Group B Streptococcus vaccine development: present status and future considerations, with emphasis on perspectives for low and middle income countries

Globally, group B Streptococcus (GBS) remains the leading cause of sepsis and meningitis in young infants, with its greatest burden in the first 90 days of life. Intrapartum antibiotic prophylaxis (IAP) for women at risk of transmitting GBS to their newborns has been effective in reducing, but not eliminating, the young infant GBS disease burden in many high income countries. However, identification of women at risk and administration of IAP is very difficult in many low and middle income country (LMIC) settings, and is not possible for home deliveries. Immunization of pregnant women with a GBS vaccine represents an alternate pathway to protecting newborns from GBS disease, through the transplacental antibody transfer to the fetus in utero. This approach to prevent GBS disease in young infants is currently under development, and is approaching late stage clinical evaluation. This manuscript includes a review of the natural history of the disease, global disease burden estimates, diagnosis and existing control options in different settings, the biological rationale for a vaccine including previous supportive studies, analysis of current candidates in development, possible correlates of protection and current status of immunogenicity assays. Future potential vaccine development pathways to licensure and use in LMICs, trial design and implementation options are discussed, with the objective to provide a basis for reflection, rather than recommendations.

Streptococcus agalactiae is also known as Lancefield's group B Streptococcus (GBS), and is a Gram-positive diplococcus, originally known for causing bovine mastitis 1 . GBS remains the leading cause of neonatal sepsis and meningitis, and is associated with significant mortality and morbidity, including long-term neurodevelopmental sequelae 2 . Disease risk is the highest during the first 3 months of life 3 , the primary target for GBS disease control efforts, but risk of invasive GBS disease increases again later in life, in particular among pregnant women and adults with underlying conditions or older age 1 .
Neonatal infections (sepsis and pneumonia) contribute importantly to deaths among children under 5 years of age globally, with the highest rates in low income countries, followed by middle income countries 4 . The etiologies of neonatal infections in low income countries are poorly characterized but GBS likely contributes to this burden. A recent systematic review showed that neonatal GBS disease incidence and case fatality rates are highest among countries in sub-Saharan Africa. However, published data from this region remain sparse and the estimated numbers are still considered underestimates 3 . In high-income countries, GBS emerged as a leading cause of neonatal infection in the 1970s for reasons that remain poorly understood. Many resource rich settings have experienced significant reductions in the incidence of early-onset disease (onset of disease during days 0-6 of life) after introduction of targeted administration of intrapartum intravenous antibiotics to women at risk of transmitting GBS to their newborns 5,6 . However, this intrapartum prophylaxis has not proven to be effective in preventing late-onset disease (disease onset during days 7-89 of life), and is not implemented in most high disease burden lowand middle-income countries (LMIC). Therefore, there has been a longstanding interest in developing a maternal vaccine against GBS to prevent disease in infants of vaccinated mothers.
Among various vaccine candidates, the glycoconjugate vaccines targeting GBS capsular polysaccharide (CPS) have been most studied, although common protein vaccines hold the appeal of broader coverage against circulating disease-causing strains. GBS vaccine development underwent an active phase in the 1990s. Although pre-clinical and early clinical studies showed promise, efforts slowed for a period, for a variety of reasons, including the strong success of intrapartum prophylaxis in reducing the earlyonset disease burden in high income countries, and concerns about the acceptance and the liability coverages for maternal immunization. Recent years have experienced a wave of new activity in GBS vaccine development. Successes in rolling out pneumococcal conjugate, rotavirus, and Haemophilus influenzae type b vaccines to the world's poorest countries through the GAVI alliance paved the way for future LMIC vaccine introductions. Finally there is a renewed interest in invigorating the maternal immunization platform, and several licensed products such as tetanus, influenza and pertussis vaccines are recommended for use among pregnant women in LMIC.
This review provides necessary background for non-GBS subject matter experts on issues of relevance to accelerating development of a GBS vaccine for LMIC. It draws almost exclusively on published literature or public information but alludes to some key activities of relevance that are anticipating publications in the near future. First we provide an overview of GBS disease and the global burden with a focus on GBS disease in infants (days 0-90 days), the primary prevention target for a maternal immunization program. This is followed by a summary of GBS diagnostics, and a review of intrapartum antibiotic prophylaxis (IAP), standards of care, strategies and impact. The next three sections provide relevant background in GBS vaccine development including a brief review of GBS virulence factors, the history of GBS vaccine development, and a review of safety and immunogenicity of current vaccine candidates, primarily from phase II studies of a trivalent glycoconjugate vaccine formulation. This section also reviews issues related to measuring serologic endpoints and the current status of establishing immune correlates of protection. The final three sections address cost-effectiveness analysis and other potential contributions of mathematical modeling to GBS vaccine decision-making; options regarding the planning and conduct of a phase III efficacy study; and different possible vaccine development pathways are presented. We conclude with a high level summary of key gaps in knowledge.

Diseases and sequelae caused by GBS and population at risk
Given the purpose of this document, information used in the next two sections ("Diseases and sequelae caused by GBS and population at risk" and "GBS disease burden and serotype distribution") is primarily from LMIC, supplemented with data from high-income countries whenever information from LMIC was not available.
Early-onset neonatal disease Definition. Although definitions for early-onset neonatal disease vary, the most common include onset of GBS disease within 72 hours of birth or days 0-6 of life 7 . See section on 'Considerations for licensure based on immune markers' for candidate definitions for a phase III trial.
Transmission. Early-onset disease is caused by vertical transmission through colonized mothers during or just before birth 8 . GBS can ascend from the vagina to the amniotic fluid after onset of labor or rupture of membranes 9 , although intrauterine infection without evidence of ruptured membranes has been reported 10,11 . GBS in the amniotic fluid can colonize the fetal skin or mucus membranes or can be aspirated into the fetal lungs, leading to an invasive infection 12,13 . Infants can also be exposed to GBS during passage through the birth canal and can become colonized at mucus membrane sites in the gastrointestinal or respiratory tracts. It has been estimated that in the absence of any intervention, approximately 50% of babies born to colonized mothers become colonized and 1-2% of them progress to develop invasive disease 14-16 .

Risk factors.
Risk factors for early-onset disease have been well described in resource-rich settings. A review of risk factors as established in United States studies showed that the strongest risk factor for neonatal disease was a positive maternal vaginal culture at delivery (Odds Ratio [OR]: 204) 17 . Other risk factors include prolonged rupture of membranes, preterm delivery, GBS bacteriuria during pregnancy, birth of a previous infant with invasive GBS disease, maternal chorioamnionitis as evidenced by intrapartum fever, young maternal age, and low levels of antibody to type-specific capsular polysaccharide antigens [18][19][20][21] . Although few risk factor analyses have been conducted in LMIC, epidemiologic characteristics of case series from these settings [22][23][24][25] , as well as a risk factor analysis of early-onset neonatal sepsis in South Africa 26 , suggest that the same risk factors play an important role in LMIC. Additionally, human immunodeficiency virus (HIV) infection in mothers has been shown to increase the risk of neonatal GBS disease. Recent studies from South Africa reported that HIV-infected women have lower GBS antibody concentrations and reduced transplacental antibody transfer compared to HIV-uninfected women 27,28 , and infants born to HIV infected mothers had lower anti-GBS surface binding antibody levels 28 . However, maternal HIV infection appears to be more of a risk for late-onset disease compared to early-onset disease.
Transmission. As with early-onset disease, development of late-onset GBS disease first requires adhesion of GBS to mucosal surfaces, followed by invasion across epithelial cells to gain entry to the bloodstream. Vertical transmission from colonized mothers can result in late-onset disease, although it is considered to play a less important role compared to early-onset disease 37 , and IAP has not impacted the late-onset disease burden in countries that provide IAP 38 . Nosocomial transmission, horizontal transmission from mother to infant after the perinatal period, and transmission from breast milk have also been described 39-42 , although it is unclear whether these are common routes of transmission 38 .

Risk factors.
Risk factors for late-onset disease are less understood than those for early-onset disease, and prevention strategies for late-onset infections have not yet been identified. Some of the identified risk factors are similar to those of early-onset disease, such as preterm delivery and maternal GBS colonization 43,44 . More recent studies have shown that preterm delivery may be a major factor for late-onset disease, with each week of decreasing gestation associated with an increased risk of late-onset disease 44,45 . Another prospective cohort study from Italy also showed that preterm infants had an increased risk for late-onset disease 46 .
As mentioned above, HIV exposure may be a greater risk for development of late-onset disease compared to early-onset disease: one study from South Africa reported that the risk ratio of the incidence of GBS disease was 1.7 (95% CI: 1.3-2.2) compared to HIVunexposed infants for early-onset disease vs. 3.2 (95% CI: 2.3-4.4) for late-onset disease 47 . Another South African study reported that the incidence of early-onset disease was similar between HIV-exposed and un-exposed (1.1 vs. 1.5; p=0.5) but there was a 4.7-fold greater risk (95% CI: 2.3 vs. 0.5; p<0.001) for late-onset disease 24 . Similar results were reported from a study conducted in Belgium 48 .

Disease onset and clinical presentation.
Studies reported different proportions of preterm infants (<37 weeks) among lateonset cases: 49% in the United States 38 , 25% in South Africa 24 , and 14% in Malawi 22 , suggesting this proportion may be lower in LMIC than in high-income countries. A study from Italy showed that term infants develop disease earlier (median 23 days, interquartile range [IQR] 15-42) compared to preterm infants (median 39 days, IQR 28-58) 46 .
The proportion of infants with late-onset disease presenting with meningitis is higher compared to infants with early-onset disease, and data from sub-Saharan Africa reported that meningitis is one of the leading clinical presentations for late-onset disease (33-59%) 22,24,25 . Data from the United States show that about 26% of infants with late-onset disease presented with meningitis, while 67% had bacteremia without a focus of infection 38 .
Disease outcomes. Because of the higher proportion of meningitis cases among infants with late-onset disease, risk of long-term neurologic sequelae may be higher among survivors of late-onset disease compared with infants surviving early-onset disease 49 . A study from South Africa showed that GBS-affected infants were >13 times more likely to have neurological sequelae at 6 months of age compared to controls, defined as abnormal Denver-II assessments (in the following domains: gross motor, fine motor, language and personal/social) or presence of hypertonia or hyper-reflexia 24 . Results from the United Kingdom showed that 22% of survivors of neonatal meningitis (≤28 days of life) had mild to moderate sequelae (e.g., isolated hydrocephalus, isolated epilepsy, mild learning problems, mild cerebral palsy), and 14% had severe sequelae (e.g., cerebral palsy, global delay, significant learning problems) at 9-10 years of age 50 . Another multi-center study from the United States described similar percentages of neurologic sequelae among GBS meningitis survivors: 25% with mild-to moderate impairment, and 19% with severe impairment at a mean age of 7 (range 3-12) years 2 .
Reported case-fatality ratios are lower compared to those of earlyonset disease 3,22,24,25,32 . In the systematic review by Edmond et al., the pooled result of all studies reporting case fatality for earlyonset disease was 12.1% (95% CI 6.2-18.3) and was 6.8% (95% CI 10.8-14.9) for late-onset disease. A more recent systematic review conducted by Sinha et al. reported that health facility-based studies from Malawi and South Africa reported case fatality ratios ranging from 20-38% for early-onset disease and 14-29% for lateonset disease (meta-analysis was not done due to heterogeneity in numerator and denominator) 51 .
Other perinatal complications (preterm delivery, stillbirth) Preterm delivery. GBS colonization during pregnancy has been associated with preterm delivery 52,53 , although the association is less clear than the association between colonization and early-onset disease. A systematic review which included 20 studies from 10 different countries summarized results by study design: results from cross-sectional studies conducted at the time of delivery had a pooled OR of 1.75 (95% CI 1.43-2.14) for preterm delivery between GBS colonized mothers and non-colonized mothers, and 1.59 (95% CI 1.03-2.44) for case-control studies that matched mothers with preterm delivery with mothers with the same gestational age, but not in labor. Whether colonization causes preterm delivery is still a matter of debate. A systematic review of cohort studies evaluating the odds of preterm delivery according to colonization status during pregnancy were inconclusive (pooled OR: 1.06; 95% CI 0.95-1.19) 54 .
Stillbirth. GBS has also been associated with spontaneous abortions and stillbirths. A retrospective study conducted in Australia which reviewed causes of spontaneous abortions (between 16 to 26 weeks gestation) among those with autopsy and microbiological cultures available showed that GBS was the most significant pathogen, often being the sole pathogen recovered, and found both in babies born to women with intact as well as ruptured membranes 55 . A study using United States population-based surveillance data showed that 24% of invasive GBS infections during pregnancy resulted in septic abortions and/or stillbirths, a higher proportion than observed for pregnancy-associated invasive infections with Streptococcus pneumoniae (8%) or group A Streptococcus (6%) 56 . Invasive GBS infections are however infrequent among pregnant women, whereas GBS colonization is much more common.
Estimating the burden of GBS-related stillbirths is challenging, even in high income countries.
Maternal pregnancy-associated and postpartum GBS disease GBS can cause urinary tract infection, chorioamnionitis, endometritis, and bacteremia in women 49 . Women during pregnancy and shortly after are at a higher risk of developing invasive GBS disease compared to non-pregnant women of the same age group 56 . Data on pregnancy-and postpartum-associated GBS disease are limited, even in resource-rich settings, and we are not aware of data from LMIC. Data from United States population-based surveillance showed that GBS bacteremia without focus was the most common presentation both during pregnancy (43%) and the postpartum period (32%), followed by chorioamnionitis (33%) in pregnant women, and endometritis (25%) in postpartum 56

