Treatment switches of disease-modifying therapies in people with multiple sclerosis: long-term experience from the German MS Registry.

Background: The spectrum of disease-modifying therapies (DMTs) for people with multiple sclerosis (PwMS) has expanded over years, but data on treatment strategies is largely lacking. DMT switches are common clinical practice. Objective: To compare switchers and non-switchers, characterize the first DMT switch and identify reasons and predictors for switching the first DMT. Methods: Data on 2722 PwMS from the German MS Registry were retrospectively analyzed regarding sociodemographic/clinical differences between 1361 switchers (PwMS discontinuing the first DMT) and non-switchers matched according to age, sex, and observation period. Frequencies of first and second DMTs were calculated and switch reasons identified. Predictors for DMT switches were revealed using univariable and multivariable regression models. Results: Switchers and non-switchers differed significantly regarding time to first DMT, education, calendar period of the first DMT start (2014-2017 versus 2018-2021), first DMT class used [mild-to-moderate efficacy (MME) versus high-efficacy (HE) DMT], time on first DMT, and disease activity at first DMT start or cessation/last follow-up. The majority of PwMS started with MME DMTs (77.1%), with the most common being glatiramer acetate, dimethyl/diroximel fumarate, and beta-interferon variants. Switchers changed treatment more often to HE DMTs (39.6%), most commonly sphingosine-1-phosphate receptor modulators, anti-CD20 monoclonal antibodies, and natalizumab. Fewer PwMS switched to MME DMTs (35.9%), with the most common being dimethyl/diroximel fumarate, teriflunomide, or beta-interferon. Among 1045 PwMS with sufficient data (76.8% of 1361 switchers), the most frequent reasons for discontinuing the first DMT were disease activity despite DMT (63.1%), adverse events (17.1%), and patient request (8.3%). Predictors for the first DMT switch were MME DMT as initial treatment [odds ratio (OR) = 2.83 (1.76-4.61), p < 0.001; reference: HE DMT], first DMT initiation between 2014 and 2017 [OR = 11.55 (6.93-19.94), p < 0.001; reference: 2018-2021], and shorter time on first DMT [OR = 0.22 (0.18-0.27), p < 0.001]. Conclusion: The initial use of MME DMTs was among the strongest predictors of DMT discontinuation in a large German retrospective MS cohort, arguing for the need for prospective treatment strategy trials, not only but also on the initial broad use of HE DMTs in PwMS.

Analysis of prototype foamy virus particle-host cell interaction with autofluorescent retroviral particles.

BACKGROUND: The foamy virus (FV) replication cycle displays several unique features, which set them apart from orthoretroviruses. First, like other B/D type orthoretroviruses, FV capsids preassemble at the centrosome, but more similar to hepadnaviruses, FV budding is strictly dependent on cognate viral glycoprotein coexpression. Second, the unusually broad host range of FV is thought to be due to use of a very common entry receptor present on host cell plasma membranes, because all cell lines tested in vitro so far are permissive. RESULTS: In order to take advantage of modern fluorescent microscopy techniques to study FV replication, we have created FV Gag proteins bearing a variety of protein tags and evaluated these for their ability to support various steps of FV replication. Addition of even small N-terminal HA-tags to FV Gag severely impaired FV particle release. For example, release was completely abrogated by an N-terminal autofluorescent protein (AFP) fusion, despite apparently normal intracellular capsid assembly. In contrast, C-terminal Gag-tags had only minor effects on particle assembly, egress and particle morphogenesis. The infectivity of C-terminal capsid-tagged FV vector particles was reduced up to 100-fold in comparison to wild type; however, infectivity was rescued by coexpression of wild type Gag and assembly of mixed particles. Specific dose-dependent binding of fluorescent FV particles to target cells was demonstrated in an Env-dependent manner, but not binding to target cell-extracted- or synthetic- lipids. Screening of target cells of various origins resulted in the identification of two cell lines, a human erythroid precursor- and a zebrafish- cell line, resistant to FV Env-mediated FV- and HIV-vector transduction. CONCLUSIONS: We have established functional, autofluorescent foamy viral particles as a valuable new tool to study FV--host cell interactions using modern fluorescent imaging techniques. Furthermore, we succeeded for the first time in identifying two cell lines resistant to Prototype Foamy Virus Env-mediated gene transfer. Interestingly, both cell lines still displayed FV Env-dependent attachment of fluorescent retroviral particles, implying a post-binding block potentially due to lack of putative FV entry cofactors. These cell lines might ultimately lead to the identification of the currently unknown ubiquitous cellular entry receptor(s) of FVs.

Subviral particle release determinants of prototype foamy virus.

Glycoproteins of several viruses have the capacity to induce release of noninfectious, capsidless particulate structures containing only the viral glycoprotein. Such structures are often called subviral particles (SVP). Foamy viruses (FVs), a special type of retroviruses with a replication strategy combining features of both orthoretroviruses and hepadnaviruses, express a glycoprotein (Env) which has the ability to induce SVP release. However, unlike human hepatitis B virus, prototype FV (PFV) naturally secretes only small amounts of SVPs, because ubiquitination of the Env protein seems to suppress the intrinsic capacity for induction of SVP release. In this study, we characterized the structural determinants influencing PFV SVP release, examined the role of specific Env ubiquitination sites in the regulation of this process, and analyzed the requirement of the cellular vacuolar protein sorting (VPS) machinery for SVP egress. We observed that the cytoplasmic and membrane-spanning domains of both the leader peptide (LP) and the transmembrane (TM) subunit harbor essential as well as inhibitory domains. Furthermore, only ubiquitination at the most N-terminal lysine residues (K(14) and K(15)) in LP reduced cell surface expression and suppressed SVP release to wild-type levels. This suggests that interaction of Env with cellular components required for SVP release suppression is effective only when Env is ubiquitinated at these lysine residues but not at others. Finally, SVP release was sensitive to dominant-negative mutants of late components, but not early components, of the cellular VPS machinery. PFV therefore differs from hepatitis B virus in using the same cellular pathway for egress of both virions and SVPs.

Inhibition of the proteasome influences murine and human dendritic cell development in vitro and in vivo.

Dendritic cells (DC) are the most potent antigen-presenting cells (APC) known today and are designated as nature's adjuvant since they are the only antigen-presenting cell type capable of inducing naïve T cell responses in vivo. In order to become potent T cell stimulators DC have to mature. This mature DC phenotype is characterized amongst other characteristics by the up-regulation of co-stimulatory molecules such as CD40, CD80, CD86 and the cell surface expression of CD83. Inhibition of their expression blocks the immune responses in vitro and in vivo, and thus represents an interesting strategy to control undesired and/or over-activated immune responses such as in autoimmune disorders, transplant rejections and allergies. Here we investigated the in vitro and in vivo effects of the proteasome inhibitor Velcade in respect to DC phenotype and DC functions in murine and human DC. Interestingly, in vitro, DC maturation as well as DC-mediated T cell stimulation and cytokine production was impaired. Furthermore, administration of the inhibitor in vivo resulted in a reduced mature phenotype of ex vivo generated murine DC. Thus, inhibition of the proteasome interferes with DC maturation and subsequently with DC-mediated T cell stimulation events.

Characterization of the prototype foamy virus envelope glycoprotein receptor-binding domain.

The foamy virus (FV) glycoprotein precursor gp130(Env) undergoes a highly unusual biosynthesis, resulting in the generation of three particle-associated, mature subunits, leader peptide (LP), surface (SU), and transmembrane (TM). Little structural and functional information on the extracellular domains of FV Env is available. In this study, we characterized the prototype FV (PFV) Env receptor-binding domain (RBD) by flow cytometric analysis of recombinant PFV Env immunoadhesin binding to target cells. The extracellular domains of the C-terminal TM subunit as well as targeting of the recombinant immunoadhesins by the cognate LP to the secretory pathway were dispensable for target cell binding, suggesting that the PFV Env RBD is contained within the SU subunit. N- and C-terminal deletion analysis of the SU domain revealed a minimal continuous RBD spanning amino acids (aa) 225 to 555; however, internal deletions covering the region from aa 397 to 483, but not aa 262 to 300 or aa 342 to 396, were tolerated without significant influence on host cell binding. Analysis of individual cysteine point mutants in PFV SU revealed that only most of those located in the nonessential region from aa 397 to 483 retained residual binding activity. Interestingly, analysis of various N-glycosylation site mutants suggests an important role of carbohydrate chain attachment to N391, either for direct interaction with the receptor or for correct folding of the PFV Env RBD. Taken together, these results suggest that a bipartite sequence motif spanning aa 225 to 396 and aa 484 to 555 is essential for formation of the PFV Env RBD, with N-glycosylation site at position 391 playing a crucial role for host cell binding.

Ubiquitination of the prototype foamy virus envelope glycoprotein leader peptide regulates subviral particle release.

Foamy virus (FV) particle egress is unique among retroviruses because of its essential requirement for Gag and Env coexpression for budding and particle release. The FV glycoprotein undergoes a highly unusual biosynthesis resulting in the generation of three particle-associated, mature subunits, leader peptide (LP), surface (SU), and transmembrane (TM), derived from a precursor protein by posttranslational proteolysis mediated by furin or furinlike proteases. Previously at least three LP products of different molecular weights were detected in purified FV particles. Here we demonstrate that the higher-molecular-weight forms gp28LP and gp38LP are ubiquitinated variants of the major gp18LP cleavage product, which has a type II membrane topology. Furthermore, we show that all five lysine residues located within the N-terminal 60-amino-acid cytoplasmic domain of gp18LP can potentially be ubiquitinated, however, there seems to be a preference for using the first three. Inactivation of ubiquitination sites individually resulted in no obvious phenotype. However, simultaneous inactivation of the first three or all five ubiquitination sites in gp18LP led to a massive increase in subviral particles released by these mutant glycoproteins that were readily detectable by electron microscopy analysis upon expression of the ubiquitination-deficient glycoprotein by itself or in a proviral context. Surprisingly, only the quintuple ubiquitination mutant showed a two- to threefold increase in single-cycle infectivity assays, whereas all other mutants displayed infectivities similar to that of the wild type. Taken together, these data suggest that the balance between viral and subviral particle release of FVs is regulated by ubiquitination of the glycoprotein LP.

Analysis and function of prototype foamy virus envelope N glycosylation.

The prototype foamy virus (PFV) glycoprotein, which is essential for PFV particle release, displays a highly unusual biosynthesis, resulting in posttranslational cleavage of the precursor protein into three particle-associated subunits, i.e., leader peptide (LP), surface (SU), and transmembrane (TM). Glycosidase digestion of metabolically labeled PFV particles revealed the presence of N-linked carbohydrates on all subunits. The differential sensitivity to specific glycosidases indicated that all oligosaccharides on LP and TM are of the high-mannose or hybrid type, whereas most of those attached to SU, which contribute to about 50% of its molecular weight, are of the complex type. Individual inactivation of all 15 potential N-glycosylation sites in PFV Env demonstrated that 14 are used, i.e., 1 out of 2 in LP, 10 in SU, and 3 in TM. Analysis of the individual altered glycoproteins revealed defects in intracellular processing, support of particle release, and infectivity for three mutants, having the evolutionarily conserved glycosylation sites N8 in SU or N13 and N15 in the cysteine-rich central "sheets-and-loops" region of TM inactivated. Examination of alternative mutants with mutations affecting glycosylation or surrounding sequences at these sites indicated that inhibition of glycosylation at N8 and N13 most likely is responsible for the observed replication defects, whereas for N15 surrounding sequences seem to contribute to a temperature-sensitive phenotype. Taken together these data demonstrate that PFV Env and in particular the SU subunit are heavily N glycosylated and suggest that although most carbohydrates are dispensable individually, some evolutionarily conserved sites are important for normal Env function of FV isolates from different species.

Prototype foamy virus envelope glycoprotein leader peptide processing is mediated by a furin-like cellular protease, but cleavage is not essential for viral infectivity.

Analogous to cellular glycoproteins, viral envelope proteins contain N-terminal signal sequences responsible for targeting them to the secretory pathway. The prototype foamy virus (PFV) envelope (Env) shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N and C terminus located in the cytoplasm. Coexpression of FV glycoprotein and interaction of its leader peptide (LP) with the viral capsid is essential for viral particle budding and egress. Processing of PFV Env into the particle-associated LP, surface (SU), and transmembrane (TM) subunits occur posttranslationally during transport to the cell surface by yet-unidentified cellular proteases. Here we provide strong evidence that furin itself or a furin-like protease and not the signal peptidase complex is responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin G immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Fully processed SU was almost completely absent in viral particles of mutants having conserved arginine residues replaced by alanines in the first furin consensus sequence, but normal processing was observed upon mutation of the second motif. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect.