An elusive, but essential goal of HIV vaccinology remains the identification of an immunogen capable of eliciting a broadly neutralizing (BN) anti-HIV immune response. A few monoclonal antibodies (Mabs) with BN activity have been identified, however. Some of these BN Mabs bind to 15-19 amino acids (aas) in the membrane proximal external region (MPER) of gp41, but immunization with MPER peptides does not elicit a BN response, probably because the MPER peptides do not form the correct tertiary structure recognized by the BN Mabs when not expressed in the context of gp41 near a membrane. We hypothesize that we can induce MPER to assume a shape that elicits a BN response if it is expressed within another, non-immunogenic scaffolding protein. However, current technology does not enable us to specifically design an MPER- scaffolding protein chimera that can drive MPER into the required shape. We propose instead to identify an MPER-scaffolding protein chimera capable of eliciting a BN immune response by using the GI mucosal immune system as a massively parallel in vivo screening device. In this screen we propose to: 1) Create a library of DNA bar-coded plasmids that include surface expression cassettes capable of placing large amounts of MPER-scaffolding proteins on the surface of Gram-negative bacteria (including trimeric autotransporter expression cassettes that express passenger proteins as trimers) so that trimers, multimers, and other aggregates of the chimeric protein form in close association with a membrane. (In this library, each member expresses a different variant MPER-scaffolding protein chimera.) 2) Feed the library to mice. 3) Use PhyloChip microarray technologies (or high throughput sequencing) to identify, via the barcodes, the members of the library that show decreased relative abundance in mouse feces over time, which we would take as initial evidence of the induction of a mucosal immune response directed against MPER-scaffolding chimeric proteins showing decreased abundance. We will also determine whether an anti-MPER immune response is induced. We will rescreen clones that initially screen positive and then test those clones individually for their ability to elicit a BN anti-HIV immune response. This proposed screen therefore offers an innovative, rapid, high- throughput approach to the identification of potential immunogens that can elicit a BN anti-HIV immune response. The approach has several additional advantages. The technology could be used to compare and evaluate any other potential immunogens for their ability to elicit a mucosal immune response, which would be helpful for many vaccine development efforts, and any immunogen identified in the screen would necessarily induce a strong mucosal immune response, a helpful characteristic for most vaccines, and HIV vaccines in particular. Immunogens found to induce a BN anti-HIV immune response in this screen would be good candidates for further development into an HIV vaccine that induced mucosal immunity.

Public Health Relevance

Zeicher, MD, PhD, Steven, L. (Contact);Fraser-Liggett, PhD, Claire, M NARRATIVE (Lay Description) An effective HIV vaccine continues to be a remote objective even though much work has been devoted to developing one. Research, however, has shown that a few monoclonal antibodies developed in the laboratory have key characteristics of the immune response that should ideally be induced by an effective HIV vaccine. These monoclonal antibodies have what is termed 'broadly neutralizing'(BN) activity, meaning that they can neutralize many different HIVs from many different regions around the world and that HIV does not rapidly become incapable of being neutralized by the antibodies. These BN monoclonal antibodies recognize a particular part of HIV called the MPER region of the HIV envelope protein, which consists of about 15 amino acids. Unfortunately, when peptides, or small proteins, consisting of these amino acids are synthesized and used to immunize animals the animals do not produce BN anti-HIV antibodies, probably because the small MPER region by itself and outside of the context of the rest of the HIV envelope protein anchored into the HIV envelope does not assume the correct shape capable of inducing a BN antibody response. We hypothesize that we can make the MPER region amino acids assume a shape that will induce a BN immune response if we express it in the context of some other non-immunogenic 'scaffolding'protein, but if this is correct we do not now know what scaffolding protein to use and where within that scaffolding protein we should put the MPER amino acids to make them fold into the correct shape to induce a BN antibody response. However, we also hypothesize that we can combine recent advances in several fields in ways that may make it possible to produce and identify proteins containing the MPER sequences that can induce BN antibodies. These advances include dramatically decreasing costs for gene synthesis, which means that we can cheaply make many different versions of proteins containing the MPER region, bacterial systems that can express large numbers of potentially immunogenic proteins on the surfaces of the bacteria, which means that we can present pools of bacteria expressing different versions of a protein to the gastrointestinal immune system of an animal and ask the animal's immune system to recognize or select the proteins that are immunogenic, and DNA 'barcoding' approaches and detection systems using microarrays, which mean that we can readily identify and quantify the bacteria bearing the plasmid that express the different antigens, including those selected by the gastrointestinal immune system. In our proposed project we will combine these approaches by feeding mice pools of bacteria expressing different MPER-containing proteins via barcoded plasmids and then screen the feces of the mice to determine the relative abundance of the different plasmids. Our hypothesis holds that if a particular scaffolding protein-MPER chimeric protein is immunongenic, then the gastrointestinal immune system of the mouse will recognize the chimeric protein and mount an immune response against that protein, and its relative abundance will decrease. We can then conduct additional experiments to determine if that chimeric protein does indeed induce a BN anti-HIV immune response and, if it does, examine its utility as a potential HIV vaccine candidate. Our proposed project, therefore, would potentially offer a rapid and innovative way to identify new candidates for an HIV vaccine that would induce a BN anti-HIV immune response. An added benefit is that any vaccine candidate identified in the screen would be able to induce a strong mucosal immune response, a highly desirable characteristic for an HIV vaccine. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Special Emphasis Panel (ZAI1-BP-A (M2))
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Shapiro, Stuart Z
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Children's Research Institute
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Jackson, Hope T; Mongodin, Emmanuel F; Davenport, Katherine P et al. (2014) Culture-independent evaluation of the appendix and rectum microbiomes in children with and without appendicitis. PLoS One 9:e95414
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