DermaVir: A Novel Topical Vaccine for HIV/AIDS

Julianna Lisziewicz,k Jeffrey Trocio, Lucia Whitman, Georg Varga, Jianqing Xu, Nyasha Bakare, Patrick
Erbacher,w Cecil Fox,z Ruth Woodward,y Phil Markham,y Suresh Arya,z Jean-Paul Behr,# and Franco Lori
Research Institute for Genetic and Human Therapy (RIGHT), Washington, District of Columbia, USA; kGenetic Immunity, LLC Washington, District of Columbia,
USA; wPolyPlus-Transfection, Illkirch, France; zMolecular Histology Labs Inc., Gaithersburg, Maryland, USA; yAdvanced BioScience Laboratories Inc., Rockville,
Maryland, USA; zNational Institutes of Health, National Cancer Institute, Bethesda, Maryland, USA; #Laboratoire de Chimie Genetique, Faculté de Pharmacie,
Illkirch, France

Human immunodeficiency virus (HIV) vaccines have the potential to improve antiretroviral drug treatment by
inducing cytotoxic killing of HIV-infected cells. Prophylactic vaccines utilize new antigens to initiate immunity;
however, in HIV-infected individuals the load of viral antigen is not the limiting factor for the restoration of immune
responses. Here we describe a novel immunization strategy with DermaVir that improves viral antigen presentation
using dendritic cells (DC). DermaVir contains a distinctive plasmid DNA expressing all HIV proteins except integ-
rase to induce immune responses with broad specificity. The DNA is formulated to a mannosilated particle to target
antigen-presenting cells and to protect the DNA from intracellular degradation. After topical application, DermaVir-
transduced cells migrate from the skin to the draining lymph node and interdigitate as DermaVir-expressing,
antigen-presenting DC. We compared the immunogenicity of topical and ex vivo DC-based DermaVir vaccinations
in naı̈ve rhesus macaques. Both vaccinations induced simian immunodeficiency virus-specific CD4 helper and CD8
memory T cells detected by an in vivo skin test and an in vitro intracellular cytokine-based assay. Topical DermaVir
vaccination represents an improvement upon existing ex vivo DC-based immunization technologies and may pro-
vide a new therapeutic option for HIV-infected patients.
Key words: cellular immunity/dendritic cells/skin/therapy/topical vaccine
J Invest Dermatol 124:160 – 169, 2005

One strategy for a new immunotherapeutic intervention tigen presentation in order to induce functional HIV-specific
against human immunodeficiency virus (HIV) infection is to T cells that could destroy HIV-infected cells.
develop a vaccine that can reconstitute HIV-specific immu- We developed a new DNA-containing vaccine, called
nity, thereby improving the efficacy of the present antiretro- DermaVir, that presents viral antigens by dendritic cells
viral regimens. The therapeutic efficacy of such a vaccine (DC), the most potent type of antigen-presenting cell (APC)
would be mediated by HIV-specific T cells, which play a (Banchereau and Steinman, 1998), to improve antigen pres-
central role in the control of virus replication (Amara and entation during therapeutic immunization of chronically HIV-
Robinson, 2002; Lisziewicz et al, 2003). Therapeutic vac- infected individuals. We have previously described an
cines are conceptually different from preventive vaccines. ex vivo vaccination strategy that uses DermaVir-transduced
Preventive vaccines must prime the immune system with autologous monocyte-derived DC for the induction of HIV-
foreign viral antigens to induce immune responses that specific T cells (Lisziewicz et al, 2001), and others have re-
protect individuals from infection. In contrast, HIV-infected cently shown control of simian immunodeficiency virus (SIV)
individuals have already developed immune responses to by ex vivo DC-based vaccination (Lu et al, 2003). DC-based
HIV, although insufficient to fully suppress virus replication vaccines using ex vivo cell manipulation are, however, in-
in the majority of patients. In these subjects, HIV is ex- herently limited by their cumbersome nature. To improve
pressed in most organs and viral load exceeds several upon this technology, we explored APC in the skin and their
thousands of viral RNA copies per milliliter in the peripheral ability to deliver the vaccine in vivo to the secondary lymph-
blood. Consequently, the load of viral antigen is not the oid organs.
limiting factor in the induction of potent immune responses. The skin is a physical barrier between the internal milieu
Since it is unlikely that additional viral antigen would provide and the external world, the latter including its pathogens.
therapeutic benefit, we focused on the improvement of an- The epidermis contains a large number of Langerhans cells
(LC) (400–1,000 per mm2), which take up and process
epicutaneous antigens and migrate to the draining lymph
nodes. Although in transit, they begin to differentiate into
Abbreviations: APC, antigen-presenting cell; DC, dendritic cell; DC that present the processed antigens to naı̈ve T cells
DTH, delayed-type hypersensitivity; HIV, human immunodeficiency (Steinman et al, 1995, 1997). The transfer of antigens to the
virus; IFN, interferon; LC, Langerhans cells; PBS, phosphate-buff-
ered saline; SHIV, simian human immunodeficiency virus; SIV, sim- lymphoid organs is critical for the induction of immune re-
ian immunodeficiency virus sponses (Zinkernagel et al, 1997). Immune responses are

Copyright r 2004 by The Society for Investigative Dermatology, Inc.

