Immunogenicity and efficacy of VLA2001 vaccine against SARS-CoV-2 infection in male cynomolgus macaques.

BACKGROUND: The fight against COVID-19 requires mass vaccination strategies, and vaccines inducing durable cross-protective responses are still needed. Inactivated vaccines have proven lasting efficacy against many pathogens and good safety records. They contain multiple protein antigens that may improve response breadth and can be easily adapted every year to maintain preparedness for future seasonally emerging variants. METHODS: The vaccine dose was determined using ELISA and pseudoviral particle-based neutralization assay in the mice. The immunogenicity was assessed in the non-human primates with multiplex ELISA, neutralization assays, ELISpot and intracellular staining. The efficacy was demonstrated by viral quantification in fluids using RT-qPCR and respiratory tissue lesions evaluation. RESULTS: Here we report the immunogenicity and efficacy of VLA2001 in animal models. VLA2001 formulated with alum and the TLR9 agonist CpG 1018™ adjuvant generate a Th1-biased immune response and serum neutralizing antibodies in female BALB/c mice. In male cynomolgus macaques, two injections of VLA2001 are sufficient to induce specific and polyfunctional CD4+ T cell responses, predominantly Th1-biased, and high levels of antibodies neutralizing SARS-CoV-2 infection in cell culture. These antibodies also inhibit the binding of the Spike protein to human ACE2 receptor of several variants of concern most resistant to neutralization. After exposure to a high dose of homologous SARS-CoV-2, vaccinated groups exhibit significant levels of protection from viral replication in the upper and lower respiratory tracts and from lung tissue inflammation. CONCLUSIONS: We demonstrate that the VLA2001 adjuvanted vaccine is immunogenic both in mouse and NHP models and prevent cynomolgus macaques from the viruses responsible of COVID-19.

Mass vaccination in response to the COVID-19 pandemic has substantially reduced the number of severe cases and hospitalizations. As the virus continues to evolve and give rise to new variants that cause local outbreaks, there is a need to develop new vaccine candidates capable of stopping the viral transmission. In this study, we explore the immune responses induced by the vaccine candidate VLA2001 in animal models. We highlight the vaccine's ability to induce an immune response capable of blocking the virus and eliminating infected cells. We show that it can protect the host from developing severe disease.

Characterization and optimization of a novel vaccine for protection against Lyme borreliosis.

Lyme borreliosis (LB) is the most common vector-borne disease in the northern hemisphere and there is no vaccine available for disease prevention. The majority of LB cases in Europe are caused by four different Borrelia species expressing six different OspA serotypes, whereas in the US only one of these serotypes is present. Immunization with the outer surface protein A (OspA) can prevent infection and the C-terminal part of OspA is sufficient for protection against infection transmitted by Ixodes ticks. Here we show that the order of the stabilized monomeric OspA fragments making up the heterodimers in our LB vaccine does not influence the induced immunogenicity and protection. Using bioinformatics analysis (surface electrostatics), we have designed an improved version of an LB vaccine which has an increased immunogenicity for OspA serotype 3 and an optimized expression and purification profile. The OspA heterodimers were highly purified with low amounts of endotoxin, host cell proteins and host cell DNA. All three proteins were at least 85% triacylated which ensured high immunogenicity. The LB vaccine presented here was designed, produced and characterized to a level which warrants further development as a second generation human LB vaccine.

Influence of elemental impurities in aluminum hydroxide adjuvant on the stability of inactivated Japanese Encephalitis vaccine, IXIARO®.

