PspA consists of five domains including a signal peptide, alpha-helical charged region, a proline-rich domain, a choline-binding domain consisting of ten amino acids repeats, and a C-terminal amino acid tail 
. Depending on the divergence of nucleotide sequences in the alpha-helical charged region, PspA is classified into three families, with no more than 50% sequence divergence within each family. The three PspA families are made up of six PspA clades that diverge from each other by no more than 20% sequence identity within each clade; family 1 (clades 1 and 2), family 2 (clades 3, 4, and 5), and family 3 (clade 6) 
Despite the great variation in the sequences of PspA, mouse and humans antibodies against PspA can be cross-reactive and cross protective against invasive disease in mice 
. The serologic cross-reactivity of PspA has been found to be strongly associated with PspA, but not restricted to family 
. Even so these antibodies can be more cross-protective than their level of cross-reactivity might suggest. Immunization of adult humans and mice with a PspA family 1 produced antibodies that could protect mice from infection with strains of PspA families 1 or 2 and from infections with strains of 3 different capsular types 
. In addition successful fusion proteins have been made between family 1 and family 2 PspAs that can elicit antibody in mice protect against challenge strains of both PspA families 
. In this study we focused on the distribution of PspA families among clinical isolates in Japan.
The Japanese strains were evenly distributed over family 1 and family 2. The proportions of the different PspA families can vary somewhat among countries. Hollingshead et al reported that the majority of PspAs in a collection of strains from Alabama fell into family 1 
. A study on invasive pneumococcal strains isolated from children less than 5 years of age in Colombia showed that 62.5% and 35.0% of strains belonged to families 1 and 2, respectively 
. In Argentina 54.4% and 41.6% of the strains belonged to family 1 and family 2, respectively, with only 4.0% of the strains isolated from children being unclassifiable 
. In Brazil, 50.5% of the isolates belonged to family 1, 43.2% were members of family 2, and 6.3% were not classified 
. In contrast, the high prevalence of PspA family 2 among pneumococci isolated from invasive pneumococcal diseases has been reported from Spain, Poland, Canada, Sweden, Germany, the USA, and France 
. A recent study of pneumococci isolates from nasopharyngeal carriage in Finnish children showed a prevalence of PspA family 1 and family 2 that was similar to our results 
. The vast majority of pneumococci isolated from the middle ear fluid or nasopharyngeal secretion samples of the Finnish children less than 2 years old were from PspA families 1 and 2 
. Prior to our study, there had been a few reports of the PspA family distribution among pneumococci in Japan or any other countries in Asia 
In contrast to the similar frequencies of PspA1 and PspA2 in Japan the frequency of different capsular serotypes was highly variable with 19F, 23F, 14, 6A, 6B, and 3 being the predominant common capsular types we observed which together accounted for 71.7% of the pneumococci isolates in this study. However, the PspA family distribution varied somewhat among serotypes. Earlier studies found that both PspA families occurred within the most common capsular serotypes, but that some serotypes were associated more strongly with one PspA family than the other 
. The capsular serotypes most strongly associated with a certain PspA family are 9N, 9V, 11A, 14, and 23F, whereas serotypes 6A, 6B, 19A, and 19F were equally associated with PspA families 1 and 2. This was most dramatic for the 24 different 23F isolates which were 25% PspA1 and 75% PspA2. In a study in France 37 different 23F strains were examined; 92% were PspA1 and 8% were PspA2 
. These findings indicate that there can be variations of distributions in PspAs in different geographic different regions although serotypes do not necessarily globally associate with certain PspAs. In some regions some capsular serotypes associated with a certain PspA family might be heavily clonal.
Based on the previously published information on PspA family distribution, there is still little information about the relationship between PspA families and antimicrobial-susceptibilities. In Japan, the rate of antimicrobial-resistant S. pneumoniae
has increased continually since around 1990 and was about 49.0% between 1998 and 2000 
. As documented in previous reports, penicillin-resistant strains were frequently identified among children younger than 2 years old 
. In our previous study most of the serotype 19F and 23F strains were classified as either PISP or PRSP, while all of serotype 3 stains were classified as PSSP in middle ear isolates 
. In this study, PRSP strains consisted equally of family 1 and 2 PspA. This means that a PspA-based vaccine would show a higher coverage of PRSP compared to the polysaccharide-based vaccines that have been available in the market.
Previous studies showed that PspA clades were independent of capsular serotypes 
. Pneumococci of the same serotype were associated with different PspA clades from the same or a different family. This means that PspA-containing vaccines may be able to improve the protective efficacy of pneumococcal vaccines compared with the currently available serotype-based vaccines and may be able to avoid the serotype replacement that has been observed with conjugate vaccines 
. The coverage of serotypes and PRSP by the 7 PCV was reported to be 62.8% and 88.0% for middle ear isolates, respectively. A PspA-based vaccine that contained representatives of PspA families 1 and 2 would potentially provide a high coverage rate because it would be cross-protective against invasive disease caused by the bulk of pneumococci infecting children and adults. It will be important however that data relating to both serotype and antibiotic resistance, similar to those reported here for Japan, should be collected in other geographical areas. Such a study would help to determine if a vaccine covering PspA families 1 and 2 would be appropriate for the geographic region in question.
In conclusion, even conjugate vaccine formulations with 13 pneumococcal capsular polysaccharides will not reach the coverage of 90% or more achieved by a vaccine containing family 1 and 2 PspA. The addition of PspA to the existing conjugate vaccine formulations may be a possible alternative for future development of pneumococcal vaccine.