Protective efficacy of intranasal cold-adapted influenza A/New Caledonia/20/99 (H1N1) vaccines comprised of egg- or cell culture-derived reassortants

Live, cold-adapted, temperature-sensitive (ca/ts) Russian influenza A vaccines are prepared in eggs by a 6:2 gene reassortment of the ca/ts donor strain A/Leningrad/134/17/57 (H2N2) (Len/17) with a current wild-type (wt) influenza A strain contributing hemagglutinin (HA) and neuraminidase (NA) genes. However, egg-derived reassortant vaccines are potentially more problematic to manufacture in large quantities than vaccines from cell-based procedures. To compare egg- and cell culture-derived reassortant vaccines, we prepared in Madin Darby canine kidney (MDCK) cells two cloned, ca/ts reassortants (25M/1, 39E/2) derived from Len/17 and a wt reference strain A/New Caledonia/20/99 (H1N1) (NC/wt). Both 25M/1 and 39E/2 reassortants preserved the ca/ts phenotype and mutations described for internal genes of the A/Len/17 parent. When compared to a commercial, egg-derived ca/ts Russian A/17/NC/99/145 (H1N1) New Caledonia vaccine (NC/145), the MDCK-derived reassortant 39E/2 vaccine conferred similar levels of protection in ferrets challenged i.n. with 7 x 10(10) pfu of NC/wt. In a dose-ranging study, the protective vaccine dose for 50% of ferrets (PD50) was less than 1.2 x 10(4) pfu for the 25M/1 vaccine derived by recombination and amplification in MDCK cells. Clonal isolates of ca/ts influenza A/New Caledonia/20/99 (H1N1) obtained by recombination and amplification entirely in MDCK cells can be highly protective i.n. vaccines.     

Genetics of cold-adapted B/Ann Arbor/1/66 influenza virus reassortants: the acidic polymerase (PA) protein gene confers temperature sensitivity and attenuated virulence

The cold-adapted B/Ann Arbor/1/66 influenza virus (ca B/AA/1/66) expresses temperature-sensitive (ts), cold-adapted (ca) and attenuation phenotypes. Reassortants which inherit one or more genes from ca B/AA/1/66 and all other genes from a virulent, wild-type influenza virus, B/Houston/1732/76, were produced and evaluated in order to identify the gene(s) responsible for the ts, ca and attenuation phenotypes. Only reassortants which inherited the PA gene from ca B/AA/1/66 expressed the ts phenotype in MDCK cells at 39 degrees C. None of the reassortants tested expressed the ca phenotype in embryonated eggs at 25 degrees C. The virulence of several reassortants was evaluated in ferrets. Inheritance of the PA gene from ca B/AA/1/66 was correlated with significant febrile attenuation and the apparent restriction of viral replication in the lower respiratory tract. Isolation of a virulent, non-ts revertant virus inheriting only the PA gene from ca B/AA/1/66 established a direct relationship between expression of the ts phenotype and attenuated virulence. Evidence for the contribution of at least one other gene from ca B/AA/1/66 to attenuation was observed. Thus, based on the methods used to determine reassortant gene compositions, these results indicate that the PA gene is primarily responsible for attenuation of ca B/AA/1/66 and its reassortants.     

Selection of a mutant S1 gene during reovirus persistent infection of L cells: role in maintenance of the persistent state

LR-7 cells, variant L cells derived from a type 3 reovirus persistently infected (p.i.) carrier culture (R. Ahmed, W. M. Canning, R. S. Kauffman, A. H. Sharpe, J. V. Hallum, and B. N. Fields, Cell 25, 325-332, 1983) were used to define the viral genes critical for maintenance of the persistent state. A cloned viral isolate (L/C virus) derived from the p.i. culture replicated normally in LR-7 cells, while wild-type (wt) viruses of the three reovirus serotypes replicated less efficiently. To identify the viral gene(s) permitting enhanced replication of L/C virus in LR-7 cells, viral reassortants were prepared by mixed infection of L cells with L/C virus and type 1 wt. Study of the one-step growth curves and final yields of large numbers of reassortants in both L cells and LR-7 cells revealed that the presence of the S1 gene from L/C virus was critical for normal viral replication in LR-7 cells. However, this phenotype was suppressed by the simultaneous presence in reassortants of both the M2 and S4 genes from the type 1 wt parent. The critical change in the S1 gene occurred by passage 13 (63 days) after initiation of the carrier culture. Although multiple mutations are present in the viral population from p.i. cultures, certain specific mutations can be identified as critical for maintenance of the persistent state.     

