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C
AU1 comparative Evaluation of Prophylactic SIV Vaccination
Modalities Administered to the Oral Cavity
AU2 cOmkar Chaudhary,
1,2
Lingyun Wang,
1,2
Deepanwita Bose,
1,2
Vivek Narayan,
1,2
Ming Te Yeh,
3
Angela Carville,
4
John D. Clements,
5
Raul Andino,
3
Pamela A. Kozlowski,
6
and Anna Aldovini
1,2
A
AU3 cbstract
Attempts to develop a protective HIV vaccine have had limited success, especially in terms of inducing
protective antibodies capable of neutralizing different viral strains. As HIV transmission occurs mainly via
mucosal surfaces, HIV replicates significantly in the gastrointestinal tract, and the oral route of vaccination is a
very convenient one to implement worldwide, we explored three SIV vaccine modalities administered orally
and composed of SIV DNA priming with different boosting immunogens, with the goal of evaluating whether
they could provide lasting humoral and cellular responses, including at mucosal surfaces that are sites of HIV
entry. Twenty-four Cynomolgus macaques (CyM) were primed with replication-incompetent SIV DNA pro-
virus and divided into three groups for the following booster vaccinations, all administered in the oral cavity:
Group 1 with recombinant SIV gp140 and Escherichia coli heat-labile toxin adjuvant dmLT, Group 2 with
recombinant SIV-Oral Poliovirus (SIV-OPV), and Group 3 with recombinant SIV-MVA. Cell-mediated re-
sponses were measured using blood, lymph node, rectal and vaginal mononuclear cells. Significant levels of
systemic and mucosal T-cell responses against Gag and Env were observed in all groups. Some SIV-specific
plasma IgG, rectal and salivary IgA antibodies were generated, mainly in animals that received SIV DNA +
SIV-MVA, but no vaginal IgA was detected. Susceptibility to infection after SIV
mac251
challenge was similar in
vaccinated and nonvaccinated animals, but acute infection viremia levels were lower in the group that received
SIV DNA +SIV-MVA. Nonvaccinated CyM maintained central memory and total CD4
+
T-cell levels in the
normal range during the 5 months of postinfection follow-up as did the vaccinated animals, precluding eval-
uation of vaccine impact on disease progression. We conclude that the oral cavity vaccination tested in these
regimens can stimulate cell-mediated immunity systemically and mucosally, but humoral response stimulation
was limited with the doses and the vaccine platforms used.
Keywords: AIDS, SIV vaccine, DNA vaccine, mucosal immunity
Introduction
N
AU4 catural transmission of HIV and SIV occurs pre-
dominantly via mucosal surfaces. Systemic dissemina-
tion occurs usually within a few days and, at that point, the
intestinal mucosa is a site of major virus replication and CD4
+
T-cell depletion in addition to lymphoid organs.
1–5
To control
both entry and systemic dissemination, an effective HIV
vaccine may need to stimulate both arms of the adaptive
immune system, eliciting cellular and humoral immunity
systemically as well as at mucosal surfaces. Among mucosal
routes, the oral route of vaccination is particularly appealing
because of its ease of administration, an issue particularly
important in settings with limited health care resources.
A few oral vaccines are in use: the attenuated oral poliovirus
vaccine (OPV), the most widely used, the typhoid, the rota-
virus, and three cholera vaccines, one based on a live-
attenuated organism, and live vaccines have been shown to be
1
Department of Medicine, Boston Children’s Hospital, Boston, Massachusetts, USA.
2
Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
3
Department of Microbiology and Immunology, UCSF, San Francisco, California, USA.
4
Biomere, Worcester, Massachusetts, USA.
5
Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA.
6
Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, New Orleans,
Louisiana, USA.
AIDS RESEARCH AND HUMAN RETROVIRUSES
Volume 00, Number 00, 2020
ªMary Ann Liebert, Inc.
DOI: 10.1089/aid.2020.0157
1
AID-2020-0157-ver9-Chaudhary_1P
Type: research-article
AID-2020-0157-ver9-Chaudhary_1P.3d 10/16/20 8:22am Page 1
on average more immunogenic than vaccines based on killed
pathogens.
6–11
These vaccines are administered in the oral
cavity, but they are swallowed and induce immunity in both
the oral cavity and the GI tract. They are safe, provide a high
level of protection, and provide evidence for oral vaccination
as an important approach to disease prevention, especially
when a pathogen enters via mucosal surfaces as is the case in
the vast majority of HIV transmissions.
It is unclear to what extent oral vaccination could protect in
the context of HIV/SIV infection.
12–14
Data from mucosal
immunization in humans indicate that responses are maximal
at the site of antigen exposure and present to a lesser degree at
other mucosal sites as well, supporting the notion of limited
compartmentalization of the mucosal immune system.
15–17
Immunization at one mucosal site can lead to an immune
response at other mucosal effector sites, as immunologically
competent cells with homing receptors specific for mucosal
sites circulate among different sites, but there are differences
in the magnitude observed at different sites. The highest
antibody (Ab) titers are usually achieved at the mucosal site
of antigen exposure and decrease at distant sites.
17–21
Al-
though HIV replicates at significant levels in the intestinal
mucosa,
1,22–24
it enters the body predominantly via the gen-
ital tract or the rectum, likely requiring more disseminated
immunity than pathogens that infect exclusively orally.
12–14
OPV is one of the most successful oral vaccines currently
used in the world.
25,26
It is very safe—in extremely rare
cases, *1 in every 2.7 million first doses of the vaccine, OPV
can cause paralysis
26
- and using a HIV-recombinant OPV
could provide a cheap way to simultaneously immunize
against HIV and poliovirus. While live-attenuated OPV has
been replaced by the less efficacious but safer, inactivated
poliovirus vaccine (IPV) in most countries, it is still in use in
many countries. Previous testing of recombinant SIV-OPV,
using a mixture of 20 recombinants expressing small SIV
fragments, indicated that excellent antibody responses can be
obtained with this approach, and partial protection from in-
fection and disease was also achieved in Cynomolgus ma-
caques (CyM).
