Reduced levels of neuraminidase of influenza A viruses correlate with attenuated phenotypes in mice

Alicia Solorzanob,1, Hongyong Zheng1, Ervin Fodor2, George G. Brownlee2, Peter Palese1 and Adolfo García-Sastre1

Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029-6574, USA1
Sir William Dunn School of Pathology, University of Oxford, Oxford , UK2

Author for correspondence: Adolfo García-Sastre. Fax +1 212 534 1684. e-mail agarcia{at}smtplink.mssm.edu


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
We have previously obtained four transfectant influenza A viruses containing neuraminidase (NA) genes with mutated base pairs in the conserved double-stranded RNA region of the viral promoter by using a ribonucleoprotein transfection system. Two mutant viruses (D2 and D1/2) which share a C-G->A-U mutation at positions 11 and 12 of the 3' and 5' ends, respectively, of the NA gene, showed an approximate 10-fold reduction of NA-specific mRNA and protein levels (Fodor et al., Journal of Virology 72, 6283–6290, 1998). These viruses have now allowed us to determine the effects of decreased NA levels on virus pathogenicity. Both D2 and D1/2 viruses were highly attenuated in mice, and their replication in mouse lungs was highly compromised as compared with wild-type influenza A/WSN/33 virus. The results highlight the importance of the level of NA activity in the biological cycle and virulence of influenza viruses. Importantly, mice immunized by a single intranasal administration of 103 infectious units of D2 or D1/2 viruses were protected against challenge with a lethal dose of wild-type influenza virus. Attenuation of influenza viruses by mutations resulting in the decreased expression of a viral protein represents a novel strategy which could be considered for the generation of live attenuated influenza virus vaccines.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Influenza viruses are enveloped, segmented negative-strand RNA viruses belonging to the family Orthomyxoviridae (Lamb & Krug, 1996 ). Among the three known types of influenza viruses, A, B and C, influenza A viruses are the most important from the point of view of human and animal pathogenicity. An influenza A virus was the causative agent of the devastating human pandemic in 1918 commonly known as ‘Spanish flu’, and influenza A virus infections continue to represent an important health problem, especially for young children and the elderly (Murphy & Webster, 1996 ). Based on the antigenicity of their external glycoproteins, haemagglutinin (HA) and neuraminidase (NA), different subtypes of influenza A viruses have been defined. At this time, only strains of H3N2 and H1N1 subtypes are circulating in the human population. Unpredictably, a new human influenza virus subtype may arise and displace the circulating strains of the old subtype (antigenic shift). These viruses can cause extensive morbidity and mortality due to the absence of neutralizing antibodies in the human population (for a review, see Webster et al., 1992 ). In general, the new pandemic viruses contain HA and/or NA genes derived from an avian influenza A virus strain, and retain most of their other genes from the circulating strain (Kawaoka et al., 1989 ; Scholtissek et al., 1978 ). In addition to antigenic shift, influenza A viruses also undergo antigenic drift. This process involves the selection of viruses bearing mutations in the HA and/or NA which change their antigenic properties (Both et al., 1983 ; Webster et al., 1982 ). Although this process is not as dramatic as antigenic shift, antigenic drift is responsible for the year-to-year variations of human influenza A and B virus strains. This drift necessitates an updating of the vaccine formulation which depends on the antigenic characteristics of the dominant strains. Due to the continuing change of the circulating strains and the limited efficacy of the currently licensed inactivated vaccines, the development of novel vaccine approaches against influenza virus represents an active research topic (Bilsel & Kawaoka, 1998 ; Palese et al., 1999 ).

The envelope of influenza A viruses contains three different transmembrane proteins, the HA, NA and M2 proteins. While the HA is responsible for the binding of the virus to neuraminic acid-containing receptors at the plasma membrane of the host cell, the NA plays a role in preventing virus aggregation after budding of the progeny viruses from the infected cell (Palese & Compans, 1976 ). This function is mediated by the enzymatic activity of the NA, which is responsible for the removal of receptors, i.e. neuraminic acids, from the virus surface. In the absence of NA activity influenza viruses remain attached to each other through HA–receptor interactions. In fact, an influenza A virus deficient in NA activity (Liu & Air, 1993 ) formed large aggregates associated with the host cell surface, but was not impaired in virus entry, replication, assembly or budding (Liu et al., 1995 ). The importance of the NA in the biological cycle of influenza viruses is also illustrated by the ability of NA inhibitors to reduce virus replication (Palese et al., 1974 ).

We have previously rescued and characterized in tissue culture four influenza A/WSN/33 viruses (D1, D2, D3 and D1/2) with altered base pairs in the conserved double-stranded region of the vRNA promoter of their NA-specific RNA segment (Fodor et al., 1998 ). The mutations did not interfere dramatically with the replication of vRNA, but the C-G->A-U (11–12') mutation in two of the transfectants (D2 and D1/2) affected their mRNA levels, most likely by interfering with the efficiency of polyadenylation. Both viruses showed an approximate tenfold reduction of NA levels and one log reduction in plaque titres on MDBK cells. In order to further characterize the role of the NA in virus pathogenicity, the virulence and replication properties of the transfectant viruses were studied in mice. Our results show that D2 and D1/2 viruses are highly attenuated in mice. In addition, single intranasal (i.n.) immunizations of mice with D2 or D1/2 viruses prevent disease and death after challenge with a lethal dose of virulent wild-type influenza virus. Therefore, these mutants are prototypes for use as live influenza virus vaccines.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cells and viruses.
Transfectant influenza viruses D1, D2, D3 and D1/2 were described previously (Fodor et al., 1998 ). These viruses are recombinant influenza A/WSN/33 (WSN) viruses containing single or double base-pair mutations in the double-stranded region of the RNA panhandle/fork structure (Fodor et al., 1995 ; Hsu et al., 1987 ) at the 3' and 5' ends of the NA-specific RNA segment. Base-pair mutations U-A->G-C (10–11'), C-G->A-U (11–12') and C-G->U-A (12–13') were introduced into the NA-specific RNA segments of D1, D2 and D3 viruses, respectively. For convenience, numbering of mutated residues starts from the 3' end and from the 5' end of the viral RNA and the 5'-end numbers are distinguished by primes. D1/2 virus contains the two base-pair changes of D1 and D2 transfectant viruses, i.e. the two base-pair mutations U-A->G-C (10–11') and C-G->A-U (11–12'). WSN wild-type and transfectant viruses D1, D2, D3 and D1/2 were grown at 37 °C in Madin–Darby bovine kidney (MDBK) cells in reinforced minimal essential medium (REM) containing 0·2% BSA. Plaque assays were performed on confluent monolayers of MDBK cells using an agar overlay in 0·2% BSA-containing REM. MDBK cells were maintained in REM containing 10% heat-inactivated foetal calf serum.

{blacksquare} Animal infections.
Mice were purchased from Taconic Farms, USA. Female BALB/c mice were used for influenza virus infections at 6–12 weeks of age. Inoculations (i.n.) were performed in mice under ether anaesthesia using 50 µl of PBS containing 106, 3x104 or 103 p.f.u. of the indicated virus. Animals were monitored daily for body weight changes, and sacrificed when observed in extremis. When appropriate, immunized animals were challenged 3 weeks post-immunization by i.n. administration of 106 p.f.u. of wild-type WSN virus. All procedures were in accord with NIH guidelines on care and use of laboratory animals.

{blacksquare} Lung titrations.
Female BALB/c mice (6–12 weeks old) were infected i.n. with 103 p.f.u. of the indicated virus. Animals were sacrificed at 3 or 6 days post-infection and their lungs were surgically removed. Lungs were homogenized in 2 ml of PBS and the homogenates were clarified by centrifugation at 3000 r.p.m. for 15 min at 4 °C. Viruses present in the supernatants were titrated by plaque assay in MDBK cells.

{blacksquare} Haemagglutination inhibition (HI) assays.
HI assays were performed as described previously (Bot et al., 1997 ). Briefly, mouse sera were treated with receptor-destroying enzyme (Sigma; cat. #C8772) to eliminate non-specific inhibitors of influenza virus-mediated haemagglutination, as described previously (Burnet & Stone, 1947 ). The HI titres are given as the highest serum dilution that was able to neutralize the haemagglutination activity of a preparation of influenza A/WSN/33 virus with a haemagglutination titre of 8. In these assays, 0·5% chicken red blood cells were used.

{blacksquare} Neuraminidase inhibition (NI) assays.
Influenza A/WSN/33 virus grown in MDBK cells (approximately 108 p.f.u./ml) was dialysed against 200 mM potassium phosphate buffer pH 6·0 containing 1 mM CaCl2. The dialysed virus preparation was used as the source for neuraminidase activity in NI assays. Neuraminidase reaction assays were carried out using 10 µl of dialysed viruses in 100 µl reaction mixtures containing 200 mM potassium phosphate buffer pH 6·0, 1 mM CaCl2, 2·8 mg/ml fetuin (Sigma) and different concentrations of sera from immunized mice. Reactions were carried out at 37 °C for 12 h, and the amount of neuraminic acids released from the fetuin was determined as previously described (García-Sastre et al., 1990 ). The NI titres represent the serum dilution that resulted in 50% inhibition of the neuraminidase activity.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
D2 and D1/2 viruses are highly attenuated in mice
Groups of five BALB/c mice were infected i.n. with 106, 3x104 or 103 p.f.u. of transfectant influenza viruses D1, D2, D3 and D1/2. As a control, mice were infected with a wild-type transfectant influenza WSN virus. This virus was previously rescued by ribonucleoprotein transfection of a wild-type NA gene (Fodor et al., 1998 ). Changes in body weight and survival rates were monitored daily. The results are shown in Fig. 1. All mice infected with wild-type virus developed signs of disease and died by day 15 post-infection. However, all mice infected with D2 and D1/2 viruses survived. Only at the higher dose (106 p.f.u.) did D2 and D1/2 virus-infected animals lose weight (10–20% of body weight was lost by day 3 post-infection), but they quickly recovered in the days following. The virulence of D1 virus was indistinguishable from that of wild-type virus. D3 virus showed a slightly attenuated phenotype in mice. As shown in Fig. 1(c) administration of 103 p.f.u. of D3 virus resulted in a statistically significant decrease in body weight loss when the mice were compared with those receiving wild-type virus (P values were 0·022, 0·007, 0·01 and 0·014 when the body weights of the animals receiving D3 and wild-type viruses were compared at days 7, 8, 9 and 10, respectively, using a t-test).



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Fig. 1. Pathogenicity of D1, D2, D3 and D1/2 viruses in mice. Groups of five mice were infected i.n. with three different doses of wild-type influenza A/WSN/33 virus and with transfectant D1, D2, D3 and D1/2 viruses:106 p.f.u. (a), 3x104 p.f.u. (b) and 103 p.f.u. (c), as indicated by the arrows. Animals were weighed daily following infection and body weights were compared with those on the day of infection. The average body weight percentages of surviving animals per group are represented. Numbers of survivors per group are indicated in the lower right corners of the graphics.

 
Impaired replication of D2 and D1/2 viruses in mouse lungs
Next, we infected i.n. groups of six BALB/c mice with 103 p.f.u. of wild-type, D1, D2, D3 or D1/2 viruses. Three days post-infection, three mice per group were sacrificed, their lungs were extracted and homogenized in 2 ml of PBS, and virus titres were measured by plaque assay in MDBK cells. Six days post-infection, the rest of the mice were also sacrificed and viral titres were determined in their lungs by the same protocol. The results are shown in Fig. 2. Wild-type and D1 viruses grew to high titres in the lungs of the infected mice (approximately 106 and 107 p.f.u./ml at days 3 and 6 post-infection, respectively). Titres in lungs of mice infected with D3 virus were approximately one and a half logs lower. By contrast, viral titres were not detectable or very low (less than 103 p.f.u./ml) in the lungs of D2- and D1/2-infected mice. The results demonstrate that replication of D2 and D1/2 viruses is highly impaired in mouse lungs.