160
124 : 1 JANUARY 2005 DERMAVIR IMMUNIZATION FOR HIV/AIDS 161

initiated in these lymphoid tissues, where antigen-loaded treatment of HIV, we wanted to use a DNA construct that
DC encounter and activate T cells (Steinman, 1991; La- mimics the expression of the wild-type virus in DC as it
nzavecchia and Sallusto, 2000). T cell activation results in occurs during primary infection (Rowland-Jones, 1999).
the generation of both effector and memory cells that play HIV-LTR-driven viral protein expression is efficient and does
distinctive roles against viral infection (von Andrian and not require codon optimization. More importantly, it results
Mackay, 2000; Masopust et al, 2001; Reinhardt et al, 2001). in processing of most viral proteins and presentation of T
Therefore, LC in the epidermis might be used as a vehicle to cell epitopes, which is a key feature of the DermaVir vac-
transfer antigens from the skin to the lymphoid organs. cine. It has been recently demonstrated that DNA vaccina-
For the treatment of HIV/AIDS, we have improved the tion induced only a few epitopes that were identical to
ex vivo DC vaccination technology with topical administra- epitopes induced by the wild-type virus (Vogel et al, 2002).
tion. DermaVir is administered to the surface of the skin with We have previously demonstrated that integrase-defective
the aim of targeting epidermal LC and transferring DNA into HIV-1 can express in DC but is not able to establish a pro-
these APC for presentation of viral antigens to T cells in ductive infection, replicate, and integrate (Lisziewicz et al,
order to induce new, functional HIV-specific T cells. The ra- 2001). To further characterize DermaVir in a relevant primate
tionale of this design is based on the following observations: model we constructed a similar integrase mutant plasmid,
(i) early treatment of primary HIV/SIV infection, where DC pSHIV(int-), encoding a mutant pathogenic simian human
are the major APC (Rowland-Jones, 1999), induced T cell- immunodeficiency virus (SHIV 89.6P) (Reimann et al, 1996;
mediated immune responses that controlled virus replica- Karlsson et al, 1997). This plasmid DNA can express
tion after treatment interruption in SIV-infected macaques both regulatory (e.g. tat, rev, nef) and structural proteins
(Lifson et al, 2000; Lori et al, 2000) and in some HIV-infected (e.g. gag, env), and is therefore expected to induce T cell-
individuals (Lisziewicz et al, 1999; Rosenberg et al, 2000); (ii) mediated immune responses with broad specificity (Fig 1).
in contrast, chronic infection, where DC are not the major
APC, is characterized by functionally impaired HIV-specific Application of DermaVir DermaVir is applied topically to
T cells (Lieberman et al, 2001) that could account for the the surface of the skin after an exfoliation procedure. Shav-
lack of T cell-mediated viral clearance and rebound of virus ing and exfoliation removes part of the stratum corneum,
replication during treatment interruption (Bonhoeffer et al, which rapidly regenerates. LC are located in the epidermis
2000; Miller et al, 2000; Deeks et al, 2001; Garcia et al, 2001; under the stratum corneum as a network of immune sen-
Lori and Lisziewicz, 2001; Ruiz et al, 2001); (iii) during es- tinels approximately 1000 cells per mm2 to protect the body
tablished infection, cell-mediated immune responses sup- against pathogens that might enter upon injury to the skin.
press SIV/HIV (Bagnarelli et al, 1994; Cao et al, 1995; Klein Disruption of the stratum corneum is required for the pen-
et al, 1995; Harrer et al, 1996; Rosenberg et al, 1997; Ogg etration of DermaVir to the epidermis and targeting of LC.
et al, 1998; Lori et al, 1999; Pitcher et al, 1999; Pontesilli The migration of LC is induced either by DermaVir applica-
et al, 1999; Schmitz et al, 1999), whereas humoral immune tion, which mimics a pathogen, or by the cytokines secreted
responses do not appear to play a major role in viral control by nearby keratinocytes upon exfoliation of the skin, or
(Wei et al, 2003). both. These ‘‘danger’’ signals are thought to trigger the LC
The design of DermaVir as a therapeutic vaccine is dis- to leave the epidermis and migrate via the lymphatic vessels
tinct from most DNA vaccines developed for the prevention to the draining lymphoid organs (Dieu-Nosjean et al, 1999).
of SIV/HIV infection in the following ways: (i) the DNA con- Histological and macroscopic evaluation of DermaVir
struct mimics viral gene expression; (ii) epitopes derived vaccination in non-human primates demonstrated that the
from the DNA antigen are presented by DC in the lymph skin exfoliation we developed for topical DermaVir admin-
nodes; (iii) the vaccine is applied topically; and (iv) the vac- istration is associated with the removal of the stratum corn-
cine induces Th-1 polarized HIV-specific T cells. eum and preservation of the epidermis (Fig 2a–d). In such
circumstances, it is unlikely that DermaVir would diffuse to
the dermis and target dermal DC; however, we do not rule
Results out this possibility. From the safety point of view, the pri-
mate study showed mild and transient erythema associated
Novel plasmid DNA in DermaVir for broad antigen pres- with skin preparation (Fig 2e–g), and lack of systemic toxic-
entation Since our goal was to develop a vaccine for the ity. The safety results in primates were supported by a study

