JVI Accepted Manuscript Posted Online 27 May 2015 J. Virol. doi:10.1128/JVI.00133-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Non-glycosylated G protein vaccine protects against homologous and heterologous RSV

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challenge while glycosylated G enhances RSV lung pathology and cytokine levels

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Sandra Fuentes, Elizabeth M Coyle, Hana Golding, and Surender Khurana*

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Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver

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Spring, MD, 20903, USA,

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Running Title: Efficacy and safety of unglycosylated RSV G vaccine

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Keywords: Respiratory Syncytial Virus, RSV, Vaccine, Recombinant, Neutralization, Antibody,

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G protein, PRNT, Infection, Affinity, Pathology, Cytokine, Bacterial, E. coli

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*

Corresponding author:

*

Surender Khurana, Ph.D.

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Division of Viral Products, Center for Biologics Evaluation and Research (CBER)

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Food and Drug Administrationa (FDA)

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10903 New Hampshire Avenue

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Silver Spring, MD, USA 20903

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Phone- 240-402-9632, Fax- (301) 595-1125

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E. mail- [email protected]

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1

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ABSTRACT

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New efforts are under way to develop a vaccine against RSV that will provide protective

25

immunity without the potential for vaccine-associated disease enhancement as observed in

26

infants following vaccination with formalin-inactivated RSV vaccine. In addition to the F fusion

27

protein, the G attachment surface protein represents a target for neutralizing antibodies and thus

28

represents an important vaccine candidate.

29

mammalian cells was shown to induce pulmonary eosinophilia upon RSV infection in mouse

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model. In the current study, we evaluated in parallel the safety and protective efficacy of RSV

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A2 recombinant unglycosylated G protein ectodomain (amino acids 67-298) expressed in E. coli

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(REG) vs. glycosylated G produced in mammalian cells (RMG) in a mouse RSV challenge

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model. Vaccination with REG generated neutralizing antibodies against RSV A2 in 7/11

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BALB/c mice, while RMG did not elicit neutralizing antibodies. Total serum binding antibodies

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against the recombinant proteins (both REG and RMG) were measured by Surface Plasmon

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Resonance (SPR) and found to be >10-fold higher for REG compared with RMG-vaccinated

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animals. Complete reduction of lung viral loads after homologous (RSV-A2) and heterologous

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(RSV-B1) viral challenge was observed in 7/8 animals vaccinated with REG but not in RMG

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vaccinated

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cytokines/chemokines were observed exclusively in animals vaccinated with RMG (but not with

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REG or PBS) after homologous or heterologous RSV challenge. This study suggests that

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bacterially produced unglycosylated G protein could be developed alone or as a component of a

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protective vaccine against RSV disease.

animals.

Furthermore,

However, glycosylated G protein expressed in

enhanced

44

2

lung

pathology

and

elevated

Th2

45

46

Importance

47

New efforts are under way to develop vaccines against RSV that will provide protective

48

immunity without the potential for disease enhancement. The G attachment protein represents an

49

important candidate for inclusion in an effective RSV vaccine.

50

evaluated the safety and protective efficacy of RSV A2 recombinant unglycosylated G protein

51

ectodomain produced in E. coli (REG) vs. glycosylated G produced in mammalian cells (RMG)

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in a mouse RSV challenge model (A2 and B1 strains). The unglycosylated G generated high

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protective immunity and no lung pathology even in animals that lacked anti-RSV neutralizing

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antibodies prior to RSV challenge. Control of viral loads correlated with antibody binding to the

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G protein. In contrast, the glycosylated G protein provided poor protection and enhanced lung

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pathology after RSV challenge. Therefore, bacterially produced unglycosylated G protein

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presents an economical approach as a protective vaccine against RSV.

