Antibody Response to the Furin Cleavable Twenty-Seven Amino Acid Peptide (p27) of the Fusion Protein in Respiratory Syncytial Virus (RSV) Infected Adult Hematopoietic Cell Transplant (HCT) Recipients.

Background: Cleavage of the inactive precursor fusion protein (F0) of respiratory syncytial virus (RSV) at two furin-recognition sites is required for membrane fusion activity, and the cleavage releases the twenty-seven amino acid peptide (p27). However, a recent study shows that p27 was an immunodominant epitope in RSV infected children, indicating that p27 was recognized as an immunogen. In the present study, we investigated the immunogenicity of p27 in an immunocompromised population of adults by measuring serum and mucosal antibody responses to p27 in samples from adult hematopoietic cell transplant (HCT) recipients. Methods: We prospectively enrolled a cohort of RSV infected HCT recipients. Serum and nasal-wash samples were obtained within the first week of RSV infection (acute) and 3 to 5 weeks post-infection (convalescent). We quantified the serum and mucosal IgG and IgA anti-p27 antibodies by a RSV/A p27 peptide enzyme-linked immunosorbent assay (ELISA) and serum and mucosal p27 like antibodies (P27LA) by a p27 competitive antibody (P27CA) assay. Results: The lower limit of detection for the ELISA and P27CA assays was 0.2 and 50 ng/mL, respectively with no cross-reaction detected with a panel of monoclonal antibodies targeting pre-fusion and post-fusion antigenic sites. P27 antibodies were detected at nanogram concentration in sera and nasal washes in the majority of RSV infected HCT recipients. However, there was no significant difference in the geometric mean antibody concentrations between the acute and convalescent sera (except for serum P27LA), between HCT recipients who shed RSV <14 days and ≥14 days, as well as between RSV/A and RSV/B infected HCT recipients. In addition, approximately 30% of HCT recipients had a 4-fold or greater decrease in mucosal IgG and IgA anti-p27 antibodies during viral clearance. Conclusion: In conclusion, in RSV naturally infected adult HCT recipients, the antibodies against p27 were detectable in both serum and nasal wash samples with higher concentration in serum than that in nasal washes. However, nearly 30% of RSV infected HCT recipients had a significant decrease in their mucosal anti-p27 antibody, suggesting that IgG and IgA anti-p27 antibodies were binding to either free viruses or RSV infected cells containing p27, and that anti-p27 antibodies in the respiratory tract were part of the mucosal antibody response in controlling RSV infection.

Antigenic Site-Specific Competitive Antibody Responses to the Fusion Protein of Respiratory Syncytial Virus Were Associated With Viral Clearance in Hematopoietic Cell Transplantation Adults.

Background: Recent studies of human sera showed that the majority of the respiratory syncytial virus (RSV) neutralizing antibodies are directed against pre-fusion conformation of the fusion (F) protein of RSV and revealed the importance of pre-fusion antigenic site Ø specific antibodies. However, detailed analysis of multiple antigenic site-specific competitive antibody responses to RSV F protein and their contribution to virus clearance in humans are lacking. Methods: We prospectively enrolled a cohort of RSV infected hematopoietic cell transplantation (HCT) adults (n = 40). Serum samples were collected at enrollment (acute, n = 40) and 14 to 60 days post-enrollment (convalescent, n = 40). Antigenic site-specific F protein antibodies were measured against pre-fusion site Ø, post-fusion site I, and sites II and IV present in both the pre-fusion and post-fusion F protein conformations utilizing four different competitive antibody assays developed with biotinylated monoclonal antibodies (mAb) D25, 131-2A, palivizumab, and 101F, respectively. The lower limit of detection were 7.8 and 1.0 µg/mL for the competitive antibody assays that measured site Ø specific response, as well as sites I, II, and IV specific responses, respectively. Neutralizing antibody titers to RSV A and B subgroups was determined by microneutralization assays. Results: The overall findings in RSV infected HCT adults revealed: (1) a significant increase in antigenic site-specific competitive antibodies in convalescent sera except for site Ø competitive antibody (p < 0.01); (2) comparable concentrations in the acute and convalescent serum samples of antigenic site-specific competitive antibodies between RSV/A and RSV/B infected HCT adults (p > 0.05); (3) significantly increased concentrations of the antigenic site-specific competitive antibodies in HCT adults who had genomic RSV detected in the upper respiratory tract for <14 days compared to those for ≥14 days (p < 0.01); and (4) statistically significant correlation between the antigenic site-specific competitive antibody concentrations and neutralizing antibody titers against RSV/A and RSV/B (r ranged from 0.33 to 0.83 for acute sera, and 0.50-0.88 for convalescent sera; p < 0.05). Conclusions: In RSV infected HCT adults, antigenic site-specific antibody responses were induced against multiple antigenic sites found in both the pre-fusion and post-fusion F conformations, and were associated with a more rapid viral clearance and neutralizing antibody activity. However, the association is not necessarily the cause and the consequence.

Comparison of Palivizumab-Like Antibody Binding to Different Conformations of the RSV F Protein in RSV-Infected Adult Hematopoietic Cell Transplant Recipients.

Background: Most respiratory syncytial virus (RSV) vaccine candidates include fusion (F) protein in different conformations. Antigenic site II found in the different F conformations is the target of palivizumab, the only US Food and Drug Administration approved monoclonal antibody (mAb). Serum palivizumab-like antibody (PLA) is a potential serologic correlate of immunity. Our objective was to determine if different conformations of F protein in a palivizumab competitive antibody (PCA) assay affect the PLA concentrations. Methods: Four PCA assays were standardized using mAbs. Each contained prefusion, postfusion, or intermediate F forms. PLA concentrations were measured in acute and convalescent sera from 22 RSV/A and 18 RSV/B-infected adult hematopoietic cell transplant (HCT) recipients. PLA concentrations were calculated using a 4-parameter logistic regression model and analyzed for statistical significance. Results: PCA assays revealed significantly greater PLA concentrations in convalescent sera; comparable increases in PLA concentration in RSV/A and RSV/B-infected HCT recipients; and significantly reduced PLA concentrations in HCT recipients who shed RSV ≥14 days. A significant positive correlation was observed between PCA assays and RSV neutralizing antibody titers. Conclusions: F protein conformation does not appear to have a measurable impact on PCA assays for measuring PLA induced by RSV/A or RSV/B infection.

Rate of decline of ferritin in patients with hemophagocytic lymphohistiocytosis as a prognostic variable for mortality.

Hemophagocytic lymphohistiocytosis (HLH) is difficult to diagnose and treat. Highly elevated ferritin is strongly associated with HLH and levels may provide a prognostic marker. A comprehensive review of ferritin data from our patients during treatment was analyzed with respect to mortality. A patient was 17 times more likely to die when percent ferritin decrease was less than 50% as compared to a 96% or greater decrease as indicated with multivariate logistic modeling. Higher maximum ferritin levels in the first 3 weeks also contributed to the odds of death (OR = 5.6; 90% CI = 1.2-24.9). Regular ferritin measurements may be useful predicting outcomes in HLH patients.