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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.
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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 Councils 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 immunodeciency 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) signicantly 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 immunodeciency 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 Dec Syndr 2017;74:e67e74)
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.
25
HIV-1/HSV-2 coinfection affects the patho-
genesis of both viruses, being associated with increased HIV-1
viral load
68
and HSV-2 shedding quantity and frequency.
911
HSV-2 plays a signicant 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 immunodeciency virus (SHIV) SF162P3
and provided some insights into possible mechanisms of
increased transmission.
13
Specically, frequency of vaginal
CD4
+
T cells expressing high level of a4b7, a gut-homing
integrin that binds gp120
14
and facilitates HIV/simian immu-
nodeciency virus (SIV) infection,
1318
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
2123
and incidence.
24,25
The development of multipurpose prevention technologies
active against HIV-1, HSV-2, and HPV vaginally and rectally
could signicantly improve global public health.
20,2628
The
Population Councils 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).
2935
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 Presidents 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 conicts 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
Copyright Ó2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
(9):54595466. doi: 10.1128/AAC.00392-16. Print 2016 Sep).
A recently completed phase I clinical trial demonstrated a favor-
able vaginal safety prole 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 Dec Syndr. 2016 Dec 15;73
(5):489496). 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).
36,3942
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-1exposed 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-certied
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 tiletaminezolazepam (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 conrmed 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
Briey, 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 = 1520, 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
Briey, 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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Copyright Ó2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
SHIV-RT and HSV-2 Cochallenge of Vaginal
and Rectal Mucosae and Antiviral Activity
of MZC
PHA/IL-2stimulated 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 mMEaglesminimum
essential medium (MEM) nonessential amino acids (Irvine
Scientic, 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
quantication of SIV gag copies and the KAPA SYBR FAST
Universal qPCR assay (Kapa Biosystems) for the quantica-
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-2positive control plasmid, a 2.6-kb
fragment of the HSV-2 UL30 gene was amplied 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 veried 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. Signicant 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 Quantica-
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
coefcient 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-2stimulated
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
signicant 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 MZCs 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):54595466. 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):54595466. 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
MZCs 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 uids of HSV-2positive
patients.
56,57
Infection with or exposure to HSV-2 can induce
apoptosis and impair dendritic cells and T cells,
5860
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):54595466. 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 ndings 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 signicantly 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 zincs 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 MZCs activity
against single-cellfree 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):54595466. 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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... Later, the tenofovir gel demonstrated the ability to act as an MPT by preventing HSV-2 acquisition among women by 51% as demonstrated by the CAPRISA 004 trial and VOICE trial [89,90]. Another promising MPT vaginal gel in development contains MIV-150 and zinc acetate dihydrate in carrageenan to prevent HIV, HSV, and HPV and is currently in phase 1 clinical trials by the Population Council [36]. The microbicides gel showed a safe vaginal profile and has been shown to inhibit SHIV-reverse transcriptase in a macaque vaginal and rectal mucosal model and demonstrated antiviral activity against HSV-2 and HPV in murine models [36]. ...
... Another promising MPT vaginal gel in development contains MIV-150 and zinc acetate dihydrate in carrageenan to prevent HIV, HSV, and HPV and is currently in phase 1 clinical trials by the Population Council [36]. The microbicides gel showed a safe vaginal profile and has been shown to inhibit SHIV-reverse transcriptase in a macaque vaginal and rectal mucosal model and demonstrated antiviral activity against HSV-2 and HPV in murine models [36]. Currently, this is the only MPT product in clinical testing that promotes the prevention of HIV in addition to two other noncurable STIs. ...
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There is a high global prevalence of HIV, sexually transmitted infections (STIs), and unplanned pregnancies. Current preventative daily oral dosing regimens can be ineffective due to low patient adherence. Sustained release delivery systems in conjunction with multipurpose prevention technologies (MPTs) can reduce high rates of HIV/STIs and unplanned pregnancies in an all-in-one efficacious, acceptable, and easily accessible technology to allow for prolonged release of antivirals and contraceptives. The concept and development of MPTs have greatly progressed over the past decade and demonstrate efficacious technologies that are user-accepted with potentially high adherence. This review gives a comprehensive overview of the latest oral, parenteral, and vaginally delivered MPTs in development as well as drug delivery formulations with the potential to advance as an MPT, and implementation studies regarding MPT user acceptability and adherence. Furthermore, there is a focus on MPT intravaginal rings emphasizing injection molding and hot-melt extrusion manufacturing limitations and emerging fabrication advancements. Lastly, formulation development considerations and limitations are discussed, such as nonhormonal contraceptive considerations, challenges with achieving a stable coformulation of multiple drugs, achieving sustained and controlled drug release, limiting drug–drug interactions, and advancing past preclinical development stages. Despite the challenges in the MPT landscape, these technologies demonstrate the potential to bridge gaps in preventative sexual and reproductive health care.
... MPTs are being designed and developed for oral [14], transdermal [15], subcutaneous (as implants) [16], and vaginal/rectal [17][18][19] use in order to meet different preferences and needs. Topical products seem particularly interesting as they typically feature reduced production cost, possibility of self-administration and discrete use, and avoidance of systemic adverse effects. ...
... Trailing on the hallmark phase 2b clinical study CAPRISA 004 demonstrating partial efficacy of a 1% tenofovir gel in preventing male-to-female transmission of HIV-1 and HSV-2, various other vaginal MPTs based on gels have been reported [47,112]. For example, in a study conducted by Calenda et al., a carrageenanbased microbicide gel showed a wide range of activity against HIV-1, HSV-2, and HPV [19]. The formulation was tested for vaginal administration in a macaque model and shown to be effective in preventing SHIV/RT and HSV-2 transmission after co-infection challenge. ...
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Introduction: Multipurpose prevention technologies (MPTs) have the potential to avert multiple concomitant sexual and reproductive health issues in women such as sexually transmitted infections and unintended pregnancy. MPTs incorporate one or more active pharmaceutical ingredients in a single product, which adds more convenience for users and may promote increased adherence. Various vaginal dosage forms/delivery systems have been studied for designing MPTs. However, several challenges remain that are mainly related with requirements of individual drugs or intended multiple applications. Areas covered: This review focuses on the emerging need and development of vaginal MPTs. It illustrates numerous examples that are currently in the pre-clinical and clinical development pipeline, highlighting the concept behind vaginal MPTs. The article also highlights the challenges associated with formulation design and development, including regulatory issues that need to be addressed. Expert opinion: Vaginal MPTs present great potential to empower women with novel, efficient and safe products for protection against sexually transmitted infections and unintended pregnancy. However, several technological issues and regulatory gaps still need to be addressed in order to meet real world needs.
... Carrageenans have also been extensively studied in vaginal administration (Pacheco-Quito et al., 2020a;Perino et al., 2019;Sánchez-Sánchez et al., 2015;Ugaonkar et al., 2015), due to their antiviral properties that inhibit the human papilloma virus (Novetsky et al., 2016;Perino et al., 2019) and offer protection against HSV-2 transmission by binding to herpes virus receptors (Boulho et al., 2017;Calenda et al., 2017;Fernández-Romero et al., 2012;Kizima et al., 2014;Pacheco-Quito et al., 2020b). There is little evidence of vaginal films based on this polymer (Gu et al., 2015;Traore et al., 2018). ...
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... SIV infections result in high viral loads, a progressive loss of CD4 + T cells, and a disease progression rate similar to that observed in HIV-1-infected humans. Rhesus macaques are used in microbicide studies for topical and systemic distribution and to evaluate API efficacy (Calenda et al., 2017;McBride et al., 2017;Zhao et al., 2017). Pig-tailed macaques have a vaginal flora similar to humans and are more susceptible to the same STIs. ...
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Although a wide variety of topical microbicides provide promising in vitro and in vivo efficacy, most of them failed to prevent sexual transmission of human immunodeficiency virus type 1 (HIV-1) in human clinical trials. In vitro, ex vivo, and in vivo models must be optimized, considering the knowledge acquired from unsuccessful and successful clinical trials to improve the current gaps and the preclinical development protocols. To date, dendrimers are the only nanotool that has advanced to human clinical trials as topical microbicides to prevent HIV-1 transmission. This fact demonstrates the importance and the potential of these molecules as microbicides. Polyanionic dendrimers are highly branched nanocompounds with potent activity against HIV-1 that disturb HIV-1 entry. Herein, the most significant advancements in topical microbicide development, trying to mimic the real-life conditions as closely as possible, are discussed. This review also provides the preclinical assays that anionic dendrimers have passed as microbicides because they can improve current antiviral treatments' efficacy. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.
... Another study demonstrated the advantage of combining carrageenan with Griffithsin, which acts as a broad-spectrum microbicide against HSV-2 and HPV in vitro and in vivo [40,41]. There is also evidence that carrageenan-based gel offers protection against HSV-2 transmission by binding to herpes virus receptors [42][43][44]. A number of reports focus on adding carrageenan to other polymers to leverage the gelling properties of carrageenan and achieve a good controlled-release profile [15,45]. ...
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Women are the most affected by genital herpes, which is one of the most common sexually transmitted infections, affecting more than 400 million people worldwide. The application of vaginal microbicides could provide a safe method of protection. Acyclovir is a safe and effective medication for vaginal administration, and numerous benefits have been observed in the treatment of primary or recurrent lesions due to genital herpes. Vaginal tablets based on a combination of the polymers iota-carrageenan and hydroxypropyl methylcellulose were developed for the controlled release of acyclovir. Swelling, mucoadhesion and drug release studies were carried out in simulated vaginal fluid. The tablets, containing a combination of iota-carrageenan and hydroxypropyl methylcellulose, have an adequate uptake of the medium that allows them to develop the precise consistency and volume of gel for the controlled release of acyclovir. Its high mucoadhesive capacity also allows the formulation to remain in the vaginal area long enough to ensure the complete release of acyclovir. These promising formulations for the prevention of genital herpes deserve further evaluation.
... Chemical compounds such as vaginal gels applied during or before sexual intercourse could also influence the effectivity of HSV-2 transmission. Incorporating microbicides into vaginal gels is a well-accepted strategy for inhibiting sexually transmitted disease transmission including HSV-2 transmission [9][10][11]. We also showed that even basic components of Open Access BMC Research Notes *Correspondence: virok.dezso.peter@med.u-szeged.hu 2 Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm sqr. 10, Szeged 6720, Hungary Full list of author information is available at the end of the article the vaginal gels, such as the gelling agent hydroxyethyl cellulose can also significantly influence the replication of sexually transmitted pathogens such as Chlamydia trachomatis [12]. ...
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Objective: Herpes simplex virus-2 (HSV-2) infections are almost exclusively sexually transmitted. The presence of vaginal gels during sexual activity may have a significant positive or negative impact on viral transmission. Therefore we investigated three off-the-shelf vaginal lubricants and one pH restoring gel to evaluate their impact on HSV-2 replication. Results: HeLa cells were infected with untreated virions and virions incubated with the particular gels. The accumulation of viral genomes was monitored by quantitative PCR (qPCR) method at 24 h post infection. Two of the tested gels had no significant effect on HSV-2 replication at the maximum applied concentration, while two had a strong inhibitory effect (~ 98% reduction of replication). The replication inhibitory effect was observed at various multiplicity of infection (MOI 0.4-6.4) and the two inhibitory gels were also capable of inhibiting the HSV-2 induced cytopathic effect on HeLa cells. The surface tension decreasing activity-an indication of detergent activity-was strongly correlated with the anti-HSV-2 activity of the gels (R2: 0.88). Our results indicate that off-the-shelf vaginal gels have a markedly different anti-HSV-2 activity that may influence HSV-2 transmission.
... Many of the world's regions with high disease prevalence are rife with malnutrition, have limited access to medical resources, and are hotbeds for non-compliance with in PubMed that utilized macaque models of human disease, 60% were published in the last ten years. These systems have also been significantly utilized in the modeling of infectious diseases, particularly HIV (~4500 publications) [14][15][16][17][18][19][20][21], HSV [22][23][24], influenza [25][26][27], S. pneumoniae [28], N. gonnorrhoeae [29], nontuberculous mycobacteria [30,31], and malaria [32,33]. These models have not only investigated pathogenesis of infection, but also related tissue pathology, vaccine and drug development and, of late, hostdirected or immunotherapeutics [34]. ...
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Animal models are important in understanding both the pathogenesis of and immunity to tuberculosis. Unfortunately, we are beginning to understand that no animal model perfectly recapitulates the human TB syndrome, which encompasses numerous different stages. Furthermore, Mtb infection is a very heterogeneous event at both the levels of pathogenesis and immunity. This review seeks to establish the current understanding of TB pathogenesis and immunity, as validated in the animal models of TB in active use today. We especially focus on the use of modern genomic approaches in these models, to determine the mechanism and the role of specific molecular pathways. Animal models have significantly enhanced our understanding of TB. Incorporation of contemporary technologies such as single cell transcriptomics, high-parameter flow cytometric immune profiling, proteomics, proteomic flow cytometry and immunocytometry etc., into the animal models in use will further enhance our understanding of TB and facilitate the development of treatment and vaccination strategies.
... Treatment of only one of the viruses could result in growth of a potentially harmful virus that was suppressed to some extent by the presence of the other virus. Antiviral treatment can be more efficient in treating superinfecting viruses if a broad spectrum antiviral is used, allowing for simultaneous treatment of both viruses ( Calenda et al., 2017 ). ...
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Molecular diagnostic techniques have revealed that approximately 43% of the patients hospitalized with influenza-like illness are infected by more than one viral pathogen, sometimes leading to long-lasting infections. It is not clear how the heterologous viruses interact within the respiratory tract of the infected host to lengthen the duration of what are usually short, self-limiting infections. We develop a mathematical model which allows for single cells to be infected simultaneously with two different respiratory viruses (superinfection) to investigate the possibility of chronic coinfections. We find that a model with superinfection and cell regeneration has a stable chronic coinfection fixed point, while superinfection without cell regeneration produces only acute infections. This analysis suggests that both superinfection and cell regeneration are required to sustain chronic coinfection via this mechanism since coinfection is maintained by superinfected cells that allow slow-growing infections a chance to infect cells and continue replicating. This model provides a possible mechanism for chronic coinfection independent of any viral interactions via the immune response.
... Tissues were washed extensively and cultured for 14 days. Infection was monitored by SIV gag qRT-PCR 47 performed on culture supernatants collected every 3-4 days and analyzed for SOFT and CUM endpoints 47,[58][59][60] . The CUM was reported. ...
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Human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP) strategies with proven in vivo efficacy rely on antiretroviral drugs, creating the potential for drug resistance and complicated treatment options in individuals who become infected. Moreover, on-demand products are currently missing from the PrEP development portfolio. Griffithsin (GRFT) is a non-antiretroviral HIV entry inhibitor derived from red algae with an excellent safety profile and potent activity in vitro. When combined with carrageenan (CG), GRFT has strong activity against herpes simplex virus-2 (HSV-2) and human papillomavirus (HPV) in vitro and in vivo. Here, we report that GRFT/CG in a freeze-dried fast dissolving insert (FDI) formulation for on-demand use protects rhesus macaques from a high dose vaginal SHIV SF162P3 challenge 4 h after FDI insertion. Furthermore, the GRFT/CG FDI also protects mice vaginally against HSV-2 and HPV pseudovirus. As a safe, potent, broad-spectrum, on-demand non-antiretroviral product, the GRFT/CG FDI warrants clinical development.
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Objective Herpes simplex virus-2 (HSV-2) infections are almost exclusively sexually transmitted. The presence of vaginal gels during the sexual activity may have a significant positive or negative impact on the viral transmission. Therefore we investigated three off-the-shelf vaginal lubricants and one pH restoring gel to evaluate their impact on HSV-2 replication. Results HeLa cells were infected with untreated virions and virions incubated with the particular gels. The accumulation of viral genomes was monitored by quantitative PCR (qPCR) method at 24 hours post infection. Two of the tested gels had no significant effect on HSV-2 replication at the maximum applied concentration, while two had a strong inhibitory effect (~ 98% reduction of replication). The replication inhibitory effect was observed at various multiplicity of infection (MOI 0.4-6.4) and the two inhibitory gels were also capable of inhibiting the HSV-2 induced cytopathic effect on HeLa cells. The surface tension decreasing activity - a sign of detergent activity - was strongly correlated with the anti-HSV-2 activity of the gels (R 2 : 0.88). Our results indicate that off-the-shelf vaginal gels have a markedly different anti-HSV-2 activity that may influence HSV-2 transmission.
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Objective: To evaluate the safety and pharmacokinetics (PK) of MIV-150 and zinc acetate in a carrageenan gel (PC-1005). Acceptability, adherence, and pharmacodynamics (PD) were also explored. Design: A 3-day open label safety run-in (n=5) preceded a placebo-controlled, double-blind trial in healthy, HIV-negative, abstinent women randomized (4:1) to vaginally apply 4 mL of PC-1005 or placebo once daily for 14d. Methods: Assessments included physical exams, safety labs, colposcopy, biopsies, cervicovaginal lavages (CVLs), and behavioral questionnaires. MIV-150 (plasma, CVL, tissue), zinc (plasma, CVL), and carrageenan (CVL) concentrations were determined with LCMS-MS, ICP-MS, and ELISA, respectively. CVL antiviral activity was measured using cell-based assays. Safety, acceptability and adherence were analyzed descriptively. PK parameters were calculated using non-compartmental techniques and actual sampling times. CVL antiviral EC50 values were calculated using a dose-response inhibition analysis. Results: Participants (n=20) ranged from 19-44 years old; 52% were Black or African American. Among those completing the trial (13/17, PC-1005; 3/3, placebo), 11/17 reported liking the gel overall; 7 recommended reducing the volume. Adverse events, which were primarily mild and/or unrelated, were comparable between groups. Low systemic MIV-150 levels were observed, without accumulation. Plasma zinc levels were unchanged from baseline. 7/7 CVLs collected 4h post-dose demonstrated antiviral (HIV, HPV) activity. High baseline CVL anti-HSV-2 activity precluded assessment of post-dose activity. Conclusion: PC-1005 used vaginally for 14d was well-tolerated. Low systemic levels of MIV-150 were observed. Plasma zinc levels were unchanged. Post-dose CVLs had anti-HIV and anti-HPV activity. These data warrant further development of PC-1005 for HIV and STI prevention.
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Women need multipurpose prevention products (MPTs) that protect against sexually transmitted infections (STIs) and provide contraception. The Population Council has developed a prototype intravaginal ring (IVR) releasing the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 (M), zinc acetate (ZA), carrageenan (CG) and levonorgestrel (LNG) (MZCL IVR) to protect against HIV, HSV-2, HPV and unintended pregnancy. Our objective was to evaluate the anti-SHIV-RT activity of MZCL IVR in genital mucosa. First, macaque vaginal tissues were challenged with SHIV-RT in the presence of (i) MIV-150 ± LNG or (ii) vaginal fluids (VF); available from studies completed earlier) collected at various time points post insertion of MZCL and MZC IVRs. Then, (iii) MZCL IVRs (vs. LNG IVRs) were inserted in non-Depo Provera-treated macaques for 24h and VF, genital biopsies, and blood were collected and tissues were challenged with SHIV-RT. Infection was monitored with one step SIV gag qRT-PCR or p27 ELISA. MIV-150 (LCMS/MS, RIA), LNG (RIA) and CG (ELISA) were measured in different compartments. Log-normal generalized mixed linear models were used for analysis. LNG did not affect the anti-SHIV-RT activity of MIV-150 in vitro. MIV-150 in VF from MZC/MZCL IVR-treated macaques inhibited SHIV-RT in vaginal mucosa in a dose-dependent manner (p<0.05). MIV-150 in vaginal tissue from MZCL IVR-treated animals inhibited ex vivo infection relative to baseline (96%; p<0.0001) and post LNG IVR group (90%, p<0.001). No MIV-150 dose-dependent protection was observed, likely because of high MIV-150 concentrations in all vaginal tissue samples. In cervical tissue, MIV-150 inhibited infection vs. baseline (99%; p<0.05). No cervical tissue was available for MIV-150 measurement. Exposure to LNG IVR did not change tissue infection level. These observations support further development of MZCL IVR as a multipurpose prevention technology to improve women's sexual and reproductive health.
