Getting to the (germinal) center of humoral immune responses to SARS-CoV-2.

Long-term protection against SARS-CoV-2 requires effective and durable immunity. In this issue of Cell, two papers closely examine germinal centers, the physiological birthplace of adaptive immunity, to quantify the specificity, breadth, magnitude, and persistence of systemic and local humoral immune responses following natural infection with, or vaccination against, SARS-CoV-2.

Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

B-cell characteristics define HCV reinfection outcome.

BACKGROUND & AIMS: In individuals highly exposed to 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 (nAbs) and Envelope 2 (E2)-specific memory B cell (MBC) responses were examined longitudinally in 15 individuals with varied reinfection outcomes. RESULTS: Broad nAb responses were associated with MBC recall, but not with clearance of reinfection. 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. IMPACT AND IMPLICATIONS: HCV continues to have a major health burden globally. Limitations in the health infrastructure for diagnosis and treatment, as well as high rates of reinfection, indicate that a vaccine that can protect against chronic HCV infection will greatly complement current efforts to eliminate HCV-related disease. With alternative approaches to testing vaccines, such as controlled human inoculation trials under consideration, we desperately need to identify the correlates of immune protection. In this study, in a small but rare cohort of high-risk injecting drug users who were reinfected multiple times, breadth of neutralisation was not associated with ultimate clearance of the reinfection event. Alternatively, characteristics of the HCV-specific B-cell response associated with B-cell proliferation were. This study indicates that humoral responses are important for protection and suggests that for genetically very diverse viruses, such as HCV, it may be beneficial to look beyond just antibodies as correlates of protection.

Discovery of a monoclonal, high-affinity CD8+ T-cell clone following natural hepatitis C virus infection.

CD8+ T cells recognizing their cognate antigen are typically recruited as a polyclonal population consisting of multiple clonotypes with varying T-cell receptor (TCR) affinity to the target peptide-major histocompatibility complex (pMHC) complex. Advances in single-cell sequencing have increased accessibility toward identifying TCRs with matched antigens. Here we present the discovery of a monoclonal CD8+ T-cell population with specificity for a hepatitis C virus (HCV)-derived human leukocyte antigen (HLA) class I epitope (HLA-B*07:02 GPRLGVRAT) which was isolated directly ex vivo from an individual with an episode of acutely resolved HCV infection. This population was absent before infection and underwent expansion and stable maintenance for at least 2 years after infection as measured by HLA-multimer staining. Furthermore, the monoclonal clonotype was characterized by an unusually long dissociation time (half-life = 794 s and koff = 5.73 × 10-4) for its target antigen when compared with previously published results. A comparison with related populations of HCV-specific populations derived from the same individual and a second individual suggested that high-affinity TCR-pMHC interactions may be inherent to epitope identity and shape the phenotype of responses which has implications for rational TCR selection and design in the age of personalized immunotherapies.

Characterization of antibody-dependent cellular phagocytosis in patients infected with hepatitis C virus with different clinical outcomes.

Early neutralizing antibodies against hepatitis C virus (HCV) and CD8 + T cell effector responses can lead to viral clearance. However, these functions alone are not sufficient to protect patients against HCV infection, thus undefined additional antiviral immune mechanisms are required. In recent years, Fc-receptor-dependent antibody effector functions, particularly, antibody-dependent cellular phagocytosis (ADCP) were shown to offer immune protection against several RNA viruses. However, its development and clinical role in patients with HCV infection remain unknown. In this study, we found that patients with chronic GT1a or GT3a HCV infection had significantly higher concentrations of anti-envelope 2 (E2) antibodies, predominantly IgG1 subclass, than patients that cleared the viruses while the latter had antibodies with higher affinities. 97% of the patients with HCV had measurable ADCP of whom patients with chronic disease showed significantly higher ADCP than those who naturally cleared the virus. Epitope mapping studies showed that patients with antibodies that target antigenic domains on the HCV E2 protein that are known to associate with neutralization function are also strongly associated with ADCP, suggesting antibodies with overlapping/dual functions. Correlation studies showed that ADCP significantly correlated with plasma anti-E2 antibody levels and neutralization function regardless of clinical outcome and genotype of infecting virus, while a significant correlation between ADCP and affinity was only evident in patients that cleared the virus. These results suggest ADCP was mostly driven by antibody titer in patients with chronic disease while maintained in clearers due to the quality (affinity) of their anti-E2 antibodies despite having lower antibody titers.

Longitudinal Characterization of Phagocytic and Neutralization Functions of Anti-Spike Antibodies in Plasma of Patients after Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Phagocytic responses by effector cells to opsonized viruses have been recognized to play a key role in antiviral immunity. Limited data on coronavirus disease 2019 suggest that the role of Ab-dependent and -independent phagocytosis may contribute to the observed immunological and inflammatory responses; however, their development, duration, and role remain to be fully elucidated. In this study of 62 acute and convalescent patients, we found that patients with acute coronavirus disease 2019 can mount a phagocytic response to autologous plasma-opsonized Spike protein-coated microbeads as early as 10 d after symptom onset, while heat inactivation of this plasma caused 77-95% abrogation of the phagocytic response and preblocking of Fc receptors showed variable 18-60% inhibition. In convalescent patients, phagocytic response significantly correlated with anti-Spike IgG titers and older patients, while patients with severe disease had significantly higher phagocytosis and neutralization functions compared with patients with asymptomatic, mild, or moderate disease. A longitudinal subset of the convalescent patients over 12 mo showed an increase in plasma Ab affinity toward Spike Ag and preservation of phagocytic and neutralization functions, despite a decline in the anti-Spike IgG titers by >90%. Our data suggest that early phagocytosis is primarily driven by heat-liable components of the plasma, such as activated complements, while anti-Spike IgG titers account for the majority of observed phagocytosis at convalescence. Longitudinally, a significant increase in the affinity of the anti-Spike Abs was observed that correlated with the maintenance of both the phagocytic and neutralization functions, suggesting an improvement in the quality of the Abs.

Total recall? Understanding the effect of antigenic distance on original antigenic sin.

Vaccination-induced antibodies are critical for protective immunity against pathogenic threats. "Original antigenic sin" (OAS), also referred to as imprinting, is the observed phenomenon whereby exposure to antigenic stimuli bias future antibody responses. This Commentary describes a recently elegant model published in Nature by Schiepers et al. which allows us to delve deeper into the processes and mechanisms of OAS than ever before.

High activation levels maintained in receptor-binding domain-specific memory B cells in people with severe coronavirus disease 2019.

The long-term health consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are still being understood. The molecular and phenotypic properties of SARS-CoV-2 antigen-specific T cells suggest a dysfunctional profile that persists in convalescence in those who were severely ill. By contrast, the antigen-specific memory B-cell (MBC) population has not yet been analyzed to the same degree, but phenotypic analysis suggests differences following recovery from mild or severe coronavirus disease 2019 (COVID-19). Here, we performed single-cell molecular analysis of the SARS-CoV-2 receptor-binding domain (RBD)-specific MBC population in three patients after severe COVID-19 and four patients after mild/moderate COVID-19. We analyzed the transcriptomic and B-cell receptor repertoire profiles at ~2 months and ~4 months after symptom onset. Transcriptomic analysis revealed a higher level of tumor necrosis factor-alpha (TNF-α) signaling via nuclear factor-kappa B in the severe group, involving CD80, FOS, CD83 and TNFAIP3 genes that was maintained over time. We demonstrated the presence of two distinct activated MBCs subsets based on expression of CD80hi TNFAIP3hi and CD11chi CD95hi at the transcriptome level. Both groups revealed an increase in somatic hypermutation over time, indicating progressive evolution of humoral memory. This study revealed distinct molecular signatures of long-term RBD-specific MBCs in convalescence, indicating that the longevity of these cells may differ depending on acute COVID-19 severity.

Evolution of the SARS-CoV-2 omicron variants BA.1 to BA.5: Implications for immune escape and transmission.

