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

GLA-AF enhances protective priming against a heterosubtypic H5N1 virus in ferrets.

<p>Animals (4/grp) injected with rH5 Indo (0.5 µg) alone or with adjuvant (SE, 20 µg GLA-AF) were challenged on d28 with 7.5×10⁵ pfu A/Vietnam/1203/04. (A) HAI titers directed against the heterologous challenge strain, A/Vietnam/1203/04. (B) Survival and the percent change in body weights per immunization group. (C) Animals (4/grp) injected with rH5 Indo (300 ng) alone or with adjuvant (SE, 20 µg GLA-AF) were challenged on d14 with 7.5×10⁵ pfu A/Vietnam/1203/04.</p

GLA-AF stimulates a Th1 antibody response.

<p>Mice (5/group) received a prime (d0)+boost (d21) immunization with rH5VN (50 ng) ± adjuvant. (A) HI titers directed against the homologous vaccine strain of virus and 2 drifted virus. (B) Total anti-H5 IgG titers measured by ELISA and the ratio of IgG2c and IgG1 isotype titers. (C) Relative production of IL-5 and IFN-γ in antigen stimulated splenocytes as measured by Luminex.</p

GLA-AF augments priming of protective immune responses in ferrets.

<p>Animals (4/group) injected once with rH5 VN (0.5 µg) alone or with adjuvant (SE, 20 µg GLA-AF) were challenged on d28 with 7.5×10⁵ pfu A/Vietnam/1203/04. Survival per immunization group was: naïve (0/4), rH5 alone (2/4), rH5+ SE (4/4), rH5+ GLA-AF (4/4). (A) Serum HI titers in d28 sera. Each bar presents the geometric mean titer and statistical difference (p<0.001) between SE and GLA-AF is indicated by the asterisk. (B) Mean percent change in body weight (+/− SEM). (C) Daily changes in viral load measured in nasal washes. The asterisk on day 5 denotes a significant difference in detectable virus between vaccine groups adjuvanted with GLA-AF and SE (p = 0.028; Fisher’s exact test). (D) Mean percent change in body temperature (+/− SEM).</p

GLA-AF enhances protection against a heterosubtypic H5N1 virus in ferrets.

<p>Animals (5/group) received a prime (d0)+boost (d21) immunization with rH5 Indo (2 µg) either alone, with SE, or with 1 µg, 5 µg, and 20 µg GLA-AF. (A) HI titers directed against the indicated strains of virus were assayed in d35 sera. Each bar presents the geometric mean titer and statistical differences (p<0.001, student’s t-test) between SE and 20 µg GLA-AF are indicated by the asterisks. (B) Survival and the percent change in body weights in animals challenged on d42 with 7.5×10⁵ pfu A/Vietnam/1203/04. All of the ferrets in this experiment had detectable virus titer in nasal washes one day after the challenge. No statistical difference in viral clearance between the GLA-AF and SE groups was observed (data not shown).</p

GLA-AF augments priming of a protective immune response.

<p>Mice (5/group) received a prime immunization on d0 with rH5 VN (50 ng) either alone, with SE, or with GLA-AF (1 µg), and were then challenged 14 days later with 1000 LD50 A/H5N1/Vietnam/1203/04. (A) Mean percent change in body weight (+/− SEM) post-challenge (representative data from three experiments). (B) Kinetics of virus clearance. Each point is the average virus titer measured in mouse lung homogenate (n = 3), where 1 ml represents approximately 20% of lung volume. Statistical differences (p<0.05) between the adjuvanted and non-adjuvanted groups on day 3 and day 5 are indicated by the asterisks. (C) Day 14 sera were assayed for HI titers directed against the clade 1 vaccine HA or 2 heterosubtypic HA antigens. Each bar presents the geometric mean titer and statistical differences (p<0.001, Fisher’s test) between SE and GLA-AF are indicated. (D) Cumulative survival of mice immunized once with 50 ng of rH5 VN or rH5 Indo antigen and then challenged 14 days later with 1000 LD50 A/H5N1/Vietnam/1203/04.</p

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, <i>expres</i>SF+ and glycoengineered <i>expres</i>SF+), 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 <i>N</i>-glycosylation patterns and structures produced by different cell types, (3) ∼20% of the <i>N</i>-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