Isolation of immunogenic neuraminidases of human influenza viruses by a combination of genetic and biochemical procedures

Gallagher, Marie R.; Bucher, Doris; Dourmashkin, R. R.; Davis, James F.; Rosenn, G; Kilbourne, Edwin D.

doi:10.1128/jcm.20.1.89-93.1984

Cited by 24 publications

(7 citation statements)

References 20 publications

(15 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…After 10 to 15 fractions (2 ml per fraction) were obtained, the elution buffer was changed to high-salt HA-eluting buffer (0.05 M Tris-hydrochloride [pH 7.5], 0.2 M NaCl, 0.1% Triton X-100). The addition of a high-salt HA-eluting buffer increased the overall yield of HA off the DEAE-Sephadex column (12). After chromatography, individual fractions were dialyzed against sodium acetate buffer with 2 mM CaCl2 for 96 h to remove any residual detergent.…”

Section: Methodsmentioning

confidence: 99%

“…NA isolated by this procedure coelutes with viral lipids and spontaneously forms liposomes after dialysis (12). Protein was quantitated by the method of Lowry et al (26).…”

Section: Methodsmentioning

confidence: 99%

“…Viruses used in this study H3 HA and N2 NA were extracted and purified from influenza virus particles as described by Gallagher et al(12) with several modifications. Virus pelleted from 200 eggs (yield, 20 to 40 mg) was suspended in 1 ml of sodium acetate buffer (0.05 M sodium acetate, 2 mM CaCl2, 0.2 mM EDTA [pH 7…”

mentioning

confidence: 99%

See 2 more Smart Citations

Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection

Johansson

Bucher

Kilbourne

1989

J Virol

Self Cite

194

View full text Add to dashboard Cite

BALB/c mice immunized with graded doses of chromatographically purified hemagglutinin (HA) and neuraminidase (NA) antigens derived from A/Hong Kong/1/68 (H3N2) influenza virus demonstrated equivalent responses when HA-specific and NA-specific serum antibodies were measured by enzyme-linked immunosorbent assays (ELISAs). Antibody responses measured by hemagglutination inhibition or neuraminidase inhibition titrations showed similar kinetic patterns, except for more rapid decline in hemagglutination inhibition antibody. Injection of mice with either purified HA or NA resulted in immunity manifested by reduction in pulmonary virus following challenge with virus containing homologous antigens. However, the nature of the immunity induced by the two antigens differed markedly. While HA immunization with all but the lowest doses of antigen prevented manifest infection, immunization with NA was infection-permissive at all antigen doses, although reduction in pulmonary virus was proportional to the amount of antigen administered. The immunizing but infection-permissive effect of NA immunization over a wide range of doses is in accord with results of earlier studies with mice in which single doses of NA and antigenically hybrid viruses were used. The demonstrable immunogenicity of highly purified NA as a single glycoprotein without adjuvant offers a novel infection-permissive approach with potentially low toxicity for human immunization against influenza virus.

show abstract

Section: Methodsmentioning

confidence: 99%

“…NA isolated by this procedure coelutes with viral lipids and spontaneously forms liposomes after dialysis (12). Protein was quantitated by the method of Lowry et al (26).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection

Johansson

Bucher

Kilbourne

1989

J Virol

Self Cite

194

View full text Add to dashboard Cite

show abstract

“…HA-free NA was purified from PR8 virus by the method of Gallagher et al . (22) . Yield of highly purified NA was very low and its use was restricted to quantitative immunoassays.…”

Section: Methodsmentioning

confidence: 99%

Class II major histocompatibility complex-restricted T cells specific for a virion structural protein that do not recognize exogenous influenza virus. Evidence that presentation of labile T cell determinants is favored by endogenous antigen synthesis.

Eisenlohr

Hackett

1989

The Journal of Experimental Medicine

View full text Add to dashboard Cite

The contribution of viral infectivity to the expression of MHC class II-restricted T cell determinants was studied. A murine I-Ed-restricted T cell hybridoma recognizing the neuraminidase (NA) glycoprotein of influenza PR8 virus was stimulated strongly by infectious virus but failed to recognize antigen introduced on noninfectious virions. Recognition correlated with the de novo synthesis of viral NA within infected APC. The effectiveness of infectious virus did not depend strictly upon the amount of NA present in cultures, since high NA concentrations could be achieved by addition of nonreplicative virus without being stimulatory for NA-specific T cells. Recognition of a determinant generated only when synthesized in murine host cells was ruled out, since, in high concentration, NA isolated from purified egg-grown virions, even if reduced and alkylated, was recognized by the T hybridoma clone. Isolated NA was recognized when added to pre-fixed APC, suggesting that this form of antigen was able to bypass the usual processing pathway of exogenous proteins. Data suggest that endogenously synthesized antigen may contribute most significantly to presentation of labile T cell determinants. In addition to NA, recognition of an I-Ed-restricted determinant of the influenza hemagglutinin (HA) molecule, shown previously to have a relatively short half-life on APC surfaces, was enhanced greatly by infectious virus. In contrast, T cell recognition of a more stably expressed I-Ed-restricted site of the same HA polypeptide was only marginally improved on infected APC.

show abstract

“…Screening for novel inhibitors of H5N1 NA requires an adequate amount of pure NA which is also active catalytically. NA is prepared conventionally by proteolytic cleavage or detergent-treatment of the viral envelope followed by subsequent purifications to obtain the catalytic NA head domain (Franç a de Barros et al, 2003;Gallagher et al, 1984;Hocart et al, 1995;Kilbourne et al, 1968;Russell et al, 2006;Seto and Rott, 1966;Takimoto et al, 1992;Ward et al, 1982). Using such methods, head domain of N1 NA was extracted from viral particles in sufficient amounts for studies of three-dimensional structure (Russell et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Avian influenza A/H5N1 neuraminidase expressed in yeast with a functional head domain

Yongkiettrakul

Boonyapakron

Jongkaewwattana

et al. 2009

Journal of Virological Methods

View full text Add to dashboard Cite

The study reports heterologous expression in Pichia pastoris of active neuraminidase derived from avian influenza virus A/Viet Nam/DT-036/2005(H5N1). A gene encoding the neuraminidase N1 head domain (residues 63-449) was fused directly in-frame with the Saccharomyces cerevisiae alpha-factor secretion signal in pPICZ(A vector. Recombinant N1 neuraminidase was expressed in P. pastoris as a 72kDa secreted, soluble protein. Glycopeptidase F treatment generated a 45kDa product, indicating that the secreted recombinant N1 neuraminidase is an N-linked glycoprotein. Kinetic studies and inhibition tests with oseltamivir carboxylate demonstrated that the recombinant N1 neuraminidase has similar K(m) and K(i) values to those of the viral N1 neuraminidase. This yeast-based heterologous expression system provided functionally active recombinant N1 neuraminidase that should be useful in anti-influenza drug screening, and also as a potential protein-based vaccine.

show abstract

Isolation of immunogenic neuraminidases of human influenza viruses by a combination of genetic and biochemical procedures

Cited by 24 publications

References 20 publications

Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection

Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection

Class II major histocompatibility complex-restricted T cells specific for a virion structural protein that do not recognize exogenous influenza virus. Evidence that presentation of labile T cell determinants is favored by endogenous antigen synthesis.

Avian influenza A/H5N1 neuraminidase expressed in yeast with a functional head domain

Contact Info

Product

Resources

About