27–29
Furthermore, pre-existing immunity to
OPV did not prevent development of immunity to a recom-
binant protein subsequently delivered by OPV.
30
One of the
shortcomings of the SIV-OPV was the apparent limited
stimulation of cell-mediated immunity, at least in CyM, but
technical limitations in analysis of T-cell responses at the
time of these experiments may have affected the evaluation.
In previous preclinical trials, we achieved significant sys-
temic and mucosal T-cell responses with a SIV or SHIV
DNA-rMVA approach after oral and intestinal immuniza-
tions.
31–35
However, IgG and IgA responses were sporadic,
short lived, and usually undetectable by the day of challenge.
Differences in systemic and vaginal anti-SIV responses were
observed in animals vaccinated orally vs. intestinally, and
these immunizations provided some protection from infec-
tion and some from disease progression. Our candidate vac-
cine, given via oral routes, resulted in two significant
outcomes: intestinal immunization provided significant pro-
tection from infection but no protection from disease pro-
gression; oral cavity immunization provided significant
protection from AIDS with >50% of the animals controlling
viremia to undetectable levels after a peak of viremia.
35
We
reasoned that it could be interesting to investigate whether a
strategy that simultaneously stimulates immunity in the oral
cavity and in the intestine could provide protection bAU5
at the
level of both infection and disease progression. The OPV is
suited for this goal, as when given orally it infects cells both
in the oral cavity and in the intestine, therefore we opted to
investigate an approach based on the combination of re-
combinant OPV and DNA immunization, where the latter is
known to be effective to induce cell-mediated immunity. This
platform was compared with two platforms, SIV DNA/MVA
and SIV DNA/protein, that we previously utilized via mu-
cosal routes.
31–35
The nonhuman primate CyM species was
selected because, at the time of this study, poliovirus, which
provides the vector used in this vaccine approach, was
thought not to be able to infect Rhesus macaques (RM)
orally.
36–38
We compared the immunogenicity and protection
of oral SIV DNA vaccine regimens boosted with either the
gp140 SIV Env protein, a SIV-recombinant attenuated oral
poliovirus (SIV-OPV) expressing SIV Gag and SIV Env,
based on an attenuated OPV vector,
39
or SIV Gag, Pol, Env
rMVA. We found that the most immunogenic approach was
the SIV DNA/MVA, while the recombinant SIV DNA/OPV
did not achieve the expected humoral responses we had an-
ticipated, although it did stimulate significant cell-mediated
immunity.
Materials and Methods
Vaccine formulations, vaccination arms, and SIV
mac251
low-dose vaginal challenge
Twenty-four female CyM, used in this study, were housed
at Biomere Biomedical Research Models, Worcester, MA,
according to an approved protocol under the guidelines es-
tablished by the Animal Welfare Act and the National In-
stitute of Health Guide for the Care and Use of Laboratory
Animals, and were divided into three groups. One animal in
Group 3 died of unrelated causes during the vaccine phase
leaving 7 animals in this group. Each animal received a total
of three DNA doses on day 1, week 8, and week 16 that
consisted of 1 mg of pVacc7 DNA ( bF1
Fig. 1A). The DNA
plasmid pVacc7 used for priming is a derivative of pVacc6 in
which SIV
mac239
Env was replaced by SIV
smE543
env and
includes a full SIVmac239 genome with multiple mutations
in the NC basic domain, in the functional domains of RT, INT,
and PR, and a stop codon at the beginning of the vpr gene.
Gene expression is under the control of the CMV promoter,
replacing the 5¢LTR, while the 3¢is replaced by a poly-
adenylation signal. The DNA sequence was confirmed by se-
quencing, and the profile of viral particles produced was
evaluated by 293T transfection and subsequent Western blot
using macaque SIV-positive sera.
35
DNA was formulated in
1 mL of 20 mM DOTAP (1,2-dioleoyl-3-trimethylammonium-
propane, cholesterol (1:1)(Encapsula Nano Sciences). On
weeks 8, 16, and 24, Group 1 animals (n=8) were immunized
with 1 mg of SIV rgp140smE543 (a.a. 23–671; Genebank No.
AAC56565; Immune Technology Corp, New York, NY) and
100 lgofE. coli double mutant heat-labile toxin (dmLT)
adjuvant.
40
Group 2 (n=8) received 5 ·10
7
pfu SIV-OPV,
and Group 3 (n=8) received 5 ·10
8
pfu SIV gag,pol,env-
expressing MVA (DR2 vector created by Dr. Bernie Moss,
National Cancer Institute).
41–44
SIV-OPV was constructed by
inserting the sequence of the entire SIV gag (nucleotides
1309–2841 of SIV
mac239
) or SIV Env (nucleotides 6860–
9499)
45
in the plasmid pSabin2-eGFP,
39
replacing GFP
2 CHAUDHARY ET AL.
AID-2020-0157-ver9-Chaudhary_1P.3d 10/16/20 8:22am Page 2
between two poliovirus protease cleavage sites. SIV gag and
Env proteins are cleaved from the OPV polyprotein by the
poliovirus protease and independently expressed intracellu-
larly. The two corresponding viruses were mixed in equal
amounts (2.5 ·10
7
each), and the mix was designated SIV-
OPV. SIV-OPV and SIV-MVA recombinant vaccines were
formulated in PBS in a final volume of 500 mL. Recombinant
MVA and OPV doses were selected based on what is the
optimal dose for each vector. DNA and boosting vaccines
were administered to the animals in the oral cavity, applied to
the mucosa between the gum and the cheek while the animals
were sedated. In the case of SIV-MVA, it was applied to the
oral cavity mucosa by scarification, as normally done for
poxvirus vaccination. Eight weeks after the last vaccination,
FIG. 1. Study overview and SIV-specific systemic IgG responses in vaccinated animals. (A) Vaccination scheme and
animal groups. Levels of antibodies to (B) SIV lysate and (C) SIV gp140 Env measured in plasma using ELISA. The left
panels show the concentrations in individual animals before vaccination and at weeks 26 and 28 (corresponding to 2 and 4
weeks after the last immunization). Bars denote the median. Panels on the right show the fold increase over prevaccination
level for each animal on week 26 when peak responses were typically observed.
