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Description, We observe previously unknown interactions between clinically important adenovirus vector capsids, platelet factor 4, and CAR., 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.

Bispecific antibodies (BsAbs) or fusion proteins (BsAbFPs) present a promising strategy for cancer immunotherapy. Numerous BsAbs targeting coinhibitory and costimulatory pathways have been developed for retargeting T cells and antigen presenting cells (APCs). It is challenging to assess the potency of BsAb that engages two different signaling pathways simultaneously in a single assay format, especially when the two antigen targets are expressed on different cells. To explore the potency of anti-PD-L1/CD40L BsAbFP, a fusion protein that binds to human CD40 and PD-L1, we engineered CHO cells as surrogate APCs that express T cell receptor activator and PD-L1, Jurkat cells with PD-1 and NFAT-luciferase reporter as effector T cells, and Raji cell with NFkB-luciferase that endogenously expresses CD40 as accessory B cells. A novel reporter gene bioassay was developed using these cell lines that allows anti-PD-L1/CD40L BsAbFP to engages both PD-1/PD-L1 and CD40/CD40L signaling pathways in one assay. As both reporters use firefly luciferase, the effects of activating both signaling pathways is observed as an increase in luminescence, either as a higher upper asymptote, a lower EC50, or both. This dual target reporter gene bioassay system reflects potential mechanism of action and demonstrated the ability of anti-PD-L1/CD40L BsAbFP to synergistically induce biological response compared to the combination of anti-PD-L1 monovalent monoclonal antibody and agonist CD40L fusion protein, or either treatment alone. The results also showed a strong correlation between the drug dose and biological response within the tested potency range with good linearity, accuracy, precision, specificity and stability indicating properties, suggesting that this “three-cell-in-one” dual target reporter gene bioassay is suitable for assessing potency, structure-function and critical quality attributes of anti-PD-L1/CD40L BsAbFP. This approach could be used for developing dual target bioassays for other BsAbs and antibodies used for combination therapy.

Control of seasonal and pandemic influenza represents a global public health challenge due to the virus's ability to circumvent protective immune responses through frequent mutation and subunit recombination. These characteristics, coupled with influenza's ability to spread rapidly during outbreaks, require continuous global surveillance, frequent vaccine reformulation, and tightly scheduled manufacturing. Egg- and cell-culture–based vaccine production methods are resource- and time-intensive, leaving little room for error in selection of vaccine components or timing of production. For example, in 2014–2015, long manufacturing lead times prevented the industry from addressing a discrepancy between the H3 virus predominantly circulating in the northern hemisphere and the H3 included in the licensed vaccine [1]. Furthermore, low vaccine effectiveness in the 2012–2013 season was attributed to mutations introduced as part of egg-based manufacturing [2]. Rapid response, high-yield methods that remain faithful to the circulating virus sequences are therefore needed to address the shortcomings of current approaches. Recombinant technologies have the potential to address such current shortcomings and include production of recombinant HA [3], virus-like particles consisting of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins [4, 5], vaccinia virus-based expression of HA and NA [6], the HA1 fragment of HA [7], or the HA2 stalk of HA [8], as well as DNA vector-based expression of multiple antigens [9]. To date, none has achieved a combination of rapid, high-yield production, potency, and ability to remain faithful to the circulating sequence that is sufficient to address the deficiencies associated with egg- and cell-based approaches. We have developed a recombinant influenza vaccine platform whereby the globular head domain of the major protective antigen, HA, is fused to the Toll-like receptor (TLR)5 agonist, flagellin (Salmonella typhimurium flagellin type 2 [STF2]) [10–12]. The binding of STF2 to TLR5 on the surface of sentinel immune cells activates the innate immune system and, in turn, enhances the adaptive immune response [10]. The flagellin moiety of the vaccine therefore serves as a built-in adjuvant. These relatively simple recombinant fusion proteins can be rapidly and inexpensively produced in standard Escherichia coli production systems at high yield. Specifically, current production yields range from 0.3 to 1 mg of purified bulk drug substance per liter of fermentation, with a cycle time of under 2 weeks (unpublished observations). In animal models and in humans, the individual HA-STF2 fusion proteins elicit HA-specific neutralizing antibodies at low microgram doses of vaccine [13]. We have reproduced these results using HA from a variety of influenza type A and B strains, and we have also determined the optimal, subtype or type-specific placement of each HA subunit within the STF2 protein sequence [12–17]. The low dose of antigen required, coupled with the rapidity and efficiency of production, further demonstrate that the platform has the capacity to address the shortcomings associated with current licensed vaccines. Extending on this work, we are developing a prototypic quadrivalent seasonal flu vaccine, VAX2012Q. We report results from a Phase I dose-ranging study designed to identify the total antigen dose and component ratio that provides optimal tolerability and immunogenicity profiles.

Previously, we demonstrated that for H1N1 and H5N1 influenza strains, the globular head of the hemagglutinin (HA) antigen fused to flagellin of Salmonella typhimurium fljB (STF2) is highly immunogenic in preclinical models and man (Song et al. (2008) [13]; Song et al. (2009) [14]; Taylor et al. (2012) [12]). Further we showed that the vaccine format, or point of attachment of the vaccine antigen to flagellin, can dramatically affect the immunogenicity and safety profile of the vaccine. However, Influenza B vaccines based on these formats are poor triggers of TLR5 and consequently are poorly immunogenic. Through rational design, here we show that we have identified a fusion position within domain 3 of flagellin that improves TLR5 signaling and consequently, immunogenicity of multiple influenza B vaccines. Our results demonstrate that, similar to influenza A strains, the protective subunit of the influenza B HA can be fused to flagellin and produced in a standard prokaryotic expression system thereby allowing for cost and time efficient production of multivalent seasonal influenza vaccines.

Background We evaluated the safety and immunogenicity profiles of 3 novel influenza vaccine constructs consisting of the globular head of the HA1 domain of the Novel H1N1 genetically fused to the TLR5 ligand, flagellin. HA1 was fused to the C-terminus of flagellin in VAX128A, replaced the D3 domain of flagellin in VAX128B and was fused in both positions in VAX128C. Methods In a dose escalation trial, 112 healthy subjects 18–49 and 100 adults ≥65 years old were enrolled in a double blind, placebo controlled clinical trial at two centers. Vaccines were administered IM at doses ranging from 0.5 to 20 μg. VAX128C was selected for second study performed in 100 subjects 18–64 years old comparing 1.25 and 2.5 μg doses. All subjects were followed for safety and sera collected pre- and post-vaccination were tested by hemagglutination-inhibition (HAI). Serum C-reactive protein and cytokine levels were also measured. Conclusions In the first study high HAI titers and high seroconversion and seroprotection rates were observed at doses ≥2.5 μg in adults 18–49. In adults ≥65 years, the vaccines doses of ≥4 μg were required to induce a ≥4-fold rise in HAI titer, 50% seroconversion and 70% seroprotection. Based on safety, VAX128A was tested up to 8 μg, VAX128B to 16 μg and VAX128C to 20 μg. Dose escalation for VAX128A was stopped at 8 μg because one subject had temperature 101.6 °F associated with a high CRP response, VAX128B was stopped at 16 μg because of a severe AE associated with a high CRP and IL-6 response. VAX128C was not stopped before reaching the 20 μg dose. In the second study VAX128C was well tolerated among 100 subjects who received 1.25 or 2.5 μg. The peak GMT was 385 (95%CI 272–546), 79% (71–87%) seroconversion and 92% (84–96%) seroprotection. Discussion Flagellin adjuvanted vaccines can be designed to minimize reactogenicity and retain immunogenicity, thereby representing a promising next generation vaccine technology.

We have previously demonstrated that the globular head of the hemagglutinin (HA) antigen fused to flagellin of Salmonella typhimurium fljB (STF2, a TLR5 ligand) elicits protective immunity to H1N1 and H5N1 lethal influenza infections in mice (Song et al., 2008, PLoS ONE 3, e2257; Song et al., 2009, Vaccine 27, 5875-5888). These fusion proteins can be efficiently and economically manufactured in E. coli fermentation systems as next generation pandemic and seasonal influenza vaccines. Here we report immunogenicity and efficacy results of three vaccine candidates in which the HA globular head of A/California/07/2009 (H1N1) was fused to STF2 at the C-terminus (STF2.HA1), in replace of domain 3 (STF2R3.HA1), or in both positions (STF2R3.2xHA1). For all three vaccines, two subcutaneous immunizations of BALB/c mice with doses of either 0.3 or 3 µg elicit robust neutralizing (HAI) antibodies, that lead to > = 2 Log(10) unit reduction in day 4 lung virus titer and full protection against a lethal A/California/04/2009 challenge. Vaccination with doses as low as 0.03 µg results in partial to full protection. Each candidate, particularly the STF2R3.HA1 and STF2R3.2xHA1 candidates, elicits robust neutralizing antibody responses that last for at least 8 months. The STF2R3.HA1 candidate, which was intermediately protective in the challenge models, is more immunogenic than the H1N1 components of two commercially available trivalent inactivated influenza vaccines (TIVs) in mice. Taken together, the results demonstrate that all three vaccine candidates are highly immunogenic and efficacious in mice, and that the STF2R3.2xHA1 format is the most effective candidate vaccine format.

Transmission of highly pathogenic avian influenza (HPAI) between birds and humans is an ongoing threat that holds potential for the emergence of a pandemic influenza strain. A major barrier to an effective vaccine against avian influenza has been the generally poor immunopotency of many of the HPAI strains coupled with the manufacturing constraints employing conventional methodologies. Fusion of flagellin, a toll-like receptor-5 ligand, to vaccine antigens has been shown to enhance the immune response to the fused antigen in preclinical studies. Here, we have evaluated the immunogenicity and efficacy of a panel of flagellin-based hemagglutinin (HA) globular head fusion vaccines in inbred mice. The HA globular head of these vaccines is derived from the A/Vietnam/1203/04 (VN04; H5N1) HA molecule. We find that replacement of domain D3 of flagellin with the VN04 HA globular head creates a highly effective vaccine that elicits protective HAI titers which protect mice against disease and death in a lethal challenge model.

The recognition of specific pathogen associated molecular patterns (PAMPs) by members of the Toll-like receptor (TLR) family is critical for the activation of the adaptive immune response. Thus, incorporation of PAMPs into vaccines should result in more potent, protective antigen-specific responses in the absence of adjuvants or complex formulations. Here we describe an influenza A vaccine that is refractory to the genetic instability of hemagglutinin and neuraminidase and includes a trigger of the innate immune response to enhance immunogenicity and efficacy. A recombinant protein comprising the TLR5 ligand flagellin fused to four tandem copies of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli and purified to homogeneity. This protein, STF2.4xM2e, retained TLR5 activity and displayed the protective epitope of M2e defined by a monoclonal antibody, 14C2. Mice immunized with STF2.4xM2e in aqueous buffer, without adjuvants or other formulation additives, developed potent M2e-specific antibody responses that were quantitatively and qualitatively superior to those observed with M2e peptide delivered in alum. The antibody response was dependent on the physical linkage of the antigen to flagellin and recognized the epitope defined by monoclonal antibody 14C2, which has been shown to protect mice from challenge with influenza A virus. Moreover, immunization with STF2.4xM2e at a dose of 0.3 microg per mouse protected mice from a lethal challenge with influenza A virus, and significantly reduced weight loss and clinical symptoms. These data demonstrate that the linkage of specific TLR ligand with influenza M2e yields a vaccine candidate that offers significant promise for widespread protection against multiple influenza A virus strains.

Among the numerous assays proposed for quantifying specific nucleic acid sequences in biological samples, PCR offers the most sensitivity and versatility. The assay for quantifying the amount of PCR products is a crucial step in any quantitative PCR method. It should be sensitive and specific, able to display a wide dynamic range, nonradioactive, easy to perform, and inexpensive. The results of the assay should also be easily digitalized. Quantification of amplicons with ELISA fulfills these criteria. It can be automated, and readers are already available in most research and clinical laboratories. This assay can be accomplished by using colorimetry, fluorometry, or luminometry, depending on the substrate used. Luminometry displays the best sensitivity and has the widest dynamic range of these three methods (Martin et al., 1995).

The recent appearance of a novel H1N1 (nH1N1) virus has highlighted the importance of rapid response to emerging pandemic threats. VaxInnate has developed an influenza vaccine production system that permits rapid response on a global scale. The vaccine comprises the protective globular head subunit of HA fused covalently to the TLR 5 ligand flagellin. Using animal models that have successfully predicted the tolerated dose range in humans we have identified an optimal position for the HA globular head. Three constructs evaluated the globular head fused to the C terminus of flagellin (C-term), in place of domain 3 (R3) and in both positions (R3.2x). The three formats were tested in mouse immunogenicity and rabbit reactogenicity models. HAI assays were performed using CA/07/2009 virus. Rabbits were monitored for body temperature, food consumption and CRP. In mice, HAI titers >1:40 were achieved with 1 μg doses of all vaccines, but R3 and R3.2x vaccines yielded higher titers. In rabbits, the C-term vaccine elicited elevated temperature and CRP levels and reduced food consumption at doses well below R3 and R3.2x. Replacement of domain 3 of flagellin with the HA globular head, either R3 or R3.2x, proves safe and immunogenic in mice and rabbits. Our clinical experience with other flagellin-linked vaccines has correlated well with the rabbit model. The results predict that the R3 or R3.2x nH1 vaccines would be both safe and efficacious in humans.