U.S. patent application number 10/530543 was filed with the patent office on 2006-05-25 for hiv vaccine formulations.
Invention is credited to Susan Barnett, John Donnelly, Derek O'Hagan.
Application Number | 20060110736 10/530543 |
Document ID | / |
Family ID | 32093869 |
Filed Date | 2006-05-25 |
United States Patent
Application |
20060110736 |
Kind Code |
A1 |
Donnelly; John ; et
al. |
May 25, 2006 |
Hiv vaccine formulations
Abstract
Provided herein are HIV vaccines comprising HIV
polypeptide-encoding DNA adsorbed to PLG and/or HIV proteins. Also
provided are methods of using these vaccines to generate immune
responses in a subject.
Inventors: |
Donnelly; John; (EMERYVILLE,
CA) ; Barnett; Susan; (San Francisco, CA) ;
O'Hagan; Derek; (Berkeley, CA) |
Correspondence
Address: |
Chiron Corporation;Intellectual Property - R440
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Family ID: |
32093869 |
Appl. No.: |
10/530543 |
Filed: |
October 7, 2003 |
PCT Filed: |
October 7, 2003 |
PCT NO: |
PCT/US03/31935 |
371 Date: |
November 7, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60416573 |
Oct 7, 2002 |
|
|
|
Current U.S.
Class: |
435/5 |
Current CPC
Class: |
A61K 2039/6093 20130101;
A61K 39/12 20130101; A61K 2039/545 20130101; A61K 2039/57 20130101;
C12N 2740/16234 20130101; A61K 39/21 20130101; A61P 37/04 20180101;
A61K 48/0041 20130101; C12N 2740/16134 20130101; A61K 48/005
20130101; A61K 48/0083 20130101; A61K 2039/55566 20130101; C07K
14/005 20130101; C12N 2740/16034 20130101; A61K 2039/53 20130101;
A61P 31/18 20180101; C12N 2740/16222 20130101; C12N 2740/16122
20130101; C12N 15/87 20130101 |
Class at
Publication: |
435/006 ;
435/005 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. An HIV DNA vaccine composition comprising a nucleic acid
expression vector comprising at least one HIV Gag- or Env-encoding
sequence; and PLG.
2. The vaccine composition of claim 1, wherein the concentration of
PLG is between about 5 and 100 fold greater than the concentration
of the nucleic acid expression vector.
3. The vaccine composition of claim 2, wherein the concentration of
nucleic acid is between about 10 .mu.g/mL and 5 mg/mL and the
concentration of the PLG is between about 100 .mu.g/mL and 100
mg/mL.
4. The vaccine composition of claim 1, wherein the nucleic acid
concentration per dose is between approximately 1 .mu.g/dose and 5
mg/dose and the PLG concentration per dose is between approximately
10 .mu.g/dose and 100 mg/dose.
5. The vaccine composition of claim 1, as set forth in Table 1 or
Table 2.
6. The vaccine composition of claim 1, as set forth in column 2 of
Table 9.
7. An HIV vaccine composition comprising oligomeric gp140
(o-gp140); and a pharmaceutically acceptable excipient.
8. The HIV vaccine of claim 7, wherein the concentration of o-gp140
is between about 0.1 and 10 mg/mL.
9. The HIV vaccine of claim 7, wherein the concentration of o-gp140
per dose is approximately 100 .mu.g/dose.
10. The HIV vaccine of claim 7, as set forth in Table 3 or Table
11.
11. The HIV vaccine of claim 7, further comprising an adjuvant.
12. The HIV vaccine of claim 11, wherein the adjuvant is MF59 or
CpG.
13. The HIV vaccine of claim 12, wherein the adjuvant is MF59 and
MF59 is as set forth in Table 4.
14. An HIV vaccine comprising an HIV Env DNA vaccine composition,
said HIV Env DNA vaccine composition comprising at least one HIV
Env-encoding sequence and PLG; an HIV Gag DNA vaccine composition,
said HIV Gag DNA vaccine composition comprising at least one HIV
Gag-encoding sequence and PLG; and an HIV vaccine composition, said
HIV vaccine composition comprising oligomeric gp140 (o-gp140) and a
pharmaceutically acceptable excipient
15. A method of generating an immune response in a subject, said
method comprising: (a) administering to the subject at least one
HIV vaccine composition, said composition comprising: (i) a nucleic
acid expression vector comprising at least one HIV Gag- or
Env-encoding sequence or (ii) an HIV oligomeric gp 140; and (b)
administering to the subject, at a time subsequent to the
administering of step (a), at least one HIV vaccine composition,
said composition comprising: (i) a nucleic acid expression vector
comprising at least one HIV Gag- or Env-encoding sequence or (ii)
an HIV oligomeric up 140.
16. A method of generating an immune response in a subject, said
method comprising: (a) administering to said subject at least one
HIV DNA vaccine composition comprising a nucleic acid expression
vector comprising at least one HIV Gag- or Env-encoding sequence;
and (b) administering to the subject, at a time subsequent to the
administering of step (a), at least one vaccine composition
comprising HIV oligomeric gp140.
17. The method of claim 16, wherein step (a) comprises multiple
administrations of said at least one HIV DNA vaccine composition
and step (b) comprises multiple administrations of said at least
one vaccine composition comprising HIV oligomeric gp 140.
18. The method of claim 17, wherein step (a) comprises two or three
administrations at one month intervals; step (b) comprises two or
three administrations at 1, 2 or 3 month intervals; and the time
between the administrations of step (a) and step (b) is 1 to 5
months.
19. The method claim 16, wherein step (a) comprises administering
at least one HIV Gag DNA vaccine and at least one HIV Env DNA
vaccine.
20. The method of claim 15 wherein step (b) comprises concurrently
administering at least one DNA vaccine comprising a nucleic acid
expression vector comprising at least one HIV Gag- or Env-encoding
sequence and at least one HIV vaccine comprising oligomeric gp
140.
21. The method of claim 20, wherein step (a) comprises
administering at least one HIV Gag DNA vaccine and at least one HIV
Env DNA vaccine.
22. The method of claim 15, wherein at least one administration is
intramuscular or intradermal.
23. A method of making oligomeric HIV Env gp140 proteins,
comprising the steps of introducing a nucleic acid encoding gp140
into a host cell; culturing the host cell under conditions such
that gp140 is expressed in the cell; and isolating oligomeric gp140
(o-gp140) protein from the host cell.
24. The method of claim 23, wherein the o-gp140 is secreted from
the cell and isolated from the cell supernatant.
25. A method of making an HIV DNA vaccine according to claim 1,
comprising the step of combining a nucleic acid expression vector
comprising a sequence encoding one or more HIV polypeptides with
aseptic PLG microparticles such that the nucleic acid binds to the
PLG microparticles to form a DNA/PLG HIV vaccine.
26. The method of claim 25, further comprising the step of
lyophilizing the DNA/PLG HIV vaccines.
27. A method of making an HIV vaccine according claim 7, comprising
combining o-gp140 with an adjuvant.
28. The HIV vaccine of claim 14, wherein the concentration of PLG
is between about 5 and 100 fold greater than the concentration of
the nucleic acid expression vector.
29. The HIV vaccine of claim 28, wherein the concentration of
nucleic acid is between about 10 .mu.g/mL and 5 mg/mL and the
concentration of the PLG is between about 100 .mu.g/mL and 100
mg/mL.
30. The HIV vaccine of claim 14, wherein the concentration of
nucleic acid per dose is between approximately 1 .mu.g/dose and 5
mg/dose and the concentration of the PLG per dose is between about
10 .mu.g/dose and 100 mg/dose.
31. The HIV vaccine of claim 14, wherein the HIV Env DNA vaccine
composition component is as set forth in Table 1 or column 2 of
Table 9.
32. The HIV vaccine of claim 14, wherein the HIV Gag DNA vaccine
composition component is as set forth in Table 2 or column 2 of
Table 9.
33. The HIV vaccine of claim 14, wherein the concentration of
o-gp140 is between about 0.1 and 10 mg/mL.
34. The HIV vaccine of claim 14, wherein the concentration of
o-gp140 per dose is approximately 100 .mu.g/dose.
35. The HIV vaccine of claim 14, wherein the HIV vaccine
composition component is as set forth in Table 3 or Table 11.
36. The HIV vaccine of claim 14, wherein the HIV vaccine
composition component further comprising an adjuvant.
37. The HIV vaccine of claim 36, wherein the adjuvant is MF59 or
CpG.
38. The HIV vaccine of claim 37, wherein the adjuvant is MF59 and
MF59 is as set forth in Table 4.
39. The method of claim 16, wherein at least one administration is
intramuscular or intradermal.
Description
TECHNICAL FIELD
[0001] The present invention relates generally to immunogenic HIV
compositions, in particular to HIV vaccines and methods of
formulating and administering these vaccines.
BACKGROUND
[0002] Acquired immune deficiency syndrome (AIDS) is recognized as
one of the greatest health threats facing modern medicine. There
is, as yet, no cure for this disease.
[0003] In 1983-1984, three groups independently identified the
suspected etiological agent of AIDS. See, e.g., Barre-Sinoussi et
al. (1983) Science 220:868-871; Montagnier et al., in Human T-Cell
Leukemia Viruses (Gallo, Essex & Gross, eds., 1984); Vilmer et
al. (1984) The Lancet 1:753; Popovic et al. (1984) Science
224:497-500; Levy et al. (1984) Science 225:840-842. These isolates
were variously called lymphadenopathy-associated virus (LAV), human
T-cell lymphotropic virus type III (HTLV-III), or AIDS-associated
retrovirus (ARV). All of these isolates are strains of the same
virus, and were later collectively named Human Immunodeficiency
Virus (HIV). With the isolation of a related AIDS-causing virus,
the strains originally called HIV are now termed HIV-1 and the
related virus is called HIV-2. See, e.g., Guyader et al. (1987)
Nature 326:662-669; Brun-Vezinet et al. (1986) Science 233:343-346;
Clavel et al. (1986) Nature 324:691-695.
[0004] Since the implementation of highly active antiretroviral
therapy (HAART) in the United States in 1996, the number of persons
diagnosed with acquired immunodeficiency syndrome (AIDS) and the
number of deaths among persons with AIDS have declined
substantially (Karon et al. (2001) Am J Public Health
91(7):1060-1068) as a result, the number of persons living with
AIDS has increased. The Centers for Disease Control (CDC) estimates
that as of Dec. 31, 2000, approximately 340,000 persons in the
United States were living with AIDS. (MMWR, Centers for Disease
Control and Prevention. HIV/AIDS Surveillence Report, 13(No.1)
2001).
[0005] Clinical trials in the US have been conducted with a limited
number of subjects and further HIV vaccine development will require
the identification of a suitable population where the HIV
seroincidence is sufficiently high to enable a distinction between
protection in the immunized population with a placebo control.
Seage III et al. (2001) Am. J. Epidemiol. 153(7):619-627; Halpern
et al. (2001) J Acquir Immune Defic Syndr 27(3):281-8.
[0006] The primary mode of transmission of HIV is through sex and
by contact with infected body fluids including blood, semen,
vaginal fluid, breast milk, and other body fluids containing blood.
In industrialized countries, the majority of cases reported in
which the person's risk is known are among men who have sex with
men. Before blood screening for antibodies to HIV was instituted,
transfusion-associated HIV was a concern in the US. (CDC. Update:
HIV-2 infection among blood and plasma donors--United States, June
1992-June 1995. MMWR, 1995. 44: p. 603-606). Other modes of
transmission include needle sharing by injection drug users,
inadvertent contact with infected blood among hospital workers, and
rare iatrogenic transmission through the re-use of contaminated
medical equipment. Higher rates of sexually transmitted infections
signal a rise in unsafe sex practices. Chen et al. (2001) Am J
Public Health 92(9):1387-1388. Heterosexual transmission of HIV-1
continues to rise, particularly among women, the young, and the
economically disadvantaged and, in fact, heterosexual transmission
is the dominant mode of transmission in the developing world. These
trends highlight the need for the development of a preventive
and/or therapeutic vaccine. Catania et al. (2001) Am J Public
Health 91(6):907-914.
[0007] Several targets for vaccine development have been examined,
including the env and Gag gene products encoded by HIV. Gag gene
products include, but are not limited to, Gag-polymerase (pol) and
Gag-protease (prot). Env gene products include, but are not limited
to, monomeric gp120 polypeptides, oligomeric gp140 polypeptides
(o-gp140) and gp160 polypeptides.
[0008] Recently, use of HIV Env polypeptides in immunogenic
compositions has been described. (see, U.S. Pat. No. 5,846,546 to
Hurwitz et al., issued Dec. 8, 1998, describing immunogenic
compositions comprising a mixture of at least four different
recombinant virus that each express a different HIV env variant;
and U.S. Pat. No. 5,840,313 to Vahlne et al., issued Nov. 24, 1998,
describing peptides which correspond to epitopes of the HIV-1 gp120
protein). In addition, U.S. Pat. No. 5,876,731 to Sia et al, issued
Mar. 2, 1999 describes candidate vaccines against HIV comprising an
amino acid sequence of a T-cell epitope of Gag linked directly to
an amino acid sequence of a B-cell epitope of the V3 loop protein
of an HIV-1 isolate containing the sequence GPGR. However, these
groups did not identify an effective HIV vaccine.
[0009] U.S. Pat. No. 6,602,705 and International Patent
Publications WO 00/39302; WO 02104493; WO 00/39303; and WO 00/39304
describe polynucleotides encoding immunogenic HIV polypeptides from
various subtypes.
[0010] Thus, there remains a need for immunogenic HIV compositions,
specifically for HIV vaccine formulations.
SUMMARY
[0011] In one aspect, the invention includes an HIV DNA vaccine
composition comprising a nucleic acid expression vector (e.g.,
plasmid, viral vector, etc.) comprising at least one HIV Gag- or
Env-encoding sequence and PLG. Preferably, the nucleic acid
expression vector is adsorbed to the PLG. In certain embodiments,
the concentration of PLG is between about 5 and 100 fold greater
than the concentration of the nucleic acid expression vector. For
example, the concentration of nucleic acid can be between about 10
.mu.g/mL and 5 mg/mL and the concentration of the PLG can be
between about 100 .mu.g/mL and 100 mg/mL and/or the nucleic acid
expression vector concentration per dose can be between
approximately 1 .mu.g/dose and 5 mg/dose and the PLG concentration
per dose can be between approximately 10 .mu.g/dose and 100
mg/dose. Specific formulations are described herein, for example,
in Table 1, Table 2, or column 2 of Table 9.
[0012] In another aspect, the invention includes an HIV vaccine
composition comprising an HIV envelope protein, for example
oligomeric gp140 (o-gp140); and a pharmaceutically acceptable
excipient. In certain embodiments, the concentration of o-gp140 is
between about 0.1 mg/mL and 10 mg/mL. Further, in certain
embodiments, the concentration of o-gp140 per dose is approximately
100 .mu.g/dose. Specific formulations of HIV protein vaccines are
also described herein, for example in Table 3 and Table 11.
[0013] In another aspect, the invention comprises an HIV vaccine
including one or more of the HIV DNA vaccines described herein
(e.g., an HIV Gag DNA vaccine as described herein and an HIV Env
DNA vaccine as described herein) and one or more of the HIV
vaccines described herein (e.g., an HIV o-gp140 preparation).
[0014] Any of the HIV vaccine compositions described herein may
further include one or more adjuvants, for example MF59 or CpG. A
particular formulation for MF59 is set forth in Table 4.
[0015] In yet another aspect, the invention includes a method of
generating an immune response in a subject, comprising (a)
administering at least one HIV vaccine composition described herein
to the subject, and (b) administering, at a time subsequent to the
administering of step (a), at least one HIV vaccine composition
described herein. In certain embodiments, the at least one HIV
vaccine composition administered in step (a) comprises an HIV DNA
vaccine (e.g., at least one HIV Gag vaccine and/or at least one HIV
Env vaccine) as described herein and the HIV vaccine composition
administered in step (b) comprises an HIV protein vaccine as
described herein. Furthermore, step (a) may comprise multiple
administrations of one or more HIV DNA vaccines as described herein
(e.g., two or three administrations at one month intervals) and
step (b) may comprise at least one administration of one or more
HIV protein vaccines as described herein (e.g., two or three
administrations at 1, 2, or 3 month intervals). Alternatively, step
(b) may comprise concurrently administering at least one HIV DNA
vaccine described herein (e.g., an HIV Gag vaccine and/or an HIV
Env vaccine) and at least one and at least one HIV protein vaccine
as described herein. The time between the administrations of step
(a) and step (b) can vary, for example between 1 to 6 months or
even longer. In any of the methods described herein, one or more
administrations may be intramuscular and/or intradermal.
[0016] In a further aspect, the invention includes a method of
making oligomeric HIV Env gp140 proteins, comprising the steps of
introducing a nucleic acid encoding gp140 into a host cell;
culturing the host cell under conditions such that gp140. is
expressed in the cell; and isolating oligomeric gp140 (o-gp140)
protein from the host cell. In certain embodiments, the o-gp140 is
secreted from the cell and isolated from the cell supernatant.
[0017] In a still further aspect, a method of making any of the HIV
DNA vaccines described herein is provided. The method comprises the
step of combining a nucleic acid expression vector comprising a
sequence encoding one or more HIV polypeptides with aseptic PLG
microparticles such that the nucleic acid expression vector binds
to the PLG microparticles to form a DNA/PLG HIV vaccine. In certain
embodiments, the method further comprises the step of lyophilizing
the DNA/PLG HIV vaccines.
