Study Of Respiratory &Flavivirus Vaccines In Volunteers

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Abstract

Vaccine for Respiratory Syncytial Virus Respiratory syncytial virus (RSV), a leading cause of severe respiratory illness in the elderly, young children and infants, is a priority for vaccine development. The primary strategy for RSV vaccine development has been to identify live attenuated RSV vaccines for intranasal administration that are satisfactorily attenuated and induce a protective immune response in the targeted age group. The live, intranasal approach is based on observations that mucosal immunity plays an important role in protection against RSV and is optimally stimulated by intranasal immunization. The original live attenuated RSV vaccine candidate was derived by passage of wild type RSV in vitro at sub-optimal temperatures, yielding the cold-passaged (cp) attenuated cpRSV. Chemical mutagenesis of cpRSV yielded further-attenuated temperature-sensitive (ts) derivatives referred to as RSVcpts viruses. One virus, designated RSVcpts248/404, was evaluated in the target group for pediatric vaccine use, the 1-2 month old infant. In this age group, intranasal immunization induced a significant antibody response and conferred protective immunity against a second vaccine dose. RSV vaccine development became much less empiric with the introduction of reverse genetic techniques that allow the production of infectious RSV entirely from cloned cDNAs. The cDNA intermediate provides a means for introducing defined mutations into infectious RSV to make ?designer? attenuated vaccine candidates. The attenuating mutations in the lineage of RSVcp and RSVcpts viruses were identified using reverse genetics, and new attenuating mutations were created by deletion of non-essential accessory genes including the small hydrophobic (SH) surface protein, the M2-2 protein involved in regulating RNA synthesis, and the NS1 and NS2 nonstructural proteins that are described further below. Reverse genetics was used to combine the attenuating mutations from the above-mentioned RSVcpts248/404 virus with a point mutation (designated 1030) from another virus along with a deletion of the SH gene. The resulting recombinant virus, called rA2cp248/404/1030delSH, was well tolerated and immunogenic in 1- to 2-month old infants, although some of the viral vaccine specimens recovered from vaccines exhibited loss of a single attenuating point mutation suggesting that greater genetic stability was desirable. Gene deletions are particularly attractive stabilizing genetic mutations because they presumably cannot undergo reversion during the limited duration of in vivo replication characteristic of RSV. Attenuation resulting from a gene deletion identifies the deleted gene product as an important virulence factor. Genes encoded by many viruses have been found to antagonize the effects of the host?s alpha/beta interferon response that functions to suppress viral growth in the early stages of infection before the adaptive immune response comes into play. Suppression of the interferon response is achieved either by a decrease in the production of interferon or by a decrease in the antiviral effects generated following interaction of interferon with its receptor, or both. The RSV non-structural proteins, NS1 and NS2, suppress the production of alpha/beta interferon and also suppress the cell?s ability to establish an antiviral state. Deletion of NS1 (delNS1) or NS2 attenuates human or bovine RSV for chimpanzees or cattle, respectively. The role of these interferon antagonists in virulence of RSV for humans remains undefined. In the current study, deletion of the NS2 gene was used to generate a vaccine candidate (rA2cpdelNS2) anticipated for use in the elderly. As RSVcpts248/404 virus had been found to be poorly infectious and immunogenic in RSV-experienced subjects, a vaccine for this population was sought that would be less attenuated than RSVcpts248/404. In previous work, deletion of the NS2 gene yielded a virus that was less attenuated in chimpanzees than the cpts248/404 virus, whereas deletion of the NS1 gene was more attenuating. Reverse genetics was used to construct the recombinant RSV vaccine candidate rA2cpdelNS2, which contains both the set of 5 mutations in the attenuated cpRSV parent and a deletion of the NS2 gene. Additionally, we evaluated two mutants as vaccine candidates for RSV seronegative children and infants. For this use, the NS2 gene was deleted from recombinant versions of two RSVcpts vaccine candidates that previously were incompletely attenuated in seronegative children (RSVcpts530/1009) or in naive infants (RSVcpts248/404). These rA2cp248/404delNS2 and rA2cp530/1009delNS2 vaccine candidates were evaluated for safety, infectivity and immunogenicity in seropositive and seronegative infants and children. At a high dose, 7log10 pfu, rA2cpdelNS2 was not shed by adults and only 13% had a serological response. The two other vaccine candidates, rA2cp248/404delNS2 and rA2cp530/1009delNS2, had greatly reduced infectivity in seronegative infants and children compared to their immediate parent strains possessing an intact NS2. Thus, deletion of the interferon antagonist protein, NS2, attenuates RSV in all ages studied. This validates the development of live respiratory virus vaccines for humans by blocking their ability to inhibit the human innate immune system. Vaccines for Dengue Viruses Studies in volunteers have been initiated with a live attenuated dengue 1 (DEN1del30) and a dengue 2 (DEN2/4del30) virus vaccine. Clinical trial materials (vaccine candidates) are being prepared for a DEN1 virus vaccine; one DEN2 virus vaccine; one DEN3 virus vaccine; and three DEN4 vaccine candidates. Vaccine for West Nile Virus Studies in volunteers have been initiated with a live attenuated West Nile virus (WN/DEN4del30) vaccine. Vaccine for Tick-borne Encephalitis Virus Studies in volunteers have been initiated with a live attenuated Tick-borne Encephalitis Virus vaccine (LGT/DEN4). Vaccines for Parainfluenza Virus Clinical trial materials (vaccine candidates) are being prepared for three live attenuated PIV3 virus vaccines, HPIV3cp45; HPIV3-PB; and B/HPIV3.