Viral delivery of a peptide-based immunomodulator enhances T cell priming during vaccination.

Modern, subunit-based vaccines have so far failed to induce significant T cell responses, contributing to ineffective vaccination against many pathogens. Importantly, while today's adjuvants are designed to trigger innate and non-specific immune responses, they fail to directly stimulate the adaptive immune compartment. Programmed cell death 1 (PD-1) partly regulates naïve-to-antigen-specific effector T cell transition and differentiation by suppressing the magnitude of activation. Indeed, we previously reported on a microbial-derived, peptide-based PD-1 checkpoint inhibitor, LD01, which showed potent T cell-stimulating activity when combined with a vaccine. Here we sought to improve the potency of LD01 by designing and testing new LD01 derivatives. Accordingly, we found that a modified version of an 18-amino acid metabolite of LD01, LD10da, improved T cell activation capability in a malaria vaccine model. Specifically, LD10da demonstrates improved antigen-specific CD8+ T cell expansion when combined prophylactically with an adenovirus-based malaria vaccine. A single dose of LD10da at the time of vaccination is sufficient to increase antigen-specific CD8+ T cell expansion in wild-type mice. Further, we show that LD10 can be encoded and delivered by a Modified Vaccinia Ankara viral vector and can enhance antigen-specific CD8+ T cell expansion comparable to that of synthetic peptide administration. Therefore, LD10da represents a promising biologic-based immunomodulator that can be genetically encoded and delivered, along with the antigen, by viral or other nucleic acid vectors to improve the efficacy and delivery of vaccines for ineradicable and emerging infectious diseases.

A Peptide-Based Checkpoint Immunomodulator Alleviates Immune Dysfunction in Murine Polymicrobial Sepsis.

ABSTRACT: Sepsis-induced immunosuppression involves both innate and adaptive immunity and is associated with the increased expression of checkpoint inhibitors, such as programmed cell-death protein 1 (PD-1). The expression of PD-1 is associated with poor outcomes in septic patients, and in models of sepsis, blocking PD-1 or its ligands with antibodies increased survival and alleviated immune suppression. While inhibitory antibodies are effective, they can lead to immune-related adverse events (irAEs), in part due to continual blockade of the PD-1 pathway, resulting in hyperactivation of the immune response. Peptide-based therapeutics are an alternative drug modality that provide a rapid pharmacokinetic profile, reducing the incidence of precipitating irAEs. We recently reported that the potent, peptide-based PD-1 checkpoint antagonist, LD01, improves T-cell responses. The goal of the current study was to determine whether LD01 treatment improved survival, bacterial clearance, and host immunity in the cecal-ligation and puncture (CLP)-induced murine polymicrobial sepsis model. LD01 treatment of CLP-induced sepsis significantly enhanced survival and decreased bacterial burden. Altered survival was associated with improved macrophage phagocytic activity and T-cell production of interferon-γ. Further, myeloperoxidase levels and esterase-positive cells were significantly reduced in LD01-treated mice. Taken together, these data establish that LD01 modulates host immunity and is a viable therapeutic candidate for alleviating immunosuppression that characterizes sepsis and other infectious diseases.

A Peptide-Based PD1 Antagonist Enhances T-Cell Priming and Efficacy of a Prophylactic Malaria Vaccine and Promotes Survival in a Lethal Malaria Model.

The blockade of programmed cell death-1 (PD1) and its ligand PDL1 has been proven to be a successful immunotherapy against several cancers. Similar to cancer, PD1 contributes to the establishment of several chronic infectious diseases, including malaria. While monoclonal antibodies (mAbs) targeting checkpoint receptors are revolutionary in cancer treatment, the immune-related adverse events (irAEs) may prevent their utilization in prophylactic and therapeutic treatments of infectious diseases. The irAEs are, in part, due to the prolonged half-life of mAbs resulting in prolonged activation of the immune system. As an alternative modality to mAbs, peptides represent a viable option because they possess a shorter pharmacokinetic half-life and offer more formulation and delivery options. Here, we report on a 22-amino acid immunomodulatory peptide, LD01, derived from a Bacillus bacteria. When combined prophylactically with an adenovirus-based or irradiated sporozoite-based malaria vaccine, LD01 significantly enhanced antigen-specific CD8+ T cell expansion. Therapeutically, LD01 treatment of mice infected with a lethal malaria strain resulted in survival that was associated with lower numbers of FOXP3+Tbet+CD4+ regulatory T cells. Taken together, our results demonstrate that LD01 is a potent immunomodulator that acts upon the adaptive immune system to stimulate T cell responses both prophylactically and therapeutically.

Novel Peptide-Based PD1 Immunomodulators Demonstrate Efficacy in Infectious Disease Vaccines and Therapeutics.

Many pathogens use the same immune evasion mechanisms as cancer cells. Patients with chronic infections have elevated levels of checkpoint receptors (e.g., programed cell death 1, PD1) on T cells. Monoclonal antibody (mAb)-based inhibitors to checkpoint receptors have also been shown to enhance T-cell responses in models of chronic infection. Therefore, inhibitors have the potential to act as a vaccine "adjuvant" by facilitating the expansion of vaccine antigen-specific T-cell repertoires. Here, we report the discovery and characterization of a peptide-based class of PD1 checkpoint inhibitors, which have a potent adaptive immunity adjuvant capability for vaccines against infectious diseases. Briefly, after identifying peptides that bind to the recombinant human PD1, we screened for in vitro efficacy in reporter assays and human peripheral blood mononuclear cells (PBMC) readouts. We first found the baseline in vivo performance of the peptides in a standard mouse oncology model that demonstrated equivalent efficacy compared to mAbs against the PD1 checkpoint. Subsequently, two strategies were used to demonstrate the utility of our peptides in infectious disease indications: (1) as a therapeutic in a bacteria-induced lethal sepsis model in which our peptides were found to increase survival with enhanced bacterial clearance and increased macrophage function; and (2) as an adjuvant in combination with a prophylactic malaria vaccine in which our peptides increased T-cell immunogenicity and the protective efficacy of the vaccine. Therefore, our peptides are promising as both a therapeutic agent and a vaccine adjuvant for infectious disease with a potentially safer and more cost-effective target product profile compared to mAbs. These findings are essential for deploying a new immunomodulatory regimen in infectious disease primary and clinical care settings.

