Skip to main content
NCBI home page
The Journal of Infectious Diseases logoLink to The Journal of Infectious Diseases
. 2022 Apr 16;226(1):177–187. doi: 10.1093/infdis/jiac148

A Recombinant Alpha-Like Protein Subunit Vaccine (GBS-NN) Provides Protection in Murine Models of Group B Streptococcus Infection 

Alyssa Brokaw 1,2, Shayla Nguyen 3, Phoenicia Quach 4,✉, Austyn Orvis 5, Anna Furuta 6,7, Bengt Johansson-Lindbom 8,9, Per B Fischer 10, Lakshmi Rajagopal 11,12,13,✉
PMCID: PMC9890916  PMID: 35429401

Abstract

Background

Group B Streptococcus (GBS) transmission during pregnancy causes preterm labor, stillbirths, fetal injury, or neonatal infections. Rates of adult infections are also rising. The GBS-NN vaccine, engineered by fusing N-terminal domains of GBS Alpha C and Rib proteins, is safe in healthy, nonpregnant women, but further assessment is needed for use during pregnancy. Here, we tested GBS-NN vaccine efficacy using mouse models that recapitulate human GBS infection outcomes.

Methods

Following administration of GBS-NN vaccine or adjuvant, antibody profiles were compared by ELISA. Vaccine efficacy was examined by comparing infection outcomes in GBS-NN vaccinated versus adjuvant controls during systemic and pregnancy-associated infections, and during intranasal infection of neonatal mice following maternal vaccination.

Results

Vaccinated mice had higher GBS-NN–specific IgG titers versus controls. These antibodies bound alpha C and Rib on GBS clinical isolates. Fewer GBS were recovered from systemically challenged vaccinated mice versus controls. Although vaccination did not eliminate GBS during ascending infection in pregnancy, vaccinated dams experienced fewer in utero fetal deaths. Additionally, maternal vaccination prolonged neonatal survival following intranasal GBS challenge.

Conclusions

These findings demonstrate GBS-NN vaccine efficacy in murine systemic and perinatal GBS infections and suggest that maternal vaccination facilitates the transfer of protective antibodies to neonates.

Keywords: group B Streptococcus, Streptococcus agalactiae, vaccine, neonate, pregnancy, maternal vaccination, surface proteins, alpha-like proteins, Rib, intranasal infection

Immunization of mice with the surface protein fusion vaccine known as GBS-NN raises antibodies that provide protection during systemic and pregnancy-associated group B streptococcal infections and promotes neonatal survival during intranasal group B streptococcal infection following maternal vaccination.

The gram-positive bacterium group B Streptococcus (GBS, Streptococcus agalactiae) is a leading cause of fetal and neonatal morbidity and mortality [1]. Systemic GBS infections in nonpregnant adults are also rising in frequency [2]. GBS commensally resides in the rectovaginal tract in approximately 25% of women [1, 3]. During pregnancy, ascending GBS infection leads to preterm labor, stillbirth, and fetal injury [1, 4]. Alternatively, aspiration of GBS-infected fluids during labor and delivery leads to neonatal pneumonia, sepsis, and meningitis [5, 6]. Perinatal GBS colonization is the primary risk factor for adverse pregnancy outcomes and neonatal invasive disease [4], and thus strategies that inhibit GBS transmission are needed.

Intrapartum antibiotic prophylaxis (IAP) lowers the risk of GBS transmission when administered to GBS-colonized mothers during labor and delivery, preventing cases of early onset disease within the first week of life. However, varied screening and IAP policies contribute to the disproportionate burden of GBS [7]. Further, IAP is administered too late to target in utero transmission and too early to target postnatal transmission. Thus, IAP cannot prevent GBS-associated preterm labor, stillbirth, and fetal injury, or late-onset disease (occurring after 7 days of age). A GBS vaccine would lead to larger reductions in disease prevalence [1].

Currently, there is no licensed vaccine to prevent GBS colonization or infection [8]. Most vaccine efforts have focused on serotype-variable capsular polysaccharide (CPS), although multivalent formulations have variable efficacy in preclinical models [9–17] and human trials [18–26]. In humans, type III CPS vaccination failed to eradicate GBS from precolonized women, but reduced rates of new acquisition by about 40% for serotype III GBS. There was no effect on nonvaccine serotypes [25]. While maternal vaccination permits CPS-specific antibody transfer to the infant [20–22], only 50%–80% of vaccinated mothers transferred protective levels [18, 20, 21, 27]. A major challenge is that an optimal formulation would incorporate all 10 CPS to provide full coverage against all encapsulated GBS strains. GBS serotype prevalence varies geographically [2, 3, 5, 27, 28], so prevalent CPS variants may suffice but may not be universally applicable. Additionally, suboptimal vaccines may select for strains expressing nonvaccine CPS [28–30]. Conserved GBS surface proteins provide alternative strategies, which may generate improved antibody potency against clinically relevant GBS strains.

MinervaX’s GBS-NN vaccine incorporates structural components of GBS alpha-like proteins (Alps). This protein family includes 6 allelic and chimeric variants, Alpha C (αC), Rib, and Alp1-4, which are associated with particular GBS serotypes and clonal complexes [31]. Adjacent to a C-terminal cell wall-anchoring motif, Alps contain tandem repeats that vary in number and sequence. The N-terminus contains domains that bind to epithelial cells [32–35] and αC N-terminus deletion attenuated virulence in neonatal mice [36], emphasizing this domain’s role in GBS pathogenesis.

In humans, low naturally occurring αC- or Rib-specific titers in maternal and neonatal serum correlated with neonatal disease progression [37]. Analysis of individual Alp domains revealed that repeat-specific antibodies are immunodominant [37, 38] and that tandem repeat number is inversely related to protection [39]. Alp N-termini are more immunogenic in the absence of tandem repeats, and N-terminal vaccination extended murine survival following lethal challenge [38]. Consequently, MinervaX’s GBS-NN vaccine was engineered to contain fused N-termini from αC and Rib (Figure 1) [38]. This prototype vaccine has completed phase 1 trials in nonpregnant women (NCT02459262, ClinicalTrials.gov), and was safe, well-tolerated, and highly immunogenic [40]. While trials to evaluate GBS-NN safety during pregnancy are underway, efficacy has not been tested.

Figure 1.

Figure 1.

Open in a new tab

Schematic of GBS-NN vaccine antigen composition. The N-terminal (N) domains from the prototype alpha-like proteins (Alps), Alpha C and Rib, were fused to create the GBS-NN peptide antigen. This antigen does not contain tandem repeat (R) or C-terminal (C) domains from either protein.

We sought to examine GBS-NN vaccine efficacy using GBS mouse models that emulate human disease outcomes. We observed robust production of vaccine-specific antibodies that recognized native forms of these antigens on clinical isolates. These antibodies were associated with reduced GBS recovery from disseminated sites following systemic infection. While GBS-NN vaccination did not confer differences in GBS colony-forming units (CFUs) in maternal and fetal tissues following vaginal challenge, pregnant immunized dams had reduced rates of intrauterine fetal death (IUFD), and maternally vaccinated dams delivered neonates that experienced improved survival following intranasal GBS challenge. These findings demonstrate that, in mice, GBS-NN vaccination elicits the production of antibodies that can protect against multiple GBS serotypes and disease outcomes. Combined with human clinical trial data [40], these findings support the need for further assessment of GBS-NN efficacy during pregnancy in human clinical trials.

METHODS

Ethics Statement

Animal experiments were approved by Seattle Children’s Research Institute’s Institutional Animal Care and Use Committee (protocol IACUC00036), and performed per the National Institutes of Health Guide for the Care and Use of Laboratory Animals, 8th ed.

Bacterial Strains and Growth Conditions

The GBS clinical isolates A909 [41] and BM110 [42] were used for this study. GBS was cultured on tryptic soy broth agar or broth (Difco Laboratories) at 37°C with 5% CO2. Overnight cultures were diluted 1:10 and grown to an optical density at 600 nm (OD600) of 0.3 for infections, or 1.0 for enzyme-linked immunosorbent assay (ELISA).

GBS-NN Vaccine Production

The GBS-NN antigen, composed through fusion of A909 αC and BM110 Rib N-termini, was generated as described [38] and stored in phosphate-buffered saline (PBS; pH 7.2) at 1.75 mg/mL. On immunization day, fresh formulations of vaccine (10 μg GBS-NN and 100 μg aluminum hydroxide [Alhydrogel; InvivoGen] in PBS) and adjuvant (100 μg aluminum hydroxide in PBS) were prepared and stored on ice until administeration.

Murine Vaccination

Six- to 8-week-old male and female wildtype (WT) C57BL/6J mice were used for vaccination studies. On day 0, 50 μL of formulated vaccine or adjuvant were injected intramuscularly. Boosters were provided on days 14 and 28.

Mouse Serum and Vaginal Lavage Collection

To assess antibody responses, retro-orbital blood was collected on days 0, 14, and 28 prior to administration of vaccine or adjuvant. Unchallenged mice were euthanized on day 48 and cardiac blood was collected. Pooled or individual blood samples were transferred to serum separator tubes (gold tubes; BD), centrifuged (6000xg, 90 seconds), and sera were stored at −80°C.

Vaginal lavage fluid was obtained by performing PBS washes with a micropipette tip (P10; Rainin). Lavage fluid was clarified by centrifugation (6000xg, 2 minutes), and samples were stored at −80°C.

ELISA and End Point Titer Calculation

High-binding, flat-bottom 96-well polystyrene plates (Costar) were incubated with GBS-NN (0.5 μg/mL in PBS; room temperature, 2 hours). Wells were washed with buffer (PBS + 0.05% Tween-20) and blocked with PBS + 1% bovine serum albumin (BSA; 37°C, 1 hour). Mouse serum was serially diluted 1:4 (1:50 to 1:51 200) in antibody diluent (PBS + 1% BSA + 0.05% Tween-20), and incubated (4°C, overnight). For vaginal lavage, serial 1:2 dilutions (1:20 to 1:640) were used. Wells were washed and biotinylated goat anti-mouse IgG (H + L) secondary antibody (1:40 000; Southern Biotech) was added (37°C, 1 hour). Wells were incubated with streptavidin-horseradish peroxidase (1:200, room temperature, 45 minutes; R&D Systems), washed, and tetramethylbenzidine peroxidase substrate and solution B were added (1:1; KPL). Sulfuric acid stop solution was added (approximately 333 mM final concentration) upon sufficient color change. Absorbances were obtained at 450 nm using a plate reader (SpectraMax i3x; Molecular Devices), and dilution curves were generated. For each plate, a cutoff curve was made by calculating the average absorbances of the preimmune (T0 or naive) serum plus 3 × the standard deviation of the controls at each dilution. The end point titer is the last dilution where a sample’s absorbance is greater than that of the cutoff curve.

To quantify binding of GBS-NN–specific antibodies to whole GBS, methods were adapted for whole GBS ELISA [43]. GBS (A909, BM110) at 5 × 108 CFU/mL in PBS was coated at 4°C overnight. The next day, wells were washed in wash buffer containing 0.02% sodium azide and remaining steps were performed as described above.

Murine Systemic Challenge Model

Vaccinated or control male and female mice were challenged intraperitoneally as described [44]. Mice were infected with approximately 1 × 108 CFU GBS (strain A909) on day 35 (relative to the vaccine schedule). At 48 hours postinfection or earlier (if exhibiting morbidity), mice were euthanized, and blood, peritoneal fluid, lungs, spleen, and brain were collected. Organs were homogenized. All samples were serially diluted and plated to enumerate GBS CFU.

Murine Pregnancy-Associated Vaginal Challenge Model

Vaccinated or control mice were challenged intravaginally during pregnancy as described [45]. The day following their final boost, female mice underwent timed pairing with isogenic males for 48 hours. Mice were monitored for signs of pregnancy including weight gain and palpation to detect pups. Pregnant mice were infected intravaginally on day 45 (relative to the vaccine schedule) with 1 × 108 CFU GBS (strain A909) using a micropipette tip (P10; Rainin). Infected mice were monitored twice daily for signs of preterm labor (vaginal bleeding or pups in cage). At 72 hours post-infection, dams were euthanized and pup viability was noted. Maternal blood, lower genital tract, uterus, and spleen were collected. The 2 left and right proximal and distal pups and placentas were also collected. Organs were homogenized. All samples were serially diluted and plated to enumerate GBS CFU.

Maternal Vaccination and Neonatal Intranasal Challenge Model

Vaccinated and control pregnant mice were monitored for litters between days 48 and 51 (relative to the vaccine schedule). Within 24 hours of birth (postnatal day 0), half of each litter was randomized into a bleed group to assess antibody transfer. Following decapitation, blood was collected into serum separator tubes, processed as described, and pooled by litter. The remaining neonates were infected intranasally with 5 × 106 CFU GBS (strain A909, 1 μL per nare). Infected pups were monitored for survival twice daily. Survivors were euthanized at postnatal day 7 and maternal blood was collected by cardiac bleed. Time-matched naive litters were bled for ELISA control serum.

Statistics

Statistical tests used are noted in the corresponding figure legend and a P value of <.05 was considered significant. Tests were performed using GraphPad Prism, version 8.4.3.

RESULTS

GBS-NN Immunization Induces a Strong Vaccine-Specific Antibody Response in Mice

To interrogate GBS-NN immunogenicity in mice, male and female C57BL/6J mice were administered 3 doses of GBS-NN vaccine (alum + GBS-NN) or adjuvant (alum + PBS) every 2 weeks. Blood was collected prior to each dose and at the study’s end point (day 48) (Figure 2A). Serum titers were compared between vaccinated and control mice who received adjuvant. We expected that vaccination would elicit vaccine-specific antibodies, which would increase in abundance over time. Hence, we performed ELISA to quantify GBS-NN–specific immunoglobulin G (IgG) titers from serum collected throughout the course of vaccination. As expected, GBS-NN–vaccinated mice produced GBS-NN–specific IgG. As represented by area under the ELISA curves, abundance of these antibodies significantly increased in the vaccinated sera over time, while no changes were observed in control sera (Figure 2B). Overall, immunized mice exhibited a substantial (580-fold) increase in GBS-NN–specific IgG titers versus controls (Figure 2C). Together, these results indicate that the GBS-NN vaccine is highly immunogenic in mice.

Figure 2.

Figure 2.

Open in a new tab

GBS-NN immunization elicits a strong vaccine-specific antibody response. A, Timeline for IM administration of full GBS-NN vaccine regimen, comprising of an initial prime followed by 2 boosts. Mice were stratified into alum + GBS-NN or an alum-only control group. Asterisks indicate serum collection. B, Serial serum samples isolated from alum control (left) or alum + GBS-NN (right) mice were analyzed by ELISA to measure GBS-NN–specific IgG. Graphs depict AUC calculated from dilution curves for each sample. Data represent the mean ± SD. Statistical differences were determined by Friedman test with Dunn multiple comparisons post hoc test (sample sizes: n = 8 alum, n = 8 alum + GBS-NN). C, An ELISA dilution curve for each mouse serum sample at T28 was used to quantify an end point titer of GBS-NN–specific IgG. Data represent the geometric mean ± SD. The dashed line denotes the limit of detection. Statistical differences were determined by Mann-Whitney test (sample sizes: n = 25 alum, n = 27 alum + GBS-NN). D, T28 serum samples isolated from alum control or alum + GBS-NN mice were analyzed by ELISA following adsorption to whole GBS (strains A909 and BM110) to quantify vaccine-specific IgG that bind native αC or Rib. Figure shows ELISA end point titers and data represent geometric mean ± SD. The dashed line denotes the limit of detection. Statistical differences were determined by Mann-Whitney test (sample sizes: A909 n = 17 alum, n = 18 alum + GBS-NN; BM110 n = 13 alum, n = 12 alum + GBS-NN). ns P > .05, * P < .05, *** P < .001, **** P < .0001. Abbreviations: AUC, area under the curve; ELISA, enzyme-linked immunosorbent assay; GBS, group B Streptococcus; IgG, immunoglobulin G; IM, intramuscular; ns, not significant.

GBS-NN–Specific Antibodies Bind to GBS Strains of Multiple CPS Serotypes

A major benefit of a GBS-NN vaccine is its potential for broader coverage of GBS clinical isolates. GBS-NN–specific antibodies should bind both αC- and Rib-expressing GBS, rendering protection against clinically relevant serotypes. We assessed reactivity of GBS-NN–specific antibodies against αC- and Rib-expressing strains representing serotype Ia (strain A909) and serotype III (strain BM110) by performing whole GBS ELISA. Serum from GBS-NN vaccinated mice bound to GBS A909 and BM110 with modest 1.5-fold difference between strains. In contrast, limited binding was observed with adjuvant control sera (Figure 2D). These data suggest that GBS-NN vaccination may protect against multiple serotypes expressing either αC or Rib.

