COLLABORATIVE PROJECTS ON MINORITY HEALTH--PROJECT III

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Sepsis with Streptococcus pneumoniae is a leading cause of death among sickle cell disease (SCD) patients. While immunization with a capsular polysaccharide vaccine can increase resistance to pneumococcal infection in older SCD patients, it is not effective in SCD patients less than 2 years of age because young children do not make adequate responses to most polysaccharide antigens. However, they do make good immune responses to most proteins. We have identified a pneumococcal surface protein (PspA) that has the potential of being a good pneumococcal vaccine for children. In mice, it is highly immunogenic and elicits antibodies that are protective against fatal infections with S. pneumoniae. PspA is a virulence factor that slows the clearance of S. pneumoniae from the blood. Although PspA is serologically variable, different PspAs are sufficiently cross-reactive that antibodies against an individual PspA can elicit protection against strains of different PspA types. Thus, a mixture of only a few PspAs (or a recombinant protein containing specific epitopes from only a few PspAs) would be needed for a vaccine. We have sequenced the pspA gene of strain Rx1. The N-terminal 49% of the inferred protein sequence is highly charged and consistent with an alpha-helix capable of forming linear coiled-coil fibers. The alpha-helical region contains the epitopes recognized by all protective monoclonal antibodies. Just C-terminal to the alpha-helical domain is a proline-rich domain followed by an anchoring domain containing ten 20 amino acid repeats. Most of the protective MAbs to Rx1 PspA recognize epitopes in the C-terminal third of the alpha-helical region. These epitopes are more cross-reactive than others in the alpha- helical region. Southern blots and the ability of specific pspA primers to amplify diverse pspAs by polymerase chain reaction (PCR) demonstrate that the portion of pspA encoding the alpha-helical domain is more variable than that encoding the C-terminal half of PspA. In the proposed studies we will sequence several pspAs and determine the portions of their alpha-helical sequences that contain epitopes capable of eliciting protective antibody in normal and SCD mice. We plan to determine whether humans make protective responses to the same epitopes by using affinity purified antibody from convalescent SCD and non-SCD patients. These studies could lead to the development of an inexpensive recombinant PspA fusion protein to be used as a vaccine against pneumococcal infection in children. We will also use the pspA sequence information to identify conserved regions for use as PCR primers to aid in the diagnosis of pneumococcal infection. This type of diagnostic capability is needed so that new vaccines and treatments can be accurately evaluated. Better diagnosis would also enable physicians to use more appropriate antibiotic treatments. An advantage of using PspA as the target of diagnostic PCR amplification is that RFLPs of the amplified products could facilitate epidemiologic studies of strains of S. pneumoniae that infect normal and SCD patients. The proposed studies will use isolates and convalescent serum from SCD patients.