Figure 1 Tat
The plasmid DNA of DermaVir. Map of the plasmid Rev
DNA, pSHIV(int-), used for DermaVir vaccination in rhe-
sus macaques. A mutation in the integrase gene has
molecularly inactivated the full-length proviral DNA. Gag Env Nef
Pol Int
Such a plasmid DNA is capable of effective gene ex- LTR LTR
pression upon transfection of 293T cells (data not
shown). Virus replication is completely impaired in the
absence of integration. LTR, gag, pol, and nef sequenc- A N S D L stop stop stop
es originate from SIVmac239 (no fill). Env, tat, and rev GCA AAT TCA GAT CTA TAG ATA GAT AGC TAG — — — —
— CCC ATC TAG
genes originate from HIV-1. Env and second exons of
tat and rev (light gray) are from the 89.6p isolate of HIV-
substitution 7 bp
1; others (dark gray) are from HXBc2.
deletion
162 LISZIEWICZ ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 2
Topical application of DermaVir.
DermaVir is applied to the surface of the
skin after a skin preparation procedure
causing the disruption of the stratum
corneum by exfoliation that allows the
vaccine to access the epidermis. Exfolia-
tion is performed by rubbing the skin 50
times with an exfoliation sponge (3M,
Heavy Duty). There is no hemorrhage,
and in most cases mild and transient ery-
thema is observed that is associated with
skin preparation, not administration of
DermaVir. (a–d) H&E-stained skin biop-
sies of two rhesus macaques, #942 and
947, prior to (a, c) and after (b, d) skin
preparation. The typical local reaction af-
ter topical DermaVir administration dem-
onstrated on clinical pictures of a rhesus
macaque (e–g). The inguinal region before
exfoliation (e), immediately following ex-
foliation and DermaVir application (f) (two
treatment areas shown in the box), and
15 min post-DermaVir application (g).
SC, stratum corneum; E, epidermis; D,
dermis.

performed in swine, an accepted animal model for treat- Table I. Detection of HIV-1 Gag mRNA by RT-PCR in the lymph
ments administered via the skin, as porcine skin is similar in nodes of mice after DermaVir vaccination
structure to human skin. Local reaction was limited to mild
Animal DNA in DermaVir (gag)copies per 106 cells
transient erythema that did not occur every time. There was
no systemic toxicity, measured by blood chemistry and he- BALB/C pGag 32,800
matology, associated with DermaVir (data not shown). BALB/C pGFP o5000
C57BL/6 pGag 64,104
Mechanism of DermaVir vaccination To activate naı̈ve T
cells, LC must undergo maturation when exposed to anti- C57BL/6 pGFP o5000
gens and migrate to the draining lymph node. Therefore, we Average control RT-PCR in different species of untreated mice was
examined DermaVir-derived gene expression using RT-PCR 4536 (SD ¼ 1610). Values represented as o5000 copies are not different
in the draining lymph nodes after topical DermaVir vacci- from the negative control mice.
nation in two different murine models; BALB/C and
C57BL/6 (Table I). DermaVir was formulated with Gag-
delta8.2, designated as pGag instead of the pSHIV(int-), DermaVir. In contrast, we found less than 4,000 copies of gag
because the LTR promoter does not result in gene expres- mRNA per 106 cells in the lymph nodes of mice vaccinated
sion in mice. pGag was a suitable reporter gene because with the same formulation containing a control DNA. This
gag mRNA can be quantified by a quantitative RT-PCR as- value was similar to that found in several untreated control
say (Bagnarelli et al, 1994). Control animals were similarly mice (average of 4,536; SD ¼ 1,610). We suspect that the
treated with DermaVir formulated with pGFP. We found over gag-specific RT-PCR reaction gives a background in the dif-
30,000 copies of gag mRNA per 106 cells in the inguinal ferent murine models, probably because of interference from
lymph node of both mice species after vaccination with endogenous retroviruses that are common in mice.
124 : 1 JANUARY 2005 DERMAVIR IMMUNIZATION FOR HIV/AIDS 163

