Genetic stability determinants of temperature sensitive, live attenuated respiratory syncytial virus vaccine candidates.

An intranasally delivered, live attenuated, temperature sensitive (ts) respiratory syncytial virus vaccine candidate, rA2cp248/404/1,030DeltaSH, exhibits a low level of genetic instability in clinical studies, in contrast to the relatively high stability of two similar candidates, cpts248/404 and rA2cp248/404DeltaSH. The latter strains, containing two ts mutations (248ts and 404ts), are partially growth restricted at 37 degrees C, whereas, rA2cp248/404/1,030DeltaSH contains an additional ts mutation (1,030ts) that increases attenuation and partially restricts virus growth at 35 degrees C. Since the maximum human airway temperature is 35.5 degrees C, we investigated whether growth restriction at 35 degrees C contributes to genetic instability of rA2cp248/404/1,030DeltaSH in vitro. We conducted in vitro passage studies with the three strains at 32 degrees C (a fully permissive growth temperature) and 35 degrees C (restrictive for only rA2cp248/404/1,030DeltaSH). Instability of the ts phenotype was observed only in rA2cp248/404/1,030DeltaSH passaged at 35 degrees C, and corresponded with reversion at the 248ts or 1,030ts mutation sites, as observed in clinical studies. This study indicates that ts mutations that partially restrict replication at physiologic temperatures may contribute to genetic instability of viruses in vivo. In vitro passage studies performed at appropriate temperatures can be used to assess genetic stability and to prioritize ts vaccine candidates for clinical evaluation.

Systematic evaluation of in vitro and in vivo adventitious virus assays for the detection of viral contamination of cell banks and biological products.

Viral vaccines and the cell substrates used to manufacture them are subjected to tests for adventitious agents, including viruses, contaminate. Some of the compendial methods (in vivo and in vitro in cell culture) were established in the mid-20th century. These methods have not been subjected to current assay validation, as new methods would need to be. This study was undertaken to provide insight into the breadth (selectivity) and sensitivity (limit of detection) of the routine methods, two such validation parameters. Sixteen viral stocks were prepared and characterized. These stocks were tested in serial dilutions by the routine methods to establish which viruses were detected by which methods and above what limit of detection. Sixteen out of sixteen viruses were detected in vitro, though one (bovine viral diarrhea virus) required special conditions to detect and another (rubella virus) was detected with low sensitivity. Many were detected at levels below 1 TCID50 or PFU (titers were established on the production cell line in most cases). In contrast, in vivo, only 6/11 viruses were detected, and 4 of these were detected only at amounts one or more logs above 1 TCID50 or PFU. Only influenza virus and vesicular stomatitis virus were detected at lower amounts in vivo than in vitro. Given the call to reduce, refine, or replace (3Rs) the use of animals in product safety testing and the emergence of new technologies for the detection of viruses, a re-examination of the current adventitious virus testing strategies seems warranted. Suggested pathways forward are offered.

Comparison of results of detection of rhinovirus by PCR and viral culture in human nasal wash specimens from subjects with and without clinical symptoms of respiratory illness.

Human rhinoviruses (HRV) cause acute upper respiratory illness. The frequency of HRV-associated illnesses appears greater when PCR assays are used to detect rhinoviruses. The present study performed PCR-based detection of HRV upon entry of subjects into respiratory syncytial virus and parainfluenza type 3 vaccine trials when subjects were symptom-free and upon subsequent development of clinical symptoms of respiratory illness during the trial. The background of HRV PCR positivity in symptom-free individuals (30/139 [22%]) was only slightly lower than in those with respiratory illness (28/77 [36%]). For subjects with multiple samples, it was estimated that HRV was detectable by PCR for approximately 100 days before, during, and after clinical symptoms were documented. PCR is a remarkably more sensitive method of detecting HRV than is tissue culture. The presence of HRV RNA may not always reflect an association with infectious virus production. The limited association of HRV RNA with illness suggests caution in assigning causality of HRV PCR positivity to clinical symptoms of respiratory illness.

The absence of enhanced disease with wild type respiratory syncytial virus infection occurring after receipt of live, attenuated, respiratory syncytial virus vaccines.

Early in the development of respiratory syncytial virus (RSV) vaccines severe disease occurred in children after receipt of formalin-inactivated RSV vaccine. Continuing efforts to develop an appropriately attenuated and immunogenic live RSV vaccine have given opportunities to assure that live vaccines are safe through surveillance of children after vaccination. In the present study, the rate of RSV-associated upper respiratory tract illness in 388 children was lower in RSV vaccinated children than in controls (14% versus 20% in a 6-24 month old group and 16% versus 25% in infants). Additionally, there was no evidence that vaccination predisposed to more severe lower respiratory tract illness. Thus infection with a series of live attenuated RSV vaccines did not result in enhanced disease upon infection with wild type RSV. The impact of RSV during this surveillance will inform the design of future efficacy studies with RSV vaccines.

The interferon antagonist NS2 protein of respiratory syncytial virus is an important virulence determinant for humans.

BACKGROUND: Respiratory syncytial virus (RSV) is targeted for vaccine development, because it causes severe respiratory tract illness in the elderly, young children, and infants. A primary strategy has been to derive live attenuated viruses for use in intranasally administered vaccines that will induce a protective immune response. In the present study, the NS2 gene, whose encoded protein antagonizes the host's interferon- alpha / beta response, was deleted from RSV vaccine candidates by use of reverse genetics. METHODS: Three NS2 gene-deleted RSV vaccine candidates were studied: rA2cp Delta NS2, rA2cp248/404 Delta NS2, and rA2cp530/1009 Delta NS2. rA2cp Delta NS2, which had the fewest attenuating mutations, was evaluated in adults and RSV-seropositive children. rA2cp248/404 Delta NS2 and rA2cp530/1009 Delta NS2 were evaluated in adults and RSV-seropositive and RSV-seronegative children. RESULTS: At a high dose (10(7.0) pfu), rA2cp Delta NS2 was not shed by adults, and only 13% of them had an immune response. The other vaccine candidates, rA2cp248/404 Delta NS2 and rA2cp530/1009 Delta NS2, had greatly decreased infectivity in RSV-seronegative children, compared with that of their immediate parent strains, which possess an intact NS2 gene. CONCLUSIONS: Deletion of the NS2 gene attenuates RSV in subjects of all ages studied. This validates the strategy of developing live respiratory tract virus vaccines in which the virus's ability to inhibit the human innate immune system is blocked. rA2cp248/404 Delta NS2 should be studied in children at a higher input titer, because it was more infectious and immunogenic than was rA2cp530/1009 Delta NS2.

Identification of a recombinant live attenuated respiratory syncytial virus vaccine candidate that is highly attenuated in infants.

BACKGROUND: Recombination technology can be used to create live attenuated respiratory syncytial virus (RSV) vaccines that contain combinations of known attenuating mutations. METHODS: Two live attenuated, recombinantly derived RSV vaccine candidates, rA2cp248/404 Delta SH and rA2cp248/404/1030 Delta SH, were evaluated in 31 adults and in 95 children >/=6 months old. rA2cp248/404/1030 Delta SH was subsequently evaluated in 44 infants 1-2 months old. These vaccine candidates share 4 attenuating genetic elements and differ only in a missense mutation (1030) in the polymerase gene. RESULTS: Both vaccines were highly attenuated in adults and RSV-seropositive children and were well tolerated and immunogenic in RSV-seronegative children. Compared with that of rA2cp248/404 Delta SH, replication of rA2cp248/404/1030 Delta SH was restricted in RSV-seronegative children (mean peak titer, 10(4.3) vs. 10(2.5) plaque-forming units [pfu]/mL), indicating that the 1030 mutation had a potent attenuating effect. Although rA2cp248/404/1030 Delta SH was well tolerated in infants, only 44% of infants who received two 10(5.3)-pfu doses of vaccine had detectable antibody responses. However, replication after administration of the second dose was highly restricted, indicating that protective immunity was induced. At least 4 of 5 attenuating genetic elements were retained in recovered vaccine viruses. CONCLUSIONS: rA2cp248/404/1030 Delta SH is the first RSV vaccine candidate to be sufficiently attenuated in young infants. Additional studies are needed to determine whether rA2cp248/404/1030 Delta SH can induce protective immunity against wild-type RSV.

Recombinant respiratory syncytial viruses lacking the C-terminal third of the attachment (G) protein are immunogenic and attenuated in vivo and in vitro.

The design of attenuated vaccines for respiratory syncytial virus (RSV) historically focused on viruses made sensitive to physiologic temperature through point mutations in the genome. These prototype vaccines were not suitable for human infants primarily because of insufficient attenuation, genetic instability, and reversion to a less-attenuated phenotype. We therefore sought to construct novel attenuated viruses with less potential for reversion through genetic alteration of the attachment G protein. Complete deletion of G protein was previously shown to result in RSV strains overly attenuated for replication in mice. Using reverse genetics, recombinant RSV (rRSV) strains were engineered with truncations at amino acid 118, 174, 193, or 213 and respectively designated rA2cpDeltaG118, rA2cpDeltaG174, rA2cpDeltaG193, and rA2cpDeltaG213. All rA2cpDeltaG strains were attenuated for growth in vitro and in the respiratory tracts of BALB/c mice but not restricted for growth at 37 degrees C. The mutations did not significantly affect nascent genome synthesis in human lung epithelial (A549) cells, but infectious rA2cpDeltaG virus shed into the culture medium was dramatically diminished. Hence, the data suggested that a site within the C-terminal 85 amino acids of G protein is important for efficient genome packaging or budding of RSV from the infected cell. Vaccination with the rA2cpDeltaG strains also generated efficacious immune responses in mice that were similar to those elicited by the temperature-sensitive cpts248/404 strain previously tested in human infants. Collectively, the data indicate that the rA2cpDeltaG strains are immunogenic, not likely to revert to the less-attenuated phenotype, and thus candidates for further development as vaccines against RSV.

Evaluation of combined live, attenuated respiratory syncytial virus and parainfluenza 3 virus vaccines in infants and young children.

We evaluated a combination respiratory syncytial virus (RSV) and parainfluenza 3 virus (PIV3) live, attenuated intranasal vaccine for safety, viral replication, and immunogenicity in doubly seronegative children 6-18 months old. RSV cpts-248/404 and PIV3-cp45 vaccines were combined in a dose of 10(5) plaque-forming units of each per 0.5-mL dose and compared with monovalent vaccines or placebo. The virus shedding pattern of RSV was not different between monovalent RSV cpts-248/404 vaccine and combination vaccine. Modest reductions in the shedding of PIV3-cp45 vaccine virus were found after the administration of RSV cpts-248/404 and PIV3-cp45 vaccine, relative to monovalent PIV3 vaccine; 16 (76%) of 21 children given combination vaccine shed PIV3-cp45 versus 11 (92%) of 12 of those given monovalent PIV3 vaccine. Both vaccines were immunogenic, and antibody responses were similar between the monovalent groups and the combination group. Combined RSV/PV3 vaccine is feasible for simultaneous administration, and further studies are warranted.

Characterization of recombinant respiratory syncytial viruses with the region responsible for type 2 T-cell responses and pulmonary eosinophilia deleted from the attachment (G) protein.

It is essential that preventative vaccines for respiratory syncytial virus (RSV) elicit balanced T-cell responses. Immune responses dominated by type 2 T cells against RSV antigens are believed to cause exaggerated respiratory tract disease and may also contribute to unwanted inflammation in the airways that predisposes infants to wheeze through adolescence. Here we report on the construction and characterization of recombinant RSV (rRSV) strains with amino acids 151 to 221 or 178 to 219 of the attachment (G) glycoprotein deleted (rA2cpDeltaG150-222 or rA2cpDeltaG177-220, respectively). The central ectodomain was chosen for modification because a peptide spanning amino acids 149 to 200 of G protein has recently been shown to prime several strains of naïve inbred mice for polarized type 2 T-cell responses, and peripheral blood T cells from most human donors recognize epitopes within this region. Quantitative PCR demonstrated that synthesis of nascent rRSV genomes in human lung epithelial cell lines was similar to that for the parent virus (cp-RSV). Plaque assays further indicated that rRSV replication was not sensitive to 37 degrees C, but pinpoint morphology was observed at 39 degrees C. Both rRSV strains replicated in the respiratory tracts of BALB/c mice and elicited serum neutralization and anti-F-protein immunoglobulin G titers that were equivalent to those elicited by cp-RSV and contributed to a 3.9-log(10)-unit reduction in RSV A2 levels 4 days after challenge. Importantly, pulmonary eosinophilia was significantly diminished in BALB/c mice primed with native G protein and challenged with either rA2cpDeltaG150-222 or rA2cpDeltaG177-220. These findings are important for the development of attenuated RSV vaccines.