Neuraminidase assays.

Anti-neuraminidase (NA) antibodies (Ab) play a role in protection against influenza and in combination with anti-HA Ab they increase the protection in mice. To control the NA content of vaccines, which should improve vaccine standardisation and may benefit vaccine efficacy, a series of questions must be addressed: 1) The antigenic characterization of NA in vaccine strains and seed lots is based on the measurement of the enzymatic (E) activity using fetuin as substrate. The antigenic profile is established by inhibiting the E activity with post infectious ferret antisera. Overnight incubation ensures sensitivity, and fetuin substrate gives specificity by detection of variant specific antibodies. Several difficulties have to be overcome, such as the low level of E activity in MDCK grown viruses, and the lability of N1. 2) The NA protein content of the vaccines (in bulk or final product) can be measured by an ELISA capture test but the lability of the NA proteins at 4 degrees C must be checked. 3) The anti NA Ab response can be measured using a neuraminidase inhibition test. --The steric hindrance by HI antibodies does not exceed a titre of 20 in human sera. --Triton treatment of viruses reduces the steric hindrance in polyclonal sera and monoclonal antibodies but unmasks epitopes. 4) The correlations between neuraminidase inhibition, neutralization and protection, has been established in the mouse model, but remains to be shown in humans. 5) The use of a small fluorescent (MUN) or chemiluminescent (NA-STAR) substrate can be used for the rapid differentiation of N1 from N2 and NB, but not for the titration of protective NI antibodies.

Suitability of MDCK cells grown in a serum-free medium for influenza virus production.

MDCK cells have been adapted to grow in a serum-free environment using Ultra-MDCK medium (BioWHITTAKER). The growth of adapted U-MDCK cells was maintained for over a year without any reduction in growth rate or modification of cell karyotype; cells were scaled up to spinner culture using several microcarriers. The cells were shown to be a very good host for influenza A and B virus replication in both the presence and absence of trypsin in the infection medium. Trypsin-independent viruses replicated to high titres (10(7)-10(8) TCID50/ml) in U-MDCK cells, after selection through serial passages without trypsin. This virus progeny exhibited uncleaved and antigenically modified haemagglutinin compared with standard viruses grown with trypsin. Finally, large amounts of influenza A and B viruses were produced in U-MDCK cells grown on microcarriers under rod-stirred conditions using selected trypsin-independent variants.

Difficulties in standardizing the neuraminidase content of influenza vaccines.

To achieve better standardization of influenza vaccines, an ELISA immunocapture assay was developed for N2 neuraminidase quantification. This sensitive and highly specific assay was successfully applied to vaccine preparations produced in embryonated hens' eggs from 1992 to 1997 and to antigenically related viral suspensions produced in MDCK cells. A study of the neuraminidase activity of prototype A/H3N2 strains stored at 4 degrees C showed the gradual development of enzymatic instability from 1994 onwards, accompanied by antigenic modifications of the antigen. As the phenomenon was also more pronounced with the recombinant, the question arose of the standard of immunity provided when such viruses are used for vaccination. The antibodies inhibiting neuraminidase activity in vaccinated subjects were monitored in parallel using both complete virus and purified N2 NA. The study revealed the existence of an interference phenomenon which resulted in the titre of the N1 antibodies being overestimated. The interference was due to anti-HA antibodies impeding access to the substrate at the enzymatic site by steric hindrance.

A sensitive and specific ELISA immunocapture assay for rapid quantitation of influenza A/H3N2 neuraminidase protein.

Both HA and NA proteins elicit antibodies which have been shown to be capable of altering the course of infection. Nevertheless, while influenza virus vaccine standardization involves hemagglutinin (HA) and neuraminidase (NA) in terms of antigenic characterization, only HA protein quantitation is undertaken. An immunocapture ELISA (EIA) is described for N2 NA quantitation, based on the use of a highly specific monoclonal antibody (MAb) for capturing NA and an anti-NA antiserum for antigen detection. The amounts of NA in samples were deduced from the standard curve established by using purified NA. The NA-EIA is specific and detects as a little as 7 ng/ml. The capture and detector antibodies directed against A/Beijing/32/92 NA were shown to react with H3N2 prototype strains used in current influenza vaccines, provided that an antigenically matched reference NA is used as standard.

