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Background: Preterm birth is a leading cause of neonatal mortality, which is often complicated by intrauterine infection and inflammation. We have established a nonhuman primate model of Group B Streptococcus (GBS, Streptococcus agalactiae) infection-associated preterm birth. Immune checkpoints are modulators of the immune response by activating or suppressing leukocyte function and are understudied in preterm birth. The objective of this study was to spatially profile changes in immune protein expression at the maternal-fetal interface during a GBS infection with a focus on immune checkpoints. Methods: Twelve nonhuman primates (pigtail macaques, Macaca nemestrina) received a choriodecidual inoculation of either: 1) 1-5 X 108 colony forming units (CFU) of hyperhemolytic/hypervirulent GBS (GBSΔcovR, N=4); 2) an isogenic/nonpigmented strain (GBS ΔcovRΔcylE, N=4); or, 3) saline (N=4). A Cesarean section was performed at preterm labor or 3 days after GBS infection or 7 days after saline inoculation. Nanostring GeoMx® Digital Spatial Profiling technology was used to segment protein expression within the amnion, chorion, and maternal decidua at the inoculation site using an immuno-oncology panel targeting 56 immunoproteins enriched in stimulatory and inhibitory immune checkpoint proteins or their protein ligands. Statistical analysis included R studio, Kruskal-Wallis, Pearson and Spearman tests. Results: Both inhibitory and stimulatory immune checkpoint proteins were significantly upregulated within the chorioamniotic membranes and decidua (VISTA, LAG3, PD-1, CD40, GITR), as well as their ligands (PD-L1, PD-L2, CD40L; all p<0.05). Immunostaining for VISTA revealed positive (VISTA+) cells, predominantly in the chorion and decidua. There were strong correlations between VISTA and amniotic fluid concentrations of IL-1β, IL-6, IL-8, and TNF-α (all p<0.05), as well as maternal placental histopathology scores (p<0.05). Conclusion: Differential regulation of multiple immune checkpoint proteins in the decidua at the site of a GBS infection indicates a major perturbation in immunologic homeostasis that could benefit the host by restricting immune-driven pathologies or the pathogen by limiting immune surveillance. Protein expression of VISTA, an inhibitory immune checkpoint, was upregulated in the chorion and decidua after GBS infection. Investigating the impact of innate immune cell expression of inhibitory immune checkpoints may reveal new insights into placental host-pathogen interactions at the maternal-fetal interface. [ABSTRACT FROM AUTHOR]

Although hemolytic lipids have been discovered from many human pathogens including Group B Streptococcus (GBS), strategies that neutralize their function are lacking. GBS is a leading cause of pregnancy-associated neonatal infections, and adult GBS infections are on the rise. The GBS hemolytic lipid toxin or granadaene, is cytotoxic to many immune cells including T and B cells. We previously showed that mice immunized with a synthetic nontoxic analog of granadaene known as R-P4 had reduced bacterial dissemination during systemic infection. However, mechanisms important for R-P4 mediated immune protection was not understood. Here, we show that immune serum from R-P4-immunized mice facilitate GBS opsonophagocytic killing and protect naïve mice from GBS infection. Further, CD4+ T cells isolated from R-P4-immunized mice proliferated in response to R-P4 stimulation in a CD1d- and iNKT cell-dependent manner. Consistent with these observations, R-P4 immunized mice lacking CD1d or CD1d-restricted iNKT cells exhibit elevated bacterial burden. Additionally, adoptive transfer of iNKT cells from R-P4 vaccinated mice significantly reduced GBS dissemination compared to adjuvant controls. Finally, maternal R-P4 vaccination provided protection against ascending GBS infection during pregnancy. These findings are relevant in the development of therapeutic strategies targeting lipid cytotoxins. Author summary: Although many pathogens encode lipid toxins that contribute to disease pathogenesis, strategies to neutralize these toxins are lacking. The hemolytic lipid toxin encoded by Group B Streptococcus (GBS) is a major virulence factor that promotes disease including preterm births, in utero fetal injury and morbidity of human neonates and adults. No strategies exist to mitigate the effects of the GBS toxin, and neutralizing antibodies have not been isolated from infected patients. The global burden of GBS disease remains high with approximately 4 million neonatal deaths occurring worldwide each year and increasing rates of infections occur in adults. Using chemical synthesis, we synthesized a derivative of GBS toxin known as R-P4. We show that vaccination with this non-toxic analog induces the production of antibodies that promotes GBS killing, confers protection to naïve mice, neutralizes the cytotoxic activity and diminishes GBS infection during pregnancy. Furthermore, we show that lipid analog mediated protection requires antigen presentation by host molecules such as CD1d and immune responses of CD1d restricted T cells such as iNKT cells. These results provide the foundation for the design of novel therapeutic strategies that target lipid cytotoxins. Despite observations that lipid toxins continue to increase the severity and rate of infections caused by pathogens such as GBS, Mycobacteria and Pseudomonas, strategies to neutralize these toxins remain undefined. Our results indicate that a nontoxic analog of one such toxin can induce protective immunity during systemic and pregnancy associated infection, through lipid antigen presentation which provides the foundation for promising future strategies to neutralize the effect of lipid toxins during human infections. [ABSTRACT FROM AUTHOR]

Group B streptococci (GBS) are bacteria that can cause preterm birth and invasive neonatal disease. Heterogeneous expression of virulence factors enables GBS to exist as both commensal bacteria and to become highly invasive. A molecular epidemiological study comparing GBS bacterial traits, genotype and host characteristics may indicate whether it is possible to predict the risk of perinatal invasive GBS disease and more accurately target intrapartum antibiotic prophylaxis. A total of 229 invasive GBS isolates from Swedish pregnant women or neonates were assessed for virulence and phenotypic traits: hemolysis zone, hemolytic pigment (Granada agar), Streptococcus B Carrot Broth (SBCB) assay, CAMP factor, and hyaluronidase activity. Genes regulating hemolytic pigment synthesis (covR/covS, abx1, stk1, stp1) were sequenced. Of the virulence factors and phenotypes assessed, a Granada pigment or SBCB score ≥ 2 captured more than 90% of EOD isolates with excellent inter-rater reliability. High enzyme activity of hyaluronidase was observed in 16% (36/229) of the invasive GBS isolates and notably, in one case of stillbirth. Hyaluronidase activity was also significantly higher in GBS isolates obtained from pregnant/postpartum individuals versus the stillbirth or neonatal invasive isolates (p < 0.001). Sequencing analysis found that abx1 (g.T106I), stk1 (g.T211N), stp1 (g.K469R) and covS (g.V343M) variants were present significantly more often in the higher (Granada pigment score ≥ 2) versus lower pigmented isolates (p < 0.001, each variant). Among the 203 higher Granada pigment scoring isolates, 22 (10.8%) isolates had 3 of the four sequence variants and 10 (4.9%) had 2 of the four sequence variants. Although heterogeneity in GBS virulence factor expression was observed, the vast majority were more highly pigmented and contained several common sequence variants in genes regulating pigment synthesis. High activity of hyaluronidase may increase risk for stillbirth and invasive disease in pregnant or postpartum individuals. Our findings suggest that testing for GBS pigmentation and hyaluronidase may, albeit imperfectly, identify pregnant people at risk for invasive disease and represent a step towards a personalized medical approach for the administration of intrapartum antibiotic prophylaxis. [ABSTRACT FROM AUTHOR]

Background: Group B Streptococcus (GBS) transmission during pregnancy causes preterm labor, stillbirths, fetal injury, or neonatal infections. Rates of adult infections are also rising. The GBS-NN vaccine, engineered by fusing N-terminal domains of GBS Alpha C and Rib proteins, is safe in healthy, nonpregnant women, but further assessment is needed for use during pregnancy. Here, we tested GBS-NN vaccine efficacy using mouse models that recapitulate human GBS infection outcomes.Methods: Following administration of GBS-NN vaccine or adjuvant, antibody profiles were compared by ELISA. Vaccine efficacy was examined by comparing infection outcomes in GBS-NN vaccinated versus adjuvant controls during systemic and pregnancy-associated infections, and during intranasal infection of neonatal mice following maternal vaccination.Results: Vaccinated mice had higher GBS-NN-specific IgG titers versus controls. These antibodies bound alpha C and Rib on GBS clinical isolates. Fewer GBS were recovered from systemically challenged vaccinated mice versus controls. Although vaccination did not eliminate GBS during ascending infection in pregnancy, vaccinated dams experienced fewer in utero fetal deaths. Additionally, maternal vaccination prolonged neonatal survival following intranasal GBS challenge.Conclusions: These findings demonstrate GBS-NN vaccine efficacy in murine systemic and perinatal GBS infections and suggest that maternal vaccination facilitates the transfer of protective antibodies to neonates. [ABSTRACT FROM AUTHOR]

Leukocyte activation within the chorioamniotic membranes is strongly associated with inflammation and preterm labor (PTL). We hypothesized that prophylaxis with a broad-spectrum chemokine inhibitor (BSCI) would downregulate the inflammatory microenvironment induced by Group B Streptococcus (GBS, Streptococcus agalactiae) to suppress PTL and microbial invasion of the amniotic cavity (MIAC). To correlate BSCI administration with PTL and MIAC, we used a unique chronically catheterized non-human primate model of Group B Streptococcus (GBS)-induced PTL. In the early third trimester (128–138 days gestation; ~29–32 weeks human pregnancy), animals received choriodecidual inoculations of either: (1) saline (N = 6), (2) GBS, 1–5 × 108 colony forming units (CFU)/ml; N = 5), or (3) pre-treatment and daily infusions of a BSCI (10 mg/kg intravenous and intra-amniotic) with GBS (1–5 × 108 CFU/ml; N = 4). We measured amniotic cavity pressure (uterine contraction strength) and sampled amniotic fluid (AF) and maternal blood serially and cord blood at delivery. Cesarean section was performed 3 days post-inoculation or earlier for PTL. Data analysis used Fisher's exact test, Wilcoxon rank sum and one-way ANOVA with Bonferroni correction. Saline inoculation did not induce PTL or infectious sequelae. In contrast, GBS inoculation typically induced PTL (4/5, 80%), MIAC and fetal bacteremia (3/5; 60%). Remarkably, PTL did not occur in the BSCI+GBS group (0/4, 0%; p = 0.02 vs. GBS), despite MIAC and fetal bacteremia in all cases (4/4; 100%). Compared to the GBS group, BSCI prophylaxis was associated with significantly lower cytokine levels including lower IL-8 in amniotic fluid (p = 0.03), TNF-α in fetal plasma (p < 0.05), IFN-α and IL-7 in the fetal lung (p = 0.02) and IL-18, IL-2, and IL-7 in the fetal brain (p = 0.03). Neutrophilic chorioamnionitis was common in the BSCI and GBS groups, but was more severe in the BSCI+GBS group with greater myeloperoxidase staining (granulocyte marker) in the amnion and chorion (p < 0.05 vs. GBS). Collectively, these observations indicate that blocking the chemokine response to infection powerfully suppressed uterine contractility, PTL and the cytokine response, but did not prevent MIAC and fetal pneumonia. Development of PTL immunotherapies should occur in tandem with evaluation for AF microbes and consideration for antibiotic therapy. [ABSTRACT FROM AUTHOR]