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647. Characterization and Development of Human Monoclonal Antibodies to Pneumococcal Serotype 3

Abstract Background Streptococcus pneumoniae is the main bacterial cause of pneumonia in the United States and globally. Although pneumococcal conjugate vaccines are highly effective against invasive pneumococcal disease, they are less effective against pneumonia, particularly in the elderly and those with immune deficiency. Given the additional challenge of antibiotic resistance, immunotherapy holds promise for treatment of pneumococcal pneumonia. The current PCV13 vaccine is less effective against serotype (ST) 3, which carries a higher risk of mortality than other vaccine-included STs. Our group has previously identified murine monoclonal antibodies (mAb) to ST3 capsular polysaccharide (PPS3) that are protective in experimental models of sepsis and pneumonia. The aim of the present study is to isolate and develop PPS3-specific human monoclonal antibodies (humAbs) as adjunctive immunotherapy for pneumonia. Methods We sorted individual PPS3-specific memory B cells from PBMCs isolated on days 0 and 7 post-vaccination from pneumococcal polysaccharide (PPS)-based vaccine (Pneumovax or Prevnar13) recipients using fluorescently labeled PPS3. Immunoglobulin heavy (Igh) and light (Igl) chains were sequenced, cloned into IgG1 and κ or λ vectors, and expressed in HEK-293 cells. PPS3 specificity was confirmed using ELISA. Results Here, we report the first 7 PPS3-specific humAbs isolated: 5 used lambda light chains and two used kappa light chains. Six of these humAbs used variable heavy 3 (VH3) Igh gene elements. Kappa humAbs used VH3-30 or VH3-7, whilst lambda humAbs used VH3-9, VH3-72 or VH3-23. Sequence analysis revealed somatic mutations in complementary determining as well as framework regions. Initial studies show that some humAbs agglutinate ST3 in vitro. Structure-function relationship studies are ongoing to identify specific determinants of PPS3 binding and biological efficacy against ST3 in vitro and in vivo. Conclusion The results of this study provide further understanding of the biology of PPS3 antibodies and may facilitate design of adjunctive immunotherapy to treat and prevent ST3 disease. Disclosures All authors: No reported disclosures.


Background. Mucosal associated invariant T (MAIT) cells are innate-like T-cells
involved in the antibacterial and fungal response by recognizing riboflavin metabolites produced by these organisms. MAIT cells are present in blood and are highly abundant in the mucosa of the liver, lungs and intestines. In murine models of urinary tract infection (UTI), MAIT cells appear to migrate to the bladder and decrease the bacterial load. It is however unknown whether MAIT cells reside in the human urogenital tract and renal tissue and whether they play a role in the first-line defense against (recurrent) UTI (RUTI).
Methods. We used a fluorescently labelled MR1-tetramer in conjunction with 14-color flowcytometry to identify and characterize MAIT cells in renal allografts after allograft failure caused by RUTI (n = 6) or rejection (n = 6) and in healthy kidney tissue surgically removed because of renal cell carcinoma (adjacent nontumorous tissue) (n = 5).
Results. The mean percentage of MAIT cells within the lymphogate was higher in the RUTI kidneys (2.24%) compared with the rejection kidneys (0.14%) and the control kidneys (0.11%) (P < 0.05).
Characterization of MAIT cells was impossible in some control samples due to MAIT cells counts <25 (predefined cutoff value), therefore the control group was excluded from further statistical analysis.
MAIT cells in RUTI kidneys appear to have a less activated profile compared with the rejection kidneys, with a lower expression of Ki67 (P < 0.01). Though the expression of the tissue resident marker CD69/CD103 was higher in 4/6 RUTI kidneys, this difference was not significant.
Conclusion. MAIT cells are present in renal tissue that is or has been subjected to an immunologic response. MAIT cells in RUTI kidneys display a more quiescent and in some samples more tissue resident phenotype than MAIT cells in rejection kidneys. These findings may suggest that (I) MAIT cells play a role in the first-line defense in the kidney and (II) that after RUTI, MAIT cells remain in renal tissue in a quiescent state. We postulate that this might be favorable in case of a second hit from an uropathogen. Background. Sequence type (ST) 17 of vancomycin-resistant Enterococcus faecium (VREF) is known to be associated with nosocomial isolates. However, there is no evidence of the effect of ST17 VREF on the patient`s clinical outcome. We conducted a retrospective cohort study to identify ST17 VREF would contribute to developing subsequent bacteremia among VREF-colonized patients.
Methods. VREF-colonized patients and its non-repetitive rectal VREF isolates were collected between March 2014 and February 2015. Subsequent bacteremia event within 1 year after colonization was reviewed from electronic medical records. STs were identified by multi-locus sequence typing. Cohort was defined as VREF with ST17 or non-ST17. Multivariable cox regression model was used to adjust effect of ST17 for developing subsequent bacteremia. If available, pulsed field gel electrophoresis (PFGE) was conducted to compare similarity between rectal and blood VREF isolates.
Results. Fifty-two patients with ST17 and 169 patients with non-ST17 VREF carriage were included in each cohort. There were six cases and 10 cases of subsequent bacteremia in cohorts ST17 and non-ST17, and 1-year VREF bacteremia free rates were 85.9% and 90.2%, respectively. There was no significant difference of subsequent bacteremia (P = 0.257) in log-rank test. However, after adjusted in multivariable models, VREF ST 17 was associated with subsequent bacteremia (adjusted relative risk, 4.02; 95% CI, 1.32-12.29, P = 0.015). Of 16 patients who had developed to subsequent VREF bacteremia, 12 VREF blood isolates could be analyzed. Only six cases (50%) of rectal and blood isolates had identical ST, whereas all available ST17 VREF cases (four cases) had identical ST and PFGE pattern (Figures 1 and 2). Patients who had identical ST isolates had shorter time difference than those who had non-identical ST isolates (P = 0.041).
Conclusion. In our study, ST17 VREF was risk factors of subsequent bacteremia and the strain that showed strong concordance between rectal and blood isolates. Further study is needed to improve clinical outcome of patients carrying VREF using genotype data of rectal VREF isolates.