Immunogenicity and efficacy of alphavirus-derived replicon vaccines for respiratory syncytial virus and human metapneumovirus in nonhuman primates.

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are major causes of illness among children, the elderly, and the immunocompromised. No vaccine has been licensed for protection against either of these viruses. We tested the ability of two Venezuelan equine encephalitis virus-based viral replicon particle (VEE-VRP) vaccines that express the hRSV or hMPV fusion (F) protein to confer protection against hRSV or hMPV in African green monkeys. Animals immunized with VEE-VRP vaccines developed RSV or MPV F-specific antibodies and serum neutralizing activity. Compared to control animals, immunized animals were better able to control viral load in the respiratory mucosa following challenge and had lower levels of viral genome in nasopharyngeal and bronchoalveolar lavage fluids. The high level of immunogenicity and protective efficacy induced by these vaccine candidates in nonhuman primates suggest that they hold promise for further development.

Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors.

Investigation of the human antibody response to influenza virus infection has been largely limited to serology, with relatively little analysis at the molecular level. The 1918 H1N1 influenza virus pandemic was the most severe of the modern era. Recent work has recovered the gene sequences of this unusual strain, so that the 1918 pandemic virus could be reconstituted to display its unique virulence phenotypes. However, little is known about adaptive immunity to this virus. We took advantage of the 1918 virus sequencing and the resultant production of recombinant 1918 haemagglutinin (HA) protein antigen to characterize at the clonal level neutralizing antibodies induced by natural exposure of survivors to the 1918 pandemic virus. Here we show that of the 32 individuals tested that were born in or before 1915, each showed seroreactivity with the 1918 virus, nearly 90 years after the pandemic. Seven of the eight donor samples tested had circulating B cells that secreted antibodies that bound the 1918 HA. We isolated B cells from subjects and generated five monoclonal antibodies that showed potent neutralizing activity against 1918 virus from three separate donors. These antibodies also cross-reacted with the genetically similar HA of a 1930 swine H1N1 influenza strain, but did not cross-react with HAs of more contemporary human influenza viruses. The antibody genes had an unusually high degree of somatic mutation. The antibodies bound to the 1918 HA protein with high affinity, had exceptional virus-neutralizing potency and protected mice from lethal infection. Isolation of viruses that escaped inhibition suggested that the antibodies recognize classical antigenic sites on the HA surface. Thus, these studies demonstrate that survivors of the 1918 influenza pandemic possess highly functional, virus-neutralizing antibodies to this uniquely virulent virus, and that humans can sustain circulating B memory cells to viruses for many decades after exposure-well into the tenth decade of life.

An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies.

We sought to develop and optimize a hybridoma-based technology for generating human hybridomas that secrete virus-specific monoclonal antibodies for clinical diagnosis and therapy. We developed a novel electrofusion protocol for efficiently fusing Epstein-Barr virus (EBV)-transformed human B cells with myeloma partners. We tested seven myeloma cell lines and achieved highest efficiency when the HMMA 2.5 line was used. We optimized the electrofusion process by improving cell treatments before and after electrofusion as well as varying cell ratios, fusion medium and other experimental parameters. Our fusion efficiency increased remarkably to 0.43%, a significant improvement over the efficiency of previous PEG-based or other electrofusion methods. Using the optimized protocol, we obtained human hybridomas that secrete fully human monoclonal antibodies against two major human respiratory pathogens: respiratory syncytial virus (RSV) and an influenza H3N2 vaccine virus strain. In conclusion, we have developed an efficient and routine approach for the generation of human hybridomas secreting functional human virus-specific monoclonal antibodies.

The role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience.

BACKGROUND: The role that human metapneumovirus (hMPV) plays in the etiology of upper respiratory tract infections (URIs) in children over a period of many years has not been evaluated previously. METHODS: By use of real-time reverse-transcriptase polymerase chain reaction, we retrospectively tested nasal wash (NW) specimens for hMPV that had been obtained from a cohort of 1532 infants and children with URIs who were prospectively followed for an average of 2.4 years during the period from 1982 to 2001. Virus genes were sequenced, and prospectively collected clinical data were analyzed. RESULTS: There were 2710 visits for URIs for which routine cultures did not reveal a viral etiology. Archival NW specimens from 2384 of these visits were available. hMPV RNA was detected in 118 (5%) of 2384 specimens. The mean age of the children with hMPV infection was 20 months, and 78% of illnesses occurred from December through May. Acute otitis media (AOM) was detected in 50% of these children. hMPV circulated each year, but the numbers of isolates detected varied by year. Reinfections with both homologous and heterologous strains occurred. Four distinct genetic lineages were present over the 20 years of surveillance, with several different lineages circulating during some seasons. CONCLUSIONS: hMPV was detected in a substantial number of children with URIs and concomitant AOM.

Progression of respiratory syncytial virus infection monitored by fluorescent quantum dot probes.

We report the use of quantum dots (QDs) to identify the presence and monitor the progression of respiratory syncytial virus (RSV) infection over time by labeling the F and G proteins. In addition, co-localization of these viral proteins was shown using confocal microscopy. The implications of these results are that QDs may provide a method for early, rapid detection of viral infection and open the door for future studies of the intricate spatial features cell trafficking of viral proteins.

Smallpox vaccination does not elevate systemic levels of prothrombotic proteins associated with ischemic cardiac events.

BACKGROUND: During the recent smallpox vaccination campaigns, ischemic cardiac complications were observed after vaccination. To examine a possible association between the smallpox vaccine and postvaccination ischemic events, we investigated alterations in levels of prothrombotic proteins (plasminogen activator inhibitor type 1 [PAI-1] and soluble CD40 ligand [sCD40L]) in recently vaccinated individuals. METHODS: Vaccinia-naive (cohort N; aged 18-32 years) and vaccinia-experienced (cohort E; aged 33-49 years) healthy adults were vaccinated with a 1 : 5 dilution of the Aventis Pasteur smallpox vaccine. Plasma levels of PAI-1 and sCD40L were measured in 30 subjects (cohort N, n=15; cohort E, n=15) at baseline and twice after vaccination (between days 7 and 9 and between days 26 and 30). RESULTS: Baseline mean PAI-1 levels significantly differed between cohorts N and E (P=.04). Within each exposure cohort, mean PAI-1 levels did not significantly change after vaccination. Baseline sCD40L levels did not differ between cohorts N and E. In cohort N, sCD40L levels significantly decreased after vaccination but returned to baseline levels within 1 month. Vaccination did not significantly alter levels of sCD40L in cohort E. CONCLUSIONS: Levels of PAI-1 and sCD40L did not significantly increase after smallpox vaccination. Vaccine-induced alterations in levels of these prothrombotic proteins do not appear to play a role in ischemic events observed after smallpox vaccination.