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Adenovirus DNA Polymerase Loses Fidelity on a Stretch of Eleven Homocytidines during Pre-GMP Vaccine Preparation

Authors :
Zara Hannoun
Edmund G. Wee
Alison Crook
Stefano Colloca
Stefania Di Marco
Tomáš Hanke
Source :
Vaccines, Vol 10, Iss 6, p 960 (2022)
Publication Year :
2022
Publisher :
MDPI AG, 2022.

Abstract

In this study, we invented and construct novel candidate HIV-1 vaccines. Through genetic and protein engineering, we unknowingly constructed an HIV-1-derived transgene with a homopolymeric run of 11 cytidines, which was inserted into an adenovirus vaccine vector. Here, we describe the virus rescue, three rounds of clonal purification and preparation of good manufacturing practise (GMP) starting material assessed for genetic stability in five additional virus passages. Throughout these steps, quality control assays indicated the presence of the transgene in the virus genome, expression of the correct transgene product and immunogenicity in mice. However, DNA sequencing of the transgene revealed additional cytidines inserted into the original 11-cytidine region, and the GMP manufacture had to be aborted. Subsequent analyses indicated that as little as 1/25th of the virus dose used for confirmation of protein expression (106 cells at a multiplicity of infection of 10) and murine immunogenicity (108 infectious units per animal) met the quality acceptance criteria. Similar frameshifts in the expressed proteins were reproduced in a one-reaction in vitro transcription/translation employing phage T7 polymerase and E. coli ribosomes. Thus, the most likely mechanism for addition of extra cytidines into the ChAdOx1.tHIVconsv6 genome is that the adenovirus DNA polymerase lost its fidelity on a stretch of 11 cytidines, which informs future adenovirus vaccine designs.

Details

Language :
English
ISSN :
10060960 and 2076393X
Volume :
10
Issue :
6
Database :
Directory of Open Access Journals
Journal :
Vaccines
Publication Type :
Academic Journal
Accession number :
edsdoj.895deba437e546f5b8ca79abe46bea1b
Document Type :
article
Full Text :
https://doi.org/10.3390/vaccines10060960