MZC gel inhibits SHIV-RT and HSV-2 in macaque vaginal mucosa and SHIV-RT in rectal mucosa

Abstract

The Population Council's microbicide gel MZC (also known as PC-1005) containing MIV-150 and zinc acetate dihydrate (ZA) in carrageenan (CG) has shown promise as a broad spectrum microbicide against HIV, HSV and HPV. Previous data show antiviral activity against these viruses in cell-based assays, prevention of vaginal and rectal SHIV-RT infection and reduction of vaginal HSV shedding in rhesus macaques and also excellent antiviral activity against HSV and HPV in murine models. Recently we demonstrated that MZC is safe and effective against SHIV-RT in macaque vaginal explants. Here we established models of ex vivo SHIV-RT/HSV-2 co-infection of vaginal mucosa and SHIV-RT infection of rectal mucosa in macaques (challenge of rectal mucosa with HSV-2 did not result in reproducible tissue infection), evaluated antiviral activity of MZC and compared qPCR and ELISA readouts for monitoring SHIV-RT infection. MZC (at non-toxic dilutions) significantly inhibited SHIV-RT in vaginal and rectal mucosa and HSV-2 in vaginal mucosa when present during viral challenge. Analysis of SHIV-RT infection and MZC activity by one-step SIV gag qRT-PCR and p27 ELISA demonstrated similar virus growth dynamics and MZC activity by both methods and higher sensitivity of qRT-PCR. Our data provide more evidence that MZC is a promising dual compartment multipurpose prevention technology candidate.

Full text

BASIC SCIENCE
MZC Gel Inhibits SHIV-RT and HSV-2 in Macaque Vaginal
Mucosa and SHIV-RT in Rectal Mucosa
Giulia Calenda, PhD,*Guillermo Villegas, MS,*Patrick Barnable, BS,*Claudia Litterst, PhD,†
Keith Levendosky, BS,*Agegnehu Gettie, MS,‡Michael L. Cooney, MS,*James Blanchard, PhD,§
José A. Fernández-Romero, PhD,*Thomas M. Zydowsky, PhD,*and Natalia Teleshova, MD, PhD*
Abstract: The Population Council’s microbicide gel MZC (also
known as PC-1005) containing MIV-150 and zinc acetate dihydrate
(ZA) in carrageenan (CG) has shown promise as a broad-spectrum
microbicide against HIV, herpes simplex virus (HSV), and human
papillomavirus. Previous data show antiviral activity against these
viruses in cell-based assays, prevention of vaginal and rectal simian–
human immunodeficiency virus reverse transcriptase (SHIV-RT)
infection, and reduction of vaginal HSV shedding in rhesus macaques
and also excellent antiviral activity against HSV and human papillo-
mavirus in murine models. Recently, we demonstrated that MZC is
safe and effective against SHIV-RT in macaque vaginal explants. Here
we established models of ex vivo SHIV-RT/HSV-2 coinfection of
vaginal mucosa and SHIV-RT infection of rectal mucosa in macaques
(challenge of rectal mucosa with HSV-2 did not result in reproducible
tissue infection), evaluated antiviral activity of MZC, and compared
quantitative polymerase chain reaction (PCR) and enzyme-linked
immunosorbent assay readouts for monitoring SHIV-RT infection.
MZC (at nontoxic dilutions) significantly inhibited SHIV-RT in
vaginal and rectal mucosas and HSV-2 in vaginal mucosa when
present during viral challenge. Analysis of SHIV-RT infection and
MZC activity by 1-step simian immunodeficiency virus gag quanti-
tative RT-PCR and p27 enzyme-linked immunosorbent assay dem-
onstrated similar virus growth dynamics and MZC activity by both
methods and higher sensitivity of quantitative RT-PCR. Our data
provide more evidence that MZC is a promising dual compartment
multipurpose prevention technology candidate.
Key Words: MIV-150, multipurpose prevention technology, SHIV-
RT, HSV-2, vaginal, rectal
(J Acquir Immune Defic Syndr 2017;74:e67–e74)
INTRODUCTION
More than 2 decades ago, an “epidemiological synergy”
between HIV-1 and other sexually transmitted infections
(STIs) increasing the risk of HIV-1 acquisition was suggested.
1
Among STIs affecting HIV transmission and pathogenesis,
noncurable STIs like herpes simplex virus 2 (HSV-2) and
human papillomavirus (HPV) deserve special attention. Recent
studies reported up to 3- and 7-fold increased risk of HIV-1
transmission with prevalent and incident HSV-2 infection,
respectively.
2–5
HIV-1/HSV-2 coinfection affects the patho-
genesis of both viruses, being associated with increased HIV-1
viral load
6–8
and HSV-2 shedding quantity and frequency.
9–11
HSV-2 plays a significant role promoting HIV transmission
and acquisition in sub-Saharan Africa, where HSV-2 may
account for 25%–35% of incident HIV infections.
12
Impor-
tantly, studies in rhesus macaques (RM) showed that vaginal
HSV-2 infection is associated with increased susceptibility to
the simian/human immunodeficiency virus (SHIV) SF162P3
and provided some insights into possible mechanisms of
increased transmission.
13
Specifically, frequency of vaginal
CD4
+
T cells expressing high level of a4b7, a gut-homing
integrin that binds gp120
14
and facilitates HIV/simian immu-
nodeficiency virus (SIV) infection,
13–18
is increased in HSV-2–
infected RM.
13
An increase of a4b7
high
CD4
+
T cells in rectal
mucosa was also observed in rectal HSV-2 infection in RM.
19
Similar to HSV-2, HPV infection is associated with an increase
of HIV-1 acquisition
20
and HIV-1 positivity is associated with
increased HPV prevalence
21–23
and incidence.
24,25
The development of multipurpose prevention technologies
active against HIV-1, HSV-2, and HPV vaginally and rectally
could significantly improve global public health.
20,26–28
The
Population Council’s gel containing 50 mM of the nonnucleoside
reverse transcriptase inhibitor MIV-150 and 14 mM zinc acetate
dihydrate (ZA) in carrageenan (CG) (MZC) demonstrated
activity against vaginal SHIV reverse transcriptase (RT) (RM),
vaginal HSV-2 (RM, mice), anorectal HSV-2 (mice), and
vaginal and anorectal HPV (mice).
29–35
MZC protects against
SHIV-RT in RM vaginal explants
36,37
and against HIV and
HSV-2 in human cervical explants (In Vitro Exposure to PC-
1005 and Cervicovaginal Lavage Fluid from Women Vaginally
Administered PC-1005 Inhibits HIV-1 and HSV-2 Infection in
Human Cervical Mucosa. Villegas G, Calenda G, Zhang S,
Mizenina O, Kleinbeck K, Cooney ML, Hoesley CJ, Creasy
GW, Friedland B, Fernández-Romero JA, Zydowsky TM,
Teleshova N. Antimicrob Agents Chemother. 2016 Aug 22;60
Received for publication March 7, 2016; accepted July 15, 2016.
From the *Population Council, New York, NY; †BioRad Laboratories,
Hercules, CA; ‡Aaron Diamond AIDS Research Center, Rockefeller
University, New York, NY; and §Tulane National Primate Research
Center, Tulane University, Covington, LA.
Supported by the US President’s Emergency Plan for AIDS Relief (PEPFAR)
through the US Agency for International Development (USAID)
Cooperative Agreement (GPO-A-00-04-00019-00, www.usaid.gov) and
from the Tulane National Primate Research Center (Primate Center base
grant P51 OD011104, http://tulane.edu/tnprc).
The authors have no funding or conflicts of interest to disclose.
G. C. and G. V. contributed equally.
Correspondence to: Natalia Teleshova, MD, PhD, Center for Biomedical
Research, Population Council, 1230 York Avenue, New York, NY 10065
(e-mail: nteleshova@popcouncil.org).
Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
J Acquir Immune Defic Syndr Volume 74, Number 3, March 1, 2017 www.jaids.com |e67
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(9):5459–5466. doi: 10.1128/AAC.00392-16. Print 2016 Sep).
A recently completed phase I clinical trial demonstrated a favor-
able vaginal safety profile of MZC
38
(First-in-Human Trial of
MIV-150 and Zinc Acetate Coformulated in a Carrageenan Gel:
Safety, Pharmacokinetics, Acceptability, Adherence, and Phar-
macodynamics. Friedland BA, Hoesley CJ, Plagianos M,
Hoskin E, Zhang S, Teleshova N, Alami M, Novak L,
Kleinbeck KR, Katzen LL, Zydowsky TM, Fernández-Romero
JA, Creasy GW. J Acquir Immune Defic Syndr. 2016 Dec 15;73
(5):489–496). The MZC gel is the only multipurpose prevention
technology product currently in clinical testing thatdemonstrates
activity against HIV and two other noncurable STIs that increase
the risk of HIV-1 transmission, HSV-2, and HPV.