[0018] In another aspect, the invention includes a method of making
an HIV protein vaccine as described herein, the method comprising
the steps of combining o-gp140 with an adjuvant.
[0019] These and other embodiments of the present invention will
readily occur to those of ordinary skill in the art in view of the
disclosure herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1A and FIG. 1B are graphs depicting the effect of PLG
microparticles on anti-Gag antibody responses induced by DNA
vaccines. FIG. 1A shows geometric mean ELISA titers of animals
immunized with plasmid DNA at weeks 0, 4 and 14, then boosted at
weeks 38 and 75 with recombinant Env protein formulated with MF59.
FIG. 1B shows geometric mean titer of animals immunized with pSINCP
DNA at weeks 0, 4 and 14, then boosted at weeks 38 and 75 with
recombinant Env protein formulated with MF59. Anti-Gag antibodies
are plotted as geometric mean ELISA titer for naked pCMV (solid
symbols) and PLG/pCMV (open symbols) and error bars represent
SEM.
[0021] FIG. 2A and FIG. 2B are graphs depicting the effect of PLG
microparticles on anti-Env antibody responses induced by DNA
vaccines. FIG. 2A shows geometric mean ELISA titers of animals
immunized with plasmid DNA at weeks 0, 4 and 14, then boosted at
weeks 38 and 75 with recombinant Env protein formulated with MF59.
FIG. 2B shows geometric mean titer of animals immunized with pSINCP
DNA at weeks 0, 4 and 14, then boosted at weeks 38 and 75 with
recombinant Env protein formulated with MF59. Anti-Env antibodies
are plotted as geometric mean ELISA titer for naked pCMV (solid
symbols) and PLG/pCMV (open symbols) and error bars represent
SEM.
[0022] FIG. 3 is a graph depicting geometric mean neutralization
titer after DNA administration.
[0023] FIG. 4 is a graph depicting the effect of Env protein
boosting on T cell responses primed by DNA vaccines.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, molecular biology, immunology and pharmacology,
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., Remington's Pharmaceutical Sciences,
18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990);
Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic
Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV
(D. M. Weir and C. C. Blackwell eds., 1986, Blackwell Scientific
Publications); Sambrook, et al., Molecular Cloning: A Laboratory
Manual (2nd Edition, 1989); Short Protocols in Molecular Biology,
4th ed. (Ausubel et al. eds., 1999, John Wiley & Sons);
Molecular Biology Techniques: An Intensive Laboratory Course, (Ream
et al., eds., 1998, Academic Press); PCR (Introduction to
Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997,
Springer Verlag); Peters and Dalrymple, Fields Virology (2d ed),
Fields et al. (eds.), B. N. Raven Press, New York, N.Y.
[0025] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entireties.
[0026] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural references unless
the content clearly dictates otherwise. Thus, for example,
reference to "an antigen" includes a mixture of two or more such
antigens.
[0027] Prior to setting forth the invention, it may be helpful to
an understanding thereof to first set forth definitions of certain
terms that will be used hereinafter.
[0028] As used herein the term "HIV polypeptide" refers to any HIV
peptide from any HIV stain or subtype, including, but not limited
to Gag, pol, env, vif, vpr, tat, rev, nef, and/or vpu; functional
(e.g., immunogenic) fragments thereof, modified polypeptides
thereof and combinations of these fragments and/or modified
peptides. Furthermore, an "HIV polypeptide" as defined herein is
not limited to a polypeptide having the exact sequence of known HIV
polypeptides. Indeed, the HIV genome is in a state of constant flux
and contains several domains that exhibit relatively high degrees
of variability between isolates. As will become evident herein, all
that is important is that the polypeptide has immunogenic
characteristics. It is readily apparent that the term encompasses
polypeptides from any of the various HIV strains and subtypes.
Furthermore, the term encompasses any such HIV protein regardless
of the method of production, including those proteins recombinantly
and synthetically produced.
[0029] Additionally, the term "HIV polypeptide" encompasses
proteins that include additional modifications to the native
sequence, such as additional internal deletions, additions and
substitutions (generally conservative in nature). These
modifications may be deliberate, as through site-directed
mutagenesis, or may be accidental, such as through naturally
occurring mutational events. All of these modifications are
encompassed in the present invention so long as the modified HIV
polypeptide functions for its intended purpose. Thus, for example,
in a vaccine composition, the modifications must be such that
immunological activity is not lost. Similarly, if the polypeptides
are to be used for diagnostic purposes, such capability must be
retained. Thus, the term also includes HIV polypeptides that differ
from naturally occurring peptides, for example peptides that
include one or more deletions (e.g., variable regions deleted from
Env), substitutions and/or insertions. Nonconservative changes are
generally substitutions of one of the above amino acids with an
amino acid from a different group (e.g., substituting Asn for Glu),
or substituting Cys, Met, His, or Pro for any of the above amino
acids. Substitutions involving common amino acids are conveniently
performed by site specific mutagenesis of an expression vector
encoding the desired protein, and subsequent expression of the
altered form. One may also alter amino acids by synthetic or
semi-synthetic methods. For example, one may convert cysteine or
serine residues to selenocysteine by appropriate chemical treatment
of the isolated protein. Alternatively, one may incorporate
uncommon amino acids in standard in vitro protein synthetic
methods. Typically, the total number of residues changed, deleted
or added to the native sequence in the mutants will be no more than
about 20, preferably no more than about 10, and most preferably no
more than about 5.
[0030] "Synthetic" polynucleotide sequences, as used herein, refers
to HIV-encoding polynucleotides (e.g., Gag- and/or Env-encoding
sequences) whose expression has been optimized, for example, by
codon substitution and inactivation of inhibitory sequences. See,
e.g., U.S. Pat. No. 6,602,705 and International Publications WO
00/39302; WO 02/04493; WO 00/39303; and WO 00/39304 for examples of
synthetic HIV-encoding polynucleotides.
[0031] "Wild-type" or "native" sequences, as used herein, refers to
polypeptide encoding sequences that are essentially as they are
found in nature, e.g., Gag and/or Env encoding sequences as found
in other isolates such as Type C isolates (e.g., Botswana isolates
AF110965, AF110967, AF110968 or AF110975 or South African
isolates).
[0032] As used herein, the term "virus-like particle" or "VLP"
refers to a nonreplicating, viral shell, derived from any of
several viruses discussed further below. VLPs are generally
composed of one or more viral proteins, such as, but not limited to
those proteins referred to as capsid, coat, shell, surface and/or
envelope proteins, or particle-forming polypeptides derived from
these proteins. VLPs can form spontaneously upon recombinant
expression of the protein in an appropriate expression system.
Methods for producing particular VLPs are known in the art and
discussed more fully below. The presence of VLPs following
recombinant expression of viral proteins can be detected using
conventional techniques known in the art, such as by electron
microscopy, X-ray crystallography, and the like. See, e.g., Baker
et al., Biophys. J. (1991) 60:1445-1456; Hagensee et al., J. Virol.
(1994) 68:4503-4505. For example, VLPs can be isolated by density
gradient centrifugation and/or identified by characteristic density
banding. Alternatively, cryoelectron microscopy can be performed on
vitrified aqueous samples of the VLP preparation in question, and
images recorded under appropriate exposure conditions.
[0033] By "particle-forming polypeptide" derived from a particular
viral protein is meant a full-length or near full-length viral
protein, as well as a fragment thereof, or a viral protein with
internal deletions, which has the ability to form VLPs under
conditions that favor VLP formation. Accordingly, the polypeptide
may comprise the full-length sequence, fragments, truncated and
partial sequences, as well as analogs and precursor forms of the
reference molecule. The term therefore intends deletions, additions
and substitutions to the sequence, so long as the polypeptide
retains the ability to form a VLP. Thus, the term includes natural
variations of the specified polypeptide since variations in coat
proteins often occur between viral isolates. The term also includes
deletions, additions and substitutions that do not naturally occur
in the reference protein, so long as the protein retains the
ability to form a VLP. Preferred substitutions are those that are
conservative in nature, i.e., those substitutions that take place
within a family of amino acids that are related in their side
chains. Specifically, amino acids are generally divided into four
families: (1) acidic--aspartate and glutamate; (2) basic--lysine,
arginine, histidine; (3) non-polar--alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan; and (4)
uncharged polar--glycine, asparagine, glutamine, cystine, serine
threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are
sometimes classified as aromatic amino acids.
[0034] An "antigen" refers to a molecule containing one or more
epitopes (either linear, conformational or both) that will
stimulate a host's immune system to make a humoral and/or cellular
antigen-specific response. The term is used interchangeably with
the term "immunogen." Normally, a B-cell epitope will include at
least about 5 amino acids but can be as small as 3-4 amino acids. A
T-cell epitope, such as a CTL epitope, will include at least about
7-9 amino acids, and a helper T-cell epitope at least about 12-20
amino acids. Normally, an epitope will include between about 7 and
15 amino acids, such as, 9, 10, 12 or 15 amino acids. The term
"antigen" denotes both subunit antigens, (i.e., antigens which are
separate and discrete from a whole organism with which the antigen
is associated in nature); as well as, killed, attenuated or
inactivated bacteria, viruses, fungi, parasites or other microbes.
Antibodies such as anti-idiotype antibodies, or fragments thereof,
and synthetic peptide mimotopes, which can mimic an antigen or
antigenic determinant, are also captured under the definition of
antigen as used herein. Similarly, an oligonucleotide or
polynucleotide that expresses an antigen or antigenic determinant
in vivo, such as in gene therapy and DNA immunization applications,
is also included in the definition of antigen herein.
[0035] For purposes of the present invention, antigens are
preferably derived from any subtype of HIV. Antigens can also be
derived from any of several known viruses, bacteria, parasites and
fungi, or tumor antigens. Furthermore, for purposes of the present
invention, an "antigen" refers to a protein that includes
modifications, such as deletions, additions and substitutions
(generally conservative in nature), to the native sequence, so long
as the protein maintains the ability to elicit an immunological
response, as defined herein. These modifications may be deliberate,
as through site-directed mutagenesis, or may be accidental, such as
through mutations of hosts that produce the antigens.
[0036] An "immunological response" to an antigen or composition is
the development in a subject of a humoral and/or a cellular immune
response to an antigen present in the composition of interest. For
purposes of the present invention, a "humoral immune response"
refers to an immune response mediated by antibody molecules, while
a "cellular immune response" is one mediated by T-lymphocytes
and/or other white blood cells. One important aspect of cellular
immunity involves an antigen-specific response by cytolytic T-cells
("CTL"s). CTLs have specificity for peptide antigens that are
presented in association with proteins encoded by the major
histocompatibility complex (MHC) and expressed on the surfaces of
cells. CTLs help induce and promote the destruction of
intracellular microbes, or the lysis of cells infected with such
microbes. Another aspect of cellular immunity involves an
antigen-specific response by helper T-cells. Helper T-cells act to
help stimulate the function, and focus the activity of; nonspecific
effector cells against cells displaying peptide antigens in
association with MHC molecules on their surface. A "cellular immune
response" also refers to the production of cytokines, chemokines
and other such molecules produced by activated T-cells and/or other
white blood cells, including those derived from CD4+ and CD8+
T-cells.
[0037] A composition or vaccine that elicits a cellular immune
response may serve to sensitize a vertebrate subject by the
presentation of antigen in association with MHC molecules at the
cell surface. The cell-mediated immune response is directed at, or
near, cells presenting antigen at their surface. In addition,
antigen-specific T-lymphocytes can be generated to allow for the
future protection of an immunized host.
[0038] The ability of a particular antigen to stimulate a
cell-mediated immunological response maybe determined by a number
of assays, such as by lymphoproliferation (lymphocyte activation)
assays, CTL cytotoxic cell assays, or by assaying for T-lymphocytes
specific for the antigen in a sensitized subject. Such assays are
well known in the art. See, e.g., Erickson et al., J. Immunol.
(1993) 151:4189-4199; Doe et al., Eur. J. Immunol. (1994)
24:2369-2376. Recent methods of measuring cell-mediated immune
response include measurement of intracellular cytokines or cytokine
secretion by T-cell populations, or by measurement of epitope
specific T-cells (e.g., by the tetramer technique) (reviewed by
McMichael, A. J., and O'Callaghan, C. A, J. Exp. Med.
187(9)1367-1371, 1998; Mcheyzer-Williams, M. G., et al, Immunol.
Rev. 150:5-21, 1996; Lalvani, A., et al, J. Exp. Med. 186:859-865,
1997).
[0039] Thus, an immunological response as used herein may be one
that stimulates the production of CTLs, and/or the production or
activation of helper T-cells. The HIV antigen(s) may also elicit an
antibody-mediated immune response. Hence, an immunological response
may include one or more of the following effects: the production of
antibodies by B-cells; and/or the activation of suppressor T-cells
and/or .gamma..delta. T-cells directed specifically to an antigen
or antigens present in the composition or vaccine of interest.
These responses may serve to neutralize infectivity, and/or mediate
antibody-complement, or antibody dependent cell cytotoxicity (ADCC)
to provide protection to an immunized host. Such responses can be
determined using standard immunoassays and neutralization assays,
well known in the art.
[0040] An "immunogenic composition" is a composition that comprises
an antigenic molecule where administration of the composition to a
subject results in the development in the subject of a humoral
and/or a cellular immune response to the antigenic molecule of
interest. The immunogenic composition can be introduced directly
into a recipient subject, such as by injection, inhalation, oral,
intranasal and mucosal (e.g., intra-rectally or intra-vaginally)
administration.
[0041] By "subunit vaccine" is meant a vaccine composition that
includes one or more selected antigens but not all antigens,
derived from or homologous to, an antigen from a pathogen of
interest such as from a virus, bacterium, parasite or fungus. Such
a composition is substantially free of intact pathogen cells or
pathogenic particles, or the lysate of such cells or particles.
Thus, a "subunit vaccine" can be prepared from at least partially
purified (preferably substantially purified) immunogenic
polypeptides from the pathogen, or analogs thereof. The method of
obtaining an antigen included in the subunit vaccine can thus
include standard purification techniques, recombinant production,
or synthetic production.
[0042] "Substantially purified" general refers to isolation of a
substance (compound, polynucleotide, protein, polypeptide,
polypeptide composition) such that the substance comprises the
majority percent of the sample in which it resides. Typically in a
sample a substantially purified component comprises 50%, preferably
80%-85%, more preferably 90-95% of the sample. Techniques for
purifnng polynucleotides and polypeptides of interest are
well-known in the art and include, for example, ion-exchange
chromatography, affinity chromatography and sedimentation according
to density.
[0043] A "coding sequence" or a sequence that "encodes" a selected
polypeptide, is a nucleic acid molecule that is transcribed (in the
case of DNA) and translated (in the case of mRNA) into a
polypeptide in vivo when placed under the control of appropriate
regulatory sequences (or "control elements"). The boundaries of the
coding,sequence are determined by a start codon at the 5' (amino)
terminus and a translation stop codon at the 3' (carboxy) terminus.
A coding sequence can include, but is not limited to, cDNA from
viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from
viral or procaryotic DNA, and even synthetic DNA sequences. A
transcription termination sequence may be located 3' to the coding
sequence.
[0044] Typical "control elements", include, but are not limited to,
transcription promoters, transcription enhancer elements,
transcription termination signals, polyadenylation sequences
(located 3' to the translation stop codon), sequences for
optimization of initiation of translation (located 5' to the coding
sequence), and translation termination sequences.
[0045] A "nucleic acid" molecule can include, but is not limited
to, procaryotic sequences, eucaryotic mRNA, cDNA from eucaryotic
mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA,
and even synthetic DNA sequences. The term also captures sequences
that include any of the known base analogs of DNA and RNA.
[0046] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. Thus, a given promoter operably linked to a
coding sequence is capable of effecting the expression of the
coding sequence when the proper enzymes are present. The promoter
need not be contiguous with the coding sequence, so long as it
functions to direct the expression thereof. Thus, for example,
intervening untranslated yet transcribed sequences can be present
between the promoter sequence and the coding sequence and the
promoter sequence can still be considered "operably linked" to the
coding sequence.
[0047] "Recombinant" as used herein to describe a nucleic acid
molecule means a polynucleotide of genomic, cDNA, semisynthetic, or
synthetic origin which, by virtue of its origin or manipulation:
(1) is not associated with all or a portion of the polynucleotide
with which it is associated in nature; and/or (2) is linked to a
polynucleotide other than that to which it is linked in nature. The
term "recombinant" as used with respect to a protein or polypeptide
means a polypeptide produced by expression of a recombinant
polynucleotide. "Recombinant host cells," "host cells," "cells,"
"cell lines," "cell cultures," and other such terms denoting
procaryotic microorganisms or eucaryotic cell lines cultured as
unicellular entities, are used interchangeably, and refer to cells
which can be, or have been, used as recipients for recombinant
vectors or other transfer DNA, and include the progeny of the
original cell which has been transfected. It is understood that the
progeny of a single parental cell may not necessarily be completely
identical in morphology or in genomic or total DNA complement to
the original parent, due to accidental or deliberate mutation.
Progeny of the parental cell which are sufficiently similar to the
parent to be characterized by the relevant property, such as the
presence of a nucleotide sequence encoding a desired peptide, are
included in the progeny intended by this definition, and are
covered by the above terms.
[0048] Techniques for determining amino acid sequence "similarity"
are well known in the art. In general, "similarity" means the exact
amino acid to amino acid comparison of two or more polypeptides at
the appropriate place, where amino acids are identical or possess
similar chemical and/or physical properties such as charge or
hydrophobicity. A so-termed "percent similarity" then can be
determined between the compared polypeptide sequences. Techniques
for determining nucleic acid and amino acid sequence identity also
are well known in the art and include determining the nucleotide
sequence of the mRNA for that gene (usually via a cDNA
intermediate) and determining the amino acid sequence encoded
thereby, and comparing this to a second amino acid sequence. In
general, "identity" refers to an exact nucleotide to nucleotide or
amino acid to amino acid correspondence of two polynucleotides or
polypeptide sequences, respectively.
