Supplementary Materials for

A replication-defective human cytomegalovirus vaccine for prevention

of congenital infection

Dai Wang*, Daniel C. Freed, Xi He, Fengsheng Li, Aimin Tang, Kara S. Cox,

Sheri A. Dubey, Suzanne Cole, Muneeswara Babu Medi, Yaping Liu, Jingyuan Xu,

Zhi-Qiang Zhang, Adam C. Finnefrock, Liping Song, Amy S. Espeseth, John W. Shiver,

Danilo R. Casimiro, Tong-Ming Fu*

*Corresponding author. Email: dai_wang@merck.com (D.W.); tong-ming_fu@merck.com (T.-M.F.)

Published 26 October 2016, Sci. Transl. Med. 8, 362ra145 (2016)

DOI: 10.1126/scitranslmed.aaf9387

This PDF file includes:

Materials and Methods

Fig. S1. Schematic diagram on the construction of epithelial-tropic AD169 virus.

Fig. S2. Effect of Shld-1 concentration on progeny production of ddFKBP fusion

mutants.

Fig. S3. Genetic map of V160, MAD169, and wild-type HCMV.

Fig. S4. Comparison of epithelial and fibroblast neutralization titers by V160 or

gB vaccines in rhesus macaques.

Fig. S5. Kinetics of anti-beMAD or anti-gB antibodies after V160 vaccination.

Fig. S6. V160 infectivity and progeny production in the absence of Shld-1.

Fig. S7. High-throughput screening of medicinal compound collection.

Fig. S8. Potency of Shld-1 and tacrolimus (FK506) in rescuing V160.

Table S1. Construction and characterization of ddFKBP fusion mutants.

Table S2. Effect of MOI on Shld-1 dependent V160 replication.

Table S3. Minimum Shld-1 and tacrolimus (FK506) concentration required to

rescue V160.

Table S4. ELISPOT responder rates to FKBP versus gB antigens.

www.sciencetranslationalmedicine.org/cgi/content/full/8/362/362ra145/DC1

Materials and Methods

Biological materials

Human ARPE-19 (CRL-2302) retinal pigmented epithelial cells, MRC-5 (CCL-171) embryonic

lung fibroblasts, primary umbilical vein endothelial cells (CRL-1730), primary aortic smooth

muscle cells (CC-2571), primary skeletal muscle cells (CC-2561), and CCF-STTG1 (CRL-1718)

astrocytoma cells were purchased from American Type Culture Collection (ATCC), and were

maintained according to the supplier's recommendations.

The AD169 strain was originally isolated from the adenoids of a 7-year-old girl (15). The virus

was passed 53 times in several types of human fibroblasts for attenuation. Merck & Co., Inc.

received the strain from Dr. H. Stern at passage 53 and passed five additional times in WI-38

human fibroblasts. This live attenuated virus has been tested clinically and the results were

recorded by Neff et al (17).

Construction and in vitro characterization of conditionally replication-defective viruses

The full-length virus genome was cloned as an infectious bacmid, bMAD169-GFP, as previously

described (35). BAC modifications were carried out by galK positive-negative selections (50).

The mutant viruses were recovered by electroporation to MRC5 or ARPE-19 cells in the medium

containing 2µM of Shld-1 when applied. The viruses were titrated by immunoplaque assay or

TCID50 assay.

Compound screening

Spectrum Collection contains 2320 compounds, including 1600 drugs from US and International

Pharmacopeia, experimental bioactives, and pure natural products (MicroSource Discovery

Systems). Fugene6 (Promega) and OptiMEM (Invitrogen) mixture at 1:80 dilution was mixed

with ddFKBP-Luciferase plasmid at a ratio of 1:4.2, and the mixture was added to MDA-MB468

cells (ATCC HTB-132) in suspension and plated at 5000 cells per well. After overnight

incubation, the library compound in triplicates was added at 10 M final concentration, and

incubated for 6 hours. Plates were then screened using a luciferase assay kit (Promega).