Figure 3
Gene expression in the lymph node after topical DermaVir vaccination in mice and monkeys. DermaVir was formulated with reporter plasmid
DNA and applied topically on the skin of mice (representative of two experiments). (a) In situ hybridization using a 35S-labeled Neo-specific sense
probe (negative control) 24 h after DermaVir application. No positive cells were detected by control in situ hybridizations. (b) In situ hybridization
using a 35S-labeled Neo-specific antisense probe on a parallel section of (a). Transduced cells expressing plasmid DNA-derived genes (white silver
grains over the cells). (c) Enlargement of (b). (d) In situ hybridization with a 35S-labeled Neo-specific antisense probe of a lymph node isolated from a
naı̈ve mouse (negative control). (e–g) Immunohistochemical staining with HIV Gag-specific antibody (KC57 FITC, Coulter, Florida) of a mouse lymph
node 72 h post-immunization. DermaVir was formulated with plasmid DNA-expressing gag. (e) Isotype control of p55 antibody (15 s exposure). (f)
p55 antibody staining (5 s exposure) on a parallel section of (d). (g) Protein expression is localized in the paracortical region of the lymph node.
Quantification of these cells in mice revealed an average of 222 (30–400) Gag-expressing cells per 0.05 mm sections, corresponding to an average
of 68 positive cells per mm2. Parallel sections stained with the isotype control gave an average of 0.6 (0–2) positive cells per section. Representative
experiments, repeated at least three times. (h) Gene expression in lymph node dendritic cells (DC) after topical DermaVir vaccination of rhesus
macaques (same methods as in mice (a–c)). Dark-field microscopic image of cells showing (white) silver grains over positive cells. Control
hybridization of a parallel section showed no positive cells (not shown). (i) A single DC expressing Neo gene encoded by the DNA used for DermaVir
formulation. Black dots are silver grains (in situ hybridization) demonstrating plasmid DNA-derived gene expression. The section was also stained
with p55 antibody (anti-human Fascin, 55K-2, Dako Corp. Carpenteria, California) which is a marker for lymph node DC (brown in the figure).
Quantitative analysis in macaques revealed 153 DC expressing DNA per 13.4 mm2 total analyzed sections (average 11 positive cells per mm2).

To visualize the RNA-expressing cells in the lymph nodes struct encoding the neomycin-phosphotransferase gene
of DermaVir-vaccinated mice, in situ hybridization experi- (Neo). Figure 3a–d demonstrate the DNA-expressing cells
ments were carried out, using another reporter DNA con- migrating into the lymph node 24 h after topical DermaVir
164 LISZIEWICZ ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

vaccination. As demonstrated in Fig 3a and d, in situ hy- same experiment in rhesus macaques. Our analysis con-
bridizations of parallel sections using a control sense probe firmed that topical DermaVir vaccination results in DNA-ex-
and sections of control mice hybridized with the antisense pressing cells located in the T cell area of the lymph
probe did not reveal any positive cells. Quantitative analysis node (Steinman et al, 1997), specifically in the paracortical
revealed 153 DC expressing DNA per 13.4 mm2 total analy- region (Fig 3h). The identity of these positive DNA-express-
zed sections (average 11 positive cells per mm2). These ing DC was demonstrated with antibody staining specific
in situ hybridization results supported the RT-PCR data for DC in the lymph node (anti-human Fascin, 55K-2,
(Table I) and suggested that topical DermaVir vaccination Dako Corp., Carpenteria, California) (Steinman et al, 1997)
can directly transduce large numbers of skin-derived cells in (Fig 3i). Control hybridization of a parallel section with
the lymph node. the sense probe did not detect positive cells (not shown).
To visualize protein expression, we formulated DermaVir Quantitative analysis revealed 153 DC expressing DNA
with pGag and analyzed the lymph nodes with an HIV Gag- per 13.4 mm2 total analyzed sections (average 11 positive
specific antibody (Fig 3e–g). Positive cells were mainly cells per mm2). The number of gene-expressing DC
located in the paracortical area, which is also referred to as after topical DermaVir vaccination was quite comparable
the T cell area known to contain the migrating LC-derived with the number of gene-expressing DC detected previ-
DC. Altogether, these results further confirmed the RT-PCR ously in the lymph node after ex vivo administration of De-
data (Table I) and demonstrated that topical DermaVir vac- rmaVir-transduced DC (Lisziewicz et al, 2001). These results
cination results in gene expression in the lymph node. indicated that after topical DermaVir vaccination gene
Since the murine epidermis differs from that of primates expression in lymph node DC could be achieved in non-
(Foster et al, 1990; Matsue et al, 1993), we performed the human primates.

DermaVir, topical DermaVir, ex vivo

Before After Before After
Macaque 800 Macaque 798
0.02 0.27 0.06 0.10

0.04 0.16 0.10 0.17

Macaque 802 Macaque 805

0.03 0.27 0.03 0.24 Figure 4

Induction of simian immunodeficien-
cy virus (SIV)-specific T cells by
DermaVir immunization. SIV-specific
T cell responses were detected in the
peripheral blood of macaques prior to
and 3 wk after the first topical (left
0.05 0.23 0.06 0.21
two columns) and ex vivo (right two
columns) DermaVir immunization.
CD8

Histograms illustrate SIV-specific T

Macaque 810 Macaque 806 cells measured as IFN-g expression in
CD8 þ and CD8
(gated on CD3) T
0.02 0.26 0.10 0.47 lymphocytes after stimulation with
whole chemically inactivated SIV anti-
gen (Lori et al, 2000; Xu et al, 2002).
Numbers represent the percentages of
IFN-g expressing CD8 þ , CD3 þ , and
CD8
, CD3 þ T cells in the quadrates.
0.03 0.21 0.09 0.42

Macaque 813 Macaque 807

0.04 0.24 0.03 0.25

0.01 0.11 0.01 0.30

IFN-γ
124 : 1 JANUARY 2005 DERMAVIR IMMUNIZATION FOR HIV/AIDS 165

Immune responses induced by DermaVir We evaluated 9

DermaVir-induced immune responses in naı̈ve rhesus mac- 8
aques, since this is a suitable animal model to study im-