GBS-NN Immunization Reduces Bacterial Burden During GBS Systemic Challenge

The studies above indicate that GBS-NN elicited a strong vaccine-specific antibody titer in our mice, confirming previous observations [38]. We next evaluated how GBS-NN immunization affects GBS dissemination in adult mice. Male and female C57BL/6J mice (n = 16–17/group) were vaccinated as described above, then infected intraperitoneally with 1 × 108 CFU of A909 GBS a week later. Mice were euthanized 48 hours postinfection, and blood, peritoneal fluid, lungs, spleen, and brain were collected and processed for CFU enumeration (Figure 3A). Following GBS challenge, vaccinated mice exhibited significantly reduced bacterial burden in the spleen, lungs, and brain versus controls, with lower CFU (albeit not significant) in the peritoneal fluid and blood (P = .2842 and .1877). Some mice in the control group were moribund or dead at necropsy (n = 4), whereas all GBS-NN vaccinated mice survived, exhibiting no signs of morbidity or mortality (Figure 3B). To correlate vaccine-specific antibodies with protection against systemic infection, we compared GBS CFU versus serum IgG titer from the same mouse. Association of these variables was assessed by Spearman correlation. Having a higher GBS-NN–specific antibody titer was moderately and significantly associated with having less GBS CFU in the spleen (P = .0425, Spearman coefficient = −0.3792), and this relationship trended towards significance in the lungs and brain (P = .0926 and .1004; Supplementary Figure 1). Thus, although sterilizing immunity (GBS clearance) did not occur, GBS-NN immunization was associated with low bacterial burden; thus vaccination promoted protection against systemic GBS. We did not perform a survival study following vaccination, as this was reported previously [38].

Figure 3.

Figure 3.

Open in a new tab

GBS-NN immunization diminishes GBS systemic infection. A, Timeline for systemic challenge of GBS following administration of GBS-NN vaccine regimen. Asterisks indicate serum collection. B, One week after the final boost, mice were systemically challenged (IP) with 108 CFU of GBS (strain A909). Bacterial burden was evaluated in blood, peritoneal fluids, lungs, spleen, and brain at 48 hours after GBS infection or earlier if mice exhibited morbidity. Data are shown as medians with circles representing values from individual mice. The Mann-Whitney test was used for comparison between groups (sample sizes: n = 17 alum, n = 16 alum + GBS-NN). ns P > .5, * P < .05. Abbreviations: CFU, colony-forming unit; GBS, group B Streptococcus; IP, intraperitoneally; ns, not significant; Pf, peritoneal fluids.

GBS-NN Maternal Immunization Prevents In Utero Fetal Demise

Because GBS-NN vaccination protected our mice against systemic GBS challenge, we hypothesized that it may also protect against ascending infection during pregnancy. To test this, vaccinated or adjuvant control female mice (n = 9/group) were mated for pregnancy. On day 45 (embryonic day 14), pregnant dams were infected intravaginally with 1 × 108 CFU of A909 GBS. Dams were euthanized 72 hours postinfection and maternal tissues (blood, lower genital tract, uterus, and spleen), plus fetal tissues (proximal and distal placenta and pups from the left and right uterine horns) were collected and processed for CFU enumeration (Figure 4A). Surprisingly, we observed no differences in GBS CFU from maternal or fetal tissues between vaccinated and adjuvant control mice (Figure 4B). However, GBS-NN vaccination led to significantly reduced IUFD rates (Figure 4C). Although GBS-NN–specific IgG titers were 15-fold higher in vaginal lavage fluids from vaccinated mice versus controls, they were nearly 100-fold lower than titers in vaccinated mouse serum (Figure 4D and Figure 2C). GBS-NN–specific mucosal IgA were not detected (data not shown). We assessed the relationship between vaccine-specific antibody titer and IUFD outcomes using Spearman correlation as described above, comparing each dam’s IUFD rate versus their matched serum and vaginal lavage titers. Interestingly, reduced IUFD rates were significantly and moderately associated with having higher GBS-NN–specific IgG titers in the serum (P = .0432, Spearman coefficient = −0.4562), but not in vaginal lavage (P = .2676; Supplementary Figure 2). Under the conditions tested, although maternal GBS-NN immunization did not lead to significant CFU reductions in reproductive or fetal tissues, vaccine-specific serum IgG helped prevent fetal demise.

Figure 4.

Figure 4.

Open in a new tab

GBS-NN immunization diminishes fetal demise. A, Timeline for pregnancy-associated vaginal GBS challenge following administration of full GBS-NN vaccine regimen. After their final boost, mice were mated and monitored for pregnancy. On day 14 of pregnancy, mice were intravaginally challenged with 108 CFU of GBS (strain A909). Dams were euthanized 72 hours postinfection and blood, lower genital tract, uterus, spleen, and proximal and distal pups and their placentas were collected, homogenized, and plated for CFU enumeration. Asterisks indicate serum and vaginal lavage collection. B, Bacterial CFU obtained from maternal (left) or fetal (right) tissues. Medians are indicated with circles representing values from individual mice. Statistical differences were determined by Mann-Whitney test (sample sizes: n = 9 alum, n = 9 alum + GBS-NN). C, Upon necropsy, fetal viability was noted to calculate percent IUFD for each dam. Data are shown as mean ± SD and statistical differences were determined by Mann-Whitney test (sample sizes: n = 9 alum, n = 9 alum + GBS-NN). D, An ELISA dilution curve for each T28 mouse vaginal lavage sample was used to quantify an end point titer of GBS-NN–specific IgG. Graph shows geometric mean values ± SD. The dashed line denotes the limit of detection. Statistical differences were determined by Mann-Whitney test (sample sizes: n = 10 alum, n = 10 alum + GBS-NN). ns P > .05, ** P < .01, *** P < .001. Abbreviations: CFU, colony-forming unit; E, embryonic day; ELISA, enzyme-linked immunosorbent assay; GBS, group B Streptococcus; IgG, immunoglobulin G; IVAG, intravaginally; IUFD, intrauterine fetal death; ns, not significant.

