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Human cytomegalovirus gH/gL/UL128/UL130/UL131A complex elicits potently neutralizing antibodies in mice

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Yingxia Wen, James Monroe, Christine Linton, Jacob Archer, Clayton W. Beard, Susan W. Barnett, Giuseppe Palladino, Peter W. Mason ∗ , Andrea Carfi ∗∗ , Anders E. Lilja 1

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Novartis Vaccines: 45 Sidney Street, Cambridge, MA 02139, United States

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Article history: Received 1 December 2013 Received in revised form 17 April 2014 Accepted 1 May 2014 Available online xxx

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Keywords: Cytomegalovirus Pentameric complex Vaccine Neutralizing antibodies

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Human cytomegalovirus (HCMV) is a member of the ␤-herpesvirus family that causes significant disease worldwide. Although evidence exists that neutralizing antibodies and cytotoxic T cell responses to HCMV antigens can prevent HCMV disease and/or infection, there are no approved vaccines to prevent HCMV disease. Over the past 10 years, multiple HCMV vaccines have been tested in man but only partial protection has been achieved in these studies. HCMV contains multiple surface-expressed glycoproteins that are critical to viral entry, including gB, the gM/gN complex, the gH/gL complex, and a pentameric gH/gL/UL128/UL130/UL131A complex. Recently we showed that viral replicon particles (VRPs) expressing the gH/gL complex elicited more potently neutralizing antibodies than VRPs expressing gB in mice. Here we compare the immunogenicity of VRPs encoding the HCMV gH/gL and pentameric complexes, as well as purified gH/gL and pentameric complexes administered in the presence or absence of the MF59 adjuvant. The results of these studies indicate that the pentameric complex elicits significantly higher levels of neutralizing antibodies than the gH/gL complex, and that MF59 significantly increases the potency of each complex. In addition, we show that animals immunized with pentamer encoding VRPs or the pentameric subunit produce antibodies that recognize a broad range of antigenic sties on the complex. Taken together, these studies support the utility of the pentameric complex in HCMV vaccine candidates. © 2014 Published by Elsevier Ltd.

1. Introduction Human cytomegalovirus (HCMV) is a member of the ␤herpesvirus family [1]. Acute infection of immunocompetent hosts is typically mild or asymptomatic, but infection of a pregnant woman can cause mortality or morbidity in the congenitally infected fetus [2]. HCMV is also a significant cause of disease in solid organ and hematopoietic stem cell transplant recipients [2]. To date, there is no licensed vaccine for prevention of HCMV

∗ Corresponding author at: Novartis Vaccines, 350 Massachusetts Ave. Mail Stop 45SS, Cambridge, MA 02139, United States. Tel.: +1 617 460 1713; fax: +1 617 871 8758. ∗∗ Corresponding author at: Novartis Vaccines, 350 Massachusetts Ave. Mail Stop 45SS, Cambridge, MA 02139, United States. Tel.: +1 617 872 8784; fax: +1 617 871 8758. E-mail addresses: [email protected] (P.W. Mason), andrea.carfi@novartis.com (A. Carfi). 1 Current address: Hookipa Biotech AG, Helmut-Qualtinger-Gasse 2, 1030 Vienna, Austria.

infection. Antivirals are standard of care for transplant recipients but have limited use and efficacy because of toxicity and viral resistance [3]. Although there are no established correlates of protection against HCMV, studies suggest that both antibodies and cytotoxic T lymphocytes specific for HCMV can prevent disease and/or infection [4]. In addition, studies with a guinea pig CMV model have demonstrated that both antibodies [5] and T cells [6] can protect against congenital infection. The primary targets of neutralizing antibodies to HCMV are the viral entry glycoproteins: gB, gM/gN, gH/gL, and gH/gL/UL128/UL130/UL131A [7–12]. gB and gH elicit a large fraction of the antibodies that neutralize infection of cultured fibroblasts [7,8,11–15]. gH associates with gL [16], and gH/gL and gB form the conserved herpesvirus fusion machinery [17] that is required for HCMV infection in fibroblasts. gH/gL can also associate with UL128, UL130, and UL131A to form a pentameric complex [18–20]. Although several functions have been ascribed to UL128, UL130 and UL131A, an abundance of data has demonstrated that the pentameric complex is required for HCMV entry into dendritic, epithelial and endothelial cells [21–23]. Importantly, antibodies

http://dx.doi.org/10.1016/j.vaccine.2014.05.004 0264-410X/© 2014 Published by Elsevier Ltd.

Please cite this article in press as: Wen Y, et al. Human cytomegalovirus gH/gL/UL128/UL130/UL131A complex elicits potently neutralizing antibodies in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.05.004

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against the pentameric complex contribute a larger fraction of the neutralizing activity of human immune globulin than do antibodies specific for gB [12]. Recent clinical trials of HCMV vaccine candidates have tested the utility of gB protein with MF59 [24–26], formulated plasmid DNA expressing gB and pp65 [27,28], and alphavirus replicon particles (VRPs) expressing gB and a pp65-IE1 fusion protein [29]. All three candidates were safe and immunogenic and the gB/MF59 and DNA vaccines showed signs of clinical efficacy [24,26,27]. However, gB only elicited responses with modest potency [28–30] and/or durability [25]. Thus, although current HCMV vaccine candidates suggest that a subunit or vectored vaccine is feasible, an antigen that elicits more potent and durable neutralizing antibody responses than gB would likely increase the protective efficacy [4]. Recently we showed that VRPs expressing the gH/gL complex elicit more potently neutralizing antibodies than VRPs expressing gB in mice [31]. In this report, we assessed the immunogenicity of recombinant, purified HCMV gH/gL and pentameric complexes, and VRPs that encode the same antigens. Our data suggest that the pentameric complex has the potential to significantly improve the clinical efficacy of future HCMV vaccine candidates.

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2. Materials and methods

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2.1. Cells and viruses

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Serum-free adapted HEK293 cells (ATCC, Manassas, VA, USA) were cultured in Gibco FreeStyle 293 expression Medium (Life Technologies Corp., Carlsbad, CA, USA). BHKV ARPE-19 cells (ATCC) and MRC-5 cells (ATCC) were cultured as previously described [31]. HCMV strains TB40-GFP (C. Sinzger, University of Tübingen, Tübingen, Germany; referred to in the remainder of this work as TB40) [32] was propagated on MRC-5 cells and VR1814 (G. Gerna, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy) [33] on ARPE-19 cells as previously described [31].

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2.2. Antibodies

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LHV606 (Novartis AG, Basel, Switzerland; a constant region variant of MSL-109) [34], 1F11, 8C15, 3G16, 13H11, 15D8var1, 7I13, 10P3, and 8I21 (A. Lanzavecchia, Institute for Research in Biomedicine, Bellinzona, Switzerland), mouse anti-His (Life Technologies), rabbit anti-gL 27-46 and rabbit anti-UL131A 90-136 (D. Johnson, OHSU, Portland, OR, USA), mouse anti-UL128 4B10 and mouse anti-UL130 3E3 (T. Shenk, Princeton University, Princeton, NJ, USA) were used as primary antibodies. HRP-linked ECL sheep anti-mouse and donkey anti-rabbit IgGs (GE Life Sciences, Piscataway, NJ, USA), IRDye 800CW goat anti-Rabbit IgG (H + L), and IRDye 680LT goat anti-Mouse IgG (H + L) were used as secondary antibodies (Li-Cor Biosciences, Lincoln, NE, USA). AlexaFluor 488conjugated monoclonal antibody to HCMV IE1 (Millipore, Billerica, MA, USA) was used in neutralization assays.

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2.3. Alphavirus replicon particles

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Genes encoding gH, gL, UL128, UL130, and UL131A from the Merlin strain of HCMV were subcloned into replicons based on the sequence of VEE strain TC-83 [35]. Expression cassettes were generated using gene synthesis and PCR cloning and inserted into appropriate restriction sites in the replicon. Schematic representations of the different replicons are shown in Fig. 1A. Alphavirus replicon particles were generated by triple electroporation of BHKV cells with in vitro-transcribed replicons and defective helper RNAs (MEGAscript® , Life Technologies), using standard methods [36]. Supernatants were collected from these cells following incubation for 24 h at 37 ◦ C, centrifuged for 3 min at 514 × g to remove cellular

debris, and the VRPs were then pelleted through a 20/50% (w/v) sucrose/phosphate-buffered saline (PBS) cushion at 153,583 × g for 2 h. These partially purified VRPs were resuspended in PBS, concentrated on 100 kDa Amicon Ultra-15 Centrifugal Filter Units (Millipore), and frozen in 0.1 ml aliquots at −80 ◦ C. VRP titers were determined by infecting BHK-V cells with serial dilutions of the stocks and immunostaining the monolayer for expression of the encoded CMV antigen by using a modification of previously described methods [31]. To purify pentameric complex from replicon-transfected cells, BHKV cells were transfected with replicon RNA using Lipofectamine 2000 (Life Technologies).

