Enhanced stability of the SARS CoV-2 spike glycoprotein following modification of an alanine cavity in the protein core.

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 Ala1016 and Ala1020 in the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 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 Ala1016/Ala1020 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 (ID50s) 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.

Characterization of a Broadly Neutralizing Monoclonal Antibody against SARS-CoV-2 Variants.

The constant mutation of SARS-CoV-2 has led to the emergence of new variants, which call for urgent effective therapeutic interventions. The trimeric spike (S) protein of SARS-CoV-2 is highly immunogenic with the receptor-binding domain (RBD) that binds first to the cellular receptor angiotensin-converting enzyme 2 (ACE2) and is therefore the target of many neutralizing antibodies. In this study, we characterized a broadly neutralizing monoclonal antibody (mAb) 9G8, which shows potent neutralization against the authentic SARS-CoV-2 wild-type (WT), Alpha (B.1.1.7), and Delta (1.617.2) viruses. Furthermore, mAb 9G8 also displayed a prominent neutralizing efficacy in the SARS-CoV-2 surrogate virus neutralization test (sVNT) against the Epsilon (B.1.429/7), Kappa (B.1.617.1), Gamma (P.1), Beta (B.1.351), and Delta Plus (1.617.2.1) RBD variants in addition to the variants mentioned above. Based on our in vitro escape mutant studies, we proved that the mutations V483F and Y489H within the RBD were involved in ACE2 binding and caused the neutralizing evasion of the virus from mAb 9G8. The development of such a cross-reactive neutralizing antibody against majority of the SARS-CoV-2 variants provides an important insight into pursuing future therapeutic agents for the prevention and treatment of COVID-19.

Limited dengue virus replication in field-collected Aedes aegypti mosquitoes infected with Wolbachia.

INTRODUCTION: Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV), is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus. METHODOLOGY/PRINCIPAL FINDINGS: Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.

The toll and Imd pathways are not required for wolbachia-mediated dengue virus interference.

Wolbachia blocks dengue virus replication in Drosophila melanogaster as well as in Aedes aegypti. Using the Drosophila model and mutations in the Toll and Imd pathways, we showed that neither pathway is required for expression of the dengue virus-blocking phenotype in the Drosophila host. This provides additional evidence that the mechanistic basis of Wolbachia-mediated dengue virus blocking in insects is more complex than simple priming of the host insect innate immune system.

Vaginal concentrations of lactic acid potently inactivate HIV.

OBJECTIVES: When Lactobacillus spp. dominate the vaginal microbiota of women of reproductive age they acidify the vagina to pH <4.0 by producing ∼1% lactic acid in a nearly racemic mixture of d- and l-isomers. We determined the HIV virucidal activity of racemic lactic acid, and its d- and l-isomers, compared with acetic acid and acidity alone (by the addition of HCl). METHODS: HIV-1 and HIV-2 were transiently treated with acids in the absence or presence of human genital secretions at 37°C for different time intervals, then immediately neutralized and residual infectivity determined in the TZM-bl reporter cell line. RESULTS: l-lactic acid at 0.3% (w/w) was 17-fold more potent than d-lactic acid in inactivating HIVBa-L. Complete inactivation of different HIV-1 subtypes and HIV-2 was achieved with ≥0.4% (w/w) l-lactic acid. At a typical vaginal pH of 3.8, l-lactic acid at 1% (w/w) more potently and rapidly inactivated HIVBa-L and HIV-1 transmitter/founder strains compared with 1% (w/w) acetic acid and with acidity alone, all adjusted to pH 3.8. A final concentration of 1% (w/w) l-lactic acid maximally inactivated HIVBa-L in the presence of cervicovaginal secretions and seminal plasma. The anti-HIV activity of l-lactic acid was pH dependent, being abrogated at neutral pH, indicating that its virucidal activity is mediated by protonated lactic acid and not the lactate anion. CONCLUSIONS: l-lactic acid at physiological concentrations demonstrates potent HIV virucidal activity distinct from acidity alone and greater than acetic acid, suggesting a protective role in the sexual transmission of HIV.

Mycolactone gene expression is controlled by strong SigA-like promoters with utility in studies of Mycobacterium ulcerans and buruli ulcer.

Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans.