Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses.

Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG(+) memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains.

Accessing the human repertoire for broadly neutralizing HIV antibodies.

The human antibody response has special significance in the ongoing efforts to develop a protective HIV vaccine. The observation that a subset of HIV infected individuals, who do not develop AIDS, have a broadly neutralizing antibody response has drawn attention to deciphering the nature of this response. It is hoped that an understanding of these protective antibodies, developed over time in response to the ongoing accumulation of mutations in the infecting virus, will facilitate the development of a vaccine that can elicit a similar response. This strategy will be greatly aided by the identification of broadly neutralizing monoclonal HIV antibodies from infected individuals. Several methods have been utilized to isolate and characterize individual antibodies from the human repertoire and each of these methods has been applied to the generation of broadly neutralizing HIV antibodies, albeit with differing rates of success. This review describes several of these methods including human hybridoma; EBV transformation; non-immortalized B cell culture; clonal sorting; and combinatorial display. Key considerations used in the comparison of different methods includes: efficiency of interrogation of an individual's entire repertoire; assay formats that can be used to screen for antibodies of interest (i.e., binding versus biological assays); and the ability to recover native antibody heavy and light chain pairs.

A molecular immunology approach to antibody humanization and functional optimization.

We introduce a new method of humanization based on a novel and immunologically relevant metric of antibody humanness, termed human string content (HSC), that quantifies a sequence at the level of potential MHC/T-cell epitopes. Use of this quantity rather than global identity as an optimization goal enables the sampling of human diversity from distinct human germline sequences across the framework and CDR regions, and allows for the generation of multiple diverse candidate sequences. As a result engineering is carried out at finer sequence resolution relative to standard CDR grafting methods, providing for the optimization of antibody properties beyond immunogenicity such as antigen affinity and solution behavior. We have applied this method to the humanization of four antibodies with different antigen specificities. The resulting variable domains differ fundamentally from CDR-grafted antibodies in that they are immunologically more human and their humanness is derived from several discrete germline sequences. Furthermore, these antibodies bind their respective antigens better than or comparable to those of the parent antibodies without the need for affinity maturation.

Drug receptor identification from multiple tissues using cellular-derived mRNA display libraries.

The use of display technologies to identify small molecule receptors from proteome libraries would provide a significant advantage in drug discovery. We have used mRNA display to select, based on affinity, proteins that bind to a drug of interest. A library of mRNA-protein fusion molecules was constructed from human liver, kidney, and bone marrow transcripts and selected using an immobilized FK506-biotin conjugate. Three rounds of selection produced full-length FKBP12 (FK506 binding protein 12 kDa) as the dominant clone. An analogous method was also used to map the minimal drug binding domain within FKBP12. Using this approach, it is anticipated that mRNA display could eventually play a key role in the discovery and characterization of new drug receptor interactions.

Generating addressable protein microarrays with PROfusion covalent mRNA-protein fusion technology.

An mRNA-protein fusion consists of a polypeptide covalently linked to its corresponding mRNA. These species, prepared individually or en masse by in vitro translation with a modified mRNA conjugate (the PROfusion process), link phenotype to genotype and enable powerful directed evolution schemes. We have exploited the informational content of the nucleic acid component of the mRNA-protein fusion to create an addressable protein microarray that self-assembles via hybridization to surface-bound DNA capture probes. The nucleic acid component not only directs the mRNA-protein fusion to the proper coordinate of the microarray, but also positions the protein in a uniform orientation. We demonstrate the feasibility of this protein chip concept with several mRNA-protein fusions, each possessing a unique peptide epitope sequence. These addressable proteins could be visualized on the microarray both by autoradiography and highly specific monoclonal antibody binding. The anchoring of the protein to the chip surface is surprisingly robust, and the system is sensitive enough to detect sub-attomole quantities of displayed protein without signal amplification. Such protein arrays should be useful for functional screening in massively parallel formats, as well as other applications involving immobilized peptides and proteins.