No abstract

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.Lactococcus lactis is a gram-positive lactic acid bacterium used for the manufacture of fermented dairy products (2). The milk fermentation process is susceptible to infection by bacteriophages found in raw milk (3,19,(32)(33)(34) or by induction of prophages from lysogenic starter strains (19). The phage infection results in lysis of the bacteria, leading to production delays, variations in the taste and texture of the products, or even complete failure of fermentation. To minimize economic losses by phage infections, a variety of precautions are used (35,36). Lactococcal phages fall into three prevalent groups of DNA homology, 936-, c2-and P335-like phages (32-34). Characteristics of these phages include a double-stranded DNA genome and a long noncontractile tail. The 936 and P335 groups have a small isometric head, while members of the c2 group have a prolate head.We describe here the generation of phage-neutralizing monoclonal single-domain antibody fragments (V H H) derived from cameloid heavy-chain antibodies. In the blood of Camelidae, a high proportion of the immunoglobulins consists of homodimers of only heavy chains, devoid of light chains (17). As described in this and other papers, it is possible to elicit good immune response in camelids against complex protein mixtures, phages, or even whole organisms (26). Genes encoding V H H fragments that bind to these complex protein mixtures can be selected easily. In such libraries of binders, there is a high probability of finding V H Hs that block essential biological processes, mainly because of the long CDR3, which can block active centers (27).It was demonstrated that after immunization of a llama with lactococcal bacteriophage p2 (936 group) the fraction of heavy-chain antibodies contained about 10-fold higher neutralizing activity than conventional antibodies. We generated a phage display library (31, 3...

Graphical abstract

Bacteriophage infection is still a persistent problem in large dairy processes despite extensive studies over the last decades. Consequently, new methods are constantly sought to prevent phage infection. In this paper, we show that phage neutralizing heavy-chain antibody fragments, obtained from Camelidae and produced at a large scale in the generally regarded as safe microorganism Saccharomyces cerevisiae, can effectively be used to impede phage induced lysis during a cheese process. The growth inhibition of the cheese starter culture by 10(5) pfu/ml cheese-milk of the small isometric-headed 936-type phage p2 was prevented by the addition of only 0.1 microg/ml (7 nM) of the neutralizing antibody fragment. The use of such antibody fragments in cheese manufacturing are a realistic and interesting option because of the small amount of antibody fragments that are needed. Moreover the antibodies are produced in a food grade microorganism and can easily be isolated from the fermentation liquid in a pure and DNA free form.

Expression of bispecific antibodies in lactobacilli resulted in slight improvement of their efficacy. Furthermore, increasing the specificity would theoretically reduce the rate of appearance of viral escape mutants and would have a broader capacity to be effective against a range of viral serotypes.