Maternal GBS-NN Immunization Results in Vertical Transmission of GBS-NN–Specific Antibodies to Neonatal Mice

To determine if GBS-NN maternal vaccination permits transfer of maternal vaccine-specific antibodies to neonates, vaccinated and adjuvant control female mice were mated for pregnancy as described. Dams were monitored until delivery (days 48–51), and half of each litter was euthanized on postnatal day 0 for serum (pooled by liter) (Figure 5A). GBS-NN–specific antibodies were quantified by ELISA, and significantly higher titers were noted from neonates born to vaccinated versus adjuvant mothers (Figure 5B). Thus, vaccine-specific maternal IgG transfer vertically to neonatal mice following maternal GBS-NN vaccination.

Figure 5.

Figure 5.

Open in a new tab

Maternal GBS-NN immunization confers neonatal protection against intranasal GBS challenge. A, Timeline for neonatal intranasal challenge following maternal GBS-NN immunization. After their final boost, mice were mated and monitored for pregnancy. Upon delivery, neonates from each litter were either euthanized for serum or intranasally challenged with 5 × 106 CFU of GBS (strain A909). Asterisks indicate maternal serum collection. B, GBS-NN–specific serum IgG end point titers from dam and neonate pairs. Data are depicted as geometric means ± SD. The dashed line denotes the limit of detection. Statistical differences were determined by Mann-Whitney test (sample sizes: n = 4 alum, n = 3 alum + GBS-NN). C, Survival of intranasally infected neonates was monitored for a week. Kaplan-Meier curve indicates survival of pups born to naive, alum control, and alum + GBS-NN immunized dams and survival ratios are shown. Statistical differences were determined using Log-rank test (sample sizes: n = 10 naive, n = 9 alum, n = 8 alum + GBS-NN). * P < .05; *** P < .001. Abbreviations: CFU, colony-forming unit; EU, euthanasia; GBS, group B Streptococcus; INAS, intranasally; ns, not significant; P, postnatal day.

Maternal GBS-NN Immunization Confers Neonatal Protection Against Intranasal GBS Challenge

To examine the functional consequence of maternal GBS-NN–specific antibodies, neonatal mice were challenged intranasally with 5 × 106 CFU of A909 GBS on postnatal day 0. Neonates were monitored for survival and euthanized after 1 week (Figure 5A). Neonates born to GBS-NN immunized dams experienced significantly improved survival following intranasal challenge, as 100% of these pups survived versus only 44% of those born to adjuvant controls (Figure 5C). Overall, these data suggest that maternal transfer of GBS-NN–specific antibodies contributes to protection against subsequent GBS neonatal infection.

DISCUSSION

GBS is a significant cause of fetal and neonatal morbidity and mortality [1], and additionally causes invasive infections in healthy, nonpregnant adults, which are increasing in prevalence [2]. Current prophylactic strategies include IAP [7], although this strategy can only restrict transmission occurring during delivery. The development and licensing of a GBS vaccine would prevent GBS transmission by other routes, resulting in greater reductions in disease burden [8].

MinervaX’s protein-based GBS-NN vaccine is promising and may confer broad protection against GBS. The formulation tested in this study contains a fusion peptide derived from αC and Rib N-termini combined with aluminum hydroxide, an adjuvant approved for use during pregnancy [46]. Phase 1 immunogenicity and safety trials exhibit GBS-NN safety in healthy nonpregnant women, eliciting robust antibody production with minimal side effects [40]. Similar trials are underway in pregnant women, but due to the disproportionate global burden of GBS and differences in screening and IAP policy, it will be difficult and expensive to pursue the corresponding efficacy trials [1, 7, 8]. Here, we evaluated GBS-NN vaccine efficacy in mice using 3 relevant GBS infection models that recapitulate common human disease manifestations.

Murine GBS-NN immunization elicited high vaccine-specific serum antibody titers, which increased with additional doses. These findings mirror human clinical trial findings [40]. GBS-NN–specific antibodies generated through vaccination bound both purified GBS-NN and GBS strains representing canonical αC- and Rib-associated serotypes [47]. Detection by GBS-NN–specific antibodies was similar between strains. While these results suggest that GBS-NN–specific antibodies may confer similar levels of protection against these and other GBS isolates, a previous study noted heightened protection against BM110 compared to A909 for mice that were either actively or passively immunized [38].

GBS-NN vaccination reduced the burden of systemic GBS upon intraperitoneal challenge. This model recapitulates dissemination profiles and disease manifestations that occur in neonates and nonpregnant adults, including GBS bloodstream invasion during sepsis, pneumonia, and meningitis. In our systemic model, GBS-NN reduced overall bacterial dissemination but did not fully clear GBS. Despite this lack of clearance, our reduced dissemination is in line with previous observations that GBS-NN vaccination led to significantly extended survival following systemic infection of mice by a lethal GBS dose [38]. In our vaginal challenge model during pregnancy, GBS burdens did not vary between vaccinated and control mice in the maternal reproductive tissues, placentas, and fetuses that were sampled. Despite this, it is exciting that fetal survival was improved for GBS-NN–vaccinated dams, who experienced significantly reduced IUFD rates versus controls. Overall, these findings suggest that the GBS-NN vaccine is partially effective in these models.

The findings in our pregnancy-associated ascending infection model contrast with observations following systemic infection; however, this reflects the disparate GBS-NN–specific antibody responses detected in serum versus vaginal fluid. To determine whether improved vaccine-specific antibody responses are achievable in the murine vaginal tract, and to test whether this may permit reductions in vaginal colonization, vertical transmission, or adverse outcomes, future studies should compare alternative formulations containing other adjuvants, such as CpG which stimulates mucosal immunity [48]. Alternately, GBS-NN–elicited immunity may be too slow to impede murine ascending infection and vertical transmission given the short distance between the vaginal tract and placenta, thus resulting in modest protection. It is possible that GBS-NN immunization may more effectively inhibit ascension and transmission in humans, where trafficking to the uterus and invasion of fetal tissues likely takes longer.

