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Recombinant Group B Streptococcus Alpha-Like Protein 3 Is an Effective Immunogen and Carrier Protein

ABSTRACT Conjugate vaccines against pathogens of multiple serotypes are optimized when all components induce functional antibody, resulting in broadened coverage. While most clinical studies of vaccines against group B Streptococcus (GBS) have evaluated conjugates composed of capsular polysaccharide (CPS) coupled to tetanus toxoid, conjugates prepared with GBS proteins as carriers have also been efficacious in animals. Here, we report that recombinant GBS alpha-like protein 3 (rAlp3) is both a strong immunogen and a viable carrier protein for type III CPS. The type III CPS-specific immunoglobulin G (IgG) titer rose from <100 to 64,000 among mice that received type III CPS coupled to rAlp3 (III-rAlp3) compared with an absence of a specific response among mice that received an uncoupled mixture. Most (94%) newborn pups born to III-rAlp-vaccinated dams survived challenge with viable type III GBS, compared with 43% survival among those born to dams that received the uncoupled mixture (P < 0.0001). A tricomponent conjugate of type III CPS, rAlp3, and a GBS recombinant beta C protein lacking its IgA binding site (III-rAlp3-rBCPΔIgA) provided protection against a serotype III strain and a serotype Ia strain bearing beta C protein. High-titered anti-rAlp3 rabbit serum opsonized Alp3-containing strains of two GBS serotypes (types V and VIII) and invasive type III strains bearing the cross-reactive Rib protein for in vitro killing by human peripheral blood leukocytes. Thus, the potential exists for the inclusion of rAlp3 in a GBS vaccine formulated to provide multiserotype coverage.

Streptococcus agalactiae, or group B Streptococcus (GBS), is an important cause of invasive infection in newborns, pregnant women, the elderly, and immunocompromised adults. The most prevalent GBS serotypes causing neonatal GBS diseases in the world (16,18,19), including the United States, Germany, and Malawi, are type Ia (ϳ14% to 30%) and type III (ϳ50% to 68%). Serotype V has emerged as the most common serotype (ϳ30%) isolated from nonpregnant adults (14,19), and serotypes VI and VIII are prevalent colonizers of pregnant women in Japan (25). Early vaccine trials with purified, uncoupled type Ia, II, and III capsule polysaccharides (CPSs) showed that these antigens evoked low and variable type-specific antibody responses in healthy adults (2,4). Later vaccine trials of conjugate vaccines created by the covalent attachment of the CPS to an immunogenic carrier protein, such as tetanus toxoid (TT), enhanced the immune response to the CPS and resulted in high levels of type-specific, functionally active antibody predominantly of the immunoglobulin G (IgG) class (1,40).
Thus far, only a few protein carriers have been used for the preparation of licensed conjugate vaccines (15). Three protein carriers, including diphtheria mutant protein cross-reactive material (CRM 197 ), TT, and diphtheria toxoid have been used in Haemophilus influenzae type b conjugate vaccines, whereas CRM 197 , TT, and diphtheria toxoid have been used in Neisseria meningitidis conjugate vaccines (15,39). Seven pneumococcal CPSs have each been coupled to CRM 197 to create the multivalent conjugate vaccine against Streptococcus pneumoniae in-fections (13). While all of these carrier proteins improved the immune response to the CPS antigens, there was concern regarding their overuse. Indeed, studies have shown that healthy adults and children vaccinated with the protein carrier subsequently have suppressed antibody responses to the CPS epitopes (6,10), and apparent epitopic suppression was observed when multiple vaccines sharing common protein epitopes were administered simultaneously (12). The use of alternative carrier proteins may reduce the potential for immune suppression in primed individuals as more conjugate vaccines are added to the routine vaccination program. Therefore, the development of alternative protein carriers that are immunogenic, protective, and easy to produce and ideally expand coverage is desired.