The first dominant SARS-CoV-2 Omicron variant BA.1 harbours 35 mutations in its Spike protein from the original SARS-CoV-2 variant that emerged late 2019. Soon after its discovery, BA.1 rapidly emerged to become the dominant variant worldwide and has since evolved into several variants. Omicron is of major public health concern owing to its high infectivity and antibody evasion. This review article examines the theories that have been proposed on the evolution of Omicron including zoonotic spillage, infection in immunocompromised individuals and cryptic spread in the community without being diagnosed. Added to the complexity of Omicron's evolution are the multiple reports of recombination events occurring between co-circulating variants of Omicron with Delta and other variants such as XE. Current literature suggests that the combination of the novel mutations in Omicron has resulted in the variant having higher infectivity than the original Wuhan-Hu-1 and Delta variant. However, severity is believed to be less owing to the reduced syncytia formation and lower multiplication in the human lung tissue. Perhaps most challenging is that several studies indicate that the efficacy of the available vaccines have been reduced against Omicron variant (8-127 times reduction) as compared to the Wuhan-Hu-1 variant. The administration of booster vaccine, however, compensates with the reduction and improves the efficacy by 12-35 fold. Concerningly though, the broadly neutralising monoclonal antibodies, including those approved by FDA for therapeutic use against previous SARS-CoV-2 variants, are mostly ineffective against Omicron with the exception of Sotrovimab and recent reports suggest that the Omicron BA.2 is also resistant to Sotrovimab. Currently two new Omicron variants BA.4 and BA.5 are emerging and are reported to be more transmissible and resistant to immunity generated by previous variants including Omicron BA.1 and most monoclonal antibodies. As new variants of SARS-CoV-2 will likely continue to emerge it is important that the evolution, and biological consequences of new mutations, in existing variants be well understood.

Human CD8 T-stem cell memory subsets phenotypic and functional characterization are defined by expression of CD122 or CXCR3.

Long-lived T-memory stem cells (TSCM ) are key to both naturally occurring and vaccine-conferred protection against infection. These cells are characterized by the CD45RA+ CCR7+ CD95+ phenotype. Significant heterogeneity within the TSCM population is recognized, but distinguishing surface markers and functional characterization of potential subsets are lacking. Human CD8 TSCM subsets were identified in healthy subjects who had been previously exposed to CMV or Influenza (Flu) virus in flow cytometry by expression of CD122 or CXCR3, and then characterized in proliferation, multipotency, self-renewal, and intracellular cytokine production (TNF-α, IL-2, IFN-γ), together with transcriptomic profiles. The TSCM CD122hi -expressing subset (versus CD122lo ) demonstrated greater proliferation, greater multipotency, and enhanced polyfunctionality with higher frequencies of triple positive (TNF-α, IL-2, IFN-γ) cytokine-producing cells upon exposure to recall antigen. The TSCM CXCR3lo subpopulation also had increased proliferation and polyfunctional cytokine production. Transcriptomic analysis further showed that the TSCM CD122hi population had increased expression of activation and homing molecules, such as Ccr6, Cxcr6, Il12rb, and Il18rap, and downregulated cell proliferation inhibitors, S100A8 and S100A9. These data reveal that the TSCM CD122hi phenotype is associated with increased proliferation, enhanced multipotency and polyfunctionality with an activated memory-cell like transcriptional profile, and hence, may be favored for induction by immunization and for adoptive immunotherapy.

Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins.

Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293TCD81KO). Clones made in 293TCD81KO cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T cells and Freestyle 293-F (293-F) cells exhibit important differences. We found that 293-F-produced sE2 harbours mostly complex-type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex-type glycans and high-mannose or hybrid-type glycans. Moreover, sE2 produced in 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293-F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.

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.

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.

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.

Chemokine-Regulated Recruitment of Antigen-Specific T-Cell Subpopulations to the Liver in Acute and Chronic Hepatitis C Infection.

BACKGROUND: In hepatitis C virus (HCV) infection, virus-specific CD8+ T cells are recruited to the liver for antiviral activity. Multiple chemokine ligands are induced by the infection, notably interferon-inducible chemokine, CXCL10. In HCV, intrahepatic T cells express chemokine receptors (CCRs), including CXCR3, CXCR6, CCR1, and CCR5, but CCR expression on antigen-specific effector and memory T cells has not been investigated. METHODS: Paired blood and liver samples were collected from subjects with chronic HCV for flow cytometric analysis of CCR expression on CD8+ T cells. Expression of these CCRs was then examined on HCV-specific CD8+ T-cell subpopulations in the blood from subjects with acute or chronic HCV. RESULTS: Relative to peripheral blood, the liver was enriched with CD8+ T cells expressing CCR2, CCR5, CXCR3, and CXCR6 either singly or in combinations. CXCR3 was preferentially expressed on HCV-specific CD8+ T cells in both acute and chronic phases of infection in blood. Both CXCR3 and CCR2 were overexpressed on HCV-specific CD8+CCR7+CD45RO+ (central memory) cells, whereas effector memory (CD8+CCR7-CD45RO+) cells expressed more CXCR6. CONCLUSIONS: CXCR3-mediated signals support the accumulation of HCV-specific CD8+ memory T cells in the infected liver, and emphasize the importance of the CXCL10/CXCR3 trafficking pathway during acute and chronic HCV infection.

Genomic variability of within-host hepatitis C variants in acute infection.

Interactions between the host immune system and the viral variants determine persistence of hepatitis C virus (HCV) infection after the acute phase of infection. This study describes the genetic variability of within-host HCV viral variants in acute infection and correlates it with host- and virus-related traits and infection outcome. Next generation sequence data (Illumina, MiSeq platform) of viral genomes from 116 incident acute infections (within 180 days of infection) were analysed to determine all the single nucleotide polymorphism (SNP) frequencies above a threshold of 0.1%. The variability of the SNPs for the full open reading frame of the genome as well as for each protein coding region were compared using mean standardized Shannon entropy (SE) values calculated separately for synonymous and nonsynonymous mutations. The envelope glycoproteins regions (E1 and E2) had the highest SE values (indicating greater variability) followed by the NS5B region. Nonsynonymous mutations rather than synonymous mutations were the main contributors to genomic variability in acute infection. The mean difference of Shannon entropy was also compared between subjects after categorizing the samples according to host and virus-related traits. Host IFNL3 allele CC polymorphism at rs12979860 (vs others) and viral genotype 1a (vs 3a) were associated with higher genomic variability across the viral open reading frame. Time since infection, host gender or continent of origin was not associated with the viral genomic variability. Viral genomic variability did not predict spontaneous clearance.

Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses.

Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3' untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and ß-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as "vesicle packets" (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools.

A molecular transmission network of recent hepatitis C infection in people with and without HIV: Implications for targeted treatment strategies.

Combining phylogenetic and network methodologies has the potential to better inform targeted interventions to prevent and treat infectious diseases. This study reconstructed a molecular transmission network for people with recent hepatitis C virus (HCV) infection and modelled the impact of targeting directly acting antiviral (DAA) treatment for HCV in the network. Participants were selected from three Australian studies of recent HCV from 2004 to 2014. HCV sequence data (Core-E2) from participants at the time of recent HCV detection were analysed to infer a network by connecting pairs of sequences whose divergence was ≤.03 substitutions/site. Logistic regression was used to identify factors associated with connectivity. Impact of targeting HCV DAAs at both HIV co-infected and random nodes was simulated (1 million replicates). Among 236 participants, 21% (n=49) were connected in the network. HCV/HIV co-infected participants (47%) were more likely to be connected compared to HCV mono-infected participants (16%) (OR 4.56; 95% CI; 2.13-9.74). Simulations targeting DAA HCV treatment to HCV/HIV co-infected individuals prevented 2.5 times more onward infections than providing DAAs to randomly selected individuals. Results demonstrate that genetic distance-based network analyses can be used to identify characteristics associated with HCV transmission, informing targeted prevention and treatment strategies.