Virus titration by TCID50 assay and immunoplaque assay

The TCID50 assay was carried out as described by Wang et al (25). The test samples were 10-

fold serially diluted in the culture medium supplemented with 10% FBS. One hundred

microliters of each serial dilution was then added to each of eight wells of a 96-wll plate

containing 1x104 ARPE-19 cells per well. The plates were then incubated at 37°C for 21 days.

The cytopathic effect (CPE) of the virus in each well was evaluated microscopically and the

TCID50 titer was determined on the basis of the number of wells displaying positive CPE. The

sensitivity of detection limit was estimated to be 1.25 TCID50/ml.

An immunoplaque assay was developed to increase the detection sensitivity of the conventional

HCMV plaque assay. Briefly, serially diluted virus samples were added to ARPE-19 cell

monolayers grown on a 6-well plate. After 2 h incubation, an agarose overlay was added and the

plates were incubated at 37°C for 21 days. The overlays were then removed, and the monolayers

were fixed at room temperature with absolute methanol for 20 min. The infected cells were

immunostained by a rabbit anti-HCMV monoclonal antibody and stained with Vectastain ABC

kits (Vector Labs).

Statistical analysis

Titers and ELISPOT responses (number of spots) were log 10 transformed first prior to any

analysis due to the distribution of the data, and the analysis results were then transformed back to

original scales for presentation purposes. All the statistical analyses were conducted using SAS

program.

The NT50 data from mice or rabbits immunized with different vaccines at different dose regimens

were used to study the vaccine immunogenicity in small animals. Analysis of variance

(ANOVA) with log transformed titers was performed to compare geometric means of different

treatment groups with Tukey’s method for pairwise testing when three or more groups were

compared to each other at an overall significance level of of 0.05.

To evaluate the immunogenicity profiles and T-cell response profiles of V160 in nonhuman

primate, repeated measures ANOVAs were used to analyze the overall treatment effects over

time for different doses of V160 with and without adjuvant formulations. For immunogenicity

study, it was a 72-week longitudinal study. For T-cell response profiles, it was a 52-week

longitudinal study. The statistical models used for the evaluation in both studies included

treatment effect, time, and an interaction term of treatment and time using either log transformed

NT50 titers or log transformed spot numbers assuming compound symmetry variance-covariance

structure of the corresponding responses at different time points. Tukey’s method was then used

to perform multiple pairwise comparisons of overall means over time by controlling Type I error

inflation at an overall significance level of of 0.05 in each study, respectively.

We analyzed the proportion of vaccine specific responders to FKBP peptides compared to gB

control to evaluate immune responses of V160 to FKBP in nonhuman primates. A rhesus

macaque was defined as a vaccine responder if the antigen-specific response of the blood sample

(1) ≥35 spots per 1x106 cells and (2) ≥ 3.5-fold higher than mock control, or ≥ 3.5-fold higher

than pre-vaccination response with ELISPOT assay. An exact Clopper-Pearson approach was

used to estimate the 95% confidence intervals of the true proportion of vaccine specific

responders, since the proportion has zero frequency in the FKBP group. The proportion

difference of vaccine specific responders between FKBP and gB control was then evaluated with

a Fisher’s exact test. If the p value for the two-sided test is less than 0.05, we will conclude the

proportions between the two groups are significantly different.

Paired two-sided t-tests were used to assess fold-rise of (1) T-cell responses, (2) antibody

responses pre and post vaccination for each monkey within individual FKBP and gB control

groups, and (3) between FKBP and gB control groups using log transformed data. If the p value

for any test is less than 0.05, the difference is considered statistically significant. Fold differences

and associated 95% confidence intervals were calculated by taking exponentiation of the

corresponding log differences from the paired t-tests. If the 95% confidence intervals of the fold

difference include 1, it indicates there is no significant change; otherwise, there is significant

change pre and post vaccination or between two groups.

Fig. S1. Schematic diagram on the construction of epithelial-tropic AD169 virus. (A)

Generation of HCMV BAC clone. A DNA fragment containing the BAC vector and a GFP

expression cassette flanked by two LoxP sites was integrated into MAD169 genome between

US28 and US29 ORFs by homologous recombination to generate BAC clone bMAD-GFP.

