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Title: The impact of vaccination against rotavirus on viral faecal shedding, genetic stability and mucosal immune response in a cohort of infants in the UK
Author: Botas Pérez, Ledicia
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2020
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Background: In July 2013, an oral live-attenuated monovalent human rotavirus G1P[8] vaccine, Rotarix® , was introduced into the United Kingdom's national immunisation programme as a two-dose regime. This vaccine is used widely on a global scale. Data on vaccine take have been reported through clinical trials assessing shedding at specific timepoints and immunogenicity by seroconversion. However, the longitudinal dynamics of shedding and mucosal antibody IgA response had not been studied. Clinical trials have also evaluated vaccine safety, however, other than reports of vaccine-related genetic variants from single-timepoints captured through clinical admissions, there is an unanswered question about genome-wide genetic stability in vaccinees. Aims: The overarching hypothesis of this thesis was that immunisation with Rotarix® would result in an evolving quasispecies through replication in vaccinees generating high-frequency variants and modulated by the mucosal secretory IgA response. To test this, the aims were i) to assess Rotarix® shedding profiles in stool of a cohort of vaccinated infants, ii) to identify any vaccine and/or novel variants in shed virus and iii) to define the infants' RV-specific copro-IgA levels. Methods: Stool samples from a cohort of vaccinated infants were collected longitudinally every other day throughout the vaccination period. Viral shedding was assessed through quantification of viral RNA extracted from faecal suspensions. Genetic variation was evaluated through next generation sequencing on an Illumina® platform, focusing on viral proteins with known function in viral entry or virulence: VP4, VP7, VP6 and NSP4. Total copro-IgA was measured using a commercial ELISA kit and RV-specific copro-IgA using an in-house ELISA. Results: All infants shed vaccine virus in faeces and patterns defined four profiles ranging from early control of vaccine virus in stool to delayed control with continued virus shedding. The maximum shedding of vaccine virus was comparable to natural infection. Some single nucleotide polymorphisms identified at low frequency in the vaccine were identified at higher frequencies in vaccine recipients, suggesting that these minority variants in cell culture were selected in infants. Novel vaccine-derived variant loci were identified from stool as a result of replication in the host, suggesting a possible effect in cell tropism, host range or immune evasion. Mutations in the outer capsid proteins VP4 and VP7 impacted on residues involved in receptor binding, trypsin cleavage, membrane fusion and neutralisation; a mutation in VP6 highlighted the importance of structural conservation of the inner capsid and all novel mutations in NPS4 suggested they may be relevant in in vivo infection. Rotavirus-specific copro-IgA differed between infants ranging from continual high levels through sporadic to no detection. High pre-vaccination specific copro-IgA levels in three infants were likely to originate from maternal antibody, although this did not appear to affect vaccine virus shedding. Three of eight infants were positive for RV-specific copro-IgA a year after vaccination, suggesting they were late immune responders or had had a recent subclinical rotavirus infection. Infants with positive RV-specific copro-IgA presented viral load control and those with protracted shedding presented undetectable or weak RV-specific copro-IgA levels. Conclusions: Shedding of RV vaccine virus in vaccine recipients suggested active virus replication over several weeks and it fell within four broad profiles. Previously identified vaccine genetic variants increased in frequency and novel variants arose after replication in the gut. Infants who could not rapidly control shedding had a weak or undetectable RV-specific copro-IgA response and a higher number of high-frequency genetic variants detectable by the end of the vaccination period. By contrast, infants who controlled shedding and presented a strong RVspecific copro-IgA response had vaccine virus variants that decreased in frequency by the end of the vaccination period, suggesting Rotarix® is stable in vaccine recipients who present a robust and early RV-specific copro-IgA response.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral