This study demonstrates that recombinant NS-LAIV(s) created directly from human swab material are effective experimental vaccines against H1N1pdm viruses. In vitro
, the NS-LAIV candidates replicated efficiently in canine MDCK cells, which produce type I IFN but lack the ISG Mx [34
]. Efficient replication in MDCK cells has important practical implications because this is an approved cell line for production of influenza A virus vaccines. Importantly, pronounced attenuation was observed in the human lung epithelial cell line A549, correlating with the induction of type I IFN and ISGs by the NS-LAIVs, and this is consistent with previous studies showing that influenza A viruses with deleted or truncated NS1 proteins are attenuated in IFN-competent cells [35
]. The results from the IFN bioassays and RT-PCR analysis show the host innate immune response was induced at much higher levels in the human lung epithelial cells infected with NS1-126 and NSΔ5 than in those infected with the WT H1N1pdm virus. This data also indicates that NS1-126 and NSΔ5 are likely to be strong inducers of IFNs and ISGs in humans and other primates. Stimulation of the innate immune system is a major advantage to the NS-LAIV strategy because this acts as a natural adjuvant and enhances the adaptive immune response of the host [15
Mice immunized with the NS-LAIVs were asymptomatic, and the tissue titers of NS1-126 and NSΔ5 viruses were dramatically lower than the titers of WT virus, indicating that they were significantly attenuated in vivo. Histopathological and immunohistochemical analysis also showed that the NS-LAIVs were severely attenuated and rapidly cleared in mice; lesions in the lungs were the smallest and most sporadic in mice inoculated with the NSΔ5, and this correlated with the level of virus replication observed for this NS-LAIV candidate. Although both NS1-126 and NSΔ5 were highly attenuated, they generated complete immune protection from lethal challenge with a mouse-adapted variant of NY1682.
Examination of the NS-LAIVs in ferrets substantiated the pronounced attenuation phenotype of the NSΔ5 candidate that was observed in mice. For example, the NSΔ5 LAIV was severely attenuated and rapidly cleared by the two hosts ( and ). Although NS1-126 and NSΔ5 LAIV candidates were markedly attenuated in ferrets, both elicited strong neutralizing antibody responses that dramatically inhibited replication in the face of a high-titer challenge, and results in rapid clearance of the challenge virus (by 3 dpc), A/Mexico/4482/2009 (H1N1pdm).
Collectively, the data from the in vitro
and in vivo
(mice, and ferrets) studies show that NS1-73 was the least attenuated LAIV candidate; and NSΔ5 was the most attenuated LAIV. NSΔ5 is a new type of NS-LAIV that has a subtle in-frame deletion (15 nt), which affects both the NS1 (residues 196-200) and NEP (residues 39-43). The NSΔ5 H1N1pdm LAIV was designed to be analogous to a naturally attenuated variant (A/SW/FJ/03) of a normally highly pathogenic H5N1 virus [33
]. The NS1 deletion in A/SW/FJ/03 (NS1 residues 191-195) was shown to reduce binding of NS1 to host cleavage and polyadenylation specificity factor (CPSF), reduce NS1 protein stability, and enhance the type I IFN response of an H5N1 virus [33
]. Our data prove that deletion of these 15nt in the NS vRNA of the quadruple reassortant H1N1pdm virus also stimulates the host innate immune response (specifically, IFN-ß, IFN-λ1, IP10, and MxA), and attenuates this virus. The fact that NSΔ5 attenuates two distantly related influenza A viruses suggests that this region of the NS gene/protein(s) is a good target for NS-LAIVs against many diverse influenza A viruses and potentially for the development of anti-viral compounds. The role of the deletion of residues 39-43 from NEP has not been elucidated, but the induction of IFN and ISGs by NSΔ5 was similar to, or slightly lower than their induction by NS-126, suggesting that the NEP mutation may also have an attenuating effect, which warrants future investigation.
LAIV safety and the potential problems associated with reassortment of LAIVs with other circulating influenza A viruses are also of paramount importance, and are challenges for the experimental approach described in this study. The currently licensed LAIVs have six internal-protein encoding vRNAs derived from the cold-adapted temperature sensitive A/Ann Arbor/6/1960 master donor strain (ca A/AA/6/60), which is an H2N2 virus that was originally isolated in 1960 and then subjected to many serial passages in vitro
]. This licensed vaccine has many point mutations in multiple vRNAs (e.g., PB1, PB2, NP) [40
] that both attenuate the virus and enhance its safety by inhibiting the rapid generation of revertants and/or single-gene reassortants that have lost the attenuating temperature sensitive phenotype, but contain wild-type HA and NA vRNAs. Although the NS-deletions used in this study are large enough so that rapid reversion to a wild-type phenotype via point mutation is highly unlikely, substitution of the NS vRNA segment via reassortment with either wild-type H1N1pdm viruses or other seasonal viruses would remove the single attenuating vRNA. This is a very serious concern, particularly in situations where the emerging strains have pandemic potential but have not yet become widespread. Therefore, we envision swapping the cis-acting segment-specific packaging signals in the vRNA segments such as the HA and NS, which was recently effective with A/PR/8/1934 (H1N1) [44
], in order to prevent reassortment and enhance safety of future NS-based LAIVs. Additional animal studies and human trials with specific NS-LAIVs such as the recent study by Wacheck et. al.
] are needed to help to define safety parameters for NS-LAIVs in humans.
A drawback of using the 6 internal-protein encoding vRNAs of ca A/AA/6/60 is that the cytotoxic T lymphocyte epitopes present in influenza A virus have mutated because of host immune pressure, which reduces immune recognition of the internal proteins expressed by currently circulating strains [45
]. Additionally, many immune evading amino acid substitutions are present when novel reassortants like the H1N1pdm virus emerge from animal reservoirs. Our strategy can be accomplished with equal, or greater, speed than typical reverse-genetics based vaccine approaches, which often require virus isolation prior to RT-PCR amplification with HA and NA specific primers. Most importantly, the LAIV approach outlined in this study has the advantage of stimulating the host's innate and adaptive immune system with the entire repertoire of proteins expressed by circulating or new emerging strains.
In summary, we rapidly generated a panel of recombinant LAIVs, directly from a patient specimen, by introducing attenuating mutations into the NS gene of an emerging pandemic influenza A virus. Our results demonstrate that two deletion mutations (NS1-126 and NSΔ5) in the NS vRNA in the unique gene constellation of the H1N1pdm virus attenuate this virus in a human lung epithelial cell line, in mice and in ferrets. A new type of NS-LAIV (NSΔ5) was designed and shown to be highly attenuated, and to elicit strong innate and adaptive immune responses, resulting in protection of mice from subsequent challenge with a lethal mouse-adapted variant of NY1682, and ferrets from challenge with A/Mexico/4482/2009. The creation of LAIVs by the rational introduction of attenuating mutations to the genome of emerging or currently circulating strains of influenza A virus could be integrated with current and future vaccine production scheme(s) and it has important potential advantages over the reassortant-based vaccine seed stock production strategies that are currently used.