DTH skin reaction (mm)

7
mune-based interventions for HIV. For these studies,
DermaVir was formulated with our prototype therapeutic 6
plasmid DNA, pSHIV(int-) (Fig 1), that is based on a SHIV 5
construct. Four monkeys were immunized topically and an- 4
other four via ex vivo immunization with DermaVir-trans-
3
duced DC as described previously (Lisziewicz et al, 2001).
SIV-specific T cells were measured in the peripheral blood 2
after whole SIV viral antigen stimulation by interferon (IFN)-g 1
production (Fig 4). The average frequency of SIV-specific 0
CD8 þ , CD3 þ , and CD8
, CD3 þ T cells prior to immuniza- # 800 # 802 # 810 # 813 # 798 # 805 # 806 # 807
tion was 0.06% and 0.07% (SD ¼ 0.03 and 0.04), respec-
tively, a characteristic background in naı̈ve macaques. After DermaVir, topical DermaVir, ex vivo
one immunization all the animals developed SIV-specific Figure 5
T cell responses. The average frequency of SIV-specific Delayed-type (type IV) simian immunodeficiency virus (SIV)-spe-
CD8 þ , CD3 þ , and CD8
, CD3 þ T cells in the topically im- cific hypersensitivity (DTH) responses in macaques immunized
munized group was 0.26% and 0.18% (SD ¼ 0.01 and 0.05), topically and ex vivo with DermaVir. DTH skin reactions (diameter,
millimeters) were detected in the skin of macaques 25 wk after the
respectively. The average frequency of SIV-specific CD8 þ , fourth topical (animals 800, 802, 810, and 813) and ex vivo (animals
CD3 þ , and CD8
, CD3 þ T cells in the ex vivo immunized 798, 805, 806, and 807) DermaVir immunization. DTH reactivity was
group was 0.27% and 0.28% (SD ¼ 0.15 and 0.11), respec- measured 72 h post-intradermal injection with 100 mL normal saline
(empty bars); 100 mL control microvesicles (gray bars), representing the
tively. These results demonstrate that topical DermaVir im-
supernatant of the parental cell line that was utilized to produce the SIV
munization was able to induce antigen-specific CD8 and antigen; and 100 mL (2 mg of p27) chemically inactivated SIVmac239
CD4 T cell-mediated immune responses in non-human pri- antigen (Arthur et al, 1998) (black bars).
mates similar to ex vivo DC vaccination.
Vaccinations were administered at months 0, 2, 5, 13,
and 21. Despite repeated immunizations, antibody respons- mune responses by DermaVir and suggested the presence
es measured after every vaccination were undetectable by of a memory T cell population in tissue reservoirs that can
ELISA in all of the animals, even after the fifth DermaVir expand upon antigenic stimulation.
vaccination. After the first vaccination, we consistently de-
tected a transient increase of SIV-specific T cells, peaking
3 wk after vaccination in the peripheral blood; however, 8 Discussion
wk after vaccination the frequency of SIV-specific cells was
undetectable in the intracellular cytokine assay. Interesting- DermaVir is a novel DNA immunization method designed to
ly, after the third vaccination we only sporadically detected improve antigen presentation and induce cytotoxic T cell
a very low amount of SIV-specific T cells in the peripheral responses for the treatment of HIV/AIDS. DermaVir is ap-
blood of the animals. Meaningful T cell responses with the plied directly to the epidermis above the basal keratinocytes
ELISPOT assay were not detected. to penetrate the skin surface and reach a network of sen-
To confirm the induction of Th-1-type cell-mediated im- tinels that serve to initiate immune responses against path-
mune responses by DermaVir, an in vivo assay was used, ogens. After DermaVir immunization, plasmid-derived gene
similar to that used to determine exposure to Mycobacte- expression occurred in lymph node DC. Gene-expressing
rium tuberculosis. When people have been exposed to DC induced T cell-mediated immune responses. Our ex-
tuberculosis, a cell-mediated immunity develops that can periments did not demonstrate cross-presentation of anti-
be detected as a local response after intradermal injection gens that might also occur. In naı̈ve macaques, DermaVir
of a small amount of tuberculin. These delayed-type hyper- effectively primed antigen-specific CD4 helper and CD8 T
sensitivity (DTH) responses are mediated by T cells, be- cells producing intracellular IFN-g after in vitro antigenic
cause they can be seen in individuals who lack immuno- stimulation. Boosting with DermaVir did not increase T cell
globulins. To perform the skin test, we used the same responses in macaques, similar to recent observations in
chemically inactivated SIV (Arthur et al, 1998) that we used DC-immunized mice (Ludewig et al, 2001). The possible
for stimulation of T cells to detect SIV-specific T cells in the explanation for these findings is that DC boosting reacti-
peripheral blood. As control antigen, we used the super- vates memory cytotoxic T lymphocytes (CTL) that can rap-
natant of the parental cell line that was utilized to produce idly eliminate the antigen-presenting DC and thereby limit
the SIV (called microvesicles). The skin test was performed the booster effect, an important mechanism to avoid exag-
5 mo after the fourth vaccination, when SIV-specific T cell gerated CTL responses. But T cell memory was maintained
responses were undetectable by intracellular cytokine as- in the reservoirs, even when SIV-specific T cell frequency
say in the peripheral blood. All monkeys immunized either was under the limit of the detection in the peripheral blood
topically or ex vivo with DermaVir developed DTH respons- (0.1%), as shown by the DTH responses to SIV antigens in
es to SIV antigen but not to the microvesicle control antigen all the animals immunized either topically or ex vivo with
(Fig 5). The reactive DTH tests in the immunized macaques DermaVir. The reactive DTH test and lack of humoral re-
further substantiated the activation of T cell-mediated im- sponse observed after repeated DermaVir immunizations
166 LISZIEWICZ ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