[Role of antineuraminidase antibodies in protection against influenza]. / Place des anticorps antineuraminidase dans la protection contre la grippe.

For improving the anti-influenza vaccination efficacy, the choice of strains carrying up dated neuraminidase antigen (NA) and the introduction of the optimal amount of NA antigen in the vaccine are critical. Monoclonal antibodies prepared against the neuraminidase N2 of A/Beijing/32/92 showed NA inhibition (NI) and neutralized (Nt) the cells infection by influenza virus either at an early stage (group 2 antibodies inhibit virus binding to cells) or at a late stage of infection (group 1 antibodies inhibit virus release). The specificity of the neutralization test is restricted to the homologous variant whereas the NI specificity is much broader. When both group 1 and group 2 antibodies are tested together, their neutralizing activity is significantly increased. The emergence in 1997 of an avian strain H5N1 in humans influenza infections at Hong Kong (Strain A/Hong-Kong/156/97) rose the threat of pandemic. The H5N1 strain carried H5 HA which is not recognized by the human immune system, but N1 might be related to other N1 antigens belonging to avian, swine and human strains. So we 1) characterized the N1 antigen from H5N1 in comparison with other known antigens, 2) we looked for anti N1 (H5N1) antibodies in humans according to the age and the vaccination status, 3) we checked the neutralizing activity of anti N1 antibodies. The N1 antigen (H5N1) appeared closely related to N1 from swine strains: Sw/31 correlated itself to the pandemic spanish virus (1918-19), and more recent swine isolates from 1982 and 1989. The anti N1 (H5N1) antibodies were present in sera collected from 75+ years old persons and these N1 antibodies were neutralizing H5N1 cells infection. Consequently, 75+ years old persons do not represent a priority group for vaccination in the case of H5N1 pandemic conditions.

Hybridoma growth in a new generation hollow fibre bioreactor: antibody productivity and consistency.

This paper analyses the performance of MAbMax(TM)/Tricentric(TM), a new generation hollow fibre bioreactor, for hybridoma growth and antibody productivity, the down stream processing of monoclonal antibody harvests throughout the run and the further control of antibody quality consistency. Handling and process parameters were optimised using a mouse hybridoma, IgG1(K) secretor, and then confirmed with several other hybridomas. Cells were kept at optimal viability during an unusually long period of time and a continuously high production of antibodies was detected over several months. Foetal bovine serum concentration was reduced to 1\% and the effects of weaning of cells from serum were monitored in terms of cell metabolism and antibody productivity. Antibody harvests collected at regular intervals throughout the run (2 to 12 weeks) were purified using affinity chromatography on a recombinant protein A/G matrix and then analysed in terms of antigen binding properties, isoelectric forms and oligosaccharide structures, in order 1) to control antibody quality consistency as a function of time and serum concentration and 2) to compare antibody characteristics as a function of culture conditions, in vitro bioreactor cultivation versus in vivo mouse ascite cultivation.

Simultaneous purification of influenza haemagglutinin and neuraminidase proteins by immunochromatography.

A new and rapid method for co-purification of haemagglutinin (HA) and neuraminidase (NA) proteins from influenza A/H3N2 viruses is described. Surface glycoproteins were first solubilized using a non-ionic detergent under high ionic strength conditions, then they were separated by chromatography on sepharose previously bound to monoclonal antibodies (MAbs) directed either against HA (IaH-chromatography) or against NA (IaN-chromatography). Depending on the protein specificity of the MAb immobilized on the column, HA or NA was bound to sepharose and the counterpart protein was free in the flow-through volume. IaH-chromatography and IaN-chromatography proved equally efficient in term of recoveries (> 75%) and purity (> or = 99%) of both HA and NA but differences appeared when considering functional and antigenic properties of pure proteins. Those properties were highly retained in IaH- and IaN-derived HA as well as in IaH-derived NA while IaN-NA was partially degraded. IaH-chromatography allowed the co-purification of HA and NA proteins in heterologous antigen-antibody system with a 50% rate of cross reactivity. IaH-HA and IaH-NA may be suitable for immunity studies, standardization of influenza vaccine and for diagnostic purposes.