Attenuation and immunogenicity in mice of temperature-sensitive influenza viruses expressing truncated NS1 proteins

Ana M. Falcón1,{dagger}, Ana Fernandez-Sesma2, Yurie Nakaya2, Thomas M. Moran2, Juan Ortín1 and Adolfo García-Sastre2

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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It was previously shown that two mutant influenza A viruses expressing C-terminally truncated forms of the NS1 protein (NS1-81 and NS1-110) were temperature sensitive in vitro. These viruses contain HA, NA and M genes derived from influenza A/WSN/33 H1N1 virus (mouse-adapted), and the remaining five genes from human influenza A/Victoria/3/75 virus. Mice intranasally infected with the NS1 mutant viruses showed undetectable levels of virus in lungs at day 3, whereas those infected with the NS1 wild-type control virus still had detectable levels of virus at this time. Nevertheless, the temperature-sensitive mutant viruses induced specific cellular and humoral immune responses similar to those induced by the wild-type virus. Mice immunized with the NS1 mutant viruses were protected against a lethal challenge with influenza A/WSN/33 virus. These results indicate that truncations in the NS1 protein resulting in temperature-sensitive phenotypes in vitro correlate with attenuation in vivo without compromising viral immunogenicity, an ideal characteristic for live attenuated viral vaccines.

{dagger}Present address: Servicio de Virología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda CP (Madrid), Spain.


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Influenza virus infections are a major cause of respiratory disease in humans. During typical interpandemic years, approximately 36 000 deaths are attributed to influenza virus infections in the United States alone (Thompson et al., 2003). Disease in humans is mainly attributed to the antigenically distinct influenza A and B types. In addition to yearly epidemics, influenza A viruses are known to cause pandemics when novel antigenic subtypes from an avian reservoir acquire the ability to infect and propagate in humans (Webster, 2002). Pandemic years are characterized by higher rates of infection, morbidity and mortality in humans due to influenza A virus. Currently, both inactivated and live attenuated (cold-adapted) vaccines against influenza are available (Palese & García-Sastre, 2002). However, these vaccines require high doses of antigen, making their use difficult on a worldwide scale. The investigation of novel approaches leading to improved influenza virus vaccines is an active area of research with potential for great benefit in human health.

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 (Madin–Darby 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. ml–1) 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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Fig. 1. Attenuation of replication in mice of NS1 mutant viruses. (a) Body weights after intranasal infection with 106 p.f.u. of viruses. (b) Viral titres in lungs after intranasal infection with 106 p.f.u. of viruses. The x axis is set at the limit of detection of the assay (10 p.f.u. ml–1).

 
Next we determined the immunogenic properties of the NS1 mutant viruses. For this purpose we measured specific cellular and humoral immune responses induced in animals intranasally inoculated with these viruses. In order to measure the induction of CD8+ specific T cells, splenocytes from groups of three mice intranasally immunized with 106 p.f.u. were collected at 6 days post-infection. CD4+ T cells were immunomagnetically depleted using anti-CD4 antibody linked to magnetic beads (Biomag selecta pure anti-mouse CD4; Polyscience). Remaining CD8+ T cells were used to perform an IFN{gamma} ELISPOT. Briefly, 96-well filter plates (Millipore) were coated with purified anti-IFN{gamma} antibody (R46A2 from BD Biosciences Pharmingen) at 5 µg per well. After incubation with the coating antibody, P815 cells previously primed with a CD8-specific NP peptide (TYQRTRALV) were plated (Murata et al., 1996). Serial dilutions of CD8+ T cells from immunized mice were then added to the plates. After a 48 h incubation at 37 °C and extensive washing, plates were incubated with anti-IFN{gamma} specific antibody linked to biotin (XGX1.2 from Pharmingen). Plates were then incubated with anti-biotin antibody linked to peroxidase (Kirkegaard and Perry Laboratories) and the assay was developed with peroxidase substrate (DakoA). Positive spots were quantified by optical microscopy. The results of the ELISPOT assay are represented in Fig. 2(a). Mice immunized with 106 p.f.u. of the mouse-adapted WSN virus developed an NP-specific immune response. Interestingly, wild-type and NS1 mutant Vic viruses induced similar levels of CD8-specific T cells to those induced by WSN virus. When we determined the amount of IFN{gamma} secreted from splenocytes (collected 10 days after immunization) after incubation for 4 days with WSN virus we also observed similar responses induced by wild-type and NS1 mutant viruses (Fig. 2b). No IL-4 was detected in these assays (data not shown). These results indicate that despite the reduced replication of NS1 mutant viruses in mice, these viruses induce influenza-virus-specific cellular immune response to levels comparable to those of wild-type virus.



