Studies in man with cold-recombinant influenza virus (h1n1) live vaccines

Two cold-recombinant influenza A (H1N1) viruses were tested in several groups of human volunteers. Only minor clinical symptoms were seen and no febrile reactions occurred. With serologically primed individuals virus shedding was low, but a high proportion showed rises in serum antibody levels after vaccination and mean titres were high. With serologically unprimed volunteers shedding was high, about 75% yielding viruses but only at low titres and for a short duration. No revertant viruses were found and there was no evidence of transmission to potentially susceptible individuals housed in close contact to the vaccinees. Serum antibody responses with unprimed volunteers were, however, low. Only about one half showed increases in serum antibody titres after vaccination and mean titres were low. Nevertheless, challenge with live attenuated virus indicated a high degree of protection based on virological evidence of infection.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38229/1/1890060110_ftp.pd

Cold-adapted recombinants of influenza a virus in MDCK cells I. Development and characterization of A/Ann Arbor/6/60 x A/Alaska /6/77 recombinant viruses

Recombinant influenza viruses made at 25 and 33[deg] in Madin-Darby canine kidney (MDCK) cells using the cold-adapted A/Ann Arbor/6/60 virus and the wild-type A/Alaska/6/77 virus were biologically and genetically analyzed. Eight recombinants were separated into two phenotypic groups based on cold-adapted (ca) and temperature-sensitive (ts) markers: ca and ts, ca and non-ts. The ca recombinants showed different degrees of cold adaptibility (DOCA) and different patterns of virus growth at 25[deg]. All recombinants contained at most three genes from the cold variant A/Ann Arbor/6/60 virus (triple-gene recombinant) and most contained two or one gene from the cold variant parent (double-gene and single-gene recombinants, respectively). Further, the same three genes, RNA2, RNA3, and RNA5 (NA) were the only ca A/Ann Arbor/6/60 genes found in the various recombinants. Two clones contained all three A/Ann Arbor/6/60 genes and were both cold-adapted (ca) and temperature-sensitive (ts). All other recombinant clones were ca and non-ts, and contained RNA2 and/or RNA5 (NA). Each set of single-gene ca recombinants correlated with a different, but specific cold-adapted characteristic exhibited by their growth curves at 25[deg]. Single-gene recombinants containing only the RNA2 of A/Ann Arbor/6/60 virus showed rapid growth early in infection and intermediate final virus yield (between the titer of virus yield for the ca A/Ann Arbor/6/60 virus and the wild-type A/Alaska/6/77 virus; while the single-gene recombinant containing only the RNA5 (NA) of A/Ann Arbor/6/60 virus showed slow growth early in infection, but a high final virus yield (equivalent to that of the ca A/Ann Arbor/6/60 parent). The double-gene recombinant containing both these genes showed both rapid growth early in infection and a high final virus yield. Thus, cold adaptation can be transferred to recombinant viruses by at least two independent genes each of which can confer the cold-adaptive property by its own pathway. The genetic basis for temperature sensitivity involves both RNA2 and RNA3, but remains ambiguous in the absence of a single-gene recombinant containing only RNA3 of the cold variant.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23988/1/0000237.pd

Cold‐Adapted Reassortants of Influenza A Virus in MDCK Cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/101846/1/mim03574.pd

Characterization of an influenza a host range mutant

A mixed infection of primary chick kidney cells at 38[deg] with A/Ann Arbor/6/60 cold adapted virus and A/Alaska/6/77 wt virus yielded a cold-reassortant virus, CR43-clone 3, which had a host range different from that of either parent. It does not produce detectable virus when grown in Madin-Darby canine kidney cells, while growing normally in primary chick kidney cells at 33[deg]. Both parents, however, grow well in either cell type at 33[deg]. Genotypic analysis of viral RNA electrophoresed in polyacrylamide gels has shown that CR43-clone 3 virus has an aberrant NS gene different from the NS gene of either parent virus. Reassortant viruses made between CR43-clone 3 virus and A/California/ 10/78 (H1N1) virus in primary chick kidney cells at 33[deg] showed the same host range restriction only if the NS gene was derived from the CR43-clone 3 virus. A mixed infection with these same parents, but in Madin-Darby canine kidney cells at 33[deg], produced reassortants that always contained the A/California/10/78 NS gene instead of the CR43clone 3 NS gene. Ferrets inoculated intranasally with the CR43-clone 3 reassortant do not become sick or infected, based on the lack of symptoms: no rhinitis, coryza, or fever; and no detectable virus recovered from nasopharyngeal swabs, turbinate, or lung tissues at 48 hr after infection. Thus, CR43-clone 3 virus contains an aberrant NS gene and manifests a restricted host range phenotype in Madin-Darby canine kidney cells and ferrets.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25081/1/0000512.pd

Development and characterization of cold-adapted viruses for use as live virus vaccines

Representative viruses from twelve RNA and two DNA virus genera have been successfully adapted to growth at sub-optimal temperature (cold-adapted). In almost every case, there was a correlation between acquisition of the cold-adaptation phenotype and loss of virulence in the normal host whether animal or man. Overall, the best method of cold adaptation to develop a live virus vaccine line appeared to be a stepwise lowering of the growth temperature allowing time for multiple lesions to occur and/or be selected. In addition, the starting virus should be a recent isolate not as yet adapted to a tissue culture host and the cold-adaptation process should then occur in a host heterologous to the virus' normal host. These viruses have been reviewed in the light of their cold-adaptation method and successful production of an attenuated line as virus vaccine candidate. Finally, detailed information is presented for the cold-adaptation process in influenza virus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25479/1/0000019.pd