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Fig. 2. Viral titres in lungs of mice infected with D1, D2, D3 and D1/2 viruses. Animals were infected i.n. with 103 p.f.u. of the indicated virus and sacrificed at days 3 or 6 post-infection. After removal and homogenization of lungs, viral titres were determined by plaque assay. Titres are shown for individual mice infected with wild-type (WT) virus (•), D1 virus ({circ}), D2 virus ({blacksquare}), D3 virus ({square}) and D1/2 virus ({blacktriangleup}).

 
Single immunization of mice with D2 and D1/2 attenuated viruses results in protection against wild-type influenza virus infection
Sera from the groups of mice which were infected i.n. with D2 or D1/2 viruses were collected 3 weeks after infection. The presence of HI antibodies against influenza WSN virus in individual sera was determined by an HI assay. HI titres were in general higher in animals immunized with the higher dose of virus (Table 1). In addition, all mice were protected against death or disease (as measured by body weight loss) when challenged with more than 1000 LD50s of wild-type WSN virus (Table 1, Fig. 3). NI antibodies (titres between 80 and 110) were also detected in sera from animals immunized with the highest doses of D2 or D1/2 viruses. These titres were approximately five times lower than those attained in animals surviving a challenge with D1 or D3 viruses. These differences most likely reflect the lower neuraminidase levels of D2 and D1/2 viruses. Nevertheless, HI antibodies alone are known to provide protection against influenza virus infections.


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Table 1. Antibody titres in sera and protection against wild-type influenza A/WSN/33 virus infection of mice immunized i.n. with D2 and D1/2 viruses

 


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Fig. 3. Immunization of mice with D2 or D1/2 viruses confers protection against a lethal challenge with wild-type influenza A/WSN/33 virus. Groups of mice were immunized i.n. with D2 (a) or D1/2 (b) viruses, using the doses indicated in the lower right corners of the graphics. Three weeks post-immunization, animals were challenged by i.n. infection with 106 p.f.u. (more than 103 LD50s) of wild-type influenza A/WSN/33 virus, as indicated by the arrows. Animals were weighed daily following challenge and their body weights were compared with those on the day of infection. Body weight percentages are shown as an average of five animals.

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
In this study, we have investigated the attenuation properties in mice of influenza A viruses which express lower levels of NA protein as compared to that of wild-type virus. For this purpose we have used transfectant influenza viruses containing mutations in the non-coding regions of their NA-specific RNA segments. In the case of D2 and D1/2 viruses, these mutations were responsible for a 10-fold decrease in the amount of the NA expression (Fodor et al., 1998 ). Since the mutations do not significantly affect the levels of viral RNA replication (Fodor et al., 1998 ) the phenotypic changes in the transfectant D2 and D1/2 viruses are most likely due to their decreased expression of the NA. The D2 and D1/2 transfectant viruses were highly attenuated in mice. By contrast D1 virus, which was previously found to contain NA levels similar to wild-type WSN virus, had pathogenic properties in mice identical to wild-type viruses. Interestingly, D3 virus showed slight levels of attenuation in mice. Although we did not find a significant change in the NA expression levels of D3 virus in tissue culture (Fodor et al., 1998 ), it is possible that subtle and difficult to detect changes in NA expression are responsible for their attenuation properties. Indeed, a 10-fold reduction in NA protein levels was responsible for at least a four log increase in the LD50 in mice of D2 and D1/2 viruses, indicating that the mouse system, and probably other animal systems, is highly sensitive to changes in NA levels of the virus.

The generation of more potent inhibitors of the NA of influenza viruses, such as zanamivir (von Itzstein et al., 1993 ), was facilitated by knowledge of the three-dimensional structure of the NA, which was determined by X-ray crystallography (Baker et al., 1987 ; Tulip et al., 1991 ; Varghese et al., 1983 , 1992 ). These novel NA inhibitors have promising therapeutic and prophylactic properties as antiviral drugs against influenza (Calfee & Hayden, 1998 ; Fleming, 1999 ; Hayden et al., 1997 ). Our results show that a 10-fold reduction of the NA levels reduces viral pathogenicity dramatically, which suggests that inhibitors of NA reducing in vivo activity by 10-fold or more may have an adequate therapeutic effect.

We have shown that D2 and D1/2 viruses, containing base-pair mutations in the non-coding regions of their NA genes, are highly attenuated in mice. Significantly, however, the D2 and D1/2 virus-immunized mice developed HI antibodies against the virus and they were protected against a lethal dose of wild-type A/WSN/33 virus corresponding to more than 1000 LD50s. Protection was achieved after a single i.n. administration of as little as 1000 p.f.u. of viruses. In our studies we have used a mouse model of influenza virus infection which requires the use of a mouse-adapted strain of influenza virus, such as WSN. Further experimentation in different animal models, e.g. ferrets, and ultimately in humans, will be required to determine the potential of human recombinant influenza A viruses with base-pair mutations in their promoters as live attenuated vaccines.

Influenza viruses with base-pair mutations are expected to be more stable than single-base mutants, since two specific mutations have to occur simultaneously in the same molecule in order to revert to a wild-type sequence. In addition, a single mutation is expected to result in further attenuation of the virus since it would disrupt the complementarity which is required for optimal transcriptional activity of the viral RNA template (Fodor et al., 1995 ; Kim et al., 1997 ; Luo et al., 1991 ; Pritlove et al., 1995 ). Indeed, the base-pair mutation in the D2 transfectant was maintained through 10 passages on MDBK cells (Fodor et al., 1998 ). It is of interest whether analogous mutations in other influenza A virus segments would affect mRNA levels and consequently protein levels in the same way as in the NA segment. Attenuation of influenza A viruses through mutations in RNA segments other than the HA- or NA-specific RNAs would result in donor strains where the HA and NA genes from recently circulating strains could be easily incorporated by reassortment.

The generation of improved vaccines against influenza viruses may be the key for the control of influenza. Current licensed influenza vaccines for humans are of the inactivated type, and it is believed that live attenuated strains such as the first-generation cold-adapted vaccines would induce higher and possibly longer lasting protection (Maassab et al., 1998 ). The development of reverse genetics techniques to genetically manipulate the genome of influenza virus (Enami et al., 1990 ; Fodor et al., 1999 ; Neumann et al., 1999 ; Pleschka et al., 1996 ) allows the rational design of attenuated influenza viruses by the introduction of specific mutations into their genomes and may lead to novel second generation live influenza virus vaccines. Mutations affecting both the amino acid sequence of the virus proteins (Castrucci et al., 1992 ; Li et al., 1999 ; Luo et al., 1993 ; Parkin et al., 1997 ; Subbarao et al., 1995 ) and the levels of replication of the RNA segments (Luo et al., 1992 ; Muster et al., 1991 ) have been used to attenuate influenza A viruses. In the present manuscript, we show a novel way leading to influenza virus attenuation in mice. The D2 and D1/2 mutations resulted in a down-regulation of mRNA and protein levels without significant effects on RNA replication (Fodor et al., 1998 ). It is noteworthy that the Sabin live poliovirus vaccines also contain attenuated viruses with mutations at the 5' non-coding regions of the viral genomes resulting in lower levels of protein expression (Gutierrez et al., 1997 ; Haller et al., 1996 ; La Monica & Racaniello, 1989 ). Mutations affecting the base-pairs of the 3' and 5' non-coding regions of the influenza virus genes might also be included in recombinant influenza virus vectors expressing foreign antigens as an additional safety control measure (García-Sastre, 1998 ).


   Acknowledgments
 
This work was supported by the National Institutes of Health (A.G.-S. and P.P.), the Max Kade Foundation (E.F.), the Medical Research Council (program grant G9523972 to G.G.B.) and an MEC pre-doctoral fellowship to A.S. from the Spanish Government. We thank Louis Nguyenvu for excellent technical assistance.


   Footnotes
 
b Present address: Department of Microbiology, University of Salamanca, Salamanca, Spain.


   References
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Received 17 August 1999; accepted 11 November 1999.