DNA and Modified Vaccinia Virus Ankara Vaccines Encoding
Multiple Cytotoxic and Helper T-Lymphocyte Epitopes of Human
Immunodeficiency Virus Type 1 (HIV-1) Are Safe but Weakly
Immunogenic in HIV-1-Uninfected, Vaccinia Virus-Naive Adults
Geoffrey J. Gorse,aMark J. Newman,bAllan deCamp,cChristine Mhorag Hay,dStephen C. De Rosa,eElizabeth Noonan,c
Brian D. Livingston,b* Jonathan D. Fuchs,fSpyros A. Kalams,gFarah L. Cassis-Ghavami,e* and the NIAID HIV Vaccine Trials Network
Division of Infectious Diseases, Allergy, and Immunology, Department of Internal Medicine, School of Medicine, Saint Louis University, St. Louis, Missouri, USAa; Pharmexa-
Epimmune, Inc., San Diego, California, USAb; Statistical Center for HIV/AIDS Research and Prevention, Fred Hutchinson Cancer Research Center, Seattle, Washington, USAc;
Infectious Diseases Division, University of Rochester Medical Center, Rochester, New York, USAd; HIV Vaccine Trials Network, Fred Hutchinson Cancer Research Center,
Seattle, Washington, USAe; San Francisco Department of Health, San Francisco, California, USAf; and Division of Infectious Diseases, Vanderbilt University Medical Center,
Nashville, Tennessee, USAg
selves elicit broad, but fairly weak, cellular and humoral immune
responses (7, 18, 40, 41, 45, 67). When used in combination with
gous prime-boost immunization regimens, they may provide a po-
These poxviruses are capable of accommodating large amounts of
foreign DNA, and some are attenuated to infect but not replicate in
human cells, resulting in expression of a large amount of foreign
protein. The use of a single viral vector to prime and boost immune
responses to a foreign antigen may not be an efficient regimen. Im-
mune responses to the vector induced after the primary vaccination
may interfere with the infection of the virus following a second vac-
et al. (72) showed that DNA priming could overcome the effect of
Like other modified vaccinia virus Ankara (MVA) vectors,
which were generated as a consequence of deletions in the MVA
genome, MVA-BN (Bavarian Nordic A/S GmbH, Martinsried,
Germany) exhibits a severely restricted host range and replicates
man cells and most transformed human cell lines (10, 12, 19, 61).
Although MVA-BN exhibits attenuated replication in these cell
types, its genes are efficiently transcribed, with the block in viral
genes have important characteristics, including a high expression
that DNA plasmid-vectored vaccines administered by them-
of vaccine antigen in MVA-infected cells (62). They induce both
cytotoxic T-lymphocyte (CTL) and antibody responses in hu-
mans (35). MVA-BN vaccines at doses ranging from 1 ? 106to
5 ? 10850% tissue culture infectious doses (TCID50) have been
administered to over 850 individuals in 12 clinical studies in
healthy adults, melanoma patients, patients with atopic dermati-
tis, and HIV-1-infected patients with no drug-related serious ad-
verse events occurring. No cases of myocarditis or pericarditis
have been observed with MVA-BN (35, 65), but the present study
clinical program with MVA-BN vaccine, the vast majority of par-
ticipants have received the vaccine via the subcutaneous route
of administration. This route of administration has shown sat-
isfactory immune responses and a good safety profile (65). T-
cell immune responses by CTL and helper T lymphocytes
(HTL) in enzyme-linked immunospot (ELISPOT) assays were
highly correlated with the results of the antibody tests (65). In
an HIV-1 prophylactic vaccine trial (HIV-NEF-003), Nef-spe-
cific cellular and humoral immune responses were induced in
subjects. After three vaccinations, 10 of 14 subjects developed
Received 24 January 2012 Accepted 24 February 2012
Published ahead of print 8 March 2012
Address correspondence to Geoffrey J. Gorse, firstname.lastname@example.org.