2.3.1. Protein expression and purification The truncated gH gene encoding the first 715 amino acids of the protein was subcloned with a C-terminal His-tag into the NheI and KpnI sites of pcDNA3.1(−)/myc-His A (Invitrogen, Life Technologies). Full-length gL, UL128, UL130, and UL131A were subcloned into the SalI and XbaI sites of the mammalian expression vector pCMVKm2 (Novartis AG). All antigens were based on the Merlin strain of HCMV. pcDNA/gH715-His and pCMVKm2/gL or pcDNA/gH715-His, pCMVKm2-gL, -UL128, -UL130 and -UL131A were co-transfected into HEK293 cells using PEI (polyethyleneimine, Polysciences Inc., Warrington, PA, USA). The gH/gL and pentameric complex were purified from cell supernatants by affinity chromatography on a Ni-NTA Superflow cartridge (Qiagen Inc., Valencia, CA, USA). Eluted proteins were concentrated and further purified by size exclusion chromatography on a Superdex 200 10/300 GL column (GE Life Sciences).

2.3.2. Vaccine immunogenicity studies Eight-to-ten-week-old female BALB/c mice (Charles River Labs, Wilmington, MA, USA) were immunized (8 mice/group) by bilateral 50 ␮l intramuscular injections in the rear quadriceps on days 0, 21, and 42 with a total dose of 106 infectious units of VRP [31,37] or with 0.1–1.6 ␮g of purified protein complex formulated with PBS or MF59 (1:1 volume ratio of antigen to MF59) as indicated. Serum samples were obtained by retro-orbital sinus bleeds on day 20 and day 41 and from bleed-outs of euthanized animals on day 63. All studies were approved by the Novartis Institutes for Biomedical Research Animal Care and Use Committee.

2.4. Serological analysis Serum samples were tested for the presence of neutralizing antibodies by HCMV microneutralization assay as previously described, except without methylcellulose overlay [31]. For capture/sandwich ELISA, plates were coated overnight with 500 ng/ml HCMV monoclonal antibodies in PBS, washed with 0.05% Tween-20 in PBS (PBST) and blocked with 1% BSA in PBS (PBSB). Serial dilutions of protein in 1% BSA and 0.1% Triton X-100 in PBS (PBSTr) were then incubated on the plates. Plates were washed with PBST and captured proteins were detected with biotinylated human anti-HCMV monoclonal antibody, followed by incubation with HRP-labeled avidin and TMB substrate. For competition ELISA, plates were coated with 500 ng/ml mouse monoclonal anti-His antibody. The plates were then washed with PBST and blocked with PBSB. Serial dilution of test sera was performed in PBST and pentameric complex was added at 250 ng/ml followed by individual biotinylated human anti-HCMV monoclonal antibodies at concentrations yielding 75% maximum signal. After incubation, plates were washed with PBST, followed by incubation with HRP-labeled avidin and TMB substrate.

Please cite this article in press as: Wen Y, et al. Human cytomegalovirus gH/gL/UL128/UL130/UL131A complex elicits potently neutralizing antibodies in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.05.004

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Fig. 1. Replicons expressing HCMV antigen complexes. (A) Schematics of replicon constructs expressing HCMV gH/gL and gH/gL/UL128/UL130/UL131A under the control of alphavirus 26S subgenomic promoters (curved arrows). Multiple antigens are expressed from a single promoter by IRES sequences between the protein-coding sequences. Sequence information for the replicons will be supplied by the authors upon request. (B) BHK cultures were seeded and infected with A527 VRPs and then 24 h later harvested, washed, fixed, permeabilized, incubated with the indicated HCMV-specific monoclonal antibody, stained with fluorescently labeled anti-human IgG, and analyzed by flow cytometry. Each panel shows an overlay of the profiles of the uninfected and infected populations obtained with each antibody, percent values indicate the proportion of each infected population that reacted with the antibody. (C) BHKV cells were transfected with A556 replicon RNA. Supernatants were collected at 48 h post transfection and His-tagged proteins were purified using Ni-affinity chromatography. Individual components of these complexes were identified by dissociation of the proteins on SDS–PAGE gels followed by immunoblotting with monoclonal antibodies specific for each of the expected proteins (indicated below each lane).

