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.Globally, group B 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 BStreptococcus (GBS), and is a Gram-positive diplococcus, originally known for causing bovine mastitis1. GBS remains the leading cause of neonatal sepsis and meningitis, and is associated with significant mortality and morbidity, including long-term neurodevelopmental sequelae2. Disease risk is the highest during the first 3 months of life3, 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 age1.4. 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 underestimates3. 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 newborns6. 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 low-and 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.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 countriesHaemophilus influenzae type b vaccines to the world’s poorest countries through theGAVI 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.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 early-onset 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, andThis 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.Given the purpose of this document, information used in the next two sections “” is primDefinition. 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 life7. See section on ‘Transmission. Early-onset disease is caused by vertical transmission through colonized mothers during or just before birth8. GBS can ascend from the vagina to the amniotic fluid after onset of labor or rupture of membranes9, although intrauterine infection without evidence of ruptured membranes has been reported11. 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 infection13. 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 disease16.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 antigens21. Although few risk factor analyses have been conducted in LMIC, epidemiologic characteristics of case series from these settings25, as well as a risk factor analysis of early-onset neonatal sepsis in South Africa26, 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 women28, and infants born to HIV infected mothers had lower anti-GBS surface binding antibody levels28. However, maternal HIV infection appears to be more of a risk for late-onset disease compared to early-onset disease.Disease onset and clinical presentation. Most cases occur within the first 24 to 48 hours31, and more than half of early-onset disease occurs in term (≥37 weeks gestation) infants: studies from sub-Saharan Africa have reported the proportion of pre-term infants among infants with early-onset disease to range from 10 to 46%25. Respiratory distress has been described as one of the most common presenting symptoms, ranging from 68% 25 to 83% 24. Early-onset disease most frequently manifests with bacteremia, and less frequently with meningitis. A study in Malawi demonstrated that about half (52%) of children with early-onset disease presented with sepsis , 17% with meningitis, and 14% with probable meningitis 22. In the US, infants with early-onset GBS disease present primarily with bacteremia (80%), meningitis (6%), or pneumonia (7%)32. A recent study from Uganda suggests that neonatal GBS infection may be an under-recognized cause of cerebral hypoxic encephalopathy33, although more data are needed to confirm the nature of the link between the two.Disease outcomes. Case fatality ratios reported in hospital-based studies from sub-Saharan Africa have ranged from 20% to 38%25, with higher case fatality among preterm infants24. A multi-country observational study conducted in Panama, Dominican Republic, and Hong Kong showed that the average number of days hospitalized ranged from 6 (Dominican Republic) to 15 (Hong Kong), with case fatality ratios ranging from 10% (Hong Kong) to 33% (Dominican Republic)34. Case fatality ratios in resource rich settings are notably lower , yet a study from the United States reported a nearly eight times higher risk of death among preterm cases compared to term cases35.Definition. Late-onset infections occur among infants aged 7–89 days of life36. In some instances the period from day 3–89 is considered7.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 disease37, and IAP has not impacted the late-onset disease burden in countries that provide IAP38. Nosocomial transmission, horizontal transmission from mother to infant after the perinatal period, and transmission from breast milk have also been described42, although it is unclear whether these are common routes of transmission38.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 colonization44. 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 disease45. Another prospective cohort study from Italy also showed that preterm infants had an increased risk for late-onset disease46.47. Another South African study reported that the incidence of early-onset disease was similar between HIV-exposed and un-exposed  but there was a 4.7-fold greater risk  for late-onset disease24. Similar results were reported from a study conducted in Belgium48.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 HIV-unexposed infants for early-onset disease vs. 3.2 (95% CI: 2.3–4.4) for late-onset diseaseDisease onset and clinical presentation. Studies reported different proportions of preterm infants (<37 weeks) among late-onset cases: 49% in the United States38, 25% in South Africa24, and 14% in Malawi22, suggesting this proportion may be lower in LMIC than in high-income countries. A study from Italy showed that term infants develop disease earlier  compared to preterm infants 46.25. 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 infection38.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%)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 disease49. 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  or presence of hypertonia or hyper-reflexia24. Results from the United Kingdom showed that 22% of survivors of neonatal meningitis (≤28 days of life) had mild to moderate sequelae , and 14% had severe sequelae  at 9–10 years of age50. 