AU11 cPostimmunization concentrations had to be
threefold higher than the preimmune concentration to be considered significant.
4C c
SIV ORAL IMMUNIZATION 3
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vaccinated animals and controls were inoculated with a low
dose (*0.2 AID
50
in a RM vaginal titration, corresponding to
100 TCID
50
) of the pathogenic SIV
mac251
virus grown in RM
PBMC (a gift from Dr. Nancy Miller and Dr. Ron Desrosiers,
stock 2010 Day 8 SIV
mac251
), administered nontraumatically
with a needleless tuberculin syringe as cell-free virus in the
vagina.
46
This virus stock is a highly diverse swarm of many
different quasispecies of CCR5-tropic viruses.
47
Challenge
was repeated weekly until RT-PCR tests in two independent
laboratories were positive for virus in plasma.
Specimens and sample processing
Blood and secretions were collected 2- and 4-weeks
postvaccination, followed by monthly collection. Rectal,
vaginal, and iliac lymph node tissues were biopsied on the
day of the first vaccination and 2 weeks after each vaccina-
tion. Plasma and peripheral blood mononuclear cell (PBMCs)
were isolated from EDTA anti-coagulated whole blood ac-
cording to the instructions of the manufacturer (Amersham
Pharmacia, Uppsala, Sweden). Mononuclear cells (MNC)
from lymph nodes and mucosal tissues were obtained from
tissue biopsies. Briefly, after Telazol anesthesia, seven to
eight biopsies/animal/time points were obtained from the
rectum and vagina and lymph nodes using sterile forceps (for
rectal and vaginal) and a small pinch biopsy device (Olympus
endoscopic biopsy forceps). MNC from tissues were obtained
with mechanical dissociation using GentleMACS dissociator
(Miltenyi Biotech, Paris, France). Suspensions were passed
through a 70 mm pore size cell-strainer and washed with 10%
FBS in RPMI.
48
PBMC and MNC are cryopreserved in FBS
with 10% DMSO. Rectal, salivary and vaginal secretions
were collected using Weck-Cel sponges (Beaver Visitec,
Waltham, MA) premoistened with 50 lL of Dulbecco’s
phosphate-buffered saline as described previously.
49
Evaluation of SIV-specific IgG in plasma samples
and IgA in rectal and vaginal secretions
Antiviral and total IgA and IgG in secretions and IgG in
plasma were measured by ELISA as described.
33,34,48,49
Briefly, for the SIV ELISAs, microtiter plates were coated
overnight with 100 ng/well SIV rgp140smE543 (Immune
Technology) or 100 lL per well of 1/400 aldrithiol-2-
inactivated SIV particles (kindly provided by Dr. Jeff Lifson,
Leidos Biomedical Research, Frederick, MD) that had been
lysed with TritonX-100 detergent. Using monoclonal anti-
bodies, the SIV lysate was found to contain gp41 but not
gp120 at the 1/400 coating dilution used. For gp140 and SIV
lysate assays, the standards were pooled serum or purified
serum IgA from SIV-infected macaques, calibrated as previ-
ously described.
32
Plates were developed using biotinylated
goat antimonkey IgA or IgG (Rockland Immunochemicals,
Gaithersburg, MD), neutravidin-labeled peroxidase and tetra-
methylbenzidene substrate (SouthenBiotech, Birmingham,
AL). Before performing IgA assays, all samples were depleted
of IgG using Protein G Sepharose as described because the
goat antimonkey IgA was found to cross-react with monkey
IgG.
49
The concentration of anti-SIV IgA or IgG in each se-
cretion was subsequently divided by the concentration of total
IgA or IgG to obtain the specific activity (ng anti-SIV antibody
per lg immunoglobulin). Specific activity or concentrations of
SIV-specific IgG in plasma were considered significant if they
were three-fold greater than those in pre-immune plasma. IgG
neutralization titers were measured as a function of Tat-
induced luciferase reporter gene expression after single round
of infection in either M7-Luc or TZM-bl cells.
50
The viruses
used in the neutralization assay were a TCLA stock of SIV-
mac251 (M7-Luc assay) and SIVmac239 (TZM-bl assay).
Titers of SIV-specific neutralizing antibodies are the plasma
dilution at which relative luminescence units were reduce
50% compared with virus control wells after subtraction of
background.
Immunophenotyping and intracellular cytokine staining
10
5
MNC and 10
6
PBMCs were incubated for 14 h with
medium (Unstimulated), 1 lg/mL pools of 15-mer SIV Gag
or SIV Env peptides. Cells incubated with 10 ng/mL PMA
(4-a-phorbol 12-myristate 13-acetate; Sigma) and 1 lg/mL
ionomycin (Sigma) or without any stimulation provides re-
spectively positive and negative controls. Cultures contained
Brefeldin A (BD GolgiPlug Cat. # 555029; BD Biosciences)
and 1 mg/mL of anti-CD49d and anti-CD28. PBMCs and
MNC were washed, stained for surface markers in the dark,
followed by fixation, permeabilization. Characterization of
CD4
+
and CD8
+
T cells in PBMC was conducted according to
previously published procedures.