GBS disease burden and serotype distribution
Challenges in estimating global disease burden Quantifying the burden of neonatal GBS disease remains a challenge even in high-income countries: clinical characteristics are non-specific and often difficult to differentiate from non-infectious causes 75 . Invasive infections are most commonly diagnosed based on isolation of GBS from a normally sterile site (e.g., blood, cerebrospinal fluid) in microbiological culture; however, sensitivity of blood culture varies depending on the bacterial load, blood collection, and culture method, and typically requires 36 to 48 hours for positive results to become available 75 . Estimating GBS disease burden in LMIC is even more difficult: a portion of births may occur outside of hospital settings; facility-born infants may be discharged quickly after birth; care seeking, particularly early in life, may be limited; access to care, particularly in rural areas may pose challenges; and health facilities may lack access to diagnostic tests or laboratory capacity or resources to diagnose GBS infection. As a result, particularly for early-onset disease, most of which occurs within the first 24-48 hours of life, GBS disease is likely underrepresented in studies from these settings 76 . Finally, incidence of neonatal GBS varies regionally 3,14 . IAP use should be considered in making regional comparisons, as IAP, an intervention known to reduce the risk of early-onset disease, is widespread in many resource-rich settings but rarely implemented in LMIC 3 .
As a result of these challenges, and the relative paucity of invasive disease data from LMIC, some researchers have focused on GBS colonization as a surrogate measure for neonatal disease. However, different studies in resource rich settings have reported similar and high maternal colonization prevalence but different neonatal disease incidence 3,14 , suggesting that the relationship between maternal colonization and newborn disease is not simple.
Estimating the invasive GBS disease burden in pregnant women is difficult due to the paucity of data from LMIC, and the common clinical practice of empiric treatment in absence of a definitive diagnosis for postpartum infections. Estimating the burden of GBSrelated stillbirths poses challenges even in high-income countries.  (Table 1). While IAP is common in the Americas it is rarely used in Africa or Southeast Asia. Incidence of early-onset disease and late-onset disease was also the highest in Africa (0.53 and 0.24 per 1,000 live births, respectively). However, only four studies were available for incidence estimates in Africa (Kenya, Malawi, Nigeria, South Africa). A more recent systematic review based on additional studies from sub-Saharan Africa reported a somewhat higher estimated incidence of neonatal disease: 1.3 cases per 1,000 births for early-onset disease (Kenya, Malawi, Mozambique, Nigeria, South Africa, Zimbabwe) and 0.73 per 1,000 births for late-onset disease (Kenya, Malawi, Mozambique, Nigeria, South Africa, Zimbabwe) 51 , although the authors believe this is still an underestimation of the actual incidence given the challenges in collecting data in these countries. A recent study of early-onset sepsis in Soweto, South Africa that used both blood culture and a real-time polymerase chain reaction test for GBS on whole blood estimated an incidence of early-onset GBS disease of 1.8 per 1000 live births, higher than the estimate of 1.3 per 1000 live births based on blood culture detections alone (Sithembiso Velaphi, SANISA study, in preparation). This difference underscores that invasive disease estimates from blood culture are minimum estimates unless they take into account blood culture sensitivity.
The low reported incidence of neonatal GBS disease from South Asia poses a puzzle: is this an accurate reflection of the disease burden, or is it a reflection of under-ascertainment due to the challenges of capturing specimens from ill newborns, particularly on day 0 of life, in this region? The Aetiology of Newborn Infections in South Asia (ANISA) 85 study attempted to fill this gap by conducting population-based surveillance and etiologic evaluation Edmond and colleagues reviewed available data on global serotype distribution. Serotype III accounted for almost half of all isolates, followed by serotypes Ia, Ib, II, and V, and, this trend was similar across all WHO regions. Five serotypes (Ia, Ib, II, III, V) accounted for more than 85% of serotypes in all regions with available data: 98% in Africa, 96% in the Americas, 93% in Europe, 89% in western Pacific, and 88% in the eastern Mediterranean 3 ( Figure 1). However, serotype studies from low-income countries and Southeast Asia were not identified in this review (Table 2).
Although not limited to neonates, a recent report from Vaccine Preventable Infections Surveillance conducted in Thailand sheds some light on serotype distribution of GBS disease in the southeast Asian region: among children aged <5 years with invasive GBS disease, serotype III was the most frequently isolated (approximately 50%), followed by Ia and Ib (approximately 13% each) 88 . In Edmond's global review, the proportion of serotype III isolates was larger for late-onset compared to early-onset disease (53% vs. 37%) 3 ( Figure 1). A more recent review from sub-Saharan Africa showed similar results: the five serotypes (Ia, Ib, II, III and V) accounted for 97% of early-onset disease and 98% of late-onset disease, and the proportion of serotype III was higher in late-onset cases (79%) than in early-onset cases (54%) 51 .
Maternal colonization. Ascertainment of maternal genital GBS colonization varies according to the specimens collected (e.g., vaginal sampling only vs. rectovaginal sampling), the culture medium, specimen transport and processing procedures and timing. In general, rectovaginal sampling has a higher yield than vaginal sampling only 89,90 , and use of selective broth media is better compared to nonselective blood agar 89 . A review estimated the prevalence of genital colonization in pregnant women to be around 13% globally, although included studies used various laboratory methods 91 . When restricted to studies which were considered to have used adequate methods (collection site including the vagina and using selective broth media), the estimated overall prevalence was 18% with regional variation: 12% in India/Pakistan, 19% in Asia/Pacific, 19% in sub-Saharan Africa, 22% in Middle East/North Africa, and 14% in the Americas (no data from Europe included) 91 . Regional variation was also reported in a multicountry cross-sectional study among pregnant women between 20 and 32 weeks gestation which used a standardized laboratory method (specimens collected from the cervix, lower vaginal wall, and urine and used selective enrichment broth) and showed that overall colonization prevalence was 11%, ranging from 8% in Manila, the Philippines, to 22% in Philadelphia, United States, which may reflect geographic differences in disease burden 92 .
GBS colonization is known to fluctuate during pregnancy, and a recent longitudinal study in South Africa reported that acquisition rates and the duration of colonization differ according to GBS serotype 93 . Serotype distribution in colonized mothers may not correlate directly with serotypes causing invasive neonatal disease, as invasiveness appear to be different according to GBS serotype 94 . However, colonization data may provide some insight into circulating GBS serotypes in regions where data from invasive disease are limited, especially Southeast Asia. A recent systematic review from sub-Saharan Africa showed that serotype III (>30%), Ia and V (both >20%) were the most frequently isolated 51 . Another study that took place on the Thai-Myanmar border showed that serotype II  Western Pacific 7 (18%) Japan (5), Australia (1), Singapore (1) was the most frequently isolated serotype (24%), followed by Ia, VI, III, and V 95 . Results from a multi-country study showed that overall, serotype III (17.8%) was the most frequently isolated serotype, followed by serotypes V (17%) and Ia (5%); however, serotype III was not isolated in two of the sites (Philadelphia, United States and Yangon, Myanmar), whereas serotype VII was the most frequently isolated serotype in Khon Kaen, Thailand, and was the only site that reported this serotype 92 . The GBS global serotype distribution appears more diverse than previously reported; a recent review of maternal GBS colonization showed significant heterogeneity across and within regions 96 . Additionally the modeling team led by the London School of Hygiene and Tropical Medicine is including a comprehensive review of maternal GBS colonization, risk of neonatal disease, neonatal disease incidence and impairment outcomes as part of their update of GBS disease burden estimates. They will also review data on GBS-related stillbirth, GBS-related preterm birth and review data on the association of GBS disease with neonatal encephalopathy. Pregnancy-associated GBS disease and stillbirths. As described above, few data are available on the incidence of invasive GBS disease among pregnant and postpartum women in low-and middle-income countries. A study from the United States showed that the incidence of invasive GBS disease was 0.04 (95% CI 0.03-0.05) per 1000 women-years for pregnant women, 0.49 (95% CI 0.36-0.64) per 1000 women-years for postpartum women, and 0.02 (95% CI 0.02-0.02) for non-pregnant women between the ages 15-44 years 56 .
A recent systematic review evaluated the incidence of GBS-related stillbirth (defined as at ≥20 weeks gestation most likely caused by GBS infection, as confirmed by a GBS-positive culture sample from the placenta and/or amniotic fluid and/or a normally sterile site) 97 . GBS-related stillbirth rates ranged from 0.04-0.9 per 1,000 births with highest reported from a small study in the United States 11 , and the proportion of stillbirths attributable to GBS infection ranged from 0 -12% 97 ; however, the review was limited by inconsistencies in stillbirth definitions and diagnostic methods and the number of studies available that met the inclusion criteria, particularly those from low-and middle-income countries to assess the burden of GBS-related stillbirth worldwide. Stillbirth data from Kenya were recently published 98 , and data from South Africa are currently being evaluated and are expected to be available, soon. These were both prospective studies that attempted to capture meaningful specimens from stillbirths for diagnostics and that applied similar, although not identical case definitions for a GBS-related stillbirth. In South Africa, preliminary estimates suggest GBS-related stillbirth incidence may be similar to that of early-onset GBS disease incidence (personal communication, Dr. Shabir Madhi).
Disease in non-pregnant adults.Annual incidence rates have been reported primarily from North America and Europe, ranging from 1.5 per 100,000 population (Spain, ages 21-100 years, 1992-1999) 73 to 7.3 per 100,000 population (United States, ages 18-105 years, 2007) 67 , and the rates tend to be higher with increasing age 35,66,67 . A population-based surveillance of invasive bacterial infections conducted in Thailand between 2010 and 2013 showed that the average annual incidence of invasive GBS disease is the highest among those aged ≥70 years (23 per 100,000 population 68 , similar to incidence reported among adults aged ≥65 years in 2005 in the United States (25.3 per 100,000 population) 35 . As seen when evaluating GBS disease incidence in other age groups or GBS colonization, geographic variation has been noted in the serotype distribution: reports from North America shows that serotype V is the most frequently isolated serotype in adult GBS disease, representing approximately 30% of the isolates as opposed to 11% for serotype III 67,72 , whereas reports from Europe show that serotype V (approximately 20%) was less frequent compared to serotype III (25-30%) 66,99 . A surveillance report from Thailand shows that among adults aged 21 years or older, serotypes Ia, II, II, V and VI represented >90% of cases, with serotype III being the most common (approximately 48%) 88 .

Diagnosis and treatment of GBS disease Diagnosis
This section summarizes clinical and laboratory methods commonly used for diagnosing GBS disease. For summary of case definitions used in published studies and discussion on candidate case definitions for phase III studies.

Newborns/Young infants
Clinical evaluation of sick children Integrated Management of Childhood Illness (IMCI) was developed jointly by WHO and the United Nations International Children's Fund (UNICEF) to promote the accurate identification and appropriate treatment of common childhood illnesses at first-level health facilities in low-income countries, where health workers rely on patients' history, and signs and symptoms to determine a course of management. Infants <2 months of age are assessed for signs of very severe disease: not feeding well, convulsions, fast breathing [≥60 breaths/min], severe chest indrawing, fever [≥37.5°C], low body temperature [<35.5°C], movement only when stimulated or no movement at all. These clinical syndromes which warrant urgent referral of young infants to hospitals are defined as possible serious bacterial infection (PSBI) 100 . Rates of PSBI among newborns young infants in LMIC can be very high (80 cases/1000 live births or higher) 101 . Even in the higher middle income country setting of South Africa, hospital admission for physician-suspected early-onset sepsis occurred at a rate of approximately 30 cases/1000 live births 15 . WHO guidance recommends that newborns presenting with signs of PSBI should be admitted to the hospital and blood cultures and lumber punctures should be obtained whenever possible before starting antibiotics 102 . Additionally, the United States Centers for Disease Control and Prevention (CDC) GBS prevention guidelines recommend a blood culture at birth for infants born to mothers with chorioamnionitis, even if the infant is well-appearing 9 .
IMCI has a separate set of algorithms for children 2 through 59 months. Children are first assessed for general danger signs (unable to drink or breastfeed, child vomits everything, lethargic or unconscious, had convulsions or actively convulsing). If stiff neck or general danger signs are present in a child with fever, administration of antibiotics and urgent referral are recommended 103 .

