B cell characteristics define HCV reinfection outcome.

BACKGROUND AND AIMS: In individuals highly exposed to hepatitis C virus (HCV), reinfection is common, suggesting that natural development of sterilising immunity is difficult. In those that are reinfected, some will develop a persistent infection, while a small proportion repeatedly clear the virus, suggesting natural protection is possible. The aim of this study was to characterise immune responses associated with rapid natural clearance of HCV reinfection. METHODS: Broad neutralising antibodies (BnAbs) and Envelope 2 (E2)-specific memory B cell (MBCs) responses were examined longitudinally in 15 subjects with varied reinfection outcomes. RESULTS: BnAb responses were associated with MBC recall, but not with reinfection clearance. Strong evidence of antigen imprinting was found, and the B cell receptor repertoire showed a high level of clonality with ongoing somatic hypermutation of many clones over subsequent reinfection events. Single cell transcriptomic analyses showed that cleared reinfections featured an activated transcriptomic profile in HCV-specific B cells that rapidly expanded upon reinfection. CONCLUSIONS: MBC quality, but not necessarily breadth of nAb responses, is important for protection against antigenically diverse variants, which is encouraging for HCV vaccine development.

Characterization of SARS-CoV-2 humoral immune response in a subject with unique sampling: A case report.

BACKGROUND: The development of vaccine candidates for COVID-19, and the administration of booster vaccines, has meant a significant reduction in COVID-19 related deaths world-wide and the easing of global restrictions. However, new variants of SARS-CoV-2 have emerged with less susceptibility to vaccine induced immunity leading to breakthrough infections among vaccinated people. It is generally acknowledged that immunoglobulins play the major role in immune-protection, primarily through binding to the SARS-COV-2 receptor binding domain (RBD) and thereby inhibiting viral binding to the ACE2 receptor. However, there are limited investigations of anti-RBD isotypes (IgM, IgG, IgA) and IgG subclasses (IgG1-4) over the course of vaccination and breakthrough infection. METHOD: In this study, SARS-CoV-2 humoral immunity is examined in a single subject with unique longitudinal sampling. Over a two year period, the subject received three doses of vaccine, had two active breakthrough infections and 22 blood samples collected. Serological testing included anti-nucleocapsid total antibodies, anti-RBD total antibodies, IgG, IgA, IgM and IgG subclasses, neutralization and ACE2 inhibition against the wildtype (WT), Delta and Omicron variants. RESULTS: Vaccination and breakthrough infections induced IgG, specifically IgG1 and IgG4 as well as IgM and IgA. IgG1 and IgG4 responses were cross reactive and associated with broad inhibition. CONCLUSION: The findings here provide novel insights into humoral immune response characteristics associated with SARS-CoV-2 breakthrough infections.

SARS-CoV-2 RBD-Specific Antibodies Induced Early in the Pandemic by Natural Infection and Vaccination Display Cross-Variant Binding and Inhibition.

The development of vaccine candidates for COVID-19 has been rapid, and those that are currently approved display high efficacy against the original circulating strains. However, recently, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged with increased transmission rates and less susceptibility to vaccine induced immunity. A greater understanding of protection mechanisms, including antibody longevity and cross-reactivity towards the variants of concern (VoCs), is needed. In this study, samples collected in Denmark early in the pandemic from paucisymptomatic subjects (n = 165) and symptomatic subjects (n = 57) infected with SARS-CoV-2 were used to assess IgG binding and inhibition in the form of angiotensin-converting enzyme 2 receptor (ACE2) competition against the wild-type and four SARS-CoV-2 VoCs (Alpha, Beta, Gamma, and Omicron). Antibodies induced early in the pandemic via natural infection were cross-reactive and inhibited ACE2 binding of the VoC, with reduced inhibition observed for the Omicron variant. When examined longitudinally, sustained cross-reactive inhibitory responses were found to exist in naturally infected paucisymptomatic subjects. After vaccination, receptor binding domain (RBD)-specific IgG binding increased by at least 3.5-fold and inhibition of ACE2 increased by at least 2-fold. When vaccination regimens were compared (two doses of Pfizer-BioNTech BNT162b2 (n = 50), or one dose of Oxford-AstraZeneca ChAdOx1 nCoV-19 followed by Pfizer-BioNTech BNT162b2 (ChAd/BNT) (n = 15)), higher levels of IgG binding and inhibition were associated with mix and match (ChAd/BNT) prime-boosting and time since vaccination. These results are particularly relevant for countries where vaccination levels are low.