Finally, maternal GBS-NN vaccination permitted maternal vaccine-specific antibody transfer to neonatal mice and enhanced neonatal survival upon GBS intranasal challenge, highlighting a protective role for maternally derived GBS-NN–specific antibodies. The intranasal route used in this study is biologically relevant and recapitulates human transmission through aspiration of GBS-infected fluids during delivery.

Overall, these data demonstrate MinervaX’s GBS-NN vaccine efficacy in 3 distinct murine models that recapitulate varying aspects of human GBS disease. The GBS-NN prototype vaccine contains 2 of 6 Alps variants, but αC and Rib expression is strongly associated with serotypes Ia/Ib/II and II/III, respectively [49, 50], which comprise >50% of maternal colonizations [3] and most cases of neonatal invasive disease [5]. Alps expression also varies by GBS clonal complex (CC). Rib is well-associated with CC17 isolates that cause strikingly high rates of neonatal sepsis and meningitis [50]; αC is common to CC12, among which serotype Ib isolates are multidrug resistant and cause fatal bloodstream infections [50]. That GBS-NN may protect against these hypervirulent strains is promising. However, MinervaX’s final formulation would include a second peptide (GBS-NN2) containing Alp1 (serotype 1a/V/VI and CC1/CC23) and Alp2/3 (III/V and CC1/CC23) N-terminal fusions [49, 50]. While these associations undergo geographical variability, GBS-NN/NN2 is predicted to target strains expressing αC, Rib, Alp1, Alp2, or Alp3. Strains expressing non-vaccine Alp4 and Alps-deficient GBS are rarely observed in geographically diverse surveillance studies [47, 49, 50]. Thus, GBS-NN/NN2 represents a promising alternative to CPS-based vaccines, which require more than 2 vaccine antigens to achieve similar coverage. GBS-NN/NN2 has completed phase 1 assessment (NCT03807245, ClinicalTrials.gov), but future studies should similarly assess efficacy of the full vaccine in mice. This formulation will soon undergo a booster follow-up study (NCT05005247), and phase 2 trials in pregnant women and their babies in Denmark, United Kingdom, South Africa, and Uganda (NCT04596878, NCT05154578). These clinical trials may finally determine whether Alps-based vaccine strategies may facilitate global reductions in GBS disease burden; however, combined with our observations of murine efficacy, the future of this vaccine looks promising.

Supplementary Material

jiac148_Supplementary_Data
Click here for additional data file. (451.9KB, zip)

Contributor Information

Alyssa Brokaw, Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA; Department of Global Health, University of Washington, Seattle, Washington, USA.

Shayla Nguyen, Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.

Phoenicia Quach, Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.

Austyn Orvis, Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.

Anna Furuta, Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA; Department of Global Health, University of Washington, Seattle, Washington, USA.

Bengt Johansson-Lindbom, MinervaX A/S,  Copenhagen, Denmark; Immunology Section, Lund University, Lund, Sweden.

Per B Fischer, MinervaX A/S,  Copenhagen, Denmark.

Lakshmi Rajagopal, Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA; Department of Global Health, University of Washington, Seattle, Washington, USA; Department of Pediatrics, University of Washington, Seattle, Washington, USA.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Author contributions. A. B. and L. R. designed the experiments. A. B. and S. N. performed the experiments with assistance from P. Q., A. O., and A. F. Data analysis was performed by A. B., S. N., and L. R., and A. B. and L. R. wrote the manuscript with input from all authors. Funding for this project was secured by L. R., A. B., B. J.-L., and P. F.

Acknowledgments. We thank Lucas Senatore, Jessica Spaulding, and Timothy Gervasi for technical support, the Seattle Children’s Research Institute’s Office of Animal Care staff for their tireless contribution to husbandry and vivarium maintenance (especially during the COVID-19 pandemic), and Connie Hughes for administrative support. Experimental timelines in all figures were created using BioRender.com.

Financial support. This work was supported by the National Institutes of Health (NIH) (grant numbers R01AI145890, R01AI152268, and R01AI133575 to L. R.); NIH training grants (grant numbers T32 AI007509 to A. B. [PI, Lee Ann Campbell] and T32 AI055396 to A. F. [PI, Ferric Fang]); Seattle Children’s Research Institute (seed funds to L. R.); and funds provided by MinervaX A/S (to P. F. and B. J.-L.).

