Development of effective preventative interventions against SARS-CoV-2, the etiologic agent of COVID-19 is urgently needed. The viral surface spike (S) protein of SARS-CoV-2 is a key target for prophylactic measures as it is critical for the viral replication cycle and the primary target of neutralizing antibodies. We evaluated design elements previously shown for other coronavirus S protein-based vaccines to be successful, e.g., prefusion-stabilizing substitutions and heterologous signal peptides, for selection of a S-based SARS-CoV-2 vaccine candidate. In vitro characterization demonstrated that the introduction of stabilizing substitutions (i.e., furin cleavage site mutations and two consecutive prolines in the hinge region of S2) increased the ratio of neutralizing versus non-neutralizing antibody binding, suggestive for a prefusion conformation of the S protein. Furthermore, the wild-type signal peptide was best suited for the correct cleavage needed for a natively folded protein. These observations translated into superior immunogenicity in mice where the Ad26 vector encoding for a membrane-bound stabilized S protein with a wild-type signal peptide elicited potent neutralizing humoral immunity and cellular immunity that was polarized towards Th1 IFN-γ. This optimized Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT04436276).

Two humanized IgG4 antibodies, natalizumab and gemtuzumab, are approved for human use, and several others, like TGN1412, are or have been in clinical development. Although IgG4 antibodies can dynamically exchange half-molecules, Fab-arm exchange with therapeutic antibodies has not been demonstrated in humans. Here, we show that natalizumab exchanges Fab arms with endogenous human IgG4 in natalizumab-treated individuals. Gemtuzumab, in contrast, contains an IgG4 core-hinge mutation that blocks Fab-arm exchange to undetectable levels both in vitro and in a mouse model. The ability of IgG4 therapeutics to recombine with endogenous IgG4 may affect their pharmacokinetics and pharmacodynamics. Although pharmacokinetic modeling lessens concerns about undesired cross-linking under normal conditions, unpredictability remains and mutations that completely prevent Fab-arm exchange in vivo should be considered when designing therapeutic IgG4 antibodies.

Oncogenic high-risk human papillomavirus (HPV) infections cause a substantial number of genital and non-genital cancers worldwide. Approximately 70% of all cervical cancers are caused by the high-risk HPV16 and 18 types. The remaining 30% can be attributed to twelve other high-risk HPV-types. Highly efficacious 2-valent, 4-valent and 9-valent L1 protein based prophylactic HPV vaccines are available however with limited cross-protection. To further increase the coverage, development of a multivalent cross-protective HPV vaccine is currently focused on the conserved N-terminus of HPV's L2 protein. We have developed a vaccine candidate based on the rare human adenovirus type 35 (HAdV35) vector that displays a concatemer of L2 protein epitopes from four different HPV-types via protein IX (pIX). A mix of two heterologous HAdV35 pIX-L2 display vectors present highly conserved linear epitopes of nine HPV-types. Each HAdV35 pIX-L2 display vector exhibits a good manufacturability profile. HAdV35 pIX-L2 display vaccine vectors were immunogenic and induced neutralizing antibodies against HPV-types included in the vaccine and cross-neutralizing antibodies against distant a HPV-type not included in the vaccine in mice. The HAdV35 pIX-L2 display vectors offer an opportunity for a multivalent HAdV-based prophylactic HPV vaccine.

a b s t r a c tA method for the quantitative determination of the protein composition of adenovirus-vector based vaccines was developed. The final method used RP-UPLC with UV absorbance detection, a C4 column (300 Å, 1.7 m, 2.1 × 150 mm), and a water-acetonitrile (ACN) gradient containing trifluoroacetic acid (TFA) as ion-pairing agent. The chromatographic resolution between the various adenovirus proteins was optimized by studying the effect of the TFA concentration and the column temperature, applying a full factorial design of experiments. A reproducible baseline separation of all relevant adenovirus proteins could be achieved within 17 min run time. Samples containing adenovirus particles could be directly injected into the UPLC system without sample pretreatment. The viruses reproducibly dissociate into proteins in the UPLC system upon contact with the mobile phase containing ACN. The new RP-UPLC method was successfully validated for protein profiling and relative quantification of proteins in vaccine products based on adenovirus vector types 26 and 35. The intermediate precision of the relative peak areas of all proteins was between 1% and 14% RSD, except for the peak assigned to protein V (26% RSD). The method proved to be stability indicating with respect to thermal and oxidation stress of the adenovirusvector based vaccine and was successfully implemented for the characterization of adenovirus-based products.