Letvin Progress Report Abstracts continued
The Letvin VDC was funded originally to develop replication-defective recombinant adenovirus (rAd) and live recombinant mycobacteria (rMyco) vectors as candidate HIV-1 vaccines. These vectors are practical to produce, and have been shown to elicit durable systemic and mucosal immune responses. A large portfolio of rAd and rMyco vectors has been constructed and evaluated. The overarching goal was to combine the most immunogenic novel rMyco and rAd vectors as immunogens in a prime/boost regimen against HIV-1 for evaluation in phase I clinical trials in the developing world. The rAd program pursued two different approaches: the Barouch group developed novel rAd vectors that evade pre-existing anti-Ad5 immunity, and the Nabel team focused on rAd vectors that target the mucosa and on improving prime/boost combinations. The rMyco team pursued three complementing strategies to select rMyco vectors with enhanced immunogenicity. Investigators from the Haynes, Lee, Jacobs, Letvin, and Porcelli laboratories have generated panels of rMyco strains that were identified on the basis of increased transgene expression/secretion, pro-apoptotic phenotype, or enhanced MHC class I presentation.
Priorities of the Letvin VDC were modified in light of the failed Merck STEP trial and of the modest success of the RV144 Thai trial. The rAd program continues with a two-pronged approach: pursuing work aimed at advancing a prototype replication-competent Ad26 (rcAd26) construct for clinical production, and using the existing portfolio of rAd vectors to continue development of optimal strategies for inducing and assessing mucosal immune responses in nonhuman primate models. Following the streamlined roadmap established for selecting candidate mycobacterial vectors for clinical development, the rMyco team is evaluating the first group of lead-candidate rBCG mutants as priming immunogens in a heterologous prime/boost vaccine regimen in nonhuman primates. The team is also evaluating the efficacy of lipid-adjuvanting of live mycobacteria, and the potential of recombinant M. Smegmatis, and auxotrophic and non-replicating mycobacteria strains as vaccines against HIV-1. Selected rMyco constructs will be evaluated as priming immunogens for use with adjuvanted Env protein or viral vector boosts for eliciting cellular and humoral immune responses to HIV-1 Env. The most immunogenic combination regimen will be advanced for clinical development.
The Letvin VDC is developing novel, recombinant adenovirus (rAd) and mycobacteria (rMyco) vectors to elicit durable systemic and mucosal immune responses against HIV-1 by three approaches: (1) The Barouch group has two parallel projects: in studying critical aspects of anti-Ad5 immunity following the results of the STEP trial, they have contributed substantially to our understanding of the biology of rAd vectors. In developing novel Ad vectors that evade pre-existing anti-Ad5 immunity, they have made substantial progress in generating both replication non-competent chimeric rAd vectors and replication competent rare serotype rAd26 vectors. (2) The Nabel team focuses on rAd vectors that target the intestinal mucosa for use in prime-boost vaccine combinations. Based on their small animal studies, they have identified rare serotype Ad28 as their lead candidate vector, and with the Letvin group, they are testing a set of nonreplicating rAd28 vectors in nonhuman primate (NHP) studies to assess the efficacy of this prime-boost regimen in an SIV challenge model. (3) The first generation rBCG-SIV vectors provided effective priming for a heterologous rAd5-SIV boost in a pilot NHP study. To optimize vector immunogenicity, the rMyco team employed genome-wide screens to identify BCG genes that affect the host’s immune responses, and molecular biology to increase transgene expression and secretion by BCG. The team is on target in generating more immunogenic second generation rBCG vectors with a streamlined roadmap towards clinical vectors production. The lead candidate rBCG-SIV vectors are on track to be tested in NHP studies in Year 4.