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

References

  • 1. Seale  AC, Bianchi-Jassir  F, Russell  NJ, et al.  Estimates of the burden of group B streptococcal disease worldwide for pregnant women, stillbirths, and children. Clin Infect Dis  2017; 65:S200–19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Francois Watkins  LK, McGee  L, Schrag  SJ, et al.  Epidemiology of invasive group B streptococcal infections among nonpregnant adults in the United States, 2008-2016. JAMA Intern Med  2019; 179:479–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Russell  NJ, Seale  AC, O’Driscoll  M, et al.  Maternal colonization with group B Streptococcus and serotype distribution worldwide: systematic review and meta-analyses. Clin Infect Dis  2017; 65:S100–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Bianchi-Jassir  F, Seale  AC, Kohli-Lynch  M, et al.  Preterm birth associated with group B Streptococcus maternal colonization worldwide: systematic review and meta-analyses. Clin Infect Dis  2017; 65:S133–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Madrid  L, Seale  AC, Kohli-Lynch  M, et al.  Infant group B streptococcal disease incidence and serotypes worldwide: systematic review and meta-analyses. Clin Infect Dis  2017; 65:S160–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Russell  NJ, Seale  AC, O’Sullivan  C, et al.  Risk of early-onset neonatal group B streptococcal disease with maternal colonization worldwide: systematic review and meta-analyses. Clin Infect Dis  2017; 65:S152–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Le Doare  K, O’Driscoll  M, Turner  K, et al.  Intrapartum antibiotic chemoprophylaxis policies for the prevention of group B streptococcal disease worldwide: systematic review. Clin Infect Dis  2017; 65:S143–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Kobayashi  M, Schrag  SJ, Alderson  MR, et al.  WHO consultation on group B Streptococcus vaccine development: report from a meeting held on 27–28 April 2016. Vaccine  2019; 37:7307–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Kasper  DL, Paoletti  LC, Wessels  MR, et al.  Immune response to type III group B streptococcal polysaccharide-tetanus toxoid conjugate vaccine. J Clin Invest  1996; 98:2308–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Baker  CJ, Paoletti  LC, Rench  MA, et al.  Use of capsular polysaccharide-tetanus toxoid conjugate vaccine for type II group B Streptococcus in healthy women. J Infect Dis  2000; 182:1129–38. [DOI] [PubMed] [Google Scholar]
  • 11. Baker  CJ, Paoletti  LC, Wessels  MR, et al.  Safety and immunogenicity of capsular polysaccharide-tetanus toxoid conjugate vaccines for group B streptococcal types Ia and Ib. J Infect Dis  1999; 179:142–50. [DOI] [PubMed] [Google Scholar]
  • 12. Paoletti  LC, Rench  MA, Kasper  DL, Molrine  D, Ambrosino  D, Baker  CJ. Effects of alum adjuvant or a booster dose on immunogenicity during clinical trials of group B streptococcal type III conjugate vaccines. Infect Immun  2001; 69:6696–701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Carboni  F, Angiolini  F, Fabbrini  M, et al.  Evaluation of immune responses to group B Streptococcus type III oligosaccharides containing a minimal protective epitope. J Infect Dis  2020; 221:943–7. [DOI] [PubMed] [Google Scholar]
  • 14. Campisi  E, Rosini  R, Romano  MR, et al.  Group B Streptococcus chimeric capsular polysaccharides as novel multivalent vaccine candidates. Glycoconj J  2021; 38:447–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Chiarot  E, Spagnuolo  A, Maccari  S, et al.  Protective effect of group B Streptococcus type-III polysaccharide conjugates against maternal colonization, ascending infection and neonatal transmission in rodent models. Sci Rep  2018; 8:2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Buurman  ET, Timofeyeva  Y, Gu  J, et al.  A novel hexavalent capsular polysaccharide conjugate vaccine (GBS6) for the prevention of neonatal group B streptococcal infections by maternal immunization. J Infect Dis  2019; 220:105–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Oldrini  D, Del Bino  L, Arda  A, et al.  Structure-guided design of a group B Streptococcus type III synthetic glycan-conjugate vaccine. Chemistry  2020; 26:7018–25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Donders  GG, Halperin  SA, Devlieger  R, et al.  Maternal immunization with an investigational trivalent group B streptococcal vaccine: a randomized controlled trial. Obstet Gynecol  2016; 127:213–21. [DOI] [PubMed] [Google Scholar]
  • 19. Leroux-Roels  G, Bebia  Z, Maes  C, et al.  Safety and immunogenicity of a second dose of an investigational maternal trivalent group B Streptococcus vaccine in nonpregnant women 4–6 years after a first dose: results from a phase 2 trial. Clin Infect Dis  2020; 70:2570–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Leroux-Roels  G, Maes  C, Willekens  J, et al.  A randomized, observer-blind phase Ib study to identify formulations and vaccine schedules of a trivalent group B Streptococcus vaccine for use in non-pregnant and pregnant women. Vaccine  2016; 34:1786–91. [DOI] [PubMed] [Google Scholar]
  • 21. Madhi  SA, Cutland  CL, Jose  L, et al.  Safety and immunogenicity of an investigational maternal trivalent group B Streptococcus vaccine in healthy women and their infants: a randomised phase 1b/2 trial. Lancet Infect Dis  2016; 16:923–34. [DOI] [PubMed] [Google Scholar]
  • 22. Madhi  SA, Koen  A, Cutland  CL, et al.  Antibody kinetics and response to routine vaccinations in infants born to women who received an investigational trivalent group B Streptococcus polysaccharide CRM197-conjugate vaccine during pregnancy. Clin Infect Dis  2017; 65:1897–904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Fabbrini  M, Rigat  F, Tuscano  G, et al.  Functional activity of maternal and cord antibodies elicited by an investigational group B Streptococcus trivalent glycoconjugate vaccine in pregnant women. J Infect  2018; 76:449–56. [DOI] [PubMed] [Google Scholar]
  • 24. Absalon  J, Segall  N, Block  SL, et al.  Safety and immunogenicity of a novel hexavalent group B Streptococcus conjugate vaccine in healthy, non-pregnant adults: a phase 1/2, randomised, placebo-controlled, observer-blinded, dose-escalation trial. Lancet Infect Dis  2021; 21:263–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Hillier  SL, Ferrieri  P, Edwards  MS, et al.  A phase 2, randomized, control trial of group B Streptococcus (GBS) type III capsular polysaccharide-tetanus toxoid (GBS III-TT) vaccine to prevent vaginal colonization with GBS III. Clin Infect Dis  2019; 68:2079–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Swamy  GK, Metz  TD, Edwards  KM, et al.  Safety and immunogenicity of an investigational maternal trivalent group B Streptococcus vaccine in pregnant women and their infants: results from a randomized placebo-controlled phase II trial. Vaccine  2020; 38:6930–40. [DOI] [PubMed] [Google Scholar]
