A broadly-neutralizing antibody against Ebolavirus glycoprotein that potentiates the breadth and neutralization potency of other antibodies.

Ebolavirus disease (EVD) is caused by multiple species of Ebolavirus. Monoclonal antibodies (mAbs) against the virus glycoprotein (GP) are the only class of therapeutic approved for treatment of EVD caused by Zaire ebolavirus (EBOV). Therefore, mAbs targeting multiple Ebolavirus species may represent the next generation of EVD therapeutics. Broadly reactive anti-GP mAbs were produced; among these, mAbs 11886 and 11883 were broadly neutralizing in vitro. A 3.0 Å cryo-electron microscopy structure of EBOV GP bound to both mAbs shows that 11886 binds a novel epitope bridging the glycan cap (GC), 310 pocket and GP2 N-terminus, whereas 11883 binds the receptor binding region (RBR) and GC. In vitro, 11886 synergized with a range of mAbs with epitope specificities spanning the RBR/GC, including 11883. Notably, 11886 increased the breadth of neutralization by partner mAbs against different Ebolavirus species. These data provide a strategic route to design improved mAb-based next-generation EVD therapeutics.

In vitro reconstitution of herpes simplex virus 1 fusion identifies low pH as a fusion co-trigger.

Membrane fusion mediated by herpes simplex virus 1 (HSV-1) is a complex, multi-protein process that is receptor triggered and can occur both at the cell surface and in endosomes. To deconvolute this complexity, we reconstituted HSV-1 fusion with synthetic lipid vesicles in vitro. Using this simplified, controllable system, we discovered that HSV-1 fusion required not only a cognate host receptor but also low pH. On the target membrane side, efficient fusion required cholesterol, negatively charged lipids found in the endosomal membranes, and an optimal balance of lipid order and disorder. On the virion side, the four HSV-1 entry glycoproteins-gB, gD, gH, and gL-were sufficient for fusion. We propose that low pH is a biologically relevant co-trigger for HSV-1 fusion. The dependence of fusion on low pH and endosomal lipids could explain why HSV-1 enters most cell types by endocytosis. We hypothesize that under neutral pH conditions, other, yet undefined, cellular factors may serve as fusion co-triggers. The in vitro fusion system established here can be employed to systematically investigate HSV-1-mediated membrane fusion.IMPORTANCEHSV-1 causes lifelong, incurable infections and diseases ranging from mucocutaneous lesions to fatal encephalitis. Fusion of viral and host membranes is a critical step in HSV-1 infection of target cells that requires multiple factors on both the viral and host sides. Due to this complexity, many fundamental questions remain unanswered, such as the identity of the viral and host factors that are necessary and sufficient for HSV-1-mediated membrane fusion and the nature of the fusion trigger. Here, we developed a simplified in vitro fusion assay to examine the fusion requirements and identified low pH as a co-trigger for virus-mediated fusion in vitro. We hypothesize that low pH has a critical role in cell entry and, potentially, pathogenesis.

Heterologous production of the D-cycloserine intermediate O-acetyl-L-serine in a human type II pulmonary cell model.

Tuberculosis (TB) is the second leading cause of death by a single infectious disease behind COVID-19. Despite a century of effort, the current TB vaccine does not effectively prevent pulmonary TB, promote herd immunity, or prevent transmission. Therefore, alternative approaches are needed. We seek to develop a cell therapy that produces an effective antibiotic in response to TB infection. D-cycloserine (D-CS) is a second-line antibiotic for TB that inhibits bacterial cell wall synthesis. We have determined D-CS to be the optimal candidate for anti-TB cell therapy due to its effectiveness against TB, relatively short biosynthetic pathway, and its low-resistance incidence. The first committed step towards D-CS synthesis is catalyzed by the L-serine-O-acetyltransferase (DcsE) which converts L-serine and acetyl-CoA to O-acetyl-L-serine (L-OAS). To test if the D-CS pathway could be an effective prophylaxis for TB, we endeavored to express functional DcsE in A549 cells as a human pulmonary model. We observed DcsE-FLAG-GFP expression using fluorescence microscopy. DcsE purified from A549 cells catalyzed the synthesis of L-OAS as observed by HPLC-MS. Therefore, human cells synthesize functional DcsE capable of converting L-serine and acetyl-CoA to L-OAS demonstrating the first step towards D-CS production in human cells.

Characterization of the SARS-CoV-2 B.1.621 (Mu) variant.

The SARS-CoV-2 B.1.621 (Mu) variant emerged in January 2021 and was categorized as a variant of interest by the World Health Organization in August 2021. This designation prompted us to study the sensitivity of this variant to antibody neutralization. In a live virus neutralization assay with serum samples from individuals vaccinated with the Pfizer/BioNTech or Moderna mRNA vaccines, we measured neutralization antibody titers against B.1.621, an early isolate (spike 614D), and a variant of concern (B.1.351, Beta variant). We observed reduced neutralizing antibody titers against the B.1.621 variant (3.4- to 7-fold reduction, depending on the serum sample and time after the second vaccination) compared to the early isolate and a similar reduction when compared to B.1.351. Likewise, convalescent serum from hamsters previously infected with an early isolate neutralized B.1.621 to a lower degree. Despite this antibody titer reduction, hamsters could not be efficiently rechallenged with the B.1.621 variant, suggesting that the immune response to the first infection is adequate to provide protection against a subsequent infection with the B.1.621 variant.

Efficacy of vaccination and previous infection against the Omicron BA.1 variant in Syrian hamsters.

The emergence of the SARS-CoV-2 Omicron (B.1.1.529) variant with a surprising number of spike mutations raises concerns about reduced sensitivity of this virus to antibody neutralization and subsequent vaccine breakthrough infections. Here, we infect Moderna mRNA-vaccinated or previously infected hamsters with the Omicron BA.1 variant. While the Moderna mRNA vaccine reduces viral loads in the respiratory tissues upon challenge with an early S-614G isolate, the vaccine efficacy is not as pronounced after infection with the Omicron variant. Previous infection with the early SARS-CoV-2 isolate prevents replication after rechallenge with either virus in the lungs of previously infected hamsters, but the Omicron variant replicates efficiently in nasal turbinate tissue. These results experimentally demonstrate in an animal model that the antigenic changes in the Omicron variant are responsible for vaccine breakthrough and re-infection.

Modeling Tubercular ESX-1 Secretion Using Mycobacterium marinum.

Pathogenic mycobacteria cause chronic and acute diseases ranging from human tuberculosis (TB) to nontubercular infections. Mycobacterium tuberculosis causes both acute and chronic human tuberculosis. Environmentally acquired nontubercular mycobacteria (NTM) cause chronic disease in humans and animals. Not surprisingly, NTM and M. tuberculosis often use shared molecular mechanisms to survive within the host. The ESX-1 system is a specialized secretion system that is essential for virulence and is functionally conserved between M. tuberculosis and Mycobacterium marinumM. marinum is an NTM found in both salt water and freshwater that is often used to study mycobacterial virulence. Since the discovery of the secretion system in 2003, the use of both M. tuberculosis and M. marinum has defined the conserved molecular mechanisms underlying protein secretion and the lytic and regulatory activities of the ESX-1 system. Here, we review the trajectory of the field, including key discoveries regarding the ESX-1 system. We highlight the contributions of M. marinum studies and the conserved and unique aspects of the ESX-1 secretion system.