Geographic patterns of koala retrovirus genetic diversity, endogenization, and subtype distributions.

Koala retrovirus (KoRV) subtype A (KoRV-A) is currently in transition from exogenous virus to endogenous viral element, providing an ideal system to elucidate retroviral-host coevolution. We characterized KoRV geography using fecal DNA from 192 samples across 20 populations throughout the koala's range. We reveal an abrupt change in KoRV genetics and incidence at the Victoria/New South Wales state border. In northern koalas, pol gene copies were ubiquitously present at above five per cell, consistent with endogenous KoRV. In southern koalas, pol copies were detected in only 25.8% of koalas and always at copy numbers below one, while the env gene was detected in all animals and in a majority at copy numbers above one per cell. These results suggest that southern koalas carry partial endogenous KoRV-like sequences. Deep sequencing of the env hypervariable region revealed three putatively endogenous KoRV-A sequences in northern koalas and a single, distinct sequence present in all southern koalas. Among northern populations, env sequence diversity decreased with distance from the equator, suggesting infectious KoRV-A invaded the koala genome in northern Australia and then spread south. The exogenous KoRV subtypes (B to K), two novel subtypes, and intermediate subtypes were detected in all northern koala populations but were strikingly absent from all southern animals tested. Apart from KoRV subtype D, these exogenous subtypes were generally locally prevalent but geographically restricted, producing KoRV genetic differentiation among northern populations. This suggests that sporadic evolution and local transmission of the exogenous subtypes have occurred within northern Australia, but this has not extended into animals within southern Australia.

SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein.

Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson's disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation. Using SARS-CoV-2 infection of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) as a COVID-19 pre-clinical model, we established the presence of virus in the brain together with microglial activation and NLRP3 inflammasome upregulation in comparison to uninfected mice. Next, utilising a model of human monocyte-derived microglia, we identified that SARS-CoV-2 isolates can bind and enter human microglia in the absence of viral replication. This interaction of virus and microglia directly induced robust inflammasome activation, even in the absence of another priming signal. Mechanistically, we demonstrated that purified SARS-CoV-2 spike glycoprotein activated the NLRP3 inflammasome in LPS-primed microglia, in a ACE2-dependent manner. Spike protein also could prime the inflammasome in microglia through NF-κB signalling, allowing for activation through either ATP, nigericin or α-synuclein. Notably, SARS-CoV-2 and spike protein-mediated microglial inflammasome activation was significantly enhanced in the presence of α-synuclein fibrils and was entirely ablated by NLRP3-inhibition. Finally, we demonstrate SARS-CoV-2 infected hACE2 mice treated orally post-infection with the NLRP3 inhibitory drug MCC950, have significantly reduced microglial inflammasome activation, and increased survival in comparison with untreated SARS-CoV-2 infected mice. These results support a possible mechanism of microglial innate immune activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson's disease in COVID-19 infected individuals, and a potential therapeutic avenue for intervention.

HTLV-1 reverse transcriptase homology model provides structural basis for sensitivity to existing nucleoside/nucleotide reverse transcriptase inhibitors.

The human T-lymphotropic virus type 1 (HTLV-1) infects millions of people globally and is endemic to various resource-limited regions. Infections persist for life and are associated with increased susceptibility to opportunistic infections and severe diseases including adult T cell leukemia/lymphoma and HTLV-1-associated myelopathy-tropical spastic paraparesis. No HTLV-1-specific anti-retrovirals have been developed and it is unclear whether existing anti-retrovirals developed for treatment of human immunodeficiency virus (HIV) have efficacy against HTLV-1. To understand the structural basis for therapeutic binding, homology modelling and machine learning were used to develop a structural model of the HTLV-1 reverse transcriptase. With this, molecular docking experiments using a panel of FDA-approved inhibitors of viral reverse transcriptases to assess their capacity for binding, and in turn, inhibition. Importantly, nucleoside/nucleotide reverse transcriptase inhibitor but not non-nucleoside reverse transcriptase inhibitors were predicted to bind the HTLV-1 reverse transcriptase, with similar affinity to HIV-1 reverse transcriptase. By strengthening the rationale for clinical testing of therapies such as tenofovir alafenamide, zidovudine, lamivudine, and azvudine for treatment of HTLV-1, this study has demonstrated the power of in silico structural biology approaches in drug design and therapeutic testing.

Koala retrovirus genetic diversity and transmission dynamics within captive koala populations.

Koala populations are currently in rapid decline across Australia, with infectious diseases being a contributing cause. The koala retrovirus (KoRV) is a gammaretrovirus present in both captive and wild koala colonies that presents an additional challenge for koala conservation in addition to habitat loss, climate change, and other factors. Currently, nine different subtypes (A to I) have been identified; however, KoRV genetic diversity analyses have been limited. KoRV is thought to be exogenously transmitted between individuals, with KoRV-A also being endogenous and transmitted through the germline. The mechanisms of exogenous KoRV transmission are yet to be extensively investigated. Here, deep sequencing was employed on 109 captive koalas of known pedigree, housed in two institutions from Southeast Queensland, to provide a detailed analysis of KoRV transmission dynamics and genetic diversity. The final dataset included 421 unique KoRV sequences, along with the finding of an additional subtype (KoRV-K). Our analysis suggests that exogenous transmission of KoRV occurs primarily between dam and joey, with evidence provided for multiple subtypes, including nonendogenized KoRV-A. No evidence of sexual transmission was observed, with mating partners found to share a similar number of sequences as unrelated koala pairs. Importantly, both distinct captive colonies showed similar trends. These findings indicate that breeding strategies or antiretroviral treatment of females could be employed as effective management approaches in combating KoRV transmission.

Micro-fusion inhibition tests: quantifying antibody neutralization of virus-mediated cell-cell fusion.

Although enveloped viruses canonically mediate particle entry through virus-cell fusion, certain viruses can spread by cell-cell fusion, brought about by receptor engagement and triggering of membrane-bound, viral-encoded fusion proteins on the surface of cells. The formation of pathogenic syncytia or multinucleated cells is seen in vivo, but their contribution to viral pathogenesis is poorly understood. For the negative-strand paramyxoviruses respiratory syncytial virus (RSV) and Nipah virus (NiV), cell-cell spread is highly efficient because their oligomeric fusion protein complexes are active at neutral pH. The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also been reported to induce syncytia formation in infected cells, with the spike protein initiating cell-cell fusion. Whilst it is well established that fusion protein-specific antibodies can block particle attachment and/or entry into the cell (canonical virus neutralization), their capacity to inhibit cell-cell fusion and the consequences of this neutralization for the control of infection are not well characterized, in part because of the lack of specific tools to assay and quantify this activity. Using an adapted bimolecular fluorescence complementation assay, based on a split GFP-Renilla luciferase reporter, we have established a micro-fusion inhibition test (mFIT) that allows the identification and quantification of these neutralizing antibodies. This assay has been optimized for high-throughput use and its applicability has been demonstrated by screening monoclonal antibody (mAb)-mediated inhibition of RSV and NiV fusion and, separately, the development of fusion-inhibitory antibodies following NiV vaccine immunization in pigs. In light of the recent emergence of coronavirus disease 2019 (COVID-19), a similar assay was developed for SARS-CoV-2 and used to screen mAbs and convalescent patient plasma for fusion-inhibitory antibodies. Using mFITs to assess antibody responses following natural infection or vaccination is favourable, as this assay can be performed entirely at low biocontainment, without the need for live virus. In addition, the repertoire of antibodies that inhibit cell-cell fusion may be different to those that inhibit particle entry, shedding light on the mechanisms underpinning antibody-mediated neutralization of viral spread.

Epidemiology of respiratory syncytial virus in a community birth cohort of infants in the first 2 years of life.

