divided by the growth of cultures in the
presence of mock antigen.
[0051] FIGS. 21A-21E show lymph node histomorphology and viral
loads at 12 weeks post-challenge.
[0052] FIG. 21A: Typical lymph node from a vaccinated macaque
showing evidence of follicular hyperplasia characterized by the
presence of numerous secondary follicles with expanded germinal
centers and discrete dark and light zones.
[0053] FIG. 21B: Typical lymph node from an infected control animal
showing follicular depletion and paracortical lymphocellular
atrophy.
[0054] FIG. 21C: A representative lymph node from an age-matched,
uninfected macaque displaying non-reactive germinal centers.
[0055] FIG. 21D: The percent of the total lymph node area occupied
by germinal centers was measured to give a non-specific indicator
of follicular hyperplasia. Data for uninfected controls are for
four age-matched rhesus macaques.
[0056] FIG. 21E: Lymph node virus burden was determined by in situ
hybridization using an antisense riboprobe cocktail that was
complementary to SHIV-89.6 gag and pol. All of the examined nodes
were inguinal lymph nodes.
[0057] FIGS. 22A-22D show temporal antibody responses following
challenge. Micrograms of total Gag (FIG. 22A) or Env (FIG. 22B)
antibody were determined using enzyme linked immunosorbent assays
(ELISAs). The titers of neutralizing antibody for 89.6 (FIG. 22C)
and 89.6P (FIG. 22D) were determined using MT-2 cell killing and
neutral red staining. Titers are the reciprocal of the serum
dilution giving 50% neutralization of the indicated viruses grown
in human PBMC. Symbols for animals are the same as in FIG. 19.
[0058] FIGS. 23A-23E show correlations and dose response curves for
the vaccine trial (FIGS. 23A and B). Inverse correlations between
peak vaccine raised IFN-.gamma. ELISPOTs and viral loads at 2 (FIG.
23A) and 3 (FIG. 23B) weeks post-challenge. Only twenty-three of
the 24 vaccinated animals are included in the correlations because
of the loss of the peak DNA/MVA ELISPOT sample for animal 3 (see
FIG. 15C). (FIG. 23C) Dose response curves for the average height
of Gag ELISPOTS at the peak DNA-MVA response (data from FIG. 15C).
(FIG. 23D) Dose response curve for the breadth of the DNA/MVA
memory ELISPOT response (data from FIG. 16B). (FIG. 23E) Dose
response curves for the peak anti-Gag antibody response post the
MVA booster (data from FIG. 22A). The different doses of DNA raised
different levels of ELISPOT and antibody responses (P<0.05). The
route of DNA inoculation had a significant effect on the antibody
(P=0.02), but not the ELISPOT response.
[0059] FIG. 24 shows anti-HA IgG raised by gene gun inoculation of
DNAs expressing HA proteins.
[0060] FIG. 25. Shows avidity of the anti HA IgG raised by the
three different HA DNA vaccines.
[0061] FIG. 26 shows protection from weight loss after virus
challenge.
[0062] FIG. 27 illustrates the importance of including Env in the
vaccine.
[0063] FIGS. 28A-28D illustrates the importance of including Env in
vaccines administered to animals challenged interectally with
SHIV-89.6P.
[0064] FIG. 29 is a schematic representation of vector DNA vaccine
constructs.
[0065] FIG. 30 shows Western blot results showing expression of
vaccine constructs in vitro.
[0066] FIG. 31 is a temporal curve of measles virus neutralizing
antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0067] This invention relates to novel vectors, novel vectors
comprising pathogen vaccine inserts, and novel methods of
immunizing patients against a pathogen. The novel immunization
methods elicit both cell-mediated and humoral immune responses that
may limit the infection, spread or growth of the pathogen and
result in protection against subsequent challenge by the
pathogen.
[0068] Classic references for DNA vaccines include the first
demonstration of the raising of an immune response (Tang, De Vit,
and Johnston, 1992); the first demonstration of cytotoxic T-cell
(Tc)-mediated immunity (Ulmer et al., 1993); the first
demonstration of the protective efficacy of intradermal (i.d.),
intramuscular (i.m.), intravenous (i.v.), intranasal (i.n.), and
gene gun (g.g.) immunizations (Fynan et al., 1993; Robinson, Hunt,
and Webster, 1993); the first use of genetic adjuvants (Xiang and
Ertl, 1995); the first use of library immunizations (Barry, Lai,
and Johnston, 1995); and the first demonstration of the ability to
modulate the T-helper type of an immune response by the method of
DNA delivery (Feltquate et al., 1997). A highly useful web site
compiling DNA vaccine information can be found at
http://www.genweb.com/Dnavax/dnavax.html.
[0069] For convenience, certain terms employed in the
specification, examples, and appended claims are collected
here.
[0070] Definitions
[0071] The term "nucleic acid" as used herein refers to any natural
and synthetic linear and sequential arrays of nucleotides and
nucleosides, for example cDNA, genomic DNA, mRNA, tRNA,
oligonucleotides, oligonucleosides and derivatives thereof. For
ease of discussion, such nucleic acids may be collectively referred
to herein as "constructs," "plasmids," or "vectors." Representative
examples of the nucleic acids of the present invention include
bacterial plasmid vectors including expression, cloning, cosmid and
transformation vectors such as, but not limited to, pBR322, animal
viral vectors such as, but not limited to, modified adenovirus,
influenza virus, polio virus, pox virus, retrovirus, and the like,
vectors derived from bacteriophage nucleic acid, and synthetic
oligonucleotides like chemically synthesized DNA or RNA. The term
"nucleic acid" further includes modified or derivatised nucleotides
and nucleosides such as, but not limited to, halogenated
nucleotides such as, but not only, 5-bromouracil, and derivatised
nucleotides such as biotin-labeled nucleotides.
[0072] The term "isolated nucleic acid" as used herein refers to a
nucleic acid with a structure (a) not identical to that of any
naturally occurring nucleic acid or (b) not identical to that of
any fragment of a naturally occurring genomic nucleic acid spanning
more than three separate genes, and includes DNA, RNA, or
derivatives or variants thereof. The term includes, but is not
limited to, the following: (a) a DNA which has the sequence of part
of a naturally occurring genomic molecule but is not flanked by at
least one of the coding sequences that flank that part of the
molecule in the genome of the species in which it naturally occurs;
(b) a nucleic acid incorporated into a vector or into the genomic
nucleic acid of a prokaryote or eukaryote in a manner such that the
resulting molecule is not identical to any vector or naturally
occurring genomic DNA; (c) a separate molecule such as a cDNA, a
genomic fragment, a fragment produced by polymerase chain reaction
(PCR), ligase chain reaction (LCR) or chemical synthesis, or a
restriction fragment; (d) a recombinant nucleotide sequence that is
part of a hybrid gene, i.e., a gene encoding a fusion protein, and
(e) a recombinant nucleotide sequence that is part of a hybrid
sequence that is not naturally occurring.
[0073] It is advantageous for some purposes that a nucleotide
sequence is in purified form. The term "purified" in reference to
nucleic acid represents that the sequence has increased purity
relative to the natural environment.
[0074] As used herein the terms "polypeptide" and "protein" refer
to a polymer of amino acids of three or more amino acids in a
serial array, linked through peptide bonds. The term "polypeptide"
includes proteins, protein fragments, protein analogues,
oligopeptides and the like. The term "polypeptides" contemplates
polypeptides as defined above that are encoded by nucleic acids,
produced through recombinant technology, isolated from an
appropriate source, or are synthesized. The term "polypeptides"
further contemplates polypeptides as defined above that include
chemically modified amino acids or amino acids covalently or
noncovalently linked to labeling ligands.
[0075] The term "fragment" as used herein to refer to a nucleic
acid (e.g., cDNA) refers to an isolated portion of the subject
nucleic acid constructed artificially (e.g., by chemical synthesis)
or by cleaving a natural product into multiple pieces, using
restriction endonucleases or mechanical shearing, or a portion of a
nucleic acid synthesized by PCR, DNA polymerase or any other
polymerizing technique well known in the art, or expressed in a
host cell by recombinant nucleic acid technology well known to one
of skill in the art. The term "fragment" as used herein may also
refer to an isolated portion of a polypeptide, wherein the portion
of the polypeptide is cleaved from a naturally occurring
polypeptide by proteolytic cleavage by at least one protease, or is
a portion of the naturally occurring polypeptide synthesized by
chemical methods well known to one of skill in the art.
[0076] The term "gene" or "genes" as used herein refers to nucleic
acid sequences (including both RNA or DNA) that encode genetic
information for the synthesis of a whole RNA, a whole protein, or
any portion of such whole RNA or whole protein. Genes that are not
naturally part of a particular organism's genome are referred to as
"foreign genes", "heterologous genes" or "exogenous genes" and
genes that are naturally a part of a particular organism's genome
are referred to as "endogenous genes".
[0077] The term "expressed" or "expression" as used herein refers
to the transcription from a gene to give an RNA nucleic acid
molecule at least complementary in part to a region of one of the
two nucleic acid strands of the gene. The term "expressed" or
"expression" as used herein also refers to the translation from
said RNA nucleic acid molecule to give a protein or polypeptide or
a portion thereof.
[0078] As used herein, the term "locus" or "loci" refers to the
site of a gene on a chromosome. Pairs of genes control hereditary
traits, each in the same position on a pair of chromosomes. These
gene pairs, or alleles, may both be dominant or both be recessive
in expression of that trait. In either case, the individual is said
to be homozygous for the trait controlled by that gene pair. If the
gene pair (alleles) consists of one dominant and one recessive
trait, the individual is heterozygous for the trait controlled by
the gene pair. Natural variation in genes or nucleic acid molecules
caused by, for example, recombination events or resulting from
mutation, gives rise to allelic variants with similar, but not
identical, nucleotide sequences. Such allelic variants typically
encode proteins with similar activity to that of the protein
encoded by the gene to which they are compared, because natural
selection typically selects against variations that alter function.
Allelic variants can also comprise alterations in the untranslated
regions of the gene as, for example, in the 3' or 5' untranslated
regions or can involve alternate splicing of a nascent transcript,
resulting in alternative exons being positioned adjacently.
[0079] The term "transcription regulatory sequences" as used herein
refers to nucleotide sequences that are associated with a gene
nucleic acid sequence and which regulate the transcriptional
expression of the gene. The "transcription regulatory sequences"
may be isolated and incorporated into a vector nucleic acid to
enable regulated transcription in appropriate cells of portions of
the vector DNA. The "transcription regulatory sequence" may
precede, but are not limited to, the region of a nucleic acid
sequence that is in the region 5' of the end of a protein coding
sequence that may be transcribed into mRNA. Transcriptional
regulatory sequences may also be located within a protein coding
region, in regions of a gene that are identified as "intron"
regions, or may be in regions of nucleic acid sequence that are in
the region of nucleic acid.
[0080] The term "coding region" as used herein refers to a
continuous linear arrangement of nucleotides that may be translated
into a protein. A full length coding region is translated into a
full length protein; that is, a complete protein as would be
translated in its natural state absent any post-translational
modifications. A full length coding region may also include any
leader protein sequence or any other region of the protein that may
be excised naturally from the translated protein.
[0081] The term "probe" as used herein, when referring to a nucleic
acid, refers to a nucleotide sequence that can be used to hybridize
with and thereby identify the presence of a complementary sequence,
or a complementary sequence differing from the probe sequence but
not to a degree that prevents hybridization under the hybridization
stringency conditions used. The probe may be modified with labels
such as, but not only, radioactive groups, biotin, or any other
label that is well known in the art.
[0082] The term "nucleic acid vector" as used herein refers to a
natural or synthetic single or double stranded plasmid or viral
nucleic acid molecule that can be transfected or transformed into
cells and replicate independently of, or within, the host cell
genome. A circular double stranded plasmid can be linearized by
treatment with an appropriate restriction enzyme based on the
nucleotide sequence of the plasmid vector. A nucleic acid can be
inserted into a vector by cutting the vector with restriction
enzymes and ligating the pieces together. The nucleic acid molecule
can be RNA or DNA.
[0083] The term "expression vector" as used herein refers to a
nucleic acid vector that may further include at least one
regulatory sequence operably linked to a nucleotide sequence coding
for the Mago protein. Regulatory sequences are well recognized in
the art and may be selected to ensure good expression of the linked
nucleotide sequence without undue experimentation by those skilled
in the art. As used herein, the term "regulatory sequences"
includes promoters, enhancers, and other elements that may control
expression. Standard molecular biology textbooks such as Sambrook
et al. eds "Molecular Cloning: A Laboratory Manual" 2nd ed. Cold
Spring Harbor Press (1989) may be consulted to design suitable
expression vectors, promoters, and other expression control
elements. It should be recognized, however, that the choice of a
suitable expression vector depends upon multiple factors including
the choice of the host cell to be transformed and/or the type of
protein to be expressed.
[0084] The terms "transformation" and "transfection" as used herein
refer to the process of inserting a nucleic acid into a host. Many
techniques are well known to those skilled in the art to facilitate
transformation or transfection of a nucleic acid into a prokaryotic
or eukaryotic organism. These methods involve a variety of
techniques, such as treating the cells with high concentrations of
salt such as, but not only a calcium or magnesium salt, an electric
field, detergent, or liposome mediated transfection, to render the
host cell competent for the uptake of the nucleic acid
molecules.
[0085] The term "recombinant cell" refers to a cell that has a new
combination of nucleic acid segments that are not covalently linked
to each other in nature. A new combination of nucleic acid segments
can be introduced into an organism using a wide array of nucleic
acid manipulation techniques available to those skilled in the art.
A recombinant cell can be a single eukaryotic cell, or a single
prokaryotic cell, or a mammalian cell. The recombinant cell can
harbor a vector that is extragenomic. An extragenomic nucleic acid
vector does not insert into the cell's genome. A recombinant cell
can further harbor a vector or a portion thereof that is
intragenomic. The term intragenomic defines a nucleic acid
construct incorporated within the recombinant cell's genome.
[0086] The term "recombinant nucleic acid" as used herein refers to
combinations of at least two nucleic acid sequences that are not
naturally found in a eukaryotic or prokaryotic cell. The nucleic
acid sequences may include, but are not limited to nucleic acid
vectors, gene expression regulatory elements, origins of
replication, sequences that when expressed confer antibiotic
resistance, and protein-encoding sequences. The term "recombinant
polypeptide" is meant to include a polypeptide produced by
recombinant DNA techniques such that it is distinct from a
naturally occurring polypeptide either in its location, purity or
structure. Generally, such a recombinant polypeptide will be
present in a cell in an amount different from that normally
observed in nature.
[0087] The term "patients," as used herein, refers to animals,
preferably mammals, and more preferably humans.
[0088] The term "immunizing" or "immunization," as used herein,
refers to the production of an immune response in a patient that
protects (partially or totally) from the manifestations of
infection (i.e., disease) caused by a pathogen. A patient immunized
by the present invention will not be infected by the pathogen or
will be infected to a lesser extent than would occur without
immunization. Immunizations may be either prophylactic or
therapeutic in nature. That is, both previously uninfected and
infected patients may be immunized with the present invention.
[0089] The term "DNA transcription unit" as used herein" refers to
a polynucleotide sequence that includes at least two components:
antigen-encoding DNA and transcriptional promoter elements. A DNA
transcription unit may optionally include additional sequences,
such as enhancer elements, splicing signals, termination and
polyadenylation signals, viral replicons, and/or bacterial plasmid
sequences. The DNA transcription unit can be produced by a number
of known methods. For example, DNA encoding the desired antigen can
be inserted into an expression vector to construct the DNA
transcription unit, as described in Maniatis et al, Molecular
Cloning: A Laboratory Manual, 2d, Cold Spring Harbor Laboratory
Press (1989), the disclosure of which is incorporated by reference
in its entirety.
[0090] The term "vaccine insert" as used herein refers to the DNA
transcription unit of a pathogen. Preferably, the vaccine insert is
a DNA transcription unit that can generate an immune responses in a
patient. For example, th evaccine insert is a pathogen vaccine
insert encoding antigens derived from any virus, bacteria, parasite
and/or fungi. Exemplary viruses include herpesvirus,
orthomyxoviruses, rhinoviruses, picornaviruses, adenoviruses,
paramyxoviruses, coronaviruses, rhabdoviruses, togaviruses,
flaviviruses, bunyaviruses, rubella virus, reovirus, measles,
hepadna viruses, Ebola, retroviruses (including human
immunodeficiency virus), and the like. Exemplary bacteria include
tuberculosis, mycobateria, spirochetes, rickettsias, chlamydia,
mycoplasma and the like. Exemplary parasites include malaria and
the like. Exemplary fungi include yeasts, molds, and the like. One
skilled in the art will appreciate that this list does not include
all potential pathogens against which a protective immune response
can be generated by the methods described herein.
[0091] The term "antigen" as used herein refers to any protein,
carbohydrate, or other moiety expressed by a pathogen that is
capable of eliciting a protective response against a pathogen. The
antigen may or may not be a structural component of the pathogen.
Also contemplated to be within the term "antigen" are encoded
antigens that can be translation products or polypeptides of
various lengths. Antigens undergo normal host cell modifications
such as glycosylation, myristoylation or phosphorylation. In
addition, they can be designed to undergo intracellular,
extracellular or cell-surface expression. Furthermore, they can be
designed to undergo assembly and release from cells.
[0092] As used herein, the term "adjuvant" means a substance added
to a vaccine to increase a vaccine's immunogenicity. The mechanism
of how an adjuvant operates is not entirely known. Some adjuvants
are believed to enhance the immune response by slowly releasing the
antigen, while other adjuvants are strongly immunogenic in their
own right and are believed to function synergistically. Known
vaccine adjuvants include, but are not limited to, oil and water
emulsions (for example, complete Freund's adjuvant and incomplete
Freund's adjuvant), Corynebacterium parvum, Bacillus Calmette
Guerin, aluminum hydroxide, glucan, dextran sulfate, iron oxide,
sodium alginate, Bacto-Adjuvant, certain synthetic polymers such as
poly amino acids and co-polymers of amino acids, saponin,
"REGRESSIN" (Vetrepharm, Athens, Ga.), "AVRIDINE" (N,
N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine), paraffin
oil, and muramyl dipeptide. Adjuvants also encompass genetic
adjuvants such as immunomodulatory molecules encoded in a
co-inoculated DNA. The co-inoculated DNA can be in the same vaccine
construct as the vaccine immunogen or in a separate DNA vector.
[0093] As used herein, the term "pharmaceutically acceptable
carrier" means a vehicle for containing the vaccine that can be
injected into a bovine host without adverse effects. Suitable
pharmaceutically acceptable carriers known in the art include, but
are not limited to, sterile water, saline, glucose, dextrose, or
buffered solutions. Carriers may include auxiliary agents
including, but not limited to, diluents, stabilizers (i.e., sugars
and amino acids), preservatives, wetting agents, emulsifying
agents, pH buffering agents, viscosity enhancing additives, colors
and the like.
[0094] The terms "selectable marker gene" as used herein refer to
an expressed gene that allows for the selection of a population of
cells containing the selectable marker gene from a population of
cells not having the expressed selectable marker gene. For example,
the "selectable marker gene" may be an "antibiotic resistance gene"
that can confer tolerance to a specific antibiotic by a
microorganism that was previously adversely affected by the drug.
Such resistance may result from a mutation or the acquisition of
resistance due to plasmids containing the resistance gene
transforming the microorganism.
[0095] The term "terminator sequence" or "terminator" as used
herein refers to nucleotide sequences that function to stop
transcription. The terms "transcription" or "transcribe" as used
herein refers to the process by which RNA molecules are formed upon
DNA templates by complementary base pairing. This process is
mediated by RNA polymerase.
[0096] The term "VLP" as used herein refers to virus-like particles
and, as used, also refers to aggregates of viral proteins.
[0097] The major immunological advantage of DNA-based immunizations
is the ability of the immunogen to be presented by both MHC class I
and class II molecules. Endogenously synthesized proteins readily
enter processing pathways for the loading of peptide epitopes onto
MHC I as well as MHC II molecules. MHC I-presented epitopes raise
cytotoxic T-cells (Tc) responses whereas MHC II-presented epitopes
raise helper T-cells (Th). By contrast, immunogens that are not
synthesized in cells are largely restricted to the loading of MHC
II epitopes and the raising of Th but not Tc. When compared with
live attenuated vaccines or recombinant viral vectors that produce
immunogens in cells and raise both Th and Tc, DNA vaccines have the
advantages of not being infectious and of focusing the immune
response on only those antigens desired for immunization. DNA
vaccines also are advantageous because they can be manipulated
relatively easily to raise type 1 or type 2 T-cell help. This
allows a vaccine to be tailored for the type of immune response
that will be mobilized to combat an infection. DNA vaccines are
also cost effective because of the ease with which plasmids can be
constructed using recombinant DNA technology, the ability to use a
generic method for vaccine production (growth and purification of
plasmid DNA), and the stability of DNA over a wide range of
temperatures.
[0098] The best immune responses are achieved using highly active
expression vectors modeled on those developed for the production of
recombinant proteins (Robinson and Pertmer, 1998). The most
frequently used transcriptional control elements include a strong
promoter. One such promoter suitable for use is the cytomegalovirus
(CMV) intermediate early promoter, although other promoters may be
used in a DNA vaccine without departing from the scope the present
invention. Other transcriptional control elements useful in the
present invention include a strong polyadenylation signal such as,
for example, that derived from a bovine growth hormone encoding
gene, or a rabbit .beta. globin polyadenylation signal (Bohm et
al., 1996; Chapman et al., 1991; Hartikka et al., 1996; Manthorpe
et al., 1993; Montgomery et al., 1993). The CMV immediate early
promoter may be used with or without intron A (Chapman et al.,
1991). The presence of intron A increases the expression of many
antigens from RNA viruses, bacteria, and parasites, presumably by
providing the expressed RNA with sequences which support processing
and function as an eukaryotic mRNA. It will be appreciated that
expression also may be enhanced by other methods known in the art
including, but not limited to, optimizing the codon usage of
prokaryotic mRNAs for eukaryotic cells (Andre et al., 1998;
Uchijima et al., 1998). Multi-cistronic vectors may be used to
express more than one immunogen or an immunogen and a
immunostimulatory protein (Iwasaki et al., 1997a; Wild et al.,
1998).
[0099] Immunogens can also be engineered to be more or less
effective for raising antibody or Tc by targeting the expressed
antigen to specific cellular compartments. For example, antibody
responses are raised more effectively by s antigens that are
displayed on the plasma membrane of cells, or secreted therefrom,
than by antigens that are localized to the interior of cells
(Boyle, Koniaras, and Lew, 1997; Inchauspe et al., 1997). Tc
responses may be enhanced by using N-terminal ubiquitination
signals which target the DNA-encoded protein to the proteosome
causing rapid cytoplasmic degradation and more efficient peptide
loading into the MHC I pathway (Rodriguez, Zhang, and Whitton,
1997; Tobery and Siliciano, 1997; Wu and Kipps, 1997). For a review
on the mechanistic basis for DNA-raised immune responses, refer to
Robinson and Pertmer, Advances in Virus Research, vol. 53, Academic
Press (2000), the disclosure of which is incorporated herein by
reference in its entirety.
[0100] The effects of different conformational forms of proteins on
antibody responses, the ability of strings of MHC I epitopes
(minigenes) to raise Tc responses, and the effect of fusing an
antigen with immune-targeting proteins have been evaluated using
defined inserts. Ordered structures such as virus-like particles
appear to be more effective than unordered structures at raising
antibody (Fomsgaard et al., 1998). This is likely to reflect the
regular array of an immunogen being more effective than a monomer
of an antigen at cross-linking Ig-receptors and signaling a B-cell
to multiply and produce antibody. Recombinant DNA molecules
encoding a string of MHC epitopes from different pathogens can
elicit Tc responses to a number of pathogens (Hanke et al., 1998b).
These strings of Tc epitopes are most effective if they also
include a Th epitope (Maecker et al., 1998; Thomson et al.,
1998).
[0101] Another approach to manipulating immune responses is to fuse
immunogens to immunotargeting or immunostimulatory molecules. To
date, the most successful of these fusions have targeted secreted
immunogens to antigen presenting cells (APC) or lymph nodes (Boyle,
Brady, and Lew, 1998). Fusion of a secreted form of human IgG with
CTLA-4 increased antibody responses to the IgG greater than
1000-fold and changed the bias of the response from complement
(C'-) dependent to C'-independent antibodies.
[0102] Fusions of human IgG with L-selectin also increased antibody
responses but did not change the C'-binding characteristics of the
raised antibody. The immunogen fused with L-selectin was presumably
delivered to lymph nodes by binding to the high endothelial
venules, which serve as portals. Fusions between antigens and
cytokine cDNAs have resulted in more moderate increases in
antibody, Th, and Tc responses (Hakim, Levy, and Levy, 1996;
Maecker et al., 1997). IL-4-fusions have increased antibody
responses, whereas IL-12 and IL-1.beta., have enhanced T-cell
responses.
[0103] Two approaches to DNA delivery are injection of DNA in
saline using a hypodermic needle or gene gun delivery of DNA-coated
gold beads. Saline injections deliver DNA into extracellular
spaces, whereas gene gun deliveries bombard DNA directly into
cells. The saline injections require much larger amounts of DNA
(100-1000 times more) than the gene gun (Fynan et al., 1993). These
two types of delivery also differ in that saline injections bias
responses towards type 1 T-cell help, whereas gene gun deliveries
bias responses towards type 2 T-cell help (Feltquate et al., 1997;
Pertmer, Roberts, and Haynes, 1996). DNAs injected in saline
rapidly spread throughout the body. DNAs delivered by the gun are
more localized at the target site. Following either method of
inoculation, extracellular plasmid DNA has a short half life on the
order of 10 minutes (Kawabata, Takakura, and Hashida, 1995; Lew et
al., 1995). Vaccination by saline injections can be intramuscular
(i.m.) or intradermal (i.d.) (Fynan et al., 1993).
[0104] Although intravenous and subcutaneous injections have met
with different degrees of success for different plasmids (Bohm et
al., 1998; Fynan et al., 1993), intraperitoneal injections have not
met with success (Bohm et al., 1998; Fynan et al., 1993). Gene gun
deliveries can be administered to the skin or to surgically exposed
muscle. Methods and routes of DNA delivery that are effective at
raising immune responses in mice are effective in other
species.
[0105] Immunization by mucosal delivery of DNA has been less
successful than immunizations using parenteral routes of
inoculation. Intranasal administration of DNA in saline has met
with both good (Asakura et al., 1997; Sasaki et al., 1998b) and
limited (Fynan et al., 1993) success. The gene gun has successfully
raised IgG following the delivery of DNA to the vaginal mucosa
(Livingston et al., 1995). Some success at delivering DNA to
mucosal surfaces has also been achieved using liposomes (McCluskie
et al., 1998), microspheres (Chen et al., 1998a; Jones et al.,
1997) and recombinant Shigella vectors (Sizemore, Branstrom, and
Sadoff, 1995; Sizemore, Branstrom, and Sadoff, 1997).
[0106] The dose of DNA needed to raise a response depends upon the
method of delivery, the host, the vector, and the encoded antigen.
The most profound effect is seen for the method of delivery. From
10 .mu.g to 1 mg of DNA is generally used for saline injections of
DNA, whereas from 0.2 .mu.g to 20 .mu.g of DNA is used for gene gun
deliveries of DNA. In general, lower doses of DNA are used in mice
(10-100 .mu.g for saline injections and 0.2 .mu.g to 2 .mu.g for
gene gun deliveries), and higher doses in primates (100 .mu.g to 1
mg for saline injections and 2 .mu.g to 20 .mu.g for gene gun
deliveries). The much lower amount of DNA required for gene gun
deliveries reflect the gold beads directly delivering DNA into
cells.
[0107] An example of the marked effect of an antigen on the raised
response can be found in studies comparing the ability to raise
antibody responses in rabbits of DNAs expressing the influenza
hemagglutinin or an immunodeficiency virus envelope glycoprotein
(Env) (Richmond et al., 1998). Under similar immunization
conditions, the hemagglutinin-expressin- g DNA raised long lasting,
high avidity, high titer antibody (.about.100 .mu.g per ml of
specific antibody), whereas the Env-expressing DNA raised only
transient, low avidity, and low titer antibody responses (<10
.mu.g per ml of specific antibody). These differences in raised
antibody were hypothesized to reflect the hemagglutinin being a
T-dependent antigen and the highly glycosylated immunodeficiency
virus Env behaving as a T-independent antigen.
[0108] Both protein and recombinant viruses have been used to boost
DNA-primed immune responses. Protein boosts have been used to
increase neutralizing antibody responses to the HIV-1 Env.
Recombinant pox virus boosts have been used to increase both
humoral and cellular immune responses.
[0109] For weak immunogens, such as the immunodeficiency virus Env,
for which DNA-raised antibody responses are only a fraction of
those in naturally infected animals, protein boosts have provided a
means of increasing low titer antibody responses (Letvin et al.,
1997; Richmond et al., 1998). In a study in rabbits, the protein
boost increased both the titers of antibody and the avidity and the
persistence of the antibody response (Richmond et al., 1998).
Consistent with a secondary immune response to the protein boost,
DNA primed animals showed both more rapid increases in antibody,
and higher titers of antibody following a protein boost than
animals receiving only the protein. However, by a second protein
immunization, the kinetics and the titer of the antibody response
were similar in animals that had, and had not, received DNA priming
immunizations.
[0110] Recombinant pox virus boosts have proved to be a highly
successful method of boosting DNA-primed CD8+ cell responses (Hanke
et al., 1998a; Kent et al., 1998; Schneider et al., 1998).
Following pox virus boosters, antigen-specific CD8+ cells have been
increased by as much as 10-fold in DNA primed mice or macaques.
Studies testing the order of immunizations reveal that the DNA must
be delivered first (Schneider et al., 1998). This has been
hypothesized to reflect the DNA focusing the immune response on the
desired immunogens. The larger increases in CD8+ cell responses
following pox virus boosts has been hypothesized to reflect both
the larger amount of antigen expressed by the pox virus vector, as
well as pox virus-induced cytokines augmenting immune responses
(Kent et al., 1998; Schneider et al., 1998).
[0111] A number of different pox viruses can be used for the pox
boost. A vaccinia virus termed modified vaccinia Ankara (MVA) has
been particularly effective in mouse models (Schneider et al.,
1998). This may reflect MVA, which is replication defective in
mammalian models, being attenuated for the ability to evade host
immune responses.
[0112] Responses raised by a DNA prime followed by pox virus boost
can be highly effective at raising protective cell-mediated immune
responses. In mice, intramuscular injections of DNA followed by
recombinant pox boosts have protected against a malaria challenge
(Schneider et al., 1998). In macaques, intradermal., but not gene
gun DNA primes, followed by recombinant pox virus boosters have
contained challenges with chimeras of simian and human
immunodeficiency viruses (Robinson et al., 1999).
[0113] DNA vaccines for immunodeficiency viruses such as HIV-1
encounter the challenge of sufficiently limiting an incoming
infection such that the inexorable long-term infections that lead
to AIDS are prevented. Complicating this is that neutralizing
antibodies is both difficult to raise and specific against
particular viral strains (Burton and Montefiori, 1997; Moore and
Ho, 1995). Given the problems with raising neutralizing antibody,
much effort has focused on raising cell-mediated responses of
sufficient strength to severely curtail infections. To date, the
best success at raising high titers of Tc have come from
immunization protocols using DNA primes followed by recombinant pox
virus boosters. The efficacy of this protocol has been evaluated by
determining the level of specific Tc using assays for cytolytic
activity (Kent et al., 1998), by staining with MHC-specific
tetramers for specific SIV Gag epitopes and by challenge with SIVs
or SHIVs (Hanke, 1999).
[0114] A number of salient findings are emerging from preclinical
trials using DNA primes and recombinant pox virus boosts. The first
is that challenge infections can be contained below the level that
can be detected using quantitative RT-PCR analyses for plasma viral
RNA (Robinson et al., 1999). The second is that this protection is
long lasting and does not require the presence of neutralizing
antibody (Robinson et al., 1999). The third is that intradermal DNA
priming with saline injections of DNA is superior to gene gun
priming for raising protective immunity (P=0.01, Fisher's exact
test) (Robinson et al., 1999).
[0115] The novel pGA vectors of the present invention have a
prokaryotic origin of replication, a selective marker gene for
plasmid selection, and a transcription cassette for eukaryotic
cells. Unique to the pGA vectors of the present invention is the
inclusion of the lambda terminator in the same transcriptional
orientation, and following, the selective marker gene. This
terminator sequence prevents read-through from the kanamycin
cassette into vaccine sequences while the plasmid is being produced
in bacteria. Prevention of transcriptional read-through stabilizes
vaccine insert sequences by limiting the exposure of secondary
structures that can be recognized by bacterial endonucleases.
