Optimization of plasmid-only rescue of highly attenuated and temperature-sensitive respiratory syncytial virus (RSV) vaccine candidates for human trials.

Respiratory syncytial virus (RSV) is the most common cause of severe bronchiolitis in infants and young children in the U.S. No licensed RSV vaccines are currently available. Established techniques for recovering RSV from cDNA utilize mammalian cells, such as HEp-2 or BSR T7/5, that are not currently suitable for vaccine manufacture. When using HEp-2 cells, co-infection with an attenuated vaccinia virus that expresses T7 RNA polymerase is also required. For human clinical trials, processes that do not require the use of helper viruses and minimize the use of animal derived materials must be developed to reduce the potential theoretical risk of transmitting adventitious agents such as BSE. RSV was generated by electroporating Vero cells from a well characterized cell bank with 6 plasmids expressing T7 RNA polymerase, the full-length anti-genomic RSV and RSV N, P, M2-1 and L. The process was optimized such that highly attenuated and temperature-sensitive RSV vaccine candidates could be recovered in a system completely free of animal derived components. Efficiencies of virus recovery ranged from 30% to 100%. Human metapneumovirus was also readily recovered, suggesting that this protocol is applicable for the production of clinical trial material of other non-segmented negative sense RNA viruses.

Plasmid-only rescue of influenza A virus vaccine candidates.

The potential threat of another influenza virus pandemic stimulates discussion on how to prepare for such an event. The most reasonable prophylactic approach appears to be the use of effective vaccines. Since influenza and other negative-stranded RNA viruses are amenable to genetic manipulation using transfection by plasmids, it is possible to outline new reverse genetics-based approaches for vaccination against influenza viruses. We suggest three approaches. First, we use a plasmid-only rescue system that allows the rapid generation of high-yield recombinant vaccine strains. Second, we propose developing second-generation live influenza virus vaccines by constructing an attenuated master strain with deletions in the NS1 protein, which acts as an interferon antagonist. Third, we suggest the use of Newcastle disease virus recombinants expressing influenza virus haemagglutinin proteins of pandemic (epizootic) strains as novel vaccine vectors for use in animals and possibly humans.

Selection in persistently infected murine cells of an MHV-A59 variant with extended host range.

Murine coronavirus MHV-A59 normally infects only murine cells in vitro and causes transmissible infection only in mice. In the 17 C1 1 line of murine cells, the receptor for MHV-A59 is MHVR, a biliary glycoprotein in the carcinoembryonic antigen (CEA) family of glycoproteins. We found that virus released from the 600th passage of 17 C1 1 cells persistently infected with MHV-A59 (MHV/pi600) replicated in hamster (BHK-21) cells. The virus was passaged and plaque-purified in BHK-21 cells, yielding the MHV/BHK strain. Because murine cells persistently infected with MHV-A59 express a markedly reduced level of MHVR (Sawicki, et al., 1995), we tested whether virus with altered receptor interactions was selected in the persistently infected culture. Infection of 17 C1 1 cells by MHV-A59 can be blocked by treating the cells with anti-MHVR MAb-CC1, while infection by MHV/BHK was only partially blocked by MAb-CC1. MHV/BHK virus was also more resistant than wild-type MHV-A59 to neutralization by purified, recombinant, soluble MHVR glycoprotein (sMHVR). Cells in the persistently infected culture may also express reduced levels of and have altered interactions with some of the Bgp-related glycoproteins that can serve as alternative receptors for MHV-A59. Unlike the parental MHV-A59 which only infects murine cells, MHV/BHK virus was able to infect cell lines derived from mice, hamsters, rats, cats, cows, monkeys and humans. However, MHV/BHK was not able to infect all mammalian species, because a pig (ST) cell line and a dog cell line (MDCK I) were not susceptible to infection. MHV/pi600 and MHV/BHK replicated in murine cells more slowly than MHV-A59 and formed smaller plaques. Thus, in the persistently infected murine cells which expressed a markedly reduced level of MHVR, virus variants were selected that have altered interactions with MHVR and an extended host range. In vivo, in mice infected with coronavirus, virus variants with altered receptor recognition and extended host range might be selected in tissues that have low levels of receptors. Depending upon the tissue in which such a virus variant was selected, it might be shed from the infected animal or eaten by a predator, thus presenting a possible means for initiating the transition of a variant virus into a new host as a model for an emerging virus disease.

The murine coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells exhibits an extended host range.

In murine 17 Cl 1 cells persistently infected with murine coronavirus mouse hepatitis virus strain A59 (MHV-A59), expression of the virus receptor glycoprotein MHVR was markedly reduced (S. G. Sawicki, J. H. Lu, and K. V. Holmes, J. Virol. 69:5535-5543, 1995). Virus isolated from passage 600 of the persistently infected cells made smaller plaques on 17 Cl 1 cells than did MHV-A59. Unlike the parental MHV-A59, this variant virus also infected the BHK-21 (BHK) line of hamster cells. Virus plaque purified on BHK cells (MHV/BHK) grew more slowly in murine cells than did MHV-A59, and the rate of viral RNA synthesis was lower and the development of the viral nucleocapsid (N) protein was slower than those of MHV-A59. MHV/BHK was 100-fold more resistant to neutralization with the purified soluble recombinant MHV receptor glycoprotein (sMHVR) than was MHV-A59. Pretreatment of 17 Cl 1 cells with anti-MHVR monoclonal antibody CC1 protected the cells from infection with MHV-A59 but only partially protected them from infection with MHV/BHK. Thus, although MHV/BHK could still utilize MHVR as a receptor, its interactions with the receptor were significantly different from those of MHV-A59. To determine whether a hemagglutinin esterase (HE) glycoprotein that could bind the virions to 9-O-acetylated neuraminic acid moieties on the cell surface was expressed by MHV/BHK, an in situ esterase assay was used. No expression of HE activity was detected in 17 Cl 1 cells infected with MHV/BHK, suggesting that this virus, like MHV-A59, bound to cell membranes via its S glycoprotein. MHV/BHK was able to infect cell lines from many mammalian species, including murine (17 Cl 1), hamster (BHK), feline (Fcwf), bovine (MDBK), rat (RIE), monkey (Vero), and human (L132 and HeLa) cell lines. MHV/BHK could not infect dog kidney (MDCK I) or swine testis (ST) cell lines. Thus, in persistently infected murine cell lines that express very low levels of virus receptor MHVR and which also have and may express alternative virus receptors of lesser efficiency, there is a strong selective advantage for virus with altered interactions with receptor (D. S. Chen, M. Asanaka, F. S. Chen, J. E. Shively, and M. M. C. Lai, J. Virol. 71:1688-1691, 1997; D. S. Chen, M. Asanaka, K. Yokomori, F.-I. Wang, S. B. Hwang, H.-P. Li, and M. M. C. Lai, Proc. Natl. Acad. Sci. USA 92:12095-12099, 1995; P. Nedellec, G. S. Dveksler, E. Daniels, C. Turbide, B. Chow, A. A. Basile, K. V. Holmes, and N. Beauchemin, J. Virol. 68:4525-4537, 1994). Possibly, in coronavirus-infected animals, replication of the virus in tissues that express low levels of receptor might also select viruses with altered receptor recognition and extended host range.