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Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections (LRTI) in infants, and toddlers and vaccines are not yet available. A pediatric RSV vaccine (ChAd155-RSV) is being developed to protect infants against RSV disease. The ChAd155-RSV vaccine consists of a recombinant replication-deficient chimpanzee-derived adenovirus (ChAd) group C vector engineered to express the RSV antigens F, N, and M2-1. The local and systemic effects of three bi-weekly intramuscular injections of the ChAd155-RSV vaccine was tested in a repeated-dose toxicity study in rabbits. After three intramuscular doses, the ChAd155-RSV vaccine was considered well-tolerated. Changes due to the vaccine-elicited inflammatory reaction/immune response were observed along with transient decreases in platelet count without physiological consequences, already reported for other adenovirus-based vaccines. In addition, the biodistribution and shedding of ChAd155-RSV were also characterized in two studies in rats. The distribution and persistence of the ChAd155-RSV vaccine candidate was consistent with other similar adenovector-based vaccines, with quantifiable levels of ChAd155-RSV observed at the injection site (muscle) and the draining lymph nodes up to 69 days post administration. The shedding results demonstrated that ChAd155-RSV was generally not detectable in any secretions or excreta samples. In conclusion, the ChAd155-RSV vaccine was well-tolerated locally and systemically.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections, and vaccines are needed to treat young children and older adults. One of GSK’s candidate vaccines for RSV contains recombinant RSVPreF3 protein maintained in the prefusion conformation. The differences in immune function of young children and older adults potentially require different vaccine approaches. For young children, anti-RSV immunity can be afforded during the first months of life by vaccinating the pregnant mother during the third trimester with unadjuvanted RSVPreF3, which results in protection of the infant due to the transplacental passage of anti-RSV maternal antibodies. For older adults with a waning immune response, the approach is to adjuvant the RSVPreF3 vaccine with AS01 to elicit a more robust immune response. The local and systemic effects of biweekly intramuscular injections of the RSVPreF3 vaccine (unadjuvanted, adjuvanted with AS01, or coadministered with a diphtheria-tetanus-acellular pertussis vaccine) was tested in a repeated dose toxicity study in rabbits. After three intramuscular doses, the only changes observed were those commonly related to a vaccine-elicited inflammatory reaction. Subsequently, the effects of unadjuvanted RSVPreF3 vaccine on female fertility, embryo-fetal, and postnatal development of offspring were evaluated in rats and rabbits. There were no effects on pregnancy, delivery, lactation, or the pre- and postnatal development of offspring. In conclusion, the RSVPreF3 vaccine was well-tolerated locally and systemically and was not associated with any adverse effects on female reproductive function or on the pre- and postnatal growth and development of offspring.

Sequential intramuscular immunization with chimeric hemagglutinins (cHA) composed of the same conserved HA stalk domain and distinct HA heads is a proposed strategy to produce a supra-seasonal universal influenza vaccine. To evaluate the local tolerance and the local and systemic effects of this strategy, two studies were performed in rabbits. In the first study, two different split virion monovalent cHA vaccines, containing cH5/1N1 and cH8/1N1, with or without AS01 or AS03, were injected at a two-week interval. In the second study, animals were given these vaccines and two weeks later an additional dose of split virion monovalent cHA vaccine containing cH11/1N1, with or without AS01 or AS03. General health status, rectal temperature, local tolerance, ophthalmology, hematology, coagulation, and blood chemistry parameters were monitored. Macroscopic and microscopic evaluations were performed three days after the last dose and after a treatment-free recovery period. The treatment-related changes included body weight loss and food consumption decrease, increases in neutrophil count, C-reactive protein and fibrinogen levels. Microscopic signs of inflammation at the injection sites and immune stimulation of the draining lymph nodes and spleen were also noticed. Most post-injection findings could be linked to the transient inflammation due to the establishment of the desired vaccine-elicited immune response, and were mainly observed in the adjuvanted groups. In conclusion, the sequential administration of different cHA vaccines was locally and systemically well-tolerated in rabbits.

Human cytomegalovirus (HCMV) remains a serious threat for immunocompromised individuals, including transplant recipients and newborns. To date, all drugs licensed for the treatment of HCMV infection and disease target the viral DNA polymerase. Although these drugs are effective, several drawbacks are associated with their use, including toxicity and emergence of drug resistance. Hence, new and improved antivirals with novel molecular targets are urgently needed. Here we report on the antiviral properties of AIC246, a representative of a novel class of low-molecular-weight compounds that is currently undergoing clinical phase II studies. The anti-HCMV activity of AIC246 was evaluated in vitro and in vivo using various cell culture assays and an engineered mouse xenograft model. In addition, antiviral properties of the drug were characterized in comparison to the current gold standard ganciclovir. We demonstrate that AIC246 exhibits excellent in vitro inhibitory activity against HCMV laboratory strains and clinical isolates, retains activity against ganciclovir-resistant viruses, is well tolerated in different cell types (median selectivity index, 18,000), and exerts a potent in vivo efficacy in a mouse xenograft model. Moreover, we show that the antiviral block induced by AIC246 is reversible and the efficacy of the drug is not significantly affected by cell culture variations such as cell type or multiplicity of infection. Finally, initial mode-of-action analyses reveal that AIC246 targets a process in the viral replication cycle that occurs later than DNA synthesis. Thus, AIC246 acts via a mode of action that differs from that of polymerase inhibitors like ganciclovir.

The efficacy of BAY 57–1293, a novel non-nucleosidic inhibitor of herpes simplex virus 1 and 2 (HSV-1 and HSV-2), bovine herpesvirus and pseudorabies virus, was studied in the guinea pig model of genital herpes in comparison with the licensed drug valaciclovir (Valtrex™). Early therapy with BAY 57–1293 almost completely suppressed the symptoms of acute HSV-2 infection, and reduced virus shedding and viral load in the sacral dorsal root ganglia by up to three orders of magnitude, resulting in decreased latency and a greatly diminished frequency of subsequent recurrent episodes. In contrast, valaciclovir showed only moderate effects in this set of experiments. When treatment was initiated late during the course of disease after symptoms were apparent, that is, a setting closer to most clinical situations, the efficacy of therapy with BAY 57–1293 was even more pronounced. Compared with valaciclovir, BAY 57–1293 halved the time necessary for complete healing. Moreover, the onset of action was fast, so that only very few animals developed new lesions after treatment commenced. Finally, in a study addressing the treatment of recurrent disease in animals whose primary infection had remained untreated BAY 57–1293 was efficient in suppressing the episodes. In summary, superior potency and efficacy of BAY 57–1293 over standard treatment with valaciclovir was demonstrated in relevant animal models of human genital herpes disease in terms of abrogating an HSV infection, reducing latency and the frequency of subsequent recurrences. Furthermore, BAY 57–1293 shortens the time to healing even if initiation of therapy is delayed.

Introduction The pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody® with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology. Methods ALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control. Results ALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration. Conclusions ALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0651-0) contains supplementary material, which is available to authorized users.

The vast majority of the world population is infected with at least one member of the human herpesvirus family. Herpes simplex virus (HSV) infections are the cause of cold sores and genital herpes as well as life-threatening or sight-impairing disease mainly in immunocompromized patients, pregnant women and newborns. Since the milestone development in the late 1970s of acyclovir (Zovirax), a nucleosidic inhibitor of the herpes DNA polymerase, no new non-nucleosidic anti-herpes drugs have been introduced. Here we report new inhibitors of the HSV helicase-primase with potent in vitro anti-herpes activity, a novel mechanism of action, a low resistance rate and superior efficacy against HSV in animal models. BAY 57-1293 (N-[5-(aminosulfonyl)-4-methyl-1,3-thiazol-2-yl]-N-methyl-2-[4-(2-pyridinyl)phenyl]acetamide), a well-tolerated member of this class of compounds, significantly reduces time to healing, prevents rebound of disease after cessation of treatment and, most importantly, reduces frequency and severity of recurrent disease. Thus, this class of drugs has significant potential for the treatment of HSV disease in humans, including those resistant to current medications.

Abstract 1063 Poster Board I-85 ALX-0681 is a humanized bivalent Nanobody®, that binds to the A1 domain of von Willebrand factor (vWF) and hence blocks its interaction with platelet receptor GPIb-IX-V. Given its mode of action, ALX-0681 could provide an alternative treatment option for thrombotic thrombocytopenic purpura (TTP), a rare and life-threatening condition characterized by systemic platelet aggregation in the microcirculation mediated by activated vWF multimers. The goal of this Phase I trial in healthy volunteers was to determine the maximum tolerated dose (MTD) or biologically effective dose (BED) and the Phase II dosing and scheduling of ALX-0681, in order to support the further clinical development of ALX-0681 in TTP patients. In total, 36 healthy volunteers were included in this randomized, placebo-controlled study to evaluate the safety of single ascending doses and multiple doses of ALX-0681 administered subcutaneously (s.c.) (Table 1). Table 1 Dosing schedule for Phase I trial with ALX-0681 Cohort Dose (mg) Number of daily doses Subjects receiving ALX-0681 Subjects receiving placebo Single dose Cohort 1 2 1 3 1 Cohort 2 4 1 3 1 Cohort 3 8 1 3 1 Cohort 4 16 1 3 1 Cohort 5 10 1 3 1 Multiple dose Cohort 6 10 7 6 2 Cohort 7 10 14 6 2 Study endpoints included safety (dose limiting toxicities, adverse events (AEs) and immunogenicity), pharmacokinetics (PK), pharmacodynamics (PD) and pharmacological efficacy of ALX-0681. The latter endpoint was addressed by measuring the ristocetin cofactor (RICO) biomarker, reflecting vWF mediated inhibition of platelet aggregation. ALX-0681 was safe and well tolerated at all dose levels (Table 2). One unrelated SAE (meniscus lesion) occurred. The number of observed signs of bleeding and bruises increased with increasing treatment duration. However, all these events were of mild intensity. No signs of immunogenicity were observed for a minimum of 45 days after the last injection. Table 2 Summary of main safety results (number (%) of subjects with event) Dose level Subjects (n) AE SAE Bleeding Hematoma at injection site Hematoma at blood sampling site Other hematoma Single dose 2 mg 3 2 (67) 0 (0) 1 (33) 0 (0) 1 (33) 0 (0) 4 mg 3 2 (67) 0 (0) 1 (33) 0 (0) 0 (0) 0 (0) 8 mg 3 3 (100) 0 (0) 0 (0) 0 (0) 1 (33) 0 (0) 16 mg 3 3 (100) 0 (0) 0 (0) 0 (0) 0 (0) 1 (33) 10 mg 3 1 (33) 0 (0) 0 (0) 0 (0) 1 (33) 0 (0) Placebo 5 3 (60) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Multiple dose 10 mg (7d) 6 6 (100) 1 (17) 5 (83) 1 (17) 0 (0) 3 (50) Placebo (7d) 2 2 (100) 0 (0) 1 (50) 0 (0) 1 (50) 0 (0) 10 mg (14d) 6 6 (100) 0 (0) 5 (83) 5 (83) 4 (67) 5 (83) Placebo (14d) 2 2 (100) 0 (0) 1 (50) 0 (0) 0 (0) 0 (0) PK analysis showed a rapid increase in ALX-0681 plasma concentration (tmax = 4-10 h post dose), followed by a slow elimination phase (t1/2 = 10-78 h). All subjects dosed with ALX-0681 at 8 mg or higher showed complete inhibition of RICO activity to < 20% with an onset of 1-6 h post dose. This inhibition was maintained until 12-360 h post dose, depending on the dose level (Table 3). Overall, 20 (74%) and 17 (63%) of ALX-0681 treated subjects experienced a drop in vWF and FVIII levels below 50% of pre-dose levels, respectively. These events were all transient and not clinically significant. Table 3 Summary of main PD results (number (%) of subjects with event) Dose level Subjects (n) RICO < 20% vWF < 50% FVIII < 50% Subjects (%) Start (h)* Stop (h)* Single dose 2 mg 3 2 (67) 2-4 12-18 3 (100) 0 (0) 4 mg 3 2 (67) 4-6 18-36 1 (33) 1 (33) 8 mg 3 3 (100) 2-4 18-48 3 (100) 3 (100) 16 mg 3 3 (100) 1-4 48 0 (0) 2 (67) 10 mg 3 3 (100) 2-6 24-36 3 (100) 3 (100) Placebo 5 0 (0) NA NA 0 (0) 0 (0) Multiple dose 10 mg (7d) 6 6 (100) 