A replication-defective human cytomegalovirus ...

SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE VACCINES

A replication-defective human cytomegalovirus vaccine for prevention of congenital infection

2016 © The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science.

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*

INTRODUCTION

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that rarely causes symptomatic infection in healthy individuals (1). However, in immunocompromised patients, including those under immunosuppression after transplantation, HCMV infection may lead to lifethreatening diseases (2, 3). Moreover, HCMV can cause congenital viral infections and is responsible for a wide range of neurodevelopmental abnormalities in children (4, 5). Development of a prophylactic vaccine is a public health priority (6). Children born to HCMV-seropositive women are ~69% less likely to suffer congenital infection than those born to seronegative mothers who acquire HCMV infection during pregnancy (7, 8). Thus, our goal when designing a vaccine was to replicate the immune responses seen in healthy seropositive individuals. Natural HCMV infection elicits broad and robust responses involving both arms of adaptive immunity. HCMV-specific T cells in healthy adults can constitute as much as 10% of the total memory CD4+ and CD8+ T cells that recognize multiple viral proteins, notably, pp65, IE1, IE2, and gB (9). HCMVspecific antibodies potently neutralize viral infection of a variety of cell types (10, 11): gB antibodies primarily prevent infection in fibroblasts, whereas most antibodies that block infection of epithelial and endothelial cells, monocytes, and placenta cytotrophoblasts target the gH/gL/ pUL128-131 pentameric complex (12–14). Although whole virus–based vaccines are more likely to produce immune responses resembling those of natural infection, live attenuated HCMV vaccines have yet to be successfully developed. The bestcharacterized live vaccines are fibroblast-adapted AD169 (15) and Towne (16). Both were safe and well tolerated in clinical studies (15–17). Efficacy of the Towne strain has been tested in renal transplant patients Merck Research Laboratories, Merck and Co. Inc., Kenilworth, NJ 07033, USA. *Corresponding author. Email: [email protected] (D.W.); tong-ming_fu@merck. com (T.-M.F.) †Present address: Janssen Research and Development, Spring House, PA 19477, USA. ‡Present address: Plasmon Analytics LLC, Berwyn, PA 19312, USA. §Present address: Sanofi Pasteur, Swiftwater, PA 18370, USA. ¶Present address: Aeras, Rockville, MD 20850, USA. Wang et al., Sci. Transl. Med. 8, 362ra145 (2016)

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(18, 19) and in seronegative women with children in day care (20). In both settings, it failed to prevent primary infection or viral reactivation. In a human challenge study, the Towne strain was less protective than natural immunity against wild-type HCMV infection (21). The reduced efficacy of Towne vaccine was at least partially attributed to its failure to elicit antibodies to the gH/gL/pUL128-131complex (22, 23). Towne virus carries a frameshift mutation in the UL130 open reading frame (ORF) (23–25). As a result, the virus is deficient in production of the pentameric complex (23, 25). A frameshift mutation in the UL131 gene was found in AD169, which also affects the pentameric complex expression (24, 25). Because the pentameric complex determines viral tropism for epithelial and endothelial cells, in contrast to clinical isolates, neither Towne nor AD169 can infect these cells efficiently (23, 24, 26). We recently reported that the restoration of the pentameric complex in AD169 virus significantly enhanced its immunogenicity (27). Here, we further improved the vaccine by rendering it conditionally replicationdefective. The control of viral replication was achieved by fusing the destabilizing domain of FK506-binding protein 12 (ddFKBP) to viral proteins IE1/2 and pUL51 (28, 29). The fusion directed these essential proteins to proteasome degradation, which effectively blocked virus progeny production. The degradation of these proteins could be reversed by a synthetic molecule termed Shield-1 (Shld-1), which specifically binds to ddFKBP (28, 30). We tested the control of vaccine virus replication by Shld-1 and its ability to elicit humoral and cell-mediated immune responses, as measured by viral neutralization and interferon-g (IFN-g) enzyme-linked immunospot (ELISPOT) assays. RESULTS

Vaccine construction and in vitro characterization A derivative from the AD169 vaccine manufactured at Merck (17), described hereafter as MAD169, was used as the parental virus to construct an infectious bacterial artificial chromosome (BAC) clone bMADGFP (green fluorescent protein) (fig. S1A). MAD169 carries attenuation markers as reported, such as the UL/b′ deletion (31, 32), UL36 substitution (33, 34), and a frameshift mutation in UL131. The UL131 mutation 1 of 8

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Congenital human cytomegalovirus (HCMV) infection occurs in ~0.64% of infants born each year in the United States and is the leading nongenetic cause of childhood neurodevelopmental disabilities. No licensed HCMV vaccine is currently available. Natural immunity to HCMV in women before pregnancy is associated with a reduced risk of fetal infection, suggesting that a vaccine is feasible if it can reproduce immune responses elicited by natural infection. On the basis of this premise, we developed a whole-virus vaccine candidate from the live attenuated AD169 strain, with genetic modifications to improve its immunogenicity and attenuation. We first restored the expression of the pentameric gH/gL/pUL128-131 protein complex, a major target for neutralizing antibodies in natural immunity. We then incorporated a chemically controlled protein stabilization switch in the virus, enabling us to regulate viral replication with a synthetic compound named Shield-1. The virus replicated as efficiently as its parental virus in the presence of Shield-1 but failed to produce progeny upon removal of the compound. The vaccine was immunogenic in multiple animal species and induced durable neutralizing antibodies, as well as CD4+ and CD8+ T cells, to multiple viral antigens in nonhuman primates.

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Immunogenicity comparison in small animals The immunogenicity of ddIE1/2, ddUL51, and V160 (ddIE1/2-ddUL51) was compared to that of beMAD. BALB/c mice were immunized with 2.5, 0.63, or 0.16 mg of purified virus at weeks 0 and 3; each microgram of vaccine contained 3 × 105 PFU of virus. Serum samples were Wang et al., Sci. Transl. Med. 8, 362ra145 (2016)

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collected after vaccination and evaluated for neutralizing activities in ARPE-19 cells. As shown in Fig. 3A, ddIE1/2, ddUL51, or V160 induced comparable neutralizing titers, calculated as reciprocal serum dilutions required to neutralize 50% input virus (NT50). The geometric mean titers (GMTs) for the 2.5 mg/dose groups were 1660, 1260, and 1160, and Tukey’s pairwise comparison revealed no difference among three viruses. The NT50 titers of the beMAD groups at the higher doses (2.5 and 0.63 mg) were slightly lower than those of other vaccines, with the GMT for the 2.5 mg/dose group calculated at 520 (P = 0.07, comparing to V160, Tukey’s pairwise analysis). Thus, the three replicationdefective viruses were as immunogenic as beMAD in mice. T cell responses induced by beMAD and V160 in mice were determined by IFN-g ELISPOT assay, using pools of overlapping peptides representing viral antigens pp65, IE1, IE2, and gB. The numbers of antigen-specific spot-forming cells (SFCs) elicited by beMAD and V160 were comparable (Fig. 3B). Next, we compared the immunogenicity of MAD169, beMAD, and V160 vaccines in rabbits (Fig. 3C). Vaccination with beMAD or V160 viruses elicited comparable neutralizing antibody titers, with respective NT50 GMT values of 1920 and 1870 (P = 1.00). In contrast, the NT50 titer elicited by MAD169 was more than 30 times lower than that elicited by beMAD derivatives expressing the pentameric complex (P < 0.0001). Vaccine immunogenicity in nonhuman primates Next, we evaluated the V160 immunogenicity in rhesus macaques. Less than 5% of the rhesus macaques had detectable neutralizing titers to HCMV, even though the entire colony had been exposed to rhesus CMV (rhCMV) (27). The kinetics of vaccine induction of the neutralizing antibodies to HCMV in monkeys were similar to those of primary responses (27), suggesting that even with chronic rhCMV infection, most monkeys were naïve to the HCMV pentameric complex. The vaccine candidate was tested in the following groups: 100 mg (3 × 107 PFU)/dose, 10 mg (3 × 106 PFU)/dose, and a formulation of 10 mg/dose with Iscomatrix, an adjuvant that has been evaluated in the clinic (37). As a control, we included a group of animals immunized with 30 mg of recombinant gB vaccine, formulated with an oil-in-water adjuvant with properties similar to those of MF59. Neutralizing activity became detectable after the first dose, and the titers peaked 4 weeks after the second and third vaccination (Fig. 4). The peak GMT for the 100 mg/ dose group was 14,500, about three times higher than that for the 10 mg/ dose group. Iscomatrix enhanced neutralizing titers, with a peak GMT of 15,800 versus 4660 for the 10 mg/dose unadjuvanted groups. Low levels of neutralizing activity were detected in the gB group, with GMTs below 200 throughout the study. At almost 1 year after vaccination, the GMTs for the 100 mg/dose group and the 10 mg/dose + Iscomatrix groups were maintained at 1400 and 3000, whereas the GMT for the 10 mg/dose unadjuvanted group had drifted to about 200. The multiple pairwise comparisons showed significantly higher titers of all V160 groups over the gB group when we assessed vaccine treatment across all time points (P < 0.01, P = 0.02, and P < 0.01 for the 100 mg/dose, 10 mg/dose, and 10 mg/dose + Iscomatrix versus gB group, respectively). The 100 mg/dose and the 10 mg/dose + Iscomatrix groups were comparable (P = 0.89), indicating that the formulation with the Iscomatrix adjuvant could induce immune responses equivalent to those of a higher vaccine dose. HCMV immune sera could neutralize virus in a variety of cells, but the titers required in epithelial cells are in general five to eight times higher than those in fibroblasts (22, 38). V160 immune sera demonstrated 2 of 8