Laboratory detection of invasive disease
Confirmation of invasive GBS disease requires isolation of GBS from a normally sterile site (e.g., blood, CSF), which is usually done by collecting cultures. Automated blood culturing systems have improved the practice of blood culture: the automated system automatically detects microbial growth by monitoring microbial CO 2 detection, and eliminates the need for manual inspection or examination. In addition, growth of aerobes and facultative anaerobes are promoted by agitating culture bottles 104 . However, a recent review reported that many studies from LMIC used manual culture methods, with lower GBS incidence rates compared to studies using automated culture methods 105 . Therefore, differences in the culture methodology used can result in variation in reported GBS disease burden.
Rates of culture-confirmed infection are typically an order of magnitude or more lower than rates of clinical sepsis, although the culture positivity rate varies according to the criteria for collecting culture and how samples were collected. It is known that the likelihood of pathogen isolation increases with the quantity of blood submitted for culture, and for neonates, at least 0.5 to 1 ml of blood is recommended 7,106 . Because of the small blood volumes that can be obtained from newborns and young infants it is also important to use pediatric rather than adult blood culture bottles. In real use many cultures contain inadequate amounts of blood 107,108 . The yield of blood culture also varies with organism density in the blood. A study in infants 0-2 months of age showed that about half of the cultures positive for GBS had a very low organism density (≤1.0 cfu/ml) 109 . Based on an estimate from a study comparing the yield of pathogens from blood culture using blood samples with various volumes and bacterial load, the sensitivity of blood culture to detect low-level GBS bacteremia (1.0 cfu/ml) could vary from 44% (0.5 ml collected) to 98% (2 ml) 110 .
CSF analysis by lumbar puncture is the gold standard to diagnose meningitis. It is considered that up to 23% of neonates with bacteremia will also have concomitant meningitis, and that up to 38% of those with meningitis will have a negative blood culture 7 . Therefore, children suspected of meningitis should undergo lumbar puncture to assess the CSF whenever possible. Laboratory methods to identify GBS are summarized in Table 3. Cloudy CSF, elevated CSF leukocyte counts, low CSF glucose (e.g., < 1.5 mmol/litre or a ratio of CSF to serum glucose of ≤0.4), elevated CSF protein (e.g., > 0.4 g/litre), and positive Gram stain results indicate presence of meningitis, and treatment should be started immediately while awaiting culture results 102 .
Once bacterial isolates suggestive of GBS are identified, various laboratory methods including culture-based methods and high sensitivity latex agglutination tests can be used for GBS identification (see Table 3). More recently, nucleic acid amplification tests (NAAT) (e.g., polymerase chain reaction) have allowed direct GBS identification from clinical samples. Some studies have used NAAT in addition to culture in order to improve the detection of cases.
Neonatal colonization It is estimated that about half of neonates exposed to GBS by their colonized mothers become colonized with GBS, and only a small proportion of those develop invasive disease. Neonatal GBS colonization results from exposure to and swallowing of GBS-infected amniotic fluid or maternal vaginal secretions.
External auditory canal cultures are more likely to yield GBS in the first 24 hours of life compared to other sites, and isolation of organisms from the ear canal is a surrogate for the degree of contamination from amniotic fluid and vaginal secretions sequestered during the birth process. After the first 48 hours of life, throat and rectal sites are the best sources for detection of GBS, and positive cultures indicate true colonization (i.e., multiplication of organisms at mucous membrane sites), not just maternal exposure 111 .

Pregnant and postpartum women
Maternal colonization Maternal colonization can be assessed by collecting swabs from the vagina and the rectum from pregnant women 9 . Swabs are inoculated into a selective broth medium, and subcultured on to an agar plate for bacterial isolation. If enriched selective broth media is used, GBS can be determined faster (see Table 3).

Chorioamnionitis
The diagnosis and reporting of chorioamnionitis varies widely. Some consider histopathologic diagnosis as the gold standard 112 . Amniotic fluid sampling and culture can be used in the diagnosis of chorioamnionitis, however, diagnosis of chorioamnionitis is often made clinically due to challenges in accessing uncontaminated amniotic fluid or placenta for culture 113,114 . Culture of the fluid may be conducted, but may have limited clinical utility due to the potential colonization of the amniotic fluid and the time it takes to obtain results 114 . In addition, the infectious etiology is often polymicrobial 115 . Fever in a pregnant women is the most important clinical sign of chorioamnionitis. Other key clinical findings associated with clinical chorioamnionitis include uterine fundal tenderness, maternal tachycardia (>100/min), fetal tachycardia (>160/min), and purulent or foul amniotic fluid 113 . The WHO reference material lists fever (≥38.0°C) in pregnant women with foul-smelling watery discharge after 22 weeks and abdominal pain as symptoms typically present among pregnant women with chorioamnionitis 116 . Endometritis. The diagnosis of endometritis is also often made clinically, and is often due to polymicrobial infection 117 . Clinically, endometritis presents as fever, uterine tenderness, abdominal pain, and a purulent lochia or a positive culture of endometrial fluid or tissue 118 . Positive blood cultures may help identify the bacterial etiology, as bacteremia may be present in up to 20% of women 118 .
In the WHO clinical guidance, fever in women after childbirth with lower abdominal pain, purulent, foul-smelling lochia and tender uterus is described as signs and symptoms typically present in women with endometritis 116 .

Stillbirths.
Identifying infection as a cause of stillbirth is challenging: it is often difficult to determine the cause of stillbirth, and organism isolation on the placenta or the surface of the fetus does not prove causality 119 . Pregnant women may be colonized with GBS, and could contaminate the fetus or the placenta after membrane rupture or vaginal contamination during delivery 119 . In a recently published systematic review of 17 studies of GBS-related stillbirths, diagnosis was made based on a range of laboratory methods: culture confirmation from placenta (eight studies), blood/CSF (12 studies), amniotic fluid (two studies), and internal organs (eight studies) 97 . Careful placental histologic examination and autopsy are considered to be more useful in identifying the cause of stillbirths 119,120 , and culture of fetal heart blood or fluid from uncontaminated fetal sites during autopsy may help identify the infectious cause 119 .

GBS typing methods
Serological methods. Serological classification of GBS is based on the identification of capsular polysaccharides and protein antigens 121 . Capsular polysaccharide is currently the most advanced glycoconjugate vaccine target, and currently ten serotypes have been described (Ia, Ib, II-IX). Several serological methods have been used for serotyping (e.g., Lancefield capillary precipitin method, double immunodiffusion 122 , coaggulutination 123 , enzyme immunoassay 124 , latex agglutination 125 ). The Lancefield capillary precipitin method is considered as the "gold standard" 1,122 . One of the most common methods for capsular polysaccharide serologic typing is the latex agglutination method, using antibodies specific for the 10 recognized capsular polysaccharides 126 . In a recent report of a multicenter external quality assessment of molecular and serological typing conducted in 14 institutions in 13 European countries, the commercially available latex agglutination method was the most widely used typing method, with a typeability value (number of accurate results/total number of tests performed) of >90% 121 . Limitations of serological methods include failure to type an isolate (~4-9% are classified as non-typeable) due to lack of or low expression of capsular polysaccharide under experimental conditions, the presence of reversible non-encapsulated variants, or, although rare, expression of a new capsular serotype 1,126 . In addition, results are dependent on the quality of the antibodies used and on the experience of the laboratory 126 .

Molecular typing methods
Serotyping As an alternative to serological serotyping methods, molecular approaches based on the detection of capsular gene typing have been developed in recent years. Molecular methods include polymerase chain reaction (PCR) in conjunction with sequencing, hybridization, or enzymatic restriction cleavage pattern analysis, and multiplex-PCR approaches 1,126-128 . These molecular approaches are attractive because they have made it possible to assign a molecular serotype to otherwise nontypeable isolates by serologic methods, and because they are reproducible, specific, easy to perform, and suited for capsular polysaccharide typing in large-scale epidemiological studies 1 . However, PCR serotyping could potentially misclassify certain serotypes 126 . Also, PCR serotyping does not reveal if the capsular polysaccharide gene locus detected is actually expressed as a polysaccharide capsule 126 . Recently, Sheppard and colleagues have conducted whole genome sequencing to determine serotype with promising results 129 . Although the method currently may not be cost-effective merely for determining serotype 129 , the whole genome sequencing platform can be used to obtain genotyping data of the strains, as described below, as well as in depth analyses of strains within clonal complexes 130 .
Genotyping Molecular typing methods have been used for further characterization of GBS and are useful in distinguishing different GBS strains in epidemiological studies 121 . Examples of methods include restriction fragment length polymorphism (RFLP) 131 , pulsed-field gel electrophoresis (PFGE) 132 , multilocus sequence-typing (MLST) 133 and more recently DNA microarraybased typing 134 . Whole genome sequencing has also enabled the investigation of large and small scale genetic changes in comprehensive collections of GBS strains, thereby permitting enhanced understanding of the diversity of the organism 135 . See Box 1 for definitions of serotype, genotype, strain, and clonal complex.

Treatment
Newborns/young infants. IMCI recommends hospitalization and intramuscular or intravenous treatment of all infants meeting the case definition for PSBI 136 . The recommended antibiotic selection for management of "serious bacterial infection" and "meningitis" in infants aged <2 months is ampicillin and gentamicin (Table 4) 102 .
To date GBS remains universally susceptible to beta lactam antibiotics so penicillin and ampicillin remain effective therapeutic agents. The 2010 CDC guidelines recommend providing antibiotic therapy pending culture results for well-appearing newborns whose mothers had suspected chorioamnionitis 9 . The WHO recommends providing prophylactic intramuscular (IM) or intravenous (IV) ampicillin and gentamicin in neonates with documented risk factors for infection (see Table 4) 102 .

Box 1. Definitions of serotype, genotype, strain, and clonal complex 1,327
Serotype: type of antigenically variable polysaccharide capsule Genotype: the genetic makeup of an organism or a group of organisms with reference to a single trait, set of traits, or an entire complex of traits Strain: a single isolate of any bacterial population and any laboratory induced variants thereof Clonal complex: a group of bacterial strains derived from a recent common ancestor that share many alleles at various phylogenetically informative loci. A clonal complex generally includes the ancestral genotype and strains with minor variation Table 4. Summary of recommended management of severe bacterial disease in young infants and perinatal infections in selected guidelines from the World Health Organization.

Prevention of neonatal infections
Give prophylactic antibiotics only to neonates with documented risk factors for infection 102 : • Membranes ruptures >18 hours before delivery • Mother had fever >38°C before delivery or during labor • Amniotic fluid was foul-smelling or purulent Give IM or IV ampicillin and gentamicin for at least 2 days and reassess; continue treatment only if there are signs of sepsis (or a positive blood culture)

Management of infants aged <2 months
Infants <2 months with any signs of very severe disease 103 : • Give first dose of intramuscular antibiotics (ampicillin 50mg/kg and gentamicin 5mg/kg [age <7 days] or 7.5mg/kg [age ≥ 7days]) • Treat to prevent low blood sugar • Refer URGENTLY to hospital • Advise mother how to keep the infant warm on the way to the hospital

Serious bacterial Infection in infants <2 months 102 :
• Admit to hospital • When possible, do a lumbar puncture and obtain blood cultures before starting antibiotics • For newborns with any signs of serious bacterial infection or sepsis, give ampicillin (or penicillin) and gentamicin as first-line antibiotic treatment* • If at greater risk of staphylococcus infection (extensive skin pustules, abscess or omphalitis in addition to signs of sepsis), give IV cloxacillin and gentamicin • The most serious bacterial infections in newborns should be treated with antibiotics for at least 7-10 days • If an infant is not improving within 2-3 days, change the antibiotic treatment or refer the infant for further management

Meningitis in infants <2 months 102 :
• The first-line antibiotics are ampicillin and gentamicin* for 3 weeks • Alternatively, give a third-generation of cephalosporin, such as ceftriaxone (50mg/kg every 12 h if <7 days of age and 75mg/kg after 1 week) or cefotaxime (50mg/kg every 12 h if <7 days or every 6-8 h if >7 days of age, and gentamicin for 3 weeks • If there are signs of hypoxaemia, give oxygen • If the infant is drowsy or unconscious, ensure that hypoglycaemia is not present; if it is, give 2ml/kg 10% glucose IV • Treat convulsions (after ensuring they are not due to hypoglycaemia or hypoxaemia) with phenobarbital • Make regular checks for hypoglycaemia *recommended ampicillin dose: 50mg/kg every 12 hours (first week of life) or every 8 hours (weeks 2-4 of life)

Septicaemia 102
Laboratory: will depend on the presentation but may include • Full blood count • Urinalysis (including urine culture) • Blood culture • Chest X-rays Treatment: • Give IV ampicillin at 50mg/kg every 6 h plus IV gentamicin 7.5mg/kg once a day for 7-10 days; alternatively, give ceftriaxone at 80-100 mg/kg IV once daily over 30-60 min for 7-10 days/ • Give oxygen if the child is in respiratory distress or shock • Treat septic shock with rapid IV infusion of 20ml/kg of normal saline or Ringer's lactate. Reassess. If the child is still in shock (fluidrefractory shock), start adrenaline or dopamine if available.

Meningitis 102
Laboratory: • Confirm the diagnosis with a lumbar puncture and examination of the CSF. If the CSF is cloudy, assume meningitis and start treatment while waiting for laboratory confirmation.
• Microscopy should indicate the presence of meningitis in the majority of cases with a white cell (polymorph) count <100/mm 3 . Confirmation can be obtained from CSF glucose (low: <1.5mmol/litre or a ratio of CSF to serum glucose of <0.4). CSF protein (high>0.4g/litre) and Gram staining and culture of CSF, where possible.
• Blood culture if available Treatment: • Ceftriaxone: 50mg/kg per dose IM or IV every 12 hours; or 100mg/kg once daily for 7-10 days administered by deep IM injection or as a slow IV injection over 30-60 min, OR • Cefotaxime: 50mg/kg per dose IM or IV every 6 hours for 7-10 days, OR • When there is no known significant resistance to chloramphenicol and β-lactam antibiotics among bacteria that cause meningitis, follow national guidelines or choose either of the following two regimens: ○ Chloramphenicol: 25mg/kg IM or IV every 6 h plus ampicillin: 50mg/kg IM or IV every 6 h for 10 days, OR ○ Chloramphenicol: 25mg/kg IM or IV every 6 h plus benzylpenicillin: 60mg/kg (100,000 U/kg) every 6 h IM or IV for 10 days.

Prevention of neonatal infections Prevention of peripartum infections
Women with group B Streptococcus colonization 100 • Intrapartum antibiotic administration to women with GBS colonization is recommended for prevention of early neonatal GBS infection (conditional recommendation based on very low-quality evidence) ○ Ampicillin or penicillin G should first be considered for treatment except where there are contraindications (e.g. allergy history) or GBS strain has been microbiologically shown to be penicillin-resistant ○ This recommendation should be implemented within the context of local policy and guidance on screening for GBS colonization.
Women in preterm prelabor rupture of membranes 100 • Antibiotic administration is recommended for women with preterm prelabor rupture of membranes (Strong recommendation based on moderate-quality evidence) ○ Erythromycin is recommended as the antibiotic choice for prophylaxis Women undergoing elective or emergency caesarean section 100,151 • Routine antibiotic prophylaxis is recommended for women undergoing elective or emergency caesarean section.
• For antibiotic prophylaxis for caesarean section, a single dose of first-generation cephalosporin or penicillin should be used in preference to other classes of antibiotics.

Managing fever in mothers during pregnancy and labor
Fever during pregnancy and labor 116 Probable diagnosis: cystitis, acute pyelonephritis, septic abortion, amnionitis, pneumonia, uncomplicated malaria, severe/ complicated malaria, typhoid Septic abortion • Begin antibiotics* as soon as possible before attempting manual vacuum aspiration

Amnionitis
• Give a combination of antibiotics until delivery -Ampicillin 2g IV every six hours -PLUS gentamicin 5mg/kg IV every 24 hours; -If the woman delivers vaginally, discontinue antibiotics postpartum; -If the woman has a caesarean section, continue antibiotics and give metronidazole 500mg IV every eight hours until the woman is fever-free for 48 hours • If metritis is suspected (fever, foul-smelling vaginal discharge), give antibiotics • If newborn sepsis is suspected, arrange for a blood culture and antibiotics *give ampicillin 2g IV very six hours PLUS gentamicin 5mg/kg IV every 24 hours PLUS metronidazole 500mg IV every eight hours until the woman is fever-free for 48 hours Chorioamnionitis 100 • A simple regimen such as ampicillin and once-daily gentamicin is recommended as first-line antibiotics for the treatment of chorioamnionitis. (Conditional recommendation based on very low-quality evidence)

Metritis
• Transfuse as necessary. Used packed cells, if available • Give a combination of antibiotics until the woman is fever-free for 48 hours: -Ampicillin 2g IV every 6 hours -PLUS gentamicin 5mg/kg IV every 24 hours -PLUS metronidazole 500mg IV every eight hours If fever is still present 72 hours after starting antibiotics, re-evaluate and revise diagnosis Postpartum endometritis 100 • A combination of clindamycin and gentamicin is recommended for the treatment of postpartum endometritis (Conditional recommendation based on very low-quality evidence).

Pregnant and postpartum women.
For treatment of chorioamnionitis, the WHO recommends ampicillin and once-daily gentamicin 100 . A combination of clindamycin and gentamicin is recommended as first-line treatment of postpartum endometritis. Use of intrapartum antibiotic prophylaxis (IAP) to prevent earlyonset neonatal disease is described in further detail in section 'Prevention of perinatal GBS disease through intrapartum antibiotic prophylaxis'.
Antimicrobial susceptibility. Globally, GBS resistance to penicillin G or ampicillin has not been reported. Thus, beta lactams are considered first-line antibiotics for GBS infection or IAP. However, isolates with increased minimum inhibitory concentrations to these antibiotics due to mutations in penicillin binding proteins have been reported primarily from Japan and North America 137-141 . Macrolide and/or clindamycin resistant strains have been increasing. There are limited invasive GBS antimicrobial susceptibility data available from LMIC. This largely reflects the relative paucity of invasive neonatal GBS disease surveillance from a majority of LMIC  [1]) showed that among GBS isolates from neonates, 100% were susceptible to penicillin, 60% (95% CI 25-91%) were susceptible to chloramphenicol, and 65% (95% CI 0-100%) to third-generation cephalosporins 142 .

Prevention of perinatal GBS disease through intrapartum antibiotic prophylaxis Intrapartum antibiotic prophylaxis
In the 1980s, clinical trials and a large observational study demonstrated that administration of intravenous ampicillin or penicillin during labor to mothers with certain risk factors for GBS transmission was highly effective (efficacy estimates of 80-100%) at preventing invasive early-onset GBS disease 143-145 . Effectiveness estimates, although often somewhat lower than estimates from trial settings due to a portion of women receiving less than the optimal prophylaxis duration (at least 4 hours of a beta lactam agent before delivery) or non-beta lactam agents, are consistent with trial findings 146,147 . Based on this evidence, penicillin or ampicillin are often the first line agents recommended for prophylaxis, with cefazolin and in narrow instances clindamycin or vancomycin as options for penicillin-allergic women. WHO recommends intrapartum antibiotic administration (first choice penicillin G or ampicillin) to women with GBS colonization based on observed clinical benefits for the neonates (see Table 4); however, the guideline development group acknowledged the challenges in implementing GBS screening and provision of IAP especially in low-resource settings 100 . WHO recommendations and feasibility in LMIC are further discussed in the two sections below.

Strategies for targeted intrapartum antibiotic prophylaxis
Because only a portion of women are at elevated risk of transmitting GBS to their infants, universal prophylaxis of all deliveries is not an optimal strategy, particularly since antibiotic exposure is associated with low but non-zero risks. The most immediate risk is maternal anaphylaxis to penicillin which is estimated to occur in four per 10,000 to 4 per 100,000 recipients 148 . In resource-rich, hospital settings, anaphylaxis-related mortality is exceedingly rare, but in low and middle income countries risks for complications from anaphylaxis, even for hospital births, may be higher. While there is no risk for anaphylaxis in the newborn, due to the very low probability of previous antibiotic exposure and the lack of transfer of maternal IgE antibodies across the placenta, intrapartum antibiotics do impact the microbiome of the maternal birth canal and thus the microbiome acquired by the newborn, particularly for vaginal births. Some studies suggest microbiome alterations, particularly at the time of birth, may result in health impacts well past the newborn period, although these have not yet been substantiated and the risks have not been quantified 149 .
Two major strategies have been employed to limit the portion of women exposed to intrapartum prophylaxis to those at most risk of transmitting GBS 9 . The risk-based strategy identifies women for antibiotic prophylaxis based on presence of known risk factors for early-onset disease including maternal fever, prolonged rupture of membranes, preterm delivery, and previous birth to an infant with invasive GBS disease and detection of GBS bacteriuria during the current pregnancy. In different countries employing the riskbased approach, variations may exist in the risk factors screened for, or in the thresholds used to identify risk, based either on local epidemiology or efforts to narrow the portion of women targeted for prophylaxis. Maternal fever is most commonly defined as ≥38°C and prolonged membrane rupture is often ≥18 hours. In contrast, the culture-based screening leads to identification of women with vaginal/rectal GBS colonization late in pregnancy, as a basis for antibiotic prophylaxis indication. Women who present at labor without a culture result are managed according to the riskbased strategy. Variants of the culture-based screening strategy also exist across countries but most recommend screening at 35-37 weeks gestation.
Both strategies have been documented to result in significant declines in invasive early-onset GBS disease, both in single hospitals and population-based analyses, in a range of resource-rich settings 5 . A population-based comparison of the two strategies in the United States found that the culture-based screening strategy was over 50% more effective than the risk-based strategy, primarily due to the high proportion of GBS positive women who received intrapartum prophylaxis and to the frequency of colonized women without any noted risk factors (18% of the delivering population in the United States) 150 .
Although current WHO recommendations do not specify a recommended approach for identifying women at risk, antibiotic prophylaxis (erythromycin) to women in preterm pre-labor rupture of membranes is recommended, as part of a strategy to improve the prognosis of infants with preterm birth (strong recommendation based on moderate-quality evidence) 100 . Antibiotic prophylaxis is not recommended for women in preterm labor with intact amniotic membranes nor for women with pre-labor rupture of membranes at term or near term (36 weeks gestation and above). The latter recommendation is based on the review of evidence from studies in women with duration of ruptured membranes less than 12 hours, and it is acknowledged that there may be a benefit from antibiotic prophylaxis in women with prolonged rupture of membranes (>18 hours) 100,151 .

Feasibility in low-and middle-income countries
Neither of the above strategies were designed in the context of LMIC and both pose implementation challenges, particularly in low-income country settings. In low income countries, safe administration of intravenous antibiotics may not always be affordable or feasible, particularly for settings where births do not occur in hospitals. Even in instances where intrapartum prophylaxis may be feasible, identifying candidates for prophylaxis poses unique barriers. The risk-based strategy has the appeal that the key variables for action can be captured at the time a woman presents for labor. However, even in middle income countries such as South Africa, capture of these variables may prove challenging in a busy labor and delivery setting. For example, in a study of over 8000 deliveries at the main public hospital serving Soweto, South Africa, less than 1% of women were noted as having intrapartum fever suggesting under-ascertainment 15 . Additionally, gestational age is not always known and clear distinctions between term and preterm deliveries may not always be straightforward. Moreover, because risk factors such as prolonged membrane rupture may evolve over the course of labor, prophylaxis may not always be administered to women who develop risk factors after admission. In resource-rich settings such as the United States, a lower proportion of women with risk factors have been noted to receive prophylaxis compared to GBS-colonized women 150 ; this may prove even more challenging in LMIC where providers care for a higher patient load. Finally, more women in LMIC than in resource-rich settings may present to facilities at a late stage in labor, leaving insufficient time for efficient prophylaxis.
While the risk-based strategy poses challenges, few LMIC are positioned to overcome the implementation and cost challenges associated with late antenatal screening. In particular, LMIC settings rarely have access to a high proportion of women at 35-37 weeks gestation, a strong microbiology laboratory network to process antenatal samples, and systems for effective communication of results to labor and delivery staff.
Non-vaccine alternatives to intrapartum antibiotic prophylaxis To date, possible alternative to intrapartum prophylaxis have not proven effective. Chlorhexidine wipes of the birth canal during labor and the newborn at birth were evaluated in a large clinical trial South Africa with no evidence of efficacy against culture-confirmed or clinical neonatal sepsis 15 . Universal administration of intramuscular penicillin to newborns within 1 hour of birth is implemented at one large center in the United States 152-154 ; however the lack of a concurrent control makes it difficult to interpret effectiveness or generalizability. This strategy also exposes all newborns to antibiotics. IM penicillin intrapartum does not achieve high enough concentrations rapidly enough, and antenatal use of oral or IM antibiotics have not shown impact 146,155-157 .