[0116] A transcription cassette as incorporated in the pGA vectors
of the present invention uses sequences from the cytomegalovirus
immediate early promoter (CMVIE) and from the bovine growth hormone
polyadenylation sequences (BGHpA) to control transcription. A
leader sequence that is a synthetic homolog of the tissue
plasminogen activator gene leader sequence (tPA) is optional in the
transcription cassette. The vectors of the present invention differ
in the sites that can be used for accepting vaccine inserts and in
whether the transcription cassette includes intron A sequences in
the CMVIE promoter. Both intron A and the tPA leader sequence have
been shown in certain instances to supply a strong expression
advantage to vaccine inserts (Chapman et al., 1991).
[0117] pGAl is a 3894 bp plasmid. pGA1 comprises a promoter (bp
1-690), the CMV-intron A (bp 691-1638), a synthetic mimic of the
tPA leader sequence (bp 1659-1721), the bovine growth hormone
polyadenylation sequence (bp1761-1983), the lambda T0 terminator
(bp 1984-2018), the kanamycin resistance gene (bp 2037-2830) and
the Co1EI replicator (bp 2831-3890). The DNA sequence of the pGA1
construct (SEQ ID NO: 1) is shown in FIG. 2. In FIG. 1, the
indicated restriction sites are single cutters useful for the
cloning of vaccine inserts. The ClaI or BspD1 sites are used when
the 5' end of a vaccine insert is cloned upstream of the tPA
leader. The NheI site is used for cloning a sequence in frame with
the tPA leader sequence. The sites listed between SmaI and B1nI are
used for cloning the 3' terminus of a vaccine insert.
[0118] pGA2 is a 2947 bp plasmid lacking the 947 bp of intron A
sequences found in pGA1. pGA2 is the same as pGA1, except for the
deletion of intron A sequences. pGA2 is valuable for cloning
sequences which do not require an upstream intron for efficient
expression, or for cloning sequences in which an upstream intron
might interfere with the pattern of splicing needed for good
expression. FIG. 3 presents a map of pGA2 with useful restriction
sites for cloning vaccine inserts, and FIG. 4 shows the DNA
sequence SEQ ID NO: 2. The use of restriction sites for cloning
vaccine inserts into pGA2 is the same as that used for cloning
fragments into pGA1.
[0119] pGA3 is a 3893 bp plasmid that contains intron A. pGA3 is
the same as pGA1 except for the cloning sites that can be used for
the introduction of vaccine inserts. In pGA3, inserts cloned
upstream of the tPA leader sequence use a Hind III site. Sequences
cloned downstream from the tPA leader sequence use sites between
the SmaI and the B1nI site. In pGA3, these sites include a BamHI
site. FIG. 5 shows the map for pGA3, and FIG. 6 shows the DNA
sequence SEQ ID NO: 3.
[0120] In view of the teachings herein, one skilled in the art will
recognize that any vaccine insert known in the art can be used in
the novel pGA constructs described herein, including but not
limited to viral pathogens like HIV, influenza, measles, herpes,
Ebola, and the like.
[0121] For example, the present invention contemplates inserts from
immunodeficiency virus, more preferably HIV, including all clades
of HIV-1 and HIV-2 and modifications thereof; influenza virus genes
including all subtypes and modifications thereof; and vaccine
inserts derived from measles genes. One skilled in the art will
appreciate that the discussion about inserts derived from
immunodeficiency virus; influenza virus; measles virus; and
modifications thereof are exemplary in nature and provided for the
sake of illustration only.
[0122] The immunodeficiency virus vaccine inserts of the present
invention were designed to express non-infectious virus like
particles (VLPs) from a single DNA. This was achieved using the
subgenomic splicing elements normally used by immunodeficiency
viruses to express multiple gene products from a single viral RNA.
Important to the subgenomic splicing patterns are (i) splice sites
and acceptors present in full length viral RNA, (ii) the Rev
responsive element (RRE) and (iii) the Rev protein. The splice
sites in retroviral RNAs use the canonical sequences for splice
sites in eukaryotic RNAs. The RRE is an .about.200 bp RNA structure
that interacts with the Rev protein to allow transport of viral
RNAs from the nucleus to the cytoplasm. In the absence of Rev, the
10 kb RNA of immunodeficiency virus undergoes splicing to the mRNAs
for the regulatory genes Tat, Rev, and Nef. These genes are encoded
by exons present between RT and Env and at the 3' end of the
genome. In the presence of Rev, the singly spliced mRNA for Env and
the unspliced mRNA for Gag and Pol are expressed in addition to the
multiply spliced mRNAs for Tat, Rev, and Nef.
[0123] The expression of non-infectious VLPs from a single DNA
affords a number of advantageous features to an immunodeficiency
virus vaccine. The expression of a number of proteins from a single
DNA affords the vaccinated host the opportunity to respond to the
breadth of T- and B-cell epitopes encompassed in these proteins.
The expression of proteins containing multiple epitopes affords the
opportunity for the presentation of epitopes by diverse
histocompatibility types. By using whole proteins, one offers hosts
of different histocompatibility types the opportunity to raise
broad-based T-cell responses. Such may be essential for the
effective containment of immunodeficiency virus infections, whose
high mutation rate supports ready escape from immune responses
(Evans et al., 1999) (Poignard et al., 1999, Evans, et al., 1995).
Just as in drug therapy, multi-epitope T-cell responses that
require multiple mutations for escape will provide better
protection than single epitope T-cell responses that require only a
single mutation for escape.
[0124] Antibody responses are often best primed by multi-valent
vaccines that present an ordered array of an epitope to responding
B-cells (Bachmann, Zinkemagel, 1997). Virus-like particles, by
virtue of the multivalency of Env in the virion membrane, will
facilitate the raising of anti-Env antibody responses. These
particles will also present non-denatured and normal forms of Env
to the immune system.
[0125] The novel vectors of the present invention can be
administered to a patient in the presence of adjuvants or other
substances that have the capability of promoting DNA uptake or
recruiting immune system cells to the site of the inoculation.
Embodiments include combining the DNA vaccine with conventional
adjuvants or genetic adjuvants. Conventional adjuvants, including
reagents that favor the stability and uptake of the DNA, recruit
immune system cells to the site of inoculation, or facilitate the
immune activation of responding lymphoid cells, include but are not
limited to oil and water emulsions (for example, complete Freund's
adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum,
Bacillus Calmette Guerin, aluminum hydroxide, glucan, dextran
sulfate, iron oxide, sodium alginate, Bacto-Adjuvant, certain
synthetic polymers such as poly amino acids and co-polymers of
amino acids, saponin, "REGRESSIN" (Vetrepharm, Athens, Ga.),
"AVRIDINE"
(N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine),
paraffin oil, and muramyl dipeptide. The present invention also
contemplates the use of genetic adjuvants such as immunomodulatory
molecules encoded in a co-inoculated DNA. The co-inoculated DNA can
be in the same vaccine construct as the vaccine immunogen or in a
separate DNA vector.
[0126] A vaccine according to the present invention can be
administered in a variety of ways including through any parenteral
or topical route. For example, an individual can be inoculated by
intravenous, intraperitoneal, intradermal, subcutaneous or
intramuscular methods. Inoculation can be, for example, with a
hypodermic needle, needleless delivery devices such as those that
propel a stream of liquid into the target site, or with the use of
a gene gun that bombards DNA on gold beads into the target site.
The vector comprising the pathogen vaccine insert can be
administered to a mucosal surface by a variety of methods including
intranasal administration, i.e., nose drops or inhalants, or
intrarectal or intravaginal administration by solutions, gels,
foams, or suppositories. Alternatively, the vector comprising the
vaccine insert can be orally administered in the form of a tablet,
capsule, chewable tablet, syrup, emulsion, or the like. In an
alternate embodiment, vectors can be administered transdermally, by
passive skin patches, iontophoretic means, and the like.
[0127] Any appropriate physiologically acceptable medium is
suitable for introducing the vector comprising the pathogen vaccine
insert into the patient. For example, suitable pharmaceutically
acceptable carriers known in the art include, but are not limited
to, sterile water, saline, glucose, dextrose, or buffered
solutions. Carriers may include auxiliary agents including, but not
limited to, diluents, stabilizers (i.e., sugars and amino acids),
preservatives, wetting agents, emulsifying agents, pH buffering
agents, viscosity enhancing additives, colors and the like.
[0128] The present invention is further illustrated by the
following examples, which are provided by way of illustration and
should not be construed as limiting. The contents of all
references, published patents and patents cited throughout the
present application are hereby incorporated by reference in their
entirety.
EXAMPLE 1
Structure and Sequence of pGA1
[0129] pGA1 as illustrated in FIG. 1 and FIG. 2 contains the Co1E1
origin of replication, the kanamycin resistance gene for antibiotic
selection, the lambda T0 terminator, and a eukaryotic expression
cassette including an upstream intron. The Co1E1 origin of
replication is a 600 nucleotide DNA fragment that contains the
origin of replication (ori), encodes an RNA primer, and encodes two
negative regulators of replication initiation. All enzymatic
functions for replication of the plasmid are provided by the
bacterial host. The original constructed plasmid that contained the
Co1E1 replicator was pBR322 (Bolivar, et al. 1977; Sutcliffe, et
al. 1978).
[0130] The kanamycin resistance gene is an antibiotic resistance
gene for plasmid selection in bacteria. The lambda T0 terminator
prevents read through from the kanamycin resistance gene into the
vaccine transcription cassette during prokaryotic growth of the
plasmid (Scholtissek and Grosse, 1987). By preventing read through
into the vaccine expression cassette, the terminator helps
stabilize plasmid inserts during growth in bacteria.
[0131] The eukaryotic expression cassette is comprised of the CMV
immediate early promoter including intron A (CMVIE-IA) and
termination sequences from the bovine growth hormone
polyadenylation sequence (BGHpA). A synthetic mimic of the leader
sequence for tissue plasminogen activator (tPA) is included as an
option within the transcription cassette. Cassettes with these
elements have proven to be highly effective for expressing foreign
genes in eukaryotic cells (Chapman et al., 1991). Cloning sites
within the transcription cassette include a Clal site upstream of
the tPA leader, a NheI site for cloning in frame with the tPA
leader, and XmnI, SmaI, RsrII, AvrII, and B1nI sites for cloning
prior to the BGHpA.
[0132] The Co1E1 replicator, the Kanamycin resistance gene and
transcriptional control elements for eukaryotic cells were combined
in one plasmid using polymerase chain reaction (PCR) fragments from
a commercial vector, pZErO-2 (Invitrogen, Carlsbad, Calif.) and a
eukaryotic expression vector , pJW4303 (Lu et al., 1997). 20 A 1853
bp fragment from pZErO2 from nt 1319 to nt 3178 included the
ColElorigin of replication and the kanamycin resistance gene. A
2040 bp fragment from pJW4303 from nt 376 to nt 2416 included the
CMVIE promoter with intron A, a synthetic homolog of the tissue
plaminogen activator leader (tPA), and the bovine growth hormone
polyadenylation site (BGHpA). Fragments were amplified by
polymerase chain reaction (PCR) with oligonucleotide primers
containing SalI sites. A ligation product with the transcription
cassettes for Kanamycin resistance from pZeRO2 and the eukaryotic
transcription cassette form pJW4303 in opposite transcriptional
orientations was identified for further development. Nucleotide
numbering for this parent for the pGA vectors was started from the
first bp of the 5' end of the CMV promoter.
[0133] The T0 terminator was introduced into this parent for the
pGA vectors by PCR amplification of a 391 bp fragment with a BamH1
restriction endonuclease site at its 5'end and an XbaI restriction
endonuclease site at its 3'end. The initial 355 bp of the fragment
were sequences in the BGHpA sequence derived from the pJW4303
transcription cassette, the next 36 bases in a synthetic
oligonuclotide introduced the T0 sequence and the XbaI site. The
introduced T0 teminator sequences comprised the nucleotide sequence
as follows:
1 5'-ATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAA-3' (SEQ ID NO:)
[0134] The T0 terminator containing BamH1-XbaI fragment was
substituted for the homologous fragment without the T0 terminator
in the plasmid created from pXeRO 2 and pJW4303. The product was
sequenced to verify the TO orientation.
[0135] A region in the eukaryotic transcription cassette between
nucleotides 1755-1845 contained the last 30bp of the reading frame
for SIV nef. This region was removed from pGA by mutating the
sequence at nt1858 and generating an Avr II restriction
endonuclease site. A naturally occurring Avr II site is located at
nt1755. Digestion with Avr II enzyme and then religation with T4
DNA ligase allowed for removal of the SIV segment of DNA between
nucleotides 1755-1845. To facilitate cloning of HIV-1 sequences,
into pGA vectors a ClaI site was introduced at bp1645 and an RsrII
site at bp 1743 using site directed mutagenesis. Constructions were
verified by sequence analyses.
EXAMPLE 2
Structure and Sequence of pGA2
[0136] pGA2, as illustrated in FIG. 3 and FIG. 4, is identical to
pGA1 except for deletion of the intron A sequences from the CMVIE
promoter. pGA2 was created from pGA1 by introducing a ClaI site 8
bp downstream from the mRNA cap site in the CMVIE promoter. The
ClaI site was introduced using oligonucleotide-directed mutagensis
using the complimentary primers
2 5'-CCGTCAGATCGCATCGATACGCCATCCACG-3' (SEQ ID NO:) and
5'-CGTGGATGGCGTATCGATGCGATCTGACGG-3' (SEQ ID NO:).
[0137] After insertion of the new ClaI site, pGA1 was digested with
ClaI to remove the 946 bp ClaI fragment from pGA1, and then
religated to yield pGA2.
EXAMPLE 3
Structure and Sequence of pGA3
[0138] pGA3 as shown in FIG. 5 and FIG. 6 is identical to pGA1
except for the introduction of a HindIII site in stead of the ClaI
site at nt 1645 and a BamHI site instead of the RsrII site at
nucleotide 1743.
EXAMPLE 4
Comparative Expression and Immunogenicity of pGA3 and pJW4303
[0139] To determine the efficacy of the pGA plasmids as vaccine
vectors, a pGA plasmid was compared to the previously described
vaccine vector pJW4303. The pJW4303 plasmid has been used for DNA
vaccinations in mice, rabbits, and rhesus macaques (Robinson et al.
1999; Robinson et al., 1997; Pertmer, et al., 1995; Feltquate, et
al. 1997; Torres, et al. 1999). Comparisons were done with pGA3
with a vaccine insert encoding the normal, plasma-membrane form of
the A/PR/8/34 (H1N1) influenza virus hemagglutinin (pGA3/H1) and
pJW4303 encoding the same fragment (pJW4303/H1). Both pGA3 and
pJW4303 contain intron A upstream of influenza H1 sequences.
[0140] The pGA3/H1 and pJW4303/H1 vaccine plasmids expressed
similar levels of H1 in eukaryotic cells, as summarized below:
3TABLE 5 In Vitro Expression Levels of HA plasmids. Relative HA
Units Plasmids Supernatant Cell Lysate PGA3/H1 0.1 .+-. 0.1 5.7
.+-. 0.6 pGA vector 0.0 .+-. 0.0 0.2 .+-. 0.1 PJW4303/H1 0.3 .+-.
0.05 4.8 .+-. 0.5 pJW4303 0.0 .+-. 0.0 0.1 .+-. 0.1
[0141] Human embryonic kidney 293T cells were transiently
transfected with 2 .mu.g of plasmid and the supernatants and cell
lysates assayed for H1 using an antigen-capture ELISA. The capture
antibody was a polyclonal rabbit serum against H1, and the
detection antibody, polyclonal mouse sera against H1. pGA3/H1
expressed slightly more H1 than pJW4303/H1 (5.8 HA units as opposed
to 5.1 H1 units (Table 6). As expected, 90% of the H1 antigen was
in the cell lysates. A comparative immunization study using
pGA3/H1, and pJW4303/H1 demonstrated comparable or better
immunogenicity for pGA3/H1 than pJW4303/H1 (FIG. 7). Immunogenicity
was assessed in BALB/c mice. In this example, mice were vaccinated
with DNA coated gold particles via gene gun. Mice were primed and
boosted with a low dose (0.1 .mu.g) or a high dose (1 .mu.g) of the
plasmid DNAs. The booster immunization was given at 4 weeks after
the priming immunization. The amount of anti-H1 IgG raised in
response to immunizations was as high or higher following
immunization with pGA3/H1 than following immunization with
pJW4303/H1 (FIG. 7). Thus the pGA vector proved to be as effective,
or more effective, than the pJW4303 vector at raising immune
responses.
EXAMPLE 5
Immunodeficiency Virus Vaccine Inserts in pGA Vectors
[0142] Immunodeficiency virus vaccine inserts expressing virus like
particles have been developed in pGA1 and pGA2. The VLP insert was
designed with dade B HIV-1 sequences so that it would match HIV-1
sequences that are endemic in the United States. Within clade B,
different isolates exhibit clustal diversity, with each isolate
having overall similar diversity from the consensus sequence for
the lade (Subbarao, Schochetman, 1996). Thus, any lade B isolate
can be used as a representative sequence for other lade B isolates.
HIV-1 isolates use different chemokine receptors as co-receptors.
The vast majority of viruses that are undergoing transmission use
the CCR-5 co-receptor (Berger, E. A., 1997). Therefore the vaccine
insert was designed to have a CCR-5 using Env.
[0143] The expression of VLPs with an R5-Env by a HIV-1 DNA vaccine
also has the advantage of supporting Env-mediated entry of
particles into professional antigen presenting cells (APCs) such as
dendritic cells and macrophages. Both dendritic cells and
macrophages express the CD4 receptor and the CCR-5 co-receptor used
by CCR-5-tropic (R5) HIV-1 Envs. By using an R5 Env in the vaccine,
the VLP expressed in a transfected non-professional APC (for
example keratinocyte or muscle cells) can gain entry into the
cytoplasm of an APC by Env-mediated entry. Following entry into the
cytoplasm of the APC, the VLP will be available for processing and
presentation by class I histocompatibility antigens. DNA-based
immunizations rely on professional APCs for antigen presentation
(Corr et al., 1996; Fu, et al., 1997; Iwasaki A, et al., 1997).
Much of DNA-based immunization is accomplished by direct
transfection of professional APC (Condon et al., 1996; Porgador et
al., 1998). Transfected muscle cells or keratinocytes serve as
factories of antigen but do not directly raise immune response
(Torres et al., 1997). By using an expressed antigen that is
assembled and released from transfected keratinocytes or muscle
cells and then actively enters professional APC, the efficiency of
the immunization may be increased.
[0144] Goals in the construction of pGA2/JS2 were (i) to achieve a
CCR-5-using lade B VLP with high expression, (ii) to produce a VLP
that was non infectious and (iii) to minimize the size of the
vaccine plasmid. Following the construction of the CCR-5-using VLP
(pGA2/JS2), a derivative of JS2 was prepared that expresses an
Env-defective VLP. This plasmid insert was designated JS5. Although
it is anticipated that this sequence will be a less effective
vaccine than the Env-containing JS2 VLP, the non-Env containing VLP
offers certain advantages for vaccination. These include the
ability to monitor vaccinated populations for infection by
sero-conversion to Env. Deletion of Env sequences also reduces the
size of the vaccine plasmid. The DNA sequence of pGA2/JS2 is shown
in FIG. 17 and that of pGA1/JS5 in FIG. 18.
[0145] To achieve a VLP plasmid with high expression, candidate
vaccines were constructed from 7 different HIV-1 sequences, as
shown in the following table:
4TABLE I Comparison of candidate vaccine inserts Ability Plasmid
Sequences to grow Expression Expression designation tested plasmid
of Gag of Env Comment BH10-VLP BH10 good good good X4 Env 6A-VLP 6A
env in poor not tested not tested BH10-VLP BAL-VLP BAL env in good
poor poor BH10-VLP ADA-VLP ADA env in good good good chosen for
vaccine, BH10-VLP renamed pGA1/JS1 CDC-A-VLP CDC-A env in good good
poor BH10-VLP CDC-B-VLP CDC-B-env in good good good not as
favorable BH10-VLP expression as ADA CDC-C-VLP CDC -C env good good
good not as favorable in BH10-VLP expression as ADA
[0146] An initial construct, pBH10-VLP, was prepared from IIIb
sequences that are stable in bacteria and have high expression in
eukaryotic cells. The BH10 sequences were obtained from the
NIH-sponsored AIDS Repository (catalog #90). The parental pBH10 was
used as the template for PCR reactions to construct pBH 10-VLP.
[0147] Primers were designed to yield a Gag-Rt PCR product (5' PCR
product) encompassing from 5' to 3' 105 bp of the 5' untranslated
leader sequence and gag and pol sequences from the start codon for
Gag to the end of the RT coding sequence. The oligonucleotide
primers introduced a ClaI site at the 5' end of the PCR product and
EcoRI and NheI sites at the 3' end of the PCR product. Sense primer
1 (5'-GAGCTCTATCGATGCAGGACTCG- GCTTGC-3' (SEQ ID NO:)) and
antisense primer 2 (5'-GGCAGGTTTTAATCGCTAGCCTA- TGCTCTCC-3' (SEQ ID
NO: )) were used to amplify the 5'PCR product.
[0148] The PCR product for the env region of HIV-1 (3'PCR product)
encompassed the vpu, tat, rev, and env sequences and the splice
acceptor sites necessary for proper processing and expression of
their respective mRNAs. An EcoRI site was introduced at the 5' end
of this product and NheI and RsrII sites were introduced into the
3' end. Sense primer 3 (5'-GGGCAGGAGTGCTAGCC-3' (SEQ ID NO:)) and
antisense primer 4 (5'-CCACACTACTTTCGGACCGCTAGCCACCC-3' (SEQ ID
NO:)) were used to amplify the 3'PCR product).
[0149] The 5' PCR product was cloned into pGA1 at the ClaI and NheI
sites and the identity of the construct confirmed by sequencing.
The 3' PCR product was then inserted into the 5' clone at the EcoRI
and NheI sites to yield pBH10-VLP. The construction of this VLP
resulted in proviral sequences that lacked LTRs, integrase, vif,
and vpr sequences (see FIG. 8A).
[0150] Because the BH1O-VLP had an X4 rather than an R5 Env,
sequences encoding six
[0151] different R5 Envs were substituted for env sequences in
BH10-VLP. This was done by cloning EcoRI to BamHI fragments
encompassing tat, rev, vpu and env coding sequences from different
viral genomes into pBH10-VLP. The resulting env and rev sequences
were chimeras for the substituted sequences and BH10 sequences (for
example see FIG. 8B). In the case of the ADA envelope, a BamHI site
was introduced into the ADA sequence to facilitate substituting an
EcoRI to BamHI fragment for the EcoRI to BamHI region of the
BH10-VLP (FIG. 8). The results of these constructions are
summarized in Table 1. Of the six sequences tested, one, the 6A-VLP
was found to be associated with poor plasmid growth in transformed
bacteria. This plasmid was not used for further vaccine development
(Table 1).
[0152] Among the plasmids exhibiting good growth in bacteria, the
best expression of the VLP
[0153] was found for the ADA-VLP (Table 1). In transient
transfections in 293T cells, the expression of the ADA-VLP was
higher than that of wt proviruses for ADA or IIIb (FIG. 9).
Expression was also higher than for a previous VLP-vaccine (dpol)
(Richmond et al., 1998) that had successfully primed cytotoxic
T-cell (Tc) responses in rhesus macaques (Kent et al., 1998).
EXAMPLE 6
Safety Mutations
[0154] Once the ADA-VLP had been identified as a favorable
candidate for further vaccine development, this plasmid was mutated
to increase its safety for use in humans. Further mutations
disabled the Zinc fingers in NC that are active in the
encapsidation of viral RNA, and added point mutations to inactivate
the activity of the viral reverse transcriptase and the viral
protease (FIG. 8). The following table summarizes the location of
the safety point mutations
5TABLE 2 Location of safety point mutations in pGA/JS2 and pGA/JS5
to inhibit viral RNA packaging and abolish reverse transcriptase
activity in vaccine constructs AMINO ACID GENE REGION FUNCTION
CHANGE.sup.1 LOCATION Gag Zn finger Viral RNA packaging C392S
1285/128 Gag Zn finger Viral RNA packaging C392S 1294/129 Gag Zn
finger Viral RNA packaging C413S 1348/135 Gag Zn finger Viral RNA
packaging C416S 1357/135 Pol RT Polymerase activity D185N 2460/246
Pol RT Strand transfer W266T 2703/2704/2 Pol RNAse H RNAse activity
E478Q 3339 .sup.1Amino acid number corresponds to individual genes
in HIV-1 BH10 sequence; .sup.2Nucleotide number in wt HIV-1 BH10
sequence
[0155] The mutations were made using a site directed mutagenesis
kit (Stratagene) following the manufacturer's protocol. All
mutations were confirmed by sequencing. Primer pairs used for the
mutagenesis were:
6 (A) C15S ZN1 5'-GGTTAAGAGCTTCAATAGCGGCAAAGAAGGGC-3' (SEQ ID NO: )
C15S ZN2 5'-GCCCTTCTTTGCCGCTATTGAAGCTCTTAACC-3' (SEQ ID NO: ) (B)
C36S ZN3 5'-GGGCAGCTGGAAAAGCGGAAAGGAAGG-3' (SEQ ID NO: ) C36S ZN4
5'-CCTTCCTTTCCGCTTTTCCAGCTGCCC-3' (SEQ ID NO: ) (C) D185N RT1
5'-CCAGACATAGTTATCTATCAATACATGAACG- ATTTGTATGTAGG-3' (SEQ ID NO: )
D185N RT2 5'-CCTACATACAAATCGTTCATGTATTGATAGATAACTATGTCTGG-3' (SEQ
ID NO: ) (D) W266T RT3 5'-GGGGAAATTGAATACCGCAAGTCAGATTTACCC-3' (SEQ
ID NO: ) W266T RT4 5'-GGGTAAATCTGACTTGCGGTATTCAATTTCCCC-3' (SEQ ID
NO: ) (E) E478Q RT5 5'-CCCTAACTAACACAACAAATCAGAAAACTCA-
GTTACAAGC-3' (SEQ ID NO: ) E478Q RT6
5'-GCTTGTAACTGAGTTTTCTGATTTGTTGTGTTAGTTAGGG-3' (SEQ ID NO: ) (F)
D25A Prt1 5'-GGCAACTAAAGGAAGCTCTATTAGCCACAGGAGC-3' (SEQ ID NO: )
D25A prt2 5'-GCTCCTGTGGCTAATAGAGCTTCCTTTAGTTGCC-3' (SEQ ID NO:
)
[0156] The ADA-VLP with the zinc finger and RT mutations was found
to express Gag and Env more effectively than the VLP plasmid
without the mutations (FIG. 10). The mutation that inactivated the
protease gene markedly reduced VLP expression (FIG. 10) and was not
included in the further development of the vaccine plasmid. The
ADA-VLP without mutations was designated JS1 and the ADA-VLP with
mutations, JS2.
EXAMPLE 7
Construction of the JS5 Vaccine Insert
[0157] The JS5 insert, a plasmid expressing Gag, RT, Tat, and Rev
was constructed from JS2 by deleting a BglII fragment in the ADA
Env (FIG. 8). This deletion removed sequences from nt 4906-5486 of
the pGA2/JS2 sequence and results in a premature stop codon in the
env gene leading to 269 out of the 854 amino acids of Env being
expressed while leaving the tat, rev, and vpu coding regions the
RRE and splice acceptor sites intact. The DNA sequence of pGA1/JS5
is shown in FIG. 18.
EXAMPLE 8
Minimizing the Size of the JS2 and JS5 Vaccine Plasmids
[0158] The JS2 and JS5 vaccine inserts were originally constructed
in pGA1, a vector that contains the .about.1 kb intron A of the
CMVIE promoter upstream of the vaccine insert. To determine whether
this intron was necessary for high levels of vaccine expression,
pGA2 vectors lacking intron A were constructed expressing the JS2
and JS5 vaccine inserts. In expression tests, pGA2 proved to have
as good an expression pattern as pGA1 for JS2 (FIG. 11). In
contrast to this result, JS5 was expressed much more effectively by
pGA1 than pGA2 (FIG. 11). For the JS5 insert, the absence of intron
A resulted in 2-3-fold lower levels of expression than in the
presence of intron A.
EXAMPLE 9
Testing for the Efficacy of the Safety Mutations in the Vaccine
Inserts JS2 and JS5
[0159] The three point mutations in RT (Table 2) completely
abolished detectable levels of reverse transcriptase activity for
JS2 and JS5. A highly sensitive reverse transcriptase assay was
used in which the product of reverse transcription was amplified by
PCR (Yamamoto, Folks, Heneine, 1996). This assay can detect reverse
trasncriptase in as few as 10 viral particles. Reverse
transcriptase assays were conducted on the culture supernatants of
transiently transfected cells. Reverse transcriptase activity was
readily detected for as few as 10 particles (4.times.10.sup.-3 pg
of p24) in the JS1 vaccine but could not be detected for the JS2 or
JS5 inserts.
[0160] The deletions and zinc finger mutations in the JS2 and JS5
vaccine inserts (Table 2) reduced the levels of viral RNA in
particles by at least 1000-fold. Particles pelleted from the
supernatants of transiently transfected cells were tested for the
efficiency of the packaging of viral RNA. The VLPs were treated
with DNase, RNA was extracted and the amount of RNA standardized by
p24 levels before RT PCR. The RT PCR reaction was followed by
nested PCR using primers specific for viral sequences. End point
dilution of the VLP RNA was compared to the signal obtained from
RNA packaged in wt HIV-1 Ba1 virus.
[0161] Packaging for both JS2 and JS5 was restricted by the
deletions in the plasmid by 500-1000-fold, as summarized below:
7TABLE 3 Packaging of viral RNA is reduced in pGA2/JS2 and pGA1/JS5
VLPs Vaccine Copies vRNA relative Construct Deletions/Mutations to
wt HIV-1 b HIV-1 bal Wt 1 pGA1/JS1 VLP Deleted: LTRs, int, vif,
.002 vpr, nef pGA1/JS2 VLP Deleted: LTRs, int, vif, .0001 vpr, nef,
Mutations in Zn fingers and RT pGA1/JS4 VLP Deleted: LTRs, int,
vif, .001 vpr, nef pGA1/JS5 VLP Deleted: LTRs, int, vif, .001 vpr,
nef, env; Mutations in Zn fingers and RT
[0162] The zinc finger mutations decreased the efficiency of
packaging for the JS2 particles a further 20-fold but did not
further affect the efficiency of packaging for the JS5 particles.
This pattern of packaging was reproducible for particles produced
in independent transfections.
EXAMPLE 10
Western Blot Analyses of Protein Expression
[0163] Western blot analyses, shown in FIGS. 12A-D, revealed the
expected patterns of expression of pGA2/JS2 and pGA1/JS5. Both
immature and mature proteins were observed in cell lysates, whereas
only the mature forms of Gag and Env were found in the
VLP-containing lysates (FIGS. 12B and 12C). Reverse transcriptase
was readily detected in cell lysates (FIG. 12D).
EXAMPLE 11
pGA2/89.6 SHIV Vector Construction
[0164] Initial immunogenicity trials have been conducted with a
SHIV-expressing VLP rather than the HIV-1-expressing vaccine
plasmids. SHIVs are hybrids of simian and human immunodeficiency
virus sequences that grow well in macaques (Li et al., 1992). By
using a SHIV, vaccines that are at least partially of HIV-1 origin
can be tested for efficacy in macaque models.
[0165] pGA2/89.6 (also designated as pGA2/M2) expresses sequences
from SHIV-89.6 (Reimann, Li, Voss, et al., 1996; Reimann, Li,
Veazey, et al., 1996). The 89.6 Env represents a patient isolate
(Collman et al., 1992). The SHIV-89.6 virus is available as a
highly pathogenic challenge stock, designated SHIV-89.6P (Reimann,
Li, Voss, et al., 1996; Reimann, Li, Veazey, et al., 1996), which
allows a rapid determination of vaccine efficacy. The SHIV-89.6P
challenge can be administered via both intrarectal and intravenous
routes. SHIV-89.6 and SHIV-89.6P do not generate cross-neutralizing
antibody.
[0166] pGA2/89.6 (FIG. 13) has many of the design features of
pGA2/JS2. Both express immunodeficiency virus VLPs: HIV-1 VLP in
the case of pGA2/JS2, while the VLP expressed by pGA2/89.6 is a
SHIV VLP. The gag-pol sequences in pGA2/89.6 are from SIV239, while
the tat, rev, and env sequences are from HIV-1-89.6. pGA2/89.6 also
differs from pGA2/JS2 in that the integrase, vif and vpr sequences
have not been deleted, nor has the reverse transcriptase gene been
inactivated by point mutations. Finally, the zinc fingers in NC
have been inactivated by a deletion and not by point mutations.
