Some intellectual property relating to PspA that is held by the UAB Research foundation includes active patents on the alpha-helical and proline-rich domains of PspA, each of which covers the use of these domains of PspA in protection-eliciting vaccines. The patents for the alpha-helical domain only covers use in a vaccine in the USA. Patents for the proline-rich domain are being pursued worldwide. Drs. Briles and Hollingshead are among the inventors on these patents and could gain monetarily if a PspA-containing vaccine were licensed for human use. Drs. Briles and Hollingshead provided advice concerning PspA family determination and assisted with manuscript preparation, but all of the data was collected the authors’ group in Japan, which has no conflicts of interest with the data obtained. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: MH NY. Performed the experiments: MH AT MK YI ST. Analyzed the data: MH NY. Contributed reagents/materials/analysis tools: MH MK. Wrote the paper: MH SKH DEB KS NY.
The protection against pneumococcal infections provided by currently available pneumococcal polysaccharide conjugate vaccines are restricted to the limited number of the serotypes included in the vaccine. In the present study, we evaluated the distribution of the pneumococcal capsular type and surface protein A (PspA) family of pneumococcal isolates from upper respiratory tract infections in Japan.
A total of 251
Among the 251 pneumococci studied, the majority (49.4%) was identified as belonging to PspA family 2, while most of the remaining isolates (44.6%) belonged to family 1. There were no significant differences between the distributions of PspA1 versus PspA2 isolates based on the age or gender of the patient, source of the isolates or the isolates’ susceptibilities to penicillin G. In contrast, the frequency of the
The vast majority of pneumococci isolated from the middle ear fluids, nasal discharges/sinus aspirates or pharyngeal secretions represented PspA families 1 and 2. Capsular serotypes were generally not exclusively associated with certain PspA families, although some capsular types showed a much higher proportion of either PspA1 or PspA2. A PspA-containing vaccine would potentially provide high coverage against pneumococcal infectious diseases because it would be cross-protective versus invasive disease with the majority of pneumococci infecting children and adults.
Currently available pneumococcal vaccines are based on capsular polysaccharides. Although the 23-valent polysaccharide vaccine (23 PPV) is immunogenic and protective in most adults, it has been shown to be poorly efficacious in children younger than 2 years of age
Pneumococcal surface protein A (PspA) is an important virulent factor expressed by all pneumococci that is essential for full virulence in invasive disease, and contributes to colonization
Since capsular type distribution is not uniform world wide, it is important to know the overall distribution of the PspA family expressed in pneumococcal strains at multiple sites around the world to make sure the PspA molecules represented in a vaccine will be effective world wide
Between January and May 2003, the Japanese Society of Infectious Disease in Otorhinolaryngology conducted the fourth nationwide surveillance of the bacterial pathogens responsible for otorhinolaryngological infections. A total of 251
Category | Sub-category | Total | Susceptibility to PCG (µg/ml) | |||
PSSP | PISP | PRSP | DRSP v.s. PSSP | |||
Gender | Female | 125 (49.8%) | 50 (19.9%) | 48 (19.1%) | 27 (10.8%) | |
Male | 126 (50.2%) | 43 (17.1%) | 56 (22.3%) | 27 (10.8%) | ||
Age | 0–2 | 92 (36.7%) | 16 (6.4%) | 50 (19.9%) | 26 (10.4%) | |
3–5 | 37 (14.7%) | 16 (6.4%) | 17 (6.8%) | 4 (1.6%) | ||
6–12 | 25 (10%) | 18 (7.2%) | 7 (2.8%) | 0 (0%) | ||
13–20 | 7 (2.8%) | 4 (1.6%) | 2 (0.8%) | 1 (0.4%) | ||
21–50 | 72 (28.7%) | 30 (12.0%) | 22 (8.8%) | 20 (8.0%) | ||
≥51 | 18 (7.2%) | 9 (3.6%) | 6 (2.4%) | 3 (1.2%) | ||
Origin | Middle ear fluids | 57 (22.7%) | 21 (8.4%) | 27 (10.8%) | 9 (3.6%) | |
Nasal discharge/Sinus aspirates | 88 (35.1%) | 37 (14.7%) | 34 (13.5%) | 17 (6.8%) | ||
Pharyngeal secretions | 106 (42.2%) | 35 (13.9%) | 43 (17.1%) | 28 (11.2%) | ||
Serotype | 1 | 2 (0.8%) | 1 (0.4%) | 0 (0%) | 1 (0.4%) | |
3 | 14 (5.6%) | 13 (5.2%) | 1 (0.4%) | 0 (0%) | ||
4 | 1 (0.4%) | 0 (0%) | 0 (0%) | 1 (0.4%) | ||
6A | 16 (6.4%) | 6 (2.4%) | 6 (2.4%) | 4 (1.6%) | ||
6B | 37 (14.7%) | 14 (5.6%) | 17 (6.8%) | 6 (2.4%) | ||
9V | 5 (2.0%) | 3 (1.2%) | 2 (0.8%) | 0 (0%) | ||
14 | 20 (8.0%) | 2 (0.8%) | 13 (5.2%) | 5 (2.0%) | ||
15B | 7 (2.8%) | 4 (1.6%) | 3 (1.2%) | 0 (0%) | ||
19A | 5 (2.0%) | 4 (1.6%) | 1 (0.4%) | 0 (0%) | ||
19F | 52 (20.7%) | 8 (3.2%) | 25 (10.0%) | 19 (7.6%) | ||
23F | 41 (16.3%) | 11 (4.4%) | 18 (7.2%) | 12 (4.8%) | ||
G23 | 6 (2.4%) | 4 (1.6%) | 2 (0.8%) | 0 (0%) | ||
Others | 45 (17.9%) | 23 (9.2%) | 16 (6.4%) | 6 (2.4%) | ||
Total | 251 (100%) | 93 (37.1%) | 104 (41.4%) | 54 (21.5%) |
G23: serogroup 23 strains except serotype 23F. PCG: penicillin G. PSSP: penicillin susceptible
comparison between ≤2 y.o. vs. ≥3 y.o.
Susceptibility to penicillin G (PCG) was tested by a broth dilution standard method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The CLSI published revised susceptibility breakpoints for penicillin and
All isolates were serotyped or serogrouped by the capsular quellung reaction method with pneumococcal capsule specific antisera (Statens Serum Institute, Copenhagen, Denmark), as recommended by the manufacturer. Strains of serotypes 4 (ATCC BAA-334) and 19F (ATCC 49619) obtained from the American Type Culture Collection 169 (ATCC, Manassas, VA, USA) were used for quality control in every reaction.
PspAs were classified into three families by PCR. Briefly, genomic DNA was extracted from pneumococcal isolates as described and stored at 4°C
The PCR conditions were 95°C for 3 min; then 30 cycles of 95°C for 1 min, 62°C for 1 min and 72°C for 3 min, and finally 72°C for 10 min. The optimal annealing temperature was 62°C. The isolates that were not initially amplified were further processed with the same cycling pattern at an annealing temperature of 58°C, or, if that also failed, of 55°C. Isolate that were not typed after the lower annealing temperatures in the family 1, 2, and 3 tests were classified as nontypeable PspA (PspA NT). An additional two tests were used to verify that the PspA NT isolates were truly pneumococcal isolates. One test was for the presence of the pneumolysin gene and another test was for the presence of the
Three microliters of the PCR products were loaded on 0.8% agarose gels, electrophoresed at 80 V for 1 h, and stained with 0.5 µg/ml ethidium bromide.
All data were statistically analyzed by using Prism 4 (GraphPad Software, Inc., La Jolla, CA, USA). A two tailed chi-square test or Fisher’s exact test (for small group sizes) was used for categorical variables to test the significance of differences between groups. A
The isolates used in this study are all clinical isolates obtained from patients with otorhinolaryngological infections as part of routine clinical diagnosis and management. The main ethical issue relates to specific consent for detailed characterization of an isolate from a clinical specimen taken from a patient on clinical ground. Because no information that would allow identification of the patients was collected in this study, this requirement was waived by the Institutional Review Board of the Ethical Committee of Wakayama Medical University. This study was therefore approved by the Institutional Review Board of the Ethical Committee of Wakayama Medical University.
The distribution of
The most common serotype was 19F (20.7%) followed by 23F (16.3%), 6B (14.7%), 14 (8.0%), 6A (6.4%) and 3 (5.6%). Among the serogroup 6 strains, we could not find the recently discovered serotype 6C and 6D strains. The distribution of
The distribution of
Category | Sub-category | Total | Macrolide resistance genes | ||||
|
|
|
None | MLR v.s. MLS | |||
Gender | Female | 99 (39.4%) | 51 (20.3%) | 39 (15.5%) | 9 (3.6%) | 26 (10.4%) | |
Male | 97 (38.6%) | 55 (21.9%) | 36 (14.3%) | 6 (2.4%) | 29 (11.6%) | ||
Age | 0–2 | 76 (30.3%) | 39 (15.5%) | 32 (12.7%) | 5 (2.0%) | 16 (6.4%) | |
3–5 | 28 (11.2%) | 14 (5.6%) | 12 (4.8%) | 2 (0.8%) | 9 (3.6%) | ||
6–12 | 14 (5.6%) | 11 (4.4%) | 3 (1.2%) | 0 (0%) | 11 (4.4%) | ||
13–20 | 4 (1.6%) | 3 (1.2%) | 0 (0%) | 1 (0.4%) | 3 (1.2%) | ||
21–50 | 58 (23.1%) | 29 (11.6%) | 22 (8.8%) | 7 (2.8%) | 14 (5.6%) | ||
≥51 | 16 (6.4%) | 10 (4.0%) | 6 (2.4%) | 0 (0%) | 2 (0.8%) | ||
Origin | Middle ear fluids | 46 (18.3%) | 34 (13.5%) | 11 (4.4%) | 1 (0.4%) | 11 (4.4%) | |
Nasal discharge/Sinus aspirates | 65 (25.9%) | 35 (13.9%) | 25 (10.0%) | 5 (2.0%) | 23 (9.2%) | ||
Pharyngeal secretions | 85 (33.9%) | 37 (14.7%) | 39 (15.5%) | 9 (3.6%) | 21 (8.4%) | ||
Serotype | 1 | 1 (0.4%) | 0 (0%) | 1 (0.4%) | 0 (0%) | 1 (0.4%) | |
3 | 10 (4.0%) | 9 (3.6%) | 1 (0.4%) | 0 (0%) | 4 (1.6%) | ||
4 | 1 (0.4%) | 0 (0%) | 1 (0.4%) | 0 (0%) | 0 (0%) | ||
6A | 13 (5.2%) | 8 (3.2%) | 4 (1.6%) | 1 (0.4%) | 3 (1.2%) | ||
6B | 27 (10.8%) | 17 (6.8%) | 7 (2.8%) | 3 (1.2%) | 10 (4.0%) | ||
9V | 4 (1.6%) | 3 (1.2%) | 0 (0%) | 1 (0.4%) | 1 (0.4%) | ||
14 | 17 (6.8%) | 10 (4.0%) | 6 (2.4%) | 1 (0.4%) | 3 (1.2%) | ||
15B | 6 (2.4%) | 5 (2.0%) | 0 (0%) | 1 (0.4%) | 1 (0.4%) | ||
19A | 1 (0.4%) | 1 (0.4%) | 0 (0%) | 0 (0%) | 4 (1.6%) | ||
19F | 49 (19.5%) | 15 (6.0%) | 30 (12.0%) | 4 (1.6%) | 3 (1.2%) | ||
23F | 36 (14.3%) | 19 (7.6%) | 14 (5.6%) | 3 (1.2%) | 5 (2.0%) | ||
G23 | 5 (2.0%) | 4 (1.6%) | 1 (0.4%) | 0 (0%) | 1 (0.4%) | ||
Others | 26 (10.4%) | 15 (6.0%) | 10 (4.0%) | 1 (0.4%) | 19 (7.6%) | ||
Total | 196 (78.1%) | 106 (42.2%) | 75 (29.9%) | 15 (6.0%) | 55 (21.9%) |
G23: serogroup 23 strains except serotype 23F. PCG: penicillin G. PSSP: penicillin susceptible
comparison between ≤2 y.o. vs. ≥3 y.o.
The distribution of
Among the 251 pneumococci isolates studied, the 49.4% were identified as belonging to family 2 (PspA2), and 44.6% to family 1 (PspA1). Thus, 94.0% of the isolates included in this study were PspA1- or PspA2-positive isolates. Eight isolates (3.2%) classified into PspA family 3 (PspA3). Four isolates (1.6%) were classified as PspA NT. Three isolates (1.2%) was identified as a PspA null strain.
Because the vast majority of PspA families were identified as PspA1 or PspA2, we further evaluated the distributions of PspA1 and PspA2 by the other parameters. There were no significant differences in the distributions of PspA1 and PspA2 based on the age and gender of the patients, the origin of the isolates (
MEF: middle ear fluid, ND/SA: nasal discharge/sinus aspirate, PS: pharyngeal secretion. Each numbers shows numbers of isolates and percentage shows in parenthesis. There is no significant differences in PspA family distribution based on sex, age and origin of isolates.
PSSP: penicillin susceptible
The distribution of PspA families based on their serotypes is shown in
G23: serogroup 23 strains except serotype 23F. Others: serotypes not included in 23 PPV. Each numbers shows numbers of isolates and percentage shows in parenthesis. *
The coverage and 95% CI of pneumococcal vaccine formulas according to serotypes and PspA families are listed in
Vaccine formulations | Number and percentable coverage of |
|||||
DRSP (n = 158) | MRSP (n = 196) | Total (n = 251) | ||||
n (%) | 95% CI | n (%) | 95% CI | n (%) | 95% CI | |
7-valent (4,6B,9V,14,18C,19F,23F) | 118 (74.7%) | 67.9%−81.5% | 134 (68.4%) | 61.9%−74.9% | 156 (62.2%) | 56.2%−68.2% |
10-valent (1,4,5,6B,7F,9V,14,18C,19F,23F) | 119 (75.3%) | 68.6%−82.0% | 135 (68.9%) | 62.4%−75.4% | 158 (62.9%) | 57.0%−68.9% |
13-valent (1,3,4,5,6A,6B,7F,9V,14,18C,19A,19F,23F) | 131 (82.9%) | 77.0%−88.8% | 159 (81.1%) | 75.6%−86.6% | 193 (76.9%) | 71.7%−82.1% |
23-valent (1,2,3,4,5,6B,7F,8,9V,9N,10A,11A,12F,14,15B,17F,18C,19A,19F,20,22F,23F,33F) | 124 (78.5%) | 72.1%−84.9% | 146 (74.5%) | 68.4%−80.6% | 177 (70.5%) | 64.9%−76.2% |
PspA (PspA1 and PspA2) | 150 (94.9%) | 91.5%−98.4% | 185 (94.4%) | 91.2%−97.6% | 236 (94.0%) | 91.1%−97.0% |
DRSP: drug resistant
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
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 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
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
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
Previous studies showed that PspA clades were independent of capsular serotypes
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.
We cordially thank the Surveillance Subcommittee, the Japan Society for Infectious Diseases in Otolaryngology, the 80 university hospitals, the affiliated hospitals, and the general practitioners who provided clinical specimens for this nationwide surveillance. We greatly thank Miss Yuki Tatsumi (Department of Otolaryngology-Head and Neck Surgery, Wakayama Medical University, Wakayama, Japan) for her technical assistance and Dr. Akihito Wada (National Institute of Infectious Disease, Tokyo, Japan) for evaluating serotype 6C and 6D.