MAB858-1 Sigma-AldrichAnti-RSV Antibody, fusion protein, all type A, B strains, clone 133-1H

Infections with human respiratory syncytial virus (HRSV) occur globally in all age groups and can have devastating consequences in young infants. We demonstrate that a vaccine based on the extracellular domain (SHe) of the small hydrophobic (SH) protein of HRSV, reduced viral replication in challenged laboratory mice and in cotton rats. We show that this suppression of viral replication can be transferred by serum and depends on a functional IgG receptor compartment with a major contribution of FcγRI and FcγRIII. Using a conditional cell depletion method, we provide evidence that alveolar macrophages are involved in the protection by SHe-specific antibodies. HRSV-infected cells abundantly express SH on the cell surface and are likely the prime target of the humoral immune response elicited by SHe-based vaccination. Finally, natural infection of humans and experimental infection of mice or cotton rats does not induce a strong immune response against HRSV SHe. Using SHe as a vaccine antigen induces immune protection against HRSV by a mechanism that differs from the natural immune response and from other HRSV vaccination strategies explored to date. Hence, HRSV vaccine candidates that aim at inducing protective neutralizing antibodies or T-cell responses could be complemented with a SHe-based antigen to further improve immune protection.

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by the fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of 'membrane-anchored' RSV F protein to elevated temperature (45-55 degrees C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of RSV F-mediated membrane fusion and have important implications for the identification of therapeutic strategies and vaccines targeting RSV F protein conformational changes.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory infection of children. Understanding RSV pathogenesis and evaluating interventions requires quantitative RSV testing. Previous studies have used the plaque assay technique. Real-time reverse transcriptase PCR (RTrtPCR) offers possible greater sensitivity, stability after freeze/thaw, and lower cost, thus facilitating multicenter studies. We developed RTrtPCR assays based upon the RSV N and F genes. The N-gene assay detected greater RSV quantity and was further evaluated. Standard curves utilized both extractions from RSV culture supernatants of known quantity and cloned purified copies of the target DNA. In vitro, the ratio of RSV subgroup A (RSV-A) genome copies to PFU was 153:1. A total of 462 samples collected quantitatively from 259 children were analyzed in duplicate by RTrtPCR. Results were compared with those of RSV plaque assays performed on fresh aliquots from the same children. Duplicate RTrtPCR results were highly correlated (r2 = 0.9964). The mean viral load from nasal washes obtained on the first study day was 5.75 +/- standard error of the mean 0.09 log PFU equivalents (PFUe)/ml. Viral load by RTrtPCR correlated with plaque assay results (r2 = 0.158; P less than 0.0001). Within individuals, upper and lower respiratory tract secretions contained similar viral concentrations. RSV-A-infected children had 1.17 log PFUe higher viral loads than did those with RSV-B (P less than 0.0001). RSV quantification by RTrtPCR of the N gene is precise and has significant, though limited, correlation with quantitative culture. The utility of the RTrtPCR quantification technique for clinical studies would be solidified after its correlation with RSV disease severity is established.

Heterogeneity in respiratory syncytial virus (RSV) disease severity likely is due to a combination of host and viral factors. Infection with RSV subgroup A is thought to produce more severe disease than RSV-B. Higher RSV loads correlate with greater disease severity in hospitalized infants. Whether subgroup-specific variations in disease severity result from differences in RSV load has not been studied. A total of 102 RSV-hospitalized infants less than 2 y of age were studied. Nasal washes were collected in a standardized manner and were cultured in less than 3 h in parallel with an RSV quantitative standard in a HEp-2 plaque assay. RSV-A (72%) was more frequent than RSV-B. Disease severity risk factors were similar between subgroups. RSV loads were similar between A and B subgroups (4.77 versus 4.68 log PFU/mL). Measures of disease severity were also similar between subgroups.