*Present address: Brian D. Livingston, MedImmune, Inc., Mountain View,
California, USA, and Farah L. Cassis-Ghavami, Swedish Medical Center, Seattle,
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
1556-6811/12/$12.00Clinical and Vaccine Immunologyp. 649–658 cvi.asm.org
T-cell responses that recognized between one and five Nef
The clinical safety and immunogenicity of a DNA vaccine (EP
HIV-1090) encoding the HIV-1-derived CTL epitopes were pre-
viously evaluated when delivered alone or simultaneously with
HIV-1-derived HTL epitopes in the form of a recombinant pro-
tein adsorbed to Alhydrogel as the adjuvant (28, 38). Only low
levels of CTL responses were observed in both clinical trials de-
Our working hypothesis was that delivery of the CTL and HTL
epitopes using different vaccine delivery formats did not result in
that increased immunogenicity could emanate from use of a het-
erologous prime-boost vaccine regimen comprised of both the
DNA and viral-vectored vaccines, wherein both vaccine compo-
nents direct the in vivo transcription, translation, processing, and
presentation of CTL and HTL epitopes by transfected cells.
those responses. The heterologous EP-1233 prime and MVA-
mBN32 boost regimen was viewed as the most promising ap-
of focusing the immune responses to the recombinant MVA on
the encoded HIV-1 epitopes.
MATERIALS AND METHODS
Study design. Our clinical trial evaluated two HIV-1 experimental vac-
cines: (i) a DNA plasmid-vectored vaccine (EP-1233) and (ii) a modified
vaccinia virus Ankara (MVA-mBN32)-vectored vaccine. This multi-
center, double-blinded, randomized, placebo-controlled phase 1 clinical
trial (HVTN protocol 067) was sponsored by the National Institute of
Allergy and Infectious Diseases (NIAID), Division of AIDS (DAIDS) and
conducted at three HIV Vaccine Trials Units (HVTU) in the United
States. The study protocol was approved by the local site institutional
review boards and was carried out in accordance with the Code of Ethics
of the World Medical Association (Declaration of Helsinki) for experi-
ments involving humans. Written informed consent was obtained from
each subject after the nature and possible consequences of the study were
fully explained. Participants were healthy adults, aged 18 to 40 years, who
were HIV-1-uninfected and did not have a self-reported history of vac-
cinia virus vaccination. Due to a possible association between unattenu-
ated vaccinia virus used for smallpox vaccination, and myopericarditis,
specific inclusion and exclusion criteria, along with enhanced cardiac
of enrolling anyone with preexisting myopericarditis or cardiovascular
disease that could increase their risk from vaccination and to detect po-
tential cardiac effects of the study vaccine.
A total of 36 volunteers were enrolled, 13 at the first site, 12 at the
second site, and 11 at the third site, with 30 randomly assigned to receive
vaccine and 6 to receive placebo. The DNA plasmid-vectored vaccine
(EP1233) was administered first; two study immunizations were given at
0 and 28 days as two 1-ml intramuscular injections in the deltoid muscles
point. The third and fourth immunizations were administered at 84 and
containing 108TCID50/ml. The placebo control recipients received the
same volume and number of injections by the same routes as the vaccine
with an ongoing evaluation of safety.
all systemic and local reactions that had occurred. The severity of a reac-
tion was defined as follows: (i) mild, transient or minimal symptoms; (ii)
moderate, notable symptoms requiring modification of activity; and (iii)
severe, incapacitating symptoms requiring bed rest and/or resulting in
loss of work or social interaction. The clinical and laboratory safety data,
including reactogenicity assessments and adverse events reported, were
reviewed weekly during the vaccination period; data on adverse events
were collected from subjects for all 12 months of study participation.
Specific cardiac assessments were conducted during the study as fol-
lows. Participants were questioned at study visits about symptoms and
and fatigue. Test results for cardiac troponin I, a biomarker for cardiac
trocardiogram (ECG) had to be normal at screening, and the test was
monary symptoms were reported during the trial, the study staff per-
formed a cardiopulmonary examination. After vaccination with MVA-
mBN32 or its placebo control, participants who developed signs and
symptoms or findings suggestive of a possible new cardiovascular condi-
congestive heart failure, ECG abnormalities, or an elevated cardiac tro-
ponin I), were evaluated with an ECG, cardiac troponin I, and creatine
kinase-MB isoenzyme (CK-MB), and an appropriate diagnostic evalua-
tion as medically indicated.
At every clinic visit, HIV-1 risk reduction counseling was provided.