2-4 168-192 5 (83) 3 (50) Placebo (7d) 2 0 (0) NA NA 0 (0) 0 (0) 10 mg (14d) 6 6 (100) 2-4 336-360 5 (83) 5 (83) Placebo (14d) 2 0 (0) NA NA 0 (0) 0 (0) * Time relative to first administration NA: not applicable In conclusion, ALX-0681 administered s.c. for up to 14 days was well tolerated and did not result in any clinically significant AEs. No local reactions, local intolerances or signs of clinically relevant bleeding were reported. The PD marker indicated complete inhibition of vWF mediated platelet aggregation following single daily s.c. injections of 10 mg, which was maintained over the 2 weeks treatment period. Multiple daily administration of s.c. injections of ALX-0681 did not result in an immunogenic reaction for a minimum of 45 days following completion of treatment. Based on the results of this study, ALX-0681 development will be advanced into a Phase II study in TTP patients to investigate the safety and efficacy of ALX-0681 in the target patient population. Disclosures: Abd-Elaziz: Ablynx NV: Consultancy. Kamphuisen:Ablynx NV: Consultancy. Lyssens:Ablynx NV: Employment. Reuvers:Ablynx NV: Consultancy. den Daas:Ablynx NV: Consultancy. Van Bockstaele:Ablynx NV: Employment. Vercruysse:Ablynx NV: Employment. Ulrichts:Ablynx NV: Employment. Baumeister:Ablynx NV: Employment. Crabbe:Ablynx NV: Employment. Compernolle:Ablynx NV: Employment. Holz:Ablynx NV: Employment.

The pseudorabies virus (PrV) gene homologous to herpes simplex virus type 1 (HSV-1) UL53, which encodes HSV-1 glycoprotein K (gK), has recently been sequenced (J. Baumeister, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 69:5560–5567, 1995). To identify the corresponding protein, a rabbit antiserum was raised against a 40-kDa glutathione S -transferase–gK fusion protein expressed in Escherichia coli . In Western blot analysis, this serum detected a 32-kDa polypeptide in PrV-infected cell lysates as well as a 36-kDa protein in purified virion preparations, demonstrating that PrV gK is a structural component of virions. After treatment of purified virions with endoglycosidase H, a 34-kDa protein was detected, while after incubation with N -glycosidase F, a 32-kDa protein was specifically recognized. This finding indicates that virion gK is modified by N-linked glycans of complex as well as high-mannose type. For functional analysis, the UL53 open reading frame was interrupted after codon 164 by insertion of a gG- lacZ expression cassette into the wild-type PrV genome (PrV-gKβ) or by insertion of the bovine herpesvirus 1 gB gene into a PrV gB − genome (PrV-gK gB ). Infectious mutant virus progeny was obtained only on complementing gK-expressing cells, suggesting that gK has an important function in the replication cycle. After infection of Vero cells with either gK mutant, only single infected cells or small foci of infected cells were visible. In addition, virus yield was reduced approximately 30-fold, and penetration kinetics showed a delay in entry which could be compensated for by phenotypic gK complementation. Interestingly, the plating efficiency of PrV-gKβ was similar to that of wild-type PrV on complementing and noncomplementing cells, pointing to an essential function of gK in virus egress but not entry. Ultrastructurally, virus assembly and morphogenesis of PrV gK mutants in noncomplementing cells were similar to wild-type virus. However, late in infection, numerous nucleocapsids were found directly underneath the plasma membrane in stages typical for the entry process, a phenomenon not observed after wild-type virus infection and also not visible after infection of gK-complementing cells. Thus, we postulate that presence of gK is important to inhibit immediate reinfection.

