Synthesis and Preclinical Evaluation of Glycoconjugate Vaccines against Group
B Streptococcus Types VI and VIII
Lawrence C. Paoletti,1Julieanne Pinel,1
Kenneth D. Johnson,1Barbara Reinap,1
Robin A. Ross,1and Dennis L. Kasper1,2
1Channing Laboratory, Department of Medicine, Brigham
and Women’s Hospital;
Genetics, Harvard Medical School, Boston, Massachusetts
2Department of Microbiology and Molecular
Group B Streptococcus (GBS) types VI and VIII are prevalent among serotypes isolated
from pregnant women in Japan. Maternal vaccination with a safe and effective GBS vaccine
has been proposed as a rational approach to prevent neonatal GBS disease. Because antibody
specific for the capsular polysaccharide (CPS) antigens of GBS is protective, vaccines were
developed with purified type VI and VIII CPS coupled to tetanus toxoid. In rabbits the newly
synthesized conjugate vaccineselicited high-titered,type-specificantibodythatwasopsonically
active in vitro. Moreover, litters born to mice actively vaccinated with the conjugate vaccines,
in contrast to uncoupled CPS or saline, were protected against an ordinarily lethal challenge
of GBS of homologous serotype. GBS types VI and VIII conjugate vaccines of the design
presented may be important components of a multivalent GBS vaccine for use in regions
where these serotypes predominate.
Group B Streptococcus (GBS) has been recognized for 125
years as the leading bacterial cause of mortality among new-
borns in the United States and as a significant contributor to
morbidity among peripartum women. With few exceptions,the
serotypes responsible for invasive GBS disease in the United
States are restricted to types Ia, Ib, II, III, and V. However, the
serotypes that cause GBS infections in many regions of the
world are not restricted to those most prevalent in the United
States . Recent studies have confirmed the prevalenceofGBS
types VI and VIII among pregnant women in Japan [2, 3],
serotypes that have not been isolated (type VI) or are found
only rarely (type VIII) in the United States .
The capsular polysaccharide (CPS) antigens of GBS are im-
portant targets of protective immunity. Conjugate vaccinespro-
duced with GBS CPS are highly immunogenic and efficacious
in animals  and safe and immunogenic in humans [6, 7]. We
applied the reductive amination coupling procedure to create
conjugate vaccines with CPS of GBS types VI and VIII. The
immunogenicity of the vaccines was tested in rabbits, and the
efficacy of the conjugate vaccines was measured in a mouse
Received 15 January 1999; revised 12 May 1999; electronically published
9 August 1999.
The contents of this publication do not necessarily reflect the views or
policies of the Department of Health and Human Services nor does the
mention of trade names, commercial products, or organizations imply en-
dorsements by the US government.
Financial support: NIH (contract AI-75326).
Reprints or correspondence: Dr. Lawrence C. Paoletti, Channing La-
boratory, 181 Longwood Ave., Boston, MA 02115 (lpaoletti@channing.
The Journal of Infectious Diseases
? 1999 by the Infectious Diseases Society of America. All rights reserved.
maternal vaccination-neonatal challenge model of GBS type VI
and type VIII disease.
Materials and Methods
and SMU 042 and type VIII strains SMU 074 and SMU 071 were
provided by C. Lachenauer, Channing Laboratory (Boston, MA)
. Type VI strain SS1214 and typeVIII strainsJM9Prague130013
and 130672 were provided by J. Jelı ´nkova ´, National Institute of
Public Health (Prague, Czech Republic).
Preparation of type VI CPS conjugate vaccine.
strain SS1214 was batch culture grown in a 20-L fermenter with
Columbia broth. Type VI CPS lot Ac was purified from harvested
and washed cells by methods described elsewhere for the purifi-
cation of GBS type III CPS . Purified type VI CPS had a relative
molecular weight of 200,000 and a 2 : 2 : 1 molar ratio of galactose
:glucose : sialic acid.
Type VI CPS (12 mg) was treated with 958 mL of 0.01 M NaIO4
in 0.5 mL of deionized water. The mixture was incubated in the
dark for 90 min at room temperature. After incubation, a drop of
ethylene glycol was added to consume the residual periodate, and
the mixture was dialyzed against water at 4?C overnight. The per-
iodate-treated type VI CPS was dried, and the degree of sialic acid
oxidation was confirmed by gas chromatography–mass spectros-
copy (GC-MS) of trimethysilyl derivatives . Mildperiodatetreat-
ment of type VI CPS resulted in the oxidation of 41% of the total
sialic acid residues. Oxidized type VI CPS (8.4 mg) was combined
with 8.6 mg of monomeric (∼150,000 Mr) tetanus toxoid (TT) in
0.5 mL of 0.2 M NaPO4buffer (pH 7.2). The coupling reaction
was initiated by the addition to the mixture of 13 mg of sodium
cyanoborohydride (Matreya, Pleasant Gap, PA). The mixture was
incubated at 37?C, andaliquots wereremovedperiodicallyformon-
itoring of the coupling reaction by gel filtration column chroma-
GBS type VI strains SMU 053, SMU 035,
GBS type VI
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