are similar to that observed in individuals previously ex- with broad specificity. HIV-LTR-driven viral protein ex-
posed to M. tuberculosis who do not develop antibody pression does not require artificial, codon-optimized se-
responses and have reactive tuberculin DTH tests. Inter- quences or heterologous promoters introduced in other
estingly, recent studies demonstrated that in M. tuberculosis DNA vaccines to achieve efficient expression (Robinson
infection DC, which are not permissive for the growth of the et al, 2002).
bacteria, take up the bacteria and induce cellular immune In DermaVir the DNA was formulated with a cationic pol-
responses (Geijtenbeek et al, 2003; Tailleux et al, 2003). ymer (PEIm) in glucose. The cationic polymer complexes
These results are in line with the concept that expression of the DNA, forming a small mannosilated particle and the
antigens within DC will generate polarized cellular respons- glucose stabilizes the complex by inhibiting aggregation
es. The fact that DNA-expressing DC in the lymph node did prior to the vaccine application. The DermaVir particle con-
not induce antibody production, regardless of the topical or tains pathogen-associated molecular patterns (e.g. man-
ex vivo DermaVir vaccination methods, could be explained nose from PEIm and CpG motifs from the plasmid DNA),
by selective antigen expression in DC in the absence and therefore might be recognized by toll like or other re-
of significant antigen expression in the local somatic cells ceptors on LC (Akira et al, 2001). PEIm plays an important
(e.g. myocytes, keratinocytes, and fibroblasts), which serve role after receptor-mediated endocytosis of DermaVir by
as antigen reservoirs for conventional DNA vaccines (Tuting breaking the endosome (Boussif et al, 1995) and facilitating
et al, 1998; Akbari et al, 1999). the trafficking of the DNA into the nucleus, where the DNA-
We have shown here in a primate model that topical encoded antigens are expressed (Pollard et al, 1998).
DermaVir vaccination is comparable with ex vivo DC-based Antigen-presenting DC in the lymph node induce T cell-
vaccination. DermaVir, administered topically or ex vivo, re- mediated anti-tumor immune responses (Banchereau and
sulted in a similar number of transduced DC in the lymph Steinman, 1998; Palucka and Banchereau, 1999; Schaden-
node and induced a similar quantity and quality of SIV-spe- dorf and Nestle, 2001). Here, we extended these observa-
cific Th1-type T cell responses. Both modalities of DermaVir tions to DC expressing viral DNA-derived antigens following
administration specifically utilize DC, cells capable of con- topical DermaVir vaccination. These results confirm and
verting naı̈ve T cells to functional cytotoxic T cells and expand the concept of using DC as preferential targets
polarizing the immune system towards T cell-mediated im- to elicit cellular immune responses (Kirk and Mule, 2000;
mune responses. Since the immune system of HIV-infected Schadendorf and Nestle, 2001). The technology described
individuals has already been primed during primary infec- here could provide the basis for novel DNA-based medi-
tion by large amounts of viral antigen, the purpose of cines. Various plasmids could be constructed to obtain the
DermaVir therapy is to improve the presentation of viral an- expression of a wide variety of tumor and viral antigens by
tigens in order to induce functional T cell responses that can DC in the lymph nodes in order to induce antigen-specific T
control viral replication. It is indeed unlikely that HIV-specific cell responses. Immune responses might be augmented by
antibodies will have a role in the control of viral load during the co-expression of recombinant cytokines (Ahuja et al,
established infection (Richman et al, 2003; Wei et al, 2003). 1999; Melero et al, 1999; Ozawa et al, 1999; Takayama et al,
Our experiments demonstrated a remarkably high effi- 1999). The technology could allow genetic manipulation of
ciency of topical gene transfer to lymph node DC. In the DC to express IL-10, TGF-b, FasL, and CTLA4Ig, for ex-
mouse model the efficacy was between 30 and 90 cells per ample, which have been suggested to enhance tolerance
mm2 and in the primate model 11 cells per mm2 (Fig 3). The and allograft survival (Lu et al, 1999). In addition, the tech-
disparity of these numbers might reflect the differences be- niques described here might be used as tools to elucidate
tween the murine and primate epidermis and lymph nodes. as yet unanswered questions in immunology, such as the
Since the average diameter of a lymph node is, however, role of lymph node DC in antigen presentation and immune
about 1.8 and 6 mm in mice and monkeys, respectively, induction.
when the size of the lymph node was taken into account, a Others have demonstrated inhibition of SIV replication
conservative estimate of the total number of positive cells (Lu et al, 2003) with DC-based ex vivo therapeutic immu-
per lymph node in the murine model was between 1,000 nization; however, these ex vivo techniques are cum-
and 4,000 cells, whereas in the primate model it was about bersome, expensive, and limited to highly specialized lab-
20,000 cells. oratories. DermaVir represents a significant advancement in
Conventional DNA vaccines designed for prevention of the field of DC-based therapeutic immunization: (i) it can be
HIV/SIV infection usually require the optimization of protein manufactured for large-scale human use; (ii) it utilizes a
expression to provide a large amount of viral antigens and needle-free, topical application; and (iii) it only requires a
thereby induce both antibody and T cell responses in hosts small amount of DNA. (iv) All pre-clinical studies with
who have not encountered such antigens. Generally, these DermaVir show no serious safety concerns. Furthermore, (v)
DNA constructs contain one or more codon-optimized HIV DermaVir suppression of viral load during chronic SIV in-
genes driven by a strong constitutive promoter (Barouch fection in non-human primates (Lisziewicz et al, in press)
and Letvin, 2000; Corbet et al, 2000; Robinson et al, 2002). has recently been observed. These features make DermaVir
Second-generation plasmid DNAs have been optimized a promising new approach to the treatment of HIV/AIDS.
for high-level expression in cell lines and improved ex-
pression has been shown to be associated with more
efficient induction of immune responses. The plasmid DNA Materials and Methods
in DermaVir was designed to express all viral genes in Plasmid DNA used for DermaVir formulation A plasmid, known
DC except integrase in order to induce HIV-specific T cells to express green fluorescent protein and neomycin phosphot-
124 : 1 JANUARY 2005 DERMAVIR IMMUNIZATION FOR HIV/AIDS 167

ransferase, pGFP (Clonetech, Palo Alto, California), was utilized RT-PCR RNA was isolated by using a TRIzol reagent (GIBCO BRL,
in most of the experiments demonstrating the mechanism of Gaithersburg, Mary Land) according to the protocol of the man-
DermaVir vaccination. A CMV-driven plasmid expressing the HIV-1 ufacturer. Quantitative RT-PCR were performed with the Roche kit,
gag protein, Gag-delta8.2 (Naldini et al, 1996), was kindly provided detection limit of 200 copies of HIV-1 RNA (Roche Diagnostic
by Inder Verma. For primate immunology experiments the proto- Systems Inc., Branchburg, New Jersey.)
type DermaVir plasmid DNA, expressing a replication-defective
virus, was used. The full-length, but integration defective, SHIV Quantitative determination of SIV-specific T cell responses in
plasmid pSHIV(int-), created by stepwise strategy starting with peripheral blood The assay was performed as previously de-
p-50 SHIV (clone KB9) and p-30 SHIV (clone 64/KB9) provided by scribed (Lori et al, 2000; Xu et al, 2002). PBMC were plated in
Joseph Sodroski of Harvard University, was used in DermaVirSHIV round-bottom 96-microtiter plates (Corning Inc., Corning, New
for all primate experiments. These clones, derivatives of SHIV 89.6, York) at 0.5 million cells per well in 0.1 mL complete RPMI-1640
are also available from the NIH Research and Reference Reagent medium containing 5 mg Zn-finger-inactivated SIV (kindly provided
Program. First, a clone of p-50 SHIV with the deletion of the internal by Jeff Lifson, NCI, Frederick, Mary Land) or mock antigen and 50
Bgl 2–Bgl 2 sites located in the pol gene was created and termed IU per mL rhIL-2 (gift from Hoffman La Roche). Cells were cultured
p-50 SHIV(dBg). The internal Bgl 2–Bgl 2 fragment was mutated by for 15 h and treated with Brefeldin A (Sigma, St Louis, Missouri) at
PCR amplification of the fragment with primers introducing muta- 10 mg per mL for an additional 3 h. Cells were collected and ali-
tions with stop codons and cloned into a separate vector and quoted into 0.5 million cells per test tube. After washing once with
termed pBg08. The mutated fragment was isolated and inserted 2 mL phosphate-buffered saline (PBS) containing 1% bovine se-
into p-50 SHIV(dBg) to obtain p-50 SHIV(int-). The Xho1–Sph1 viral rum albumin (BSA), cells were suspended in 0.1 mL PBS/1% BSA
fragment ( 6.5 kb) from p-50 SHIV(int-) and Sph1–Not1 viral frag- and stained with CD8-Phycoerythrin-Cyanine 5 (PC5; Immunotech,
ment ( 4.0 Kb) from p-30 SHIV clones were isolated and cloned Marseille, France) and CD3 fluorescein isothiocyanate (FITC; BD
into a pBluescript (Stratagene Inc., La Jolla, California) vector PharMingen, San Diego, California) fluorescent antibodies for 15
backbone to obtain the pSHIV(int-) clone. The sequence of the min at room temperature. After washing, cells were fixed with 2%
junctions and of the integrase gene region of this clone was paraformaldehyde, pH 7.4 for 10 min and washed with PBS/1%
checked. It contained small deletions, frame shift, and three sep- BSA, and then permeabilized with 0.1 mL 0.1% saponin in PBS/
arated stop codons in the integrase gene open reading frame. 1% BSA for 5 min and stained with IFN-g-R-Phycoerythrin con-
It also contained stop codons in the other reading frame in this jugated (IFN-g-PE; BD PharMingen) antibody for 15 min at room
region. SIVmac239 sequence: (nt 4696) 50 -AGATCTAGGGACTTG temperature. After intracellular staining, cells were washed twice
GCAAATGGATTGTACCCAT-30 (nt 4729). pSHIV(int-) sequence: 50 - with 1 mL PBS and then re-suspended in a 0.5 mL 1% parafor-
AGATCTATAGATAGATAGCTAGCCCAT-30 . maldehyde PBS buffer. Samples were analyzed by FACS (EPICS
XL-MCL, Coulter, Miami, Florida). A total of 50,000 events were
acquired. Gated CD3 þ CD8 þ cells were examined for staining
Topical and ex vivo DermaVir immunization DermaVir is for-
with IFN-g.
mulated to make a approximately 100 nm particle containing DNA,
PEIm, and glucose and the particle size was determined by light Detection of SIV-specific T cell responses in vivo A skin test
