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Human Vaccines & Immunotherapeutics
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Comparison of intradermal and intramuscular delivery
followed by in vivo electroporation of SIV Env DNA in
macaques
Viraj Kulkarnia, Margherita Rosatib, Jenifer Beara, Guy R Pilkingtona, Rashmi Jalaha, Cristina
Bergamaschia, Ashish K Singhb, Candido Aliceaa, Bhabadeb Chowdhuryb, Gen-Mu Zhangab,
Eun-Young Kimc, Steven M Wolinskyc, Wensheng Huangd, Yongjun Guand, Celia LaBranchee,
David C Montefiorie, Kate E Broderickf, Niranjan Y Sardesaif, Antonio Valentinb, Barbara K
Felbera & George N Pavlakisb
a Human Retrovirus Pathogenesis Section; Vaccine Branch; Center for Cancer Research;
National Cancer Institute; Frederick, MD USA
b Human Retrovirus Section; Vaccine Branch; Center for Cancer Research; National Cancer
Institute; Frederick, MD USA
c Division of Infectious Diseases; The Feinberg School of Medicine; Northwestern University;
Chicago, IL USA
d Institute of Human Virology; Department of Microbiology and Immunology; University of
Maryland School of Medicine; Baltimore, MD USA
e Department of Surgery; Laboratory for AIDS Vaccine Research and Development; Duke
University Medical Center; Durham, NC USA
f Inovio Pharmaceuticals, Inc.; Blue Bell, PA USA
Published online: 28 Jun 2013.
To cite this article: Viraj Kulkarni, Margherita Rosati, Jenifer Bear, Guy R Pilkington, Rashmi Jalah, Cristina Bergamaschi,
Ashish K Singh, Candido Alicea, Bhabadeb Chowdhury, Gen-Mu Zhang, Eun-Young Kim, Steven M Wolinsky, Wensheng Huang,
Yongjun Guan, Celia LaBranche, David C Montefiori, Kate E Broderick, Niranjan Y Sardesai, Antonio Valentin, Barbara K Felber
& George N Pavlakis (2013) Comparison of intradermal and intramuscular delivery followed by in vivo electroporation of SIV
Env DNA in macaques, Human Vaccines & Immunotherapeutics, 9:10, 2081-2094, DOI: 10.4161/hv.25473
To link to this article: http://dx.doi.org/10.4161/hv.25473
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RESEARCH PAPER
www.landesbioscience.com Human Vaccines & Immunotherapeutics 2081
Human Vaccines & Immunotherapeutics 9:10, 2081–2094; October 2013; © 2013 Landes Bioscience
RESEARCH PAPER
*Correspondence to: Barbara K Felber, Email: felberb@mail.nih.gov; George N Pavlakis, Email: pavlakig@mail.nih.gov
Submitted: 05/20/2013; Accepted: 06/20/2013
http://dx.doi.org/10.4161/hv.25473
Introduction
DNA is a compelling vaccine vehicle because of its simplicity,
scalability, and lack of immunity against the vector. Different
DNA delivery methods are being tested, including intramuscular
DNA delivery by in vivo electroporation (IM/EP) (reviewed
in refs. 1–3); Dermavir,4 liposome delivery with Vaxfectin®,5
biojector or intradermal EP (ID/EP)6,7 or gene gun.8
The development of intramuscular DNA injection followed
by in vivo electroporation9-14 brought a significant change in the
efficiency of DNA delivery, especially to higher mammals like
macaques and humans. In the field of HIV/SIV vaccine, several
groups showed that IM/EP delivery led to increased immune
responses (several fold over those induced by conventional needle
and syringe).15- 18 The magnitude of the DNA induced immune
responses could be further augmented by the inclusion of IL-12
A panel of SIVmac251 transmitted Env sequences were tested for expression, function and immunogenicity in mice and
macaques. The immunogenicity of a DNA vaccine cocktail expressing SIVmac239 and three transmitted SIVmac251 Env
sequences was evaluated upon intradermal or intramuscular injection followed by in vivo electroporation in macaques
using sequential vaccination of gp160, gp120 and gp140 expressing DNAs. Both intra dermal and intramuscular vaccination
regimens using the gp160 expression plasmids induced robust humoral immune responses, which further improved
using the gp120 expressing DNAs. The responses showed durability of binding and neutralizing antibody titers and high
avidity for >1 y. The intradermal DNA delivery regimen induced higher cross-reactive responses able to neutralize the
heterologous tier 1B-like SIVsmE660_CG7V. Analysis of cellular immune responses showed induction of Env-specic
memory responses and cytotoxic granzyme B+ T cells in both vaccine groups, although the magnitude of the responses
were ~10x higher in the intramuscular/electroporation group. The cellular responses induced by both regimens were
long lasting and could be detected ~1 y after the last vaccination. These data show that both DNA delivery methods are
able to induce robust and durable immune responses in macaques.
Comparison of intradermal and intramuscular
delivery followed by in vivo electroporation
of SIV Env DNA in macaques
Viraj Kulkarni1, Margherita Rosati2, Jenifer Bear1, Guy R Pilkington1, Rashmi Jalah1, Cristina Bergamaschi1, Ashish K Singh2,
Candido Alicea1, Bhabadeb Chowdhury2, Gen-Mu Zhang1,2, Eun-Young Kim3, Steven M Wolinsky3, Wensheng Huang4,
Yongjun Guan4, Celia LaBranche5, David C Monteori5, Kate E Broderick6, Niranjan Y Sardesai6, Antonio Valentin2,
Barbara K Felber1,*, and George N Pavlakis2,*
1Human Retrovi rus Pathogenesis Sec tion; Vaccine Branch; Center fo r Cancer Research; Nationa l Cancer Institute; Freder ick, MD USA; 2Human Retrov irus Section; Vaccine
Branch; Center f or Cancer Research; Natio nal Cancer Institute; Frede rick, MD USA; 3Divisio n of Infectious Diseas es; The Feinberg Schoo l of Medicine; Northwe stern University;
Chicago, IL USA ; 4Institute of Human Virol ogy; Departmen t of Microbiology and I mmunology; Univers ity of Maryland Sc hool of Medicine; Balt imore, MD USA; 5Depart ment of
Surgery ; Laboratory for AI DS Vaccine Research and Devel opment; Duke Universi ty Medical Center; D urham, NC USA; 6Inovio Pharm aceuticals, Inc.; Blu e Bell, PA USA
Keywords: intradermal, intramuscular, in vivo electroporation, DNA vaccine, HIV, SIVsmE660, SIVmac239, SIVmac251,
transmitted Env, binding antibody, neutralizing antibody, avidity, transitional memory, effector memory
Abbreviations: EP, electroporation; ID, intradermal; IM, intramuscular; GzmB, GranzymeB; TM, transitional memory;
EM, effector memory
DNA as adjuvant in mice and macaques.18-25 Importantly, in
macaques, the combination of optimized DNA delivery by IM/
EP and the inclusion of IL-12 DNA led to robust cellular and
humoral immune responses,15, 18, 2 4 -2 8 which resulted not only in
quantitative improvement,18,24,25 but also in improved quality
of the responses, e.g., increase of antigen-specific cytotoxic T
cells and greater breadth of the neutralizing antibody (NAb)
responses.24 Similarly, ID/EP delivery of DNA vaccines yielded
improved humoral immune responses and led to protection from
challenge as judged by lethality, viremia, and morbidity in two
independent macaque models testing a multivalent small pox
vaccine and a multivalent avian influenza (A/H5N1 vaccine).29, 30
DNA as the only vaccine component had been considered
poorly immunogenic in humans, until recent results dem-
onstrated that in vivo electroporation is more efficient (more
responders, and higher, longer-lasting immunity) than DNA
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2082 Human Vaccines & Immunotherapeutics Volume 9 Issue 10
35014 (M766) and mac251_9 share a characteristic V1-V2
region found in several of our infected animals. Of note, the Env
sequence 35014 was independently identified by B. Keele et al.33
(GenBank accession #JQ086004) and also named M766, and
is also referred to with this name in this report. Env mac251_15
and mac251_2 contain a different V1-V2 region. In addition, we
included the previously reported transmitted Env CR 2.RU.3R134
as a sequence with different V1V2 from a macaque infected with
a different SIVmac251 stock.
The phylogenetic tree analysis shows the relation among the
selected SIVmac251 Env sequences, which share 4.7–6.8% AA
difference with SIVmac239 (Fig. 1C). We also include two heter-
ologous Env sequences from SIVsmE660 (CG7V and CG7G)34
which differ by ~20% from the SIVmac251 sequences and are
used in neutralization assays (see Fig. 2). We generated a col-
lection of vectors producing gp160, the trimeric gp140 and the
soluble gp120 of the different Envs from expression-optimized
cDNAs, which were cloned into the mammalian expression
vector CMVkan (Table 1). The Env plasmids were tested for
expression upon transient transfection and Western immunoblot
assay (Fig. 3A). The newly identified SIVmac251 Env proteins
were expressed at levels similar to mac239 Env. Two of the Envs
(mac251_2 and mac251_9) showed poor cleavage of gp160, with
no production of gp41, although no apparent AA changes sur-
rounding the furin cleavage site (AA 525) were noted. In addition
to the gp160 Env protein, we also generated plasmids (Fig. 3B)
expressing the trimeric gp140 (AA 1 to 686) lacking the mem-
brane spanning domain (MSD) and the soluble gp120 (A A 1 to
525). Figure 3C shows that uncleaved gp140 and gp120 were
efficiently secreted and accumulated in the extracellular com-
partment. Similar data were obtained for the other Env proteins
(not shown) and the plasmids are listed in Table 1. The sequences
were further tested for function using the pseudotype infection
assay (Fig. 3D). All sequences produced functional pseudotyped
virions, except for the 2 Env with processing defects.
To test the immunogenicity of the identified SIVmac251 Env
sequences, groups of BALB/c mice were vaccinated with four
different DNAs expressing gp160 from mac239, 251_15, 251_2,
and 251_9 by the conventional needle and syringe intramuscular
injection method (Fig. 4A). All Env vectors induced robust cel-
lular immune responses with similar levels as SIVmac239 Env,
including the 2 non-functional Env (mac251_2 and mac251_9)
(Fig. 4B). Analysis of humoral immunity by ELISA showed the
induction of robust bAb titers, except for the 2 Env which showed
impaired gp160 processing (Fig. 4C). Together, these data led to
the selection of 4 newly identified Env for the immunogenicity
study in macaques (see below).
Production of SIVmac251 Env proteins from mammalian
cells. Another goal of this work has been to select representa-
tive sequences able to efficiently produce SIV Env proteins in
mammalian cells. We tested this by the generation and screen-
ing of stable overproducing HEK293 cells. Stable high producer
HEK293 cell clones using env DNAs expressing the gp120
form of SIVmac251 35014 (M766) gp120 and the gp140 form
of SIVmac251 35014_7 were generated. In addition, we also
generated cell lines producing gp140 forms of SIVmac239 and
delivery by needle/syringe also in man,31 corroborating the find-
ings of macaque studies from us and others. Another trial, using
HIV gag DNA and IM/EP DNA delivery showed that inclu-
sion of IL-12 DNA is advantageous, resulting in both increased
frequency of responders and level of Gag-specific immunity.32
Together, these studies showed that the findings obtained in the
macaque model could be well translated to humans, which is an
encouraging step in the development of a DNA-based vaccine
against AIDS.
This report is a pilot study in macaques to compare a
SIVmac251 derived DNA vaccine based on the selection of trans-
mitted Env clones with varying sequences, and delivered by two
different routes (intradermal and intramuscular) followed by
in vivo electroporation. The vaccine candidate and the delivery
routes were designed to test whether they might induce broader
immunity compared with SIVmac239. Both vaccination regi-
mens induced potent long-lasting immune responses with IM/
EP vaccination inducing higher levels of cellular responses, and
ID/EP vaccination inducing broader humoral responses.
Results
Identification and characterization of transmitted SIVmac251
Env sequences. The Env sequence from the infectious molecu-
lar clone SIVmac239 has been frequently used as antigen to test
immunogenicity and potency of SIV vaccines in macaques. We
wished to test the hypothesis that a selection of transmitted Env
clones with varying sequences might induce broader immunity
compared with SIVmac239 Env. Toward this goal, we identified
full-length Env sequences present in the plasma early during the
peak of primary viremia from macaques infected via the mucosal
route with low dose SIVmac251. To obtain a more comprehen-
sive picture of the virus variants present in the SIVmac251 stock
and the infected animals, we also interrogated by Single Genome
Amplification (SGA) and sequencing the variable regions V1&V2
region (AA 53–260 following the numbering of SIVmac239 con-
taining V1 [AA 113–168] and V2 [AA 168–211] Fig. 1A and B),
since V1 is known to encompass most of the diversity of the SIV
Env. We noted that, despite the diversity of the stock (ref. 33 and
our unpublished analysis), viruses sharing a narrow selection of
Env sequences were found to replicate in the majority of the ana-
lyzed animals. The Env sequences were closely related to those
found in the inoculum and represented a single inferred founder
virus in 7 of 9 rhesus macaques.
From the collection of sequences, we selected Env sequences
with distinct V1-V2 regions for further analysis. Comparison
of SIVmac239 to these sequences revealed numerous changes
throughout Env in addition to the highly variable V1 region
(AA 113–168) (Fig. 1A). A cartoon of SIVmac239 indicating the
location of the variable (V) and constant (C) regions is shown
in Figure 1B. In contrast to V1 and the variable V4 (AA 402–
432), comparison to mac239 Env showed that the transmitted
sequences share several changes to the same A A in V2, V3 (AA
311–344) as well as in C1-C4 and they did not show changes in
V5 (AA 459–484) and C5, whereas numerous AA changes are
found in gp41 (AA 526–879). Of these sequences, Env 35014_7,
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www.landesbioscience.com Human Vaccines & Immunotherapeutics 2083
blue and, as a representative example, the production of 35014
(M766) gp120 Env overtime is shown (Fig. 5A). A strong band
corresponding to secreted gp120 could be detected in addition
to a ~60 kDa band corresponding to albumin, introduced from
the seed culture as part of the complete culture medium that
SIVsmE660_CG7V. Selected high producer cell clones were
grown in serum-free media in a Hollow Fiber bioreactor that
allows high-density growth in defined serum-free medium.
Analysis of a 6 μl aliquots from the supernatants of daily har-
vests (20 ml) was performed on gels stained with Coomassie
Figure 1. Amino acid alignment of transmitted SIVmac251 Env sequences. (A) The identied SIVmac251 sequences and the repor ted SIVmac251
sequence CR2.RU.3R1 are compared with SIVmac239. (B) Cartoon depicts the location of the signal peptide, the variable ( V) and conserved (C) regions,
and gp41 of SIVmac239. The numbering follows SIVmac239 sequence. (C) Phylogenetic tree analysis of the SIVmac239 and the dierent SIVmac251
sequences. The tree also included 2 SIVsmE660 env CG7G and CG7V that are used in the neutralization assays.
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2084 Human Vaccines & Immunotherapeutics Volume 9 Issue 10
contained fetal calf serum. The culture medium was changed
daily and typically after 7–10 d, albumin was no longer detect-
able. High level of Env production persisted for > 15 weeks of
continuous cell growth. The estimated Env production is ~3
mg/20 ml/day using purified SIV gp120 Env protein as standard.
Supernatants collected after the 1st week of culturing the cells in
the Hollow Fiber bioreactor were enriched for Env and were used
for protein isolation using standard lectin column purification.
Similar stable daily production was obtained for the other Env
proteins (not shown). After purification, the proteins were ana-
lyzed on non-denaturing gels and stained with Coomassie blue,
which revealed high quality of the produced monomeric SIVmac
35014 (M766) gp120 as well as stable trimeric forms of SIVmac
35014_7 and SIVsmE660_CG7V gp140 Env (Fig. 5B).
Vaccination via intradermal/EP and intramuscular/EP
route induces robust humoral immune responses. We compared
the immunogenicity of a combination of SIV Env DNA in rhesus
macaques using intradermal (ID) and intramuscular (IM) DNA
delivery followed by in vivo electroporation. As vaccine, we fol-
lowed a sequential immunization scheme (outlined in Fig. 6A)
using a mixture of Env DNAs expressing gp160 (EP1–3), gp120
(EP4) or gp140 (EP5). Macaques received the same DNA vac-
cine (1 mg/animal) via intradermal/EP (n = 3) or intramuscular/
EP (n = 2). Similar levels (reciprocal endpoint titer log 4.5–5)
of SIVmac251 binding Ab (bAb) were found in the plasma
(Fig. 6B) using the ID/EP or the IM/EP delivery method. The
bAb showed similar longevity during the 11 weeks of follow-up
(EP3wk2 to EP4) with a decline of < 0.5 log. Vaccination with
DNAs expressing the secreted gp120 forms of Env increased the
bAb endpoint titers in both groups (EP4wk2) to levels slightly
(~0.5 log) higher than the levels obtained upon vaccination with
DNAs expressing the gp160 forms of Env. This is likely due
to the more efficient production of gp120 from these plasmids
compared with the gp160 vectors (see Fig. 3C). Subsequent vac-
cination with plasmids expressing the trimeric gp140 showed
similar bAb levels, likely due to the fact that maximal levels were
obtained using the gp120 DNAs mixture. Together, these data
show that both DNA delivery systems efficiently induced robust
systemic binding Ab levels in the vaccinated macaques.
We added 3 animals to the IM/EP group, which were vac-
cinated with a 2 mg dose using the gp160 forms only of the same
four Env DNAs administered separately (using a slightly differ-
ent schedule with vaccinations at 0, 4, 12 weeks). These animals
showed similar reciprocal endpoint bAb titers of ~log 5 (not
shown) and allowed us to include the data from this group (after
gp160 DNA vaccination) in this study. The responses from the 1
and 2 mg dose IM/EP groups trended to be higher (~1.5 log) com-
pared those we reported from macaques (n = 8) which received
0.5 mg gp160 env DNA administered via IM/EP.24 These data
indicated that higher humoral responses could be obtained using
the IM/EP delivery by increasing the env DNA dose, with 1 mg
reflecting saturating levels in the rhesus macaque.
Intradermal DNA delivery induced more robust broadly
neutralizing antibodies. We next tested the neutralization capa-
bility of the vaccine-induced Env antibodies (Fig. 2) induced
upon ID/EP (Fig. 2, left panel) and IM/EP (Fig. 2, middle
Figure 2. Induction of neutralizing antibody in macaques immunized
via the intradermal (left panel) and the intramuscular (middle and
right panels) route followed by EP. Neutralizing antibody titers were
determined against SIVmac251-TCLA measured in M7-Luc cells (A) and
against pseudotyped viruses containing the following Env: SIVmac251
35014 (M766) (B) and 35014_7 (C), the heterologous SIVsmE660 Env
CG7G (D) and CG7V (E) were measured using the TZM-bl assay. The NAb
measurements of a group of macaques which received 3 vaccinations
of the same DNAs (total 2 mg) expressing the gp160 forms of Env via the
IM/EP deliver y is included (right panel). Neutralization titers shown are
the log of the reciprocal dilution of sample that reduced the signal by
50% compared with virus in the absence of sample.
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The plasma samples were also tested for their ability
to neutralize the heterologous SIVsmE660 Env (Fig. 2D
and E) CG7G and CG7V which differ by ~20% from the
SIVmac251 Envs present in the vaccine mixture (see also Fig.
1C). The Ab from both the ID/EP and the IM/EP groups
were able to neutralize the tier 1A-like SIVsmE660_CG7G
(Fig. 2D) to similar extent. Interestingly, we noted a more robust
neutralization capability to the tier 1B-like SIVsmE660_CG7V
by the antibodies induced by the ID /EP protocol, especially after
vaccination with gp120 forms of Env (EP4wk2) (Fig. 2E, left
panel) with more responders and higher titers. Of note, CG7G
and CG7V Env are 99% identical and differ by 4 AA in gp120
(1AA lies in the N-terminal portion [C1], 2 AA lie in V1, 1 AA
lies in C5) and 5 A A lie in gp41. Despite this high homology
in gp120, we noted a distinct neutralization pattern by these 2
Env in the pseudotype assay. This observation is reminiscent
to the finding using the different highly homologous gp120
proteins from SIVmac251, which we described above. Thus, it is
interesting to note that the gp120 proteins differing by very few
AA (3 A A in the SIVmac251 Envs or 4 AA in the E660 Envs)
can have such a profound distinct characteristics in the NAb
assay. This finding is agreement with a previous observations
of a single AA change in V2 of HIV Env35 and of truncation of
SIV g p4136 that also altered neutralizability.
To understand whether the inclusion of several Env in the
DNA vaccine cocktail induced Ab with broader crossreactivity,
we compared the results shown in Figure 2 to our previously
reported data of macaques vaccinated with mac239 gp160
DNA.24 Using the same panel of Env, we did not find broader
crossreactivity in macaques vaccinated with a mixture of different
SIVmac251 Env vs. only SIVmac239 Env. More importantly
however, we noted a trend that the ID/EP delivery is more potent,
inducing more consistent and higher tier NAb to the difficult to
neutralize E660_CG7V. Thus, the delivery method to the skin
panel). We also added the results from the group that received
IM/EP vaccination with the 2 mg DNA dose (Fig. 2, right panel)
which shows similar data as the 1 mg group (Fig. 2, middle panel,
compare gp160 DNA vaccination only). The neutralization capa-
bility was measured against a panel of SIV Env variants including
the homologous T cell-adapted (TCLA) SIVmac251 (Fig. 2A),
two transmitted SIVmac251 Env 35014 (M766) (Fig. 2B),
35014_7 (Fig. 2C) as well as mac251_15 (not shown) and CR2.
RU.3R1 (not shown). Plasma samples collected 2 weeks after the
gp160 (EP3wk2), gp120 (EP4wk2) and gp140 (EP5wk2) DNA
vaccinations were analyzed. Overall, similar NAb titers were
detected using ID/EP (left panel) and the IM/EP vaccination
regimens (Fig. 2A–C) upon vaccination with different forms of
Env (gp160, gp120, gp140).
We noted a distinct difference in neutralization of the trans-
mitted Env 35014_7 (Fig. 2C) compared with Env_35014
(M766 ) (Fig. 2B), with the latter being more easy to neutralize
(see also Tabl e 1) as shown by the higher NAb titers measured in
both vaccine groups. We noted this difference also upon analysis
of macaques vaccinated with SIVmac239 Env only (unpublished
observation). Thus, inclusion of 35014_7 Env in the vaccine mix-
ture did not improve the ability to neutralize 35014_7 Env pseu-
dotyped virions. It is noteworthy that the mature gp120 forms
of 35014_7 and M766 differ by only 3 AA (1 AA lies in the
N-terminal portion [C1], 2 AA lie in V4, while the remaining
8 AA changes lie in gp41; Fig. 1A). Interestingly, these 3 AA
changes in gp120 generated an Env protein with profoundly dif-
ferent behavior in the TZM-bl neutralization assay. We tested
the ability of another 2 Env (mac251_15 used in all mixtures
and CR2.RU.3R1, included in gp120 and gp140 mixtures) to
be neutralized. None of the plasma samples from the vaccinated
macaques showed any neutralizing activity against these Envs,
which like SIVmac239, appear to constitute a group of Env that
are difficult to neutralize (Table 1).
Table1. SIV Env and functional characteristics
SIV Plasmid
name
GenBank
accession #
Optimized env DNA expres-
sion plasmids (plasmid code) gp120/gp41
production from
gp160 DNA
Function in
pseudotype
assay
Ability of Env to
be neutralized
(TZM-bl assay)
Evaluation of
neutralizability of
Env
(TZM-bl assay)
gp160 gp140 gp120
mac 251
mac239 M33262 99S 237S 17 3S yes yes no difficult to neutralize
35014
(M76634)
KF003433
(JQ08600434)221S 241S 246S yes yes yes tier 1A-like
35014 _ 7 KF003434 220S 234S 230S yes yes yes tier 1B-like
251_9 K F003437 219S poor gp41
cleavage no
251_15 KF003436 217S 240S 229S yes yes no difficult to neutralize
251_ 2 KF0 03435 218 S poor gp41
cleavage no
CR2.
RU.3R1 FJ57 8 0 4 434 2 31S 242S 223S yes ND no difficult to neutralize
smE660 CG7G FJ57 838 134 251S 254S yes ND yes tier 1A-like
CG7V FJ 57 8 428 34 252S 255S yes ND Yes (ID/EP group) tier 1B-like
ND, not determined.
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2086 Human Vaccines & Immunotherapeutics Volume 9 Issue 10
appeared to have an important feature to
induce antibodies with better breadth.
Longevity of humoral immune
responses. To address the persistence of the
humoral immune responses, the animals
were monitored for 14–20 mo after the
last vaccination (Fig. 7). The bAb titers to
SIVmac239 showed an initial decline during
the first ~2 mo after the last vaccination,
which was followed by similar persistence
in the intradermal/EP and intramuscular/
EP groups (Fig. 7A). We also monitored
the avidity of the SIVmac239 bAb (Table
2). The avidity index of sham DNA
injected animals or pre-samples were below
detection limit (assigned to 0.1%). The
mac239 bAb showed an avidity index with
a range of 15–47% at 2 weeks after the last
vaccination, and these values did not change
significantly overtime, indicating that the
quality of the vaccine-induced antibody
responses was maintained over time. In
addition, we also found persistence of
NAbs to SIVmac_35014 (M766) (Fig. 7B)
and the heterologous SIVsmE660_CG7G
(Fig. 7C) using both of the vaccination
methods. Together, long-term follow-up
showed that both ID/EP and the IM/EP
DNA delivery methods induced Env-specific
antibodies with similar potent durability.
Cellular immune responses induced
upon intradermal and intramuscular
delivery of Env DNA plasmids. The
induction of Env-specific cellular immune
responses was monitored upon stimulation
of PBMC with SIVmac239 Env peptide
pool (15-mer overlapping by 11 AA)
and measured by intracellular cytokine
staining and flow cytometry. The two
Figure 3. Characterization of dierent SIV
mac251 Env proteins. (A) Western immunoblot
analysis of the expression of SIVmac239 Env and
the dierent SIVmac251 Envs upon transient
transfection of HEK293 cells. (B) Cartoon depict-
ing gp160, gp140 and gp120 Env. (C) Western im-
munoblot analysis the gp160, gp140 and gp120
Env of SIVmac251_35014 (M766) upon transient
transfection of HEK293 cells. Cell-associated and
extra-cellular fractions are shown. (D) Pseu-
dotyped recombinant HIV-luc virus reporter
(NL4–3.LucR−E−) viruses were generated using
the SIVmac239 Env and the indicated SIVmac251
Env. Infectivity was tested in modied CEM-X174
cells that contain the HIV-LTR promoter linked
to GFP stably integrated. GFP expression, which
serves as read-out for infection, was measured
by FACS analysis.
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www.landesbioscience.com Human Vaccines & Immunotherapeutics 2087
IM/EP vaccinated macaques.24 Thus, the delivery route (e.g.,
ID/EP) or inclusion of IL-12 cytokine DNA contributed sig-
nificantly to the induction of humoral responses with improved
breadth.
In contrast to the humoral responses, we found a significant
difference in the quantity of the cellular immune responses, with
the ID/EP delivery inducing lower responses. Despite this, both
vaccine regimens induced Env-specific transitional and effector
memory T cells and the cells contained the cytotoxic marker
granzyme B, a desired vaccine-induced T-cell response. Thus,
the cellular responses although different in magnitude, showed
the necessary potency as T-cell vaccine. As a matter of fact, the
potency of the vaccine reponses induced upon IM/EP DNA
delivery has been tested upon subsequent challenge of macaques
with pathogenic SIV25,26,2 8,48,49 and with SHIV50 and showed sig-
nificant reduction of viremia.
Different EP delivery methods developed by Inovio have
been reported which showed improved immunity including
HIV/SIV DNAs in macaques using intradermal low-current
EP,51 or using intradermal/subcutaneous EP,52 as well as devices
that deliver DNA simultaneously to both skin and muscle of
mice and guinea pigs53 and intradermal route in mice.54 Here,
we directly compared the immune responses induced upon IM
IM/EP immunized animals showed robust responses with
a frequency of 0.1–0.6% of Env-specific IFN-γ+ T cells
(Fig. 8A). Similar responses were measured in the 3 macaques
that were vaccinated only with gp160 DNAs (Fig. 8D). The
cellular responses induced against SIV Env were mediated
typically by both CD4+ and CD8+ T cells and, importantly,
these responses were ~10-fold higher in the IM/EP vaccinated
macaques (Fig. 8A and D) compared with the ID/EP
immunized animals (Fig. 8F). Detailed analysis of the
Env-specific responses of both groups of macaques showed
induction of transitional (CD28+CD95+CCR7−) and effector
(CD28-CD95+CCR7−) memory T cells (Fig. 8B, E and G)
containing the cytotoxic marker granzyme B (GzmB) (Fig. 8C
and H). Importantly, the T cell responses were also long-lasting
and could be detected for >1 y after the last vaccination. Together
these data demonstrate that both vaccination protocols induced
durable cytotoxic cellular immune responses although the IM/
EP delivery route induced higher levels of Env-specific T cell
responses.
Discussion
We have a long-standing interest in optimizing DNA-based
vaccine approaches, and thus, we have taken sequential steps
to improve efficacy of this vaccine vehicle including RNA or
codon optimization of the DNA expressing the immunogen,
optimization of expression vectors, antigen design, choice of
DNA adjuvants such as IL-12 and IL-15, and DNA delivery
methods.15,20,24-28,37-46 Our recent data on the combination of SIV
DNA and inactivated SIV particles, used as protein source in
the same vaccine regimen, revealed great potency to augment
humoral responses.28 To improve Env immunogenicity, we
describe herein the testing of a panel of transmitted SIV env
DNA and establishment of HEK293 cell lines stably producing
trimeric gp140 and soluble gp120 Env proteins, which will allow
us to further expand on this study by using purified Env proteins
instead of virus particles.
In this report, we tested different DNA-only delivery meth-
ods and compared the immunogenicity of the same cocktail of
expression-optimized SIV env DNAs using 2 different routes of
vaccine delivery in macaques: intradermal/EP and intramuscu-
lar/EP. We did not find significant differences in magnitude of
the humoral responses using these two DNA delivery methods.
Importantly, both vaccine regimens induced potent long-lasting
humoral responses, detectable for > 1 y. The IM/EP DNA deliv-
ery method was previously reported to induce long-lasting sys-
temic responses to HIV/SIV immunogens in macaques.24, 26 -28, 47
To test the breadth of the responses, we used a panel of SIVmac
and SIVsm Env sequences in pseudotype neutralization assays.
The ability to neutralize the tier 1B-like SIVsmE660_CG7V
Env served as a measure of neutralization strength. Interestingly,
we found more potent NAb to the heterologous, more difficult to
neutralize SIVsm E660_CG7V in macaques vaccinated via the
ID/EP route. These data are reminiscent of our previous report
where we found the presence of NAb to E660_CG7V only upon
inclusion of IL-12 DNA as vaccine adjuvant in the SIVmac239
Figure 4. Immunogenicity of Env DNAs in mice. (A) Outline of vaccina-
tion study of BALB/c mice. Mice (n = 5/group) were vaccinated via the
IM route (needle and syringe) at 0 and 4 weeks with SIV Env DNA for-
mulated in PBS, and were sacriced 2 week s after the 2nd vaccination.
Spleen and blood were analyzed. (B) Splenocytes were stimulated with
an Env peptide pool (15-mer, 11 AA overlap) and the IFN-γ producing T
cells were measured by ELISPOT assay. (C) The Env-specic binding an-
tibodies titers were measured by ELISA using serial dilutions of pooled
plasma samples.
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2088 Human Vaccines & Immunotherapeutics Volume 9 Issue 10
Materials and Methods
Identification of transmitted SIVmac251 Env
sequences. The SIVmac251 stocks were generated
by propagating the original SIVmac251 isolate
in monkey peripheral blood mononuclear cells.56
Two stocks were used to infect rhesus macaques
(stock 2000: animals 35003, 35014, 33390; and
stock 06: animals M761, M766, M752, M757,
M764, M741) using low dose inocula. Plasma
samples collected at the peak of infection were
used for RNA extraction by PureLink viral
RNA isolation kit (Invitrogen). The full-length
env (2.86 kb) or the V1-V2 region (586 bp)
of the env gene were reverse transcribed with
Thermoscript (Invitrogen) using reverse outer
primer and amplified by nested PCR using High
Fidelity Platinum Taq Polymerase (Invitrogen)
and performed single genome sequencing assay
(SGA) as described.57 Approximately 10,000
copies of genomic RNA were amplified for each
sample at SGA. Thermal cycling conditions for
full-length env genes were as follows: an initial 1
min denaturation at 94 °C, followed by 35 cycles
of 94 °C for 15 sec, 55 °C for 30 sec, and 68 °C for
3 min; and 68 °C for 20 min extension. Thermal
cycling conditions for env V1-V2 were as follows:
an initial 1 min denaturation at 94 °C, followed
by 35 cycles of 94 °C for 30 sec, 54 °C for 30
sec, and 68 °C for 80 sec; and 68 °C for 3 min
extension. The primer position (corresponding
to SIVmac251 GenBank Accession
numberM19499) and sequence for full-length
env genes were: external sense primer: SIV251_
REV_outF 5'-CACATGCTAT TGTAAAAAGT GTTGCTA-3'
(6433→6459) and external antisense primer: SIV251 NEF_outR,
5'-TGTAATAAAT CCCTTCCAGT CC-3' (9440←9461),
internal sense primer: SIV251_REVinF, 5'-ACCATTGCCA
GTTTTGTTTT CTTA-3' (6459→6482), and internal
antisense primer: SIV251NEF_innerR, 5'-ATACCCCTAC
CA AGTCATCA TCT-3' (9337←9359). The PCR products were
sequenced with six sequencing primers as follows: SIV251_6532F
5'-TCCGAAAAAG GCTAAGGCTA ATAC-3', SIV251_6928F
5' TGAGACAGAT AGATGGGGAT TGACA-3', SIV251_8079R
5'-TTTATAATCT CCCA ACTCCA ATCG-3', SIV251_7888F
5'-AGACAAATAA TCAACACTTG GCATAAA-3',
SIV251_8404F 5'-AATGCTTGGG GATGTGCGTT TAG-
3', and SIV251_9359R 5'-ATACCCCTAC CA AGTCATCA
TCT-3'. The primer position and sequence for V1-V2 env were:
external sense primer: SIV251_V1–2_outF 5'-CTGCTTATCG
CCATCTTGCT TTTA-3' (6601→6624) and external
antisense primer: SIV251 V1–2_outR, 5'-CGTAATTCCT
TCTATGCCGT TCC-3' (7851←7873), internal sense
primer: T7-SIV251V1–2 inF, 5'-TAATACG ACT C ACTATA-
GGGGA ACA AC TCAG-3' (T7 + 6733→6755), and internal
antisense primer: M13R-SIV251V1–2inR, 5'-CAGGAAACAG
vs. ID delivery followed by EP of the same DNA mixtures into
macaques. Our data suggest that a combination of ID and IM
delivery is most effective in eliciting high humoral and cellular
responses. Indeed, such a delivery device has been developed and
shown superior results than either of the routes53 and should be
tested in the macaque model.
Mucosal dissemination is critical for an HIV/SIV vaccine,
since this is the route of natural infection. Although it has not
been tested yet for the intradermal/EP delivery method, the use of
intramuscular/EP elicited robust, long-lasting systemic immune
responses that disseminate into mucosal sites in vaccinated
macaques.27, 2 8 It was further shown that IM/EP did not increase
the activation state of SIV-specific CD4+ T cells in the gastro-
intestinal mucosa, thus no increase in target cells for HIV
infection was induced by this delivery method.55 Overall, our
study demonstrates that both routes of immunization followed
by EP induce robust and durable immune responses in macaques.
Thus, a combination of immune responses induced via ID and
IM delivery routes may be able to further improve immunity,
and to reduce virus acquisition. Further studies will be needed to
evaluate the efficacy of these two delivery systems upon challenge
with pathogenic SIV.
Figure 5. Production and purication of SIV Env protein from stable HEK293 cells. (A)
Samples of culture media from Fibercell grown HEK293 cells expressing SIV env protein
were assessed for purity on Coomassie blue stained SDS PAGE gels (10% TGX gel, Bio-Rad).
(B) Puried SIVsmE660_CG7V gp140, SIVmac gp140 (35014_7) and gp120 (35014/M766)
proteins were assessed for purity and quality on non-denaturing gel (10% TGX gel) and
visualized by Coomassie blue staining.
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www.landesbioscience.com Human Vaccines & Immunotherapeutics 2089
CTAT-GACC ACCACC T TAGA AC-3' (7349←7365+M13R ).
Internal sense and antisense primers were flanking with T7 and
M13R sequence at the 5'end of the primers, respectively, so that
the sequencing was performed with T7 and M13R primers.
PCR products were amplified for sequencing using Applied
Biosystem’s BigDye Terminator v1.1 Ready Reaction mix
(Applied Biosystems). Sequencing products were detected using
Applied Biosystems 3730xl DNA analyzer. Sequencing products
were detected using Applied Biosystems 3730xl DNA analyzer.
Sequence alignments and phylogenetic analysis were performed
with the CUSTALW method using the Lasergene software
package (DNASTAR).
DNA vectors and in vitro expression. Selected Env sequences
were RNA-optimized and cloned into the CMVkan vector
comprising the CMV promoter, the bovine growth hormone
polyadenyation signal and the kanamycin gene in the plasmid
backbone.37 All env sequences a re submitted to GenBank a nd listed
in Table 1. GenBank submission for 35014 and 251_2 represent
optimize d consensus sequences. A ll plasmid DNAs were produced
in E. coli DH10B (Invitrogen) grown at 32 °C and endotoxin-
free DNA was purified (Qiagen). The DNAs were resuspended
in sterile water (Hospira, Inc.). Env production was measured in
supernatant and cell-associated fractions (1/200 of fraction) from
transfected HEK293 cells using Western immunoblot (1:5000
dilution of pooled sera from SIVmac251 infected macaques,
followed by 1:10,000 dilution of HRP-labeled anti-monkey
antiserum (Fitzgerald Industries International Inc.). The bands
were visualized using the enhanced chemiluminescence (ECL)
plus western blotting detection system (GE HealthCare).
Env protein production from stable HEK293 cells. Linearized
Env expression plasmids, purified using Nucleotide Removal Kit
Figure 7. Persistence of humoral responses in macaques vaccinated
via the intradermal and the intramuscular route. The animals were
monitored for 14–20 mo after the last vaccination. Humoral immune
responses were measured in the macaques that received DNA via the
intradermal (n = 3) and the intramuscular route (n = 5, with 3 macaques
receiving 1 mg DNA and 2 macaques receiving 2 mg DNA). (A) Endpoint
binding Ab titers to SIVmac239, (B) NAb titers to SIVmac_35014 (M766)
and (C) NAb to the heterologous SIVsmE660_CG7G (tier 1A-like) are
shown as log of reciprocal dilution of plasma that produces a 50%
reduction in signal compared with wells receiving no sample.
Figure 6. Analysis of humoral immune responses upon vaccination of
macaques with env DNA. (A) Outline of vaccination study of macaques
indicating the sequential immunization with DNA mixtures expressing
a cocktail of multivalent Env gp160 (EP1-EP3), gp120 (EP4) and gp140
(EP5). (B) Endpoint SIVmac251 Env binding Ab titers are shown for the
animals vaccinated ID/EP and the IM/EP DNA delivery regimen using
the same DNA mix tures.
Table2. Avidity of SIVmac239 Env binding antibody over time
Avidity measurements after last Env DNA vaccination
(in month)
Animal 0.5 1 3 6 10 14 20
ID/EP
M694 23.3 30.0 49.6
M710 30.3 31. 8 21. 3
M084 21.1 16. 8 ND
IM/EP
M092 21.1 32 .7 60.7
M095 47. 4 31.2 62.6
M705 25.9 20.4 20.3 30.8 35.7 32.5
M781 15. 3 14 . 2 12.8 26.2 29.1 28.8
M782 37. 0 39.9 42 .2 43.7 52.3 41.1
ND, not determined.
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2090 Human Vaccines & Immunotherapeutics Volume 9 Issue 10
Figure 8. For gure legend, see page 2091.
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www.landesbioscience.com Human Vaccines & Immunotherapeutics 2091
shallow ID DNA delivery using a novel 4 × 4 minimally invasive
needle array.51,61 This ID device differs from other invasive EP
devices in that the electrodes are minimally invasive (make con-
tact with the skin surface but do not penetrate) and also operate
at significantly lower voltages (15–25 V). The electrode configu-
ration is designed to achieve threshold electric fields over a wider
area at lower applied voltages. Specifically, the EP applicator con-
sists of gold-plated stainless steel needle electrodes with trocar
grinds at 1.5 mm spacing in a 4 × 4 array pattern. The surface
device was built with attachment cord for linkage to the ELGEN
1000 pulse generator (Inovio Pharmaceuticals). The following
Env DNA were used: SIVmac239 and SIVmac 251_15 DNAs
as gp160, gp140, gp120; SIVmac 35014 (M766) was included in
the gp160 and gp140 mixtures; SIVmac 35014_7 was included
in gp160 and gp120 mixtures; and SIVmac CR2.RU.3R1 was
included in the gp120 and gp140 mixtures. For ID vaccination,
macaques M084, M694 and M710 received a mixture of 1 mg
DNA (0.25 mg of each plasmid) resuspended in total of 0.25 ml
of sterile water, injected as 5 × 50 μl bleps on 3 places (2 × 50 μl
closely spaced [2× ]; 1 × 50 μl injected separately) on the back of
the animal, followed by EP. For the IM vaccination (macaques
M092 and M095) 1 mg of the env DNA mixture was resuspended
in 0.6 ml water and injected at 2 sites (0.3 ml each, left and right
internal thighs) followed by EP. The IM/EP was performed
using the ELGEN Twin Injector (Inovio Pharmaceuticals) which
consists of an outer housing with an inner wagon carrying two
standard 1 ml syringes with 21 g needles, 4 mm apart.62 A gear-
ing system presses the piston of the syringes when the wagon
slides forward in order to inject DNA during the insertion. The
needles subsequently serve as electrodes. The needles penetrate
the full depth of the targeted muscle, distributing the DNA in
a columnbular fashion throughout the muscle and co-locate the
electrical field with the delivered plasmid. The device operates at
an applied voltage of 60 and pulses twice, both of 60 ms dura-
tion. The ELGEN Twin Injector is directly linked to the ELGEN
1000 pulse generator (Inovio Pharmaceuticals). Macaques
(M705, M781, M782) received 3 vaccinations with 0.5 mg
DNA each of the same 4 gp160 DNAs resuspended separately in
0.3 ml each and injected at 4 different sites (arms and thighs).
Blood samples were collected at each vaccination and at various
time points throughout the course of vaccination to measure cel-
lular and humoral immune responses.
Humoral immune responses. The end-point binding
antibody titers to SIVmac251 gp120 were measured by ELISA
(Advanced BioScience Laboratory, Inc.) and titers greater than
the mean +3SD of normal plasma at OD450 were considered
positive. Antibody titers to SIVmac239 and antibody avidity
upon treatment with 1.5 M sodium thiocyanate (NaSCN;
Sigma-Aldrich) were measured as described.63,6 4 Neutralizing
(Qiagen), were used to transfected HEK293 cells together with
pRSVhygro as selection marker. Stable high producer HEK293
cell clones were generated using Env plasmids expressing
SIVmac251_35014 (M766) gp120 (plasmid 246S), 35014_7
gp140 (plasmid 234S) and SIVsmE660_CG7V gp120 (plasmid
261S). The following clones were among the highest producers
of 35014 (M766) gp120 (clone 36), 35014_7 gp140 (clone 47),
SIVsmE660_CG7V gp120 (clone X). Env proteins were produced
from selected stable clones grown in serum-free media in a Hollow
Fiber bioreactor that allows high-density growth without any
animal components (Fibercell®) as described.58 Supernatants (6 μl
of daily harvests of 20 ml) were monitored on gels stained with
Coomassie blue. Serial dilutions of the samples were analyzed,
together with purified SIV gp120 protein (a gift from E. Chertova)
to calibrate, after staining of the gels with Emerald Green.
Virus infectivity assay. Pseudovirus preparations were gener-
ated upon cotransfection of HEK293 cells with 5 μg pNL4–3.
LucR-E- (NIH AIDS Research and Reference reagent program,
Division of AIDS, NIAID;59) and 50 ng of the different SIV Env
expressing plasmids using the calcium phosphate method. Eight
hours following transfection, the culture medium was replaced
with fresh complete DMEM containing 10% FBS. Twenty-
four hours later, the cell supernatants were collected and 1 ml of
cell-free supernatant was added to 3 to 5 million of CEMx174-
GFP, an indicator cell line that contains the HIV-LTR promoter
linked to GFP stably integrated.60 After a 2-h incubation, 1 ml of
medium was added and the cells were incubated for 48 h. GFP
expression in infected cells was monitored by flow cytometry.
DNA vaccination of mice. Female BALB/c (6 to 8 weeks
old) were obtained from Charles River Laboratories, Inc. and
were housed at the National Cancer Institute in a temperature-
controlled, light-cycled facility. The mice were immunized by
intramuscular injection (week 0 and 4) with 100 μg plasmids
expressing the indicated Env gp160 plasmids. Two weeks after
the last vaccination, spleens and plasma were collected to mea-
sure cellular and humoral immune responses as described below.
DNA vaccination of macaques. This study was performed in
accordance with the Guide for the Care and Use of Laboratory
Animals of the National Institutes of Health. Rhesus macaques
were housed and handled in accordance with the standards of the
Association for the Assessment and Accreditation of Laboratory
Animal Care International at the Advanced BioScience
Laboratories Inc., and were approved by the Institutional Animal
Care and Use Committee (OLAW assurance number A3467-01
and USDA Certificate number 51-R-0059). Macaques received
a mixture of 4 env plasmids DNA (1 mg) via intradermal route
(ID) or via intramuscular route (IM) route followed by in vivo
electroporation (Inovio Pharmaceuticals, Inc.). The ID EP was
performed using the surface EP device (SEP) which allows for
Figure 8 (See opposite page). Comparison of magnitude and longevity of cellular responses in macaques vaccinated via the intramuscular (A–C,
D–E) and the intradermal (F–H) route. Cellular immune responses were measured in PBMC upon stimulation with a SIVmac239-specic peptide pool
(15-mer overlapping by 11 AA) at 2 weeks after vaccination with gp160 (EP3wk2), gp120 (EP4wk2) and gp140 (EP5wk2) (A–C, F–H). Animals in D–E
were monitored af ter the 3rd gp160 DNA vaccinations. All animals were monitored for ~1 y and the cellular responses were measured at EP5wk57 and
EP3wk43, respectively. The responses are shown as % Env-specic IFN-γ+ T cells among CD4+ and CD8+ (A, D, F); transitional (TM; CD28+CD95+CCR7−)
and eector memory (EM; CD28−CD95+CCR7−) (B, E, G) T cells. The frequency of the Env-specic IFN-γ+ granzyme B+ T cells is shown (C, H). The ma-
caques of panels B and E were not analyzed for this function. Asterisk indicates that the sample scored negative for the assay.
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2092 Human Vaccines & Immunotherapeutics Volume 9 Issue 10
Disclosure of Potential Conflicts of Interest
GNP and BKF are inventors on US Government-owned patents
and patent applications related to DNA vaccines and gene expres-
sion optimization that have been licensed to several companies.
There are no further patents, products in development or mar-
keted products to declare. KEB and NS are employed by Inovio
Pharmaceuticals, Inc. as such receive salary, and bonuses and
stock options as compensation.
BKF, GNP, AV: designed, coordinated the study, analyzed the
data, and wrote the paper. VK, MR, JB, GRP, RJ, CB, AKS,
CA, BC, G-MZ: performed experiments and analyzed the data.
E-YK, SMW: performed Env identification study. WH, YG: per-
formed binding Ab and avidity assays. CL, DCM: performed
and analyzed NAb assays. KEB, NYS: contributed electropora-
tion delivery methods and electroporation devices to access IM
and ID tissues for immunization.
Acknowledgments
We are grateful to D Weiss, J Treece, I Kalisz, R Pal and staff
at Advanced BioScience Laboratories, Inc., Rockville, for their
expert help. We thank A von Gegerfelt, B Keele, JJS Cadwell, and
E Chertova for discussions, R Desrosiers, R Pal, and NIAID for
SIVmac251 virus and macaque plasma samples, and T Jones for
editorial a ssistance. pNL4 -3.LucR-E- wa s provided by NI H AIDS
Research and Reference reagent program, Division of AIDS,
NIAID. This work was supported by the Intramural Research
Program of the National Cancer Institute, National Institutes of
Health (NCI/NIH) and by NIH HHSN 27201100016C.
antibody titers were determined using the M7-luc assay for the
TCLA-SIVmac251/H9 and the TZM-bl assay for the transmitted
SIVmac251 35014_7 and 35014 (M766), and the heterologous
SI Vsm E 66 0 _ C G7G a nd SIVsmE 6 6 0 _ C G7 V.65
Cellular immune responses. Mouse splenocytes were incu-
bated with a pool of overlapping 15-mer peptides (1 μg/ml final
concentration) spanning the entire gp160 region of SIVmac239
(Infinity Inc. Biotech Research and Resource) and ELIspot assays
were performed as described.38 Env-specific spots were calculated
by subtracting the cut-off value and adjusted to the number of
spot-forming cells per million splenocytes.
The frequency of antigen-specific T cells from vaccinated
macaques was determined upon stimulation with the Env pep-
tide pool followed by intracellular cytokine staining and multi-
parametric flow cytometry as described.28 The antibody panel
for surface staining consisted of: CD3-APCCy7, CD4-AmCyan,
CD95-FITC (BD PharMingen), CD8-AF-405 (Invitrogen),
CD28-PerCP Cy5.5 (BioLegend), CCR7-APC (R&D Systems
Inc.) and CD45RA-AF700 (ABD Serotec). The intracellular
staining was performed using IFN-γ-PE Cy7 (BD PharMingen)
and granzyme B-PE (Invitrogen) antibodies. PBMC were cul-
tured in medium without peptide pools as negative control or
stimulated with phorbol myristate acetate (PMA) and calcium
ionophore (Sigma) as positive control. Positive samples had a fre-
quency of IFN-γ positivity in the peptide-stimulated sample that
was > 2 fold higher than the frequency obtained in unstimulated
(minus peptide) medium only control sample. At least 105 T cells
from each sample were acquired on an LSR II flow cytometer
(BD Biosciences) and the data were analyzed using FlowJo soft-
ware (Tree Star, Inc.).
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