B00003Characterization of Monoclonal Antibodies directed against major epitopes of the F and G proteins of Bovine Respiratory Syncytial Virus
Bontems S., Matheise J-P., Walravens K., Didembourg C., Knott I. and J-J. Letesson
Lab. Immunologie-Microbiologie, U.R. Biologie Moléculaire, Facultés Notre-Dame de la Paix, rue de Bruxelles 61, 5000 Namur, Belgium
Abstract:
Bovine Respiratory Syncytial Virus (BRSV) is one of the major causes of lower respiratory tract diseases in young cattle. The fusion (F) and attachment (G) glycoproteins are the major targets of humoral protective response against RSV. In this study, we describe the characterization of two monoclonal antibodies (MAbs) directed against the F protein and one against the G protein. The G-specific Mab detected only BRSV strains classified in the A and A/B subgroups, indicating a discriminating pattern of reactivity among BRSV strains. Furthermore, competitive binding assays with other discriminating MAbs located the epitope recognized by this Mab near to the major immunodominant region of the G protein. The two F-specific MAbs recognized all the BRSV strains tested, but only one of these reacted with strain A2 of HRSV, the human counterpart of BRSV. Competitive studies with a panel of MAbs directed to the F protein showed that the epitopes recognized by both MAbs are related to the major neutralizing site of the F protein. According to these data, the MAbs described in this study could be consider as helpful tools for a better understanding of the antigenic structure of BRSV.
Introduction:
BRSV is one of the major causes of lower tract diseases in young cattle. Due to their location at the surface of the virus, the fusion (F) and the attachment protein (G) are the major targets of humoral protective response against RSV.
BRSV isolates have been divided into 2 distinct subgroups (A and B) and one intermediate (A/B) based on their reactivity with Monoclonal Antibodies (MAbs) directed against the G protein (1) (2).
In this study, we describe the characterization of two MAbs against the F protein and one against the G protein.
Materials and Methods:
Viral strains used during this study were: RB94 (3), Bov-X (4), WBH (CVI, Lelystad) and NMK7 (5) of BRSV and the HRSV A2 strain (6), the human counterpart of BRSV. Strains were grown on Vero cells.
Antibodies: MAbs 20, 57 and 61 are directed against the G protein (1). MAb 20 is specific of subgroup A and A/B strains, while MAbs 57 and 61 only recognize subgroup A strains. MAbs AK13A2 and AL11C2 are directed against the F protein (7). 1D2G8 and 6B7 MAbs are negative control antibodies. The anti-proteins were kindly provided by G Taylor (1) and J.P.M. Langedijk (8)
Fusions: Balb/c mice were several times injected with either F or G protein of the RB94 strain of BRSV and fusions were performed with the SP20-Ag 14 myeloma
Screening: Specificity of hybridomas was evaluated in ELISA on several antigens: a lysate of cell culture infected with RB94 and purified F and G proteins.
Competition binding studies: each Mab was conjugated with peroxidase. Bidirectional binding competition assays were performed on a lysate of cell culture infected with RB94 strain. The percentage of binding was calculated by the following formula: (DO/DOmax)x100, where DO is the specific optical density for wells with the competitor antibody and DOmax is the mean of the optical density for wells without competitor antibody (7).
Results:
Two MAbs directed against the F protein (3H4 and 2D6) and one against the G protein (3A11) were identified by the screening of hybridomas.
The reactivity of those MAbs was tested on a panel of RSV strains. 3H4 (anti-protein F) recognized all bovine strains tested as well as A2 strain, while 2D6 (anti-protein F) recognized all bovine strains but not the human strain. 3A11 (anti-protein G) recognized only bovine strains which belong to A and A/B subgroups (see figure 1).
Competition binding studies were performed using a panel of MAbs directed against F or G protein previously characterised.
As shown in figure 2, only MAb 20 prevented the fixation of MAb 3A11 (anti-G protein). Reciprocal competition assays gave similar results (data not show).
Figure 3 shows that MAb 3H4 competed with MAb 2D6 (anti-F protein). Competition was also observed with MAbs AK13A2 and AL11C2 (see figure 4). Reciprocal blocking activities were also observed (data not show).
Discussion and
Conclusions:
We produced and characterized two MAbs (3H4 and 2D6) directed against the F protein of BRSV and one (3A11) against the G protein.
3A11 recognized only A and A/B strains of BRSV, indicating a discriminating pattern of reactivity as described for MAb 20 (1). Furthemore, those antibodies showed a reciprocal level of competition, suggesting the recognition of the same antigenic site on the G protein. MAb 20 reacted with a peptide containing the sequence 174-185 which corresponds to a major immunodominant region of the G protein of HRSV (8). According to these data, 3A11 could be specific to an important antigenic site of the G protein of BRSV.
We also characterized two anti-protein F MAbs, one specific for BRSV strains (2D6) and another reacting with bovine and humain strains of RSV (3H4). We observe reciprocal blocking activities with this antibodies and with previously characterized antibodies (AK13A2 and AL11C2). MAbs AK13A2 and AL11C2 were assigned to the 251-285 peptide of the F protein (Matheise et al., not published results), a region included in the major neutralization site of the RSV (9).
The MAbs describe in this study could be useful tools for epidemiological studies and for a better understanding of the antigenic structure of the RSV.
References:
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Comments:
Address questions and comments about this abstract to Sebastien Bontems ( Jean-Philippe.Matheis@fundp.ac.be).