Respiratory syncytial virus (RSV) is the most common virus identified in children hospitalised with acute respiratory infections. However, less is known about RSV in community settings. This report describes RSV epidemiology in the community, including acute illness episodes, healthcare burden, and risk factors in Australian children during the first 2-years of life. A community-based, birth cohort from Brisbane, Australia, followed children until their second birthday. Parents completed daily respiratory symptom and illness-burden diaries. Weekly parent-collected nasal swabs were analysed for RSV by real-time polymerase chain reaction assays. Serum RSV-neutralising antibodies were assayed at age 3 years. Overall, 158 children provided 11,216 swabs, of which 104 were RSV-positive (85 incident episodes). RSV incidence in the first 2 years of life was 0.46 (95% CI = 0.37-0.58) episodes per child-year. Incidence increased with age and formal childcare attendance and was highest in autumn. Of 82 episodes linked with symptom data, 60 (73.2%) were symptomatic, 28 (34.1%) received community-based medical care, and 2 (2.4%) led to hospitalisation. Viral load was higher in symptomatic than asymptomatic infections. In 72 children, RSV-specific antibody seroprevalence was 94.4% at age 3 years.Conclusion: RSV incidence increased after age 6-months with approximately three-quarters of infections symptomatic and most infections treated in the community. What is known •RSV is a major cause of hospitalisation for acute lower respiratory infections in infants and young children, especially in the first 6 months of life. •However, limited data exist on the overall burden in young children at the community level. What is new •RSV incidence in the community increases after age 6 months, and by 3 years, most children have been infected. •About one-quarter of RSV infections were asymptomatic in children aged < 2 years, and approximately 60% of children with RSV-related symptoms had a healthcare contact of any kind with most managed within the community.

Analysis of phylogenetic diversity and in vitro adherence characteristics of respiratory syncytial virus and Streptococcus pneumoniae clinical isolates obtained during pediatric respiratory co-infections.

Respiratory syncytial virus (RSV) and Streptococcus pneumoniae are frequently co-associated during acute respiratory infections, particularly amongst infants and young children. In this study, we aimed to identify strains of RSV and serotypes/sequence types of S. pneumoniae associated with co-infections within a cohort of paediatric patients, and to assess RSV-mediated adhesion of pneumococcal isolates. The RSV glycoprotein sequence was determined for 58 RSV-positive samples and molecular serotyping and MLST was used to analyse 26 pneumococcal isolates. We also compared 23 pneumococcal isolates for their adherence to RSV-infected or mock-infected airway epithelia cells using immunofluorescence microscopy and automated particle counting. The tight association between RSV and S. pneumoniae was also visualized using scanning electron microscopy. This study did not identify any statistically significant trend in the strains of RSV and S. pneumoniae associated with co-infections. Furthermore, almost all isolates (22 of 23) showed significantly increased adherence to RSV-infected cells. The level of adherence did not appear to correlate with pneumococcal strain or sequence type, and isolates obtained from RSV-infected patients displayed a similar level of adherence as those from RSV-negative patients. The absence of particular S. pneumoniae or RSV strains associated with co-infection, together with the near ubiquitous presence of RSV-mediated adhesion throughout the pneumococcal clinical isolates, may indicate that the mechanisms governing the association with RSV are of sufficient importance to be maintained across much of the species.

The Heptad Repeat C Domain of the Respiratory Syncytial Virus Fusion Protein Plays a Key Role in Membrane Fusion.

