Haemagglutinin substitutions N125D, D127E, D222G and R223Q improve replicative fitness and vaccine effectiveness of an A/H1N1pdm09 live attenuated influenza vaccine virus by enhancing α-2,6 receptor binding.

During 2013-14 and 2015-16, A/H1N1pdm09 live attenuated influenza vaccine (LAIV) viruses replicated inefficiently in primary human nasal epithelial cells (hNEC). This led to reduced vaccine effectiveness (VE) in quadrivalent formulations, mediated by inter-strain competition. By mutating the haemagglutinin (HA) protein, we aimed to enhance hNEC replication of a novel A/H1N1pdm09 vaccine strain to overcome competition and improve VE. Combinations of N125D, D127E, D222G and R223Q substitutions were introduced to the HA protein of A/Slovenia/2903/2015 (A/SLOV15). A/SLOV15 S13, containing all four HA substitutions, produced approximately 1000-fold more virus than parental V1 during hNEC infection. Immunogenicity in ferrets was increased by approximately 10-fold, without compromising yield in eggs or antigenic match to wild-type (wt) reference strains. Despite S13 and V1 being antigenically similar, only S13 protected ferrets from wt virus shedding and fever post-challenge. Crucially, these data suggested that enhanced fitness allowed S13 to overcome inter-strain competition in quadrivalent LAIV (QLAIV). This improved efficacy was later validated by real-world VE data. S13 displayed increased binding avidity to a mammalian-like α-2,6 receptor analogue (6-SLN), relative to V1, while maintaining avian-like 3-SLN avidity. In silico modelling of the HA receptor binding site revealed additional interactions in the S13:6-SLN binding network and a mild increase in 6-SLN binding energy, indicating a possible mechanism for increased α-2,6 receptor-binding avidity. These data confirm that rational HA mutagenesis can be used to optimise hNEC replication and VE for A/H1N1pdm09 LAIV viruses.

Sequential Delivery of Live Attenuated Influenza Vaccine and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in the Ferret Model Can Reduce SARS-CoV-2 Shedding and Does Not Result in Enhanced Lung Pathology.

Cocirculation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses could pose unpredictable risks to health systems globally, with recent studies suggesting more severe disease outcomes in coinfected patients. The initial lack of a readily available coronavirus disease 2019 (COVID-19) vaccine has reinforced the importance of influenza vaccine programs during the COVID-19 pandemic. Live attenuated influenza vaccine (LAIV) is an important tool in protecting against influenza, particularly in children. However, it is unknown whether LAIV administration influences the outcomes of acute SARS-CoV-2 infection or disease. To investigate this, quadrivalent LAIV was administered to ferrets 3 days before or after SARS-CoV-2 infection. LAIV administration did not exacerbate the SARS-CoV-2 disease course or lung pathology with either regimen. In addition, LAIV administered before SARS-CoV-2 infection significantly reduced SARS-CoV-2 replication and shedding in the upper respiratory tract. This study demonstrated that LAIV administration in close proximity to SARS-CoV-2 infection does not exacerbate mild disease and can reduce SARS-CoV-2 shedding.

Defining the root cause of reduced H1N1 live attenuated influenza vaccine effectiveness: low viral fitness leads to inter-strain competition.

In the 2013-14 and 2015-16 influenza seasons, reduced vaccine effectiveness (VE) was observed for the H1N1 component of the FluMist quadrivalent live attenuated influenza vaccine (QLAIV) in the USA, leading to loss of Advisory Committee on Immunization Practices recommendation. Here we demonstrate in ferrets that 2015-16A/H1N1pdm09 vaccine strain A/Bolivia/559/2013 (A/BOL13) is outcompeted in trivalent (TLAIV) and QLAIV formulations, leading to reduced protection from wild-type challenge. While monovalent (MLAIV) A/BOL13 provided significant protection from wild-type virus shedding and fever at doses as low as 3.0 log10 fluorescent focus units (FFU), it failed to provide a similar level of protection in TLAIV or QLAIV formulation, even at a 6.0 log10 FFU dose. Conversely, clinically effective H1N1 strain A/New Caledonia/20/1999 provided significant protection in MLAIV, TLAIV, and QLAIV formulations. In conclusion, reduced A/BOL13 replicative fitness rendered it susceptible to inter-strain competition in QLAIV, contributing to its reduced VE in the 2015-16 season.

