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Uncoating of the metastable HIV-1 capsid is a tightly regulated disassembly process required for release of the viral cDNA prior to nuclear import. To understand the intrinsic capsid disassembly pathway and how it can be modulated, we have developed a single-particle fluorescence microscopy method to follow the real-time uncoating kinetics of authentic HIV capsids in vitro immediately after permeabilizing the viral membrane. Opening of the first defect in the lattice is the rate-limiting step of uncoating, which is followed by rapid, catastrophic collapse. The capsid-binding inhibitor PF74 accelerates capsid opening but stabilizes the remaining lattice. In contrast, binding of a polyanion to a conserved arginine cluster in the lattice strongly delays initiation of uncoating but does not prevent subsequent lattice disassembly. Our observations suggest that different stages of uncoating can be controlled independently with the interplay between different capsid-binding regulators likely to determine the overall uncoating kinetics. Viruses need to enter their host’s cells in order to replicate their genetic material and produce more copies of the virus. A protein shell called a capsid protects the virus during this journey. But the structure of the capsid presents a mystery. How can this protein shell be strong enough to remain intact as it enters a host cell, and yet quickly open up to release the viral genome after replication? Unlike the capsids of many other viruses, those of HIV have irregular structures that rapidly fall apart once removed from the virus. This has thwarted attempts to study intact HIV capsids in order to understand how they work. However, we do know that HIV hijacks a range of molecules produced by the invaded host cell. Dissecting their effects on the capsid is key to understanding how the capsid disassembles. Márquez et al. have now developed a method that can visualize individual HIV capsids – and how they disassemble – in real time using single-molecule microscopy. This revealed that capsids differ widely in their stability. The shell remains closed for a variable period of time and then collapses catastrophically as soon as it loses its first subunit. Using the new technique, Márquez et al. also found that a small molecule drug called PF74 causes the capsid to crack open rapidly, but the remaining shell is then stabilized against further disassembly. These observations reconcile seemingly contradictory observations made by different research groups about how this drug affects the stability of the capsid. The method developed by Márquez et al. enables researchers to measure how molecules produced by host cells interact with the viral capsid, a structure that is fundamental for the virus to establish an infection. It could also be used to test the effects of antiviral drugs that have been designed to target the capsid. The new technique has already been instrumental in related research by Mallery et al., which identifies a molecule found in host cells that stabilizes the capsid of HIV.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antibody neutralization response and its evasion by emerging viral variants and variant of concern (VOC) are unknown, but critical to understand reinfection risk and breakthrough infection following vaccination. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus–cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 reverse transcription polymerase chain reaction (RT-PCR)–confirmed Coronavirus Disease 2019 (COVID-19) individuals with detailed demographics and followed up to 7 months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization, was associated with COVID-19 severity. A subgroup of “high responders” maintained high neutralizing responses over time, representing ideal convalescent plasma donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants and VOC. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal responders and vaccine monitoring and design.

This study investigates the humoral and cellular immune responses and health-related quality of life measures in individuals with mild to moderate long COVID (LC) compared to age and gender matched recovered COVID-19 controls (MC) over 24 months. LC participants show elevated nucleocapsid IgG levels at 3 months, and higher neutralizing capacity up to 8 months post-infection. Increased spike-specific and nucleocapsid-specific CD4 + T cells, PD-1, and TIM-3 expression on CD4 + and CD8 + T cells were observed at 3 and 8 months, but these differences do not persist at 24 months. Some LC participants had detectable IFN-γ and IFN-β, that was attributed to reinfection and antigen re-exposure. Single-cell RNA sequencing at the 24 month timepoint shows similar immune cell proportions and reconstitution of naïve T and B cell subsets in LC and MC. No significant differences in exhaustion scores or antigen-specific T cell clones are observed. These findings suggest resolution of immune activation in LC and return to comparable immune responses between LC and MC over time. Improvement in self-reported health-related quality of life at 24 months was also evident in the majority of LC (62%). PTX3, CRP levels and platelet count are associated with improvements in health-related quality of life. Post-acute sequelae of COVID (PASC) or long-COVID can affect a proportion of those infected but this is not well understood. Here the authors perform a single cell transcriptomics analysis of immune cells from long-COVID patients at 24 months and find that cell changes observed at 3 and 8 months do not persist to 24 months.

Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Guérin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilised, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum. This vaccine formulation, BCG:CoVac, induced high-titre SARS-CoV-2 neutralising antibodies (NAbs) and Th1-biased cytokine release by vaccine-specific T cells, which correlated with the early emergence of T follicular helper cells in local lymph nodes and heightened levels of antigen-specific plasma B cells after vaccination. Vaccination of K18-hACE2 mice with a single dose of BCG:CoVac almost completely abrogated disease after SARS-CoV-2 challenge, with minimal inflammation and no detectable virus in the lungs of infected animals. Boosting BCG:CoVac-primed mice with a heterologous vaccine further increased SARS-CoV-2-specific antibody responses, which effectively neutralised B.1.1.7 and B.1.351 SARS-CoV-2 variants of concern. These findings demonstrate the potential for BCG-based vaccination to protect against major SARS-CoV-2 variants circulating globally.

Current vaccines against SARS-CoV-2 substantially reduce mortality, but protection against infection is less effective. Enhancing immunity in the respiratory tract, via mucosal vaccination, may provide protection against infection and minimise viral spread. Here, we report testing of a subunit vaccine in mice, consisting of SARS-CoV-2 Spike protein with a TLR2-stimulating adjuvant (Pam 2 Cys), delivered to mice parenterally or mucosally. Both routes of vaccination induce substantial neutralising antibody (nAb) titres, however, mucosal vaccination uniquely generates anti-Spike IgA, increases nAb in the serum and airways, and increases lung CD4 + T-cell responses. TLR2 is expressed by respiratory epithelia and immune cells. Using TLR2 deficient chimeric mice, we determine that TLR2 expression in either compartment facilitates early innate responses to mucosal vaccination. By contrast, TLR2 on hematopoietic cells is essential for optimal lung-localised, antigen-specific responses. In K18-hACE2 mice, vaccination provides complete protection against disease and sterilising lung immunity against SARS-CoV-2, with a short-term non-specific protective effect from mucosal Pam 2 Cys alone. These data support mucosal vaccination as a strategy to improve protection in the respiratory tract against SARS-CoV-2 and other respiratory viruses. Current vaccines against SARS-CoV-2 reduce mortality but are less effective in preventing infection. Here the authors show that intranasal vaccination with a subunit vaccine including an TLR2-stimulating adjuvant induces strong neutralising antibody and T-cell responses against SARS-CoV-2 in the lungs that protect against infection.

Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR -related cell surface receptor called leucine-rich repeat-containing protein 15 ( LRRC15 ). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans . Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.

Graves' disease is a self-limiting autoimmune thyroid disorder caused by stimulating antibodies to the thyroid-stimulating hormone receptor. It usually affects middle-aged females in the fourth to sixth decade of life. It is distinguished by keratopathy, chemosis, proptosis, and eyelid swelling, in addition to ocular discomfort. A total of 3-5% of cases present with a severe form of Graves' orbitopathy, which manifests with diminution of vision, optic nerve compression, optic neuropathy, and exposure keratopathy. We describe a case of a 34-year-old female patient who presented with the chief complaint of rapid deterioration of vision over a period of three months in the right eye. Ocular examination revealed proptosis, widened palpebral aperture, elevation of intra-ocular pressure (IOP) in the upgaze, restricted eye movements, and signs of optic nerve compression. Findings were confirmed on a CT scan of the orbit. The unusual presentation in this case was that she had rapid, significant deterioration of vision in the right eye, with a progression of proptosis more marked in the contralateral eye. This underlies the importance of thoroughly examining for any possible orbital apex syndrome in both eyes, not just the eye with marked proptosis. The patient, being reluctant for orbital decompression, was prescribed IV methylprednisolone 1 g for three consecutive days, which reduced her proptosis and improved her vision. This acted as a temporary measure to increase the duration of the surgical window until the time the patient undergoes the surgery.

Abstract Purpose: The purpose of this study was to evaluate face mask-associated factors causing dry eye among medical students. Methodology: This was a cross-sectional study conducted on undergraduate medical and dental students, of all phases, while they were attending offline classes and were required to wear face masks in accordance with the government regulations. Sociodemographic data, ocular and medical history, face mask-wearing practices, screen usage, and quantification of symptoms using the modified Ocular Surface Disease Index (OSDI) questionnaire were collected. Objective tests were conducted in students having dry eye. The association of quantitative variables was done using ANOVA, Mann-Whitney, and Kruskal-Wallis test, whereas the Chi-square test was done for qualitative variables. Multivariate logistic regression was used to identify the risk factors for varying severity of dry eye. Results: The mean age of the 410 students was 21 ± 1.6 years. According to the OSDI, 39.51% (162/410) of students had dry eyes, 23.41% (96/410) had mild dry eye, 8.78% (36/410) had moderate dry eye, and 7.32% (30/410) had severe dry eye. Face mask-associated factors which were significantly linked to dry eye were N95 masks, loose-fit masks, and 6-8 h of continuous mask use. The Schirmer's test and tear film break-up time were performed on 29 and 20 students, respectively, mean values being 19.25 ± 5.29 mm and 10.15 ± 1.41 s for nonsevere and 6.53 ± 1.55 mm and 5.3 ± 0.98 s for severe dry eye, respectively. Conclusion: It is important to educate medical students and create awareness regarding \"face mask-appropriate behavior\" to reduce the chances of dry eye secondary to face masks use.

The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein subunit and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3–4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity next to Ala 1016 and Ala 1020 in the 3–4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala 1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. S glycoprotein membrane fusion function was retained with Ala 1016 /Ala 1020 cavity-filling mutations associated with improved recombinant S2P-FHA thermostability, however 2 mutants, A1016L and A1016V/A1020I, lacked ability to mediate entry of S-HIV-1 pseudoparticles into 293-ACE2 cells. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited neutralizing antibody with 50%-inhibitory dilutions (ID 50 s) in the range 2,700–5,110 for ancestral and Delta-derived viruses, and 210–1,744 for Omicron BA.1. The antigens elicited antibody specificities directed to the receptor-binding domain (RBD), N-terminal domain (NTD), fusion peptide and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S2P-FHA-like ectodomain oligomers in the absence of an external trimerization motif (T4 foldon), thus representing an alternative approach for stabilizing oligomeric S glycoprotein vaccines.

Critical to facilitating SARS-CoV-2 point-of-care (POC) testing is assurance that viruses present in specimens are inactivated onsite prior to processing. Here, we conducted experiments to determine the virucidal activity of commercially available Viral Transport Mediums (VTMs) to inactivate SARS-CoV-2. Independent testing methods for viral inactivation testing were applied, including a previously described World Health Organization (WHO) protocol, in addition to a buffer exchange method where the virus is physically separated from the VTM post exposure. The latter method enables sensitive detection of viral viability at higher viral titre when incubated with VTM. We demonstrate that VTM formulations, Primestore® Molecular Transport Medium (MTM) and COPAN eNAT™ completely inactivate high-titre SARS-CoV-2 virus (>1 × 107 copies/mL) and are compatible with POC processing. Furthermore, full viral inactivation was rapidly achieved in as little as 2 min of VTM exposure. We conclude that adding certain VTM formulations as a first step post specimen collection will render SARS-CoV-2 non-infectious for transport, or for further in-field POC molecular testing using rapid turnaround GeneXpert platforms or equivalent.