An insect cell derived respiratory syncytial virus (RSV) F nanoparticle vaccine induces antigenic site II antibodies and protects against RSV challenge in cotton rats by active and passive immunization

Raghunandan, Rama; Lü, Huanzhang; Zhou, Bin; Xabier, Mimi Guebre; Massare, Michael J.; Flyer, David C.; Fries, Louis; Smith, Gale; Glenn, Gregory M.

doi:10.1016/j.vaccine.2014.09.030

Cited by 62 publications

(42 citation statements)

References 48 publications

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“…Such a complex array of competition-binding groups was unexpected, because the site II mAb palivizumab, which is used in prophylactic treatment, also bidirectionally competed with the nonneutralizing mAb 12I1. A palivizumabcompetition assay designed to detect the presence of site II antibodies in immune serum by competing with palivizumab (7,8) has been proposed as a correlate of immunity for an RSV postfusion F protein vaccine candidate. Indeed, we repeated the competition using published palivizumab competition assay protocols (7), where biotinylated palivizumab was spiked into control mAbs, as well as donor serum.…”

Section: Resultsmentioning

confidence: 99%

“…Site II is the target of palivizumab (5), a prophylactic treatment licensed for use in high-risk infants during the RSV season. An RSV F protein subunit vaccine candidate comprising aggregates of the postfusion conformation of RSV F is being tested currently in clinical trials (6), and serum antibody competition with palivizumab has been proposed as a potential serologic correlate of immunity for that vaccine (7,8). We and others have isolated and studied RSV F-specific mAbs using murine hybridomas (9), sorted macaque B cells (10), and transformed human B cells or human antibody gene phage-display libraries (11,12).…”

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confidence: 99%

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Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein

Mousa

Sauer

Sevy

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Palivizumab was the first antiviral monoclonal antibody (mAb) approved for therapeutic use in humans, and remains a prophylactic treatment for infants at risk for severe disease because of respiratory syncytial virus (RSV). Palivizumab is an engineered humanized version of a murine mAb targeting antigenic site II of the RSV fusion (F) protein, a key target in vaccine development. There are limited reported naturally occurring human mAbs to site II; therefore, the structural basis for human antibody recognition of this major antigenic site is poorly understood. Here, we describe a nonneutralizing class of site II-specific mAbs that competed for binding with palivizumab to postfusion RSV F protein. We also describe two classes of site II-specific neutralizing mAbs, one of which escaped competition with nonneutralizing mAbs. An X-ray crystal structure of the neutralizing mAb 14N4 in complex with F protein showed that the binding angle at which human neutralizing mAbs interact with antigenic site II determines whether or not nonneutralizing antibodies compete with their binding. Fine-mapping studies determined that nonneutralizing mAbs that interfere with binding of neutralizing mAbs recognize site II with a pose that facilitates binding to an epitope containing F surface residues on a neighboring protomer. Neutralizing antibodies, like motavizumab and a new mAb designated 3J20 that escape interference by the inhibiting mAbs, avoid such contact by binding at an angle that is shifted away from the nonneutralizing site. Furthermore, binding to rationally and computationally designed site II helixloop-helix epitope-scaffold vaccines distinguished neutralizing from nonneutralizing site II antibodies.F protein | human respiratory syncytial virus | neutralizing antibodies R espiratory syncytial virus (RSV) is a highly contagious human pathogen, infecting the majority of infants before age 2 y, and is the leading cause of viral bronchiolitis and viral pneumonia in infants and children (1, 2). RSV remains a top priority for vaccine development, as thousands of deaths are recorded worldwide each year because of complications from infection (3). To date, there is no licensed RSV vaccine. A major focus of RSV vaccine development has been inclusion of the RSV fusion (F) protein, a class I fusion glycoprotein that is synthesized as a precursor and cleaved into two disulfide-linked fragments upon maturation into a trimer (4). Although the RSV virion contains two additional surface proteins, the highly-glycosylated attachment (G) protein and the small hydrophobic protein, the F protein is highly conserved among strains of RSV strains and is the major target of protective neutralizing antibodies.The F protein is known to adopt at least two major conformations: the metastable prefusion conformation and the postfusion conformation. Following attachment of the virion to a cell by the G protein, the F protein undergoes a dramatic structural rearrangement, resulting in fusion of the viral and cell membranes, and in cultured cells causes...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein

Mousa

Sauer

Sevy

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Most recent RSV vaccine development efforts are focused on the F protein, which is relatively conserved among strains (3) and can be expressed in mammalian and insect cells (14,18,19,41). The G attachment protein, expressed on the virus surface, also represents an important target of protective immunity.…”

Section: Discussionmentioning

confidence: 99%

“…Vaccines based on recombinant proteins in different cell substrates have been pursued as well (3,14). Earlier, RSV F glycoprotein (PFP-2) formulated in alum was well tolerated in clinical trials but only modestly immunogenic in adults, pregnant women, and the elderly (15).…”

Section: Importancementioning

confidence: 99%

Nonglycosylated G-Protein Vaccine Protects against Homologous and Heterologous Respiratory Syncytial Virus (RSV) Challenge, while Glycosylated G Enhances RSV Lung Pathology and Cytokine Levels

