Newcastle Disease Virus‐Like Particles: Preparation, Purification, Quantification, and Incorporation of Foreign Glycoproteins

McGinnes, Lori W.; Morrison, Trudy G.

doi:10.1002/9780471729259.mc1802s30

Cited by 28 publications

(41 citation statements)

References 31 publications

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“…For Western analysis, proteins in the polyacrylamide gels were transferred to polyvinylidene difluoride membranes using dry transfer (iblot; ThermoFisher/Invitrogen). Proteins were detected in the blots using anti-RSV HR2 peptide antibody, anti-NDV F tail antibody, or anti-RSV G protein antibody as previously described (23,26,36,37).…”

Section: Methodsmentioning

confidence: 99%

Effect of Previous Respiratory Syncytial Virus Infection on Murine Immune Responses to F and G Protein-Containing Virus-Like Particles

Cullen

Schmidt

Morrison

2019

J Virol

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Most individuals are infected with respiratory syncytial virus (RSV) by age two, but infection does not result in long-term protective immunity to subsequent infections. Previous RSV infection may, however, impact responses to an RSV vaccine. The goal of these studies was to explore the effect of previous RSV infection on murine antibody responses to RSV F and G protein-containing virus-like particles (VLP), comparing responses to those resulting from VLP immunization of RSV-naive animals. These studies showed that after RSV infection, immunization with a single dose of VLPs containing a conformation-stabilized prefusion F protein stimulated high titers of neutralizing antibodies (NA), while an immunization with post-F-containing VLPs or a second RSV infection only weakly stimulated NA, even though total anti-F protein IgG antibody levels in both VLP-immunized animals were similar. Furthermore, single pre-F or post-F VLP immunization of animals previously infected (primed) with RSV resulted in total anti-F antibody titers that were 10- to 12-fold higher than titers after a VLP prime and boost of RSV-naive animals or after two consecutive RSV infections. The avidities of serum antibodies as well as numbers of splenic B cells and bone marrow cells after different immunization protocols were also assessed. The combined results show that RSV infection can quite effectively prime animals for the production of protective antibodies that can be efficiently activated by a pre-F VLP boost but not by a post-F VLP boost or a second RSV infection.IMPORTANCEHumans may experience repeated infections caused by the same serotype of respiratory syncytial virus (RSV), in contrast to infections with most other viruses, indicating that immune memory responses to RSV are defective. However, the effects of any residual but nonprotective immunity on responses to RSV vaccines are not clear. This study demonstrates that a VLP vaccine candidate containing a stabilized prefusion F protein can robustly stimulate protective immunity in animals previously infected with RSV, while a second RSV infection or a postfusion F-containing VLP cannot. This result shows that a properly constructed immunogen can be an effective vaccine in animals previously infected with RSV. The results also suggest that the defect in RSV memory is not in the induction of that memory but rather in its activation by a subsequent RSV infection.

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Section: Methodsmentioning

confidence: 99%

Effect of Previous Respiratory Syncytial Virus Infection on Murine Immune Responses to F and G Protein-Containing Virus-Like Particles

Cullen

Schmidt

Morrison

2019

J Virol

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“…Silver staining of proteins in the polyacrylamide gels was accomplished as recommended by the manufacturer (Pierce). Quantification of NP, M, different forms of F/F, and H/G proteins in the polyacrylamide gels was accomplished by Western blotting of the proteins as well as protein standards as previously described (27,33). For Western analysis, proteins in the polyacrylamide gels were transferred to polyvinylidene difluoride (PVDF) membranes using dry transfer (iblot; Invitrogen).…”

Section: Cellsmentioning

confidence: 99%

“…At 24 h posttransfection, heparin was added to the cells at a final concentration of 10 g/ml (26) to inhibit rebinding of released VLPs to cells. At 48, 72, and 96 h posttransfection, cell supernatants were collected, and VLPs were purified by sequential pelleting and sucrose gradient fractionation as previously described (26,27,33). Concentrations of proteins in the purified VLPs were determined by silver-stained polyacrylamide gels and by Western analysis using marker proteins for standard curves (27,33).…”

Section: Cellsmentioning

confidence: 99%

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Murine Immune Responses to Virus-Like Particle-Associated Pre- and Postfusion Forms of the Respiratory Syncytial Virus F Protein