Production of PfEMP1-Specific Human Monoclonal Antibodies from Naturally Immune Individuals.

Plasmodium falciparum parasites express variable surface antigens on the infected erythrocyte surface allowing adhesion to human host receptors on the blood and endothelial cells, which can result in immune evasion. One of the most studied and key antigens in adhesion is the highly polymorphic PfEMP1. However, despite the vast variation in the PfEMP1 antigens, they are the main targets of naturally acquired immunity and are therefore promising candidates for malaria vaccine development. Generating PfEMP1-specific human monoclonal antibodies from naturally immune individuals will help to determine the best targets of protection from clinical disease. Immortalization of human B cells is one of the oldest and most efficient techniques to generate human monoclonal antibodies. Nevertheless, most protocols require flow cytometry-based cell sorting, which can be a limiting factor for many laboratories. This chapter describes an efficient protocol for the generation of PfEMP1-specific human monoclonal antibodies from malaria immune individuals that can be performed without the use of advanced cell-sorting techniques.

Enhancing neutralization of Plasmodium falciparum using a novel monoclonal antibody against the rhoptry-associated membrane antigen.

The pathogenesis of malaria is associated with blood-stage infection and there is strong evidence that antibodies specific to parasite blood-stage antigens can control parasitemia. This provides a strong rational for applying blood-stage antigen components in a multivalent vaccine, as the induced antibodies in combination can enhance protection. The Plasmodium falciparum rhoptry-associated membrane antigen (PfRAMA) is a promising vaccine target, due to its fundamental role in merozoite invasion and low level of polymorphism. Polyclonal antibodies against PfRAMA are able to inhibit P. falciparum growth and interact synergistically when combined with antibodies against P. falciparum reticulocyte-binding protein 5 (PfRh5) or cysteine-rich protective antigen (PfCyRPA). In this study, we identified a novel PfRAMA-specific mAb with neutralizing activity, which in combination with PfRh5- or PfCyRPA-specific mAbs potentiated the neutralizing effect. By applying phage display technology, we mapped the protective epitope to be in the C-terminal region of PfRAMA. Our results confirmed previous finding of synergy between PfRAMA-, PfRh5- and PfCyRPA-specific antibodies, thereby paving the way of testing these antigens (or fragments of these antigens) in combination to improve the efficacy of blood-stage malaria vaccines. The results emphasize the importance of directing antibody responses towards protective epitopes, as the majority of anti-PfRAMA mAbs were unable to inhibit merozoite invasion of erythrocytes.

O-linked α2,3 sialylation defines stem cell populations in breast cancer.

We pursued the hypothesis that specific glycans can be used to distinguish breast cancer stem cells (CSCs) and influence their function. Comparison of CSCs and non-CSCs from multiple breast cancer models revealed that CSCs are distinguished by expression of α2,3 sialylated core2 O-linked glycans. We identified a lectin, SLBR-N, which binds to O-linked α2,3 sialic acids, that was able to enrich for CSCs in vitro and in vivo. This O-glycan is expressed on CD44 and promotes its interaction with hyaluronic acid, facilitating CD44 signaling and CSC properties. In contrast, FUT3, which contributes to sialyl Lewis X (sLeX) production, is preferentially expressed in the non-CSC population, and it antagonizes CSC function. Collectively, our data indicate that SLBR-N can be more efficient at enriching for CSCs than CD44 itself because its use avoids the issues of CD44 splicing and glycan status. These data also reveal how differential glycosylation influences CSC fate.

Strain-Dependent Inhibition of Erythrocyte Invasion by Monoclonal Antibodies Against Plasmodium falciparum CyRPA.

The highly conserved Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is a key target for next-generation vaccines against blood-stage malaria. PfCyRPA constitute the core of a ternary complex, including the reticulocyte binding-like homologous protein 5 (PfRh5) and the Rh5-interacting protein (PfRipr), and is fundamental for merozoite invasion of erythrocytes. In this study, we show that monoclonal antibodies (mAbs) specific to PfCyRPA neutralize the in vitro growth of Ghanaian field isolates as well as numerous laboratory-adapted parasite lines. We identified subsets of mAbs with neutralizing activity that bind to distinct sites on PfCyRPA and that in combination potentiate the neutralizing effect. As antibody responses against multiple merozoite invasion proteins are thought to improve the efficacy of blood-stage vaccines, we also demonstrated that combinations of PfCyRPA- and PfRh5 specific mAbs act synergistically to neutralize parasite growth. Yet, we identified prominent strain-dependent neutralization potencies, which our results suggest is independent of PfCyRPA expression level and polymorphism, demonstrating the importance of addressing functional converseness when evaluating blood-stage vaccine candidates. Finally, our results suggest that blood-stage vaccine efficacy can be improved by directing the antibody response towards defined protective epitopes on multiple parasite antigens.

Acquisition and decay of IgM and IgG responses to merozoite antigens after Plasmodium falciparum malaria in Ghanaian children.

Developing a vaccine against Plasmodium falciparum malaria has been challenging, primarily due to high levels of antigen polymorphism and a complex parasite lifecycle. Immunization with the P. falciparum merozoite antigens PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5 has been shown to give rise to growth inhibitory and synergistic antisera. Therefore, these five merozoite proteins are considered to be promising candidates for a second-generation multivalent malaria vaccine. Nevertheless, little is known about IgG and IgM responses to these antigens in populations that are naturally exposed to P. falciparum. In this study, serum samples from clinically immune adults and malaria exposed children from Ghana were studied to compare levels of IgG and IgM specific for PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5. All five antigens were found to be specifically recognized by both IgM and IgG in serum from clinically immune adults and from children with malaria. Longitudinal analysis of the latter group showed an early, transient IgM response that was followed by IgG, which peaked 14 days after the initial diagnosis. IgG levels and parasitemia did not correlate, whereas parasitemia was weakly positively correlated with IgM levels. These findings show that IgG and IgM specific for merozoite antigens PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5 are high in children during P. falciparum malaria, but that the IgM induction and decline occurs earlier in infection than that of IgG.

Differentiation of Cancer Stem Cells through Nanoparticle Surface Engineering.

Cancer stem cells (CSCs) are a crucial therapeutic target because of their role in resistance to chemo- and radiation therapy, metastasis, and tumor recurrence. Differentiation therapy presents a potential strategy for "defanging" CSCs. To date, only a limited number of small-molecule and nanomaterial-based differentiating agents have been identified. We report here the integrated use of nanoparticle engineering and hypothesis-free sensing to identify nanoparticles capable of efficient differentiation of CSCs into non-CSC phenotypes. Using this strategy, we identified a nanoparticle that induces CSC differentiation by increasing intracellular reactive oxygen species levels. Importantly, this unreported phenotype is more susceptible to drug treatment than either CSCs or non-CSCs, demonstrating a potentially powerful strategy for anticancer therapeutics.

Anti-envelope antibody responses in highly exposed seronegative individuals may be associated with protection from HCV infection.

In rare cases, individuals with a history of long-term injecting drug use remain seronegative and aviraemic, despite prolonged and likely repeated exposure to Hepatitis C virus (HCV) through high-risk behaviour. We describe anti-HCV Envelope (E) antibody responses in a prospective cohort of carefully defined highly exposed but uninfected subjects (HESN) and comparison subjects who were also high risk and uninfected, but rapidly became HCV infected (Incident). Longitudinally collected samples from HESN cases (n = 22) were compared to Incident controls (n = 22). IgG, IgM and IgA from sera were tested by ELISA to genotype 1a and 3a E glycoproteins, and recombinant genotype 1a E2 antigen. IgG subclass isotyping was performed for those positive for IgG. Virus-neutralizing activity was assessed on HCV pseudoparticles, and HCV E-specific B cells analysed using flow cytometry. A significant minority of HESN cases (n = 10; 45%) had anti-E, predominantly in the IgG2 subclass, which was not found in the pre-infection time point of the Incident cases (n = 1; 5%). A subset of the HESN subjects also had neutralizing activity and HCV-specific B cells detected significantly more than Incident cases pre-infection. In conclusion, the HESN phenotype is associated with IgG2 anti-E antibodies, neutralization activity and HCV E-specific memory B cells. These findings suggest that HESN subjects may be resistant to HCV infection through humoral immune-mediated mechanisms.

Envelope-Specific IgG3 and IgG1 Responses Are Associated with Clearance of Acute Hepatitis C Virus Infection.

Hepatitis C virus (HCV) can be cleared naturally in a subset of individuals. However, the asymptomatic nature of acute HCV infection makes the study of the early immune response and defining the correlates of protection challenging. Despite this, there is now strong evidence implicating the humoral immune response, specifically neutralising antibodies, in determining the clearance or chronicity outcomes of primary HCV infection. In general, immunoglobulin G (IgG) plays the major role in viral neutralisation. However, there are limited investigations of anti-HCV envelope protein 2 (E2) isotypes (IgM, IgG, IgA) and IgG subclasses (IgG1-4) in early HCV infection. In this study, using a rare cohort of 14 very recently HCV-infected individuals (4-45 days) with varying disease outcome (n = 7 clearers), the timing and potency of anti-HCV E2 isotypes and IgG subclasses were examined longitudinally, in relation to neutralising antibody activity. Clearance was associated with anti-E2 IgG, specifically IgG1 and IgG3, and appeared essential to prevent the emergence of new HCV variants and the chronic infection outcome. Interestingly, these IgG responses were accompanied by IgM antibodies and were associated with neutralising antibody activity in the subjects who cleared infection. These findings provide novel insights into the early humoral immune response characteristics associated with HCV disease outcome.

Alveolar progenitor cells in the mammary gland are dependent on the ß4 integrin.

Understanding how progenitor cell function is regulated in the mammary gland is an important developmental problem that has significant implications for breast cancer. Although it had been assumed that the expression the α6ß4 integrin (ß4) is restricted to the basal lineage, we report that alveolar progenitor cells in the mouse mammary gland also express this integrin based on analysis of single cell RNA-Seq data. Subsequent experiments using a mouse mammary epithelial cell line (NMuMG) confirmed this finding and revealed that ß4 is essential for maintaining progenitor function as assessed by serial passage mammosphere assays. These data were substantiated by analyzing the alveolar progenitor population isolated from nulliparous mouse mammary glands. Based on the finding that the alveolar progenitor cells express Whey Acidic Protein (WAP), WAP-Cre mice were crossed with itgß4flox/flox mice to generate conditional knock-out of ß4 in alveolar progenitor cells. These itgß4flox/floxWAP-Cre+ mice exhibited significant defects in alveologenesis and milk production during pregnancy compared to itgß4flox/floxWAP-Cre- mice, establishing a novel role for the ß4 integrin in alveolar progenitor function and alveologenesis.

B cell immunodominance in primary hepatitis C virus infection.

BACKGROUND & AIMS: Neutralising antibodies (NAbs) play a key role in clearance of HCV. NAbs have been isolated and mapped to several domains on the HCV envelope proteins. However, the immunodominance of these epitopes in HCV infection remains unknown, hindering efforts to elicit optimal epitope-specific responses. Furthermore, it remains unclear which epitope-specific responses are associated with broad NAb (bNAb) activity in primary HCV infection. The aim of this study was to define B cell immunodominance in primary HCV, and its implications on neutralisation breadth and clearance. METHODS: Using samples from 168 patients with primary HCV infection, the antibody responses targeted 2 immunodominant domains, termed domains B and C. Genotype 1 and 3 infections were associated with responses targeted towards different bNAb domains. RESULTS: No epitopes were uniquely targeted by clearers compared to those who developed chronic infection. Samples with bNAb activity were enriched for multi-specific responses directed towards the epitopes antigenic region 3, antigenic region 4, and domain D, and did not target non-neutralising domains. CONCLUSIONS: This study outlines for the first time a clear NAb immunodominance profile in primary HCV infection, and indicates that it is influenced by the infecting virus. It also highlights the need for a vaccination strategy to induce multi-specific responses that do not target non-neutralising domains. LAY SUMMARY: Neutralising antibodies will likely form a key component of a protective hepatitis C virus vaccine. In this work we characterise the predominant neutralising and non-neutralising antibody (epitope) targets in acute hepatitis C virus infection. We have defined the natural hierarchy of epitope immunodominance, and demonstrated that viral genotype can impact on this hierarchy. Our findings highlight key epitopes that are associated with broadly neutralising antibodies, and the deleterious impact of mounting a response towards some of these domains on neutralising breadth. These findings should guide future efforts to design immunogens aimed at generating neutralising antibodies with a vaccine candidate.

Clearance of hepatitis C virus is associated with early and potent but narrowly-directed, Envelope-specific antibodies.

Hepatitis C virus (HCV) is one of very few viruses that are either naturally cleared, or alternatively persist to cause chronic disease. Viral diversity and escape, as well as host adaptive immune factors, are believed to control the outcome. To date, there is limited understanding of the critical, early host-pathogen interactions. The asymptomatic nature of early HCV infection generally prevents identification of the transmitted/founder (T/F) virus, and thus the study of host responses directed against the autologous T/F strain. In this study, 14 rare subjects identified from very early in infection (4-45 days) with varied disease outcomes (n = 7 clearers) were examined in regard to the timing, breadth, and magnitude of the neutralizing antibody (nAb) response, as well as evolution of the T/F strain. Clearance was associated with earlier onset and more potent nAb responses appearing at a mean of 71 days post-infection (DPI), but these responses were narrowly directed against the autologous T/F virus or closely related variants. In contrast, a delayed onset of nAbs (mean 425 DPI) was observed in chronic progressors that appear to have targeted longitudinal variants rather than the T/F strain. The nAb responses in the chronic progressors mapped to known CD81 binding epitopes, and were associated with rapid emergence of new viral variants with reduced CD81 binding. We propose that the prolonged period of viremia in the absence of nAbs in these subjects was associated with an increase in viral diversity, affording the virus greater options to escape nAb pressure once it emerged. These findings indicate that timing of the nAb response is essential for clearance. Further investigation of the specificities of the early nAbs and the factors regulating early induction of protective nAbs is needed.

Real-time imaging of integrin ß4 dynamics using a reporter cell line generated by Crispr/Cas9 genome editing.

The ability to monitor changes in the expression and localization of integrins is essential for understanding their contribution to development, tissue homeostasis and disease. Here, we pioneered the use of Crispr/Cas9 genome editing to tag an allele of the ß4 subunit of the α6ß4 integrin. A tdTomato tag was inserted with a linker at the C-terminus of integrin ß4 in mouse mammary epithelial cells. Cells harboring this tagged allele were similar to wild-type cells with respect to integrin ß4 surface expression, association with the α6 subunit, adhesion to laminin and consequent signaling. These integrin ß4 reporter cells were transformed with YAP (also known as YAP1), which enabled us to obtain novel insight into integrin ß4 dynamics in response to a migratory stimulus (scratch wound) by live-cell video microscopy. An increase in integrin ß4 expression in cells proximal to the wound edge was evident, and a population of integrin ß4-expressing cells that exhibited unusually rapid migration was identified. These findings could shed insight into integrin ß4 dynamics during invasion and metastasis. Moreover, these integrin ß4 reporter cells should facilitate studies on the contribution of this integrin to mammary gland biology and cancer.This article has an associated First Person interview with the first author of the paper.

