Anna Lengyel

Grouping of monoclonal antibodies to adenovirus hexons by their cross-reactivity

SummaryThirty two mouse ascitic fluids containing monoclonal antibodies (MAbs) directed against the hexon of human adenovirus type 1 were examined. The type and degree of cross-reactivity (CR) of the MAbs were determined by ELISA and hemagglutination methods with 10 heterologous hexon types. The similarity and the dissimilarity of the MAbs was also characterized by the correlation coefficient calculated from their reactivity values. On the basis of these, the 32 MAbs could be divided into five groups and fourteen individual MAbs, which altogether recognized 19 distinct epitopes. One of the recognized epitopes is the genus specific epitope of adenovirus hexons; the others are interspecies specific ones which can be found on the surface of the different hexon types in characteristic, mosaic-like combinations. The type and degree of CR of the MAbs lead to the conclusion that there exists a close antigenic relationship among the members of subgenus C of human adenoviruses and there is also a definite antigenic relationship between subgenera C and D. Hexons belonging to the oncogenic subgenera A and B display a much looser antigenic relationship with subgenus C.

Retrospective detection of a subclinical hepatitis A virus (HAV) epidemic affecting juvenile cohorts of the Hungarian population

Sero-epidemiological surveys of serum samples taken in 1982, 1987, 1994 and 1999 have been performed with hepatitis A virus-specific (HAV-specific) serological tests. Results obtained during these surveys show that the proportion of seropositive blood donors decreased from 69% to 18% within 17 years. The authors have recognised a (mainly subclinical) epidemic, affecting about 115000 teenagers in 1992-1994 in Hungary, is a threatening phenomenon. It was calculated that only about 3600 clinical diseases were associated with the epidemic, recognised retrospectively from the findings of the four sero-epidemiological surveys. Epidemiological data indicated that the excess clinical diseases caused by HAV concentrated in the southern counties of Hungary, which have been affected by the social and military activities between 1992 and 1994. Due to the decrease of subjects seropositive for HAV, sera from preselected or actively immunised donors will be required in the future and vaccination against HAV with killed virus is likely to be recommended for risk groups. Furthermore, health authorities might promote active immunisation of young children against HAV infection; for that, promotion of manufacturing combination vaccines of HAV/HBV/DPT or, for certain countries, HAV/DPT would be desirable.

Comparative studies of adenovirus hexon antigens

Purified soluble hexon antigens of type 5, 7, and 8 adenoviruses, belonging to three different subgroups have been studied. The electrophoretic mobility of the three hexons was found to be different in immunoelectrophoresis, and the calculation of absolute electrophoretic mobility gave different values too. Significant dissimilarities were observed in the elution patterns of the hexons in anionexchange chromatography. However, no essential differences between the molecular weights of the hexons were demonstrable, they appeared to be with all three types about 300,000. Determination of the smallest hexon quantities demonstrable by immunodiffusion, immuno-osmophoresis and complement fixation (CF) reaction, showed the CF reaction to be the most sensitive. 0.02 μg of type 5 and type 8 hexons and 0.08 μg of type 7 hexon were detectable using homologous anti-hexon sera. The anti-hexon sera neutralized the cytopathic effect of the homologous adenovirus types only. In the immunodiffusion experiments any two antigens of the three proved to be identical, examined with the third (heterologous) serum. Using homologous serum they revealed only a partial identity. Absorption experiments were successful only when homologous antigen was used, even excess amount of heterologous hexons failed to remove the antibodies from the anti-hexon sera. These results demonstrate the presence of closely connected group-, subgroup or type-specific components in the hexons of adenoviruses, and definite differences in the physico-chemical properties of hexons belonging to different serotypes.

Delineation of antigenic determinants of adenovirus hexons by means of monoclonal antibodies.

60 hexon-specific mouse hybridoma clones were selected by using crystallized hexon capsomers of human adenovirus type 1 as immunizing and selecting antigen. The reactivities of the monoclonal antibodies were tested with purified hexon preparations from 10 human adenovirus types by enzyme-linked immunosorbent assay and passive hemagglutination techniques. The reactivity patterns delineated a total of 41 adenovirus-type-1-related determinants present on a number of heterologous hexons in different interspecies combinations. The pattern of interspecies specificities did not coincide with the current grouping of adenoviruses into subgenera.

Antigenic homogeneity among the adenovirus hexon types of subgenus C

SummaryAntigenic relationships of hexons of human adenovirus (Ad h) types 1, 2, 5 and 6 of subgenus C were studied with 61 monoclonal antibodies (MAbs) raised against human Ad h1, Ad h35 and bovine adenovirus 2. The reactivity pattern (RP) and the titers of the MAbs were determined in indirect ELISA. In previous experiments with hexons of different subgenera 49 MAbs displayed numerous different intertype specificities besides genus specific and type specific ones. With the four hexon types of subgenus C all MAbs gave identical RPs except the type specific ones. Data reveal the existence of a remarkable homogeneity in the antigenic structure among the hexon types of subgenus C defined by the presence of identical or closely related intertype specific epitopes on the surface of the hexons. The possible significance of the results in the experimental gene therapy is discussed.

Determination of different antigenic sites on the adenovirus hexon using monoclonal antibodies

SummaryEighteen mouse ascitic fluids containing monoclonal antibodies (MAbs) directed against crystallized hexon of adenovirus (AV) type I were used to map the antigenic structure of the capsomer in reciprocal competitive binding ELISA. With the help of peroxidase-labelled MAbs at least nine epitopes (epitope clusters) located on three distinct antigenic sites were identified on the hexon. Epitope on antigenic site I recognized by two MAbs could be the genus specific antigenic determinant based on the broad reactivity patterns of the MAbs. Epitopes on the antigenic site II recognized by fifteen MAbs could be divided into seven epitope clusters according to the competition patterns. Antigenic site III recognized by one MAb completely differs from the antigenic site I and on the basis of one-way blocking with all the MAbs specific for antigenic site II, should be also different from the latter one. The data suggest that the seven epitope clusters of antigenic site II contain partially overlapping epitopes and may be a part of a large single immunodominant antigenic region on AV1 hexon as well as on hexons of heterologous types.

Differentiation of adenovirus hexon epitopes with monoclonal antibodies by gel diffusion assays

Nine monoclonal antibodies (MAbs) directed against crystallized human adenovirus type 1 (Ad h 1) hexon were tested with purified homologous and heterologous hexon preparations by gel diffusion. Six MAbs formed a single line with the homologous hexon in a 2-well pattern, and 3 MAbs formed lines only in biclonal combinations with an appropriate MAb. All of the 6 precipitating MAbs formed a continuous line of complete identity when tested simultaneously against homologous and different heterologous hexons. With Ad h 1 hexon a line of double partial identity (double spur) was formed when some pair combinations of 2 MAbs were placed in 2 juxtaposed wells. Other MAbs in the adjacent wells formed a line of identity. The MAbs could be divided into 2 antibody groups (groups A and B) based on this phenomenon. Members of antibody groups A and B apparently identified 2 sterically distinct epitopes: one of them is presumably the genus-specific epitope of the hexons (group A) and the other(s) should be intertype-specific epitope(s). Thus, the gel diffusion method can be used for selecting pairs of MAbs for their specificity to sterically independent epitopes. Mixtures of 2 MAbs belonging to the different antibody groups formed double lines with Ad h 1 hexons. Members of group A showed some helper effect to the members of group B for their precipitin line formation.

Antigenic relationship between human and animal adenovirus hexons determined by means of monoclonal antibodies directed against bovine adenovirus type 2 hexon

SummaryEight monoclonal antibodies (MAbs) directed against bovine adenovirus (BAV) 2 subtype B hexon were studied with 12 different hexon types of human adenoviruses (AV) belonging to 5 different subgenera using indirect ELISA, passive hemagglutination (HA), and gel diffusion assays. Two hexon types of animal origin (BAV3 and SAV16) were investigated, too. The reactivity of the MAb IV.F3 was the broadest, i.e. in ELISA and HA experiments it reacted with all hexon types studied. Based on these results as well as on the results of gel diffusion assays, this MAb should recognize the genus specific epitope of adenovirus hexons. Three MAbs (CA12, III.B11, and A12) could recognize different epitopes showing intersubgenus or intertype specificities. In spite of the fact, that all the eight MAbs proved to be bound by the hexon of ORT/111 (BAV2 subtype B) blotted onto nitrocellulose filter, four of the eight MAbs (BB7, BH5, II.A9, and IV.F5) failed to react with any human, and animal hexon types used in the present experiments. The results suggest that a gradient of antigenic relationship may exist between BAV2 hexon and the hexons of human serotypes.

Multiple copies of identical epitopes on the adenovirus hexon

A double monoclonal antibody (MAb) sandwich enzyme-linked immunosorbent assay (double MAb ELISA), which uses the same MAb as solid-phase immunosorbent (capture MAb) and as detector MAb (peroxidase-labeled), was developed to quantify the specific epitopes of adenovirus hexon. Four MAbs directed against crystallized adenovirus type 1 (Ad h 1) hexon were tested by this assay with homologous and different heterologous hexons. The lowest reacting concn with the homologous and heterologous hexon types both in direct and double MAb ELISA was determined and compared. At least two copies of four different epitopes were identified by the MAbs. Evidence is presented that more than one copy of identical or closely related epitopes exist on the homologous as well as on the heterologous hexon molecules. However, their presence could be detected only in higher concn of hexon preparations of subgenera A, B and D.

Cross‐reactivity between human adenoviruses in delayed‐type hypersensitivity

The aim of this study was to determine the antigen responsible for the induction of delayed‐type hypersensitivity (DTH) by human adenoviruses (Ads). The estimation of DTH was based on measurement of the extent of swelling of the hind footpads of mice. CsCl density gradient‐purified human Ad serotype 6 (Ad6) induced DTH in a dose‐dependent manner. In Ad6‐sensitized mice, DTH could be elicited by serotypes belonging to the same species of human Ads (types 1 and 5) and by a serotype (type 3) belonging to another species. Latex particles coated with purified hexon antigen prepared from Ad5 had the capacity to sensitize mice and elicit a DTH reaction. We suggest that, for serotypes belonging to species C, the cross‐reactive highly conserved T cell epitope of the hexon protein might be responsible for the DTH induction, and furthermore the same epitope might result in the cross‐reactivity between serotypes 3 and 6. The possible importance of these data is discussed in relation to human gene therapy through the application of Ad vectors.