U.S. patent application number 16/396676 was filed with the patent office on 2019-10-31 for hiv vaccination compositions.
The applicant listed for this patent is ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATEUNIVERSITY. Invention is credited to Bertram Jacobs, Karen Kibler, Lydia Meador, Tsafrir Mor.
Application Number | 20190328864 16/396676 |
Document ID | / |
Family ID | 68291997 |
Filed Date | 2019-10-31 |
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United States Patent
Application |
20190328864 |
Kind Code |
A1 |
Mor; Tsafrir ; et
al. |
October 31, 2019 |
HIV VACCINATION COMPOSITIONS
Abstract
Disclosed are compositions for generating an immune response
against human immunodeficiency virus (HIV) and their methods of
uses.
Inventors: |
Mor; Tsafrir; (Tempe,
AZ) ; Jacobs; Bertram; (Tempe, AZ) ; Meador;
Lydia; (Tempe, AZ) ; Kibler; Karen;
(Scottsdale, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA
STATEUNIVERSITY |
Scottsdale |
AZ |
US |
|
|
Family ID: |
68291997 |
Appl. No.: |
16/396676 |
Filed: |
April 27, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62663391 |
Apr 27, 2018 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/575 20130101;
C12N 15/8257 20130101; C12N 2740/16034 20130101; A61K 39/12
20130101; A61K 2039/5258 20130101; C12N 2740/16234 20130101; A61K
39/21 20130101; C12N 2740/16134 20130101 |
International
Class: |
A61K 39/21 20060101
A61K039/21 |
Claims
1. A composition comprising: a vaccinia virus-like particle (VLP),
wherein the vaccinia VLP presents Gag, a fragment of gp41, or both;
and a plant-produced HIV VLP, wherein the plant-produced HIV VLP
presents Gag, a fragment of gp41, or both.
2. The composition of claim 1, wherein the plant-produced HIV VLP
is isolated from plant tissue transformed with a plant expression
vector comprising a T-DNA region that comprises: a first nucleic
acid sequence encoding Gag and a first promoter region upstream of
the first nucleic acid sequence encoding Gag; and/or a second
nucleic acid sequence encoding a fragment of gp41 and a second
promoter upstream of the second nucleic acid sequence encoding a
fragment of gp41.
3. The composition of claim 1, wherein the vaccinia VLP is isolated
from a mammalian cell transfected with at least one replicating but
highly attenuated vaccinia virus vector selected from the group
consisting of: a first replicating but highly attenuated vaccinia
virus vector comprising a third nucleic acid sequence encoding Gag,
a second replicating but highly attenuated vaccinia virus vector
comprising a fourth nucleic acid sequence encoding a fragment of
gp41, and a third replicating but highly attenuated vaccinia virus
vector comprising the third nucleic acid sequence encoding Gag and
the fourth nucleic acid sequence encoding fragment of gp41.
4. The composition of claim 1, wherein the fragment of gp41 is
dgp41.
5. The composition of claim 2, wherein the plant expression vector
is a tobacco mosaic virus-based vector.
6. The composition of claim 1, wherein the vaccinia VLP and the
plant-produced HIV VLP is in an amount sufficient to induce an
immune response against human immunodeficiency virus (HIV) when
administered to a mammalian subject.
7. The composition of claim 2, wherein the plant expression vector
is a geminivirus-based vector, the T-DNA region of the plant
expression vector further comprises a nucleic acid sequence
encoding at least one replication gene.
8. The composition of claim 7, wherein the geminivirus-based vector
is a bean yellow mosaic virus-based vector, the nucleic acid
sequence encoding at least one replication gene that encodes
Rep/RepA.
9. The composition of claim 8, wherein the first promoter region
and the second promoter region comprise a nucleic acid sequence
encoding the cauliflower mosaic virus 35S promoter (P35).
10. The composition of claim 9, wherein the first promoter region
and the second promoter region further comprise two translation
enhancer binding sites downstream of nucleic acid sequence encoding
P35.
11. The composition of claim 7, wherein the T-DNA region of the
plant expression vector further comprises a nucleic acid sequence
encoding a silencing suppressor protein, wherein the nucleic acid
sequence encoding the silencing suppressor protein is upstream of
the first nucleic acid sequence encoding Gag and the first promoter
region upstream of the first nucleic acid sequence encoding Gag and
upstream of the second nucleic acid sequence encoding a fragment of
gp41 and a second promoter upstream of the second nucleic acid
sequence encoding a fragment of gp41.
12. The composition of claim 10, wherein the T-DNA region of the
plant expression vector further comprises a pair of long intergenic
regions, wherein the pair of long intergenic regions flank a
portion of the T-DNA region that does not comprise the nucleic acid
sequence encoding the silencing suppressor protein and does
comprise the at least one replication gene, the first nucleic acid
sequence encoding Gag and the first promoter region upstream of the
first nucleic acid sequence encoding Gag; and/or the second nucleic
acid sequence encoding a fragment of gp41 and a second promoter
upstream of the second nucleic acid sequence encoding a fragment of
gp41.
13. The composition of claim 7, wherein the T-DNA region of the
plant expression vector further comprises a nucleic acid sequence
encoding barley .alpha.-amylase signal peptide, wherein the nucleic
acid sequence encoding barley .alpha.-amylase signal peptide is
upstream of the first nucleic acid sequence encoding a fragment of
gp41 and downstream of the second promoter region.
14. The composition of claim 12, wherein the second nucleic acid
sequence encoding the fragment of gp41 comprises the nucleic acid
sequence of dgp41.
15. The composition of claim 3, wherein the replicating but highly
attenuated vaccinia virus vector is NYVAC.
16. A method of generating an immune response in a mammalian
subject against HIV comprising: administering a vaccinia VLP to the
mammalian subject, wherein the vaccinia VLP presents Gag, a
fragment of gp41, or both; and administering a plant-produced HIV
VLP to the mammalian subject, wherein the plant-produced HIV VLP
presents, a fragment of gp41, or both, wherein the mammalian
subject is administered the vaccinia VLP and the plant-produced HIV
VLP in an amount sufficient to generate an HIV immune response in
the mammalian subject.
17. The method of claim 16, wherein the mammalian subject is
administered the vaccinia VLP presenting both Gag and dgp41, and is
also administered the plant-produced HIV VLP presenting both Gag
and a fragment of gp41.
18. The method of claim 16, wherein the mammalian subject is
administered the vaccinia VLP at least 30 days prior to the
administration of the plant-produced HIV VLP.
19. A replicating but highly attenuated vaccinia virus vector
comprising: a thymidine kinase (TK) locus, the TK locus comprising
homologous recombination arms; and a nucleic acid sequence encoding
Gag or dgp41, wherein the nucleic acid sequence encoding Gag or
dgp41 is between the homologous recombination arms of the TK
locus.
20. The replicating but highly attenuated vaccinia virus vector of
claim 19, wherein the vector is NYVAC-KC.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
provisional patent application 62/663,391, filed Apr. 27, 2018, the
entirety of the disclosure of which is hereby incorporated by this
reference.
INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED
[0002] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 3,256 byte
ASCII (text) file named "SeqList" created on Apr. 25, 2019.
TECHNICAL FIELD
[0003] The disclosure relates to vaccination strategies against
human immunodeficiency virus (HIV), including methods of generating
an immune response against HIV, vectors, compositions, and
vaccination kits for use in the methods of generating an immune
response against HIV.
BACKGROUND
[0004] Despite the success of antiretroviral treatment, human
immunodeficiency virus (HIV) still causes millions of new
infections every year, primarily in regions of the world with
limited access to healthcare, making the need for a vaccine more
apparent every year. To date, even the most successful HIV-1
vaccine clinical trial, the Phase III Thai Trial (also referenced
herein as "RV144 Thai Trial" or "Thai Trial") had only modest and
short-lived efficacy (Rerks-Ngarm et al., 2009). The Thai Trial
used a non-replicating canarypox viral vector (ALVAC) carrying
recombinant genes for Gag, Pol and the surface subunit of the
envelope (ENV) protein (gp120) followed by an AIDSVAX protein boost
consisting of recombinant gp120 B/E produced in Chinese hamster
ovary (CHO) cells. While ALVAC and AIDSVAX showed no protection
when tested individually, their combination in RV144 has been shown
to be modestly protective, providing valuable information on immune
protection correlates. The most direct immune protection correlates
pertain to antibody (Ab) responses against the V1-V2 loop of gp120.
From these studies the importance of polyfunctional,
non-neutralizing Abs (Haynes et al., 2012; Yates et al., 2014;
Chung et al., 2014; Liao et al., 2013) has been shown. This
short-lived humoral response faded over time and did not provide
long-lasting protective advantage over the placebo arm (Yates et
al., 2014). The results of the RV144 trial indicated the strength
of a prime-boost approach integrating on a live vector with a
subunit protein vaccine. The modest efficacy of the trial, however,
also suggests that the particular combination of the specific
antigens requires further study (Haynes et al., 2012; Yates et al.,
2014; Corey et al., 2015; McMichael and Koff, 2014; Prentice et
al., 2015). Accordingly, there is a significant need for new
vaccination strategies against HIV.
SUMMARY
[0005] The disclosure relates to the immunogenicity of virus-like
particles (VLPs) produced by a replicating but highly attenuated
vaccinia virus vector, a tobacco mosaic virus-based vector, a
geminivirus-based vector, or combinations thereof. In certain
embodiments, the disclosure is directed to composition comprising a
vaccinia VLP and a plant-produced HIV VLP in sufficient amounts to
generate an immune response in a mammalian subject against human
immunodeficiency virus (HIV). The tobacco mosaic virus-based vector
or the geminivirus-based vector expresses Gag, a fragment of gp41,
or Gag and a fragment of gp41. In some aspects, geminivirus-based
vector further comprises p19. The replicating but highly attenuated
vaccinia virus vector expresses Gag, or a fragment of gp41 in
certain embodiments, and may be based on NYVAC-KC. In some aspects,
the fragment of gp41 is a membrane anchored truncated gp41
presenting the membrane proximal external region with its conserved
broadly neutralizing epitopes in the perfusion conformation.
[0006] Exemplary embodiments also include a replicating but highly
attenuated vaccinia virus vector expressing Gag or a fragment of
gp41 and a tobacco mosaic virus-based vector or a geminivirus-based
vector expressing Gag, a fragment of gp41, or Gag and a fragment of
gp41 are disclosed along with VLPs produced from these vectors. The
VLPs produced from the replicating but highly attenuated vaccinia
virus vector are a vaccinia VLP presenting Gag, a fragment of gp41,
or Gag and a fragment of gp41. The VLPs produced from the tobacco
mosaic virus-based vector and the geminivirus-based vector may be
plant-produced HIV VLPs presenting Gag and/or a fragment of
gp41.
[0007] Also disclosed herein are methods of purifying VLPs from
plants infiltrated with agrobacterium transformed with the tobacco
mosaic virus-based vector or the geminivirus-based vector.
[0008] The disclosure also relates to a composition or a kit for
generating VLPs for generating an immune response in a mammalian
subject against human immunodeficiency virus (HIV) and a
vaccination composition or kit against HIV comprising the described
VLPs. In some embodiments, the composition or kit for generating
VLPs for generating an immune response in a mammalian subject
against HIV comprise a first replicating but highly attenuated
vaccinia virus vector expressing Gag and a second replicating but
highly attenuated vaccinia virus vector expressing a fragment of
gp41. In other embodiments, the compositions comprise a replicating
but highly attenuated vaccinia virus vector expressing Gag and a
fragment of gp41. In some aspects, in addition to the
aforementioned vaccinia virus vector or vectors, the compositions
further comprise a vector for expression of HIV virus-like
particles (VLP) in plants. In certain specific aspects the VLP is
plant expressed. In exemplary aspects, the plant expression vectors
are tobacco mosaic virus-based or geminivirus-based. The HIV
virus-like particles (VLP) in plants comprise Gag and a fragment of
gp41. Accordingly, the plant expression vectors express Gag, dgp41,
or Gag and dgp41.
[0009] The disclosure is further related to methods of generating
an immune response against HIV in a mammalian subject, wherein the
subject is administered the vaccinia VLPs or the plant-produced HIV
VLP. In some implementations, the mammalian subject is administered
the vaccinia VLP and then the mammalian subject is administered the
plant-produced HIV VLP at least 30 days later. In some aspects, the
administration of the plant-produced HIV VLP is accompanied with
administration of the vaccinia VLPs. In some implementations, the
subject is administered a combination of the vaccinia VLPs and the
plant-produced HIV VLP.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0011] FIGS. 1A-1E depict, in accordance with certain embodiments,
the generation of recombinant vaccinia viruses. Plasmids were
cloned to contain either full-length HIV-1 Gag (FIG. 1A) or
deconstructed-gp41 (FIG. 1B) as pTM 815 and 816, respectively. The
genes are located between homologous recombination arms for the
vaccinia virus (VACV) thymidine kinase (TK) locus under the control
of a synthetic early/late (E/L) promoter. Also included is the
neomycin resistance gene fused to GFP (GFP-neoR). (C and D)
SDS-PAGE shows full-length Gag (FIG. 1C) or dgp41 (FIG. 1D)
expression in BSC-40's of NYVAC-KC-Gag (Lane 2) and dgp41 (Lane 3)
from the final P2 virus stock used in animal experiments. Parental
NYVAC-KC shows no expression of either protein (Lane 1). FIG. 1E
shoes the SDS-PAGE of ammonium sulfate precipitations from media of
BSC-40 cells infected with: NYVAC-KC (WT, Lane 1), Gag (Lane 2),
dgp41 (Lane 3), or a co-infection of Gag/dgp41 viruses (G/d, Lane
4). Samples were tested for both Gag (top) and dgp41 (bottom)
expression.
[0012] FIGS. 2A-2D depict, in accordance with certain embodiment,
electron microscopy of NYVAC-KC infected BSC-40s. BSC-40 cells were
infected at an MOI of 5 and harvested 24 hpi and prepared for
transmission electron microscopy. Cells were infected with either
NYVAC-KC (FIG. 2A), NYVAC-KC-Gag (B), NYVAC-KC-dgp41 (FIG. 2C), or
a co-infection with an MOI of 5 for each NYVAC-KC-Gag and
NYVAC-KC-dgp41 (FIG. 2D). All stages of VACV replication are
present: mature virions (white arrow), immature virions (black
triangle), immature virions with genome incorporated (white
triangle), crescent formation (black arrow), and `unfilled/empty`
virions (FIG. 2E). Budding HIV-1 Gag VLPs (black star) are also
seen in those cells infected with NYVAC-KC-Gag. In certain
infections, an accumulation of cytoplasmic `junk` (J) is visible.
Other cellular features: mitochondria (M), nucleus (N). Scale bar:
500 nm for all images, except (FIG. 2B-2) and (FIG. 2D-2) images
are 100 nm for HIV-1 VLPs.
[0013] FIGS. 3A-3C depict, in accordance with certain embodiments,
a mouse immunization schedule. FIG. 3A shows the time line of
treatment. Six-week-old C57BL/6 mice were immunized at day 0, 45,
and 90 with different combinations of recombinant vaccinia virus
(represented as an image of a green-enveloped particle) and
plant-produced HIV-1 VLPs (represented as an image of a poxvirus).
Vaccinia virus doses (VV) consisted of 1.times.10.sup.6 pfu of
equally mixed NYVAC-KC-Gag and NYVAC-KC-dgp41 delivered i.m., while
plant-produced VLPs were mixed with Ribi adjuvant and delivered
i.p. with 2 .mu.g p24 and 1.2 .mu.g MPER. Serum, fecal, and vaginal
lavage samples were collected once every 2 weeks (indicated by
arrows). Serum samples were analyzed for antigen-specific IgG to
p24 Gag (FIG. 3B) or the MPER region of dgp41 (FIG. 3C), shown over
the length of the experiment as the ELISA OD 490 nm for the 1:50
dilution (lowest tested).
[0014] FIGS. 4A-4C depict, in accordance with certain embodiments,
systemic and mucosal antibody production. Endpoint (Day 97) samples
were analyzed for serum IgG (FIG. 4A) and mucosal IgA in fecal
(FIG. 4B) and vaginal lavage (FIG. 4C) samples. Data are shown for
both p24-specific (left) and MPER-specific (right) antibodies. Raw
OD 490 nm values are shown for the lowest dilution factor tested
(1:50). Statistically significant differences (p<0.05) are
indicated with an asterisk (*). Each marker represents a single
animal.
[0015] FIGS. 5A-5C depict, in accordance with certain embodiments,
endpoint serum IgG titers and isotyping results. FIG. 5A shows
endpoint titers, which were calculated as the reciprocal of the
dilution factor that had background level of OD 490 nm (<0.1).
Isotypes were determined by ELISA for antigen-specific IgG1 (FIG.
5B) or IgG2a (FIG. 5C) for both p24 (left) and MPER (right) serum
IgG. OD 490 nm readings are shown and clearly indicate a bias
towards IgG1 production for both groups. Only endpoint (Day 97)
serum samples were tested for these two groups because they had the
highest responses. (*p<0.05; **p<0.01).
[0016] FIG. 6 depicts, in accordance with certain embodiments,
anti-VACV responses in serum at endpoint. Day 97 endpoint serum was
analyzed by ELISA for anti-vector responses with endpoint titers
calculated as for Gag and dgp41-specific serum IgG. The group which
received a total of 3 doses of NYVAC-KC vectors shows significantly
higher titers of VV-specific antibodies compared with any other
group (purple triangles). (***p<0.001; ****p<0.0001).
[0017] FIGS. 7A-7F depict, in accordance with certain embodiments,
depicts Gag-specific CD8 T cell responses. One week after the final
immunization, peak CD8 T cell responses were measured by
intracellular cytokine staining and flow cytometry for 5
immunodominant ZM96 Gag epitopes: AMQ8 (FIG. 7A), EVK10 (FIG. 7B),
LRS8 (FIG. 7C), VIP8 (FIG. 7D), and YSP8 (FIG. 7E). Number of
IFN-.gamma..sup.+ CD8.sup.+ T cells in the spleen is shown for each
peptide individually and the total response in the spleen (FIG. 7F)
was calculated by adding the individual responses together for each
group. The group boosted with VV and VLPs (purple) was
significantly higher than the VV/Tris group (red) for two peptides
and the overall response. (* p<0.05; ** p<0.01).
[0018] FIGS. 8a-8d depicts, in accordance with certain embodiments,
the T-DNA regions of tobacco mosaic virus (TMV) and
geminivirus-based transient expression vectors. Four transient
deconstructed viral expression vectors were compared in this study.
FIG. 8a shows a TMV-based vector, pTM 602, that transiently
expresses dgp41 after recombining with two other plasmids as part
of the ICON expression system (Marillonnet et al., 2004; Kessans et
al., 2013). FIGS. 8b-8d show vectors based on the geminivirus bean
yellow dwarf virus (BYDV) that express dgp41 in a replicon bordered
by LIRs in pTM 924 (FIG. 8b) and simultaneously with the silencing
suppressor protein p19 in pTM 925 (FIG. 8c). These vectors contain
the barley .alpha.-amylase signal peptide 5' of dgp41 for targeting
to the secretory pathway which is under control of the cauliflower
mosaic virus 35S promoter (P35) followed by 2 translation enhancer
binding sites (2e). ICON systems recombine to add the signal
peptide in planta and express dgp41 under the control of the Nos
promoter. Additionally, the last geminivirus vector contains two
replicons separated by an SIR and LIR for simultaneous expression
of Gag and dgp41 in addition to p19 in pTM 901 (FIG. 8d). The size
of the full plasmid is indicated. For replicon amplification,
geminivirus-based systems also contain the viral replication
proteins C1 and C2 (Rep and RepA, respectively). SIR--short
intergenic region; BAA--barley .alpha.-amylase; T-DNA--transfer
DNA; P--promoter, LIR--long intergenic region; 2e--2 enhancer
binding sites
[0019] FIGS. 9a-9d depict, in accordance with certain embodiments,
observations of leaf necrosis over a seven-day kinetics time course
experiment. Four transient expression constructs were analyzed for
expression of dgp41 alone in Gag transgenic N. benthamiana (FIG.
9a-9c) or simultaneously transient expression of Gag and dgp41 in
WT plants (FIG. 9d). Leaf samples were collected in triplicate over
seven days and images scanned to monitor leaf necrosis. For FIG.
9a, the TMV-based vector expressing dgp41 displays onset of
necrosis around 5 dpi. Geminivirus-based transient expression of
dgp41 in Gag plants displays necrosis more rapidly, around 3 dpi,
without the silencing suppressor protein p19 (Gemini) (FIG. 9b)
than the Gemini vector which expresses dgp41 and p19 (Gemini +p19)
from the same construct and shows onset of necrosis around 4 dpi
(FIG. 9c). WT plants infiltrated with the dual-replicon Gemini
vector (Dual Gem. +p19) simultaneously express Gag, dgp41, and p19
from the same construct, results in visible necrosis around 4 dpi
(FIG. 9d). dpi--days post infiltration; OD--optical density
[0020] FIGS. 10A-10D depict, in accordance with certain
embodiments, the expression kinetics of Gag and dgp41 with TMV and
geminivirus-based vectors. Homogenized leaf tissue was analyzed in
triplicate for Gag (FIG. 10A) and dgp41 (FIG. 10B) expression by
immunoblot over the seven-day time course and percent of peak
expression day calculated through band intensity. Stable Gag
expression and transient dgp41 expression was assessed for three
vectors in Gag transgenic N. benthamiana: TMV, Gemini, and Gemini
+p19. A fourth dual-replicon Gemini vector +p19 transiently
expresses Gag, dgp41, and p19 simultaneously in wild-type plants.