58 59

3

In the current study, we

60 61

INTRODUCTION

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Respiratory syncytial virus (RSV) is the leading cause of virus mediated lower

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respiratory tract illness (LRI) in infants and children worldwide. In the United States, RSV is a

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major cause of morbidity second only to influenza virus (1). In infants, more than 2%

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hospitalizations are attributable to RSV infection annually (1). Although traditionally regarded as

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a pediatric pathogen, RSV can cause life-threatening pulmonary disease in bone marrow

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transplant recipients and immunocompromised patients (2, 3). In developing countries, most

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RSV-mediated severe disease occurs in infants younger than 2 years and results in significant

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infant mortality (4). Among the elderly, RSV is also a common cause of severe respiratory

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infections that requires hospitalization (4).

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Although the importance of RSV as a respiratory pathogen has been recognized for over

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50 years, a vaccine is not yet available because of several problems inherent in RSV vaccine

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development. These barriers to development include the very young age of the target population,

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recurrent infections in spite of prior exposure, and history of enhanced disease in young children

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that were immunized with a formaldehyde inactivated RSV (FI-RSV) vaccine in the 1960s (3, 5).

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Subsequent studies with samples from these children showed poor functional antibody responses

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with low neutralization or fusion-inhibition titers (6, 7). There was also evidence for deposition

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of immune complexes in the small airways (8), however the mechanism of the FI-RSV vaccine

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induced enhanced disease is poorly understood. Animal models of the FI-RSV vaccine

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associated enhanced respiratory disease (VAERD) suggested a possible combination of poor

4

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functional antibody responses and Th2-biased hyper cytokine release leading to eosinophilic

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infiltration in the lungs (9, 10).

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RSV live-attenuated vaccines (LAV) are an attractive vaccine modality for young

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children. It presents to the immune system many viral genes with potential protective targets

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including the F and G membrane proteins. Using reverse genetics, attenuating mutations were

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incorporated into RSV A2 in different combinations, and this has been explored extensively with

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emphasis on reaching a good balance between safety and immunogenicity (11). However,

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stability of the engineered mutations is an important technical challenge (12). A recent RSV

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LAV candidate (rA2cp248/404/1030deltaSH) was found to be safe in infants but poorly

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immunogenic (13). However, new RSV LAV vaccine candidates are being evaluated.

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Vaccines based on recombinant proteins in different cell substrates have been pursued as

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well (3, 14). Earlier, RSV F glycoprotein (PFP-2) formulated in alum was well tolerated in

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clinical trials but only modestly immunogenic in adults, pregnant women, and in the elderly (15).

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A mixture of F, G, and M recombinant proteins was tested in subjects >65 years and was found

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to induce >4-fold increase in serum neutralizing activity in 58% of subjects with low pre-vaccine

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titers (16). Recently, the structures of the F protective target recognized by the MAbs

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palivizumab and 101F were resolved, as well as the pre-fusion form of the F protein trimer,

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leading to structure-based design of stabilized F protein vaccine candidates (17-19). The G

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protein was also evaluated as vaccine candidate in preclinical studies. Nanoparticle vaccine

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encompassing the RSV G protein CX3C chemokine motif protected BALB/c mice from RSV

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challenge (20). A subunit vaccine based on the central conserved region of the G attachment

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surface glycoprotein was fused to the albumin-binding domain from streptococcal protein G,

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produced in prokaryotic cells, and formulated with an alum-based adjuvant. After promising 5

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results in murine models, challenge studies in rhesus macaques showed no reduction of viral

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loads, and studies in human adults showed a relatively low capacity for inducing neutralizing

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antibodies (21, 22). Ideally, an optimally effective RSV vaccine must protect against

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antigenically divergent groups A and B RSV strains.

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In the current study, we evaluate side-by-side the immunogenicity, safety and protective

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capacity of a recombinant RSV G protein ectodomain (amino acids 67-298) vaccine produced

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either as an unglycosylated protein in E. coli prokaryotic system (REG) or as a fully glycosylated

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protein in mammalian cells (RMG) in a mouse challenge model using homologous RSV A2 and

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heterologous RSV B1 virus strains. We demonstrate that the unglycosylated REG generated

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higher protective immunity against both homologous and heterologous RSV strains while the

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glycosylated RMG immunogen resulted in enhanced lung pathology and increased cytokine

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levels in lungs following virus challenge.

