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

To gain insight into the intertypic incompatibility between typeThe genomes of influenza A and B viruses each consist of eight single-stranded RNA segments of negative polarity. Both types of viruses have two envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA is responsible for viral binding to cellular sialic acid-containing receptors, followed by fusion between envelope and cell endosomal membranes, while NA is responsible for viral release from infected cells through removal of sialic acids from cellular glycoconjugates and viral glycoproteins (11). Type A viruses are further divided into subtypes based on HA (H1 to H15) and NA (N1 to N9) antigenicities. In cells infected with two different type A viruses, intratypic reassortants possessing various combinations of gene segments are produced (24). However, intertypic reassortants between type A and B viruses have not been detected in nature, although both viruses are cocirculating in human populations.Attempts to generate reassortants between type A and B viruses have been unsuccessful (9, 15, 21). With reverse genetics, Muster et al. (17) produced a mutant type A virus whose NA noncoding regions were replaced with those of the nonstructural (NS) gene of type B virus. This result suggests that at least the noncoding regions of the type B NS segment are compatible with influenza A viral components at the level of RNA transcription and replication. However, this mutant virus replicated more slowly than wild-type WSN virus in cell culture and was attenuated in mice (17). Animals infected with the chimeric virus were resistant to challenge with the wild-type virus, suggesting that such manipulation of the influenza virus genome might be useful in producing live attenuated vaccine strains.Recently, we established a plasmid-based reverse genetics system (18) that has provided a powerful tool for generating recombinant influenza viruses. To gain insight into the intertypic incompatibility between type A and B viruses, we used this technology to study the functionality of chimeric (A/B) HAs and then evaluated the potential of the chimeric (A/B) HA viruses as live vaccines. MATERIALS AND METHODSCells. 293T human embryonic kidney cells and COS-7 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum and antibiotics. Madin-Darby canine kidney (MDCK) cells were grown in minimal essential medium (MEM) with 5% newborn calf serum and antibiotics. Cells were maintained at 37°C in 5% CO 2 .Construction of plasmids. The generation of plasmid constructs for viral RNA production (pPolI), containing the HA genes of wild-type A/WSN/33 (H1N1, A/WSN) (pPolI-WSN-HA) and wild-type B/Lee/40 (B/Lee) (pPolI-B-HA) flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator, was described in a previous publication (18). A series of chimeric (A/B) HA pPolI constructs (Fig. 1) were produced with these wild-type HA constructs, PCR amplification with ProofStart polymerase (Qiagen), and ligation. Primer sequences will...

Section: Discussionmentioning

confidence: 99%

Generation of Influenza A Viruses with Chimeric (Type A/B) Hemagglutinins

Horimoto

Takada

Iwatsuki‐Horimoto

et al. 2003

“…Seasonal live attenuated influenza vaccine (LAIV) viruses are reassortant viruses that contain six genes from a coldadapted (ca), temperature-sensitive (ts), attenuated (att) master donor virus (A/Ann Arbor/6/1960) (AA/60 ca) and the HA and NA glycoproteins of the wild-type (wt) virus to which the immune response is targeted (12)(13)(14)(15)(16)(17). These vaccines are safe, infectious, immunogenic, nontransmissible, genetically stable, and efficacious (15)(16)(17).…”

mentioning

confidence: 99%

The Matrix Gene Segment Destabilizes the Acid and Thermal Stability of the Hemagglutinin of Pandemic Live Attenuated Influenza Virus Vaccines

O’Donnell

Vogel

Matsuoka

et al. 2014

The threat of future influenza pandemics and their potential for rapid spread, morbidity, and mortality has led to the development of pandemic vaccines. We generated seven reassortant pandemic live attenuated influenza vaccines (pLAIVs) with the hemagglutinin (HA) and neuraminidase (NA) genes derived from animal influenza viruses on the backbone of the six internal protein gene segments of the temperature sensitive, cold-adapted (ca) A/Ann Arbor/60 (H2N2) virus (AA/60 ca) of the licensed seasonal LAIV. The pLAIV viruses were moderately to highly restricted in replication in seronegative adults; we sought to determine the biological basis for this restriction. Avian influenza viruses generally replicate at higher temperatures than human influenza viruses and, although they shared the same backbone, the pLAIV viruses had a lower shutoff temperature than seasonal LAIV viruses, suggesting that the HA and NA influence the degree of temperature sensitivity. The pH of HA activation of highly pathogenic avian influenza viruses was greater than human and low-pathogenicity avian influenza viruses, as reported by others. However, pLAIV viruses had a consistently higher pH of HA activation and reduced HA thermostability compared to the corresponding wild-type parental viruses. From studies with single-gene reassortant viruses bearing one gene segment from the AA/60 ca virus in recombinant H5N1 or pH1N1 viruses, we found that the lower HA thermal stability and increased pH of HA activation were associated with the AA/60 M gene. Together, the impaired HA acid and thermal stability and temperature sensitivity likely contributed to the restricted replication of the pLAIV viruses we observed in seronegative adults. IMPORTANCEThere is increasing evidence that the HA stability of influenza viruses depends on the virus strain and host species and that HA stability can influence replication, virulence, and transmission of influenza A viruses in different species. We investigated the HA stability of pandemic live attenuated influenza vaccine (pLAIV) viruses and observed that the pLAIV viruses consistently had a less stable HA than the corresponding wild-type influenza viruses. The reduced HA stability and temperature sensitivity of the pLAIV viruses may account for their restricted replication in clinical trials.

“…The final vaccine strains contain a mixed genome consisting of six genome segments inherited from the master strain (ensuring the attenuation and ca phenotype of the vaccine virus) and two genome segments encoding the viral surface antigens hemagglutinin (HA) and neuraminidase, derived from the current epidemic viruses (30,36,56).…”

mentioning

confidence: 99%

Live Attenuated Influenza Virus Expressing Human Interleukin-2 Reveals Increased Immunogenic Potential in Young and Aged Hosts

Ferko

Kittel

Romanova

et al. 2006

Despite the reported efficacy of commercially available influenza virus vaccines, a considerable proportion of the human population does not respond well to vaccination. In an attempt to improve the immunogenicity of live influenza vaccines, an attenuated, cold-adapted (ca) influenza A virus expressing human interleukin-2 (IL-2) from the NS gene was generated. Intranasal immunization of young adult and aged mice with the IL-2-expressing virus resulted in markedly enhanced mucosal and cellular immune responses compared to those of mice immunized with the nonrecombinant ca parent strain. Interestingly, the mucosal immunoglobulin A (IgA) and CD8؉ T-cell responses in the respiratory compartment could be restored in aged mice primed with the IL-2-expressing virus to magnitudes similar to those in young adult mice. The immunomodulating effect of locally expressed IL-2 also gave rise to a systemic CD8 ؉ T-cell and distant urogenital IgA response in young adult mice, but this effect was less distinct in aged mice. Importantly, only mice immunized with the recombinant IL-2 virus were completely protected from a pathogenic wild-type virus challenge and revealed a stronger onset of virus-specific CD8 ؉ T-cell recall response. Our findings emphasize the potential of reverse genetics to improve the efficacy of live influenza vaccines, thus rendering them more suitable for high-risk age groups.