Recombinant Haemagglutinin Derived From the Ciliated Protozoan Tetrahymena thermophila Is Protective Against Influenza Infection

Abstract

Current influenza vaccines manufactured using eggs have considerable limitations, both in terms of scale up production and the potential impact passaging through eggs can have on the antigenicity of the vaccine virus strains. Alternative methods of manufacture are required, particularly in the context of an emerging pandemic strain. Here we explore the production of recombinant influenza haemagglutinin using the ciliated protozoan Tetrahymena thermophila. For the first time we were able to produce haemagglutinin from both seasonal influenza A and B strains. This ciliate derived material was immunogenic, inducing an antibody response in both mice and non-human primates. Mice immunized with ciliate derived haemagglutinin were protected against challenge with homologous influenza A or B viruses. The antigen could also be combined with submicron particles containing a Nod2 ligand, significantly boosting the immune response and reducing the dose of antigen required. Thus, we show that Tetrahymena can be used as a manufacturing platform for viral vaccine antigens.

Keywords: adjuvant; influena virus; nanoparticle; protozoa; vaccine manufacture.

Figure 1

Production and characterization of recombinant rHA expressed in Tetrahymena thermophila. (A) Schematic of production and purification; see Table S1 for details of conditions used for different rHA. Each purified rHA was analyzed by SDS-PAGE and silver staining (B,D) or Western blot (C,E). A/Cal, ciliate derived rHA A/California/07/09; B/Bri, ciliate derived rHA B/Brisbane/60/2008; PC, positive control either A/California/07/09 antigen (12/168, NIBSC) or B/Brisbane/60/2008 (13/234, NIBSC) as appropriate; M, molecular weight marker. (F) Example haemagglutination assay of rHA A/Cal, samples two-fold diluted, (G) Example haemagglutination value for rHA A/Cal.

Figure 2

Ciliate produced rHA induces an antibody response in mice. BALB/c mice were immunized three times at 3 weekly intervals with either 15 μg A/Cal (A–C) or 30 μg B/Bri (D–F) by the intramuscular (i.m.) or subcutaneous (s.c.) routes. A/Cal-specific and B/Bri-specific IgG were evaluated in sera collected before immunization (week -1) and at weeks 3, 6 and 8, by ELISA. Values are reported as geometric mean titers (GMT) ± 95% CI of 6 mice per group (A,D). Sera for haemagglutination inhibition (HAI: B,E) and microneutralization (MN: C,F) analysis were collected at week 8. Symbols represents individual mice and bars represents the mean of n = 6 animals (B,C,E,F). ^**p < 0.01 between i.m. at week 8 and other time points, ^##p < 0.01 between s.c. at week 8 and other time points by ANOVA and Tukey post test for multiple comparisons.

Figure 3

Ciliate produced rHA induces an antibody response in NHP. NHP were immunized three times at 3 weekly intervals with 45 μg each of A/NC (A), A/Uru (B), B/Jia (C), and B/Mal (D) by the intramuscular (i.m.) or subcutaneous (s.c.) routes, n = 2 animals per route. Blood samples for ELISA (A–D) and haemagglutination inhibition (E) analysis were collected.

Figure 4

Ciliate produced rHA protects against infection with H1N1 influenza. Mice were immunized three times at 3 weekly intervals with 15 or 1.5 μg ciliate derived A/Cal, or 15 μg control inactivated influenza antigen (IIV) by the intramuscular (i.m.) route. Two weeks after the final immunization, anti-H1N1 antibody titer was measured by ELISA (A). Mice were then challenged with pH1N1 by the intranasal route. Weight change (B), viral load (C), and lung cell number (D) were assessed after infection. Points represent individual animals (A,C,D) or mean (B) of n = 5 mice per group, ^***p < 0.001 by ANOVA and post test.

Figure 5

Ciliate produced rHA protects against infection with B/Brisbane influenza. Mice were immunized three times at 3 weekly intervals with 30 or 3 μg ciliate derived B/Bri by the intramuscular (i.m.) route. Two weeks after the final immunization, anti-B/Bri antibody titer was measured by ELISA (A). Mice were then challenged with B/Bri by the intranasal route. Weight change (B), viral load (C), lung cell number (D), and clinical score (E) were assessed after infection. Points represent individual animals (A,C,D,E) or mean (B) of n = 5 mice per group, ^**p < 0.01, ^***p < 0.001 by ANOVA and post test.

Figure 6

Combination of antigen with PLA-Nod2 particles enables dose sparing. Mice were immunized three times at 3 weekly intervals with 1.5 or 0.015 μg ciliate derived rHA A/Cal by the intramuscular (i.m.) or subcutaneous (s.c.) routes; responses were compared to 0.015 μg antigen loaded onto PLA-Nod2 particles or empty PLA particles. Anti-H1 antibody was measured by ELISA (A) or HAI (B). Mice were then challenged with A/Cal by the intranasal route. Weight change over time (C) and at d4 (D), clinical score (E), lung cell number (F), and viral load (G) were assessed after infection of n = 5 mice per group, ^**p < 0.001 by ANOVA and post test. Points represent individual animals (A,D,E,F,G) or mean (B,C) of n = 5 mice per group, ^*p < 0.05, ^**p < 0.01 by ANOVA and post test.