26
Here we aimed to establish vaginal and rectal explant
SHIV-RT/HSV-2 coinfection models for microbicide testing
and assess the activity of MZC against both viruses in these
models. Traditionally, infection with HIV and SIV/SHIV in
explants is monitored by p24 and p27 enzyme-linked
immunosorbent assays (ELISAs).
36,39–42
Here we explored
whether analysis of accumulation of viral RNA can be used as
an alternative for p27 ELISA.
MATERIALS AND METHODS
Macaques
Naive, SHIV-RT–, SIV-, and HSV-1–exposed uninfected/
infected Chinese and Indian RMs (Macaca mulatta)were
used. Macaques were housed at the Tulane National Primate
Research Center (TNPRC, Covington, LA), accredited by the
Association for Assessment and Accreditation of Laboratory
Animal Care (AAALAC no. 000594). The use of macaques was
approved by the Animal Care and Use Committee of the TNPRC
(OLAW assurance no. A4499-01), and animal care complied
with the regulations in the Animal Welfare Act
43
and the Guide
for the Care and Use of Laboratory Animals.
44
All vaginal and
rectal biopsy procedures were performed by board-certified
veterinarians (American College of Laboratory Animal Medi-
cine). The biopsies were collected not more often than every 4
weeks. Anesthesia was administered before and during all
procedures, and analgesics were provided afterward as pre-
viously described.
29,45
Necropsy tissues were available from 9
animals. These animals were euthanized using methods consis-
tent with recommendations of the American Veterinary Medical
Association Guidelines for Euthanasia. The animals were
anesthetized with tiletamine–zolazepam (each at 4 mg/kg intra-
muscularly) and given buprenorphine (0.01 mg/kg intramuscu-
larly) followed by an overdose of pentobarbital sodium. Death
was confirmed by auscultation of the heart and pupillary dilation.
Gels
The components of MZC and CG gels are summarized
in Table 1.
Viral Stocks
SHIV-RT was generated from the original stock pro-
vided by Disa Böttiger (Medivir AB, Sweden)
46
using
phytohaemagglutinin (PHA)/interleukin-2 (IL-2)-activated
macaque peripheral blood mononuclear cells and titered in
CEMx174 cells before use.
29
HSV-2 strain G was generated as described previ-
ously.
47
Briefly, HSV-2 strain G was propagated in Vero cells
American Type Culture Collection (ATCC)
48
as described
earlier,
49
and the viral titer in plaque-forming units (pfu)/mL
was obtained by plaque formation assay on monolayer
cultures of Vero cells.
50
Macaque Tissue Processing
Vaginal mucosa (n = 2, 3 ·5 mm biopsies procured at
each collection time; or necropsy tissues) was obtained from
RM. Tissues were transported overnight and cut into 3 ·3mm
explants as described previously
36,51
before viral challenge
(below). Rectal mucosa (n = 15–20, 1.5 ·1.5 mm biopsies
procured at each collection time) was processed for viral
challenge (below) at TNPRC immediately after collection.
Tissue Viability
To assess tissue viability after overnight (;18 hours)
exposure to MZC or CG gels, MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed
as previously described.
36,51
Comparison of SHIV-RT Growth Kinetics
Using Quantitative RT Polymerase Chain
Reaction and p27 ELISA
To compare SHIV-RT infection readout methods in
vaginal explants, tissues were processed and challenged with
SHIV-RT as previously described.
36
Briefly, vaginal explants
were stimulated [5 mg/mL PHA (Sigma Aldrich) and 100
U/mL IL-2 (NCI BRB Preclinical Repository, Frederick,
MD)] for 48 hours and then challenged with 10
4
TCID
50
SHIV-RT per explant for ;18 hours. After washout, explants
were cultured in the presence of IL-2 for 14 days.
36
Super-
natants were collected on days 0, 3, 7, 11, and 14, and
infection levels were analyzed by SIV gag one-step quanti-
tative RT polymerase chain reaction (qRT-PCR) and p27
ELISA. To compare SHIV-RT infection readout methods in
rectal biopsies, supernatants from SHIV-RT/HSV-2 cochal-
lenge experiments (below) were used. Controls included
10 mM 3TC or 10 mM 3TC/100 mg/mL Acyclovir.
TABLE 1. Components of MZC and CG Gels
Components
Gel Formulation
MZC (Batch No.
130605A1005TR)
CG (Batch No.
130613A525TR)
MIV-150 0.00184 wt% (50 mM) —
ZA 0.3 wt% (14 mM) —
CG 2.8 wt% (28 mg/mL) 2.6 wt% (26 mg/mL)
Propylene glycol 2 wt% 2 wt%
Methylparaben 0.2 wt% 0.2 wt%
Sodium acetate 0.131 wt% (10 mM) 0.131 wt% (10 mM)
Calenda et al J Acquir Immune Defic Syndr Volume 74, Number 3, March 1, 2017
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SHIV-RT and HSV-2 Cochallenge of Vaginal
and Rectal Mucosae and Antiviral Activity
of MZC
PHA/IL-2–stimulated vaginal explants and unstimulated
rectal biopsies were cochallenged with 10
4
TCID
50
SHIV-RT
and 10
6
pfu HSV-2 per explant for ;18 (vaginal) or 4 hours
(rectal). To test the antiviral activity of MZC, viral challenge was
done in the presence of 1:300 and/or 1:100 diluted MZC and CG
gels vs. untreated (medium) and 3TC/Acyclovir controls. After
washout, vaginal explants were cultured in the presence of IL-2
for 14 days.
36
Rectal biopsies (n = 4) were placed on 12-mm
diameter Gelfoam sponges (Ethicon, Somerville, NJ) presoaked
in complete DMEM (cDMEM) (DMEM Cellgro Mediatech)
containing 10% fetal bovine serum (Gibco; Life Technologies,
Grand Island, NY), 100 U/mL penicillin, 100 mg/mL strepto-
mycin (Cellgro Mediatech), and 100 mMEagle’sminimum
essential medium (MEM) nonessential amino acids (Irvine
Scientific, Santa Ana, CA) at 37°C, 5% CO
2
,foratleast
30 minutes and cultured for 14 days (no IL-2). Rectal biopsies
were cultured in cDMEM to which 80 mg/mL gentamicin had
been added (Gibco). Supernatants from both vaginal and rectal
tissues were collected on days 0, 3, 7, 11, and 14, and levels of
infections were analyzed by qPCR and/or ELISA.
qPCR and qRT-PCR
Five microliters of tissue culture supernatants were
analyzed using the KAPA SYBR FAST Universal one-Step
qRT-PCR kit (Kapa Biosystems, Wilmington, MA) for the
quantification of SIV gag copies and the KAPA SYBR FAST
Universal qPCR assay (Kapa Biosystems) for the quantifica-
tion of HSV pol copies with the Viia 7 real-time PCR system
(Applied Biosystems, Carlsbad, CA).
Primers for SIV were 59-GGTTGCACCCCCTATGA-
CAT-39(SIV667gag Fwd) and 59-TGCATAGCCGCTT-
GATGGT-39(SIV731gag Rev). Results were analyzed by the
standard curve method, using SIV
mac1A11
DNA obtained from
Dr. Paul Luciw through the National Institutes of Health AIDS
Reagent Program, Division of AIDS, National Institute of
Allergy and Infectious Diseases, National Institutes of Health,
52
as the standard (
37
; A Novel Microbicide/Contraceptive Intra-
vaginal Ring Protects Macaque Genital Mucosa against SHIV-
RT Infection Ex Vivo. Villegas G, Calenda G, Ugaonkar S,
Zhang S, Kizima L, Mizenina O, Gettie A, Blanchard J, Cooney
ML, Robbiani M, Fernández-Romero JA, Zydowsky TM,
Teleshova N. PLoS One. 2016 Jul 18;11(7):e0159332. doi:
10.1371/journal.pone.0159332. eCollection 2016).
Primers for HSV-2 pol were 59-GCTCGAGTGC-
GAAAAAACGTTC-39(HSV-2pol Fwd) and 59-
TGCGGTTGATAAACGCGCAGT-39(HSV-2pol Rev).
13,53
For the generation of HSV-2–positive control plasmid, a 2.6-kb
fragment of the HSV-2 UL30 gene was amplified by PCR using
the iProof High-Fidelity DNA Polymerase (BioRad, Hercules,
CA). Genomic DNA template was prepared from HSV-2 stock,
and primers 59-GACGAGCGCGACGTCCTC-39(Fwd) and 59-
TCGTCGTAAAACAGCAGGTC-39(Rev) were used. The
PCR product was cloned into the pCR Blunt II TOPO vector
(Life Technologies) and the construct verified by sequencing.
p27 ELISA
p27 content in tissue culture supernatants was measured
by RETRO-TEK SIV p27 Antigen ELISA kit (ZeptoMetrix,
Buffalo, NY).
Statistical Analyses
The sensitivity of paired p27 ELISA and SIV gag qRT-
PCR results was compared using McNemar test and interrater
agreement (Kappa) statistics using GraphPad calculators
available online (graphpad.com).
