Humoral and Mucosal Antibody Response to RSV Structural Proteins in RSV-Infected Adult Hematopoietic Cell Transplant (HCT) Recipients.

Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract infection in infants, the elderly, and immunocompromised patients. RSV antibodies play a role in preventing reinfection and in clearance of RSV, but data regarding the levels of viral protein-specific antibodies elicited and their contribution to patient recovery from RSV-induced disease are limited. We prospectively enrolled a cohort of RSV-infected adult hematopoietic cell transplant (HCT) recipients (n = 40). Serum and nasal-wash samples were obtained at enrollment (acute samples) and convalescence (convalescent samples). We measured (1) humoral IgG and mucosal IgA binding antibody levels to multiple RSV proteins (F, G, N, P, and M2-1) by Western blot (WB); (2) neutralizing antibody (Nt Ab) titers by microneutralization assay; and (3) palivizumab-like antibody (PLA) concentrations by an ELISA-based competitive binding assay developed in the lab. Finally, we tested for correlations between protein-specific antibody levels and duration of viral shedding (normal: cleared in <14 days and delayed: cleared ≥14 days), as well as RSV/A and RSV/B subtypes. Convalescent sera from HCT recipients had significantly higher levels of anti-RSV antibodies to all 5 RSV structural proteins assayed (G, F, N, P, M2-1), higher Nt Abs to both RSV subtypes, and higher serum PLAs than at enrollment. Significantly higher levels of mucosal antibodies to 3 RSV structural proteins (G, N, and M2-1) were observed in the convalescent nasal wash versus acute nasal wash. Normal viral clearance group had significantly higher levels of serum IgG antibodies to F, N, and P viral proteins, higher Nt Ab to both RSV subtypes, and higher PLA, as well as higher levels of mucosal IgA antibodies to G and M2-1 viral proteins, and higher Nt Ab to both RSV subtypes compared to delayed viral clearance group. Normal RSV clearance was associated with higher IgG serum antibody levels to F and P viral proteins, and PLAs in convalescent serum (p < 0.05). Finally, overall antibody levels in RSV/A- and/B-infected HCT recipients were not significantly different. In summary, specific humoral and mucosal RSV antibodies are associated with viral clearance in HCT recipients naturally infected with RSV. In contrast to the humoral response, the F surface glycoprotein was not a major target of mucosal immunity. Our findings have implications for antigen selection in the development of RSV vaccines.

Viral Load of Severe Acute Respiratory Syndrome Coronavirus 2 in Adults During the First and Second Wave of Coronavirus Disease 2019 Pandemic in Houston, Texas: The Potential of the Superspreader.

BACKGROUND: During the coronavirus disease 2019 pandemic, a minority of index cases are associated with a majority of secondary cases suggesting that superspreaders could drive the pandemic. We identified a phenotype in individuals with extremely high viral load who could act as superspreaders. METHODS: Data were analyzed from individuals tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from 18 March through 15 August 2020. Outcomes were compared using contingency table and quantile regression to test the equality of medians between the pandemic waves and by viral load groups. RESULTS: Of the 11 564 samples tested, 1319 (11.4%) were positive for SARS-CoV-2. An increase in weekly median viral load occurred in the second wave of the SARS-CoV2 pandemic. This population was more likely to be women, outpatients, and symptomatic and to have an extremely high or high viral load. In patients with multiple reverse-transcription polymerase chain reaction-positive test results, the durations of viral shedding were comparable between individuals with asymptomatic/mild and mild/moderate illness severity. CONCLUSIONS: We detected a small group of individuals with extremely high SARS-CoV-2 viral loads and mild illness. We believe that these individuals' characteristics could be consistent with the superspreader phenomenon and that greater awareness of the social dynamics of these individuals is needed to understand the spread of SARS-CoV-2.

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.

Lactate dehydrogenase and caspase activity in nasopharyngeal secretions are predictors of bronchiolitis severity.

BACKGROUND: Bronchiolitis is the leading cause of hospitalization in infants. Biomarkers of disease severity might help in clinical management. OBJECTIVE: To determine the clinical predictiveness of NW-LDH, NW-caspase 3/7, and NW-LDH/NW-caspase 3/7 ratio in bronchiolitis. METHODS: Previously healthy children less than 24 months of age with bronchiolitis were recruited from the Texas Children's emergency room and intensive care unit from October 2010 to April 2011. Demographic, clinical information, and NW samples were obtained at enrollment. NW samples were analyzed for respiratory viruses, caspase 3/7, and LDH. RESULTS: A viral pathogen was detected in 91·6% of 131 children, with the most common being respiratory syncytial virus and human rhinovirus. A single infection was found in 61·8% of subjects and co-infection in 29·8%. Children admitted to ICU had significantly higher NW-LDH than children sent home from the ER or admitted to the general floor (P = 0·02). Children infected with RSV had the highest NW-LDH concentration (P = 0·03) compared with other viral infections. NW-LDH and NW-caspase were significantly correlated (r = 0·77, P < 0·0001). The univariate models showed NW-LDH and NW-LDH/NW- caspase 3/7 ratio were directly associated with hospitalization. Mutivariate regression analyses suggested a complex interaction between the biomarkers, demographics, and disposition. CONCLUSIONS: NW-LDH, NW-caspase 3/7 and NW-LDH/NW-caspase 3/7 ratio and their interactions with demographic factors are predictive of bronchiolitis severity and can help distinguish children requiring ICU-level care from those admitted to the general floor, or discharged home from the emergency center.