Rapidly produced SAM(®) vaccine against H7N9 influenza is immunogenic in mice

Abstract

The timing of vaccine availability is essential for an effective response to pandemic influenza. In 2009, vaccine became available after the disease peak, and this has motivated the development of next generation vaccine technologies for more rapid responses. The SAM(®) vaccine platform, now in pre-clinical development, is based on a synthetic, self-amplifying mRNA, delivered by a synthetic lipid nanoparticle (LNP). When used to express seasonal influenza hemagglutinin (HA), a SAM vaccine elicited potent immune responses, comparable to those elicited by a licensed influenza subunit vaccine preparation. When the sequences coding for the HA and neuraminidase (NA) genes from the H7N9 influenza outbreak in China were posted on a web-based data sharing system, the combination of rapid and accurate cell-free gene synthesis and SAM vaccine technology allowed the generation of a vaccine candidate in 8 days. Two weeks after the first immunization, mice had measurable hemagglutinin inhibition (HI) and neutralizing antibody titers against the new virus. Two weeks after the second immunization, all mice had HI titers considered protective. If the SAM vaccine platform proves safe, potent, well tolerated and effective in humans, fully synthetic vaccine technologies could provide unparalleled speed of response to stem the initial wave of influenza outbreaks, allowing first availability of a vaccine candidate days after the discovery of a new virus.

Keywords: RNA vaccine; SAM vaccine; influenza; vaccine platform.

Figure 1

Comparative mouse immunogenicity study of a SAM (H1/LNP) vaccine. Groups of 12 mice were vaccinated i.m. on days 0 and 21 (3-week interval, 3wk) or 0 and 56 (8-week interval, 8wk), with the SAM (H1/LNP) vaccine (0.1 µg and 1.0 µg), subunit (0.1 µg, +/− MF59 adjuvant) or PBS (0). H1-specific antibodies were quantified in sera 3 weeks and 2 weeks after the first and second vaccination, respectively. (A) Total IgG titers; (B) HI titers; (C) VN titers (pooled sera). *P<0.05, **P<0.01 vs. H1N1, one-way ANOVA non-parametric Dunn's multicomparison test. Data are from individual mice (depicted as dots) and the GMT are solid lines. For determination of GMT, titers below the limit of detection were assigned a value of 10. ANOVA, analysis of variance; i.m., intramuscularly; ns, not significant; PBS, phosphate-buffered saline.

Figure 2

Timeline from electronic gene sequence posting to production of RNA prior to formulation with the LNP delivery system. GISAID, Global Initiative for Sharing All Influenza Data; PCR, polymerase chain reaction.

Figure 3

RNA quality and confirmation of antigen expression. (A) The SAM vector encoding the H7 antigen was analyzed on a denaturing agarose gel. (B) A SAM vector encoding the H7 antigen was transiently transfected into BHK cells. Cell lysates were separated by SDS–PAGE, and blotted to nitrocellulose membranes. H7 HA was visualized by Western blot using a H7-specific polyclonal antibody. A SAM vector encoding eGFP served as an NC. eGFP, enhanced green fluorescent protein; NC, negative control; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Figure 4

Comparative mouse immunogenicity study of a SAM (H7/LNP) vaccine. Groups of 6–8 mice were vaccinated i.m. on days 0 and 21, with unformulated SAM (H7) RNA (1.0 µg), a SAM (H7/LNP) vaccine (1.0 µg) or SAM (H1/LNP) vaccine (1.0 µg). HA-specific antibodies were quantified in sera 14 days after the first and second vaccinations. (A) H7-specific HI titers; (B) H1-specific HI titers; (C) H7N9 VN titers. **P<0.01, ***P<0.001 vs. H7 RNA, one-way ANOVA non-parametric Dunn's multicomparison test. (D) Kinetics of H7 HI titers after vaccination. Mice were vaccinated on day 0 and 56 with a SAM (H7/LNP) vaccine (1.0 µg), and H7-specific HI titers were measured in sera 13, 21, 35 and 55 days after the first immunization, and 14 days post second vaccination; **P<0.01, ****P<0.0001 vs. day 13. Data are from individual mice (depicted as dots) and the GMTs are solid lines. For determination of GMT, titers below the limit of detection were assigned a value of 10. ANOVA, analysis of variance; i.m., intramuscularly; ND, not determined.