1 Centro Nacional de Biotecnología, CSIC, 28049 Madrid, Spain
2 Department of Microbiology, Mount Sinai School of Medicine, Box 1124, 1 Gustave L. Levy Place, New York, NY 10029, USA
Correspondence
Adolfo García-Sastre
adolfo.garcia-sastre{at}mssm.edu
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ABSTRACT |
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Present address: Servicio de Virología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda CP (Madrid), Spain.
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MAIN TEXT |
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Reverse genetics techniques to engineer mutant influenza viruses containing defined sequences were first developed in the early 1990s (Enami et al., 1990). These techniques allowed the exchange of one of the eight genes of influenza virus for a plasmid-derived mutant gene. In the late 1990s, new techniques were developed that allowed the rescue of influenza viruses completely derived from plasmid sequences (Fodor et al., 1999
; Neumann et al., 1999
). This technical breakthrough opened the possibility to rationally attenuate influenza viruses through the generation of recombinant virus mutants containing attenuation markers. These recombinant viruses might be suitable live vaccines against influenza if proven safely attenuated and immunogenic.
An attractive influenza A virus gene for use as a target for the inclusion of attenuating mutations leading to vaccine strains is the NS1 gene. This gene encodes a non-structural protein abundantly expressed in infected cells that appears to be involved in several functions during influenza virus replication, including the down-regulation of the antiviral type I IFN-mediated response (García-Sastre et al., 1998; Talon et al., 2000a
; Noah et al., 2003
), the inhibition of host mRNA processing (Fortes et al., 1994
; Lu et al., 1994
; Qiu & Krug, 1994
), the enhancement of viral mRNA translation (Aragón et al., 2000
), the regulation of apoptosis (Schultz-Cherry et al., 2001
; Zhirnov et al., 2002
) and the control of viral RNA replication (Falcón et al., 2004
). Importantly, the NS1 gene encodes an accessory virulence factor whose deletion results in viral attenuation (García-Sastre et al., 1998
). In fact, mouse-adapted NS1 mutant influenza A/PR/8/34 viruses have been proven to be attenuated and immunogenic in mice (Talon et al., 2000b
; Ferko et al., 2004
).
Recently, Falcón et al. (2004) described the generation and characterization in vitro of two recombinant influenza viruses expressing mutant NS1 proteins derived from the human influenza A virus strain A/Victoria/3/75 (Vic). These viruses, Vic-NS1-110 and Vic-NS1-81, encoded C-terminally truncated forms of the NS1 protein of 110 and 81 aa, instead of the 238 aa wild-type protein. The recombinant Vic viruses contained all viral RNAs derived from the Vic virus strain, except for the HA, NA and M segments, which were derived from the influenza A/WSN/33 H1N1 virus strain (WSN). Interestingly, these NS1 mutant viruses displayed a temperature-sensitive phenotype, with kinetics of virus replication in MDCK (MadinDarby canine kidney) cells similar to those of wild-type virus when grown at 32 °C, but unable to replicate at the non-permissive temperature of 39 °C. This phenotype correlated with a defect in late steps of virus replication (accumulation of viral RNA, synthesis of late viral proteins and nucleocytoplasmic export of viral ribonucleoproteins) at the non-permissive temperature (Falcón et al., 2004
). In this study, we investigated whether NS1 mutations conferring a temperature-sensitive phenotype in vitro also confer attenuation in vivo using a mouse model of influenza virus infection. We also analysed the ability of the temperature-sensitive NS1 mutant to induce humoral and cellular immune responses and to confer protection against influenza virus infection in the same animal model.
