Camelid VHHs are widely known to have highly favourable properties as antigen binding agents for research and commercial applications, but the poor accessibility, large size, expense and difficult handling that characterizes the use of camels and llamas has significantly limited their general use by scientists. Alpacas are more widely available, less expensive to maintain, smaller and more tractable than camels and llamas and their use as a source of immune B cells for VHH library construction could expand the accessibility of VHHs to many more laboratories. In a recent publication, the first reporting the use of alpacas as a VHH source (Rothbauer et al., 2006
), the authors commented on the distinct utility of the alpaca model. Here we show that alpacas appear to be at least as useful as camels and llamas as a source for immune HcAbs and we provide the information necessary to guide researchers in the preparation of VHH libraries that accurately represent the VHH repertoire of the animal.
More than 50 random alpaca HcAb cDNAs were sequenced through the entire VHH coding region to aid in the design of PCR primers. The goal is to use primers that amplify the vast majority of alpaca VHH coding DNAs to produce a DNA pool that closely represents the VHH repertoire of an immunized animal. A highly complex library created from this pool will thus contain a more diverse variety of VHH clones with the ability to bind the immunogen. Obviously, the larger the number of different VHH clones that bind the target of interest will increase the likelihood of finding a VHH with the specific properties that are most important to the researcher. The type of desirable properties being sought might include high affinity, target neutralization, target specificity, stability, high level of functional expression in E. coli
and others. We were able to design a single primer pool (AlpVh-F1) homologous to the beginning of the FR1 domain that consistently amplified 23/23 diverse alpaca VHH cDNAs in combination with a primer pool homologous to the CH2 domain. Similar PCR using an FR1 primer commonly used to amplify camel and llama VHHs efficiently amplified only about half of the cDNA and failed to amplify several clones. Since llamas are much more closely related to alpacas than to camels (Stanley et al., 1994
), it seems likely that VHH primers designed from alpaca cDNAs will improve the quality of VHH libraries prepared from llama B cell cDNA.
Sequencing of the entire VHH leader sequence coding DNA for 25 random cDNAs demonstrated that a primer can be designed from this region as a means to “pre-amplify” the VHH and VH coding DNA prior to amplification of the DNA that will be used for library construction. This would be of distinct value when only a limited amount of B cell cDNA is available. In that case, the cDNA can first be amplified with a combination of a leader sequence primer and a CH2 primer to generate a product of about 600 bp. This DNA can be purified and then used as the template to amplify the VHH coding DNA for phage display library construction. When B cell cDNA is not limiting, the preferred strategy is to directly amplify the cDNA in two separate reactions with Alp-Vh-F1 forward primer in combination with a reverse primer that is specific to either the short hinge or the long hinge to produce a product of about 400 bp for library construction.
For this study, we used tissue from the lymph node draining the immunization site as the source of cDNA synthesis for a VHH library. Lymph node tissue is not difficult to obtain and is expected to yield cDNA that is more enriched in immunogen-specific VHHs than peripheral blood lymphocytes (Basalp and Yucel, 2003
), although either source can be used for VHH library construction (Saerens et al., 2004
). The ease with which sufficient B cells can be obtained from camelids makes it readily feasible to use these animals successively as a source of VHHs for multiple immunogens.
As a demonstration of the utility of the immunized alpaca as a source of VHHs against an antigen, we prepared neutralizing VHHs against ovine TNFα. Tumour necrosis factor (TNF) plays critical roles in the initiation, maintenance and resolution of various immune responses. In particular, its overproduction has been implicated in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis and Crohn’s disease (Ruuls and Sedgwick, 1999
). Anti-TNF therapy can ameliorate much of the pathology from a number of intestinal diseases in humans and animals (Abuzakouk et al., 2002
) (Marini et al., 2003
) (Lawrence et al., 1998
) (Liesenfeld et al., 1999
). In the sheep, expression of TNFα is associated with increased chronic intestinal inflammation from parasite infection (Pernthaner et al., 2005
). Based on the success of human anti-TNFα therapies, we hypothesize that neutralization of TNFα will diminish the intestinal pathology associated with nematode infection, Johne’s and other diseases. Availability of anti-ovTNFα VHHs that can be produced economically at large scale, as reported here, will allow its testing as a therapeutic agent in sheep.