In Vitro Neutralisation of Rotavirus Infection by Two Broadly Specific Recombinant Monovalent Llama-Derived Antibody Fragments

Rotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (referred to as Anti-Rotavirus Proteins or ARP1) derived from a heavy chain antibody of a llama immunised with rotavirus was able to neutralise rotavirus infection in a mouse model system. In the present work we investigated the specificity and neutralising activity of two llama antibody fragments, ARP1 and ARP3, against 13 cell culture adapted rotavirus strains of diverse genotypes. In addition, immunocapture electron microscopy (IEM) was performed to determine binding of ARP1 to clinical isolates and cell culture adapted strains. ARP1 and ARP3 were able to neutralise a broad variety of rotavirus serotypes/genotypes in vitro, and in addition, IEM showed specific binding to a variety of cell adapted strains as well as strains from clinical specimens. These results indicated that these molecules could potentially be used as immunoprophylactic and/or immunotherapeutic products for the prevention and/or treatment of infection of a broad range of clinically relevant rotavirus strains

Llama Antibodies against a Lactococcal Protein Located at the Tip of the Phage Tail Prevent Phage Infection

Bacteriophage p2 belongs to the most prevalent lactococcal phage group (936) responsible for considerable losses in industrial production of cheese. Immunization of a llama with bacteriophage p2 led to higher titers of neutralizing heavy-chain antibodies (i.e., devoid of light chains) than of the classical type of immunoglobulins. A panel of p2-specific single-domain antibody fragments was obtained using phage display technology, from which a group of potent neutralizing antibodies were identified. The antigen bound by these antibodies was identified as a protein with a molecular mass of 30 kDa, homologous to open reading frame 18 (ORF18) of phage sk1, another 936-like phage for which the complete genomic sequence is available. By the use of immunoelectron microscopy, the protein is located at the tip of the tail of the phage particle. The addition of purified ORF18 protein to a bacterial culture suppressed phage infection. This result and the inhibition of cell lysis by anti-ORF18 protein antibodies support the conclusion that the ORF18 protein plays a crucial role in the interaction of bacteriophage p2 with the surface receptors of Lactococcus lactis

Western blots.

<p>Western blots were performed with either [a] monoclonal mouse anti-VP6 or [b] ARP1-biotin. Concentrated cell adapted rotaviruses were run on an SDS-PAGE gel, transferred to a nitrocellulose membrane, and blotted with appropriate antibodies.</p

Immune EM with rotavirus strains of different genotypes from clinical samples or cultured reference rotavirus strains.

<p>Two different antibody batches were used, one batch was used at 0.14 mg/ml and the results are shown in italics, the second batch was used at 0.15 mg/ml. The number of virus particles seen in 5 grid squares was determined and the results expressed as particles/grid square. Specific reactivity was inferred from an increase (≥4 fold) in the number of particles/grid square in the grid coated with ARP1 when compared to the control grid.</p

Neutralisation of cell culture adapted rotavirus strains in MA104 cells.

<p>Neutralisation experiments were performed using rotavirus-specific antibody fragments ARP1 and ARP3 and control antibody VHH R2. Bars represent percentage of infected cells (fffu) compared with absence of any antibody. A 95% confidence interval is shown by the error bar. A dashed line indicating 50% reduction in fffu is highlighted, and the antibody concentration at which this is achieved is boxed. Concentration of ARP or VHH R2 is expressed in µg/ml on the X-axis in each graph.</p

Infection neutralisation of different rotavirus strains with rotavirus-specific antibody fragments ARP1 and/or ARP3.

<p>A summary of neutralisation of different tissue-culture adapted rotavirus strains with ARP1 and/or ARP3, with the concentration of antibody required to achieve a 50% reduction in fffu. VHH R2, a non-related llama antibody fragment, was used as a control.</p