[0049] Two or more polynucleotide sequences can be compared by
determining their "percent identity." Two or more amino acid
sequences likewise can be compared by determining their "percent
identity." The percent identity of two sequences, whether nucleic
acid or peptide sequences, is generally described as the number of
exact matches between two aligned sequences divided by the length
of the shorter sequence and multiplied by 100. An approximate
alignment for nucleic acid sequences is provided by the local
homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2:482-489 (1981). This algorithm can be extended to use
with peptide sequences using the scoring matrix developed by
Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff
ed., 5 suppl. 3:353-358, National Biomedical Research Foundation,
Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res.
14(6):6745-6763 (1986). An implementation of this algorithm for
nucleic acid and peptide sequences is provided by the Genetics
Computer Group (Madison, Wis.) in their BestFit utility
application. The default parameters for-this method are described
in the Wisconsin Sequence Analysis Package Program Manual, Version
8 (1995) (available from Genetics Computer Group, Madison, Wis.).
Other equally suitable programs for calculating the percent
identity or similarity between sequences are generally known in the
art.
[0050] For example, percent identity of a particular nucleotide
sequence to a reference sequence can be determined using the
homology algorithm of Smith and Waterman with a default scoring
table and a gap penalty of six nucleotide positions. Another method
of establishing percent identity in the context of the present
invention is to use the MPSRCH package of programs copyrighted by
the University of Edinburgh, developed by John F. Collins and Shane
S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain
View, Calif.). From this suite of packages, the Smith-Waterman
algorithm can be employed where default parameters are used for the
scoring table (for example, gap open penalty of 12, gap extension
penalty of one, and a gap of six). From the data generated, the
"Match" value reflects "sequence identity." Other suitable programs
for calculating the percent identity or similarity between
sequences are generally k)own in the art, such as the alignment
program BLAST, which can also be used with default parameters. For
example, BLASTN and BLAST? can be used with the following default
parameters: genetic code=standard; filter=none; strand=both;
cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences;
sort by=HIGH SCORE; Databases=non-redundant,
GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss
protein+Spupdate+PIR. Details of these programs can be found at the
following internet address:
http://www.ncbi.nlm.gov/cgi-bin/BLAST.
[0051] One of skill in the art can readily determine the proper
search parameters to use for a given sequence in the above
programs. For example, the search parameters may vary based on the
size of the sequence in question Thus, for example, a
representative embodiment of the present invention would include an
isolated polynucleotide having X contiguous nucleotides, wherein
(i) the X contiguous nucleotides have at least about 50% identity
to Y contiguous nucleotides derived from any of the sequences
described herein, (ii) X equals Y, and (iii) X is greater than or
equal to 6 nucleotides and up to 5000 nucleotides, preferably
greater than or equal to 8 nucleotides and up to 5000 nucleotides,
more preferably 10-12 nucleotides and up to 5000 nucleotides, and
even more preferably 15-20 nucleotides, up to the number of
nucleotides present in the full-length sequences described herein
(e.g., see the Sequence Listing and claims), including all integer
values falling within the above-described ranges.
[0052] The polynucleotides described herein include related
polynucleotide sequences having about 80% to 100%, greater than
80-85%, preferably greater than 90-92%, more preferably greater
than 95%, and most preferably greater than 98% sequence (including
all integer values faling within these described ranges) identity
to the sequences disclosed herein (for example, to the claimed
sequences or other sequences of the present invention) when the
sequences of the present invention are used as the query
sequence.
[0053] Two nucleic acid fragments are considered to "selectively
hybridize" as described herein. The degree of sequence identity
between two nucleic acid molecules affects the efficiency and
strength of hybridization events between such molecules. A
partially identical nucleic acid sequence will at least partially
inhibit a completely identical sequence from hybridizing to a
target molecule. Inhibition of hybridization of the completely
identical sequence can be assessed using hybridization assays that
are well known in the art (e.g., Southern blot, Northern blot,
solution hybridization, or the like, see Sambrook, et al., supra or
Ausubel et al., supra). Such assays can be conducted using varying
degrees of selectivity, for example, using conditions varying from
low to high stringency. If conditions of low stringency are
employed, the absence of non-specific binding can be assessed using
a secondary probe that lacks even a partial degree of sequence
identity (for example, a probe having less than about 30% sequence
identity with the target molecule), such that, in the absence of
non-specific binding events, the secondary probe will not hybridize
to the target.
[0054] When utilizing a hybridization-based detection system, a
nucleic acid probe is chosen that is complementary to a target
nucleic acid sequence, and then by selection of appropriate
conditions the probe and the target sequence "selectively
hybridize," or bind, to each other to form a hybrid molecule. A
nucleic acid molecule that is capable of hybridizing selectively to
a target sequence under "moderately stringent" typically hybridizes
under conditions that allow detection of a target nucleic acid
sequence of at least about 10-14 nucleotides in length having at
least approximately 70% sequence identity with the sequence of the
selected nucleic acid probe. Stringent hybridization conditions
typically allow detection of target nucleic acid sequences of at
least about 10-14 nucleotides in length having a sequence identity
of greater than about 90-95% with the sequence of the selected
nucleic acid probe. Hybridization conditions useful for
probe/target hybridization where the probe and target have a
specific degree of sequence identity, can be determined as is known
in the art (see, for example, Nucleic Acid Hybridization: A
Practical Approach, editors B. D. Hames and S. J. Higgins, (1985)
Oxford; Washington, D.C.; IRL Press).
[0055] With respect to stringency conditions for hybridization, it
is well known in the art that numerous equivalent conditions can be
employed to establish a particular stringency by varying, for
example, the following factors: the length and nature of probe and
target sequences, base composition of the various sequences,
concentrations of salts and other hybridization solution
components, the presence or absence of blocking agents in the
hybridization solutions (e.g., formamide, dextran sulfate, and
polyethylene glycol), hybridization reaction temperature and time
parameters, as well as, varying wash conditions. The selection of a
particular set of hybridization conditions is selected following
standard methods in the art (see, for example, Sambrook, et al.,
supra or Ausubel et al., supra).
[0056] A first polynucleotide is "derived from" second
polynucleotide if it has-the same or substantially the same
basepair sequence as a region of the second polynucleotide, its
cDNA, complements thereof, or if it displays sequence identity as
described above.
[0057] A first polypeptide is "derived from" a second polypeptide
if it is (i) encoded by a first polynucleotide derived from a
second polynucleotide, or (ii) displays sequence identity to the
second polypeptides as described above.
[0058] Generally, a viral polypeptide is "derived from" a
particular polypeptide of a virus (viral polypeptide) if it is (i)
encoded by an open reading frame of a polynucleotide of that virus
(viral polynucleotide), or (ii) displays sequence identity to
polypeptides of that virus as described above.
[0059] "Encoded by" refers to a nucleic acid sequence which codes
for a polypeptide sequence, wherein the polypeptide sequence or a
portion thereof contains an amino acid sequence of at least 3 to 5
amino acids, more preferably at least 8 to 10 amino acids, and even
more preferably at least 15 to 20 amino acids from a polypeptide
encoded by the nucleic acid sequence. Also encompassed are
polypeptide sequences which are immunologically identifiable with a
polypeptide encoded by the sequence.
[0060] "Purified polynucleotide" refers to a polynucleotide of
interest or fragment thereof that is essentially free, e.g.,
contains less than about 50%, preferably less than about 70%, and
more preferably less than about 90%, of the protein with which the
polynucleotide is naturally associated. Techniques for purifying
polynucleotides of interest are well-known in the art and include,
for example, disruption of the cell containing the polynucleotide
with a chaotropic agent and separation of the polynucleotide(s) and
proteins by ion-exchange chromatography, affinity chromatography
and sedimentation according to density.
[0061] By "nucleic acid immunization" is meant the introduction of
a nucleic acid molecule encoding one or more selected antigens into
a host cell, for the in vivo expression of an antigen, antigens, an
epitope, or epitopes. The nucleic acid molecule can be introduced
directly into a recipient subject, such as by injection,
inhalation, oral, intranasal and mucosal administration, or the
like, or can be introduced ex vivo, into cells which have been
removed from the host. In the latter case, the transformed cells
are reintroduced into the subject where an immune response can be
mounted against the antigen encoded by the nucleic acid
molecule.
[0062] "Gene transfer" or "gene delivery" refers to methods or
systems for reliably inserting DNA of interest into a host cell.
Such methods can result in transient expression of non-integrated
transferred DNA, extrachromosomal replication and expression of
transferred replicons (e.g., episomes), or integration of
transferred genetic material into the genomic DNA of host cells.
Gene delivery expression vectors include, but are not limited to,
vectors derived from alphaviruses, pox viruses and vaccinia
viruses. When used for immunization, such gene delivery expression
vectors may be referred to as vaccines or vaccine vectors.
[0063] "T lymphocytes" or "T cells" are non-antibody producing
lymphocytes that constitute a part of the cell-mediated arm of the
immune system. T cells arise from immature lymphocytes that migrate
from the bone marrow to the thymus, where they undergo a maturation
process under the direction of thymic hormones. Here, the mature
lymphocytes rapidly divide increasing to very large numbers. The
maturing T cells become immunocompetent based on their ability to
recognize and bind a specific antigen. Activation of
immunocompetent T cells is triggered when an antigen binds to the
lymphocyte's surface receptors.
[0064] The term "transfection" is used to refer to the uptake of
foreign DNA by a cell. A cell has been "transfected" when exogenous
DNA has been introduced inside the cell membrane. A number of
transfection techniques are generally known in the art. See, e.g.,
Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989)
Molecular Cloning, a laboratory manual, Cold Spring Harbor
Laboratories, New York, Davis et al. (1986) Basic Methods in
Molecular Biology, Elsevier, and Chu et al (1981) Gene 13:197. Such
techniques can be used to introduce one or more exogenous DNA
moieties into suitable host cells. The term refers to both stable
and transient uptake of the genetic material, and includes uptake
of peptide- or antibody-linked DNAs.
[0065] Transfer of a "suicide gene" (e.g., a drug-susceptibility
gene) to a target cell renders the cell sensitive to compounds or
compositions that are relatively nontoxic to normal cells. Moolten,
F. L. (1994) Cancer Gene Ther. 1:279-287. Examples of suicide genes
are thymidine kinase of herpes simplex virus (HSV-tk), cytochrome
P450 (Manome et al. (1996) Gene Therapy 3:513-520), human
deoxycytidine kinase (Manome et al. (1996) Nature Medicine
2(5):567-573) and the bacterial enzyme cytosine deaminase Dong et
al. (1996) Human Gene Therapy 7:713-720). Cells that express these
genes are rendered sensitive to the effects of the relatively
nontoxic prodrugs ganciclovir (HSV-tk), cyclophosphamide
(cytochrome P450 2B1), cytosine arabinoside (human deoxycytidine
kinase) or 5-fluorocytosine (bacterial cytosine deaminase). Culver
et al. (1992) Science 256:1550-1552, Huber et al. (1994) Proc.
Natl. Acad. Sci. USA 91:8302-8306.
[0066] A "selectable marker" or "reporter marker" refers to a
nucleotide sequence included in a gene transfer vector that has no
therapeutic activity, but rather is included to allow for simpler
preparation, manufacturing, characterization or testing of the gene
transfer vector.
[0067] A "specific binding agent" refers to a member of a specific
binding pair of molecules wherein one of the molecules specifically
binds to the second molecule through chemical and/or physical
means. One example of a specific binding agent is an antibody
directed against a selected antigen.
[0068] By "subject" is meant any member of the subphylum chordata,
including, without limitation, humans and other primates, including
non-human primates such as chimpanzees and other apes and monkey
species; farm animals such as cattle, sheep, pigs, goats and
horses; domestic mammals such as dogs and cats; laboratory animals
including rodents such as mice, rats and guinea pigs; birds,
including domestic, wild and game birds such as chickens, turkeys
and other gallinaceous birds, ducks, geese, and the like. The term
does not denote a particular age. Thus, both adult and newborn
individuals are intended to be covered. The system described above
is intended for use in any of the above vertebrate species, since
the immune systems of all of these vertebrates operate
similarly.
[0069] By "pharmaceutically acceptable" or "pharmacologically
acceptable" is meant a material which is not biologically or
otherwise undesirable, i.e., the material may be administered to an
individual in a formulation or composition without causing any
undesirable biological effects or interacting in a deleterious
manner with any of the components of the composition in which it is
contained.
[0070] By "physiological pH" or a "pH in the physiological range"
is meant a pH in the range of approximately 7.2 to 8.0 inclusive,
more typically in the range of approximately 7.2 to 7.6
inclusive.
[0071] As used herein, "treatment" refers to any of (i) the
prevention of infection or reinfection, as in a traditional
vaccine, (ii) the reduction or elimination of symptoms, and/or
(iii) the substantial or complete elimination of the pathogen in
question. Treatment may be effected prophylactically (prior to
infection) or therapeutically (following infection).
[0072] "Nucleic acid expression vector" refers to an assembly that
is capable of directing the expression of a sequence or gene of
interest. The nucleic acid expression vector may include a promoter
that is operably linked to the sequences or gene(s) of interest.
Other control elements may be present as well. Nucleic acid
expression vectors include, but are not limited to, plasmids, viral
vectors, alphavirus vectors (e.g., Sindbis), eukaryotic layered
vector initiation systems (see, e.g., U.S. Pat. No. 6,342,372),
retroviral vectors, adenoviral vectors, adeno-associated virus
vectors and the like. See, also, U.S. Pat. No. 6,602,705 for a
description of various nucleic acid expression vectors. Expression
cassettes-maybe contained within a nucleic acid expression vector.
The vector may also include a bacterial origin of replication, one
or more selectable markers, a signal that allows the construct to
exist as single-stranded DNA (e.g., a M13 origin of replication), a
multiple cloning site, and a "mammalian" origin of replication
(e.g., a SV40 or adenovirus origin of replication).
[0073] "Packaging cell" refers to a cell that contains those
elements necessary for production of infectious recombinant
retrovirus that are lacking in a recombinant retroviral vector.
Typically, such packaging cells contain one or more expression
cassettes which are capable of expressing proteins which encode
Gag, pol and env proteins.
[0074] "Producer cell" or "vector producing cell" refers to a cell
that contains all elements necessary for production of recombinant
retroviral vector particles.
[0075] In addition, the following is a partial list of
abbreviations used herein: TABLE-US-00001 .mu.g microgram AIDS
acquired immune deficiency syndrome APC antigen presenting cell
CCR5 chemokine receptor 5 CD4+ cluster of differeniation 4 receptor
CD8+ cluster of differeniation 8 receptor CDC centers for disease
control CHO cells Chinese hamster ovary cells CMV cytomegalovirus
ConA Concanvalim A CRF case report form CRF's circulating
recombinant forms CTAB cetyltrimetylamnonium bromide CTL cytotoxic
T lymphocyte Cv cromium DEAE Diethylaminoethyl DNA deoxyribonucleic
acid DTH delayed type hypersensitivity ELISA enzyme-linked
immunosorbent assay ELISPOT enzyme-linked immunospot assay ENV
envelope FIGE field inversion gel electrophoresis GAG
group-specific antigen GLP good laboratory practices gp
glycoprotein HAART highly active antiretroviral therapy HAP
hydroziapatic HBsAg hepatitis B surface antigen HCV hepatitis C
virus HIV/HIV-1 human immunodeficiency virus/Type 1 hr hour HSV
herpes simplex virus IFN interferon IFN.gamma. interferon gamma IM
intramuscular IND investigational new drug IV intravenous Kb
kilobase kD kilodalton Kg kilogram mg milligram mL milliliter MF59
oil-in-water emulsion adjuvant NaCl sodium chloride NIAID National
Institute of Allergy and Infectious Disease NIH National Institutes
of Health o- or O- oligomeric PCR polymerase chain reaction PEG
polyethylene glycol PLG cationic poly-lactide-coglycolide pSIN
sindbis virus vector PVA poly(vinyl alcohol) REV viral protein -
involved in regulation of viral expression SAE serious adverse
event SHIV simian human immunodefiency virus SP resin modified
polyester-carbonate resin
[0076] General Overview
[0077] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
formulations or process parameters as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments of the invention
only, and is not intended to be limiting.
[0078] Although a number of methods and materials similar or
equivalent to those described herein can be used in the practice of
the present invention, the preferred materials and methods are
described herein.
[0079] The present invention relates to methods and compositions
for the development of immunogenic compositions (e.g., vaccines)
for HIV. For example, an HIV vaccine as described herein may
include three or more components. Vaccines as described herein may
be intended for intramuscular injection. In certain embodiments,
two nucleic acid components are formulated onto (adsorbed onto)
cationic poly-lactide-coglycolide (PLG) microparticles and
administered as priming immunizations. In addition to the DNA
components, a protein composition is also administered in one or
more boosting immunizations. The protein component typically
comprises at least one HIV polypeptide, for example, a CHO
cell-produced, recombinant oligomeric envelope protein with a
deletion in the V2 region mixed with the MF-59 adjuvant.
