The First-in-Human Synthetic Glycan-Based Conjugate Vaccine Candidate against Shigella.

Shigella, the causative agent of shigellosis, is among the main causes of diarrheal diseases with still a high morbidity in low-income countries. Relying on chemical synthesis, we implemented a multidisciplinary strategy to design SF2a-TT15, an original glycoconjugate vaccine candidate targeting Shigella flexneri 2a (SF2a). Whereas the SF2a O-antigen features nonstoichiometric O-acetylation, SF2a-TT15 is made of a synthetic 15mer oligosaccharide, corresponding to three non-O-acetylated repeats, linked at its reducing end to tetanus toxoid by means of a thiol-maleimide spacer. We report on the scale-up feasibility under GMP conditions of a high yielding bioconjugation process established to ensure a reproducible and controllable glycan/protein ratio. Preclinical and clinical batches complying with specifications from ICH guidelines, WHO recommendations for polysaccharide conjugate vaccines, and (non)compendial tests were produced. The obtained SF2a-TT15 vaccine candidate passed all toxicity-related criteria, was immunogenic in rabbits, and elicited bactericidal antibodies in mice. Remarkably, the induced IgG antibodies recognized a large panel of SF2a circulating strains. These preclinical data have paved the way forward to the first-in-human study for SF2a-TT15, demonstrating safety and immunogenicity. This contribution discloses the yet unreported feasibility of the GMP synthesis of conjugate vaccines featuring a unique homogeneous synthetic glycan hapten fine-tuned to protect against an infectious disease.

Immunochemical and biophysical characterization of inactivated Sabin poliovirus products: Insights into rapid quality assessment tools.

The aim of this study was to demonstrate the strength of combining immunochemical and biophysical analysis tools for assessing the quality of Sabin inactivated poliovirus vaccine (Sabin-IPV) bulk products. We assessed Sabin-IPV serotypes 1, 2 and 3 from six different manufacturers and evaluated their comparability through biosensor analysis and biophysical characterization methods, including tryptophan fluorescence and asymmetrical flow field-flow fractionation - multi-angle light scattering analysis. These methods enabled us to assess antigenic as well as conformational and structural integrity profiles, respectively. Based on Sabin-IPV samples that were subjected to accelerated storage conditions, we revealed that existing immunochemical methods exhibit remarkably similar trends to the results obtained by the biophysical characterization methods. While the results underpin that the comparability of Sabin-IPV bulk products of different manufacturers is weak, information about their quality can rapidly be obtained by using both immunochemical and biophysical methods. Furthermore, the study highlights that quality assessment of Sabin-IPV can be obtained through biophysical techniques can complement the assessments performed with monoclonal antibodies and suggests that similar techniques could be employed to characterize other enteroviruses.

Characterisation of tetanus monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay.

Batch release testing for human and veterinary tetanus vaccines still relies heavily on methods that involve animals, particularly for potency testing. The quantity and quality of tetanus antigen present in these products is of utmost importance for product safety and clinical effect. Immunochemical methods that measure consistency of antigen content and quality, potentially as an indicator of potency, could be a better choice and negate the need for an in vivo potency test. These immunochemical methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against tetanus with a view to select antibodies that can be used for development of an in vitro potency immunoassay. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined using an in-house cell-based neutralisation assay to support prior in vivo potency data that was available for some, but not all, of the antibodies. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of "superiority" of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development.

Characterisation of diphtheria monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay.

Immunoassays are used for routine potency assessment of several vaccines, in some cases having been specifically developed as alternatives to in vivo potency tests. These methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against diphtheria with a view to select antibodies that can be used for development of an in vitro potency immunoassay for diphtheria vaccines. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined by a cell-based toxin neutralisation test and diphtheria toxin-domain recognition was determined by Western blotting. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of "superiority" of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development.

Safety and immunogenicity of a synthetic carbohydrate conjugate vaccine against Shigella flexneri 2a in healthy adult volunteers: a phase 1, dose-escalating, single-blind, randomised, placebo-controlled study.

BACKGROUND: Shigella remains in the top four pathogens responsible for moderate to severe diarrhoea in children below 5 years of age. The shigella O-specific polysaccharide (O-SP) is a promising vaccine target. We developed a conjugate vaccine prototype incorporating a unique well defined synthetic oligosaccharide hapten, chemically designed for optimal antigenic, conformational, structural, and functional mimicry of the O-SP from Shigella flexneri 2a (SF2a). We aimed to assess the safety, tolerability, and immunogenicity of this original synthetic oligosaccharide-based vaccine candidate, SF2a-TT15, conceived to drive the antibody response towards the key protective determinants of the native lipopolysaccharide antigen, in a first-in-human phase 1 study. METHODS: We did a first-in-human, dose-escalating, single-blind, observer-masked, randomised, placebo-controlled study at the Clinical Research Center of Tel Aviv Sourasky Medical Center (Israel). Participants were healthy adults aged 18-45 years with low titres of serum SF2a-specific IgG antibodies. 64 eligible participants were assigned to one of two cohorts. 32 participants in each of the two cohorts were randomly assigned via computer-generated algorithm in a stepwise manner to receive the 2 µg (cohort 1) and 10 µg oligosaccharide dose (cohort 2) of the SF2a-TT15 vaccine candidate non-adjuvanted or adjuvanted with aluminium hydroxide (alum) or matching placebos. The vaccine was administered as three single intramuscular injections into the arm, 28 days apart. The primary outcome was the incidence and severity of adverse events, which were assessed in the intention-to-treat safety population analysis including all participants who were randomly assigned and received at least one vaccine or placebo injection. The immunogenicity endpoints were secondary outcomes and were analysed in all participants who were randomly assigned, received all of the assigned injections before the time of the immunogenicity assessment, and provided blood samples for immunological follow-up (per-protocol immunogenicity analysis). The study is registered with ClinicalStudies.gov, NCT02797236 and is completed. FINDINGS: Of 203 volunteers initially screened, 64 participants were enrolled between Sept 20, 2016, and Sept 26, 2017. In each of the two cohorts, 12 participants received the adjuvanted vaccine, 12 received the non-adjuvanted vaccine and eight received the matching placebo (four each). The SF2a-TT15 glycoconjugate was well tolerated at both doses. No serious or severe adverse events occurred. Overall, seven (88%) of eight to 12 (100%) of 12 in each group of volunteers had one adverse event or more after receiving the study agents with the majority of adverse events, 300 (98%) of 307, considered mild in intensity. Of the seven adverse events defined as moderate in severity, one (nausea) was suspected to be related to the vaccine candidate. At all post-immunisation days and for both oligosaccharide doses, whether adjuvanted or not, SF2a-TT15 induced significantly higher serum IgG anti-SF2a lipopolysaccharide geometric mean titres (GMTs) as compared with baseline or with the corresponding GMTs in placebo recipients (p<0·01). After one injection, the non-adjuvanted 10 µg oligosaccharide dose induced a 27-times increase in IgG GMT (5080 vs 189) and the non-adjuvanted 2 µg oligosaccharide dose induced a five-times increase (1411 vs 283), compared with baseline. Alum enhanced the specific IgG response at 2 µg oligosaccharide dose after the third injection (GMTs 3200 vs 1176, p=0.045). INTERPRETATION: SF2a-TT15 was safe and well tolerated and induced high titres of anti-SF2a LPS IgG antibodies. These results support further evaluation of this original synthetic oligosaccharide-protein conjugate vaccine candidate for safety, immunogenicity, and protective efficacy in target populations. FUNDING: The European Union Seventh Framework Programme.

Antigenic fingerprinting of diphtheria toxoid adsorbed to aluminium phosphate.

The antigenicity of alum-adsorbed diphtheria toxoid (DTd) was determined in combination vaccines, containing DTd, tetanus toxoid and inactivated poliovirus. A panel of monoclonal antibodies was used, covering five epitopes, distributed over the antigen. The resulting antigenic fingerprint of DTd demonstrates consistency of adsorption at antigen level in final product combination vaccines. The antigenic quality of DTd alone, adsorbed to aluminium phosphate, was also determined and compared with pre-adsorbed toxoid (starting material as well as toxoid desorbed from aluminium phosphate). Some epitopes became less accessible after adsorption, while others became relatively better exposed. Some epitopes disappeared almost completely upon adsorption, but were re-established after desorption of the antigen. The results indicate that DTd is adsorbed to aluminium phosphate in a preferred orientation and not randomly.

Immunization with Small Amyloid-ß-derived Cyclopeptide Conjugates Diminishes Amyloid-ß-Induced Neurodegeneration in Mice.

BACKGROUND: Soluble oligomeric (misfolded) species of amyloid-ß (Aß) are the main mediators of toxicity in Alzheimer's disease (AD). These oligomers subsequently form aggregates of insoluble fibrils that precipitate as extracellular and perivascular plaques in the brain. Active immunization against Aß is a promising disease modifying strategy. However, eliciting an immune response against Aß in general may interfere with its biological function and was shown to cause unwanted side-effects. Therefore, we have developed a novel experimental vaccine based on conformational neo-epitopes that are exposed in the misfolded oligomeric Aß, inducing a specific antibody response. OBJECTIVE: Here we investigate the protective effects of the experimental vaccine against oligomeric Aß1-42-induced neuronal fiber loss in vivo. METHODS: C57BL/6 mice were immunized or mock-immunized. Antibody responses were measured by enzyme-linked immunosorbent assay. Next, mice received a stereotactic injection of oligomeric Aß1-42 into the nucleus basalis of Meynert (NBM) on one side of the brain (lesion side), and scrambled Aß1-42 peptide in the contralateral NBM (control side). The densities of choline acetyltransferase-stained cholinergic fibers origination from the NBM were measured in the parietal neocortex postmortem. The percentage of fiber loss in the lesion side was determined relative to the control side of the brain. RESULTS: Immunized responders (79%) showed 23% less cholinergic fiber loss (p = 0.01) relative to mock-immunized mice. Moreover, fiber loss in immunized responders correlated negatively with the measured antibody responses (R2 = 0.29, p = 0.02). CONCLUSION: These results may provide a lead towards a (prophylactic) vaccine to prevent or at least attenuate (early onset) AD symptoms.

Measuring Poliovirus Antigenicity by Surface Plasmon Resonance. Application for Potency Indicating Assays.

The D-antigen ELISA is the commonly accepted test for release of inactivated poliovirus containing vaccines. However, this test has a few drawbacks regarding the many variations in the method to quantify the D-unit. The result may depend on method and reagents used which makes standardization of inactivated polio vaccines, based on D-units, to a real challenge. This chapter describes a surface plasmon resonance based method to quantify D-units. The advantage of the calibrated D-antigen assay is the decrease in test variations because no labels, [no incubation times] and no washing steps are necessary. For standardization of both IPV and Sabin IPV, the calibration free concentration analysis could be an improvement as compared to ELISA or other SPR methods because this method combines quantity (particle concentration) and quality (antigenicity) in one assay.

A Synthetic Carbohydrate Conjugate Vaccine Candidate against Shigellosis: Improved Bioconjugation and Impact of Alum on Immunogenicity.

Conjugation chemistry is among the most important parameters governing the efficacy of glycoconjugate vaccines. High robustness is required to ensure high yields and batch to batch reproducibility. Herein, we have established a robust bioconjugation protocol based on the thiol-maleimide addition. Major variables were determined and acceptable margins were investigated for a synthetic pentadecasaccharide-tetanus toxoid conjugate, which is a promising vaccine candidate against Shigella flexneri serotype 2a infection. The optimized process is applicable to any thiol-equipped hapten and provides an efficient control of the hapten:carrier ratio. Moreover, comparison of four S. flexneri 2a glycoconjugates only differing by their pentadecasaccharide:tetanus toxoid ratio confirmed preliminary findings indicating that hapten loading is critical for immunogenicity with an optimal ratio here in the range of 17 ± 5. In addition, the powerful influence of alum on the immunogenicity of a Shigella synthetic carbohydrate-based conjugate vaccine candidate is demonstrated for the first time, with a strong anti-S. flexneri 2a antibody response sustained for more than one year.

A mucosal adjuvant for the inactivated poliovirus vaccine.