underlies AD169’s deficiency in infecting epithelial cells (23–25), and the frameshift was repaired by deleting one adenine in the first exon of UL131 to create B(b)AC-derived, epithelial-tropic MAD169 GFPexpressing clone, designated as beMAD-GFP (fig. S1C). This clone was then modified by replacing the GFP with a cre recombinase (35) to create a self-excisable BAC, beMAD. We further attenuated the virus by fusing ddFKBP to a panel of 12 essential genes individually to restrict its replication to a single cycle (table S1). All proteins selected were nonstructural (36), so the ddFKBP fusion proteins are unlikely to be packaged in mature viral particles. The viral constructs were named after the genes to which ddFKBP was fused. Because IE1 and IE2 are expressed from alternatively spliced, major immediate-early transcripts and share the first three exons, both were produced as ddFKBP fusion proteins. Among the recombinant viruses, ddIE1/2, ddUL51, ddUL52, ddUL84, ddUL79, and ddUL87 were readily rescued. The ddUL37x1, ddUL77, and ddUL53 viruses produced small plaques (table S1), indicating impaired growth; increasing Shld-1 concentration to 10 mM did not improve their growth. The ddUL56 and ddUL105 mutants could not be recovered. Efficient replication of all ddFKBP mutants depended on the Shld-1 concentration, albeit to varying degrees (fig. S2). In general, lower concentrations of Shld-1 reduced the titer of progeny virus. Among these ddFKBP viruses, only ddUL51 and ddUL52 absolutely required Shld-1 for replication. Other viruses, ddIE1/2, ddUL84, ddUL79, and ddUL87, could produce lower but detectable progeny in the absence of Shld-1 (table S1 and fig. S2), suggesting that addition of ddFKBP to these ORFs did not completely abrogate their functions. Because the steps of HCMV replication occur in a specific temporal order, we hypothesized that the stringency of viral replication control could be improved if replication was blocked or attenuated at two different stages of the viral life cycle. To test this hypothesis, we constructed a double-tagged virus in which the ddFKBP was fused to the N termini of the IE1/2 and pUL51 proteins. As shown in Fig. 1A, at a multiplicity of infection (MOI) of 0.01 plaque-forming unit (PFU) per cell, no progeny of the ddIE1/2-ddUL51 virus could be detected if the Shld-1 concentration was below 0.1 mM at day 7 after infection. In contrast, after infection with the ddUL51 virus, low levels of progeny virus could be found in the presence of 0.05 mM Shld-1 (fig. S2). Because the ddIE1/2-ddUL51 virus showed the most stringent dependency on Shld-1, it was selected as the candidate and named hereafter as V160 (fig. S3). The growth kinetics of V160 in the presence of 2 mM Shld-1 were comparable to those of its parental virus beMAD in ARPE-19 cells, with both showing peak titers of ~1 × 107 PFU/ml around day 11. In the absence of Shld-1, no progeny virus could be detected in the supernatants (Fig. 1B). The stringency of V160 replication control by Shld-1 was also tested in different types of human cells including fibroblasts (MRC-5), umbilical vein endothelial cells, aortic smooth and skeletal muscle cells, and neuronal astrocytoma cells (Fig. 2). In all cell types tested, no V160 production could be detected in the absence of Shld-1, suggesting that the conditional replication of the vaccine is not cell type–specific.

SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE similar patterns (fig. S4), whereas the gB immune sera in our rhesus study showed low neutralizing activities in both cell types, consistent with previous observations (22, 39). To ensure that the gB vaccine was immunogenic, we tested the immune sera for binding activities to purified HCMV virions or recombinant gB, and the result confirmed that the gB vaccine was immunogenic in monkeys (fig. S5). We tested peripheral blood mononuclear cells (PBMCs) collected before and after vaccination in the IFN-g ELISPOT assay, using pools of peptides from pp65, IE1, IE2, and gB (Fig. 5). Before vaccination, the number of antigen-specific cells was comparable to that elicited by mock antigen, except in two monkeys that showed responses to gB (Fig. 5B). V160 vaccination elicited T cell responses to both structural (pp65) and regulatory antigens (IE1 and IE2). Iscomatrix adjuvant led to a significantly higher response to pp65 and IE1 than did the unadjuvanted 10 mg/dose treatment (Fig. 5B versus Fig. 5C; P = 0.03

Virus titer (log10 TCID50/ml)

Conditional replication of V160 vaccine virus Three studies were conducted to assess the stringency of the ddFKBP/ Shld-1 mechanism on control of productive V160 infection. First, we evaluated the MOI effect on virus growth in the absence of Shld-1. ARPE-19 cells were infected at MOIs ranging from 0.01 to 10 PFU per cell. After culturing in the absence of Shld-1, cell-free virus was collected at various time points after infection, and the titers were determined by TCID50 in the presence of Shld-1. No cell-free progeny virus could be detected at any time point after infection, regardless of the initial MOI tested (table S2). Second, we determined the minimum concentration of Shld-1 required to rescue V160 progeny production. ARPE-19 cells were infected at MOIs ranging from 0.01 to 10 PFU per cell and then cultured in a medium containing various concentrations of Shld-1. The supernatants were collected after infection, and infectivity was measured in the presence of Shld-1. Rescued replication was defined by presence of detectable infectious progeny. An Shld-1 concentration of 50 nM or greater was necessary for viral replication, at an MOI above 0.01 PFU per cell, in ARPE-19 cells (table S3). At an MOI of 0.1 PFU/ml, greater than 25 nM Shld-1 was needed to rescue V160 replication in MRC-5 cells. Last, we refined the viral culture scheme with two rounds of amplification, attempting to detect Shld-1–independent virions from an inoculum of ~1 × 109 PFUs of V160. The inoculum was plated onto ARPE-19 cells, at an MOI of 2 PFU per cell, statistically capable of infecting 86% of cells (fig. S6A). At day 21 after infection, the supernatants were harvested and added to fresh ARPE-19 monolayers, and these were incubated for an additional 21 days. No visible plaques could be detected in the V160 inoculum, although this expansion procedure

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Fig. 1. Conditional growth of V160 (ddIE1/2-ddUL51) in ARPE-19 cells. (A) Production of V160 progeny virus at different concentrations of Shld-1. ARPE-19 cells were infected at an MOI of 0.01 PFU per cell with V160 for 1 hour and then incubated in a medium containing 0, 0.05, 0.1, 0.5, or 2 mM Shld-1. At day 7 after infection, the culture supernatant was collected, and the virus was quantified by median tissure culture infectious dose (TCID50) assay on ARPE-19 cells in the presence of 2 mM Shld-1. (B) Growth comparison of V160 with beMAD virus. ARPE-19 cells were infected with V160 at an MOI of 0.01 PFU per cell and incubated in the absence (○) or presence (●) of 2 mM Shld-1. Cells infected with beMAD at an MOI of 0.01 PFU per cell were included as a control (D). Progeny virus was collected at the indicated time points after infection and quantified by TCID50 assay on ARPE-19 cells supplemented with 2 mM Shld-1. The experiments were conducted twice, and the representative data are shown.

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Days after infection Fig. 2. Conditional replication of V160 in different cell types. Human fibroblasts (MRC-5), endothelial cells [human umbilical cord endothelial cell (HUVEC)], muscle cells [aortic smooth muscle cell (AoSMC) and skeletal muscle cell (SKMC)], or neuronal cells (CCF-STTG1) were infected with V160 at an MOI of 0.1 PFU per cell, except for CCFSTTG1, which were infected at a MOI of 5 PFU per cell. After 1 hour, the cells were washed twice and incubated in the absence or presence of 2 mM Shld-1. Progeny virus was collected at the indicated time points after infection and quantified by TCID50 assay on ARPE-19 cells supplemented with 2 mM Shld-1. The experiments were conducted twice, and the representative data are shown. Wang et al., Sci. Transl. Med. 8, 362ra145 (2016)

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A

and P = 0.04, repeated-measures ANOVA). The gB vaccine group showed modest responses to gB peptides after vaccination but not to other antigens (Fig. 5D). PBMCs from the 100 mg/dose and the 10 mg/dose + Iscomatrix groups were further analyzed by intracellular IFN-g cytokine staining (Fig. 6). One naïve monkey, included as a negative control, showed minimal responses to HCMV antigens but responded to superantigen staphylococcal enterotoxin B (SEB), as expected. All vaccinated monkeys from the 100 mg/dose and 10 mg/dose + Iscomatrix groups responded to viral antigens, with comparable CD4+ and CD8+ T cell responses when stimulated with peptides from pp65, IE1, and IE2; only CD4+ responses were observed after stimulation with purified virions.

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Fig. 3. Immunogenicity of V160 in mice and rabbits. (A) Six-week-old female BALB/c mice (n = 10 per group) were immunized at weeks 0 and 3 with beMAD, ddIE1/ 2, ddUL51, and V160 (ddIE1/2-ddUL51) vaccines, at doses ranging from 0.16 to 2.5 mg. Serum samples were collected at week 6 and analyzed for HCMV neutralization in ARPE-19 cells. Lines indicate the GMT of NT50 in each group. Tukey’s method for multiple pairwise comparisons was conducted for the groups at the 2.5 mg/dose with n = 10 per group (P = 0.81 for ddIE1/2 versus ddUL51, P = 0.68 for ddIE1/2 versus V160, and P = 0.99 for ddUL51 versus V160). (B) Spleen cells were pooled from three mice in the 2.5 mg/dose groups at week 10. Cellular responses to pp65, IE1, IE2, and gB were determined by IFN-g ELISPOT assay. (C) New Zealand White rabbits (n = 4 per group) were immunized at weeks 0, 3, and 8, with 10 mg of MAD169, beMAD, or V160 vaccines. Sera were collected at week 11 and analyzed for HCMV neutralization in ARPE-19 cells. Tukey’s method for multiple pairwise comparisons revealed a P value of 1.00 for beMAD versus V160 and a P value of