Fuentes

Coyle

Golding

et al. 2015

J Virol

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New efforts are under way to develop a vaccine against respiratory syncytial virus (RSV) that will provide protective immunity without the potential for vaccine-associated disease enhancement such as that observed in infants following vaccination with formalin-inactivated RSV vaccine. In addition to the F fusion protein, the G attachment surface protein is a target for neutralizing antibodies and thus represents an important vaccine candidate. However, glycosylated G protein expressed in mammalian cells has been shown to induce pulmonary eosinophilia upon RSV infection in a mouse model. In the current study, we evaluated in parallel the safety and protective efficacy of the RSV A2 recombinant unglycosylated G protein ectodomain (amino acids 67 to 298) expressed in Escherichia coli (REG) and those of glycosylated G produced in mammalian cells (RMG) in a mouse RSV challenge model. Vaccination with REG generated neutralizing antibodies against RSV A2 in 7/11 BALB/c mice, while RMG did not elicit neutralizing antibodies. Total serum binding antibodies against the recombinant proteins (both REG and RMG) were measured by surface plasmon resonance (SPR) and were found to be >10-fold higher for REG-than for RMG-vaccinated animals. Reduction of lung viral loads to undetectable levels after homologous (RSV-A2) and heterologous (RSV-B1) viral challenge was observed in 7/8 animals vaccinated with REG but not in RMG-vaccinated animals. Furthermore, enhanced lung pathology and elevated Th2 cytokines/chemokines were observed exclusively in animals vaccinated with RMG (but not in those vaccinated with REG or phosphate-buffered saline [PBS]) after homologous or heterologous RSV challenge. This study suggests that bacterially produced unglycosylated G protein could be developed alone or as a component of a protective vaccine against RSV disease. IMPORTANCENew efforts are under way to develop vaccines against RSV that will provide protective immunity without the potential for disease enhancement. The G attachment protein represents an important candidate for inclusion in an effective RSV vaccine. In the current study, we evaluated the safety and protective efficacy of the RSV A2 recombinant unglycosylated G protein ectodomain produced in E. coli (REG) and those of glycosylated G produced in mammalian cells (RMG) in a mouse RSV challenge model (strains A2 and B1). The unglycosylated G generated high protective immunity and no lung pathology, even in animals that lacked anti-RSV neutralizing antibodies prior to RSV challenge. Control of viral loads correlated with antibody binding to the G protein. In contrast, the glycosylated G protein provided poor protection and enhanced lung pathology after RSV challenge. Therefore, bacterially produced unglycosylated G protein holds promise as an economical approach to a protective vaccine against RSV.

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“…The vast majority of the reports of tests of vaccine potential concern heterologous VLPs or nanoparticles carrying the hRSV F and/or G protein, in part because these systems are established or efficient and because hRSV particle assembly is poorly understood. The heterologous systems include Newcastle disease virus-, Sendai virus-, or baculovirus-based VLPs; nanoparticles; and gold-based nanorods and have shown encouraging results in the BALB/c mouse model (6)(7)(8)(9)(10)(11)(12) and humans (13,14). In comparison, authentic hRSV VLPs structurally resemble wild-type (wt) virions and also incorporate some of the internal hRSV proteins (5), features that may be advantageous for vaccine purposes.…”

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confidence: 99%

The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation

Meshram

Baviskar

Ognibene

et al. 2016

J Virol

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Virus-like particles (VLPs) are attractive as a vaccine concept. For human respiratory syncytial virus (hRSV), VLP assembly is poorly understood and appears inefficient. Hence, hRSV antigens are often incorporated into foreign VLP systems to generate anti-RSV vaccine candidates. To better understand the assembly, and ultimately to enable efficient production, of authentic hRSV VLPs, we examined the associated requirements and mechanisms. In a previous analysis in HEp-2 cells, the nucleoprotein (N), phosphoprotein (P), matrix protein (M), and fusion protein (F) were required for formation of filamentous VLPs, which, similar to those of wild-type virus, were associated with the cell surface. Using fluorescence and electron microscopy combined with immunogold labeling, we examined the surfaces of transfected HEp-2 cells and further dissected the process of filamentous VLP formation. Our results show that N is not required. Coexpression of P plus M plus F, but not P plus M, M plus F, or P plus F, induced both viral protein coalescence and formation of filamentous VLPs that resembled wild-type virions. Despite suboptimal coalescence in the absence of P, the M and F proteins, when coexpressed, formed cell surface-associated filaments with abnormal morphology, appearing longer and thinner than wild-type virions. For F, only the carboxy terminus (Fstem) was required, and addition of foreign protein sequences to Fstem allowed incorporation into VLPs. Together, the data show that P, M, and the F carboxy terminus are sufficient for robust viral protein coalescence and filamentous VLP formation and suggest that M-F interaction drives viral filament formation, with P acting as a type of cofactor facilitating the process and exerting control over particle morphology. IMPORTANCEhRSV is responsible for >100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on resemblance to virus or viral components, can induce protective immunity. For hRSV, few reports are available concerning authentic VLP production or testing, in large part because VLP production is inefficient and the mechanisms underlying particle assembly are poorly understood. Here, we took advantage of the cell-associated nature of RSV particles and used high-resolution microscopy analyses to examine the viral proteins required for formation of wild-type-virus-resembling VLPs, the contributions of these proteins to morphology, and the domains involved in incorporation of the antigenically important viral F protein. The results provide new insights that will facilitate future production of hRSV VLPs with defined shapes and compositions and may translate into improved manufacture of live-attenuated hRSV vaccines. H uman respiratory syncytial virus (hRSV) is an important viral agent of respiratory tract disease in infants, children, immunosuppressed individuals, and the elderly (1-3). In pursuit of a vaccine to prevent hRSV disease, a virus-like particle (...

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An insect cell derived respiratory syncytial virus (RSV) F nanoparticle vaccine induces antigenic site II antibodies and protects against RSV challenge in cotton rats by active and passive immunization

Cited by 62 publications

References 48 publications

Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein

Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein

Nonglycosylated G-Protein Vaccine Protects against Homologous and Heterologous Respiratory Syncytial Virus (RSV) Challenge, while Glycosylated G Enhances RSV Lung Pathology and Cytokine Levels

The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation

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