Cold chain and virus-free chloroplast-made booster vaccine to confer immunity against different poliovirus serotypes

Abstract

The WHO recommends complete withdrawal of oral polio vaccine (OPV) type 2 by April 2016 globally and replacing with at least one dose of inactivated poliovirus vaccine (IPV). However, high-cost, limited supply of IPV, persistent circulating vaccine-derived polioviruses transmission and need for subsequent boosters remain unresolved. To meet this critical need, a novel strategy of a low-cost cold chain-free plant-made viral protein 1 (VP1) subunit oral booster vaccine after single IPV dose is reported. Codon optimization of the VP1 gene enhanced expression by 50-fold in chloroplasts. Oral boosting of VP1 expressed in plant cells with plant-derived adjuvants after single priming with IPV significantly increased VP1-IgG1 and VP1-IgA titres when compared to lower IgG1 or negligible IgA titres with IPV injections. IgA plays a pivotal role in polio eradication because of its transmission through contaminated water or sewer systems. Neutralizing antibody titres (~3.17-10.17 log₂ titre) and seropositivity (70-90%) against all three poliovirus Sabin serotypes were observed with two doses of IPV and plant-cell oral boosters but single dose of IPV resulted in poor neutralization. Lyophilized plant cells expressing VP1 stored at ambient temperature maintained efficacy and preserved antigen folding/assembly indefinitely, thereby eliminating cold chain currently required for all vaccines. Replacement of OPV with this booster vaccine and the next steps in clinical translation of FDA-approved antigens and adjuvants are discussed.

Keywords: bioencapsulation; chloroplast transformation; human infectious diseases; molecular farming; mucosal immunity; oral delivery.

Figure 1

Creation and characterization of transplastomic tobacco lines expressing native and codon‐optimized CTB‐VP1. (a) tobacco chloroplast transformation vectors containing CTB‐VP1 expression cassettes. Prrn, rRNA operon promoter; aadA, aminoglycoside 3'‐adenylyltransferase gene; PpsbA, promoter and 5'‐UTR of the psbA gene; CTB , coding sequence of nontoxic cholera B subunit; VP1, coding sequence for poliovirus VP1 gene; TpsbA, 3'‐UTR of the psbA gene; trnI, isoleucyl‐tRNA; trnA, alanyl‐tRNA; (b) Southern blot analysis of native and codon‐optimized CTB‐VP1 transplastomic tobacco lines. Afl III‐digested wild type (WT) and transformed (line 1, 2, 3 and 4) genomic DNA was probed with DIG‐labelled flanking sequence digested with Bam HI/BglII. UTR, untranslated region.

Figure 2

Characterization of CTB‐VP1 transplastomic lines. (a) Western blot of CTB‐VP1 native (N) or codon optimized (CO) and untransformed plant extracts loaded at indicated total protein (top) or serial dilution (bottom) and probed with anti‐CTB antibody. CTB (5 ng) was loaded as a positive control. (b) Western blot of CTB‐VP1 in four independent transplastomic lines and wild type (WT) (1 μg/lane) controls probed with anti‐CTB or anti‐VP1 and protein standards. Antibodies used (dilution factor): rabbit anti‐CTB polyclonal antibody 1 : 10 000 dilution; rabbit anti‐VP1 polyclonal antibody 1 : 1000 dilution. (c) Comparison of CTB‐VP1 in lyophilized (L) or frozen (F) leaf samples; 10 mg ground powder was extracted in 300 μL of extraction buffer. 1× is 1 μL of ground extract. (d) CTB‐VP1 stability after storage of lyophilized leaves at ambient temperature for 4 or 8 months (e) GM1 binding assay of native (N) and codon‐optimized CTB‐VP1 (CO). F: fresh; L: lyophilized; WT: untransformed; BSA (1%, w/v), bovine serum albumin. Data are means ± SD of three independent experiments.

Figure 3

Evaluation of serum VP1‐IgG1 and VP1‐IgA antibody titres after oral or subcutaneous vaccination. Antibody responses of mice (n = 10/group) vaccinated with single or two doses of inactivated poliovirus vaccine (IPV) or VP1 bioencapsulated in plant cells. (a–d) VP1‐IgG1 antibody titres at different time points: (a, b) weekly boosts and sera samples collected on days 29 and 57; (c, d) monthly boosts and samples collected on days 87 and 117; (e–h) VP1‐IgA antibody titres at different time points: (e, f) weekly boosts and sera samples collected on days 29 and 57; (g, h) monthly boosts with sera samples collected on days 87 and 117. Group 1: untreated; group 2: 2 doses of IPV; group 3: IPV single dose; group 6: IPV prime, boosted with native VP1 or group 9: codon‐optimized VP1 in plant cells with adjuvants (saponin/squalene); group 10: same as group 9 but without IPV priming. Results are shown as individual reciprocal endpoint antibody titres and mean ± SEM. One‐way ANOVA showed significant differences between groups (P < 0.0001) and post hoc comparisons by t‐test showed significant differences between specific treatment groups and respective control group. (**P < 0.05, ***P < 0.01, ****P < 0.001, NS not significant).

