GLA-AF, an emulsion-free vaccine adjuvant for pandemic influenza.

The ongoing threat from Influenza necessitates the development of new vaccine and adjuvant technologies that can maximize vaccine immunogenicity, shorten production cycles, and increase global vaccine supply. Currently, the most successful adjuvants for Influenza vaccines are squalene-based oil-in-water emulsions. These adjuvants enhance seroprotective antibody titers to homologous and heterologous strains of virus, and augment a significant dose sparing activity that could improve vaccine manufacturing capacity. As an alternative to an emulsion, we tested a simple lipid-based aqueous formulation containing a synthetic TLR4 ligand (GLA-AF) for its ability to enhance protection against H5N1 infection. GLA-AF was very effective in adjuvanting recombinant H5 hemagglutinin antigen (rH5) in mice and was as potent as the stable emulsion, SE. Both adjuvants induced similar antibody titers using a sub-microgram dose of rH5, and both conferred complete protection against a highly pathogenic H5N1 challenge. However, GLA-AF was the superior adjuvant in ferrets. GLA-AF stimulated a broader antibody response than SE after both the prime and boost immunization with rH5, and ferrets were better protected against homologous and heterologous strains of H5N1 virus. Thus, GLA-AF is a potent emulsion-free adjuvant that warrants consideration for pandemic influenza vaccine development.

Clinical vaccine development for H5N1 influenza.

H5N1 is a highly pathogenic avian influenza virus that can cause severe disease and death in humans. H5N1 is spreading rapidly in bird populations and there is great concern that this virus will begin to transmit between people and cause a global crisis. Vaccines are the cornerstone strategy for combating avian influenza but there are complex challenges for pandemic preparedness including the unpredictability of the vaccine target and the manufacturing requirement for rapid deployment. The less-than-optimal response against the 2009 H1N1 pandemic unmasked the limitations associated with influenza vaccine production and in 2010, the President's Council of Advisors on Science and Technology re-emphasized the need for new recombinant-based vaccines and adjuvants that can shorten production cycles, maximize immunogenicity and satisfy global demand. In this article, the authors review the efforts spent in developing an effective vaccine for H5N1 influenza and summarize clinical studies that highlight the progress made to date.

Comparative glycomics analysis of influenza Hemagglutinin (H5N1) produced in vaccine relevant cell platforms.

Hemagglutinin (HA) is the major antigen in influenza vaccines, and glycosylation is known to influence its antigenicity. Embryonated hen eggs are traditionally used for influenza vaccine production, but vaccines produced in mammalian and insect cells were recently licensed. This raises the concern that vaccines produced with different cell systems might not be equivalent due to differences in their glycosylation patterns. Thus, we developed an analytical method to monitor vaccine glycosylation through a combination of nanoLC/MS(E) and quantitative MALDI-TOF MS permethylation profiling. We then used this method to examine glycosylation of HAs from two different influenza H5N1 strains produced in five different platforms, including hen eggs, three different insect cell lines (High Five, expresSF+ and glycoengineered expresSF+), and a human cell line (HEK293). Our results demonstrated that (1) sequon utilization is not necessarily equivalent in different cell types, (2) there are quantitative and qualitative differences in the overall N-glycosylation patterns and structures produced by different cell types, (3) ∼20% of the N-glycans on the HAs produced by High Five cells are core α1,3-fucosylated structures, which may be allergenic in humans, and (4) our method can be used to monitor differences in glycosylation during the cellular glycoengineering stages of vaccine development.

Pandemic influenza vaccine: characterization of A/California/07/2009 (H1N1) recombinant hemagglutinin protein and insights into H1N1 antigen stability.

