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Surface modifications of influenza proteins upon virus inactivation by ?-propiolactone.

ABSTRACT: Inactivation of intact influenza viruses using formaldehyde or ?-propiolactone (BPL) is essential for vaccine production and safety. The extent of chemical modifications of such reagents on viral proteins needs to be extensively investigated to better control the reactions and quality of vaccines. We have evaluated the effect of BPL inactivation on two candidate re-assortant vaccines (NIBRG-121xp and NYMC-X181A) derived from A/California/07/2009 pandemic influenza viruses using high-resolution FT-ICR MS-based proteomic approaches. We report here an ultra performance LC MS/MS method for determining full-length protein sequences of hemagglutinin and neuraminidase through protein delipidation, various enzymatic digestions, and subsequent mass spectrometric analyses of the proteolytic peptides. We also demonstrate the ability to reliably identify hundreds of unique sites modified by propiolactone on the surface of glycoprotein antigens. The location of these modifications correlated with changes to protein folding, conformation, and stability, but demonstrated no effect on protein disulfide linkages. In some cases, these modifications resulted in suppression of protein function, an effect that correlated with the degree of change of the modified amino acids' side chain length and polarity.

SUBMITTER: She YM

PROVIDER: S-EPMC4265195 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Surface modifications of influenza proteins upon virus inactivation by β-propiolactone.

She Yi-Min YM Cheng Keding K Farnsworth Aaron A Li Xuguang X Cyr Terry D TD

Proteomics 20131201 23-24

Inactivation of intact influenza viruses using formaldehyde or β-propiolactone (BPL) is essential for vaccine production and safety. The extent of chemical modifications of such reagents on viral proteins needs to be extensively investigated to better control the reactions and quality of vaccines. We have evaluated the effect of BPL inactivation on two candidate re-assortant vaccines (NIBRG-121xp and NYMC-X181A) derived from A/California/07/2009 pandemic influenza viruses using high-resolution F ...[more]

PMID: 24123778

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Project description:Beta-propiolactone (BPL) is an inactivating agent that is widely used in the vaccine industry. However, its effects on vaccine protein antigens and its mechanisms of action remain poorly understood. Here we present cryo-electron microscopy (cryo-EM) structures of BPL-treated coxsackievirus A16 (CVA16) mature virions and procapsids at resolutions of 3.9 Å and 6.5 Å, respectively. Notably, both particles were found to adopt an expanded conformation resembling the 135S-like uncoating intermediate, with characteristic features including an opened 2-fold channel, the externalization of the N terminus of VP1 capsid protein, and the absence of pocket factor. However, major neutralizing epitopes are very well preserved on these particles. Further biochemical analyses revealed that BPL treatment impairs the abilities of CVA16 particles to bind to the attachment receptor heparan sulfate and to a conformation-dependent monoclonal antibody in a BPL dose-dependent manner, indicating that BPL is able to modify surface-exposed amino acid residues. Taken together, our results demonstrate that BPL treatment may induce alteration of the overall structure and surface properties of a nonenveloped viral capsid, thus revealing a novel mode of action of BPL.IMPORTANCE Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. It is recognized that BPL inactivates viral infectivity through modification of viral nucleic acids. However, its effect on viral proteins remains largely unknown. Here, we present high-resolution cryo-EM structures of BPL-treated coxsackievirus A16 (CVA16) mature virions and procapsids, which reveals an expanded overall conformation and characteristic features that are typical for the 135S-like uncoating intermediate. We further show that the BPL concentration affects the binding of inactivated CVA16 particles to their receptor/antibody. Thus, BPL treatment can alter the overall structure and surface properties of viral capsids, which may lead to antigenic and immunogenic variations. Our findings provide important information for future development of BPL-inactivated vaccines.

Project description:Whole inactivated viral particles have been successfully used as vaccines for some viruses, but procedures historically used for inactivation can denature virion proteins. Results have been inconsistent, with enhancement of disease rather than protection seen in some notable instances following vaccination. We used the compound 2,2'-dithiodipyridine (aldrithiol-2; AT-2) to covalently modify the essential zinc fingers in the nucleocapsid (NC) protein of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) virions, thereby inactivating infectivity. The inactivated virus was not detectably infectious in vitro (up to 5 log units of inactivation). However, in contrast to virions inactivated by conventional methods such as heat or formalin treatment, viral and host cell-derived proteins on virion surfaces retained conformational and functional integrity. Thus, immunoprecipitation of AT-2-treated virions was comparable to precipitation of matched untreated virus, even when using antibodies to conformational determinants on gp120. AT-2 inactivated virions bound to CD4(+) target cells and mediated virus-induced, CD4-dependent "fusion from without" comparably to native virions. However, viral entry assays demonstrated that the viral life cycle of AT-2-treated virions was arrested before initiation of reverse transcription. The major histocompatibility complex (MHC) class II molecules on the surface of AT-2-treated virions produced from MHC class II-expressing cells retained the ability to support class II-dependent, superantigen-triggered proliferative responses by resting T lymphocytes. These findings indicate that inactivation via this method results in elimination of infectivity with preservation of conformational and functional integrity of virion surface proteins, including both virally encoded determinants and proteins derived from the host cells in which the virus was produced. Such inactivated virions should provide a promising candidate vaccine antigen and a useful reagent for experimentally probing the postulated involvement of virion surface proteins in indirect mechanisms of HIV-1 pathogenesis.

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Surface modifications of influenza proteins upon virus inactivation by ?-propiolactone.

Publications

Surface modifications of influenza proteins upon virus inactivation by β-propiolactone.

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