[0080] Pharmaceutic Compositions
[0081] In a preferred embodiment, the HIV vaccines described herein
includes multiple (e.g., three or more) components intended for
administration (e.g., intramuscularly) in a 6-9 month, or even
longer, time period. The components may be given concurrently or at
different time points. For example, two nucleic acid "priming"
immunizations may be given, where each priming immunization
includes include two separate preparations of DNA encoding Gag
protein(s) (e.g., p55 Gag from HIV-1 SF2), and/or Env protein(s)
(e.g., an oligomeric, V2-deleted, gp140 envelope protein from HIV-1
SF162), both formulated on PLG microparticles. The nucleic acids
will typically be provided separately in unit dose vials containing
between 1 .mu.g to 10 mg of DNA and between 10 .mu.g and 100 mg of
PLG (e.g., 1 mg of DNA and 25 mg of PLG microparticles). The
DNA-containing doses are typically stored in lyophilized form and
vials are generally reconstituted in the field. It should be noted
that each unit dose vial will typically contain more DNA (or
protein) than is actually administered to the patient. The final
dosage typically consists of 1 mg in 0.5 mL each of Gag and Env
DNA. The DNA components of the vaccine are intended to prime
antibody, CD4 and CD8 T cell responses to HIV antigens (e.g., Gag
and Env).
[0082] As noted above, the immunogenic systems (vaccines) described
herein also comprise at least one protein component, typically an
HIV polypeptide from any isolate or strain of HIV. For example, in
certain embodiments, the protein component comprises a recombinant
oligomeric envelope protein from the SF162 strain of HIV-1. Protein
monomers of HIV Env maybe truncated to an approximate molecular
size of 140 kD (e.g., to improve solubility) and the V2 loop may be
at least partially removed. The resulting oligomeric molecule
resembles the envelope structure of HIV closely. Removal of the V2
variable loop exposes conserved epitopes involved in receptor
and/or co-receptor binding. Macaques primed with naked DNA vaccines
encoding oligomeric V2-deleted gp140 from the subtype B (CCR5)
primary isolate SF162, and boosted with the corresponding
recombinant protein, produced antibodies capable of neutralizing a
range of distinct subtype B primary isolates. Barnett et al. (2001)
J Virol. 75(12):5526-40; Srivastava et al. (2002) J Virol.
(6):2835-47; Srivastava et al. (2003) J. Virol.
77(20):11244-11259.
[0083] Based on the quantities of passively administered antibodies
required to protect macaques and the magnitude and breadth of the
neutralization titers seen in macaque studies, suggest that the
antibodies induced by vaccines described herein are likely to
provide protection from infection in a proportion of animals.
Mascola et al. (1999) J Virol. 73(5): 4009-18. The amount of
protein per does can vary from microgram to milligram amounts. In
certain embodiments, the protein is provided such that the
dose-administered is approximately 100 micrograms in unit dose
vials containing envelope protein in sodium citrate buffer, pH 6.0
without preservative.
[0084] The protein and/or nucleic acid compositions described
herein may also comprise a pharmaceutically acceptable carrier. The
carrier should not itself induce the production of antibodies
harmful to the host. Pharmaceutically acceptable carriers are well
known to those in the art. Suitable carriers are typically large,
slowly metabolized macromolecules such as proteins,
polysaccharides, polylactic acids, polyglycolic acids, polymeric
amino acids, amino acid copolymers, lipid aggregates (such as oil
droplets or liposomes), and inactive virus particles. Examples of
particulate carriers include those derived from polymethyl
methacrylate polymers, as well as microparticles derived from
poly(lactides) and poly(lactide-co-glycolides), known as PLG. See,
e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee et al.
(1997) J Microencapsul. 14(2):197-210; O'Hagan et al. (1993)
Vaccine 11(2): 149-54. Such carriers are well known to those of
ordinary skill in the art. Additionally, these carriers may
function as immunostimulating agents ("adjuvants"). Furthermore,
the antigen may be conjugated to a bacterial toxoid, such as toxoid
from diphtheria, tetanus, cholera, etc., as well as toxins derived
from E. coli.
[0085] Pharmaceutically acceptable salts can also be used in
compositions of the invention, for example, mineral salts such as
hydrochlorides, hydrobromides, phosphates, or sulfates, as well as
salts of organic acids such as acetates, proprionates, malonates,
or benzoates. Especially useful protein substrates are serum
albumins, keyhole limpet hemocyanin, immunoglobulin molecules,
thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well
known to those of skill in the art. Compositions of the invention
can also contain liquids or excipients, such as water, saline,
glycerol, dextrose, ethanol, or the like, singly or in combination,
as well as substances such as wetting agents, emulsifying agents,
or pH buffering agents. Liposomes can also be used as a carrier for
a composition of the invention, such liposomes are described
above.
[0086] Briefly, with regard to viral particles,
replication-defective vectors (also referred to above as particles)
may be preserved either in crude or purified forms. Preservation
methods and conditions are described in U.S. Pat. No.
6,015,694.
[0087] Further, the compositions described herein can include
various excipients, adjuvants, carriers, auxiliary substances,
modulating agents, and the like. Preferably, the compositions will
include an amount of the antigen sufficient to mount an
immunological response. An appropriate effective amount can be
determined by one of skill in the art. Such an amount will fall in
a relatively broad range that can be determined through routine
trials and will generally be an amount on the order of about 0.1
.mu.g to about 1000 .mu.g (e.g., antigen and/or particle), more
preferably about 1 .mu.g to about 300 .mu.g, of
particle/antigen.
[0088] As noted above, one or more of the components may further
comprise one or more adjuvants. Preferred adjvuants to enhance
effectiveness include of the composition includes, but are not
limited to: (1) aluminum salts (alum), such as aluminum hydroxide,
aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water
emulsion formulations (with or without other specific
immunostimulating agents such as muramyl peptides (see below) or
bacterial cell wall components), such as for example (a) MF59.TM.
(International Publication No. WO 90/14837; Chapter 10 in Vaccine
design: the subunit and adjuvant approach, eds. Powell &
Newman, Plenus Press, 1995), containing 5% Squalene, 0.5% Tween 80,
and 0.5% Span 85 (optionally containing various amounts of MTP-PE)
formulated into submicron particles using a microfluidizer, (b)
SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked
polymer L121, and thr-MDP (see below) either microfluidized into a
submicron emulsion or vortexed to generate a larger particle size
emulsion, and (c) Ribi.TM. adjuvant system (RAS), (Ribi Immunochem,
Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or
more bacterial cell wall components fiom the group consisting of
monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell
wall skeleton (CWS), preferably MPL+CWS (Detox.TM.); (3) saponin
adjuvants, such as QS21 or Stimulon.TM. (Cambridge Bioscience,
Worcester, Mass.) may be used or particle generated therefrom such
as ISCOMs (immunostimulating complexes), which ISCOMS maybe devoid
of additional detergent (see, e.g., WO 00/07621); (4) Complete
Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5)
cytokines, such as interleukins (IL-1, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-12 (WO 99/44636), IL16, etc.), interferons (e.g., gamma
interferon), macrophage colony stimulating factor (M-CSF), tumor
necrosis factor (TNF), beta chemokines (MIP, 1-alpha, 1-beta
Rantes, etc.), etc.; (6) monophosphoryl lipids A (MPL) or
3-O-deacylated MPL (3dMPL) e.g., GB-222021, EP-A-0689454,
optionally in the substantial absence of alum when used with
pneumococcal saccharides e.g., WO 00/56358; (7) combinations of
3dMPL with, for example, QS21 and/or oil-in-water emulsions e.g.,
EP-A-0835318, EP-A-0735898, EP-A-0761231; (8) oligonucleotides
comprising CpG motifs (Roman et al., Nat. Med., 1997, 3:849-854;
Weiner et al., PNAS USA, 1997, 94:10833-10837; Davis et al. J.
Immunol., 1998, 160:870-876; Chu et al., J. Exp. Med., 1997,
186:1623-1631; Lipford et al. Eur. J Immunol. 1997, 27:2340-2344;
Moldoveanu et al., Vaccine, 1988, 16:1216-1224, Krieg et al.,
Nature, 1995, 3742:546-549; Klinman et al., PNAS USA, 1996,
93:2879-2883: Ballas et al., J Immunol., 1996, 157:1840-1845;
Cowdery et al., J Immunol., 1996, 156:4570-4575; Halpern et al.,
Cell. Immunol., 1996, 167:72-78; Yamamoto et al., Jpn. J Cancer
Res., 1988, 79:866-873; Stacey et al., J Immunol, 1996,
157:2116-2122; Messina et al., J Immunol., 1991, 147:17591764; Yi
et al., J Immunol., 1996, 157:4918-4925; Yi et al., J Immunol.,
1996, 157:5394-5402; Yi et al., J Immunol., 1998, 160:4755-4761;
and Yi et al., J Immunol., 1998, 1605:5898-5906; International
patent applications WO96/02555, WO98/16247, WO98/18810, WO98/401005
WO98/55495, WO98/37919 and WO98/52581) i.e. containing at least one
CG dinucleotide, with 5 methylcytosine optionally being used in
place of cytosine; (8) a polyoxyethylene ether or a polyoxyethylone
ester e.g. WO 99/52549; (9) a polyoxyethylene sorbitan ester
surfactant in combination with an octoxynol (WO 01/21207) or a
polyoxyethylene alkyl ether or ester surfactant in combination with
at least one additional non-ionic surfactant such as an octoxynol
(WO 01/21152); (10) a saponin and an immunostimulatory
oligonucleotide (e.g., a CpG oligonucleotide) (WO 00/62800); (11)
an immunostimulant and a particle of metal salt e.g. WO 00/23105;
(12) a saponin and oil-in-water emulsion e.g., WO 99/11241; (13) a
saponin (e.g., QS21)+3dMPL=IL-12 (optionally+a sterol) e.g., WO
98/57659; (14) other substances that act as immunostimulating
agents to enhance the effectiveness of the composition. Alum
(especially aluminum phosphate and/or hydroxide) and MW59.TM. are
preferred
[0089] Muramyl peptides include, but are not limited to,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1'-2'-dipalmitoyl-s-
n-glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
[0090] Administration of the pharmaceutical compositions described
herein may be by any suitable route (see, e.g., Section C).
Particularly preferred is intramuscular or mucosal (e.g., rectal
and/or vaginal) administration. Dosage treatment may be a single
dose schedule or a multiple dose schedule. A multiple dose schedule
is one in which a primary course of vaccination may be with 1-10
separate doses, followed by other doses given at subsequent time
intervals, chosen to maintain and/or reinforce the immune response,
for example at 1 to 6 months for a second dose, and if needed, a
subsequent dose(s) after several months. The dosage regimen will
also, at least in part, be determined by the potency of the
modality, the vaccine delivery employed, the need of the subject
and be dependent on the judgment of the practitioner.
[0091] In certain embodiments, the protein component is mixed
before administration with a proprietary oil-in-water emulsion
adjuvant, MF59C.1 (hereafter referred to as MF59) (See, e.g,
International Publication No. WO 90/14837). Various subunit
antigens (e.g., HCV E2, HIV gp120, HBsAg, CMV gB, and HSV 2 gD)
have been combined with MF59 adjuvant and administered to over
18,000 human subjects to date with an excellent safety and
tolerability profile. The protein booster is intended to amplify
the primary antibody and CD4+ T cell responses in breadth and
duration and to provide a balanced response in both the humoral and
cellular compartments of the immune system, capable to achieve the
prevention of HIV-1 infection.
[0092] As noted above, MF59 adjuvant has been extensively evaluated
in clinical trials with a number of different subunit antigens,
including those derived from influenza, herpes simplex virus 2
(HSV), human immunodeficiency virus (HIV), cytomegalovirus (CMV),
and hepatitis B virus (HBV) and is generally well tolerated with
minimal local and systemic adverse reactions that are transient and
of mild-to-moderate severity. Over 12,000 subjects have received
influenza virus vaccines combined with MF59 adjuvant emulsion in
more than 30 clinical studies. Only two patients had serious
adverse effects. Moreover, the incidence of adverse effects depend
upon the antigen used.
[0093] Prime-Boost Regimes
[0094] In certain embodiments, multiple administrations (e.g.,
prime-boost type administration) will be advantageously employed.
For example, nucleic acid constructs expressing one or more HIV
antigen(s) of interest are administered. Subsequently, the same
and/or different HIV antigen(s) are administered, for example in
compositions comprising the polypeptide antigen(s) and a suitable
adjuvant. Alternatively, antigens are administered prior to the
DNA. Multiple polypeptide and multiple nucleic acid administrations
(in any order) may also be employed.
[0095] As described herein, one exemplary prime-boost regime
described herein includes two or more administrations of
DNAs-encoding one or more HIV antigens followed by one or more
administrations of HIV polypeptide antigens themselves. For
example, two or more administrations of HIV Gag and HIV Env DNA/PLG
compositions (e.g. separate Gag and Env) may be followed by one or
more administration of HIV Env protein. HIV-1 DNA constructs are
able to stimulate the cellular and humoral arms of the immune
system and elicit immune responses capable of preventing HIV-1
infection in chimpanzees. Boyer et al., (1997) Nat Med 3:526-532.
Adsorption of DNA onto the surface of PLG microparticles improves
DNA uptake by the antigen presenting cells (APCs), and enhance
cellular and humoral immune responses. O'Hagan et al. (2001) J
Virol. 75(19):9037-43. PLG is particularly preferred to deliver DNA
because the polymer is biodegradable, biocompatible and has been
used to develop several drug delivery systems. Okada et al. (1997)
Adv Drug Deliv Rev 28(1):43-70. In certain embodiments, the ratio
of DNA:PLG is between about 1 and 16 w/w % (or any value
therebetween).
[0096] The "booster" component comprises an HIV protein from any
HIV strain or subtype, for example a recombinant oligomeric
envelope protein from the subtype B strain (e.g., SF2, SF162, etc.)
and/or subtype C strain (Botswana strains and/or South African
strains such as TV1). See, e.g., Scriba et al. (2001) AIDS Res Hum
Retroviruses 17(8):775-81; Scriba et al. (2002) AIDS Res Hum
Retroviruses 18(2):149-59; Treurnicht et al. (2002) J Med Virol.
68(2):141-6. The protein monomers of the Env protein may be
truncated and the V2 loop partially removed to increase the
exposure of conserved epitopes that are more efficient to elicit
cross-reactive neutralizing antibody. Without being bound by one
theory, it appears that the protein booster is intended to amplify
the primary antibody and CD4+ T cell responses in breadth and
duration. Barnett et al. (2001) J Virol 75(12):5526-40; Cherpelis
et al. (2001) J. Virol. 75(3):1547-50. The concentration of protein
in each dose may vary from approximately 1 .mu.g to over 1000 .mu.g
(or any value therebetween), preferably between about 10 .mu.g and
500 .mu.g, and even more preferably between about 30 .mu.g and 300
.mu.g.
[0097] To date, HIV vaccines as described herein have demonstrated
a strong record of safety in preclinical studies and clinical
trials. See, also, Example 4 below. No evidence of vaccine-related
immunodeficiency has been reported. Toxicology studies conducted in
mice and rabbits with the HIV vaccine demonstrated that the vaccine
was very well tolerated. Findings were consistent with studies
conducted with other viral subunit vaccines or with MF59 adjuvant.
Reversible local (intramuscular) inflammation is the only notable
change seen with such vaccines (see Example 4).
[0098] The goal of the HIV vaccine development program is to
demonstrate the safety and efficacy of a novel DNA-prime plus
recombinant protein-boost HIV vaccine, that is capable of eliciting
a combination of broad humoral and cellular responses, and
preventing HIV infection or the development of advanced HIV
disease/AIDS.
[0099] Sources of HIV Antigens
[0100] Polynucleotide sequences (e.g., for use in nucleic acid
expression constructs) can be obtained using recombinant methods,
such as by screening cDNA and genomic libraries from cells
expressing the gene, or by deriving the gene from a vector known to
include the same. Furthermore, the desired gene can be isolated
directly from cells and tissues containing the same, using standard
techniques, such as phenol extraction and PCR of cDNA or genomic
DNA. See, e.g., Sambrook et al., supra, for a description of
techniques used to obtain and isolate DNA. The gene of interest can
also be produced synthetically, rather than cloned. The nucleotide
sequence can be designed with the appropriate codons for the
particular amino acid sequence desired. In general, one will select
preferred codons for the intended host in which the sequence will
be expressed. The complete sequence is assembled from overlapping
oligonucleotides prepared by standard methods and assembled into a
complete coding sequence. See, e.g., Edge, Nature (1981) 292:756;
Nambair et al., Science (1984) 223:1299; Jay et al., J. Biol. Chem.
(1984) 259:6311; Stemmer, W. P. C., (1995) Gene 164:49-53.
[0101] Next, the gene sequence encoding the desired antigen can be
inserted into a vector as described for example, in U.S. Pat. No.
6,602,705 and International Patent Publications WO 00/39302; WO
02/04493; WO 00/39303; and WO 00/39304, which describe suitable
exemplary nucleic acid expression vectors and methods of obtaining
additional vectors useful in the compositions and methods,
described herein.
[0102] Expression constructs (e.g., plasmids) typically include
control elements operably linked to the coding sequence, which
allow for the expression of the gene in vivo in the subject
species. For example, typical promoters for mammalian cell
expression include the SV40 early promoter, a CMV promoter such as
the CMV immediate early promoter; the mouse mammary tumor virus LTR
promoter, the adenovirus major late promoter (Ad MLP), and the
herpes simplex virus promoter, among others. Other nonviral
promoters, such as a promoter derived from the murine
metatlothionein gene, will also find use for mammalian expression.
Typically, transciption termination and polyadenylation sequences
will also be present, located 3' to the translation stop codon.
Preferably, a sequence for optimization of initiation of
translation, located 5' to the coding sequence, is also present.
Examples of transcription terminator/polyadenylation signals
include those derived from SV40, as described in Sambrook et al.,
supra, as well as a bovine growth hormone terminator sequence.
[0103] Enhancer elements may also be used herein to increase
expression levels of the mammalian constructs. Examples include the
SV40 early gene enhancer, as described in Dijkema et al., EMBO J.
(1985) 4:761, the enhancer/promoter derived from the long terminal
repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et
al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and elements
derived from human CMV, as described in Boshart et al., Cell (1985)
41:521, such as elements included in the CMV intron A sequence.
[0104] Furthermore, HIV polypeptide-encoding nucleic acids can be
constructed which include a chimeric antigen-coding gene sequences,
encoding, e.g., multiple antigens/epitopes of interest, for example
derived from one or more viral isolates. Alternatively,
multi-cistronic cassettes (e.g., bi-cistronic cassettes) can be
constructed allowing expression of multiple antigens from a single
mRNA using the EMCV IRES, or the like.
[0105] Further, the HIV antigens (and polynucleotides encoding
these antigens) used in the claimed formulations may be obtained
from one or more subtypes of HIV. There are three distinct branches
in the phylogenetic tree of HIV-1 sequences, among these, the M
(main) viruses account for almost all of the human infections
worldwide. The M-group viruses have been divided into 9 distinct
genetic subtypes or clades (A through K). Worldwide, the subtypes A
and C account for most of the infections, these subtypes are most
common in southern Africa and India, The subtype B is dominant in
the American continent, Australia and Europe. Malim et al. (2001)
Cell 104(4): 469-72. These subtypes are followed in frequency by
newer circulating recombinant forms (CRFs). HIV-1 displays an
unprecedented genetic diversity within a subtype and even within a
single individual. Kwong et al. (2000) J Virol. 74(4): 1961-72.
This diversity is simply enormous when compared to the diversity
found in viruses for which effective vaccines have been developed.
Moore et al. (2001) J Virol. 75(13): 5721-9. Thus, though vaccines
described herein are typically developed based on dominant genetic
subtypes, for HIV, effective vaccines against a specific subtype
can be readily generated using the teachings herein.
INDUSTRIAL APPLICABILITY
[0106] The discovery that HIV was the etiological agent of AIDS in
1983-84 raised hopes for the rapid development of a vaccine. More
than 40 candidate HIV vaccines have already been tested in phase I
and II clinical trials, and the first phase II trials are now under
way in the United States and Thailand. Esparza, J. (2001) Bull
World Health Organ. 79(12): 1133-7. However, a major impediment for
the development of the vaccine has been the lack of scientific
evidence on the immunological correlates of protection against HIV
and AIDS. Clerici et al. (1996) Immunol Lett 51(1-2):69-73. Even
though most HIV infected individuals develop broad immunological
responses against the virus, these responses are incapable of
eliminating the infection or preventing disease progression. This
problem is further complicated by the fact that HIV strains vary
significantly in different parts of the world. HIV exhibits
extensive genetic sequence heterogeneity, particularly in the genes
encoding for viral envelope proteins. Different subtype viruses can
combine among themselves, generating additional circulating
recombinant forms (CRFs). McCutchan et al. (1996) J. Virol.
70(6):3331-3338.
[0107] Using vaccination to induce a specific anti HIV-1 immune
response that is more effective than the natural response to the
HIV-1 infection has proven difficult to achieve. In most of the
infections for which vaccines are effective, viremia or bacteremia
is a critical phase that permits the immune system to contain the
pathogen before it reaches the target organ. Ada et al. (2001) New
Engl. J Med. 345:1042-1053. Thus, it has been postulated that the
lack of adequate immune control of HIV-1 is likely due to several
factors, including HIV-1's ability to infect and deplete CD4+
cells, the main target during the initial phase of viremia (Greene
et al. (2002) Nat Med. 8(7):673-80); HIV-1's ability to mutate the
sequence of its surface antigens rapidly, the fact that HIV-1 is a
weak immunogen that has the ability to mask surface epitopes that
would otherwise be recognized by neutralizing antibodies; and/or
the fact that HIV can evade cellular immune responses and establish
latent infection at sites that are, inaccessible to the immune
system (Gotch et al. (2000) Curr Opin Infect Dis 13(1):13-17).
[0108] Further, although most licensed vaccines elicit both
cellular and antibody responses, little is understood about how
these known vaccines actually protect against infection. It has
been postulated that functional antibody responses can eliminate
the inoculum either by killing bacteria, inactivating viruses or
neutralizing toxins. Plotkin et al. (2001) Pediatr Infect Dis J.
20(1):63-75. However, previously, the HIV vaccines tested have not
been able to elicit adequate titers of HIV-1 specific broad
neutralizing antibodies against diverse primary isolates of
HIV-1.
[0109] Among the scientific community, there is general agreement
that in order to be successful, an HIV/AIDS vaccine should i)
induce antibodies able to neutralize a broad range of primary
isolates, ii) induce a durable CD8+ mediated cytotoxic response
against a variety of strains, and iii) induce a strong CD4+ T cell
response to sustain the CTL activity. See, e.g., Mascola et al.
(1999) J Virol 73(5): 4009-18. Passively administered antibodies
alone can protect macaques against both mucosal and IV challenges
with pathogenic SHIV. See, e.g., Mascola et al. (2001) Curr Opin
Immunol 13(4):489-95. There is, however, skepticism that broadly
cross-reactive neutralizing antibodies can be elicited in humans by
immunization. This has led some investigators to abandon efforts to
include envelope in their vaccines and promote vaccines that rely
exclusively on cellular immunity for protection. See, also, Kaul et
al. (2001) J Clin Invest 107(3): 341-9). However, such vaccines are
unlikely to protect from infection and may be expected to limit
disease progression.
[0110] Thus, the compositions and methods described herein
preferably elicit a combination of humoral (neutralizing antibody)
and cellular (CD4+ T cells and CD8+ T Cells) responses, although
humoral or cellular responses individually may be sufficient. The
priming regimen is preferably based on nucleic acid vectors (e.g.,
pCMV or pSIN) that comprise Gag and/or Env HIV genes, respectively.
DNA-based vaccines are attractive because they are flexible and
relatively simple to produce. Their distribution may be simplified
because DNA itself is very durable when properly stored.
Immunization with DNA encoding antigenic proteins elicits both
antibody and cell-mediated immune responses. DNA immunization has
provided protective immunity in various animal models. See, e.g.,
Donnelly et al. (1997) Life Sci. 60(3):163-72. A DNA vaccine
encoding a malaria antigen was tolerated relatively well by 20
volunteers, with only few and mild local reactogenicity and
systemic symptoms. Wang et al. (1998) Science 282(5388):476-80. A
DNA-based vaccine containing HIV-1 Env and Rev genes was
administered to 15 asymptomatic HIV-infected patients who were not
using antiviral drugs. The vaccine induced no local or systemic
reactions, and no laboratory abnormalities were detected.
Specifically, no patient developed autoimmune antibodies. MacGregor
et al. (1998) J Infect Dis 178(1):92-100. Ongoing Phase 1 clinical
trials show that therapeutic vaccinations indeed boost anti-HIV-1
immune responses in humans. Ugen et al. (1998) Vaccine
16(19):1818-21.
[0111] The boost component of the compositions and methods
described herein typically includes an HIV protein (e.g., a HIV
envelope gp140 protein that has a deletion of the V2 loop, thus
exposing conserved epitopes). The HIV protein vaccines described
herein generally comprise subunit rccombinant antigens and are
predicted to be both well tolerated and immunogenic (humoral and
cellular) in view of the safety and efficacy date obtained with
non-recombinant HIV protein vaccines.
[0112] Formulations and Administration
[0113] As noted above, the compositions are preferably administered
using a "prime-boost" approach, for example, two priming injections
(e.g., each including two separate preparations of DNA encoding p55
Gag from HIV-1 SF2, and oligomeric, V2 loop-deleted, gp140 envelope
protein from HIV-1 SF162, both formulated on PLG microparticles
(Env or Gag PLG/DNA)) are administered. The boost composition
comprises a protein, for example an antigen is composed of a
recombinant oligomeric, V2 loop-deleted, gp140 envelope protein
(HIV o-gp140) in combination with MF59 adjuvant. The protein is
typically mixed with the adjuvant shortly before injection.
[0114] The DNA vaccines may be provided in 5.0 mL Type I glass
vials containing 1.4 mg of DNA and 35 mg of PLG microparticles per
vial, in lyophilized form. HIV o-gp140 antigen is supplied as a
liquid in 3-mL Type I glass vials containing 140 .mu.g in 0.35
mL/vial. MF59 adjuvant is supplied in 3-mL Type I glass vials
containing 0.7 mL/vial. Generally, the dose of DNA and protein
actually administered to the subject is less than contained in the
vial, for example approximately 1.0 mg of DNA is typically
administered to the subject when the vial contains 1.4 mg.
Similarly, approximately 100 .mu.g of protein is typically
administered to the subject when each unit dose vial contains 140
.mu.g of protein.
[0115] Any suitable delivery mode can be used for the nucleic acids
and polypeptides. Liposomes can also be used for delivery of these
molecules. For a review of the use of liposomes as carriers for
delivery of nucleic acids, see, Hug and Sleight, Biochim. Biophys.
Acta. (1991) 1097:1-17; Straubinger et al., in Methods of
Enzymology (1983), Vol. 101, pp. 512-527. Liposomal preparations
for use in the present invention include cationic (positively
charged), anionic (negatively charged) and neutral preparations,
with cationic liposomes particularly preferred. Cationic liposomes
have been shown to mediate intracellular delivery of plasmid DNA
(Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416);
mRNA (Malone et al., Proc. Natl. Acacd. Sci. USA (1989)
86:6077-6081); and purified transcription factors (Debs et al., J.
Biol. Chem. (1990) 265:10189-10192), in functional form. Cationic
liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are available under the trademark Lipofectin, from GIBCO BRL, Grand
Island, N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA
(1987) 84:7413-7416). Other commercially available lipids include
(DDAB/DOPE) and DOTAP/DOPE (Boerhinger).
[0116] Similarly, anionic and neutral liposomes are readily
available, such as, from Avanti Polar Lipids (Birmingham, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios. Methods for making liposomes using these materials are well
known in the art.
[0117] The DNA and/or protein antigen(s) can also be delivered in
cochleate lipid compositions similar to those described by
Papahadjopoulos et al., Biochem. Biophys. Acta. (1975) 394:483-491.
See, also, U.S. Pat. Nos. 4,663,161 and 4,871,488.
[0118] The vaccine components may also be encapsulated, adsorbed
to, or associated with, particulate carriers. Such carriers present
multiple copies of a selected antigen to the immune system and
promote trapping and retention of antigens in local lymph nodes.
The particles can be phagocytosed by macrophages and can enhance
antigen presentation through cytokine release. Examples of
particulate carriers include those derived from polymethyl
methacrylate polymers, as well as microparticles derived from
poly(lactides) and poly(lactide-co-glycolides), known as PLG. See,
e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J P, et
al., J Microencapsul. 14(2):197-210, 1997; O'Hagan D T, et al.,
Vaccine 11(2):149-54, 1993. Suitable microparticles may also be
manufactured in the presence of charged detergents, such as anionic
or cationic detergents, to yield microparticles with a surface
having a net negative or a net positive charge. For example,
microparticles manufactured with anionic detergents, such as
hexadecyltrimethylammonium bromide (CTAB), i.e. CTAB-PLG
microparticles, adsorb negatively charged macromolecules, such as
DNA. (see, e.g., Int'l Application Number PCT/US99/17308). Methods
of making and using PLG particles to deliver nucleic acids are
described in International Patent Publications WO 98/33487; WO
00/06123; WO 02/26212; and WO 02/26209.
[0119] Polymers such as polylysine, polyarginine, polyornithine,
spermine, spermidine, as well as conjugates of these molecules, may
also be used for transferring a nucleic acid of interest.
[0120] Additionally, biolistic delivery systems employing
particulate carriers such as gold and tungsten, are especially
useful for delivering nucleic acid vectors of the present
invention. The particles are coated with the nucleic acid(s) to be
delivered and accelerated to high velocity, generally under a
reduced atmosphere, using a gun powder discharge from a "gene gun."
For a description of such techniques, and apparatuses useful
therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006;
5,100,792; 5,179,022; 5,371,015; and 5,478,744. Also, needle-less
injection systems can be used (Davis, H. L., et al, Vaccine
12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).
[0121] The compositions described herein may either be prophylactic
(to prevent infection) or therapeutic (to treat disease after
infection). The compositions will comprise a "therapeutically
effective amount" of the gene of interest such that an amount of
the antigen can be produced in vivo so that an immune response is
generated in the individual to which it is administered. The exact
amount necessary will vary depending on the subject being treated;
the age and general condition of the subject to be treated; the
capacity of the subject's immune system to synthesize antibodies;
the degree of protection desired; the severity of the condition
being treated; the particular antigen selected and its mode of
administration, among other factors. An appropriate effective
amount can be readily determined by one of skill in the art Thus, a
"therapeutically effective amount" will fall in a relatively broad
range that can be determined through routine trials.
[0122] The compositions will generally include one or more
"pharmaceutically acceptable excipients or vehicles" such as water,
saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol,
etc. Additionally, auxiliary substances, such as wetting or
emulsifying agents, pH buffering substances, and the like, may be
present in such vehicles. Certain facilitators of nucleic acid
uptake and/or expression can also be included in the compositions
or coadministered, such as, but not limited to, bupivacaine,
cardiotoxin and sucrose.
[0123] Once formulated, the compositions of the invention can be
administered directly to the subject (e.g., as described above) or,
alternatively, delivered ex vivo, to cells derived from the
subject, using methods such as those described above. For example,
methods for the ex vivo delivery and reimplantation of transformed
cells into a subject are known in the art and can include, e.g.,
dextran-mediated transfection, calcium phosphate precipitation,
polybrene mediated transfection, lipofectamine and LT-1 mediated
transfection, protoplast fusion, electroporation, encapsulation of
the polynucleotide(s) (with or without the corresponding antigen)
in liposomes, and direct microinjection of the DNA into nuclei.
[0124] Direct delivery of polynucleoides and polypeptides in vivo
will generally be accomplished, as described herein, by injection
using either a conventional syringe or a gene gun, such as the
Accell.RTM. gene delivery system (PowderJect Technologies, Inc.,
Oxford, England). The constructs can be injected either
subcutaneously, epidermally, intradermally, intramucosally such as
nasally, rectally and vaginally, intraperitoneally, intravenously,
orally or, preferably, intramuscularly. Dosage treatment may be a
single dose schedule or a multiple dose schedule. Administration of
nucleic acids may also be combined with administration of peptides
or other substances.
[0125] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way. Efforts have been made to ensure
accuracy with respect to numbers used (e.g., amounts, temperatures,
etc.), but some experimental error and deviation should, of course,
be allowed for.
EXPERIMENTAL
Example 1
Vaccine Manufacturing Process and Release
[0126] A. PLG/DNA HIV Vaccines
[0127] For PLG/DNA priming immunization with nucleic acid, plasmid
DNA (Env or Gag) was adsorbed onto biodegradable polymer
microparticles (PLG) essentially as follows. To manufacture the DNA
vaccines, E. coli (strain DH5) was transformed with plasmids
encoding the HIV Env and Gag genes. A modified alkaline
lysis-method was used to isolate plasmid DNA from chromosomal DNA,
proteins, and other cellular debris. Plasmid DNA was concentrated
by precipitation using PEG 8000. The plasmids were then purified by
two chromatography steps and transferred by ultrafiltration into
formulation buffer.
[0128] PLG microparticles were produced by an aseptic manufacturing
process. See, e.g., U.S. Pat. Nos. 5,603,960; 6,534,064 and
6,573,238; Gupta et al. (1998) Adv Drug Deliv Rev. 32(3):225-246;
O'Hagan (1998) J Pharm Pharmacol. 50(1):1-10. In particular, PLG
(dissolved in methylene chloride) was homogenized with formulation
buffer and CTAB (cation surfactant) solution under high speed and
high shear of mixing to form a stable emulsion. The removal of
methylene chloride by nitrogen purge causes PLG to form
microparticles, due to the tendency of the cationic surfactant to
stay at the PLG interface. These positively charged microparticles
bind with negatively charged DNA to form the PLG/DNA immunogen.
[0129] B. HIV o-gp140 Antigen
[0130] The recombinant, oligomeric HIV gp-140 (o-gp140) was
prepared essentially as described in Srivastava et al. (2003) J
Virol. 77(20):11244-11259. Following fermentation of the host
cells, the cell culture supernatant was harvested, filtered,
concentrated, and purified.
[0131] The purified o-gp140 protein fraction was further treated to
remove adventitious viruses. The first of these steps was viral
inactivation at pH 3.5 for 1 hour. The sample was then concentrated
and diafiltered into a buffer at pH 4 in preparation for cation
capture using SP resin, which captures o-gp140 and allows many
viruses to flow through. The o-gp140 was eluted, concentrated and
diafiltered into formulation buffer. This formulated bulk product
was then filtered through a Ultipor.RTM. VF grade DV50 virus
removal membrane followed by filtration through a 0.2 .mu.m
membrane.
[0132] C. MF59 Adjuvant
[0133] MF59 adjuvant (MF59C.1) is an oil-in-water emulsion with a
squalene internal oil phase and a citrate buffer external aqueous
phase. See, e.g., U.S. Pat. Nos. 6,299,884 and 6,086,901; Ott et
al. "MF59--Design and Evaluation of a Safe and Potent Adjuvant for
Human Vaccines," Vaccine Design: The Subunit and Adjuvant Approach
(Powell, M. F. and Newman, M. J. eds.) Plenum Press, New York, pp.
