Antivirals for Broader Coverage against Human Coronaviruses.

Coronaviruses (CoVs) are enveloped positive-sense single-stranded RNA viruses with a genome that is 27-31 kbases in length. Critical genes include the spike (S), envelope (E), membrane (M), nucleocapsid (N) and nine accessory open reading frames encoding for non-structural proteins (NSPs) that have multiple roles in the replication cycle and immune evasion (1). There are seven known human CoVs that most likely appeared after zoonotic transfer, the most recent being SARS-CoV-2, responsible for the COVID-19 pandemic. Antivirals that have been approved by the FDA for use against COVID-19 such as Paxlovid can target and successfully inhibit the main protease (MPro) activity of multiple human CoVs; however, alternative proteomes encoded by CoV genomes have a closer genetic similarity to each other, suggesting that antivirals could be developed now that target future CoVs. New zoonotic introductions of CoVs to humans are inevitable and unpredictable. Therefore, new antivirals are required to control not only the next human CoV outbreak but also the four common human CoVs (229E, OC43, NL63, HKU1) that circulate frequently and to contain sporadic outbreaks of the severe human CoVs (SARS-CoV, MERS and SARS-CoV-2). The current study found that emerging antiviral drugs, such as Paxlovid, could target other CoVs, but only SARS-CoV-2 is known to be targeted in vivo. Other drugs which have the potential to target other human CoVs are still within clinical trials and are not yet available for public use. Monoclonal antibody (mAb) treatment and vaccines for SARS-CoV-2 can reduce mortality and hospitalisation rates; however, they target the Spike protein whose sequence mutates frequently and drifts. Spike is also not applicable for targeting other HCoVs as these are not well-conserved sequences among human CoVs. Thus, there is a need for readily available treatments globally that target all seven human CoVs and improve the preparedness for inevitable future outbreaks. Here, we discuss antiviral research, contributing to the control of common and severe CoV replication and transmission, including the current SARS-CoV-2 outbreak. The aim was to identify common features of CoVs for antivirals, biologics and vaccines that could reduce the scientific, political, economic and public health strain caused by CoV outbreaks now and in the future.

A phase 3, multicenter, randomized, double-blind study to evaluate the interchangeability of V114, a 15-valent pneumococcal conjugate vaccine, and PCV13 with respect to safety, tolerability, and immunogenicity in healthy infants (PNEU-DIRECTION).

BACKGROUND: Pneumococcal disease (PD) remains a major health concern globally. In children, pneumococcal conjugate vaccines (PCVs) provide protection against PD from most vaccine serotypes, but non-vaccine serotypes contribute to residual disease. V114 is a 15-valent PCV containing all 13 serotypes in Prevnar 13™ (PCV13) and public health important serotypes 22F and 33F. This phase 3 study evaluated safety and immunogenicity of mixed PCV13/V114 regimens using a 3 + 1 dosing schedule when changing from PCV13 to V114 at doses 2, 3, or 4. METHODS: 900 healthy infants were randomized equally to 5 intervention groups. PCVs were administered in a 3-dose infant series at 2, 4, and 6 months of age followed by a toddler dose at 12-15 months along with concomitant routine vaccines. Safety was evaluated as the proportion of participants with adverse events (AEs). Immunoglobulin G (IgG) responses to the 15 serotypes in V114 were measured at 30 days post-dose 3 and 30 days post-dose 4 (PD4). RESULTS: Frequencies of injection-site and systemic AEs were generally comparable across all intervention groups. At 30 days PD4 (primary endpoint), IgG geometric mean concentrations (GMCs) for the 13 shared serotypes were generally comparable between mixed V114/PCV13 and 4-dose regimens of PCV13 or V114. In mixed regimens at 30 days PD4, a toddler dose of V114 was sufficient to achieve IgG GMCs comparable to a 4-dose regimen of V114 for serotype 22F, while at least one infant dose was needed in addition to the toddler dose to achieve IgG GMCs comparable to a 4-dose regimen of V114 for serotype 33F. CONCLUSIONS: V114 was well tolerated with a generally comparable safety profile to PCV13. For 13 shared serotypes, both mixed regimens and the V114 4-dose regimen induced generally comparable antibody responses to 4-dose regimen with PCV13. Study results support interchangeability of V114 with PCV13 in infants. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03620162; EudraCT: 2018-001151-12.

Epitope mapping of antibodies induced with a conserved rhinovirus protein generating protective anti-rhinovirus immunity.

Human rhinovirus (RV) infections are the principle cause of common colds and precipitate asthma and chronic obstructive pulmonary disease (COPD) exacerbations. Currently there is no vaccine for RV which is largely due to the existence of ∼160 serotypes/strains. We demonstrated previously that immunising mice with highly conserved VP4 and VP2 regions of the RV polyprotein (RV-A16 VP0) generated cross-reactive immunity to RV in vivo. The current study investigated and mapped the epitopes of RV-A16 VP0 that are targets for antibodies in serum samples from VP0 immunisation and RV challenge studies in mice. Recombinant capsid proteins, peptide pools and individual peptides spanning the immunogen sequence (RV-A16 VP0) were assessed for IgG binding sites to identify epitopes. We found that peptide pools covering the C-terminus of VP4, the N-terminus of VP2 and the neutralising NIm-II site within VP2 were bound by serum IgG from immunised mice. The NIm-II site peptide pool blocked IgG binding to the immunogen RV-A16 VP0 and individual peptides within the pool binding IgG were further mapped. Thus, we have identified immunodominant epitopes of RV vaccine candidate RV-A16 VP0, noting that strong IgG binding antibodies were observed that target a key neutralising epitope that is highly variable amongst RV serotypes.

Enhanced replication of R5 HIV-1 isolates in†vitro by a small-molecule reagent targeting HIV-1 protease.

CHEMICAL ENHANCEMENT: Designed to target HIV-1 protease, a novel γ-hydroxyphosphonate has been found to significantly enhance viral replication in a panel of clinically relevant R5 HIV-1 isolates. This unexpected result constitutes the first instance of a small molecule capable of doing this, and it has implications for the preparation and use of R5 isolates in vaccine and drug development.

Loss and spontaneous recovery of forelimb evoked potentials in both the adult rat cuneate nucleus and somatosensory cortex following contusive cervical spinal cord injury.

Varying degrees of neurologic function spontaneously recovers in humans and animals during the days and months after spinal cord injury (SCI). For example, abolished upper limb somatosensory potentials (SSEPs) and cutaneous sensations can recover in persons post-contusive cervical SCI. To maximize recovery and the development/evaluation of repair strategies, a better understanding of the anatomical locations and physiological processes underlying spontaneous recovery after SCI is needed. As an initial step, the present study examined whether recovery of upper limb SSEPs after contusive cervical SCI was due to the integrity of some spared dorsal column primary afferents that terminate within the cuneate nucleus and not one of several alternate routes. C5-6 contusions were performed on male adult rats. Electrophysiological techniques were used in the same rat to determine forelimb evoked neuronal responses in both cortex (SSEPs) and the cuneate nucleus (terminal extracellular recordings). SSEPs were not evoked 2 days post-SCI but were found at 7 days and beyond, with an observed change in latencies between 7 and 14 days (suggestive of spared axon remyelination). Forelimb evoked activity in the cuneate nucleus at 15 but not 3 days post-injury occurred despite dorsal column damage throughout the cervical injury (as seen histologically). Neuroanatomical tracing (using 1% unconjugated cholera toxin B subunit) confirmed that upper limb primary afferent terminals remained within the cuneate nuclei. Taken together, these results indicate that neural transmission between dorsal column primary afferents and cuneate nuclei neurons is likely involved in the recovery of upper limb SSEPs after contusive cervical SCI.

Use of environmentally enriched housing for rats with spinal cord injury: the need for standardization.

A variety of rehabilitation methods that increase social interaction and locomotor activity are reported to yield positive benefits in humans and animals with spinal cord injury (SCI). Environmental enrichment often incorporates group housing, increased cage size, and objects to increase social interaction and stimulate locomotor activity of animals. Others have reported that adult rats housed in enriched environments immediately after moderate contusion thoracic SCI show improvements in locomotion, but not in neurotransmission through or anatomy at the SCI site. In the present study, in contrast to previous reports, environmental enrichment did not improve the locomotion of rats with contusion thoracic SCI. Furthermore, as in previous reports, improvements were not observed for either electrophysiologic measures of neurotransmission through (transcranial magnetic motor-evoked potentials) and caudal to (magnetic-evoked interlimb reflex) the injury site or the amount of spared white matter at the epicenter. Determining the effectiveness of environmental enrichment to improve locomotor recovery in the SCI model requires standardization of housing procedures, outcome measures, and analyses.

Crystal structure of tobacco etch virus protease shows the protein C terminus bound within the active site.

Tobacco etch virus (TEV) protease is a cysteine protease exhibiting stringent sequence specificity. The enzyme is widely used in biotechnology for the removal of the affinity tags from recombinant fusion proteins. Crystal structures of two TEV protease mutants as complexes with a substrate and a product peptide provided the first insight into the mechanism of substrate specificity of this enzyme. We now report a 2.7A crystal structure of a full-length inactive C151A mutant protein crystallised in the absence of peptide. The structure reveals the C terminus of the protease bound to the active site. In addition, we determined dissociation constants of TEV protease substrate and product peptides using isothermal titration calorimetry for various forms of this enzyme. Data suggest that TEV protease could be inhibited by the peptide product of autolysis. Separate modes of recognition for native substrates and the site of TEV protease self-cleavage are proposed.

The mechanism of Mycobacterium tuberculosis alkylhydroperoxidase AhpD as defined by mutagenesis, crystallography, and kinetics.

AhpD, a protein with two cysteine residues, is required for physiological reduction of the Mycobacterium tuberculosis alkylhydroperoxidase AhpC. AhpD also has an alkylhydroperoxidase activity of its own. The AhpC/AhpD system provides critical antioxidant protection, particularly in the absence of the catalase-peroxidase KatG, which is suppressed in most isoniazid-resistant strains. Based on the crystal structure, we proposed recently a catalytic mechanism for AhpD involving a proton relay in which the Glu118 carboxylate group, via His137 and a water molecule, deprotonates the catalytic residue Cys133 (Nunn, C. M., Djordjevic, S., Hillas, P. J., Nishida, C., and Ortiz de Montellano, P. R. (2002) J. Biol. Chem. 277, 20033-20040). A possible role for His132 in subsequent formation of the Cys133-Cys130 disulfide bond was also noted. To test this proposed mechanism, we have expressed the H137F, H137Q, H132F, H132Q, E118F, E118Q, C133S, and C130S mutants of AhpD, determined the crystal structures of the H137F and H132Q mutants, estimated the pKa values of the cysteine residues, and defined the kinetic properties of the mutant proteins. The collective results strongly support the proposed catalytic mechanism for AhpD.

Chaperonin assisted overexpression, purification, and characterisation of human PP2A methyltransferase.

Protein phosphatase 2A (PP2A) is a ubiquitous phosphatase found in many eukaryotic cell types and is involved in regulating a number of intracellular signalling pathways. Its activity, in turn, is regulated through covalent modification, involving phosphorylation and methylation reactions. The effect of phosphorylation on the activity of the protein is well known, but the effects of methylation have only recently been documented and the mechanistic details of methylation are lacking. Methylation, which occurs on the catalytic subunit of PP2A, is catalysed by PP2A methyltransferase (PP2Amt). Here, we present a method for the large-scale purification of human PP2Amt using an Escherichia coli host, coexpressing the chaperonins GroEL and GroES. Purified PP2Amt was identified by peptide mass mapping using MALD-MS and peptide sequencing using ESI-LC-MS/MS. The CD spectrum indicated that purified PP2Amt was folded, with about one-third of the protein adopting an alpha-helical conformation. Analytical gel filtration estimated the molecular weight to be 34kDa, equivalent to the monomeric form of the protein. Further CD analysis showed that in the presence and absence of the ligand S-adenosylhomocysteine, the thermal denaturation profiles were biphasic. However, the transition midpoints shifted to a higher temperature in the presence of ligand, indicating stabilisation of ligand-bound PP2Amt compared to the apo-form. We also report on the progress made in determining the structure of PP2Amt, using both X-ray crystallography and NMR spectroscopy.

The crystal structure of Mycobacterium tuberculosis alkylhydroperoxidase AhpD, a potential target for antitubercular drug design.

The resistance of Mycobacterium tuberculosis to isoniazid is commonly linked to inactivation of a catalase-peroxidase, KatG, that converts isoniazid to its biologically active form. Loss of KatG is associated with elevated expression of the alkylhydroperoxidases AhpC and AhpD. AhpD has no sequence identity with AhpC or other proteins but has alkylhydroperoxidase activity and possibly additional physiological activities. The alkylhydroperoxidase activity, in the absence of KatG, provides an important antioxidant defense. We have determined the M. tuberculosis AhpD structure to a resolution of 1.9 A. The protein is a trimer in a symmetrical cloverleaf arrangement. Each subunit exhibits a new all-helical protein fold in which the two catalytic sulfhydryl groups, Cys-130 and Cys-133, are located near a central cavity in the trimer. The structure supports a mechanism for the alkylhydroperoxidase activity in which Cys-133 is deprotonated by a distant glutamic acid via the relay action of His-137 and a water molecule. The cysteine then reacts with the peroxide to give a sulfenic acid that subsequently forms a disulfide bond with Cys-130. The crystal structure of AhpD identifies a new protein fold relevant to members of this protein family in other organisms. The structural details constitute a potential platform for the design of inhibitors of potential utility as antitubercular agents and suggest that AhpD may have disulfide exchange properties of importance in other areas of M. tuberculosis biology.