Aluminum hydroxide is a critical raw material in the production of many vaccines. It is used as an adjuvant in the formulation of the final bulk vaccine, and for this it must meet the specifications of the European Pharmacopeia Monograph. We investigated whether vaccine stability was affected by the presence of trace amounts of elemental impurities in commercially available aluminum hydroxide. The content of residual elemental impurities in commercially available aluminum hydroxide was determined by selective and sensitive inductively coupled-plasma mass spectrometry and inductively coupled plasma atomic emission spectroscopy. We found significant differences between different suppliers, but also between different lots from the same supplier. Inactivated Japanese encephalitis vaccine, IXIARO(®), was used to study the effect of residual metals in aluminum hydroxide on antigen stability. We propose that antigen degradation occurred via a pathway involving the metal-catalyzed, auto-oxidation of a process-related impurity (sulfite). Thus, sulfite auto-oxidation resulted in antigen degradation when residual Cu was present at elevated concentrations in aluminum hydroxide.

A nonadjuvanted transcutaneous tetanus patch is effective in boosting anti-tetanus toxoid immune responses.

Dry tetanus toxoid (TTx) patches were formulated without any adjuvant, with excipients to impart antigen stabilization and to enhance skin delivery. The booster effects of the TTx patches were assessed using a guinea pig model. The study revealed significant rises in TTx IgG titers induced by the TTx patches after a low-dose subcutaneous (s.c.) prime with TTx adsorbed to aluminum hydroxide. The TTx patch can therefore be considered an effective alternative to a subcutaneous booster.

Continuous matrix assisted refolding of alpha-lactalbumin by ion exchange chromatography with recycling of aggregates combined with ultradiafiltration.

Continuous matrix assisted refolding (MAR) can be achieved on a solid support by using a continuous chromatographic system. Recycling the aggregate fraction, simultaneously formed during a refolding reaction, can further increase the refolding yield. Due to the nature of this reaction, aggregates are the main reason for a refolding yield below stoichiometric conversion. A preparative continuous annular chromatographic system (P-CAC) equipped with an ion exchange resin was used to continuously refold the model protein alpha-lactalbumin. For this purpose, this protein was denatured, reduced and adsorbed on the ion exchange resin. Elution was performed with or without redox reagents in the buffer system permitting fast formation of the native disulfide bonds. In the case redox reagents were present, the protein refolds then during its residence time on the matrix. However, aggregate formation is also increased and refolding yields are lower. Tightly bound aggregates were removed from the column by 2M guanidinium hydrochloride. In order to increase the system yield, this aggregate fraction was recycled after lowering the conductivity by ultradiafiltration and adjustment of the protein concentration by dilution. For on-column refolding, recycling of aggregates at a recycling rate of 0.17 increased the system yield from 25% to 30%. An algorithm was developed to show interdependencies of the single influencing parameters. The operability of the system was demonstrated but limitations due to instability of the P-CAC, especially inhomogeneous flow and peak wobbling, have to be considered.

Matrix assisted refolding of proteins by ion exchange chromatography.

Two different approaches of matrix assisted refolding have been evaluated and compared to conventional refolding by dilution. Bovine alpha-lactalbumin was used for the studies as model protein. It was adsorbed under denaturing conditions on an ion exchange matrix and refolding was completed on the column prior to elution or, depending on the buffer system, in the eluate. Agarose based chromatography matrices showed high capacities for the denatured alpha-lactalbumin. A positive effect on the yield of refolded protein by the matrix could be observed for Fractogel EMD DEAE and a negative for Toyopearl DEAE 650M, DEAE Sepharose FF and Q Sepharose FF. In the case of Fractogel EMD DEAE the ion exchange surface might act as a folding helper. This property may be caused by the grafted polymers. For Source 30Q only a marginal negative influence on the refolding kinetics was observed, thus the ion exchanger is only a mean for removal of chaotropic agents. Refolding on the column is characterized by a low yield but high productivity due to significant reduction of refolding time.

Application of monoliths for plasmid DNA purification development and transfer to production.

