Evaluation of Influenza A/Hong Kong/123/77 (H1N1) ts-1A2 and Cold-Adapted Recombinant Viruses in Seronegative Adult Volunteers

Two attenuated influenza A donor viruses, the A/Udorn/72 ts-1A2 and the A/Ann Arbor/6/60 cold-adapted (ca) viruses, are being evaluated for their ability to reproducibly attenuate each new variant of influenza A virus to a specific and desired level by the transfer of one or more attenuating genes. Each of these donor viruses has been able to attenuate influenza A viruses belonging to the H3N2 subtype by the transfer of one or more attenuating genes. To determine whether these two donor viruses could attenuate a wild-type virus that belonged to a different influenza A subtype, ts-1A2 and ca recombinants of a wild-type virus representative of the A/USSR/77 (H1N1) Russian influenza strain were prepared and evaluated in adult doubly seronegative volunteers at several doses. The recombinants derived from both donor viruses were attenuated for the doubly seronegative adults. Less than 5% of infected vaccinees developed a febrile or systemic reaction, whereas five of six recipients of wild-type virus developed such a response. The 50% human infectious dose (HID₅₀) for each recombinant was approximately 10^5.0 50% tissue culture infective doses. The virus shed by the ts-1A2 and ca vaccinees retained the ts or ca phenotype, or both. This occurred despite replication of the recombinant viruses for up to 9 days. No evidence for transmission of the ca or ts-1A2 recombinant virus to controls was observed. A serum hemagglutination inhibition response was detected in less than 50% of the infected vaccinees. However, with the more sensitive enzyme-linked immunosorbent assay, a serological response was detected in 100% of the ca vaccinees given 300 HID₅₀ and approximately 70% of ca or ts vaccinees who received 10 to 32 HID₅₀ of virus. These results indicate that the recombinants derived from both donor viruses were satisfactorily attenuated and were stable genetically after replication in doubly seronegative adults although they induced a lower serum hemagglutination inhibition response than that found previously for H3N2 ts and ca recombinants.     

Characterization of Reverse Genetics-Derived Cold-Adapted Master Donor Virus A/Leningrad/134/17/57 (H2N2) and Reassortants with H5N1 Surface Genes in a Mouse Model

Live attenuated influenza vaccines (LAIV) offer significant advantages over subunit or split inactivated vaccines to mitigate an eventual influenza pandemic, including simpler manufacturing processes and more cross-protective immune responses. Using an established reverse genetics (rg) system for wild-type (wt) A/Leningrad/134/1957 and cold-adapted (ca) A/Leningrad/134/17/1957 (Len17) master donor virus (MDV), we produced and characterized three rg H5N1 reassortant viruses carrying modified HA and intact NA genes from either A/Vietnam/1203/2004 (H5N1, VN1203, clade 1) or A/Egypt/321/2007 (H5N1, EG321, clade 2) virus. A mouse model of infection was used to determine the infectivity and tissue tropism of the parental wt viruses compared to the ca master donor viruses as well as the H5N1 reassortants. All ca viruses showed reduced replication in lungs and enhanced replication in nasal epithelium. In addition, the H5N1 HA and NA enhanced replication in lungs unless it was restricted by the internal genes of the ca MDV. Mice inoculated twice 4 weeks apart with the H5N1 reassortant LAIV candidate viruses developed serum hemagglutination inhibition HI and IgA antibody titers to the homologous and heterologous viruses consistent with protective immunity. These animals remained healthy after challenge inoculation with a lethal dose with homologous or heterologous wt H5N1 highly pathogenic avian influenza (HPAI) viruses. The profiles of viral replication in respiratory tissues and the immunogenicity and protective efficacy characteristics of the two ca H5N1 candidate LAIV viruses warrant further development into a vaccine for human use.     

Temperature-Sensitive Mutants of Influenza A Virus: Transfer of the Two Temperature-Sensitive Lesions in the Udorn/72-ts-1A2 Virus to the A/Hong Kong/123/77 (H1N1) Wild-Type Virus

The influenza A/Udorn/72-ts-1A2 virus possesses temperature-sensitive mutations in the genes coding for the P1 and P3 polymerase proteins. It is being evaluated as a donor of its attenuating temperature-sensitive genes to produce recombinant live vaccine strains of epidemic variants of influenza A virus. Transfer of the P1 and P3 genes to two viruses within the H3N2 subtype of influenza A virus (i.e., the A/Victoria/3/75 and A/Alaska/6/77 viruses) conferred on each variant the following properties: (i) 37°C shutoff temperature for plaque formation, (ii) almost complete restriction of viral replication in the lungs, (iii) a 100-fold restriction of viral replication in the nasal turbinates, and (iv) genetic stability after replication in hamsters. This study was undertaken to determine whether the transfer of the two ts-1A2 temperature-sensitive genes into a virus belonging to the H1N1 subtype (i.e., the A/Hong Kong/123/77 virus) would result in a restriction of replication in vitro and in vivo comparable to that observed with the previously studied H3N2 recombinant viruses in hamsters. This was found to be the case. In addition, infection of hamsters with the A/Hong Kong/77-ts-1A2 virus induced significant resistance to infection with wild-type A/Hong Kong/77 virus. Thus, the two ts-1A2 temperature-sensitive genes attenuated influenza A viruses belonging to two distinct subtypes to a specific and predictable level. An unexpected genetic interaction was observed between several A/Hong Kong/77-ts-1A2 segregants bearing the group 5 (P1) temperature-sensitive lesion. One interpretation of these results is that intracistronic complementation occurred between these segregants.     

ts P1 and P3 genes are responsible for satisfactory level of attenuation of ts-1A2 recombinants bearing H1N1 or H3N2 surface antigens of influenza a virus

To produce live, temperature-sensitive (ts) influenza A virus vaccines, the A/Udorn/72-ts-1A2 donor strain, which has is lesions in the genes coding for the P3 and P1 polymerase proteins, was mated with three wild-type viruses: A/Victoria/3/75 (H3N2), A/Alaskal6/77 (H3N2), and A/Hong Kong/123/77 (H1N1). From each mating a vaccine strain was selected which contained the two ts genes from the 1A2 parent and the hemagglutinin and neuraminidase genes from the wild-type parent. The three ts-1A2 recombinant viruses were given to adult seronegative volunteers and each was found to be satisfactorily attenuated. The parental origin of genes in each ts-1A2 recombinant was identified to determine which genes from the A/Udorn/72-ts-1A2 parent were responsible for the attenuation of the wild-type influenza A viruses. Only the ts-1A2 P3 and P1 genes were required to produce a satisfactory level of attenuation of the wild-type viruses for adults.     

Characterization of DNA complementary to nucleotide sequences adjacent to poly(A) at the 3'-terminus of the avian sarcoma virus genome.

The temperature-sensitive influenza virus A/Hong Kong/68 (H3N2) ts-1[E] has been used as a prototype live attenuated influenza virus vaccine. Using recently developed techniques to map the genome of influenza viruses and to “genotype” influenza virus recombinants, the temperature-sensitive lesions in the virus were identified. These defects, responsible for the attenuation of the virus, are located in the genes for the P3 protein and the nucleoprotein and are associated with virus-specific RNA synthesis. Hong Kong/68 (H3N2) ts-1[E] virus can also serve as a donor of “attenuation characteristics” for the selection of recombinant strains which have different surface antigens and may be used as vaccine strains in the future. The temperature-sensitive mutations of Hong Kong/68 (H3N2) ts-1[E] virus were previously transferred to recombinant viruses carrying the HO hemagglutinin. The RNAs of 8 of these temperature-sensitive recombinants were analyzed. One of these viruses, R1, classified in group 1 of the Hong Kong mutant virus set was found to possess a ts defect only in the P3 protein. R8, a member of group 2 of the Hong Kong mutant virus set had a ts mutation in the nucleoprotein.     

The cold adapted and temperature sensitive influenza A/Ann Arbor/6/60 virus, the master donor virus for live attenuated influenza vaccines, has multiple defects in replication at the restrictive temperature

We have previously determined that the temperature sensitive (ts) and attenuated (att) phenotypes of the cold adapted influenza A/Ann Arbor/6/60 strain (MDV-A), the master donor virus for the live attenuated influenza A vaccines (FluMist^®), are specified by the five amino acids in the PB1, PB2 and NP gene segments. To understand how these loci control the ts phenotype of MDV-A, replication of MDV-A at the non-permissive temperature (39 °C) was compared with recombinant wild-type A/Ann Arbor/6/60 (rWt). The mRNA and protein synthesis of MDV-A in the infected MDCK cells were not significantly reduced at 39 °C during a single-step replication, however, vRNA synthesis was reduced and the nuclear–cytoplasmic export of viral RNP (vRNP) was blocked. In addition, the virions released from MDV-A infected cells at 39 °C exhibited irregular morphology and had a greatly reduced amount of the M1 protein incorporated. The reduced M1 protein incorporation and vRNP export blockage correlated well with the virus ts phenotype because these defects could be partially alleviated by removing the three ts loci from the PB1 gene. The virions and vRNPs isolated from the MDV-A infected cells contained a higher level of heat shock protein 70 (Hsp70) than those of rWt, however, whether Hsp70 is involved in thermal inhibition of MDV-A replication remains to be determined. Our studies demonstrate that restrictive replication of MDV-A at the non-permissive temperature occurs in multiple steps of the virus replication cycle.     

Genetic factors associated with loss of the temperature-sensitive phenotype of the influenza A/Alaska/77-ts-1A2 recombinant during growth in vivo

The influenza A/Alaska/77-ts-1A2 vaccine candidate strain, which has a 37° shutoff temperature for plaque formation and temperature-sensitive (ts) lesions on the P1 and P3 genes, was administered intranasally to a seronegative child who subsequently remained well. However, starting 7 days after inoculation, A/Alaska/77 virus with a 40° shutoff temperature (ts⁺) was shed. To determine whether an extragenic suppressor mutation was responsible for this loss of ts phenotype of the vaccine candidate virus, the ts⁺ isolate was mated with wild-type virus, and ts recombinant progeny were sought. Approximately 25% of the progeny were ts, suggesting that a suppressor mutation was present in the child's ts⁺ isolate. Each of 62 ts progeny viruses had the ts P3 gene. The extragenic suppressor of this ts gene was localized to the P2 gene. The ts P3 gene of the A/Alaska/77-ts-1A2 recombinant specified a 37–38° shutoff temperature for plaque formation, whereas the ts P3 gene in the ts segregants derived from the ts⁺ isolate had a 39° shutoff temperature. Hence, the ts P3 gene in the ts⁺ isolate developed an intragenic mutation affecting its level of temperature sensitivity. In addition, the P3 RNA of the ts⁺ virus migrated more slowly in polyacrylamide gel than the P3 RNA of its A/Alaska/77-ts-1A2 parent. We failed to isolate ts P1 segregants from the ts⁺ isolate, indicating that the P1 ts mutation had reverted or undergone intragenic suppression. The ability of ts mutants to escape from their ts phenotype by suppression and other mutations may limit their usefulness in immunoprophylaxis since attenuation of ts mutants of influenza virus appears to be a function of the ts phenotype. Additionally, suppressor mutations provide new opportunities for studying the nature of protein-protein interactions.     

Temperature-sensitive mutants of influenza virus XVI. Transfer of the two ts lesions present in the Udorn/72-ts- 1 A2 donor virus to the Victoria/3/75 wild-type virus

Previously, the influenza A/Hong Kong/68-ts-1[E] virus and its recombinants (38° shutoff temperature of plaque formation) were shown to be insufficiently attenuated for persons who lacked immunity to both the hemagglutinin and neuraminidase surface glycoproteins, i.e., doubly seronegative individuals. To meet the need for immunization of such individuals, a virus more defective than the ts-1[E] recombinants was produced. The resulting virus, designated Udorn/72-ts-1A2, possessed is mutations in genes represented by complementation-recombination groups 1 and 5 and was more restricted in replication in vitro at 37° and in vivo in the hamster's lungs and nasal turbinates than the Udorn/72ts-1[E] virus. These properties suggested that the Udorn/72 ts-1A2 virus might serve as a donor of its two ts lesions to new variants of influenza A virus to produce attenuated strains for use in doubly seronegative individuals. The Udorn/72-ts-1A2 virus was mated with the influenza A/Victoria/3/75 wild-type virus, and six is recombinant viruses bearing the Vic/75 hemagglutinin were isolated. Five of these Vic/75-ts-1A2 recombinants had a 37° shutoff temperature and two ts lesions like their Udorn/72-s-1A2 parent. Each of these clones replicated to low titer or not at all in the hamster's lungs and were 100-fold restricted in the nasal turbinates like their ts parental virus. Each of 158 isolates recovered from hamsters infected with the parental Udorn/72-ts-1A2 virus or one of its Vic/75 ts double lesion recombinants retained the ts phenotype. One Vic/75-ts-1A2 recombinant clone that had a 38°hutoff temperature, and only the group 1 ts lesion replicated in the hamster's lungs to a level intermediate between that of the parental ts-1A2 and wild-type virus. All isolates recovered from hamsters infected with this recombinant were also ts. Infection of hamsters with a Vic/75-ts-1A2 recombinant, bearing the group 1 and group 5 ts mutations, induced resistance to homologous wild-type virus challenge. Since the parental and double lesion recombinant ts-1A2 viruses had similar restriction of replication in vitro at 37° and in vivo, it is likely that the two ts-1A2 is lesions will effect a similar level of attenuation following transfer into other antigenic variants of influenza A virus.     

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.     

Temperature sensitive mutants of influenza virus. IX. Genetic and biological characterization of TS-1[E] lesions when transferred to a 1972 (H3N2) influenza A virus.

The temperature-sensitive (ts) mutant recombinant influenza A/Hong Kong/1968-ts-1[E] (H3N2) previously was shown to possess properties desirable for a live attenuated vaccine. We investigated the regularity of transfer of these desirable properties by recombination to a newly emerged wild-type influenza A (H3N2) variant, A/Udorn/307/1972. Examination of Udorn/72-ts-1[E] recombinant clones confirmed that the ts-1[E] donor contained two independently segregating ts lesions, each of which segregated independently of the hemagglutinin and neuraminidase genes. Transfer of both of these ts lesions to a new recombinant was associated with a specific shutoff temperature in vitro of 38°, a moderate restriction of growth in the respiratory tract of hamsters, and infrequent reversion to ts+ phenotype in vivo. The absence of one of the two ts lesions in a Udorn/72-ts-1[E] recombinant clone resulted in a higher in vitro shutoff temperature (39°), less growth restriction in hamsters, and less genetic stability with regard to reversion to the ts+ phenotype. These results supported the hypothesis that well-defined ts lesions can be utilized as specific markers of attenuation which can be transferred to new, potentially epidemic, antigenic variants of influenza A virus.     

用8质粒病毒拯救系统产生冷适应减毒的重组A型人流感病毒的研究

目的 利用流感病毒8质粒病毒拯救系统,产生冷适应减毒的重组A型人流感病毒,建立以冷适应流感病毒株为拯救骨架的反向遗传学技术平台.方法 以冷适应、温度敏感、减毒的A/Ann Arbor/6/60(H2N2)流感病毒株作为拯救病毒的骨架,人工合成了该病毒株的6个内部基因片段,即PB2、PB1、PA、NP、M和NS,同时引入5个氨基酸突变作为标签.6个基因片段通过与改造后的转录载体pAD3000连接,构建6个基因的拯救载体,经测序获得序列准确的拯救质粒:pMDV-A-PB2、pMDV-A-PB1、pMDV-A-PA、pMDV-A-NP、pMDV-A-M、pMDV-A-NS.结果 6质粒与PR8的表面基因HA和NA进行"6+2"组合的病毒拯救,8个重组质粒共转染COS-1细胞,成功拯救出了具有血凝活性的冷适应减毒的重组A型人流感病毒,命名为rMDV-A.鸡胚尿囊液中重组病毒的血凝(HA)效价为1∶2 9～1∶2 10.结论 构建的A/Ann Arbor/6/60的6个内部基因的病毒骨架拯救系统,为深入研究冷适应减毒人流感病毒的基因功能和新型疫苗研发提供了试验材料.     

Temperature-Sensitive mutants of influenza virus. VII. Transfer of the TS-1[E] lesions to a wild-type influenza A virus with the HON1 surface antigens.

The nature of the genetic defect(s) present in the influenza A/Hong Kong/1968-ts-1[E] (H3N2) candidate vaccine virus, was analyzed by transferring them by genetic recombination to a 1943 HON1 wild-type virus. Three classes of ts viruses bearing the HO hemagglutinin were identified. One class consisted of two clones of virus, R1 and R8, which underwent complementation-recombination with each other but not with the Hong Kong/1968 (H3N2)-ts-1[E] parent virus. The second class consisted of clones of virus, such as R4 and R11, that failed to undergo complementation-recombination with R1, with R8, or with the ts-1[E] parent. These data indicated that the parent Hong Kong ts-1[E] virus possessed two ts lesions that segregated independently of each other and were presumably on different segments of the influenza A virus genome. The third class of ts mutants consisted of a spontaneously occurring ts mutant, clone R9; this clone underwent complementation-recombination with the ts-1[E] parent and other HO-ts recombinants and was shown to belong to a complementation group different than clones R1 and R8. The ts defects present in the Hong Kong-ts-1[E] donor virus segregated independently of the genes that coded for the epidemiologically important surface antigens, i.e., the hemagglutinin and the neuraminidase glycoproteins. The implications of these findings for the development of a live attenuated influenza virus vaccine are discussed.     

Demonstration of a non-temperature-sensitive growth-restricting mutation in a ts mutant of influenza a virus: Implications for live virus vaccine development

Temperature-sensitive (ts) mutants of influenza A virus have been developed for use in a live virus vaccine with the expectation that the ts lesions would determine the level of attenuation. Since chemical mutagenesis can also induce non-ts lesions which restrict growth, an effort was made to detect such non-ts lesions in three representative ts mutants of influenza A virus. ts⁺ “revertants” isolated from ts viruses and ts⁺ recombinant viruses produced by a mating of two complementing ts viruses were examined for their ability to replicate in the hamster lung. One virus, ts-304, which was partially restricted in replication in vitro at the permissive temperature, yielded ts⁺ revertants and recombinants that were restricted in growth in the hamster lung. This indicated that ts-304 contained both a ts lesion and a non-ts lesion which restricted growth in the lung, but that the ts lesion exerted a greater suppressive effect. In constrast to ts viruses which manifest a greater degree of suppression of replication in lungs (37°) than in nasal turbinates (32–34°), recombinant or revertant viruses possessing only a non-ts lesion were equally restricted in replication in these two organs. A non-ts lesion was not found in the Hong Kong/68-ts1[E] candidate vaccine virus. This virus was not restricted in growth in vitro at permissive temperatures and the two ts lesions present in this virus appeared to be the sole determinants of growth restriction in the hamster lung. An additional virus, the ts-2[C] virus, which also was not restricted in replication at permissive temperature, did not possess a growth-restricting lesion on its non-ts genes. Therefore the ts-2[C] virus is a useful probe to detect non-ts lesions in viruses suspected of having such a mutation.     

Molecular studies of temperature-sensitive replication of the cold-adapted B/Ann Arbor/1/66, the master donor virus for live attenuated influenza FluMist vaccines

Cold-adapted (ca) B/Ann Arbor/1/66 is the master donor virus for influenza B (MDV-B) vaccine component of live attenuated influenza FluMist^® vaccine. The six internal protein gene segments of MDV-B confer the characteristic cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) phenotypes to the reassortant vaccine strains that contain the HA and NA RNA segments from the circulating wild type strains. Previously, we have mapped the loci in the NP, PA and M genes that determine the ca, ts and att phenotypes of MDV-B. In this report, the ts mechanism of MDV-B was described by comparing replication of MDV-B with its wild type counterpart at permissive and restricted temperatures. We showed that the PA and NP proteins of MDV-B are defective in RNA polymerase function at the restricted temperature of 37 °C resulting in greatly reduced viral RNA and protein synthesis. In addition, the two M1 residues, Q159 and V183 that are unique to MDV-B, contribute to reduced virus replication at temperatures greater than 33 °C, possibly due to the reduced M1 membrane association and its reduced virion M1 incorporation. Thus, the previously identified MDV-B loci not only reduce viral polymerase function at the restricted temperature but also affect virus assembly and release.     

Temperature-sensitive mutants of influenza A virus: Evaluation of the Alaska/77-ts-1 A 2 temperature-sensitive recombinant virus in seronegative adult volunteers

Summary An influenza A virus recombinant bearing the surface antigens of the A/Alaska/6/77 (H 3 N 2) wild type virus and the twots genes of the A/Udorn/72-ts-1 A 2 (H 3 N 2) virus was evaluated for attenuation, antigenicity, and transmissibility in 28 adult volunteers all of whom possessed a preinoculation serum hemagglutination-inhibiting (HAI) antibody titer of 1:8 and 18 of whom also possessed a serum neuraminidase-inhibiting (NI) antibody titer of 1:4. The Alaska/77-ts-1 A 2 recombinant, which had a 37° C shutoff temperature for plaque formation andts mutations on the genes thought to code for the P1 and P3 polymerase proteins, infected 71 percent of the vaccinees when administered at a dose of 10^6.5 TCID₅₀. Only 3 percent of the vaccinees developed symptoms in contrast to 50 percent of volunteers who received 10^4.2 TCID₅₀ of wild type virus. Vaccinees shed virus for a shorter interval and at a lower titer than the volunteers who received wild type virus. Eachts-1 A 2 isolate retained thets phenotype indicating that the recombinant was stable genetically in seronegative adults. An immunological response, as measured by a rise in serum HAI and/or NI antibody, was detected in 71 percent of the vaccinees and 87 percent of the recipients of wild type virus. Transmission of vaccine virus to susceptible contacts was not observed. The twots-1 A 2ts genes have now been transferred to two variants within the H 3 N 2 subtype, the Vic/75 and Alaska/77 viruses, and have rendered the viruses satisfactorily attenuated for adults. The level of infectivity of the Alaska/77-ts-1 A 2 virus appeared to be low, however.This work supported in part by contracts N01-A 1-42553 and N01-A1-22503 sponsored by the National Institute of Allergy and Infectious Diseases.     

Acquisition of the ts phenotype by a chemically mutagenized cold-passaged human respiratory syncytial virus vaccine candidate results from the acquisition of a single mutation in the polymerase (L) gene

A cold-passaged (cp) temperature-sensitive (ts) mutant of human respiratory syncytial virus designated RSV cpts-248 was previously derived by random chemical mutagenesis of the non-ts mutant cp-RSV that possesses one or more host range mutations. We previously demonstrated in rodents and seronegative chimpanzees that the cpts-248 virus is more attenuated than cp-RSV and is more stable genetically than previously isolated RSV ts mutants. In the present study, we determined that the acquisition of the ts phenotype and the increased attenuation of the cpts-248 virus are associated with a single nucleotide substitution at nucleotide 10,989 that results in a change in the coding region (amino acid position 831) of the polymerase gene. The identification of this attenuating ts mutation is important because cpts-248 was used as the parent virus for the generation of a number of further attenuated mutants that are currently being evaluated as candidate vaccine strains in clinical trials in infants. Furthermore, technology now exists to rationally design new vaccine candidates by incorporating multiple attenuating mutations, such as the one identified here, into infectious viruses that are genetically stable and appropriately attenuated.     

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.     

The hemagglutinin protein of influenza A/Vietnam/1203/2004 (H5N1) contributes to hyperinduction of proinflammatory cytokines in human epithelial cells

Live attenuated influenza A/Vietnam/1203/04 (H5N1) (VN04 cold adapted [ca]) and A/Hong Kong/213/03 (H5N1) (HK03 ca) vaccine viruses were compared with the A/New Caledonia/20/99 (H1N1) (NC99 ca) seasonal vaccine virus for induction of host gene expression in infected human epithelial cells. Levels of proinflammatory cytokines and interferon-related genes were significantly upregulated in VN04 ca virus-infected A549 cells compared to cells infected with the HK03 ca and NC99 ca viruses as examined by microarray analysis and confirmed by quantitative RT-PCR and ELISA assays. Further mapping studies demonstrated that the hemagglutinin (HA) protein of the VN04 ca virus contributed to the hyperinduction of cytokines. The inactivated viruses could also induce the production of the cytokines and chemokines, albeit at a much lower level than live viruses. Compared to HK03 ca virus, VN04 ca virus differs by 9 amino acids including an additional glycosylation site at residue 158N of the HA protein and a shortened stalk in the neuraminidase (NA) protein. Increased cytokine production by HK03 ca virus was only observed when HK03 ca virus acquired an additional glycosylation in the HA protein and when its NA protein was replaced by that of VN04. Thus, our data indicate that the HA protein and its interaction with the NA protein play a role in triggering cytokine responses. The full implications of cytokine induction in vaccine virus-induced immune responses remain to be explored.     

Effect of murine cytomegalovirus infection on mitogen responses in genetically resistant and susceptible mice.

Temperature-sensitive (ts) reassortant vaccine strains derived from the A/Udorn/72 ts-1A2 donor virus were not sufficiently stable genetically in humans. We therefore sought to produce a new, more stable donor virus. We had previously identified a stable ts virus with a ts P3 gene and in the current study identified another relatively stable single-lesion ts virus with a ts mutation in the NP gene. A new ts reassortant virus was constructed by mating these two single mutants and by isolating three reassortant progeny, clones 20, 53, and 55, that contained both a ts P3 and a ts NP gene. These reassortant progeny possessed a 37 to 38°C shutoff temperature and were as restricted in their replication in hamster lungs as the A/Udorn/72 ts-1A2 virus. All isolates from the lungs and nasal turbinates of hamsters were temperature sensitive. An in vitro stress test was used to determine whether the new ts P3 ts NP reassortant virus would undergo loss of its ts phenotype after replication at semipermissive temperature. Clone 20 and 55 reassortants underwent progressive loss of their ts phenotype in vitro, although at a rate slightly less than that of the A/Udorn/72 ts-1A2 virus. The level of genetic stability after replication in vivo was assessed in cyclophosphamide-treated hamsters in which virus replication continued for up to 15 days. Again, both the A/Udorn/72 ts-1A2 and the new ts P3 ts NP reassortant clone 55 manifested a progressive loss of temperature sensitivity after 7 days of replication. Clone 55 virus lost temperature sensitivity significantly less rapidly than the A/Udorn/72 ts-1A2 virus. These results indicated that, although the new ts P3 ts NP reassortant virus was more stable than the A/Udorn/72 ts-1A2 virus, it nevertheless underwent progressive loss of temperature sensitivity after replication in vitro and in vivo. Therefore, it does not appear to be a satisfactory donor virus. This experience plus that gained earlier with other ts mutants of influenza A virus suggest that influenza A virus mutants that rely solely upon their ts phenotype for attenuation are unlikely to exhibit the phenotypic stability required of a vaccine virus. Other genetic techniques are needed to produce more stable influenza A virus strains.     

Temperature-sensitive mutants of influenza A virus. Transfer of the twots-1 A 2ts lesions present in the Udorn/72-ts-1 A 2 donor virus to the influenza A/Alaska/6/77 (H 3 N 2) wild type virus

Summary The Udorn/72-ts-1 A 2 temperature-sensitive influenza A virus has a 37° C shutoff temperature and ats mutation on the genes coding for the P1 and P3 proteins. Thists donor virus was produced with the expectation that the transfer of its twots genes would regularly and predictably attenuate each new variant of influenza A virus. It had previously been mated with the A/Victoria/75 (H 3 N 2) virus and five Vic/75-ts-1 A 2 recombinants were isolated that had bothts-1 A 2ts genes andin vitro andin vivo genetic and biological properties similar to their Udorn/72-ts-1 A 2 parent. The present study was designed to determine if the acquisition of the twots-1 A 2ts genes would also confer a specific level of attenuation on the influenza A/Alaska/6/77 (H 3 N 2) wild type virus.Fifteen recombinant Alaska/77-ts-1 A 2 viruses were isolated and characterized genetically for the number and location ofts mutations. These clones were also studied for their level of replication and genetic stability in hamsters. Four recombinants possessed both of thets-1 A 2 mutations and had a 37° C shutoff temperature for plaque formation. Two recombinants possessed only ats P1 gene and had either a 38° C or 39° C shutoff temperature. The remaining nine clones had thets P3 gene and a shutoff temperature of 37° C, 38° C or 39° C.Each of the four 37° C shutoff temperature recombinants that possessed bothts P1 and P3 genes were restricted at least 10,000-fold in replication in the hamster's lung and approximately 100-fold in the nasal turbinates compared to the level of replication of wild type virus in these sites. All isolates from these animals retained thets phenotype. The level of replicationin vivo of thets P1 and P3 segregants was related to their shutoff temperature of plaque formationin vitro, e.g. the 38° Cts P3 segregant was less restricted in replication in the lungs than a 37° Cts P3 segregant. All isolates from animals infected with thets P3 segregants werets, whereas a low level of genetic instability was detected for one of thets P1 segregants. Since ten independentts-1 A 2 recombinants (one Udorn/72, 5 Victoria/75 and 4 Alaska/77) with bothts-1 A 2 mutations exhibited the same genetic and biologic properties, it is likely that thesets P1 and P3 genes were the prime determinants of attenuation and could effect a similar level of attenuation in other influenza A viruses within the H 3 N 2 subtype.With 2 FiguresThe work of J. G. M. constitutes partial fulfillment of the requirements for a Doctor of Philosophy degree from George Washington University, Washington, D. C.