The eradication of poliovirus from the majority of the world has been achieved through the use of two vaccines: the inactivated poliovirus vaccine (IPV) and the live-attenuated oral poliovirus vaccine (OPV). Both vaccines are effective at preventing paralytic poliomyelitis, however, they also have significant differences. Most importantly for this work is the risk of revertant virus from OPV, the greater cost of IPV, and the low mucosal immunity induced by IPV. We and others have previously described the use of an alphavirus-based adjuvant that can induce a mucosal immune response to a co-administered antigen even when delivered at a non-mucosal site. In this report, we describe the use of an alphavirus-based adjuvant (GVI3000) with IPV. The IPV-GVI3000 vaccine significantly increased systemic IgG, mucosal IgG and mucosal IgA antibody responses to all three poliovirus serotypes in mice even when administered intramuscularly. Furthermore, GVI3000 significantly increased the potency of IPV in rat potency tests as measured by poliovirus neutralizing antibodies in serum. Thus, an IPV-GVI3000 vaccine would reduce the dose of IPV needed and provide significantly improved mucosal immunity. This vaccine could be an effective tool to use in the poliovirus eradication campaign without risking the re-introduction of revertant poliovirus derived from OPV.

Antigen sparing with adjuvanted inactivated polio vaccine based on Sabin strains.

Six different adjuvants, each in combination with inactivated polio vaccine (IPV) produced with attenuated Sabin strains (sIPV), were evaluated for their ability to enhance virus neutralizing antibody titres (VNTs) in the rat potency model. The increase of VNTs was on average 3-, 15-, 24-fold with adjuvants after one immunization (serotypes 1, 2, and 3, respectively). Also after a boost immunization the VNTs of adjuvanted sIPV were on average another 7-20-27 times higher than after two inoculations of sIPV without adjuvant. The results indicate that it is feasible to increase the potency of inactivated polio vaccines by using adjuvants.

Inactivated polio vaccine development for technology transfer using attenuated Sabin poliovirus strains to shift from Salk-IPV to Sabin-IPV.

Industrial-scale inactivated polio vaccine (IPV) production dates back to the 1960s when at the Rijks Instituut voor de Volksgezondheid (RIV) in Bilthoven a process was developed based on micro-carrier technology and primary monkey kidney cells. This technology was freely shared with several pharmaceutical companies and institutes worldwide. In this contribution, the history of one of the first cell-culture based large-scale biological production processes is summarized. Also, recent developments and the anticipated upcoming shift from regular IPV to Sabin-IPV are presented. Responding to a call by the World Health Organization (WHO) for new polio vaccines, the development of Sabin-IPV was continued, after demonstrating proof of principle in the 1990s, at the Netherlands Vaccine Institute (NVI). Development of Sabin-IPV plays an important role in the WHO polio eradication strategy as biocontainment will be critical in the post-OPV cessation period. The use of attenuated Sabin strains instead of wild-type Salk polio strains will provide additional safety during vaccine production. Initially, the Sabin-IPV production process will be based on the scale-down model of the current, and well-established, Salk-IPV process. In parallel to clinical trial material production, process development, optimization and formulation research is being carried out to further optimize the process and reduce cost per dose. Also, results will be shown from large-scale (to prepare for future technology transfer) generation of Master- and Working virus seedlots, and clinical trial material (for phase I studies) production. Finally, the planned technology transfer to vaccine manufacturers in low and middle-income countries is discussed.

A cyclic undecamer peptide mimics a turn in folded Alzheimer amyloid ß and elicits antibodies against oligomeric and fibrillar amyloid and plaques.

The 39- to 42-residue amyloid ß (Aß) peptide is deposited in extracellular fibrillar plaques in the brain of patients suffering from Alzheimer's Disease (AD). Vaccination with these peptides seems to be a promising approach to reduce the plaque load but results in a dominant antibody response directed against the N-terminus. Antibodies against the N-terminus will capture Aß immediately after normal physiological processing of the amyloid precursor protein and therefore will also reduce the levels of non-misfolded Aß, which might have a physiologically relevant function. Therefore, we have targeted an immune response on a conformational neo-epitope in misfolded amyloid that is formed in advance of Aß-aggregation. A tetanus toxoid-conjugate of the 11-meric cyclic peptide Aß(22-28)-YNGK' elicited specific antibodies in Balb/c mice. These antibodies bound strongly to the homologous cyclic peptide-bovine serum albumin conjugate, but not to the homologous linear peptide-conjugate, as detected in vitro by enzyme-linked immunosorbent assay. The antibodies also bound--although more weakly--to Aß(1-42) oligomers as well as fibrils in this assay. Finally, the antibodies recognized Aß deposits in AD mouse and human brain tissue as established by immunohistological staining. We propose that the cyclic peptide conjugate might provide a lead towards a vaccine that could be administered before the onset of AD symptoms. Further investigation of this hypothesis requires immunization of transgenic AD model mice.

Characterization and standardization of Sabin based inactivated polio vaccine: proposal for a new antigen unit for inactivated polio vaccines.

GMP-batches of Sabin-IPV were characterized for their antigenic and immunogenic properties. Antigenic fingerprints of Sabin-IPV reveal that the D-antigen unit is not a fixed amount of antigen but depends on antibody and assay type. Instead of the D-antigen unit we propose standardization of IPV based on a combination of protein amount for dose and D-antigenicity for quality of the vaccine. Although Sabin-IPV type 2 is less immunogenic than regular wild type IPV type 2, the immunogenicity (virus neutralizing titers) per microgram antigen for Sabin-IPV type 2 is in the same order as for wild type serotypes 1 and 3. The latter observations are in line with data from human trials. This suggests that a higher dose of Sabin-IPV type 2 to compensate for the lower rat immunogenicity may not be necessary.

Physicochemical studies on the stability of influenza haemagglutinin in vaccine bulk material.

The relative unknown conformational stability of monovalent bulks of influenza virus haemagglutinin (HA) from three different strains (B/Guangdong, A/New Caledonia and A/Panama) was investigated with fluorescence and circular dichroism (CD) spectroscopy. Various stress conditions (concentration of denaturant, freeze-thawing, pH and temperature) affected the spectroscopic properties of the haemagglutinin proteins differently. Unfolding experiments revealed a poor stability of Guangdong haemagglutinin (GD-HA) in comparison with New Caledonia (NC-HA) and Panama haemagglutinin (P-HA). Freeze-thawing altered the secondary and tertiary structure of Guangdong haemagglutinin and only the tertiary structure of Panama haemagglutinin. From pH 4.6-9.2 the tertiary structures of Guangdong, New Caledonia and Panama haemagglutinin were all affected to a different extent. The secondary structure was only altered at low pH. Incubation of haemagglutinin at 60 degrees C resulted in denaturation of the protein and a dramatic change of the fluorescence spectrum, indicative of oxidised tryptophan (Trp). In conclusion, fluorescence and circular dichroism spectroscopy are highly suitable techniques to monitor the stability of haemagglutinin in a straightforward and fast way.

Stability of mono- and trivalent meningococcal outer membrane vesicle vaccines.

The stability during storage of outer membrane vesicles (OMVs) of Neisseria meningitidis group B was studied. Three types of OMVs were compared for their stability, containing either one (monovalent) or three different PorA subtypes (trivalent), the latter with and without class 4 outer membrane protein (OMO, RmpM). Aqueous formulations were stored freeze-dried (4 degrees C), frozen (-70 degrees C) and in liquid form at 4, 37 and 56 degrees C. Physico-chemical properties and immunogenicity of the OMVs as well as PorA conformation and antigenicity (P1.7-2,4, the subtype present in all formulations) were monitored during 1 year. At -70 or 4 degrees C, the structure and immunogenicity of OMVs was preserved. Storage of OMVs at high temperatures (37 or 56 degrees C) induced destruction of the OMV structure and denaturation of PorA, followed by chemical degradation. Immunogenicity decreased or was lost completely. Changes observed in the fluorescence spectra of degraded OMVs were also seen in tryptophan (Trp) and tyrosine (Tyr) derivatives incubated at 56 degrees C, indicating the occurrence of chemical degradation of tryptophan and tyrosine residues in PorA. Trivalent OMVs were slightly more stable at 37 degrees C than monovalent OMVs as assessed by in vitro methods, but these differences did not result in differences in the immunogenicity. The stability of trivalent OMVs was not affected by the presence of RmpM. Both trivalent and monovalent OMVs could be freeze-dried with preservation of their immunogenicity. In conclusion, OMVs are sensitive to elevated temperatures, but are stable in the frozen or freeze-dried state or when stored at 4 degrees C in the liquid state.