Molecular Methods and Bioinformatic Tools for Adjuvant Characterization by High-Throughput Sequencing.

Adjuvants in vaccine formulations are designed to enhance immune responses against a target antigen or pathogen. The ability of these vaccines to induce activation and differentiation of mature naïve B cells to produce pathogen-specific antibodies (immunoglobulins; Ig) helps guarantee long-lived humoral immunity. This process involves clonal expansion of antigen-specific B cells, genomic rearrangement of Ig heavy (IgH) and light (IgL) loci, somatic hypermutation (SHM), and clonal selection for affinity-matured antibody, resulting in a vast but directed repertoire of B cells expressing highly specific antibody proteins. High-throughput sequencing of the IgH and IgL complementary determining regions (CDRs) derived from various B cell populations provides an unprecedented way to observe dynamic responses of the humoral immune repertoire in response to vaccination. However, applying high-throughput sequencing (HTS) methodologies to multi-armed in vivo experiments requires careful coordination of sample preparation with downstream bioinformatics, particularly with regard to issues of quantitation, sequence fidelity, bar-coding, and multiplexing strategies. Here, we overview strategies of high-throughput sequencing and analysis of the adaptive immune complex loci applied to multi-armed, multiplexed experiments.

Selection of therapeutic H5N1 monoclonal antibodies following IgVH repertoire analysis in mice.

The rapid rate of influenza virus mutation drives the emergence of new strains that inflict serious seasonal epidemics and less frequent, but more deadly, pandemics. While vaccination provides the best protection against influenza, its utility is often diminished by the unpredictability of new pathogenic strains. Consequently, efforts are underway to identify new antiviral drugs and monoclonal antibodies that can be used to treat recently infected individuals and prevent disease in vulnerable populations. Next Generation Sequencing (NGS) and the analysis of antibody gene repertoires is a valuable tool for Ab discovery. Here, we describe a technology platform for isolating therapeutic monoclonal antibodies (MAbs) by analyzing the IgVH repertoires of mice immunized with recombinant H5N1 hemagglutinin (rH5). As an initial proof of concept, 35 IgVH genes were selected using a CDRH3 search algorithm and co-expressed in a murine IgG2a expression vector with a panel of germline murine kappa genes. Culture supernatants were then screened for antigen binding. Seventeen of the 35 IgVH MAbs (49%) bound rH5VN1203 in preliminary screens and 8 of 9 purified MAbs inhibited 3 heterosubtypic strains of H5N1 virus when assayed by HI. Two of these MAbs demonstrated prophylactic and therapeutic activity in virus-challenged mice. This is the first example in which an NGS discovery platform has been used to isolate anti-influenza MAbs with relevant therapeutic activity.

Targeting TLRs expands the antibody repertoire in response to a malaria vaccine.

Vaccination with an isolated antigen is frequently not sufficient to elicit a protective immune response. The addition of adjuvants to the antigen can increase the magnitude and breadth of the response generated, but quantification of this increase as a function of adjuvant has been intractable. We have directly determined the variation of the immunoglobulin G variable-chain repertoire of an entire organism as a function of vaccination. Using the well-established Plasmodium vivax antigen, PvRII, and massively parallel sequencing, we showed that the use of a Toll-like receptor (TLR) agonist in the vaccine formulation increased the diversity of the variable region sequences in comparison to the use of an oil-in-water emulsion adjuvant alone. Moreover, increased variable domain diversity in response to the use of TLR agonist-based adjuvants correlated with improved antigen neutralization. The use of TLR agonists also broadened the range of polymorphic variants against which these antibodies could be effective. In addition, a peptide microarray demonstrated that inclusion of adjuvants changed the profile of linear epitopes from PvRII that were recognized by serum from immunized animals. The results of these studies have broad implications for vaccine design--they may enable tailored adjuvants that elicit the broad spectrum of antibodies required to neutralize drifted and polymorphic pathogen strains as well as provide a method for rapid determination of correlates of adjuvant-induced humoral immunity.

TWEAK, a member of the TNF superfamily, is a multifunctional cytokine that binds the TweakR/Fn14 receptor.

The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) was initially described as a member of the tumor necrosis factor (TNF) superfamily in 1997. TWEAK is a cell surface-associated type II transmembrane protein, but a smaller, biologically active form can also be shed into the extracellular milieu. There is one receptor currently known to bind TWEAK with physiological affinity, and it is a type I transmembrane protein that is referred to in the literature as either TWEAK receptor (TweakR) or fibroblast growth factor-inducible 14 (Fn14). TweakR/Fn14 is the smallest member of the TNF receptor (TNFR) superfamily described to date, and it appears to signal via recruitment of several different TNFR-associated factors. TWEAK has multiple biological activities, including stimulation of cell growth and angiogenesis, induction of inflammatory cytokines, and under some experimental conditions, stimulation of apoptosis. In this report, we summarize the results from recent studies focused on the TWEAK cytokine. Although these studies have contributed a significant amount of new information, numerous questions still remain regarding the role of TWEAK in both normal physiology and the pathogenesis of human disease.