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MATERIAL AND METHODS

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Cell, Viruses and Plasmids

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Vero cells (CCL-81) and A549 cells (CCL-185) were obtained from the ATCC. 293-Flp-

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in cells (R750-07) were obtained from Invitrogen. Vero cells, A549 cells and 293-Flp-In cells

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were grown in EMEM medium, F12K medium and D-MEM (high glucose) respectively. All cell

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lines were supplemented with 10% heat inactivated fetal bovine serum (FBS) and 1X

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Penicillin/Streptomycin (P/S) and L-glutamine and maintained in an incubator at 37°C and 5%

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CO2. 6

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RSV virus, strains A2 (NR-12149) and B1 (NR-4052) were obtained from BEI

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Resources, NIAID, NIH. All virus stocks were prepared by infecting a sub-confluent A549 cell

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monolayers with virus in F12K media with L-glutamine supplemented with 2 % FBS and 1X P/S

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(infection media). Virus was collected 3-5 days post-infection (dpi) by freeze-thawing cells twice

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and combining with supernatant. Virus harvests were cleared of cell debris by centrifugation at

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3,000 rpm for 15 min. Virus stocks to be used in challenged studies were pelleted by

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centrifugation at 7,000 rpm overnight. Pelleted virus was resuspended in F12K media

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supplemented with 50mM HEPES and 100mM MgSO4, aliquoted and frozen at -80°C until

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needed. Virus titers were determined by plaque assay in Vero cells.

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Codon optimized RSV-G coding DNA for E. coli and mammalian cells were chemically

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synthesized. A NotI and PacI site was used for cloning the RSV A2 G ectodomain coding

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sequence (67-298) in the T7 based pSK expression vector (23) for bacterial expression and the

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pSecR vector for mammalian expression (24) to express G protein in E. coli and in 293Flp-In

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cells, respectively.

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Purified RSV A2 F protein encoding amino acids 22-529 and fused to a polyhistidine tag

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produced in insect cells with endotoxin levels 100-fold higher in the lungs of RMG immunized mice compared to placebo

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(PBS), while IL-4 levels in REG immunized mice were comparable to the PBS control (Figure

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6A). IFN-γ, a Th1 cytokine, was only ~2 fold higher in RMG immunized mice than placebo or

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REG immunized mice (Figure 6E). As a consequence, the ratio of Th2/Th1 (IL-4/IFNγ)

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cytokines was significantly higher in the lungs of mice immunized with RMG immunogen

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compared to placebo or REG immunized mice following RSV infection (Figure 6I). The Th2

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cytokines IL-5, and IL-13, known to be involved in eosinophil recruitment, airway hyper-

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responsiveness and mucus production (32, 36), were found at significantly elevated levels in

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mice immunized with RMG when compared to placebo. In contrast, in the lungs of REG

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immunized mice, levels of IL-5 and IL-13 were at similar range to those of the placebo control

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lungs (Figure 6B-D). We also identified significant production of IL-6, a proinflammatory

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cytokine often associated with increased C-reactive protein (CRP) and elevation in body

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temperature. The chemokines Eotaxin, MCP-1 and MIP-1α, previously found to be involved in

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RSV induced enhanced lung pathology (37), were also observed at significantly higher levels in

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the lungs of RSV-challenged RMG-immunized mice compared to the REG-immunized or PBS

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control mice (Figure 6F-H). The elevated levels of cytokines and chemokines in the RMG

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immunized mice following viral challenge seems to be immune mediated and were independent 19

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of lung viral loads following RSV viral challenge as similar or higher viral loads were observed

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in the PBS control mice (Fig. 4).

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Together, this study demonstrated that RMG vaccination induces enhanced lung cellular

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infiltration, as measured by both histopathology and cytokine levels after RSV challenge. At the

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same time, RMG vaccine does not elicit strong protective immunity. On the other hand, the

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vaccination with the unglycosylated REG did not induce enhanced lung pathology and did confer

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protection against both homologous and heterologous RSV strains even in the absence of a

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PRNT-neutralizing antibody response.