Background Interleukin-6 (IL-6) is a pleiotropic cytokine inducing a wide range of biological activities via its receptor, which can either be soluble (sIL-6R) or membrane-bound (mIL-6R). Blocking of IL-6R results in clinical benefit in rheumatoid arthritis as demonstrated by the marketed IL-6R inhibitor tocilizumab (TCZ). Signalling via the mIL-6R (“classical pathway”) is confined to selected cell types due to the restricted expression of mIL-6R. However, IL-6 can also activate cells through sIL-6R in a process known as trans-signalling. Unwanted pharmacology associated with IL-6 pathway inhibition has been linked to inhibition of mIL-6R. Preferential inhibition of sIL-6R could therefore provide higher therapeutic efficacy with a better side effect profile compared to equivalent inhibition of both IL-6R forms (1). Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy chain-only antibodies, naturally occurring in the Camelidae family. ALX-0061 is a bispecific anti-IL-6R Nanobody engineered to have an extended half-life in vivo by targeting human serum albumin (HSA), in combination with a high target affinity and potency using a single anti-IL-6R building block. Objectives ALX-0061 was extensively characterised using in vitro systems: biological activity and affinity for both sIL-6R and mIL-6R were assessed and compared to TCZ. Methods Biological activity of ALX-0061 and TCZ was analysed in a cell-based assay for mIL-6R, ELISA-based neutralisation assays for sIL-6R, and cell-binding and cell-signalling (mIL-6R) experiments in whole blood from human donors using flow cytometry. Due to very tight target binding, the affinity of ALX-0061 for sIL-6R could not be accurately determined via surface plasmon resonance. Consequently, the more sensitive GyrolabTM platform was used to assess affinity for both receptors. For the KD determination on mIL-6R, free compound concentrations were measured in the supernatant, after pre-incubation of mIL-6R-transfected cells with a constant compound concentration. Results Flow cytometry experiments demonstrated that ALX-0061 binds to mIL-6R expressed on peripheral blood leukocyte populations with expected pharmacology. ALX-0061 specifically neutralised sIL-6R with a 10-fold higher in vitro potency compared to TCZ, while the (apparent) affinity of ALX-0061 for sIL-6R (0.20 pM) was 2000-fold superior compared to TCZ (462 pM). In the mIL-6R-driven cell-based assay, however, in vitro potencies were similar for ALX-0061 and TCZ, with the latter one showing avid binding due to its bivalency. In addition, TCZ showed a 3-fold higher affinity for mIL-6R (160 pM) compared to sIL-6R, while the affinity of ALX-0061 was 45-fold lower for mIL-6R (9 pM) compared to sIL-6R. Conclusions ALX-0061 demonstrates in vitro a preferential biological activity profile for sIL-6R with a lesser activity for mIL-6R, while TCZ has a higher preference for mIL-6R. Preferential inhibition of sIL-6R trans-signalling by ALX-0061 could provide superior therapeutic efficacy with a better side effect profile than TCZ. ALX-0061 is currently in clinical development. Analysis of a phase I/II study demonstrated a strong efficacy and an attractive safety profile. References Waetzig G.H. & Rose-John S., Expert Opin Ther Targets (2012) 16(2) Disclosure of Interest None Declared