[0167] pGA1/Gag-Pol was also constructed to allow evaluation of the
protective efficacy of a Gag-Pol expressing vector with the
Gag-Pol-Env expresssing pGA2/89.6. This vector was constructed from
pGA1/JS5 and pGA2/89.6 (FIG. 13).
EXAMPLE 12
Comparison of the Expression of pGA2/89.6 SHIV Plasmid with
pGA2/JS2 Expression
[0168] Both pGA2/89.6 and pGA1/Gag-Pol expressed similar levels of
Gag as pGA2/JS2. Comparative studies for expression were performed
on transiently transfected 293T cells. Analyses of the lysates and
supernatants of transiently transfected cells revealed that both
plasmids expressed similar levels of capsid antigen (FIG. 14). The
capsid proteins were quantified using commercial antigen capture
ELISA kits for HIV-1 p24 and SIV p27.
EXAMPLE 13
pGA2/89.6 SHIV Vaccine Protocol
[0169] A rhesus macaque model was used to investigate the ability
of systemic DNA priming followed by a recombinant MVA (rMVA)
booster to protect against a mucosal challenge with the SHIV-89.6P
challenge strain (Amara et al, 2001).
[0170] The DNA component of the vaccine (pGA2/89.6) was made as
described in Example 11 and and expressed eight immunodeficiency
virus proteins (SIV Gag, Pol, Vif, Vpx, and Vpr and HIV Env, Tat,
and Rev) from a single transcript using the subgenomic splicing
mechanisms of immunodeficiency viruses. The rMVA booster (89.6-MVA)
was provided by Dr. Bernard Moss (NIH) and expresses both the HIV
89.6 Env and the SIV 239 Gag-Pol, inserted into deletion II and
deletion III of MVA respectively, under the control of vaccinia
virus early/late promoters (Wyatt and Moss, unpublished results).
The 89.6 Env protein was truncated for the C-terminal 115 amino
acids of gp41. The modified H5 promoter controlled the expression
of both foreign genes.
[0171] The vaccination trial compared i.d. and i.m. administration
of the DNA vaccine and the ability of a genetic adjuvant, a plasmid
expressing macaque GM-CSF, to enhance the immune response raised by
the vaccine inserts. Vaccination was accomplished by priming with
DNA at 0 and 8 weeks and boosting with rMVA at 24 weeks. For
co-delivery of a plasmid expressing GM-CSF, 1-100 .mu.l i.d.
inoculation was given with a solution containing 2.5 mg of
pGA2/89.6 and 2.5 mg per ml of pGM-CSF.
[0172] I.d. and i.m. deliveries of DNA were compared for two doses,
2.5 mg and 250 .mu.g of DNA. Four vaccine groups of six rhesus
macaques were primed with either 2.5 mg (high-dose) or 250 .mu.g
(low-dose) of DNA by intradermal (i.d.) or intramuscular (i.m.)
routes using a needleless jet injection device (Bioject, Portland
Oreg.). The 89.6-MVA booster immunization (2.times.10.sup.8 pfu)
was injected with a needle both i.d. and i.m. A control group
included two mock immunized animals and two naive animals. The
vaccination protocol is summarized as follows:
8TABLE 4 Vaccination Trial Group, Prime at Boost at (# macaque) 0
and 8 weeks Immunogen 24 weeks Immunogen 1 (6) i.d. bioject 2.5 mg
VLP DNA i.d. + i.m. MVA gag-pol-env 2 (6) i.m. bioject 2.5 mg VLP
DNA i.d. + i.m. MVA gag-pol-env 3 (6) i.d bioject 250 ug VLP DNA
i.d. + i.m MVA gag-pol-env 4 (6) i.m. bioject 250 ug VLP DNA i.d. +
i.m. MVA gag-pol-env 5 (6) i.d. bioject 2.5 mg gag-pol DNA i.d. +
i.m. MVA gag-pol 6 (6) i.d. bioject 250 ug gag-pol DNA i.d. + i.m.
MVA gag-pol 7 (6) i.d bioject 250 ug VLP DNA + 250 i.d. + i.m. MVA
gag-pol-env ug GM-CSF DNA 8 (5) i.d. bioject 2.5 mg control DNA
i.d. + i.m. control MVA i.d. + i.m. control control MVA MVA 9 (4)
i.d., bioject 250 ug control DNA + i.d. + i.m. MVA gag-pol-env 250
ug GM-CSF DNA 10 (6) i.d. + i.m. MVA gag-pol-env i.d. + i.m. MVA
gag-pol-env VLP DNA expresses all SHIV-89.6 proteins except Nef,
truncated for LTRs, 2.sup.nd ZN++ finger, mutated to express cell
surface Env; gag-pol DNA expresses SIV mac 239 gag-pol; MVA
gag-pol-env expresses 89.6 truncated env and SIV mac 239 gag-pol;
MVA gag-pol expresses SIVmac239 gag-pol; MVA dose is 1 .times.
10.sup.8 pfu
[0173] Animals were challenged seven months after the rMVA booster
to test whether the vaccine had generated long-term immunity.
Because most HIV-1 infections are transmitted across mucosal
surfaces, an intrarectal challenge was administered to test whether
the vaccine could control a mucosal immunodeficiency virus
challenge. Briefly, the challenge stock (5.7.times.10.sup.9 copies
of viral RNA per ml) was produced by one i.v. followed by one
intrarectal passage in rhesus macaques of the original SHIV-89.6P
stock. Lymphoid cells were harvested from the intrarectally
infected animal at peak viremia, CD8-depleted and
mitogen-stimulated for stock production. Prior to intrarectal
challenge, fasted animals were anesthetized (ketamine, 10 mg/kg)
and placed on their stomach with the pelvic region slightly
elevated. A feeding tube [8 Fr (2.7 mm).times.16 inches (41 cm),
Sherwood Medical, St. Louis, Mo.] was inserted into the rectum for
a distance of 15-20 cm. Following insertion of the feeding tube, a
syringe containing 20 intrarectal infectious doses in two ml of
RPMI-1640 plus 10% fetal bovine serum (FBS) was attached to the
tube and the inoculum slowly injected into the rectum. Following
delivery of the inoculum, the feeding tube was flushed with 3.0 ml
of RPMI without fetal calf serum and then slowly withdrawn. Animals
were left in place, with pelvic regions slightly elevated, for a
period of ten minutes following the challenge.
EXAMPLE 14
Vaccine-raised T-cell Responses
[0174] DNA priming followed by rMVA boosting generated high
frequencies of virus-specific T cells that peaked at one week
following the rMVA booster, as shown in FIG. 15. The frequencies of
T cells recognizing the Gag-CM9 epitope were assessed using
Mamu-A*01-tetramers; and the frequencies of T cells recognizing
epitopes throughout Gag and Env, using pools of overlapping Gag and
Env peptides and an enzyme linked immunospot (ELISPOT) assay.
[0175] For tetramer analyses, approximately 1.times.10.sup.6 PBMC
were surface stained with antibodies to CD3 (FN-18, Biosource
International, Camarillo, Calif.), CD8 (SKi, Becton Dickinson, San
Jose, Calif.), and Gag-CM9 (CTPYDINQM)-Mamu-A*01 tetramer
conjugated to FITC, PerCP and APC respectively, in a volume of 100
.mu.l at 8-10.degree. C. for 30 min. Cells were washed twice with
cold PBS containing 2% FBS, fixed with 1% paraformaldehyde in PBS
and analyses acquired within 24 hrs. on a FACScaliber (Becton
Dickinson, San Jose, Calif.). Cells were initially gated on
lymphocyte populations using forward scatter and side scatter and
then on CD3 cells. The CD3 cells were then analyzed for CD8 and
tetramer-binding cells. Approximately 150,000 lymphocytes were
acquired for each sample. Data were analyzed using FloJo software
(Tree Star, Inc. San Carlos, Calif.).
[0176] For IFN-.gamma. ELISPOTs, MULTISCREEN 96 well filtration
plates (Millipore Inc. Bedford, Mass.) were coated overnight with
anti-human IFN-.gamma. antibody (Clone B27, Pharmingen, San Diego,
Calif.) at a concentration of 2 .mu.g/ml in sodium bicarbonate
buffer (pH 9.6) at 8-10.degree. C. Plates were washed two times
with RPMI medium then blocked for one hour with complete medium
(RPMI containing 10% FBS) at 37.degree. C. Plates were washed five
more times with plain RPMI medium and cells were seeded in
duplicate in 100 .mu.l complete medium at numbers ranging from
2.times.10.sup.4 to 5.times.10.sup.5 cells per well. Peptide pools
were added to each well to a final concentration of 2 .mu.g/ml of
each peptide in a volume of 100 .mu.l in complete medium. Cells
were cultured at 37.degree. C. for about 36 hrs under 5% CO.sub.2.
Plates were washed six times with wash buffer (PBS with 0.05%
Tween-20) and then incubated with 1 .mu.g of biotinylated
anti-human IFN-y antibody per ml (clone 7-86-1, Diapharma Group
Inc., West Chester, Ohio) diluted in wash buffer containing 2% FBS.
Plates were incubated for 2 hrs at 37.degree. C. and washed six
times with wash buffer. Avidin-HRP (Vector Laboratories Inc,
Burlingame, Calif.) was added to each well and incubated for 30-60
min at 37.degree. C. Plates were washed six times with wash buffer
and spots were developed using stable DAB as substrate (Research
Genetics Inc., Huntsville, Ala.). Spots were counted using a stereo
dissecting microscope. An ovalbumin peptide (SIINFEKL) was included
as a control in each analysis. Background spots for the ovalbumin
peptide were generally <5 for 5.times.10.sup.5 PBMC s. This
background when normalized for 1.times.10.sup.6 PBMC is <10.
Only ELISPOT counts of twice the background (>20) were
considered significant. The frequencies of ELISPOTs are approximate
because different dilutions of cells have different efficiencies of
spot formation in the absence of feeder cells (34). The same
dilution of cells was used for all animals at a given time point,
but different dilutions were used to detect memory and peak
effector responses.
[0177] Simple linear regression was used to estimate correlations
between post-booster and post-challenge ELISPOT responses, between
memory and post-challenge ELISPOT responses, and between log viral
loads and ELISPOT frequencies in vaccinated groups. Comparisons
between vaccine and control groups were performed by means of
2-sample t-tests using log viral load and log ELISPOT responses.
Comparisons of ELISPOTs or log viral loads between A*01 and non
A*01 macaques were done using 2-sample t-tests. Two-way analyses of
variance were used to examine the effects of dose and route of
administration on peak DNA/MVA ELISPOTs, memory DNA/MVA ELISPOTs,
and on logarithmically transformed Gag antibody data.
[0178] Gag-CM9 tetramer analyses were restricted to macaques that
expressed the Mamu-A*01 histocompatibility type, whereas ELISPOT
responses did not depend on a specific histocompatibility type.
Temporal T cell assays were designed to score both the acute (peak
of effector cells) and long-term (memory) phases of the T cell
response (FIG. 15A). As expected, the DNA immunizations raised low
levels of memory cells that expanded to high frequencies within one
week of the rMVA booster (FIG. 15). In Mamu-A*01 macaques, cells
specific to the Gag-CM9 epitope expanded to frequencies as high as
19% of total CD8 T cells (see animal 2 FIG. 15B). This peak of
specific cells underwent a >10-fold contraction into the DNA/MVA
memory pool (FIGS. 15A and B). ELISPOTs for three pools of Gag
peptides also underwent a major expansion (frequencies up to 4000
spots for 1.times.10.sup.6 PBMC) before contracting into the
DNA/MVA memory response (FIG. 15C). The frequencies of ELISPOTs
were the same in macaques with and without the A*01
histocompatibility type (P>0.2.). At both peak and memory phases
of the vaccine response, the rank order for the height of the
ELISPOTs in the different vaccine groups was 2.5 mg i.d >2.5 mg
i.m. >250 .mu.g i.d. >250 .mu.g i.m. (FIG. 15C). The
IFN-.gamma.-ELISPOTs included both CD4 and CD8 cells (work in
progress). Gag-CM9-specific CD8 cells had good lytic activity
following restimulation with peptide (data not shown).
[0179] Impressively, in the outbred population of animals, pools of
peptides throughout Gag and Env stimulated IFN-.gamma.-ELISPOTs
(FIG. 16A). The breadth of the cellular response was tested at 25
weeks after the rMVA boost, a time when vaccine-raised T cells were
in memory. Seven out of 7 pools of Gag peptides and 16 out of 21
pools of Env peptides were recognized by T cells in vaccinated
animals. Of the five Env pools that were not recognized, two have
been recognized in a macaque DNA/MVA vaccine trial at the U.S.
Centers for Disease Control (data not shown). The remaining three
(pools 19-21) had been truncated in our immunogens (Amara et al,
2001, submitted) and served as negative controls. Gag and Env
ELISPOTs had overall similar frequencies in the DNA/MVA memory
response (FIG. 16B). The greatest breadth of response was in
high-dose i.d. DNA-primed animals where on average 10 peptide pools
(4.5 Gag and 5.3 Env) were recognized. The rank order of the
vaccine groups for breadth was the same as for the peak DNA/MVA
response: 2.5 mg i.d. >2.5 mg i.m. >250 .mu.g i.d. >250
.mu.g i.m. (FIG. 16B).
EXAMPLE 15
Challenge and Protection Against AIDS
[0180] The highly pathogenic SHIV-89.6P challenge was administered
intrarectally at 7 months after the rMVA booster, when
vaccine-raised T cells were in memory (FIG. 15).
[0181] Determination of SHIV Copy Number:
[0182] Viral RNA from 150 .mu.l of ACD anticoagulated plasma was
directly extracted with the QIAamp Viral RNA kit (Qiagen), eluted
in 60 .mu.l AVE buffer, and frozen at -80.degree. C. until SHIV RNA
quantitation was performed. 5 .mu.l of purified plasma RNA was
reverse transcribed in a final 20 .mu.l volume containing 50 mM
KCI, 10 mM Tris-HCl, pH 8.3, 4 mM MgCl.sub.2, 1 mM each dNTP, 2.5
.mu.M random hexamers, 20 units MultiScribe RT, and 8 units RNase
inhibitor. Reactions were incubated at 25.degree. C. for 10 min.,
followed by incubation at 42.degree. C. for 20 min. and
inactivation of reverse transcriptase at 99.degree. C. for 5 min.
The reaction mix was adjusted to a final volume of 50 .mu.l
containing 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 4 mM MgCl.sub.2, 0.4
mM each dNTP, 0.2 .mu.M forward primer, 0.2 .mu.M reverse primer,
0.1 .mu.M probe and 5 units AmpliTaq Gold DNA polymerase (all
reagents from Perkin Elmer Applied Biosystems, Foster City,
Calif.). The primer sequences within a conserved portion of the SIV
gag gene are the same as those described previously (Staprans, S.,
et al., 1996).
[0183] A Perkin Elmer Applied Biosystems 7700 Sequence Detection
System was used with the PCR profile: 95.degree. C. for 10 min.,
followed by 40 cycles at 93.degree. C. for 30 sec., 59.5.degree. C.
for 1 min. PCR product accumulation was monitored using the 7700
sequence detector and a probe to an internal conserved gag gene
sequence, where FAM and Tamra denote the reporter and quencher
dyes. SHIV RNA copy number was determined by comparison to an
external standard curve consisting of virion-derived SIVmac239 RNA
quantified by the SIV bDNA method (Bayer Diagnostics, Emeryville,
Calif.). All specimens were extracted and amplified in duplicate,
with the mean result reported. With a 0.15-ml plasma input, the
assay has a sensitivity of 10.sup.3 copies RNA/ml plasma, and a
linear dynamic range of 10.sup.3 to 10.sup.8 RNA copies
(R.sup.2=0.995). The intra-assay coefficient of variation is
<20% for samples containing >10.sup.4 SHIV RNA copies/ml, and
<25% for samples containing 10.sup.3-10.sup.4 SHIV RNA
copies/ml. In order to more accurately quantitate low SHIV RNA copy
number in vaccinated animals at weeks 16 and 20, the following
modifications to increase the sensitivity of the SHIV RNA assay
were made: 1) Virions from .ltoreq.1 ml of plasma were concentrated
by centrifugation at 23,000 g, 10.degree. C. for 150 minutes and
viral RNA was extracted; 2) A one-step RT-PCR method was used.