51
After the permeabiliza-
tion, cells were intracellularly stained for cytokine expression
with anticytokine antibodies for 1 h in the dark according to
previously described procedures.
52
The following antibodies
were used in this study: anti-CD3-pacific blue/PerCp-Cy5.5
(clone SP34–2), anti-CD4-Amcyan (clone L200), anti-CD8-
APC-Cy7 (clone RPA-T8), anti-TNFa-PE (clone MAb11),
anti-IFNc-Alexa Fluor-700 (clone B27), IL-2-APC (clone
MQ1–17H12), anti-CD95-FITC (clone DX2), and anti-
CD28-Pe-Cy5 (clone CD28.2). Gates for all antibodies were
estimated in Fluorescence Minus One (FMO) staining sam-
ples. A viability dye (VIVID, LIVE/DEAD kit; Invitrogen)
was added to the antibody cocktail to exclude dead cells. The
acquisition of cells was done on LSRII flow cytometry using
FACSDIVA software. The data were analyzed using FlowJO
version 10.5.2 software (TreeStar, Ashland, OR). Data for
peptide-stimulated populations are reported as percentage,
determined after subtracting the percentage of positive cells
detected in unstimulated cells for each sample. Evaluation of
single, double, or triple-positive cells was carried out using
FlowJo Boolean gate.
Viral load quantitation
Plasma SIV RNA levels were measured by real-time RT-
PCR assay in Dr. Lifson’s facility as described.
53
The Lif-
son assay has a threshold sensitivity of 30 copy equivalents
per milliliter. Interassay variation is <25% (coefficient of
variation). Mean viral loads were calculated by transform-
ing the number in its logarithmic value and averaging the
logarithmic values of all the animals of the group at one
specific time point.
Euthanasia
Animals were euthanized because of closure of the study,
or earlier if they developed signs and symptoms consistent
with the definition of AIDS. AIDS was defined as being SIV
+
(detectable viremia) and experiencing one of the following
4 CHAUDHARY ET AL.
AID-2020-0157-ver9-Chaudhary_1P.3d 10/16/20 8:22am Page 4
criteria: 1—weight loss >15% in 2 weeks or >30% in 2
months; 2—documented opportunistic infection; 3—per-
sistent anorexia >3 days without explicable cause; 4—severe,
intractable diarrhea, 5—progressive neurologic signs, 6—
significant cardiac and/or pulmonary signs, 7—loss of CD4
+
T cells <200 or 10%.
Statistical analysis
Calculations and statistical analyses were performed
using the GraphPad Prism version 7 software. Two-tailed
Fisher’s exact test was used to compare the frequency of
IgA responses between groups. Between-group compari-
sons were carried out by two-tailed, t-test or Mann–
Whitney test, and among four groups one-way ANOVA and
Mann–Whitney test were used. Results of statistical ana-
lyses were considered significant if they produced pvalues
£.05. Display of multicomponent distributions was per-
formed with SPICE v5.2 (freely available from http://exon
.niaid.nih.gov/spice/).
54
Results
Vaccine data
Oral vaccination can stimulate systemic and mucosal anti-
SIV responses. We reasoned that the combination of DNA,
an excellent stimulator of T-cell responses, and SIV-OPV
could provide a very interesting vaccine platform to explore
and compare to what we accomplished using SIV DNA+
SIV-MVA via the oral route and with a vaccine composed of
SIV DNA+SIVgp140, which constitutes
AU6 ca more heavily
explored approach to SIV and HIV immunization. During the
vaccination phase of the study, the systemic and mucosal
antibody responses were measured in plasma and secretions.
The oral vaccination elicited systemic IgG responses to SIV
antigens in only two of eight Group 1 and one of eight Group
2 animals on weeks 26 and 28, corresponding to 2 and 4
weeks after the last vaccination (Fig. 1B, C). Plasma IgG
antibodies against SIV lysate or gp140 Env were more fre-
quently observed in Group 3 animals immunized with SIV
DNA/rMVA (Fig. 1B, C). These IgG responses were nega-
tive in neutralization assays. A similar trend was observed for
rectal IgA responses (
F2 cFig. 2A, B). The oral cavity vaccination
stimulated rectal IgA responses to SIV antigens in only two
Group 1 and Group 2 animals, whereas five of the seven
animals in Group 3 had detectable anti-SIV or -gp140 Env
IgA in rectal secretions. The same number of Group 3 ani-
mals also developed salivary IgA antibodies to SIV antigens,
and these responses were detected in 50% of Group 1 animals
that were orally boosted with SIV gp140 and dmLT adjuvant.
However, only three of the eight Group 2 animals boosted
with SIV-OPV had SIV-specific salivary IgA antibodies
(Fig. 2A, B), and none of the animals in any group were found
to have SIV-specific IgA in vaginal secretions (not shown).
Thus, the SIV-OPV boost failed to stimulate the significant
antibody responses we expected from an OPV-based ap-
proach. These results indicate that the oral cavity immuni-
zation is a suitable route to induce systemic, salivary, and
rectal humoral responses using diverse approaches, but ap-
propriate doses and formulations need to be systematically
evaluated to identify those that more consistently generate
antibodies.
Vaccine-induced systemic and mucosal SIV-specific
CD4
+
and CD8
+
T-cell responses
As anti-SIV cell-mediated responses, in particular CD8
+
T-cell responses, are critical in viremia control and long-term
protection from disease progression, we evaluated the levels
and breath of vaccine-induced cell-mediated immunity.