(B) Rescue of the epithelial tropism of bMAD-GFP. One adenine nucleotide was removed

from the first exon of UL131 in bMAD-cre to repair the frame shift mutation of UL131,

which is sufficient to rescue the viral tropism to epithelial cells (25). (C) Construction of self-

excisable BAC. The GFP ORF in bMAD-GFP was subsequently replaced by a cre

recombinase ORF to generate beMAD.

Fig. S2. Effect of Shld-1 concentration on progeny production of ddFKBP fusion mutants. ARPE-19 cells were infected at MOI of 0.01 pfu/cell with ddFKBP fusion mutants for one hour,

washed twice with fresh medium, and incubated in the growth medium containing 0, 0.05, 0.1,

0.5 or 2 µM of Shld-1. Seven days post infection, the cell free virus was collected from

supernatant, and virus titers were determined by TCID50 assay on ARPE-19 cells in the

presence of 2 µM of Shld-1. The experiments were conducted twice, and the representative data

are shown.

Fig. S3. Genetic map of V160, MAD169, and wild-type HCMV. Notable changes in MAD169

include deletion of ULb’ and duplication of RL fragment, point mutations in genes RL5A, RL13,

UL36 and UL131. V160 was engineered using MAD169 as parental virus, in which the UL131

mutation was repaired and ddFKBP was fused in frame to the N-termini of UL123 exon1 and

UL51. Symbols: * mutation, d ddFKBP

Fig. S4. Comparison of epithelial and fibroblast neutralization titers by V160 or gB

vaccines in rhesus macaques. The week 28 sera as shown in Fig. 4 were analyzed by HCMV

micro-neutralization assay on ARPE-19 or MRC-5 cells. Numbers represent the GMT for the

corresponding groups.

Fig. S5. Kinetics of anti-beMAD or anti-gB antibodies after V160 vaccination. Serum

samples collected as shown in Fig. 4 were analyzed using purified beMAD virions (panel A) or

recombinant gB (panel B) as antigen in ELISA. The lines represent GMT for the indicated

groups with standard errors.

Fig. S6. V160 infectivity and progeny production in the absence of Shld-1. (A) Experimental

outline for detection of replication-competent virus from 1x109 pfu of the V160 vaccine virus in

the absence of Shld-1. The virus inoculum was plated onto ninety 150-cm2 flasks of ARPE-19

cells, at MOI of 2 pfu/cell. At day 21 post-infection, the supernatants were harvested and added

to fresh ARPE-19 monolayers cultured on ninety 10-cm Petri dishes, through two rounds of

absorption with 1 hour for each infection. After absorption, an agarose overlay was added and

the plates were incubated for an additional 21 days. The plates were then immunostained by a

gB-specific monoclonal antibody and examined for virus plaque formation under a light box. (B)

Immunostaining of ARPE-19 monolayer infected by V160 or beMAD. ARPE-19 cells were

inoculated with day 21 harvests as described in (A) and the infected cells were detected by

immunostaining with a gB specific monoclonal antibody and examined under microscope.

Fig. S7. High-throughput screening of medicinal compound collection. The library was

screened for compounds that can mimic Shld-1 in MDA-MB468 cells expressing ddFKBP-

luciferase. (A) Compounds were added at 10 M to cells expressing ddFKBP-luciferase for six

hours and the luciferase activity [presented as ACT% (x-axis)] is measured and plotted against

the cell viability (y-axis). The average signal for each compound was normalized as luciferase

activity relative to the Shld-1 control on each assay plate. (B) Rapamycin (sirolimus) and its

derivatives, including everolimus, pimecrolimus, temsirolimus, deforolimus, and tacrolimus

(FK506), were tested in titrations, with Shld-1 as the positive control. EC50 for Shld-1 is

calculated as 0.18 M; EC50 for tacrolimus (FK506) is 0.88 M. The compound library (n=2320

entities) was obtained from MicroSource.

