Abstract

ChAdOx1 nCov-19 and Ad26.COV2.S are approved vaccines inducing protective immunity against SARS-CoV-2 infection in humans by expressing the Spike protein of SARS-CoV-2. We analyzed protein content and protein composition of ChAdOx1 nCov-19 and Ad26.COV2.S by biochemical methods and by mass-spectrometry. Four out of four tested lots of ChAdOx1 nCoV-19 contained significantly higher than expected levels of host cell proteins (HCPs) and of free viral proteins. The most abundant contaminating HCPs belonged to the heat-shock protein (HSP) and cytoskeletal protein families. The HCP content exceeded the 400 ng specification limit per vaccine dose, as set by the European Medicines Agency (EMA) for this vaccine, by at least 25-fold and the manufacturer's batch-release data in some of the lots by several hundred-fold. In contrast, three tested lots of the Ad26.COV2.S vaccine contained only very low amounts of HCPs. As shown for Ad26.COV2.S production of clinical grade adenovirus vaccines of high purity is feasible at an industrial scale. Correspondingly, purification procedures of the ChAdOx1 nCov-19 vaccine should be modified to remove protein impurities as good as possible. Our data also indicate that standard quality assays, as they are used in the manufacturing of proteins, have to be adapted for vectored vaccines.

Data availability

All data supporting the findings of this study are available within this paper. An overview of protein identifications and quantifications based on LC/MS analysis is shown in the source data (Fig. 2 - Source Data 1, Fig. 3 - Source Data 1, and Fig. 4 - Source Data 1).LC/MS-raw data and search results have been deposited at the Mass Spectrometry Interactive Virtual Environment(MassIVE; https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp) data lake and are publicly available under ID MSV000089634.

The following data sets were generated

Article and author information

Author details

  1. Lea Krutzke

    Department of Gene Therapy, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4092-4131
  2. Reinhild Rösler

    Core Unit Mass Spectrometry and Proteomics, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Ellen Allmendinger

    Department of Gene Therapy, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Tatjana Engler

    Department of Gene Therapy, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Sebastian Wiese

    Core Unit Mass Spectrometry and Proteomics, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Stefan Kochanek

    Department of Gene Therapy, University of Ulm, Ulm, Germany
    For correspondence
    stefan.kochanek@uni-ulm.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7494-1602

Funding

German Federal Ministry of Education and Research and Federal States of Germany Grant Innovative Hochschule"" (FKZ3IHS024D)

  • Lea Krutzke
  • Reinhild Rösler
  • Ellen Allmendinger
  • Tatjana Engler
  • Sebastian Wiese
  • Stefan Kochanek

German Research Foundation (SFB1074)

  • Lea Krutzke
  • Reinhild Rösler
  • Ellen Allmendinger
  • Tatjana Engler
  • Sebastian Wiese
  • Stefan Kochanek

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: Animal experiments were approved by the Animal Care Commission of the Government Baden-Württemberg. Reference number: TVA #1508.

Copyright

© 2022, Krutzke et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 14,532
    views
  • 1,331
    downloads
  • 30
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Lea Krutzke
  2. Reinhild Rösler
  3. Ellen Allmendinger
  4. Tatjana Engler
  5. Sebastian Wiese
  6. Stefan Kochanek
(2022)
Process- and product-related impurities in the ChAdOx1 nCov-19 vaccine
eLife 11:e78513.
https://doi.org/10.7554/eLife.78513

Share this article

https://doi.org/10.7554/eLife.78513

Further reading

    1. Immunology and Inflammation
    2. Microbiology and Infectious Disease
    Malika Hale, Kennidy K Takehara ... Marion Pepper
    Research Article

    Pseudomonas aeruginosa (PA) is an opportunistic, frequently multidrug-resistant pathogen that can cause severe infections in hospitalized patients. Antibodies against the PA virulence factor, PcrV, protect from death and disease in a variety of animal models. However, clinical trials of PcrV-binding antibody-based products have thus far failed to demonstrate benefit. Prior candidates were derivations of antibodies identified using protein-immunized animal systems and required extensive engineering to optimize binding and/or reduce immunogenicity. Of note, PA infections are common in people with cystic fibrosis (pwCF), who are generally believed to mount normal adaptive immune responses. Here, we utilized a tetramer reagent to detect and isolate PcrV-specific B cells in pwCF and, via single-cell sorting and paired-chain sequencing, identified the B cell receptor (BCR) variable region sequences that confer PcrV-specificity. We derived multiple high affinity anti-PcrV monoclonal antibodies (mAbs) from PcrV-specific B cells across three donors, including mAbs that exhibit potent anti-PA activity in a murine pneumonia model. This robust strategy for mAb discovery expands what is known about PA-specific B cells in pwCF and yields novel mAbs with potential for future clinical use.

    1. Microbiology and Infectious Disease
    Ziyu Wen, Pingchao Li ... Caijun Sun
    Research Article

    The persistence of latent viral reservoirs remains the major obstacle to eradicating human immunodeficiency virus (HIV). We herein found that ICP34.5 can act as an antagonistic factor for the reactivation of HIV latency by herpes simplex virus type I (HSV-1), and thus recombinant HSV-1 with ICP34.5 deletion could more effectively reactivate HIV latency than its wild-type counterpart. Mechanistically, HSV-ΔICP34.5 promoted the phosphorylation of HSF1 by decreasing the recruitment of protein phosphatase 1 (PP1α), thus effectively binding to the HIV LTR to reactivate the latent reservoirs. In addition, HSV-ΔICP34.5 enhanced the phosphorylation of IKKα/β through the degradation of IκBα, leading to p65 accumulation in the nucleus to elicit NF-κB pathway-dependent reactivation of HIV latency. Then, we constructed the recombinant HSV-ΔICP34.5 expressing simian immunodeficiency virus (SIV) env, gag, or the fusion antigen sPD1-SIVgag as a therapeutic vaccine, aiming to achieve a functional cure by simultaneously reactivating viral latency and eliciting antigen-specific immune responses. Results showed that these constructs effectively elicited SIV-specific immune responses, reactivated SIV latency, and delayed viral rebound after the interruption of antiretroviral therapy (ART) in chronically SIV-infected rhesus macaques. Collectively, these findings provide insights into the rational design of HSV-vectored therapeutic strategies for pursuing an HIV functional cure.