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Vaccines derived from chimpanzee adenovirus Y25 (ChAdOx1), human adenovirus type 26 (HAdV-D26), and human adenovirus type 5 (HAdV-C5) are critical in combatting the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic. As part of the largest vaccination campaign in history, ultrarare side effects not seen in phase 3 trials, including thrombosis with thrombocytopenia syndrome (TTS), a rare condition resembling heparin-induced thrombocytopenia (HIT), have been observed. This study demonstrates that all three adenoviruses deployed as vaccination vectors versus SARS-CoV-2 bind to platelet factor 4 (PF4), a protein implicated in the pathogenesis of HIT. We have determined the structure of the ChAdOx1 viral vector and used it in state-of-the-art computational simulations to demonstrate an electrostatic interaction mechanism with PF4, which was confirmed experimentally by surface plasmon resonance. These data confirm that PF4 is capable of forming stable complexes with clinically relevant adenoviruses, an important step in unraveling the mechanisms underlying TTS.

It is known that physical linkage of TLR ligands and vaccine antigens significantly enhances the immunopotency of the linked antigens. We have used this approach to generate novel influenza vaccines that fuse the globular head domain of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, flagellin. These fusion proteins are efficiently expressed in standard E. coli fermentation systems and the HA moiety can be faithfully refolded to take on the native conformation of the globular head. In mouse models of influenza infection, the vaccines elicit robust antibody responses that mitigate disease and protect mice from lethal challenge. These immunologically potent vaccines can be efficiently manufactured to support pandemic response, pre-pandemic and seasonal vaccines.

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DNA vaccines are widely recognized for their ability to elicit cellular as well as humoral immune responses to microbial antigens (23). An important characteristic of DNA vaccines is that they can mimic characteristics of live vaccines due to their ability to induce the de novo production of microbial antigens in both transiently transfected nonimmune cells and antigenpresenting cells in the vaccine inoculation site and nearby draining lymph nodes (12,14,25). The promise of intramuscularly injected DNA vaccines has yet to be realized in the clinic, since a number of recent human trials investigating this approach have not yielded the strength and breadth of responses obtained previously in animal models (4,26,49). This may be in part due to the need for better delivery systems, since a recent human trial employing particle-mediated, intracellular delivery ("gene gun") of a hepatitis B DNA vaccine to the epidermis resulted in 100% seroconversion to protective titers and the induction of strong cellular responses (38). In addition to improved delivery, any DNA vaccine, with or without a specific delivery system, can potentially benefit from the inclusion of an adjuvant to augment the strength or quality of a given response. To this end, several investigators have demonstrated the ability to modulate the responses to both naked DNA and particle-based DNA vaccines by inclusion of vectors encoding a variety of cytokines and chemokines (23,24,36,40,46).Our efforts to identify a safe and potent adjuvant for particle-mediated DNA vaccines have recently focused on cholera toxin (CT) and the related Escherichia coli heat-labile enterotoxin (LT). CT and LT are two of the most powerful adjuvants known, due to their ability to elicit robust mucosal and systemic responses via the mucosal and parenteral routes (37, 50). CT and LT are members of the AB 5 class of bacterial toxins and are 80% homologous in their primary structure with essentially identical tertiary structures. These toxins exist as hexamers in which the pentameric B subunit oligomer contains the cellular receptor binding function and is linked to a single A subunit containing an ADP ribosyltransferase activity that is associated with both toxicity and adjuvant effects. Proteolytic cleavage of a trypsin-sensitive site in the A subunit is required for activation of enzymatic activity that is contained in the N-terminal A 1 subunit. This activity results in the ADP ribosylation of several G proteins involved in signal transduction. ADP ribosylation of G s , the regulator of adenylate cyclase, results in the permanent activation of adenylate cyclase and accumulation of cellular cyclic AMP (cAMP) levels.Although CT and LT are closely related molecules, important differences exist in the adjuvant effects that they exhibit (3,37,50). Use of CT as an adjuvant generally results in the induction of antigen-specific Th2 cytokine production, while LT elicits both Th1 and Th2 cytokines (30,51). This immuno-* Corresponding author. Mailing address: PowderJect Vaccines, Inc., 5...