Please cite this article in press as: Wen Y, et al. Human cytomegalovirus gH/gL/UL128/UL130/UL131A complex elicits potently neutralizing antibodies in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.05.004

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3. Results 3.1. In vitro characterization of alphavirus replicons expressing HCMV antigen complexes Multiple replicons expressing the HCMV gH/gL and pentameric complexes were constructed using multiple 26S promoters and/or IRES sequences (Fig. 1A) using standard molecular biology methods. Expression of full-length gH/gL by the A160 replicon was confirmed by immunoblot (data not shown). To show that the A527 replicon produced the pentameric complex antigens, A527infected cells were analyzed by flow cytometry and shown to bind conformation-dependent human monoclonal antibodies specific for the gH, UL128, UL128/UL130/UL131A, UL130/UL131A, or gH/gL/UL128/UL130 protein complexes [8] (Fig. 1B). To further characterize complexes expressed by replicons we constructed A556, which encodes a pentameric complex with a Histagged gH lacking the transmembrane region. Ni2+ affinity-purified proteins from media of A556-transfected cells were shown by immunobloting (Fig. 1C) to contain gL, UL128, UL130, and UL131A. To measure the amount of antigen produced by the repliconinfected cells, BHK cells were infected with VRPs expressing soluble gH/gL (A531 replicon, Fig. 1A) and gH/gL/UL128/UL130/UL131A (A555, Fig. 1A) complexes. The culture media were harvested for

capture ELISA to quantify secreted antigen, and the cell monolayers were fixed for immunostaining to count infected cells. The gH/gLand pentamer-encoding VRPs expressed gH/gL and the pentameric complex at 250–300 ng/1000 infected cells. 3.2. Purification and characterization of gH/gL and gH/gL/UL128/UL130/UL131A complexes A secreted form of the HCMV gH/gL complex, generated by replacing the gH C-terminal transmembrane domain with a His-tag to facilitate purification, was purified from mammalian cell supernatants and evaluated by SDS–PAGE and immunoblotting. This analysis revealed the presence of two forms of gH/gL with molecular weights consistent with monomeric (M) and dimeric (D) gH/gL heterodimers (Fig. 2A, left panels). Size exclusion chromatography (SEC) allowed separation of these two forms of the complex (Fig. 2A, right panel). Notably, when monomeric and dimeric species were analyzed by SDS–PAGE after boiling in the presence of reducing agent, complete dissociation into gH and gL was achieved, whereas no dissociation of gH from gL and only partial dissociation of the gH/gL dimer of heterodimers was observed in absence of reducing agent (Fig. 2A). Together these data suggest that not only gH and gL are linked through disulfide bonds, but intermolecular disulfides are also formed between the gH/gL dimers of heterodimers.

Fig. 2. Expression and purification of gH/gL and gH/gL/UL128/UL130/UL131A protein complexes. SDS–PAGE with A) coomassie blue staining and immunoblots of the complex between the C-terminally His-tagged gH ectodomain and the full-length gL co-expressed in HEK293. Left panels, coomassie blue staining for Ni2+ -affinity-purified gH/gL; middle panels, immunoblot for Ni-affinity-purified gH/gL using anti-His (red) and anti-gL (green) antibodies; right panels, coomassie blue staining for monomeric gH/gL heterodimer (M in figure) and dimeric gH/gL heterodimer (D) separated by size exclusion chromatography (SEC). B) gH ectodomain with a C-terminal His-tag and full-length Q5 forms of gL, UL128, UL130 and UL131A were co-expressed in HEK293 cells and co-purified as a complex containing all five proteins. SDS–PAGE under reducing conditions and immunoblot (using anti-His, anti-gL, anti-UL128, anti-UL130 and anti-UL131A antibodies to detect gH, gL, UL128, UL130 and UL131A, respectively) are shown.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 3. Purified gH/gL and pentameric complexes bind to conformation-dependent neutralizing antibodies. Purified protein complexes were added to plates coated with the indicated capturing antibodies. 3G16 and 13H11 are specific for gH, 15D8, 1F11, 10P3, 8C15, 7I13 and 8I21 for different substructures of gH/gL/UL128/UL130/UL131A [8]. Captured proteins were detected with 13H11. ND, not done.