Participants were monitored for incidents of social harm related to their
participation in the protocol. Three commercially available enzyme-
linked immunosorbent assay (ELISA) kits were used to detect HIV-1 in-
Before the first study injection, 14 days after each study injection, and
at months 9 and 12, blood specimens were obtained by venipuncture for
safety assessments and to assess immune responses to vaccination. Pe-
ripheral blood mononuclear cells (PBMC) were purified from sodium
heparin anti-coagulated whole blood within 8 h of venipuncture and
ing was done on all study subjects with available samples, as described
previously (59). Briefly, the sequence based typing strategy we used que-
reported as HLA types according to the nomenclature outlined in the
International ImMunoGenetics HLA database (www.ebi.ac.uk/imgt/hla
Vaccines. (i) DNA plasmid vaccine EP-1233. EP-1233 (Pharmexa-
Epimmune, Inc.) was a DNA plasmid-vectored vaccine encoding 21 CTL
and 18 HTL epitopes from Gag, Pol, Vpr, Nef, Rev, and Env. Epitope
selection criteria and vaccine design and construction were described in
based on HLA peptide-binding motifs using 128 HIV-1 sequences; ap-
proximately half of which were clade B and the remainder from other
clades. Potential epitopes were initially selected based on supertype bind-
ing properties of the peptides to soluble HLA-A2, HLA-A3, and HLA-B7
HLA superfamily class I or the HLA-DR1,4,7 supertype molecules. Sub-
sequent testing included immunogenicity assessments using HLA trans-
genic mice and antigenicity measured using PBMC obtained from HIV-
1-infected donors. Based on HLA types the vaccines were predicted to
induce CD8 and CD4 responses in ca. 85 and 100% of the general popu-
also encodes the universal HTL pan-DR epitope (PADRE, amino acid
sequence AKFVAAWTLKAA), which has high binding affinity to a wide
helper-facilitated immunity in mice (1, 16) and lymphoproliferation in
Gorse et al.
cvi.asm.org Clinical and Vaccine Immunology
to increase the magnitude and duration of the CTL responses (37).
The EP-1233 DNA vaccine was developed from two sets of HIV-1
epitopes that were fused into a single contiguous reading frame; the N
C terminus was composed of the HTL epitopes. Amino acid spacers were
was assembled from overlapping synthetic oligonucleotides using Pfu
DNA polymerase. The 5-prime end of the 843-bp sequence contains a
consensus Kozak sequence to support translation of the gene product. A
consensus Ig-? signal sequence was fused to the 5-prime end of the pro-
endoplasmic reticulum. A stop codon was included at the 3-prime end of
construct was cloned into the HindIII and XbaI sites of the pMB75.6
vector backbone. The EP-1233 DNA vaccine plasmid encodes only two
open reading frames: the kanamycin resistance gene and the synthetic
HIV-1 epitope product.
EP-1233 bulk drug substance was produced in E. coli strain DH5?
using high-density fermentation and purified from bacterial lysate mate-
rial by ethanol precipitation and anion-exchange column chromatogra-
phy by Althea Technologies, Inc. (San Diego, CA). The DNA vaccine was
formulated with a biocompatible adhesive polymer polyvinylpyrrolidone
1233 was a clear colorless solution containing 2 mg of DNA vaccine/ml.
saline with vaccine and in the placebo control for the DNA vaccine. The
PVP binds to DNA, stabilizes and protects it in vivo, facilitates the disper-
cells, thus resulting in increased in vivo gene expression and vaccine im-
munogenicity (5, 43, 44).
(ii) MVA-mBN32 vaccine. The modified vaccinia virus Ankara-HIV
polyepitope (MVA-mBN32) vaccine encodes the same 21 CTL and 18
HTL HIV-1 epitopes but under separate promoters. The HTL gene was
cloned into the intergenic region I4L/I5L of the MVA-BN genome, and
the CTL gene was cloned into the deletion II site. Both recombinant in-
serts are under the control of the vaccinia virus early/late promoter p7.5.
The MVA-mBN32 clinical trial material was manufactured by Bavar-
ian Nordic (Berlin, Germany) on primary chicken embryonic fibroblasts
derived from specific pathogen-free eggs in a bioreactor culture using
serum-free medium. The virus suspension was harvested and then sub-
jected to ultrasonication, concentration, and purification by serial filtra-
tion steps. The purified harvest (bulk drug substance) was formulated in
Tris-buffered saline and contained approximately 2 ? 108TCID50/ml.
