Griffithsin protects mice from genital herpes by preventing cell-to-cell spread

Abstract

Griffithsin, which binds N-linked glycans on gp120 to prevent HIV entry, has the most potent HIV-1 inhibitory activity described for any antiviral lectin and is being developed for topical preexposure prophylaxis. The current studies were designed to further assess its potential by exploring its activity against herpes simplex virus 2 (HSV-2), a cofactor for HIV acquisition, in vitro and in a murine model. Safety was evaluated by examining its impact on epithelial barrier integrity in polarized cultures and testing whether repeated intravaginal dosing potentiates the susceptibility of mice to genital herpes. Griffithsin displayed modest inhibitory activity against HSV-2 if present during viral entry but completely blocked plaque formation if present postentry, reduced plaque size, and prevented cell-to-cell spread. These in vitro findings translated to significant protection against genital herpes in mice treated with 0.1% griffithsin gel. Griffithsin, but not placebo gel, prevented viral spread (visualized with a luciferase-expressing virus), significantly reduced disease scores, and resulted in greater survival (P < 0.05, log rank test). Protection persisted when HSV-2 was introduced in seminal plasma. Although griffithsin triggered a small decline in transepithelial electrical resistance in polarized cultures, this did not translate to any significant increase in the ability of HIV to migrate from the apical to the basolateral chamber nor to an increase in susceptibility to HSV-2 in mice treated with griffithsin gel for 7 days. These findings demonstrate that griffithsin inhibits HSV-2 by a unique mechanism of blocking cell-to-cell spread and support its further development for HIV and HSV-2 prevention.

Fig 1

Griffithsin inhibits HSV-2 infection post entry. (A) CaSki, VK2/E6E7, and 3T3 cells were exposed to GRFT or PRO 2000 for 15 min, followed by challenge with HSV-2(4674) for 1 h. The cells were washed and then overlaid with medium, and plaques were counted after 24 to 48 h; results are presented as PFU/well as percentages of control (no drug) and are means + SEM from 2 to 4 experiments conducted in duplicate. (B) Immature moDCs were exposed to GRFT overnight and then challenged with HSV-2(333)ZAG (MOI of 1 PFU/cell) for 1 h at 37°C. Infection was assessed by quantifying GFP expression (10,000 live event acquisition) by flow cytometry 4 hpi. Results are presented as percentage of GFP⁺ cells and are means + SEM from six (pretreat) independent experiments. To monitor effects of GRFT on the release of viral progeny, the DCs were exposed to GRFT either before or after initial entry, and supernatants were harvested 24 hpi and overlaid onto Vero cells; viral plaques were counted after Giemsa staining and are presented as percentages relative to DCs infected in the absence of GRFT from 2 independent experiments, each conducted in duplicate. (C) CaSki or VK2/E6E7 cells were first exposed to HSV-2 for 1 h, washed with PBS, and then cultured in medium supplemented with the indicated concentration of GRFT; results are presented as percentages of the control and are the means + SEM from 3 experiments performed in duplicate. (D) Representative photograph of viral plaques in VK2/E6E7 cells when GRFT was added postentry. (E) EC₅₀ and EC₉₀ (μg/ml) were calculated for GRFT using data from panels A to C and Fig. 2A. Asterisks indicate P values of <0.05 compared to no drug (t test).

Fig 2

Griffithsin inhibits cell-to-cell spread of HSV-2. (A) One-step growth of HSV was quantified by infecting CaSki cells with an MOI of 5 and then adding GRFT or ACV to the overlay media. At 4, 8, and 16 hpi, supernatants and cells were harvested and, after cells were freeze-thawed three times, viral growth was quantified by determining the titers of the supernatants (solid lines) or cells (dotted lines) on Vero cells. (B) To assess multistep growth, CaSki cells were infected with an MOI of 0.01, and drug was added to overlay media as described for panel A, and then at 12, 24, 36, 48, and 72 hpi, supernatants and cells were again harvested and titers were determined on Vero cells. Data shown are from two experiments conducted in duplicate. Asterisks indicate significance compared to no drug using two-way ANOVA. (C) CaSki cells were infected with HSV-2 at an MOI of 1 with GRFT, ACV, or no drug (control) added to the overlay media. Cells were harvested 8 or 12 hpi, and DNA was extracted and run in real-time PCR. Data are presented as means ± SEM of log change in ICP4 DNA levels relative to infected cells without drug, controlling for RPLPO as the housekeeping gene. Asterisk represents significance compared to HSV without drug using unpaired t test, P < 0.05. (D) Cells were infected with HSV-2 for 4 to 5 h, after which time they were trypsinized and cocultured with uninfected cells in medium supplemented with pooled human IgG and GRFT for 48 h to allow plaques to form. Data are shown as means + SEM of percentage of virus control from three independent experiments. Asterisks indicates significance from virus control (no GRFT) using unpaired t test, P < 0.05.

Fig 3

Griffithsin protects mice from genital herpes infection. Medroxyprogesterone acetate-treated mice received a single dose of 0.1% GRFT or Carbopol gel intravaginally (20 μl). Fifteen minutes later, the mice were intravaginally inoculated with an LD₅₀ (A) or LD₉₀ (B) of HSV-2(4674) diluted in either PBS or pooled human seminal plasma (SP). Mice were scored for disease daily and were sacrificed if the score reached 4. Additional controls included mice that received no gel prior to challenge with virus in PBS. The number of mice in each group is indicated in the figure legend of the Kaplan-Meier survival curves; asterisks indicate P values of <0.05 (log rank test) (A and B). Disease scores for the 20 mice treated with Carbopol or GRFT gel and challenged with an LD₅₀ of HSV-2 in PBS are shown in panel C, with average daily disease scores indicated above each bar. The asterisks denote that the difference in disease scores was significantly lower for GRFT- than for Carbopol-treated mice at each time point (P < 0.05; t test). Genital tract tissue was harvested 1 and 7 days postinfection, weighed, and homogenized, and supernatants were cultured on Vero cells to determine the amount of infectious HSV-2 present. Plaques were counted after 48 h; each dot represents an individual animal, and the line indicates mean ± SEM; day 1, P = 0.12; day 7, P = 0.07, unpaired t test.

Fig 4

GRFT treatment prevents viral dissemination in mice. Medroxyprogesterone-treated mice received a single dose of GRFT or Carbopol gel and 15 min later were intravaginally infected with HSV-2-MS-luciferase (10⁶ PFU in 30 μl). Mice were imaged on days 1 and 4 postinfection, 15 min after receiving an intraperitoneal dose of d-luciferin. Total flux from identical ROIs were compared to total flux from ROI on the control mouse pretreated with medroxyprogesterone, not infected with HSV-2, 15 min after intraperitoneal injection of d-luciferin.

Fig 5

HSV induces proinflammatory and interferon responses in the genital tract independent of griffithsin treatment. (A) Concentrations of cytokines and chemokines were measured in vaginal washes on days 1 and 2 postinfection. Results are presented as pg/ml in vaginal washes and are means + SEM from 2 to 4 pools of washes from 2 to 5 mice per pool. Asterisks represent significance compared to untreated, uninfected mice using unpaired t test. (B) Gene expression was measured by qRT-PCR on genital tract tissue harvested from 2 to 10 mice on days 1 and 3 postinfection; results are normalized to β-actin and expressed as means + SEM log change relative to control untreated, uninfected mice (*, P < 0.05).

Fig 6

Griffithsin is not cytotoxic but reduces transepithelial electrical resistance in a dual-chamber model. (A) Cells were exposed to GRFT for 48 h (CaSki, VK2/E6E7, 3T3) or overnight (moDCs), and cell viability was assessed by MTS; results are means + SEM from at least 2 independent experiments. (B) HEC-1-A cells were cultured in Transwells (0.5 × 10⁵ to 1 × 10⁵ cells/insert), and TER was monitored daily. After the TER reached a plateau (4 or 5 days), cells were exposed to 0.1 or 1 mg/ml GRFT, 0.1% (vol/vol) N-9, or medium alone for 18 h. After removal of drug by washing, HIV-1_BaL (40 ng p24) and JT-CCR5 cells (10⁵ cells/well) were added to the upper and lower chambers, respectively. The TER was monitored prior to drug addition (t = −1 day) and then daily after addition of HIV. (C) Supernatants were collected from the basolateral chambers at the indicated times and tested for p24 content by enzyme-linked immunosorbent assay (ELISA). Results are means ± SD from at least two independent experiments, where each condition was tested in duplicate. Controls included wells in which no drug was added (medium), no cells were added, and no HIV was added. Asterisks indicate significance compared to medium (P < 0.05) using two-way ANOVA with Tukey's multiple comparisons test.

Fig 7

Repeat exposure to griffithsin does not increase susceptibility to HSV-2 in a murine model. Mice were pretreated with medroxyprogesterone acetate; 5 days later, mice received one 20-μl dose of 0.1% GRFT, Carbopol, or 4% N-9 gel intravaginally daily for 7 days. On the eighth day, a vaginal wash was taken to remove residual drug and to test for residual anti-HIV activity in a TZM-bl assay; results are means (SEM) from 3 pools of vaginal washes, each pool containing washes from 2 mice, conducted in triplicate. (A) Data are plotted as percentages of virus control. Asterisk indicates P values of <0.05 compared to washes from Carbopol-treated mice (unpaired t test). Mice were then challenged with HSV-2(4674) diluted in 30 μl PBS at LD₁₀ (B), LD₅₀ (C), or LD₉₀ (D), with 10 to 20 mice per group. Mice were scored for disease daily and were sacrificed if indicated by disease scores. Kaplan-Meier survival curves are shown; asterisks indicate P values of <0.05 (log rank test), compared to Carbopol treatment.

Fig 8

Griffithsin does not induce DC maturation or induce apoptosis. (A) Immature moDCs were exposed to medium (mock) or LPS (10 μg/ml) for 24 h at 37°C, and then one set each was stimulated with LPS (10 μg/ml) for 1 h. The cells were then analyzed by flow cytometry for surface marker CD83 expression. Data are mean percentages of CD83⁺ + SEM from six (24 h) and two (+LPS) independent experiments. (B) Immature moDCs were exposed to the indicated concentrations of GRFT overnight and analyzed by flow cytometry for percentage of cells that are annexin V⁺/7AAD⁻ (early apoptosis) and annexin V⁺/7AAD⁺ (late apoptosis). Data are means + SEM from two independent experiments. Asterisks indicate significance compared to mock-treated cells under each condition (P < 0.05, ANOVA).