Respiratory syncytial virus (RSV) mediates host cell entry through the fusion (F) protein, which undergoes a conformational change to facilitate the merger of viral and host lipid membrane envelopes. The RSV F protein comprises a trimer of disulfide-bonded F1 and F2 subunits that is present on the virion surface in a metastable prefusion state. This prefusion form is readily triggered to undergo refolding to bring two heptad repeats (heptad repeat A [HRA] and HRB) into close proximity to form a six-helix bundle that stabilizes the postfusion form and provides the free energy required for membrane fusion. This process can be triggered independently of other proteins. Here, we have performed a comprehensive analysis of a third heptad repeat region, HRC (amino acids 75 to 97), an amphipathic α-helix that lies at the interface of the prefusion F trimer and is a major structural feature of the F2 subunit. We performed alanine scanning mutagenesis from Lys-75 to Met-97 and assessed all mutations in transient cell culture for expression, proteolytic processing, cell surface localization, protein conformation, and membrane fusion. Functional characterization revealed a striking distribution of activity in which fusion-increasing mutations localized to one side of the helical face, while fusion-decreasing mutations clustered on the opposing face. Here, we propose a model in which HRC plays a stabilizing role within the globular head for the prefusion F trimer and is potentially involved in the early events of triggering, prompting fusion peptide release and transition into the postfusion state.IMPORTANCE RSV is recognized as the most important viral pathogen among pediatric populations worldwide, yet no vaccine or widely available therapeutic treatment is available. The F protein is critical for the viral replication process and is the major target for neutralizing antibodies. Recent years have seen the development of prefusion stabilized F protein-based approaches to vaccine design. A detailed understanding of the specific domains and residues that contribute to protein stability and fusion function is fundamental to such efforts. Here, we present a comprehensive mutagenesis-based study of a region of the RSV F2 subunit (amino acids 75 to 97), referred to as HRC, and propose a role for this helical region in maintaining the delicate stability of the prefusion form.

Streptococcus pneumoniae colonization of the nasopharynx is associated with increased severity during respiratory syncytial virus infection in young children.

BACKGROUND AND OBJECTIVE: Respiratory syncytial virus (RSV) is the most significant cause of acute respiratory infection (ARI) in early life. RSV and other respiratory viruses are known to stimulate substantial outgrowth of potentially pathogenic bacteria in the upper airways of young children. However, the clinical significance of interactions between viruses and bacteria is currently unclear. The present study aimed to clarify the effect of viral and bacterial co-detections on disease severity during paediatric ARI. METHODS: Nasopharyngeal aspirates from children under 2 years of age presenting with ARI to the emergency department were screened by quantitative PCR for 17 respiratory viruses and the bacterial pathogens Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. Associations between pathogen detection and clinical measures of disease severity were investigated. RESULTS: RSV was the most common virus detected, present in 29 of 58 samples from children with ARI (50%). Detection of S. pneumoniae was significantly more frequent during RSV infections compared to other respiratory viruses (adjusted effect size: 1.8, P: 0.03), and co-detection of both pathogens was associated with higher clinical disease severity scores (adjusted effect size: 1.2, P: 0.03). CONCLUSION: Co-detection of RSV and S. pneumoniae in the nasopharynx was associated with more severe ARI, suggesting that S. pneumoniae colonization plays a pathogenic role in young children.

Proteoform-Specific Insights into Cellular Proteome Regulation.

Knowledge regarding compositions of proteomes at the proteoform level enhances insights into cellular phenotypes. A strategy is described herein for discovery of proteoform-specific information about cellular proteomes. This strategy involved analysis of data obtained by bottom-up mass spectrometry of multiple protein OGE separations on a fraction by fraction basis. The strategy was exemplified using five matched sets of lysates of uninfected and human respiratory syncytial virus-infected A549 cells. Template matching demonstrated that 67.3% of 10475 protein profiles identified focused to narrow pI windows indicative of efficacious focusing. Furthermore, correlation between experimental and theoretical pI gradients indicated reproducible focusing. Based on these observations a proteoform profiling strategy was developed to identify proteoforms, detect proteoform diversity and discover potential proteoform regulation. One component of this strategy involved examination of the focusing profiles for protein groups. A novel concordance analysis facilitated differentiation between proteoforms, including proteoforms generated by alternate splicing and proteolysis. Evaluation of focusing profiles and concordance analysis were applicable to cells from a single and/or multiple biological states. Statistical analyses identified proteoform variation between biological states. Regulation relevant to cellular responses to human respiratory syncytial virus was revealed. Western blotting and Protomap analyses validated the proteoform regulation. Discovery of STAT1, WARS, MX1, and HSPB1 proteoform regulation by human respiratory syncytial virus highlighted the impact of the profiling strategy. Novel truncated proteoforms of MX1 were identified in infected cells and phosphorylation driven regulation of HSPB1 proteoforms was correlated with infection. The proteoform profiling strategy is generally applicable to investigating interactions between viruses and host cells and the analysis of other biological systems.

Human T-lymphotropic virus type 1 and antiretroviral therapy: practical considerations for pre-exposure and post-exposure prophylaxis, transmission prevention, and mitigation of severe disease.

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus associated with substantial risk of secondary (often life-threatening) disease for the estimated 10 million to 20 million people infected globally. Despite a clear need, no HTLV-1-specific vaccine or antiretroviral therapy has been developed to date. Instead, existing public and primary health-care interventions inadequately focus on infection prevention and management of secondary diseases. In this Personal View, we discuss the evidence that exists to support the sensitivity of HTLV-1 to antiretroviral therapies approved by the US Food and Drug Administration for the treatment of HIV-1, how this sensitivity is affected by clinically relevant virological and immunological features, and additional practical considerations for the use of antiretroviral therapies in the context of HTLV-1.

Integrated molecular and immunological features of human T-lymphotropic virus type 1 infection and disease progression to adult T-cell leukaemia or lymphoma.

The human T-lymphotropic virus type 1 (HTLV-1) retrovirus infects 10-20 million people globally, with endemic regions in southwestern Japan, the Caribbean basin, Africa, and central Australia. HTLV-1 is associated with lifelong infection and immune suppression, resulting in a range of serious sequalae, including adult T-cell leukaemia or lymphoma (ATLL) in 5% of cases. To date, there are no preventive or curative treatments for HTLV-1 and treatment outcomes for ATLL remain generally poor. Depending on the disease subtype, overall survival is 8-55 months. Recent advancements in the past decade have identified genetic, molecular, and immunological events occurring throughout the lives of individuals infected with HTLV-1 and of those who progress to ATLL. In addition, updated guidelines for clinical management have been published. With the aim of focusing research efforts on the development of treatments for both HTLV-1 infections and ATLL, we have conceptualised a four-step disease model for HTLV-1-associated ATLL: (1) viral exposure, (2) establishment of chronic infection, (3) cellular transformation and evolution, and (4) disease presentation and management. For each stage we describe the clinical features, molecular and immunological factors involved, potential biomarkers of disease progression, and the therapeutic applicability of individual targets. We also discuss emerging concepts and novel treatment approaches. Our hope is that this model will promote research interest and guide the testing of new treatments for this neglected virus and its associated rare cancer.

Structure and antigenicity of divergent Henipavirus fusion glycoproteins.

In August 2022, a novel henipavirus (HNV) named Langya virus (LayV) was isolated from patients with severe pneumonic disease in China. This virus is closely related to Mòjiang virus (MojV), and both are divergent from the bat-borne HNV members, Nipah (NiV) and Hendra (HeV) viruses. The spillover of LayV is the first instance of a HNV zoonosis to humans outside of NiV and HeV, highlighting the continuing threat this genus poses to human health. In this work, we determine the prefusion structures of MojV and LayV F proteins via cryogenic electron microscopy to 2.66 and 3.37 Å, respectively. We show that despite sequence divergence from NiV, the F proteins adopt an overall similar structure but are antigenically distinct as they do not react to known antibodies or sera. Glycoproteomic analysis revealed that while LayV F is less glycosylated than NiV F, it contains a glycan that shields a site of vulnerability previously identified for NiV. These findings explain the distinct antigenic profile of LayV and MojV F, despite the extent to which they are otherwise structurally similar to NiV. Our results carry implications for broad-spectrum HNV vaccines and therapeutics, and indicate an antigenic, yet not structural, divergence from prototypical HNVs.

