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The specification and differentiation of eighteen intertype specific (IT) epitopes of adenovirus hexons defined by monoclonal antibodies are given by two groups of hexon types: on which they are and on which they are not present. The close specificity relationship among some groups of epitopes determined by pairwise analysis according to their presence on the hexon types indicate that they could be continuous (sequential), overlapping or discontinuous (topographic) epitopes. Based on the identification of the hydrophilic regions and the localization of beta-turns sixteen IT epitope sites were predicted on human adenovirus (HAdV) type 2 and nineteen on HAdV-41 hexon's amino acid (aa) sequences beside the type and genus specific ones. The 16 predicted IT epitopes on HAdV-2 hexon show good coincidency with the 14 IT epitopes demonstrated with monoclonal antibodies (MAbs) on this hexon type. The predicted number of common epitopes between HAdV types 2 and 41 also corresponds well with the eight IT epitopes determined by MAbs, but the 17 predicted non-common epitopes indicate the possibility of the existence of more epitopes on these hexon types. The location of the predicted epitopes of HAdV-2 were determined by alignment of their sequence numbers on the three dimensional ribbon representation of the hexon subunit. Most of the predicted IT epitopes were found between the type and genus specific epitopes i.e. in the "upper" regions of pedestal domains P1 and P2 orientated toward the virion surface and in the "lower" part of loop 1 region orientated inside the virion. Two peptides representing potential IT epitopes were synthesized corresponding to residues 309-320 from the "lower" part of loop 1 and 399-408 from the "upper" part of P1 region of HAdV-2 hexon. Antibodies raised against the peptide-carrier conjugates recognized different purified native hexon types in ELISA.

For the investigation of epitope composition of different adenovirus hexon types sixty-one mouse ascitic fluids containing monoclonal antibodies (MAbs) developed in three different panels were used. The distinction and marking of the different epitopes recognized by the MAbs were carried out by the determination of the composite cross-reactivity pattern, the titer and the correlation coefficient of all the 61 MAbs with 21 different hexon types representing all the six human subgenera, as well as different bovine and simian adenoviruses. The distinct epitopes were marked by two numbers referring the homologous hexon type to which the MAbs were directed and the serial number of the epitope specified by the different members of the given panel of the MAbs. The three panels of MAbs recognized 22 epitopes on the 21 hexon types among them a genus and three type specific ones and 18 different bi- and multilateral intertype (IT) specific epitopes that grouped adenoviruses within the genus, independently from the subgenus they belong to. Considering that the type specific epitope could be present only on the homologous hexon type, the largest number of the different epitopes distinguishable by the MAbs used could be 20 on the homologous hexon and 19 on the heterologous ones. It was found that the total number of IT specific epitopes on the hexons varied between 2 and 18. The distribution of the distinct specific epitopes on the different hexon types was different, as expected. The antigenic structure of the individual hexon types were characterized by the determination of their IT specific epitope spectrum. By pairwise analysis ten human hexon types formed three epitope clusters (types 4 and 19; types 8, 9, 9/13 and 10; as well as all types of subgenus C) showing identical epitope spectra. No clustering was found with human type 7, 12, 13, 18, 26, 27, 35 and 41, as well as with bovine and simian adenovirus hexons studied. However, they displayed a closer or looser antigenic relationship among each other and to members of the epitope clusters. The degree of antigenic relationship could be expressed by the similarity/dissimilarity percentage calculated from the number of the identical and different epitopes present on any two given hexon types.

Antigenic 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.

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.

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.

Summary Eighteen 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.

Thirty 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.

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.

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 l 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 l hexons. Members of group A showed some helper effect to the members of group B for their precipitin line formation.

In der Zonenelektrophorese wurde das Verhalten der Komponenten von sechs Adenovirusstammen untersucht. Das Hamagglutinin von drei, das Virion von vier und das Hexon-Antigen von allen Stammen besas die gleiche elektrophoretische Beweglichkeit. Die Komponenten der Prototypstamme 9 und 23 liesen sich nicht voneinander trennen. Hamagglutinin und Virion konnten bei einem Typ 9 und dem Typ 9–15-Stamm, sowie bei den Prototypen 15 und 19 getrennt gewonnen werden. Eine Beziehung zwischen der elektrophoretischen Beweglichkeit des Hamagglutinins und dem serologischen Typ scheint nicht zu bestehen.

A panel of 37 monoclonal antibodies (MAbs) directed against adenovirus type 35 (AV35) hexon was studied by indirect enzyme-linked immunosorbent assay (ELISA) and passive hemagglutination (HA) methods. Nine heterologous hexon types and the homologous type were used to determine the reactivity pattern (RP) of the MAbs and to study the antigenic relationship among the different hexon types. Eleven types of RPs were shown using ELISA and seven types were shown using the HA test. In the case of six MAbs, the RPs were identical in both assay systems; 31 MAbs showed some differences when the results of the two methods were compared. The common epitopes of the different hexon types studied seem to be characterized as genus, subgenus, intersubgenus, and intertype specificities. The type-specific determinant of AV35 hexon could be detected by several MAbs. The antigenic relationship seems to be closer between the two oncogenic subgenera (A and B) of adenoviruses, whereas the antigenic relationship to AV35 hexon is somewhat looser for subgenera D, and E. Hexon types of subgenus C showed the greatest differences in antigenic structure compared with the AV35 hexon.

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.

Lipid changes of human erythrocytes treated with adenovirus type 8, 9 and 10 have been studied with thin-layer chromatography. It has been established that each of the virus types induced several qualitative changes, which were manifested either in the disappearance of spots or in the development of new ones, as well as in the changes of Rf values and staining characteristics. These changes varied with the individual types and were different from those observed with influenza virus. The decrease of aminophosphatides and other amino acid-containing lipids characterized the effect of type 8 adenovirus. Erythrocytes treated with type 9 adenovirus displayed the highest number of spots, while the decrease of unsaturated lipids and neuraminic acid characterized the effect of type 10 adenovirus.