Formulation Approach that Enables the Coating of a Stable Influenza Vaccine on a Transdermal Microneedle Patch.

A trivalent influenza split vaccine was formulated at high concentration for coating on the transdermal microneedle system. Monovalent vaccine bulks of three influenza strains, two influenza A strains, and one B strain were diafiltrated, concentrated, and lyophilized. The lyophilized powder of each vaccine strain was separately reconstituted and subsequently combined into a coating formulation of high concentration trivalent vaccine. The formulation process converted the monovalent vaccine bulks with low hemagglutinin (HA) concentrations 0.1 mg/mL into a viscous, emulsion containing HA at ~50 mg/mL. This physically stable emulsion demonstrated viscosity 1 poise and 30° contact angle for effective, homogeneous coating on each microneedle. Evaluation of the vaccine antigen HA by SRID and SDS-PAGE/Western blot showed that HA remained stable throughout the vaccine transdermal microneedle system manufacturing process and 1-year ambient storage (25°C). Anti-influenza antibody responses were evaluated by ELISA and hemagglutination inhibition (HAI) assay after primary and booster immunization with the vaccine-coated transdermal microneedle systems at either 25-µg or 40-µg total HA. The results showed the induction of serum anti-influenza IgG and anti-HA neutralizing antibodies after primary immunization and significant titer rises after booster immunization for both doses, indicating the dry-coated trivalent vaccine delivered by transdermal microneedle system elicited both primary and recall antibody responses against all three antigen strains. The study demonstrates that the transdermal microneedle system provides an attractive alternative for influenza vaccine delivery with key advantages such as preservative-free and room-temperature storage.

Mannosylation of LNP Results in Improved Potency for Self-Amplifying RNA (SAM) Vaccines.

Mannosylation of Lipid Nanoparticles (LNP) can potentially enhance uptake by Antigen Presenting Cells, which are highly abundant in dermal tissues, to improve the potency of Self Amplifying mRNA (SAM) vaccines in comparison to the established unmodified LNP delivery system. In the current studies, we evaluated mannosylated LNP (MLNP), which were obtained by incorporation of a stable Mannose-cholesterol amine conjugate, for the delivery of an influenza (hemagglutinin) encoded SAM vaccine in mice, by both intramuscular and intradermal routes of administration. SAM MLNP exhibited in vitro enhanced uptake in comparison to unglycosylated LNP from bone marrow-derived dendritic cells, and in vivo more rapid onset of the antibody response, independent of the route. The increased binding antibody levels also translated into higher functional hemagglutinin inhibition titers, particularly following intradermal administration. T cell assay on splenocytes from immunized mice also showed an increase in antigen specific CD8+ T responses, following intradermal administration of MLNP SAM vaccines. Induction of enhanced antigen specific CD4+ T cells, correlating with higher IgG2a antibody responses, was also observed. Hence, the present work illustrates the benefit of mannosylation of LNPs to achieve a faster immune response with SAM vaccines and these observations could contribute to the development of novel skin delivery systems for SAM vaccines.

Conserved peptides enhance immune efficiency of inactive vaccines against emerging avian influenza viruses in chicken.

Avian influenza viruses (AIVs) such as H5N1 and H7N9 are a great threat to poultry economics and public health. Vaccination can effectively inhibit the spread of AIV in poultry, which is also a viable strategy for controlling virus transmission from poultry to human. Adjuvants that are commonly used in current inactivated vaccines to provide stronger anti-AIV immune responses are often limited in their capacity to quantitatively induce both humoral and cellular immune responses. Herein, we assessed the levels of immune responses generated by a vaccine formulation comprising inactivated H5N1 antigen and synthetic peptides covering conserved CD4+, CD8+ T cell, and B cell epitopes. We found that the synthetic peptides enhanced the antibody responses against conserved influenza virus antigen M2e. Notably, the hemagglutination inhibition test results indicated that the peptides significantly augmented the antibody responses of inactivated H5N1 antigen even in the 1/10 or 1/5 dose group, in the identical antibody level as antigen alone used at the full dose. This indicates that the peptide can significantly reduce the use of inactivated virus, lowering the cost of the vaccine. Moreover, the peptides increased the transcript levels of interleukin-4 and interferon-γ cytokines in chicken peripheral blood mononuclear cells, which may facilitate both humoral and cellular immune responses. Our data suggest that this peptide combined with inactivated H5N1 antigen enhances both the humoral and cellular immune responses, which may benefit the prediction and design of synthetic peptide-based adjuvants for vaccines in chicken.

Augmenting Influenza-Specific T Cell Memory Generation with a Natural Killer T Cell-Dependent Glycolipid-Peptide Vaccine.

The development of a universal vaccine for influenza A virus (IAV) that does not require seasonal modification is a long-standing health goal, particularly in the context of the increasing threat of new global pandemics. Vaccines that specifically induce T cell responses are of considerable interest because they can target viral proteins that are more likely to be shared between different virus strains and subtypes and hence provide effective cross-reactive IAV immunity. From a practical perspective, such vaccines should induce T cell responses with long-lasting memory, while also being simple to manufacture and cost-effective. Here we describe the synthesis and evaluation of a vaccine platform based on solid phase peptide synthesis and bio-orthogonal conjugation methodologies. The chemical approach involves covalently attaching synthetic long peptides from a virus-associated protein to a powerful adjuvant molecule, α-galactosylceramide (α-GalCer). Strain-promoted azide-alkyne cycloaddition is used as a simple and efficient method for conjugation, and pseudoproline methodology is used to increase the efficiency of the peptide synthesis. α-GalCer is a glycolipid that stimulates NKT cells, a population of lymphoid-resident immune cells that can provide potent stimulatory signals to antigen-presenting cells engaged in driving proliferation and differentiation of peptide-specific T cells. When used in mice, the vaccine induced T cell responses that provided effective prophylactic protection against IAV infection, with the speed of viral clearance greater than that seen from previous viral exposure. These findings are significant because the vaccines are highly defined, quick to synthesize, and easily characterized and are therefore appropriate for large scale affordable manufacture.

A Design of Experiment approach to predict product and process parameters for a spray dried influenza vaccine.

Spray dried vaccine formulations might be an alternative to traditional lyophilized vaccines. Compared to lyophilization, spray drying is a fast and cheap process extensively used for drying biologicals. The current study provides an approach that utilizes Design of Experiments for spray drying process to stabilize whole inactivated influenza virus (WIV) vaccine. The approach included systematically screening and optimizing the spray drying process variables, determining the desired process parameters and predicting product quality parameters. The process parameters inlet air temperature, nozzle gas flow rate and feed flow rate and their effect on WIV vaccine powder characteristics such as particle size, residual moisture content (RMC) and powder yield were investigated. Vaccine powders with a broad range of physical characteristics (RMC 1.2-4.9%, particle size 2.4-8.5µm and powder yield 42-82%) were obtained. WIV showed no significant loss in antigenicity as revealed by hemagglutination test. Furthermore, descriptive models generated by DoE software could be used to determine and select (set) spray drying process parameter. This was used to generate a dried WIV powder with predefined (predicted) characteristics. Moreover, the spray dried vaccine powders retained their antigenic stability even after storage for 3 months at 60°C. The approach used here enabled the generation of a thermostable, antigenic WIV vaccine powder with desired physical characteristics that could be potentially used for pulmonary administration.

Chemical Platforms for Peptide Vaccine Constructs.

Knowledge of the sequences and structures of proteins from pathogenic microorganisms has been put to great use in the field of protein chemistry for the development of peptide-based vaccines. These vaccine constructs include chemically tailored, shorter peptidic fragments that can induce high immunogenicity, thus shunning the allergenic and nonimmunogenic part of the antigens. Based on this concept, several different chemistries have been pursued to obtain novel platforms onto which antigenic epitopes can be tethered, with the aim to achieve a higher antibody response. In this regard, here we attempt to summarize the chemical strategies developed for the presentation of peptide epitopes.

A GFP-tagged nucleoprotein-based aggregation assay for anti-influenza drug discovery and antibody development.

Influenza is a viral pandemic that affects millions of people worldwide. Seasonal variations due to genetic shuffling and antigenic drifts in the influenza viruses have necessitated continual updating of therapeutics. The growing resistance to current influenza drugs has increased demand for new antivirals. The highly conserved nature of NP, a multi-functional viral protein that is serotypically distinct and abundantly expressed during infection, has led to its use in developing universal biotherapeutics and vaccines that could be effective against the virus, irrespective of its strain variations. Compounds causing aggregation of NP have recently been shown to be potent antivirals but require the development of new high-throughput assays capable of screening compounds with similar modes of action. Here, we describe the development of a new bioassay for the Influenza A nucleoprotein (NP). The assay was developed to quantify ligand-induced aggregation of a GFP-tagged NP and was validated with aggregation-inducing compounds such as nucleozin and a NP-specific antibody. The new NP-GFP aggregation assay can be performed with partially purified or mixtures of proteins and is amenable to a high-throughput format. Using this assay, we demonstrate the potential of a new anti-NP polyclonal antibody that we have obtained from chicken. This cost-effective high-yield source of anti-NP IgY has potential for large-scale production and development of therapeutic antibodies. The simplicity, speed and flexibility of this assay make it an invaluable tool for timely development of effective antivirals that can help to control future epidemics.

Continuous purification of influenza virus using simulated moving bed chromatography.

Continuous size exclusion chromatography for the separation of cell culture-derived influenza virus from contaminating proteins was established successfully. Therefore, an open loop simulated moving bed (SMB) setup with one column per zone was applied. Several operating conditions were tested and overall trends were found to be in agreement with expectations derived from theory. Furthermore, the separation performance was compared to an optimized conventional batch chromatography. The yield of influenza virus in the product fraction, based on a hemagglutination assay, was 70% (SMB) and 80% (batch), respectively. The amount of contaminating protein per product was 0.61µgkHAU(-1) (SMB) compared to 0.29µgkHAU(-1) (batch). This corresponds to a reduction of the respective amount in the feed solution by 60% and 80%, respectively. For both processes, the estimated amount of total protein per vaccine dose would meet the level required for manufacturing of human influenza vaccines prepared in cell cultures. Depending on the strategy chosen for sanitization and equilibration of columns the calculated overall productivity for the SMB process was up to 3.8 times higher compared to the batch mode. SMB, therefore, has the potential to replace single column discontinuous chromatography in order to design more efficient purification trains for production of cell culture-derived influenza vaccines.

Toward animal cell culture-based influenza vaccine design: viral hemagglutinin N-glycosylation markedly impacts immunogenicity.

The glycoproteins hemagglutinin (HA) and neuraminidase are the major determinants of host range and tissue tropism of the influenza virus. HA is the most abundant protein in the virus particle membrane and represents the basis of most influenza vaccines. It has been reported that influenza virus HA N-glycosylation markedly depends on the host cell line used for virus production. However, little is known about how differential glycosylation affects immunogenicity of the viral proteins. This is of importance for virus propagation in chicken eggs as well as for innovative influenza vaccine production in mammalian cell lines. In this study, we investigated the impact of the differential N-glycosylation patterns of two influenza A virus PR/8/34 (H1N1) variants on immunogenicity. Madin-Darby canine kidney cell-derived and Vero cell-derived glycovariants were analyzed for immunogenicity in a TCR-HA transgenic mouse model. Next-generation pyrosequencing validated the congruence of the potential HA N-glycosylation sites as well as the presence of the HA peptide recognized by the TCR-HA transgenic T cells. We show that differential HA N-glycosylation markedly affected T cell activation and cytokine production in vitro and moderately influenced IL-2 production in vivo. Cocultivation assays indicated that the difference in immunogenicity was mediated by CD11c(+) dendritic cells. Native virus deglycosylation by endo- and exoglycosidases dramatically reduced cytokine production by splenocytes in vitro and markedly decreased HA-specific Ab production in vivo. In conclusion, this study indicates a crucial importance of HA N-glycosylation for immunogenicity. Our findings have implications for cell line-based influenza vaccine design.

