Changes in the cis-trans isomer selectivity of a reversed-phase liquid chromatography column during use with acidic mobile phase conditions.

Reversed-phase liquid chromatography (RPLC) is the analytical tool of choice for monitoring process-related organic impurities and degradants in pharmaceutical materials. Its popularity is due to its general ease-of-use, high performance, and reproducibility in most cases, all of which have improved as the technique has matured over the past few decades. Nevertheless, in our work we still occasionally observe situations where RPLC methods are not as robust as we would like them to be in practice due to variations in stationary phase chemistry between manufactured batches (i.e., lot-to-lot variability), and changes in stationary phase chemistry over time. Over the last three decades several models of RPLC selectivity have been developed and used to quantify and rationalize the effects of numerous parameters (e.g., effect of bonded phase density) on separation selectivity. The Hydrophobic Subtraction Model (HSM) of RPLC selectivity has been used extensively for these purposes; currently the publicly available database of column parameters contains data for 750 columns. In this work we explored the possibility that the HSM could be used to better understand the chemical basis of observed differences in stationary phase selectivity when they occur - for example, lot-to-lot variations or changes in selectivity during column use. We focused our attention on differences and changes in the observed selectivity for a pair of cis-trans isomers of a pharmaceutical intermediate. Although this is admittedly a challenging case, we find that the observed changes in selectivity are not strongly correlated with HSM column parameters, suggesting that there is a gap in the information provided by the HSM with respect to cis-trans isomer selectivity specifically. Further work with additional probe molecules showed that larger changes in cis-trans isomer selectivity were observed for pairs of molecules with greater molecular complexity, compared to the selectivity changes observed for simpler molecules. These results do not provide definitive answers to questions about the chemical basis of changes in stationary phase chemistry that lead to observed differences in cis-trans isomer selectivity. However, the results do provide important insights about the critical importance of molecular complexity when choosing probe compounds and indicate opportunities to develop improved selectivity models with increased sensitivity for cis-trans isomer selectivity.

Derivatization and determination of residual N,N-Carbonyldiimidazole by LC for an in-process control test.

For the robust analysis of N,N-Carbonyldiimidazole (CDI), its derivatization into a more stable compound may be needed. Herein, the reaction of CDI with N-benzylmethylamine followed by LC-UV quantitative analysis was explored. Reaction conditions as well as LC method feasibility were demonstrated by qualification of selectivity from other impurities and reagents, linearity across a range of 0.05-0.15%w/w, spike and recovery across a range of 0.05-0.15%w/w, reaction reproducibility with various samples, reagents and analytical chemists, and sample stability of over 24 h. Rapid and quantitative derivatization of residual CDI was achieved at 0.1% w/w relative to the synthetic product under consideration. A fit-for-purpose limit test using a RPLC-UV method as an in-process control for the reaction completion of product, at scale, was successfully implemented and executed.

Rapid development of a bromochloropyridine regioisomer purity method enabled by strategic LC screening.

With the intent to provide aligned, impactful, and efficient strategies for liquid chromatography method development, tier-based stationary/mobile phase screening workflows have been implemented in the Chemical Process Development department at Bristol Myers Squibb. These workflows are utilized as tools that enable more rapid method generation for early to mid-stage clinical development programs. An illustrative example of applying this approach was the method development for 3-bromo-2-chloropyridine and six of its positional isomeric impurities. Several parameters (gradient time, flow rate, column geometry, particle size, temperature, and solvent effects) were evaluated to achieve a baseline resolved separation for this challenging mixture. The impact that the screening workflows have regarding timesavings, effort, and resourcing to develop and optimize this LC method will be discussed.

ICTV Virus Taxonomy Profile: Parvoviridae.

Members of the family Parvoviridae are small, resilient, non-enveloped viruses with linear, single-stranded DNA genomes of 4-6 kb. Viruses in two subfamilies, the Parvovirinae and Densovirinae, are distinguished primarily by their respective ability to infect vertebrates (including humans) versus invertebrates. Being genetically limited, most parvoviruses require actively dividing host cells and are host and/or tissue specific. Some cause diseases, which range from subclinical to lethal. A few require co-infection with helper viruses from other families. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Parvoviridae, which is available at www.ictv.global/report/parvoviridae.

White paper on microbial anti-cancer therapy and prevention.

In this White Paper, we discuss the current state of microbial cancer therapy. This paper resulted from a meeting ('Microbial Based Cancer Therapy') at the US National Cancer Institute in the summer of 2017. Here, we define 'Microbial Therapy' to include both oncolytic viral therapy and bacterial anticancer therapy. Both of these fields exploit tumor-specific infectious microbes to treat cancer, have similar mechanisms of action, and are facing similar challenges to commercialization. We designed this paper to nucleate this growing field of microbial therapeutics and increase interactions between researchers in it and related fields. The authors of this paper include many primary researchers in this field. In this paper, we discuss the potential, status and opportunities for microbial therapy as well as strategies attempted to date and important questions that need to be addressed. The main areas that we think will have the greatest impact are immune stimulation, control of efficacy, control of delivery, and safety. There is much excitement about the potential of this field to treat currently intractable cancer. Much of the potential exists because these therapies utilize unique mechanisms of action, difficult to achieve with other biological or small molecule drugs. By better understanding and controlling these mechanisms, we will create new therapies that will become integral components of cancer care.

Optimizing the Targeting of Mouse Parvovirus 1 to Murine Melanoma Selects for Recombinant Genomes and Novel Mutations in the Viral Capsid Gene.

Combining virus-enhanced immunogenicity with direct delivery of immunomodulatory molecules would represent a novel treatment modality for melanoma, and would require development of new viral vectors capable of targeting melanoma cells preferentially. Here we explore the use of rodent protoparvoviruses targeting cells of the murine melanoma model B16F10. An uncloned stock of mouse parvovirus 1 (MPV1) showed some efficacy, which was substantially enhanced following serial passage in the target cell. Molecular cloning of the genes of both starter and selected virus pools revealed considerable sequence diversity. Chimera analysis mapped the majority of the improved infectivity to the product of the major coat protein gene, VP2, in which linked blocks of amino acid changes and one or other of two apparently spontaneous mutations were selected. Intragenic chimeras showed that these represented separable components, both contributing to enhanced infection. Comparison of biochemical parameters of infection by clonal viruses indicated that the enhancement due to changes in VP2 operates after the virus has bound to the cell surface and penetrated into the cell. Construction of an in silico homology model for MPV1 allowed placement of these changes within the capsid shell, and revealed aspects of the capsid involved in infection initiation that had not been previously recognized.

Atomic Resolution Structure of the Oncolytic Parvovirus LuIII by Electron Microscopy and 3D Image Reconstruction.

LuIII, a protoparvovirus pathogenic to rodents, replicates in human mitotic cells, making it applicable for use to kill cancer cells. This virus group includes H-1 parvovirus (H-1PV) and minute virus of mice (MVM). However, LuIII displays enhanced oncolysis compared to H-1PV and MVM, a phenotype mapped to the major capsid viral protein 2 (VP2). This suggests that within LuIII VP2 are determinants for improved tumor lysis. To investigate this, the structure of the LuIII virus-like-particle was determined using single particle cryo-electron microscopy and image reconstruction to 3.17 Å resolution, and compared to the H-1PV and MVM structures. The LuIII VP2 structure, ordered from residue 37 to 587 (C-terminal), had the conserved VP topology and capsid morphology previously reported for other protoparvoviruses. This includes a core ß-barrel and α-helix A, a depression at the icosahedral 2-fold and surrounding the 5-fold axes, and a single protrusion at the 3-fold axes. Comparative analysis identified surface loop differences among LuIII, H-1PV, and MVM at or close to the capsid 2- and 5-fold symmetry axes, and the shoulder of the 3-fold protrusions. The 2-fold differences cluster near the previously identified MVM sialic acid receptor binding pocket, and revealed potential determinants of protoparvovirus tumor tropism.

Cryo-EM maps reveal five-fold channel structures and their modification by gatekeeper mutations in the parvovirus minute virus of mice (MVM) capsid.

In minute virus of mice (MVM) capsids, icosahedral five-fold channels serve as portals mediating genome packaging, genome release, and the phased extrusion of viral peptides. Previous studies suggest that residues L172 and V40 are essential for channel function. The structures of MVMi wildtype, and mutant L172T and V40A virus-like particles (VLPs) were solved from cryo-EM data. Two constriction points, termed the mid-gate and inner-gate, were observed in the channels of wildtype particles, involving residues L172 and V40 respectively. While the mid-gate of V40A VLPs appeared normal, in L172T adjacent channel walls were altered, and in both mutants there was major disruption of the inner-gate, demonstrating that direct L172:V40 bonding is essential for its structural integrity. In wildtype particles, residues from the N-termini of VP2 map into claw-like densities positioned below the channel opening, which become disordered in the mutants, implicating both L172 and V40 in the organization of VP2 N-termini.

The MVMp P4 promoter is a host cell-type range determinant in vivo.

The protoparvovirus early promoters, e.g. P4 of Minute Virus of Mice (MVM), play a critical role during infection. Initial P4 activity depends on the host transcription machinery only. Since this is cell-type dependent, it is hypothesized that P4 is a host cell-type range determinant. Yet host range determinants have mapped mostly to capsid, never P4. Here we test the hypothesis using the mouse embryo as a model system. Disruption of the CRE element of P4 drastically decreased infection levels without altering range. However, when we swapped promoter elements of MVM P4 with those from equivalent regions of the closely related H1 virus, we observed elimination of infection in fibroblasts and chondrocytes and the acquisition of infection in skeletal muscle. We conclude that P4 is a host range determinant and a target for modifying the productive infection potential of the virus - an important consideration in adapting these viruses for oncotherapy.

In Vivo Examination of Mouse APOBEC3- and Human APOBEC3A- and APOBEC3G-Mediated Restriction of Parvovirus and Herpesvirus Infection in Mouse Models.

UNLABELLED: APOBEC3 knockout and human APOBEC3A and -3G transgenic mice were tested for their ability to be infected by the herpesviruses herpes simplex virus 1 and murine herpesvirus 68 and the parvovirus minute virus of mice (MVM). Knockout, APOBEC3A and APOBEC3G transgenic, and wild-type mice were equally infected by the herpesviruses, while APOBEC3A but not mouse APOBEC3 conferred resistance to MVM. No viruses showed evidence of cytidine deamination by mouse or human APOBEC3s. These data suggest that in vitro studies implicating APOBEC3 proteins in virus resistance may not reflect their role in vivo IMPORTANCE: It is well established that APOBEC3 proteins in different species are a critical component of the host antiretroviral defense. Whether these proteins also function to inhibit other viruses is not clear. There have been a number of in vitro studies suggesting that different APOBEC3 proteins restrict herpesviruses and parvoviruses, among others, but whether they also work in vivo has not been demonstrated. Our studies looking at the role of mouse and human APOBEC3 proteins in transgenic and knockout mouse models of viral infection suggest that these restriction factors are not broadly antiviral and demonstrate the importance of testing their activity in vivo.

Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding.

Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins.

Complementation for an essential ancillary non-structural protein function across parvovirus genera.

Parvoviruses encode a small number of ancillary proteins that differ substantially between genera. Within the genus Protoparvovirus, minute virus of mice (MVM) encodes three isoforms of its ancillary protein NS2, while human bocavirus 1 (HBoV1), in the genus Bocaparvovirus, encodes an NP1 protein that is unrelated in primary sequence to MVM NS2. To search for functional overlap between NS2 and NP1, we generated murine A9 cell populations that inducibly express HBoV1 NP1. These were used to test whether NP1 expression could complement specific defects resulting from depletion of MVM NS2 isoforms. NP1 induction had little impact on cell viability or cell cycle progression in uninfected cells, and was unable to complement late defects in MVM virion production associated with low NS2 levels. However, NP1 did relocate to MVM replication centers, and supports both the normal expansion of these foci and overcomes the early paralysis of DNA replication in NS2-null infections.

Genome sequence of tumor virus x, a member of the genus protoparvovirus in the family parvoviridae.

The orphan parvovirus tumor virus X (TVX) has potent oncolytic activity. Compared to other viruses from the species Rodent protoparvovirus 1, TVX has a 111 nucleotide deletion in its nonstructural (NS) gene, a 24 nucleotide insertion in VP1, and a 93 nucleotide repeat initiating from the C-terminus of the capsid gene.

Profiling of glycan receptors for minute virus of mice in permissive cell lines towards understanding the mechanism of cell recognition.

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galß1-4GlcNAc motif (Neu5Acα2-3Galß1-4GlcNAc or 3'SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3'SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3'SIA-Le(X) identified in a previous glycan microarray screen.

The family Parvoviridae.

A set of proposals to rationalize and extend the taxonomy of the family Parvoviridae is currently under review by the International Committee on Taxonomy of Viruses (ICTV). Viruses in this family infect a wide range of hosts, as reflected by the longstanding division into two subfamilies: the Parvovirinae, which contains viruses that infect vertebrate hosts, and the Densovirinae, encompassing viruses that infect arthropod hosts. Using a modified definition for classification into the family that no longer demands isolation as long as the biological context is strong, but does require a near-complete DNA sequence, 134 new viruses and virus variants were identified. The proposals introduce new species and genera into both subfamilies, resolve one misclassified species, and improve taxonomic clarity by employing a series of systematic changes. These include identifying a precise level of sequence similarity required for viruses to belong to the same genus and decreasing the level of sequence similarity required for viruses to belong to the same species. These steps will facilitate recognition of the major phylogenetic branches within genera and eliminate the confusion caused by the near-identity of species and viruses. Changes to taxon nomenclature will establish numbered, non-Latinized binomial names for species, indicating genus affiliation and host range rather than recapitulating virus names. Also, affixes will be included in the names of genera to clarify subfamily affiliation and reduce the ambiguity that results from the vernacular use of "parvovirus" and "densovirus" to denote multiple taxon levels.

Parvoviruses: Small Does Not Mean Simple.

