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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 HAreceptor 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 (1112') 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animal infections.
Mice were purchased from Taconic Farms, USA. Female BALB/c mice were used for influenza virus infections at 612 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.
Lung titrations.
Female BALB/c mice (612 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.
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.
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bilsel, P. & Kawaoka, Y. (1998). New approaches to vaccination. In Textbook of Influenza, pp. 422-434. Edited by K. G. Nicholson, R. G. Webster & A. J. Hay. Oxford: Blackwell Science.
Bot, A., Antohi, S., Bot, S., García-Sastre, A. & Bona, C. (1997). Induction of humoral and cellular immunity against influenza virus by immunization of newborn mice with a plasmid bearing a hemagglutinin gene. International Immunology 9, 1641-1650.[Abstract]
Both, G. W., Sleigh, M. J., Cox, N. J. & Kendal, A. P. (1983). Antigenic drift in influenza virus H3 hemagglutinin from 1968 to 1980: multiple evolutionary pathways and sequential amino acid changes at key antigenic sites. Journal of Virology 48, 52-60.[Medline]
Burnet, F. M. & Stone, J. D. (1947). The receptor-destroying enzyme of V. cholerae. Australian Journal of Experimental Medical Sciences 25, 227-233.
Calfee, D. P. & Hayden, F. G. (1998). New approaches to influenza chemotherapy. Neuraminidase inhibitors. Drugs 56, 537-553.[Medline]
Castrucci, M. R., Bilsel, P. & Kawaoka, Y. (1992). Attenuation of influenza A virus by insertion of a foreign epitope into the neuraminidase. Journal of Virology 66, 4647-4653.[Abstract]
Enami, M., Luytjes, W., Krystal, M. & Palese, P. (1990). Introduction of site-specific mutations into the genome of influenza virus. Proceedings of the National Academy of Sciences, USA 87, 3802-3805.[Abstract]
Fleming, D. M. (1999). Treating influenza with zanamivir. Lancet 353, 668-669.[Medline]
Fodor, E., Pritlove, D. C. & Brownlee, G. G. (1995). Characterization of the RNA-fork model of virion RNA in the initiation of transcription in influenza A virus. Journal of Virology 69, 4012-4019.[Abstract]
Fodor, E., Palese, P., Brownlee, G. G. & García-Sastre, A. (1998). Attenuation of influenza A virus mRNA levels by promoter mutations. Journal of Virology 72, 6283-6290.
Fodor, E., Devenish, L., Engelhardt, O. G., Palese, P., Brownlee, G. G. & García-Sastre, A. (1999). Rescue of influenza A virus from recombinant DNA. Journal of Virology 73, 9679-9682.
García-Sastre, A. (1998). Negative-strand RNA viruses: applications to biotechnology. Trends in Biotechnology 16, 230-235.[Medline]
García-Sastre, A., Villar, E., Hannoun, C. & Cabezas, J. A. (1990). Sialidase activity in rimantadine-resistant and -sensitive influenza A viruses. Enzyme 43, 207-211.[Medline]
Gutierrez, A. L., Denova-Ocampo, M., Racaniello, V. R. & del Angel, R. M. (1997). Attenuating mutations in the poliovirus 5' untranslated region alter its interaction with polypyrimidine tract-binding protein. Journal of Virology 71, 3826-3833.[Abstract]
Haller, A. A., Stewart, S. R. & Semler, B. L. (1996). Attenuation stemloop lesions in the 5' noncoding region of poliovirus RNA: neuronal cell-specific translation defects. Journal of Virology 70, 1467-1474.[Abstract]
Hayden, F. G., Osterhaus, A. D., Treanor, J. J., Fleming, D. M., Aoki, F. Y., Nicholson, K. G., Bohnen, A. M., Hirst, H. M., Keene, O. & Wightman, K. (1997). Efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenzavirus infections. GG167 Influenza Study Group. New England Journal of Medicine 337, 874-880.
Hsu, M. T., Parvin, J. D., Gupta, S., Krystal, M. & Palese, P. (1987). Genomic RNAs of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle. Proceedings of the National Academy of Sciences, USA 84, 8140-8144.[Abstract]
Kawaoka, Y., Krauss, S. & Webster, R. G. (1989). Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics. Journal of Virology 63, 4603-4608.[Medline]
Kim, H. J., Fodor, E., Brownlee, G. G. & Seong, B. L. (1997). Mutational analysis of the RNA- fork model of the influenza A virus vRNA promoter in vivo. Journal of General Virology 78, 353-357.[Abstract]
Lamb, R. A. & Krug, R. M. (1996). Orthomyxoviridae. The viruses and their replication. In Fields Virology, pp. 1353-1395. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
La Monica, N. & Racaniello, V. R. (1989). Differences in replication of attenuated and neurovirulent polioviruses in human neuroblastoma cell line SH-SY5Y. Journal of Virology 63, 2357-2360.[Medline]
Li, S., Liu, C., Klimov, A., Subbarao, K., Perdue, M. L., Mo, D., Ji, Y., Woods, L., Hietala, S. & Bryant, M. (1999). Recombinant influenza A virus vaccines for the pathogenic human A/Hong Kong/97 (H5N1) viruses. Journal of Infectious Diseases 179, 1132-1138.[Medline]
Liu, C. & Air, G. M. (1993). Selection and characterization of a neuraminidase-minus mutant of influenza virus and its rescue by cloned neuraminidase genes. Virology 194, 403-407.[Medline]
Liu, C., Eichelberger, M. C., Compans, R. W. & Air, G. M. (1995). Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly, or budding. Journal of Virology 69, 1099-1106.[Abstract]
Luo, G. X., Luytjes, W., Enami, M. & Palese, P. (1991). The polyadenylation signal of influenza virus RNA involves a stretch of uridines followed by the RNA duplex of the panhandle structure. Journal of Virology 65, 2861-2867.[Medline]
Luo, G., Bergmann, M., García-Sastre, A. & Palese, P. (1992). Mechanism of attenuation of a chimeric influenza A/B transfectant virus. Journal of Virology 66, 4679-4685.[Abstract]
Luo, G., Chung, J. & Palese, P. (1993). Alterations of the stalk of the influenza virus neuraminidase: deletions and insertions. Virus Research 29, 141-153.[Medline]
Maassab, H. F., LaMontagne, J. R. & DeBorde, D. C. (1998). Live influenza virus vaccines. In Vaccines, pp. 435-457. Edited by S. A. Plotkin & E. A. Mortimer. Philadelphia: Saunders.
Murphy, B. R. & Webster, R. G. (1996). Orthomyxoviruses. In Fields Virology, pp. 1397-1445. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
Muster, T., Subbarao, E. K., Enami, M., Murphy, B. R. & Palese, P. (1991). An influenza A virus containing influenza B virus 5' and 3' noncoding regions on the neuraminidase gene is attenuated in mice. Proceedings of the National Academy of Sciences, USA 88, 5177-5181.[Abstract]
Neumann, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Gao, P., Hughes, M., Perez, D. R., Donis, R., Hoffmann, E., Hobom, G. & Kawaoka, Y. (1999). Generation of influenza A viruses entirely from cloned cDNAs. Proceedings of the National Academy of Sciences, USA 96, 9345-9350.
Palese, P. & Compans, R. W. (1976). Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA): mechanism of action. Journal of General Virology 33, 159-163.[Abstract]
Palese, P., Schulman, J. L., Bodo, G. & Meindl, P. (1974). Inhibition of influenza and parainfluenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA). Virology 59, 490-498.[Medline]
Palese, P., Muster, T., Zheng, H., ONeill, R. & García-Sastre, A. (1999). Learning from our foes: a novel vaccine concept for influenza virus. Archives of Virology 15, 1-8.
Parkin, N. T., Chiu, P. & Coelingh, K. (1997). Genetically engineered live attenuated influenza A virus vaccine candidates. Journal of Virology 71, 2772-2778.[Abstract]
Pleschka, S., Jaskunas, R., Engelhardt, O. G., Zürcher, T., Palese, P. & García-Sastre, A. (1996). A plasmid-based reverse genetics system for influenza A virus. Journal of Virology 70, 4188-4192.[Abstract]
Pritlove, D. C., Fodor, E., Seong, B. L. & Brownlee, G. G. (1995). In vitro transcription and polymerase binding studies of the termini of influenza A virus cRNA: evidence for a cRNA panhandle. Journal of General Virology 76, 2205-2213.[Abstract]
Scholtissek, C., Rohde, W., Von Hoyningen, V. & Rott, R. (1978). On the origin of the human influenza virus subtypes H2N2 and H3N2. Virology 87, 13-20.[Medline]
Subbarao, E. K., Park, E. J., Lawson, C. M., Chen, A. Y. & Murphy, B. R. (1995). Sequential addition of temperature-sensitive missense mutations into the PB2 gene of influenza A transfectant viruses can effect an increase in temperature sensitivity and attenuation and permits the rational design of a genetically engineered live influenza A virus vaccine. Journal of Virology 69, 5969-5977.[Abstract]
Tulip, W. R., Varghese, J. N., Baker, A. T., van Donkelaar, A., Laver, W. G., Webster, R. G. & Colman, P. M. (1991). Refined atomic structures of N9 subtype influenza virus neuraminidase and escape mutants. Journal of Molecular Biology 221, 487-497.[Medline]
Varghese, J. N., Laver, W. G. & Colman, P. M. (1983). Structure of the influenza virus glycoprotein antigen neuraminidase at 2·9 resolution. Nature 303, 35-40.[Medline]
Varghese, J. N., McKimm-Breschkin, J. L., Caldwell, J. B., Kortt, A. A. & Colman, P. M. (1992). The structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor. Proteins 14, 327-332.[Medline]
von Itzstein, M., Wu, W. Y., Kok, G. B., Pegg, M. S., Dyason, J. C., Jin, B., Van Phan, T., Smythe, M. L., White, H. F., Oliver, S. W., Colman, P. M., Varghese, J. N., Ryan, D. M., Woods, J. M., Bethell, R. C., Hotham, V. J., Cameron, J. M. & Penn, C. R. (1993). Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature 363, 418-423.[Medline]
Webster, R. G., Laver, W. G., Air, G. M. & Schild, G. C. (1982). Molecular mechanisms of variation in influenza viruses. Nature 296, 115-121.[Medline]
Webster, R. G., Bean, W. J., Gorman, O. T., Chambers, T. M. & Kawaoka, Y. (1992). Evolution and ecology of influenza A viruses. Microbiological Reviews 56, 152-179.[Abstract]
Received 17 August 1999;
accepted 11 November 1999.