The Letvin VDTC comprises investigators from the Beth Israel Deaconess Medical Center (BIDMC), Duke, Einstein, and the NIAID Vaccine Research Center (VRC). We are developing recombinant adenovirus (rAd) and mycobacteria (rMyco) vector-based vaccines against HIV-1 by three approaches: (1) The Barouch group at BIDMC has made substantial progress towards the original project goal of generating novel, replication non-competent and competent rare serotype rAd vectors that evade pre-existing anti-Ad5 immunity. Following the termination of the STEP trial, they initiated a series of studies that are contributing to the understanding of the immunologic basis of anti-vector immunity. (2) The Nabel team at the VRC has developed a set of prototype chimeric rAd vectors that target the intestinal mucosa. Vector immunogenicity was tested in mice by direct delivery to the small intestine; these vectors elicited both systemic and mucosal immune responses that can be enhanced by intramuscular boost. With the Letvin group at BIDMC, they are designing nonhuman primate studies to test this prime-boost regimen in an SIV challenge model. To help address issues raised by the STEP trial data, the Nabel group has analyzed the effect of naturally occurring anti-Ad5 immunity on rAd5 vaccine vectors. Surprisingly, they found that natural infection by Ad5 and vaccination with rAd5 vectors generated neutralizing antibodies (NAbs) that target to different proteins of the viral capsid. They are continuing to analyze the differences in the anti-Ad5 NAbs. (3) The rMyco team is developing vectors with improved immunogenicity. The Haynes/Lee group at Duke has generated recombinant M. bovis Bacille Calmette-Guerin (rBCG) vectors in both episomal and integrated forms that are optimized for transgene cell-surface expression and secretion. Mutant rMyco libraries were generated from the optimized vectors by random transposon-insertion mutagenesis. The Duke group has identified about 200 mutants with improved transgene secretion and/or expression, which are being tested for enhanced immunogenicity in mice. The Jacobs/Larsen group is developing novel strains of rMyco with improved immunogenicity and safety. They have generated rBCG and recombinant M. smegmatis (rMsmeg) mutants with deletions in the homologous genetic loci identified on M. tuberculosis (MTb) that are responsible for immune evasion. They have also screened over 5,000 transposon-insertion mutants of BCG and identified 13 that mediate enhanced apoptosis. The ability of these mutant strains to inhibit macrophage apoptosis is being assessed. Using different approaches, the Letvin and Porcelli groups have identified over 100 rMyco mutants that elicit increased antigen presentation in the MHC class I and/or class II pathways; 20 mutants have been sequenced and characterized. The team is benchmarking the novel mutants with industry-standard rMyco vectors. Mutations will be combined to generate a set of rMyco vectors that are optimized for vaccine-elicited immune responses. The Letvin VDTC will test the novel rMyco and rAd vectors in prime-boost combinations in preclinical studies to assess their effectiveness as vaccines against HIV-1.
The Letvin VDTC is developing recombinant adenovirus (rAd) and mycobacteria (rMyco) vectors that will elicit durable systemic and mucosal immune responses against HIV-1 by three approaches:
- The Barouch group focuses on novel rAd vectors that evade pre-existing anti- Ad5 immunity. They have also begun to investigate critical aspects of anti-Ad5 immunity that are related to the results of the STEP study.
- The Nabel team is developing rAd vectors that target the intestinal mucosa for use in prime-boost vaccine combinations. They have shown that rAd41 has enteric tropism in mice, and are characterizing the enteric tropism of the low seroprevalent rAd35/41 fiber chimeric vectors.
- The Haynes, Jacobs/Larsen, Porcelli and Letvin Team is developing novel rMyco vectors with improved immunogenicity. The Haynes group is improving vector expression by appending strong promoters and signal peptides to the transgene as well as screening for clones with enhanced secretion. The Jacobs/Larsen group is developing novel strains of mycobacteria with improved safety and immunogenicity. They are generating mutants in M. bovis Bacille Calmette-Guerin (BCG) and M. smegmatis (Msmeg) with deletions in homologous genetic loci identified on M. tuberculosis that are responsible for immune evasion. These mutants will be tested for improved ability to stimulate T cell responses and antigen-specific memory. The Letvin and Porcelli groups have identified rBCG and rMsmeg mutants that elicit an increased capacity to process and present antigens in the MHC class I and class II pathways. These novel rMyco vector-based vaccines will be developed as a priming strategy in prime-boost vaccination regimens for immunotherapy or prevention of disease caused by HIV.
The goal of the Letvin VDTC is to develop novel recombinant adenovirus (rAd) and mycobacteria (rMyco) vectors that are immunogenic in the setting of anti-vector immunity and that elicit durable systemic and mucosal immune responses. 3 different approaches are employed:
- The Barouch group focuses on rAd vectors that evade pre-existing anti-Ad5 immunity. They have initiated three parallel vector construction programs: (i) generation of improved novel serotype rAd vectors, (ii) construction of an expanded set of chimeric rAd5 vectors, and (iii) development of replication-competent rare serotype rAd vectors. They have also begun to explore Ad-specific vector immunity in greater detail given the potential association of pre-existing anti-Ad5 immunity and HIV-1 acquisition risk in vaccinees.
- The Nabel group focuses on novel rAd vectors that target the mucosa for use in prime-boost vaccine combinations to stimulate mucosal immune response. In collaboration with GenVec, the initial focus is on rAd41, which demonstrates enteric tropism. rAd41 vectors encoding HIV gp140B have been constructed and shown to generate T-cell and humoral responses in mice. Modifications of the Ad41 fiber genes that preserve the functionally relevant structure of the fibers and allow their incorporation into different rAd serotypes are being generated and evaluated. They have constructed rAd35/41 fiber chimeric vectors with the long and short fibers of Ad41 and rare serotype Ad35. These low seroprevalent chimeric rAd vectors are being characterized for their enteric tropism for targeting the mucosa.
- The Haynes, Jacobs and Letvin team has demonstrated that rMyco vaccine constructs elicited durable systemic and mucosal immune responses in mice. They are developing novel rBCG and rMsmeg vectors with improved immunogenicity. The Haynes group has developed a flow cytometry-based assay to detect transgene products expressed on the surface of de-lipidated rBCG using an anti-HIV-1 Env monoclonal antibody. Populations of rBCG expressing CONS Env have been sorted into clones of high expressers for expansion and re-analysis. Compared to the unsorted population, the sorted rBCG clones expressing high levels of HIV-1 Env induced greater HIV-1 Env p18-specific tetramer staining in mouse PBMC and ELISPOT on splenocytes. They have increased the expression of the group M HIV-1 gp140 CF CON-S Env transgene in rMsmeg and rBCG using the heat shock protein 60 (hsp60) promoter and the twin arginine signal peptide. Immunogenicities of these vectors are being assessed in mice. The Einstein group is developing novel strains of BCG and Msmeg with improved safety and immunogenicity. From previous studies, they have identified multiple genetic loci on M. tuberculosis that are responsible for immune evasion, and that deletion of one or more of these loci can enhance the ability of the bacterium to generate protective immune responses against its antigens. Deletion mutants at homologous loci in BCG and Msmeg are being constructed that will be tested for improved ability to stimulate T cell responses and antigen specific memory. Along with the Letvin group, screening procedures have been developed to select and isolate randomly generated mutants of BCG and Msmeg that have an increased capacity to deliver their antigens into the MHC class I and class II antigen processing and presentation pathways. These studies will lead to optimized live attenuated rMyco vectors that will be safe and practical vehicles for the expression of selected HIV antigens. These novel rMycobacterial vector-based vaccines will be developed as a priming strategy in prime-boost vaccination regimens for immunotherapy or prevention of disease caused by HIV.
We are developing novel recombinant adenovirus (rAd) and mycobacteria (rMyco) vectors that are immunogenic in the setting of anti-vector immunity and that elicit durable systemic and mucosal immune responses. 3 different approaches are employed: (1) rAd vectors that evade pre-existing anti-Ad5 immunity: the Barouch group is constructing novel serotype rAd vectors and an expanded set of chimeric rAd5 vectors that express novel hexon loops. (2) rAd vectors for use in prime-boost vaccine combinations to stimulate mucosal immune responses: the Nabel group is focused on chimeric rAd 41 vectors, which target the intestinal mucosa. (3) rMyco vectors as vaccines for AIDS: the Haynes, Jacobs and Letvin team has generated recombinant Mycobacterium bovis Bacille Calmette-Guerin (rBCG) with increased HIV-1 Env expression. In mice, the rBCG elicited durable systemic and mucosal immune responses. However, this prototype vaccine did not induce adequate immunogenicity in nonhuman primates. Three new sets of studies have been initiated to improve the rMyco vectors as vaccines. First, the recombinant M. smegmatis (rM. Smeg) constructs were evaluated in mice and showed that the vaccine-elicited cellular immune responses have a significant bias towards CD8+ T cells with a memory phenotype and function. Second, libraries of mutant rBCG and rM. Smeg are being generated to isolate clones with increased surface expression and antigen processing and presentation. Third, a novel mutant M. Smeg has been defined, which demonstrated increased immunogenicity and protective efficacy in mice. This M. Smeg mutant is being evaluated as a vaccine vector for HIV antigens.