Virulence factors of GBS
GBS disease typically progresses from bacterial colonization, penetration of placental or epithelial barriers, and immune evasion preventing clearance of GBS from the bloodstream. In the case of meningitis, the ability to cross the endothelial blood-brain barrier is also needed 158,159 . GBS expresses a number of virulence factors, which play different roles in these steps (summarized in Table 5), but one of the most prominent and best-studied is the capsular polysaccharide (CPS), which protects the bacteria from opsonization and subsequent phagocytosis and intracellular killing 160,161 . More recently, multilocus sequence typing (MLST) analysis has shown that sequence type (ST) 17 is associated with enhanced invasiveness in neonates independent of capsular serotype although most ST17 isolates are CPS type III 165 . ST17 displays meningeal tropism, and has been referred to as the hypervirulent clone 166 .
It has been hypothesized that the GBS isolates causing invasive GBS disease in neonates worldwide emerged from a few successful clonal lineages, and virulence factor identification to date has focused on elements common across these clones 161 . Factors under investigation to date include the C5a peptidase, the AlphaC-like surface protein family, the Sip-protein, and pilus islands, all of which have different roles in the infection process 161 , and have been investigated as vaccine targets. Pili mediate GBS resistance to cationic antimicrobial peptides (AMPs), which are components of the host innate immune system that play a critical role in combating bacterial infections 167 , and also facilitate adherence and attachment of the pathogen to host mucosal cells. More recently, a surface-anchored adhesion protein called hypervirulent GBS adhesion (HvgA) was identified from comparative expression analysis between clones of different virulence. HvgA is considered to be a specific virulence factor of hypervirulent ST17 168 . In a manner similar to that of pili, HvgA mediates both colonization and invasion in the intestine, which appears to be a prerequisite for meningitis in the neonatal mouse model 161 .

GBS vaccine development
Biological rationale for a vaccine Prevention of neonatal GBS disease has been the primary focus for GBS vaccine development. Most cases of early-onset neonatal and young infant disease occur within the first 24 hours. Therefore, maternal immunization rather than direct vaccination of newborns is required to prevent neonatal and young infant disease. In animal models, passive immunization (e.g., transferring sera of animals exposed to GBS disease) and active immunization (e.g., mouse maternal vaccination-neonatal pup challenge model) have been shown to be protective against development of GBS disease (see below for details on animal models). In humans, transplacental transfer of protective maternal antibodies against GBS was first reported by Baker and colleagues 169 . Their study showed that mothers whose infants developed invasive GBS disease from serotype III had significantly lower levels of serum IgG levels to CPS III compared to mothers whose infants were exposed to type III but did not develop disease. Subsequent studies reported similar findings with other GBS serotypes 170,171 and the association of low maternal GBS CPS specific IgG levels and the risk of GBS disease in their infants was further described 19 . Attempts have been made to identify a threshold that would confer protection against GBS disease for vaccine development.  176 . This is based on the assumption that infants born ≥35 weeks gestation would have acquired sufficient concentrations of maternal antibodies, which would protect the infant from GBS disease for the first 6 weeks of their life (translating to two half-lives of antibody decay). The optimal timing of maternal immunization that would maximize protection against young infants requires further investigation.
Results from a phase II randomized controlled trial have shown that the III-TT vaccine delayed the acquisition of vaginal and rectal GBS III (NCT00128219) 178 . Another study reported an association between increased serum CPS IgG levels and reduced homotypic GBS rectovaginal acquisition 179 . If the vaccine reduces maternal colonization, maternal vaccination could further reduce the risk of neonatal disease by reducing exposure to GBS in the first months of life.
Newborn and young infant response to natural GBS infection Opsonization, followed by phagocytosis (ingestion of invading microorganism) and intracellular killing are the main mechanisms of host defense against GBS infection 180 . Opsonization requires the deposition of specific antibody and complement on the bacterial surface, and antibody and complement do not kill GBS in the absence of phagocytes 181 . Type III GBS-CPS was shown to prevent activation of the alternative complement pathway but this effect can be overcome by the presence of a sufficient amount of CPS antibody 182,183 .
Immaturity of the immune system makes neonates more susceptible to infections: neutrophils have a small storage pool at birth, and are less responsive to chemoattractants than later in life. Neonatal monocytes, which mature into macrophages, are impaired in their capacity for killing intracellular GBS 184 . Newborns have an impaired ability to form antibodies in general, and are particularly deficient in their ability to mount antibody responses against polysaccharide antigens 185 . Altogether, their capacity for GBS CPS antigenspecific protection is determined largely by the placental transfer of maternal IgG antibodies 186 . Therefore, the goal of maternal immunization is to induce GBS-specific antibody levels in the mother to achieve antibody levels in the child that would confer protection during the first 3 months of life.
Animal models GBS disease models. Pre-clinical studies using animal models are important to obtain sufficient data on safety, immunogenicity and potential efficacy of candidate vaccines before proceeding to clinical trials. A wide range of animal models has been used to study GBS-host interactions and to provide means to test potential therapies and vaccine approaches. A sampling, rather than a comprehensive review, is provided below.
Mice have been commonly used to model GBS infections. The earliest animal models studies of GBS infections date to the 1930s 122 .
In later studies, intraperitoneal or intravenous models of GBS infection in adult or neonatal mice were developed to simulate human infections 187,188 . In some cases, oral inoculation has been used as a means of inducing systemic infection in mice 189 . Notably, in both mice and rats, there appears to be an age-related decrease in susceptibility to invasive GBS infection 190,191 .
A large number of other animal model systems have been explored, including chicken embryo 192 , rabbits 193 , sheep 194-196 , piglet 197 , and non-human primates 198,199 . Some of these models (especially the large animal models) have been used to provide insights that are difficult or impossible to study in mice. The sheep [194][195][196] and piglet models 197 are of particular relevance for the study of hemodynamic changes in host animals during GBS sepsis. Non-human primate models of GBS infection have been used sparingly, but they are of particular utility in modeling newborn infections and host responses in vivo 198,199 .

Animal models of GBS colonization.
Fewer studies have used animals to model asymptomatic GBS carriage, despite the importance of the carrier state for maintenance of GBS in the population and the role of maternal colonization as the major risk factor for neonatal disease. Most recent work in this area has used murine models of vaginal or gastrointestinal colonization.
Vaginal colonization models have allowed determination of specific bacterial or host factors involved in carriage in the absence of invasive disease [200][201][202][203][204][205][206][207][208] . Gastrointestinal GBS colonization has been modeled in gnotobiotic mice and used as a means to understand the role of surface proteins in GBS carriage 209 . Neonatal mice have also been used as a model for gastrointestinal carriage, especially as a prelude to invasive disease 168 . Oral colonization of infant rats has been used to examine the utility of antibiotics to decrease mucosal bacterial load 210 .

Animal models of ascending infection and/or perinatal GBS transmission.
To examine the role of ascending infection in adverse pregnancy outcomes (e.g., preterm delivery, stillbirths), animal models simulating human infections have been explored. Examples include intracervical GBS inoculation of rabbits 211-213 , murine intravaginal/intrauterine/intraperitoneal inoculation 214-216 ; catheterization and intraamniotic instillation of GBS has been used to model chorioamnionitis in non-human primates and to study its effects on fetal lung tissue 217-220 . However, initial attempts to create an ascending infection animal model secondary to chronic vaginal colonization, which is a better simulation of human infection, were unsuccessful 213 . Recently, Randis and colleagues have developed a model of GBS ascending infection during pregnancy secondary to vaginal colonization using pregnant mice. This model may shed light on the role of bacterial virulence factors such as beta-hemolysin/cytolysin in causing adverse pregnancy outcomes associated with maternal GBS colonization 203 .

Preclinical studies of GBS vaccines in animal models
a. Passive immunization Animal models have been used to examine the effect of antibody delivery (passive immunization) on invasive GBS disease in vivo. The first studies used generation of antibodies in rabbits followed by passive protection of mice exposed to systemic GBS infection 122,221 . Subsequent studies used hyperimmune serum or purified antibody preparations to provide protection to neonatal experimental animals 199 .  [233][234][235] . However, GBS strains isolated from human infections may be highly adapted to their human host, and results obtained from mouse models must be interpreted with caution 236 . For example, human GBS isolates may express surface proteins that specifically interact with the human hosts but not with other animals [236][237][238] . In addition, the shorter gestational period of mice (19-22 days) should be taken into account to measure the timing of vaccination and passive protection in neonates 239 .

b. Active immunization
The structure and function of antibodies induced by vaccination and the kinetics of maternal antibody transfer to the fetus are most similar between human and non-human primates. Baboon models have been used in preclinical GBS vaccine studies 229,230,240 . As in mouse models, these studies showed that GBS conjugate vaccine induced CPS-specific antibodies 230,240 , and there was a correlation between maternal and infant baboon serum antibody levels 230 .
Differences have been noted in the kinetics of antibody responses and waning between humans and baboons 241 .

History of GBS vaccine development
Polysaccharide vaccines. GBS capsular polysaccharide (CPS) has been the primary target for vaccine development. In the 1930s studies demonstrated that CPS-specific rabbit sera could be used to protect mice against lethal challenge with GBS 242 . The first purified type III CPS vaccine underwent phase I testing in healthy adults in 1978 243 , and subsequently type Ia and II CPS vaccines were tested. Type II CPS was found to be the most immunogenic, while type Ia and III showed an immune response in about half of the recipients 244 . Most adults (nearly 90%) had very low serum concentrations of CPS specific antibodies before immunization, which was considered to indicate immunologic naivety to GBS polysaccharides, and was a partial predictor for a poor immune response 244, 245 . Favorable safety of CPS vaccines was shown on a small scale in non-pregnant adults and among pregnant women 245,246 , and infant antibody levels in cord serum correlated with maternal antibody levels at delivery 246 .

Glycoconjugate vaccines. Immunogenicity of polysaccharides is enhanced by covalent conjugation with a carrier protein. Glycoconjugate vaccines have been developed for Haemophilus influenzae type b (Hib), Neisseria meningitidis and Streptococcus pneumoniae.
Unlike T-cell-independent B-cell activation by non-conjugated polysaccharide antigens, glycoconjugate vaccines have the potential to induce both B-and T-cell memory and produce a stronger and highly functional IgG response through antibody class switching 160 .
The first GBS glycoconjugate vaccine trial conducted in humans involved a GBS III CPS-tetanus toxoid (III-TT) glycoconjugate 160,247 . Healthy non-pregnant women were recruited and randomized to receive III-TT, type III CPS vaccine, or placebo 247 . Results showed that the highest dose of III-TT produced higher levels of type III CPS-specific antibody measured two weeks after vaccination, and that the proportion of recipients achieving a ≥4-fold rise in antibody concentration was higher among those who received III-TT compared to those who received unconjugated type III CPS vaccine 247 , suggesting that the glycoconjugated vaccines are able to induce a more robust immune response compared to polysaccharide-only vaccines. Following this first trial, phase I trials of monovalent Ia, Ib, II and V-TT conjugates showed immunogenicity of a single dose suggesting no need for addition of an adjuvant 241,248,249 . In another randomized controlled study in healthy non-pregnant women, receipt of GBS III-TT was associated with protection against future acquisition of type-specific GBS colonization, with 36% vaccine efficacy for vaginal acquisition and 43% efficacy for rectal acquisition compared to controls who received tetanus and diphtheria toxoids (clinicaltrials.gov NCT00128219) 178 .
To achieve broader coverage against the GBS serotypes causing disease in humans, several multivalent vaccines have been developed and tested in humans. The immune response in subjects who received a bivalent vaccine containing II-TT and III-TT glycoconjugates did not differ statistically from the antibody responses to monovalent vaccines 250

Protein-based vaccines.
Polysaccharide-based vaccines typically only provide protection against CPS types included in the vaccine or closely related serotypes, and may be vulnerable to serotype replacement/switching. Therefore, efforts have been made to identify proteins common to all GBS as the basis of a vaccine that would confer broad protection against GBS 250 .
Until whole genome sequences of two GBS strains became available in 2002, only a limited number of proteins involved in GBS pathogenesis were identified as potential vaccine candidates 250 . Rib and alpha are among the GBS surface proteins that have been studied extensively as possible vaccine targets 257,258 . Recently, Miner-vaX, a privately held Danish biotech company, has initiated phase I clinical trials with a protein vaccine based on a fusion of the N-terminal portion of two surface proteins, AlphaC and Rib (GBS-NN) (NCT02459262) 259 . MinervaX expects that GBS-NN will protect against up to 95% of GBS isolates, given the broad expression of AlphaC and Rib as well as cross-reactive proteins 259 .
During the past decades, the application of recombinant DNA techniques and the availability of complete bacterial genomes have allowed use of genome-based vaccinology to identify new protein vaccine candidates 250 . Investigators from GSK used reverse vaccinology to identify a conserved sequence encoding components of pili proteins on the bacterial surface. A vaccine based on a combination of these proteins conferred protection against different GBS strains in a mouse model 260 . However, coverage against all GBS strains was not possible due to antigenic variation associated with the pilin subunits 250,260 . Structural vaccinology was successfully applied to design an optimized BP-2a protein, a subunit of the backbone protein of the GBS pili known to have high gene variability 250 . The protective capacity of a BP-2a variant is restricted to a small region (D3), and each variant fused into a single recombinant chimeric construct expressed in Escherichia coli which conferred strong protection against all six strains expressing a BP-2a variant in challenged mice 235 .

Evidence for immune correlates of protection GBS-specific antibody concentration and correlates of protection.
Sero-epidemiological studies showed some evidence in favor of an association between low maternal GBS CPS specific IgG levels and the risk of GBS disease in offspring. Associations between maternal GBS surface-protein antibody concentrations and invasive disease in their infants have not been as clearly established: among the surface proteins studied so far (surface immunogenic protein [Sip], resistance to proteases immunity group B [Rib], AlphaC protein, BetaC protein, fibrinogen-binding protein A, GBSimmunogenic bacterial adhesion, and pilus-island surface protein antibodies), limited data suggest that antibodies against alphaC and Rib proteins may provide protection against invasive neonatal GBS disease 258,261-265 .

Evidence from sero-epidemiological studies.
Most of the earlier studies comparing capsular antibody concentrations between cases and controls were done using a small sample size (e.g., ≈10-50 cases total per capsular serotype). More recent studies with larger sample sizes (e.g., >50-300 cases total per capsular serotype) have attempted to identify a serotype-specific IgG level in mothers that would confer protection against infant disease due to the same serotype [266][267][268][269] . A summary of studies published after 2000 is shown in Table 6. Both studies by Lin and colleagues were case-control studies using data collected from multiple study sites in the United States 266,269 . Maternal and cord serum samples were collected from enrolled participants after delivery and antibody levels were compared between cases (neonates who developed early-onset disease and their mothers) and controls (neonates who remained healthy despite being colonized with the same serotype and their mothers). The case-control study by Baker and colleagues was also a multi-center study in the United States and compared maternal serum samples from cases (those whose infants developed early-onset disease due to specific serotypes) matched by age and ethnicity with those from controls (those who were colonized with the sample capsular serotypes but whose infants did not develop disease) 267 . The study by Matsubara and colleagues was conducted at a single institution in Japan and compared serum antibody levels of pregnant women with serotype VIII colonization with stored serum samples from four mother-and-neonate pairs with earlyonset serotype VIII infection 270 . Dangor and colleagues conducted a matched case-control study in South Africa; cases were infants with laboratory-confirmed invasive GBS disease within <90 days of age, and controls were age-matched healthy infants, whose mothers were colonized with the same GBS CPS serotypes as cases. Maternal and infant serum from cases were compared with those of controls (or cord serum in case of controls of early-onset disease 268 . The results showed that in general, there was an inverse relationship between maternal serotype-specific IgG levels and the risk of their infants developing GBS disease ( Functional antibody concentrations and other potential endpoints of relevance. While the above studies showed evidence of an association between antibodies and risk of invasive infection, some infants developed disease despite having high antibody levels. Measurement of functional antibodies rather than overall antibody concentrations may be important to shed further light on immune correlates of protection, as total antibody levels might include inactive antibodies 272-274 . An example of this is the opsonophagocytosis killing assay (OPkA) 182,275 , which mimics the in vivo process of the killing of the bacterium by host effector cells following opsonization by specific antibodies. Antibody-mediated bacterial killing has also been shown to protect infants from GBS disease and may be a more useful marker than purely measuring antibody quantity via an enzyme-linked immunosorbent assay (ELISA)-type assay 267 . Functional antibody assessed by OPkA appears to correlate more closely with protection from GBS colonization, a precursor to disease in infants, than CPS-specific antibody concentration 276 . However, OPkA assays are laborious to perform and require large volumes of test sera. This is a critical issue in studies where sample volume is at a premium, such as in neonatal studies. Other assays, including an antibody-mediated complement C3b/iC3b deposition assay 28,277 have been developed that are less labor intensive and less variable as they do not rely on human phagocytes and require small serum volumes. Avidity assays have also been explored but results indicate no significant difference in median avidity between antibodies induced by unconjugated or conjugated vaccines with a large range of values obtained for both vaccines 278 .

Status of assay and reagent standardization efforts
Different assay methods, antigen constructs and standard quantitation for serotype-specific antibody levels 267,269 have made comparison across studies challenging 272 . Different specific antibody concentrations that could be associated with protection from disease have been defined. However, these vary across studies and by GBS serotypes (Table 6), and there has been significant controversy regarding appropriate laboratory methods to derive such thresholds reliably 279 .
Historically, the radioantigen binding assay (RABA) has been seen as the gold standard for the quantification of anti-GBS antibody as it measures antibody in its native state 169 . However, the RABA has low sensitivity towards the lower limit of quantification and is unable to identify immunoglobulin of different isotypes and subclasses as so offers an incomplete picture of immunoglobulin concentration. Several more sensitive isotypespecific ELISA have subsequently been developed and have been used in the majority of vaccine studies to date; however, the estimated antibody concentration required to reduce the risk of GBS disease varied 266,267,269,270,[280][281][282][283][284] .These assay methods vary, resulting in difficulties in extrapolating data between studies. More recently, studies have used Luminex or Bioplex platforms in order to improve the sensitivity and throughput of these assays and The suggested threshold was >1 μg/ml but OR of early onset GBS disease in neonates was calculated using maternal serum capsular polysaccharide-specific IgG concentrations at delivery of ≥0.5μg/ml compared to those with <0.1 μg/ml in a logistic regression model allow multiplexing. However, none of these ELISA or Luminex assays provide information on the ability of the antibodies to neutralize GBS. Therefore, an ELISA alone may not be sufficient in predicting protective immunity from GBS infection 272 . A possible solution to this may be the development of an effective functional antibody assay that could be used as an in vitro correlate of protection, such as OPkA.
However, to achieve this goal for GBS, assay standardization is required for each GBS antigen of relevance and for each serotype (Table 6. It is also possible that proposed thresholds might vary depending on study population differences (e.g., higher prevalence of HIV positive patients in the study 268 ). Efforts to standardize quantitative and functional immunoassays are needed for phase II and phase III GBS vaccine studies using immunogenicity endpoints.

Vaccine development pathway
The development of a GBS vaccine as considered here is unique in that the primary target population is pregnant women, as opposed to vaccines that WHO currently recommends in pregnant women (e.g., tetanus toxoid, inactivated trivalent influenza vaccine, acellular pertussis vaccine) which were not developed nor licensed to target pregnant women 285-287 . The anticipated vaccine development pathway will likely begin with preclinical studies relying on animal models to assess the immunogenicity and safety of the product. Potential adverse outcomes in both mothers and their offspring are evaluated, including reproductive and developmental toxicity associated with the product 288 . Upon favorable pre-clinical evaluation, first time in human studies are conducted in healthy adults. Phase I testing could start in non-pregnant women of childbearing age, in a limited number of participants (e.g., <100) 288 . Phase II studies of up to several hundred subjects per trial typically provide more information on common local and systemic reactions and immunogenicity evaluations of dose range and dose schedule 288 . Evaluation in pregnant women would typically only start upon favorable evaluation in non-pregnant women. In addition to adding to information on adverse events among mothers, phase II trials in pregnant women can provide initial information about safety effects in newborns, as well as information about IgG antibody transfer ratios to the newborn and duration/decay of these antibodies over time (see following section on endpoints of relevance in immunogenicity studies). Phase III trials would typically have a large enough sample size to provide data supportive of licensure 288 . Phase III pivotal licensure studies most classically include a well-defined primary clinical endpoint, but alternative pathways to licensure are being discussed in the case of GBS vaccines, considering the possibility of establishing a regulatory acceptable immune correlate of protection. Post-licensure evaluations may play a critical role in characterizing rarer safety events and effectiveness under real-world conditions, as well as in special populations of interest.

Safety of vaccination during pregnancy
Vaccines targeting maternal immunization during pregnancy must demonstrate favorable safety for the mother, the developing fetus and the newborn. Upon request by the WHO Strategic Advisory Group of Experts (the senior WHO vaccine governance board), the WHO Global Advisory Committee on Vaccine Safety (GACVS) recently reviewed safety data on existing vaccines for maternal immunization in pregnancy 289,290 . The GACVS concluded that there is no evidence of adverse pregnancy outcomes from the vaccination of pregnant women with currently licensed inactivated virus, bacterial, or toxoid vaccines. They concluded that pregnancy should not preclude women from immunization with these vaccines if medically indicated. As described in the previous paragraph, WHO currently recommends administration of tetanus toxoid, inactivated trivalent influenza vaccine, and acellular pertussis vaccine to pregnant women, although none of these vaccines were licensed for use in pregnant women [285][286][287] . Conjugate vaccines (either licensed or investigational), when conjugated with different carrier proteins (e.g., TT, DT, CRM 197 ), as well as vaccine formulations including alum and oil-in-water emulsions as adjuvants have been used in pregnant women, and favorable safety has been documented 174,252,291,292 . Further considerations on safety evaluation of GBS vaccine candidates are presented in following sections.

Current GBS vaccine candidates in development
Review of existing candidates CPS-based vaccines have been the most extensively studied among vaccine candidates, and trivalent glycoconjugate vaccine candidates have gone through phase I and II trials. Currently, there are no plans for these trivalent vaccine candidates to move on to phase III studies.
GBS protein vaccines using other target antigens 293 and polysaccharide vaccines conjugated with different carriers (e.g., GBS80 pilus protein, peptide) 233,294 have been tested in animal models. GBS-NN is undergoing phase I evaluation (NCT02459262). A summary of candidate vaccines is shown in Table 7.
Safety data from phase I and II studies Non-pregnant women. Multiple polysaccharide and protein conjugate GBS vaccines have been tested in healthy non-pregnant women, although the number of volunteers included was usually small (e.g., ≤30 in each vaccine group). Earlier studies testing vaccine dose-response have shown local pain or mild redness which seemed to be more frequent upon immunization with higher doses 241,247,248 . More recently, a phase Ib randomized, observer-blind and placebo-controlled trial of a trivalent (serotypes Ia, Ib, III) GBS CPS-CRM 197 conjugate vaccine was conducted among healthy nonpregnant women (NCT01150123) 295 . In this study, approximately 40 women were enrolled in each vaccine group, which consisted of different dosing schedule (e.g., one dose vs. two doses) and different use of adjuvants (no adjuvant, use of Al(OH) 3 , or MF59 [either half dose or full dose]). Results showed that local reactogenicity was increased in those who received vaccines with adjuvants (range: 40-42% in placebo group, 75-88% in vaccine group without adjuvants, 93-100% in those with Al(OH) 3 , 83-100% in those with half dose MF59, and 93-100% in those with full dose MF59); the proportion of solicited systematic reactions was less frequent (58-65% in the placebo groups, 50-85% across vaccine groups). Serious adverse reactions were similar among the vaccine and the placebo groups (5-11% in placebo group, 0-5% in vaccine group without adjuvants, 0-15% in those with Al(OH) 3 , 0-8% in those with half dose MF59, and 5-15% in those with full dose MF59), but none of them were considered related to vaccination, and there were no deaths or premature withdrawals due to adverse events (NCT01150123) 295 .
Pregnant women and newborns. The first phase I trial that used a glycoconjugate vaccine among pregnant women was conducted with III-TT vaccine with a saline placebo control group 175 . A total of 30 participants were enrolled, and no vaccine-associated serious adverse events were observed. Mild to moderate pain at the injection site occurred in 70% of the vaccine recipients compared to 40% in placebo recipients; 10% had redness at the injection site in the vaccine group compared to 0 in the placebo group. Obstetrical complications, mostly related to need for cesarean section or postpartum fever, occurred in 35% of vaccine and 70% of placebo recipients. All neonates had an uncomplicated hospital course in both groups. Results from a phase II randomized, observer-blind, multicenter study using trivalent (Ia, Ib, III) GBS polysaccharide-CRM conjugate vaccine among pregnant women has been published recently (NCT01446289) 174 . A total of 86 women at 24-35 weeks gestation were enrolled, of whom 51 were assigned to the vaccine group. Reports of solicited adverse reactions were similar between the groups, with 54% of the vaccine group vs. 53% in the placebo group reporting at least one solicited reaction. Reported rates of systematic reactions were similar, although more participants in the vaccine group reported local adverse reactions (40% in the vaccine group vs. 24% in the placebo group). All women gave birth to single, live born neonates, and obstetric outcomes were similar between the two groups. No infant deaths occurred during the study period, and serious adverse events were reported in 24% of the vaccine and 31% of the placebo group infants.
Immunogenicity Endpoints of relevance. The phase I/II trials using investigational trivalent GBS conjugate vaccines quantified GBS serotypespecific antibody levels using ELISA and reported as geometric mean concentrations (GMC) (NCT 01446289, NCT01150123, NCT01412801). None of these studies evaluated antibody functionality, but earlier GBS conjugate vaccine studies reported Opsonophagocytic assay (OPA) evaluation 175,241,247,248 . An ongoing phase I trial of GBS-NN is using both ELISA and OPA to measure immunogenicity.

