Clin Exp Vaccine Res.  2013 Jan;2(1):58-65.

Inactivated pep27 mutant as an effective mucosal vaccine against a secondary lethal pneumococcal challenge in mice

• Choi SY
• Tran TD
• Briles DE
• Rhee DK

Abstract

PURPOSE
A pep27 mutant may be able to elicit mucosal immunity against pneumococcal diseases, and could be employed as an inexpensive attenuated vaccine. However, this particular mutant contains an erythromycin-resistance marker. The purpose of the current study is to develop a markerless pep27 mutant and assess whether this inactivated mutant is able to induce mucosal immunity.
MATERIALS AND METHODS
Mice were vaccinated intranasally with the inactivated markerless pep27 mutant every 2 weeks for a total of three times, after which time serum samples were analyzed for antibody titers. The mice were then challenged with a lethal D39 strain and their survival time was measured. The cross-reactivity of the antisera against pep27 was also compared to other mutant serotypes.
RESULTS
Intranasal immunization of mice with the inactivated markerless pep27 mutant provides effective protection and rapidly cleared bacterial colonization in vivo. Moreover, antisera raised against the pep27 mutant may cross-react with several other serotype strains.
CONCLUSION
Intranasal immunization with the inactivated pep27 mutant may be able to provide mucosal immunity, and could represent an efficient mucosal vaccine.

Keyword

Streptococcus pneumoniae; pep27 mutant; Inactivated vaccine; No antibiotic resistance marker; Mucosal immunity

MeSH Terms

Animals
Colon
Immune Sera
Immunity, Mucosal
Immunization
Mice
Sprains and Strains
Streptococcus pneumoniae
Immune Sera

Figure

• 

  Fig. 1 Construction of a markerless THpep27 mutant. (A) Transcription of the pep27 locus and insertion and orientation of the Cheshire cassette. The numbers represent nucleotide sequence numbers in the D39 wild type (D39WT) genome. A THpep27 mutant with a pep27 deletion was constructed. (B) Identification of the THpep27 mutant. Streptococcus pneumoniae D39 and its isogenic THpep27 mutant were cultured in THY broth until mid-log phase, bacterial RNA was extracted, and mRNA levels of pep27 and 16s RNA were analyzed by reverse transcription polymerase chain reaction.

• 

  Fig. 2 Cross-reactivity of the pep27 mutant antisera with heterologous strains. Strains grown in THY broth were lysed, and cell lysates (10 µg/mL) of each pneumococcal strain were adsorbed onto titer plates. Antisera raised against the live pep27 mutant (THpep27) in CD1 mice after three i.n. immunizations was used as antisera for titer determination. The antigens used in this study included: D39 (type 2), EF3030 (type 19F), TIGR4 (type 4), BG7322 (type 6B), WU2 (type 3), R6 (non-encapsulated type 2). Student's t-test is indicated as follows: **p<0.01, ***p < 0.001.

• 

  Fig. 3 Protection from lethal challenge after intranasal immunization with the inactivated THpep27 mutant. (A) Mice were intranasally (i.n.) immunized with inactivated THpep27 mutant (1 × 107 colony-forming unit [CFU]) at two-week intervals a total of three times. Sera were collected by bleeding the mice 10 days after the final immunization, and antibody titer to THpep27 lysate was measured by enzyme-linked immunosorbent assay (10 µg/mL lysate protein). (B) Groups of 10 mice were i.n. immunized with either live or inactivated THpep27 mutant (1 × 107) three times, then i.n. challenged 2 weeks after the third immunization with approximately 1 × 107 CFU of the virulent D39 strain. Total mouse Inactivated markerless pep27 mutant as a mucosal vaccine survival was tracked every day and analyzed using GraphPad Prism. NS, not significant. **p < 0.01, ***p < 0.001.

• 

  Fig. 4 Decrease in colonization of lethal D39 strain at the early phase of infection after immunization with the inactivated THpep27 mutant in vivo. Groups of 10 mice were intranasally (i.n.) immunized with inactivated THpep27 mutant (1 × 107) three times, then i.n. challenged 2 weeks after the third immunization with approximately 1×107 colony-forming unit (CFU) of the virulent D39 strain. Three mice from each group were sacrificed at 3, 6, and 12 hours after infection, after which time the number of recovered bacteria was quantified by plating on blood agar. Significant difference were analyzed by two-way ANOVA. Orange color, control; blue color, inactivated THpep27. *p < 0.05.

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