The demand of high-purity plasmid DNA (pDNA) for gene-therapy and genetic vaccination is still increasing. For the large scale production of pharmaceutical grade plasmids generic and economic purification processes are needed. Most of the current processes for pDNA production use at least one chromatography step, which always constitutes as the key-step in the purification sequence. Monolithic chromatographic supports are an alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. Anion-exchange chromatography is the most popular chromatography method for plasmid separation, since polynucleotides are negatively charged independent of the buffer conditions. For the implementation of a monolith-based anion exchange step into a pDNA purification process detailed screening experiments were performed. These studies included supports, ligand-types and ligand-densities and optimization of resolution and productivity. For this purpose model plasmids with a size of 4.3 and 6.9 kilo base pairs (kbp) were used. It could be shown, that up-scaling to the production scale using 800 ml CIM Convective Interaction Media radial flow monoliths is possible under low pressure conditions. CIM DEAE was successfully implemented as intermediate step of the cGMP pDNA manufacturing process. Starting from 2001 fermentation aliquots pilot scale purification runs were performed in order to prove scale-up and to predict further up-scaling to 8 1 tube monolithic columns. The analytical results obtained from these runs confirmed suitability for pharmaceutical applications.

Folding and refolding of proteins in chromatographic beds.

The correct folding of solubilized recombinant proteins is of key importance for their production in industry. On-column refolding of proteins is mainly achieved by three methods: size-exclusion chromatography, ion exchange chromatography and affinity chromatography using immobilized metal chelates. The principles of these methods were first laid down in the 1990s, but many recent improvements have been made to these processes including sophisticated changes to the mobile phase composition and the recycling of aggregates to improve yield. Advances have also been made in the use of immobilized metal affinity chromatography and by mimicking the natural folding process with artificial chaperones.

Continuous matrix-assisted refolding of proteins.

A refolding reactor was developed for continuous matrix-assisted refolding of proteins. The reactor was composed of an annular chromatography system and an ultrafiltration system to recycle aggregated proteins produced during the refolding reaction. The feed solution containing the denatured protein was continuously fed to the rotating bed perfused with buffer promoting folding of the protein. As the protein passed through the column, it was separated from chaotropic and reducing agents and the refolding process took place. Native proteins and aggregates could be continuously separated due to different molecular size. The exit stream containing aggregates was collected, concentrated by ultrafiltration and recycled to the feed solution. The high concentrations of chaotropic and reducing agents in the feed solution enabled dissociation of the recycled aggregates and consequently were fed again to the refolding reactor. When the initial feed mixture of denatured protein is used up, only buffer-containing chaotropic agents and recycled aggregates are fully converted to native protein. This process resulted in a stoichiometric conversion from the denatured protein to its correctly folded native state. The system was tested with bovine alpha-lactalbumin as model protein. Superdex 75 PrepGrade was used as size-exclusion medium. The yield of 30% active monomer in the batch process was improved to 41% at a recycling rate of 65%. Assuming that the aggregates can be redissolved and recycled into the feed stream in a quantitative manner, a refolding yield close to 100% is possible. The method can be also applied to other chromatographic principles suited for the separation of aggregates.

Absence of SV40 in Austrian tumors correlates with low incidence of mesotheliomas.

Between 1955 and 1963 millions of people were worldwide vaccinated with polio-vaccines that were contaminated with the simian virus 40 (SV40). This tumor-inducing virus has subsequently been detected in several human tumors. In Austria, polio mass vaccination started in winter 1961/62 with a presumably SV40-free British vaccine. Thus, we hypothesized that the Austrian population should be SV40-free. We used a polymerase chain reaction-based (PCR) method to search for SV40 sequences in DNA that was extracted from 14 giant cell tumors, ten osteosarcomas and eight mesotheliomas. SV40 was easily detected in two bone tumor DNAs from Italy, and one from the USA, and in one SV40 positive cell line. In parallel experiments all Austrian samples tested consistently negative. Our findings support the notion that: 1) polio vaccination is the main source for SV40 in human populations, 2) Austria was not exposed to SV40, and 3) its absence correlates with the low incidence of mesotheliomas in Austria.