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Fig. 2. Immunogenicity in mice of NS1 mutant viruses. (a) Numbers of IFN{gamma}-secreting, NP-peptide-specific CD8+ T cells in spleen at day 6 after intranasal immunization with 106 p.f.u. of viruses. No spots were detected using splenocytes from naïve animals. (b) Levels of IFN{gamma} secreted after in vitro stimulation with influenza virus of splenocytes isolated from mice at day 10 after intranasal immunization with 106 p.f.u. of viruses. Limit of detection, 0·5 ng ml–1. (c) Levels of influenza-virus-specific IgG antibodies in sera from mice at week 3 after intranasal immunization. WSN-specific IgG levels in naïve animals were undetectable. Limit of detection, 0·1 µg ml–1. The error bars indicate SD (n=8).

 
To determine the ability of the NS1 mutant viruses to induce humoral responses, we immunized mice with 106 or 3x104 p.f.u. of wild-type, Vic-NS1-110 or Vic-NS1-81 viruses. Sera from immunized animals were collected at 3 weeks post-immunization. As controls, we also immunized mice with a 50 % lethal dose of mouse-adapted WSN virus (103 p.f.u.). Detection of influenza-virus-specific IgG antibodies in the sera of immunized mice was performed by ELISA. Since the recombinant Vic viruses have the HA and NA antigens derived from WSN virus, we used ELISA plates coated with WSN virus (25 µg ml–1), and subsequently incubated with dilutions of sera from immunized mice. Anti mouse-IgG specific antibodies linked to biotin were used for detection (RD Systems) followed by incubation with streptavidin linked to peroxidase. The assays were developed using ABTS substrate for peroxidase (Roche). ELISA plates were read at 450 nm in an ELISA reader. Concentrations of antibodies were calculated using a standard curve generated with a monoclonal antibody (2G9) recognizing the HA protein of WSN virus influenza virus. As seen in Fig. 2(c), immunizations with 106 p.f.u. of wild-type and Vic-NS1-110 viruses were highly effective in inducing a humoral immune response against the homologue H1N1 WSN virus. Although Vic-NS1-81 virus-immunized mice also developed antibodies against influenza virus, the response was reduced by approximately 50 %. A similar reduction was observed in mice immunized with a lower dose (3x104 p.f.u.) of the recombinant viruses.

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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Fig. 3. Mice immunized with NS1 mutant viruses are protected against lethal influenza virus challenge. Groups of eight mice were intranasally immunized with a high dose (HD, 106 p.f.u.) or low dose (LD, 3x104 p.f.u.) of the indicated NS1 mutant viruses, and subsequently challenged with a lethal dose of influenza A/WSN/33 virus. (a) Percentage survival after challenge. (b) Body weights after challenge.

 
The results presented in this report clearly show that NS1 mutants Vic-NS1-110 and Vic-NS1-81 are not only temperature-sensitive in tissue culture but attenuated in the murine system in vivo. These results are in line with other publications that have shown a role for NS1 as a virulence factor and the possibility of using NS1 mutant viruses as master strains for vaccine development (Talon et al., 2000b; Ferko et al., 2001, 2004; Takasuka et al., 2002). However, the viruses used before for these assays were adapted to grow in mice or chick embryos. Of note, the temperature-sensitive phenotype of the Vic-NS1-110 and Vic-NS1-81 mutant viruses allows their replication at the permissive temperature in MDCK cells, a vehicle under consideration for influenza virus vaccine preparation (Falcón et al., 2004). In addition, the mutations present in these viruses are insertions that lead to frameshifts in the NS1 ORF and therefore are likely to be stable. Indeed, no revertant virus could be obtained after plating more than 3x106 p.f.u. of these mutants at the restrictive temperature (data not shown).

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{gamma} 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.


   ACKNOWLEDGEMENTS
 
We gratefully acknowledge Richard Cádagan for excellent technical assistance and Luis Martínez-Sobrido for advice and help in all experiments. A. M. F. was a fellow of the Programa de Formación de Personal Investigador, Ministerio de Ciencia y Tecnología, and of the European Molecular Biology Organization (EMBO) short-term programme. This work was partly supported by NIH grants AI46954 and AI48204 to A. G.-S., AI62623 to A. F.-S. and T. M. M., and by the Programa Sectorial de Promoción General del Conocimiento (grants PB97-1160 and BMC01-1223) and the Comunidad de Madrid (grants 08.2/0025/98 and 08.2/0012.1/2001).


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Received 23 February 2005; accepted 15 June 2005.