277-296 (1995). Two nonionic surfactants, sorbitan trioleate and
polysorbate 80, serve to stabililize the emulsion. The safety of
the MF59 adjuvant has been demonstrated in animals and in humans in
combination with a number of antigens. See, e.g., Higgins et al.,
"MF59 Adjuvant Enhances the Immunogenicity of Influenza Vaccine in
Both Young and Old Mice," Vaccine 14(6):478-484 (1996).
Example 2
Vaccine Composition
[0134] The components of the PLG/DNA priming vaccines, o-gp140
boost antigen, and MF59 adjuvant are provided in the following
tables. TABLE-US-00002 TABLE 1 PLG DNA (Env) Vaccine Composition
Quantity Quantity per dose Component per mL* (maximum dose) Poly
(D,L-Lactide-co-glycolide) 50.0 mg 25.0 mg Plasmid DNA (Env) 2.0 mg
1.0 mg Hexadecyltrimethylammonium Bromide 0.5 mg 0.25 mg Mannitol,
USP, EP 44 mg 22 mg Sucrose, USP/NF 14.7 mg 7.35 mg EDTA, Disodium
salt Dihydrate, USP 0.37 mg 0.28 mg Sodium Citrate Dihydrate,
USP/EP 1.4 mg 1.10 mg Citric Acid Monohydrate, USP/EP 0.04 mg 0.02
mg Water for Injection qs qs *following reconstitution
[0135] TABLE-US-00003 TABLE 2 PLG DNA (Gag) Vaccine Composition
Quantity Quantity per dose Component per mL* (maximum dose) Poly
(D,L-Lactide-co-glycolide) 50.0 mg 25.0 mg Plasmid DNA (Gag) 2.0 mg
1.0 mg Hexadecyltrimethylammonium Bromide 0.5 mg 0.25 mg Mannitol,
USP, EP 44 mg 22 mg Sucrose, USP/NF 14.7 mg 7.35 mg EDTA, Disodium
salt Dihydrate, USP 0.37 mg 0.18 mg Sodium Citrate Dihydrate,
USP/EP 1.4 mg 0.70 mg Citric Acid Monohydrate, USP/EP 0.04 mg 0.02
mg Water for Injection qs qs *following reconstitution
[0136] TABLE-US-00004 TABLE 3 HIV o-gp140 Autigen Composition
Quantity per dose Component Quantity per mL (100 .mu.g) o-gp140 0.4
mg 0.1 mg Sodium citrate, dihydrate 2.75 mg 0.69 mg Citric acid,
monohydrate 0.15 mg 0.04 mg Sodium chloride 17.53 mg 4.38 mg Water
for Injection qs qs
[0137] TABLE-US-00005 TABLE 4 MF59C.1 Adjuvant Composition
Component Quantity per mL Quantity per dose Squalene 39 mg 9.75 mg
Polysorbate 80 4.7 mg 1.18 mg Sorbitan trioleate 4.7 mg 1.18 mg
Sodium citrate, dihydrate, USP 2.68 mg 0.66 mg Citric acid,
monohydrate, USP 0.17 mg 0.04 mg Water for Injection qs qs
[0138] The schedule for vaccination injections is to inject at
multiple time points (e.g., at 5 or 6 different time points),
administered at 0, 1, 2, 6, 9 and possibly 12 months. Several
immunization schedules are evaluated to maximize the immune
response. These schedules may include vaccinations at 4 or 5
timepoints, according to any schedule, for example as set forth
below. All vaccinations will be administered by intramuscular
injection in the outpatient setting. Table 5 shows an exemplary
immunization protocol. TABLE-US-00006 TABLE 5 Protocol Of
Immunization STUDY AGENTS A: Clade B Gag + Env DNA/PLG
microparticles, dose indicated below B: Clade B gp140 Env protein,
100 .mu.g DNA Dose Gag/ Month (day) Env Protein 0 1 2 4 6 9 12* #
(.mu.g) Dose (0) (28) (56) (112) (168) (236) (365) 1 1000/ 100
.mu.g A A A B B B 1000 2 1000/ 100 .mu.g A A A + B B B 1000 3 1000/
100 .mu.g A A A + B B B 1000 4 1000/ 100 .mu.g A A B B B 1000 #
schedule *If needed to sustain an immunologic response
Example 3
Handling and Storage
[0139] To prepare the DNA/PLG vaccine for administration, one vial
of each DNA/PLG (Env or Gag) is reconstituted by drawing 0.7 mL
Water for Injection into a syringe and adding it to each of the two
vials. The vials are swirled vigorously for up to two minutes. The
mixing is complete when the suspensions appear uniform, milky, and
fully dispersed. The reconstituted solutions are administered
without further preparation to deliver the highest DNA/PLG dose
(1000 .mu.g). To prepare the 500-.mu.g, and 250-.mu.g doses, use a
new syringe to add an additional 0.7 mL or 2.1 mL of 0.9% NaCl
solution (Normal Saline), respectively, to the all ready
reconstituted vials, and swirl to mix. Using a new syringe, draw up
0.5 mL of the Env PLG/DNA mixture, and then 0.5 mL of the Gag
DNA/PLG mixture, into the same syringe. The total DNA dose, in a
combined volume of 1 mL, can then be administered intramuscularly
(IM) into the deltoid muscle.
[0140] HIV o-gp140 antigen will be mixed before administration with
MF59 adjuvant. To prepare the vaccine dose for administration, mix
the contents of the MF59 vial by repeated gentle swirling and
inversion (not vigorous shaking) and then withdraw 0.35 mL into a
1-mL sterile syringe. Inject this adjuvant into the 3 mL vial
containing the thawed HIV o-gp 140 antigen and mix by gentle
swirling. Use a new syringe to draw up 0.5 mL of the mixture, which
can then be administered intramuscularly (IM) into the deltoid. The
final vaccine has a milky white opacity. The injection should be
given shortly after addition of the adjuvant.
[0141] The thawed HIV o-gp140 antigen is stable at 2.degree. to
8.degree. C. for 8 hours. Antigen that has been thawed for over 8
hours (even with refrigeration), is not preferred, as it may have
reduced potency.
[0142] Individuals receiving placebo will receive 0.5 mL of
calcium- and magnesium-free phosphate-buffered saline. Supplied as
a clear, colorless solution in vials containing a volume to deliver
a 1 mL dose. The vials must be stored in a refrigerator at 2 to
8.degree. C.
[0143] A. Vaccine Storage Conditions
[0144] The lyophilized DNA/PLG vaccines are stored at 2-8.degree.
C. HIV o-gp140 are stored frozen below -60.degree. C. and the MF59
adjuvant is stored in a refrigerator at 2.degree. to 8.degree. C.
MF59 should not be frozen.
Example 4
Animal Studies
[0145] A nonclinical safety assessment program was designed to
support the clinical administration of three intramuscular (IM)
doses of the HIV DNA vaccine formulation followed by three IM doses
of the HIV Protein vaccine formulation. One clinical dose (1.0 mL)
of the DNA vaccine formulation contains 1 mg Env-DNA, 1 mg Gag-DNA,
and 50 mg PLG whereas one clinical dose (0.5 mL) of the HIV Protein
Vaccine contains 0.1 mg/0.25 mL Env protein plus 0.25 mL MF59.
[0146] The following GLP studies were conducted to assess whether
integration into host genomic DNA occurs and to characterize tissue
localization and persistence of the HIV DNA vaccine formulation
when administered as a single IM injection to New Zealand. White
rabbits and BALB/c mice, respectively. These studies are further
described below in Section A titled "An Integration Study with
DNA-PLG Formulations after a Single Intramuscular Injection to New
Zealand White Rabbits" and Section B titled "Single Dose
Biodistribution Study of HIV DNA Vaccine Formulations in BALB/c
Mice."
[0147] As described in further detail below, in these studies,
toxicity was evaluated based on viability, clinical observations,
body weights, and macroscopic postmortem examinations. Physical
examinations and dermal scoring of injection sites were also
performed in the mouse biodistribution study. Results of these
studies demonstrated that administration of a single dose of the
Env-DNA vaccine formulation resulted in no integration into the
rabbit genomic DNA and good tolerability in New Zealand White
rabbits and BALB/c mice. The analysis of mouse tissues for
distribution of the HIV DNA vaccine formulation was also
performed.
[0148] In addition, the following GLP toxicology study was
conducted to assess the systemic and local tolerability of the HIV
vaccine formulation when administered to New Zealand White rabbits
via IM injection. (See, Section C below, titled "Multiple-Dose
Intramuscular Injection Toxicity Study with HIV DNA Vaccine
Formulation in New Zealand White Rabbits"). In this study, animals
received four doses, two weeks apart, of the HIV DNA vaccine
formulation followed by four doses, two weeks apart, of the HIV
Protein vaccine formulation. The first HIV Protein vaccine dose was
administered on the same day as the last HIV DNA vaccine dose. A
recovery period of two weeks was included in the study design.
Rabbits received the planned clinical dose (1 mL HIV DNA
vaccine/dose; 0.5 mL HIV Protein vaccine/dose) by the clinical
route of administration (IM). However, rabbits received four doses
each of the HIV DNA vaccine and the HIV Protein vaccine, exceeding
the intended clinical regimen (three doses each) by one dose. The
rabbit dosing regimen was condensed relative to the clinical
regimen (monthly), however, rabbit immunogenicity studies have
demonstrated that an every two-week regimen is appropriate from an
immunological standpoint.
[0149] In this study, toxicity was evaluated based on clinical
signs, dermal scoring of injection sites, body weights and
temperatures, food consumption, ophthalmoscopy, clinical pathology
(hematology, serum chemistry, and coagulation including
fibrinogen), organ weights, and macroscopic postmortem and
histopathological examinations. Analysis of serum for antibodies
(anti-nuclear and Env- and Gag-antibodies) was also performed.
Under the conditions of this study and based on the available
preliminary data (terminal organ weights, macroscopic evaluation
and histology pending), no systemic or local effects related to the
administration of the HIV vaccine formulation were identified.
[0150] The safety and persistence at the injection site of the HIV
DNA vaccine formulation was further assessed in the following
non-GLP studies, described in further detail below in Section D
titled "Exploratory DNA/PLG Local Irritation Tolerance Study in
Male New Zealand White Rabbits" and Section B titled "Single Dose
Intramuscular and Multiple-Dose (Two) Mouse Immunogenicity Study
with PCR Injection Site Assessment."
[0151] The single dose study was conducted to evaluate the
potential local irritant effects of various concentrations of
DNA/PLG in New Zealand White male rabbits when admininstered by a
single IM injection. Potential toxicity was evaluated based on
clinical signs, dermal scoring of injection sites, body weight,
comprehensive macroscopic examination, and microscopic evaluation
of injection sites. Under the conditions of this study, various
concentrations of DNA/PLG were well tolerated when administered to
male New Zealand White rabbits as a single IM injection.
[0152] The multiple-dose immunogenicity study assessed the presence
of Gag-DNA PLG at the IM injection site four and eight weeks
post-last dose in female BALB/c mice that received two
administrations (Days 0 and 28) of Gag-DNA PLG formulations.
Results demonstrated that the PLG formulations were comparable to a
naked-DNA control with regard to persistence and that the amount
remaining in the injection site 4 and 8 weeks post-last dose was
insignificant (approximately 10.sup.-7% of the infected
amount).
[0153] A. An Integration Study with DNA-PLG Formulations after a
Single Intramuscular Injection to New Zealand White Rabbits
[0154] To assess the integration of the HIV DNA-PLG vaccine
formulation (Env-DNA PLG and Gag-DNA PLG) into the host genomic DNA
when administered via a single IM injection to New Zealand White
rabbits the following studies were performed. The study consisted
of three groups of 2 animals/sex/group. On Day 0, treated rabbits
received a single IM injection (0.5 mL/leg) of either the Env-DNA
PLG or the Gag-DNA PLG in each hind leg. (See, Table 6). Control
rabbits received no injection. All animals were necropsied on Day
29. TABLE-US-00007 TABLE 6 Experimental Study Design Treatment
Number of Animals Dose.sup.a Volume.sup.b Total Necropsy.sup.c
Group Material DNA (mg) (mL) M F M F 1 Control 0 0 2 2 2 2 2
Env-DNA PLG 2 1 2 2 2 2 3 Gag-DNA PLG 2 1 2 2 2 2 .sup.aGroup 2 and
3 animals received a dose of 1 mg DNA, 25 mg PLG/0.5 mL/leg in each
hind leg for a total dose/animal of 2 mg DNA, 50 mg PLG. Dosing
occurred on Day 0 of the study. .sup.bGroup 2 and 3 animals
received a volume of 0.5 mL/leg for a total volume/animal of 1 mL.
.sup.cNecropsy was performed 30 days post-dosing (Day 29)
[0155] Potential toxicity was evaluated based on viability
observations for mortality and general condition, body weights, and
a comprehensive postmortem macroscopic examination. In addition,
injection sites were collected at necropsy for Polymerase Chain
Reaction (PCR) analysis to evaluate the integration of the DNA
vaccine into the rabbit genomic DNA. Additional tissues (see Table
7) were also collected for potential PCR analysis in the event of
positive integration results at the injection site. For the PCR
analysis, DNA was extracted from the rabbit tissue, quantitated,
and subjected to field inversion gel electrophoresis (FIGE) to
separate the rabbit genomic DNA from the extrachromosomal plasmid
DNA. DNA of a size greater than 17 kb was excised and purified from
the gel. Both the extracted and the FIGE purified DNAs (1 .mu.g)
were analyzed using a quantitative PCR assay to assess the
integration of the target sequence (plasmid vector Env-DNA PLG) in
each preparation. DNA extracted from tissues of control animals was
pooled according to sex; DNA from treated animals was not pooled
but analyzed separately. TABLE-US-00008 TABLE 7 Tissues collected
for PCR analysis Bone marrow (sternum, femur) Lungs (with mainstem
bronchi) Brain (medulla, pons, cerebrum, Lymph nodes
(submandibular) cerebellum) Kidneys Ovaries Injection Sites Spleen
Liver Testes
[0156] There were no deaths and no treatment-related adverse
effects on clinical signs and body weights. No treatment related
changes were noted in the macroscopic examination either. Results
of the PCR integration analysis revealed no integration of the
Env-DNA PLG into the host genomic DNA (see Table 8). Because no
integration occurred at the injection sites, no additional tissues
were evaluated. TABLE-US-00009 TABLE 8 Quantitative PCR assay
results of injection sites Env-DNA PLG (copies/.mu.g DNA) SAMPLE
Extracted DNA.sup.a FIGE Purified DNA.sup.b Control Male LLD LLD
Male # 2020 2637 LLD Male # 2021 2364 LLD Control Female LLD LLD
Female # 2520 33890 LLD Female # 2521 19814 LLD
.sup.aquantification the target sequence in genomic DNA prior to
field inversion gel electrophoresis (extrachromosomal plasmid DNA
plus genomic DNA) .sup.bquantification of the target sequence in
genomic DNA purified by field inversion gel electrophoresis
(genomic DNA only) LLD = lower that the limit of detection of the
assay (5 copies/.mu.g DNA)
[0157] In conclusion, a single IM dose of either Env-DNA PLG or
Gag-DNA PLG containing a total of 2 mg of DNA and 50 mg of PLG were
well tolerated in New Zealand White rabbits. No treatment-related
adverse effects were noted and no integration of plasmid vector
Env-DNA into rabbit genomic DNA obtained from the injection sites
was detected.
[0158] B. Single Dose Biodistribution Study of HIV DNA Vaccine
Formulations in BALB/c Mice
[0159] To assess the tissue localization and persistence of the HIV
DNA PLG vaccine formulations (Env-DNA PLG and Gag-DNA PLG) after a
single administration via IM injection to BALB/c mice, the
following studies were performed. The study included five groups of
15 animals/sex/group. On Day 1, treated mice received a single IM
injection of either a high or a low dose of Env-DNA PLG or Gag-DNA
PLG in the right biceps femoris area. Control mice received no
injection. Five animals/sex/group were necropsied one week (Day 8),
two months (Day 61), or three months (Day 91) post-dosing. (Table
9).
[0160] Potential toxicity was evaluated based on viability
observations for mortality and general condition, physical
examinations, body weights, dermal Drazie scoring of injection
sites, and a comprehensive postmortem macroscopic examination. In
addition, selected tissues (see Table 10) were collected at each
necropsy for PCR analysis to evaluate the biodistribution and
persistence of the DNA vaccine into mouse tissues. For the PCR
analysis, DNA was extracted from each mouse tissue, quantitated,
subjected to PCR amplification using a fluorescence probe, and
followed by fluorescence detection. Of the collected tissues, only
tissues from the Env-DNA PLG treated rabbits were analyzed.
TABLE-US-00010 TABLE 9 Experimental Study Design Dose volume Number
of Animals/Sex Group and Dose (.mu.L/ Day 8 Day 61 Day 91 Treatment
Level.sup.a dose).sup.a Total Necropsy Necropsy Necropsy 1 0 0 15 5
5 5 (Control) None 2 10 .mu.g 20 15 5 5 5 Env-DNA DNA PLG 0.25 mg
PLG 3 100 .mu.g 50 15 5 5 5 Env-DNA DNA PLG 2.5 mg PLG 4 10 .mu.g
20 15 5 5 5 Gag-DNA DNA PLG 0.25 mg PLG 5 100 .mu.g 50 15 5 5 5
Gag-DNA DNA PLG 2.5 mg PLG .sup.aDosing occurred on Day 1 of the
study.