The dose used in the present study was 108TCID50, corresponding to 0.5
Tris-buffered sterile saline was the control preparation for the MVA-
prime-boost schedules in rabbit studies by SRI (Menlo Park, CA) to de-
termine biodistribution, clearance and general safety to support Phase 1
on the clinical supplies included appearance, pH, DNA concentration,
DNA integrity, and immunogenicity measured using HLA-A2 transgenic
Immunologic assays. (i) ICS assay. PBMC were thawed and rested
overnight, suspended in R10 (RPMI 1640 [Gibco-BRL, NY] containing
10% fetal calf serum [Gemini Bioproducts, CA], 2 mM L-glutamine
[Gibco-BRL], 100 U of penicillin G/ml, 100 ?g of streptomycin sulfate/
ml), and incubated at 37°C in 5% CO2prior to stimulation. A minimum
cell viability of 66% measured after the overnight incubation on the day
following the thawing procedure was required for use in the intracellular
cytokine staining (ICS) assays. In the presence of brefeldin A (10 ?g/ml;
Sigma, St. Louis, MO) and 1 ?g each of anti-CD28 and anti-CD49d anti-
bodies (BD Biosciences, San Jose, CA)/ml, PBMC were stimulated for 6 h
with (i) the HTL peptide pool consisting of the 18 HTL epitopes found in
the vaccines, (ii) the CTL peptide pool consisting of the 21 CTL epitopes
found in the vaccines, and (iii) the PADRE HTL epitope. For the flow
cytometric analysis, the specimens were collected from 96-well plates us-
ing a high-throughput sampler (BD Biosciences) device on a BD LSRII
flow cytometer and then analyzed using FlowJo software (Treestar, Inc.,
OR) and LabKey Flow (58).
The eight-color ICS protocol was previously validated (36) for detec-
CD3?CD8?and CD3?CD4?HIV-1-specific T cells. In addition, both
The cells were first stained with violet live/dead fixable dead cell stain
(Invitrogen/Molecular Probes, Eugene, OR) (47) and then fixed, perme-
abilized, and stained intracellularly with the following antibody reagents:
CD8 peridinin chlorophyll protein complex-cyanin 5.5 (PerCP-Cy),
IFN-? PE-Cy7, IL-2 PE, TNF-? Alexa 700, and IL-4 allophycocyanin
(APC). All antibodies, except the CD3 PE-TR (Beckman-Coulter, Mar-
seille, France) and perforin (Tepnel/Diaclone, Stamford, CT) were ob-
with a perforin antibody that was conjugated to Alexa 647 (Invitrogen
Corp., Carlsbad, CA) in the laboratory. At the time of the present study
the analyses of IL-4, perforin, and TNF-? were not validated.
Positive responses and criteria for evaluable responses were deter-
mined as previously described (36) and were based on background mea-
were applied separately for CD3?CD4?and CD3?CD8?cells, the total
number of specimens included in each ICS analysis could differ between
the CD4?and CD8?T-cell evaluations.
(ii) IFN-? ELISPOT assay. PBMC samples (2 weeks after the fourth
CTL peptide pool by ICS were further tested by ELISPOT assay to deter-
mine the specific peptides they responded to. ELISPOT assays were per-
formed using a standardized, validated, bulk IFN-? ELISPOT assay (21,
42) for each of the 21 CTL peptides representing the CTL epitopes in the
(iii) Vaccinia virus-specific anti-MVA IgG antibody assay. Vaccinia
virus-specific antibodies were measured by using an automated direct
Maxisorb plates (Nunc, Wiesbaden, Germany) coated with a preparation
of MVA-BN-infected CEF cell lysate in coating buffer (200 mM Na2CO3
[pH 9.6]), as described previously (66).
Statistical methods. As part of the trial design, early evaluation of the
immunogenicity response data determined whether the trial would stop
after the first group was enrolled and completed follow-up. A determina-
immunization and 2 weeks after the fourth immunization stopped the
trial after participants were enrolled in the first group. The sample size of
30 vaccine and 6 placebo recipients adequately allowed us to evaluate
observing at least one serious adverse event, if the true rate of such an
event was at least 8%.
To summarize the T-cell response data, positive response rates to any
peptide pool were reported. The positivity of the ICS responses was de-
termined by a one-sided Fisher exact test applied to each peptide pool-
specific response versus the negative control response with a discrete
Bonferroni adjustment for the multiple comparisons. Peptide pools
with adjusted P values of ? 0.00001 were considered positive (36).