Peak expression day was determined for each vector. dpi--days post
infiltration. Each data point represents mean.+-.SEM.
[0021] FIGS. 11a-11c depict, in accordance with certain
embodiments, optimization of purification technique to minimize
rubisco in VLP extractions. Two strategies were tested for
efficiency in extracting VLPs with minimal remaining rubisco in
each sample. The first strategy utilized sequential ammonium
sulfate precipitations at 20% (20% SN and P) and 40% (40% SN and
P), where rubisco is expected to pellet around 25%. The second
strategy employed pH changes to pH 5.5 (5.5 P) followed by pH 5.0
(5.0 SN and P) where rubisco is expected to pellet at pH 5.5. An
additional 40% ammonium sulfate precipitation was performed on the
pH 5.0 SN to pellet any remaining VLPs (5.0-40% SN and P). Lanes
were loaded with equivalent TSP as shown by Coomassie staining
(FIG. 11a). Expression of Gag (FIG. 11b) and dgp41 (FIG. 11c) in
each fraction was analyzed by immunoblot. SN--supernatant;
P--pellet; TSP--total soluble protein
[0022] FIGS. 12a-12d depict, in accordance with certain
embodiments, purification of HIV VLPs for TMV and geminivirus-based
vectors. Six-week-old N. benthamiana Gag transgenic plants were
infiltrated to transiently express dgp41 with TMV (FIG. 12a),
Gemini (FIG. 12b), or Gemini +p19 (FIG. 12c). In FIG. 12d, WT
plants were infiltrated with a dual-replicon Gemini +p19 to
transiently express Gag and dgp41. Leaf tissue was harvested on
peak expression day and VLPs were extracted in 20 g batches
(T--total extract) through 20% ammonium sulfate precipitation
(SN--20% ammonium sulfate supernatant; P--pellet) and density
gradient centrifugation with step-wise 10-20-50% iodixanol layers
(Top--sample after centrifugation; 10% iodixanol; 20% iodixanol;
50% iodixanol). 5 .mu.g total soluble protein (TSP), as determined
by Bradford assay, was loaded into each lane and analyzed by
Coomassie staining (left), and for Gag (middle) and dgp41 (right)
expression. VLPs are almost completely pelleted with 20% ammonium
sulfate with little to no detectable protein remaining in the SN
and isolate to the 20/50% barrier of the density gradient.
Purifications representative of three independent extractions for
each vector are shown.
[0023] FIG. 13 show, in accordance with certain embodiments,
VLP-induced Gag and dgp41-specific serum IgG levels over the course
of 52 days. C57BL/6 mice were injected i.p. at Day 0 and Day 45
with VLPs, indicated by arrows. Anti-p24 (Gag) and anti-MPER
(dgp41) serum IgG is shown over time as the ELISA OD 490 nm for the
1:50 dilution. At the Day 52 endpoint, all animals have detectable
levels of both Gag and dgp41 specific antibodies.
[0024] FIGS. 14a-14d depict, in accordance with certain
embodiments, the expression of Gag and dgp41 with TMV and
geminivirus-based vectors. Leaf samples pictured in FIGS. 9a-9d
were homogenized for each vector, and 20 .mu.g TSP was loaded in
each lane (dpi 1-7), as measured by Bradford assay. Three vectors
were compared for dgp41 expression in Gag transgenic N.
benthamiana: (FIG. 14a) TMV, (FIG. 14b) Gemini, and (FIG. 14c)
Gemini +p19. A fourth dual replicon Gemini vector +p19 transiently
expresses Gag, dgp41, and p19 simultaneously in WT plants (FIG.
14d). Coomassie-stained gels show equal TSP across lanes (left) and
specific immunoblot for Gag (middle) and dgp41 (right) proteins was
used to determine expression levels and identify peak expression
day. Images shown are representative of triplicate leaf samples
processed for each vector. dpi--days post infiltration; TSP--total
soluble protein; WT--wild-type
DETAILED DESCRIPTION
[0025] Detailed aspects and applications of the disclosure are
described below in the following drawings and detailed description
of the technology. Unless specifically noted, it is intended that
the words and phrases in the specification and the claims be given
their plain, ordinary, and accustomed meaning to those of ordinary
skill in the applicable arts.
[0026] In the following description, and for the purposes of
explanation, numerous specific details are set forth in order to
provide a thorough understanding of the various aspects of the
disclosure. It will be understood, however, by those skilled in the
relevant arts, that embodiments of the technology disclosed herein
may be practiced without these specific details. It should be noted
that there are many different and alternative configurations,
devices and technologies to which the disclosed technologies may be
applied. The full scope of the technology disclosed herein is not
limited to the examples that are described below.
[0027] The singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a step" includes reference to one or more of
such steps.
[0028] As used herein, the term "virus-like particle" or "VLP"
refers to multiple protein structure that mimic the organization
and conformation of authentic native viruses but lack the viral
genome. In some embodiments, expression of viral structural
proteins, for example capsid or envelope proteins, result in the
self-assembly of VLPs. In other embodiments, a viral core is
required to facilitate the assembly of the VLP when fragments of a
protein are the desired presentation targets at the surface of the
VLP. Viral cores used in the design of VLPs include bacteriophage
MS2, adeno-associated virus, adenovirus, and tobacco mosaic
virus.
[0029] As referenced herein, the nucleic acid sequence of Gag has
at least 95% identity with the nucleic acid sequence of Accession
No. AY805330 or Accession No. JX534517.
[0030] As used herein, the term "dgp41" refers to a membrane
anchored truncated gp41 presenting the membrane proximal external
region with its conserved broadly neutralizing epitopes in the
prefusion conformation. As referenced herein, the nucleic acid
sequence of dgp41 has at least 95% identity with the nucleic acid
sequence of Accession No. AF075722, Accession No. AY805330, or
Accession No. JX534518.
[0031] The disclosure relates to a vaccination strategy against
human immunodeficiency virus (HIV) using VLPs presenting Gag and/or
dgp41. VLPs are safe, yet immunogenic, components of several
vaccines and candidate vaccines for multiple infectious diseases
(for a review see Kushnir et al., 2012). The potential for success
of VLP-based vaccines is indicated by the widespread use of the
human papillomavirus (HPV) vaccines Gardasil.RTM. (Merck and Co,
2006) and Cervarix.RTM. (GlaxoSmithKline, 2014). To date, many
plant-produced vaccines have been tested in animal studies for
immunogenicity for both human and veterinary diseases (Rybicki,
2010; Scotti and Rybicki, 2013). Additionally, VLPs, but not
soluble subunit vaccines, are more likely to properly induce innate
and low affinity B cells to transport and display antigen to induce
Ab responses (Bessa et al., 2012; Link et al., 2012).
[0032] In some aspects, the disclosure is directed to a replicating
but highly attenuated vaccinia virus for expressing Gag and/or
dgp41. Upon infection of mammalian cells with the vaccinia virus,
VLPs presenting Gag and/or dgp41 are produced. In other aspects,
the disclosure is directed to a HIV VLP produced from a tobacco
mosaic virus-based vector (magnICON) or a geminivirus-based vector.
The tobacco mosaic virus-based vector and geminivirus-based vector
are designed for expression of the HIV VLP in plants. Accordingly,
the disclosure also relates to optimized methods of purifying the
HIV VLP from plants to reduce the amount of protein contaminants,
for example rubisco (Ribulose-1,5-bisphosphate
carboxylase/oxygenase). The disclosure is also encompasses vector
compositions for producing vaccinia VLPs and plant-produced HIV
VLPs that generate an immune response against HIV in a mammalian
subject, particularly HIV-1.
[0033] In other aspects, the disclosure is directed to methods of
generating an immune response against HIV, particularly HIV-1, in a
mammalian subject using the VLPs described herein.
[0034] In some implementations, the methods of generating an immune
response against HIV in a mammalian subject comprises administering
the VLP described herein to the mammalian subject on two separate
occasions, for example, at least 30 days apart. In certain
implementations, the two administrations of the VLP are 45 days
apart. In one implementation, the VLP administered in both
instances are plant-produced HIV VLPs. Plant-produced HIV VLPs are
immunogenic in mice and can elicit antibodies specific to the MPER
broadly neutralizing antibody (bnAb) target in the dgp41 protein
(FIG. 13). In one aspect, two weeks after the first dose of
Gag/dgp41 VLPs produced using the TMV-based vector was administered
to murine subjects, p24-specific IgG as detectable in the serum
while MPER-specific antibodies were below detection limit (FIG. 6).
One week after the second immunization, a 100% response rate was
observed for both p24- and MPER-specific serum IgG.
[0035] In a particular embodiment, the method of generating an
immune response against HIV in a mammalian subject comprising
exposing the mammalian subject to a VLP presenting Gag and/or a
fragment of gp41 (for example, dgp41) on three separate occasions,
wherein each exposure is at least 30 days apart, for example 45
days apart. In some aspects, the first exposure comprises
administration of attenuated vaccinia virus expressing Gag and/or
dgp41 to the mammalian subject, which results in exposure to
vaccinia VLPs presenting Gag and/or dgp41, while the second
exposure and third exposure comprise administration of a
plant-produced HIV VLP to the mammalian subject. In some
implementations, an adjuvant is administered with the
plant-produced HIV VLP. In certain embodiments, the HIV VLPs
administered in the second and third exposure are produced from a
plant transformed with a tobacco mosaic virus-based vector
(magnICON) or a geminivirus-based vector expressing Gag and/or
dgp41. In particular implementations, the second exposure and third
exposure further comprise administration of attenuated vaccinia
virus expressing Gag and/or dgp41. In certain implementations, the
amount of plant-produced HIV VLP administered to a mammalian
subject per exposure that sufficient to ultimately generate an HIV
immune response in the mammalian subject is between 1.5 and 3 .mu.g
Gag (p24) and between 0.75 and 2.5 .mu.g dpg41 (MPER), between 1.75
and 2.25 .mu.g Gag and between 0.95 and 1.45 .mu.g dpg41, or
preferably about 2 .mu.g Gag and about 1.2 .mu.g dpg41. In some
aspects, the molar ratio of dpg41 to Gag is at least 1.7 or between
1.7-11.8. In some aspects, the total amount of Gag and dpg41
administered per exposure is between 60 and 400 .mu.g for each
antigen, between 100 and 350 .mu.g for each antigen, between 150
and 300 .mu.g for each antigen, between 250 and 250 .mu.g for each
antigen, or preferably 300 .mu.g for each antigen. Thus, in some
aspects, the disclosure relates to a composition comprising a
plant-produced HIV VLPs and vaccinia VLP.
[0036] This vaccine strategy is based on two discoveries. The first
is that replicating vectors, in addition to their excellent
facility in eliciting T cell responses, are expected to increase
antigen load, resulting in improved immunogenicity. The second is
that VLPs improve presentation of relevant neutralizing
determinants to the immune system. In particular, the inventors
developed replicating vaccinia virus vectors based on NYVAC-KC that
express Gag and dgp41 of HIV-1 as matching antigens to the
plant-produced VLPs for prime/boosting purposes. The HIV membrane
protein gp41 contains the bnAb target known as the MPER. This
region requires the context of a membrane in order to elicit the
partially auto-reactive bnAbs found in HIV-infected patients
(Verkoczy et al., 2010, 2013; Haynes et al., 2005; Zhang et al.,
2016). In some implementations, the method further comprises
administering to the subject a plant-produced HIV VLP. Accordingly,
in some embodiments, the methods comprise administering together
the combination of NYVAC-KC-Gag/dgp41 replicating vaccinia virus
vectors in a regimen closely mimicking the RV144 clinical
trial.
[0037] Despite the poor priming capacity of the vaccinia VLP (VV)
in terms of eliciting antibody responses against Gag and gp41, upon
boosting with plant-derived HIV VLPs at Day 45, antibody (Ab)
production spiked with another increase after the final
immunization at Day 97 (FIG. 3B-3C). Endpoint anti-p24 Ab titers
reached significant levels for serum IgG in the group boosted with
just VLPs (Group 2) or the combination of VV+VLPs (Group 3) (FIG.
5A), and all groups boosted with any combination of VV and VLPs
(Groups 2-4) reached significance for MPER-specific serum IgG (FIG.
4A). As noted above, due to the lack of detectable Abs prior to
boosting with VLPs, it seems that the plant-produced VLPs
administered at a relatively low dose, are largely responsible for
boosting the Ab responses following sub-responsive priming. In the
groups with the highest responses, these antibodies were shown to
be entirely IgG1 with no detectable IgG2a (FIG. 5B-5C). The
inventors also discovered that plant-produced HIV VLPs stimulate a
predominant Th2 response through activation of M2b macrophages,
thus stimulating strong B cell Ab production.
[0038] In some aspects, the method induces systemic and mucosal
antibody responses to Gag and dgp41 with a bias towards IgG1. For
example, the methods of generating an immune response against HIV
in a mammalian subject produce robust Gag-specific CD8 T cell
responses. Gag-specific CD8 T cell responses were highest in the
group boosted with VV+VLPs (Group 3), reaching 12.7% of CD8 T cells
expressing IFN-.gamma. in response to the five Gag peptides (FIG.
7). While inventors do not have a non-replicating vector in this
study, these responses are higher than MVA-induced Gag-specific CD8
T cells (<0.5%) in humans after two doses (Keefer et al., 2011).
Additionally, results here show higher Gag-specific CD8 T cell
responses than seen in mice after DNA-NYVAC or DNA-MVA prime-boost
regimen which elicit <2% GPN/Env-specific CD8 T cells (<0.5%
Gag specific) and 12-15% GPN/Env-specific CD8 T cells (5.5%
Gag-specific), respectively (Gomez et al., 2012b; Garcia-Arriaza et
al., 2013). Thus, replication plays a role in increasing T cell
responses. Results from NYVAC HIV vaccines have shown proficiency
for eliciting polyfunctional T cell responses (i.e.
antigen-specific T cells expressing multiple cytokines) which are
primarily effector memory T cells (Harari et al., 2008, 2012; Gomez
et al., 2012b; Garcia-Arriaza et al., 2011). Accordingly,
replicating vectors such as NYVAC-KC, may have additional
beneficial roles in the prevention of HIV infection due to the
association of polyfunctional T cells in the periphery (Betts et
al., 2006). Additionally, it is important to note than when Env is
included in the MVA or NYVAC vector, T cell responses are skewed to
be primarily Env-specific with minimal Gag recognition (Harari et
al., 2008, 2012; Gomez et al., 2012b; Garcia-Arriaza et al., 2013;
Garcia et al., 2011; Mooij et al., 2009). Env-specific cytotoxic T
lymphocyte (CTL) responses were not protective in clinical trials
nor correlated with improved disease in natural infection
(McMichael and Koff, 2014), while Gag-specific responses have been
correlated with improved CD4 count and reduced viral load (Kiepiela
et al., 2007; Koup et al., 1994; Stephenson et al., 2012; Jiao et
al., 2006; Ogg et al., 1998; Brander and Walker, 1999) and have
epitopes which are less prone to CTL escape (Goulder and Watkins,
2004).
[0039] As shown in the animal study of Example 1, the described
VLPs produced from the vaccinia virus vector and the plant-produced
HIV VLPs work in concert to elicit Gag-specific CD8 T cell
responses and both systemic and mucosal antibodies to Gag and dgp41
peptides in the immunization group which most closely mimics the
Thai Trial. The vaccina virus vector is developed from the NYVAC
strain previously rendered replication incompetent in human cells
by deletion of 18 open reading frames from the genome of Copenhagen
(Cop), its parental strain (Tartaglia et al., 1992a). NYVAC-KC
contains a reinsertion of two host-range genes, K1L and C7L, which
allows the virus to replicate in human tissue, thus improving
immunogenicity while remaining highly attenuated (Kibler et al.,
2011; Quakkelaar et al., 2011).
1. Gag
[0040] Because of the surface exposure, immunogenicity, and the
critical roles during target-cell infection of HIV envelope protein
(Env/gp120), this protein has been a natural target for vaccine
development since the early days of HIV-1 research (Burton and
Mascola, 2015; Mascola and Haynes, 2013). However, targeting gp120
in the development of an HIV vaccine has proven to be far from
straightforward. gp120 is not highly conserved, and many critical
neutralization targets are hidden or are only exposed upon
conformational change during viral entry, thus limiting the
effectiveness of any NAb response to this antigen (Pancera et al.,
2014; Decker et al., 2005; Labrijn et al., 2003). Thus, gp120
functions as a highly mutable decoy with most of its functionally
important immunogenic sites conformationally occluded or shielded
by glycans. In fact, monomeric preparations of gp120, as well as
various preparations aimed at presentation of gp120 trimers, have
repeatedly failed to induce protective immune responses in animal
models or humans, with the sole and modest exception of the RV144
Thai Trial (also referenced herein as "Thai Trial") (McGuire et
al., 2014; Jacob et al., 2015; Moore et al., 2015; Haynes et al.,
2016).
[0041] The Thai Trial demonstrated the strength of prime/boost
vaccination approach by effectively combining two components that
were previously shown to be ineffective on their own: a live
(albeit non-replicating) canarypox viral vector (ALVAC) and a
soluble protein boost (AIDSVAX). The low efficacy of the trial left
much to be desired for widespread use as a vaccine while providing
the conceptual basis for further improvement (Rerks-Ngarm et al.,
2009). Protein engineering has attempted to resolve many of the
issues of targeting gp120, including the design of SOSIP trimeric
gp140 variants of Env to make a more structurally accurate target
necessary for eliciting specific types of NAbs, including a design
to specifically target nAb germline B cells (Jardine et al., 2013;
Billington et al., 2007; Du et al., 2009; Sellhorn et al., 2012;
Wan et al., 2009). To date, no successful clinical trials
incorporating an engineered gp140 antigen have been conducted.
[0042] An optimized nucleic acid sequence encoding Gag for VLP
formation was previously described (Kessans et al., 2013). As
described herein, the optimized Gag sequence is suitable for VLP
formation using a vaccinia virus vector, tobacco mosaic virus-based
vector, and geminivirus-based vector. In some aspects, the nucleic
acid sequence of the optimized Gag comprises the nucleic acid
sequence of Accession No. JX534517. In other aspects, the nucleic
acid sequence of the optimized Gag has at least 95% identity with
the nucleic acid sequence of Accession No. JX534517. In some
embodiments, the
2. gp41
[0043] Whereas many studies have examined the immunogenicity of
Gag, far fewer studies have examined gp41. The gp41 protein of HIV
is the transmembrane portion of the HIV surface spike and contains
the highly immunogenic membrane proximal external region (MPER), a
target of many broadly neutralizing antibodies (bnAbs) (Purtscher
et al., 1994; Zwick et al., 2001; Huang et al., 2012). gp41
contains highly immunogenic determinants that induce production of
Abs that are among the first to arise during acute HIV-1 infection
but are of very limited protective value, showing little or no
antiviral activities (Burton and Mascola, 2015; Liao et al., 2011;
Bonsignori et al., 2012). These immunodominant epitopes are located
in a region of the protein spanning the two heptad repeat domains
and in particular within the loop that connects them (Zolla-Pazner,
2004) and are exposed on the gp41 "stump" in its six-helical bundle
conformation upon removal of the gp120 subunit (Burton and Mascola,
2015). Still, gp41 was found to be the target of a number of
broadly neutralizing Abs (bnAbs) directed against conformational
(35O22 binding at the gp41-gp120 interface) (Huang et al., 2014)
and linear epitopes (2F5, 4E10, Z13 and 10E8 that recognize closely
situated sites within the membrane proximal external region, MPER)
(Parker et al., 2001; Cardoso et al., 2005; Nelson et al., 2007;
Huang et al., 2012).
[0044] Beyond neutralization, anti-MPER Abs, including the
above-mentioned bnAbs, were found to exhibit other potent
anti-HIV-1 activities including transcytosis blockade (Tudor et
al., 2012; Shen et al., 2010; Matoba et al., 2004, 2008, 2009),
Ab-dependent cell-mediated cytotoxicity (ADCC) (Tudor and Bomsel,
2011; Hessell et al., 2007) and curbing of dendritic cell-mediated
trans-infection (Tudor et al., 2012; Sagar et al., 2012;
Magerus-Chatinet et al., 2007). Moreover, passive immunization with
these bnAbs provided impressive protection against mucosal
transmission of a simian-HIV hybrid (SHIV) in the macaque model
(Baba et al., 2000; Hessell et al., 2010; Klein et al., 2013a).