Analysis of tissue viability (MTT assay) was performed
as described earlier
36
using a log-normal generalized linear
mixed model predicting the weight-normalized optical den-
sity (OD)
570
of each replicate.
Analysis of gel activity against SHIV-RT and HSV-2
was performed as described previously
36,37,39,40,54
using
a log-normal generalized linear mixed model with SOFT or
CUM as the response and gel treatment as the predictor. For
the experiments using rectal biopsies, a random intercept for
each animal was included. For the vaginal experiments,
a random intercept was included for each biopsy, noting that
2 animals contributed 2 biopsies.
All analyses were performed with SAS V9.4 and SAS/
STAT V13.1 with a= 0.05. Significant Pvalues of ,0.05
(*), ,0.01 (**), and #0.001 (***) are indicated.
RESULTS
SIV gag qRT-PCR and p27 ELISA Demonstrate
Similar SHIV-RT Growth Kinetics in Explants
With qRT-PCR Being a More Sensitive
Infection Readout
To determine the feasibility of one-step SIV gag qRT-
PCR for analysis of SHIV-RT infection in vaginal and rectal
tissue cultures, a comparison with p27 ELISA was carried out.
Supernatants from vaginal (n = 7 experiments) and
rectal (n = 8 experiments) tissues challenged with SHIV-RT
(vaginal) or SHIV-RT and HSV-2 (rectal) were analyzed by
both ELISA and qRT-PCR at 5 time points postchallenge
(days 0, 3, 7, 11, and 14). 3TC and 3TC/Acyclovir controls
were available in n = 2 vaginal tissue experiments and in all
n = 8 rectal tissue experiments, respectively. The results show
similar SHIV-RT growth kinetics by both methods (Fig. 1). A
total of n = 45 (vaginal) and n = 80 (rectal) data points were
collected. In vaginal tissue culture supernatants, 8/45 samples
had a positive readout (values $Lower Limit of Quantifica-
tion (LLOQ)) by ELISA, whereas 42/45 samples had
a positive readout by qRT-PCR. In rectal tissue supernatants,
positive readout was obtained in 28/80 samples by ELISA
and in 65/80 by qRT-PCR. McNemar test for matched pairs
showed higher sensitivity of qRT-PCR vs. ELISA (P,
0.0001, both vaginal and rectal tissue experiments). Examin-
ing statistical agreement by Kappa analysis, we found a Kappa
coefficient of 0.03 for vaginal and 0.22 for rectal super-
natants, indicating poor and fair strength of agreement,
respectively (Table 2). These results further emphasize higher
sensitivity of qRT-PCR vs. ELISA.
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MZC Protects Macaque Vaginal Mucosa
Against SHIV-RT and HSV-2 Coinfection
Stimulated vaginal explants were cochallenged with
SHIV-RT (10
4
TCID
50
/explant) based on our published
data
36,40,51
and with HSV-2 (10
6
pfu/explant) based on
titration experiments demonstrating robust infection with
this challenge dose (not shown). Controls included cochal-
lenged tissues cultured in the presence of 3TC and
Acyclovir. Figure 2A provides representative examples of
SHIV-RT and HSV-2 growth curves. Reproducible tissue
infection was achieved with both viruses (Fig. 2B). In this
system, no enhancement of SHIV-RT infection was de-
tected in the presence of HSV-2 compared with SHIV-
RT alone.
We previously demonstrated that MZC gel is active
against single SHIV-RT infection in PHA/IL-2–stimulated
vaginal explants.
36
In this study, we aimed to determine
whether MZC is active against SHIV and HSV-2 in a high-
dose SHIV-RT/HSV-2 cochallenge model. Stimulated tis-
sues were exposed to SHIV-RT and HSV-2 in the presence
of nontoxic (Fig. 3A) 1:100 or 1:300 diluted MZC or CG
(placebo) gel. Of note, one outlier in 1:100 MZC group was
detected by MTT assay (Fig. 3A). The outlier was not
excluded from the analysis as the data from explants in
medium and CG 1:100 groups from the same donor were
within the viable range.
To allow a direct comparison with our previous
work on MZC in the single infection model,
36
p27 ELISA
was used as a readout. MZC (1:100 and 1:300 dilutions)
strongly inhibited SHIV-RT infection relative to untreated
(medium) and CG controls (SOFT/CUM, 90%–99%
inhibition, P,0.0001/0.05) (Fig. 3B). MZC and CG at
1:100 dilution (SOFT/CUM, 99% inhibition, P,0.0001)
and CG at 1:300 dilution (SOFT/CUM, .90% inhibition,
P,0.05) inhibited HSV-2 vs. untreated control, pointing
to CG-mediated activity of MZC against HSV-2.