A method for detecting hepatitis C envelope specific memory B cells from multiple genotypes using cocktail E2 tetramers.

Hepatitis C (HCV) is a rapidly mutating RNA virus, with a strong propensity to cause chronic infection and progressive liver disease. Recent evidence has shown that early appearance of neutralizing antibodies in primary infection is associated with clearance. Little is known about the characteristics of HCV-specific B cells and their correlation with outcomes in primary infection, as there is a lack of sensitive tools for HCV-specific B cells which are present at very low frequency. We describe the development and optimisation of tetramer staining for flow cytometric detection of HCV-specific B cells using a cocktail of two recombinant HCV Envelope-2 (rE2) glycoproteins (from genotype 1a and 3a; Gt1a and Gt3a) and streptavidin dyes. The optimal weight to weight (w/w) ratio of streptavidin-phycoerythrin (PE) and rE2 proteins were determined for sensitive detection using HCV E2-specific hybridoma cell lines and peripheral blood mononuclear cells (PBMC) from HCV-infected individuals. In a cross-sectional set of PBMC samples collected from 33 subjects with either chronic infection or previous clearance, HCV E2-specific B cells (CD19+CD20+CD10-IgD-tetramer+) were detected in 29 subjects (87.8%), with a mean frequency of 0.45% (0.012-2.20%). To validate the specificity of tetramer staining, 367 HCV E2-specific B cells were single cell sorted from 9 PBMC samples before monoclonal antibodies (mAbs) were synthesised, with 87.5% being reactive to E2 via ELISA. Of these mAbs, 284 and 246 clones were reactive to either Gt1a or Gt3a E2 proteins, respectively. This is a sensitive and robust method for future studies investigating B cell responses against the HCV Envelope protein.

Broadly neutralizing antibodies from an individual that naturally cleared multiple hepatitis C virus infections uncover molecular determinants for E2 targeting and vaccine design.

Cumulative evidence supports a role for neutralizing antibodies contributing to spontaneous viral clearance during acute hepatitis C virus (HCV) infection. Information on the timing and specificity of the B cell response associated with clearance is crucial to inform vaccine design. From an individual who cleared three sequential HCV infections with genotypes 1b, 1a and 3a strains, respectively, we employed peripheral B cells to isolate and characterize neutralizing human monoclonal antibodies (HMAbs) to HCV after the genotype 1 infections. The majority of isolated antibodies, designated as HMAbs 212, target conformational epitopes on the envelope glycoprotein E2 and bound broadly to genotype 1-6 E1E2 proteins. Further, some of these antibodies showed neutralization potential against cultured genotype 1-6 viruses. Competition studies with defined broadly neutralizing HCV HMAbs to epitopes in distinct clusters, designated antigenic domains B, C, D and E, revealed that the selected HMAbs compete with B, C and D HMAbs, previously isolated from subjects with chronic HCV infections. Epitope mapping studies revealed domain B and C specificity of these HMAbs 212. Sequential serum samples from the studied subject inhibited the binding of HMAbs 212 to autologous E2 and blocked a representative domain D HMAb. The specificity of this antibody response appears similar to that observed during chronic infection, suggesting that the timing and affinity maturation of the antibody response are the critical determinants in successful and repeated viral clearance. While additional studies should be performed for individuals with clearance or persistence of HCV, our results define epitope determinants for antibody E2 targeting with important implications for the development of a B cell vaccine.