To date, several GBS protein carriers that confer protection in animals have been described, including the alpha C protein (ACP) (17,29), beta C protein (BCP) (32), Rib (29,45), Sip (9), C5a peptidase (11), and surface-exposed antigens GBS67, GBS80, and GBS104 (36). The majority of GBS isolates express one or more of a family of surface-anchored proteins that form ladder-like patterns on Western blot due to a large number of repeat units. These proteins include ACP, Rib, alphalike protein 2 (Alp2), and alpha-like protein 3 (Alp3) (31). The Rib protein is present in most type III strains, and Alp3 is present on most type V and type VIII strains (22,31). The Alp3 protein has N-terminal regions that are highly similar to those of the ACP and BCP, and its tandem repeats are essentially identical to the repeat region of the Rib protein (24). This tandem repeat region in Alp3 might constitute the antigenic site that was also present in the Rib protein (34,33). Except for the number of repeats, the sequence of Alp3 is 98% identical to the sequence of R28 from S. pyogenes, and anti-bodies against R28 confer protective immunity against S. pyogenes disease in mice (44). Rabbit antiserum to a GBS purified alpha-like protein, later named Alp2, conferred protection against an alpha-like protein-expressing GBS type V strain in a neonatal mouse model of GBS disease (26). The ability of an alpha-like protein to elicit antibody-mediated protection in experimental animals provided the rationale to investigate whether Alp3 could be used as an immunogen and/or as a protein carrier for CPS antigens in the development of GBS vaccines.
We have recently demonstrated that a recombinant BCP and a recombinant BCP modified to eliminate its human IgAbinding site (rBCP ⌬IgA ) were not only effective immunogens but also strong carriers for the type III CPS, creating vaccines with greater serotype coverage than a conjugated vaccine prepared with a non-GBS protein carrier (47). In this report, we describe the expression and purification of a recombinant Alp3 (rAlp3) and demonstrate its immunogenicity in mice and its ability to be a good carrier protein for GBS type III CPS. Type III CPS conjugate vaccines prepared with rAlp3 (III-rAlp3) elicited IgG in adult female mice, which provided protection against lethal GBS challenges to their newborn pups. In addition, rabbit serum to rAlp3 opsonized GBS for in vitro killing by human peripheral blood leukocytes, further confirming the potential of this protein as a viable vaccine antigen.
Protein expression and purification. To prepare each recombinant protein, a 1-liter culture of each E. coli transformant was incubated at 37°C in Luria-Bertani medium (Difco, Sparks, MD) supplemented with 100 g/ml of ampicillin (Sigma, St. Louis, MO) with shaking at 200 rpm to an optical density at 600 nm of 0.6. Protein expression was induced with 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) (Invitrogen, Carlsbad, CA) for 3 h at 37°C. Histidine-tagged recombinant proteins were purified by His-bind Fractogel chromatography (Novagen) as described previously (47).
Generation of GBS conjugate vaccines. GBS type III CPS was purified from GBS strain M781 as previously described (46). To create aldehydes on the sialic acid moieties of type III CPS, 15.2 mg of pure type III CPS was combined with 4 M freshly prepared sodium m-periodate (Sigma) in 1 ml of distilled water. The resulting oxidized CPS was purified, and the degree of sialic acid oxidation was determined by pulsed amperometric detection as described previously (47).
A III-rAlp3 conjugate was created by combining 5 mg of oxidized type III CPS with 5 mg of rAlp3 in 1 ml of phosphate-buffered saline (PBS) (Gibco, Grand Island, NY). A tricomponent conjugate vaccine consisting of III-rAlp3-rBCP ⌬IgA was generated by combining 5 mg of oxidized type III CPS with 2.5 mg of rAlp3 and 2.5 mg of rBCP ⌬IgA in 1 ml PBS. Sodium cyanoborohydride (Matreya, Pleasant Gap, PA) was added to each mixture to promote the covalent attachment of the protein(s) to the oxidized CPS. The conjugation procedure, column purification of the conjugates, and biochemical analyses of the vaccines were performed as described previously (47).
ELISAs. Specific IgG levels elicited by each vaccine component (type III CPS, rAlp3, and rBCP ⌬IgA ) were measured by enzyme-linked immunosorbent assay (ELISA) as described previously (47). Briefly, microtiter plates were coated with the following concentration of primary antigen: 0.2 g/ml rAlp3, 0.2 g/ml rBCP ⌬IgA , or 1.0 g/ml type III CPS-human serum albumin in 0.1 M sodium carbonate buffer (pH 9.8). Mouse sera from each vaccine group were pooled and serially twofold diluted across wells of the coated microtiter plates. Phosphataselabeled goat anti-mouse IgG (diluted 1:3,000) was used as the secondary antibody. ELISA titers were reported as the reciprocal of the highest dilution corresponding to an optical density at 405 nm of Ն0.2 after 30 min of development at 37°C with 1 mg/ml phosphatase substrate (Sigma) dissolved in substrate buffer (1 M Tris, 0.3 mM MgCl 2 [pH 9.8]).
Antigenicity of coupled and uncoupled rAlp3. Competitive inhibition of the binding of mouse antiserum (diluted 1:10,000) to rAlp3-coated microtiter plates was assessed by using rAlp3, II-rAlp3, and III-rAlp3-rBCP ⌬IgA as inhibitors at concentrations ranging from 1 to 1,000 ng/ml; competitive inhibition in rBCP ⌬IgA -coated microtiter plates was assessed by using rBCP ⌬IgA and III-rAlp3-rBCP ⌬IgA as inhibitors. Percent inhibition was calculated as [(absorbance at 405 nm without inhibitor Ϫ absorbance at 405 nm with inhibitor)/absorbance at 405 nm without inhibitor] ϫ 100; the percentage was plotted against the log 10 of the inhibitor concentration to generate inhibition curves.
Rabbit antiserum to rAlp3. A New Zealand White rabbit was given three 200-g doses of rAlp3 mixed with incomplete Freund's adjuvant at 21-day intervals. The rAlp3-specific IgG titer was determined with the use of the rAlp3 ELISA described above and goat anti-rabbit IgG-alkaline phosphatase conjugate as the secondary antibody.
Whole-cell GBS Western blot analysis. A 1-ml culture of GBS grown overnight was pelleted by centrifugation, washed with 1 ml PBS (Gibco, Grand Island, NY), and resuspended in 200 l PBS. A 10-l aliquot of resuspended cells was mixed with 15 l loading buffer (NuPAGE LDS sample buffer and NuPAGE sample reducing agent; Invitrogen) and heated at 70°C for 10 min, and proteins were separated on NuPAGE Novex Bis-Tris gels (Invitrogen) according to the manufacturer's protocol. Western blot analysis of separated GBS proteins was performed with mouse anti-rAlp3 serum followed by phosphatase-labeled goat anti-mouse IgG.
Functionality of rAlp3 antiserum. The functionality of rabbit antiserum to rAlp3 was evaluated using an in vitro opsonophagocytosis assay as described previously (5). Briefly, 2.5 l of rabbit serum collected on day 57 following vaccination with rAlp3 was diluted in 47.5 l of minimum essential medium (Invitrogen) to yield a final assay dilution of 1:100 when combined with 25 l of baby rabbit complement (Cedarlane, Burlington, NC), 150 l of human peripheral blood leukocytes, and 25 l of GBS. Pooled rabbit antiserum to GBS type V CPS-CRM 197 was used as a positive control. Viable GBS cells were enumerated by quantitative plate counts at time zero and after a 60-min incubation at 37°C with mixing. The difference in the number of GBS CFU was calculated and expressed as the average of two determinations per GBS strain.
Statistics. Fisher's exact test was used to compare the efficacies of GBS vaccines (Instat, version 3.0a; Graphpad Software, San Diego, CA) at a significance level of 0.05.

Composition of the conjugate vaccines.
Approximately 34% of sialic acid residues on the type III CPS were oxidized by sodium periodate. Oxidized type III CPS was conjugated to either rAlp3 alone or both rAlp3 and rBCP ⌬IgA . Purified III-rAlp3 conjugate was composed of 32% (wt/wt) protein and 63% (wt/wt) carbohydrate, while the III-rAlp3-rBCP ⌬IgA conjugate was composed of 32% (wt/wt) protein and 59% carbohydrate. The presence of rAlp3 in purified conjugate vaccines was confirmed by Western blot analysis (Fig. 1).
Antigenicity of coupled recombinant GBS proteins. Following covalent coupling to oxidized type III CPS, the antigenicity of rAlp3 remained intact, as determined by inhibition ELISA using uncoupled rAlp3 and mouse anti-III-rAlp3-rBCP ⌬IgA serum ( Fig. 2A). Similarly, the antigenicity of rBCP ⌬IgA was also retained following coupling to type III CPS in the presence of rAlp3 (Fig. 2B). These data imply that the conjugation procedure did not destroy antigenic epitopes of rAlp3 when used alone or in combination with rBCP ⌬IgA .
Immunogenicity of vaccines in mice. The immunogenicity of uncoupled rAlp3 and two conjugate vaccines containing rAlp3 (III-rAlp3 and III-rAlp3-rBCP ⌬IgA ) in mice was evaluated. An uncoupled mixture (IIIϩrAlp3ϩrBCP ⌬IgA ) served as a control vaccine. Conjugate vaccines containing rAlp3 either as a single carrier protein or as one of two carrier proteins induced high levels of type III CPS-specific IgG in outbred mice following a single dose (Table 1). A second dose of conjugate vaccines at day 21 served to improve these titers 30-to 60-fold, with no additional increases in titer following the third dose. As expected, uncoupled rAlp3, either alone or admixed with rBCP ⌬IgA , did not induce type III CPS-specific antibody.
High-titer IgG specific to rBCP ⌬IgA was also elicited in mice that received vaccines containing this protein either coupled to type III CPS or admixed with type III CPS and rAlp3 (Table 2). Similarly, rAlp3 was highly immunogenic in mice following a single dose, with subsequent increases in specific IgG titers after each of two booster doses ( Table 3). The immunogenicity of rAlp3 remained high whether coupled to type III CPS alone or in combination with type III CPS and rBCP ⌬IgA .
Efficacy of vaccines in neonatal mice. Female mice that received GBS vaccines or saline were mated, and their litters were challenged with viable GBS. Most (94%) pups born to III-rAlp3-vaccinated dams and all 20 pups born to dams that received the combination III-rAlp3-BCP ⌬IgA vaccine survived   (Table 4). Interestingly, 44% survival and 43% survival were observed among pups born to dams that received uncoupled rAlp3 (rAlp3 or IIIϩrAlp3ϩrBCP ⌬IgA , respectively) and challenged with strain M781, a rate significantly greater (P Ͻ 0.001) than those for pups born to dams that received saline. The tricomponent III-rAlp3-BCP ⌬IgA vaccine was immunogenic in female mice, and antibody to this vaccine provided protection to 69% (25 survived of 36 challenged) of their pups against challenge with GBS type Ia strain A909 bearing BCP (Table 4). Significant (P Ͻ 0.005) reduction in survival was observed among pups born to dams that received an uncoupled tricomponent mixture (IIIϩrAlp3ϩBCP ⌬IgA ).
None of the vaccines tested were highly efficacious against challenge with GBS strain CJB111, with the highest survival rate of 39% afforded by III-rAlp3 (Table 4). This surprising finding led us to analyze whole GBS cell extracts for the expression of Alp3 among these GBS strains. Despite possessing the alp3 gene, GBS strain CJB111 did not express Alp3 as revealed by a lack of whole-cell reactivity with rAlp3 antisera (Fig. 3). The lack of Alp3 expression provided a possible explanation for the poor efficacy of the rAlp3-containing vaccines against challenge with CJB111, a type V strain that also does not express the Rib protein or BCP.