Long-term safety and immunogenicity of an MF59-adjuvanted spike glycoprotein-clamp vaccine for SARS-CoV-2 in adults aged 18-55 years or ≥56 years: 12-month results from a randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND: We previously demonstrated the safety and immunogenicity of an MF59-adjuvanted COVID-19 vaccine based on the SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a molecular clamp using HIV-1 glycoprotein 41 sequences. Here, we describe 12-month results in adults aged 18-55 years and ≥56 years. METHODS: Phase 1, double-blind, placebo-controlled trial conducted in Australia (July 2020-December 2021; ClinicalTrials.govNCT04495933; active, not recruiting). Healthy adults (Part 1: 18-55 years; Part 2: ≥56 years) received two doses of placebo, 5 µg, 15 µg, or 45 µg vaccine, or one 45 µg dose of vaccine followed by placebo (Part 1 only), 28 days apart (n = 216; 24 per group). Safety, humoral immunogenicity (including against virus variants), and cellular immunogenicity were assessed to day 394 (12 months after second dose). Effects of subsequent COVID-19 vaccination on humoral responses were examined. FINDINGS: All two-dose vaccine regimens were well tolerated and elicited strong antigen-specific and neutralising humoral responses, and CD4+ T-cell responses, by day 43 in younger and older adults, although cellular responses were lower in older adults. Humoral responses waned by day 209 but were boosted in those receiving authorised vaccines. Neutralising activity against Delta and Omicron variants was present but lower than against the Wuhan strain. Cross-reactivity in HIV diagnostic tests declined over time but remained detectable in most participants. INTERPRETATION: The SARS-CoV-2 molecular clamp vaccine is well tolerated and evokes robust immune responses in adults of all ages. Although the HIV glycoprotein 41-based molecular clamp is not being progressed, the clamp concept represents a viable platform for vaccine development. FUNDING: This study was funded by the Coalition for Epidemic Preparedness Innovations, the National Health and Medical Research Council of Australia, and the Queensland Government.

Peptide-Based Vaccine against SARS-CoV-2: Peptide Antigen Discovery and Screening of Adjuvant Systems.

The SARS-CoV-2 virus has caused a global crisis, resulting in 0.5 billion infections and over 6 million deaths as of March 2022. Fortunately, infection and hospitalization rates were curbed due to the rollout of DNA and mRNA vaccines. However, the efficacy of these vaccines significantly drops a few months post immunization, from 88% down to 47% in the case of the Pfizer BNT162 vaccine. The emergence of variant strains, especially delta and omicron, have also significantly reduced vaccine efficacy. We propose peptide vaccines as a potential solution to address the inadequacies of the current vaccines. Peptide vaccines can be easily modified to target emerging strains, have greater stability, and do not require cold-chain storage. We screened five peptide fragments (B1-B5) derived from the SARS-CoV-2 spike protein to identify neutralizing B-cell peptide antigens. We then investigated adjuvant systems for efficient stimulation of immune responses against the most promising peptide antigens, including liposomal formulations of polyleucine (L10) and polymethylacrylate (PMA), as well as classical adjuvants (CFA and MF59). Immune efficacy of formulations was evaluated using competitive ELISA, pseudovirion neutralization, and live virus neutralization assays. Unfortunately, peptide conjugation to L10 and PMA dramatically altered the secondary structure, resulting in low antibody neutralization efficacy. Of the peptides tested, only B3 administered with CFA or MF59 was highly immunogenic. Thus, a peptide vaccine relying on B3 may provide an attractive alternative to currently marketed vaccines.

Diversity and transmission of koala retrovirus: a case study in three captive koala populations.

Koala retrovirus is a recently endogenized retrovirus associated with the onset of neoplasia and infectious disease in koalas. There are currently twelve described KoRV subtypes (KoRV-A to I, K-M), most of which were identified through recently implemented deep sequencing methods which reveal an animals' overall KoRV profile. This approach has primarily been carried out on wild koala populations around Australia, with few investigations into the whole-population KoRV profile of captive koala colonies to date. This study conducted deep sequencing on 64 captive koalas of known pedigree, housed in three institutions from New South Wales and South-East Queensland, to provide a detailed analysis of KoRV genetic diversity and transmission. The final dataset included 93 unique KoRV sequences and the first detection of KoRV-E within Australian koala populations. Our analysis suggests that exogenous transmission of KoRV-A, B, D, I and K primarily occurs between dam and joey. Detection of KoRV-D in a neonate sample raises the possibility of this transmission occurring in utero. Overall, the prevalence and abundance of KoRV subtypes was found to vary considerably between captive populations, likely due to their different histories of animal acquisition. Together these findings highlight the importance of KoRV profiling for captive koalas, in particular females, who play a primary role in KoRV exogenous transmission.