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) years2.32. In the systematic review by Edmondet al., the pooled result of all studies reporting case fatality for early-onset 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 Sinhaet 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 late-onset disease 51.Reported case-fatality ratios are lower compared to those of early-onset diseasePreterm delivery. GBS colonization during pregnancy has been associated with preterm delivery53, 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 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 membranes55. 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 withStreptococcus pneumoniae (8%) or group AStreptococcus (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.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 group56. 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 postpartum56. Pneumonia and puerperal sepsis have also been reported35. Unlike influenza, invasive GBS infection during pregnancy or the postpartum period was not associated with a longer hospital stay, an indicator of disease severity, or increased mortality risk, compared to non-pregnant women56. Most (81%) of these pregnancy-associated invasive infections occurred in absence of additional underlying conditions35.GBS can cause urinary tract infection, chorioamnionitis, endometritis, and bacteremia in women58. Some studies have suggested that GBS bacteriuria during pregnancy, possibly an indicator for heavy colonization60, may be associated with an increased risk for adverse obstetric outcomes, such as habitual abortion, intrauterine growth restriction, preterm labor, chorioamnionitis and premature rupture of membranes61. However, other studies have shown that asymptomatic GBS bacteriuria during pregnancy correlates poorly with GBS genital cultures at 35–37 weeks62 or at delivery63. Both GBS vaginal colonization and bacteriuria are commonly asymptomatic among pregnant women.Vaginal GBS colonization is considered to be a risk for maternal chorioamnionitis and postpartum endometritis67. Results from population surveillance from the United States showed a doubling of invasive GBS cases among non-pregnant adults (≥18 years) between 1990 and 200767. The only multi-province surveillance for invasive GBS disease among non-pregnant adults in LMIC that we are aware of comes from Thailand, where GBS was the leading pathogen in an invasive bacterial diseases surveillance system68.An increasing incidence of invasive GBS disease has been reported among non-pregnant adults in recent years, primarily from high-income countries where surveillance for invasive GBS disease among all ages has been conducted69. A study from South Africa showed that soft tissue abscesses and pneumonia accounted for 70% of the presentations and reported an overall 35% mortality among all identified GBS cases70. Another study from Malaysia showed that skin and soft-tissue infections accounted for >70% of all GBS infections71. The majority of disease occurs in people with significant underlying conditions, particularly diabetes mellitus. The proportion of subjects with diabetes reported in population-based surveillance for invasive disease ranged from 20% in Canada72 to ≥40% in the United States of non-pregnant adults aged ≥15 years67. Estimates of the prevalence of diabetes among adults with invasive GBS infections from other countries come primarily from single institution studies, and vary from 28% in Soweto, South Africa to 71% in Malaysia74. Other conditions associated with increased risk of invasive GBS disease among non-pregnant adults include atherosclerotic cardiovascular disease, obesity, cancer, heart failure, and renal disease67. Age is also a risk factor; data from the United States, Europe and Thailand have shown that incidence rates for invasive GBS are highest among adults aged ≥65 years68.Skin- and soft- tissue infections are one of the most frequent clinical presentations in adults, although clinical syndromes associated with invasive GBS infections are variable, including bacteremia, pneumonia, bone and joint infections, urosepsis, endocarditis, meningitis, and intravenous catheter infections75. Invasive infections are most commonly diagnosed based on isolation of GBS from a normally sterile site  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 available75. 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 settings76. Finally, incidence of neonatal GBS varies regionally14. 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 LMIC3.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 causes14, suggesting that the relationship between maternal colonization and newborn disease is not simple.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 incidence77. However, various definitions of stillbirth have been used, making comparisons difficult between countries, or even within the same country80. In addition, stillbirths are not reported in national surveillance systems in about 90 countries81, and even where stillbirth is included in vital reporting systems, the causes of stillbirth are generally not recorded82. Diagnostic procedures may not be conducted or may not be available, and even if performed, there may be difficulties in producing valid results, as pathologic changes could have occurred before the time of investigation. Because GBS is a common colonizing organism of the birth canal, distinguishing the presence of GBS  due to colonization from a direct role of GBS in fetal death adds further challenge in identifying the cause of death82.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 GBS-related stillbirths poses challenges even in high-income countries. First, there is inconsistency in the definition used for stillbirth. For international comparison, the World Health Organization (WHO) defines stillbirth as a baby born with no signs of life at or after 28 weeks gestationInfants aged <90 days. Neonatal sepsis (in infants aged <1 month) is one of the leading causes of neonatal deaths globally83, and among the WHO regions, the burden is the highest in the African region, where it was estimated to cause 5.3 deaths per 1,000 live births in 201284.3 . This difference underscores that invasive disease estimates from blood culture are minimum estimates unless they take into account blood culture sensitivity.A group at the London School of Hygiene and Tropical Medicine is updating the global GBS disease burden estimates, focusing on neonatal and maternal disease; results are expected in 2017. The most recent systematic review and meta-analysis currently available of invasive GBS disease in infants aged <90 days was published in 2012. This review showed that incidence of GBS disease was the highest in Africa , followed by the Americas , and lowest in southeast Asia 3 . While I85 study attempted to fill this gap by conducting population-based surveillance and etiologic evaluation of possible serious infections among newborns <60 days of age in the community in catchment areas in Bangladesh, Pakistan and India86. The study faced some anticipated challenges in registering babies on day 0 of life  and in capturing specimens from babies who died, particularly on day 0 of life. The study nevertheless included samples from over 970 babies with possible serious infection onset on day 0 of life. At all study sites, presence of GBS was assessed by blood culture, and by PCR on whole blood and PCR on nasopharyngeal/oropharyngeal samples on both ill babies and healthy controls. Colonization was detected, providing evidence that GBS is present in this region, consistent with other studies in Bangladesh and India87. However, culture-confirmed infections were rare among babies with possible serious infection in the ANISA study; culture-confirmed GBS infections were identified at the Sylhet site (Bangladesh) and Vellore site (India). Fuller results are under preparation for publication.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)3 3  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 Mediterranean3 . However3 . Althoug 37%)3 . A more Maternal colonization. Ascertainment of maternal genital GBS colonization varies according to the specimens collected , the culture medium, specimen transport and processing procedures and timing. In general, rectovaginal sampling has a higher yield than vaginal sampling only90, and use of selective broth media is better compared to nonselective blood agar89. A review estimated the prevalence of genital colonization in pregnant women to be around 13% globally, although included studies used various laboratory methods91. 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 multi-country cross-sectional study among pregnant women between 20 and 32 weeks gestation which used a standardized laboratory method  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 burden92.93. Serotype distribution in colonized mothers may not correlate directly with serotypes causing invasive neonatal disease, as invasiveness appear to be different according to GBS serotype94. 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 isolated51. Another study that took place on the Thai-Myanmar border showed that serotype II was the most frequently isolated serotype (24%), followed by Ia, VI, III, and V95. 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 , whereas serotype VII was the most frequently isolated serotype in Khon Kaen, Thailand, and was the only site that reported this serotype92. The GBS global serotype distribution appears more diverse than previously reported; a recent review of maternal GBS colonization showed significant heterogeneity across and within regions96. 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.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 serotypePregnancy-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 years56.97. GBS-related stillbirth rates ranged from 0.04–0.9 per 1,000 births with highest reported from a small study in the United States11, 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 published98, 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 .A recent systematic review evaluated the incidence of GBS-related stillbirth 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 73 to 7.3 per 100,000 population 67, and the rates tend to be higher with increasing age67. 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 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 III72, whereas reports from Europe show that serotype V (approximately 20%) was less frequent compared to serotype III (25–30%)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.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 infantsClinical evaluation of sick childrenIntegrated 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 births15. 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 antibiotics102. 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-appearing9.103.IMCI has a separate set of algorithms for children 2 through 59 months. Children are first assessed for general danger signs . If stiff neck or general danger signs are present in a child with fever, administration of antibiotics and urgent referral are recommendedLaboratory detection of invasive disease Confirmation of invasive GBS disease requires isolation of GBS from a normally sterile site , 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 CO2 detection, and eliminates the need for manual inspection or examination. In addition, growth of aerobes and facultative anaerobes are promoted by agitating culture bottles104. 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 methods105. Therefore, differences in the culture methodology used can result in variation in reported GBS disease burden.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 blood108. 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.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 recommended7. Therefore, children suspected of meningitis should undergo lumbar puncture to assess the CSF whenever possible. Laboratory methods to identify GBS are summarized ine.g., > 0.4 g/litre), and positive Gram stain results indicate presence of meningitis, and treatment should be started immediately while awaiting culture results102.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 culturee.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.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. More reNeonatal 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 , not just maternal exposure111.Pregnant and postpartum womenMaternal colonization Maternal colonization can be assessed by collecting swabs from the vagina and the rectum from pregnant women9. 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 , fetal tachycardia (>160/min), and purulent or foul amniotic fluid113. 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 chorioamnionitis116.Endometritis. The diagnosis of endometritis is also often made clinically, and is often due to polymicrobial infection117. Clinically, endometritis presents as fever, uterine tenderness, abdominal pain, and a purulent lochia or a positive culture of endometrial fluid or tissue118. Positive blood cultures may help identify the bacterial etiology, as bacteremia may be present in up to 20% of women118. 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 endometritis116.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 causality119. Pregnant women may be colonized with GBS, and could contaminate the fetus or the placenta after membrane rupture or vaginal contamination during delivery119. 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 stillbirths120, and culture of fetal heart blood or fluid from uncontaminated fetal sites during autopsy may help identify the infectious cause119.Serological methods. Serological classification of GBS is based on the identification of capsular polysaccharides and protein antigens121. Capsular polysaccharide is currently the most advanced glycoconjugate vaccine target, and currently ten serotypes have been described . Several serological methods have been used for serotyping . The Lancefield capillary precipitin method is considered as the “gold standard”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 polysaccharides126. 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  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 serotype126. In addition, results are dependent on the quality of the antibodies used and on the experience of the laboratory126.Molecular typing methodsSerotyping 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 approaches128. 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 studies1. However, PCR serotyping could potentially misclassify certain serotypes126. Also, PCR serotyping does not reveal if the capsular polysaccharide gene locus detected is actually expressed as a polysaccharide capsule126. Recently, Sheppard and colleagues have conducted whole genome sequencing to determine serotype with promising results129. Although the method currently may not be cost-effective merely for determining serotype129, 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 complexes130.Genotyping Molecular typing methods have been used for further characterization of GBS and are useful in distinguishing different GBS strains in epidemiological studies121. 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 microarray-based typing134. 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 organism135. SeeBox 1. Serotype: type of antigenically variable polysaccharide capsuleGenotype: 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 traitsStrain: a single isolate of any bacterial population and any laboratory induced variants thereofClonal 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 variationNewborns/young infants. IMCI recommends hospitalization and intramuscular or intravenous treatment of all infants meeting the case definition for PSBI136. The recommended antibiotic selection for management of “serious bacterial infection” and “meningitis” in infants aged <2 months is ampicillin and gentamicin  or intravenous (IV) ampicillin and gentamicin in neonates with documented risk factors for infection  to prevent early-onset 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 America141. 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. A systematic review on community-acquired neonatal and infant sepsis in developing countries  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 cephalosporins142.145. 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 findings147. 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  at preventing invasive early-onset GBS diseaseates see; however148. 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 quantified149.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 recipients9. 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 risk-based 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 risk-based strategy. Variants of the culture-based screening strategy also exist across countries but most recommend screening at 35–37 weeks gestation.Two major strategies have been employed to limit the portion of women exposed to intrapartum prophylaxis to those at most risk of transmitting GBS5. 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.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 settings100. 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)151.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 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 women150; 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.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-ascertainmentWhile 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.15. Universal administration of intramuscular penicillin to newborns within 1 hour of birth is implemented at one large center in the United States154; 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 impact157.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 sepsis159. GBS expresses a number of virulence factors, which play different roles in these steps  have been identified164.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 needed165. ST17 displays meningeal tropism, and has been referred to as the hypervirulent clone166.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 III161. 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 process161, 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 infections167, 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 ST17168. 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 model161.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 clonese.g., transferring sera of animals exposed to GBS disease) and active immunization  have been shown to be protective against development of GBS disease . In humans, transplacental transfer of protective maternal antibodies against GBS was first reported by Baker and colleagues169. 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 serotypes171 and the association of low maternal GBS CPS specific IgG levels and the risk of GBS disease in their infants was further described19. Attempts have been made to identify a threshold that would confer protection against GBS disease for vaccine development.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 : maternal IgG is endocytosed within placental syncytiotrophoblast cells and bound to FcRn in the acidic environment of the endosome, then released to the fetal side of the syncytiotrophoblast when exposed to physiological pHet al. using data from the United States estimated that third trimester maternal immunization could prevent approximately 46% of all late-onset GBS infections, given that 63.3% of infants who developed late-onset disease were born ≥35 weeks gestation, and of those born ≥35 weeks gestation, approximately 72% of infections occurred during the first 6 weeks of life176. 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 . The optimal timing of maternal immunization that would maximize protection against young infants requires further investigation.A study by Baker178. Another study reported an association between increased serum CPS IgG levels and reduced homotypic GBS rectovaginal acquisition179. 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.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)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 phagocytes181. 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 antibody183.Opsonization, followed by phagocytosis (ingestion of invading microorganism) and intracellular killing are the main mechanisms of host defense against GBS infection184. Newborns have an impaired ability to form antibodies in general, and are particularly deficient in their ability to mount antibody responses against polysaccharide antigens185. Altogether, their capacity for GBS CPS antigen-specific protection is determined largely by the placental transfer of maternal IgG antibodies186. 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.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 GBSGBS 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.122. In later studies, intraperitoneal or intravenous models of GBS infection in adult or neonatal mice were developed to simulate human infections188. In some cases, oral inoculation has been used as a means of inducing systemic infection in mice189. Notably, in both mice and rats, there appears to be an age-related decrease in susceptibility to invasive GBS infection191.Mice have been commonly used to model GBS infections. The earliest animal models studies of GBS infections date to the 1930s192, rabbits193, sheep196, piglet197, and non-human primates199. Some of these models  have been used to provide insights that are difficult or impossible to study in mice. The sheep196 and piglet models197 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 responsesin vivo199.A large number of other animal model systems have been explored, including chicken embryoAnimal 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.208. Gastrointestinal GBS colonization has been modeled in gnotobiotic mice and used as a means to understand the role of surface proteins in GBS carriage209. Neonatal mice have also been used as a model for gastrointestinal carriage, especially as a prelude to invasive disease168. Oral colonization of infant rats has been used to examine the utility of antibiotics to decrease mucosal bacterial load210.Vaginal colonization models have allowed determination of specific bacterial or host factors involved in carriage in the absence of invasive diseaseAnimal models of ascending infection and/or perinatal GBS transmission. To examine the role of ascending infection in adverse pregnancy outcomes , animal models simulating human infections have been explored. Examples include intracervical GBS inoculation of rabbits213, murine intravaginal/intrauterine/intraperitoneal inoculation216; catheterization and intraamniotic instillation of GBS has been used to model chorioamnionitis in non-human primates and to study its effects on fetal lung tissue220. However, initial attempts to create an ascending infection animal model secondary to chronic vaginal colonization, which is a better simulation of human infection, were unsuccessful213. 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 colonization203.Preclinical studies of GBS vaccines in animal modelsa. Passive immunization Animal models have been used to examine the effect of antibody delivery (passive immunization) on invasive GBS diseasein vivo. The first studies used generation of antibodies in rabbits followed by passive protection of mice exposed to systemic GBS infection221. Subsequent studies used hyperimmune serum or purified antibody preparations to provide protection to neonatal experimental animals199.b. Active immunization Active immunization formulations used in experimental animals have included whole killed GBS, purified bacterial components , or conjugate vaccine preparations. Most studies have used systemic vaccination , though some investigations of mucosal vaccine delivery  have been reported227.228. The most frequently used model to evaluate the efficacy of GBS vaccines is the mouse maternal vaccination-neonatal pup challenge model. In this model, female mice are actively vaccinated, and their offspring are challenged with GBS230. Maternal IgG is transferred to the pups similar to maternal antibody transfer in humans, and most pups of mothers vaccinated with a range of conjugate formulations have survived challenge232. This model continues to be used to test new vaccine targets235. 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 caution236. For example, human GBS isolates may express surface proteins that specifically interact with the human hosts but not with other animals238. 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 neonates239.Most vaccine studies have used protection from invasive infection as their outcome, but reports of potential effects of vaccination on GBS colonization have also been presented240. As in mouse models, these studies showed that GBS conjugate vaccine induced CPS-specific antibodies240, and there was a correlation between maternal and infant baboon serum antibody levels230. Differences have been noted in the kinetics of antibody responses and waning between humans and baboons241.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 studiesPolysaccharide 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 GBS242. The first purified type III CPS vaccine underwent phase I testing in healthy adults in 1978243, 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 recipients244. 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 response245. Favorable safety of CPS vaccines was shown on a small scale in non-pregnant adults and among pregnant women246, and infant antibody levels in cord serum correlated with maternal antibody levels at delivery246.Glycoconjugate vaccines. Immunogenicity of polysaccharides is enhanced by covalent conjugation with a carrier protein. Glycoconjugate vaccines have been developed forHaemophilus influenzae type b (Hib),Neisseria meningitidis andStreptococcus 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 switching160.247. Healthy non-pregnant women were recruited and randomized to receive III-TT, type III CPS vaccine, or placebo247. 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 vaccine247, 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 adjuvant249. 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 178.The first GBS glycoconjugate vaccine trial conducted in humans involved a GBS III CPS-tetanus toxoid (III-TT) glycoconjugate251. Novartis (now GSK) has developed a trivalent  glycoconjugate vaccine conjugated to a CRM197 carrier and conducted several phase I and II clinical trials in healthy non-pregnant and pregnant women .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 vaccines250. CRM197, a nontoxic mutant of diphtheria toxin (DT), is another carrier protein, and has been used in the investigational trivalent GBS vaccine that went through phase II studies 252. A study showed equivalent immunogenicity of CPS V-TT and CPS V- CRM197 against the target GBS antigen249.Initially, GBS vaccine studies have used tetanus toxoid (TT) as the carrier protein, but there have been concerns about possible immune interference and adverse events upon subsequent vaccination with TT-conjugated vaccines®) and DT  have shown to induce immunity against TT or DT, vaccines using CRM197 as the carrier protein  have not shown to induce immunity against DT253. The interest of inducing protection against tetanus through a TT-conjugated GBS vaccine could be considered where maternal and neonatal tetanus remain a concern.While vaccines conjugated to TT  (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 proteins259.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 candidates250. 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 model260. However, coverage against all GBS strains was not possible due to antigenic variation associated with the pilin subunits260. 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 variability250. 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 inEscherichia coli which conferred strong protection against all six strains expressing a BP-2a variant in challenged mice235.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 candidatesGBS-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 , limited data suggest that antibodies against alphaC and Rib proteins may provide protection against invasive neonatal GBS disease265.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 . More recent studies with larger sample sizes  have attempted to identify a serotype-specific IgG level in mothers that would confer protection against infant disease due to the same serotype269. A summary of studies published after 2000 is shown in269. 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 . 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 early-onset serotype VIII infection270. 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 275, which mimics thein 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 assay267. 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 concentration276. 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 assay277 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 vaccines278.269 have made comparison across studies challenging272. Different specific antibody concentrations that could be associated with protection from disease have been defined. However, these vary across studies and by GBS serotypes  has been seen as the gold standard for the quantification of anti-GBS antibody as it measures antibody in its native statee.g., higher prevalence of HIV positive patients in the study268). Efforts to standardize quantitative and functional immunoassays are needed for phase II and phase III GBS vaccine studies using immunogenicity endpoints.However, to achieve this goal for GBS, assay standardization is required for each GBS antigen of relevance and for each serotype  which were not developed nor licensed to target pregnant women287. 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 product288. 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 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 schedule288. 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 licensure288. 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.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 , when conjugated with different carrier proteins , 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 documented292. Further considerations on safety evaluation of GBS vaccine candidates are presented in following sections.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 pregnancyCPS-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.293 and polysaccharide vaccines conjugated with different carriers 294 have been tested in animal models. GBS-NN is undergoing phase I evaluation (NCT02459262). A summary of candidate vaccines is shown inGBS protein vaccines using other target antigensNon-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 . Earlier studies testing vaccine dose-response have shown local pain or mild redness which seemed to be more frequent upon immunization with higher doses248. More recently, a phase Ib randomized, observer-blind and placebo-controlled trial of a trivalent  GBS CPS-CRM197 conjugate vaccine was conducted among healthy non-pregnant women (NCT01150123)295. In this study, approximately 40 women were enrolled in each vaccine group, which consisted of different dosing schedule  and different use of adjuvants 3, or MF59 [either half dose or full dose]). Results showed that local reactogenicity was increased in those who received vaccines with adjuvants 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 . Serious adverse reactions were similar among the vaccine and the placebo groups 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 group175. 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  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.Endpoints of relevance. The phase I/II trials using investigational trivalent GBS conjugate vaccines quantified GBS serotype-specific antibody levels using ELISA and reported as geometric mean concentrations (GMC) . None of these studies evaluated antibody functionality, but earlier GBS conjugate vaccine studies reported Opsonophagocytic assay (OPA) evaluation248. An ongoing phase I trial of GBS-NN is using both ELISA and OPA to measure immunogenicity.Evidence from Phase I and II trialsa. Non-pregnant women  A phase Ib/II trial in which 40 non-pregnant women received two doses of trivalent GBS vaccine  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 , and remained higher a year after the first dose  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 disease175. A phase II placebo-controlled trial using a single dose of trivalent  GBS polysaccharide-CRM197 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  ranged from 0.68 to 0.81 across the three serotypes . 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).HIV-infected mothers and their newborns. A non-randomized phase II open-label study using the trivalent  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  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.300 and two recent analyses302.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 cost-effectiveness have been published to date, including four before the current era of GBS vaccine development300 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 . This study also emphasized the need for additional information on key model parameters.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 analysis302 focused on the conjugate trivalent vaccine  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 and75% 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 CDC-sponsored cost-effectiveness analysis for the United States is in progress, with results anticipated in late 2016.Two more recently published cost-effectiveness analysesThe 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 . 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.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.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  and the 13-valent pneumococcal conjugate vaccine (PCV13) were licensed based on non-inferiority trials compared against PCV7 using serological end-points304. Use of immunogenicity bridging studies comparing new vaccine products with those with established clinical efficacy is an accepted licensure pathway for inactivated seasonal influenza vaccine306. 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.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 protectionTrial with a disease endpointGBS disease GBS invasive disease in young infants would likely be viewed as a relevant primary efficacy endpoint , 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  on protection. Factors that influence the extent of protection, such as when maternal vaccination occurs in relation to the birth of the child , the gestational age at birth  and the chronological age of the infant (antibody levels will wane over the first 2–3 months of life), may also need to be characterized76. 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 conducted76. 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. Seeoint see76. GiveColonization 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 decrease76. However, other factors may play a role, as only a small proportion of colonized neonates develop disease. Further considerations on case definitions are provided inTrial 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 developed274. The Prentice Criteria  is significantly related to vaccination status3. The substitute endpoint is significantly related to protections against the clinical endpoint4. The full effect of the vaccine on the frequency of the clinical endpoint is explained by the substitute endpoint, as it lies on the sole causal pathwayThe 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  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.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).The Prentice Criteria are not the only approach to evaluation of a substitute endpoint. The Qin framework147. GBS isolation by culture is considered the reference standard. Automated culture methods yield higher detection rates compared to manual culture methods308; minimizing time between collection and inoculation of blood culture bottles, using pediatric bottles for young infants, and maximizing blood volumes are important for optimal results105. For GBS meningitis, in addition to positive CSF culture, case definitions have included detection of GBS antigens in CSF 46, detection by PCR46, and GBS positive blood culture with CSF findings consistent with meningitis47. 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 disease147. 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 GBS311. 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 culture312. 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 IMCI103 is sensitive but not specific: the sensitivity is estimated to be 85% and the specificity of 75% based on an experienced pediatrician’s assessment101. South Africa has used a more specific definition including both clinical and laboratory signs15. Use of chest X-rays may be considered if pneumonia is one of the outcomes of interest. Candidate case definitions are summarized inIsolation 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 disease76 , 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 , 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 births288. 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.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 status313. In 2014 WHO held a consultation jointly with the Brighton Collaboration to facilitate harmonization of existing key terms to support monitoring of vaccine safety in pregnant women and newborn children314. Key terms were chosen for discussion based on (1) frequency, (2) severity of health outcome, (3) public health relevance, and (4) measurability in different settings. The Global Alignment of Immunization Safety Assessment in pregnancy (GAIA) established working groups to review the evidence from the WHO-Brighton Collaboration landscape analysis, and has developed a set of interim case definitions according to the Brighton Collaboration process and format315. As of September 2015, the working group has developed 10 case definitions currently undergoing peer-review316.One of the challenges in assessing safety of maternal immunization has been a lack of standard definitions for maternal immunization adverse events319. 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 low320. 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 placebo76.There is no regulatory or ethical prohibition on studies in pregnancy321. 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 community321. 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 guidelines116. 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.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 care76; availability of clinical and laboratory infrastructure for optimal capture of PSBI cases for specimen collection, processing, and identification of GBS from collected specimens105; proportion of home deliveries; access to care supportive of rapid clinical sepsis diagnosis and collection of appropriate specimens close to disease onset76; 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 infants76. Clinical management study algorithms can support standardized collection of safety events and endpoints of interest according to defined case definitions.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 , which aPotential challenges. Several review articles have summarized challenges in conducting studies that involve pregnant women in low- and middle-income countries322. Reaching women during the early stages of their pregnancy may be challenging in societies where women are reluctant about revealing their pregnancy early322, and may miss the window of enrollment and vaccine administration. In addition, accurate estimation of gestational age, which is important in assessing pregnancy outcomes , 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 follow-up visit after delivery is not customary.323, and that this applies also to vaccines intended to protect primarily the offspring.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)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.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.Regulatory considerations from the FDA on the clinical development of vaccines indicated for use in pregnancy have been presented elsewheree.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 account324. 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 obstacles325. Target product profiles (TPP) list desirable characteristics, features, and attributes of a candidate vaccine, and have been long used by biotechnological and pharmaceutical companies326.Safety evaluations would be conducted considering vaccine effects on both pregnant women and their infants, taking into account background rates of common pregnancy complications  where the burden of disease is at its greatest whereas most of the available data are from resource rich countries. In addition, the review covers other important topics often omitted from other overviews such as the increased risk of neonatal GBS disease in HIV-positive mothers, the use of protein based vaccines, reverse vaccinology and the necessary use of surrogate endpoints in vaccine trials. Importantly, they have addressed the problem that logistically, because of the estimated sample size for a phase III randomised controlled trial using a clinical endpoint of invasive neonatal disease, the different burden of disease across geographic regions dictates that trials may have to be performed in LMICs. This has its own problems, not the least of which is the acceptability of local standards of care in the context of such a trial. Finally, a minor criticism would be the omission of a discussion on the association between black race and GBS rectal colonisation, which is particularly pertinent bearing in mind the burden of disease in Africa and in African American women in the USA. In addition, while the use is mentioned, the role of rapid detection of GBS carriage using automated PCR techniques that might decrease the unnecessary use of intrapartum antibiotic prophlyxis during labour merits some emphasis.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This is an excellent review article summarizing the status of GBS vaccine development for global use. This manuscript, in a logical and practical way, lays out the case for further development and testing of a GBS vaccine to be given to mothers primarily for the protection of their neonates. The burden of maternal and neonatal disease is thoroughly discussed and highlights the paucity of epidemiological data in many resource limited settings. This is the critical data needed to determine the contribution of GBS to overall neonatal morbidity and mortality in low and middle income countries where a candidate vaccine is most likely to be beneficial. There is an excellent list and discussion of previous GBS vaccine trials and outcomes. Even more useful is the discussion of challenges going forward with GBS vaccine trials, including a review of potential trial endpoints, challenges of any maternal vaccination program, and potential ethical and regulatory hurdles.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.