Phase 1 Safety and Immunogenicity Study of a Quadrivalent Seasonal Flu Vaccine Comprising Recombinant Hemagglutinin-Flagellin Fusion Proteins.

Background. We evaluated the safety and immunogenicity of VAX2012Q, a quadrivalent influenza vaccine comprising 4 hemagglutinin subunits fused to flagellin. Methods. In this dose-ranging, open-label study, healthy adults (18-40 years) were divided into 7 cohorts for evaluation of 5 dose levels and 3 component ratios. Dose levels were as follows: (1) 1 mcg per component of VAX128C (H1N1), VAX181 (H3N2), VAX173 (B-YAM), and VAX172 (B-VIC), respectively; (2) 2 mcg per component, respectively; (3) 2, 4, 4, and 4 mcg of each component, respectively; (4) 2, 4, 6, and 6 mcg of each component, respectively; and (5) 3 mcg per component, respectively. Tolerability and immunogenicity data were analyzed. Results. Three hundred sixteen subjects received VAX2012Q (309 per protocol). At all dose levels, 54% to 65% of subjects reported mild injection site pain, the most common local reaction. Moderate injection site pain increased at dose levels 2 through 5 (22%-42%, compared with 20% at dose level 1). Systemic symptoms were mostly mild to moderate with moderate symptoms increasing in dose levels 3 and 4. Three dose level 3 subjects (6%) reported severe, transient chills and or fever. Mean fold rises in hemagglutination inhibition titers ranged from 2.5 to 6.9 despite high baseline titers. Mean seroprotection rates were ≥90% and mean seroconversion rates were ≥40% for all strains in all groups postvaccination. Conclusions. VAX2012Q elicited immune responses at all dose levels with no significant safety concerns. Doses of 2 or 3 mcg per component provided a favorable balance of tolerability and immunogenicity.

Immunogenicity and efficacy of flagellin-envelope fusion dengue vaccines in mice and monkeys.

The envelope (E) protein of flaviviruses includes three domains, EI, EII, and EIII, and is the major protective antigen. Because EIII is rich in type-specific and subcomplex-specific neutralizing epitopes and is easy to express, it is particularly attractive as a recombinant vaccine antigen. VaxInnate has developed a vaccine platform that genetically links vaccine antigens to bacterial flagellin, a Toll-like receptor 5 ligand. Here we report that tetravalent dengue vaccines (TDVs) consisting of four constructs, each containing two copies of EIII fused to flagellin (R3.2x format), elicited robust and long-lived neutralizing antibodies (geometric mean titers of 200 to 3,000), as measured with a 50% focus reduction neutralization test (FRNT50). In an immunogenicity study, rhesus macaques (n = 2) immunized subcutaneously with 10 µg or 90 µg of TDV three or four times, at 4- to 6-week intervals, developed neutralizing antibodies to four dengue virus (DENV) serotypes (mean post-dose 3 FRNT50 titers of 102 to 601). In an efficacy study, rhesus macaques (n = 4) were immunized intramuscularly with 16 µg or 48 µg of TDV or a placebo control three times, at 1-month intervals. The animals that received 48-µg doses of TDV developed neutralizing antibodies against the four serotypes (geometric mean titers of 49 to 258) and exhibited reduced viremia after DENV-2 challenge, with a group mean viremia duration of 1.25 days and 2 of 4 animals being completely protected, compared to the placebo-treated animals, which all developed viremia, with a mean duration of 4 days. In conclusion, flagellin-EIII fusion vaccines are immunogenic and partially protective in a nonhuman primate model.

A rationally designed form of the TLR5 agonist, flagellin, supports superior immunogenicity of Influenza B globular head vaccines.

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.

Antibody recognition of the pandemic H1N1 Influenza virus hemagglutinin receptor binding site.

Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines.

Flagellin-HA vaccines protect ferrets and mice against H5N1 highly pathogenic avian influenza virus (HPAIV) infections.

In order to meet the global demand for rapid production of pandemic influenza vaccines, we have developed a recombinant fusion vaccine platform in which the globular head of hemagglutinin (HA) antigen is genetically fused to bacterial flagellin (a TLR5 ligand). These flagellin-HA fusion vaccine candidates elicit highly protective immunity against a lethal challenge with 2009 pandemic H1N1 (Liu, et al. PLoS ONE 2011; 6:e20928) or H5N1 influenza A/Vietnam/1203/04 (A/VN) infections in mice (Song, et al. Vaccine 2009;27:5875-88). Here we provide the first evidence showing that two A/VN vaccine candidates elicited HA-specific IgG, reduced nasal virus shedding, and conferred full protection against a lethal A/VN infection in ferrets. Furthermore, we show that similar flagellin-HA vaccine candidates of two other H5N1 HPAIV are immunogenic and/or efficacious in mice. Vaccines of A/Indonesia/5/05 (A/IN) induced significant HAI titers to homologous and heterologous A/Anhui/1/05 (A/AN) H5N1 viruses. Two subcutaneous immunizations with doses of either 0.3 µg or 3 µg of A/IN candidates resulted in ≥ 2.5 log(10) unit reduction in day 5 lung virus titer and 90-100% protection against a lethal A/IN challenge in mice. Both R3.HA5 IN and R3.2xHA5 IN vaccines elicited robust neutralizing antibody responses that last for at least 9 months and demonstrated a significant anamnestic antibody response upon further booster immunization. Finally, we found that two vaccine candidates of A/AN induced significant HAI titers in mice. Taken together, our recombinant flagellin-HA platform has been successfully used to generate potent H5N1 HPAIV vaccine candidates. These promising preclinical results justify the advancement of these candidates into the clinic.

Development of VAX128, a recombinant hemagglutinin (HA) influenza-flagellin fusion vaccine with improved safety and immune response.

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.

Immunogenicity and efficacy of flagellin-fused vaccine candidates targeting 2009 pandemic H1N1 influenza in mice.

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.

Safety and immunogenicity of a recombinant hemagglutinin influenza-flagellin fusion vaccine (VAX125) in healthy young adults.

BACKGROUND: The need for worldwide seasonal and pandemic vaccine production has increased interest in the development of innovative technologies for influenza vaccine production. We evaluated a novel influenza vaccine consisting of the globular head of the HA1 domain of the A/Solomon Islands/3/2006 (H1N1) influenza virus (VAX125) genetically fused to the TLR5 ligand, flagellin, and produced in E. coli. METHODS: 128 healthy adult subjects 18-49 years old were enrolled in a clinical trial conducted in three stages at a single center. Stage 1 was an open-label, dose escalation study in which the VAX125 vaccine was administered intramuscularly (im) at doses of 0.1 µg, 0.3 µg, 1 µg, 2 µg, 3 µg, 5 µg and 8 µg to groups of 8 subjects each. Stage 2 was a double-blind, placebo-controlled study in which subjects were randomized to receive 1.0 µg and 2.0 µg VAX125 vaccine doses or placebo, with 16 subjects per group. Finally, an additional 24 subjects received a 0.5 µg dose of VAX125 in stage 3, which was a non-randomized, open label study. In all parts subjects were followed for adverse events and sera was tested by hemagglutination-inhibition (HAI) and microneutralization (MN) against egg-grown virus on days 0, 7, 14, and 28. Serum C-reactive protein (CRP), cytokine levels, and anti-flagellin antibody were also assessed. RESULTS: Vaccine was generally well tolerated and there were no serious adverse events. Pain at the injection site was the most common local adverse event, and was mild or moderate in intensity. Systemic symptoms after vaccination include fatigue and headache, and two subjects, who received either 3 or 8 µg, had moderately severe systemic symptoms accompanied by substantial increases in serum CRP. Serum antibody responses against SI were seen by HAI and MN in most study subjects, with the geometric mean titer of post vaccination antibody increasing in a dose-dependent fashion. Overall, four-fold or greater serum HAI responses were seen in 61 of 96 (64%) subjects who received doses of 0.5 µg or greater, including in 46 of 72 subjects who received doses from 0.5 µg to 2 µg. CONCLUSIONS: The globular head of the influenza HA expressed in a prokaryotic system was able to induce a functional antibody response against native virions. Vigorous responses were seen at relatively low doses of HA antigen suggesting that the addition of flagellin provided a substantial adjuvanting effect. The high levels of immune response at low doses of antigen and the relative ease of production associated with E. coli expression suggests that this approach may represent an effective strategy for enhancing the global influenza vaccine supply.

Superior efficacy of a recombinant flagellin:H5N1 HA globular head vaccine is determined by the placement of the globular head within flagellin.

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.

Efficacious recombinant influenza vaccines produced by high yield bacterial expression: a solution to global pandemic and seasonal needs.

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.

Potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza M2e to the TLR5 ligand flagellin.

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.

Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity.

Recognition of specific pathogen associated molecular patterns (PAMPs) is mediated primarily by members of the Toll-like receptor (TLR) family. Stimulation through these receptors results in quantitative and qualitative changes in antigen presentation and cellular activation, thereby linking innate and adaptive immunity. Consequently, the incorporation of TLR-ligands into vaccines could result in more potent and efficacious vaccines. To test this hypothesis, we employed a recombinant fusion protein strategy using the TLR5 ligand flagellin fused to specific antigens to promote protective immunity. These purified recombinant fusion proteins demonstrated potent TLR5-specific NF-kappaB dependent activity in vitro. Immunization of mice with the recombinant-flagellin-OVA fusion protein STF2.OVA resulted in potent antigen-specific T and B cell responses that were equal to or better than responses induced by OVA emulsified in Complete Freund's adjuvant. These included rapid and consistent antigen-specific IgG(1) and IgG(2a) antibody responses that were detectable within 7 days of immunization, and the development of protective CD8 T cell responses. Moreover, the enhanced immunogenicity to OVA is dependant on the direct fusion to flagellin, as co-delivery of OVA with flagellin unlinked failed to augment antigen-specific responses in vivo. Similar results were obtained using a recombinant fusion protein comprised of flagellin and a novel polypetide sequence containing two immuno-protective epitopes derived from the Listeria monocytogenes antigens p60 and listeriolysin O. Animals immunized with this recombinant protein demonstrated significant antigen-specific CD8 T cell responses and protection upon challenge with virulent L. monocytogenes. We conclude that immunization with PAMP:antigen fusion proteins induce rapid and potent antigen-specific responses in the absence of supplemental adjuvants. Collectively our data demonstrate that PAMP:antigen fusion proteins offer significant promise for developing recombinant protein vaccines.

Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex.

SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. Here we present the crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex, which shows that Cul1 is an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1-F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1-F boxSkp2 and Rbx1 subunits, holding them over 100 A apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold.