In this study we demonstrated that recombinant single-domain llama antibody fragments (VHH) directed against the VP6 protein of RV are ideal reagents for RV diagnosis by ELISA. Furthermore, three of the four selected binders were able to neutralize RV infectivity in vitro. To our knowledge, this is the first report of VHH fragments directed to the inner capsid protein VP6 of RV with broad neutralization properties. Furthermore, two VHH conferred 60% protection against RV-induced diarrhea in a neonatal mouse model.
The immunization schedule for the llama was adapted from previous work (39
). It was, however, modified according to the monitoring of the number of ASC specific to RV circulating in the blood, a parameter more appropriate for monitoring the active B-cell response to the antigen of interest than the serum antibody titer. The results obtained suggest that a long interval between the last two boosters promoted a higher number of specific ASC, from which the RNA for the VHH library was then extracted. Further experiments with a larger number of llamas and different antigens are needed to generalize this finding. In addition, we confirmed for this llama, as previously described for other animals, that higher amounts of specific ASC are among the circulating lymphocytes at 4 days than at 7 days after the final booster (54
The goal of our strategy based on immunizing the llama with VP6 protein and using the whole virus for binder selection was to obtain VHH molecules directed to VP6 epitopes shared by all group A RVs and accessible in the intact viral particle. The results obtained from different assays (phage ELISA, ELISAs with different RVs, Western blotting, and VP6-mediated blocking VN assays) indicates that the selected VHH are specific to VP6. The four selected binders (2KA4, 2KD1, 3A6, and 3B2) are most likely directed against conformational epitopes of VP6, given that a strong signal is detected at the corresponding molecular weight of the VP6 trimers and other oligomers in Western blotting under nonreducing conditions. The four VHH recognize RVs from different subgroup specificities, different G/P-type combinations, and different species of origin, by ELISA, indicating that they are directed against shared epitopes that are different from the subgroup-specific ones. Our single-domain antibody fragments are therefore excellent, inexpensive alternatives to the use of mouse monoclonal antibodies for the design of group A RV diagnosis tests and for VP6 or virus affinity purification. We found, furthermore, that bivalent VHH molecules have the advantage over monovalent VHH that they can be used as direct coating for RV capture in ELISA (eliminating the need for additional capture antibodies).
RV neutralization in vitro has been documented for antibodies directed against the outer capsid proteins VP4 and VP7, and it is serotype specific (29
). Most conventional antibodies directed against VP6 have not been reported to have extracellular neutralizing activity in vitro (20
). For instance, even a monoclonal antibody to VP6 (RV-133) binding both double- and triple-layer particles which induces partial decapsidation did not neutralize RV in vitro (56
). On the other hand, a secretory IgA monoclonal antibody to VP6 was reported to mediate protection by intracellular neutralization during transcytosis in mice and shown to inhibit RV replication and transcription (3
In agreement with the general statement that antibodies to VP6 do not have neutralizing activity in vitro, the baculovirus recombinant VP6 derived from BRV C486, used in our study, was reported to induce partial protection against RV challenge in a neonatal mouse model but did not induce the production of neutralizing antibodies in the vaccinated dams (44
). In contrast, monospecific rabbit antiserum to this protein, as well as monoclonal antibodies to a VP6 peptide corresponding to the region from amino acids 40 to 60 of SA11 simian RV, had been reported to have very low neutralization activity in vitro (45
). Our discovery of anti-VP6 VHH antibodies with neutralizing activity is in concordance with the latter observation. The recent finding that VP6 is involved in RV cell entry via its binding to the cellular protein hsp70 (22
) might be related to the presence of neutralizing epitopes in VP6. Due to their small size, VHH directed to VP6 can, therefore, be expected to access these neutralizing epitopes more easily than the conventional anti-VP6 antibodies, and this highlights the potential use of VHH as excellent tools for studying the role of VP6 in virus-cell interactions.
Interestingly, three of the four monovalent anti-VP6 VHH were able to neutralize all of the RV strains tested independently of their G and P types, demonstrating broad neutralization activity in vitro. In addition, the preincubation of VHH with recombinant VP6 strongly reduced this neutralization activity. This suggests that binding of the VHH to VP6 of the infectious TLP causes neutralization of virus infectivity in a polyreactive way. The capacity of the monomeric VHH to have neutralizing activity could be related to the small size of the VHH molecule. This hypothesis is supported by our observation that the bivalent VHH Biv3A6 and Biv2KD1 showed lower VN activity than their monovalent counterpart.
Several mechanisms could be responsible for the neutralizing activity of the obtained VHH. Their binding could block VP6 interaction with a cellular receptor or induce a conformational change in the viral particle, interfering with virus attachment to the cell.
This hypothesis is supported by previous data demonstrating that VP4 and VP6 bind the hsp70 cellular protein (22
). On the other hand, if the VHH-virus complex can attach and enter the cell, virus decapsidation or transcriptional activity and mRNA exit might be blocked. Further studies are needed to elucidate the mechanism behind this neutralizing behavior.
Recently, the generation of a VHH library from lymphocytes of a llama immunized with the RV strain RRV was reported. The binders selected from that library showed high serotype-specific neutralizing activity in vitro (57
). The four VP6 specific binders characterized in the present study show similar neutralizing capacity with the advantage of neutralizing group A RV strains of different subgroups and serotypes because they are directed against a highly conserved protein. Interestingly, the highest neutralizing titer was found against the human RV strain (Wa, SbII, PG1), which is the most common strain associated with gastroenteritis in human infants worldwide (29
The intragastric administration of the VHH 3B2 induced partial protection against RV-induced diarrhea caused by both BRV C486 and by a highly infectious murine RV EC in a neonatal mouse model. Furthermore, for mice treated with VHH 3B2 and challenged with murine EC RV, there was a delay of 2 days in the onset of diarrhea compared to the nontreated group.
In the group treated with the monovalent VHH 2KD1, only 27% of the pups were protected against diarrhea caused by BRV C486. However, after challenge with the RV murine strain, 60% of the pups were protected, and no virus was detected in the intestine homogenates at 96 h postchallenge. This observation is hard to explain but is in agreement with the high neutralization capacity of this VHH. Pups were euthanized at 96 h postinoculation, given that the peak of intestinal virus shedding in suckling mice was reported to be at that point (2
). Considering that the virus shedding pattern trough time is highly variable, the absence of virus detection in the 2KD1 VHH-treated group could also be due to a modification in the peak of virus shedding (2
). In the present study, challenge of RV was performed by the intragastric route without the need for premixing the virus with the VHH, in contrast to a previous report (57
The results obtained indicate that the 2KD1 and 3B2 binders are polyreactive tools that could be applied for the preventive or therapeutic treatment of RV-associated diarrhea, avoiding the need to prepare a cocktail of different antibodies directed to the common VP7 and VP4 types. Following these preliminary results obtained in mice, it will be interesting to evaluate the protective capacity of the selected VHH against the virulent HRV Wa in a gnotobiotic pig model for RV infection and disease.
Finally, we described here for the first time a broad neutralization activity of VP6-specific VHH in vitro. The engineered VHH could be applied in group A RV diagnosis and represent excellent tools for the study of RV-cell interaction. Furthermore, if future protection studies are successful, heterologous passive protection treatments based on recombinant llama VP6 VHH might offer promising alternative strategies to prevent RV-induced diarrhea in premature infants and provide other means to complement RV vaccination to reduce diarrhea severity and associated deaths.