Absolute SHIV RNA copy numbers were determined by comparison to the
same SIVmac239 standards. These changes provided a reliable
quantitation limit of 300 SHIV RNA copies/ml, and gave SHIV RNA
values that were highly correlated to those obtained by the first
method used (r 0.91, p<0.0001).
[0184] Challenge Results:
[0185] The challenge infected all of the vaccinated and control
animals. However, by two weeks post-challenge, titers of plasma
viral RNA were at least 10-fold lower in the vaccine groups
(geometric means of 1.times.10.sup.7 to 5.times.10.sup.7) than in
the control animals (geometric mean of 4.times.10.sup.8) (FIG.
19A). By 8 weeks post-challenge, both high-dose DNA-primed groups
and the low-dose i.d. DNA-primed group had reduced their geometric
mean loads to about 1000 copies of viral RNA per ml. At this time
the low-dose i.m. DNA-primed group had a geometric mean of
6.times.10.sup.3 copies of viral RNA and the non-vaccinated
controls, a geometric mean of 2.times.10.sup.6. By 20 weeks
post-challenge, even the low-dose i.m. group had reduced its
geometric mean copies of viral RNA to 1000. At this time, the
unvaccinated controls were succumbing to AIDS. Among the 24
vaccinated animals, only one animal, in the low dose i.m. group,
had intermittent viral loads above 1.times.10.sup.4 copies per ml
(FIG. 19D).
[0186] The rapid reduction of viral loads protected the vaccinated
macaques against the loss of CD4 cells and the rapid onset of AIDS
(FIGS. 19B, 19C, 19E). By 5 weeks post-challenge, all of the
non-vaccinated controls had undergone the profound depletion of CD4
cells that is characteristic of SHIV-89.6P infections (FIG. 19B).
All of the vaccinated animals maintained their CD4 cells with the
exception of animal 22 (see above), which underwent a slow CD4
decline (FIG. 19E). By 23 weeks post-challenge, three of the four
control animals had succumbed to AIDS (FIG. 19C). These animals had
variable degrees of enterocolitis with diarrhea, cryptosporidiosis,
colicystitis, enteric campylobacter infection, spenomegaly,
lymphadenopathy, and SIV-associated giant cell pneumonia. In
contrast, all 24 vaccinated animals have maintained their
health.
[0187] Intracellular Cytokine Assays:
[0188] Approximately 1.times.10.sup.6 PBMC were stimulated for one
hour at 37.degree. C. in 5 ml polypropylene tubes with 100 .mu.g of
Gag-CM9 peptide (CTPYDINQM) per ml in a volume of 100 .mu.l RPMI
containing 0.1% BSA and anti-human CD28 and anti-human CD49d
(Pharmingen, Inc. San Diego, Calif.) costimulatory antibodies (1
.mu.g/ml). 900 .mu.l RPMI containing 10% FBS and monensin (10
.mu.g/ml) was added and the cells cultured for an additional 5 hrs
at 37.degree. C. at an angle of 5 degrees under 5% CO.sub.2. Cells
were surface stained with antibodies to CD8 conjugated to PerCP
(clone SK1, Becton Dickinson) at 8.degree.-10.degree. C. for 30
min., washed twice with cold PBS containing 2% FBS, fixed and
permeabilized with Cytofix/Cytopern solution (Phanningen, Inc.).
Cells were then incubated with antibodies to human CD3 (clone
FN-18, Biosource International, Camarillo, Calif.) and IFN-.gamma.
(Clone B27, Pharmingen) conjugated to FITC and PE, respectively, in
Perm wash solution (Pharmingen) for 30 min at 4.degree. C. Cells
were washed twice with Perm wash once with plain PBS, resuspended
in 1% para-formaldehyde in PBS. Approximately 150,000 lymphocytes
were acquired on the FACScaliber and analyzed using FloJo
software.
[0189] Proliferation Assay:
[0190] Approximately 0.2 million PBMC were stimulated with
appropriate antigen in triplicate in a volume of 200 .mu.1 for five
days in RPMI containing 10% FCS at 37.degree. C. under 5% CO.sub.2.
Supernatants from 293T cells transfected with the DNA expressing
either SHIV-89.6 Gag and Pol or SHIV-89.6 Gag, Pol and Env were
used directly as antigens. Supernatants from mock DNA (vector
alone) transfected cells served as negative controls. On day six
cells were pulsed with 1 .mu.Ci of tritiated-thymidine per well for
16-20 hrs. Cells were harvested using an automated cell harvester
(TOMTEC, Harvester 96, Model 1010, Hamden, Conn.) and counted using
a Wallac 1450 MICROBETA Scintillation counter (Gaithersburg, Md.).
Stimulation indices are the counts of tritiated-thymidine
incorporated in PBMC stimulated with 89.6 antigens divided by the
counts of tritiated-thymidine incorporated by the same PBMC
stimulated with mock antigen.
[0191] Post-challenge T Cell Results:
[0192] Containment of the viral challenge was associated with a
burst of antiviral T cells (FIG. 15; FIG. 20A). At one-week post
challenge, the frequency of tetramer+ cells in the peripheral blood
had decreased, potentially reflecting the recruitment of specific T
cells to the site of infection (FIG. 20A). However, by two weeks
post-challenge, tetramer+ cells in the peripheral blood had
expanded rapidly, to frequencies as high, or higher, than after the
MVA booster (FIGS. 15, 20A). The majority of the tetramer+ cells
produced IFN-y in response to a 6-hour stimulation with peptide
Gag-CM9 (FIG. 20B) and did not have the "stunned" IFN-.gamma.
negative phenotype sometimes observed in chronic viral infections.
The post-challenge burst of T cells contracted concomitant with the
decline of the viral load. By 12 weeks post-challenge,
virus-specific T cells were present at approximately one tenth of
their peak height (FIGS. 15A, 20A, and data not shown). The height
of the peak DNA/MVA-induced ELISPOTs presaged the height of the
post-challenge T cell response as measured by ELISPOTs (r=+0.79,
P<0.0001). In contrast to the vigorous secondary response in the
vaccinated animals, the naive animals mounted a modest primary
response (FIGS. 15B, 15C and 20A). Tetramer+ cells peaked at less
than 1% of total CD8 cells (FIG. 20A), and IFN-y-producing T cells
were present at a mean frequency of about 300 as opposed to the
much higher frequencies of 1000 to 6000 in the vaccine groups (FIG.
15C) (P<0.05). The tetramer+ cells in the control group, like
those in the vaccine group, were largely IFN-.gamma. producing
following stimulation with the Gag-CM9 peptide (FIG. 20B). By 12
weeks post challenge, 3 of the 4 controls had undetectable levels
of IFN-.gamma.-producing T cells (data not shown). This rapid loss
of anti-viral CD8 cells in the presence of high viral loads may
reflect the lack of CD4 help.
[0193] T cell proliferative responses demonstrated that
virus-specific CD4 cells had survived the challenge and were
available to support the antiviral immune response (FIG. 20C). At
12 weeks post-challenge, mean stimulation indices for Gag-Pol-Env
or Gag-Pol proteins ranged from 35 to 14 in the vaccine groups but
were undetectable in the control group. Consistent with the
proliferation assays, intracellular cytokine assays demonstrated
the presence of virus-specific CD4 cells in vaccinated but not
control animals (data not shown). The overall rank order of the
vaccine groups for the magnitude of the proliferative response was
2.5 mg i.d. >2.5 mg i.m. >250 .mu.g i.d. >250 .mu.g
i.m.
[0194] Preservation of Lymph Nodes:
[0195] At 12 weeks post-challenge, lymph nodes from the vaccinated
animals were morphologically intact and responding to the infection
whereas those from the infected controls had been functionally
destroyed (FIG. 5). Nodes from vaccinated animals contained large
numbers of reactive secondary follicles with expanded germinal
centers and discrete dark and light zones (FIG. 5A). By contrast,
lymph nodes from the non-vaccinated control animals showed
follicular and paracortical depletion (FIG. 5B), while those from
unvaccinated and unchallenged animals displayed normal numbers of
minimally reactive germinal centers (FIG. 5C). Germinal centers
occupied <0.05% of total lymph node area in the infected
controls, 2% of the lymph node area in the uninfected controls, and
up to 18% of the lymph node area in the vaccinated groups (FIG.
5D). The lymph node area occupied by germinal centers was about two
times greater for animals receiving low-dose DNA priming than for
those receiving high-dose DNA priming, suggesting more vigorous
immune reactivity in the low-dose animals (FIG. 5D). At 12 weeks
post-challenge, in situ hybridization for viral RNA revealed rare
virus-expressing cells in lymph nodes from 3 of the 24 vaccinated
macaques, whereas virus-expressing cells were readily detected in
lymph nodes from each of the infected control animals (FIG. 5E). In
the controls, which had undergone a profound depletion in CD4 T
cells, the cytomorphology of infected lymph node cells was
consistent with a macrophage phenotype (data not shown).
[0196] Temporal Antibody Response:
[0197] ELISAs for total anti-Gag antibody used bacterial produced
SIV gag p27 to coat wells (2 .mu.g per ml in bicarbonate buffer).
ELISAs for anti-Env antibody used 89.6 Env produced in transiently
transfected 293T cells captured with sheep antibody against Env
(catalog number 6205; International Enzymes, Fairbrook Calif.).
Standard curves for Gag and Env ELISAs were produced using serum
from a SHIV-89.6-infected macaque with known amounts of anti-Gag or
anti-Env IgG. Bound antibody was detected using goat anti-macaque
IgG-PO (catalog # YNGMOIGGFCP, Accurate Chemical, Westbury, N.Y.)
and TMB substrate (Catalog # T3405, Sigma, St. Louis, Mo.). Sera
were assayed at 3-fold dilutions in duplicate wells. Dilutions of
test sera were performed in whey buffer (4% whey and 0.1% tween 20
in 1.times. PBS). Blocking buffer consisted of whey buffer plus
0.5% non-fat dry milk. Reactions were stopped with 2M
H.sub.2SO.sub.4 and the optical density read at 450 nm. Standard
curves were fitted and sample concentrations were interpolated as
.mu.g of antibody per ml of serum using SOFTmax 2.3 software
(Molecular Devices, Sunnyvale, Calif.).
[0198] Results showed that the prime/boost strategy raised low
levels of anti-Gag antibody and undetectable levels of anti-Env
antibody (FIG. 22). However, post-challenge, antibodies to both Env
and Gag underwent anamnestic responses with total Gag antibody
reaching heights approaching one mg per ml and total Env antibody
reaching heights of up to 100 .mu.g per ml (FIGS. 22A and B).
[0199] By two weeks post-challenge, neutralizing antibodies for the
89.6 immunogen, but not the SHIV-89.6P challenge were present in
the high-dose DNA-primed groups (geometric mean titers of 352 in
the i.d. and 303 in the i.m. groups) (FIG. 22C). By 5 weeks
post-challenge, neutralizing antibody to 89.6P had been generated
(geometric mean titers of 200 in the high-dose i.d. and 126 in the
high-dose i.m. group) (FIG. 22D) and neutralizing antibody to 89.6
had started to decline. Thus, priming of an antibody response to
89.6 did not prevent a B cell response leading to neutralizing
antibody for SHIV-89.6P. By 16 to 20 weeks post-challenge,
antibodies to Gag and Env had fallen in most animals (FIGS. 22A and
B). This would be consistent with the control of the virus
infection.
[0200] T Cells Correlate with Protection.
[0201] The levels of plasma viral RNA at both two and three weeks
post-challenge correlated inversely with the peak pre-challenge
frequencies of DNA/MVA-raised IFN-.gamma. ELISPOTs (r=-0.53,
P=0.008 and r=-0.70, P=0.0002 respectively) (FIG. 23A).
[0202] Importantly, these correlations were observed during the
time the immune response was actively reducing the levels of
viremia. At later times post-challenge, the clustering of viral
loads at or below the level of detection precluded correlations.
Correlations also were sought between viral load and post-challenge
ELISPOT, proliferative, and neutralizing antibody responses. The
levels of IFN-.gamma. ELISPOTS at two weeks post-challenge
correlated with the viral load at 3 weeks post-challenge (r=-0.51,
P=0.009) (data not shown). Post-challenge proliferative and
neutralizing antibody responses did not correlate with viral
loads.
[0203] Dose and Route:
[0204] The dose of DNA had significant effects on both cellular and
humoral responses (P<0.05) while the route of DNA administration
had a significant effect only on humoral responses (FIGS. 23C-E).
The intradermal route of DNA delivery was about 10 times more
effective than the intramuscular route for generating antibody to
Gag (P=0.02) (FIG. 23E). Within our data set, i.d. DNA injections
were about 3 times more effective at priming the height and breadth
of virus-specific T cells (FIGS. 23C and D). However, these
differences were not significant (height, P=0.2; breadth, P=0.08).
Interestingly, the route and dose of DNA had no significant effect
on the level of protection. At 20 weeks post-challenge, the
high-dose DNA-primed animals had slightly lower geometric mean
levels of viral RNA (7.times.10.sup.2 and 5.times.10.sup.2) than
the low-dose DNA-primed animals (9.times.10.sup.2 and
1.times.10.sup.3). The animal with the highest intermittent viral
loads (macaque 22) was in the low dose i.m.-primed group (FIG.
19D). Thus, the low dose i.m.-primed group, which was slow to
control viremia (FIG. 19A), may have poorer long term protection.
The breadth of the response did not have an immediate effect on the
containment of viral loads, but with time may affect the frequency
of viral escape.
[0205] These results clearly demonstrate that a multiprotein
DNA/MVA vaccine can raise a memory immune response capable of
controlling a highly virulent mucosal immunodeficiency virus
challenge. Our excellent levels of viral control are more favorable
than have been achieved using only DNA or rMVA vaccines (Egan et
al., 2000; I. Ourmanov et al., 2000) and comparable to those
obtained for DNA immunizations adjuvanted with interleukin-2
(Barouch et al., 2000). All of these previous studies have used
more than three vaccine inoculations, none have used mucosal
challenges, and most have challenged at peak effector responses and
not allowed a prolonged post vaccination period to test for "long
term" efficacy as was done in our study. Our results also
demonstrate for the first time that vaccine-raised T cells, as
measured by IFN-y ELISPOTs, are a correlate for the control of
viremia. This relatively simple assay can now be used for
preclinical evaluation of DNA and MVA immunogens for HIV-1, and
should be able to be used as a marker for the efficacy of clinical
trials in humans.
[0206] The DNA/MVA vaccine did not prevent infection. Rather, the
vaccine controlled the infection, rapidly reducing viral loads to
near or below 1000 copies of viral RNA per ml of blood.
Containment, rather than prevention of infection, affords the virus
the opportunity to establish a chronic infection (Chun et al.,
1998). Nevertheless, by rapidly reducing viral loads, a
multiprotein DNA/MVA vaccine will extend the prospect for long-term
non-progression and limit HIV transmission.
EXAMPLE 16
Gag-Pol Vaccine Trial
[0207] A trial using Gag-Pol rather than Gag-Pol-Env expressing
immunogens was conducted to determine the importance of including
Env in the vaccine (see FIG. 27 for constructs). A vaccine that did
not include Env would have certain advantages in the field, such as
the ability to screen for anti-Env antibody as a marker for
infection. This trial used pGA1/Gag-Pol and a rMVA expressing the
Gag-Pol sequences of SIV239 (MVA/Gag-Pol) supplied by Dr. Bernard
Moss (NIH-NIAID)
[0208] The "Gag-Pol" immunogens were administered using the
schedule described in Example
[0209] 13 above for the "Gag-Pol-Env" (pGA2/89.6 MVA/89.6)
immunogens (see Table 4, Groups 5 and 6). The same doses of DNA,
2.5 mg and 250 .mu.g, were used to prime a high dose and a low dose
group and administration was via an intradermal route. As in the
previous vaccine trial described in examples 13-15, two to three
mamu A*01 macaques were included in each trial group. T cell
responses were followed for those specific for the p11c-m epitope
using the p11c-m tetramers and using ELISPOTs stimulated by pools
of overlapping peptides, as described in the above Examples.