Figure 4

Faecal IgA antibody titres after oral and subcutaneous vaccination. Levels of VP1 and CTB‐specific IgA antibody titres in faecal extracts obtained from mice (n = 10/group) after 3‐month vaccination. Faecal pellets were collected at day 10 after oral boosting. ELISA titres are shown (a) VP1‐specific IgA antibody titre. (b) CTB‐specific IgA antibody titre. Group 1: untreated; group 2: 2 doses of inactivated poliovirus vaccine (IPV); group 3: IPV single dose; group 6: IPV prime, boosted with native VP1 or group 9: codon‐optimized VP1 in plant cells with adjuvants (saponin/squalene); group 10: same as group 9 but without IPV priming. Antibody titres are defined as the reciprocal of the highest dilution above the cut‐off, which was three times the mean background. All samples were tested in triplicate. Results are shown as individual reciprocal endpoint antibody titres and mean ± SEM. One‐way ANOVA showed significant differences between groups (P < 0.0001) and post hoc comparisons by t‐test showed significant differences between specific treatment groups and the control group. (***P < 0.01, ****P < 0.001, NS not significant).

Figure 5

Evaluation of serum CTB‐IgG1 antibody titres after oral vaccination. Cholera toxin B subunit was coated on ELISA plates and probed with individual heat‐inactivated sera samples (starting with a 1 : 50 dilution). CTB‐IgG1 antibody titres at different time points: (a, b) weekly boosts and sera samples collected on days 29 and 57; (c) monthly boosts and samples collected on day 117. Group 1: untreated; group 2: 2 doses of inactivated poliovirus vaccine (IPV); group 3: IPV single dose. group 6: IPV prime, boosted with native VP1 protein with adjuvants; group 9: IPV prime, boosted with codon‐optimized VP1 protein with adjuvants; group 10: boosted with codon‐optimized VP1 with adjuvants but without IPV priming. Saponin and squalene were used as adjuvants. Results are shown as individual reciprocal endpoint antibody titre and mean ± SEM. One‐way ANOVA showed significant differences between groups (P < 0.0001) and post hoc comparisons by t‐test showed significant differences between specific treatment groups and the control group. (**P < 0.05, ***P < 0.01, ****P < 0.001, NS not significant).

Figure 6

Determination of poliovirus‐neutralizing titres and seropositivity rate of Sabin 1‐, 2‐ and 3‐neutralizing titres after subcutaneous inactivated poliovirus vaccine (IPV) or oral VP1 boosting. Virus‐neutralizing antibody titres of 117 days sera from mice (n = 10/group) boosted with native or codon‐optimized CTB‐VP1 antigens adjuvanted with saponin only (groups 4 and 7), squalene only (groups 5 and 8) or both (groups 6, 9 and 10); mice with two doses of IPV (group 2) or single IPV dose (group 3); and untreated mice (group 1). Individual titres for each mouse were plotted, and the bar represents the mean neutralizing titre ± SEM. The serum dilution of a reciprocal titre at which no virus neutralization was detected was recorded as the log₂ (titre) of 2.5. Poliovirus‐neutralizing antibodies against all three Sabin strains, (a) Sabin 1, (b) Sabin 2 and (c) Sabin 3. One‐way ANOVA showed significant differences between groups (P < 0.0001) and post hoc comparisons by t‐test showed significant differences between specific treatment groups and group 3 – single IPV dose (***P < 0.01, ****P < 0.001, NS not significant). The seropositivity rate of poliovirus‐neutralizing antibodies as determined by the number of mice with seroprevalence (neutralizing antibody log₂ (titre) ≥3) with the total number of mice in each group boosted with the native or codon‐optimized CTB‐VP1 (groups 4–10), or, IPV two doses (group 2) at day 1 and day 30 or IPV single dose (group 3). The seropositivity rate of neutralizing titres against Sabin strains 1, 2 and 3 (d–f) are shown.