Virus-specific T-cell responses were measured by intracel-
lular staining and flow cytometric analysis at multiple time
points, and are reported as percentage of antigen-specific
CD4
+
and CD8
+
T-cells producing TNFa, IFNc, and IL-2 in
MNC after stimulation with SIV Gag or Env peptide. The
analysis of the systemic SIV-specific immune responses,
measured during immunization in PBMC and reported as the
sum of anti-Gag and anti-Env percentages, revealed that all
three vaccine modalities could stimulate significant anti-SIV
cell-mediated responses in PBMCs and that, although con-
sistently slightly higher in Group 3, these differences were
not statistically significant ( bF3
Fig. 3A).
We also evaluated bAU7
long-term magnitude and functionality
of these anti-SIV responses, measuring the fraction of SIV-
specific CD4
+
and CD8
+
T cells producing TNFa, IFNc, and
IL-2 as single, double, or triple-positive cells, on week 32, 2
months after the last immunization, when the immune re-
sponse has usually contracted to the memory level (Fig. 3B).
On the day of challenge, Group 3 maintained levels of anti-
SIV CD4
+
T-cell immune responses *2-fold higher than the
other groups; the same was true for anti-SIV CD8
+
T-cell
responses when both Groups 2 and 3 were compared with
Group 1. We found that these responses were mainly
monofunctional and predominantly made of cells producing
IFNcin all groups (Fig. 3B). However, variable percentages
of multifunctional responses, varying between 8.7% and
38.15% of the total when both CD4
+
and CD8
+
T-cells were
considered, were present in the different groups.
We evaluated the ability of the vaccine candidate to induce
SIV-specific T-cell responses in tissues during the time
course of the immunization regimen by investigating the
percentages of SIV-specific mucosal MNC present in ingui-
nal lymph nodes, rectal and vaginal biopsies, collected during
immunization. Samples were collected at five time points, on
day of first immunization, 2 weeks after second, third and
fourth immunization, when the responses were likely to reach
peak, and 8 weeks after the last immunization, when the
effector component of the response is usually substantially
reduced. Oral vaccine administration stimulated CD4
+
and
CD8
+
T-cell responses at all examined sites with variable
magnitude that increased over the course of the immuniza-
tions. When SIV-specific responses were analyzed in rectal,
vaginal, and lymph node MNC on week 26, 2 weeks after the
last immunization, significant levels of Gag and Env specific
for CD4
+
and CD8
+
T-cell responses were observed in all
vaccinated groups in all tissues ( bF4
Fig. 4A). The differences in
SIV-specific immune responses did not reach significance
when compared between the groups, although rectal and
vaginal responses were consistently higher than those in
lymph node MNC. On week 32, 8 weeks after last immuni-
zation, the responses had contracted to a frequency varying
from *0.2% to 0.4% for the individual cytokines with T cells
producing IL-2, or IFNcor TNFasimilarly represented in
SIV-specific rectal and vaginal CD4
+
and CD8
+
T cells and
values varying between 0.6% and 1.2% (Fig. 4A, B). When
SIV ORAL IMMUNIZATION 5
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multifunctionality of antigen-specific responses was inves-
tigated with Boolean analysis on the same time point sam-
ples, a large fraction of the response, ranging from 70% to
90% of the total, was characterized by monofunctional cells
producing each of the three cytokines tested. However,
polyfunctional responses were detectable in all tissues with
frequency varying from *10% to 32% of the total (Fig. 4C).
Analysis of SIV-specific CD4
+
and CD8
+
T-cell
responses memory and effector subsets
We compared the central memory (C
M
) and effector
memory (E
M
) subset fractions of the cell-mediated response
in blood and tissue MNC at peak after the last immunization
(week 26) and 2 months after the last immunization (week
32). On week 26, C
M
or E
M
varied within a twofold range
among the three immunization regimens, both being consis-
tently lower in Group 1 that received boosting immunizations
not including a SIV-Gag antigen but only SIV Env (
F5 cFig. 5A).
However, differences between groups did not reach statistical
significance, probably because of the small size of the groups
and of the variability in the magnitude of the responses
among animals within the same groups. On week 32, the SIV
responses were also of comparable magnitude in all three
groups, although reduced compared with week 26 peak re-
sponses (Fig. 5A). When the distribution between the C
M
and
E
M
CD4
+
and CD8
+
T cells was evaluated for each animal
within each vaccination group, the average group C
M
/E
M
ratio of SIV-specific cells present in tissue compartments on
week 32 was higher compared with week 26 for most com-
parisons ( p<.05), supporting a larger contraction of the E
M
than the C
M
immune response since its peak and the persis-
tence of significant C
M
antigen-specific responses.
When the fraction of antigen-specific responses producing
the same cytokine alone or in combination was compared
with each of the other two cytokines in C
M
and E
M
of SIV-
specific CD4
+
and CD8
+
T cells, IL-2 was the predominantly
expressed cytokine in CD4
+
/E
M
+
and CD8
+
/E
M
T cells of
FIG. 2. Rectal and salivary IgA responses in vaccinated animals. Shown are the postimmunization fold increases in
specific activity (ng of specific antibody per lg total IgA) to (A) SIV lysate and (B) SIV gp140 Env measured in rectal and
salivary secretions. Fold increases were calculated by dividing the week 26 or 28 specific activity by that measured on week
0. To be considered significant, the specific activity had to exceed the dashed line (representing the mean +3SD of negative
controls) in the graphs. Bars represent the median for each group.
4C c
6 CHAUDHARY ET AL.
AID-2020-0157-ver9-Chaudhary_1P.3d 10/16/20 8:22am Page 6
Group 2 (Fig. 5B, p<.05) and the multifunctional C
M
CD4
+
T-cell SIV Gag+Env-specific responses were a significantly
larger percentage than that of the E
M
CD4
+
fraction ( p<.05).
The other two groups had similar levels of C
M
and E
M
antigen-specific multifunctional responses, supporting a
more prolonged survival of E
M
responses in these groups and
in CD8
+
C
M
and E
M
responses the largest fraction of antigen-
specific cells secreted IFNc(p<.05).
When all the above data are considered together, we
concluded that all vaccine modalities given in the oral cavity
were similarly effective in stimulating cell-mediated T-cell
responses at multiple sites, including sites of HIV exposure,
but much less effective at stimulating humoral responses. The
vaccination utilizing the SIV DNA-MVA modality produced
more consistent humoral results, although some low-level
responses were observed in a couple of animals of the other
two groups as well. These data indicate that the oral cavity
can be explored as a site to stimulate broadly distributed
responses but higher doses of vaccine, more immunizations,
or different formulations might be necessary.
FIG. 3. Circulating cell-mediated total percentages of SIV Gag and SIV Env-specific T-cell responses during immuni-
zation phase. (A) Geometric means of each vaccinated group is shown during the immunization phase as percentage of
CD4
+
T-cells and CD8
+
T-cells producing IFNc, TNFa, and IL-2, measured in PBMC by ICS upon stimulation with Gag or
Env peptide pools. The graphs show the total SIV-specific T-cell responses (Gag plus Env) for vaccinated groups: SIV-
DNA+SIV-gp140 vaccinated animals are represented in blue (Group 1), SIV-DNA+SIV-OPV in brown (Group 2), SIV-
DNA+SIV-MVA in green (Group 3). Statistical significance between groups was tested with one-way ANOVA assay
(p<.05). (B) Qualitative analysis of systemic SIV Gag and Env-specific cell-mediated responses for each vaccinated group
2 weeks on the first day of challenge (week 32), 8 weeks after last immunization. Pie graphs representing the diversity of
CD4
+
(left panels) and CD8
+
T-cell responses (right panels) with different colors representing proportionally to percentages
of the production of IFNc, TNFa, and IL-2 alone and the simultaneous production of ‡2 cytokines combined are shown. The
total mean percentage of SIV-specific multifunctional responses for each group is shown on the upper left side of each pie.
ICS, intracellular cytokine staining; PBMC, peripheral blood mononuclear cell.
4C c
SIV ORAL IMMUNIZATION 7
AID-2020-0157-ver9-Chaudhary_1P.3d 10/16/20 8:22am Page 7
FIG. 4. Tissue SIV-specific cell-mediated
T-cell responses during immunization. SIV
Gag+Env-specific (A) CD4
+
and (B) CD8
+
T-cell responses were detected in MNC
isolated from vaginal and rectal and lymph
node biopsies during the immunization
protocol, and the group average is re-
presented as a bar graph for each group.
Each bar represents the sum of Gag- and
Env-specific T-cells producing IFNc, IL-2,
and TNFaevaluated as individual cytokines
after stimulation with Gag or Env peptide
pools. (C) Functionality of lymph node,
vaginal and rectal SIV-specific T-cell re-
sponses on the first day of challenge (week
32). Pie graphs illustrate the diversity of
Gag+Env-specific CD4
+
T-cells and CD8
+
T-cells producing IFNc, TNFa, and IL-2 and
colors are proportionate to the percentages
of single, double, or triple positives for each
group. The SIV-specific T-cell mean per-
centage of the total CD4
+
or CD8
+
T cells is
shown for each group beneath each pie. The
total mean percentage of SIV-specific mul-
tifunctional responses for each group is
shown on the upper left side of each pie.
MNC, mononuclear cell.
b4C
AID-2020-0157-ver9-Chaudhary_1P.3d 10/16/20 8:22am Page 8
8
FIG. 5. Central memory (CD95
+
/CD28
+
) and effector memory (CD95
+
/CD28
-
) SIV-specific cell-mediated T-cell re-
sponses during the immunization protocol. (A) SIV Gag+Env-specific CD4
+
and CD8
+
T-cell responses were detected in
PBMC and in MNC isolated from lymph node, vaginal and rectal biopsies 2 weeks after last immunization (week 26) and on
first day of challenge (week 32), and the group average is represented as a bar graph for each group. Each bar represents the
sum of anti-C
M
and E
M
Gag +Env-specific T-cells producing IFNc, IL-2, and TNFaevaluated as individual cytokines.
CD3
+
T cells were gated within total PMBC, CD4
+
and CD8
+
T cells were gated within the CD3
+
population, and
percentages of cytokine
+
cells were estimated in these populations for each cytokine and added to provide the total.
(B)
AU12 cFunctionality of PBMC SIV-specific T-cell responses on first day of challenge (week 32). Pie graphs illustrate the
diversity of Gagmen-specific CD4
+
T-cells and CD8
+
T-cells producing IFNc, TNFa, and IL-2 as percentages of single,
double, or triple positives for each group. The SIV-specific T-cell mean percentage of the total CD4
+
or CD8
+
T cells is
shown for each group beneath each pie.
4C c
AID-2020-0157-ver9-Chaudhary_1P.3d 10/16/20 8:22am Page 9
9
Resistance to challenge and disease progression
To evaluate levels of protection provided by the vaccina-
tion, animals were vaginally challenged with repeated low
doses of SIV
mac251
(
F6 cFig. 6). No significant differences were
observed when the median number of challenges was com-
pared among groups, indicating that the levels of immune
responses present in the vaginal tissues were not sufficient to
provide protection (Fig. 6A). When viral loads were evalu-
ated, early control of viremia from peak levels to week 8 was
observed in Group 3 compared with other groups. However,
after that, all three groups equally controlled viremia
(Fig. 6B). The level of viremia control observed in the control
group was significantly higher than that previously observed
in infected RM
32,33,35
and viremia was at least half log lower
than what is normally observed for CyM and *2 logs of what
was observed in another study where SIV-OPV was inves-
tigated in CyM.