Understanding the Determinants of BnAb Induction in Acute HCV Infection.

Despite recent advances in curative therapy, hepatitis C virus (HCV) still remains a global threat. In order to achieve global elimination, a prophylactic vaccine should be considered high priority. Previous immunogens used to induce broad neutralising antibodies (BnAbs) have been met with limited success. To improve immunogen design, factors associated with the early development of BnAbs in natural infection must first be understood. In this study, 43 subjects identified with acute HCV were analysed longitudinally using a panel of heterogeneous HCV pseudoparticles (HCVpp), to understand the emergence of BnAbs. Compared to those infected with a single genotype, early BnAb development was associated with subjects co-infected with at least 2 HCV subtypes during acute infection. In those that were mono-infected, BnAbs were seen to emerge with increasing viral persistence. If subjects acquired a secondary infection, nAb breadth was seen to boost upon viral re-exposure. Importantly, this data highlights the potential for multivalent and prime-boost vaccine strategies to induce BnAbs against HCV in humans. However, the data also indicate that the infecting genotype may influence the development of BnAbs. Therefore, the choice of antigen will need to be carefully considered in future vaccine trials.

Limited naturally occurring escape in broadly neutralizing antibody epitopes in hepatitis C glycoprotein E2 and constrained sequence usage in acute infection.

Broadly neutralizing antibodies have been associated with spontaneous clearance of the hepatitis C infection as well as viral persistence by immune escape. Further study of neutralizing antibody epitopes is needed to unravel pathways of resistance to virus neutralization, and to identify conserved regions for vaccine design. All reported broadly neutralizing antibody (BNAb) epitopes in the HCV Envelope (E2) glycoprotein were identified. The critical contact residues of these epitopes were mapped onto the linear E2 sequence. All publicly available E2 sequences were then downloaded and the contact residues within the BNAb epitopes were assessed for the level of conservation, as well as the frequency of occurrence of experimentally-proven resistance mutations. Epitopes were also compared between two sequence datasets obtained from samples collected at well-defined time points from acute (<180days) and chronic (>180days) infections, to identify any significant differences in residue usage. The contact residues for all BNAbs were contained within 3 linear regions of the E2 protein sequence. An analysis of 1749 full length E2 sequences from public databases showed that only 10 out of 29 experimentally-proven resistance mutations were present at a frequency >5%. Comparison of subtype 1a viral sequences obtained from samples collected during acute or chronic infection revealed significant differences at positions 610 and 655 with changes in residue (p<0.05), and at position 422 (p<0.001) with a significant difference in variability (entropy). The majority of experimentally-described escape variants do not occur frequently in nature. The observed differences between acute and chronically isolated sequences suggest constraints on residue usage early in infection.

Incident Hepatitis C Virus Genotype Distribution and Multiple Infection in Australian Prisons.

Hepatitis C virus (HCV) is a highly diverse pathogen that is classified into seven distinct genotypes. Simultaneous or sequential reinfection with multiple HCV genotypes is recognized in high-risk populations, such as injecting drug users (IDUs). Multiple infection is of clinical concern as different genotypes have various sensitivities to current antiviral therapies. Therefore, a better understanding of the frequency of multiple infection and of the genotypes currently being transmitted is clinically relevant. An Australian cohort of IDUs (n = 123), identified with primary incident infection, was followed for multiple infection by regular HCV RNA testing between 2005 and 2013. A total of 354 samples were tested. Sequencing of primary incident infections revealed that genotype 3a was the most common circulating genotype, followed by genotype 1a. Examination of longitudinally collected samples identified complex patterns of multiple infection, including reinfection and superinfection. In those with multiple infection, there was no apparent evidence of homotypic immunity conferring protection against reinfection of the same subtype. This study revealed frequent multiple infection in a high-risk prisoner cohort, illustrating the complex nature of HCV infection and reinfection and highlighting the need for pan-genotypic antiviral therapies.