Clinical formulation development of Plasmodium falciparum malaria vaccine candidates based on Pfs48/45, Pfs230, and PfCSP.

Two malaria transmission-blocking vaccine (TBV) candidates, R0.6C and ProC6C, have completed preclinical development including the selection of adjuvants, Alhydrogel® with or without the saponin based adjuvant Matrix-M™. Here, we report on the final drug product (formulation) design of R0.6C and ProC6C and evaluate their safety and biochemical stability in preparation for preclinical and clinical pharmacy handling. The point-of-injection stability studies demonstrated that both the R0.6C and ProC6C antigens are stable on Alhydrogel in the presence or absence of Matrix-M for up to 24 h at room temperature. As this is the first study to combine Alhydrogel and Matrix-M for clinical use, we also evaluated their potential interactions. Matrix-M adsorbs to Alhydrogel, while not displacing the > 95 % adsorbed protein. The R0.6C and ProC6C formulations were found to be safe and well tolerated in repeated dose toxicity studies in rabbits generating high levels of functional antibodies that blocked infection of mosquitoes. Further, the R0.6C and ProC6C drug products were found to be stable for minimally 24 months when stored at 2-8 °C, with studies ongoing through 36 months. Together, this data demonstrates the safety and suitability of the L. lactis expression system as well as supports the clinical testing of the R0.6C and ProC6C malaria vaccine candidates in First-In-Human clinical trials.

First in man study: Bcl-Xl_42-CAF®09b vaccines in patients with locally advanced prostate cancer.

Background: The B-cell lymphoma-extra-large (Bcl-XL) protein plays an important role in cancer cells' resistance to apoptosis. Pre-clinical studies have shown that vaccination with Bcl-XL-derived peptides can induce tumor-specific T cell responses that may lead to the elimination of cancer cells. Furthermore, pre-clinical studies of the novel adjuvant CAF®09b have shown that intraperitoneal (IP) injections of this adjuvant can improve the activation of the immune system. In this study, patients with hormone-sensitive prostate cancer (PC) received a vaccine consisting of Bcl-XL-peptide with CAF®09b as an adjuvant. The primary aim was to evaluate the tolerability and safety of IP and intramuscular (IM) administration, determine the optimal route of administration, and characterize vaccine immunogenicity. Patients and methods: Twenty patients were included. A total of six vaccinations were scheduled: in Group A (IM to IP injections), ten patients received three vaccines IM biweekly; after a three-week pause, patients then received three vaccines IP biweekly. In Group B (IP to IM injections), ten patients received IP vaccines first, followed by IM under a similar vaccination schedule. Safety was assessed by logging and evaluating adverse events (AE) according to Common Terminology Criteria for Adverse Events (CTCAE v. 4.0). Vaccines-induced immune responses were analyzed by Enzyme-Linked Immunospot and flow cytometry. Results: No serious AEs were reported. Although an increase in T cell response against the Bcl-XL-peptide was found in all patients, a larger proportion of patients in group B demonstrated earlier and stronger immune responses to the vaccine compared to patients in group A. Further, we demonstrated vaccine-induced immunity towards patient-specific CD4, and CD8 T cell epitopes embedded in Bcl-XL-peptide and an increase in CD4 and CD8 T cell activation markers CD107a and CD137 following vaccination. At a median follow-up of 21 months, no patients had experienced clinically significant disease progression. Conclusion: The Bcl-XL-peptide-CAF®09b vaccination was feasible and safe in patients with l hormone-sensitive PC. In addition, the vaccine was immunogenic and able to elicit CD4 and CD8 T cell responses with initial IP administration eliciting early and high levels of vaccine-specific responses in a higher number og patients. Clinical trial registration: https://clinicaltrials.gov, identifier NCT03412786.

A novel adjuvant formulation induces robust Th1/Th17 memory and mucosal recall responses in Non-Human Primates.

After clean drinking water, vaccination is the most impactful global health intervention. However, development of new vaccines against difficult-to-target diseases is hampered by the lack of diverse adjuvants for human use. Of particular interest, none of the currently available adjuvants induce Th17 cells. Here, we develop and test an improved liposomal adjuvant, termed CAF®10b, that incorporates a TLR-9 agonist. In a head-to-head study in non-human primates (NHPs), immunization with antigen adjuvanted with CAF®10b induced significantly increased antibody and cellular immune responses compared to previous CAF® adjuvants, already in clinical trials. This was not seen in the mouse model, demonstrating that adjuvant effects can be highly species specific. Importantly, intramuscular immunization of NHPs with CAF®10b induced robust Th17 responses that were observed in circulation half a year after vaccination. Furthermore, subsequent instillation of unadjuvanted antigen into the skin and lungs of these memory animals led to significant recall responses including transient local lung inflammation observed by Positron Emission Tomography-Computed Tomography (PET-CT), elevated antibody titers, and expanded systemic and local Th1 and Th17 responses, including >20% antigen-specific T cells in the bronchoalveolar lavage. Overall, CAF®10b demonstrated an adjuvant able to drive true memory antibody, Th1 and Th17 vaccine-responses across rodent and primate species, supporting its translational potential.

Unusual Self-Assembly of the Recombinant Chlamydia trachomatis Major Outer Membrane Protein-Based Fusion Antigen CTH522 Into Protein Nanoparticles.

Sexually transmitted Chlamydia trachomatis infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ctrachomatis major outer membrane protein (MOMP) is highly immunogenic but is a challenging vaccine candidate by being an integral membrane protein, and the immunogenicity depends on a correctly folded structure. We investigated the biophysical properties of the recombinant MOMP-based fusion antigen CTH522, which is tested in early human clinical trials. It consists of a truncated and cysteine-free version of MOMP fused to 4 variable domains from serovars D-G. In the native state, CTH522 did not exist as a monomer but showed an unusual self-assembly into nanoparticles with a negative zeta potential. In contrast to the ß-barrel structure of MOMP, native CTH522 contained no well-defined secondary structural elements, and no thermal transitions were measurable. Chemical unfolding resulted in monomers that upon removal of the denaturant self-assembled into higher order structures, comparable to the structure of the native protein. The conformation of CTH522 in nanoparticles is thus not entirely random and contains structural elements stabilized via denaturant-disruptable hydrophobic interactions. In conclusion, CTH522 has an unusual quaternary structure of supramolecular self-assemblies.

Protein adsorption and displacement at lipid layers determined by total internal reflection fluorescence (TIRF).

In many drug delivery systems such as liposomes, the adsorption of interstitial proteins upon administration can have a huge effect on the elimination, release, and stability of the delivery system. For example, it is assumed that PEGylated liposomes prevent the adsorption of opsonins and thereby prolong the circulation time in vivo, and EMEA guidelines recommend that more than 80% of the protein antigen is adsorbed in the formulation of adjuvant systems. However, few methods exist to elucidate this protein adsorption. The present study indicates that total internal reflection fluorescence (TIRF) is a possible method to examine the adsorption and exchange of proteins at lipid surfaces. In the TIRF set-up, a lipid layer can be formed [exemplified with dimethyldioctadecylammonium bromide (DDA) and D-(+)-trehalose 6,6'-dibehenate (TDB)] whereafter protein (i.e., ovalbumin or an antigen, Ag85B-ESAT-6) is adsorbed, and these proteins can subsequently be displaced by the abundant interstitial protein (i.e., serum albumin).