Long-term evaluation of mucosal and systemic immunity and protection conferred by different polio booster vaccines

Abstract

Oral polio vaccine (OPV) and Inactivated Polio Vaccine (IPV) have distinct advantages and limitations. IPV does not provide mucosal immunity and introduction of IPV to mitigate consequences of circulating vaccine-derived polio virus from OPV has very limited effect on transmission and OPV campaigns are essential for interrupting wild polio virus transmission, even in developed countries with a high coverage of IPV and protected sewer systems. The problem is magnified in many countries with limited resources. Requirement of refrigeration for storage and transportation for both IPV and OPV is also a major challenge in developing countries. Therefore, we present here long-term studies on comparison of a plant-based booster vaccine, which is free of virus and cold chain with IPV boosters and provide data on mucosal and systemic immunity and protection conferred by neutralizing antibodies. Mice were primed subcutaneously with IPV and boosted orally with lyophilized plant cells containing 1μg or 25μg polio viral protein 1 (VP1), once a month for three months or a single booster one year after the first prime. Our results show that VP1-IgG1 titers in single or double dose IPV dropped to background levels after one year of immunization. This decrease correlated with >50% reduction in seropositivity in double dose and <10% seropositivity in single dose IPV against serotype 1. Single dose IPV offered no or minimal protection against serotype 1 and 2 but conferred protection against serotype 3. VP1-IgA titers were negligible in IPV single or double dose vaccinated mice. VP1 antigen with two plant-derived adjuvants induced significantly high level and long lasting VP1-IgG1, IgA and neutralizing antibody titers (average 4.3-6.8 log2 titers). Plant boosters with VP1 and plant derived adjuvants maintained the same level titers from 29 to 400days and conferred the same level of protection against all three serotypes throughout the duration of this study. Even during period, when no plant booster was given (∼260days), VP1-IgG1 titers were maintained at high levels. Lyophilized plant cells expressing VP1 can be stored without losing efficacy, eliminating cold chain. Virus-free, cold-chain free vaccine is ready for further clinical development.

Keywords: Bioencapsulated plant cells; Oral delivery; Polio viral protein 1 (VP1); Poliovirus.

Fig. 1

Design of long-term in vivo polio vaccine study. (A) Female CD-1 mice were randomly divided into 10 groups. Lyophilized plant cells expressing 1 μg or 25 μg of viral protein 1 (VP1) and plant-derived adjuvants (Saponin and/or Squalene) were used in this study. (B) All groups of mice except group 1 and group 10 were subcutaneously primed with IPV on day 0, and Group 2 mice were boosted with the same IPV 30 days after priming. Group 4–10 were orally boosted with lyophilized plant cells expressing VP1 once a week for 8 weeks, followed by once a month for three months. One year after priming, group 4–10 mice were boosted once a month for two times.

Fig. 2

Kinetic antibody response of groups of mice after oral and/or subcutaneous vaccination. Serum VP1-IgG1 (A and B) and VP1-IgA (C and D) antibody titers were assayed by direct ELISA in 96 well plates pre-coated with purified VP1 protein (10 μg/ml). Antibody titers from six groups of mice are shown: untreated group, single or two doses of IPV, priming with IPV and oral boosting with 1 μg or 25 μg plant VP1 protein with two adjuvants (saponin/squalene), and oral boosting with VP1 formulation but without IPV priming at different time points: 29, 57, 87, 117, 360, 370 and 400 days after priming. Statistical analysis (by Student’s t-test) (GraphPad Prism version 6) are noted with ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 2

Kinetic antibody response of groups of mice after oral and/or subcutaneous vaccination. Serum VP1-IgG1 (A and B) and VP1-IgA (C and D) antibody titers were assayed by direct ELISA in 96 well plates pre-coated with purified VP1 protein (10 μg/ml). Antibody titers from six groups of mice are shown: untreated group, single or two doses of IPV, priming with IPV and oral boosting with 1 μg or 25 μg plant VP1 protein with two adjuvants (saponin/squalene), and oral boosting with VP1 formulation but without IPV priming at different time points: 29, 57, 87, 117, 360, 370 and 400 days after priming. Statistical analysis (by Student’s t-test) (GraphPad Prism version 6) are noted with ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 3

Determination of poliovirus neutralizing antibodies against all three Sabin strains after subcutaneous IPV or oral VP1 boosting. (A) Sabin 1 at 42, 56, 84, 360 and 370 days after prime, (B) Sabin 2 and (C) Sabin 3 at 84, 360 and 370 days after prime. Data are from seven groups: CTB-VP1 antigens (1 μg or 25 μg) adjuvanted with saponin and squalene (group 6, 9 and 10), or squalene only (group 8), two doses of IPV (group 2) or single IPV dose (group 3); and untreated mice (group 1). Each dot represents the mean neutralizing titer ± SEM. The serum dilution of a reciprocal titer at which no virus neutralization was detected was recorded as the log₂ (titer) of 2.5. The Student’s t-test (GraphPad Prism version 6) was used for statistical analysis.

Fig. 4

Determination of seropositivity rate of Sabin 1, 2 and 3 neutralizing titers after subcutaneous IPV or oral VP1 boosting. The seropositivity rate of poliovirus-neutralizing antibodies are determined by the number of mice with seroprevalence (neutralizing antibody log₂(titer) ⩾3) with the total number of mice in each group boosted with 1 μg or 25 μg CTB-VP1 (Groups 4–10), or, IPV two doses (Group 2) at day 1 and day 30 or IPV single dose (Group 3). The kinetic change of seropositivity rate of neutralizing titers against Sabin strains 1, 2 and 3 at 42, 57, 117, 360 and 370 days after priming are shown.