406 407

DISCUSSION

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New efforts are under way to develop subunit vaccines against RSV that will provide

409

protective immunity without the potential for disease enhancement, as was observed in infants

410

following vaccination with the formalin-inactivated RSV (FI-RSV) vaccine in the 1960s (5, 38-

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40). Most recent RSV vaccine development efforts are focused on the F protein, which is more

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conserved among strains (3) and can be expressed in mammalian and insect cells (14, 18, 19,

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41). The G attachment protein expressed on the virus surface also represents an important target

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of protective immunity. However, in earlier studies, glycosylated G protein expressed in

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mammalian cells or recombinant vaccinia virus expressing G protein was shown in mouse

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models to induce pulmonary eosinophilia upon RSV infection (36, 42, 43). Therefore, we

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decided to evaluate side by side the immunogenicity and safety of recombinant unglycosylated G

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protein ectodomain (amino acids 67-298) expressed in E. coli (REG) and a fully glycosylated G 20

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ectodomain produced in 293-Flp-In mammalian cells (RMG) in a mouse RSV-challenge model.

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The FI-RSV model was not included in the current study since it has been documented in

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previous studies, demonstrating immune response imbalance, levels of lung cytokines and

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cellular infiltrates were associated with enhanced lung pathology. The targets of the non-

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neutralizing antibodies were not completely deciphered. Furthermore, F and G subunit vaccines

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studied early on have a history of disease enhancement, albeit to a lesser extent than FI-RSV (3).

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Previous studies in which glycosylated G protein was shown to be protective in mice

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typically used three vaccinations with > 10 μg protein per dose (57–60). In this study, we

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immunized mice with only two vaccinations of a lower dose of the G proteins (5 μg/dose) to

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differentiate the impact of glycosylation on virus neutralization and disease enhancement

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phenomenon after RSV infection. Two vaccinations with REG generated neutralizing antibodies

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against RSV A2 in 7/11 animals, while the glycosylated RMG did not elicit neutralizing

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antibodies as measured in the PRNT. Furthermore, total binding antibodies against the

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recombinant proteins (both REG and RMG) were found to be >10-fold higher in the REG

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immunized mice sera compared with RMG-vaccinated animals. The REG immune sera also

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demonstrated broader epitope recognition spanning the entire G-ecotdomain compared with

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RMG immune sera. Homologous and heterologous protection was evaluated by measuring lung

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viral loads after challenge with either RSV A2 (homologous) or RSV B1 (heterologous) strains.

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Complete control of viral loads was observed in the majority of animals vaccinated with REG

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and challenged with either RSV strain. In contrast, the majority of RMG-vaccinated animals did

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not control virus replication after homologous or heterologous virus challenge. There were

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animals with low or no serum neutralization titers that controlled lung viral loads very well. This 21

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was most evident in animals challenged with the heterologous RSV B1 strain. It has been shown

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before that anti-G antibodies may neutralize virus in vivo but not in the in vitro PRNT (29).

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Antibody dependent cellular cytotoxicity (ADCC) and/or cell mediated immunity may also play

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a role in virus clearance after RSV challenge. However, previous studies showed that BALB/c

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mice immunized with either FI-RSV or recombinant vaccinia virus that expresses the

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glycosylated RSV G attachment protein generated RSV specific CD4+ T cells but did not prime

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CD8+ T cells (44, 45). In our study, a strong inverse correlation was observed between the total

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anti-G binding serum antibody titers (measured by SPR) and lung viral loads after RSV

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challenge. Therefore, it is likely that anti-G binding antibodies that are protective in vivo do not

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neutralize RSV in the PRNT as was observed for anti-G MAb 131-2G (29). Therefore, this study

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identifies that for a RSV-G protein based vaccine, the correlate of protection is mediated by total

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G-binding antibodies as measured by SPR may provide a reasonably good predictor of control of

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viral replication after intranasal RSV challenge. Additional studies with patients-derived RSV

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isolates (as they become available) are planned to confirm and expand our findings. These

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analytical tools could also further evaluated in pre-clinical and clinical studies of other RSV

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vaccines containing both F and G proteins, including live attenuated vaccines currently under

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investigation in children (11, 12).

