SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 immunogenicity in baboons and protection in mice.

The COVID-19 pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd protection and control the spread of SARS-CoV-2. Here, we report the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) from the full-length spike (S) protein that is stable in the prefusion conformation. NVX-CoV2373 S form 27.2-nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicit high titer anti-S IgG that blocks hACE2 receptor binding, neutralize virus, and protects against SARS-CoV-2 challenge with no evidence of vaccine-associated enhanced respiratory disease. NVX-CoV2373 also elicits multifunctional CD4+ and CD8+ T cells, CD4+ follicular helper T cells (Tfh), and antigen-specific germinal center (GC) B cells in the spleen. In baboons, low-dose levels of NVX-CoV2373 with Matrix-M was also highly immunogenic and elicited high titer anti-S antibodies and functional antibodies that block S-protein binding to hACE2 and neutralize virus infection and antigen-specific T cells. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2373 with Matrix-M (NCT04368988).

Transfer of a mitochondrial DNA fragment to MCOLN1 causes an inherited case of mucolipidosis IV.

A patient with mucolipidosis-IV heterozygous for two mutations in MCOLN1 expressed only her father's cDNA mutation c.1207C>T predicting an R403C change in mucolipin. She inherited a 93bp segment from mitochondrial NADH dehydrogenase 5 (MTND5) from her mother that was inserted in-frame prior to the last nucleotide of exon 2 of MCOLN1 (c.236_237ins93). This alteration abolished proper splicing of MCOLN1. The splice site at the end of the exon was not used due to an inhibitory effect of the inserted segment, resulting in two aberrant splice products containing stop codons in the downstream intron. These products were eliminated via nonsense-mediated decay. This is the first report of an inherited transfer of mitochondrial nuclear DNA causing a genetic disease. The elimination of the splice site by the mitochondrial DNA requires a change in splicing prediction models.

Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans.

BACKGROUND: Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder characterized by severe neurologic and ophthalmologic abnormalities. Recently the MLIV gene, MCOLN1, has been identified as a new member of the transient receptor potential (TRP) cation channel superfamily. Here we report the cloning and characterization of the mouse homologue, Mcoln1, and report a novel splice variant that is not seen in humans. RESULTS: The human and mouse genes display a high degree of synteny. Mcoln1 shows 91% amino acid and 86% nucleotide identity to MCOLN1. Also, Mcoln1 maps to chromosome 8 and contains an open reading frame of 580 amino acids, with a transcript length of approximately 2 kb encoded by 14 exons, similar to its human counterpart. The transcript that results from murine specific alternative splicing encodes a 611 amino acid protein that differs at the c-terminus. CONCLUSIONS: Mcoln1 is highly similar to MCOLN1, especially in the transmembrane domains and ion pore region. Also, the late endosomal/lysosomal targeting signal is conserved, supporting the hypothesis that the protein is localized to these vesicle membranes. To date, there are very few reports describing species-specific splice variants. While identification of Mcoln1 is crucial to the development of mouse models for MLIV, the fact that there are two transcripts in mice suggests an additional or alternate function of the gene that may complicate phenotypic assessment.

Characterization of the native form of anthrax lethal factor for use in the toxin neutralization assay.

The cell-based anthrax toxin neutralization assay (TNA) is used to determine functional antibody titers of sera from animals and humans immunized with anthrax vaccines. The anthrax lethal toxin is a critical reagent of the TNA composed of protective antigen (PA) and lethal factor (LF), which are neutralization targets of serum antibodies. Cytotoxic potency of recombinant LF (rLF) lots can vary substantially, causing a challenge in producing a renewable supply of this reagent for validated TNAs. To address this issue, we characterized a more potent rLF variant (rLF-A) with the exact native LF amino acid sequence that lacks the additional N-terminal histidine and methionine residues present on the commonly used form of rLF (rLF-HMA) as a consequence of the expression vector. rLF-A can be used at 4 to 6 ng/ml (in contrast to 40 ng/ml rLF-HMA) with 50 ng/ml recombinant PA (rPA) to achieve 95 to 99% cytotoxicity. In the presence of 50 ng/ml rPA, both rLF-A and rLF-HMA allowed for similar potencies (50% effective dilution) among immune sera in the TNA. rPA, but not rLF, was the dominant factor in determining potency of serum samples containing anti-PA antibodies only or an excess of anti-PA relative to anti-rLF antibodies. Such anti-PA content is reflected in immune sera derived from most anthrax vaccines in development. These results support that 7- to 10-fold less rLF-A can be used in place of rLF-HMA without changing TNA serum dilution curve parameters, thus extending the use of a single rLF lot and a consistent, renewable supply.

A novel fusion protein containing the receptor binding domains of C. difficile toxin A and toxin B elicits protective immunity against lethal toxin and spore challenge in preclinical efficacy models.

