PspA Family Distribution, unlike Capsular Serotype, Remains
Unaltered following Introduction of the Heptavalent Pneumococcal
Christina M. Croney,aMamie T. Coats,a,dMoon H. Nahm,a,bDavid E. Briles,a,cand Marilyn J. Craina,c
Departments of Microbiology,aPathology,band Pediatrics,cUniversity of Alabama at Birmingham, Birmingham, Alabama, USA, and Department of Biological Sciences,
Alabama State University, Montgomery, Alabama, USAd
ingitis. Pneumococcal infections are estimated to cause 826,000
duction of the heptavalent pneumococcal conjugate vaccine
(PCV7) led to almost complete elimination of invasive pneumo-
coccal disease (IPD) caused by the seven PCV capsular types (4,
6B, 9V, 14, 18C, 19F, and 23F) causing IPD prior to the introduc-
tion of that vaccine. Subsequently, an increase in the incidence of
IPD caused by non-PCV7 capsular types has been observed (11,
21). In 2010, a new 13-valent vaccine was introduced to provide
protection against the original PCV7 serotypes plus an additional
In the European Union, serotypes 3 and 19A cause 2.5% and ap-
proximately 15% of IPD cases, respectively. A recent report indi-
cates that PCV13 is expected to cover no more than 68% of IPD
those covered by the vaccine (18). Since there are ?90 known
capsular serotypes (5), continuing to increase the number of se-
closing the gap in PCV coverage and countering future serotype
A potential strategy to reduce serotype replacement could be
the inclusion of protein vaccine immunogens that could provide
protection that is not dependent on antibody responses to capsu-
lar polysaccharides. One candidate protein antigen is the cross-
protective protein antigen pneumococcal surface protein A
(PspA). Prior to the use of PCV7, this cell surface-associated pro-
tein virulence factor (13) was found on virtually all clinically rel-
evant strains of pneumococci (6), and almost all strains express
one of 2 major serologic/sequence families. Prior to the licensure
of strains reported from studies of 3 collections comprising more
treptococcus pneumoniae is a major cause of morbidity and
a PspA-containing vaccine should include representatives of each
of these two major families (1, 12, 23). Strains of the most com-
mon seven capsular types before the year 2000 almost exclusively
expressed either PspA family 1 or family 2. For many of the other
capsular types, the numbers of strains examined were so few that
little information could be gained about whether they were also
se, it was possible that some of the pneumococcal clones that ex-
panded in frequency following the use of PCV7 might already
have expressed PspAs that were not within families 1 or 2.
In this study, we present data on the PspA family and capsular
serotype distribution of 157 isolates from IPD collected between
2002 and 2010 at a children’s hospital. Our study examined the
proportion of IPD isolates with serotypes not included in PCV13
and the distribution of two major PspA families. Forty percent of
strains in this collection were not covered by PCV13. We found,
isolates, overall. These findings indicate that a vaccine containing
PspA molecules from these two families would still have the po-
tential to cover IPD isolates following the introduction of PCV7
and, probably, PCV13.
Received 6 December 2011 Returned for modification 15 February 2012
Accepted 19 April 2012
Published ahead of print 25 April 2012
Address correspondence to Marilyn J. Crain, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
June 2012 Volume 19 Number 6 Clinical and Vaccine Immunologyp. 891–896 cvi.asm.org
lumbia, Canada, October 2010.)
MATERIALS AND METHODS
2010 were collected prospectively. The site of isolation, clinical disease
diagnosis, date of culture, antimicrobial susceptibilities, and patient de-
mographic data associated with each strain were retrieved from the elec-
tronic medical record under an approved protocol of the Institutional
informed consent. IPD for this study was defined as infection with Strep-
cerebrospinal fluid (CSF), pleural fluid, sputum, peritoneal fluid, and
bone or joint aspirates. Seven clinical disease categories were considered:
bacteremia, bacteremic pneumonia (bacteremia in association with a
bacterial pneumonia), complicated pneumonia (chest X ray with pneu-
fluid), pneumonia (chest X ray with pneumonia and pneumococci iso-
lated from sputum or bronchoalveolar lavage fluid), mastoiditis (pneu-
mococci obtained at surgery), meningitis (cerebrospinal fluid indices
and/or blood), and other IPD, including endocarditis (vegetations on
echocardiogram and pneumococci in blood cultures) and bone or joint
infection (compatible clinical diagnosis with pneumococci isolated from
bone or joint aspirate). Two hundred thirty pneumococcal isolates were
to receiving multiple copies of an isolate, loss of a viable sample, multiple
analyses included 157 IPD isolates.