Virulence signatures: microarray-based approaches to discovery and analysis.

Rapid, accurate, and sensitive detection of biothreat agents requires a broad-spectrum assay capable of discriminating between closely related microbial or viral pathogens. Moreover, in cases where a biological agent release has been identified, forensic analysis demands detailed genetic signature data for accurate strain identification and attribution. To date, nucleic acid sequences have provided the most robust and phylogentically illuminating signature information. Nucleic acid signature sequences are not often linked to genomic or extrachromosomal determinants of virulence, a link that would further facilitate discrimination between pathogens and closely related species. Inextricably coupling genetic determinants of virulence with highly informative nucleic acid signatures would provide a robust means of identifying human, livestock, and agricultural pathogens. By means of example, we present here an overview of two general applications of microarray-based methods for: (1) the identification of candidate virulence factors; and (2) the analysis of genetic polymorphisms that are coupled to Bacillus anthracis virulence factors using an accurate, low cost solid-phase mini-sequencing assay. We show that microarray-based analysis of gene expression can identify potential virulence associated genes for use as candidate signature targets, and, further, that microarray-based single nucleotide polymorphism assays provide a robust platform for the detection and identification of signature sequences in a manner independent of the genetic background in which the signature is embedded. We discuss the strategy as a general approach or pipeline for the discovery of virulence-linked nucleic acid signatures for biothreat agents.

Statistical analysis of microarray data: a Bayesian approach.

The potential of microarray data is enormous. It allows us to monitor the expression of thousands of genes simultaneously. A common task with microarray is to determine which genes are differentially expressed between two samples obtained under two different conditions. Recently, several statistical methods have been proposed to perform such a task when there are replicate samples under each condition. Two major problems arise with microarray data. The first one is that the number of replicates is very small (usually 2-10), leading to noisy point estimates. As a consequence, traditional statistics that are based on the means and standard deviations, e.g. t-statistic, are not suitable. The second problem is that the number of genes is usually very large (approximately 10,000), and one is faced with an extreme multiple testing problem. Most multiple testing adjustments are relatively conservative, especially when the number of replicates is small. In this paper we present an empirical Bayes analysis that handles both problems very well. Using different parametrizations, we develop four statistics that can be used to test hypotheses about the means and/or variances of the gene expression levels in both one- and two-sample problems. The methods are illustrated using experimental data with prior knowledge. In addition, we present the result of a simulation comparing our methods to well-known statistics and multiple testing adjustments.

DNA sequence conservation between the Bacillus anthracis pXO2 plasmid and genomic sequence from closely related bacteria.

BACKGROUND: Complete sequencing and annotation of the 96.2 kb Bacillus anthracis plasmid, pXO2, predicted 85 open reading frames (ORFs). Bacillus cereus and Bacillus thuringiensis isolates that ranged in genomic similarity to B. anthracis, as determined by amplified fragment length polymorphism (AFLP) analysis, were examined by PCR for the presence of sequences similar to 47 pXO2 ORFs. RESULTS: The two most distantly related isolates examined, B. thuringiensis 33679 and B. thuringiensis AWO6, produced the greatest number of ORF sequences similar to pXO2; 10 detected in 33679 and 16 in AWO6. No more than two of the pXO2 ORFs were detected in any one of the remaining isolates. Dot-blot DNA hybridizations between pXO2 ORF fragments and total genomic DNA from AWO6 were consistent with the PCR assay results for this isolate and also revealed nine additional ORFs shared between these two bacteria. Sequences similar to the B. anthracis cap genes or their regulator, acpA, were not detected among any of the examined isolates. CONCLUSIONS: The presence of pXO2 sequences in the other Bacillus isolates did not correlate with genomic relatedness established by AFLP analysis. The presence of pXO2 ORF sequences in other Bacillus species suggests the possibility that certain pXO2 plasmid gene functions may also be present in other closely related bacteria.

Bacillus anthracis pXO1 plasmid sequence conservation among closely related bacterial species.

The complete sequencing and annotation of the 181.7-kb Bacillus anthracis virulence plasmid pXO1 predicted 143 genes but could only assign putative functions to 45. Hybridization assays, PCR amplification, and DNA sequencing were used to determine whether pXO1 open reading frame (ORF) sequences were present in other bacilli and more distantly related bacterial genera. Eighteen Bacillus species isolates and four other bacterial species were tested for the presence of 106 pXO1 ORFs. Three ORFs were conserved in most of the bacteria tested. Many of the pXO1 ORFs were detected in closely related Bacillus species, and some were detected only in B. anthracis isolates. Three isolates, Bacillus cereus D-17, B. cereus 43881, and Bacillus thuringiensis 33679, contained sequences that were similar to more than one-half of the pXO1 ORF sequences examined. The majority of the DNA fragments that were amplified by PCR from these organisms had DNA sequences between 80 and 98% similar to that of pXO1. Pulsed-field gel electrophoresis revealed large potential plasmids present in both B. cereus 43881 (341 kb) and B. thuringiensis ATCC 33679 (327 kb) that hybridized with a DNA probe composed of six pXO1 ORFs.