[0210] Following immunization, vaccine recipients showed anti-Gag T
cell responses similar to those observed in the Gag-Pol-Env vaccine
trial. Animals were challenged intrarectally with SHIV-89.6P at 7.5
months following the rMVA booster (FIG. 28). In contrast to the
Gag-Pol-Env vaccine protocol, which protected animals against the
rapid loss of CD4 cells, the Gag-Pol animals uniformly lost CD4
cells (FIGS. 28B and 28D ). This loss was most pronounced in the
group receiving the low dose i.d. DNA prime. Consistent with the
loss of CD4 cells, the Gag-Pol DNA-immunized groups were also less
effective at reducing their viral loads than the Gag-Pol-Env groups
(FIGS. 28A and 28C). Geometric mean viral loads for these groups
were 10-100-fold higher at 3 weeks post challenge and 10 fold
higher at 5 weeks post challenge. These results demonstrate that
the Env gene plays an important role in protecting CD4 cells and
reducing the levels of viral RNA in challenged animals. The results
also show that Gag-Pol-Env DNA/MVA vaccines function more
effectively than Gag-Pol DNA/MVA vaccines in protecting recipients
against a virulent challenge.
EXAMPLE 17
Measles Inserts
[0211] A DNA vaccine expressing a fusion of measles H and the C3d
component of complement was used to determine if vaccination could
achieve earlier and more efficient anti-H antibody responses. In
prior studies in mice by Dempsey et al., the fusion of two or three
copies of C3d to a model antigen, hen egg lysozyme increased the
efficiency of immunizations by more than 1000-fold (Dempsey et al,
1996). This resulted in more rapid appearance of hemagglutination
inhibition (HI) activity and protective immunity (Ross et al, 2000
and Ross et al., 2001).
[0212] In the human immune system, one consequence of complement
activation is the covalent attachment of the C3d fragment of the
third complement protein to the activating protein. C3d in turn
binds to CD21 on B lymphocytes, a molecule with B cell stimulatory
functions that amplify B lymphocyte activation. In a measles H-C3d
fusion protein, the H moiety of the fusion would bind to anti-H Ig
receptors on B cells and signal through the B cell receptor, while
the C3d moiety of the fusion would bind to CD21 and signal through
CD19. In this hypothesis, a B cell responding to an H-C3d fusion
protein would undergo more effective signaling than a B cell
responding to H alone. Mice vaccinated with DNA expressing a
secreted H-fused to three copies of C3d (sH-3C3d) generated a more
rapid appearance and higher levels of neutralizing antibody
activity than DNA expressing sH only.
[0213] Plasmid DNA: pTR600, a eukaryotic expression vector, was
constructed to contain two copies of the cytomegalovirus
immediate-early promoter (CMV-IE) plus intron A (IA) for initiating
transcription of eukaryotic inserts and the bovine growth hormone
polyadenylation signal (BGH poly A) for termination of
transcription. The vector contains a multi-cloning site for the
easy insertion of gene segments and the Col El origin of
replication for prokaryotic replication and the Kanamycin
resistance gene (Kanr) for selection in antibiotic media (FIG.
29A).
[0214] Hemagglutinin (H) cDNA sequences from the Edmonton strain
and C3d sequences were cloned as previously described and
transferred into the pTR600 vaccine vector using unique restriction
endonuclease sites (FIG. 29B). The secreted version was generated
by deleting the transmembrane and cytoplasmic domains of H. This
was accomplished using PCR to clone a fragment of the H gene in
frame with an N-terminal synthetic mimic of the tissue plasminogen
activator (tpA) leader sequence (Torres, et al, 2000).
[0215] The vectors expressing sH-C3d fusion proteins were generated
by cloning three tandem repeats of the mouse homologue of C3d in
frame at the 3' end of the sH gene as previously described
(Dempsey, 1996; Ross et al, 2000; and Ross et al, 2001). The
construct design was based upon Dempsey et al. and used sequences
from pSLG-C3d. Linkers composed of two repeats of 4 glycines and a
serine {(G.sub.4S).sub.2} were fused at the junctures of H and C3d
and between each C3d repeat. Potential proteolytic cleavage sites
between the junctions of C3d and the junction of sH and C3d were
mutated by using Bam HI and Bgl II fusion to mutate an Arg codon to
a Gly codon.
[0216] The plasmids were amplified in Escherichia coli strain,
DH5a, purified using anion-exchange resin columns (Qiagen,
Valencia, CA) and stored at -20.degree. C. in dH.sub.2O. Plasmids
were verified by appropriate restriction enzyme digestion and gel
electrophoresis. Purity of DNA preparations was determined by
optical density reading at 260 nm and 280 nm.
[0217] Mice and DNA immunizations: Six to 8 week old BALB/c mice
(Harlan Sprague Dawley, Indianapolis, Ind.) were used for
inoculations. Briefly, mice were anesthetized with 0.03-0.04 ml of
a mixture of 5 ml ketamine HCl (100 mg/ml) and 1 ml xylazine (20
mg/ml). Mice were immunized with two gene gun doses containing 0.5
.mu.g of DNA per 0.5 mg of approximately 1-.mu.m gold beads
(DeGussa-Huls Corp., Ridgefield Park, N.J.) at a helium pressure
setting of 400 psi.
[0218] Transfections and Expression Analysis:
[0219] The human embryonic kidney cell line 293T (5.times.10.sup.5
cells/transfection) was transfected with 2 .mu.g of DNA using 12%
lipofectamine according to the manufacture's guidelines (Life
Technologies, Grand Island, N.Y.). Supernatants were collected and
stored at -20.degree. C. Quantitative antigen capture ELISAs for H
were conducted as previously described (Cardoso et al, 1998).
[0220] For western hybridization analysis, 15 .mu.l of supernatant
or cell lysate was diluted 1:2 in SDS sample buffer (Bio-Rad,
Hercules, CA) and loaded onto a 10% polyacrylamide/SDS gel. The
resolved proteins were transferred onto a nitrocellulose membrane
(Bio-Rad, Hercules, Calif.) and incubated with a 1:1000 dilution of
polyclonal rabbit anti-HA antisera in PBS containing 0.1% Tween 20
and 1% nonfat dry milk. After extensive washing, bound rabbit
antibodies were detected using a 1:2000 dilution of horseradish
peroxidase-conjugated goat anti-rabbit antiserum and enhanced
chemiluminescence (Amersham, Buckinghamshire, UK).
[0221] Antibody Assays:
[0222] A quantitative ELISA was performed to assess anti-H specific
IgG levels. Briefly, Ltk-cells constitutively expressing the H
protein of MV (24) were grown in 96-well plates. Antisera dilutions
were incubated with the intact cells expressing H antigen. The
anti-H antibodies were allowed to bind to the cells for 30 min
following which the cells were fixed in acetone (80%). The specific
antibody responses were detected with biotinylated anti-mouse IgG
antibodies and the streptavidine-phosphatase alkaline system
(Sigma). Antibody binding to Ltk-cells not expressing H antigen was
used to standardize the system. The results were expressed as the
endpoint dilution titer.
[0223] Neutralization Assays.
[0224] Neutralization assays were conducted on Vero cells grown in
six well plates (25). Briefly, 100-200 p.f.u. of the Edmonton
strain of measles virus were mixed with serial dilution of sera,
incubated for 1 h at 37.degree. C. and then inoculated onto plates.
Plates were incubated at 37.degree. C. for 48 h and containing
either the sH or sH-3C3d compared to transmembrane-associated forms
of the antigen. Human 293T cells were transiently transfected with
2 .mu.g of plasmid and both supernatants and cell lysates were
assayed for H using an antigen capture ELISA. Approximately 75% of
the H protein was secreted into the supernatant for both sH-DNA and
sH-3C3d-DNA transfected cells. As expected, .about.99% of the H
antigen was detected in the cell lysate of cells transfected with
plasmids expressing transmembrane form of H.
[0225] Antibody Response to Measles H DNA Immunizations:
[0226] The sH-3C3d expressing DNA plasmids raised higher titers of
ELISA antibody than sH DNA. BALB/c mice were vaccinated by DNA
coated gold particles via gene gun with either a 0.1 .mu.g or a 1
.mu.g inoculum. At 4 and 26 weeks post vaccination, mice were
boosted with the same dose of DNA given in the first immunization.
The temporal pattern for the appearance of anti-H antibody showed a
faster onset in mice vaccinated with the C3d fusion expressing DNA
compared to mice vaccinated with sH DNA. Good titers of antibody
were raised by the first immunization . These were boosted by the
2.sup.nd and 3.sup.rd immunizations. following the third
immunization, titers were 5-6 times higher in the sH-3C3D
vaccinated mice than in those vaccinated with sH DNA.
[0227] Neutralization Assays:
[0228] Examination of the serum for MV neutralization showed titers
up to 1700 after the second inoculation of 0.1 .mu.g of sH-3C3d
expressing DNA. Neutralizing antibody studies performed on Vero
cells detected higher titers of neutralizing activity against the
prototype MV Edmonton strain in mouse sera elicited by the sH-3C3d
constructs than in the sera of mice vaccinated with plaques were
counted. Neutralization titers are defined as the reciprocal
dilution of sera required to reduce plaque formation by 50% or 90%.
Preimmune sera served as negative controls.
[0229] Results:
[0230] Two hemagglutinin-expressing vaccine plasmids were
constructed in the pTR600 vector to express either a secreted form
of H (sH) from the Edmonston strain or a C3d-fusion of the secreted
form of H (sH-3C3d) (FIG. 29). The sH represented the entire
ectodomain of H, but excluded the transmembrane and cytoplasmic
region. The cloning placed the N-terminal synthetic mimic of the
tissue plasminongen activator (tPA) leader sequence in frame with
the H sequence. The tPA leader and H sequences were fused
immediately 3' to the transmembrane domain of H. The sH-3C3d fusion
protein was generated by cloning three tandem repeats of the mouse
homologue of C3d in frame with the secreted H gene (FIG. 29B). The
proteolytic cleavage sites, found at the junction between each C3d
molecule as well as the junction between the H protein and the
first C3d coding region, were destroyed by mutagenesis.
[0231] Western blot analyses revealed sH and sH-3C3d proteins of
the expected sizes. Using a rabbit polyclonal antibody to MV H
antisera, western blot analysis showed a broad band of .about.70 kD
corresponding to the secreted form of H in the supernatant of
transfected cells. A higher molecular weight band at .about.190 kD
is consistent with the projected size of the sH-3C3d fusion protein
(FIG. 30). No evidence for the proteolytic cleavage of the sH-C3d
fusion protein was seen by western analysis.
[0232] Measles virus H was expressed at slightly lower levels by
plasmids sH expressing DNA. Mice vaccinated with sH-3C3d expressing
plasmids had a sharp rise in neutralizing antibody levels that
reached a plateau by week 14. In contrast, it took a third
vaccination with sH expressing DNA to elicit detectable levels of
neutralizing antibodies. After 28 weeks post-vaccination, sera from
mice vaccinated with sH-3C3d-DNA had neutralizing titers (>250)
that could reduce plaque formation of MV infection by 90%.
[0233] The increase in height of the antibody response to H was
7-15 fold higher in mice vaccinated with the C3d protein expressing
constructs compared to mice vaccinated with DNA expressing sH only.
The increase in antibody response with DNA expressing sH-3C3d is
even more intriguing, since this plasmid expressed .about.60% as
much protein as plasmid expressing sH only.
[0234] In addition to the increase in the overall antibody level,
there was a faster onset of antibodies that could specifically
neutralize MV in an in vitro infection assay. After the second
immunization, detectable levels of neutralizing antibodies were
observed in mice vaccinated with DNA expressing sH-3C3d. The titer
of the neutralizing antibody peaked at week 14 (1700 for 50% plaque
reduction), which are substantially above the minimum correlate for
protection (>120 for 50% plaque reduction). In contrast, mice
vaccinated with sH expressing DNA had low levels of neutralizing
antibody even after the third vaccination (180 for 50% plaque
reduction) (FIG. 31).
EXAMPLE 18
Influenza Inserts with and without-C3d
[0235] Plasmid vector construction and purification procedures have
been previously described for JW4303 (Torres, et al. 1999; Pertmer
et al. 1995; Feltquate et al. 1997). In brief, influenza
hemagglutinin (HA) sequences from A/PR/8/34 (H1N1) were cloned into
either the pJW4303 or pGA eukaryotic expression vector using unique
restriction sites.
[0236] Two versions of HA, a secreted(s) and a transmembrane (tm)
associated, have been previously described (Torres et al. 1999;
Feltquate et al.,1997). Vectors expressing sHA or tmHA in pJW4303
were designated pJW/sHA and pJW/tmHA respectively and the vectors
expressing sHA, tmHA, or sHA-3C3d in pGA were designated pGA/sHA,
pGA/tmHA, and pGA/sHA-3C3d respectively.
[0237] Vectors expressing HA-C3d fusion proteins were generated by
cloning three tandem repeats of the mouse homolog of C3d and
placing the three tandem repeats in-frame with the secreted HA
gene. The construct designed was based upon Dempsey et al (1996).
Linkers composed of two repeats of 4 glycines and a serine
[(G.sub.4S).sub.2] were fused at the joints of each C3d repeat. The
pGA/sHA-3C3d plasmid expressed approximately 50% of the protein
expressed by the pGA/sHA vector. However, the ratio of sHA-3C3d
found in the supernatant vs. the cell lysate was similar to the
ratio of antigen expressed by pGA/sHA. More than 80% of the protein
was secreted into the supernatant. In western analysis, a higher
molecular weight band was detected at 120 kDa and represented the
sHA-3C3d fusion protein. Therefore, the sHA-3C3d fusion protein is
secreted into the supernatant as efficiently as the sHA
antigen.
[0238] Mice and DNA Immunizations.
[0239] Six to 8 week old BALB/c mice (Harlan Sprague Dawley,
Indianapolis, Ind.) were used for inoculations. Mice, housed in
microisolator units and allowed free access to food and water, were
cared for under USDA guidelines for laboratory animals. Mice were
anesthetized with 0.03-0.04 ml of a mixture of 5 ml ketamine HCl
(100 mg/ml) and 1 ml xylazine (20 mg/ml). Gene gun immunizations
were performed on shaved abdominal skin using the hand held Accell
gene delivery system and immunized with two gene gun doses
containing 0.5 .mu.g of DNA per 0.5 mg of approximately 1-.mu.m
gold beads (DeGussa-Huls Corp., Ridgefield Park, N.J.) at a helium
pressure setting of 400 psi.
[0240] Influenza Virus Challenge.
[0241] Challenge with live, mouse-adapted, influenza virus
(A/PR/8/34) was performed by intranasal instillation of 50 .mu.l
allantoic fluid, diluted in PBS to contain 3 lethal doses of virus,
into the nares of ketamine-anesthetized mice. This method leads to
rapid lung infections and is lethal to 100% of non-immunized mice.
Individual mice were challenge at either 8 or 14 weeks after
vaccination and monitored for both weight loss and survival. Data
were plotted as the average individual weight in a group, as a
percentage of pre-challenge weight, versus days after
challenge.
[0242] Antibody Response to the HA DNA Immunization Protocol:
[0243] The tmHA and sHA-3C3d expressing DNA plasmids raised higher
titers of ELISA antibody than the sHA DNA. BALB/c mice were
vaccinated by DNA coated gold particles via gene gun with either a
0.1 .mu.g or 1 .mu.g dose inoculum. At 4 weeks post vaccination,
half of the mice in each group were boosted with the same dose of
DNA given in the first immunization. Total anti-HA IgG induced by
the sHA-3C3d- and tmHA-expressing plasmids were similar in the
different experimental mouse groups and 3-5 times higher then the
amount raised by the sHA expressing plasmids (FIG. 24). In
addition, the amount of anti-HA antibody elicited increased
relative to the amount of DNA used for vaccination in a dose
dependent manner (FIGS. 24E-24F). Overall, the dose response curves
and temporal pattern for the appearance of anti-HA antibody were
similar in the mice vaccinated with tmHA-DNA or sHA-3C3d-DNA, but
lower and slower, in the mice vaccinated with sHA-DNA. As expected,
the booster immunization both accelerated and increased the titers
of antibodies to HA.
[0244] Avidity of Mouse HA Antiserum.
[0245] Sodium thiocyanate (NaSCN) displacement ELISAs demonstrated
that the avidity of the HA-specific antibody generated with
sHA-3C3d expressing DNA was consistently higher than antibodies
from sHA-DNA or tmHA-DNA vaccinated mice (FIG. 25). The avidity of
specific antibodies to HA was compared by using graded
concentrations NaSCN, a chaotropic agent, to disrupt
antigen-antibody interactions. The binding of antibodies with less
avidity to the antigen is disrupted at lower concentrations of
NaSCN than that of antibodies with greater avidity to the antigen.
