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Identification of a Universal Group B Streptococcus Vaccine by Multiple Genome Screen
Domenico Maione,1*Immaculada Margarit,1*Cira D. Rinaudo,1Vega Masignani,1Marirosa Mora,1Maria Scarselli,1Hervé Tettelin,2Cecilia Brettoni,1Emilia T. Iacobini,1Roberto Rosini,1Nunzio D'Agostino,1Lisa Miorin,1Scilla Buccato,1Massimo Mariani,1Giuliano Galli,1Renzo Nogarotto,1Vincenzo Nardi Dei,1Filipo Vegni,1Claire Fraser,2Giuseppe Mancuso,3Giuseppe Teti,3Lawrence C. Madoff,4Lawrence C. Paoletti,4Rino Rappuoli,1Dennis L. Kasper,4John L. Telford,1Guido Grandi1
Group B Streptococcus (GBS) is a multiserotype bacterial pathogenrepresenting a major cause of life-threatening infections innewborns. To develop a broadly protective vaccine, we analyzedthe genome sequences of eight GBS isolates and cloned and tested312 surface proteins as vaccines. Four proteins elicited protectionin mice, and their combination proved highly protective againsta large panel of strains, including all circulating serotypes.Protection also correlated with antigen accessibility on thebacterial surface and with the induction of opsonophagocyticantibodies. Multigenome analysis and screening described hererepresent a powerful strategy for identifying potential vaccinecandidates against highly variable pathogens.
1 Chiron srl, Via Fiorentina 1, 53100 Siena, Italy. 2 Institute for Genome Research, 9712 Medical Center Drive, Rockville, MD 20850, USA. 3 Department of Pathology and Experimental Microbiology, University of Messina Medical School, 98125 Messina, Italy. 4 Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02125, USA.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: guido_grandi{at}chiron.com
Group B Streptococcus (GBS) is the foremost cause of life-threateningbacterial infections in newborns (1). In about 80% of cases,neonatal GBS infection is acquired during delivery by directmother-to-baby transmission of the pathogen, which colonizesthe anogenital mucosa of 25 to 40% of healthy women (2). Despitethe introduction of intrapartum antibiotic prophylaxis, in theUnited States GBS still causes 2500 cases of infection and 100deaths annually among newborns in the first 3 months of life(3). About half of these cases occur in the first week afterbirth. Thus, it is commonly believed that effective vaccinationwill be the only way to reduce the incidence of GBS diseaseover the long term. The rationale for GBS vaccine developmentis supported by the observation that the risk of neonatal infectionis inversely proportional to the maternal amounts of specificantibodies to the capsular polysaccharide (CPS) antigen thatsurrounds GBS (4, 5), the implication being that protectiveimmunoglobulin G (IgG) antibodies are transferred from the motherto the baby through the placenta.
As a first approach to vaccine development, CPS-tetanus toxoidconjugates against all nine GBS serotypes were shown to induceCPS-specific IgG that is functionally active against GBS ofthe homologous serotype (6). Clinical phase 1 and phase 2 trialsof conjugate vaccines prepared with CPS from GBS types Ia, Ib,II, III, and V revealed that these preparations are safe andhighly immunogenic in healthy adults (7). Although these vaccinesare likely to provide coverage against the majority of GBS serotypesthat currently cause disease in the United States, they do notoffer protection against pathogenic serotypes that are moreprevalent in other parts of the world (e.g., serotypes VI andVIII, which predominate among GBS isolates from Japanese women)(8). Hence, a universal protein-based vaccine against GBS ishighly desirable. To date, a few potential protective antigenshave been described. These include the tandem repeat–containing and ß antigens of the C protein complex (9) and Rib(10); surface immunogenic protein, Sip (11); and C5a-ase, aserine protease that inactivates complement factor C5a (12).However, of these proteins, only Sip and C5a-ase are conservedat the gene level in the majority of GBS isolates (11, 13),and no systematic analysis on the extent of cross-protectionis available.
To identify possible antigens suitable for use in a universalGBS vaccine, we compared the genome sequences of eight GBS strainsbelonging to serotypes Ia (515 and A909), Ib (H36B), II (18RS21),III (COH1 and NEM316), and V (2603 and CJB111), which representthe most important disease-causing serotypes (14). This analysisidentified a "core" genome of 1811 genes (80% of each genome)shared by all strains and a "variable" genome of 765 genes thatwere not present in all strains. Computer algorithms were thenused to select, within the two subgenomes, the genes encodingputative surface-associated and secreted proteins. Among thepredicted surface-exposed proteins, 396 were core genes and193 were variable genes. Of these 589 proteins, 312 were successfullyexpressed in Escherichia coli either as soluble His-tagged fusionsor soluble glutathione S-transferase fusions.