Phenotypic properties resulting from directed gene segment reassortment between wild-type A/Sydney/5/97 influenza virus and the live attenuated vaccine strain

Widespread use of a live-attenuated influenza vaccine (LAIV) in the United States (licensed as FluMist) raises the possibility that vaccine viruses will contribute gene segments to the type A influenza virus gene pool. Progeny viruses possessing new genotypes might arise from genetic reassortment between circulating wild-type (wt) and vaccine strains, but it will be difficult to predict whether they will be viable or exhibit novel properties. To begin addressing these uncertainties, reverse-genetics was used to generate 34 reassortant viruses derived from wt influenza virus A/Sydney/5/97 and the corresponding live vaccine strain. The reassortants contained different combinations of vaccine and wt PB2, PB1, PA, NP, M, and NS gene segments whereas all strains encoded wt HA and NA glycoproteins. The phenotypes of the reassortant strains were compared to wt and vaccine viruses by evaluating temperature-sensitive (ts) plaque formation and replication attenuation (att) in ferrets following intranasal inoculation. The results demonstrated that the vaccine virus PB1, PB2, and NP gene segments were dominant when introduced into the wt A/Sydney/5/97 genetic background, producing recombinant viruses that expressed the ts and att phenotypes. A dominant attenuated phenotype also was evident when reassortant strains contained the vaccine M or PA gene segments, even though these polypeptides are not temperature-sensitive. Although the vaccine M and NS gene segments typically are not associated with temperature sensitivity, a number of reassortants containing these vaccine gene segments did exhibit a more restricted ts phenotype. Overall, no reassortant strains were more virulent than wt, and in fact, 33 of the 34 recombinant viruses replicated less efficiently in infected ferrets. These results suggest that genetic reassortment between wt and vaccine strains is unlikely to produce viruses having novel properties that differ substantially from either progenitor, and that the likely outcome of reassortment will be attenuated viruses.     

Phenotypic and genetic analyses of the heterogeneous population present in the cold-adapted master donor strain: A/Leningrad/134/17/57 (H2N2)

For the past three decades the cold-adapted (ca) and temperature sensitive (ts) master donor strain, A/Leningrad/134/17/57 (H2N2) has been successfully used as the basis for the live attenuated reassortant influenza A vaccine. This donor strain was developed from A/Leningrad/134/57 (H2N2) wild-type (wt) virus following 17 passages in eggs at 25 degrees C. Our detailed investigation has revealed that the A/Leningrad/134/17/57 (Len/17) master donor stock is a mixed population comprised of numerous variants of the ca/ts Len/17 influenza virus. We have identified these variants to exhibit a broad range in their temperature sensitive phenotype when assayed on Madin-Darby canine kidney (MDCK) cells at 37 degrees C. A selection of these variant clones has been fully characterized by sequencing in order to understand the variability in the ts phenotype. This study has also addressed the feasibility of using cell culture technology for the propagation and subsequent manufacturing of the cold-adapted influenza vaccine (CAIV), particularly with respect to retaining the defined mutations that contribute toward the ca/ts phenotype.     

Characterization of the polypeptides synthesized in cells infected with a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture

Summary The intracellular synthesis of virus-specific polypeptides in cells infected with the wild-type virus of HVJ (HVJ-W) (haemagglutinating virus of Japan—the Sendai strain of parainfluenza 1 virus) and with a temperature-sensitive(ts) mutant (HVJ-pB) derived from an HVJ carrier culture has been analysed by polyacrylamide gel electrophoresis. At the permissive temperature (32° C), all of the known virus structural polypeptides were identified in cells infected with each strain of virus and in addition to the non-structural polypeptides B and C, another polypeptide at the region with a molecular weight of 26,000 to 27,000 (26 to 27K) could be detected in infected cells. At the non-permissive temperature (38° C), the synthesis of the polypeptide M was markedly restrained in cells infected with HVJ-pB, while other major virus polypeptides were present in approximately comparable amounts to cells infected with the wild-type virus. A non-structural polypeptide with a molecular weight of 105K was dominant ints mutant infected cells at higher temperatures and disappeared after temperature-shift from 38° to 32° C. The production of the non-structural polypeptides B and 27K was also temperature-sensitive. The molecular weights of the polypeptides B, M and 27K in HVJ-pB infected cells were larger than those of the corresponding polypeptides in HVJ-W infected cells. The synthesis of the M protein in HVJ-pB infected cells started just after lowering the incubation temperature and the newly made M protein was successfully incorporated into virus particles.With 4 Figures     

Cold-adapted reassortants of influenza A virus: Pathogenicity of A/Ann Arbor/6/60×A/Alaska/6/77 reassortant virusesIn vivo andIn vitro

Summary Cold-adapted reassortants of A/Ann Arbor/6/60×A/Alaska/6/77 viruses made in MDCK cells have recently been assessed genotypically and for temperature-sensitive and cold-adapted phenotypes. These reassortants were used to infect ferrets and hamsters and to inoculate organ cultures of hamster tracheal rings, in order to assess their degree of virulence. Virulence in the three model systems corresponded quite well, and a correlation between loss of virulence and particular A/AA/6/60 genes present in the reassortants was noted. Two different reassortants containing either RNA 2 or RNA 5 (NA gene) alone from A/AA/6/60 showed little attenuation from the wild-type parent. A reassortant containing both RNA2 and the NA gene from A/AA/6/60 and all remaining wild-type genes showed some small decrease in virulence compared to the wild-type virus. However a reassortant containing these two A/AA/6/60 genes and RNA 3 as an additional gene from this parent, had a level of attenuation comparable to that of the cold-adapted virus.With 1 Figure     

Herpes simplex virus activates expression of a cellular gene by specific binding to the cell surface

The herpes simplex virus (HSV) type 1 mutant ts1204 attaches to the cell surface at 38.5 degrees but fails to penetrate the plasma membrane. A striking feature of human fetal lung cells infected with ts1204 at 38.5 degrees was the presence of enhanced amounts of a 56,000 molecular weight host protein, p56. Studies with protein and RNA synthesis inhibitors suggested that binding of the mutant virus to cells activated expression of the cellular gene encoding p56 and not an intermediary protein. Evidence presented in this paper supports the idea that p56 is induced by a specific interaction between ts1204 virions and the cell surface.     

Genetic mapping of the cold-adapted phenotype of B/Ann Arbor/1/66, the master donor virus for live attenuated influenza vaccines (FluMist)

Cold adapted (ca) B/Ann Arbor/1/66 is the master donor virus for the influenza B (MDV-B) vaccine component of the live attenuated influenza vaccine (FluMist). The six internal genes contributed by MDV-B confer the characteristic cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) phenotypes to the vaccine strains. Previously, it has been determined that the PA and NP segments of MDV-B control the ts phenotype while the att phenotype requires the M segment in addition to PA and NP. Here, we show that the PA, NP and PB2 segments are responsible for the ca phenotype of MDV-B when examined in chicken cell lines. Five loci in three RNA segments, R630 in PB2, M431 in PA and A114, H410 and T509 in NP, are sufficient to allow efficient virus growth at 25 degrees C. Substitution of these five amino acids with wt (wild type) residues completely reverted the MDV-B ca phenotype. Conversely, introduction of these five ca amino acids into B/Yamanashi/166/98 imparted the ca phenotype to this heterologous wt virus. In addition, we also found that the MDV-B M1 gene affected virus replication in chicken cells at 33 and 37 degrees C. Recombinant viruses containing the two MDV-B M1 residues (Q159, V183) replicated less efficiently than those containing wt M1 residues (H159, M183) at 33 and 37 degrees C, implicating the role of the MDV-B M segment to the att phenotype. The complexity of the multigenic signatures controlling the ca, ts and att phenotypes of MDV-B provides the molecular basis for the observed genetic stability of the FluMist vaccines.     

Effect of undiluted passage on the polypeptides of measles virus.

Measles virus induces a large polypeptide (L; mol. wt. 180 K), a large glycopolypeptide (H; mol. wt. 80 K), a nucleocapsid associated polypeptide (P; mol. wt. 70 K), a nucleocapsid polypeptide (N; mol. wt. 60 K), a second glycopolypeptide (F0; mol. wt. 60 K), a matrix or membrane polypeptide (M; mol. wt. 37 K) and a small polypeptide (S; mol. wt. 15 K). The second glycopolypeptide (F0) appears to be cleaved in purified measles virus. Defective interfering particles accumulate during passage of measles virus leading to a decrease in the amounts of virus-specific protein synthesized in infected cells. Even in the best preparations of purified measles virus, host proteins are always detected and these become more predominant in preparations with low infectivity.     

Peculiarities of obtaining and characterization of a cold-adapted A/PR/8/34 influenza virus variant

The dynamics of alterations in biological properties of A/PR/8/34 virus strain was studied in the process of prolonged adaptation to the growth in chick embryos (CE) at a lowered (25 degrees C) cultivation temperature. The variant selected after 59 passages and subsequent cloning at the above temperature retained the high reproductive capacity in CE at optimal temperature (34 degrees C) - a characteristic of the original strain. Unlike to the latter, it showed a distinctly reduced ability to reproduce at 40 degrees C and a lower level of pathogenicity for white mice and CE. Analysis of genes of the cloned cold-adapted A/PR/8/34 strain detected 5 ts mutations in genes 1, 3, 5, 6 and 7 coding for P3, P2, NP, NA and M proteins, respectively.     

A single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature

The spike (S) glycoprotein of coronavirus is responsible for receptor binding and membrane fusion. A number of variants with deletions and mutations in the S protein have been isolated from naturally and persistently infected animals and tissue cultures. Here, we report the emergence and isolation of two temperature sensitive (ts) mutants and a revertant in the process of cold-adaptation of coronavirus infectious bronchitis virus (IBV) to a monkey kidney cell line. The complete sequences of wild type (wt) virus, two ts mutants, and the revertant were compared and variations linked to phenotypes were mapped. A single amino acid reversion (L294-to-Q) in the S protein is sufficient to abrogate the ts phenotype. Interestingly, unlike wt virus, the revertant grows well at and below 32 degrees C, the permissive temperature, as it carries other mutations in multiple genes that might be associated with the cold-adaptation phenotype. If the two ts mutants were allowed to enter cells at 32 degrees C, the S protein was synthesized, core-glycosylated and at least partially modified at 40 degrees C. However, compared with wt virus and the revertant, no infectious particles of these ts mutants were assembled and released from the ts mutant-infected cells at 40 degrees C. Evidence presented demonstrated that the Q294-to-L294 mutation, located at a highly conserved domain of the S1 subunit, might hamper processing of the S protein to a matured 180-kDa, endo-glycosidase H-resistant glycoprotein and the translocation of the protein to the cell surface. Consequently, some essential functions of the S protein, including mediation of cell-to-cell fusion and its incorporation into virions, were completely abolished.     