BACKGROUND: The recent H1N1 influenza pandemic illustrated the shortcomings of the vaccine manufacturing process. The A/California/07/2009 H1N1 pandemic influenza vaccine or A(H1N1)pdm09 was available late and in short supply as a result of delays in production caused by low yields and poor antigen stability. Recombinant technology offers the opportunity to shorten manufacturing time. A trivalent recombinant hemagglutinin (rHA) vaccine candidate for seasonal influenza produced using the baculovirus expression vector system (BEVS) was shown to be as effective and safe as egg-derived trivalent inactivated vaccine (TIV) in human clinical studies. In this study, we describe the characterization of the A/California/07/2009 rHA protein and compare the H1N1 pandemic rHA to other seasonal rHA proteins. RESULTS: Our data show that, like other rHA proteins, purified A/California/07/2009 rHA forms multimeric rosette-like particles of 20-40 nm that are biologically active and immunogenic in mice as assayed by hemagglutination inhibition (HAI) antibody titers. However, proteolytic digest analysis revealed that A/California/07/2009 rHA is more susceptible to proteolytic degradation than rHA proteins derived from other seasonal influenza viruses. We identified a specific proteolytic site conserved across multiple hemagglutinin (HA) proteins that is likely more accessible in A/California/07/2009 HA, possibly as a result of differences in its protein structure, and may contribute to lower antigen stability. CONCLUSION: We conclude that, similar to the recombinant seasonal influenza vaccine, recombinant A(H1N1)pdm09 vaccine is likely to perform comparably to licensed A(H1N1)pdm09 vaccines and could offer manufacturing advantages.

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.

Characterization of a recombinant influenza vaccine candidate using complementary LC-MS methods.

Influenza vaccination is recognized as the most effective method for reducing morbidity and mortality due to seasonal influenza. To improve vaccine supply and to increase flexibility in vaccine manufacturing, cell culture-based vaccine production has emerged to overcome limitations of egg-based production. The switch of production system and the need for annual re-evaluation of vaccines for the effectiveness due to frequent viral antigenic changes call for methods for complete characterization of the hemagglutinin (HA) antigens and the final vaccine products. This study describes advanced liquid chromatography-mass spectrometry (LC-MS) methods for simultaneous identification of HA proteins and process-related impurities in a trivalent influenza candidate vaccine, comprised of purified recombinant HA (rHA) antigens produced in an insect cell-baculovirus expression vector system (BEVS). N-linked glycosylation sites and glycoforms of the three rHA proteins (corresponding to influenza A subtypes H1N1 and H3N2 and B virus, respectively) were profiled by peptide mapping using reversed-phase (RP) LC-MS(E) (data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode). The detected site-specific glycoforms were further confirmed and quantified by hydrophilic interaction LC (HILIC)-multiple reaction monitoring (MRM) assays. LC-MS(E) was used to characterize the vaccine candidate, providing both protein identities and site-specific information of glycosylation and degradations on each rHA protein. HILIC-MRM methodology was used for rapid confirming and quantifying site-specific glycoforms and potential degradations on each rHA protein. These methods can contribute to the monitoring of vaccine quality especially as it pertains to product comparability studies to evaluate the impact of production process changes.

A recombinant West Nile virus envelope protein vaccine candidate produced in Spodoptera frugiperda expresSF+ cells.

In this study, a recombinant truncated West Nile virus envelope protein antigen (rWNV-E) was produced in serum-free cultures of the expresSF+ insect cell line via baculovirus infection. This production system was selected based on its use in the production of candidate human and animal vaccine antigens. A defined fermentation and purification process for the rWNV-E antigen was established to control for purity and immunogenicity of each protein batch. The material formulated with aluminum hydroxide was stable for greater than 8months at 4 degrees C. The recombinant vaccine candidate was evaluated for immunogenicity and protective efficacy in several animal models. In mouse and hamster WNV challenge models, the vaccine candidate induced viral protection that correlated with anti-rWNV-E immunogenicity and WNV neutralizing antibody titers. The rWNV-E vaccine candidate was used to boost horses previously immunized with the Fort Dodge inactivated WNV vaccine and also to induce WNV neutralizing titers in naïve foals that were at least 14weeks of age. Furthermore, the vaccine candidate was found safe when high doses were injected into rats, with no detectable treatment-related clinical adverse effects. These observations demonstrate that baculovirus-produced rWNV-E can be formulated with aluminum hydroxide to produce a stable and safe vaccine which induces humoral immunity that can protect against WNV infection.

Staking out novelty on the genomic frontier.

Recent advances in genomics include global assessment and classification of genome content, high-throughput biological pathway construction, systematic identification of previously unpredicted genes and the in vitro creation of novel motifs with biological function not found in nature (extra-genomic gene discovery). The ability to make global surveys of transcriptomes has given rise to fields such as pharmacogenomics and toxicogenomics. These applications of genomics technologies, with conventional drug assessment methodologies, will lead to more tolerable drugs and a better understanding of clinical populations. Integration of pathway mapping, using proteomics married to expression, will also significantly affect how new therapeutics are discovered as cross-biological cross-pathway interactions lead to novel drug targets and better predictions of drug tolerance.