scattering using a Zetasizer (Malvern Instrument, Orsay, France). was developed to detect SIV-specific T cell responses in vivo. SIV
For topical DermaVir application, a skin preparation procedure was and control antigens in 0.1 mL physiological salt were intradermally
developed to allow vaccine penetration to the epidermis. Hair was injected. DTH skin reactions were recorded as the diameter in milli-
removed by shaving and the skin was exfoliated with an exfoliating meters by a blinded operator 72 h later. The following purified sol-
sponge (3M Scotch-Brite Heavy Duty Scrub Sponge 3M Corp., St. uble antigens were used: 2 mg (p27) Zn-finger-inactivated SIVmac239
Paul, Minnesota), followed by tape stripping (Fig 2). This procedure (Arthur et al, 1998); microvesicles as control (the supernatant of the
results generally in transient and mild erythema but not in hem- SupT1 cell line used to produce SIVmac239); and normal saline so-
orrhage and eschar formation. 0.2 mL DermaVir was applied on lution for suspension of antigens (Zinc-inactivated-SIVmac239 and
approximately 40 cm2 prepared skin at four locations: the left and microvesicles were kindly provided by Jeff Lifson, NCI).
right upper inguinal region and left and right axillary region. After
30 min of contact time under general anesthesia, DermaVir solution Detection of antibody responses The native SIV p27 used was
dried and the animals were returned to the cage. For mice, 0.2 mL purified from conditioned media from infected mammalian cells
DermaVir was applied on approximately 20 cm2 area on the dor- (Hut 78) that secrete SIV env and gag proteins. The recombinant
sum. In all ex vivo experiments, 106 ex vivo DermaVir-transduced HIV-1 89.6p gp140 protein was prepared from (293) mammalian
DC were injected subcutaneously at the same four locations as cells expressing the gene. To prepare ELISA plates, SIV p27 was
previously described (Lisziewicz et al, 2001). added at 50 ng per well, and gp140 at 50 ng per well. Binding
antibody ELISA titers are routinely presented as the reciprocal of
Animals All animal experiments were performed under protocols the serum dilution at which the adsorbance of the test serum is
approved by an Institutional Animal Care and Use Committee. twice that of negative control serum (Zhao et al, 2003; Patterson
Four- to 6-wk-old female BALB/C mice were used. The mice were et al, 2004).
anesthetized using methoxyflurane. In non-human primate studies,
rhesus macaques were sedated with ketamine xylazine and placed
on a circulating water heating pad for the duration of the immu- We thank N. Miller and M. Ussery (NIAID) for the support of the animal
nization procedure. For the in situ hybridization experiment, the studies (NIH, NIADS, DAIDS, Contract # NO1-Al-15430), J. Newsome
draining lymph node was surgically removed 24 h after DermaVir for his contribution to the murine experiments, and D. Zinn for technical
assistance. We thank J. Lifson and L. Arthur for providing the Zn-finger-
vaccination.
inactivated SIV. We are grateful to S. Petrocchi and T. Battle for their
editorial assistance.
In situ hybridization In situ hybridization was conducted using
standard protocols (Fox and Cottler-Fox, 1993a, b). Riboprobes DOI: 10.1111/j.0022-202X.2004.23535.x
were 33P labeled and were determined to detect 20–30 copies per
Manuscript received December 22, 2003; revised June 30, 2004;
cell of HIV gag RNA, although in the case of Neo probes the sen- accepted for publication July 20, 2004
sitivity was somewhat less. The slides were exposed for 5 d be-
fore development and examination by dark-field microscopy. Address correspondence to: Julianna Lisziewicz, Research Institute for
Immunohistochemistry was performed using protocols recom- Genetic and Human Therapy (RIGHT), Washington, District of Colum-
mended by the supplier of the primary antibodies. bia 20007, USA. Email: [email protected]
168 LISZIEWICZ ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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