Fig. S8. Potency of Shld-1 and tacrolimus (FK506) in rescuing V160. ARPE-19 cells were

infected with V160 at MOI of 3 pfu/cell, and then incubated with growth medium supplemented

with indicated concentrations of Shld-1 or tacrolimus. Cell-free progeny virus was collected at

the indicated time points after infection, and was quantified by TCID50 assay on ARPE-19 cells

in the medium containing 2 µM of Shld-1. The experiments were conducted twice, and the

representative data are shown.

Table S1. Construction and characterization of ddFKBP fusion mutants.

Target genes Fusion position Growth and plaque phenotype (+Shld-1)

Immediate early genes

IE1/2 N terminal WT-like

UL37 N terminal Small plaque*

DNA replication

UL44 C terminal Small plaque*

UL105 C terminal No plaque

DNA packaging and cleavage

UL51 N terminal WT-like

UL52 N terminal WT-like

UL56 C terminal No plaque

UL77 C terminal Small plaque*

Nuclear egress

UL53 C terminal WT-like

Multifunctional regulatory

UL84 C terminal WT-like

Unknown functions

UL79 N terminal WT-like

UL87 N terminal WT-like

Small plaque*, <20% of the wild type plaque size

Table S2. Effect of MOI on Shld-1 dependent V160 replication.

Days post

infection

V160 virus titers by TCID50 after infection with the MOI (pfu/cell)

10 3 1 0.3 0.1 0.1(Shld-1)

3 N.D.* N.D. N.D. N.D. N.D. N.D.

6 N.D. N.D. N.D. N.D. N.D. 1.68x102

9 N.D. N.D. N.D. N.D. N.D. 1.00x103

16 N.D. N.D. N.D. N.D. N.D. 1.49x105

*N.D., not detectable

ARPE-19 Cells were infected with V160 at MOI of 10, 3, 1, 0.3 and 0.1 PFU per cell for 1 hour, washed with citrate

buffer for one minute to inactivate unpenetrated virus, and then cultured in the absence of Shld-1. An infection of

MOI of 0.1 with 0.25 μM Shld-1 was included as positive control. Cell-free virus was collected at the indicated time

points after infection, and infectious virus was quantified by TCID50 assay on ARPE-19 cells in medium containing

2 μM Shld-1.

Table S3. Minimum Shld-1 and tacrolimus (FK506) concentration required to rescue V160.

ARPE19 MRC5

MOI (pfu/cell) 10 3 1 0.3 0.1 0.01 0.1

Shld-1 (nM) >50 >50 >50 >50 >50 >100 >25

Tacrolimus (nM) N.T.* >1000 >1000 >1000 N.T. N.T. N.T.

*N.T., not tested

ARPE-19 cells were infected at MOI of 10, 3, 1, 0.3, 0.1 and 0.01 pfu/cell. MRC5 cells were infected at MOI of 0.1

pfu/cell. The infected cells were then washed with a citrate buffer for 1 min to inactivate unpenetrated virus.

Following infection, the cells were cultured in the medium containing 2, 0.25, 0.1, 0.05, 0.025 and 0.01 µM of Shld-

1. The culture supernatants were collected at day 3, 6, 9 and 19 days post infection, and the titers were determined

by TCID50 assay. The minimum concentration was determined by highest concentration of Shld-1 in which no

progeny virus could be detected at all 4 time points.

Table S4. ELISPOT responder rates to FKBP versus gB antigens. FKBP peptides gB peptides

Number of responder of total cohort 0 out of 49 24 out of 49

Responder rate (95% CI) (%) 0 (0.0 – 7.3) 49.0 (34.4 – 63.7)

Note: a vaccine-specific responder was defined if an animal was a responder with (1) ≥ 35 SFC per 106 PBMC and

(2) ≥3.5-fold elevation to antigen peptides over the mock control after vaccination but a non-responder before

vaccination, or the animal was responders after and before vaccination but with the SFC ratio of after and before

≥3.5. The responder rate and 95% confidence interval were estimated with Clopper-Pearson (exact) method for

binomial data.