Another group of female mice were vaccinated as described above, and their pups were challenged with GBS type V strain H1A00001 and type VIII strain JM9130013, both of which possess the alp3 gene and express Alp3 (Fig. 3). While pooled sera from dams of each of the four vaccine groups (rAlp3, III-rAlp3, III-rAlp3-BCP ⌬IgA , and IIIϩrAlp3ϩBCP ⌬IgA ) had high rAlp3-specific IgG titers (Ͼ1,000,000), the survival among litters was poor: from 0% to 3% for H1A00001 challenge and from 0% to 26% with JM9130013 challenge. These sera were also not functionally active against these GBS strains in the opsonophagocytosis assay (data not shown).
Rabbit antiserum to rAlp3. To explore the functional activity of rAlp3 antiserum further, a rabbit was vaccinated with rAlp3 admixed with incomplete Freund's adjuvant. The titer of rAlp3-specific IgG rose from Ͻ100 before vaccination to 656,100 on days 42 and 50 and to 1,968,300 on day 57.
Opsonophagocytosis of GBS strains by rabbit serum raised to rAlp3. The in vitro opsonophagocytosis assay was used to further explore the number of GBS serotypes that would be susceptible to the antibody-mediated killing of GBS in vivo. GBS type V strain H1A00001, which possesses alp3 and expresses Alp3 (Fig. 3), was opsonized for killing by human peripheral blood leukocytes in the presence of complement (Table 5). GBS type VIII strain JM9130013, which bears Alp3, was also killed in this assay, in sharp contrast to the lack of opsonization and killing of type V strain CJB111 and type Ia strain A909, both which lack Alp3 ( Fig. 3 and Table 5). Interestingly, cells of GBS type III strain M781, which lacks alp3 but possesses the anti-rAlp3 cross-reactive Rib protein (Fig. 3), were opsonized and killed in this assay ( Table 5).
The latter findings led us to explore if rAlp3 antiserum would be functionally active against other GBS type III strains. Rabbit antiserum to rAlp3 promoted the killing of six (86%) of seven type III strains isolated from newborns with invasive GBS disease by Ն0.6 log 10 CFU (Fig. 4). Western blot analysis of whole-cell proteins from these strains showed that all but strain KMP114, which was not opsonized and killed, expressed cross-reactive proteins, presumably Rib (Fig. 4); the nonopsonized strain possessed alp3 but did not express Alp3. Rabbit serum to rAlp3 (diluted 1:10,000) reacted with colony blots of all GBS strains used in this study, with the exception of CJB111, A909, and KMP114.

DISCUSSION
Conjugate vaccines against pathogens of multiple serotypes are optimized when all components induce functional antibody, resulting in broadened coverage. The search for a protein that would serve as a serotype-independent vaccine against GBS infections has been ongoing since Lancefield and colleagues reported protection of mice against GBS infection by antibody specific to both polysaccharide and protein antigens (27). Several GBS surface proteins, including ACP, BCP, C5a peptidase, Rib, and Sip, have been tested as independent vaccines or as carrier proteins for GBS CPSs (31). As standalone vaccines, GBS proteins have not progressed beyond animal testing. Preclinical studies with Sip demonstrated that this antigen would be an ideal vaccine, as it was conserved among serotypes, was highly immunogenic, and protected mice against challenge with most GBS serotypes (9,38). Unfortunately, however, sera from most women and newborns tested including newborns with invasive GBS disease had naturally occurring antibody to Sip (37), a result that raised concerns about the use of this protein as a vaccine. The presence of naturally occurring human maternal antibody to GBS BCP also failed to correlate with neonatal protection (23) despite promising studies of this protein as a vaccine in animals (33).  Recently, a combination of Sip and three newly described GBS pilus proteins has demonstrated efficacy against several GBS serotypes in mice (36), results that provided a renewed interest in a GBS protein-based vaccine. Because of the preclinical and clinical successes with GBS CPS-protein conjugate vaccines (20), we chose to examine the role of the Alp3 protein not as a stand-alone vaccine per se but as a carrier protein for CPSs. As it was shown to be a strong immunogen in animals, rAlp3, like other GBS proteins (31), was a good carrier for type III CPS, as revealed by efficacy in mice. Surprisingly, in this animal model, uncoupled rAlp3 conferred partial protection against a GBS type III strain lacking Alp3. GBS Alp3 possessed at least two antigenic determinants: Alp2/Alp3 and Alp3/Rib common sites (34). Repeat regions of Alp3 and of Rib match by 99% and are likely sites for the Alp3/Rib serological cross-reactivity (24). This cross-reactivity was also reflected in the ability of rAlp3 rabbit serum to opsonize Rib protein-expressing type III strain M781 for killing in vitro, a result that explains the Ͼ40% protection against challenge with strain M781 in newborn mouse pups born to dams that received rAlp3. Indeed, the degree of opsonically mediated killing of GBS strains with this antiserum correlated with the expression of Alp3-cross-reactive proteins, as measured among a panel of invasive GBS isolates.