[0161] TABLE-US-00011 TABLE 10 Tissues Collected for PCR Analysis
Bone marrow (both femurs) Lung Brain Lymph nodes (mandibular)
Kidneys Ovaries Injection Site (right biceps femoris) Spleen Liver
Testes
[0162] There were no deaths that could be associated with
administration of the test articles and no treatment-related
adverse effects on clinical signs and body weights. No erythema or
edema was seen at the injection sites. No treatment related changes
were noted in the macroscopic examination.
[0163] In conclusion, a single IM dose of either Env-DNA PLG or
Gag-DNA PLG containing up to 100 .mu.g of DNA and up to 2.5 mg of
PLG was well tolerated in BALB/c mice. No treatment-related adverse
effects were noted.
[0164] C. Multiple-Dose Intramuscular Injection Toxicity Study with
HIV DNA Vaccine Formulation in New Zealand White Rabbits
[0165] To assess the local and systemic toxicity of the HIV Vaccine
formulation in New Zealand White rabbits after repeated
administration and to determine the reversibility of findings, the
following studies were conducted. Two groups of 8 animals/sex/group
were used. Treated rabbits received four doses of the HIV DNA
vaccine formulation (Env- and Gag-DNA PLG) given every other week
followed by four doses of the HIV Protein Vaccine formulation, also
given every other week. The last HIV DNA vaccine dose and the first
HIV Protein vaccine dose were administered on the same day (Day
43). Doses were administered via IM injections into the quadricep
leg muscle and legs were alternated except on Day 43 when both legs
were injected. Control animals received four IM injections of
saline solution followed by four IM injections of MF59. Four
animals/sex/group were necropsied three days (Day 88, main
necropsy) or two weeks post-dosing (Day 99, recovery necropsy).
Table 11 describes the experimental design.
[0166] Potential toxicity was evaluated based on clinical signs,
dermal scoring of injection sites, body temperature, body weight,
food consumption, ophthalmic examination, clinical pathology
(hematology, coagulation, and serum chemistry parameters), terminal
organ weights, comprehensive macroscopic examination, and
microscopic evaluation of selected tissues. TABLE-US-00012 TABLE 11
Experimental Study Design GROUP 1.sup.a Control (dose volume) DAY
OF STUDY Treatment 1 15 29 43 57 71 85 88 99 Saline 1 mL 1 mL 1 mL
1 mL none none none Main Recovery Control Necropsy.sup.e
Necropsy.sup.e MF59 none none none 0.5 mL 0.5 mL 0.5 mL 0.5 mL
Control.sup.b GROUP 2.sup.a DNA Vaccine (dose volume) Env- &
1.sup.st 2.sup.nd 3.sup.rd 4.sup.th Gag-DNA dose dose dose dose
PLG.sup.c (1 mL) (1 mL) (1 mL) (1 mL) none none none Env Protein
none none none 1.sup.st 2.sup.nd 3.sup.rd 4.sup.th Dose.sup.d dose
dose dose dose (0.5 mL) (0.5 mL) (0.5 mL) (0.5 mL) .sup.a16 animals
(8 M + 8 F) .sup.cconsists of 0.25 mL of MF59 plus 0.25 mL saline
.sup.cconsists of 0.5 mL Env-DNA PLG (2 mg DNA, 50 mg PLG/mL) plus
0.5 mL Gag-DNA PLG (2 mg DNA, 50 mg PLG/mL) .sup.dconsists of 0.25
mL of Env Protein (0.4 mg/mL) plus 0.25 mL MF59 .sup.e4
animals/sex/group
[0167] The animals were observed twice daily for mortality and
morbidity and once daily for signs of toxicity. In addition,
detailed observations were made predose, 4 hr post-dose on each
dosing day, weekly, and at each necropsy. Injection sites were
assessed for signs of irritation and graded based on a modified
Draize score prior to dosing and 24 and 48 hr after each injection.
Body temperatures were taken pretreatment, prior to each dose, and
24 hr after each dose. Body weights were recorded pre-treatment,
weekly thereafter, and at necropsy. Food consumption was assessed
weekly. The ophthalmology evaluation was performed pre-treatment
and prior to each necropsy. Blood samples for hematology, serum
chemistry, and coagulation (including fibrinogen) analysis were
collected pre-treatment, pre-dose on Days 29 and 57, and on Days 87
and 99. Additional blood samples were taken pre-treatment, pre-dose
on Days 15, 43, 71, and on Days 87 and 99 for antibody
(anti-nuclear and Env- and Gag-antibodies) analysis. At each
necropsy, a complete macroscopic examination and microscopic
evaluation of selected tissues (see Table 12) were performed. Organ
weight data on selected organs (Table 13) were also collected. In
addition, selected tissues were collected for possible assessment
of distribution of the DNA vaccine into host tissues by PCR
analysis (Table 14). TABLE-US-00013 TABLE 12 Histopathology Tissue
List Eyes Kidneys Femur with bone marrow (including knee joint)
Liver Gall Bladder Lung and bronchi Lesions (if any) Optic nerve
Lymph nodes (inguinal, lumbar, mesenteric, and Spleen popliteal)
Injection Sites Thymus
[0168] TABLE-US-00014 TABLE 13 Organ Weights List Adrenals Heart
Spleen Brain Kidneys Testis Epididymis Liver Thymus Gall Bladder
Ovaries
[0169] TABLE-US-00015 TABLE 14 Tissues Collected for Potential PCR
Analysis Brain Spleen Lung Mandibular lymph node Liver Injection
Sites Ovaries/Testis Kidney Bone marrow
[0170] Preliminary data (up to Day 84) revealed no deaths that
could be associated with administration of the test articles and no
treatment-related adverse effects on clinical signs, body weights,
food consumption, and body temperature. Dermal scoring of the
injection sites revealed occasional instances of edema or erythema
in a few animals from both the control and treated group. Although
the incidence of these dermal irritation, reactions was slightly
higher in Group 2 (HIV Vaccine treatment) animals, the findings
were mild in severity (very slight to slight) and completely
resolved by the next observation period. Available preliminary data
(up to Day 57) for clinical pathology demonstrated that there were
no treatment-related effects on hematology, coagulation, or
clinical chemistry parameters.
[0171] In conclusion, under the conditions of this study and based
on the available preliminary data, no systemic effects related to
the administration of the HIV vaccine formulation were identified.
Local effects consisted of occasional instances of very slight to
slight erythema or edema at the injection sites, which appeared
fully resolved by the next observation period. Four IM injections
of the HIV DNA vaccine given every other week, followed by four IM
injections of the HIV Protein vaccine, also given every other week,
were well tolerated by New Zealand White rabbits.
[0172] D. Exploratory DNA/PLG Local Irritation Tolerance Study in
Male New Zealand White Rabbits--Single Dose Intramuscular
[0173] To assess the potential local irritant effects of various
concentrations of DNA/PLG in New Zealand White male rabbits when
administered by a single IM injection, the followings studies were
performed using two groups of 9 male rabbits each. On Day 1, each
rabbit received a 0.5 mL IM injection of the test and control
articles. Three rabbits/group were necropsied one day (Day 2), one
week (Day 8), or two weeks post-dosing (Day 15). Experimental
design is depicted in Table 15.
[0174] Potential toxicity was evaluated based on clinical signs,
dermal scoring of injection sites, body weight, comprehensive
macroscopic examination, and microscopic evaluation of injection
sites. TABLE-US-00016 TABLE 15 Experimental Design Necropsy Day -
No. No. of Treatment.sup.a of animals Group Males IM Site 1 IM Site
2 IM Site 3 IM Site 4 2 8 15 1 9 Saline 100 mg 1% DNA + 100 mg 1%
DNA + 100 mg 3 3 3 PLG/PVA PLG (DF) PLG (RF) 2 9 0.1% 100 mg 2% DNA
+ 50 mg 4% DNA + 25 mg 3 3 3 CTBA PLG/PVA PLG (DF) PLG (DF)
.sup.aInjection volume = 0.5 mL DF = Development Formulation RF =
Research Formulation
[0175] There was no mortality and no treatment-related effects on
body weight. Apparent bruising of the injection sites was observed
sporadically in 4/9 and 5/9 rabbits in Groups 1 and 2,
respectively, during days 1-4. Bruising was noted at all injection
sites except injection site #2. This finding of slight bruising at
the injection sites is consistent with IM injections. Results of
the dermal Draize scoring of the injection sites are presented in
Table 16. Very slight edema was noted in two Group 1 rabbits (IM
sites 3 and 4) on Days 13 to 15 and in one Group 2 rabbit (IM site
4) on Days 13 to 14. Postmortem macroscopic findings were limited
to the injection sites and consisted of red firm areas, tan areas,
hemorrhage on fascia overlying muscle, and subcutaneous hemorrhagic
areas. These findings were more prevalent on Day 2.
Histopathological examination of the injection sites revealed the
characteristic response to needle trauma (muscle fiber degeneration
and hemorrhage) in the saline treated sites. Evaluation of the test
article treated sites revealed, on Day 2, minimal to mild
treatment-related inflammation that was similar for all
formulations. On Day 8, granulomatous changes were the predominant
findings and there was no difference between the formulations.
These granulomatous changes are consistent with know responses to
PLG microspheres and/or the regenerative process. By Day 15, the
histological changes were partially [1% DNA+100 mg PLG (development
and research formulations), 2% DNA+50 mg PLG, 4% DNA+25 mg PLG] or
fully resolved (100 mg PLG/PVA). See, also Table 16. TABLE-US-00017
TABLE 16 Dermal Irritation Results Group Test/Control Article
Identification Findings 1 Saline None 100 mg PLG/PVA None 1% DNA +
100 mg PLG (DF) Very slight edema in 1 rabbit on Days 13-15. 1% DNA
+ 100 mg PLG (RF) Very slight edema in 1 rabbit on Days 13-15. 2
0.1% CTAB None 100 mg PLG/PVA None 2% DNA + 50 mg PLG (DF) None 4%
DNA + 25 mg PLG (DF) Very slight edema in 1 rabbit on Days 13-14.
DF = Development Formulation RF = Research Formulation
[0176] In conclusion, various concentrations of DNA/PLG were well
tolerated when administered to male New Zealand White rabbits as a
single IM injection. Injection site findings were most
frequent/strongest (mild to minimal) on day 2 and were partially to
fully resolved by the end of the recovery period.
[0177] E. Multiple-Dose (Two) Mouse Immunogenicity Study with PCR
Injection Site Assessment
[0178] To assess immunogenicity and persistence of Gag-DNA PLG
formulations at the IM injection sites, ten female BALB/c mice per
group were treated as outlined in Table 17. Animals were dosed on
days 0 and 28 and IM injection sites were harvested 4 and 8 weeks
post-last dose. The formulations tested in this study were similar
to the formulation used in the toxicology studies. TABLE-US-00018
TABLE 17 Experimental Design No of Necropsy - No of mice Group mice
Treatment.sup.a Main.sup.b Recovery.sup.c 1 10 1 .mu.g Gag-DNA, 24
.mu.g PLG 5 5 2 10 10 .mu.g Gag-DNA, 240 .mu.g PLG 5 5 3 10 10
.mu.g Gag-DNA 5 5 .sup.aAdministered by IM injection on Days 0 and
28; .sup.bFour weeks post-last dose; .sup.cEight weeks post-last
dose
[0179] Results of the PCR analysis of injection sites are presented
in Table 18. Results showed that the DNA-PLG formulations were
comparable to the naked-DNA control with regard to persistence.
Although the Gag-DNA was still detectable at the injection site 4
and 8 weeks post-last dose, the amount remaining was insignificant
(approximately 10.sup.-7% of the amount of DNA injected).
TABLE-US-00019 TABLE 18 PCR Analysis of Injection Sites Group and
Mean DNA Standard % from Treatment Time copy number.sup.c Deviation
Time 0 1 0 1.6 .times. 10.sup.11 0 100 1 .mu.g Gag-DNA, Main 470.6
378.9 2.9 .times. 10.sup.-7 24 .mu.g PLG Necropsy.sup.a Recovery
178.4 74.5 1.1 .times. 10.sup.-7 Necropsy.sup.b 2 0 1.6 .times.
10.sup.12 0 100 10 .mu.g Gag-DNA, Main 1061.4 432.7 6.6 .times.
10.sup.-8 240 .mu.g PLG Necropsy.sup.a Recovery 209 108.0 1.3
.times. 10.sup.-8 Necropsy.sup.b 3 0 1.6 .times. 10.sup.12 0 100 10
.mu.g Gag-DNA Main 473 108.7 3.0 .times. 10.sup.-8 Necropsy.sup.a
Recovery 66.3 22.9 4.1 .times. 10.sup.-8 Necropsy.sup.b .sup.aFour
weeks post-last dose .sup.bEight weeks post-last dose .sup.cThe
mean DNA copy number at time 0 was estimated based on the number of
copies/.mu.g of DNA injected.
CONCLUSIONS
[0180] Under the conditions of these studies, single and/or
multiple administrations of the HIV vaccine formulation was well
tolerated in animal models (New Zealand White rabbits and BALB/c
mice) and, in addition, the formulations elicited potent immune
responses. In the multiple-dose rabbit study, the HIV vaccine
formulation produced no treatment-related adverse effects on
clinical observations, body weights and temperatures, food
consumption, and clinical pathology (hematology, coagulation, and
clinical chemistry). Dermal scoring of injection sites revealed
occasional instances of very slight to slight erythema or edema,
which appeared to be reversible. These findings at the injection
site are consistent with those observed in a single-dose local
tolerance rabbit study. In the latter, histopathological evaluation
revealed treatment-related minimal to mild inflammation at the
injection site, which partially or fully resolved by the end of the
recovery period. In further studies, PCR analysis of the injection
sites demonstrated that the Env-DNA PLG did not integrate into the
host genomic DNA and that the Gag-DNA PLG did not persist at the
injection sites after 4 or 8 weeks.
[0181] In the multiple-dose rabbit study, animals received the
planned clinical dose (1 mL HIV DNA vaccine, 0.5 mL HIV Protein
vaccine/dose) by the clinical route of administration (IM).
However, rabbits received four doses each of the HIV DNA vaccine
and the HIV Protein vaccine, exceeding the intended clinical
regimen (three doses each) by one dose. Further, on a body weight
basis, the dose in rabbits (approximately 2.5 Kg) was approximately
24 times higher than the same dose in humans (approximately 60 Kg).
Therefore, administration of the clinical dose and regimen to
normal human subjects is expected to be well tolerated.
[0182] In addition, the vaccine formulations were shown to be
immunogenic as high titers of antibodies Gag and Env were
observed.
Example 5
Enhanced Potency of Plasmid DNA/PLG Microparticle HIV Vaccines in
Rhesus Macaques Using a Prime-Boost Regimen with Recombinant
Proteins
[0183] The following study was conducted to determine the effect of
PLG-mediated delivery on inmmunogencity.
[0184] A. Preparation of Vectors, Protein, PLG
[0185] HIV vaccines as described herein were evaluated in rhesus
macaques as follows. Plasmids pCMVKm2.GagMod.SF2 and
pCMVKm2.o-gp140.SF162 were prepared essentially as described in
U.S. Pat. No. 6,602,705. Sindbis constructs were prepared by
excising the gag and env inserts from pCMVKm2 constructs and
ligating them into pSINCP (a modified version of pSIN1.5, as
describe essentially in Hariharan et al. (1998) J Virol
72(2):950-8).
[0186] Recombinant Env protein o-gp140SF162.DELTA.V2 was produced
in CHO cells and purified essentially as described in Srivastava et
al. (2003) J Virol. 77(20):11244-11259.
[0187] Cationic PLG microparticles were prepared as follows. The
microparticles were prepared using an IKA homogenizer at high speed
to emulsify 10 ml of a 5% w/v polymer solution in methylene
chloride with 1 mL of PBS. The primary emulsion was then added to
50 ml of distilled water containing CTAB (0.5% w/v). This resulted
in the formation of a water-in-oil-in-water emulsion that was
stirred at 6000 rpm for 12 hours at room temperature, allowing the
methylene chloride to evaporate. The resulting microparticles were
washed four times in distilled water by centrifugation at 10,000 g
and freeze dried. The DNA was adsorbed onto PLG-CTAB microparticles
by incubating 1 mg of DNA in 1 ml of 1.times. TE buffer with 100 mg
of microparticles overnight at 4.degree. C. with gentle rocking.
The microparticles were then pelleted by centrifugation at 10,000
rpm for 10 minutes, washed with 1.times. TE buffer, re-centrifuged,
and suspended in 5 ml of deionized water and freeze dried. The size
distribution of the microparticles was determined using a particle
size analyzer (Master sizer, Malvern Instruments, UK).
[0188] DNA constructs were adsorbed onto PLG particles are
described above. Similarly, HIV p55 gag protein was adsorbed onto
anionic PLG microparticles as follows. Microparticles were prepared
by homogenizing 10 ml of 6% w/v polymer solution in methylene
chloride with 40 ml of distilled water containing SDS (1% w/v) at
high speed. using a 10 mm probe. This resulted in an oil-in-water
emulsion, which was stirred at 1000 rpm for 12 hours at room
temperature, and the methylene chloride was allowed to evaporate.
The resulting microparticles were filtered through 38 .mu.m mesh,
washed 3 times in distilled water, and freeze-dried. The size
distribution of the microparticles was determined using a particles
size analyzer (Master sizer, Malvern Instruments, UK).