Parvoviruses are small, rugged, nonenveloped protein particles containing a linear, nonpermuted, single-stranded DNA genome of ∼5 kb. Their limited coding potential requires optimal adaptation to the environment of particular host cells, where entry is mediated by a variable program of capsid dynamics, ultimately leading to genome ejection from intact particles within the host nucleus. Genomes are amplified by a continuous unidirectional strand-displacement mechanism, a linear adaptation of rolling circle replication that relies on the repeated folding and unfolding of small hairpin telomeres to reorient the advancing fork. Progeny genomes are propelled by the viral helicase into the preformed capsid via a pore at one of its icosahedral fivefold axes. Here we explore how the fine-tuning of this unique replication system and the mechanics that regulate opening and closing of the capsid fivefold portals have evolved in different viral lineages to create a remarkably complex spectrum of phenotypes.

Distinct host cell fates for human malignant melanoma targeted by oncolytic rodent parvoviruses.

The rodent parvoviruses are known to be oncoselective, and lytically infect many transformed human cells. Because current therapeutic regimens for metastatic melanoma have low response rates and have little effect on improving survival, this disease is a prime candidate for novel approaches to therapy, including oncolytic parvoviruses. Screening of low-passage, patient-derived melanoma cell lines for multiplicity-dependent killing by a panel of five rodent parvoviruses identified LuIII as the most melanoma-lytic. This property was mapped to the LuIII capsid gene, and an efficiently melanoma tropic chimeric virus shown to undergo three types of interaction with primary human melanoma cells: (1) complete lysis of cultures infected at very low multiplicities; (2) acute killing resulting from viral protein synthesis and DNA replication, without concomitant expansion of the infection, due to failure to export progeny virions efficiently; or (3) complete resistance that operates at an intracellular step following virion uptake, but preceding viral transcription.

Parvoviral left-end hairpin ears are essential during infection for establishing a functional intranuclear transcription template and for efficient progeny genome encapsidation.

The 121-nucleotide left-end telomere of Minute Virus of Mice (MVM) can be folded into a Y-shaped hairpin with short axial ears that are highly conserved within genus Parvovirus. To explore their potential role(s) during infection, we constructed infectious plasmid clones that lacked one or other ear. Although these were nonviable when transfected into A9 cells, excision of the viral genome and DNA amplification appeared normal, and viral transcripts and proteins were expressed, but progeny virion production was minimal, supporting the idea of a potential role for the ears in genome packaging. To circumvent the absence of progeny that confounded further analysis of these mutants, plasmids were transfected into 293T cells both with and without an adenovirus helper construct, generating single bursts of progeny. These virions bound to A9 cells and were internalized but failed to initiate viral transcription, protein expression, or DNA replication. No defects in mutant virion stability or function could be detected in vitro. Significantly, mutant capsid gene expression and DNA replication could be rescued by coinfection with wild-type virions carrying a replication-competent, capsid-gene-replacement vector. To pinpoint where such complementation occurred, prior transfection of plasmids expressing only MVM nonstructural proteins was explored. NS1 alone, but not NS2, rescued transcription and protein expression from both P4 and P38 promoters, whereas NS1 molecules deleted for their C-terminal transactivation domain did not. These results suggest that the mutant virions reach the nucleus, uncoat, and are converted to duplex DNA but require an intact left-end hairpin structure to form the initiating transcription complex.

Parvovirus evades interferon-dependent viral control in primary mouse embryonic fibroblasts.

Engagement of innate viral sensors elicits a robust antiviral program via the induction of type I interferons (IFNs). Innate defense mechanisms against ssDNA viruses are not well defined. Here, we examine type I IFN induction and effectiveness in controlling a ssDNA virus. Using mouse embryonic fibroblasts (MEFs), we found that a murine parvovirus, minute virus of mice (MVMp), induced a delayed but significant IFN response. MEFs deficient in mitochondrial antiviral signaling protein (MAVS) mounted a wild-type IFN response to MVMp infection, indicating that RIG-I-dependent RNA intermediate recognition is not required for innate sensing of this virus. However, MVMp-induced IFNs, as well recombinant type I IFNs, were unable to inhibit viral replication. Finally, MVMp infected cells became unresponsive to Poly (I:C) stimulation. Together, these data suggest that the MVMp efficiently evades antiviral immune mechanisms imposed by type I IFNs, which may in part explain their efficient transmission between mice.

Toll-like receptor 9 in plasmacytoid dendritic cells fails to detect parvoviruses.

Toll-like receptor 9 (TLR9) recognizes genomes of double-stranded DNA (dsDNA) viruses in the endosome to stimulate plasmacytoid dendritic cells (pDCs). However, how and if viruses with single-stranded DNA (ssDNA) genomes are detected by pDCs remain unclear. Here we have shown that despite the ability of purified genomic DNA to stimulate TLR9 and despite the ability to enter TLR9 endosomes, ssDNA viruses of the Parvoviridae family failed to elicit an interferon (IFN) response in pDCs.