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The Ni-affinity purified pentameric complex was also analyzed by SDS–PAGE and immunoblotting. Antibodies against gL, UL128, UL130, UL131A and the His-tag on gH, confirmed the presence of all five components of the complex (Fig. 2B). All five proteins coeluted in one peak when further purified by SEC, consistent with the formation of an intact complex (data not shown). 3.3. Human neutralizing monoclonal antibodies bind the purified protein complexes To characterize the presentation of neutralizing epitopes on the purified complexes, we tested the binding of conformationdependent neutralizing monoclonal antibodies against gH [8,34] and gH/gL/UL128/UL130/UL131A [8] in a sandwich ELISA. Antibodies specific for epitopes formed by gH/gL/UL128/UL130/UL131A captured the pentameric complex (Fig. 3, 15D8, 1F11, 10P3, 8C15, 7I13 and 8I21). 8I21, specific for an epitope on gH/gL/UL128/UL130, bound only the pentameric complex and not gH/gL. The gH antibody 3G16 captured both gH/gL and the pentameric complex and allowed detection by the gH antibody 13H11 (Fig. 3) suggesting simultaneous presentation of the 3G16 and 13H11 epitopes on both the gH/gL and pentameric complexes. These results confirmed that purified gH/gL and pentameric complexes were well folded and recognized by neutralizing antibodies. 3.4. Purified subunit pentamer and alphavirus replicon particles expressing the pentameric complex elicit potently and broadly neutralizing antibodies in mice To assess the neutralizing antibody response to the subunit antigens, mice were immunized with different doses of purified gH/gL and pentameric complexes with or without MF59 adjuvant. Microneutralization assays were then used to evaluate the titers of antibodies present in mouse immune sera that could block infection of cultured epithelial cells (ARPE-19) with the HCMV strain TB40 in absence of complement. gH/gL formulated with MF59 was administered at doses of 0.1 and 1 ␮g and showed a clear but non-linear dose response (Fig. 4). MF59 markedly improved the neutralizing antibody response to the purified gH/gL complex, since 1 ␮g gH/gL in MF59 elicited 9–10 fold higher titers of neutralizing antibodies than 1 ␮g of gH/gL in PBS (Fig. 4). Neutralizing antibody titers in mice immunized with monomer or dimer of gH/gL heterodimer were comparable, suggesting that gH/gL dimerization did not negatively affect exposure or structure of the neutralizing antibody

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Fig. 4. Neutralization titers in terminal sera from mice immunized three times with purified gH/gL or pentameric complexes with or without MF59. Neutralization assays were performed using VR1814 HCMV strain to infect epithelial cells (ARPE-19). These data are from a single experiment that was performed using all of the conditions of antigen and adjuvant shown. Subsets of these antigens/conditions were retested in other experiments. By combining the data shown with data from three additional experiments using the same immunization regimen, we determined that MF59-adjuvanted pentameric complex was statistically superior to MF59-adjuvanted gH/gL (p < 0.05 using the Mann–Whitney test, indicated by the “*”). By combining the data shown with data from three additional experiments using the same immunization regimen, we determined that MF59-adjuvanted gH/gL was statistically superior to unadjuvanted gH/gL (p < 0.05 using the Mann–Whitney test, indicated by the “**”). By combining the data shown with data from three additional experiments using the same immunization regimen, we determined that MF59-adjuvanted pentameric complex was statistically superior to unadjuvanted pentameric complex (p < 0.05 using the Z test, indicated by the “***”).

binding sites (Fig. 4). When we compared the neutralizing antibodies elicited by purified gH/gL to purified gB, we found that the relative potency of gH/gL was 10–20 fold higher (supplementary materials, Fig. S1), consistent with our previously reported differences in the relative potency of VRPs expressing these antigens [31]. In this study, mice were also immunized with purified pentameric complex (at doses of 0.16 and 1.6 ␮g of to compensate for the ∼60% greater molecular weight compared to gH/gL). Similar to gH/gL, the addition of MF59 adjuvant increased the neutralizing antibody response of the pentameric antigen (Fig. 4). There was a slight, non-linear dose response when comparing 0.16–1.6 ␮g of the pentameric antigen in MF59 (Fig. 4, data bars 9 and 10). Overall, the neutralizing titers elicited by the purified pentameric antigen appeared to be 3- to 4-fold greater than gH/gL when approximately equal molar amounts of each complex were used (Fig. 4). Neutralizing antibody responses elicited by the purified pentamer and gH/gL were also measured in rabbit and cotton rats. In these studies, the pentamer elicited 10–90 fold greater neutralizing titers than the gH/gL antigen (Supplementary materials, Fig. S2). To compare the neutralizing antibody responses to recombinant and VRP-expressed gH/gL and gH/gL/UL128/UL130/UL131A, BALB/c mice were immunized with VRPs expressing the fulllength antigens (selected as the best mimic of the authentic virally encoded antigen) or with the purified soluble protein complexes formulated with MF59. Sera from the immunized mice were tested in microneutralization assays to measure antibodies that can block infection of epithelial cells (ARPE-19) and fibroblasts (MRC-5) with the HCMV strain TB40. The gH/gL and pentameric antigens, delivered as subunit or VRP vaccines, elicited antibodies that strongly neutralized infection of epithelial cells and the addition of complement to the assay did not increase neutralization (Fig. 5A). For either vaccine modality, the pentameric antigen elicited responses that neutralized infection of epithelial cells 3–7 fold more potently than gH/gL (Fig. 5A). However, gH/gL and pentamer elicited very similar

Please cite this article in press as: Wen Y, et al. Human cytomegalovirus gH/gL/UL128/UL130/UL131A complex elicits potently neutralizing antibodies in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.05.004

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Fig. 5. The pentameric complex elicits broadly and potently neutralizing antibodies in mice. A) Neutralizing titers in sera from mice immunized with VRPs expressing gH/gL or pentamer or with purified gH/gL or pentameric complexes with MF59. Neutralization assays were performed using TB40 and VR1814 strains to infect epithelial cells (ARPE-19) and fibroblasts (MRC-5) in the presence or absence of complement. B) Comparison of potency of sera for neutralization of infection of epithelial cells and fibroblasts. The ratio of neutralizing titers determined on epithelial cells and fibroblasts in panel A is graphed for the TB40 and VR1814 virus strains. The sera were tested with two CMV strains, VR1814 and TB40, and the data showed a similar trend.

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titers of neutralizing antibodies (which were independent of complement addition) when the sera were tested in virus neutralization assays using fibroblasts as target cells (Fig. 5A). To test the breadth of these neutralizing responses, we evaluated the immune sera in assays using another low passage HCMV strain, VR1814. For all the sera in this panel, infection of epithelial cells with VR1814 was neutralized to a similar level as was infection of epithelial cells with TB40 (Fig. 5A). In contrast, infection of fibroblasts with VR1814 was generally more sensitive to serum neutralization than with TB40 (Fig. 5A). Similar to neutralization of TB40 (Fig. 5A), addition of complement did not contribute to neutralizing VR1814 infection of either cell type (data not shown). We believe that the strain-specific differences we have observed in these studies are related to the biology of these strains in cell culture, and/or differences in glycoprotein content on the virion envelopes of the different viral strains. The mouse immune sera raised against the pentameric complex neutralized infection of fibroblasts very well. The pentameric complex is not required for infection of fibroblasts and, consequently, antibodies specific for this complex should have little or no effect when used to block infection on these cells [8]. To assess the contribution of gH/gL- and pentamer-specific antibodies to neutralization, we calculated the ratio of neutralizing titers on epithelial cells and fibroblasts for each sample (Fig. 5B). gH/gL sera neutralized TB40 infection of epithelial cells 30–60 times better than fibroblasts (Fig. 5B). In contrast, sera raised against the

pentameric complex blocked infection of epithelial cells 200–500 times more efficiently than fibroblasts. These observations were not complement dependent (Fig. 5B). Further, gH/gL sera blocked VR1814 infection of fibroblasts and epithelial cells with comparable potency, whereas sera raised against the pentameric complex inhibited VR1814 infection of epithelial cells 20–80 times more efficiently than fibroblasts (Fig. 5B). Taken together, these data suggest that pentameric complex-specific antibodies neutralize infection of epithelial cells more potently than gH/gL antibodies. 3.5. Specificity of antibodies elicited by gH/gL and gH/gL/UL128/UL130/UL131A antigens To test the specificity of the neutralizing antibodies generated by immunization with gH/gL or the pentameric complex, as subunit or VRP, we incubated sera with purified antigens before performing in vitro virus neutralization assay in epithelial cells. The gH/gL complex competed efficiently only in sera from animals immunized with gH/gL while the pentameric complex generated very high competition of neutralization activity in all sera tested (Fig. 6A). Overall these findings suggested that most of the in vitro neutralizing activity generated by immunization with gH/gL is directed to epitopes that are accessible also when gH/gL is part of the pentameric complex. Conversely, immunization with the pentameric complex generated in vitro neutralizing activity directed to epitopes that are present only in the pentameric complex.

Please cite this article in press as: Wen Y, et al. Human cytomegalovirus gH/gL/UL128/UL130/UL131A complex elicits potently neutralizing antibodies in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.05.004

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Fig. 6. Inhibition of the in vitro neutralization activity of mouse sera on epithelial cells (ARPE-19, panel A) or fibroblast cells (Panel B, MRC-5) by soluble protein competitor. Sera from mice immunized with either pentameric complex or gH/gL complex in the form of recombinant protein or VRP expressed antigen were incubated with or without soluble protein competitor before testing for in vitro neutralization activity. The competition activity is calculated as the ratio of the reduced neutralization titer in the presence of competitor to the baseline neutralization titer without competitor and expressed as percentage. The similar neutralization inhibition conferred by gH/gL or pentameric proteins on sera elicited by gH/gL or pentameric antigens demonstrate the specificity of the sera raised by our two platforms.