Cullen

Schmidt

Kenward

et al. 2015

J Virol

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Virus IMPORTANCEThe development of vaccines for respiratory syncytial virus has been hampered by a lack of understanding of the requirements for eliciting high titers of neutralizing antibodies. The results of this study suggest that particle-associated RSV F protein containing mutations that stabilize the structure in a prefusion conformation may stimulate higher titers of protective antibodies than particles containing F protein in a wild-type or postfusion conformation. These findings indicate that the prefusion F protein assembled into VLPs has the potential to produce a successful RSV vaccine candidate.H uman respiratory syncytial virus (RSV) is the most significant cause of acute viral respiratory disease in infants and young children (1). There are from 34 to 65 million RSV infections resulting in acute lower respiratory disease requiring hospitalization and 160,000 to 199,000 deaths per year worldwide (2). Elderly populations are also at significant risk for serious RSV disease. In the United States, the virus accounts for 10,000 deaths and 14,000 to 60,000 hospitalizations per year among individuals more than 64 years of age (3-5). Indeed, RSV infection of this population is at least as significant as influenza virus infections. RSV infections result in high mortality rates in immunocompromised populations, particularly stem cell transplant recipients (6) and individuals with cardiopulmonary diseases (7). Despite the significance of RSV disease in different populations, there are no vaccines available.Failure to develop a licensed RSV vaccine is not due to lack of effort as numerous vaccine candidates have been characterized in preclinical and clinical studies spanning 5 decades (summarized in references 8 to 9). While many problems have uniquely hindered RSV vaccine development, a major hurdle has been a lack of understanding of requirements for generation of protective immunity to RSV infection. Many vaccine candidates are protective in animal models and, while stimulating antibody responses in humans, have failed to induce high levels of neutralizing antibodies and protection from virus challenge in human trials (reviewed in references 10 and 11). Although there are likely many reasons for these observations, one important issue has been a lack of clear understanding of the most effective form of the RSV antigens, particularly the F protein, for stimulating potent neutralizing antibodies.The paramyxovirus F protein is folded into a metastable conformation and upon fusion activation refolds, through a series of conformational intermediates, into the postfusion conformation, which is structurally very different from the prefusion form (12)(13)(14)(15)(16)(17)(18)(19). While it is logical to assume that the prefusion form of F protein should be more effective in stimulating optimally neutralizing antibodies, recent structural studies have shown that the postfusion form of the F protein contains at least some epitopes recognized by neutralizing monoclonal antibodies (17, 18). Thus, it has been a...

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“…Quantifications of NP, M, RSV G protein, and RSV F proteins in VLPs or in soluble F or G protein preparations were accomplished after their separation in polyacrylamide gels followed by silver staining (Pierce Silver Stain, ThermoFisher) or Western blots of the proteins in parallel with protein standards, as previously described [35].…”

Section: Quantification Of Np M H/g and Vlp Associated F Proteins mentioning

confidence: 99%

Comparisons of Antibody Populations in Different Pre-Fusion F VLP-Immunized Cotton Rat Dams and Their Offspring

et al. 2020

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Respiratory syncytial virus (RSV) infection poses a significant risk for infants. Since the direct vaccination of infants is problematic, maternal vaccination may provide a safer, more effective approach to their protection. In the cotton rat (CR) model, we have compared the immunization of pregnant CR dams with virus-like particles assembled with the prototype mutation stabilized pre-fusion F protein, DS-Cav1, as well two alternative mutation stabilized pre-fusion proteins (UC-2 F, UC-3 F) and showed that the alternative pre-fusion F VLPs protected the offspring of immunized dams significantly better than DS-Cav1 F VLPs (Blanco, et al. J. Virol. 93: e00914). Here, we have addressed the reasons for this increased protection by characterizing the specificities of antibodies in the sera of both immunized dams and their offspring. The approach was to measure the levels of total anti-pre-F IgG serum antibodies that would block the binding of representative pre-fusion specific monoclonal antibodies to soluble pre-fusion F protein targets. Strikingly, we found that the sera in most offspring of DS-Cav1 F VLP-immunized dams had no mAb D25-blocking antibodies, although their dams had robust levels. In contrast, all offspring of UC-3 F VLP-immunized dams had robust levels of these D25-blocking antibodies. Both sets of pup sera had significant levels of mAb AM14-blocking antibodies, indicating that all pups received maternal antibodies. A lack of mAb D25-blocking antibodies in the offspring of DS-Cav1 F VLP-immunized dams may account for the lower protection of their pups from challenge compared to the offspring of UC-3 F VLP-immunized dams.Vaccines 2020, 8, 133 2 of 15 to infectious, attenuated, or vector viruses, since they do not contain a genome and do not produce a spreading infection. Our VLPs are based on the core proteins of Newcastle disease virus (NDV), NP and M proteins, and they are assembled with the RSV F and G protein ectodomains fused to the transmembane and cytoplasmic domains of the NDV F and HN proteins, respectively.There has been a resurgence of interest and activity in RSV vaccine development due to the ground-breaking studies of McLellan, et al. who succeeded in solving the crystal structure of the RSV pre-fusion F protein and identifying a set of mutations in the F protein, termed DS-Cav1, which stabilized the pre-fusion form of the F protein [20,21]. We have reported that VLPs assembled with the DS-Cav1 mutant F protein stimulate, in mice and in cotton rats, neutralizing antibody titers much higher than those induced by VLPs assembled with the post-fusion F protein or wild-type F protein [16,22]. Furthermore, the immunization of cotton rat dams with DS-Cav1 F VLPs protected their offspring from RSV challenge [14].Since the description of DS-Cav1 F protein, a number of other laboratories and companies have identified different sets of mutations that reportedly stabilize the pre-fusion F protein [23][24][25][26][27][28][29]. A very important question for vaccine development is whether the different m...

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Newcastle Disease Virus‐Like Particles: Preparation, Purification, Quantification, and Incorporation of Foreign Glycoproteins

Cited by 28 publications

References 31 publications

Effect of Previous Respiratory Syncytial Virus Infection on Murine Immune Responses to F and G Protein-Containing Virus-Like Particles

Effect of Previous Respiratory Syncytial Virus Infection on Murine Immune Responses to F and G Protein-Containing Virus-Like Particles

Murine Immune Responses to Virus-Like Particle-Associated Pre- and Postfusion Forms of the Respiratory Syncytial Virus F Protein

Comparisons of Antibody Populations in Different Pre-Fusion F VLP-Immunized Cotton Rat Dams and Their Offspring

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