Anti-MPER Abs with anti-viral activities were also described in
mucosal secretions of highly-exposed but seronegative individuals
(Kaul et al., 1999, 2001; Pastori et al., 2000; Devito et al.,
2000a, 2000b; Tudor et al., 2009). Lastly, anti-MPER Abs that were
passively passed through breast milk from infected mothers to their
uninfected babies were correlated with protection (Diomede et al.,
2012; Pollara et al., 2015).
[0045] Despite their long-standing promise, anti-MPER bnAbs are
naturally rare and are notoriously difficult to elicit. For
example, less than 25% of HIV-1-infected patients develop broad and
potent nAbs, which even if present require .about.2-4 years to
produce (Burton and Mascola, 2015; Mascola and Haynes, 2013; Haynes
et al., 2016; Mascola and Montefiori, 2010). Several explanations
have been suggested, including a lengthy maturation process
involving many somatic mutations and extensive affinity maturation
(Klein et al., 2013b; Kepler et al., 2014), and clonal deletion of
the B-cell lines secreting such bnAbs that were shown to be
partially autoreactive (Verkoczy et al., 2010, 2011, 2013).
Interestingly, many of the autoreactivity targets are lipids that
can be found in both the plasma membrane of the host cell and in
the viral envelope (Haynes et al., 2005; Alam et al., 2007, 2009;
Irimia et al., 2016).
[0046] It is widely accepted that the membrane milieu of the MPER
region plays a major role in the functional immunogenicity of gp41
(Alam et al., 2009; Chen et al., 2014). Both the metastable native
conformation of the gp120-gp41 trimer and the highly stable
post-fusion conformations do not expose the neutralizing epitopes
(Frey et al., 2008, 2010; Buzon et al., 2010; Pancera et al.,
2014), and fail to efficiently elicit bnAbs (Williams et al., 2015;
Sanders et al., 2015; Crooks et al., 2015; Davis et al., 2009;
Decker et al., 2005; Labrijn et al., 2003) or engage the germline B
cell precursors for specific bnAbs (McGuire et al., 2014; Hoot et
al., 2013; Jardine et al., 2013).
[0047] An effective MPER-based antigen therefore requires
presentation of the MPER within a context of a membrane and
exposure of the region in a conformation mimicking the putative
pre-fusion intermediate. In part, this is because many of the
apparent interactions between the amphipathic peptide and the
membrane affect the ability of the region to interact with bnAbs
(Haynes et al., 2005; Verkoczy et al., 2010; Williams et al., 2015;
Haynes et al., 2016; Zhang et al., 2016). This antigenic region
when presented correctly on a membrane has strong potential as a
vaccine component. Displaying the MPER in a virosome successfully
elicited mucosal and systemic Abs with transcytosis-blocking
activity in a Phase I clinical trial (Leroux-Roels et al., 2013).
Bomsel and co-workers used a gp41 peptide (residues 649-684)
spanning the MPER and part of the C-terminal heptad repeat to
decorate liposomes ("virosome"), and the virosome elicited
protective responses against SHIV infection in the macaque model
(Bomsel et al., 2011). VLPs displaying gp41 can elicit both
systemic and mucosal Abs to this region (Kessans et al., 2016) and
transcytosis blocking Abs to this region are achievable (Matoba et
al., 2008).
[0048] As described herein, a fragment of gp41 that comprises a
membrane anchored truncated gp41 presenting the membrane proximal
external region with its conserved broadly neutralizing epitopes in
the prefusion conformation is suitable for triggering an immune
response against HIV. In some aspects, the fragment of gp41 is
dgp41. In certain embodiments, the nucleic acid sequence of dgp41
comprises the nucleic acid sequence of Accession No. JX534518. In
other embodiments, the nucleic acid sequence of dgp41 has at least
95% identity with the nucleic acid sequence of Accession No.
JX534518. In some aspects, the nucleic acid sequence of dgp41 is
the nucleic sequence of the digestion fragment pTM 602 using the
restriction enzymes NcoI and SacI.
3. Vaccinia VLP
[0049] Live recombinant vectors based on viruses belonging to a
wide range of families such as adenoviridae, poxviridae, and
herpesviridae have been previously tested for their immunogenicity
(Rerks-Ngarm et al., 2009; Hansen et al., 2013a; Huang et al.,
2015; Buchbinder et al., 2008; Tartaglia et al., 1992b). The
relative success of ALVAC within the context of RV144 increased the
focus on poxviruses (see Gomez et al., 2012a; Pantaleo et al.,
2010; Jacobs et al., 2009). ALVAC is based on canarypox, which like
other avipoxviruses is naturally attenuated in humans due to its
restricted replication in non-avian cells (Taylor and Paoletti,
1988; Taylor et al., 1988) accounting for ALVAC's high safety
profile (Team AVEGP, 2001; Nitayaphan et al., 2004). Similarly,
when developing vaccinia-based vaccine vectors, efforts were
initially focused on strains that are non-replicating in human
cells (e.g. MVA and NYVAC), due to safety concerns over potential
complications with VACV infection in immune-compromised
individuals.
[0050] Live viral vectors are appealing as vaccine vehicles for the
expression of HIV-1 antigens due to their proficiency for eliciting
T cell responses. For example, a cytomegalovirus (CMV) vector was
recently shown in nonhuman primates to clear an established SIV
infection with dependency on CD8 T cells recognizing non-canonical
epitopes on major histocompatibility complex (MHC) II instead of
MHC I (Hansen et al., 2013a, 2013b). Poxviruses also stand out
amongst other viral vectors [see reviews (Gomez et al., 2012a;
Pantaleo et al., 2010; Jacobs et al., 2009)] as attested by the
Thai Trial that employed a canarypox-based vector and was the first
and only HIV-1 vaccine clinical trial to show efficacy (Rerks-Ngarm
et al., 2009; Haynes et al., 2012). The canarypox-based viral
vector used in the trial, ALVAC, is not capable of replication in
mammalian cells (Taylor and Paoletti, 1988; Taylor et al., 1988).
Inability to replicate in vivo in humans makes the virus safer to
use as a vaccine or a vaccine vector. However, as described herein,
replication could enhance immunogenicity by increasing antigen load
as the virus replicates in vivo.
[0051] Intensive research led to partial uncoupling of attenuation
and replication in NYVAC-based vectors showing enhanced
immunogenicity without compromising their safety. Specifically,
reinsertion of two host-range genes yielded NYVAC-KC. This strain
is capable of replicating in human cells and displays enhanced
immune activation, yet it remains highly attenuated in a mouse
model of pathogenesis (Kibler et al., 2011; Quakkelaar et al.,
2011), thus giving this particular vector many desirable traits of
a vaccine vector and was chosen for use in the work presented here.
NYVAC was originally designed as a highly attenuated vaccine vector
by specifically deleting 18 open reading frames from its parental
strain (Cop), thus preventing its replication in humans (Tartaglia
et al., 1992b). NYVAC has been tested as an HIV vaccine vector and
was shown to induce robust, polyfunctional CD4 and CD8 T cell
responses to Env and Gag-Pol-Nef (GPN) antigens in vaccinated
individuals (Harari et al., 2008, 2012). Because mucosal immune
responses are thought to be particularly important in protection
against HIV-1, it was significant to note that such responses could
be detected in the gut of NYVAC-immunized patients (Perreau et al.,
2011). More recently, intensive research led to partial uncoupling
of attenuation and replication in NYVAC-based vectors showing
enhanced immunogenicity without compromising their safety.
Specifically, reinsertion of two host-range genes yielded NYVAC-KC.
The NYVAC-KC vector is a replicating, but safe, live viral vector
with which induces higher Gag-specific CD8 T cell responses than
with non-replicating poxviral vaccine vectors, although a direct
comparison has yet to be performed. NYVAC-KC is capable of
replicating in human cells and displays enhanced immune activation,
yet it remains highly attenuated in a mouse model of pathogenesis
(Kibler et al., 2011; Quakkelaar et al., 2011), thus giving this
particular vector many desirable traits of a vaccine vector and was
chosen for use in the vaccine strategy described herein.
[0052] In some embodiments, the vaccine design strategy disclosed
herein utilized the replicating vaccinia strain NYVAC-KC to express
the full-length Gag or a fragment gp41 of HIV, for example a
membrane anchored truncated gp41 presenting the membrane proximal
external region with its conserved broadly neutralizing epitopes in
the prefusion conformation (dgp41). As is well understood in the
art, recombinant vaccinia virus is produced by inserting foreign
genes into the thymidine kinase (TK) gene using homologous
recombination. This is achieved by a recombination plasmid
containing the TK locus with the foreign gene inserted between the
homologous recombination arms. Accordingly, the vaccinia vector
described herein comprises a nucleic acid encoding Gag or a
fragment of gp41 (for example, dgp41) in the TK locus between the
homologous recombination arms.
[0053] Vaccinia VLPs presenting Gag, a fragment of gp41 (for
example, dgp41), or both can be produced from the described
vaccinia vector by transfecting mammalian cells with these vectors
and infecting the transformed mammalian cells with a replicating
but highly attenuated vaccinia virus (for example NYVAC).
Transfecting the mammalian cells with a vaccinia vector expressing
Gag produces VLPs presenting Gag upon viral infection, while
transfecting the mammalian cells with vaccinia vector expressing
dgp41 produces VLPs presenting dgp41 upon viral infection. If the
mammalian cells are transfected with both a vaccinia vector
comprising Gag and a vaccinia vector comprising dgp41, however,
then VLPs presenting Gag and dgp41 are produced. These VLPs can be
administered to a mammalian subject to prime an immune response
against HIV, for example, when used in combination with the HIV
VLPs described herein. In some implementations, NYVAC-KC for
expression of Gag and NYVAC-KC for expression of dgp41 of HIV-1 are
used as matching antigens to the plant-produced VLPs for
prime/boosting purposes. Accordingly, the disclosure relates to
compositions comprising a replicating and highly-attenuated
vaccinia virus vector expressing Gag and/or replicating and
highly-attenuated vaccinia virus expressing a fragment of gp41 of
HIV along with a construct for the expression of HIV VLP in plants.
The vaccine strategy targets gp41 of HIV.
[0054] Infecting susceptible cells with NYVAC-KC-Gag with or
without NYVAC-KC-dgp41 releases, respectively, -100 nm Gag or
Gag/dgp41 VLPs into the extracellular medium (FIG. 1E). Such
particles can be seen by TEM budding out of the cell (FIGS. 2B-2
and D-2). dgp41 is detected in the medium only when co-expressed
with Gag (FIG. 1E), a result compatible with the notion the two
proteins assemble at the plasma membranes of cells in culture to
form VLPs. Although other groups have shown that VLPs expressing
HIV-1 proteins produced by other VACV-based vectors elicit both
humoral and cellular immunity (Chen et al., 2005; Goepfert et al.,
2011; Perdiguero et al., 2015), the experiments disclosed herein
did not detect any antibodies in any test group until after the
first plant-derived VLP immunization (FIG. 3B-C). Without being
limiting to any one mechanism, one explanation of the difference in
experimental results is that production of Gag/dgp41 VLPs in vivo
necessitates co-infection of the same cell by the two viruses.
While this is achievable under cell culture conditions where high
MOI can be ensured, but co-infection of the same cell by the two
viruses is unlikely to occur in an animal.
[0055] Accordingly, in another embodiment, the vaccinia virus
vector co-expresses Gag and dgp41 from the same locus to ensure in
vivo Gag/dgp41 VLP formation. As such, in some aspects, the
vaccinia virus vector comprises a nucleic acid encoding Gag and a
fragment of gp41 (for example, dgp41) in the TK locus between the
homologous recombination arms. VLPs produced from infecting
mammalian cells with this vaccinia virus vector, which would
express Gag and dgp41, are also suitable for priming an immune
response against HIV, for example, when used in combination with
the HIV VLPs described herein.
[0056] Another explanation for the limited functionality of the
dual vectors is the poor replication and/or spread in mice. In some
aspects, poor replication and/or spread of the vectors is due to
the cytotoxicity represented by protein precipitates in the
cytoplasm and nuclear condensation in NYVAC-KC-Gag-, dgp41- or
co-infected cells (FIG. 2B-2D). The toxicity seen with TEM strongly
correlates to reduced plaque sizes and lower viral titers noted
during viral selection. Gp41 is known to have a toxic cytoplasmic
tail (Postler and Desrosiers, 2013; Micoli et al., 2006), so it can
be responsible for the toxic effects seen in FIGS. 2A-2D. The
cytotoxicity of full-length gp160 has largely limited the use of
gp41 in vaccine candidates despite the appealing, highly conserved
target region of the MPER. Additionally, this is consistent with
published data that NYVAC expressing HIV Gag-Pol-Nef induces
extensive apoptosis (Gomez et al., 2007). In one implementation,
the toxicity of gp41 cytoplasmic tail is reduced by using other
vaccinia virus strains that produces vaccine candidates with
full-length gp160 in its native structurally accurate
conformation.
4. Plant-Produced HIV VLPs
[0057] Plant production of recombinant proteins and pharmaceuticals
offers multiple advantages, including lack of contamination by
mammalian pathogens, less expensive scale-up, speed of expression,
and platform flexibility (Horn et al., 2003; Ma et al., 2005;
Daniell et al., 2009; Rybicki, 2009, 2010; Egelkrout et al., 2012).
Many different types of VLPs have been produced in plants, for
review see (Chen and Lai, 2013; Scotti and Rybicki, 2013). Several
plant-made products have been tested in human clinical trials (Ma
et al., 2005; Yusibov et al., 2014) and one product has been
approved by the FDA for treatment of Gaucher disease (Zimran et
al., 2011). Economic viability of plant production relies heavily
on oftentimes poor yields, and downstream processing costs to
remove host proteins accounts for .about.80% of production
expenditures (Egelkrout et al., 2012; Wilken and Nikolov,
2012).
[0058] Several transient deconstructed viral-based vectors for
rapid, high level protein expression in plants are currently
available. The tobacco mosaic virus (TMV)-based magnICON system has
been extensively used to express recombinant proteins in plants
since its invention and was the first to provide gram-levels of
antigen (Marillonnet et al., 2004; Gleba et al., 2005; Marillonnet
et al., 2005). However, other expression systems based on other
plant viruses such as geminiviruses (Gemini) (Mor et al., 2003;
Huang et al., 2009) and Cowpea mosaic virus (CPMV)-based pEAQ
vectors (Sainsbury et al., 2009; Peyret and Lomonossoff, 2013) are
less well characterized. The TMV system requires simultaneous
delivery of three plasmids by Agrobacterium tumefaciens
infiltration (agroinfiltration) to recombine in planta within the
nucleus and the TMV movement protein transfers amplified mRNA to
surrounding cells (Marillonnet et al., 2004). However, Gemini
vectors are single plasmid delivery yet lack a movement protein and
are known to induce gene silencing (Seemanpillai et al., 2003;
Rodriguez-Negrete et al., 2009; Aregger et al., 2012) but this can
be suppressed using the Tomato bushy stunt virus p19 protein
(Silhavy et al., 2002; Voinnet et al., 2003; Garabagi et al.,
2012). Furthermore, multiple proteins can be delivered on the same
plasmid and expressed in separate replicons established by short
and long intergenic regions (SIR/LIR) (Huang et al., 2009). After
transfer to the nucleus, Gemini DNA is amplified via rolling-circle
mechanism by the C1/C2 (Rep/RepA) proteins (Gutierrez, 1999; Mor et
al., 2003).
[0059] HIV VLPs have been produced in plants with low yield,
particularly of full-length Gag, being a common theme (Meyers et
al., 2008; Kessans et al., 2013). Yield can be increased by
expressing Gag in transgenic chloroplasts (Scotti et al., 2009). An
efficient production method for enveloped VLPs in the
tobacco-relative Nicotiana benthamiana, consisting of Gag and
deconstructed-gp41 (dgp41: MPER, transmembrane domain, and
full-length cytoplasmic tail) was developed (Kessans et al., 2013).
Such VLPs display the MPER of gp41 without steric hindrance from
gp120, without the immunodominant epitopes on both Env subunits,
and with a higher antigen load per VLP than what exists in an HIV
virion (Kessans et al., 2016). A similar construct has been shown
by us to be a likely trimer with its bnAb epitopes exposed (Gong et
al., 2015). When administered to mice, these VLPs elicit both serum
IgG and mucosal IgA to Gag and dgp41 antigens (Kessans et al.,
2016).
[0060] The described plant-produced HIV VLPs comprising full length
Gag and a fragment of gp41 have the advantage of displaying the
MPER of gp41 in the native context of a Gag matrix, providing an
immunogenic platform for humoral responses to both antigens
(Kessans et al., 2013, 2016). Additionally, HIV VLPs have been
shown to boost T cell responses following a heterologous prime
(Chapman et al., 2013; Chege et al., 2008; Pillay et al., 2010).
CD8 T cell responses, often targeting the Gag protein, are known to
be associated with reduced viral load, making this a key target for
protective T cell immunity (Kiepiela et al., 2007; Koup et al.,
1994; Mudd et al., 2012; Stephenson et al., 2012; Jiao et al.,
2006). Plant-produced VLPs show proficiency for eliciting high Ab
titers and may boost T cell responses primed by the NYVAC-KC
vectors.
[0061] The primary roadblocks to plant-produced pharmaceuticals
revolve around both purification and low yield/expression levels
(Wilken and Nikolov, 2012). Recombinant protein often accounts for
only 0.7-7% of total soluble protein (TSP) (Egelkrout et al., 2012)
whereas rubisco is the most prominent soluble protein, and the most
abundant protein in the world, at 30-50% of the plant TSP
(Spreitzer and Salvucci, 2002; Feller et al., 2008). Removal of
rubisco has plagued the plant-produced pharmaceutical field;
however, recent developments show great promise at removing rubisco
with simple chromatography and precipitation methods (Buyel et al.,
2013; Buyel and Fischer, 2014; Arfi et al., 2016). One such study
reports the ability to remove up to 92% of rubisco from plant
extractions using polyethylene glycol (PEG) precipitation (Arfi et
al., 2016). Described herein is a method for removing the primary
large and small subunit bands of rubisco from our VLP preparations
by simple ammonium sulfate precipitation (FIG. 4). The method
comprises sequential ammonium sulfate precipitation of supernatant
of homogenized recombinant plant tissue with starting concentration
of 20% ammonium sulfate and an ending concentration at 40% ammonium
sulfate. In one exemplary implementation, the supernatant of
homogenized recombinant plant tissue is precipitated first at 20%
ammonium sulfate followed by precipitation at 40% ammonium sulfate.
The VLPs are pelleted at 36,000.times.g for 30 min at 4.degree. C.
between fractions. These are critical advancements because the
economic advantage of using plants over other expression systems is
touted by downstream processing accounting for up to 80% of
production costs (Wilken and Nikolov, 2012).
[0062] a. Tobacco Mosaic Virus-Based Vector
[0063] The tobacco mosaic virus (TMV)-based magnICON system has
been extensively used to express recombinant proteins in plants
since its invention and was the first to provide gram-levels of
antigen (Marillonnet et al., 2004; Gleba et al., 2005; Marillonnet
et al., 2005). In some aspects, a tobacco mosaic virus-based vector
encoding Gag and/or dgp41 is used to produce the HIV VLP.
Accordingly, the tobacco mosaic virus-based vector comprises a
T-DNA region comprising a nucleic acid sequence encoding Gag,
dgp41, or both Gag and dgp41. In some embodiments, expression of
Gag, dgp41, or a nucleic acid sequence encoding both Gag and dpf41
is driven by the nopaline synthase (NOS) promoter. In some
embodiments, expression of Gag, dgp41, or a nucleic acid sequence
encoding both Gag and dpf41 is driven by the cauliflower mosaic
virus 35S promoter (P35). In some aspects, a nucleic acid sequence
encoding an attB site is upstream of the nucleic acid sequence
encoding Gag, dgp41, or both Gag and dgp41 and is downstream of the
promoter.
[0064] The TMV system requires simultaneous delivery of three
plasmids by Agrobacterium tumefaciens infiltration
(agroinfiltration) to recombine in planta within the nucleus and
the TMV movement protein transfers amplified mRNA to surrounding
cells (Marillonnet et al., 2004).
[0065] b. Geminivirus-Based Vector
[0066] In other aspects, a geminivirus-based vector encoding Gag
and/or dgp41 is used to produce the HIV VLP. Geminivirus-based
vectors are single plasmid delivery that lack a movement protein
and are known to induce gene silencing (Seemanpillai et al., 2003;
Rodriguez-Negrete et al., 2009; Aregger et al., 2012) but this can
be suppressed using the Tomato bushy stunt virus p19 protein
(Silhavy et al., 2002; Voinnet et al., 2003; Garabagi et al.,
2012). Furthermore, multiple proteins can be delivered on the same
geminivirus-based plasmid and expressed in separate replicons
established by short and long intergenic regions (SIR/LIR) (Huang
et al., 2009). After transfer to the nucleus, Gemini DNA is
amplified via rolling-circle mechanism by the C1/C2 (Rep/RepA)
proteins (Gutierrez, 1999; Mor et al., 2003).
[0067] In some aspects, the geminivirus-based vector is a vector
based on bean yellow dwarf virus (BYDV). Accordingly, the
geminivirus-based vector comprises a T-DNA region comprising at
least one replicon gene and a nucleic acid sequence encoding Gag
and/or a nucleic acid encoding dgp41, wherein the at least one
replicon gene is downstream of the nucleic acid sequence encoding
Gag and the nucleic acid encoding dgp41. Where the T-DNA region of
the geminivirus-based vector comprises a nucleic acid sequence
encoding Gag and a nucleic acid encoding dgp41, in some
embodiments, the nucleic acid sequence encoding dgp41 is upstream
of the nucleic acid sequence encoding Gag. In some embodiments, the
T-DNA region comprises a nucleic acid encoding Rep/RepA. In certain
embodiments, the BYDV short intergenic region is upstream of
Rep/RepA.
[0068] In some embodiments of the geminivirus-based vector, Gag and
dgp41 expression is under the control of P35. Thus, in certain
embodiments of the geminivirus-based vector, the T-DNA region
further comprises a promoter upstream of the nucleic acid sequence
encoding Gag and the nucleic acid encoding dgp41, where the
promoter region comprises a nucleic acid sequence encoding P35. In
some aspects, the promoter region further comprises two translation
enhancer binding sites (2e), which are downstream of the nucleic
acid sequence encoding P35. In certain embodiments, the
geminivirus-based vector further comprises a barley .alpha.-amylase
signal peptide upstream of the nucleic acid encoding dgp41 but
downstream of the promoter region.
[0069] In some aspects, the T-DNA region of the geminivirus-based
vector also comprises two BYDV long intergenic regions. One long
intergenic region is upstream of the promoter region, and one long
intergenic region is downstream of the at least one replicon gene.
Where the T-DNA region of the geminivirus-based vector comprises a
nucleic acid sequence encoding Gag controlled by one promoter
region and a nucleic acid encoding dgp41 controlled by another
promoter region, the long intergenic regions flank the portion of
the T-DNA region containing the nucleic acid sequence encoding Gag,
the nucleic acid encoding dgp41, and the at least one replication
gene.
[0070] In particular embodiments, the T-DNA region of the
geminivirus-based vector further comprises a nucleic acid sequence
encoding a silencing suppressor protein. In some aspects, the
silencing suppressor protein is p19, for example, tomato bushy
stunt virus p19. The expression of the silencing suppressor protein
by the geminivirus-based vector delays the onset of plant cell
necrosis when compared with plant cells transformed with
geminivirus-based vectors lacking a nucleic acid sequence encoding
the silencing suppressor protein. The nucleic acid sequence
encoding the silencing suppressor protein is upstream of the
nucleic acid sequence encoding Gag and the nucleic acid encoding
dgp41 and is upstream of the region flanked by the long intergenic
regions. In some aspects, expression is silencing suppressor
protein is controlled by a promoter downstream of the nucleic acid
sequence encoding the silencing suppressor protein. In certain
embodiments, the promoter is NOS.
[0071] Illustrative, Non-Limiting Example in Accordance with
Certain Embodiments
[0072] The disclosure is further illustrated by the following
examples that should not be construed as limiting. The contents of
all references, patents, and published patent applications cited
throughout this application, as well as the figures, are
incorporated herein by reference in their entirety for all
purposes.
1. Prime/Boost Anti-HIV-1 Vaccination Strategy
[0073] a. Materials and Methods
[0074] i. Cloning pGNR Plasmids for Virus Recombination
[0075] Construction of synthetic genes encoding Gag (from subtype C
R5 HIV-1 isolate 1084i, GenBank #AY805330; synthetic construct
GenBank #JX534517) and dgp41 (MPER derived from the B-clade MN
isolate, GenBank #AF075722, transmembrane and C-terminal domains
from the 1084i isolate #AY805330; synthetic construct # JX534518)
was previously described (Kessans et al., 2013). The genes were
cloned into the pGNR plasmid, which harbors a neoGFP selection
cassette (neomycin resistance gene fused to GFP, known as pGNR)
between homologous recombination arms for the vaccinia TK locus.
Gag and dgp41 genes were amplified from pTM 488 (Gag) and pTM 602
(dgp41) [described in (Kessans et al., 2013)] into TOPO pCR-2.1
vectors (Invitrogen). Cloning was achieved using AccuStart Taq DNA
polymerase HiFi PCR kit (Quanta Biosciences) with primers oTM 664
(SEQ ID NO. 1: 5'-ACTAGTATGGGAGCTAGAGCCTCT-3') and 665 1 (SEQ ID
NO. 2: 5'-CCCGGGTTATTGAGAGGAAG-3') for Gag and oTM 666 (SEQ ID NO.
3: 5'-ACTAGTATGGGATCTCAAACTCAACAA-3') and 1667 (SEQ ID NO. 4:
5'-CCCGGGTTATTGCAAAGCA G-3') for dgp41 for the addition of 5'
flanking Spel and 3' flanking XmaI sites, denoted in italics.
Ligation reactions were electroporated into DH5 a Escherichia coli
and plated onto LB +ampicillin plates. Gene insertion was confirmed
by colony PCR using GoTaq Green Master Mix (Promega). Plasmids were
extracted using the E.Z.N.A. mini-prep kit (Omega) and sequences
verified using backbone-specific primers M13-forward and
M13-reverse. The TOPO plasmids were designated pTM 813 (Gag) and
pTM 814 (dgp41). Both pTM 813, 814, and pGNR were digested with
Spel and XmaI (NEB) and fragments separated via gel electrophoresis
and extracted using QIAquick Gel Extraction Kit (Qiagen), then
ligated into the pGNR backbone using T4 DNA ligase (Promega).
Ampicillin resistant DH5.alpha. E. coli colonies were screened by
colony PCR and plasmids extracted as above. Sequences were
confirmed with pGNR backbone-specific primers oTM 686 (SEQ ID NO.
5: 5'-CCCACCCGCTTTTTATAGTAA-3') and 687 (SEQ ID NO. 6:
5'-CGGTTTATCTAACGACACAACA-3'). Sequence-verified pGNR plasmids were
named pTM 815 (Gag) and pTM 816 (dgp41).
[0076] ii. Cell Lines and Viruses
[0077] Monkey kidney BSC-40 cells were grown in DMEM (Corning
Cellgro) with 5% FBS plus gentamycin and 2 mM L-glutamine. Baby
hamster kidney BHK cells were grown in MEM (Corning Cellgro) with
5% FBS plus gentamycin. Generation of the parental vaccinia virus
(VACV) strain NYVAC-KC was described previously (Kibler et al.,
2011).
[0078] iii. In Vivo Recombination (IVR)
[0079] Simultaneous transfection/infection [in vivo recombination,
IVR (Kibler et al., 1997; Brandt and Jacobs, 2001)] was performed
with pTM 815 (Gag) and pTM 816 (dgp41) and NYVAC-KC to insert Gag
and dgp41 into the vaccinia virus TK locus. 500 ng of plasmid DNA
was transfected using Lipofectamine and Plus.TM. Reagent
(Invitrogen) per manufacturer's protocol. This was immediately
followed by infection with NYVAC-KC at an MOI=0.01 in 35 mm2 dishes
of BSC-40 cells. Recombination was allowed to proceed for 24 h
followed by addition of G418 antibiotic (500 .mu.g/mL). Cells were
harvested and lysed at 48 h post infection (hpi). The IVR was used
to infect 100 mm2 dishes of BSC-40 cells for selection of
individual antibiotic-resistant plaques for subsequent expression
screening (note: a mutation in the GFP gene prevented use of
fluorescent screening). This process was repeated for multiple
rounds of antibiotic selection before a >98% pure virus was
isolated as measured by immunoplaque assay.
[0080] iv. Expression Screening
[0081] Individual antibiotic-resistant plaques were grown in 60 mm2
dishes of BSC-40 cells to CPE and harvested in 1.times.SDS sample
buffer [50 mM Tris-Cl pH 6.8, 2% SDS, 0.1% bromophenol blue, 10%
glycerol, 100 mM .beta.-mercaptoethanol, 1.times. protease
inhibitor cocktail III (Research Products International Corp.,
Prospect, Ill.)] and then centrifuged through a QiaShredder
(Qiagen) for NYVAC-KC-Gag plaques. For NYVAC-KC-dgp41, cells were
lysed with RIPA lysis buffer [1% NP40, 0.1% SDS, 0.5% sodium
deoxycholate, 1.times. protease inhibitor cocktail III, in
1.times.PBS without calcium or magnesium (Corning)]. RIPA lysates
were mixed with an equal volume of 2.times.SDS sample buffer. Cell
lysates were screened using SDS-PAGE as previously described
(Kessans et al., 2013). Briefly, boiled samples were run on 12%
polyacrylamide gels under denaturing conditions, transferred to
nitrocellulose membranes (Bio-Rad) and probed with either Gag or
dgp41 antibodies and anti-rabbit or anti-human IgG-HRP,
respectively. Proteins were detected via chemiluminescence
(ImmunoCruz Luminol Reagent, Santa Cruz).
[0082] v. Immunoplaque Assays
[0083] Immunoplaque assays were performed in 6-well dishes by
infecting BSC-40s with 50 pfu from cell lysates of individual
plaques. Once plaques were visible, the cells were fixed with 1:1
acetone:methanol for 30 min at .about.20.degree. C., washed with
PBS, then incubated with anti-p24 Gag polyclonal rabbit serum or
2F5 (obtained through the NIH AIDS Reagent Program, Division of
AIDS, NIAID, NIH: from Dr. Hermann Katinger) at 1:1000 in 3%
FBS-PBS. Secondary biotinylated antibodies for anti-rabbit IgG or
anti-human IgG from Vectastain ABC Kits (Vector Laboratories, Inc.)
were used for detection with DAB peroxidase substrate (Vector
Laboratories, Inc.) per manufacturer's protocol. After counting
positive plaques, cells were stained with Coomassie blue dye to
count the number of negative plaques. Percentages were calculated
by dividing positive plaques by total plaques per well. The plaque
with the highest percentage of positives was used for further
purification until a plaque with >98% purity was identified and
used to grow stocks.
[0084] vi. Virus Stocks
[0085] The final NYVAC-KC-Gag and NYVAC-KC-dgp41 plaques with
>98% purity determined by immunoplaque assay were grown to CPE
in BHK cells in a 60 mm2 dish for the first passage (P1) stock. The
P1 stock was titered in BSC-40s and used to infect five T150 flasks
of BHK cells at an MOI=0.01 for the P2 stock. Cells were harvested
at CPE, subjected to freeze-thaw and sonication, then cell lysates
were placed on a 36% sucrose pad and centrifuged at 22,000 rpm for
80 min at 4.degree. C. with an SW28 rotor to pellet virions.
Partially purified virions were resuspended in 10 mM Tris-HCl pH
9.0, their titer determined in BSC-40s, and were stored in aliquots
at -80.degree. C. until further use.
[0086] vii. Vaccinia In Vitro VLP Production
[0087] BSC-40 cells seeded in 100 mm2 dishes were infected with
each of the vectors at an MOI=5 for a total MOI=10. At 24 hpi,
medium and cells were harvested by centrifugation (700.times.g for
5 min). VLPs in the clarified medium were subjected to 40% ammonium
sulfate precipitation and resuspended in 1.times.PBS (140 mM NaCl,
2 mM KCl, 10 mM Na2HPO4, 1 mM KH2PO4, pH 7.4). Aliquots were boiled
and analyzed by SDS-PAGE for presence of Gag and dgp41 as described
above.
[0088] viii. Expression in Plants of VLPs and their
Purification
[0089] HIV-1 Gag/dgp41 VLPs were expressed and purified as
previously described (Kessans et al., 2013). Briefly, Gag
transgenic N. benthamiana plants were infiltrated with
Agrobacterium tumefaciens harboring pTM 602 with the dgp41 gene and
leaf tissue was harvested 4-6 days post infiltration (dpi). Leaf
tissue was crushed in a blender in extraction buffer (25 mM sodium
phosphate, 100 mM NaCl, 1 mM EDTA, 50 mM sodium ascorbate, 1 mM
PMSF, pH 7.8). VLPs were precipitated with 40% ammonium sulfate and
resuspended in 1.times.PBS (140 mM NaCl, 2 mM KCl, 10 mM Na2HPO4, 1
mM KH2PO4, pH 7.4), then purified via iodixanol density gradient
centrifugation. The VLP-containing 20% iodixanol fraction was
concentrated on a 300 kDa molecular weight cut-off membrane
(Sartorius) and quantified via immunoblot for immunizations as
described in (Kessans et al., 2016). A typical total yield of a
highly enriched VLP preparation in terms of its HIV-1 protein
constituents was 275 mg/kg and 92 mg/kg for Gag and dgp41,
respectively.
[0090] ix. Antibodies
[0091] The following antibodies were used for the indicated assays:
anti-p24 Gag polyclonal rabbit serum (Kessans et al., 2013); human
anti-MPER 2F5 (AIDS Reagent Program and the kind gift of Morgane
Bomsel); goat anti-human IgG-HRP (Sigma) for immunoblot; goat
anti-rabbit IgG-HRP (Santa Cruz) for immunoblot; rabbit anti-human
IgG-HRP (Santa Cruz) for ELISAs; rabbit anti-mouse IgG-HRP
(Calbiochem) for ELISAs; mouse IgA-kappa (ICL Labs) for ELISAs;
goat anti-mouse IgA (Sigma) for ELISAs.
[0092] x. Transmission Electron Microscopy (TEM)
[0093] BSC-40 cells grown in T75 flasks were infected with each of
the vectors at an MOI=5 (for a total MOI=10) and harvested by
trypsinization 24 hpi. Cells were pelleted between all wash steps
at 700.times.g for 5 min at 4.degree. C. until embedded in agarose
prior to secondary fixation. Pelleted cells were washed twice in
1.times.PBS (140 mM NaCl, 2 mM KCl, 10 mM Na2HPO4, 1 mM KH2PO4, pH
7.4). For primary fixation, cells were placed in 2% glutaraldehyde
in PBS for 15 min at room temperature followed by a second
incubation with fresh fixative for 1 h at 4.degree. C. Fixed cells
were resuspended in 1% agarose and washed three times in PBS for 30
min each at room temperature. Cells were then fixed with 1% osmium
tetroxide in PBS for 1 h at room temperature followed by four
15-min washes in water. A 0.2% uranyl acetate solution in water was
used to stain en bloc overnight at 4.degree. C. followed by three
15-min washes in water. An ethanol series from 20% to 100%
(anhydrous) was used for dehydration, increasing the ethanol
concentration by 20% every 10 min with three incubations in
anhydrous ethanol. Spurr's resin was gradually introduced through
4- to 8-h incubations with 1:3, 1:1, and 3:1 ratios of resin:100%
ethanol followed by three incubations in 100% resin. Cells were
divided into several blocks of Spurr's resin and polymerized at
60.degree. C. for 36 h. Sections were cut at 70 nm thick and were
placed on formvar-coated copper slot grids followed by post-stain
treatment with 2% uranyl acetate and 3% Sato's lead citrate before
imaging.
[0094] xi. Mouse Immunizations
[0095] All animal experiments were done with approval from the
Arizona State University Institutional Animal Care and Use
Committee.
[0096] Six-week-old C57BL/6 mice (Jackson Laboratories) were split
into five groups. Three groups (n=5) received virus prime (VV)
followed by two boosts of either mock (Group 1), VLP (Group 2), or
VV+VLP (Group 3). Group 4 (n=3) received no virus prime and two VLP
boosts and Group 5 served as the naive control (n=1). NYVAC-KC-Gag
and NYVAC-KC-dgp41 P2 stocks, each at a dose of 5.times.10.sup.5
pfu, were mixed together for a total dose of 1.times.10.sup.6 pfu
per mouse. Virus immunizations prepared in Tris HCl pH 9.0 were
injected intramuscularly (i.m.) into the thigh. Plant-produced,
gradient-purified VLPs were prepared in 1.times.PBS pH 7.4 with 2
.mu.g p24 and 1.2 .mu.g MPER and mixed with Ribi adjuvant (Sigma)
per manufacturer's protocol to reach a final concentration of 2%
oil [described previously in (Kessans et al., 2016)]. Mock mice
were injected i.m. with Tris HCl pH 9.0 buffer in equivalent
volumes. VACV and vehicle injections were delivered i.m. while VLPs
were delivered intraperitoneally (i.p).
[0097] Three immunizations were given to mice at days 0, 45, and
90. Serum, fecal, and vaginal lavage samples were collected as
previously described (Kessans et al., 2016) every 2 weeks at days
0, 14, 28, 42, 56, 80, and at the endpoint of day 97 for analysis
of Ab production. One week post the final boost (day 97), spleens
were collected for analysis of CD8 T-cell responses as described
below.
[0098] Animals were monitored every 2-3 days for weight loss and
other signs of illness, as a measure of vector safety.
[0099] xii. ELISA Detection of IgG and IgA
[0100] Antibody production was measured via ELISA as previously
described (Kessans et al., 2016). Briefly, serum, fecal, and
vaginal lavage samples were analyzed for IgG (serum) or IgA
(mucosal sites) specific for Gag p24 or gp41 MPER regions. Samples
were analyzed in threefold serial dilutions starting at 1:50, 1:2,
and 1:5 for serum, fecal, and vaginal lavage samples, respectively.
Endpoint titers were calculated as the reciprocal of the dilution
factor at which OD 490 nm was equal to background levels
(OD490<0.1) as previously described (Matoba et al., 2008;
Kessans et al., 2016). IgG1 and IgG2a isotypes were detected with
the same ELISA protocol, but with goat anti-mouse IgG1-HRP or goat
anti-mouse IgG2a-HRP secondary antibodies (Santa Cruz
Biotechnology) instead of total IgG. Purified mouse IgG1 kappa
chain or mouse IgG2a kappa chain primary antibodies (Sigma) were
used as controls.
[0101] For anti-VACV ELISAs, 96-well plates were coated with
10.sup.7 pfu/well of sucrose-cushion purified NYVAC-KC in
1.times.PBS, and the plates were overnight at 37.degree. C. Wells
were fixed in 2% paraformaldehyde in 1.times.PBS for 10 min at
4.degree. C. to inactivate the virus. Wells were washed with buffer
(150 mM NaCl, 0.5% Tween-20) then blocked with 5% nonfat milk in
1.times.PBST. Plates were overlaid with serially diluted serum
samples and detected as previously described (Matoba et al., 2008;
Kessans et al., 2016).
[0102] xiii. Intracellular Cytokine Staining and Flow Cytometry
[0103] Spleens were harvested in Hank's medium (Corning Cellgro)
and cells were strained through a 70 m filter for resuspension in
complete RPMI [cRPMI: 10% FBS, penicillin (100 units/mL),
streptomycin (100 .mu.g/mL), 2 mM L-glutamine]. Red blood cells
were lysed with ACK Lysing Buffer (Gibco) and splenocytes were
resuspended in cRPMI at a concentration of 2.times.107 cells/mL.
Cells were plated at 1.times.106 cells/well and incubated for 5 h
in the presence of Golgi Plug (BD Biosciences) and 1 .mu.g of one
of five different immunodominant ZM96 Gag CD8 epitopes: LRSLYNTV
(LRS8, SEQ ID NO. 7), VIPMFTAL (VIP8, SEQ ID NO. 8), AMQMLKDT
(AMQ8, SEQ ID NO. 9), YSPVSILDI (YSP9, SEQ ID NO. 10), EVKNWMTDTL
(EVK10, SEQ ID NO. 11) (synthesized by GenScript and resuspended
according to manufacturer's protocol). Cells were stained with
fluorescently conjugated antibodies: CD8-Pacific Blue,
TNF.alpha.-FITC, and IFN.gamma.-PE (BD Biosciences). Fixation and
permeabilization was performed with a Cytofix/Cytoperm Kit (BD
Biosciences) with final resuspension in FACS Buffer (1% FBS in
1.times.PBS). Samples were analyzed by flow cytometry on an LSR
Fortessa. Data was analyzed using FlowJo Data Analysis Software
(FlowJo, LLC; Ashland, Oreg.).
[0104] xiv. Statistics
[0105] All statistical analyses were performed in GraphPad Prism
Software (GraphPad Prism Software Inc.; La Jolla, Calif.). All data
were analyzed using a one-way ANOVA multiple comparison with Dunn's
post-test. Significance cut-off was defined as p<0.05.
[0106] b. Generation of Recombinant Vaccinia Virus Vectors
[0107] NYVAC-KC-Gag and NYVAC-KC-dgp41 viruses were generated
through in vitro recombination (see Materials and Methods) (FIGS.
1A and B). Each gene was under control of a synthetic early/late
VACV promoter, ensuring the genes were expressed at all stages of
viral replication to maximize antigen production. Second round
individual plaques were screened for expression via immunoblot by
probing for either Gag or dgp41 expression. Immunoplaque assays for
either Gag or dgp41 were performed to identify the plaque with
highest purity for further selection. This process was repeated
until a plaque with >98% purity was identified and then used to
grow a P2 stock. The final sucrose-pad purified P2 stock show Gag
and dgp41 expression in BSC-40 cell lysates at the proper size
(FIGS. 1C and D). It was noted during selection that viruses
recombining with dgp41 plasmids had reduced plaque size and grew to
much lower titers, suggesting that the HIV-1 gene makes the viral
vector cytotoxic (data not shown).