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We also tested the inhibition by soluble proteins of the in vitro neutralization activity of sera from mice immunized with gH/gL or pentamer on fibroblast cells. Sera from mice immunized with pentameric complex or gH/gL complex in the presence of MF59 were incubated with or without soluble protein competitor before testing for in vitro neutralization activity. Both gH/gL and pentameric proteins inhibited the neutralization of sera of mice immunized with either gH/gL or pentameric antigens to the same extent. This result demonstrates that both antigens elicit antibodies that neutralize infection in fibroblasts by interfering with gH/gL activity and that antibodies binding to epitopes present only on the pentamer

complex do not contribute to neutralization of fibroblast infection (Fig. 6B). To further analyze the response of immunized animals, we tested the ability of polyclonal animal serum to compete with the binding of individual human neutralizing monoclonal antibodies to the pentameric complex. These studies showed that sera from animals immunized with the pentameric complex compete with the binding of monoclonal antibodies that identify six of seven epitopes unique to the pentameric complex and two additional epitopes that are present also on gH/gL [6] (Supplementary materials, Fig. S3), suggesting structural integrity of both purified

Please cite this article in press as: Wen Y, et al. Human cytomegalovirus gH/gL/UL128/UL130/UL131A complex elicits potently neutralizing antibodies in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.05.004

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recombinant subunit and pentameric complex that is expressed in vivo after immunization with VRP.

4. Discussion Using two different vaccine approaches, we showed that mice immunized with the pentameric complex elicited very high titers of antibodies that neutralized HCMV infection of epithelial cells. These results are consistent with recent data showing that vaccinia virus (MVA) vectors can be used to express the rhesus CMV pentameric complex and elicit potent neutralizing responses in non-human primates [38]. To enable the efficient delivery of gH/gL and pentameric antigens for vaccination, we engineered alphavirus replicons to express these complexes in situ, and we prepared purified recombinant protein antigens. VRPs were compared to purified gH/gL and pentameric complexes, alone or formulated with MF59, in immunization experiments in mice. Overall, the neutralizing antibody response elicited by the pentameric protein was significantly higher than the gH/gL protein in mice (4–7-fold). In addition, in single studies in rabbits and cotton rats, we observed that immunization with the pentamer antigen results in 10- and 90- fold greater neutralizing responses over gH/gL, respectively. Of note, the difference in neutralizing responses between pentamer and gH/gL complexes we observe in rabbits is comparable to the differences in responses recently reported between those elicited by a HCMV virus, with restored expression of the pentameric complex, and an attenuated AD169 virus lacking the complex [39]. MF59 increased the potency of both recombinant protein complexes resulting in neutralizing responses that were comparable to the corresponding VRPs. Dimers of gH/gL and monomers of gH/gL were equivalent in eliciting neutralizing antibodies in mice, suggesting that the dimerization does not result in masking of gH/gL neutralizing sites for the HCMV strains tested. To date there are no reports showing that gH/gL forms disulfide linked dimers of gH/gL heterodimers on the surface of the CMV virion. In addition none of the herpes virus gH/gL complexes described so far has been shown to homodimerize on the viral surface. We believe the dimerization through intermolecular disulfide bonds is an artifact caused by the unpaired Cys on gH/gL. Sera from mice immunized with the pentameric antigens neutralized infection of fibroblasts with potencies comparable to sera from mice immunized with gH/gL, suggesting that gH/gL epitopes presented on both complexes elicited antibodies that could interfere with gH/gL function. Furthermore, both complexes inhibited the ability of sera raised to either complex to neutralize infection on fibroblasts. However, gH/gL only partially inhibited the virus neutralization activity in epithelial cells of sera of mice immunized with pentamer. Thus, immunization with the pentamer can simultaneously raise pentamer-specific antibodies that potently neutralize infection of epithelial cells and gH/gL antibodies that neutralize infection of a wider range of cell types [8]. HCMV vaccine candidates have shown promise in recent clinical trials [24,26,27], but none thus far have elicited neutralizing antibody titers that are quantitatively equivalent to convalescent sera [25,28,29]. We showed here that the pentameric complex, as a purified subunit vaccine or expressed in situ by alphavirus VRPs, elicited strongly neutralizing antibodies in mice. Using the same neutralization assay employed in these studies to evaluate human sera, we have shown convalescent sera titers (1:10,000–1:22,000 without complement [31]) in the same range as those seen in mice vaccinated three times with pentameric protein (>200,000) or pentamer-expressing VRPs (>50,000), or cotton rat vaccinated three times with pentameric protein (1:60,589) and rabbits vaccinated two times with pentameric protein (1:6224). Thus, we