  • 27. Heyderman  RS, Madhi  SA, French  N, et al.  Group B Streptococcus vaccination in pregnant women with or without HIV in Africa: a non-randomised phase 2, open-label, multicentre trial. Lancet Infect Dis  2016; 16:546–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Jisuvei  SC, Osoti  A, Njeri  MA. Prevalence, antimicrobial susceptibility patterns, serotypes and risk factors for group B Streptococcus rectovaginal isolates among pregnant women at Kenyatta National Hospital, Kenya; a cross-sectional study. BMC Infect Dis  2020; 20:302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Bellais  S, Six  A, Fouet  A, et al.  Capsular switching in group B Streptococcus CC17 hypervirulent clone: a future challenge for polysaccharide vaccine development. J Infect Dis  2012; 206:1745–52. [DOI] [PubMed] [Google Scholar]
  • 30. Neemuchwala  A, Teatero  S, Athey  TB, McGeer  A, Fittipaldi  N. Capsular switching and other large-scale recombination events in invasive sequence type 1 group B Streptococcus. Emerg Infect Dis  2016; 22:1941–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Lindahl  G, Stalhammar-Carlemalm  M, Areschoug  T. Surface proteins of Streptococcus agalactiae and related proteins in other bacterial pathogens. Clin Microbiol Rev  2005; 18:102–27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Baron  MJ, Bolduc  GR, Goldberg  MB, Auperin  TC, Madoff  LC. Alpha C protein of group B Streptococcus binds host cell surface glycosaminoglycan and enters cells by an actin-dependent mechanism. J Biol Chem  2004; 279:24714–23. [DOI] [PubMed] [Google Scholar]
  • 33. Bolduc  GR, Baron  MJ, Gravekamp  C, Lachenauer  CS, Madoff  LC. The alpha C protein mediates internalization of group B Streptococcus within human cervical epithelial cells. Cell Microbiol  2002; 4:751–8. [DOI] [PubMed] [Google Scholar]
  • 34. Bolduc  GR, Madoff  LC. The group B streptococcal alpha C protein binds alpha1beta1-integrin through a novel KTD motif that promotes internalization of GBS within human epithelial cells. Microbiology (Reading)  2007; 153:4039–49. [DOI] [PubMed] [Google Scholar]
  • 35. Baron  MJ, Filman  DJ, Prophete  GA, Hogle  JM, Madoff  LC. Identification of a glycosaminoglycan binding region of the alpha C protein that mediates entry of group B streptococci into host cells. J Biol Chem  2007; 282:10526–36. [DOI] [PubMed] [Google Scholar]
  • 36. Li  J, Kasper  DL, Ausubel  FM, Rosner  B, Michel  JL. Inactivation of the alpha C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus. Proc Natl Acad Sci USA  1997; 94:13251–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Larsson  C, Lindroth  M, Nordin  P, Stalhammar-Carlemalm  M, Lindahl  G, Krantz  I. Association between low concentrations of antibodies to protein alpha and Rib and invasive neonatal group B streptococcal infection. Arch Dis Child Fetal Neonatal Ed  2006; 91:F403–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Stålhammar-Carlemalm  M, Waldemarsson  J, Johnsson  E, Areschoug  T, Lindahl  G. Nonimmunodominant regions are effective as building blocks in a streptococcal fusion protein vaccine. Cell Host Microbe  2007; 2:427–34. [DOI] [PubMed] [Google Scholar]
  • 39. Gravekamp  C, Kasper  DL, Madoff  LC. Immunization with a single-repeat alpha C protein may prevent escape of lower repeat mutants of group B Streptococcus. Adv Exp Med Biol  1997; 418:855–7. [DOI] [PubMed] [Google Scholar]
  • 40. Fischer  P, Pawlowski  A, Cao  D, et al.  Safety and immunogenicity of a prototype recombinant alpha-like protein subunit vaccine (GBS-NN) against group B Streptococcus in a randomised placebo-controlled double-blind phase 1 trial in healthy adult women. Vaccine  2021; 39:4489–99. [DOI] [PubMed] [Google Scholar]
  • 41. Lancefield  RC, McCarty  M, Everly  WN. Multiple mouse-protective antibodies directed against group B streptococci. Special reference to antibodies effective against protein antigens. J Exp Med  1975; 142:165–79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Stalhammar-Carlemalm  M, Stenberg  L, Lindahl  G. Protein rib: a novel group B streptococcal cell surface protein that confers protective immunity and is expressed by most strains causing invasive infections. J Exp Med  1993; 177:1593–603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Elder  BL, Boraker  DK, Fives-Taylor  PM. Whole-bacterial cell enzyme-linked immunosorbent assay for Streptococcus sanguis fimbrial antigens. J Clin Microbiol  1982; 16:141–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Armistead  B, Herrero-Foncubierta  P, Coleman  M, et al.  Lipid analogs reveal features critical for hemolysis and diminish granadaene mediated group B Streptococcus infection. Nat Commun  2020; 11:1502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Vornhagen  J, Quach  P, Boldenow  E, et al.  Bacterial hyaluronidase promotes ascending GBS infection and preterm birth. mBio  2016; 7:e00781-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Mold  M, Shardlow  E, Exley  C. Insight into the cellular fate and toxicity of aluminium adjuvants used in clinically approved human vaccinations. Sci Rep  2016; 6:31578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47. McGee  L, Chochua  S, Li  Z, et al.  Multistate, population-based distributions of candidate vaccine targets, clonal complexes, and resistance features of invasive group B streptococci within the United States, 2015–2017. Clin Infect Dis  2021; 72:1004–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48. Iho  S, Maeyama  J, Suzuki  F. CpG oligodeoxynucleotides as mucosal adjuvants. Hum Vaccin Immunother  2015; 11:755–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Shabayek  S, Abdalla  S, Abouzeid  AM. Serotype and surface protein gene distribution of colonizing group B Streptococcus in women in Egypt. Epidemiol Infect  2014; 142:208–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. Meehan  M, Cunney  R, Cafferkey  M. Molecular epidemiology of group B streptococci in Ireland reveals a diverse population with evidence of capsular switching. Eur J Clin Microbiol Infect Dis  2014; 33:1155–62. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

jiac148_Supplementary_Data
Click here for additional data file. (451.9KB, zip)

Articles from The Journal of Infectious Diseases are provided here courtesy of Oxford University Press

RESOURCES