[0108] Gag is known to be sufficient and necessary for HIV-1 VLP
formation and budding into the medium (Garoff et al., 1998). To
test whether BSC-40-infected cells support VLP formation and
budding, culture medium was analyzed for the presence of Gag and
dgp41. Indeed, Gag protein was detected in culture medium of cells
either infected with NYVAC-KC-Gag or co-infected with NYVAC-KC-Gag
and NYVAC-KC-dgp41 (FIG. 1E, Lanes 2 and 4). In contrast, dgp41 was
detected only in the medium from co-infected cells, not in medium
from cells that were infected with NYVAC-KC-dgp41 alone (FIG. 1E,
Lane 4).
[0109] c. NYVAC-KC-Gag/Dgp41 Show Cytotoxicity and In Vitro VLP
Production
[0110] These results indicate that export of dgp41 to the medium
depends on expression and export of Gag, suggesting that Gag VLPs
and Gag/dgp41 VLPs are released, respectively, from cells that
express Gag alone or co-express Gag and dgp41. Inventors used TEM
to test this possibility. BSC-40 cells that were infected (MOI=5)
with NYVAC-KC, NYVAC-KC-Gag, NYVAC-KC-dgp41, or co-infected with
Gag/dgp4, were processed for TEM at 24 hpi.
[0111] In cells infected with the parental NYVAC-KC strain, viral
replication factories are clearly visible and all stages of viral
replication are easily identified (FIG. 2A). Mature virions
(indicated by white arrows) are seen both intracellularly (FIG.
2A-1) and extracellularly (FIG. 2A-2). Immature virions with and
without incorporated genomes are also abundantly visible in this
section (white and black triangles, respectively).
[0112] NYVAC-KC-Gag-, dgp41-, or co-infected cells have
disorganized viral factories and mature viral particles are rare
(FIG. 2B-D). Many early crescent formations are visible (black
arrow), along with what appears to be "unfilled" or empty virion
shells (indicated by "E"). The rarity of mature virus particles
corresponds well to the smaller plaques typical of these strains
and their reduced titers, as noted above. Instead, these infected
cells appear to have large protein aggregates or precipitates in
their cytoplasm ("junk" indicated by "J"), mimicking a phenotype
associated with palmitate deficiency (Greseth and Traktman, 2014).
Additionally, NYVAC-KC-dgp41-infected cells demonstrate nuclear
condensation (FIG. 2C-2, "nucleus" indicated by "N"), a classic
sign of apoptosis (Duprez et al., 2009). Furthermore, the
mitochondria (indicated by "M") in NYVAC-KC-Gag- and
NYVAC-KC-dgp41-infected cells appear to be malformed, potentially
indicating loss of structural integrity, which can also indicate
occurrence of apoptosis (Li and Dewson, 2015). This phenotype seems
far more drastic than that of NYVAC-KC-infected cells.
[0113] Importantly, in cells that were infected with NYVAC-KC-Gag
(FIG. 2B-2) or co-infected with NYVAC-KC-Gag and NYVAC-KC-dgp41
(FIG. 2D-2), particles of approximately 100 nm in diameter are seen
budding at the cell surface (black stars). Similar structures were
not observed in any of the sections of cells expressing by itself
dgp41. Based on their size, and appearance, the particles are
likely to be HIV-1 Gag or Gag/dgp41 particles budding out of the
cell. This identification is supported by our observation that Gag
and dgp41 accumulated in the media of these infected cells (FIG.
1E).
[0114] i. Mouse Immunizations
[0115] Six-week-old C57BL/6 mice were separated into five groups in
order to assess different combinations of VACV and VLP immunization
regimens (FIG. 3A). Mice were given a total of three immunizations
separated by 45 days to allow for memory responses to develop prior
to boosting. Three groups (n=5) were primed with the combination of
NYVAC-KC-Gag and NYVAC-KC-dgp41 (denoted in all figures and text as
VV), followed by two boosts with either vehicle (Group 1), VLPs
alone (Group 2), or a combination of VV and VLPs (Group 3). A
fourth group (n=3) received a mock (vehicle) prime followed by
boosts with VLPs. The fifth group served as a naive control
(n=1).
[0116] Monitoring of weight revealed that none of the mice in any
group lost weight (data not shown). This indicates little to no
pathogenicity of the virus and is consistent with previous
virulence data for NYVAC-KC (Kibler et al., 2011) and general
safety of the plant-derived VLPs (Kessans et al., 2016).
[0117] d. Serum IgG Responses to Gag and MPER
[0118] Antigen-specific serum IgG responses were measured by ELISA
using p24 subunit or MPER peptide to detect Gag and dgp41 Ab
responses, respectively. Throughout, OD 450 nm values for the
lowest dilution tested (1:50) are shown both over time (FIG. 3B-C)
and for individual animals at the endpoint, Day 97 (FIG. 4A).
Additionally, endpoint titers were calculated for the final serum
samples and are shown in FIG. 5A as the reciprocal of the dilution
where the OD 450 nm fell below background levels (OD<0.1).
[0119] Serum IgG responses against both p24 and MPER antigens
remained undetectable in all groups until the first sample (Day 55)
after the first boost. Titers increased gradually and were boosted
after the final immunization with VLPs (FIG. 3B-C), reaching
significant levels at the endpoint analysis (Day 97) for both
anti-p24 Gag and anti-MPER responses (FIG. 4A). Endpoint Gag
responses were highest in the group boosted with a combination of
VV and VLPs (VV/VV+VLPs), while the group boosted with VLPs alone
(VV/VLPs) also showed significant titers which were more variable
responses among animals (FIG. 5A). Anti-MPER serum IgG responses
were lower than Gag titers but showed a similar trend, reaching
significant levels in all groups receiving VLPs regardless of
prime/boost regimen (FIG. 4A). However, the endpoint titer
calculations showed no difference between groups, consistent with
low Ab responses (FIG. 5A). It is interesting to note that
plant-produced VLPs appear to be largely responsible for Ab
stimulation because no detectable levels of antibodies were present
in the first 45 days after a single dose of VV and only reached
detectable levels after the second immunization in those groups
that received VLPs.
[0120] e. Mucosal IgA Responses to Gag and MPER
[0121] Gastrointestinal IgA responses to the dgp41 (MPER) antigen
(as determined by measuring the secreted Ab levels in fecal
samples) were low but detectable (FIG. 4B), whereas anti-MPER IgA
levels in vaginal secretions were below our detection level
irrespective of treatment (FIG. 4C). Fecal anti-MPER IgAs were
higher in groups that were immunized using the prime/boost VV/VLPs
regimen than groups that were vaccinated with either VV or VLPs
alone. Contrasting the two groups, those mice that were boosted
with a combination of VV and VLPs exhibited marginally higher
levels of fecal IgAs than those boosted with VLPs alone, but their
fecal IgA levels were significantly higher than the group that was
primed with VV only. Interestingly, levels of fecal and vaginal
anti-p24 Gag IgAs were too low for detection in all groups. This
contrasts sharply with the generally much higher titers of serum
anti-p24 Gag IgGs as compared to serum anti-MPER IgGs.
[0122] f. IgG Isotyping
[0123] The two major IgG isotypes are IgG1 and IgG2a; the former is
consistent with stimulation of a Th2 response for B cell
activation, whereas the latter would be indicative of a Th1
response, which primarily results in inflammatory cytokine
production and killing of intracellular pathogens (Snapper and
Paul, 1987; Szabo et al., 2003). Results indicated that the most
important immunogens contributing to the elicitation of serum IgGs
are the plant-derived VLPs. To further substantiate this
conclusion, we determined the Ab isotypes contributing to this
response, as protein-based vaccines (i.e. immune-complexes) usually
elicit IgG1 (Mosser and Edwards, 2008; Martinez and Gordon,
2014).
[0124] To this end, serum samples from the endpoint for the two
groups which showed the highest serum IgG responses (VV/VLP and
VV/VV+VLP) were analyzed for their IgG isotype content by
isotype-specific ELISA (FIGS. 5A and B, respectively). Both groups
show high levels of antigen-specific (either anti-p24 or anti-MPER)
IgG1, but levels of IgG2a were too low for detection.
[0125] g. Anti-Vector Antibody Responses
[0126] Having substantiated the contribution of the Gag/dgp41 VLPs
for the elicitation of serum Ab responses against the two HIV-1
antigens, the question arose whether or not the viral vector
induced production of anti-vector (i.e. anti-VV) serum Abs. Mice
belonging to Group 3 that received a total of three doses of VV
(prime plus two boosts) showed significant levels of anti-VV serum
IgG at the endpoint (Day 97, FIG. 6). However, a single dose of VV
at Day 0 was not enough to induce a potent anti-vector response
(Group 1).
[0127] h. Gag-Specific CD8 T Cell Responses
[0128] A major rationale to include Gag in an HIV-1 vaccine,
especially a live-vectored one, is its excellent ability to induce
cellular responses. To test induction of CD8 T cell responses,
inventors tested the ability of peptides corresponding to known Gag
T-cell epitopes to stimulate proliferation of CD8 T-cells among
splenocytes obtained from vaccinated animals. One week after the
final boost, splenocytes were harvested and stimulated with five
CD8 ZM96 Gag peptides that were previously determined to be
dominant in C57BL/6 mice (Chowell et al., 2015). Peptides were
assessed individually in order to obtain a better resolution of
their responses. CD8 T cells were analyzed for IFN-.gamma. or
TNF-.alpha. production in response to peptide stimulation using
cytokine staining and flow cytometry. Little to no TNF-.alpha.
production was seen in the flow cytometry analysis (data not
shown); however, IFN-.gamma. production was detectable in several
groups. The group boosted with both VV and VLPs showed significant
production of IFN-.gamma. with two of the five Gag peptides, AMQ8
and EVK10, at 2.6.times.105 CD8.sup.+ IFN-.gamma.+ T cells (3.5% of
CD8.sup.+ T cells) and 1.91.times.105 CD8+IFN-.gamma.+ T cells
(2.4%), respectively (FIGS. 7A and B). The other three peptides,
LRS8, VIP8, and YSP9 showed a similar trend as AMQ8 and EVK10 but
with slightly lower responses of CD8+IFN-.gamma.+ T cells at
1.73.times.105 (2.4%), 1.61.times.105 (2.1%), and 1.67.times.105
cells (2.3%), respectively, but failed to reach significance (FIG.
7C-E). When responses from all five peptides are added together,
the VV/VV+VLPs group has a total CD8 Gag-specific mean response of
9.53.times.105 CD8+IFN-.gamma.+ T cells (12.7% of CD8 T cells)
(FIG. 7F). The total Gag-specific CD8 responses of the other groups
show minute differences between groups at 8.0% in Tris/VLP, 5.6% in
VV/VLP, and 4.2% in VV/Tris. This suggests that plant-derived
Gag/dgp41 VLPs may be providing some component of the T cell
response and potentially boosting the initial VV-primed T cell
responses as previously described for other Gag-containing VLPs
(Williamson and Rybicki, 2015).
2. Transient, Deconstructed Tobacco Mosaic Virus- and
Geminivirus-Based Vectors for Enhancing Expression and Yield of
HIV-1 Gag/Dgp41 Virus-Like Particles
[0129] a. Experimental Procedures
[0130] i. Cloning Geminivirus-Based Vectors
[0131] The TMV-based vector, pTM 602, was previously described
(Kessans et al., 2013). Geminivirus-based vectors were generously
provided by Dr. Hugh Mason (Arizona State University) for cloning
dgp41 and Gag. Three vectors were generated: pTM 924 (Gemini), pTM
925 which contains p19 (Gemini +p19), and pTM 901 (Dual Gem. +p19),
a dual-replicon vector for simultaneous expression of two genes
from the same backbone which also contains p19.
[0132] For cloning into pTM 924 and pTM 925, dgp41 was digested
from pTM 602 using NcoI and SacI. The barley .alpha.-amylase (BAA)
signal peptide was digested from pTM 796 using XbaI and SacI. The
geminivirus backbone for pTM 924 is pTM 890 (formerly pBYR2fb)
which was opened with XbaI and SacI. A triple ligation with dgp41,
BAA, and the opened pTM 890 backbone using T4 DNA Ligase (Promega)
yielded pTM 924. To derive pTM 925, the backbone pTM 800 was
linearized using XbaI and SacI and the BAA-dgp41 insert was derived
from pTM 924 by digesting with XbaI and SacI. The backbone and
BAA-dgp41 fragment was ligated to yield pTM 925. Colonies were
screened and sent for sequencing to confirm no mutations.
[0133] The dual-replicon vector, pTM 901, requires cloning into the
XbaI/SacI cut site first due to the presence of a KpnI cut site in
the multiple cloning site because the second cut site requires
digestion with BsrGI and KpnI. BAA-dgp41 was excised from pTM 924
using XbaI and SacI while pTM 900 (formerly pBYR27p) was linearized
using the same restriction sites. After confirming BAA-dgp41
insertion into the first cloning site, Gag was amplified from pTM
488 via PCR using AccuStart Taq DNA Polymerase HiFi (Quanta
Biosciences) using oTM 776 (SEQ ID NO. 12:
5'-GAGATGTACAATGGGAGCTAGAGCCTCT-3') and oTM 777 (SEQ ID NO. 13:
5'-GAGAGGTACCTTATTGAGAGGAAGGGT-3') for addition of a 5' BsrGI and
3' KpnI cut site, shown in italics, and ligated to a TOPO TA
backbone (Invitrogen). The Gag gene contained a BsrGI cut site
which was removed via mutagenesis using whole plasmid replication
with the QuikChange II Site-Directed Mutagenesis Kit (Agilent
Technologies) per manufacturer's protocol. The first reaction
changed a TTG codon to CTG using primers oTM 827 (SEQ ID NO. 14:
5'-GAGGAGCTTAGGTCTCTGTACAACACAGTGGCT-3') and oTM 828 (SEQ ID NO.
15: 5'-AGCCACTGTGTTGTACAGAGACCTAAGCTCCTC-3') where the mutated
nucleotide is underlined. However, this introduces a non-synonymous
codon so an additional change from CTG to CTC was performed using
oTM 829 (SEQ ID NO. 16: 5'-GAGGAGCTTAGGTCTCTCTACAACACAGTGGCT-3')
and oTM 830 (SEQ ID NO. 17:
5'-AGCCACTGTGTTGTAGAGAGACCTAAGCTCCTC-3'). Upon sequence
confirmation this plasmid was named pTM 923. From there, pTM 923
and pTM 901 with BAA-dgp41 inserted were both digested using BsrGI
and KpnI and then ligated together to derive the complete pTM 901.
Sequences were confirmed for both Gag and dgp41 prior to use.
[0134] Each vector was transformed into GV3101 Agrobacterium
tumefaciens and colony-screened to confirm transformation. An
isolated colony was grown and used to start infiltration cultures
for each of the three Geminivirus-based vectors.
[0135] ii. Kinetics Time Course
[0136] Six-week old Nicotiana benthamiana plants were vacuum
infiltrated with an OD600=0.1 of Agrobacterium tumefaciens
resuspended in infiltration buffer (10 mM MES, 10 mM magnesium
sulfate heptahydrate, pH 5.5). Bacteria harboring pTM 602, 924, or
925 were infiltrated into Gag transgenic plants while pTM 901 was
used in wild-type plants. Leaves were collected in triplicate every
day for one week following infiltration. Collected leaves were
scanned to monitor necrosis prior to flash-freezing 200 mg tissue
samples in liquid nitrogen for expression analysis. Upon
time-course completion, frozen tissue samples were homogenized in
1.times.SDS sample without dye (60 mM Tris-HCl, 100 mM DTT, 1.6%
SDS (w/v), 5% glycerol) buffer using a ceramic bead in a Fast
Prep-24 (MP Biomedicals, Solon, Ohio) machine twice for 30 s each.
Homogenized samples were clarified by centrifugation at
14,000.times.g for 10 min at 4.degree. C. Supernatant was collected
and analyzed by Bradford assay (Bio-Rad) to determine total soluble
protein (TSP) concentration. 20 .mu.g TSP was loaded for each leaf
sample into their respective lanes on a 12% polyacrylamide gel for
SDS-PAGE followed by immunoblot as previously described (Kessans et
al., 2013). Immunoblots for Gag were probed with primary polyclonal
anti-p24 rabbit serum and secondary goat anti-rabbit IgG-HRP (Santa
Cruz), while dgp41 blots were probed with primary human anti-gp41
2F5 (AIDS Reagents Program) and secondary goat anti-human IgG-HRP
(Sigma). Coomassie staining was performed to ensure equal protein
loading for each lane.
[0137] To quantify expression over time, ImageJ software (Schneider
et al., 2012) was used to measure band density. For each replicate,
the peak day (highest density) for Gag and dgp41 was identified and
other day band densities expressed as a percentage of the peak
day.
[0138] iii. Optimization of VLP Purification
[0139] Two strategies for removing rubisco from VLP preparations
were tested. In the first experiment, flash-frozen leaf tissue
harvested on peak expression day was homogenized in a blender in
extraction buffer (25 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA,
50 mM sodium ascorbate, 1 mM PMSF, pH 7.8), strained through
miracloth, and insoluble protein was removed by centrifugation at
14,000.times.g for 20 min at 4.degree. C. The supernatant was
collected and subjected to sequential ammonium sulfate
precipitation at 20% followed by 40% with VLPs pelleted at
36,000.times.g for 30 min at 4.degree. C. between fractions. To
remove rubisco using pH changes, after pelleting insoluble
proteins, the supernatant was titrated to a pH of 5.5 to pellet
rubisco followed by adjustment to pH 5.0. After each pH change
protein was pelleted at 36,000.times.g for 30 min at 4.degree. C.
The remaining pH 5.0 supernatant was subjected to a 40% ammonium
sulfate precipitation to pellet all remaining VLPs and rubisco. All
pellets were resuspended in 1.times.PBS (140 mM NaCl, 2 mM KCl, 10
mM Na2HPO4, 1 mM KH2PO4, pH 7.4). Samples were analyzed via
Bradford assay and protein loaded equally into each lane for
Coomassie and SDS-PAGE as above.
[0140] iv. Large-Scale VLP Production and Purification
[0141] Six-week old N. benthamiana plants were infiltrated at
OD600=0.1 with each of the four vectors. At peak expression day, as
determined by the kinetics time course, leaf material was harvested
in 20 g batches and flash frozen in liquid nitrogen and stored at
-80.degree. C. until extraction. VLPs were purified using 20%
ammonium sulfate precipitation described above. Following
precipitation VLPs were resuspended in 1.times.PBS and separated
via iodixanol density gradient as previously described (Kessans et
al., 2013). Purification progress was monitored by immunoblot by
loading 5 .mu.g total soluble protein (TSP) per lane for each step
of the extraction and density gradient. Gels were used for
Coomassie staining, Gag expression, or dgp41 expression as
described above and in (Kessans et al., 2013).
[0142] v. VLP Quantification
[0143] VLP-containing fractions were concentrated on a 300 kDa
molecular weight cut-off membrane (Sartorius) and quantified via
immunoblot for total Gag and dgp41 content as previously described
(Kessans et al., 2016). Ammonium sulfate pellets were directly
quantified without concentrating each sample. Briefly, g/mL amounts
for both proteins were calculated by comparing serial dilutions of
concentrated VLP gradient fractions and ammonium sulfate pellets to
known concentrations of purified p24-CTA2 or CTB-MPER using ImageJ
software to determine band density (Schneider et al., 2012). The
total calculated p24 and MPER protein amount was then divided by
kilograms of fresh leaf weight (FW) to determine g/kg yield for Gag
and dgp41. Molar ratio of MPER to p24 was determined by relative
protein sizes (MPER=15 kDa; p24=24 kDa) for a molecular weight
ratio of 1.6 p24 to MPER.
[0144] vi. Mouse Immunizations
[0145] All animal studies were conducted under the approval of the
Arizona State University Institutional Animal Care and Use
Committee under protocol number 15-1386R. Four to six-week old
C57BL/6 mice (Jackson Laboratories; n=3) were injected i.p. with 2
.mu.g p24 and 1.2 g MPER gradient-purified VLPs mixed with Ribi
adjuvant (Sigma) per manufacturer's protocol. Serum was collected
every 2 weeks for analysis of p24 and MPER-specific IgG by ELISA as
previously described (Kessans et al., 2016). Results are reported
as OD 490 nm over time for the 1:50 dilution (lowest dilution
tested).
[0146] b. Construction of Transient Geminivirus Vectors
[0147] The development of tobacco mosaic virus (TMV)-based
vectors--known as MagnICON vectors--for transgene expression in
plants has been extensively described (Marillonnet et al., 2004;
Gleba et al., 2005; Marillonnet et al., 2005). A TMV vector for
transiently expressing dgp41 for making HIV-1 VLPs (referred herein
as TMV) was previously described (Kessans et al., 2013) (FIG. 8a).
Two Geminivirus vectors (referred herein as Gemini and Gemini+p19)
were cloned to transiently express dgp41 with or without the
silencing suppressor protein p19 (FIGS. 8b-8c). Geminiviruses are
known to induce gene silencing both during natural infection and
with expression of transgenes (Seemanpillai et al., 2003;
Rodriguez-Negrete et al., 2009; Aregger et al., 2012). The p19
protein has been shown to increase expression of recombinant
proteins in plants by preventing gene silencing, thus the inclusion
in transient expression vectors is hypothesized to increase yield
(Voinnet et al., 2003; Garabagi et al., 2012). A third Geminivirus
vector (referred herein as Dual Gemini+p19), and the last tested in
this study, was designed to co-express Gag and dgp41 from the same
T-DNA fragment, but in separate Geminivirus replicons separated by
short and long intergenic regions (LIR/SIR) (FIG. 8d). These
replicons are amplified by the replication proteins C1/C2 in the
nucleus after Agrobacterium-mediated T-DNA transfer (Gutierrez,
1999; Mor et al., 2003; Huang et al., 2009). Expression in all
Geminivirus vectors is under control of the Cauliflower mosaic
virus (CMV) 35S promoter with two enhancer (2e) binding sites to
improve translation efficiency.
[0148] c. Geminivirus vectors display accelerated expression
[0149] Following their introduction into Agrobacterium tumefaciens
cells, agroinfiltration was used to introduce the four vectors were
infiltrated into six-week old Nicotiana benthamiana plants and
expression kinetics was analyzed over the course of seven days.
Three of the vectors (TMV, Gemini, and Gemini+p19) were infiltrated
into Gag transgenic plants to transiently express dgp41 for VLP
production while the fourth vector (Dual Gemini+p19) was
infiltrated into wild-type (WT) plants for transient co-expression
of Gag and dgp41. Triplicate leaf samples were collected and
monitored for necrosis over seven days (FIG. 9). All
Geminivirus-based vectors display necrosis earlier than the TMV
vector (3-4 days post infection [dpi] vs. 6 dpi). The inclusion of
p19 with Geminivirus vectors appears to delay the onset of necrosis
by 1-2 days. However, all Geminivirus infiltrated plants had severe
necrosis at 7 dpi that prevented further sampling.
[0150] Quantitative analysis of Gag and dgp41 expression reveals
distinct differences between TMV and Geminivirus vectors (FIGS.
10a-10b). Gemini and Gemini+p19 vectors display a gradual decrease
in transgenic Gag levels over the seven days and including p19 in
the vectors reduced this decrease by 26.5% on average after 2 dpi
(FIGS. 10a, 14b, and 14c, compare red squares with green
triangles). In some aspects, Gag level is increased due to reduced
gene silencing leading to a decrease in necrosis. The TMV vector
maintains steady levels in Gag expression with variability across
plants and no clear peak day (FIGS. 10a and 14a, blue circles).
Transient expression of Gag by the Dual Gemini+p19 vector shows a
clear peak at 2 dpi followed by a steady decline with little to no
expression after 4 dpi (FIGS. 10a and 14d), correlating with onset
of necrosis (FIG. 9).
[0151] Transient expression of dgp41 in the TMV vector shows
initial expression at 3 dpi with a steady increase over time
peaking at 7 dpi (FIGS. 10b and 14a). The Gemini and Gemini+19
vectors drove expression much earlier with significant accumulation
already at 2 dpi with peak expression between 2 and 4 dpi (FIGS.
10b, 14b, and 14c). Interestingly, there is a decrease in dgp41
expression in both vectors at 5 dpi despite equivalent protein
loading into the gels (FIGS. 13a-13d) and this is far more drastic
in the vector without p19. Dual Gemini +p19 initiates detectable
expression at 2 dpi with a steady decline similar to that seen with
Gag expression by the same vector (FIGS. 10b and 14d). Based on the
combined necrosis and Gag/dgp41 expression levels during the time
course, peak expression day was determined to be 4 dpi for TMV and
2-3 dpi for all Gemini vectors.
[0152] d. Removal of Rubisco in VLP Preparations
[0153] Two strategies were employed in an effort to reduce rubisco
in VLP preparations. Rubisco bands were detected by Coomassie,
staining for large bands around 50 and 25 kDa corresponding to the
large and small subunits of rubisco, respectively. Gag (55 kDa) and
dgp41 (23 kDa) were detected by western blot. A majority of VLPs
were found to pellet with addition of 20% ammonium sulfate as
determined by Gag and dgp41 presence (FIG. 4b-c, 20% P) while the
major rubisco band around 50 kDa remains in the 20% supernatant
(SN) (FIG. 1a). The rubisco can be precipitated with further
increase to 40% ammonium sulfate (FIG. 4a, 40% P) along with any
remaining Gag/dgp41 VLPs (FIGS. 11b and 11c). The second strategy
utilized pH changes where a majority of the VLPs pellet at pH 5.5
and a further reduction to pH 5.0 did not result in detection of
Gag or dgp41 (FIGS. 11b and 11c, pH 5.5 & 5.0 P). Ammonium
sulfate was added to a concentration of 40% in the remaining pH 5.0
SN and a large band around 50 kDa was visible in the Coomassie with
no detectable Gag and little dgp41, indicating this likely
represents a majority of the rubisco (FIGS. 11a-11c, 5.0-40% P).
Based on these data, the 20% ammonium sulfate precipitation appears
cleaner with fewer protein contaminants, including rubisco, and was
therefore chosen as the method of purification for subsequent
experiments.
[0154] e. VLP Purification and Yield
[0155] Large-scale purifications were performed for each of the
four vectors harvesting at peak expression day and processing in 20
g batches. VLPs for all samples were primarily isolated to the 20%
Optiprep fraction with some large aggregates accumulating in the
50% fraction (FIGS. 12a-12d). Comparison of Gag and dgp41 band
intensity on equivalently loaded gels indicates that Gemini +p19
and Dual Gemini +p19 vectors qualitatively have the highest yield
of both Gag and dgp41 (FIGS. 12c and 12d). After running density
gradients, both the 20% ammonium sulfate pellets and the 20%
Optiprep gradient fractions were quantified by immunoblot as
previously described (Kessans et al., 2016).
[0156] Total expression of Gag reached a maximum around 1 mg/kg
fresh leaf weight for both Dual Gemini +p19 transient expression
and with the TMV vector in stable transgenics (Table 1). Transient
expression of Gag and dgp41 was lowest in the Gemini vector without
p19 at 0.62 mg/kg and 0.82 mg/kg, respectively (Table 1). The
highest transient dgp41 expression was seen with the Dual Gemini
+p19 at 5.5 mg/kg (Table 1). Both Geminivirus vectors which include
p19 had higher dgp41 expression than the TMV-based vector.
Interestingly, though the Gag yield in the VLP gradient fraction
was similar for the TMV and Dual Gemini +p19, but the TMV vector
had lower dgp41 yield (Table 2). A similar phenomenon was seen with
the Gemini .+-.p19 vectors where inclusion of p19 during expression
resulted in an increase in dgp41 yield within the VLP fraction
(Table 2). Furthermore, the higher dgp41 expression in both the
Gemini +p19 and Dual Gemini +p19 vectors corresponds to a higher
molar ratio of MPER:p24 with an average of 2.8 and 3.3,
respectively (Table 2). This indicates more dgp41 per Gag in each
VLP particle, thus presenting a better immunogen with more
neutralization targets.
TABLE-US-00001 TABLE 1 Total Gag and dgp41 expression from ammonium
sulfate precipitation. Infiltrated Transient Gag Yield dgp41 Yield
Vector Plant Line Expression (mg p24/kg) (mg MPER/kg) TMV 1.04 .+-.
0.59 3.45 .+-. 2.29 Gemini Gag dgp41 0.62 .+-. 0.16 0.82 .+-. 0.52
Gemini 0.85 .+-. 0.34 4.62 .+-. 1.52 (+p19) Dual Gem. WT Gag &
dgp41 1.09 .+-. 0.38 5.46 .+-. 1.49 (+p19)
Protein expression is expressed as mg of p24 or MPER per kg fresh
leaf weight for Gag and dgp41, respectively. Triplicate samples
were processed for each vector and reported here as mean with
standard error.
TABLE-US-00002 TABLE 2 HIV VLP yield with transient expression in
N. benthamiana. Infiltrated Transient Gag Yield dgp41 Yield
MPER:p24 Vector Plant Line Expression (.mu.g p24/kg) (.mu.g
MPER/kg) (molar) TMV 343 .+-. 131 33 .+-. 16 0.74-5.2 Gemini Gag
dgp41 175 .+-. 68 11 .+-. 3 0.47-1.34 Gemini (+p19) 130 .+-. 30 57
.+-. 37 0.61-9.8 Dual Gem. (+p19) WT Gag & dgp41 322 .+-. 92 84
.+-. 38 1.7-11.8
Levels of protein are expressed as g p24 or MPER for Gag and dgp41,
respectively, per kg fresh leaf weight. Gag and dgp41 yield were
determined in triplicate by quantitative immunoblot from density
gradient VLP-enriched fractions and reported here as mean with
standard error. Molar ratio of MPER to p24 is calculated as amount
of dgp41 MPER to p24 Gag based on molecular weight ratio of 1.6
(p24=24 kDa/MPER=15 kDa).
[0157] f. VLPs Elicit Gag and Dgp41-Specific Antibodies in Mice
[0158] C57BL/6 mice were immunized with Gag/dgp41 VLPs produced
using the TMV-based vector at Day 0 and 45. Two weeks after the
first dose, p24-specific IgG is detectable in the serum while
MPER-specific antibodies were below detection limit (FIG. 13). On
week after the second immunization, a 100% response rate was
observed for both p24- and MPER-specific serum IgG. This indicates
the plant-produced VLPs are immunogenic in mice and can elicit
antibodies specific to the MPER bnAb target in the dgp41
protein.
REFERENCES CITED
[0159] Alam S M, McAdams M, Boren D, Rak M, Scearce R M, Gao F,
Camacho Z T, Gewirth D, Kelsoe G, Chen P, Haynes B F. The role of
antibody polyspecificity and lipid reactivity in binding of broadly
neutralizing anti-HIV-1 envelope human monoclonal antibodies 2F5
and 4E10 to glycoprotein 41 membrane proximal envelope epitopes. J
Immunol. 2007; 178:4424-4435. [PubMed: 17372000] [0160] Alam S M,
Morelli M, Dennison S M, Liao H X, Zhang R, Xia S M, Rits-Volloch
S, Sun L, Harrison S C, Haynes B F, Chen B. Role of HIV membrane in
neutralization by two broadly neutralizing antibodies. Proc Natl
Acad Sci USA. 2009; 106:20234-20239. [PubMed: 19906992] [0161]
Aregger M, Borah B K, Seguin J, Rajeswaran R, Gubaeva E G, Zvereva
A S, Windels D, Vazquez F, Blevins T, Farinelli L, Pooggin M M
(2012) Primary and secondary siRNAs in geminivirus-induced gene
silencing. PLoS Pathog 8: e1002941 [0162] Arfi Z A, Hellwig S,
Drossard J, Fischer R, Buyel J F (2016) Polyclonal antibodies for
specific detection of tobacco host cell proteins can be efficiently
generated following RuBisCO depletion and the removal of
endotoxins. Biotechnol J 11: 507-518 [0163] Baba T W, Liska V,
Hofmann-Lehmann R, Vlasak J, Xu W, Ayehunie S, Cavacini L A, Posner
M R, Katinger H, Stiegler G, Bernacky B J, Rizvi T A, Schmidt R,
Hill L R, Keeling M E, Lu Y, Wright J E, Chou T C, Ruprecht R M.
Human neutralizing monoclonal antibodies of the IgG1 subtype
protect against mucosal simian-human immunodeficiency virus
infection. Nat Med. 2000; 6:200-206. [PubMed: 10655110] [0164]
Bessa J, Zabel F, Link A, Jegerlehner A, Hinton H, Schmitz N, Bauer
M, Kundig T M, Saudan P, Bachmann M F (2012) Low-affinity B cells
transport viral particles from the lung to the spleen to initiate
antibody responses. Proc Natl Acad Sci USA 109: 20566-20571 [0165]
Betts M R, Nason M C, West S M, De Rosa S C, Migueles S A, Abraham
J, Lederman M M, Benito J M, Goepfert P A, Connors M, Roederer M,
Koup R A. HIV nonprogressors preferentially maintain highly
functional HIV-specific CD8+ T cells. Blood. 2006; 107:4781-4789.
[PubMed: 16467198] [0166] Billington J, Hickling T P, Munro G H,
Halai C, Chung R, Dodson G G, Daniels R S. Stability of a
receptor-binding active human immunodeficiency virus type 1
recombinant gp140 trimer conferred by intermonomer disulfide
bonding of the V3 loop: differential effects of protein disulfide
isomerase on CD4 and coreceptor binding. J Virol. 2007;
81:4604-4614. [PubMed: 17301129] [0167] Bomsel M, Tudor D, Drillet
A S, Alfsen A, Ganor Y, Roger M G, Mouz N, Amacker M, Chalifour A,
Diomede L, Devillier G, Cong Z, Wei Q, Gao H, Qin C, Yang G B,
Zurbriggen R, Lopalco L, Fleury S. Immunization with HIV-1 gp41
subunit virosomes induces mucosal antibodies protecting nonhuman
primates against vaginal SHIV challenges. Immunity. 2011;
34:269-280. [PubMed: 21315623] [0168] Bonsignori M, Alam S M, Liao
H X, Verkoczy L, Tomaras G D, Haynes B F, Moody M A. HIV-1
antibodies from infection and vaccination: insights for guiding
vaccine design. Trends Microbiol. 2012; 20:532-539. [PubMed:
22981828] [0169] Brander C, Walker B D. T lymphocyte responses in
HIV-1 infection: implications for vaccine development. Curr Opin
Immunol. 1999; 11:451-459. [PubMed: 10448136] [0170] Brandt T A,
Jacobs B L. Both carboxy- and amino-terminal domains of the
vaccinia virus interferon resistance gene, E3L, are required for
pathogenesis in a mouse model. J Virol. 2001; 75:850-856. [PubMed:
11134298] [0171] Briggs J A, Krausslich H G (2011) The molecular
architecture of HIV. J Mol Biol 410: 491-500 [0172] Buchbinder S P,
Mehrotra D V, Duerr A, Fitzgerald D W, Mogg R, Li D, Gilbert P B,
Lama J R, Marmor M, Del Rio C, McElrath M J, Casimiro D R,
Gottesdiener K M, Chodakewitz J A, Corey L, Robertson M N, Step
Study Protocol, T. Efficacy assessment of a cell-mediated immunity
HIV-1 vaccine (the Step Study): a double-blind, randomised,
placebo-controlled, test-of-concept trial. Lancet. 2008;
372:1881-1893. [PubMed: 19012954] [0173] Burton D R, Mascola J R.
Antibody responses to envelope glycoproteins in HIV-1 infection.
Nat Immunol. 2015; 16:571-576. [PubMed: 25988889] [0174] Buyel J F,
Fischer R (2014) Generic chromatography-based purification
strategies accelerate the development of downstream processes for
biopharmaceutical proteins produced in plants. Biotechnol J 9:
566-577 [0175] Buyel J F, Woo J A, Cramer S M, Fischer R (2013) The
use of quantitative structure-activity relationship models to
develop optimized processes for the removal of tobacco host cell
proteins during biopharmaceutical production. J Chromatogr A 1322:
18-28 [0176] Buzon V, Natrajan G, Schibli D, Campelo F, Kozlov M M,
Weissenhorn W. Crystal structure of HIV-1 gp41 including both
fusion peptide and membrane proximal external regions. PLoS Pathog.
2010; 6:e1000880. [PubMed: 20463810] [0177] Cardoso R M, Zwick M B,
Stanfield R L, Kunert R, Binley J M, Katinger H, Burton D R, Wilson
I A. Broadly neutralizing anti-HIV antibody 4E10 recognizes a
helical conformation of a highly conserved fusion-associated motif
in gp41. Immunity. 2005; 22:163-173. [PubMed: 15723805] [0178]
Chapman R, Stutz H, Jacobs W Jr, Shephard E, Williamson A L.
Priming with recombinant auxotrophic BCG expressing HIV-1 Gag, R T
and Gp120 and boosting with recombinant MVA induces a robust T cell
response in mice. PLoS One. 2013; 8:e71601. [PubMed: 23977084]
[0179] Chege G K, Shephard E G, Meyers A, van Harmelen J,
Williamson C, Lynch A, Gray C M, Rybicki E P, Williamson A L. HIV-1
subtype C Pr55gag virus-like particle vaccine efficiently boosts
baboons primed with a matched DNA vaccine. J Gen Virol. 2008;
89:2214-2227. [PubMed: 18753231] [0180] Chen J, Frey G, Peng H,
Rits-Volloch S, Garrity J, Seaman M S, Chen B. Mechanism of HIV-1
neutralization by antibodies targeting a membrane-proximal region
of gp41. J Virol. 2014; 88:1249-1258. [PubMed: 24227838] [0181]
Chen Q, Lai H (2013) Plant-derived virus-like particles as
vaccines. Human vaccines & immunotherapeutics 9: 26-49 [0182]
Chen X, Rock M T, Hammonds J, Tartaglia J, Shintani A, Currier J,
Slike B, Crowe J E Jr, Marovich M, Spearman P. Pseudovirion
particle production by live poxvirus human immunodeficiency virus
vaccine vector enhances humoral and cellular immune responses. J
Virol. 2005; 79:5537-5547. [PubMed: 15827168] [0183] Chowell D,
Krishna S, Becker P D, Cocita C, Shu J, Tan X, Greenberg P D,
Klavinskis L S, Blattman J N, Anderson K S. TCR contact residue
hydrophobicity is a hallmark of immunogenic CD8+ T cell epitopes.
Proc Natl Acad Sci USA. 2015; 112:E1754-E1762. [PubMed: 25831525]
[0184] Chung A W, Ghebremichael M, Robinson H, Brown E, Choi I,
Lane S, Dugast A S, Schoen M K, Rolland M, Suscovich T J, Mahan A
E, Liao L, Streeck H, Andrews C, Rerks-Ngarm S, Nitayaphan S, de
Souza M S, Kaewkungwal J, Pitisuttithum P, Francis D, Michael N L,
Kim J H, Bailey-Kellogg C, Ackerman M E, Alter G. Polyfunctional
Fc-effector profiles mediated by IgG subclass selection distinguish
RV144 and VAX003 vaccines. Sci Transl Med. 2014; 6:228ra238. [0185]
Corey L, Gilbert P B, Tomaras G D, Haynes B F, Pantaleo G, Fauci A
S. Immune correlates of vaccine protection against HIV-1
acquisition. Sci Transl Med. 2015; 7:310rv317. [0186] Crooks E T,
Tong T, Chakrabarti B, Narayan K, Georgiev I S, Menis S, Huang X,
Kulp D, Osawa K, Muranaka J, Stewart-Jones G, Destefano J, O'Dell
S, LaBranche C, Robinson J E, Montefiori D C, McKee K, Du S X,
Doria-Rose N, Kwong P D, Mascola J R, Zhu P, Schief W R, Wyatt R T,
Whalen R G, Binley J M. Vaccine-elicited tier 2 HIV-1 neutralizing
antibodies bind to quaternary epitopes involving glycan-deficient
patches proximal to the CD4 binding site. PLoS Pathog. 2015;
11:e1004932. [PubMed: 26023780] [0187] Daniell H, Singh N D, Mason
H, Streatfield S J (2009) Plant-made vaccine antigens and
biopharmaceuticals. Trends Plant Sci 14: 669-679 [0188] Davis K L,
Gray E S, Moore P L, Decker J M, Salomon A, Montefiori D C, Graham
B S, Keefer M C, Pinter A, Morris L, Hahn B H, Shaw G M. High titer
HIV-1 V3-specific antibodies with broad reactivity but low
neutralizing potency in acute infection and following vaccination.
Virology. 2009; 387:414-426. [PubMed: 19298995] [0189] Decker J M,
Bibollet-Ruche F, Wei X, Wang S, Levy D N, Wang W, Delaporte E,
Peeters M, Derdeyn C A, Allen S, Hunter E, Saag M S, Hoxie J A,
Hahn B H, Kwong P D, Robinson J E, Shaw G M. Antigenic conservation
and immunogenicity of the HIV coreceptor binding site. J Exp Med.