conclude that the pentameric antigen may enable an efficacious HCMV vaccine and should be included in the next generation of vaccine candidates alone or in combination with gB. Contributors YW, GP, PWM, AC and AEL conceived of the study and designed experiments; JA cloned the plasmid and replicon constructs; CWB oversaw VRP production; JM and GP carried out immunological assays, CL and YW carried out subunit purification and analyses; AEL coordinated the animal studies; PWM, AC, YW and AEL wrote the manuscript with contributions from CWB, SWB and GP; SWB oversaw the project. Conflict of interest All authors were employed by Novartis Vaccines at the time they completed work described in this publication; Novartis Vaccines funded all of this research, and some of the authors own Novartis stock. Acknowledgments We thank Kyoko Uehara for initial pentamer purification experiments, Jimna Cisto and Karen Matsuoka for mammalian protein expression, Armin Hekele for key input into replicon construction and critical review of data, Amanda McCann and Alix Faguet for VRP production, Kara Balabanis and Pu Zhang for flow cytometry, Rebecca Loomis and Michael Franti for cloning expression vectors, Mary Schaefer, Gib Otten and members of the Novartis Institute for Biomedical Research (NIBR) Laboratory Animal Services for their help in completing the animal studies. We thank A. Feire (NIBR), C. Sinzger (University of Tübingen), and G. Gerna (Fondazione IRCCS Policlinico San Matteo), D. Johnson (OHSU, OR, USA), and T. Shenk (Princeton University, NJ, US) for providing critical reagents. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vaccine.2014.05.004. References [1] Mocarski ES, Shenk T, Pass RF. Cytomegaloviruses. In: Knipe DM, Howley PM, editors. Fields virology. Lippincott Williams & Wilkins; 2007. p. 2699–772. [2] Britt W. Manifestations of human cytomegalovirus infection: proposed mechanisms of acute and chronic disease. Curr Top Microbiol Immunol 2008;325:417–70. [3] Lischka P, Zimmermann H. Antiviral strategies to combat cytomegalovirus infections in transplant recipients. Curr Opin Pharmacol 2008;8:541–8. [4] Lilja AE, Mason PW. The next generation recombinant human cytomegalovirus Q4 vaccine candidates-Beyond gB. Vaccine 2012. [5] Schleiss MR, Bourne N, Stroup G, Bravo FJ, Jensen NJ, Bernstein DI. Protection against congenital cytomegalovirus infection and disease in guinea pigs, conferred by a purified recombinant glycoprotein B vaccine. J Infect Dis 2004;189:1374–81. [6] Schleiss MR, Lacayo JC, Belkaid Y, McGregor A, Stroup G, Rayner J, et al. Preconceptual administration of an alphavirus replicon UL83 (pp65 homolog) vaccine induces humoral and cellular immunity and improves pregnancy outcome in the guinea pig model of congenital cytomegalovirus infection. J Infect Dis 2007;195:789–98. [7] Britt WJ, Vugler L, Butfiloski EJ, Stephens EB. Cell surface expression of human cytomegalovirus (HCMV) gp 55-116 (gB): use of HCMV-recombinant vaccinia virus-infected cells in analysis of the human neutralizing antibody response. J Virol 1990;64:1079–85. [8] Macagno A, Bernasconi NL, Vanzetta F, Dander E, Sarasini A, Revello MG, et al. Isolation of human monoclonal antibodies that potently neutralize human cytomegalovirus infection by targeting different epitopes on the gH/gL/UL128131A complex. J Virol 2010;84:1005–13.

Please cite this article in press as: Wen Y, et al. Human cytomegalovirus gH/gL/UL128/UL130/UL131A complex elicits potently neutralizing antibodies in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.05.004

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Please cite this article in press as: Wen Y, et al. Human cytomegalovirus gH/gL/UL128/UL130/UL131A complex elicits potently neutralizing antibodies in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.05.004

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UL131A complex elicits potently neutralizing antibodies in mice.

Human cytomegalovirus (HCMV) is a member of the β-herpesvirus family that causes significant disease worldwide. Although evidence exists that neutrali...
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