The effective concentration of NaSCN required to release 50% of
antiserum (ED.sub.50) collected at 8 weeks after vaccination from
sHA-DNA or tmHA-DNA boosted mice (0.1 .mu.g dose or 1 .mu.g dose)
was .about.1.20 M (FIG. 25A). In contrast, antiserum from mice
vaccinated and boosted with sHA-3C3d-DNA had an ED.sub.50 of
.about.1.75 M (FIG. 25B). At the time of challenge (14 weeks after
vaccination), the ED.sub.50 had increased to 1.8 M for antibodies
from both sHA-DNA and tmHA-DNA vaccinated mice (FIG. 25C).
Antibodies from mice vaccinated with sHA-3C3d-DNA had increased to
an ED.sub.50 of .about.2.0 M (FIG. 25D). These results suggest that
the antibody from sHA-3C3d-DNA vaccinated mice had undergone more
rapid affinity maturation than antibody from either sHA-DNA or
tmHA-DNA vaccinated mice. The difference between the temporal
avidity maturation of antibody for sHA-3C3d and tmHA was
independent of the level of the raised antibody. Both of these
plasmids had similar temporal patterns for the appearance of
antibody and dose response curves for the ability to raise antibody
(FIG. 25).
[0246] Hemagglutinin-inhibition (HI) titers.
[0247] Hemagglutination-inhibition assays (HI) were performed to
evaluate the ability of the raised antibody to block binding of
A/PR/8/34 (H1N1) to sialic acid. The HI titers were measured from
serum samples harvested from mice at 8 and 14 weeks after
vaccination. All boosted mice had measurable HI titers at week 14
regardless of the dose or vaccine given. The highest titers (up to
1:1200) were recorded for the sHA-3C3d-DNA vaccinated mice.
Nonboosted mice showed more variation in HI titers. Nonboosted mice
vaccinated with a 0.1 .mu.g dose of either sHA-DNA or tmHA-DNA
expressing plasmids had low HI titers of 1:10. In contrast, mice
vaccinated with sHA-3C3d-DNA had titers greater than 1:640. The
only vaccinated mice that had a measurable HI titer (1:160) at week
8 were boosted mice vaccinated with 1 .mu.g dose sHA-3C3d-DNA.
These results indicate that C3d, when fused to sHA, is able to
stimulate specific B cells to increase the avidity maturation of
antibody and thus the production of neutralizing antibodies to
HA.
[0248] Protective Efficacy to Influenza Challenge.
[0249] Consistent with eliciting the highest titers of HI antibody,
the sHA-3C3d DNA raised more effective protection than the sHA or
tmHA DNAs. To test the protective efficacy of the various HA-DNA
vaccines, mice were challenged with a lethal dose of A/PR/8/34
influenza virus (HlNl) and monitored daily for morbidity (as
measured by weight loss) and mortality. Weight loss for each animal
was plotted as a percentage of the average pre-challenge weight
versus days after challenge (FIG. 26). Virus-challenged naive mice
and pGA vector only vaccinated mice showed rapid weight loss with
all the mice losing >20% of their body weight by 8 days
post-challenge (FIG. 26). In contrast, PBS mock-challenged mice
showed no weight loss over the 14 days of observation. All boosted
mice survived challenge, 14 weeks after vaccination, regardless of
the dose of DNA plasmid administered. However, boosted mice
vaccinated with a 0.1 .mu.g dose of sHA-DNA did drop to 92% of
their initial body weight at 8 days post-challenge before
recovering (FIG. 26). In contrast, when 1 .mu.g dose, boosted mice
were challenged at 8 weeks after vaccination, the only mice to
survive challenge were sHA-3C3d- and tmHA-DNA vaccinated mice,
albeit with greater weight loss than was observed from mice
challenged at 14 weeks after vaccination. The only 0.1 .mu.g dose,
boosted mice to survive challenge at 8 weeks after vaccination were
the sHA-3C3d vaccinated mice (FIG. 26).
[0250] Among the nonboosted, 0.1 .mu.g dose immunizations, only the
sHA-3C3d-DNA vaccinated mice survived challenge at 14 weeks after
vaccination (FIG. 26). All mice administered a single DNA
vaccination lost weight. However, of these, the sHA-3C3d-DNA
vaccinated mice lost the least weight and these mice were the only
mice to survive the lethal challenge (FIG. 26). These results
demonstrate the that 3C3d protein, when fused to HA, increased the
efficiency of a DNA vaccine, allowing for the reduction in dose of
DNA and the number of vaccinations needed to afford protection to a
lethal influenza virus challenge.
EXAMPLE 19
HIV gp120-C3d Fusion Constructs
[0251] In this study, a similar approach to that described in
Example 18 was used to fuse three copies of murine C3d to the
carboxyl terminus of HIV Env gp120 subunit. Using DNA vaccination,
BALB/c mice were inoculated and assayed for enhanced immune
responses. The fusion constructs induced higher antibody responses
to Env and a faster onset of avidity maturation than did the
respective wild-type gp 120 sequences. These results suggest that
the efficacy of DNA vaccines for raising antibody can be
significantly improved by fusing proteins with C3d.
[0252] Plasmid DNA:
[0253] pGA was constructed as described in Example 1 above to
contain the cytomegalovirus immediate-early promoter (CMV-IE) plus
intron A (IA) for initiating transcription of eukaryotic inserts
and the bovine growth hormone polyadenylation signal (BGH poly A)
for termination of transcription. HIV envelope sequences from the
isolates ADA, IIIB, and 89.6, encoding almost the entire gp120
region, and C3d sequences were cloned into the pGA vaccine vector
using unique restriction endonuclease sites. The gp120 segment
encoded a region from amino acid 32 to 465 and ended with the amino
acid sequence VAPTRA. The first 32 amino acids were deleted from
the N-terminus of each sgp 120 and replaced with a leader sequenced
from the tissue plasminogen activator (tpA). The vectors expressing
sgp120-C3d fusion proteins were generated by cloning three tandem
repeats of the mouse homologue of C3d in frame with the sgp120
expressing DNA. The construct design was based upon Dempsey et al
(1996). Linkers composed of two repeats of 4 glycines and a serine
{(G.sub.4S).sub.2} were fused at the junctures of HA and C3d and
between each C3d repeat. Potential proteolytic cleavage sites
between the junctions of C3d and the junction of 3C3d were mutated
by ligating Bam HI and Bgl II restriction endonuclease sites to
mutate an Arg codon to a Gly codon.
[0254] The plasmids were amplified in Escherichia coli
strain-DH5.alpha., purified using anion-exchange resin columns
(Qiagen, Valencia, Calif.) and stored at -20.degree. C. in
dH.sub.2O. Plasmids were verified by appropriate restriction enzyme
digestion and gel electrophoresis. Purity of DNA preparations was
determined by optical density reading at 260 nm and 280 nm.
[0255] Mice and DNA Immunizations:
[0256] Six to 8 week old BALB/c mice (Harlan Sprague Dawley,
Indianapolis, Ind.) were vaccinated as described in Example 17
above. Briefly, mice were immunized with two gene gun doses
containing 0.5 .mu.g of DNA per 0.5 mg of approximately 1-.mu.m
gold beads (DeGussa-Huls Corp., Ridgefield Park, N.J.) at a helium
pressure setting of 400 psi.
[0257] Transfections and expression analysis and western
hybridization experiments were conducted as described in Example
17, except that the nitrocellulose membranes were incubated with a
1:1000 dilution of polyclonal human HIV-infected patient antisera
in PBS containing 0.1% Tween 20 and 1% nonfat dry milk. After
extensive washing, bound human antibodies were detected using a
1:2000 dilution of horseradish peroxidase-conjugated goat
anti-human antiserum and enhanced chemiluminescence (Amersham,
Buckinghamshire, UK).
[0258] ELISA and Avidity Assays:
[0259] An endpoint ELISA was performed to assess the titers of
anti-Env IgG in immune serum using purified HIV-1-IIIB gp120
CHO-expressed protein (Intracell) to coat plates as described
(Richmond et al., 1998). Alternatively, plates were coated with
sheep anti-Env antibody (International Enzymes Inc., Fallbrook,
Calif.) and used to capture sgp120 produced in 293T cells that were
transiently transfected with sgp120 expression vectors. Mouse sera
from vaccinated mice was allowed to bind and subsequently detected
by anti-mouse IgG conjugated to horseradish peroxidase. Endpoint
titers were considered positive that were two fold higher than
background. Avidity ELISAs were performed similarly to serum
antibody determination ELISAs up to the addition of samples and
standards. Samples were diluted to give similar concentrations of
specific IgG by O.D. Plates were washed three times with 0.05%
PBS-Tween 20. Different concentrations of the chaotropic agent,
sodium thiocyanate (NaSCN) in PBS, were then added (0M, 1 M, 1.5 M,
2 M, 2.5 M, and 3 M NaSCN). Plates were allowed to stand at room
temperature for 15 minutes and then washed six times with PBS-Tween
20. Subsequent steps were performed similarly to the serum antibody
determination ELISA and percent of initial IgG calculated as a
percent of the initial O.D. All assays were done in triplicate.
[0260] Neutralizing Antibody Assays:
[0261] Antibody-mediated neutralization of HIV-1 IIIB and 89.6 was
measured in an MT-2 cell-killing assay as described previously
(Montefiori et al., 1988). Briefly, cell-free virus (50 .mu.l
containing 10.sup.8 TCID.sub.50 of virus) was added to multiple
dilutions of serum samples in 100 .mu.l of growth medium in
triplicate wells of 96-well microtiter plates coated with
poly-L-lysine and incubated at 37.degree. C. for 1 h before MT-2
cells were added (10.sup.5 cells in 100 .quadrature.l added per
well). Cell densities were reduced and the medium was replaced
after 3 days of incubation when necessary. Neutralization was
measured by staining viable cells with Finter's neutral red when
cytopathic effects in control wells were >70% but less than
100%. Percentage protection was determined by calculating the
difference in absorption (A.sub.540) between test wells
(cells+virus) and dividing this result by the difference in
absorption between cell control wells (cells only) and virus
control wells (virus only). Neutralizing titers are expressed as
the reciprocal of the plasma dilution required to protect at least
50% of cells from virus-induced killing.
[0262] Results:
[0263] Env was expressed at overall similar levels by plasmids
containing either the secreted form of the antigen, but at a
two-four-fold lower level by the sgp120-C3d expressing plasmids.
Human 293T cells were transiently transfected with 2 .mu.g of
plasmid and both supernatants and cell lysates were assayed for
gp120 using an antigen capture ELISA. The sgp120 constructs
expressed from 450 to 800 ng per ml, whereas the 3C3d fusions
expressed from 140 to 250 ng per ml. Approximately 90% of the Env
protein was present in the supernatant for both sgp120 and
sgp120-3C3d-DNA transfected cells (data not shown). The
approximately 2-fold differences in the levels of expression of the
different sgp120s is likely a reflection in differences in the Env
genes as well as differences in the efficiency that the capture and
detection antibodies recognized the different Envs.
[0264] Western blot analyses revealed sgp120 and sgp120-3C3d
proteins of the expected sizes. Using human patient polyclonal
antisera, western blot analysis showed the expected broad band of
115-120 kD corresponding to gp120. A higher molecular weight band
at 240 kD was consistent with the projected size of the sgp120-3C3d
fusion protein. Consistent with the antigen-capture assay, intense
protein bands were present in the supernatants of cells transfected
with sgp120-DNA, whereas less intense bands were present in the
supernatants of cells transfected with sgp120-3C3d-DNA (data not
shown). No evidence for the proteolytic cleavage of the sgp120-C3d
fusion protein was seen by western analysis.
[0265] Antibody Response to Env gp120 DNA Immunizations:
[0266] The sgp120-3C3d expressing DNA plasmids raised higher titers
of ELISA antibody than the sgp 120 DNA. BALB/c mice were vaccinated
by DNA coated gold particles via gene gun with a 1 .mu.g dose
inoculum. Mice were vaccinated at day 1 and then boosted at 4, 14,
and 26 weeks with the same DNA given in the first immunization.
When sera were assayed on gp120-IIB-coated plates, mice vaccinated
with the DNAs expressing the C3d fusion proteins had anti-Env
antibodies 3-7 times higher then the amount of antibody raised by
the counterpart sgp120 expressing plasmids. Among the C3d
constructs, mice vaccinated with sgp120-(IIIB)-3C3d had the highest
levels of antibody and mice vaccinated with sgp120-(ADA)-3C3d
expressing DNA had the lowest levels of anti-Env antibodies. The
temporal pattern for the appearance of anti-Env antibody revealed
titers being boosted at each of the inoculations for all constructs
tested.
[0267] Differences in the levels of the antibody raised by the
different Envs appeared to be determined by the specificity of the
raised antibody. Using an alternative ELISA protocol, in which
antibody was captured on the homologous Env, all of the C3d-fusions
appeared to raise similar levels of antibody. In this assay, sheep
anti-Env antibody was used to capture transiently produced sgp120
proteins. This assay revealed low, but similar levels of antibody
raised by each of the sgp120-3C3d constructs. The lower levels of
antibody detected in this assay are likely to reflect the levels of
transfection-produced Env used to capture antibody being lower than
in the assays using commercially produced IIIB gp120 to coat
plates. As expected using either ELISA method, booster
immunizations were necessary to achieve even the most modest
antibody response.
[0268] Avidity of Mouse Env Antiserum:
[0269] Sodium thiocyanate (NaSCN) displacement ELISAs demonstrated
that the avidity of the antibody generated with sgp120-3C3d
expressing DNA was consistently higher than that from sgp120-DNA
vaccinated mice. Avidity assays were conducted on sera raised by
sgp120-(IIIB) and sgp120-(IIIB)-3C3d because of the type
specificity of the raised antisera and the commercial availability
of the IIIB protein (but not the other proteins) for use as capture
antigen. The avidity of specific antibodies to Env was compared by
using graded concentrations NaSCN, a chaotropic agent, to disrupt
antigen-antibody interaction. Results indicatedthat the antibody
from sgp120-3C3d-DNA vaccinated mice underwent more rapid affinity
maturation than antibody from sgp120-DNA vaccinated mice.
[0270] Env-3C3d Expressing Plasmids Elicit Modest Neutralizing
Antibody:
[0271] Neutralizing antibody studies performed on MT-2 cells
detected higher titers of neutralizing activity in the sera
generated by the gp120-3C3d constructs than in the sera generated
by the sgp120 constructs. Sera were tested against two syncytium
inducing, IIIB (X4) and 89.6 (X4R5) viruses. Mice vaccinated with
sgp120-3C3d expressing plasmids had very modest levels of
neutralizing antibody to the homologous strain of HIV tested by the
protection of MT-2 cells from virus-induced killing as measured by
neutral red uptake. Titers of neutralizing antibody raised by the
gp120-expressing DNAs were at the background of the assay.
[0272] The results of this study showed that fusions of HIV-1 Env
to three copies of murine C3d enhanced the antibody response to Env
in vaccinated mice. Mice vaccinated with any of the three DNA
plasmids expressing sgp120 sequence had low or undetectable levels
of antibody after 4 vaccinations (28 weeks post-prime).
[0273] In contrast, mice vaccinated with DNA expressing the fusion
of sgp120 and 3C3d proteins elicited a faster onset of antibody (3
vaccinations), as well as higher levels of antibodies.
[0274] In contrast to the enhancement of antibody titers and
avidity maturation of antibodies to Env, the amount of
neutralizing-antibody elicited in the vaccinated mice was low. Mice
vaccinated with plasmids expressing sgp120 had low levels of
neutralizing antibody that were only modestly increased in mice
vaccinated with sp120-3C3d expressing plasmids. However, the levels
of neutralizing antibodies did apparently increase after the fourth
immunization. The poor titers of neutralizing antibody could have
reflected an inherent poor ability of the sgp120-3C3d fusion
protein to raise neutralizing antibody because of the failure to
adequately expose neutralizing epitopes to responding B cells. The
intrinsic high backgrounds for HIV-1 neutralization assays in mouse
sera also may have contributed to the poor neutralization titers.
The results demonstrate the effectiveness of C3d-fusions as a
molecular adjuvant in enhancing antibody production and enhancing
antibody maturation. In addition, the neutralizing antibody
response to Env was modestly increased in mice vaccinated with
C3d-fusion vaccines. Similar to results seen in Examples 17 and 18,
using secreted versions of HA from measles and influenza viruses,
C3d-enhanced antibody responses were achieved with plasmids
expressing only half as much protein as plasmids expressing
non-fused sgp120.
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References