Recombinant cold-adapted attenuated influenza A vaccines for use in children: reactogenicity and antigenic activity of cold-adapted recombinants and analysis of isolates from the vaccinees

Reactogenicity and antigenic activity of recombinants obtained by crossing cold-adapted donor of attenuation A/Leningrad/134/47/57 with wild-type influenza virus strains A/Leningrad/322/79(H1N1) and A/Bangkok/1/79(H3N2) were studied. The recombinants were areactogenic when administered as an intranasal spray to children aged 3 to 15, including those who lacked or had only low titers of pre-existing anti-hemagglutinin and anti-neuraminidase antibody in their blood. After two administrations of vaccines at a 3-week interval, both strains induced antibody in 75 to 95% of the children. On coinfection of chicken embryos with both recombinants, only weak interference was observed. Administration to children of the bivalent vaccine containing H1N1 and H3N2 recombinants induced efficient production of antibody to H1 and H3 hemagglutinins and N1 and N2 neuraminidases without adverse reactions. The recombinants studied were genetically stable as judged by retention of the temperature-sensitive phenotypes and a lack of reversion of the genes carrying temperature-sensitive mutations in all of the reisolates from vaccinated children.     

Variations in the temperature range for reproduction of virulent and attenuated cold-adapted influenza A viruses

The informativity of RCT40, RCT37.5, and RCT25 markers for differentiation of wild type strains from ca-recombinants obtained on the basis of ca donors of attenuation was studied. The RCT25 marker and express RCT37.5 marker (determined within 24 hours) were found to be universal characteristics differentiating ca-recombinants from virulent viruses.     

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.     

Conditions for production of thermosensitive attenuated influenza virus recombinants.

Recombination and cross-reactivation between virulent influenza viruses and a cold-adapted thermosensitive vaccine strain regularly produced genetically stable attenuated recombinants, the selection of which was based on the thermosensitivity marker. This marker, correlating with the safety of the recombinants for man was inherited independently on the properties of the haemagglutinin and neuraminidase surface antigens. There was no relationship between the thermosensitivity of the resulting recombinants and the decrease in the optimal temperature of the neuraminidase activity (OTNA marker). This indicates a separate localization in the viral genome of the mutation damages responsible for the expression of ts and OTNA markers.     

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 development of live attenuated cold-adapted influenza virus vaccine for humans

A procedure to attenuate live influenza virus of type A and type B was developed using adaptation of the virus to grow at 25 degrees C (cold adaptation; ca). Through a series of stepwise passages, two stable mutants were obtained and designated as 'Master' strains, one for type A influenza virus (A/Ann Arbor/6/60-H2N2) and one for type B influenza virus (B/Ann Arbor/1/66). These mutants were used in genetic reassortment using either the classical method or more recently described reverse genetics to update the relevant surface antigens of the circulating strains of influenza virus. The derivation is based on the concept of 6/2 where 6 signifies the six internal genes of the master strain and 2 refers to the two genes coding for the two surface glycoproteins HA and NA of the circulating influenza virus. The advantages of this vaccine were demonstrated to be (1) proper level of attenuation, (2) non-transmissibility, (3) genetic stability, (4) presence of the ca and ts markers and (5) immunogenicity involving both local and the cell-mediated immune responses. The clinical trials in infants, children, adults and elderly have provided the necessary data for eventual licensing of this vaccine. The ease of administration (intranasal) safety and high efficacy make this vaccine suitable to prevent influenza virus infection in all age groups.     

Safety and efficacy of live attenuated, cold-adapted, influenza vaccine-trivalent

This article describes the efficacy, immunogenicity, and safety of CAIV-T. This vaccine has the potential to significantly contribute to the control of influenza infection and influenza-associated illnesses, including febrile otitis media and lower respiratory disease. When compared with inactivated vaccine, CAIV-T has significant advantages in convenience of administration. The high efficacy of CAIV-T and its efficacy in children against a significantly drifted strain of H3N2 (A/Sydney), a strain not contained in the vaccine, are compelling observations for use of the vaccine in children. Effectiveness in adults was demonstrated using the same vaccine strain against the drifted H3N2 strain. The proposed vaccine administration schedule for healthy individuals aged 9 to 49 years is a single dose administered annually before the winter. For children aged 5 to 8 years, two doses are recommended the first year they are immunized with CAIV-T to ensure protection against all strains contained in the vaccine. Thereafter, a single annual revaccination is sufficient.     

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.     

Structure-based design of NS2 mutants for attenuated influenza A virus vaccines

We previously characterised the matrix 1 (M1)-binding domain of the influenza A virus NS2/nuclear export protein (NEP), reporting a critical role for the tryptophan (W78) residue that is surrounded by a cluster of glutamate residues in the C-terminal region that interacts with the M1 protein (Akarsu et al., 2003). To gain further insight into the functional role of this interaction, here we used reverse genetics to generate a series of A/WSN/33 (H1N1)-based NS2/NEP mutants for W78 or the C-terminal glutamate residues and assessed their effect on virus growth. We found that simultaneous mutations at three positions (E67S/E74S/E75S) of NS2/NEP were important for inhibition of influenza viral polymerase activity, although the W78S mutant and other glutamate mutants with single substitutions were not. In addition, double and triple substitutions in the NS2/NEP glutamine residues, which resulted in the addition of seven amino acids to the C-terminus of NS1 due to gene overlapping, resulted in virus attenuation in mice. Animal studies with this mutant suggest a potential benefit to incorporating these NS mutations into live vaccines.     

Plaquing characteristics of influenza A virus recombinants of defined genetic composition

Summary The plaque size and morphology of twenty-two influenza A virus recombinants representing seven distinct families were analyzed on MDCK cells. By examination of the genetic composition of the recombinants no relationship could be established between any gene, including those coding for the surface antigens, and the plaque size and morphology.With 1 Figure     

Molecular mechanisms of reversions to the ts+ -phenotype of cold-adapted influenza virus A strains--attenuation donors for live influenza reassortant vaccines

A ts+ revertant of cold-adapted (ca) strain A/Leningrad/134/47/57--the attenuation donor for live influenza reassortant vaccines--was obtained by passages of the ca strain in chick embryos at nonpermissive temperatures. The ts+ revertant acquired the ability to grow in chick embryos at 40 degrees C and lost the capacity to reproduce there at 25 degrees C. A complementation-recombination test using the fowl plague virus (FPV0 ts-mutants showed the loss of the ts-phenotype in the RNA-segments of ts+ revertants' genome coding for PB2, NP, and NS (NS2) proteins. However, PCR-restriction analysis revealed a true reversion in RNA-segment coding for PB2 protein only. All the investigated mutations in the ts+ revertant genome were preserved. This phenomenon could be explained by the appearance of intragenic and extragenic suppression mutations in the ts+ revertant genome. The data of the complementation-recombination test suggest that reversion of ts-phenotype occurs more frequently due to extra- or intragenic suppression rather than as a result of a true mutation loss. Estimation of the genetic stability of vaccine ca strains of influenza virus should be based on the combined use of PCR-restriction and complementation tests.     

Universal influenza vaccines: developments, prospects for use

The review analyzes the developments of genetic engineering influenza vaccines based on the conservative epitopes of viral surface proteins, such as hemagglutinin, neuraminidase, ectodomain of matrix protein M2. It estimates the capacity of the vaccines to induce an immune response to a wide variety of influenza viruses, considers ways to increase the immunogenicity and protective properties of the vaccines, based on the conservative epitopes of viral surface proteins, and prospects for their use to prevent influenza.     

Response of seronegative and seropositive adult volunteers to live attenuated cold-adapted reassortant influenza A virus vaccine.

The infectivity and immunogenicity of live attenuated A/Washington/897/80 cold-adapted reassortant virus vaccine was evaluated in seronegative (hemagglutination inhibition titer, less than or equal to 1:4) and seropositive (hemagglutination inhibition titer, greater than 1:4) adult volunteers. The vaccine was efficient in infecting seronegative volunteers (94%). Moreover, 51% of seropositive vaccinees were infected by the virus. After live virus vaccination, greater than 83% of both seronegative and seropositive vaccinees achieved a level of nasal wash antibody previously associated with resistance to infection with influenza A virus. These findings indicate that both seronegative and seropositive vaccinees can benefit from live virus vaccination.     

Cold-adapted variants of influenza virus A: I. Comparison of the genetic properties of ts mutants and five cold-adapted variants of influenza virus A

The genetic properties of seven cold-adapted variants of influenza virus A were compared with those of nine 5-fluorouracil (5-FU)-induced ts mutants. The 5-FU mutants had previously been placed into seven complementation-recombination groups; five of the seven cold-adapted variants also had the ts phenotype, and all five were shown to share the group 1 lesion. Three of the cold variants also had additional ts lesions.     

Reassortants of influenza B viruses for use in vaccines: An evaluation

Summary Three slightly different procedures for the preparation of influenza B virus reassortants from B/Lee/40 and B/Lyon/79, and B/Johannesburg/58 and B/Hong Kong/82 parental viruses are described. Following cloning procedures in eggs or allantois-on-shell cultures in the presence of antisera to B/Lee or B/Johannesburg, twenty-eight putative reassortants were obtained. Using SDS-PAGE to determine the migration rates of virion polypeptides, eighteen isolates from mixed infections of B/Lee with B/Lyon, and three isolates from mixed infections of B/Johannesburg with B/Hong Kong, were found to be reassortants. All reassortants possessed surface proteins derived from either B/Lyon or B/Hong Kong and one to three internal polypeptides from either B/Lee or B/Johannesburg.None of the reassortants showed a growth capacity in embryonated eggs as high as that of their B/Lee or B/Johannesburg parent viruses.The procedures used, and the influenza B reassortants thus generated, would appear to offer no major advantages over the existing methods and influenza B strains at present employed for the preparation of inactivated influenza virus vaccines.With 1 Figure     

Homotypic and heterotypic immunity of influenza A viruses induced by recombinants of the cold-adapted master strain A/Ann Arbor/6/60-ca

Summary The cold-adapted (ca) influenza A virus A/Ann Arbor/6/60-ca when administered intranasally to mice in two doses 3 weeks apart induces solid immunity to challenge 3 weeks later with heterotypic influenza A wild-type viruses (17). In the present study heterotypic immunity against viruses from different sub-types was shown to be relatively short-lived, having declined significantly 9 weeks after vaccination and being completely absent by 21 weeks. On the other hand, immunity against challenge viruses with surface antigens similar to the ca vaccinating virus or to other H3N2 viruses remained high, even in the absence of detectable serum haem-agglutination-inhibiting antibody. Both short- and long-term immunity induced by ca viruses was unaffected by earlier priming experiences with other wild-type or ca viruses. These results suggest that at least two mechanisms are involved in respiratory immunity to influenza viruses.With 5 FiguresSupported by a grant from the Commonwealth Serum Laboratories Parkville, Victoria 3052, Australia.