FIGURE 1. SIV gag qRT-PCR and p27 ELISA demonstrate similar SHIV-RT growth kinetic in explants. PHA/IL-2–stimulated
macaque vaginal explants were challenged with 10
4
TCID
50
SHIV-RT for ;18 hours. After washout, tissues were cultured (3
explants per condition) for 14 days. A summary of n = 7 SHIV-RT challenge experiments [mean 6standard error of the mean
(SEM)] is shown. 3TC controls were included in 2 of 7 experiments. Rectal explants were challenged with 10
4
TCID
50
SHIV-RT and
10
6
pfu HSV-2 vs. 3TC/Acyclovir control for 4 hours. After washout, tissues were cultured (4 biopsies per well; single well per
condition) for 14 days. A summary of n = 8 experiments (mean 6SEM) is shown. SHIV-RT infection kinetics were followed by
one-step SIV gag qRT-PCR and p27 ELISA.
TABLE 2. Readouts Obtained in Vaginal and Rectal Tissue
Supernatants by ELISA and PCR Assay
Positive (PCR) Negative (PCR)
Vaginal
Positive (ELISA) 8 0
Negative (ELISA) 34 3
Rectal
Positive (ELISA) 28 0
Negative (ELISA) 37 15
Matched pairs were analyzed by McNemar test and Kappa analysis.
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MZC Protects Rectal Mucosa Against SHIV-
RT Infection
Rectal biopsies were cochallenged with 10
4
TCID
50
SHIV-RT and HSV-2 10
3
–10
6
pfu per biopsy vs. 3TC/Acyclovir
controls. The SHIV-RT challenge dose was chosen based on
studies in vaginal tissues (above) and resulted in reproducible
SHIV-RT infection (Fig. 1). In contrast, no productive HSV-2
infection was detected in rectal mucosa as similar HSV pol copy
numbers were detected in cultures with and without Acyclovir.
A differential with Acyclovir control was observed only in 1 out
of 10 experiments using 10
6
pfu challenge dose (not shown). We
chose to test gel activity in the SHIV-RT (10
4
TCID
50
)and
HSV-2 (10
6
pfu) cochallenge settings to mimic possible real-life
HIV-1/HSV-2 coexposure scenario.
Tissues were challenged in the presence of nontoxic
(Fig. 4A) 1:100 dilution of MZC for 4 hours vs. untreated
(medium), CG, and 3TC/Acyclovir controls. MZC afforded
significant protection against SHIV-RT relative to untreated
and CG controls (SOFT/CUM, 92%–98% inhibition, P,
0.0001) (Fig. 4B). Similar anti-SHIV-RT activity (SOFT/
CUM, 97%–98% inhibition, P,0.0001) was detected by
analysis of the same data set by qRT-PCR (not shown). As
expected, HSV-2 failed to infect the rectal tissues (not shown).
DISCUSSION
In this study, we introduced ex vivo vaginal SHIV-RT/
HSV-2 coinfection and rectal SHIV-RT infection models.
These models were used to test MZC’s activity. We also
explored methodological aspects of monitoring tissue infection
ex vivo. Here we compared the sensitivity of SIV gag one-step
qRT-PCR and p27 ELISA methods to monitor SHIV-RT
infection in vaginal and rectal explants.
A recent study by Rollenhagen et al
55
and data from our
group (In Vitro Exposure to PC-1005 and Cervicovaginal
Lavage Fluid from Women Vaginally Administered
PC-1005 Inhibits HIV-1 and HSV-2 Infection in Human
Cervical Mucosa. Villegas G, Calenda G, Zhang S,
Mizenina O, Kleinbeck K, Cooney ML, Hoesley CJ, Creasy
GW, Friedland B, Fernández-Romero JA, Zydowsky TM,
Teleshova N. Antimicrob Agents Chemother. 2016 Aug
22;60(9):5459–5466. doi: 10.1128/AAC.00392-16. Print
2016 Sep) demonstrated enhancement of ex vivo cervical
HIV-1 infection in the HSV-2 coinfection settings. The
elevated tissue HIV-1 infection coincided with increased
numbers of CD4
+
CCR5
+
CD38
+
T cells and reduced anti-
HIV-1 activity of low-dose Tenofovir (1 mg/mL).
55
In contrast to data in human cervical tissue, no
enhancement of SHIV-RT infection by HSV-2 in stimulated
macaque vaginal mucosa was seen in the current study. The
same result was obtained when unstimulated tissues were
cochallenged with the same viral doses (not shown). It is worth
pointing out that in this study and in the human ectocervical
tissue coinfection model (In Vitro Exposure to PC-1005 and
Cervicovaginal Lavage Fluid from Women Vaginally Admin-
istered PC-1005 Inhibits HIV-1 and HSV-2 Infection in
Human Cervical Mucosa. Villegas G, Calenda G, Zhang S,
Mizenina O, Kleinbeck K, Cooney ML, Hoesley CJ, Creasy
GW, Friedland B, Fernández-Romero JA, Zydowsky TM,
Teleshova N. Antimicrob Agents Chemother. 2016 Aug 22;60
(9):5459–5466. doi: 10.1128/AAC.00392-16. Print 2016 Sep),
FIGURE 2. Macaque vaginal tissue is susceptible to SHIV-RT/HSV-2 infections after cochallenge. PHA/IL-2–stimulated macaque
vaginal explants were challenged with 10
4
TCID
50
SHIV-RT and/or 10
4
TCID
50
HSV-2 (3 explants per condition) vs. 3TC/Acy
controls for ;18 hours. After washout, tissues were cultured for 14 days in the presence of IL-2. A, Representative examples of
SHIV-RT and HSV-2 growth after single challenge or cochallenge are shown. Infections were monitored by one-step SIV gag qRT-
PCR and HSV-2 pol qPCR. B, Summaries of 7 experiments (SOFT and CUM analyses of SHIV-RT and HSV-2 infection) are shown.
Dotted lines represent input virus (mean) after washout at day 0.
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we resorted to a high HSV-2 viral challenge dose (10
6
pfu/
explant) to assure reproducible HSV-2 infection and to test
MZC’s activity under stringent conditions. Of note, 10
6
pfu
(;10
7
copies of DNA) HSV-2 per explant highly exceeds the
amount of HSV-2 shed in the genital fluids of HSV-2–positive
patients.
56,57
Infection with or exposure to HSV-2 can induce
apoptosis and impair dendritic cells and T cells,
58–60
potentially
affecting tissue susceptibility to SHIV-RT. Although we
cannot exclude these effects of HSV-2 in our tissue models,
SHIV-RT infection after cochallenge of vaginal and rectal
tissues was robust and reproducible. In our human cervical
tissue explant model, HIV-1
BaL
/HSV-2 cochallenge results in
;60% frequency of productive HIV-1
BaL
vs. 100% after HIV-
1
BaL
only challenge (In Vitro Exposure to PC-1005 and
FIGURE 3. MZC under nontoxic dilutions inhibits SHIV-RT/HSV-2 coinfection in macaque vaginal explants. A, MTT assay was
performed on tissues exposed to 1:100 and 1:300 diluted MZC and CG (2–3 explants per condition) for ;18 hours. 1:10 diluted
gynol served as a toxicity control. Each symbol indicates a donor, and the mean 6standard error of the mean (SEM) of the
log
10
(OD
570
/g) for each condition is shown. B, PHA/IL-2–stimulated explants were challenged with 10
4
TCID
50
SHIV-RT and 10
6
pfu HSV-2 in the presence of 1:100 and 1:300 diluted gels (3 explants per condition) vs. untreated (medium) and 3TC/Acy
controls for ;18 hours. Then the tissues were washed and cultured for 14 days in the presence of IL-2. Infections were monitored
at 0, 3, 7, 11, 14 days of culture by p27 ELISA and HSV-2 pol qPCR. Summary of 5–10 experiments (mean 6SEM of SOFT and
CUM analyses) is shown. Dotted lines represent input virus (mean) after washout at day 0.
FIGURE 4. MZC under nontoxic dilutions inhibits SHIV-RT infection in SHIV-RT/HSV-2 cochallenged macaque rectal explants. A,
MTT assay was performed on tissues exposed to 1:100 diluted MZC and CG (3 explants per condition) for ;18 hours. 1:10
diluted gynol served as a toxicity control. Each symbol indicates a donor, and the mean 6standard error of the mean (SEM) of the
log
10
(OD
570
/g) for each condition is shown (B). Explants were challenged with 10
4
TCID
50
SHIV-RT and 10
6
pfu HSV-2 immersed
in medium containing 1:100 diluted gels (3 explants per condition) vs. untreated (medium) and 3TC/Acy controls for 4 hours.
Then the tissues were washed, transferred to Gelfoam sponges, and cultured for 14 days. SHIV-RT infection was measured at 0, 3,
7, 11, 14 days of culture by p27 ELISA. Summary of 8 experiments (mean 6SEM of SOFT and CUM analyses) is shown. Dotted
lines represent input virus (mean) after washout at day 0.
