Neutralising antibody responses in acute hepatitis C and SARS-CoV-2 infection

Neutralising antibodies (nAbs) are capable of blocking viral entry to host cells. Understanding the nAb response in early infection informs preventative and therapeutic interventions. The acute phase of hepatitis C virus (HCV) infection is characterised by two bottlenecks of viral diversity, one at the initiation of infection (selection of transmitted/founder (T/F) variants), and another within 100 days of infection (emergence of fit variants against host immunity). Three out of four patients with HCV develop chronic infection which is associated with a delayed nAb response against variants emerging from the second bottleneck. However, the ability for HCV T/F variants to induce nAbs is poorly understood. This first part of this thesis aimed to characterise T/Fs using next-generation sequencing, and determine if the immunogenicity of T/Fs was associated with infection outcome. Approximately one in four primary HCV infections were found to originate from a single T/F virus and in those with spontaneous clearance of infection, T/Fs have a stronger binding affinity towards cluster of differentiation (CD) -81, a key receptor for HCV cell entry. T/Fs of spontaneous clearers were also more susceptible to neutralisation compared to chronic progressors. The second part of this thesis describes how the techniques utilised to study nAb responses in HCV were adapted to study Severe acute respiratory syndrome (SARS) coronavirus (CoV) -2, hoping to unravel the comparable nAb protection in coronavirus disease 2019 (COVID-19) infection. A new assay to study SARS-CoV-2 neutralisation was optimised and used to characterise the longitudinal nAb responses in individuals recovering from COVID-19 infection, and up to six months post-infection. In the majority, nAb responses (titers and neutralisation capacity) were evident, but declined modestly, with most being above background at six months post-infection. This decline was associated with older age, but not with gender or illness severity. In summary, this thesis examined the role of viral and nAb traits associated with two different viruses and their varied infection outcomes. The findings contribute to better understanding of interactions between virus and host antibody responses, which is critical for vaccine design

Genomic characterization of hepatitis C virus transmitted founder variants with deep sequencing

Transfer of hepatitis C virus (HCV) infection from a donor to a new recipient is associated with a bottleneck of genetic diversity in the transmitted viral variants. Existing data suggests that one, or very few, variants emerge from this bottleneck to establish the infection (transmitted founder [T/F] variants). In HCV, very few T/F variants have been characterized due to the challenges of obtaining early infection samples and of high throughput viral genome sequencing. This study used a large, acute HCV, deep-sequenced dataset from first viremia samples collected in nine prospective cohorts across four countries, to estimate the prevalence of single T/F viruses, and to identify host and virus-related factors associated with infections initiated by a single T/F variant. The short reads generated by Illumina sequencing were used to reconstruct viral haplotypes with two haplotype reconstruction algorithms. The haplotypes were examined for random mutations (Poisson distribution) and a star-like phylogeny to identify T/F viruses. The findings were cross-validated by haplotype reconstructions across three regions of the genome (Core-E2, NS3, NS5A) to minimize the possibility of spurious overestimation of single T/F variants. Of 190 acute infection samples examined, 54 were very early acute infections (HCV antibody negative, RNA positive), and single transmitted founders were identified in 14 (26%, 95% CI: 16–39%) after cross validation across multiple regions of the genome with two haplotype reconstruction algorithms. The presence of a single T/F virus was not associated with any host or virus-related factors, notably viral genotype or spontaneous clearance. In conclusion, approximately one in four new HCV infections originates from a single T/F virus. Resolution of genomic sequences of single T/F variants is the first step in exploring unique properties of these variants in the infection of host hepatocytes

Long-term persistence of neutralizing memory B cells in SARS-CoV-2

Considerable concerns relating to the duration of protective immunity against SARS-CoV-2 have been raised, with evidence of antibody titres declining rapidly after infection and reports of reinfection. Here we monitored antibody responses against SARS-CoV-2 receptor binding domain (RBD) for up to six months after infection. While antibody titres were maintained, half of the cohort's neutralising responses had returned to background. However, encouragingly in a selected subset of 13 participants, 12 had detectable RBD-specific memory B cells and these generally increased out to 6 months. Furthermore, we were able to generate monoclonal antibodies with SARS-CoV-2 neutralising capacity from these memory B cells. Overall our study suggests that the loss of neutralising antibodies in plasma may be countered by the maintenance of neutralising capacity in the memory B cell repertoire

SARS-CoV-2 Omicron variant escapes neutralizing antibodies and T cell responses more efficiently than other variants in mild COVID-19 convalescents.

Coronavirus disease 2019 (COVID-19) convalescents living in regions with low vaccination rates rely on post-infection immunity for protection against re-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluate humoral and T cell immunity against five variants of concern (VOCs) in mild-COVID-19 convalescents at 12 months after infection with ancestral virus. In this cohort, ancestral, receptor-binding domain (RBD)-specific antibody and circulating memory B cell levels are conserved in most individuals, and yet serum neutralization against live B.1.1.529 (Omicron) is completely abrogated and significantly reduced for other VOCs. Likewise, ancestral SARS-CoV-2-specific memory T cell frequencies are maintained in >50% of convalescents, but the cytokine response in these cells to mutated spike epitopes corresponding to B.1.1.529 and B.1.351 (Beta) VOCs were impaired. These results indicate that increased antigen variability in VOCs impairs humoral and spike-specific T cell immunity post-infection, strongly suggesting that COVID-19 convalescents are vulnerable and at risk of re-infection with VOCs, thus stressing the importance of vaccination programs