Characterization and comparison of novel adjuvants for a prefusion clamped MERS vaccine.

Various chemical adjuvants are available to augment immune responses to non-replicative, subunit vaccines. Optimized adjuvant selection can ensure that vaccine-induced immune responses protect against the diversity of pathogen-associated infection routes, mechanisms of infectious spread, and pathways of immune evasion. In this study, we compare the immune response of mice to a subunit vaccine of Middle Eastern respiratory syndrome coronavirus (MERS-CoV) spike protein, stabilized in its prefusion conformation by a proprietary molecular clamp (MERS SClamp) alone or formulated with one of six adjuvants: either (i) aluminium hydroxide, (ii) SWE, a squalene-in-water emulsion, (iii) SQ, a squalene-in-water emulsion containing QS21 saponin, (iv) SMQ, a squalene-in-water emulsion containing QS21 and a synthetic toll-like receptor 4 (TLR4) agonist 3D-6-acyl Phosphorylated HexaAcyl Disaccharide (3D6AP); (v) LQ, neutral liposomes containing cholesterol, 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and QS21, (vi) or LMQ, neutral liposomes containing cholesterol, DOPC, QS21, and 3D6AP. All adjuvanted formulations induced elevated antibody titers which where greatest for QS21-containing formulations. These had elevated neutralization capacity and induced higher frequencies of IFNƔ and IL-2-producing CD4+ and CD8+ T cells. Additionally, LMQ-containing formulations skewed the antibody response towards IgG2b/c isotypes, allowing for antibody-dependent cellular cytotoxicity. This study highlights the utility of side-by-side adjuvant comparisons in vaccine development.

Nucleocapsid Specific Diagnostics for the Detection of Divergent SARS-CoV-2 Variants.

Since the start of the COVID-19 pandemic, multiple waves of SARS-CoV-2 variants have emerged. Of particular concern is the omicron variant, which harbors 28 mutations in the spike glycoprotein receptor binding and N-terminal domains relative to the ancestral strain. The high mutability of SARS-CoV-2 therefore poses significant hurdles for development of universal assays that rely on spike-specific immune detection. To address this, more conserved viral antigens need to be targeted. In this work, we comprehensively demonstrate the use of nucleocapsid (N)-specific detection across several assays using previously described nanobodies C2 and E2. We show that these nanobodies are highly sensitive and can detect divergent SARS-CoV-2 ancestral, delta and omicron variants across several assays. By comparison, spike-specific antibodies S309 and CR3022 only disparately detect SARS-CoV-2 variant targets. As such, we conclude that N-specific detection could provide a standardized universal target for detection of current and emerging SARS-CoV-2 variants of concern.

A platform technology for generating subunit vaccines against diverse viral pathogens.

The COVID-19 pandemic response has shown how vaccine platform technologies can be used to rapidly and effectively counteract a novel emerging infectious disease. The speed of development for mRNA and vector-based vaccines outpaced those of subunit vaccines, however, subunit vaccines can offer advantages in terms of safety and stability. Here we describe a subunit vaccine platform technology, the molecular clamp, in application to four viruses from divergent taxonomic families: Middle Eastern respiratory syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), Lassa virus (LASV) and Nipah virus (NiV). The clamp streamlines subunit antigen production by both stabilising the immunologically important prefusion epitopes of trimeric viral fusion proteins while enabling purification without target-specific reagents by acting as an affinity tag. Conformations for each viral antigen were confirmed by monoclonal antibody binding, size exclusion chromatography and electron microscopy. Notably, all four antigens tested remained stable over four weeks of incubation at 40°C. Of the four vaccines tested, a neutralising immune response was stimulated by clamp stabilised MERS-CoV spike, EBOV glycoprotein and NiV fusion protein. Only the clamp stabilised LASV glycoprotein precursor failed to elicit virus neutralising antibodies. MERS-CoV and EBOV vaccine candidates were both tested in animal models and found to provide protection against viral challenge.