29,55,56
C
M
CD4
+
T-cell counts, which usually
decline earlier after infection than total CD4
+
T-cell counts
in RM, recovered after the declined observed 2 week post-
infection to levels comparable with those preinfection and
remained stable during the postinfection follow-up in all
groups (Fig. 6C, group average, Fig. 6E, individual absolute
counts/mm
3
). Total CD4
+
T-cell counts declined *10% 2
weeks after infection in all groups and remained at that level
for the remaining of the follow-up (Fig. 6D, group percentage
FIG. 6. Low-dose vaginal SIV
mac251
challenge outcome. (A)
AU13 cThe Kaplan–Meier graph represents the number of non-
traumatic challenges received for each vaccinated group until a positive infection was detected in plasma. The nontraumatic
challenges were stopped after 26 challenges when all the animals had become positive. The dotted line intersects the point
in each curve corresponding to 50% of the animals being infected. (B) Average viral loads in the groups. (C) PBMC C
M
(CD95
+
/CD28
+
) CD4
+
T-cell percentages postinfection. (D) PBMC total CD4
+
T-cell percentages postinfection. (E, F)
Absolute number of PBMCs C
M
(E) and total
78
CD4
+
T-cells during the course of the infection.
4C c
10 CHAUDHARY ET AL.
AID-2020-0157-ver9-Chaudhary_1P.3d 10/16/20 8:22am Page 10
averages, Fig. 6F, individual absolute counts/mm
3
,p=ns
when time 0 values were compared with those of week 20
postinfection), indicating that the levels of viremia were
compatible with preservation of these cells for the length of
the follow-up in vaccinated group and controls. Although a
milder course of SIV infection in CyM versus RM has been
reported, preservation of CD4
+
T cells occurred in these
animals at levels that were not statistically different than
when noninfected, unlike what was observed by others in
SIV-infected CyM, where significant decrease to <50% of
preinfection value in both C
M
and total CD4
+
T cells has been
observed [Fig. 2 in
55
]. The unusual resistance of these control
animals to SIV-mediated CD4
+
T-cell decline reduced the
ability to reveal any level protection from the vaccination, if
any had been induced.
Discussion
As of today, only one vaccine modality tested in clinical
trials and administered intramuscularly (i.m.) achieved par-
tial protection (31.2% efficacy), the RV-144 ALVAC-HIV
(v CP1521) plus AIDSVAX,
57
AU8 cnot only supporting the fea-
sibility of achieving protection but also requiring further
improvement. However, the recent trial HVTN702 in South
Africa, based on the same modality of vaccination, was ter-
minated because of lack of efficacy.
58
This trial utilized
MF59 instead of Alum, and the difference in outcome was
predicted in a preclinical SIV
mac251
trial in RM that compared
side by side the two adjuvants, supporting the different
contributions of each vaccine adjuvant to the vaccine out-
come and the need to test each variable independently.
59
Achievement of appropriate antibody responses, in particular
neutralizing antibody, is considered the holy grail of the
successful HIV vaccine [
60–62
and references therein].
In previous studies in RM, we have shown that mucosal
immunization stimulating cell-mediated immune responses
can achieve protection from disease progression but did not
protect from acquisition of infection, although in some cases
infection was delayed.
32–34,63
Intranasal vaccination was
more efficient in eliciting cellular and humoral virus-specific
responses at mucosal sites than the same regime administered
systemically (i.m.) and provided better protection from dis-
ease progression after rectal or vaginal challenge. SIV-
specific CD4
+
and CD8
+
IFNcproducing T cells present at
the time of challenge correlated with the subsequent control
of the viremia and longer survival of these animals. However,
it did not stimulate significant humoral responses in the cir-
culation or vaginal mucosa. In a recent study, where HIV-
MVA followed by recombinant trimeric HIV gp120 with
dmLT was administered with a pressurized, needle-free in-
jection devise both buccally and sublingually in RM, Jones
et al. found that this immunization strategy provided an ef-
fective route to induce immunity and partial protection
against rectal SHIV challenge.
64
The study reported here was initiated with the goal of using
a vector based on OPV, a virus known to be excellent at
stimulating long-lived antibody responses and in particular
mucosal IgA, to improve the stimulation of humoral immu-
nity and the oral cavity route as easily accessible and prac-
tical in resource-limited settings. The importance of mucosal
IgA responses is highlighted in humans by the detection of
HIV-specific IgA in semen or vaginal secretions of some
cohorts of HIV-1 resistant, heavily exposed but seronegative
sex workers, which has been interpreted as indication that
local IgA, induced by viral exposure, can protect during
subsequent exposures.
65–68
Secretory IgA antibodies have
been shown to inhibit host entry and dissemination of path-
ogens into systemic compartment, and this mechanism,
combined with others, such as antibody-dependent cellular
cytotoxicity (ADCC) by FcaR
+
tissue macrophages, could
work for HIV as well.
69–71
An additional modality capable of inducing significant
antibodies is the use of a purified protein as priming and/or
boosting tool of antipathogen responses. Comparative studies
in humans and NHP that utilized protein immunogens effi-
ciently internalized at all mucosal surfaces established that
local immunization stimulates mucosal IgA responses often
limited to the vaccination site or of less magnitude at more
distant sites (reviewed in
72
). These differential antibody re-
sponses may be less apparent with replicating vaccines. For
example, a replication-competent adenovirus type 5 vector
with SIV genes was recently reported to induce SIV-specific
mucosal IgA responses in multiple mucosal tissues regardless
of whether it was administered to female macaques by the
sublingual, rectal, vaginal, or nasal +intratracheal mucosal
routes.
73
The protein dose used here did not provide satis-
factory results and may have been too low, as uptake of to-
pically applied proteins in the oral cavity is inefficient.
Antigens formulated in conjunction with adjuvants, as we did
in this immunization, avoid inducing tolerance and failure to
stimulate an immune response is dose- or antigen depen-
dent.
74–76
Achieving immune responses with oral protein
immunization may require testing multiple doses, formula-
tions, and methods of application to find the dose high enough
to induce the optimal response.
The approaches used here and compared with the
DNA/MVA platform previously used did not consistently
stimulate the desired humoral responses but were able to
stimulate cell-mediated responses. As the last boost did not
include the SIV DNA component and managed to increase
the levels of cell-mediated responses, it provided the evi-
dence that these different boosting modalities were immu-
nogenic on their own. In the case of the gp140 boost,
increasing the amount of protein, and perhaps adjuvant, could
work in a more significant and consistent way. In the case of
the SIV-OPV vectors, a few issues could have affected the
outcome. The vaccination was done with sedated animals that
were not swallowing, possibly limiting the spreading of it to
the entire gastrointestinal tract. Alternatively, the recombi-
nant SIV-OPV stability in vivo may have been more limited
than what was observed in vitro, with reduced in vivo viral
replication, and the possibility that a higher dose could have
provided better results. The OPV recombinant previously
used carried much smaller inserts than those employed here,
and that feature may have favored better in vivo replica-
tion.
27–29
Despite these shortcomings, this study shows that
the oral cavity can be considered a useful route of vaccination,
as all platforms achieved significant levels of cell-mediated
immunity and sporadic levels of humoral immunity, leaving
open the possibility of improvement for the latter if more
doses, formulations, and schedules are evaluated. Additional
studies should aim at improving vaccine platforms used via
this route, as it is highly amenable to its employment in
resource-limited environments. An unexpected result at the
SIV ORAL IMMUNIZATION 11
AID-2020-0157-ver9-Chaudhary_1P.3d 10/16/20 8:22am Page 11
time of challenge was the viremia control observed in the naive
animals, being better than that normally observed in CyM.
This species was selected because previous results indicated
that RM could not be infected orally with OPV. No significant
decline of C
M
CD4
+
T cells or total CD4
+
T cells was observed
during the 5 months after infection. This occurrence prevented
the evaluation of the vaccination on the preservation of the
immune system. In addition to species-specific issues,
AU9 cit is
possible that the virus stock used in this study may not have
had the same virulence as other related similar stocks. As RMs
have been recently shown to be infectable orally
77
and post-
infection data in these species have been more consistent in our
hands, future experiments in this species with additional re-
combinant SIV- and SHIV-OPV will be pursued.
Acknowledgments
We thank Dr. David Montefiori (Duke University, Dur-
ham, NC) for carrying out the neutralization assay on plasma
samples, Dr. Jeff Lifson (Leidos Biomedical Research, Inc.,
Frederick National Laboratory, Frederick, MD 21702) for
evaluating plasma SIV viral loads, Robert L. Wilson for
analyses of antibody responses, and Olga Nichols for pro-
duction of the SIV-MVA in the LSU Health Sciences Vector
Core Facility.
Authors’ Contribution
Omkar Chaudhary obtained PBMC and MNC from blood
and tissues, carried out analysis of immune response in
samples, prepared the first partial draft of the article and
graphs for the figures reporting the cell-mediated immune
responses, contributed to some statistical analysis, and re-
vised final draft of the article.
Lingyun Wang obtained PBMC and MNC from blood and
tissues, carried out qualitative evaluation of SIV plasma viral
loads weekly after challenge, prepared graphs for some fig-
ures, contributed to some statistical analysis, and revised final
draft of the article.
Deepanwita Bose obtained PBMC and MNC from blood
and tissues, carried out analysis of immune responses in a
subset of samples and evaluated CD4+T cell counts, pre-
pared graphs for some figures, contributed to some statistical
analysis, and revised final draft of the article.
Vivek Narayan constructed and tested SIV-OPV recom-
binants, and revised final draft of the article.
Ming Te Yeh provided the pSabin2-eGFP vector, advised all
technical aspects of oral poliovirus vaccine work and titrated
the SIV-OPV virus stocks, revised final draft of the article.
Angela Carville supervised vaccinations, sampling and
animal care at the Biomere facility, revised final draft of the
article.
John D. Clements provided the double mutant heat-labile
toxin (dmLT) adjuvant, critically contributed to the inter-
pretation of the technical and intellectual content and to the
editing of the article.
Raul Andino provided the OPV vector, supervised Dr
Yeh’s work, and critically revised final version of the article.
Pamela A. Kozlowski provided the SIV-MVA, supervised
evaluation of antibody responses, composed the corre-
sponding figures, critically contributed to the interpretation
of the technical and intellectual content and to the editing of
the article.
Anna Aldovini designed the experiments, secured funding,
coordinated, supervised, and troubleshooted all aspects of the
execution, interpreted the data, wrote the article, and edited
the figures.
Author Disclosure Statement
No competing financial interests exist.
Funding Information
This work was supported by Grant NIH-R01DE026325
to AA.
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Address correspondence to:
Anna Aldovini
Department of Medicine
Boston Children’s Hospital
Enders 861, 300 Longwood Avenue
Boston, MA 02115
USA
E-mail: anna.aldovini@childrens.harvard.edu
14 CHAUDHARY ET AL.
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AUTHOR QUERY FOR AID-2020-0157-VER9-CHAUDHARY_1P
AU1: Please note that gene symbols in any article should be formatted as per the gene nomenclature. Thus, please make
sure that gene symbols, if any in this article, are italicized.
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vaccinated group until a positive infection was detected in plasma.’’ has been edited for clarity. Please check.
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