458

An important safety consideration for the use of subunit and non-replicating candidate

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vaccines against RSV is enhanced Th2 cytokine response and its potential, to increase disease

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severity upon virus challenge. The RSV G glycoprotein in particular has been implicated as an

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RSV antigen that promotes Th2 CD4+ T lymphocytes and induces eosinophilic infiltrates in the

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lungs after RSV challenge (31, 32, 36, 46-49). In the current study, the glycosylated RMG

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vaccinated animals but not PBS or REG-vaccinated animals demonstrated Th2-biased cytokine 22

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and elevated chemokine responses in the lungs that correlated with significant lung

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histopathology after either RSV-A2 or RSV-B1 challenge. The higher number of eosinophils

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(100-fold) and neutrophils (10-fold) cellular infiltration in the RMG vaccinated mice following

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RSV challenge recapitulated the lung pathology previously observed in models of enhanced

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RSV disease (33, 34). The enhanced lung pathology in the RMG immunized mice following

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viral challenge seems to be immune mediated since similar or higher viral loads were observed

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in the PBS control mice that showed lower cytokine levels and less lung pathology.

471

Both purified recombinant G proteins were identical in terms of their primary amino acid

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sequence and were administered in combination with the same adjuvant, Emulsigen. The only

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difference between the REG and RMG proteins was the presence of glycosylation as N- and O-

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linked glycans in RMG protein. Therefore, the observed shift towards higher Th2/Th1 ratio after

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RMG-vaccination could not be simply attributed to binding of the CX3C motif within CCD to

476

the CX3CR1 (fractalkine receptor) on several cell types, as previously suggested (51-53).

477

Instead, the enhanced Th2 and chemokine levels could possibly be induced by the high level of

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O-linked sugars characteristic of mammalian cell expressed glycosylated RMG and also of the

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native virus-associated G attachment protein., A role for processing by different glycosylation

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specific antigen presenting cell subsets and routes of immunization that could influence the

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subsequent Th2 vs. Th1 cytokines has been reported and should be further investigated (54, 55,

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56). Carbonylation of protein is one of the several factors that have been proposed to be

483

responsible for induction of Th2 disease in this model. However, we did not observe any

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difference in carbonyl content of the REG and RMG proteins used in our studies.

23

485

Our findings are in agreement with previous studies using E. coli produced fusion

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protein, BBG2Na that contained the central conserved region of A2 G gene (amino acids 130-

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230) (G2Na) fused to the albumin-binding domain of streptococcal protein G protein (BB)

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formulated with aluminum adjuvant (57-60). In comparison with BBG2Na, our REG encompass

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the entire G-ectodomain with no “foreign” sequences or fusion protein that may influence the

490

immune response to the irrelevant target (BB rather than G2Na) during vaccinations. Most of the

491

studies with BBG2Na used 20 μg of the vaccine in a series of three vaccinations. In contrast, in

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our study, only two vaccinations with 5 μg REG protein/dose elicited strong protection. Also,

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REG-immunization induced antibodies with much epitope repertoire compared with BBG2Na.

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In summary, our study for the first time compared side by side fully glycosylated and

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unglycosylated G proteins for immunogenicity and safety in a murine RSV challenge model. The

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combination of low virus neutralizing activity, lower G-binding antibody titers, and enhanced

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lung pathology observed in the RMG vaccinated animals is in agreement with previous studies

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using various forms of glycosylated G proteins (34) (31, 32, 48, 50). In contrast, the lack of

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enhanced lung pathology after REG vaccination was an unexpected encouraging finding that

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provides supports for further development of this vaccine approach. It also provided data

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supporting the feasibility of developing simple recombinant RSV G-based vaccine produced in

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E. coli expression system that elicits protective antibodies against both homologous and

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heterologous RSV challenge. The bacterial production system for a G protein based vaccine

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provides an economical, simple and rapid alternative to cell-based subunit vaccines as was

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previously demonstrated for influenza HA1 proteins from multiple virus strains (25, 61-63), and

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could be applied for expression of multiple G proteins from circulating RSV strains. Such a

24

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multi-component G immunogen could be tested alone or in combination with F- subunit vaccines

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for effective protection against RSV disease.

