Hemagglutinin Content Research Articles

Quantification of haemagglutinin of influenza Tween-ether split vaccines by immunodiffusion

The haemagglutinin content of monovalent influenza whole virus and Tween-ether split vaccines derived therefrom, were assayed comparatively using single radial immunodiffusion (SRID, the only test recommended for influenza vaccines by the European Pharmacopoeia Commission), quantitative SDS-polyacrylamide gel electrophoresis and immunization of guinea pigs. If SRID was performed with split vaccines, reduced haemagglutinin values were consistently recorded which were 50-25% of values obtained before disruption of virions. If, however, disruption was conducted in the presence of excess detergent thus preventing aggregate formation of solubilized haemagglutinin, test values comparable to those of whole virus vaccines were obtained. In agreement with these results, immunization experiments revealed that whole virus and the corresponding split vaccines exhibited comparable immunogenicity in guinea pigs. From SDS-polyacrylamide gel electrophoresis and densitometer tracings obtained by scanning the gels after staining with either Coomassie Blue or fluorescein isothiocyanate-labelled concanavalin A it was calculated that about 90% of whole virus HA2 was recovered in Tween-ether split vaccines. From our experiments we conclude that precise quantification of solubilized haemagglutinin is not achievable by the single radial immunodiffusion test alone. Aggregate formation of solubilized haemagglutinin frequently occurs when the applied detergent is removed and, therefore, a physico-chemical method including an effective disaggregation procedure like SDS treatment in combination with PAGE is recommended.

Standardization of Inactivated H5N2 Influenza Vaccine and Efficacy against Lethal A/Chicken/Pennsylvania/1370/83 Infection

The hemagglutinin concentration of beta-propiolactone-inactivated influenza vaccine containing A/Duck/N.Y./189/82 (H5N2) virus was measured by single-radial-immunodiffusion (SRD) test. After administration of vaccine to chickens in Freund's complete adjuvant, vaccine efficacy was assessed by challenge with lethal A/Chicken/Penn./1370/83 (H5N2) virus. SRD potency values correlated with post-vaccination antibody levels and protection against infection.

Studies on a temperature-sensitive mutant of fowl plague virus having a mutation in gene 7 coding for the M protein.

A fowl plague virus (FPV) temperature-sensitive mutant, ts 303/1 having a ts mutation in gene 7 coding for the matrix (M) protein has been obtained. The mutant induced synthesis of virus-specific RNA and polypeptides as well as ribonuclear protein (RNP) formation in cells under non-permissive conditions; however, haemagglutinin cleavage was reduced, functionally active haemagglutinin and neuraminidase were absent and virions were not formed. In mutant-infected cells at 36 degrees C haemagglutinin cleavage was also reduced and virions formed had an altered NP:M ratio as well as a decreased haemagglutinin content. A population of virions formed under these conditions was heterogeneous both in morphology and in buoyant density. The data obtained suggest that a mutation in the M proteins of orthomyxoviruses can affect processing of the haemagglutinin and impair final stages of virion morphogenesis.

Investigations on the trypsin inhibitor, hemagglutinin, phytic and tannic acid contents of cowpea Vigna unguiculata

Ten cultivated varieties of mature dry beans Vigna unguiculata were analysed for trypsin inhibitor (TI) and hemagglutinating activities, phytic acid, phytic acid-phosphorus and tannic acid. The respective concentrations were: 19·6–28·2 TUI mg −1 protein, ∗ ∗ Trypsin units inhibited (TUI) per mg protein, as defined by Kakade et al. (1969). 33·5–98·9 HU mg −1 protein, † † Hemagglutinating units (HU) per mg protein, as defined by Liener (1955). 280–331 mg 100 g −1, 131–200 mg 100 g −1 and 0·42–0·78 g 100 g −1 dry weight. Phytic acid-phosphorus as a percentage of total phosphorus was highest in Farv-13 (49·9) and lowest in Samaru local (29·8). Considerable variability in hemagglutinating activity was evident among the different varieties as indicated by the high percentage co-efficient of variation. Some of these differences may be genetic and may provide an opportunity for the genetic development of cowpea strains with superior protein quality, low in hemagglutinin content.

The quantification of the haemagglutinin content of influenza whole virus and Tween-ether split vaccines

Monovalent whole virus and Tween-ether split vaccines prepared from influenza A/Bangkok, A/Brazil and B/Singapore were assayed for haemagglutinin content using single radial immunodiffusion (SRID), quantitative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunization of guinea pigs. When SRID was performed with split vaccines, haemagglutinin values were consistently recorded which were in the range of 50 to 25% of the values obtained before disruption of virions. When, however, disruption was conducted in the presence of excess detergent, thus preventing aggregate formation of solubilized haemagglutinin, test values comparable with those of whole virus vaccines were obtained. In agreement with these results, immunization experiments revealed that whole virus and corresponding split vaccines exhibited comparable immunogenicity in guinea pigs. Additionally it could be calculated from SDS-PAGE and densitometer tracings, obtained by scanning the gels after staining with either Coomassie blue or FITC-Con A, that 90 to 95% of whole virus HA2 was recovered in Tween-ether split vaccines. On the basis of these findings we conclude that precise quantification of Tween-ether split vaccines is not possible by the SRID test alone. As aggregate formation of solubilized haemagglutinin occurs, we suggest that either a physico-chemical method including a disaggregation procedure, such as SDS treatment, or immunological evaluation of the original whole virus preparation before disruption of virions should be applied as an additional criterion for quantification of influenza Tween-ether split vaccines.

Influenza Immunization in Children and Young Adults

Clinical reactions and hemagglutinating-inhibiting (HAI) antibody responses to recent whole-virus and split-product influenza vaccines were studied in 168 children and young adults. The subjects initially received a monovalent vaccine, followed one month later by a trivalent preparation. The reactogenicity of whole-virus and split-product vaccines with an equivalent hemagglutinin content was similar except in the youngest age (6 to 36 months) group in which the whole-virus preparation was more reactogenic. The whole-virus vaccines were more immunogenic, especially in subjects who were previously unprimed (preimmunization HAI antibody titer, less than 5). In these subjects, the geometric mean titers of HAI antibody wee significantly higher after vaccination with whole-virus vaccines than the split-product vaccines. Specific IgM antibody was found more frequently after vaccination with whole-virus vaccines (34%) than after split-product vaccines (11%).

Toxicity of Kidney Beans (Phaseolus Vulgaris) With Particular Reference to Lectins

Abstract: As a follow-up to recent outbreaks of food poisoning (nausea, vomiting, diarrhea) associated with raw or undercooked red kidney beans, a study was conducted on the lethality and heat destruction of the implicated lectin causative agent present in the beans. Agglutinating activity towards trypsinized rabbit erythrocytes was used as an in vitro assay for lectin content. Only the 3 varieties of P. vulgaris (red and white kidney beans, rose coco bean) contained large amounts of lectins. Soaking overnight removed 20-70% of the lectin. Red and white kidney beans caused 100% mortality to various sets of rats when fed at 10% or more of the diet for up to 14 days. Complete destruction of the hemagglutinin content of the beans (due to lectin) was generally attained by heating for up to 20 minutes at 100 degrees Centigrade. Relevant literature on the toxic effect of these beans is reviewed. (wz)

Biochemical Evaluation of Twelve Broad Bean Cultivars

The proximate composition, lipoxygenase, hemagglutinin and trypsin inhibitor contents of twelve broad bean cultivars grown in Iraq were determined. Two bean cultivars were found to contain about 15mg/g phytic acid. The average moisture content was 9.7%, crude fat 1.0%, protein 27.3%, ash 3.2% and crude fibre 6.1%. Except for protein, which showed a range of 24.2% to 29.2%, the other components in the cultivars did not show much variation. A wide variation, however, was noted in hemagglutinin, trypsin inhibitor and lipoxygenase activities. The oil from the cultivar Cyprus contained 78% unsaturated and 21% saturated fatty acids. The major unsaturated fatty acid was linoleic while palmitic was the major saturated fatty acid. The very potent lipoxygenase activity could be almost completely destroyed by a 7min blanch in boiling water.

Quantitative analysis of the protein composition of influenza A and B viruses using high resolution SDS polyacrylamide gels

The structural polypeptide composition of a number of influenza A and B viruses was analysed using a high resolution SDS polyacrylamide gel (SDS PAGE) system. The haemagglutinin (HA) content of six influenza A viruses of three antigenic subtypes was relatively constant with a mean of 34·7 ± 3·4%. A similar quantitation was observed for the HA of influenza A recombinant viruses (31·9 ± 4·1%) and influenza B viruses (32·8 ± 3·3%). The percentage content of matrix protein (M), nucleoprotein (NP) and neuraminidase and polymerase polypeptides (NA + P) in the virion was 33·5 ± 6·7, 22·6 ± 4·5 and 7·3 ± 2·3 respectively. The SDS PAGE technique appears to provide a reproducible quantitative assay of the relative proportions of the structural proteins in influenza viruses used as standard preparations for potency estimations of inactivated influenza vaccines.

New developments in the measurement of the hemagglutinin content of influenza virus vaccines by single-radial-immunodiffusion

A variety of detergents were compared as solubilizing agents for the measurement of the hemagglutinin (HA) content of influenza vaccines by the single-radial-immunodiffusion (SRID) assay. The surfactant, Emulphogene (BC-720), was found to be generally the most satisfactory of the detergents tested against a variety of vaccine strains. In particular, the zone size for B/Hong Kong and A/USSR/77 strains was found to be unaffected by a wide range of BC-720 concentrations (1–4%) in contrast to the variable reaction size obtained with different concentrations of sodium sarkosyl sulfate (SSS). Little difference in zone size was observed between SSS and BC-720 detergents when testing A/Texas/77 antigens. The action of BC-720 was relatively mild as shown by the lack of adverse effect on previously solubilized A/USSR/77 and B/Hong Kong/73 HA.

Cytotoxic effect of two legumes in epithelial cells of the small intestine

The previously known high toxicity and hemagglutinin concentration found in Escumite bean (Phaseolous acutifolius), widely consumed in the south-east of Mexico, induced us to study the cytotoxic effect of these beans on the epithelial cells of the small intestine of rats. Another, less toxic leguminosae, chick-pea (Cicer arietinum), was used for comparison. In vitro tests showed that the viability of the cells was much more affected by the Escumite bean extract than by the chick-pea extract (P<0.001). The cellularity was much diminished when the epithelial cells were treated with Escumite extract (P<0.001), but not with chick-pea extract. In vivo experiments confirmed these findings. The present results suggest that the deleterious effect of some toxicants present in the raw legume is due to an interaction with intestinal epithelium, which, in turn, may cause a reduced absorption of nutrients.

Comparative antigenicity and immunogenicity of 1976 influenza virus vaccines: Results of mouse protection experiments

During the 1976 National Immunization Program large scale clinical studies of A/New Jersey/76, A/Victoria/75 and B/Hong Kong influenza virus vaccines were performed. These vaccines have now been tested in mice for their ability to induce serum HI antibody and to confer resistance to challenge infection. Vaccines were compared on the basis of their hemagglutinin content, rather than their CCA titer. The results indicate that sub-unit influenza A vaccines are less antigenic than equal doses of whole virus vaccine given to non-primed mice but that the relative antigenicity of a specific manufacturer's sub-unit vaccine may vary depending on the virus strain. Influenza B vaccines were of similar antigenicity in mice, regardless of the method of manufacture. A good correlation between serum HI antibody titer and resistance to challenge infection was found for all vaccines. Comparison of the results of these mouse protection experiments and the findings from the human studies with the same vaccines indicates that only in some general areas are mouse responses an indicator of human responses.

Inactivated influenza vaccine efficacy: diminished antigenicity of split-product vaccines in mice.

Groups of 60 to 120 mice were given a single intraperitoneal inoculation of varying dilutions of commercially prepared and licensed bivalent (A/England and B/Mass) and monovalent (A/Aichi or B/Hong Kong) inactivated influenza vaccines, and their antibody responses at 14 days were quantitated by hemagglutination inhibition tests. Split-product vaccines prepared by the treatment of A/England, B/Mass, and B/Hong Kong whole virus with Tween-80 and either tributylphosphate or ether produced significantly lower mean antibody titers than did equivalent whole-virus preparations. The rates of seroconversion (<1:8 to >/=1:8) at the various dilutions tested were also significantly reduced when these split-product vaccines were given. When the antigen content of all vaccines was quantitated by the chick cell agglutination test, between 10 and 100 times more split-product antigen than whole-virus antigen was required to produce seroconversion in 50% of the mice tested. Differences between split-product and whole-virus A/Aichi vaccines were less marked. These data point out the need to consider factors other than hemagglutinin content alone in determining the immunogenicity of inactivated influenza vaccines.

Identification and Quantitation of the Components of Polyvalent Inactivated Influenza Virus Vaccines by Immunodiffusion

One of the basic problems in the standardization of inactivated polyvalent influenza virus vaccines has been the determination of the relative potency of the individual strain components. The chicken cell agglutination test measures reliably the total hemagglutinin content of these vaccines. With immunodiffusion techniques, it is now possible to quantitate each strain component of polyvalent vaccines. Routine application of these techniques would serve as an interim procedure to assess antigenic potency of individual strain components of commercial vaccines until improved tests are developed.

Primary and secondary immune response kinetics in the Syrian hamster.

Numbers of direct and indirect plaque-forming cells (pfc) per 10&lt;sup&gt;6&lt;/sup&gt; spleen cells, total and mercaptoethanol-resistant hemolysin and hemagglutinin activity have been determined in adult male Syrian hamsters at different times after primary and secondary intraperitoneal injection of 1&amp;#215;10&lt;sup&gt;10&lt;/sup&gt; sheep red blood cells (SRBC). Only a high antigen dose was sufficient for stimulating a uniform immune response. The kinetics of the primary response at the cellular and serum level corresponds in general to that of the mouse and other laboratory animals. In the secondary response the direct pfc/10&lt;sup&gt;6&lt;/sup&gt; spleen cells and total serum hemolysin and hemagglutinin concentrations were higher than in the primary response and the pfc and serum antibody curves declined rapidly again after reaching their peaks.

Identification and Quantitation of the Components of Polyvalent Inactivated Influenza Virus Vaccines by Immunodiffusion

One of the basic problems in the standardization of inactivated polyvalent influenza virus vaccines has been the determination of the relative potency of the individual strain components. The chicken cell agglutination test measures reliably the total hemagglutinin content of these vaccines. With immunodiffusion techniques, it is now possible to quantitate each strain component of polyvalent vaccines. Routine application of these techniques would serve as an interim procedure to assess antigenic potency of individual strain components of commercial vaccines until improved tests are developed.

Variations in the Haemagglutinin Content of Crude Mouse Brain Antigens Containing Louping Ill Virus

The haemagglutinin content of crude mouse brain antigens prepared from 55 field isolations of louping ill (LI) virus was examined. While all of the preparations contained good complement-fixing antigens, less than one third had haemagglutinating activity. Determination of the haemagglutinin content of such antigens is of limited application from a diagnostic view-point.

The British reference preparation for influenza virus haemagglutinin.

The two methods for measuring the haemagglutinin content of an influenza virus suspension are the haemagglutinating (HA) and chick cell agglutinating (CCA) techniques and both measure the same biological activity. With the establishment of an international reference preparation for influenza virus haemagglutinin (type A), however, it seems logical to express the haemagglutinin content of influenza vaccines in international units. Accordingly a collaborative study was arranged in order to obtain agreement on the number of units to be assigned to a British reference preparation for influenza haemagglutinin. It was agreed that the preparation contains 190 i.u. per ampoule and 1 i.u. is contained in 0.0622 mg. of the dried material.

Measurement of Rubella Antibody by Hemagglutination Inhibition

Abstract The variables which affect the qualitative and quantitative aspects of the interaction between fowl erythrocytes and rubella virus were defined and evaluated. The sensitivity of the hemagglutination reaction is at least tenfold greater at pH 6.0 to 6.5 than it is at pH 7.2, the pH of dextrose-gelatin-Veronal which is widely used as diluent for rubella hemagglutination (HA) and hemagglutination inhibition (HAI) tests. In the presence of 0.001 M Ca++ ion the reaction is two to eight times more sensitive than it is in the absence of Ca++ ion. The development of agglutinated and non-agglutinated RBC patterns that are stable and clearly distinguishable from one another is critically dependent upon the concentrations of gelatin and/or albumin in the diluent. The diluents employed in previously published HAI test procedures differ widely with respect to pH, Ca++, gelatin and/or albumin concentration. The lack of comparability of HAI test results among laboratories may in part reflect these differences in diluent composition. In an effort to correct such difficulties, optimal conditions for the agglutination of fowl RBC by rubella virus were defined. A 0.14 M NaCl solution buffered with N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid (HEPES) at pH 6.05 to 6.15, containing 0.001 M CaCl2, 1.0% bovine serum albumin and 0.00025% gelatin was shown to provide conditions for a maximally sensitive, stable and clear test. Under these conditions the outcome of the hemagglutination test is solely a function of the concentration of hemagglutinin in the test specimen.

Preliminary Studies on the Development of a Bovine Parainfluenza 3 Virus Vaccine

SUMMARY The propagation of hovine parainfluenza 3 (PI3) virus, and the preparation of a PI3 vaccine inactivated with perchlorethylene and adsorbed to aluminium hydroxide, is described. The vaccine stimulated a satisfactory haemagglutination-inhibition antibody response in hamsters, guinea-pigs and colostrum-deprived calves. A single dose in hamsters enabled these animals to resist a challenge with virulent PI3 virus that caused pneumonia in unvaccinated controls. Inactivation with formalin and beta-propiolactone was also examined, but did not prove satisfactory owing to the destructive effect on haemagglutinin content.