Active immunization of mice with rAlp3 or rAlp3-containing vaccines did not provide protection against challenge with Alp3-expressing GBS strains H1A00001 and JM9130013, nor did antisera to these vaccines mediate opsonic killing. However, high-titered rabbit rAlp3 antisera mediated the opsonization and killing of these strains, a finding that raises the possibility that the Alp3/Rib common site may, under certain conditions, be partially concealed by other surface antigens such as the CPS.
As both Alp3 and Rib proteins occur with high frequency in GBS, usually in strains of different capsular antigen types (8,22), the Alp3/Rib common site will be expressed more often antiserum. The alp3 gene was PCR amplified with use of specific primers (42). As expected, strain M781, which contains rib but not alp3, expressed Rib that cross-reacted with rAlp3 antiserum. Surprisingly, strain CJB111 did not express Alp3 despite possessing the gene for this protein, and as expected, strain A909 neither possessed alp3 nor expressed the protein.  on February 12, 2021 by guest http://cvi.asm.org/ than either of the two proteins alone. Indeed, expression of the Alp3/Rib common site occurs frequently in GBS, notably in pathogenic serotype III, V, and VIII strains (22,35), and in group A streptococci that express the Alp3-like protein R28 (43,44), suggesting that the inclusion of the Alp3/Rib common region in a vaccine might provide at least partial protection against a broader range of streptococci. The ability of rAlp3 antisera to opsonize invasive GBS type III strains bearing the cross-reactive proteins, presumably Rib, is of particular importance given the correlation of low maternal levels of Rib antibody with neonatal GBS disease (28). BCP, encoded by the bac gene, is found on nearly all strains of GBS serotype Ib as well as on some isolates of types Ia, II, and V (31), and approximately 23% of GBS strains contained the bac gene according to bac-specific PCR analysis (21). Our recently developed rBCP ⌬IgA demonstrated a higher degree of efficacy against a BCP-positive GBS type Ia strain when coupled to type III CPS and rAlp3 than as a component of an uncoupled mixture, despite evoking similar levels of maternal IgG. Mechanisms for this enhanced efficacy are currently unknown but may include an effect of size, given the generation of large polymers due to conjugation or the exposure of protective epitopes by conjugation.
In summary, our data suggest that rAlp3 is a good carrier for GBS CPS, and antiserum to this protein is functionally active against GBS strains bearing Alp3 or the cross-reactive Rib protein. In theory, a conjugate vaccine containing type Ia CPS, rBCP ⌬IgA , and rAlp3 could provide coverage against multiple GBS serotypes, serotypes Ia (from the CPS), Ib (from BCP), III (from the cross-reactivity of rAlp3 with Rib), and V and VIII (from rAlp3) or most serotypes causing early-and lateonset neonatal GBS disease in the United States (14,19).

ACKNOWLEDGMENTS
We thank Francesco Berti, Novartis Vaccines and Diagnostics, Siena, Italy, for measuring the degree of CPS oxidation and Karen Puopolo, Channing Laboratory, for providing the six clinical GBS isolates.
This work was supported by PHS grants U01AI-060603, R01AI 053191 (L.C.P.), and R01AI38424 (L.C.M.) from the National Institute of Allergy and Infectious Diseases.
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