We first intranasally infected groups of eight 6-week-old female BALB/c mice with 106 p.f.u. of either wild-type Vic, mutant Vic-NS1-110 or Vic-NS1-81 viruses, and determined their ability to induce disease in mice by daily monitoring the body weight of the infected mice for 10 days. As expected, none of the mice infected, including those infected with wild-type Vic virus, showed decreases in body weight (Fig. 1a). This is typical for human influenza virus strains, which only become pathogenic in mice after several rounds of mouse adaptation. We therefore determined virus titres in lungs as indicative of viral attenuation (Fig. 1b
). Lungs were collected from mice intranasally infected with 106 p.f.u. of wild-type Vic, mutant Vic-NS1-110 or Vic-NS1-81 viruses at days 3 and 6 post-infection. As a comparison, mice were intranasally infected with the mouse-adapted influenza WSN virus. Wild-type Vic virus was detected (3x104 p.f.u. ml1) in lungs of infected mice at day 3, but the virus was cleared by day 6, in contrast with the mouse-adapted WSN virus, which was still present at significant levels at day 6. However, no viruses were detected in lungs of animals infected with Vic-NS1-110 and Vic-NS1-81 viruses, indicating that the replication of these viruses is severely compromised in the lungs. This suggests that Vic NS1 mutant viruses would also be more compromised in replicating in the lower respiratory tract of humans than the wild-type Vic virus and therefore would not induce lower respiratory symptoms, i.e. severe disease. However, it remains to be seen whether attenuation mediated by NS1 modification of influenza viruses in the mouse model translates to attenuation in humans.
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Finally, in order to analyse levels of protection induced after immunization with the Vic-NS1 mutant viruses, groups of eight mice were immunized with a high dose (106 p.f.u.) and a low dose (3x104 p.f.u.) of mutant Vic-NS1-110 or Vic-NS1-81 viruses. Three weeks after immunization, mice were challenged by intranasal infection with a lethal dose (104 p.f.u., corresponding to 10 LD50s) of homologous H1N1 WSN virus, containing the same HA and NA genes as the recombinant Vic viruses. In all cases, immunized animals were protected against death (Fig. 3a) as well as against severe disease, as monitored by body weight loss (Fig. 3b
). Animals immunized with the lower virus doses showed slightly greater decreases in body weight, but all animals started to recover weight by day 4 post-challenge. Vic-NS1-110 virus was slightly more efficient in protecting against body weight loss than Vic-NS1-81 (see Fig. 3b
).
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In spite of the reduced replication of the NS1 mutant viruses at non-permissive temperature, they induced almost the same levels of cellular and humoral responses in inoculated animals (Fig. 2) and the animals developed full protection against a subsequent lethal infection with WSN virus (Fig. 3
), Vic-NS1-110 virus being slightly more efficient than Vic-NS1-81 virus. These results would suggest that the mutant viruses are able to replicate to low levels in the upper respiratory tract of the inoculated animals, at places where the body temperature may be reduced, while they are unable to replicate in the lungs (Fig. 1
). Furthermore, a Th1 immune response was induced upon infection with the mutant viruses, characterized by the secretion of IFN
and the absence of IL-4 after splenocyte restimulation (Fig. 2b
and data not shown). Hence, the immune system appears to be primed in much the same way as in a normal infection but most of the pathogenicity is avoided. It also might be that the mutations in the NS1 protein result in enhanced ability of the Vic-NS1 mutant viruses to induce a pro-inflammatory response, as previously seen in mouse-adapted strains of influenza A viruses (López et al., 2003
; Stasakova et al., 2005
). For instance, mouse dendritic cells infected with influenza A/PR/8/34 viruses lacking the NS1 gene induce higher levels of pro-inflammatory cytokines and undergo a more robust maturation than those infected with wild-type viruses (López et al., 2003
; Stasakova et al., 2005
). This might compensate for an expected decrease in immunogenicity as compared to wild-type virus, due to their lower levels of replication in vivo and therefore of antigen expression.
In summary, our results indicate that mutant influenza viruses that are temperature-sensitive as a result of a C-terminal deletion in NS1 protein can be considered as potential master strains for preparation of attenuated live vaccines, as they (i) grow efficiently in tissue culture, (ii) contain an easily monitored genetic marker, (iii) induce efficient cellular and humoral immune responses and (iv) do not produce disease but confer protection in inoculated animals.
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ACKNOWLEDGEMENTS |
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Received 23 February 2005;
accepted 15 June 2005.