[0189] 50 mg of lyophilized SDS blank particles were incubated with
0.5 mg of p55gag protein in 10 ml 25 mM Borate buffer, pH 9, with
6M Urea. 50 mg lyophilized DSS blank microparticles were incubated
with 0.5 mg of gp120 protein in 10 mL PBS. Particles were left on a
lab rocker, (Aliquot mixer, Miles Laboratories) at room temperature
for 5 hours. The microparticles were separated from the incubation
medium by centrifugation, and the SDS pellet was washed once with
Borate buffer with 6 M urea, then three times with distilled water,
and lyophilized.
[0190] The loading level of protein adsorbed to microparticles was
determined by dissolving 10 mg of the microparticles in 2 ml of 5%
SDS-0.2 M sodium hydroxide solution at room temperature. Protein
concentration was measured by BCA protein assay (Pierce Rockford,
Ill.). The Zeta potential for both blank and adsorbed
microparticles was measured using a Malvern Zeta analyzer (Malvern
Instruments, UK).
[0191] B. Vaccination
[0192] Rhesus immunization studies were undertaken to evaluate two
DNA vaccine vectors and a cationic PLG microparticle DNA delivery
system in a prune-boost regimen with recombinant proteins. Groups
of 5 rhesus macaques were immunized by intramuscular injection.
injection on weeks 0, 4 and 14 with DNA vaccines encoding HIV SF2
Gag (0.5 mg) and HIV SF162 gp140 Env (1.0 mg) with or without
adsorption to PLG microparticles. The animals were boosted with
yeast-derived p55 Gag protein adsorbed onto anionic PLG
microparticles (Gag/PLG) on week 29. Finally, the animals were
boosted with CHO cell-derived oligomeric gp140 Env protein with a
deleted V2 region administered with the oil-in-water MF59 adjuvant
(Env/MF59) on weeks 38 and 75.
[0193] Immunogenicity of the vaccine compositions was assessed at
various times after each immunization by quantitative and
qualitative measurements of antibody (ELISA, neutralization) and T
cell responses (lymphoproliferation, intracellular cytokine
staining, CTL).
[0194] C. Antibody Assays
[0195] The antibody responses against Env and Gag proteins were
measured by an enzyme-linked immunosorbent assay (ELISA). For both
ELISA's, Nunc Maxisorp plates were coated overnight at 4.degree. C.
with 50 .mu.g of 5 .mu.g/ml of Env protein or Gag protein in PBS,
pH 7.0. The coated wells were blocked for 1 hr at 37.degree. C.
with 150 .mu.l of 5% goat serum (Gibco BRL, Grand Island, N.Y.) in
phosphate-buffered saline (PBS). Serum samples were initially
diluted 1:25 or 1:100 in the Blocking buffer followed by three-fold
serial dilution. The bound antibodies were detected with
horseradish peroxidase-conjugated goat anti-monkey IgG (Southern
Biotechnology Associates, Inc, diluted 1:5,000 with the blocking
buffer) and incubated for 1 hour at 37.degree. C. For development,
3,3', 5,5' tetramethylbenzidine (TMB) was incubated for 15 minutes
according to the manufacturer's instructions, and the reaction was
stopped by adding 2 N HCL. The assay plates were then read on an
ELISA plate reader at an absorbance wavelength of 450 nm. A serum
standard was included on each microtiter plate, and a reference
value of the standard was used for the normalization of the sample
ELISA titers. The titers represent the inverse of the serum
dilution, giving an optical density of 0.5. Virus neutralizing
antibodies were assessed against homologous HIV-1 SF162 virus,
using standard techniques.
[0196] D. Purification of Rhesus PBMC and Derivation of B
Lymphoblastoid Cell Lines (B-LCL)
[0197] Rhesus peripheral blood mononuclear cells (PBMC) were
separated from heparinized whole blood on Ficoll-Hypaque gradients.
To derive rhesus B-lymphoblastoid cell lines, PBMC were exposed to
Herpesvirus papio-containing culture supernatant from the 594S cell
line in the presence of 0.5 .mu.g/ml Cyclosporin A (Sigma). Rhesus
PBMC were cultured at 2-3.times.10.sup.6 per well in 1.5 ml in
24-well plates for 8 days in AIM-V:RPMI 1640 (50:50) culture medium
(Gibco) supplemented with 10% heat-inactivated fetal bovine serum
(AR10). Antigen-specific cells were stimulated by the addition of a
pool of either gag or env peptides (10.7 .mu.g/ml total peptide).
Recombinant human IL-7 (15 ng/ml, R&D Systems, Minneapolis,
Minn.) was added at the initiation of culture. Human rIL2
(Proleukin, 20 IU/ml, Chiron) was added on days 1, 3, and 6.
[0198] E. .sup.51Cr-Release Assay for CTL Activity
[0199] Autologous B-LCL were infected with recombinant vaccinia
viruses (rVV) expressing gag (rVVgag-pol.sub.SF2) or env (rVVgp160
env.sub.SF162), then labeled overnight with
Na.sub.2[.sup.51Cr]O.sub.4 (NEN, Boston, Mass.; 10 .mu.Ci per
2.5.times.10.sup.5 B-LCL) and washed. Recombinant VV infected,
.sup.51Cr-labeled B-LCL were added (2500 per round bottom well) to
duplicate wells containing 3-fold serial dilutions of cultured
PBMC. Unlabeled B-LCL (1.times.10.sup.5 per well) were added to
inhibit non-specific cytolysis. After4 h, 50 .mu.l of culture
supernatant was harvested, added to Lumaplates (Packard, Meriden,
Conn.) and counted with a Wallac Microbeta TriLux liquid
scintillation counter (Perkin Elmer Life Sciences, Boston, Mass.).
.sup.51Cr released from lysed targets was normalized by the
formula: Percent specific .sup.51Cr release=100%.times.(mean
experimental release-spontaneous release)/(maximum
release-spontaneous release), where spontaneous release=mean counts
per minute (cpm) released from target cells in the absence of PBMC
and maximum release=mean cpm released from target cells in the
presence of 0.1% Triton X-100. A response was scored as positive if
the net specific lysis (antigen-specific minus non-specific lysis)
was greater than or equal to 10% at two consecutive PBMC
dilutions.
[0200] F. Lymphoproliferation Assay
[0201] 2.times.10.sup.5 PBMC were incubated in flat bottom
microtiter wells in a volume of 0.2 ml AR10 in the absence or
presence of p55 Gag protein (3 .mu.g/ml) or a pool of Env peptides
(16 .mu.g/ml). Six replicate cultures were established. After 4
days incubation [.sup.3H]-thymidine ([.sup.3H]TdR, Amersham,
Piscataway, N.J.) was added (1 .mu.Ci/well). Following overnight
incubation, cultures were harvested onto glass microfiber filters.
Cellular uptake of [.sup.3H]TdR was measured with a Microbeta
TriLux liquid scintillation counter (Perkin Elmer).
[0202] G. Intracellular Cytokine Staining and Flow Cytometry
[0203] Rhesus PBMC were incubated overnight at 37.degree. C. in the
absence or presence of antigen (gag peptide pool, 30 .mu.g/ml, or
env peptide pool, 30 .mu.g/ml). Anti-CD28 (1 .mu.g/ml, Pharmingen,
San Diego, Calif.) was added as a source of costimulation and
Brefeldin A (1:1000, Pharmingen) was added to prevent cytokine
secretion. After overnight incubation PBMC were stained for cell
surface CD4 (anti-CD4 allophycocyanin conjugate, clone SK3, Becton
Dickinson, San Jose, Calif.) and CD8 (anti-CD8.alpha. PerCP
conjugate, clone SK1, Becton Dickinson), permeabilized with
Cytofix/Cytoperm (Pharmingen), and then stained for intracellular
IFN-.gamma. (monoclonal antibody 4S.B3, phycoerythrin conjugate,
Pharmingen) and TNF-.alpha. (MAb11, FITC conjugate, Pharmingen).
Stained cells were analyzed with a FACSCalibur.TM. flow cytometer
(Becton Dickinson).
[0204] H. Comparison of DNA Vaccine Vectors
[0205] Immunogenicity of DNA vectors without PLG was evaluated. For
anti-Gag antibodies, neither vector (pCMV or pSINCP) was effective
when given in saline as a primary immunization regimen. However,
boosting of animals primed with naked gag DNA using Gag/PLG protein
antigen rapidly induced significant antibody responses. Similarly,
Env/MF59 protein rapidly boosted anti-Env antibodies. At no time
was there a significant difference in the antibody titers induced
by pCMV or pSINCP.
[0206] Helper T cell responses were measured by both
lymphoproliferation (LPA) and intracellular cytokine staining
(ICS). Peripheral blood mononuclear cells (PBMC) were stimulated
with recombinant p55 gag protein or with a pool of synthetic env
peptides. As with antibody responses, the naked pCMV and pSINCP DNA
vaccines were not very effective at inducing LPA or ICS responses.
However, for Gag LPA responses, pSINCP seemed to be generally more
potent. Statistical significance between the pSINCP and pCMV groups
was reached at weeks 20 and 27 (p=0.018, 0.023, respectively).
[0207] Similarly, pSINCP seemed to be more effective at inducing
Env LPA responses. Significantly higher LPA responses between
groups were observed during DNA priming at weeks 20, 24, and 27
(p=0.028, 0;022, and 0.044, respectively), as well as after the Env
protein boost at week 44 (p=0.016).
[0208] To quantify T cell responses further, PBMC were stimulated
overnight with antigen and then stained the PBMC with PE-conjugated
anti-IFN-.gamma. mAb and FITC-conjugated anti-TNF-.alpha. mAb
(intracellular). PBMC were counterstained with APC-conjugated
anti-CD4 and PerCP-conjugated anti-CD8 and analyzed by flow
cytometry for cytokine-positive cells, particularly for
IFN-.gamma./TNF-.alpha.-double positive cells, which were the most
prevalent antigen-specific cells. No significant differences in
frequencies of antigen-specific T cells were seen between groups of
animals receiving pSINCP and pCMV.
[0209] For measurement of CTL, PBMC were cultured in the presence
of a pool of gag peptides or env peptides, IL2, and IL-7. On day 8,
PBMC cultures were harvested, serially diluted, and added to
microtiter wells containing .sup.51Cr-labeled autologous B-LCL that
had been infected the day before with recombinant vaccinia vectors
that expressed gag (rVVgagpol.sub.SF2) or
env-(rVVgp160env.sub.SF162). pCMV appeared more potent at inducing
Gag CTL responses than pSINCP, with a greater number of responses
over the course of the study. Neither DNA vaccine was effective at
inducing Env CTL.
[0210] In summary, both pCMV and pSINCP naked DNA vaccines induced
antibody and T cell responses against HIV Gag and Env.
[0211] I. PLG Microparticle Delivery of DNA Vaccines
[0212] Animals were also immunized as described above with DNA/PLG
compositions to evaluate immunogenicity of DNA vaccines adsorbed to
PLG microparticles. Adsorption of the HIV DNA vaccines onto
cationic PLG microparticles was effective at enhancing immune
responses, particularly for antibodies. PLG delivery markedly
increased antibody titers in macaques receiving either pCMV or
pSINCP. During the DNA priming phase, anti-gag titers were
significantly higher in the PLG groups compared to naked DNA at
every time point measured (p=0.0003 to 0.04), with peak titers
.about.1000-fold higher (FIG. 1). These differences were maintained
after the protein boost, where pCMV/PLG and pSINCP/PLG were
.about.10- to 25-fold higher compared to naked DNA (p=0.02 to
0.04). Anti-Env antibody responses were also significantly higher
in the PLG groups, but only at the peak response after DNA priming
(2 and 6 weeks post second DNA) (P=0.003 to 0.015). Thereafter and
during protein boosting, the anti-env titers were similar in all
groups. For the PLG/DNA vaccine groups, peak antibody responses
were observed after the second DNA immunization, whereas 3
immunizations were required for peak responses by naked DNA.
[0213] The PLG/DNA vaccines induced helper T cell responses against
Gag and Env, as measured by LPA and ICS. By LPA, the magnitudes of
the responses in the naked and PLG/DNA groups generally were
similar, but when grouped (pCMV+pSINCP), PLG had significantly
higher responses at 6 weeks for Gag and 16 weeks for Env, compared
to naked DNA (p=0.05). The frequencies of cytokine production by
CD4 T cells, as measured by ICS, showed enhanced responses in the
Gag PLG group (pCMV+pSINCP groups combined) versus naked DNA at 2
weeks post second DNA (p<0.05). No differences were observed for
the Env DNA vaccines. CD8 T cells responses were measured by ICS
and .sup.51Cr release. By ICS, the responses were generally low and
no differences were seen among the groups. By .sup.51Cr release of
cultured PBMC, good CTL responses were detected against Gag, but
not against Env. The total number of Gag CTL responses was 24 in
the PLG groups and 18 in the naked DNA groups over the course of
the study, with an apparent earlier onset of anti-Gag CTL in the
pCMV/PLG group (3 of 5 animals at 2 weeks post first DNA).
[0214] In summary, PLG delivery of HIV DNA vaccines was effective
at inducing antibody and cellular immune responses. Moreover, PLG
significantly enhanced immunogenic responses as compared to naked
DNA. Particularly strong enhancement of antibody responses was
observed for both the pCMV and pSINCP DNA vaccines. For Gag, this
was true during both the DNA priming and protein boosting phases of
the study. Cellular immune responses also were enhanced in some
cases by PLG during DNA priming, as seen by earlier onset,
increased magnitude, and increased frequency of responses.
[0215] J. Protein Boosting
[0216] The animals were boosted with recombinant Gag protein
adsorbed onto anionic PLG microparticles at 29 weeks, then with
recombinant Env in MF59 adjuvant at 38 and 75 weeks (15, 24, and 51
weeks, respectively, after the last DNA immunization). Antibody
titers were boosted markedly in all groups (FIGS. 1, 2). After
boosting with gag protein the anti-gag antibody titers were
approximately tenfold higher in the animals primed with
PLG/CTAB-DNA than those primed with naked DNA. The anti-gag titers
equaled (DNA/PLG) or exceeded (DNA/saline) the peak titers achieved
by DNA priming. For Env, titers in all groups were significantly
boosted above peak titers after DNA priming (p=0.0002 to 0.02)
(FIG. 2). The second Env protein boost restored antibody titers to
levels seen after the first Env protein boost. Virus-neutralizing
antibody responses were not detected in any animals after DNA
vaccine priming. However, increasing titers were observed after one
and two protein booster immunizations, with overall geometric mean
titers of 8 and 64, respectively (p=0.00071) (FIG. 3). At both of
these time points, the titers were not statistically different
among the various vaccine groups.
[0217] T cell responses also appeared to be boosted after protein
immunization. For Gag, mean SI increased 4- to 7-fold over baseline
after protein boosting, with the number of responders increasing
from 7 to 14 (out of 20). However, the magnitude of the responses
was not higher than those seen at the peak after DNA priming.
[0218] After Env protein boosting, mean SI increased 11- to 25-fold
over baseline and these responses were higher than those measured
after DNA priming. By ICS, little or no increases were observed
after Gag protein boosting, but substantial increases in the
proportion of cells secreting IFN-.gamma. and TNF-.alpha. were seen
after each Env protein boosting. Furthermore, the overall magnitude
of the ICS response was higher after the second compared to the
first protein boost (p=0.0008) (FIG. 4), with responses approaching
4% of CD4 T cells in some animals. As expected, CTL responses were
not boosted by protein immunization.
[0219] In summary, boosting DNA-primed macaques with recombinant
Gag and Env proteins resulted in rapid and significant enhancement
of antibody and T cell responses. In some cases, the magnitude of
these responses was markedly higher than achieved after DNA
priming.
[0220] Thus, DNA/PLG vaccines as described herein induce strong
immune responses in rhesus macaques, with particular enhancement of
antibody responses and an effect on helper and cytotoxic T cells.
The effectiveness of boosting DNA/PLG-primed macaques with
recombinant protein was also established, including strong Th1-type
cytokine production from T cells after Env protein boosting.
Example 6
Human Studies
[0221] Based on data from previous HIV vaccine trials (with other
products), the rate of serious adverse experiences in the placebo
controls is approximately 3.5%. Extensive safety data on the use of
other recombinant glycoprotein antigens with MF59 indicate that
such vaccine antigens, when administered with MF59, are very safe
and generally well tolerated. Additionally, these vaccines have
elicited a strong antibody response against the particular
antigens.
[0222] An exemplary protocol for human studies is shown below in
Table 19. Although exemplified with regard to subtype B, it will
readily apparent that the protocol can also be used as is, or with
modifications, for other strains or subtypes of HIV. TABLE-US-00020
TABLE 19 Human Protocol STUDY AGENTS A: Clade B Gag + Env DNA/PLG
microparticles, dose indicated below (.mu.g) B: Clade B gp140 Env
protein, 100 .mu.g P: Placebo: PBS Immunization Schedule in Months
(Days) DNA Protein 6 9 Group #/grp dose dose 0 (0) 1 (28) 2 (56) 4
(112) (168) (236) PART ONE 1 10 250/250 100 .mu.g A A A B B 2
Placebo P P P P P 2 10 500/500 100 .mu.g A A A B B 2 Placebo P P P
P P 3 10 1000/1000 100 .mu.g A A A B B 2 Placebo P P P P P PART TWO
4 20 1000/1000 100 .mu.g A A A B B 4 Placebo P P P P P 5 30
1000/1000 100 .mu.g A A A + B B 6 Placebo P P P P 6 30 1000/1000
100 .mu.g A A A + B B 6 Placebo P P P P 7 30 1000/1000 100 .mu.g A
A B B 6 Placebo P P P P TOTAL: 168
* * * * *
References