In vivo active aldosterone synthase inhibitors with improved selectivity: lead optimization providing a series of pyridine substituted 3,4-dihydro-1H-quinolin-2-one derivatives.

Pyridine substituted naphthalenes (e.g., I-III) constitute a class of potent inhibitors of aldosterone synthase (CYP11B2). To overcome the unwanted inhibition of the hepatic enzyme CYP1A2, we aimed at reducing the number of aromatic carbons of these molecules because aromaticity has previously been identified to correlate positively with CYP1A2 inhibition. As hypothesized, inhibitors with a tetrahydronaphthalene type molecular scaffold (1-11) exhibit a decreased CYP1A2 inhibition. However, tetralone 9 turned out to be cytotoxic to the human cell line U-937 at higher concentrations. Consequent structural optimization culminated in the discovery of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones (12-26), with 12, a bioisostere of 9, being nontoxic up to 200 microM. The investigated molecules are highly selective toward both CYP1A2 and a wide range of other cytochrome P450 enzymes and show a good pharmacokinetic profile in vivo (e.g., 12 with a peroral bioavailability of 71%). Furthermore, isoquinoline derivative 21 proved to significantly reduce plasma aldosterone levels of ACTH stimulated rats.

Novel aldosterone synthase inhibitors with extended carbocyclic skeleton by a combined ligand-based and structure-based drug design approach.

Pharmacophore modeling of a series of aldosterone synthase (CYP11B2) inhibitors triggered the design of compounds 11 and 12 by extending a previously established naphthalene molecular scaffold (e.g., present in molecules 1 and 2) via introduction of a phenyl or benzyl residue in 3-position. These additional aromatic moieties have been hypothesized to fit into the newly identified hydrophobic pharmacophore feature HY3. Subsequent docking studies in our refined CYP11B2 protein model have been performed prior to synthesis to estimate the inhibitory properties of the proposed molecules. While phenyl-substituted compound 11 (IC50 > 500 nM) did not dock under the given pharmacophore constraint (i.e., the Fe(heme)-N(ligand) interaction), benzyl-substituted compound 12 (IC50 = 154 nM) was found to exploit a previously unexplored subpocket of the inhibitor binding site. By structural optimization based on the pharmacophore hypothesis, 25 novel compounds were synthesized, among them highly potent CYP11B2 inhibitors (e.g., 17, IC50 = 2.7 nM) with pronounced selectivity toward the most important steroidogenic and hepatic CYP enzymes.

Overcoming undesirable CYP1A2 inhibition of pyridylnaphthalene-type aldosterone synthase inhibitors: influence of heteroaryl derivatization on potency and selectivity.

Recently, we reported on the development of potent and selective inhibitors of aldosterone synthase (CYP11B2) for the treatment of congestive heart failure and myocardial fibrosis. A major drawback of these nonsteroidal compounds was a strong inhibition of the hepatic drug-metabolizing enzyme CYP1A2. In the present study, we examined the influence of substituents in the heterocycle of lead structures with a naphthalene molecular scaffold to overcome this unwanted side effect. With respect to CYP11B2 inhibition, some substituents induced a dramatic increase in inhibitory potency. The methoxyalkyl derivatives 22 and 26 are the most potent CYP11B2 inhibitors up to now (IC50 = 0.2 nM). Most compounds also clearly discriminated between CYP11B2 and CYP11B1, and the CYP1A2 potency significantly decreased in some cases (e.g., isoquinoline derivative 30 displayed only 6% CYP1A2 inhibition at 2 microM concentration). Furthermore, isoquinoline derivative 28 proved to be capable of passing the gastrointestinal tract and reached the general circulation after peroral administration to male Wistar rats.

Imidazolylmethylbenzophenones as highly potent aromatase inhibitors.

Suppression of tumor and plasma estrogen levels by inhibition of aromatase is one of the most effective treatments for postmenopausal breast cancer patients. Starting from an easy, synthetically accessible, benzophenone scaffold, a new class of potent aromatase inhibitors was synthesized, endowed with high selectivity with respect to 17 alpha-hydroxylase/17,20-lyase (CYP17). Compounds 1b and 1d proved to be among the most potent inhibitors described so far.