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Mucosal immunization with a killed whole-cell pneumococcal vaccine, given with enterotoxin-related adjuvants, has been shown to confer multi-serotype protection against colonization of the nasopharynx and middle ear in mice. However, because novel mucosal immunization strategies may be difficult to implement, here we evaluated subcutaneous injection. Strain RM200 was engineered to be capsule-negative, autolysin-negative, and to express a non-toxic mutant pneumolysoid. Liter-scale and 60-l Good Manufacturing Practice (GMP) cultures were grown in bovine-free soy-based medium, killed with chloroform or beta-propiolactone, and injected into C57Bl/6 mice without or with aluminum adjuvant. The adjuvant Al(OH)(3) strongly increased responses, particularly if pre-treated with phosphate. Protection was found in several tested model infections: nasal colonization with a serotype 6B strain and fatal aspiration-sepsis with strains of serotype 3 and 5. Protection against colonization was mechanistically dependent on the presence of CD4+ T cells at the time of challenge; in contrast, in the type 3 aspiration-sepsis model, CD4+ T cells were not required for protection at the time of challenge, suggesting that antibody alone was sufficient to protect against death in this model. Rabbits receiving sequential intramuscular injections in a pilot toxicity study displayed local reactogenicity at injection sites but no clinical signs. The rabbit antiserum thus produced was active in an in vitro phagocytic killing assay and passively protected mice in the type 3 aspiration-sepsis model. Approval is being sought for human trials of this vaccine.

Cholera toxin B subunit (CTB) is responsible for CT holotoxin binding to the cell and has been described as a mucosal adjuvant for vaccines. In this work, the ctxB gene was genetically fused to the psaA gene from Streptococcus pneumoniae, a surface protein involved in its colonization in the host that is also considered a vaccine antigen candidate against this pathogen. The CTB-PsaA fusion protein was expressed in Escherichia coli, and the purified protein was used for intranasal immunization experiments in Balb/C mice. CTB-PsaA was able to induce both systemic and mucosal antibodies evaluated in serum, saliva, and in nasal and bronchial wash samples, showing that CTB-PsaA is a promising molecule to be investigated as S. pneumoniae vaccine antigen candidate.

Streptococcus pneumoniae is one of the most important human pathogens and improvement of the currently used polysaccharide vaccines is being pursued. We constructed DNA vaccine vectors containing either the full-length psaA (pneumococcal surface adhesin A) or a truncated pspA (pneumococcal surface protein A--pspA') gene. Both constructs showed transient expression of the antigens in vertebrate cells and induced significant antibody response to the pneumococcal antigens in BALB/c mice injected intramuscularly (i.m.). Fusion with an N-terminal cytoplasmatic SV40 T-antigen (CT-Ag), which was previously shown to stabilize poorly expressed antigens through association with Hsp73, also induced anti-PspA antibody response. The induction of antibodies with a low IgG1:IgG2a ratio and elevated gamma interferon (IFN-gamma) production by spleen cells elicited by DNA vaccination indicate preferential priming of Th1 immunity. Since induction of antibodies against both PsaA and PspA was previously shown to correlate with protection against fatal infection with S. pneumoniae and cell-mediated immune responses could contribute to protection, further evaluation of PsaA and PspA as antigens for a DNA vaccine against S. pneumoniae could be promising.