The role of RNA segment 1 in an in vitro host restriction occurring in an avian influenza virus mutant

A temperature sensitive mutant, ts C47, derived from A/FPV/Rostock/34 and with a ts mutation in RNA segment 8, fails to form plaques in MDCK cells. From data obtained with reassortant viruses using the human influenza isolate A/FM/1/47 it was apparent that more than one mutation contributed to the temperature-sensitive (ts) and host range (hr) phenotypes of ts C47, and the phenotype of reassortants containing RNA segment 1 from A/FM/1/47 indicated that this segment was involved. A single nucleotide substitution at nucleotide 1961, resulting in valine instead of methionine in the predicted amino acid sequence of polypeptide PB2, was found in RNA segment 1 of ts C47, but this mutation did not segregate with the attenuated phenotype on gene reassortment. The following conclusions are drawn: (a) that ts C47 has at least two mutations in addition to that already known to exist in RNA segment 8, one of which (that in RNA segment 1) does not contribute to the observed ts hr phenotypes and (b) that the hr phenotype can be suppressed by substitution of RNA segment 1 by that of another strain.     

Control of protein synthesis in herpesvirus-infected cells: analysis of the polypeptides induced by wild type and sixteen temperature-sensitive mutants of HSV strain 17.

The polypeptides induced in cells infected with a Glasgow isolate of HSV-I (17 syn+) have been characterized by SDS polyacrylamide gel electrophoresis. Study of the kinetics of synthesis in three cell lines has detected a total of 52 polypeptides, 33 of which can be identified in polypeptide profiles of purified virions. These include six low mol. wt. polypeptides that have not been previously reported. Several polypeptides were labelled with glucosamine in infected BHK cells. The different polypeptide patterns obtained at permissive (31 degrees C) and nonpermissive (38 degrees C) temperature in cells infected with 16 temperature-sensitive (ts) mutants are reported. The effect of multiplicity of infection (m.o.i.) on the polypeptide profile has been examined for two of the DNA -ve mutants: below ten, the profile varied with the m.o.i. whereas above ten it was constant. All mutants were therefore examined at an m.o.i. of approx. 20. Mutants from the same complementation group showed very similar profiles. A number of general conclusions concerning control of protein synthesis in HSV infected cells can be made: (I) As most of the 16 ts mutants affected the synthesis of several or many polypeptides it follows that a large proportion of genome specifies controlling functions. (2) The high frequency with which some polypeptides were affected suggests they are at or near the terminus of biosynthetic pathways which are under multiple control. (3) Conversely, some polypeptides were affected with a low frequency suggesting that their synthesis is not dependent on the expression of many virus functions. (4) Several individual ts mutations lead to the synthesis of increased amounts of different large polypeptides. (5) Analysis of every band detectably affected by at least one ts mutation has disclosed nine classes of dependence relationship between polypeptide synthesis and the DNA phenotype of the mutants, illustrating that this relationship is complex and different for different polypeptides. (6) The inhibition of host protein synthesis by the virus may not be a simple single step process.     

Studies on the temperature sensitivity of influenza A virus reassortants nonpathogenic for chicken

Influenza A virus reassortants which are nonpathogenic for chickens are like mammalian influenza A viruses in that they are temperature sensitive for growth at 41 degrees C. We have investigated the mechanism of this temperature sensitivity using reassortants between the two highly pathogenic strains A/FPV/Rostock/34 (FPV, H7N1) and A/turkey/England/63 (TE, H7N3). These reassortants show a strict correlation between the pathogenicity for chickens and the constellation of the genes coding for the ribonucleoprotein complex, RNP. Evidence is presented which shows that all viral components are synthesized in sufficient amounts and that the block in the viral replication cycle at the nonpermissive temperature is a late one affecting virus maturation. It is suggested that the RNP, although still enzymatically functional, may lose its ability to interact normally with viral surface components, thus interfering with the process of virus maturation. Some of the nonpathogenic reassortants which possessed the neuraminidase of TE showed an interesting temperature-dependent phenomenon: the haemagglutinin synthesized at the elevated temperature could only agglutinate erythrocytes at 20 degrees C, when the neuraminidase was inhibited or the infected cells vigorously disrupted by ultrasonication. This phenomenon is possibly not directly related to the temperature-sensitive block.     

Ts mutations in the genome of cold-adapted variants of human influenza virus A/Krasnodar/101/59 (H2N2) at different passage levels and their reproduction in lungs of hamsters

Human influenza virus A/Krasnodar/101/59 (H2N2) was passaged in chick fibroblast cultures in the presence of trypsin at suboptimal temperature. The virus which underwent 16 passages at 28 degrees C possessed cold-adapted (ca) and temperature sensitive (ts) phenotypes and formed larger plaques at the optimal temperature (33 degrees C). Its reproduction in the lungs of hamsters was decreased as evidenced by approximately 2.5 log10 lower titres; only one of 9 virus isolates from the lungs of hamsters acquired the ts +/- phenotype, although it had retained a ca phenotype. Recombination of this variant with ts mutants of fowl plague virus (FPV) revealed a ts mutation only in gene 4 of this variant coding for haemagglutinin (HA). The virus which had had 25 passages at 28 degrees C possessed the same properties as the previous variant, but all eight virus isolates from the lungs of hamsters retained the ts phenotype; the genome of this variant contained ts mutations in genes 1, 3, 4, 5 and 6. The mutation found in gene 8 was not a ts mutation. The virus, which underwent 25 passages at 28 degrees C and additional 15 passages at 27 degrees C, formed large plaques and alike to the previous variants it possessed the ca and ts phenotypes; however, its reproduction in the lungs of hamsters was decreased by 4.0 log10 and occurred in the lungs only of 4 out 16 infected animals. This variant contained ts mutations in genes 1, 3, 4, 5, 6 and 7 and a non-ts mutation in gene 8.     

Multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (FluMist) derived from cold-adapted A/Ann Arbor/6/60

FluMist influenza A vaccine strains contain the PB1, PB2, PA, NP, M, and NS gene segments of ca A/AA/6/60, the master donor virus-A strain. These gene segments impart the characteristic cold-adapted (ca), attenuated (att), and temperature-sensitive (ts) phenotypes to the vaccine strains. A plasmid-based reverse genetics system was used to create a series of recombinant hybrids between the isogenic non-ts wt A/Ann Arbor/6/60 and MDV-A strains to characterize the genetic basis of the ts phenotype, a critical, genetically stable, biological trait that contributes to the attenuation and safety of FluMist vaccines. PB1, PB2, and NP derived from MDV-A each expressed determinants of temperature sensitivity and the combination of all three gene segments was synergistic, resulting in expression of the characteristic MDV-A ts phenotype. Site-directed mutagenesis analysis mapped the MDV-A ts phenotype to the following four major loci: PB1(1195) (K391E), PB1(1766) (E581G), PB2(821) (N265S), and NP(146) (D34G). In addition, PB1(2005) (A661T) also contributed to the ts phenotype. The identification of multiple genetic loci that control the MDV-A ts phenotype provides a molecular basis for the observed genetic stability of FluMist vaccines.     

Live cold-adapted influenza A vaccine produced in Vero cell line

The African green monkey kidney (Vero) cell line was used as a substrate for the development of a live cold-adapted (ca) reassortant influenza vaccine. For that purpose, a new master strain was generated by an adaptation of the wild type (wt) A/Singapore/1/57 virus to growth at 25 degrees C in a Vero cell line. The resulting cold-adapted (ca) muster strain A/Singapore/1/57ca showed temperature sensitive (ts) phenotype and was attenuated in animal models and protective in the challenge experiments in ferrets. Two vaccine candidates of influenza A(H1N1) and A(H3N2) subtypes (6/2 reassortants) inheriting six genes coding internal proteins from the new master strain and the surface antigens hemagglutinin (HA) and neuraminidase (NA) from the epidemic viruses were obtained by a standard method of genetic reassortment. All steps of the vaccine preparation were done exclusively in Vero cells, including the isolation of the epidemic viruses. Both vaccine strains were used for immunization of young adult volunteers in a limited clinical trial and appeared to be safe, well tolerated and immunogenic after intranasal administration.