Multiplex assays for serotype detection. Strains were typed serologi-
cally and/or by PCR for all 93 known pneumococcal capsular types. Cap-
sular serotyping was performed using a multiplex immunoassay with
monoclonal antibodies specific for each of the following serotypes as de-
19A, 2, 6C, 6D, 8, 9N, 10A/39, 11A/D/F, 11E, 12F/B, 15B/C, 17F/A, 20,
93 known serotypes that also included detection of autolysin and cpsA.
7F/A strains were furthered typed by agglutination with factor sera (Stat-
ens Serum Institut, Denmark). Strains were then grouped according to
whether their capsular serotype was included or not included as an anti-
gen in PCVs, including (i) the original heptavalent PCV (PCV7), (ii) the
capsule antigens for the 7 serotypes in PCV7 plus six additional serotypes
with typeable capsules (nonvaccine type [NVT]), and (iv) nontypeable
capsules (NT). All strains NT for capsule were also observed to have cpsA
(24), which is known to be present in the capsule loci of strains of almost
all capsular types (24).
confirmed to be S. pneumoniae through testing for optochin sensitivity,
bile solubility, and the presence of the pneumolysin gene (ply). Strains
preparation by inoculation with 100 ?l deionized water, boiling for 15
was examined by PCR. The following primers were used to amplify a
348-bp region in Ply: ply F=, 5=-ATTTCTGTAACAGCTACCAACGA-3=,
and ply R=, 5=-GAATTCCCTGTCTTTTCAAAGTC-3=.
as previously described by Hollingshead et al. (13). For isolates not type-
able by PCR, a dot blot assay was performed (23), and this result was
considered to define the PspA family.
Statistical analysis. Statistical analyses between groups were per-
formed using the ?2test, the ?2test for trend, or Fisher’s exact test. A P
value of ?0.05 was considered to be significant. All tests were performed
in GraphPad InStat, version 5.0 (GraphPad, La Jolla, CA).
Patient demographics and serotype distribution of isolates
from 2002 to 2010. The population was 62.4% male, 50.3% Cau-
casian, and 26.8% African American. Of 157 IPD isolates col-
lected, 64/157 (40.8%) were obtained from children less than 24
months of age, 43 (27.4%) were from children aged 24 to 60
age and capsular serotype distribution (P ? 0.0664). Among the
three age groups, patients ?60 months of age had the greatest
diversity in capsular serotype distribution (the highest number of
prevalent serotypes and change over time within age group); 50%
of strains from this group were non-PCV13 capsular types. Al-
though the highest percentage (41%) of serotype 19A isolates was
found among children ?24 months old, there was no significant
difference in the occurrence of serotype 19A between any of the
significant. In order to capture the seasonality of pneumococcal
Between 2002 and 2010, the PCV7 capsular types causing IPD
virtually disappeared. Although the proportion of non-PCV cap-
sular types differed from year to year, overall, 39.5% of the 157
IPD isolates failed to express PCV13 capsular types. A total of 17
different non-PCV13 capsular types were observed (Fig. 1). Two
not shown). This increase of serotype 19A strains largely ac-
counted for the increase of PCV13 strains.
Serotype distribution also varied by clinical disease (P ?
0.0013) (Fig. 2). Meningitis and “other IPD” (i.e., endocarditis,
TABLE 1 Demographics of patients with IPD at Children’s of Alabama
Characteristic Overall no. (%)
No. (%) of isolates of a
Age 157 (100)
aPCV13, all serotypes included in the 13-valent vaccine; NVT, encapsulated and
nonencapsulated nonvaccine types.
Croney et al.
cvi.asm.org Clinical and Vaccine Immunology
associated than bacteremia with nonvaccine serotypes (P ?
0.0176 and P ? 0.0071, respectively). The remaining clinical dis-
ease categories (bacteremia, bacteremic pneumonia, complicated
pneumonia, pneumonia, and mastoiditis) trended toward being
more likely to be caused by PCV13 serotypes.
FIG 1 Serotype distribution of IPD isolates by period of isolation. (A) Serotype distributions for IPD isolates are shown for the years 2002 through 2010. Each
seasonal period spans from July 1 through June 30 of the second year. N, total isolates from the indicated period; PCV7, capsular serotypes in the heptavalent
pneumococcal conjugate vaccine; PCV13, capsular serotypes in the 13-valent PCV that are not included in PCV7; NVT, typeable serotypes not included in
PCV13 (nonvaccine types); NT, nontypeable isolates. Overall, 60.5% of IPD cases were caused by serotypes included in Prevnar 13 (PCV13). (B) Raw serotype
data of all IPD isolates. N, total number of isolates for indicated serotype. Serotype 19A accounted for 32.5% of all IPD isolates.