Calenda et al J Acquir Immune Defic Syndr Volume 74, Number 3, March 1, 2017
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Cervicovaginal Lavage Fluid from Women Vaginally Admin-
istered PC-1005 Inhibits HIV-1 and HSV-2 Infection in
Human Cervical Mucosa. Villegas G, Calenda G, Zhang S,
Mizenina O, Kleinbeck K, Cooney ML, Hoesley CJ, Creasy
GW, Friedland B, Fernández-Romero JA, Zydowsky TM,
Teleshova N. Antimicrob Agents Chemother. 2016 Aug 22;60
(9):5459–5466. doi: 10.1128/AAC.00392-16. Print 2016 Sep).
A more physiologically relevant, low-dose SHIV-RT/HSV-2
cochallenge model would be needed to explore whether and
how coinfection drives mucosal SHIV-RT infection in mac-
aques. We were unable to infect rectal mucosa with HSV-2 ex
vivo. In vivo rectal HSV-2 infection was previously reported in
9 out of 11 SIV-infected macaques that were challenged
rectally with 2 ·10
8
pfu HSV-2.
19
However, in naive animals,
the frequency of infection after the same-dose HSV-2 chal-
lenge is 55%.
61
As epithelial cells represent the primary target
for HSV-2, loss of the single-layer columnar epithelium during
the culture period
42
could have contributed to the lack of ex
vivo infection in rectal biopsies.
Our side-by-side comparison indicates that 1-step SIV
gag qRT-PCR using tissue culture supernatants can be used
as an alternative to p27 ELISA to monitor tissue infection and
product activity. The data indicate similar SHIV-RT growth
kinetics and MZC activity as detected by both assays. The
1-step SIV gag qRT-PCR has proven to be a more sensitive
method than p27 ELISA. Our results are in agreement with
the findings of Janocko et al,
62
who demonstrated the
feasibility of qRT-PCR as a readout of HIV infection.
62
Also
in agreement with this report,
62
qRT-PCR did not shorten the
time to detect evidence of infection. Overall, the qRT-PCR
approach offers increased sensitivity and high dynamic range.
The assay requires only a small volume of the supernatant
(5 mL) and is time and cost effective.
MZC gel protected against SHIV-RT at 1:100 (;0.18 mg/
mL MIV-150) and 1:300 (;0.06 mg/mL MIV-150) dilutions
and against HSV-2 at 1:100 dilution in vaginal mucosa. The
activity against HSV-2 was CG mediated. Similarly, the gel at
1:100 dilution also protected against SHIV-RT in rectal mucosa.
It is important to note that previous studies demonstrated that
the combination of CG and zinc acetate results in antiviral
synergy against HSV-2.
47
The HSV-2 mouse model has shown
that under stringent conditions, the combination of CG and zinc
protects significantly while CG alone does not protect against
HSV-2 infection.
31,47,63
The use of undiluted formulations in
a mouse model allowed to appreciate the advantage of the CG/
zinc combination when compared with CG alone. In our explant
system, CG alone provides strong inhibition (even after diluting
the gel) that masks zinc’s contribution.
The results demonstrating potent activity of MZC against
coinfection of vaginal mucosa with SHIV-RT and HSV-2 add
to our previously published data showing potent MZC’s activity
against single-cell–free or cell-associated SHIV-RT challenge of
macaque vaginal mucosa.
36,37
These results are also consistent
with the potent activity of MZC against coinfection with HIV-
1
BaL
and HSV-2 in human cervical mucosa (In Vitro Exposure
to PC-1005 and Cervicovaginal Lavage Fluid from Women
Vaginally Administered PC-1005 Inhibits HIV-1 and HSV-2
Infection in Human Cervical Mucosa. Villegas G, Calenda G,
Zhang S, Mizenina O, Kleinbeck K, Cooney ML, Hoesley CJ,
Creasy GW, Friedland B, Fernández-Romero JA, Zydowsky
TM, Teleshova N. Antimicrob Agents Chemother. 2016 Aug
22;60(9):5459–5466. doi: 10.1128/AAC.00392-16. Print 2016
Sep), suggesting that ex vivo activity testing in macaque
mucosa may predict results in human mucosa. Overall, our
data support further development of MZC as a potential broad-
spectrum vaginal and rectal on-demand microbicide.
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Calenda et al J Acquir Immune Defic Syndr Volume 74, Number 3, March 1, 2017
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