Each purified soluble protein was next used to immunize groupsof adult female mice. At the end of the immunization schedule,these were mated, and the resulting offspring (<48 hoursof age) were challenged with a dose of GBS calculated to kill80 to 90% of the pups (14). For conserved antigens, a virulentsero-type III strain (COH1) was used for challenge; antigensthat were absent from the COH1 strain were tested with one ofthe sequenced strains known to carry the corresponding gene.This systematic screening identified four antigens capable ofsignificantly increasing the survival rate among challengedinfant mice. One of these antigens—GBS322 (SAG0032), whichencoded the previously described Sip protein (11)—waspart of the core genome. The other three antigens—GBS67(SAG1408), GBS80 (SAG0645), and GBS104 (SAG0649)—werepresent in the variable portion of the subgenome. The proportionof mice protected against challenge with strains carrying oneof these proteins varied from 43% in the case of GBS104 to 80%in the case of GBS80.
The four proteins were purified to homogeneity (14) and thentested in mice in the active maternal immunization–neonatalpup challenge model described above with the use of six GBSstrains. Each antigen elicited protection against more thanone strain but not against all strains (Table 1). As expected,whenever the corresponding gene was absent from the challengestrain, the antigen was not protective. However, in a few cases,protection was not conferred even though the challenge straincarried the antigen-coding gene. To test whether this is dueto variability in antigen expression and/or surface exposure,we assessed antigen expression on the surface of each challengestrain by fluorescence-activated cell sorting analysis usingmouse sera specific for each of the four protective antigens.The levels of surface expression, as measured by antibody bindingto viable bacteria, were variable and correlated with the protectiveactivity of the antigen (Table 1). From the data accumulatedup to this point, we estimated that an antigen was protectiveif antigen-specific antibody binding resulted in a >fivefoldincrease in fluorescence intensity over that in pre-immune controls.We then tested a combination of all four antigens in the samemouse model with the use of a panel of 12 challenge strainsthat represented the major pathogenic GBS serotypes and thatbelong to eight Multi Locus Sequence Types (MLST) (15). Thecombination of the four antigens was highly protective againstall 12 strains (Table 2), with protection ranging from 59% to100%—comparable to that conferred to mice vaccinated withCPS-tetanus toxoid glycoconjugates and challenged with homologousstrains (16).
Table 1. Protection conferred by four antigens against six GBS strains assessed by active maternal immunization/neonatal pup challenge model. Female mice received three doses (days 1, 21, 35) of either 20 µg antigen or phosphate-buffered saline (PBS) combined with Freund's adjuvant. Mice were then mated, and the resulting offspring challenged with a dose of GBS calculated to kill 80 to 90% of the pups. Survival of pups was monitored for 2 days after challenge. Fluorescence given in -fold difference between cells stained with immune sera versus pre-immune sera. Protection values calculated as [(% dead in control – % dead in vaccine)/% dead in control] x 100. ND, not determined.
Table 2. Protection against 12 GBS strains by a four-antigen combination. Experiments were performed as in Table 1 except that mice were vaccinated with a mixture of 15 µg of each protein (a total of 60 µg). Protection P values were less than 0.0001.
GBS strain
Serotype
Vaccine (alive/treated)
PBS (alive/treated)
Protection
515
Ia
39/40
6/40
97.0%
DK1
Ia
50/50
8/38
82.5%
7357B
Ib
49/60
5/46
79.4%
DK21
II
25/34
17/48
59.3%
5401
II
35/40
3/37
86.4%
3050
II
48/48
1/30
100%
COH1
III
36/36
7/40
100%
M781
III
30/40
4/39
72.0%
2603V/R
V
27/33
10/35
75.0%
CJB111
V
25/28
4/46
88.2%
JM9130013
VIII
37/39
5/40
94.2%
SMU071
VIII
44/50
18/50
81.2%
Total
445/498
88/498
87.0%
Lastly, we assayed the in vitro opsonophagocytic activity (17)of sera from mice immunized with the single antigens and withthe four-antigen combination. Sera were incubated with the highlyencapsulated GBS type V strain CJB111, which expresses all fourantigens, and bacterial killing was measured in the presenceof both polymorphonuclear leukocytes (PMNs) and rabbit complement.All sera promoted opsonophagocytosis and killing of GBS by PMNs(Fig. 1), and killing was both PMN- and complement-dependent(14). However, the bacteria were most efficiently killed whenopsonized with sera from mice vaccinated with the combinationof four protein antigens, suggesting that the four proteinswork additively as potent immunogens. Taken together, the protectionin mice and the opsonophagocytic activity of the mouse serasuggest that a vaccine based on these four antigens may confereffective protection in humans also.
Fig. 1. Opsonophagocytic activity of sera specific for vaccine antigens. Live GBS bacteria of strain CJB111 were incubated for 1 hour with human PMNs in the presence of baby rabbit complement and specific antisera. The log10 of the difference between bacterial colony forming units at time = 0 and time = 1 hour are shown. Values for preimmune sera are negative because of bacterial growth during the assay. The antigens used are recorded above each bar. Shaded bars represent specific immune sera; open bars, the corresponding preimmune sera from the same animals. Error bars indicate standard deviation.
[View Larger Version of this Image (24K GIF file)]
At least two major conclusions can be drawn from this work.First, multistrain genome analysis and screening constitutean effective new approach to identifying vaccine candidatesthat can provide broad protective activity when used in combination.Of the four antigens identified, none could be classified asuniversal because, in a fraction of GBS strains, either theircoding gene was absent or their surface accessibility was negligible.Therefore, a genome screen of a single strain (18) would nothave led to the identification of all four antigens but wouldhave identified only those that, by coincidence, were sufficientlyexpressed in the strain used for challenge in the mouse model.
Despite the absence of universal antigens, it is clear thatappropriate combinations of protective antigens—each effectiveagainst overlapping populations of isolates—can conferunexpectedly broad serotype-independent protection. In fact,the four-antigen vaccine used in this work protected mice against12 virulent strains belonging to all nine major GBS serotypes.To estimate the strain coverage of the vaccine, we analyzedthe surface expression of the four antigens on a total of 37GBS isolates. We found that at least one of the antigens washighly accessible to antibodies (>fivefold shift in fluorescence)in 32 out of the 37 strains tested, which corresponds to 87%of circulating strains assuming that these strains sufficientlyreflect the variability in the population.
A second conclusion from this work is that the extent of surfaceaccessibility of antigens may vary from strain to strain, evenif the antigens' coding genes are conserved (Table 1). Suchvariability may be due to differences in gene expression, antigenmasking by other cellular components (e.g., CPS), protein degradation,or other factors. For instance, we found that the surface accessibilityof the protein Sip was dependent on the presence of the polysaccharidecapsule (table S2). In line with this, the protective antigenswe identified were effective only against those strains in whichthe antigens were sufficiently exposed on the bacterial surface.From a practical point of view, variability in surface antigenexpression highlights the importance of upfront rational selectionof strains to be used in protection models. The strains shouldbe selected not only because they carry the gene for the antigenunder examination, but also in light of the amount of expressionand accessibility of the antigen itself. Between 30 and 40%of the genes of all bacteria sequenced so far belong to hypotheticalor unknown families. Because our approach selects antigens independentof their function, it was likely that some protective antigenswould have no assigned function. This is the case for all fourprotective antigens described herein. GBS322 contains a LysMdomain, which is found in a variety of enzymes involved in bacterialcell-wall degradation and may have a general peptidoglycan-bindingfunction. GBS67, GBS80, and GBS104 all contain LPXTG (Leu-Pro-X-Thr-Gly,where X is any amino acid) motifs associated with covalent linkageto the cell wall (19). Indeed, we have recently found that allthree proteins are components of pilus-like structures neverdescribed before in GBS (20).
In GBS and probably other bacterial pathogens that adopt thestrategy of gene variability to escape the immune system, universalprotective protein antigens are unlikely to exist. However,some protein antigens are conserved in sufficiently large subpopulationsof GBS that in combination they can be broadly protective. Thesuccessful use of multistrain genome analysis and screeningdescribed here for GBS provides the basis for the potentialdevelopment of universal protein-based vaccines against otherimportant and highly variable pathogens such as Group A Streptococcusand S. pneumoniae.
21. D. Maione and I. Margarit contributed equally to this work. We thank M. Tortoli, S. Torricelli, G. Volpini, and the animal care facility at Chiron srl for expert technical assistance and G. Corsi for artworks. This work was supported, in part, by grant AI-060603 (L.C.P.) from the NIH National Institute of Allergy and Infectious Diseases. D.K. is a paid consultant for Chiron Corporation.
Received for publication 18 January 2005. Accepted for publication 27 April 2005.
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To whom correspondence should be addressed. E-mail: 
2500 cases of infection and 100 deaths annually among newborns in the first 3 months of life (
and ß antigens of the C protein complex (