2005; 201:1407-1419. [PubMed: 15867093] [0190] Devito C, Broliden
K, Kaul R, Svensson L, Johansen K, Kiama P, Kimani J, Lopalco L,
Piconi S, Bwayo J J, Plummer F, Clerici M, Hinkula J. Mucosal and
plasma IgA from HIV-1-exposed uninfected individuals inhibit HIV-1
transcytosis across human epithelial cells. J Immunol. 2000b;
165:5170-5176. [PubMed: 11046049] [0191] Devito C, Hinkula J, Kaul
R, Lopalco L, Bwayo J J, Plummer F, Clerici M, Broliden K. Mucosal
and plasma IgA from HIV-exposed seronegative individuals neutralize
a primary HIV-1 isolate. AIDS. 2000a; 14:1917-1920. [PubMed:
10997395] [0192] Diomede L, Nyoka S, Pastori C, Scotti L, Zambon A,
Sherman G, Gray C M, Sarzotti-Kelsoe M, Lopalco L. Passively
transmitted gp41 antibodies in babies born from HIV-1 subtype
C-seropositive women: correlation between fine specificity and
protection. J Virol. 2012; 86:4129-4138. [PubMed: 22301151] [0193]
Du S X, Idiart R J, Mariano E B, Chen H, Jiang P, Xu L, Ostrow K M,
Wrin T, Phung P, Binley J M, Petropoulos C J, Ballantyne J A,
Whalen R G. Effect of trimerization motifs on quaternary structure,
antigenicity, and immunogenicity of a noncleavable HIV-1 gp140
envelope glycoprotein. Virology. 2009; 395:33-44. [PubMed:
19815247] [0194] Duprez L, Wirawan E, Vanden Berghe T, Vandenabeele
P. Major cell death pathways at a glance. Microbes Infect. 2009;
11:1050-1062. [PubMed: 19733681] [0195] Egelkrout E, Rajan V,
Howard J A (2012) Overproduction of recombinant proteins in plants.
Plant Sci 184: 83-101 [0196] Feller U, Anders I, Mae T (2008)
Rubiscolytics: fate of Rubisco after its enzymatic function in a
cell is terminated. J Exp Bot 59: 1615-1624 [0197] Fischer R,
Schillberg S, Hellwig S, Twyman R M, Drossard J (2012) GMP issues
for recombinant plant-derived pharmaceutical proteins. Biotechnol
Adv 30: 434-439 [0198] Frey G, Chen J, Rits-Volloch S, Freeman M M,
Zolla-Pazner S, Chen B. Distinct conformational states of HIV-1
gp41 are recognized by neutralizing and non-neutralizing
antibodies. Nat Struct Mol Biol. 2010; 17:1486-1491. [PubMed:
21076402] [0199] Frey G, Peng H, Rits-Volloch S, Morelli M, Cheng
Y, Chen B. A fusion-intermediate state of HIV-1 gp41 targeted by
broadly neutralizing antibodies. Proc Natl Acad Sci USA. 2008;
105:3739-3744. [PubMed: 18322015] [0200] Garabagi F, Gilbert E,
Loos A, McLean M D, Hall J C (2012) Utility of the P19 suppressor
of gene-silencing protein for production of therapeutic antibodies
in Nicotiana expression hosts. Plant Biotechnol J 10: 1118-1128
[0201] Garcia F, Bernaldo de Quiros J C, Gomez C E, Perdiguero B,
Najera J L, Jimenez V, Garcia-Arriaza J, Guardo A C, Perez I,
Diaz-Brito V, Conde M S, Gonzalez N, Alvarez A, Alcami J, Jimenez J
L, Pich J, Arnaiz J A, Maleno M J, Leon A, Munoz-Fernandez M A,
Liljestrom P, Weber J, Pantaleo G, Gatell J M, Plana M, Esteban M.
Safety and immunogenicity of a modified pox vector-based HIV/AIDS
vaccine candidate expressing Env, Gag, Pol and Nef proteins of
HIV-1 subtype B (MVA-B) in healthy HIV-1-uninfected volunteers: a
phase I clinical trial (RISVAC02). Vaccine. 2011; 29:8309-8316.
[PubMed: 21907749] [0202] Garcia-Arriaza J, Arnaez P, Gomez C E,
Sorzano C O, Esteban M. Improving adaptive and memory immune
responses of an HIV/AIDS vaccine candidate MVA-B by deletion of
vaccinia virus genes (C6L and K7R) blocking interferon signaling
pathways. PLoS One. 2013; 8:e66894. [PubMed: 23826170] [0203]
Garcia-Arriaza J, Najera J L, Gomez C E, Tewabe N, Sorzano C O,
Calandra T, Roger T, Esteban M. A candidate HIV/AIDS vaccine
(MVA-B) lacking vaccinia virus gene C6L enhances memory
HIV-1-specific T-cell responses. PLoS One. 2011; 6:e24244. [PubMed:
21909386] [0204] Garoff H, Hewson R, Opstelten D J. Virus
maturation by budding. Microbiol Mol Biol Rev. 1998; 62:1171-1190.
[PubMed: 9841669] [0205] GlaxoSmithKline I. Product
Monograph--Cervarix. 2014 [0206] Gleba Y, Klimyuk V, Marillonnet S
(2005) Magnifection--a new platform for expressing recombinant
vaccines in plants. Vaccine 23: 2042-2048 [0207] Goepfert P A,
Elizaga M L, Sato A, Qin L, Cardinali M, Hay C M, Hural J, DeRosa S
C, DeFawe O D, Tomaras G D, Montefiori D C, Xu Y, Lai L, Kalams S
A, Baden L R, Frey S E, Blattner W A, Wyatt L S, Moss B, Robinson H
L, National Institute of A, Infectious Diseases HIVVTN. Phase 1
safety and immunogenicity testing of DNA and recombinant modified
vaccinia Ankara vaccines expressing HIV-1 virus-like particles. J
Infect Dis. 2011; 203:610-619. [PubMed: 21282192] [0208] Gomez C E,
Naj era J L, Jimenez E P, Jimenez V, Wagner R, Graf M, Frachette M
J, Lilj estrom P, Pantaleo G, Esteban M. Head-to-head comparison on
the immunogenicity of two HIV/AIDS vaccine candidates based on the
attenuated poxvirus strains MVA and NYVAC co-expressing in a single
locus the HIV-1BX08 gp120 and HIV-1(IIIB) Gag-Pol-Nef proteins of
clade B. Vaccine. 2007; 25:2863-2885. [PubMed: 17113200] [0209]
Gomez C E, Perdiguero B, Garcia-Arriaza J, Esteban M. Poxvirus
vectors as HIV/AIDS vaccines in humans. Hum Vaccin Immunother.
2012a; 8:1192-1207. [PubMed: 22906946] [0210] Gomez C E, Perdiguero
B, Najera J L, Sorzano C O, Jimenez V, Gonzalez-Sanz R, Esteban M.
Removal of vaccinia virus genes that block interferon type I and II
pathways improves adaptive and memory responses of the HIV/AIDS
vaccine candidate NYVAC-C in mice. J Virol. 2012b; 86:5026-5038.
[PubMed: 22419805] [0211] Gong Z, Martin-Garcia J M, Daskalova S M,
Craciunescu F M, Song L, Dorner K, Hansen D T, Yang J H, LaBaer J,
Hogue B G, Mor T S, Fromme P. Biophysical characterization of a
vaccine candidate against HIV-1: the transmembrane and membrane
proximal domains of HIV-1 gp41 as a maltose binding protein fusion.
PLoS One. 2015; 10:e0136507. [PubMed: 26295457] [0212] Goulder P J,
Watkins D I. HIV and SIV CTL escape: implications for vaccine
design. Nat Rev Immunol. 2004; 4:630-640. [PubMed: 15286729] [0213]
Greseth M D, Traktman P. De novo fatty acid biosynthesis
contributes significantly to establishment of a bioenergetically
favorable environment for vaccinia virus infection. PLoS Pathog.
2014; 10:e1004021. [PubMed: 24651651] [0214] Gutierrez C (1999)
Geminivirus DNA replication. Cellular and Molecular Life Sciences
56: 313-329 [0215] Hansen S G, Piatak M Jr, Ventura A B, Hughes C
M, Gilbride R M, Ford J C, Oswald K, Shoemaker R, Li Y, Lewis M S,
Gilliam A N, Xu G, Whizin N, Burwitz B J, Planer S L, Turner J M,
Legasse A W, Axthelm M K, Nelson J A, Fruh K, Sacha J B, Estes J D,
Keele B F, Edlefsen P T, Lifson J D, Picker L J. Immune clearance
of highly pathogenic SIV infection. Nature. 2013a; 502:100-104.
[PubMed: 24025770]
[0216] Hansen S G, Sacha J B, Hughes C M, Ford J C, Burwitz B J,
Scholz I, Gilbride R M, Lewis M S, Gilliam A N, Ventura A B,
Malouli D, Xu G, Richards R, Whizin N, Reed J S, Hammond K B,
Fischer M, Turner J M, Legasse A W, Axthelm M K, Edlefsen P T,
Nelson J A, Lifson J D, Fruh K, Picker L J. Cytomegalovirus vectors
violate CD8+ T cell epitope recognition paradigms. Science. 2013b;
340:1237874. [PubMed: 23704576] [0217] Harari A, Bart P A, Stohr W,
Tapia G, Garcia M, Medjitna-Rais E, Burnet S, Cellerai C, Erlwein
O, Barber T, Moog C, Liljestrom P, Wagner R, Wolf H, Kraehenbuhl J
P, Esteban M, Heeney J, Frachette M J, Tartaglia J, McCormack S,
Babiker A, Weber J, Pantaleo G. An HIV-1 clade C DNA prime, NYVAC
boost vaccine regimen induces reliable, polyfunctional, and
long-lasting T cell responses. J Exp Med. 2008; 205:63-77. [PubMed:
18195071] [0218] Harari A, Rozot V, Cavassini M, Bellutti Enders F,
Vigano S, Tapia G, Castro E, Burnet S, Lange J, Moog C, Garin D,
Costagliola D, Autran B, Pantaleo G, Bart P A. NYVAC immunization
induces polyfunctional HIV-specific T-cell responses in
chronically-infected, ART-treated HIV patients. Eur J Immunol.
2012; 42:3038-3048. [PubMed: 22930439] [0219] Haynes B F, Fleming
J, St Clair E W, Katinger H, Stiegler G, Kunert R, Robinson J,
Scearce R M, Plonk K, Staats H F, Ortel T L, Liao H X, Alam S M.
Cardiolipin polyspecific autoreactivity in two broadly neutralizing
HIV-1 antibodies. Science. 2005; 308:1906-1908. [PubMed: 15860590]
[0220] Haynes B F, Gilbert P B, McElrath M J, Zolla-Pazner S,
Tomaras G D, Alam S M, Evans D T, Montefiori D C, Karnasuta C,
Sutthent R, Liao H X, DeVico A L, Lewis G K, Williams C, Pinter A,
Fong Y, Janes H, DeCamp A, Huang Y, Rao M, Billings E, Karasavvas
N, Robb M L, Ngauy V, de Souza M S, Paris R, Ferrari G, Bailer R T,
Soderberg K A, Andrews C, Berman P W, Frahm N, De Rosa S C, Alpert
M D, Yates N L, Shen X, Koup R A, Pitisuttithum P, Kaewkungwal J,
Nitayaphan S, Rerks-Ngarm S, Michael N L, Kim J H.
Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N
Engl J Med. 2012; 366:1275-1286. [PubMed: 22475592] [0221] Haynes B
F, Shaw G M, Korber B, Kelsoe G, Sodroski J, Hahn B H, Borrow P,
McMichael A J. HIV-host interactions: implications for vaccine
design. Cell Host Microbe. 2016; 19:292-303. [PubMed: 26922989]
[0222] Hessell A J, Hangartner L, Hunter M, Havenith C E, Beurskens
F J, Bakker J M, Lanigan C M, Landucci G, Forthal D N, Parren P W,
Marx P A, Burton D R. Fc receptor but not complement binding is
important in antibody protection against HIV. Nature. 2007;
449:101-104. [PubMed: 17805298] [0223] Hessell A J, Rakasz E G,
Tehrani D M, Huber M, Weisgrau K L, Landucci G, Forthal D N, Koff W
C, Poignard P, Watkins D I, Burton D R. Broadly neutralizing
monoclonal antibodies 2F5 and 4E10 directed against the human
immunodeficiency virus type 1 gp41 membrane-proximal external
region protect against mucosal challenge by simian-human
immunodeficiency virus SHIVBa-L. J Virol. 2010; 84:1302-1313.
[PubMed: 19906907] [0224] Hoot S, McGuire A T, Cohen K W, Strong R
K, Hangartner L, Klein F, Diskin R, Scheid J F, Sather D N, Burton
D R, Stamatatos L. Recombinant HIV envelope proteins fail to engage
germline versions of anti-CD4bs bNAbs. PLoS Pathog. 2013;
9:e1003106. [PubMed: 23300456] [0225] Horn M E, Pappu K M, Bailey M
R, Clough R C, Barker M, Jilka J M, Howard J A, Streatfield S J
(2003) Advantageous features of plant-based systems for the
development of HIV vaccines. J Drug Target 11: 539-545 [0226] Huang
J, Kang B H, Pancera M, Lee J H, Tong T, Feng Y, Imamichi H,
Georgiev I S, Chuang G Y, Druz A, Doria-Rose N A, Laub L, Sliepen
K, van Gils M J, de la Pena A T, Derking R, Klasse P J, Migueles S
A, Bailer R T, Alam M, Pugach P, Haynes B F, Wyatt R T, Sanders R
W, Binley J M, Ward A B, Mascola J R, Kwong P D, Connors M. Broad
and potent HIV-1 neutralization by a human antibody that binds the
gp41-gp120 interface. Nature. 2014; 515:138-142. [PubMed: 25186731]
[0227] Huang J, Ofek G, Laub L, Louder M K, Doria-Rose N A, Longo N
S, Imamichi H, Bailer R T, Chakrabarti B, Sharma S K, Alam S M,
Wang T, Yang Y, Zhang B, Migueles S A, Wyatt R, Haynes B F, Kwong P
D, Mascola J R, Connors M. Broad and potent neutralization of HIV-1
by a gp41-specific human antibody. Nature. 2012; 491:406-412.
[PubMed: 23151583] [0228] Huang Y, Follmann D, Nason M, Zhang L,
Huang Y, Mehrotra D V, Moodie Z, Metch B, Janes H, Keefer M C,
Churchyard G, Robb M L, Fast P E, Duerr A, McElrath M J, Corey L,
Mascola J R, Graham B S, Sobieszczyk M E, Kublin J G, Robertson M,
Hammer S M, Gray G E, Buchbinder S P, Gilbert P B. Effect of
rAd5-vector HIV-1 preventive vaccines on HIV-1 acquisition: a
participant-level meta-analysis of randomized trials. PLoS One.
2015; 10:e0136626. [PubMed: 26332672] [0229] Huang Z, Chen Q, Hjelm
B, Arntzen C, Mason H (2009) A DNA replicon system for rapid
high-level production of virus-like particles in plants.
Biotechnology and bioengineering 103: 706-714 [0230] Irimia A,
Sarkar A, Stanfield R L, Wilson I A. Crystallographic
identification of lipid as an integral component of the epitope of
HIV broadly neutralizing antibody 4E10. Immunity. 2016; 44:21-31.
[PubMed: 26777395] [0231] Jacob R A, Moyo T, Schomaker M, Abrahams
F, Grau Pujol B, Dorfman J R. Anti-V3/glycan and Anti-MPER
neutralizing antibodies, but not anti-V2/glycan site antibodies,
are strongly associated with greater anti-HIV-1 neutralization
breadth and potency. J Virol. 2015; 89:5264-5275. [PubMed:
25673728] [0232] Jacobs B L, Langland J O, Kibler K V, Denzler K L,
White S D, Holechek S A, Wong S, Huynh T, Baskin C R. Vaccinia
virus vaccines: past, present and future. Antivir Res. 2009;
84:1-13. [PubMed: 19563829] [0233] Jardine J, Julien J P, Menis S,
Ota T, Kalyuzhniy O, McGuire A, Sok D, Huang P S, MacPherson S,
Jones M, Nieusma T, Mathison J, Baker D, Ward A B, Burton D R,
Stamatatos L, Nemazee D, Wilson I A, Schief W R. Rational HIV
immunogen design to target specific germline B cell receptors.
Science. 2013; 340:711-716. [PubMed: 23539181] [0234] Jiao Y, Xie
J, L T, Han Y, Qiu Z, Zuo L, Wang A. Correlation between
gag-specific CD8 T-cell responses, viral load, and CD4 count in
HIV-1 infection is dependent on disease status. J Acquir Immune
Defic Syndr. 2006; 42:263-268. [PubMed: 16763520] [0235] Kaul R,
Plummer F, Clerici M, Bomsel M, Lopalco L, Broliden K. Mucosal IgA
in exposed, uninfected subjects: evidence for a role in protection
against HIV infection. AIDS. 2001; 15:431-432. [PubMed: 11273233]
[0236] Kaul R, Trabattoni D, Bwayo J J, Arienti D, Zagliani A,
Mwangi F M, Kariuki C, Ngugi E N, MacDonald K S, Ball T B, Clerici
M, Plummer F A. HIV-1-specific mucosal IgA in a cohort of
HIV-1-resistant Kenyan sex workers. AIDS. 1999; 13:23-29. [PubMed:
10207541] [0237] Keefer M C, Frey S E, Elizaga M, Metch B, De Rosa
S C, Barroso P F, Tomaras G, Cardinali M, Goepfert P, Kalichman A,
Philippon V, McElrath M J, Jin X, Ferrari G, Defawe O D, Mazzara G
P, Montefiori D, Pensiero M, Panicali D L, Corey L, Network NHVT. A
phase I trial of preventive HIV vaccination with heterologous
poxviral-vectors containing matching HIV-1 inserts in healthy
HIV-uninfected subjects. Vaccine. 2011; 29:1948-1958. [PubMed:
21216311] [0238] Kepler T B, Liao H X, Alam S M, Bhaskarabhatla R,
Zhang R, Yandava C, Stewart S, Anasti K, Kelsoe G, Parks R, Lloyd K
E, Stolarchuk C, Pritchett J, Solomon E, Friberg E, Morris L, Karim
S S, Cohen M S, Walter E, Moody M A, Wu X, Altae-Tran H R, Georgiev
I S, Kwong P D, Boyd S D, Fire A Z, Mascola J R, Haynes B F.
Immunoglobulin gene insertions and deletions in the affinity
maturation of HIV-1 broadly reactive neutralizing antibodies. Cell
Host Microbe. 2014; 16:304-313. [PubMed: 25211073] [0239] Kessans S
A, Linhart M D, Matoba N, Mor T. Biological and biochemical
characterization of HIV-1 Gag/dgp41 virus-like particles expressed
in Nicotiana benthamiana. Plant Biotechnol J. 2013; 11:681-690.
[PubMed: 23506331] [0240] Kessans S A, Linhart M D, Meador L R,
Kilbourne J, Hogue B G, Fromme P, Matoba N, Mor T S. Immunological
characterization of plant-based HIV-1 Gag/Dgp41 virus-like
particles. PLoS One. 2016; 11:e0151842. [PubMed: 26986483] [0241]
Kibler K V, Gomez C E, Perdiguero B, Wong S, Huynh T, Holechek S,
Arndt W, Jimenez V, Gonzalez-Sanz R, Denzler K, Haddad E K, Wagner
R, Sekaly R P, Tartaglia J, Pantaleo G, Jacobs B L, Esteban M.
Improved NYVAC-based vaccine vectors. PLoS One. 2011; 6:e25674.
[PubMed: 22096477] [0242] Kibler K V, Shors T, Perkins K, Zeman C,
M P B, Biesterfeldt J, Langland J O, Jacobs B L. Double-stranded
RNA is a trigger for apoptosis in vaccinia virus-infected cells. J
Virol. 1997; 71:1992-2003. [PubMed: 9032331] [0243] Kiepiela P,
Ngumbela K, Thobakgale C, Ramduth D, Honeyborne I, Moodley E, Reddy
S, de Pierres C, Mncube Z, Mkhwanazi N, Bishop K, van der Stok M,
Nair K, Khan N, Crawford H, Payne R, Leslie A, Prado J, Prendergast
A, Frater J, McCarthy N, Brander C, Learn G H, Nickle D, Rousseau
C, Coovadia H, Mullins J I, Heckerman D, Walker B D, Goulder P.
CD8+ T-cell responses to different HIV proteins have discordant
associations with viral load. Nat Med. 2007; 13:46-53. [PubMed:
17173051] [0244] Klein F, Diskin R, Scheid J F, Gaebler C, Mouquet
H, Georgiev I S, Pancera M, Zhou T, Incesu R B, Fu B Z,
Gnanapragasam P N, Oliveira T Y, Seaman M S, Kwong P D, Bjorkman P
J, Nussenzweig M C. Somatic mutations of the immunoglobulin
framework are generally required for broad and potent HIV-1
neutralization. Cell. 2013b; 153:126-138. [PubMed: 23540694] [0245]
Klein F, Mouquet H, Dosenovic P, Scheid J F, Scharf L, Nussenzweig
M C. Antibodies in HIV-1 vaccine development and therapy. Science.
2013a; 341:1199-1204. [PubMed: 24031012] [0246] Koup R A, Safrit J
T, Cao Y, Andrews C A, McLeod G, Borkowsky W, Farthing C, Ho D D.
Temporal association of cellular immune responses with the initial
control of viremia in primary human immunodeficiency virus type 1
syndrome. J Virol. 1994; 68:4650-4655. [PubMed: 8207839] [0247]
Kushnir N, Streatfield S J, Yusibov V. Virus-like particles as a
highly efficient vaccine platform: diversity of targets and
production systems and advances in clinical development. Vaccine.
2012; 31:58-83. [PubMed: 23142589] [0248] Labrijn A F, Poignard P,
Raja A, Zwick M B, Delgado K, Franti M, Binley J, Vivona V,
Grundner C, Huang C C, Venturi M, Petropoulos C J, Wrin T, Dimitrov
D S, Robinson J, Kwong P D, Wyatt R T, Sodroski J, Burton D R.
Access of antibody molecules to the conserved coreceptor binding
site on glycoprotein gp120 is sterically restricted on primary
human immunodeficiency virus type 1. J Virol. 2003; 77:10557-10565.
[PubMed: 12970440] [0249] Leroux-Roels G, Maes C, Clement F, van
Engelenburg F, van den Dobbelsteen M, Adler M, Amacker M, Lopalco
L, Bomsel M, Chalifour A, Fleury S. Randomized phase I: safety,
immunogenicity and mucosal antiviral activity in young healthy
women vaccinated with HIV-1 Gp41 P1 peptide on virosomes. PloS One.
2013; 8:e55438. [PubMed: 23437055] [0250] Li M X, Dewson G.
Mitochondria and apoptosis: emerging concepts. F1000Prime Rep.
2015; 7:42. [PubMed: 26097715] [0251] Liao H X, Bonsignori M, Alam
S M, McLellan J S, Tomaras G D, Moody M A, Kozink D M, Hwang K K,
Chen X, Tsao C Y, Liu P, Lu X, Parks R J, Montefiori D C, Ferrari
G, Pollara J, Rao M, Peachman K K, Santra S, Letvin N L, Karasavvas
N, Yang Z Y, Dai K, Pancera M, Gorman J, Wiehe K, Nicely N I,
Rerks-Ngarm S, Nitayaphan S, Kaewkungwal J, Pitisuttithum P,
Tartaglia J, Sinangil F, Kim J H, Michael N L, Kepler T B, Kwong P
D, Mascola J R, Nabel G J, Pinter A, Zolla-Pazner S, Haynes B F.
Vaccine induction of antibodies against a structurally
heterogeneous site of immune pressure within HIV-1 envelope protein
variable regions 1 and 2. Immunity. 2013; 38:176-186. [PubMed:
23313589] [0252] Liao H X, Chen X, Munshaw S, Zhang R, Marshall D
J, Vandergrift N, Whitesides J F, Lu X, Yu J S, Hwang K K, Gao F,
Markowitz M, Heath S L, Bar K J, Goepfert P A, Montefiori D C, Shaw
G C, Alam S M, Margolis D M, Denny T N, Boyd S D, Marshal E, Egholm
M, Simen B B, Hanczaruk B, Fire A Z, Voss G, Kelsoe G, Tomaras G D,
Moody M A, Kepler T B, Haynes B F. Initial antibodies binding to
HIV-1 gp41 in acutely infected subjects are polyreactive and highly
mutated. J Exp Med. 2011; 208:2237-2249. [PubMed: 21987658] [0253]
Link A, Zabel F, Schnetzler Y, Titz A, Brombacher F, Bachmann M F
(2012) Innate immunity mediates follicular transport of particulate
but not soluble protein antigen. Journal of immunology 188:
3724-3733 [0254] Ma J K, Chikwamba R, Sparrow P, Fischer R, Mahoney
R, Twyman R M (2005) Plant-derived pharmaceuticals--the road
forward. Trends Plant Sci 10: 580-585 [0255] Magerus-Chatinet A, Yu
H, Garcia S, Ducloux E, Terris B, Bomsel M. Galactosyl ceramide
expressed on dendritic cells can mediate HIV-1 transfer from
monocyte derived dendritic cells to autologous T cells. Virology.
2007; 362:67-74. [PubMed: 17234232] [0256] Marillonnet S, Giritch
A, Gils M, Kandzia R, Klimyuk V, Gleba Y (2004) In planta
engineering of viral RNA replicons: Efficient assembly by
recombination of DNA modules deliveredy by Agrobacterium.
Proceedings of the National Academy of Sciences 101: 6852-6857
[0257] Marillonnet S, Thoeringer C, Kandzia R, Klimyuk V, Gleba Y
(2005) Systemic Agrobacterium tumefaciens-mediated transfection of
viral replicons for efficient transient expression in plants. Nat
Biotechnol 23: 718-723 [0258] Martinez F O, Gordon S. The M1 and M2
paradigm of macrophage activation: time for reassessment.
F1000Prime Rep. 2014; 6:13. [PubMed: 24669294] [0259] Mascola J R,
Haynes B F. HIV-1 neutralizing antibodies: understanding nature's
pathways. Immunol Rev. 2013; 254:225-244. [PubMed: 23772623] [0260]
Mascola J R, Montefiori D C. The role of antibodies in HIV
vaccines. Annu Rev Immunol. 2010; 28:413-444. [PubMed: 20192810]
[0261] Matoba N, Griffin T A, Mittman M, Doran J D, Alfsen A,
Montefiori D C, Hanson C V, Bomsel M, Mor T S.
Transcytosis-blocking abs elicited by an oligomeric immunogen based
on the membrane proximal region of HIV-1 gp41 target
non-neutralizing epitopes. Curr HIV Res. 2008; 6:218-229. [PubMed:
18473785] [0262] Matoba N, Kajiura H, Cherni I, Doran J D, Bomsel
M, Fujiyama K, Mor T S. Biochemical and immunological
characterization of the plant-derived candidate human
immunodeficiency virus type 1 mucosal vaccine CTB-MPR. Plant
Biotechnol J. 2009; 7:129-145. [PubMed: 19037902] [0263] Matoba N,
Magerus A, Geyer B C, Zhang Y, Muralidharan M, Alfsen A, Arntzen C
J, Bomsel M, Mor T S. A mucosally targeted subunit vaccine
candidate eliciting HIV-1 transcytosis-blocking Abs. Proc Natl Acad
Sci USA. 2004; 101:13584-13589. [PubMed: 15347807] [0264] McGuire A
T, Glenn J A, Lippy A, Stamatatos L. Diverse recombinant HIV-1 Envs
fail to activate B cells expressing the germline B cell receptors
of the broadly neutralizing anti-HIV-1 antibodies PG9 and 447-52D.
J Virol. 2014; 88:2645-2657. [PubMed: 24352455]
[0265] McMichael A J, Koff W C. Vaccines that stimulate T cell
immunity to HIV-1: the next step. Nat Immunol. 2014; 15:319-322.
[PubMed: 24646598] [0266] Merck, Co. I. Prescribing
Information--Gardasil. 2006 [0267] Meyers A, Chakauya E, Shephard
E, Tanzer F L, Maclean J, Lynch A, Williamson A L, Rybicki E P
(2008) Expression of HIV-1 antigens in plants as potential subunit
vaccines. BMC Biotechnol 8: 53 [0268] Micoli K J, Mamaeva O, Piller
S C, Barker J L, Pan G, Hunter E, McDonald J M. Point mutations in
the C-terminus of HIV-1 gp160 reduce apoptosis and calmodulin
binding without affecting viral replication. Virology. 2006;
344:468-479. [PubMed: 16229872] [0269] Mooij P,
Balla-Jhagjhoorsingh S S, Beenhakker N, van Haaften P, Baak I,
Nieuwenhuis I G, Heidari S, Wolf H, Frachette M J, Bieler K,
Sheppard N, Harari A, Bart P A, Liljestrom P, Wagner R, Pantaleo G,
Heeney J L. Comparison of human and rhesus macaque T-cell responses
elicited by boosting with NYVAC encoding human immunodeficiency
virus type 1 clade C immunogens. J Virol. 2009; 83:5881-5889.
[PubMed: 19321612] [0270] Moore P L, Williamson C, Morris L.
Virological features associated with the development of broadly
neutralizing antibodies to HIV-1. Trends Microbiol. 2015;
23:204-211. [PubMed: 25572881] [0271] Mor T S, Moon Y, Palmer K E,
Mason H S (2003) Geminivirus vectors for high-level expression of
foregin proteins in plant cells. Wiley Periodicals Biotechnology
and Bioengineering 81: 430-437 [0272] Mosser D M, Edwards J P.
Exploring the full spectrum of macrophage activation. Nat Rev
Immunol. 2008; 8:958-969. [PubMed: 19029990] [0273] Mudd P A,
Martins M A, Ericsen A J, Tully D C, Power K A, Bean A T,
Piaskowski S M, Duan L, Seese A, Gladden A D, Weisgrau K L, Furlott
J R, Kim Y I, Veloso de Santana M G, Rakasz E, Capuano S 3rd,
Wilson N A, Bonaldo M C, Galler R, Allison D B, Piatak M Jr, Haase
A T, Lifson J D, Allen T M, Watkins D I. Vaccine-induced CD8+ T
cells control AIDS virus replication. Nature. 2012; 491:129-133.
[PubMed: 23023123] [0274] Nelson J D, Brunel F M, Jensen R, Crooks
E T, Cardoso R M, Wang M, Hessell A, Wilson I A, Binley J M, Dawson
P E, Burton D R, Zwick M B. An affinity-enhanced neutralizing
antibody against the membrane-proximal external region of human
immunodeficiency virus type 1 gp41 recognizes an epitope between
those of 2F5 and 4E10. J Virol. 2007; 81:4033-4043. [PubMed:
17287272] [0275] Nitayaphan S, Pitisuttithum P, Karnasuta C,
Eamsila C, de Souza M, Morgan P, Polonis V, Benenson M, VanCott T,
Ratto-Kim S, Kim J, Thapinta D, Garner R, Bussaratid V, Singharaj
P, el-Habib R, Gurunathan S, Heyward W, Birx D, McNeil J, Brown A
E, Thai AVEG. Safety and immunogenicity of an HIV subtype B and E
prime-boost vaccine combination in HIV-negative Thai adults. J
Infect Dis. 2004; 190:702-706. [PubMed: 15272397] [0276] Ogg G S,
Jin X, Bonhoeffer S, Dunbar P R, Nowak M A, Monard S, Segal J P,
Cao Y, Rowland-Jones S L, Cerundolo V, Hurley A, Markowitz M, Ho D
D, Nixon D F, McMichael A J. Quantitation of HIV-1-specific
cytotoxic T lymphocytes and plasma load of viral RNA. Science.
1998; 279:2103-2106. [PubMed: 9516110] [0277] Pancera M, Zhou T,
Druz A, Georgiev I S, Soto C, Gorman J, Huang J, Acharya P, Chuang
G Y, Ofek G, Stewart-Jones G B, Stuckey J, Bailer R T, Joyce M G,
Louder M K, Tumba N, Yang Y, Zhang B, Cohen M S, Haynes B F,
Mascola J R, Morris L, Munro J B, Blanchard S C, Mothes W, Connors
M, Kwong P D. Structure and immune recognition of trimeric
pre-fusion HIV-1 Env. Nature. 2014; 514:455-461. [PubMed: 25296255]
[0278] Pantaleo G, Esteban M, Jacobs B, Tartaglia J. Poxvirus
vector-based HIV vaccines. Curr Opin HIV AIDS. 2010; 5:391-396.
[PubMed: 20978379] [0279] Parker C E, Deterding L J, Hager-Braun C,
Binley J M, Schulke N, Katinger H, Moore J P, Tomer K B. Fine
definition of the epitope on the gp41 glycoprotein of human
immunodeficiency virus type 1 for the neutralizing monoclonal
antibody 2F5. J Virol. 2001; 75:10906-10911. [PubMed: 11602730]
[0280] Pastori C, Barassi C, Piconi S, Longhi R, Villa M L,
Siccardi A G, Clerici M, Lopalco L. HIV neutralizing IgA in exposed
seronegative subjects recognise an epitope within the gp41
coiled-coil pocket. J Biol Regul Homeost Agents. 2000; 14:15-21.
[PubMed: 10763886] [0281] Perdiguero B, Gomez C E, Cepeda V,
Sanchez-Sampedro L, Garcia-Arriaza J, Mejias-Perez E, Jimenez V,
Sanchez C, Sorzano C O, Oliveros J C, Delaloye J, Roger T, Calandra
T, Asbach B, Wagner R, Kibler K V, Jacobs B L, Pantaleo G, Esteban
M. Virological and immunological characterization of novel
NYVAC-based HIV/AIDS vaccine candidates expressing clade C trimeric
soluble gp140(ZM96) and Gag(ZM96)-Pol-Nef(CN54) as virus-like
particles. J Virol. 2015; 89:970-988. [PubMed: 25355891] [0282]
Perreau M, Welles H C, Harari A, Hall O, Martin R, Maillard M,
Dorta G, Bart P A, Kremer E J, Tartaglia J, Wagner R, Esteban M,
Levy Y, Pantaleo G. DNA/NYVAC vaccine regimen induces HIV-specific
CD4 and CD8 T-cell responses in intestinal mucosa. J Virol. 2011;
85:9854-9862. [PubMed: 21775454] [0283] Peyret H, Lomonossoff G P
(2013) The pEAQ vector series: the easy and quick way to produce
recombinant proteins in plants. Plant molecular biology 83: 51-58
[0284] Pillay S, Shephard E G, Meyers A E, Williamson A L, Rybicki
E P. HIV-1 sub-type C chimaeric VLPs boost cellular immune
responses in mice. J Immune Based Ther Vaccin. 2010; 8:7. [0285]
Pollara J, McGuire E, Fouda G G, Rountree W, Eudailey J, Overman R
G, Seaton K E, Deal A, Edwards R W, Tegha G, Kamwendo D, Kumwenda
J, Nelson J A, Liao H X, Brinkley C, Denny T N, Ochsenbauer C,
Ellington S, King C C, Jamieson D J, van der Horst C, Kourtis A P,
Tomaras G D, Ferrari G, Permar S R. Association of HIV-1
envelope-specific breast milk IgA responses with reduced risk of
postnatal mother-to-child transmission of HIV-1. J Virol. 2015;
89:9952-9961. [PubMed: 26202232] [0286] Postler T S, Desrosiers R
C. The tale of the long tail: the cytoplasmic domain of HIV-1 gp41.
J Virol. 2013; 87:2-15. [PubMed: 23077317] [0287] Prentice H A,
Tomaras G, Geraghty D E, Apps R, Fong Y, Ehrenberg P K, Rolland M,
Kijak G H, Krebs S J, Nelson W, DeCamp A, Shen X, Yates N L,
Zolla-Pazner S, Nitayaphan S, Rerks-Ngarm S, Kaewkungwal J,
Pitisuttihum P, Ferrari G, McElrath M J, Montefiori D, Bailer R T,
Koup R A, O'Connell R J, Robb M L, Michael N L, Gilbert P B, Kim J
H, Thomas R. HLA class I I genes modulates vaccine-induced antibody
responses to affect HIV-1 acquisition. Sci Transl Med. 2015;
7:296ra112. [0288] Purtscher M, Trkola A, Gruber G, Buchacher A,
Predl R, Steindl F, Tauer C, Berger R, Barrett N, Jungbauer A,
Katinger H (1994) A broadly neutralizing human monoclonal antibody
against gp41 of Human Immunodeficiency Virus Type 1. AIDS Research
and Human Retroviruses 10: 1651-1658 [0289] Quakkelaar E D, Redeker
A, Haddad E K, Harari A, McCaughey S M, Duhen T, Filali-Mouhim A,
Goulet J P, Loof N M, Ossendorp F, Perdiguero B, Heinen P, Gomez C
E, Kibler K V, Koelle D M, Sekaly R P, Sallusto F, Lanzavecchia A,
Pantaleo G, Esteban M, Tartaglia J, Jacobs B L, Melief C J.
Improved innate and adaptive immunostimulation by genetically
modified HIV-1 protein expressing NYVAC vectors. PLoS One. 2011;
6:e16819. [PubMed: 21347234] [0290] Rerks-Ngarm S, Pitisuttithum P,
Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, Premsri N, Namwat C,
de Souza M, Adams E, Benenson M, Gurunathan S, Tartaglia J, McNeil
J G, Francis D P, Stablein D, Birx D L, Chunsuttiwat S,
Khamboonruang C, Thongcharoen P, Robb M L, Michael L, Kunasol P,
Kim J H. Vaccination with ALVAC and AIDSVAX to Prevent HIV-1
Infection in Thailand. N Engl J Med. 2009 (doi:NEJMoa0908492)(pii)
(10.1056/NEJMoa0908492). [0291] Rodriguez-Negrete E A,
Carrillo-Tripp J, Rivera-Bustamante R F (2009) RNA silencing
against geminivirus: complementary action of posttranscriptional
gene silencing and transcriptional gene silencing in host recovery.
Journal of virology 83: 1332-1340 [0292] Rosales-Mendoza S,
Rubio-Infante N, Govea-Alonso D O, Moreno-Fierros L (2012) Current
status and perspectives of plant-based candidate vaccines against
the human immunodeficiency virus (HIV). Plant Cell Rep 31: 495-511
[0293] Rosales-Mendoza S, Rubio-Infante N, Monreal-Escalante E,
Govea-Alonso D O, Garcia-Hernindez A L, Salazar-Gonzalez J A,
Gonzalez-Ortega O, Paz-Maldonado LMT, Moreno-Fierros L (2013)
Chloroplast expression of an HIV envelop-derived multiepitope
protein: towards a multivalent plant-based vaccine. Plant Cell,
Tissue and Organ Culture (PCTOC) 116: 111-123 [0294] Rybicki E P.
Plant-produced vaccines: promise and reality. Drug Discov Today.
2009; 14: 16-24 [0295] Rybicki E P. Plant-based vaccines against
viruses. Virology J. 2014; 11:205 [0296] Rybicki E P. Plant-made
vaccines for humans and animals. Plant Biotechnol J. 2010;
8:620-637. [PubMed: 20233333] [0297] Sagar M, Akiyama H, Etemad B,
Ramirez N, Freitas I, Gummuluru S. Transmembrane domain membrane
proximal external region but not surface unit-directed broadly
neutralizing hiv-1 antibodies can restrict dendritic cell-mediated
HIV-1 trans-infection. J Infect Dis. 2012; 205:1248-1257. [PubMed:
22396600] [0298] Sainsbury F, Thuenemann E C, Lomonossoff G P
(2009) pEAQ: versatile expression vectors for easy and quick
transient expression of heterologous proteins in plants. Plant
biotechnology journal 7: 682-693 [0299] Sala F, Rigano M M,
Barbante A, Basso B, Walmsley A M, Castiglione S (2003) Vaccine
antigen production in transgenic plants: strategies, gene
constructs and perspectives. Vaccine 21: 803-808 [0300] Sanders R
W, van Gils M J, Derking R, Sok D, Ketas T J, Burger J A, Ozorowski
G, Cupo A, Simonich C, Goo L, Arendt H, Kim H J, Lee J H, Pugach P,
Williams M, Debnath G, Moldt B, van Breemen M J, Isik G,
Medina-Ramirez M, Back J W, Koff W C, Julien J P, Rakasz E G,
Seaman M S, Guttman M, Lee K K, Klasse P J, LaBranche C, Schief W
R, Wilson I A, Overbaugh J, Burton D R, Ward A B, Montefiori D C,
Dean H, Moore J P. HIV-1 VACCINES. HIV-1 neutralizing antibodies
induced by native-like envelope trimers. Science. 2015;
349:aac4223. [PubMed: 26089353] [0301] Schneider C A, Rasband W S,
Eliceiri K W (2012) NIH Image to ImageJ: 25 years of image
analysis. Nature Methods 9: 671-675 [0302] Scotti N, Alagna F,
Ferraiolo E, Formisano G, Sannino L, Buonaguro L, De Stradis A,
Vitale A, Monti L, Grillo S, Buonaguro F M, Cardi T (2009)
High-level expression of the HIV-1 Pr55gag polyprotein in
transgenic tobacco chloroplasts. Planta 229: 1109-1122 [0303]
Scotti N, Rybicki E P. Virus-like particles produced in plants as
potential vaccines. Expert Rev Vaccin. 2013; 12:211-224. [0304]
Seemanpillai M, Dry I, Randles J, Rezaian A (2003) Transcriptional
Silencing of Geminiviral Promoter-Driven Transgenes Following
Homologous Virus Infection. Molecular Plant-Microbe Interactions
16: 429-438 [0305] Sellhorn G, Kraft Z, Caldwell Z, Ellingson K,
Mineart C, Seaman M S, Montefiori D C, Lagerquist E, Stamatatos L.
Engineering, expression, purification, and characterization of
stable clade A/B recombinant soluble heterotrimeric gp140 proteins.
J Virol. 2012; 86:128-142. [PubMed: 22031951] [0306] Shen R,
Drelichman E R, Bimczok D, Ochsenbauer C, Kappes J C, Cannon J A,
Tudor D, Bomsel M, Smythies L E, Smith P D. GP41-specific antibody
blocks cell-free HIV type 1 transcytosis through human rectal
mucosa and model colonic epithelium. J Immunol. 2010;
184:3648-3655. [PubMed: 20208001] [0307] Silhavy D, Molnar A,
Lucioli A, Szittya G, Hornyik C, Tavazza M, Burgyan J (2002) A
viral protein suppresses RNA silencing and bin