Evidence from Phase I and II trials a. Non-pregnant women (NCT01193920, NCT01150123)
A phase Ib/II trial in which 40 non-pregnant women received two doses of trivalent GBS vaccine (Ia, Ib, III, 20/20/20μg) showed that compared to the placebo group, the geometric mean concentration (GMC) of antibody measured by ELISA a month after the second vaccination was significantly higher for all measured serotypes (serotype Ia 40 μg/mL in vaccine group vs. 0.88 in placebo group; serotype Ib 5.3 vs. 0.25; serotype III 11 vs. 0.61), and remained higher a year after the first dose (serotype Ia 15 μg/mL in vaccine group vs. 0.86 in placebo group; serotype Ib 5.28 vs. 0.4; serotype III 7.03 vs. 0.3) (clinicaltrials.gov: NCT01193920) 296 . In a study by Leroux-Roels and colleagues comparing vaccine groups with different antigen concentration, adjuvants, and dosing schedule (NCT01150123) 295 , results showed that all vaccine groups had a higher GMC compared to placebo groups at both 61 days and 361 days after vaccination; a higher dose level, the presence of aluminum hydroxide adjuvant or a second dose did not significantly increase antibody concentration. The exception was a higher GMC against serotype III one year vaccination in the group having received a second dose. When stratified by antibody concentrations at baseline, women who had undetectable antibody concentrations had lower antibody responses than those with detectable antibodies at baseline.

b. Pregnant women and newborn (NCT01446289)
The aforementioned phase I trial using III-TT vaccine in pregnant women reported that 19 of 20 recipients had 4-fold increases in III CPS-specific IgG after vaccination relative to pre-vaccination levels, infant cord levels were approximately 70% of maternal values at delivery, and opsonophagocytic killing measured in sera of infants born to vaccine-but not placebo-recipients persisted until 2 months of age, suggesting the potential to protect against both early-and late-onset GBS infant disease 175 . A phase II placebocontrolled trial using a single dose of trivalent (Ia, Ib, III, 5/5/5μg) GBS polysaccharide-CRM 197 conjugate vaccine administered to pregnant women at 24-35 weeks gestation was conducted in Belgium and in Canada (NCT01446289) 174 . Levels of antibodies against serotypes Ia, Ib, and III at delivery were respectively 16-, 23-and 20-fold higher than pre-vaccination. Of note, those with baseline antibody concentrations below the lower limit of detection had lower antibody responses compared to those with higher antibody levels at baseline. Infants born to vaccinated mothers had significantly increased antibody levels at birth, which persisted above placebo group levels at least 3 months after birth. Antibody concentrations decreased after birth and by day 91 were 22-25% of the levels measured at birth but were still 5-8.5 fold higher than those observed in the placebo group. There was only one (2%) preterm infant in the vaccine group, and there was no clear relationship between time from vaccination to delivery and maternal or neonatal antibody concentrations at birth for any of the serotypes. GBS-specific antibody ratios between vaccinated mother and infant (calculated as the paired ratio between the GBS-specific antibody concentration measured in the cord blood of the neonate to those measured in maternal sera at birth) ranged from 0.68 to 0.81 across the three serotypes (serotype Ia: 0.81, serotype Ib: 0.77, serotype III: 0.68). Currently, an extension study is underway to examine the safety and immunogenicity of a second dose of the trivalent vaccine administered in non-pregnant women after a time interval close to inter-pregnancy interval (NCT02690181).

Safety and immunogenicity evidence among special populations HIV-infected mothers and their newborns. A non-randomized
phase II open-label study using the trivalent (Ia, Ib, III) GBS polysaccharide-CRM conjugate vaccine was conducted in Malawi and South Africa among 270 pregnant women aged 18-40 years between 24-35 weeks gestation with or without HIV infection (NCT01412801) 252 . There was no control group. Enrolment stratification ensured that about half of the HIV-infected women were in a low CD4 cell count category [50-350 cells/μL] or high CD4 cell count category [>350 cells/μL]. Results showed that immune response to vaccines as well as serotype-specific antibody levels in infants at birth were lower in HIV-infected mothers and their infants. In mothers, the fold change in antibody concentrations was higher for the HIV-uninfected group than the HIV-infected groups, and those with undetectable antibody levels at baseline had lower antibody concentrations post-vaccination compared to those with detectable antibody concentration at baseline. Transfer ratios (infant geometric mean antibody concentration in blood collected within 72 hours of birth divided by maternal geometric mean antibody concentration in blood collected at delivery) were similar across all three groups (0.49-0.72).
Rates of women reporting at least one solicited adverse reaction were highest in the HIV-uninfected group (67%), compared with HIV-infected women with a low CD4 cell count (44%) or high CD4 cell count (59%). Local reactions (most frequently injection site pain) were reported by 18-39% of women across the groups, and systematic reactions were reported by 40-59% of women (fatigue and headache were most frequent). Adverse events were reported by 74-78%, of which 7-23% were deemed to be caused by study vaccination. None of the reported serious adverse events (reported by 28-32% of women) or adverse events reported in infants (41-49%) were deemed to be caused by vaccination.

Cost-effectiveness evaluation for low and middle income countries
GBS vaccine cost-effectiveness assessments may shed light on the potential investment case for GBS vaccines before phase III trials have been completed. Six analyses of GBS vaccine costeffectiveness have been published to date, including four before the current era of GBS vaccine development 297-300 and two recent analyses 301,302 .
The older studies evaluated cost-effectiveness in resource-rich settings (three in the United States and one in the United Kingdom). These documented the value of variants of screening-or risk-based intrapartum prophylaxis compared to 'doing nothing' and also assessed the potential value for a vaccine with assumed efficacy levels against GBS disease-causing serotypes, either as a maternal immunization strategy or as a vaccine delivered to adolescent females. The UK analysis 300 found that if a vaccine was available, the most cost-effective prevention strategy would include vaccination of all pregnant women, in combination with IAP for all preterm deliveries and a subset of term deliveries with risk factors (19% of all women treated). This study also emphasized the need for additional information on key model parameters.
Two more recently published cost-effectiveness analyses 301,302 focused on the conjugate trivalent vaccine (serotypes Ia, Ib, III) in clinical development at the time, assuming a single dose of GBS vaccine would be recommended during each pregnancy. The Oster analysis evaluated the addition of universal vaccination of pregnant women to the screening-based IAP strategy in the United States. Assuming a vaccine cost of $100 per dose and 75% vaccine efficacy against included serotypes among term deliveries and a reduced efficacy among preterm deliveries, this analysis found that the cost-effectiveness of maternal immunization may be comparable to other recently approved vaccines in the United States. A CDCsponsored cost-effectiveness analysis for the United States is in progress, with results anticipated in late 2016.
The Kim analysis focused on the upper middle-income country of South Africa. This decision-analytic model simulated the natural history of GBS transmission from mothers to infants and compared four strategies: do nothing, risk factor-based IAP, maternal GBS vaccination, and vaccination plus risk factor-based IAP. National and hospital-based GBS prevention policies in South Africa are consistent with variants of the risk factor-based IAP approach, although group of women eligible is quite narrow and implementation is limited. This analysis assumed a vaccine price per dose of 10-30 U.S. dollars (USD) and vaccine efficacy against included serotypes of 50-90% among term infants with a reduction among preterm infants. The most influential parameters in one-way sensitivity analyses were vaccine price per dose and early onset GBS disease incidence. This analysis concluded that maternal immunization would lead to important reductions in the burden of infant GBS disease and be considered very cost-effective (range 416-3,545 in 2010 USD/DALY averted comparing vaccination to doing nothing; range 461-5,491 2010 USD/DALY averted comparing vaccination to risk factor-based IAP). Notably, vaccination plus risk factor-based IAP was more effective and consistently very cost effective. Risk factor based IAP alone was also very cost effective but prevented only a small burden of infant GBS disease.
Sinha and team are also in the process of conducting a GBS vaccine cost-effectiveness analysis for GAVI-eligible low-income sub-Saharan African countries. Thirty seven countries in the region were clustered into four groups based on 24 measures of economic development, general health resources, and past success in public health programs. A decision-analytic model was built to compare a natural history arm ('do nothing') with maternal immunization as part of antenatal care. Risk factor-based IAP was not included in this assessment due to expert opinion that this was not feasible for these low income birth settings. Results are expected in late 2016.

Mathematical modeling related to GBS vaccines
Mathematical models, can inform decision-making related to vaccine development and implementation in several ways. For example, disease transmission models can shed light on the impact of varying key aspects of vaccine delivery such as age at vaccination, dosing schedules and method of delivery (e.g., vaccine campaigns versus incorporation into routine schedules). Models can also clarify the potential impact of a vaccine on unvaccinated members of the population (herd immunity) and predict potential unintended consequences of vaccine introduction such as an increased age at first infection, or the potential for replacement disease due to strains not included in the vaccine candidate. Finally, mathematical models can often highlight influential parameters where there would be value in a strengthened evidence base to allow for more accurate estimates.
In the context of maternal immunization for GBS, mathematical modeling to date is extremely limited. Some of the costeffectiveness models developed have included a natural history arm that estimates disease burden based on a variety of maternal risk factors 301 . A non-dynamic compartmental model that estimates GBS-related outcomes based on maternal GBS colonization and risk of neonatal disease is under development as part of a global GBS disease burden estimation activity led by the London School of Hygiene and Tropical Medicine. It is possible that models could prove useful to better understand the impact of maternal vaccination timing on the preventable portion of newborn disease, particularly since earlier vaccination may offer protection to later preterm deliveries, depending on antibody transfer ratios and decay rates. If conjugate GBS vaccines have an appreciable effect on reducing acquisition of GBS colonization with vaccine-included serotypes, models may also help assessing the consequences of reduced exposure of the newborn to GBS. Models may also help predict the impact of maternal immunization across LMIC settings with different prevalence of maternal HIV infection, levels of home vs facility deliveries, and optimal window for vaccination considering also antenatal care seeking behaviors. Models could contribute to the understanding of the potential impact of GBS vaccination on the burden of GBS-related stillbirth and preterm delivery.

Considerations about options to generate pivotal licensure data
The present document intends to provide an overview of available options and a framework for future reflection and should not be interpreted as guidance or recommendations.

Trial design options
Double-blind individually randomized controlled trial designs generate most robust data and minimize risks of bias. A relevant clinical endpoint 288 supportive of efficacy evaluation provides the most direct evidence of the potential health impact. However, low baseline disease incidence may lead to very large sample size requirements for vaccine efficacy evaluation. A potential alternative option may be to use immunologic correlates of protection as the primary endpoint 76,272 . Correlates of protection have been used for licensure in future generations or variants of a licensed product, or in instances where direct efficacy against disease is not readily feasible and correlates of protection are well-established. The evidence supporting recognition of a correlate of protection may be derived from an efficacy trial, which provides the opportunity for nested immunogenicity evaluations and detailed analysis of the relationship between immune and clinical endpoints. Alternatively, as in the case of GBS, immunological correlate of protection may be inferred from sero-epidemiological studies.
Correlates of protection have indeed been used for licensure of meningococcal, pneumococcal conjugate, and inactivated influenza vaccines. Group C meningococcal conjugate vaccine was licensed in the United Kingdom based on immunogenicity studies without efficacy data. These compared serum bactericidal assay titers induced by the experimental vaccine with a licensed serogroup C polysaccharide vaccine, which had an established evidence of efficacy and correlates of protection 274,303 . The 10-valent pneumococcal conjugate vaccine (PCV10) and the 13-valent pneumococcal conjugate vaccine (PCV13) were licensed based on non-inferiority trials compared against PCV7 using serological end-points 304 . Use of immunogenicity bridging studies comparing new vaccine products with those with established clinical efficacy is an accepted licensure pathway for inactivated seasonal influenza vaccine 305,306 . If licensure is granted based on a primary immunogenicity endpoint, there may be a regulatory requirement for post-licensure evaluations of effectiveness against disease endpoints. The optimal design of post-licensure trials need careful considerations as the inclusion of a non-vaccinated study arm may be deemed ethically unacceptable. Alternative case control, cluster randomized or ecological studies are possible.

Possible study endpoints
Trial with a disease endpoint GBS disease GBS invasive disease in young infants would likely be viewed as a relevant primary efficacy endpoint (see Table 8) 76 . Given that GBS-related stillbirths have similar pathophysiology as neonatal GBS disease (ascending infection from a colonizedmother), using a composite disease endpoint that includes GBS-related stillbirths is a possibility, which could help reduce the study sample size. Subgroup analyses may be used to assess the influence of various maternal factors (e.g., HIV infection, malaria, malnutrition, maternal age, multiparity) on protection. Factors that influence the extent of protection, such as when maternal vaccination occurs in relation to the birth of the child (allowing sufficient time for a maternal antibody response), the gestational age at birth (placental transfer will be less in those born prematurely) and the chronological age of the infant (antibody levels will wane over the first 2-3 months of life), may also need to be characterized 76 . Analyses of vaccine serotype-specific efficacy and efficacy stratified by term vs. preterm, early-onset disease vs. late-onset disease, and serotype-specific efficacy could be conducted 76 . Other endpoints of public health interest such as prevention of prematurity, stillbirths, hospitalization, and mortality could be considered but interpreted carefully in the context of multiple statistical testing and statistical power. See Table 8 for summary of candidate case definitions. • Requires sites to have a standardized specimen collection and processing procedure.
• Knowledge of colonization status before delivery may necessitate IAP.
• Indirect measurement of disease endpoint: a lack of reduction of colonization may not mean lack of protection against invasive infant disease CSF: cerebrospinal fluid, GBS: group B streptococcus, IAP: intrapartum antibiotic prophylaxis, LMIC: low-and middle-income countries, PSBI: probable severe bacterial infection, WHO: World Health Organization Colonization Newborn GBS colonization or exposure from colonized mothers is a precursor to GBS disease. The demonstration of vaccine efficacy against maternal and newborn colonization may argue for a protective effect of GBS vaccination. If vaccination reduces vaginal GBS colonization with the targeted invasive strains at the time of delivery, the risk of developing early-onset disease and potentially late-onset disease by strains targeted by the vaccine would likely decrease 76 . However, other factors may play a role, as only a small proportion of colonized neonates develop disease. Further considerations on case definitions are provided in Table 8.

Trial with immunologic correlates of protection.
For glycoconjugate GBS vaccines, evidence from immune-epidemiological studies suggest that maternally-transmitted, functional IgG antibodies against GBS capsular polysaccharides, as measured by a quality-assured opsonophagocytic assay in serum from neonates and/or young infants may constitute a candidate substitute endpoint (see immune correlates of protection section). Further evidence is needed to evaluate the possible role for immune markers of protection induced by protein vaccine candidate in the licensure pathway.

Considerations for licensure based on immune markers
While associations between antibody concentrations and risk of disease have been observed, the strength and nature of these associations require further investigation and continued assay standardization efforts. Several analytical frameworks for validating immune markers as substitute endpoints for protection against clinical disease have been developed 274 . The Prentice Criteria (see Box 2), originally designed for randomized-controlled trial data, but extended by others to observational designs 274 , can be used to evaluate potential substitute endpoints.
The evidence base to evaluate whether a substitute endpoint fulfils the Prentice Criteria would most typically come from a trial with a clinical disease endpoint and nested immune marker study. For a GBS candidate vaccine, evidence for these criteria may need to be gleaned from a range of experimental and observational studies. The first criterion, that protection against the clinical endpoint is significantly associated with vaccine receipt, may derive from animal challenge studies. Evidence for the second criterion (the immune marker is significantly related to vaccination status) would likely derive from phase II studies. Evidence for the third (the substitute endpoint is significantly related to protection against the clinical endpoint) would likely derive from sero-epidemiological observational cohort and case-control studies. Evidence for the fourth criterion may come largely from existing knowledge about immune response and protection among young infants in the first 3 months of life.
The Prentice Criteria are not the only approach to evaluation of a substitute endpoint. The Qin framework 307 can also be applied. This framework distinguishes associations between immune markers and clinical disease endpoints into three classes, and within these also offers more options for causal inference frameworks that can be applied. This framework also highlights whether a substitute endpoint is specific to a single population (the data derived just from one population) or whether it is general (meaning it has been observed in multiple populations).

Endpoint case definitions
Isolation of GBS from a normally sterile site, such as blood or CSF in an infant with possible sepsis or meningitis, is a widely used definition for young infant invasive GBS disease 22,24,32,37,38,46,146,147 . GBS isolation by culture is considered the reference standard. Automated culture methods yield higher detection rates compared to manual culture methods 105,308 ; minimizing time between collection and inoculation of blood culture bottles, using pediatric bottles for young infants, and maximizing blood volumes are important for optimal results 105 . For GBS meningitis, in addition to positive CSF culture, case definitions have included detection of GBS antigens in CSF (e.g., latex agglutination) 24,46 , detection by PCR 46 , and GBS positive blood culture with CSF findings consistent with meningitis 22,46,47 . As described, onset of disease during days 0-2 or 0-6 of life is commonly used for early-onset disease and onset during days 7-89 is used for late-onset disease 3,24,32,38,46,147 . Due to challenges in surveillance for invasive disease, some young infant studies have developed case definitions for probable GBS infection capturing infants with clinical sepsis and surface colonization with GBS 309-311 . Because surface colonization of young infants can be common, however, such definitions have limited specificity. Recently, some studies have used PCR on whole blood in addition to blood culture 95,312 . This can enhance detection but blood samples from healthy controls provide an important context: a low percentage of healthy controls have been documented with positive PCR on blood in both South Africa and South Asia (SANISA and ANISA unpublished studies). Another option for newborn disease is clinical sepsis. Several definitions have been used. PSBI, as defined by IMCI 103 is sensitive but not specific: the sensitivity is estimated to be 85% and the specificity of 75% based on an experienced pediatrician's assessment 101 . South Africa has used a more specific definition including both clinical and laboratory signs 15 . Use of chest X-rays may be considered if pneumonia is one of the outcomes of interest. Candidate case definitions are summarized in Table 8.

Sample size
The number of young infant GBS cases at single institutions is relatively small, depending on the number of annual deliveries and the disease incidence rate. A trial with a disease endpoint would likely necessitate a multi-center trial. An efficacy trial conducted in settings where standards of care include screening-guided IAP would lead to very large sample size requirements. If acceptable, trial conduct in a high incidence setting where screening-based IAP is not implemented as standard of care would reduce the sample size requirement. Adequate infection risk management in study participants would need to be discussed with relevant authorities and institutional review boards (IRBs), in consideration of local recommendations and WHO recommendations. Acceptability may be higher if favorable safety has already been established in a significant number of individuals. At a site with an incidence of 2.0 per 1000 live births for neonatal GBS disease <90 days of age, approximately 34-44,000 pregnant women will need to be enrolled (assuming that 75-85% of neonatal GBS disease are caused by vaccine serotypes, 70-80% are eligible, and 90% power to detect vaccine efficacy of 75% against vaccine serotypes), whereas >100,000 pregnant women will be needed in countries such as Europe and North America where IAP has reduced the incidence of early-onset disease to markedly less than 1 per 1000 live births 76 (Table 9. For conjugate vaccines this number will vary depending on the GBS serotypes contained in the candidate vaccine and the serotype distribution at the study site. A licensure trial based on an established immune correlate of protection would require a smaller sample size and the total pre-licensure exposed population would likely be determined by safety characterization requirements.
Considerations for safety evaluation are described in "safety considerations" and "regulatory considerations and potential licensure pathways for low-and middle-income countries".

Safety considerations
Evaluation of safety for a vaccine that will be specifically approved for use in pregnant women is unique given that: (1) the safety of both the mother and the fetus/child will need to be considered, and (2) complications of pregnancy may occur even in pregnancies considered as "low risk" regardless of the vaccination status 288 . Therefore, the relative risk of common adverse pregnancy outcomes in the study population should be determined. Sample sizes must be adequate considering baseline incidence of adverse pregnancy outcomes and may not be finalized until phase II safety data are available. Detection of rare adverse outcomes require large sample size. Baseline studies can be useful to determine sample size needs, which should be discussed in advance with regulators.
One of the challenges in assessing safety of maternal immunization has been a lack of standard definitions for maternal immunization adverse events 313  Ethical and standard of care considerations There is no regulatory or ethical prohibition on studies in pregnancy 317-319 . However, the concept of maternal vaccination, which may potentially pose harm to both the mother and the infant, may not be well received in countries where uptake of vaccines currently recommended for pregnant women by WHO is low 320 . If a randomized-controlled study is designed, an important consideration is whether the control group should receive another vaccine that is currently recommended by WHO rather than placebo 76 .
Controversies exist surrounding whether trials in low-and middle-income countries with a high burden of GBS disease should offer universal screening and IAP to their participants, the worldwide "best available" standard of care 321 . Arguments against this have been presented: provision of care that is not sustainable at the study site could produce results that are more generalizable in higher-income countries and have little social value for the host community 321 . Additionally, provision of a standard of care normally not available could coerce pregnant women into trial participation. Authors have suggested that study sites should adhere to local recommendations, in consideration of WHO guidelines 100,102,116 . The acceptability of a trial under local standards of care may be dependent on the benefit risk assessment and the available safety data on the candidate vaccine. Whatever the approach, GCP trials should be authorized and under oversight by local IRBs and recognized authorities, with participant agreement documented through an informed consent process. Considerations about research center characteristics in low-and middle-income countries Phase III studies with clinical outcomes as endpoints would likely need to be conducted in geographical locations with a high burden of neonatal invasive GBS disease (Table 9), which are likely to be in LMIC. Important trial site characteristics are reviewed in Table 10. Important aspects include the presence of experienced clinical-trialists and established Ethics Review Committees (ERC) and Regulatory Authorities (RA) oversight, to ensure the highest compliance with Good Clinical Practice standards 76 ; availability of clinical and laboratory infrastructure for optimal capture of PSBI cases for specimen collection, processing, and identification of GBS from collected specimens 105 ; proportion of home deliveries; access to care supportive of rapid clinical sepsis diagnosis and collection  of appropriate specimens close to disease onset 76 ; capacity to assess gestational age, provide sufficient medical care and to identify and respond to adverse events in both vaccinated pregnant women and their newborn infants 76 . Clinical management study algorithms can support standardized collection of safety events and endpoints of interest according to defined case definitions.
Potential challenges. Several review articles have summarized challenges in conducting studies that involve pregnant women in low-and middle-income countries 320,322 . Reaching women during the early stages of their pregnancy may be challenging in societies where women are reluctant about revealing their pregnancy early 322 , and may miss the window of enrollment and vaccine administration. In addition, accurate estimation of gestational age, which is important in assessing pregnancy outcomes (e.g., preterm), is often a challenge in resource-limited settings. Measurements that are typically used, such as based on last menstrual period or measurement of fundal height, often do not provide consistent results. It is important for the participants to deliver their infants at predictable locations affiliated with the study and to be able to follow through on the follow-up visits to assess study-specific adverse events. However, this can be challenging in settings where regular followup visit after delivery is not customary.

Regulatory considerations and potential licensure pathways for low-and middle-income countries
The regulatory considerations for products seeking an indication for use in pregnant women differ between already-licensed products and new products seeking licensure expressly for use among pregnant women. While there are several examples of already-licensed products with public health recommendations for use during pregnancy, there are no products yet that have achieved licensure for the specific indication of use during pregnancy. Respiratory syncytial virus (RSV) vaccines may represent the first pathogen class of vaccines that gain an initial indication for immunization of pregnant women as at least one RSV vaccine is ahead of GBS vaccines in their development timelines. Early dialogue between vaccine developers and regulators can play a particularly important role for maternal immunization product development. Major regulatory authorities, as well as the Council for International Organizations of Medical Sciences (CIOSM) in collaboration with WHO, have agreed that pregnant women should be presumed eligible for participation in research studies (CIOMS Guideline 17) 323 , and that this applies also to vaccines intended to protect primarily the offspring.
Licensure in the United States and Europe can be requested through due Food and Drug Administration (FDA) and European Medicine Agency (EMA) processes respectively. The Article 58 pathway provides a collaborative review framework between the EMA and WHO, for products not intended to be used in Europe. Submissions can be done as specified by relevant national regulatory authorities in LMICs. The African Vaccine Regulatory Forum (AVAREF) is a collaborative forum of regulators from different African countries, constituted to enable information sharing between African NRAs. The Developing Country Vaccine Regulators' Network (DCVRN) may also facilitate steps in regulatory processes in LMICs that are members.
Regulatory considerations from the FDA on the clinical development of vaccines indicated for use in pregnancy have been presented elsewhere 288 . In addition to evidence to support safety and effectiveness claims, maternal immunization submission packages may need to include information on potential immune interference in the infant, due to the transfer of maternal antibodies to the vaccine antigen or to carrier proteins that may share epitopes with carriers used in the infant vaccine series. The role of immunological correlates of protection will need to be clarified, especially with regards to the primary licensure. Relevant quality-assured immunogenicity endpoints may also be used to bridge across populations, for instance when considering generalizability across LMIC or between LMIC and resource-rich settings.
Safety evaluations would be conducted considering vaccine effects on both pregnant women and their infants, taking into account background rates of common pregnancy complications (e.g., pre-eclampsia, miscarriage/spontaneous abortion, stillbirth, preterm delivery). Pregnancy and neonatal outcomes, serious adverse events, new onset maternal medical conditions. The duration of safety follow-up for pregnant women and for newborns needs to be determined. Phase II data may be needed for optimal determination of Phase III sample size requirement for safety evaluation. Multiple factors including accumulated safety data associated with the product to date, safety signals, and the overall benefit to risk ratio assessment would likely be taken into account 324 . See previous section for considerations on safety in phase III trials. Lastly, the need for post-approval investigations should be reflected on. To maximize chances of success for a candidate vaccine, vaccine developers should plan ahead to overcome potential post-approval obstacles 325 . Target product profiles (TPP) list desirable characteristics, features, and attributes of a candidate vaccine, and have been long used by biotechnological and pharmaceutical companies 326 .

Areas for future research
We briefly summarize some of the leading scientific gaps relevant to GBS vaccine development and areas for future research based on the section topics addressed in this briefing document (Table 11).