Adjuvant solution for pandemic influenza vaccine production.

Extensive preparation is underway to mitigate the next pandemic influenza outbreak. New vaccine technologies intended to supplant egg-based production methods are being developed, with recombinant hemagglutinin (rHA) as the most advanced program for preventing seasonal and avian H5N1 Influenza. Increased efforts are being focused on adjuvants that can broaden vaccine immunogenicity against emerging viruses and maximize vaccine supply on a worldwide scale. Here, we test protection against avian flu by using H5N1-derived rHA and GLA-SE, a two-part adjuvant system containing glucopyranosyl lipid adjuvant (GLA), a formulated synthetic Toll-like receptor 4 agonist, and a stable emulsion (SE) of oil in water, which is similar to the best-in-class adjuvants being developed for pandemic flu. Notably, a single submicrogram dose of rH5 adjuvanted with GLA-SE protects mice and ferrets against a high titer challenge with H5N1 virus. GLA-SE, relative to emulsion alone, accelerated induction of the primary immune response and broadened its durability against heterosubtypic H5N1 virus challenge. Mechanistically, GLA-SE augments protection via induction of a Th1-mediated antibody response. Innate signaling pathways that amplify priming of Th1 CD4 T cells will likely improve vaccine performance against future outbreaks of lethal pandemic flu.

Exploring synergies between academia and vaccine manufacturers: a pilot study on how to rapidly produce vaccines to combat emerging pathogens.

BACKGROUND: In spring 2009, a new swine-origin influenza A (H1N1) virus emerged in Mexico. During the following weeks the virus spread worldwide, prompting the World Health Organization to declare the first influenza pandemic of the 21st century. Sustained human-to-human transmission and severe disease progression observed in some patients urged public health authorities to respond rapidly to the disease outbreak and vaccine manufacturers to develop pandemic influenza vaccines for mass distribution. With the onset of the pandemic we began to explore the potential of academic/industrial collaboration to accelerate the production of vaccines during an outbreak of an emerging virus by combining the use of an academic BSL-4 laboratory with the expertise of a commercial vaccine manufacturer. METHODS AND RESULTS: To obtain virus seed stocks used for the production of a vaccine to combat the pandemic H1N1 2009 influenza virus (H1N1pdm), we followed various strategies: (i) optimization of cell culture conditions for growth of wild-type H1N1pdm isolates; (ii) classical reassortment of H1N1pdm and standard influenza vaccine donor strain PR8; and (iii) generation of corresponding reassortant viruses using reverse genetics. To ensure a rapid transition to production, the entire potential seed stock development process was carried out in a certified canine kidney suspension cell line (MDCK 33016-PF) under Good Manufacturing Practice (GMP) conditions. CONCLUSIONS: The outcome of this study indicates that a combination of different experimental strategies is the best way to cope with the need to develop vaccines rapidly in the midst of an emerging pandemic.

Ultrasonic synthetic technique to manufacture a pHEMA nanopolymeric-based vaccine against the H6N2 avian influenza virus: a preliminary investigation.

This preliminary study investigated the use of poly (2-hydroxyethyl methacrylate) (pHEMA) nanoparticles for the delivery of the deoxyribonucleic acid (DNA) vaccine pCAG-HAk, which expresses the full length hemagglutinin (HA) gene of the avian influenza A/Eurasian coot/Western Australian/2727/1979 (H6N2) virus with a Kozak sequence which is in the form of a pCAGGS vector. The loaded and unloaded nanoparticles were characterized using field-emission scanning electron microscopy. Further characterizations of the nanoparticles were made using atomic force microscopy and dynamic light scattering, which was used to investigate particle size distributions. This preliminary study suggests that using 100 µg of pHEMA nanoparticles as a nanocarrier/adjuvant produced a reduction in virus shedding and improved the immune response to the DNA vaccine pCAG-HAk.

[Immunogenicity of inactivated subunit adsorbed monovalent vaccine against influenza A/California/7/2009 (H1N1) strain].

The immunogenicity of Pandeflu subunit vaccine against influenza A/California/7/2009 (H1N1) was evaluated in 70 healthy volunteers aged 18 to 60 years. The vaccine was intramuscularly injected twice at an interval of 28 days. Each dose (0.5 ml) contains A(HIN1) influenza virus hemagglutinin (15 +/- 2.2 microg), aluminum hydroxide (Denmark) (0.475 +/- 0.075 microg), and the preservative thiomerosal (merthiolate) (50 +/- 7.5 microg). The level of antibodies was determined in the microneutralization assay. After administration of two doses of the vaccine at a 28-day interval, the geometric mean antibody titer (GMAT) reached 1:21.1 with a further increase to 1:30 (the baseline GMAT) was 1:6.1). The frequencies of seroconversion and seroprotection were 71.4 and 59.2%, respectively; the antibody increase factor was 4.92, which meets the CPMP criteria. The administration of the vaccine did not result in adverse reactions in the postvaccination period.

Vaccine design of hemagglutinin glycoprotein against influenza.

Influenza viruses continue to cause annual epidemics and pose the threat of a deadly global pandemic. Vaccination has remained the best approach for prevention and control of influenza infection. However, current influenza vaccines are only effective against closely-matched circulating strains, and therefore must be updated and administered every year. In this review, we discuss recent developments in the search for better influenza vaccines, especially using the major virus surface glycoprotein hemagglutinins (HAs). Understanding how glycans on HAs affect the immune response and knowledge of how broadly neutralizing antibodies are induced will pave the way for a cross-protective influenza vaccine that does not require frequent updates or annual immunizations.

Characterization of a branched lipopeptide candidate vaccine against influenza A/Puerto Rico 8/34 which is recognized by human B and T-cell immune responses.

The use of synthetic peptides as immunogens represents an exciting alternative to traditional vaccines. However, to date most of these synthetic peptides are not highly immunogenic. The lack of immunogenicity might be addressed by conjugation between T or B cell epitopes with universal or immunodominant T-helper epitopes. The construction of lipidated peptides, branched peptides, or designs combining both of these elements might enhance the immunogenicity, as they might target Toll-Like Receptors and/or mimic the 3-dimensional structure of epitopes within the native protein. Herein, a recognized peptide immunogen based on the hemagglutinin protein of A/Puerto Rico/8/34 was chosen as a backbone and modified to evaluate if the construction of branched peptides, lipidation, the addition of cysteine residues, or mutations could indeed alter epitope reactivity. Screening the different designs with various antibody binding and cellular assays revealed that combining a branched design with the addition of lipid moieties greatly enhanced the immunoreactivity.

A rapid Flp-In system for expression of secreted H5N1 influenza hemagglutinin vaccine immunogen in mammalian cells.