FIG 2 Serotype distribution of pneumococci causing clinical disease. N, number of isolates; PCV13, serotypes included in PCV13; NVT, all pneumococci not
expressing the serotypes included in the 13-valent vaccine (nonvaccine types), including both typeable isolates and those with nontypeable capsules. Serotype
distribution versus disease manifestation overall was statistically significant (P ? 0.0013, ?2test for trend; all groups were compared as PCV13 versus NVT).
Individual P values were obtained by setting bacteremia as the reference group and comparing with the disease of interest using Fisher’s exact test. Bacteremic
pneumonia was defined as a patient with a chest X ray (interpreted by a pediatric imaging specialist) for bacterial pneumonia and pneumococci isolated from a
blood culture, complicated pneumonia as a chest X ray with pneumonia with effusion and pneumococci isolated from pleural fluid or pleural space, and
pneumonia as a chest X ray positive for pneumonia and culture positive for pneumococci. “Other IPD” included endocarditis and bone or joint infection.
PspA and Capsule Diversity following PCV7
June 2012 Volume 19 Number 6cvi.asm.org 893
ers for families 1, 2, and 3 (13, 23) were able to ascertain the PspA
(40.8%) were PspA family 1, 87 (55.4%) were PspA family 2, and
2 (1.3%) were PspA family 3, while 4 (2.5%) had nontypeable
PspA (Table 2). A shift from majority PspA family 1 to family 2
occurred (P ? 0.0052) during the study period; however, there
among the strains with PCV13 serotypes versus those with NVT
serotypes overall (P ? 0.1777) (Table 3). When we looked at in-
dividual serotypes versus the overall population distribution, se-
rotypes 7F, 6C, 22F/A, and 23A were found to be significantly
different from the population as a whole in terms of PspA family
type (P ? 0.01, 0.002, 0.005, and 0.03, respectively). Eleven of 13
7F strains expressed PspA family 2. For capsular types 6C, 22F/A,
and 23A, 100% of the 4 to 7 strains in each group expressed PspA
family 1. Among the PCV13 and NVT strains, the percentages of
strains expressing either family 1 or family 2 PspA were virtually
family 3 strains, both had NT capsules. Of the four strains that
expressed NT PspA, one also had a nontypeable capsule.
When PCV7 was licensed in 2000, 81% of cases of serious pneu-
mococcal disease in the United States were caused by pneumo-
cocci with one of the vaccine capsule types. PCV7 was highly ef-
fective at reducing IPD caused by vaccine serotypes and reduced
the incidence in children ?5 years of age by about 75% by 2002
(11, 21). The incidence of disease by PCV7 isolates continued to
decrease through 2007; however, the overall incidence of IPD
failed to decrease significantly below the 2002 level due to an in-
crease in IPD caused by non-PCV7 capsule type strains (18).
PCV13 was anticipated to have lower coverage for pneumococcal
capsular serotypes in childhood IPD (67.8% in children ?5 years
support this expectation. Although the relative capsular serotype
distribution of our IPD isolates from Alabama children varied
considerably from year to year, IPD coverage by the recently li-
censed PCV13 among these strains collected from 2002 to 2010
was only 60.5% overall, even prior to the introduction of the vac-
cine in 2010. This relatively high proportion of non-PCV13 sero-
cine introduction probably reflects the unmasking of previously
carried strains or new serotypes entering the niche originally oc-
cupied by the PCV7 strains. It should be noted that the serotype
coverage by PCV13 in our strain collection had not yet been af-
fected by PCV13 use since it was not in general clinical use until
after the collection was closed.
The multiplex serotyping assay used in this study was capable of
typing pneumococci of all 93 currently known serotypes (5). None-
required the inclusion of an additional 17 or more (total of 30)
capsular types. Based on our data, these 17 capsular types not
covered by PCV13 would provide coverage of 1.6% to 13% of the
is limited to 157 isolates from a relatively small geographic region
size from a larger geographic area or more diverse region would
distribution of serotypes (18).
TABLE 2 Frequencies of PspA families by characteristics of S. pneumoniae isolates
No. (%) of isolates expressinga:
Total no. (%)
(n ? 157)
2 (3.1) 64 (40.8)
50 (31.8)2 (4.0)
2 (4.3)3 (6.5)
Period of isolationc
48 (30.6) 2 (4.2)
aPspA, pneumococcal surface protein A; NT, PspA nontypeable.
bOther IPD was defined as endocarditis and bone or joint isolates.
cP ? 0.0052, ?2test for trend, period of isolation versus PspA family 1 or 2.
Croney et al.
cvi.asm.org Clinical and Vaccine Immunology
reactive protection-eliciting pneumococcal protein antigens (22)
or to develop a protein-only vaccine consisting of several protec-
tion-eliciting protein antigens. A vaccine containing proteins
from both PspA families 1 and 2 would cover 96% of IPD isolates
family distribution from global isolates of pneumococci collected
prior to the effect of PCV7 on capsular type distribution (12, 23).
Our present results indicate that among the non-PCV7 strains
which have expanded to fill the niche following mass immuniza-
tion with PCV7, only 4% failed to express PspA family 1 or 2. To
see whether this result can be generalized for strains around the
world following the introduction of PCVs, it will be necessary to
study additional isolates.
Immunization of healthy adults with a single PspA family 2
molecule resulted in the production of antibody that was able to
passively protect mice infected with pneumococcal strains ex-
pressing either family 1 or family 2 PspAs (3). However, it was
previously shown that PspAs exhibited the strongest cross-reac-
tivity with PspAs of the same family. Thus, an effective PspA vac-
cine was expected to require epitopes from a single family 1 PspA
and one or two family 2 PspAs to cover all strains (3, 15, 20). This
could be achieved by mixing PspAs together (3, 20), making fu-
or using PspA as a carrier for one or more capsular polysaccha-
ecules may make it possible to select individual molecules with
wide cross-reactivity with other PspAs (14). Finally, recent work
has shown that the relatively conserved proline-rich domain,
which lacks any alpha-helical epitopes and is common to all fam-
infection in mice (7). The proline-rich domain of PspA alone has
not yet been studied in clinical trials (3).
Several experiments have shown that mixtures of different
pneumococcal proteins can, in some cases, elicit much more pro-
tection than the individual proteins in the mixtures (2, 4, 17).
conjugate vaccines include pneumolysoid, PspC (CbpA), PcpA,
and PhtD (9, 17, 19, 22). Regardless of how new vaccines are
cal serotypes associated with invasive disease will be critical to
identify the best vaccine strategy for further improvement in the
control of pneumococcal disease.
Janice King and Evida Dennis for help with maintenance of the isolate
from the National Institute of Diabetes and Digestive and Kidney Dis-
eases, grant 5TL1 RR025775-04 from the National Center for Research
Resources, and the Howard Hughes Medical Institute through the Med
into Grad Initiative to University of Alabama at Birmingham.
The contents of this paper are solely the responsibility of the au-
thors and do not represent the views of HHMI, NIAID, NIDDK,
NCRR, or NIH.
Potential conflicts of interest include the fact that David E. Briles is a
consultant for Sanofi Pasteur and the PATH Foundation. The University
of Alabama at Birmingham (UAB) holds intellectual property rights re-
UAB also holds intellectual property rights for monoclonal antibodies
used in this study. C.M.C., M.H.N., D.E.B., and M.J.C. are employees
1. Brandileone MC, et al. 2004. Typing of pneumococcal surface protein A
(PspA) in Streptococcus pneumoniae isolated during epidemiological sur-
2. Briles DE, et al. 2000. Intranasal immunization of mice with a mixture of
the pneumococcal proteins PsaA and PspA is highly protective against
nasopharyngeal carriage of Streptococcus pneumoniae. Infect. Immun. 68:
3. Briles DE, et al. 2000. Immunization of humans with rPspA elicits anti-
bodies, which passively protect mice from fatal infection with Streptococ-
TABLE 3 Capsular serotype and PspA family type for all isolates
No. (%) of isolates expressingb:
2 (66.7)1 (33.3)
4 (57.1)2 (28.6)1 (14.3)
aThe first 7 serotypes are in PCV7, the next 6 are the additional serotypes in PCV13,
and the last 17 serotypes are in neither PCV7 nor PCV13.
bPspA, pneumococcal surface protein A; NT, nontypeable.
cP ? 0.05, Fisher’s exact test, individual serotype versus overall distribution of PspA
family 1 or 2.
PspA and Capsule Diversity following PCV7
June 2012 Volume 19 Number 6cvi.asm.org 895
A and pneumolysin are protective against pneumonia in a murine model
of pulmonary infection with Streptococcus pneumoniae. J. Infect. Dis. 188:
5. Calix JJ, Nahm MH. 2010. A new pneumococcal serotype, 11E, has a
variably inactivated wcjE gene. J. Infect. Dis. 202:29–38.
6. Crain MJ, et al. 1990. Pneumococcal surface protein A (PspA) is serolog-
ically highly variable and is expressed by all clinically important capsular
serotypes of Streptococcus pneumoniae. Infect. Immun. 58:3293–3299.
7. Daniels CC, et al. 2010. The proline-rich region of pneumococcal surface
proteins A and C contains surface-accessible epitopes common to all
pneumococci and elicits antibody-mediated protection against sepsis. In-
fect. Immun. 78:2163–2172.
8. Darrieux M, et al. 2007. Fusion proteins containing family 1 and family 2
PspAs elicit protection against Streptococcus pneumoniae that correlates
with antibody-mediated enhancement of complement deposition. Infect.
9. Glover DT, Hollingshead SK, Briles DE. 2008. Streptococcus pneu-
moniae surface protein PcpA elicits protection against lung infection and
fatal sepsis. Infect. Immun. 76:2767–2776.
10. Hathaway LJ, Stutzmann Meier P, Battig P, Aebi S, Muhlemann K.
2004. A homologue of aliB is found in the capsule region of nonencapsu-
lated Streptococcus pneumoniae. J. Bacteriol. 186:3721–3729.
11. Hicks LA, et al. 2007. Incidence of pneumococcal disease due to non-
pneumococcal conjugate vaccine (PCV7) serotypes in the United States
during the era of widespread PCV7 vaccination, 1998-2004. J. Infect. Dis.
12. Hollingshead SK, et al. 2006. Pneumococcal surface protein A (PspA)
collected in seven countries. J. Med. Microbiol. 55:215–221.
13. Hollingshead SK, Becker R, Briles DE. 2000. Diversity of PspA: mosaic
Infect. Immun. 68:5889–5900.
14. Moreno AT, et al. 2010. Immunization of mice with single PspA frag-
ments induces antibodies capable of mediating complement deposition
on different pneumococcal strains and cross-protection. Clin. Vaccine
15. Nabors GS, et al. 2000. Immunization of healthy adults with a single
recombinant pneumococcal surface protein A (PspA) variant stimulates
broadly cross-reactive antibodies to heterologous PspA molecules. Vac-
16. O’Brien KL, et al. 2009. Burden of disease caused by Streptococcus pneu-
moniae in children younger than 5 years: global estimates. Lancet 374:
17. Ogunniyi AD, Grabowicz M, Briles DE, Cook J, Paton JC. 2007.
combinations of virulence proteins of Streptococcus pneumoniae. Infect.
18. Pilishvili T, et al. 2010. Sustained reductions in invasive pneumococcal
disease in the era of conjugate vaccine. J. Infect. Dis. 201:32–41.
19. Posfay-Barbe KM, et al. 2011. Immunity to pneumococcal surface pro-
20. Roche H, Ren B, McDaniel LS, Hakansson A, Briles DE. 2003. Relative
roles of genetic background and variation in PspA in the ability of anti-
bodies to PspA to protect against capsular type 3 and 4 strains of Strepto-
coccus pneumoniae. Infect. Immun. 71:4498–4505.
21. Singleton RJ, et al. 2007. Invasive pneumococcal disease caused by non-
vaccine serotypes among Alaska native children with high levels of 7-va-
lent pneumococcal conjugate vaccine coverage. JAMA 297:1784–1792.
22. Tai SS. 2006. Streptococcus pneumoniae protein vaccine candidates:
properties, activities and animal studies. Crit. Rev. Microbiol. 32:139–
23. Vela Coral MC, et al. 2001. Pneumococcal surface protein A of invasive
fect. Dis. 7:832–836.
24. Yother J. 2011. Capsules of Streptococcus pneumoniae and other bacte-
25. Yu J, et al. 2005. Rapid multiplex assay for serotyping pneumococci with
monoclonal and polyclonal antibodies. J. Clin. Microbiol. 43:156–162.
26. Yu J, Lin J, Kim KH, Benjamin WH, Jr, Nahm MH. 2011. Development
moniae. Clin. Vaccine Immunol. 18:1900–1907.
Croney et al.
cvi.asm.orgClinical and Vaccine Immunology