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Acknowledgement: We thank Dr. Judy Beeler and Dr. Haruhiko Murata for their thorough

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review of the manuscript. We thank Swati Verma and Nitin Verma for technical support.

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719

FIGURE LEGENDS

720

Figure 1: Purification of recombinant G protein from E. coli and 293 cells.

721

(A) Schematic representation of the RSV G protein. RSV G protein purified from E. coli

722

(REG) inclusion bodies is lacking the cytoplasmic and transmembrane domains (CT-TM) and is

723

not glycosylated, while the RSV G protein secreted from 293-Flp-In cells (RMG) is

724

glycosylated. N–linked glycosylation sites, as predicted by NetNGlyc 1.0 software, are

725

represented above the bar and predicted O-linked glycosylation sites, as predicted by NetOGlyc

726

4.0 software, are represented below the bar in the full length RSV G and RMG proteins. REG

727

was purified from E. coli inclusion bodies as described in materials and methods. RMG was

728

purified from the clarified supernatant of 293-Flip-In cells stably expressing the RSV G protein.

729

Both REG (B) and RMG (C) were purified through a Ni-NTA column and the final products

730

were detected as a single band in western blot under reducing conditions using Mab 131-2G (D)

731

SPR interaction profile of REG and RMG binding to the protective MAb 131-2G that targets the

732

central conserved domain of RSV G. (E-F) Superdex S-200 gel filtration chromatography of

733

RSV G protein produced in bacterial system (REG) (E) and mammalian system (RMG) (F). The

734

panels present superimposed elution profiles of purified RSV G proteins (red lines) overlaid with

735

calibration standards (grey lines).

736 737

Figure 2: Neutralizing antibody response following RSV G (REG, RMG) and F protein

738

immunization

31

739

(A) Schematic representation of mice immunization and challenge schedule. BALB/c mice

740

were immunized i.m. with 5 µg of REG, RMG or F proteins of RSV-A2 virus strain with

741

Emulsigen adjuvant, or with PBS control, on days 0 and 20.

742

immunization, blood was collected from the tail veins. 14 days after the second immunization,

743

mice were challenged intranasally with 106 pfu of either RSV-A2 or RSV-B1 viral strains (6-11

744

mice per group). Mice were sacrificed on days 2 or 4 post-challenge wherein lungs and blood

745

were collected. (B-C) Serum from individual mice collected on day 10 post second immunization

746

was tested for neutralization in a plaque reduction neutralization test (PRNT) against the

747

homologous RSV-A2 strain (B) or heterologous RSV-B1 strain (C). Neutralizing antibody titers

748

represent 50% inhibition of plaque numbers. The average for each group is represented with a

749

horizontal line. Pre-vaccination (day 0) serum samples from all the animals tested negative in

750

PRNT (data not shown). Statistical significance was tested with one-way ANOVA and

751

Bonferroni multiple comparisons tests. *** p < 0.0001, ** p < 0.001, *p < 0.05.

Ten days after the second

752 753

Figure 3: Surface Plasmon Resonance analysis of post-vaccination sera to REG, RMG and

754

different antigenic regions within RSV G

755

(A-B) The same individual post-vaccination mice sera from Fig. 3 were tested for total

756

antibody binding to the REG protein (A) or RMG protein (B) by Surface Plasmon Resonance

757

(SPR). (C) Antigenic peptides representing amino acids 66-90, 90-110, 148-178, 169-207 and

758

236-263 of the RSV G protein were chemically synthesized and tested for binding to serum

759

antibodies from REG or RMG immunized mice in a real time SPR kinetics experiment. Total

760

antibody binding is represented as SPR resonance units. Statistical significance was tested with 32

761

one-way ANOVA and Bonferroni multiple comparisons tests. *** p < 0.0001, ** p < 0.001, *p