Antibodies targeting the Clostridium difficile toxin A and toxin B confer protective immunity to C. difficile associated disease in animal models and provided protection against recurrent C. difficile disease in human subjects. These antibodies are directed against the receptor binding domains (RBD) located in the carboxy-terminal portion of both toxins and inhibit binding of the toxins to their receptors. We have constructed a recombinant fusion protein containing portions of the RBD from both toxin A and toxin B and expressed it in Escherichia coli. The fusion protein induced high levels of serum antibodies to both toxins A and B capable of neutralizing toxin activity both in vitro and in vivo. In a hamster C. difficile infection model, immunization with the fusion protein reduced disease severity and conferred significant protection against a lethal dose of C. difficile spores. Our studies demonstrate the potential of the fusion protein as a vaccine that could provide protection from C. difficile disease in humans.

Prediction of response of mutated alpha-galactosidase A to a pharmacological chaperone.

OBJECTIVE: To examine the relationship between types and locations of mutations of the enzyme alpha-galactosidase (Gal) A in Fabry disease and the response to the pharmacological chaperone 1-deoxygalactonojirimycin (DGJ). METHODS: T cells grown from normal individuals or from patients with Fabry disease were tested for response to treatment with DGJ by increased activity of alpha-Gal A. RESULTS: Cells from normal controls responded with a 28% increase in alpha-Gal A activity, whereas response in Fabry individuals was mutation dependent ranging from no increase to fully normal activity. Nine truncation mutations (all nonresponsive) and 31 missense mutations were tested. Three groups of missense mutations were categorized: responders with activity more than 25% of normal, nonresponders, with less than 7% and an intermediate response group. In normal cells and in responders an increase in the mature lysosomal form of alpha-Gal A was observed after DGJ treatment. Nonresponders showed little or no protein with or without DGJ. The intermediate response group showed an increase in band intensity but incomplete processing of the enzyme to the mature form. CONCLUSION: Mapping the missense mutations to the structure of alpha-Gal A identified several factors that may influence response. Mutations in regions that are not in alpha-helix or beta-sheets, neither involved in disulfide bonds nor with an identified functional or structural role were more likely to respond. Predictability is, however, not precise and testing of each mutation for response to pharmacological chaperone therapy is necessary for Fabry disease and related lysosomal storage disorders.

Screening for pharmacological chaperones in Fabry disease.

As a prerequisite for clinical trials of pharmacological chaperone therapy (PCT) for Fabry disease, we developed a rapid screening assay for enhancement of endogenous alpha-galactosidase A (alpha-Gal A) in patient-derived cells. We used a T-cell based system to screen 11 mutations causing Fabry disease for enhanceability using 1-deoxygalactonojirimycin (DGJ). When patient-derived T-cells were grown in the presence of DGJ, alpha-Gal A activity increased to more than 50% of normal in several mutations but was unaffected in others. In addition to the mutation R301Q, reported previously, A97V, R112H, R112C, A143T, and L300P were enhanceable, but R356W, G132R, A143P, R220X, and 30delG were not. The level of alpha-Gal A activity achieved provides a basis for the therapeutic trial of DGJ in patients with similarly enhanceable enzyme. This assay method has general utility in other disorders in assessing the degree of enhancement of activity of mutated proteins by PCT.

Molecular pathophysiology of mucolipidosis type IV: pH dysregulation of the mucolipin-1 cation channel.

Mucolipidosis type IV (MLIV) is an autosomal recessive neurogenetic disorder characterized by developmental abnormalities of the brain and impaired neurological, ophthalmologic and gastric function. Large vacuoles accumulate in various types of cells in MLIV patients. However, the pathophysiology of the disease at the cellular level is still unknown. MLIV is caused by mutations in a recently described gene, MCOLN1, encoding mucolipin-1 (ML1), a 65 kDa protein whose function is also unknown. ML1 shows sequence homology and topological similarities with polycystin-2 and other transient receptor potential (Trp) channels. In this study, we assessed both, whether ML1 has ion channel properties, and whether disease-causing mutations in MCOLN1 have functional differences with the wild-type (WT) protein. ML1 channel function was assessed from endosomal vesicles of null (MCOLN1(-/-)) and ML1 over-expressing cells, and liposomes containing the in vitro translated protein. Evidence from both preparations indicated that WT ML1 is a multiple subconductance non-selective cation channel whose function is inhibited by a reduction of pH. The V446L and DeltaF408 MLIV causing mutations retain channel function but not the sharp inhibition by lowering pH. Atomic force imaging of ML1 channels indicated that changes in pH modified the aggregation of unitary channels. Mutant-ML1 did not change in size on reduction of pH. The data indicate that ML1 channel activity is regulated by a pH-dependent mechanism that is deficient in some MLIV causing mutations of the gene. The evidence also supports a novel role for cation channels in the acidification and normal endosomal function.