Positivity of the individual peptide IFN-? ELISPOT responses was deter-
mined by a one-sided bootstrap test of the null hypothesis that the exper-
imental well responses were twice those of the background (? ? 0.05). A
Westfall-Young (69) approach was used to adjust for the multiple com-
parisons across individual peptides. Peptides with adjusted one-sided P
HIV-1 Polyepitope DNA and MVA-Vectored Vaccines
May 2012 Volume 19 Number 5cvi.asm.org 651
the experimental and negative control wells had to exceed 50 spot-form-
ing cells (SFC) per 106PBMC for the response to be positive. Vaccinia
virus-specific antibody titers were calculated by linear regression and de-
fined as the serum dilution that resulted in an optical density of 0.30
(endpoint titers). A titer of 50 was the lowest reliably detectable antibody
level (assay cutoff), and when this titer was reached, the subject was con-
sidered seropositive. Antibody titers below the cutoff titer in the assay
were assigned an arbitrary value of 25 for the purpose of calculations.
and R were used for all analyses.
Clinical trial registration number. This study was performed under
clinical trial registration number NCT00428337.
Description of study population: demographics, HLA genetic
2007 and September 2007. Demographics did not differ between
study groups (Table 1). The HLA supertype A2, A3, and B7 pheno-
types were each present in 40, 51, and 46%, respectively, of the 35
study subjects for whom HLA typing was done (Table 1). Sixteen
two subjects were positive for all three, and 31 (88.6%) of the 35
in Fig. 1. All but 4 of the 36 subjects received all study treatment
injections; these four subjects, who were all vaccine recipients,
and two who did not receive the second MVA-mBN32 injection.
The reasons were either loss to follow-up or lack of availability to
receive the vaccine at the protocol-specified study time in three
fourth. Two of the four subjects who did not receive all study
complete all study visits.
Safety assessment. Local injection site erythema and indura-
tion were more frequent in the 3 days after injection with MVA-
mBN32 than with EP-1233 DNA vaccine, and erythema and in-
duration of moderate severity were more common after the
second MVA-mBN32 injection than after the first (Table 2). Res-
olution of the injection site induration after the first MVA-
mBN32 injection was delayed past 3 days in six subjects (resolu-
tion at days 8, 9, 13, 23, 40, and 57 postvaccination), and
resolution after the second MVA-mBN32 injection was delayed
past 3 days in seven subjects (resolution at days 5, 6, 7, 9, 10, 10,
after injection was least frequent (70%) after the second EP-1233
DNA vaccination and most frequent (96%) after the second
and second immunizations with the placebo control preparation
consisted of mild pain and/or tenderness and was similar to that
site signs and symptoms during the 3 days after the third and
fourth immunizations with placebo control preparation included
infrequent mild pain and/or tenderness and erythema.
The proportions of subjects with systemic reactogenicity
between those observed after EP-1233 DNA vaccine and MVA-
mBN32 vaccine injections (Table 2). Only one subject experi-
enced fever during the 3 days postvaccination (grade 1, 37.7 to
38.6°C), which was after the second MVA-mBN32 vaccination.
Systemic reactogenicity during the 3 days after control injections
was less than after either vaccine preparation.
the time of the first MVA-mBN32 vaccine injection, 19 (63.3%)
site ecchymoses (five definitely and one probably related), four
were injection site erythema (two definitely and two probably re-
probably related), four were injection site pruritus (all definitely
related), two were an aspartate aminotransferase elevation (both
possibly related), three were injection site exfoliation (one defi-
nitely and two probably related), two were injection site swelling
ably related). From the time of the first placebo control injection
for MVA-mBN32, one adverse event was thought to be definitely
pepsia, dyspnea, fatigue, and palpitations. All of these were deter-
TABLE 1 Demographics, vaccinations, and HLA genetic profile
No. of subjects (%)a
(n ? 30)
(n ? 6)
Median age (yr)
Age range (yr)
Vaccination received by
MHC class I HLA supertypeb
aExcept as noted otherwise in column 1.
bMHC class I HLA molecular typing was performed for a total of 35 subjects: 29
vaccine and 6 placebo control recipients.
Gorse et al.
cvi.asm.orgClinical and Vaccine Immunology
mBN32 vaccine and had a QTc interval prolongation (435 ms) that
was 83 ms over baseline on the ECG done 3 weeks after the fourth
vaccination. This adverse event was considered severe, because the
interval was at least 60 ms greater than baseline, but was deemed
unrelated to the study by the site investigator. The adverse event re-
baseline interval. This subject reported use of trazodone and bupro-
pion during this time and may have used methamphetamine. The
Assessment of the injection sites of MVA-mBN32 recipients
resulted in reports of unsolicited local injection site adverse reac-
tions that consisted of discrete subcutaneous nodules of mild se-
verity in eight subjects after the first and in six subjects after the
Of the six subjects with a subcutaneous nodule after the second
MVA-mBN32 injection, four had the same type of finding after
the first MVA-mBN32 injection. Onset was zero to 8 days after
vaccination and the range of days to resolution was three to 92
days. None were reported to be painful or tender, and the sizes
HIV-1 Polyepitope DNA and MVA-Vectored Vaccines
May 2012 Volume 19 Number 5cvi.asm.org 653
varied from about 2 mm to 4.5 cm. A uniform method for assess-
injection site, so it was not standardized.
Study subjects were assessed for social impact events pro-
spectively throughout the study. Two subjects reported three
personal relationship events of minimal impact on quality of
life, such as family members expressing concern for their
health. Despite staff assistance, two events remained unre-
solved. There were no reports of problems with employment,
education, medical/dental care, health/life insurance, travel/
immigration, a military/other government agency, or HIV an-
tibody testing outside of the study.
vaccine were T-cell, not B-cell epitopes and were not chosen to
induce HIV-1-specific antibodies.
Immunogenicity. (i) Cellular immune response. HIV-1-spe-
cific immunogenicity was assessed by ICS assay with PBMC sam-
ples obtained on day 98, which was 2 weeks after the third immu-
nization (n ? 34), and on day 182, which was 2 weeks after the
fourth immunization (n ? 30). Cellular immune responses were
detected in 3 (12%) of 25 subjects who received vaccine when
measured 2 weeks after the fourth immunization (Table 3 and
and the positivity assessments were based only on these two cyto-
kines. TNF-? and IL-4 were also examined after the third immu-
one vaccine recipient with a positive CD4?T-cell response at this
time point. None of the vaccine-induced cells produced IL-4.
TNF-? was coproduced by a large proportion of cells, although
few cells produced TNF-? without either IFN-? or IL-2. Because
a marker of cytotoxic potential. The responder at the earlier time
representation of IFN-?, IL-2, and TNF-? was observed, without
the expression of perforin. Two other vaccine recipients had
CD8?T-cell responses at this later time point. For one subject, a
large proportion of the responding cells expressed perforin (Fig.
produced by few of the CD8?T cells for either one. Thus, al-
cells with cytotoxic potential were induced.
PBMC from all three responders in the ICS assay at day 182
were further tested using the IFN-? ELISPOT assay to determine
the specific peptides among the 21 CTL epitopes in the CTL pep-
tide pool that were responsible for cytokine induction. Only one
of the three responders (study subject 067-025) had a positive
response measured using the IFN-? ELISPOT assay, and this was
to only one CTL epitope peptide, Gag 271 (single-letter amino
ground-adjusted mean SFC per 2 ? 105PBMC.
(ii) Vaccinia virus-specific antibodies to MVA. Four subjects
had detectable antibodies to MVA prior to first immunization (2
Two weeks after the first and second MVA-mBN32 immuniza-
detectable antibodies. Both MVA-mBN32 vaccine recipients with
TABLE 2 Local injection site and systemic symptoms within 3 days of vaccination with either EP-1233 DNA or MVA-mBN32 vaccines
Symptom or diagnosis
No. of subjects (% of total evaluated)
Day 0 (n ? 30) Day 28 (n ? 30)Day 84 (n ? 28)Day 168 (n ? 26)
MildModerate SevereMild ModerateSevereMild ModerateSevereMildModerate Severe
Pain and/or tenderness
Malaise and/or fatigue
Maximum of all systemic
12 (40)1 (3)09 (30)2 (7)07 (25) 3 (11)0 11 (42)3 (12)0
TABLE 3 Cellular immune responses measured by ICS for IL-2 and/or
Study treatment group Study day
No. of samples
Placebo control recipients 98
aThat is, the number of subjects with detectable IL-2 and/or IFN-? cytokine
production by CD3?CD4?and/or CD3?CD8?T cells.