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Our recent Phase 1 trial demonstrated that PC-1005 gel containing 50 μM MIV-150, 14 mM zinc acetate dihydrate and carrageenan (CG) applied daily vaginally for 14 days is safe and well tolerated. Importantly, cervicovaginal lavages (CVLs) collected 4h or 24h after last gel application inhibited HIV-1 and HPV in cell-based assays in a dose-dependent manner (MIV-150 for HIV-1 and CG for HPV). Herein we aimed to determine the anti-HIV and anti-HSV-2 activity of PC-1005 in human cervical explants after in vitro exposure to the gel and to CVLs from participants in the Phase 1 trial. Single HIV-1 BaL infection and HIV-1 BaL /HSV-2 co-infection explant models were utilized. Co-infection with HSV-2 enhanced tissue HIV-1 BaL infection. In vitro exposure to PC-1005 protected cervical mucosa against HIV-1 BaL (up to 1:300 dilution) in a single challenge and co-challenge models. CG gel (PC-525) provided some barrier effect against HIV-1 BaL at the 1:100 dilution in a single challenge model, but not in the co-challenge model. Both PC-1005 and PC-525 at the 1:100 dilution inhibited HSV-2 infection, pointing to a CG-mediated protection. MIV-150 and CG in CVLs inhibited HIV (single challenge or co-challenge models) and HSV-2 infections in explants in a dose-dependent manner (p< 0.05). Stronger inhibition of HIV-1 infection by CVLs collected 4h post last gel administration was observed when compared to infection detected in the presence of baseline CVLs. The anti-HIV and anti-HSV-2 activity of PC-1005 gel in vitro and CVLs in human ectocervical explants supports the further development of PC-1005 gel as a broad-spectrum on-demand microbicide.
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Prevalent HSV-2 infection increases the risk of HIV acquisition both in men and women even in asymptomatic subjects. Understanding the impact of HSV-2 on the mucosal microenvironment may help to identify determinants of susceptibility to HIV. Vaginal HSV-2 infection increases the frequency of cells highly susceptible to HIV in the vaginal tissue of women and macaques and this correlates with increased susceptibility to vaginal SHIV infection in macaques. However, the effect of rectal HSV-2 infection on HIV acquisition remains understudied. We developed a model of rectal HSV-2 infection in macaques in combination with rectal SIVmac239Δnef (SIVΔnef) vaccination and our results suggest that rectal HSV-2 infection may increase the susceptibility of macaques to rectal SIVmac239 wild-type (wt) infection even in SIVΔnef-infected animals. Rectal SIVΔnef infection/vaccination protected 7 out of 7 SIVΔnef-infected macaques from SIVmac239wt rectal infection (vs 12 out of 16 SIVΔnef-negative macaques), while 1 out of 3 animals co-infected with SIVΔnef and HSV-2 acquired SIVmac239wt infection. HSV-2/SIVmac239wt co-infected animals had increased concentrations of inflammatory factors in their plasma and rectal fluids and a tendency toward higher acute SIVmac239wt plasma viral load. However, they had higher blood CD4 counts and reduced depletion of CCR5+ CD4+ T cells compared to SIVmac239wt-only infected animals. Thus, rectal HSV-2 infection generates a pro-inflammatory environment that may increase susceptibility to rectal SIV infection and may impact immunological and virological parameters during acute SIV infection. Studies with larger number of animals are needed to confirm these findings.
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Extensive preclinical evaluation of griffithsin (GRFT) has identified this lectin as a promising broad-spectrum microbicide. We set out to explore the antiviral properties of a GRFT and carrageenan (CG) combination product against herpes simplex virus-2 (HSV-2) and human papillomavirus (HPV) as well as determine GRFT mechanism of action (MOA) against both viruses. We performed the experiments in different cell lines, using time-of-addition and temperature dependence experiments to differentiate inhibition of viral attachment from entry and viral receptor internalization. Surface plasmon resonance (SPR) was used to assess GRFT binding to viral glycoproteins and immunoprecipitation and immunohistochemistry were used to identify the specific glycoprotein involved. We determined the antiviral activity of GRFT against HSV-2 to be EC 50 =230nM and provided the first evidence that GRFT has moderate anti-HPV activity (EC 50 =0.429-1.39μM). GRFT blocks entry but not adsorption of HSV-2 and HPV to target cells. The combined analyses of SPR, immunoprecipitation and immunohistochemistry for HSV-2, suggest that GRFT may block viral entry by binding to HSV-2 glycoprotein D. Cell-based assays suggest anti-HPV activity through α 6 integrin internalization. The GRFT/CG combination product but not GRFT or CG alone, reduced HSV-2 vaginal infection in mice when given an hour before challenge (p=0.0352). While GRFT significantly protected mice against vaginal HPV when dosed during and after HPV16 pseudovirus challenge (p
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Transmission of both cell-free and cell-associated immunodeficiency viruses has been demonstrated directly in multiple animal species and possibly occurs in humans, as suggested by genotyping the infecting virus in acutely infected women and HIV in semen from their partners. Therefore, a microbicide may need to block both mechanisms of HIV transmission to achieve maximum efficacy. To date, most of the preclinical evaluation of candidate microbicides has been performed using cell-free HIV. New models of mucosal transmission of cell-associated HIV are needed to evaluate candidate microbicide performance. The MIV-150/zinc acetate/carrageenan (MZC) gel protects Depo-Provera-treated macaques against cell-free SHIV-RT infection when applied vaginally up to 8h before challenge. We recently demonstrated the potent activity of MZC gel against cell-free SHIV-RT in macaque vaginal explants. In the current study, we established a cell-associated SHIV-RT infection model of macaque vaginal tissues and tested the activity of MZC gel in this model. MZC gel protected tissues against cell-associated SHIV-RT infection when present at the time of viral exposure or when applied up to 4d prior to viral challenge. These data support clinical testing of the MZC gel. Overall, our ex vivo model of cell-associated SHIV-RT infection in macaque vaginal mucosa complements the cell-free infection models providing tools for prioritization of products that block both modes of HIV transmission. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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The ex vivo mucosal explant model is frequently used to test the efficacy of microbicides that have the potential for preventing HIV-1 transmission. The conventional assessment of product efficacy has been the extent of HIV-1 p24 suppression in supernatant fluids sampled up to Day 14 after HIV-1 challenge ex vivo. The purpose of this study was to determine if measurement of HIV-1 nucleic acids by real-time PCR and HIV-1 integration by Alu-gag PCR provides advantages with regard to monitoring HIV-1 infection in explants. Rectal biopsies from HIV-1-negative individuals were challenged with 1 x 105 virions/mL of HIV-1BaL or HIV-1CH077 ex vivo. HIV-1 RNA and HIV-1 p24 in supernatant fluids and HIV-1 nucleic acids and integrated provirus in individual biopsies were measured at Days 1-14 after infection. HIV-1 RNA and proviral DNA were measured by quantitative real-time PCR (qRT-PCR) while integrated virus was detected by Alu-gag PCR. Real-time PCR assays detecting HIV-1 DNA and RNA performed similarly provided that the infecting virus sequences were a good match with the sequences of the assay primers and probes. Increased HIV-1 nucleic acid levels and DNA integration were measurable on Days 11 and 14 after infection. The magnitude of explant infection was similar after challenge with HIV-1BaL and HIV-1CH077 although the trajectory of infection was delayed in the HIV-1CH077-infected biopsies. In the majority of experiments, qRT-PCR did not appreciably shorten the time necessary to detect evidence of HIV-1 infection.
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Human tissue explants are a valuable tool to study the interactions between host and infectious agents. They reliably mimic many important aspects of tissue cytoarchitecture and functions and allow us the investigation of the mechanisms of microbial pathogenesis under controlled laboratory conditions. One of the advantages of this system is that, unlike isolated cells, infection of tissue blocks with HIV-1 does not require exogenous stimulation with mitogens or activating factors. Here we describe a protocol to infect with HIV-1 human lymphoid tissue from tonsils and cervico-vaginal tissue and maintain them in culture in a non-polarized setting. These ex vivo infected tissues can be used as fruitful models to study HIV-1 pathogenesis and HIV-1 vaginal transmission, respectively, as well as an efficient platform for testing anti-HIV therapeutic and preventative strategies.