BACKGROUND: Continuing transmissions of highly pathogenic H5N1 viruses in poultry and humans underscores the need for a rapid response to potential pandemic in the form of vaccine. Recombinant technologies for production of immunogenic hemagglutinin (HA) could provide an advantage over the traditional inactivated vaccine manufacturing process. Generation of stably transfected mammalian cells secreting properly folded HA proteins is important for scalable controlled manufacturing. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a Flp-In based 293 stable cell lines through targeted site-specific recombination for expression of secreted hemagglutinin (HA) proteins and evaluated their immunogenicity. H5N1 globular domain HA1(1-330) and HA0(1-500) proteins were purified from the supernatants of 293 Flp-In stable cell lines. Both proteins were properly folded as confirmed by binding to H5N1-neutralizing conformation-dependent human monoclonal antibodies. The HA0 (with unmodified cleavage site) was monomeric, while the HA1 contained oligomeric forms. Upon rabbit immunization, both HA proteins elicited neutralizing antibodies against the homologous virus (A/Vietnam/1203/2004, clade 1) as well as cross-neutralizing antibodies against heterologous H5N1 clade 2 strains, including A/Indonesia/5/2005. These results exceeded the human antibody responses against the inactivated sub-virion H5N1 vaccine. CONCLUSIONS/SIGNIFICANCE: Our data suggest that the 293 Flp-In system could serve as a platform for rapid expression of HA immunogens in mammalian cells from emerging influenza strains.

Structural basis of influenza virus neutralization.

Although seasonal influenza vaccines play a valuable role in reducing the spread of virus at the population level, ongoing viral evolution to evade immune responses remains problematic. No current vaccines elicit enduring protection in the face of emerging and re-emerging influenza viruses that are rapidly undergoing antigenic drift. Eliciting broadly cross-neutralizing antibody (nAb) responses against influenza virus is a crucial goal for seasonal and pandemic influenza vaccine preparation. Recent three-dimensional structure information obtained from crystallization of influenza antigens in complex with nAbs has provided a framework for interpreting antibody-based viral neutralization that should aid in the design of vaccine immunogens. Here, we will review current knowledge of the structure-based mechanisms contributing to the neutralization and neutralization escape of influenza viruses. We will also explore the potential for this structure-based approach to overcome the obstacles in developing the highly desired "universal" influenza vaccine.

The development of vaccine viruses against pandemic A(H1N1) influenza.

Wild type human influenza viruses do not usually grow well in embryonated hens' eggs, the substrate of choice for the production of inactivated influenza vaccine, and vaccine viruses need to be developed specifically for this purpose. In the event of a pandemic of influenza, vaccine viruses need to be created with utmost speed. At the onset of the current A(H1N1) pandemic in April 2009, a network of laboratories began a race against time to develop suitable candidate vaccine viruses. Two approaches were followed, the classical reassortment approach and the more recent reverse genetics approach. This report describes the development and the characteristics of current pandemic H1N1 candidate vaccine viruses.

An integrated bioinformatics approach to the characterization of influenza A/H5N1 viral sequences by microarray data: Implication for monitoring H5N1 emerging strains and designing appropriate influenza vaccines.

In order to characterize A/H5N1 viral sequences, a bioinformatics approach accurately identified viral sequences from discovery of a sequence signature, which provided enough distinctive information for sequence identification. Eight highly pathogenic H5N1 viral isolations were collected from different areas of Thailand between 2003 and 2006, and were used for analysis of H5N1 genotypic testing with a semiconductor-based oligonucleotide microarray. All H5N1 samples and H1N1, H4N8 negative controls were correctly subtyped. Sensitivity of the eight oligonucleotide probes, with optimized cut-offs, ranged from 70% (95% CI 65-75) to 87% (95% CI 84-91), and the corresponding Kappa values ranged from 0.76 (95% CI 0.72-0.80) to 0.86 (95% CI 0.83-0.89). Semi-conductor-based oligonucleotide array and oligonucleotide probes corresponded well when detecting H5N1. After fully correcting the subtype from the result of microarray signal intensity, the microarray output method combined with bioinformatics tools, identified and monitored genetic variations of H5N1. Capability of distinguishing different strains of H5N1 from Thailand was the outstanding feature of this assay. Ninety percent of HA and NA (4/5) genes were sequenced correctly, in accordance with previous examinations performed by classical diagnostic methods. The low-medium-high bioinformatics resolutions were able to predict an epidemic strain of H5N1. This study also showed the advantage of using a large genotypic database to predict the epidemic strain of H5N1. However, the monitoring protocol of this new strain has been recommended for further study with a large-scale sample.