G.C. Schild

An improved single-radial-immunodiffusion technique for the assay of influenza haemagglutinin antigen: Application for potency determinations of inactivated whole virus and subunit vaccines

An improved single-radial-diffusion technique for the assay of influenza haemagglutinin antigen is described. The modified method enables the results of assays of antigen to be obtained more rapidly and with greater precision than previously. The use of immunoplates containing varied, pre-selected concentrations of anti-haemagglutinin antibody allows accurate assays to be performed over a wide range of antigen concentrations. Concentrations of haemagglutinin as low as 40 i.u./ml could be assayed with accuracy and reproducibility using immunoplates containing low antibody levels. The method is applicable to the accurate determination of haemagglutinin concentrations over the ranges likely to be present in inactivated influenza vaccines. In tests on ‘whole virus’ antigen preparations, it was found that the ratio between haemagglutination titre (i.u./ml) and haemagglutinin antigen activity (μg/ml) determined by single-radial-diffusion was relatively constant for antigens containing a given strain but showed variation between strains (range 16·5–26·8 i.u./μg HA activity). For the subunit vaccines examined this ratio showed a large degree of variation (range 1·4–16·6 i.u./μg HA activity) and in general was considerably lower than for whole virus antigens. These findings support the conclusion that techniques based on the agglutination of erythrocytes may provide data on vaccine potency which are not directly comparable from strain to strain for ‘whole virus’ vaccines and that these methods are entirely inappropriate to potency assays of split-product or subunit vaccines. In contrast, single-radial-diffusion may be of value for assays of both ‘whole virus’ vaccines and those containing disrupted virions.

Alterations in the hemagglutinin associated with adaptation of influenza B virus to growth in eggs

In 1943 Burnet reported on changes in the hemagglutinating properties of human influenza virus which occurred during adaptation of the virus to growth in chicken eggs. Only recently has direct evidence been presented that these changes affect the antigenic properties of the virus. Schild et al. (G. C. Schild, J. S. Oxford, J. C. deJong, and R. G. Webster (1983), Nature (London) 303, 706-709) demonstrated that egg adaptation of influenza B virus selects variants which are antigenically distinct from virus grown from the same source in mammalian cells. The molecular changes in the hemagglutinin (HA) of influenza B virus associated with adaptation to growth in eggs have now been identified. A specific glycosylation site at the distal tip of the HA of influenza B virus grown exclusively in mammalian cell culture is lost or altered during egg adaptation. Since the HA functions in adsorption of virus to cells, it is concluded that removal or modification of an oligosaccharide structure at this position is required for influenza B virus to attach to and infect the allantois cells of the egg and that this has important implications for the antigenic configuration of the molecule.

Clinical studies of monovalent inactivated whole virus and subunit A/USSR/77 (H1N1) vaccine: serological responses and clinical reactions

The clinical acceptability and antibody responses to graded doses of whole virus, aqueous surface antigen and adsorbed surface antigen influenza A/USSR/92/77 (H1N1) virus vaccines were assessed in 1335 healthy volunteers in a double-blind, multi-centre study conducted in the U.K. in February and March 1978. Before vaccination the presence of haemagglutination-inhibiting (HI) and neuraminidase-inhibiting (NI) serum anti-bodies to the vaccine virus was infrequent and in low titres in volunteers aged ≤ 25 years but was more frequent in older persons. In volunteers aged ≤ 25 years, who were sero-negative for HI antibody before vaccination, the HI antibody responses following one dose of vaccine were generally of low frequency and titre for all doses and types of vaccine. Nevertheless, whole virus or subunit vaccines with high antigenic content (≥ 47 μg haemagglutinin (HA) per dose) induced HI antibody responses in at least 60% of recipients. Following two doses of whole virus, aqueous or adsorbed surface antigen vaccine containing at least 9 μg HA per dose, serum HI titres of ≥ 40 were detected in 69–100% of recipients. In volunteers aged ≥ 26 years, who received a single dose of any of the test vaccines containing at least 3 μg HA, the HI antibody responses were frequent and post vaccine titres of ≥ 40 were detected in 80% of vaccinees. A ‘shallow’ dose response effect over a wide range of antigen concentrations of whole virus (5–94 μg HA per dose) or subunit vaccines was noted for each age group. Both the geometric mean HI antibody titres and proportion of vaccinees developing HI titres ≥ 40 were similar for each age group when comparable doses of vaccine were given by the subcutaneous and intradermal routes. A preliminary study of the NI antibody responses to vaccination indicated that all the whole virus and subunit vaccines stimulated serum NI antibodies in a proportion of recipients. All vaccines were clinically well tolerated.

A plant-produced influenza subunit vaccine protects ferrets against virus challenge

Background  Influenza A viruses are of major concern for public health, causing worldwide epidemics associated with high morbidity and mortality. Vaccines are critical for protection against influenza, but given the recent emergence of new strains with pandemic potential, and some limitations of the current production systems, there is a need for new approaches for vaccine development.

Monoclonal antibodies which block cellular receptors of poliovirus.

The isolation and properties of monoclonal antibodies which specifically inhibit the binding of poliovirus types 1, 2 and 3 to cells are described. The antibodies were of the IgG class and blocked infection of cells by all strains of the three poliovirus serotypes, but by none of a wide range of other viruses examined, including nine human enteroviruses. The antibodies prevented poliovirus growth in all susceptible human and primate cells tested. We conclude that the antibodies are directed against the receptor site for poliovirus which is distinct from those required by other picornaviruses, and which seems to be antigenically well conserved between cells of human and primate origin.

Studies with inactivated equine influenza vaccine: 1. Serological responses of ponies to graded doses of vaccine

Serological responses to three bivalent aqueous equine influenza vaccines of different potency and an adjuvanted bivalent vaccine containing inactivated A/equine/Prague/56 (H7N7) and A/equine/Miami/63 (H3N8) viruses, were examined in seronegative ponies. Potencies of the vaccines, measured by single-radial-diffusion tests, ranged from 4 to 56 micrograms of haemagglutinin (HA) antigen activity/virus strain per dose. Serological responses to vaccination were examined by haemagglutination-inhibition (HI) and single-radial-haemolysis (SRH) tests. Four weeks after a primary dose, HI responses to both vaccine viruses were barely detectable; after a second dose the HI responses to A/Miami/63 virus were low or undetectable but HI responses to A/Prague/56 virus were higher (17/20 ponies with titres greater than or equal to 1:16). In contrast SRH tests revealed dose-related antibody responses to both virus strains after one and two vaccine doses; levels after the second dose were 2- to 5-fold higher than after the primary dose. Highest post-vaccination antibody titres were obtained with the adjuvanted vaccine which contained 2- to 4-fold less antigen (13-23 micrograms HA) than the most potent aqueous vaccine. Post-vaccination antibody reacted well in SRH tests with recent antigenic variants of equine influenza virus. A remarkable finding was the high rate of decline in antibody, detected by HI or SRH tests, following one or two doses of vaccine. Even in animals with the highest post-vaccine antibody levels 2-4 weeks after a booster dose, antibody levels had declined to low or indetectable levels 14 weeks later. The low antibody titres detected at 14-32 weeks after vaccination were nevertheless vaccine dose-related.

Studies with inactivated equine influenza vaccine. 2. Protection against experimental infection with influenza virus A/equine/Newmarket/79 (H3N8).

Forty ponies immunized with inactivated virus vaccine containing A/equine/Miami/63 (H3N8) virus and six unvaccinated, seronegative ponies were experimentally challenged with a representative of recent equine H3N8 virus isolates, A/equine/Newmarket/79. All unvaccinated ponies became infected as judged by virus excretion, febrile responses and antibody responses, but only two of the vaccinated ponies were fully protected. Pre-challenge antibody levels to A/Newmarket/79 virus detected by single radial haemolysis (SRH) correlated well with the degree of clinical protection but the levels required for complete protection (SRH zones greater than 65 mm2) were high. The importance of these results in relation to conventional vaccination procedures against equine influenza is discussed.

The specificity of the anti-haemagglutinin antibody response induced in man by inactivated influenza vaccines and by natural infection.

The anti-haemagglutinin antibody response in adult human volunteers to inactivated whole virus or tween ether split influenza A/Victoria/75 (H3N2) and A/Scotland/74 (H3N2) virus vaccines was investigated using antibody absorption and single-radial-haemolysis (SRH) techniques. The concentrations of haemagglutinin (HA), nucleoprotein (NP) and matrix (M) antigens measured by single radial diffusion (SRD) and rocket immunoelectrophoresis were similar for both the whole virus and split vaccines. Whole virus and split vaccines induced crossreactive (CR) antibody in 87% of vaccinees. Strain specific (SS) antibody to A/Hong Kong/1/68 of the homologous virus was induced less frequently than CR antibody. Higher anti-haemagglutinin antibody titres were detected in persons receiving the split virus vaccines than in those receiving the whole virus vaccines. No antibody to the type-specific matrix protein was detectable, but 33% of volunteers developed an antibody rise to type-specific nucleoprotein antigen. The specificity of the anti-haemagglutinin antibody response in human adults to natural infection with A/Port Chalmers/73 (H3N2) virus was similar to that induced by inactivated vaccines in that a high proportion of subjects developed CR anti-haemagglutinin antibody, which reacted with A/Hong Kong/68 virus and the homologous A/Port Chalmers/73 virus, and SS antibody for A/Hong Kong/68 virus but SS antibody for A/Port Chalmers/73 virus was infrequently stimulated by natural infection.

A graded-dose study of inactivated, surface antigen influenza B vaccine in volunteers: reactogenicity, antibody response and protection to challenge virus infection

One hundred and nineteen volunteers were divided into five groups, and each volunteer inoculated subcutaneously with an aqueous subunit B/Hong Kong/73 vaccine containing 40, 20, 10, or 5 micrograms of HA or saline alone in a 0.5 ml volume. The incidence of reactions was recorded 24 h after inoculation. One month following immunization the serum HI antibody to B/Hong Kong/73 virus was measured; each volunteer was inoculated intranasally with live, attenuated influenza B (RB77) virus; and the incidence of infection by the challenge virus was determined by HI antibody response. The results showed that the incidence of reactions to all doses of vaccine were relatively low, the severity mild, and the duration short. However, the incidence of reactions was highest for those given 40 micrograms HA and least for those given 5 micrograms HA. The serum HI antibody responses to vaccine showed a dose-response relationship. For volunteers given 40 micrograms HA, 22 (96%) showed a fourfold rise in antibody titre and all volunteers had antibody titres of greater than 40 following immunization: for volunteers given 5 micrograms HA the g.m.t. increased from 16.6 to 86.1; and for those given 10 and 20 micrograms HA the response was intermediate. Following challenge, the lowest incidence of infection was seen in volunteers given the highest dose of vaccine. However, all doses of vaccine induced some protection against challenge virus infection, and the incidence of infection was directly related to the serum antibody titre at the time of challenge. The 50% protection titre of serum HI antibody was estimated as 15 to 20.

Protection against experimental infection with influenza virus A/equine/Miami/63 (H3N8) provided by inactivated whole virus vaccines containing homologous virus.

Thirty-one ponies immunized with inactivated virus vaccine containing A/equine/Miami/63 (H3N8) virus and six seronegative ponies were experimentally challenged with the homologous virus strain. All 6 unvaccinated ponies and 11 out of 31 vaccinated ponies became infected. A clear relationship between pre-challenge antibody, measured by single radial haemolysis (SRH), and protection was demonstrated as judged by virus excretion, febrile responses and antibody responses. Those ponies with SRH antibody levels greater than 74 mm2 were completely protected against challenge infection by the intranasal route.