Gorse et al.
cvi.asm.org Clinical and Vaccine Immunology
a reciprocal antibody titer of 50, and both experienced a 4-fold
increase in antibody titer. The anti-MVA antibody GMT was at
least 4-fold greater at 14 days after the second MVA-mBN32 im-
munization (GMT ? 694) and statistically higher than the anti-
body GMT measured 14 days after the first MVA-mBN32 immu-
nization (GMT ? 115.5) (P ? 0.0001). At 3 months after the
second MVA-mBN32 immunization the antibody GMT declined
to 167.1 (Fig. 3).
This phase 1 study tested the safety and immunogenicity of a DNA
plasmid and recombinant MVA-vectored vaccines, both encoding
genes that are restricted by the HLA-A2, -A3, and -B7 HLA super-
family allelic products, defined HTL epitopes of HIV-1 within Env,
Gag, Pol, and Vpr genes, and the PADRE HTL epitope. This repre-
sents the first clinical trial of these second-generation multiepitope
a DNA plasmid-vectored vaccine, EP HIV-1090, that encoded only
cine (38). The recombinant polypeptide protein induced polyfunc-
tional CD4?helper T-cell responses in two-thirds of subjects after
two immunizations, but the EP HIV-1090 vaccine was minimally
EP-1233 and MVA-mBN32 in our study were both safe and
HIV-1 Polyepitope DNA and MVA-Vectored Vaccines
May 2012 Volume 19 Number 5cvi.asm.org 655
well tolerated with more local injection site reactogenicity, and
more adverse events were observed after administration of the
longation after MVA vaccination in one subject may have been
related to trazodone, bupropion, or methamphetamine use re-
ported by the subject (17, 49). Amphetamines are associated with
acute and chronic cardiotoxicities, but unlike trazodone and
(50); however, this subject did not have other reasons to invoke
this as the cause. Systemic reactions were infrequent and mild in
vectored vaccine to the MVA-BN32 vaccine, but were more com-
mon than among the placebo control recipients. The subcutane-
ous nodules at the MVA-mBN32 injection site were a different
phenomenon than injection site induration, resolved more
slowly, were clinically not very significant and have not been re-
ported in studies employing other MVA vaccines.
A low rate of IL-2- and IFN-?-secreting CD4?and CD8?T-
cell responses was detected by ICS in the present study; all four
positive specimens from the three vaccine responders had T-cell
TNF-? production and perforin by the CD8?T cells. One partic-
ipant’s cells from one time point produced IL-2, IFN-?, and
TNF-? in response to the HTL peptide pool and PADRE. Cells
from one subject whose CD8?T cells produced IFN-?, TNF-?,
and perforin in response to the CTL peptide pool in the ICS assay
also produced IFN-? to one HIV-1 Gag peptide that is a CTL
epitope in the ELISPOT assay.
Low levels of CTL and HTL responses induced by this prime-
boost vaccination regimen were not due to lack of an immune
response to MVA. Antibodies to MVA-BN were induced by the
MVA-mBN32 vaccine. Also, the historical and clinical assess-
ments to exclude persons who previously had been vaccinated
with vaccinia virus were in general successful since only two vac-
at low titer to MVA at day 0. The subjects with detectable anti-
MVA antibody before vaccination did not have T-cell cytokine
highest level after the second MVA-mBN32 vaccination com-
first vaccination by 3 months after the second vaccination.
Clinical studies in the literature have reported use of the plasmid
DNA/recombinant MVA prime-boost vaccination strategy consist-
ing of other constructs expressing HIV-1 proteins with demonstra-
tion of a good safety profile, lack of cases of myopericarditis and
various rates of CD4?and CD8?T-cell immune responses to the
HIV-1 components (13, 27, 29, 30, 32, 48, 53). The dose, route of
administration, quality of immune priming achieved with the DNA
plasmid vector, sensitivity of the assays used to measure vaccine-in-
duced T-cell responses, and time after booster immunization when
peak response is measured all may affect observed response rates to
similar to that of others based largely on the use of defined CTL
epitopes (13, 27, 32). We included several design features which dif-
ferentiate the product tested here and which we believed would in-
est affinity HLA-supertype epitopes, the use of spacers to optimize
epitope processing and presentation, the inclusion of the universal
PADRE HTL epitope as an adjuvant and formulation with PVP to
augment cellular uptake of the plasmid DNA. Despite these features
We now believe the observed poor levels of immunogenicity in
suboptimal match between the vaccine design and the DNA and
MVA vectors with respect to how the epitopes are delivered to the
immune system. Most recent experimental evidence indicates a
dominant role for the cross-presentation pathway following
intramuscular vaccine immunogen delivery using DNA plas-
mid vectors, although some data support a minor role for di-
rect antigen presentation in vivo by transfected myocytes (14,
22, 51, 54, 57). Vaccinia viruses and MVA can infect profes-
may be able to mediate vaccine immunogen presentation
through the direct priming pathway. However, this infection
can impact dendritic cell maturation and function, and evi-
dence points to the dominant use of the cross-presentation
pathway for the induction of cellular immune responses to the
transgene and viral proteins for vaccinia virus vaccines and
MVA-based vectors (20, 23, 39, 56). A recent report of high
levels of HIV-1-specific CD4?(77%) and CD8?(42%) T-cell
responses to other DNA plasmid and recombinant MVA-vec-
tored vaccines that encode noninfectious virus-like particles of
HIV-1 and were administered in a heterologous prime-boost
regimen as in our study supports the need for emphasis on
FIG 3 The vaccinia virus-specific IgG antibody titers measured by ELISA are
plotted for individual vaccine and placebo control group study subjects by
study time point. Study subjects first received two immunizations with the
DNA plasmid-vectored vaccine (EP1233) or placebo control on study days 0
and 28 and then received the recombinant modified vaccinia virus Ankara
(MVA)-vectored vaccine (MVA-mBN32) or placebo control, as third and
fourth immunizations on study days 84 and 168. Closed symbols represent
anti-MVA antibody titers classified as positive for antibody in the assay. Open
symbols that represent subjects without detectable anti-MVA antibody in the
ELISA are plotted as an antibody titer of 25. The geometric mean titer (GMT)
of positive antibody titers is shown as a horizontal line within each box. The
boxes represent the interquartile range and the whiskers extend to the data
point which is no more than 1.5 times the interquartile range.
Gorse et al.
cvi.asm.org Clinical and Vaccine Immunology
optimizing the HIV-1 insert to achieve a desirable cellular im-
mune response (26).
the substrate and accumulation of amounts sufficient to induce
responses, whereas direct presentation is efficient when unstable
or rapidly degraded proteins are presented (46, 55, 63, 71). The
EP1233 and the MVA-mBN32 vaccines in our study both encode
spacer sequences designed to optimize intracellular processing of
individual epitopes within transfected or infected cells (5, 28).
Although it is feasible that this design could support efficient in-
results from our clinical trial indicate otherwise. Overall, our cur-
phase 1 clinical trial result suggest that these vector platforms are
not well-suited for use with T-cell epitope-based vaccines (9, 25,
51, 64). Vaccine delivery methods that efficiently deliver epitope-
be investigated to further develop the concept of epitope-based
Conclusions. In summary, our study demonstrated higher re-
actogenicity and adverse event rates with MVA-mBN32 com-
pared to the DNA plasmid-vectored vaccine, but the MVA was
still well-tolerated. These vaccines given in high dose and prime-
boost sequence did not elicit adequate HIV-1-specific T-cell im-
mune responses and further optimization of the HIV-1 T-cell
epitope-based constructs expressed by DNA plasmids and MVA-
vectored vaccines is needed.
This study was supported by grants from the NIAID to Saint Louis Uni-
versity (U01AI-48021), to Pharmexa-Epimmune (contract N01AI-
30031), to the HIV Vaccine Trials Network (U01AI-46747), and to the
following clinical study sites: University of Rochester, Rochester, NY
(U01AI-069511); Vanderbilt University, Nashville, TN (CTSA grant UL1
RR024975 from the NCRR/NIH); and the San Francisco Department of
Health, San Francisco, CA (1 U01 AI-069496-01).
We thank all past and present members of the HVTN-067 team whose
sity. We thank Scharla G. Estep, Pharmacist, Pharmaceutical Affairs Branch
and trial conduct. We also thank Danielle Harden for statistical assistance at
SCHARP, Cathy Bunce at the University of Rochester, and the study staff at
Vanderbilt University and the HIV Research Section at the San Francisco
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