Immunomodulation by the Commensal Microbiome During Immune-Targeted Interventions: Focus on Cancer Immune Checkpoint Inhibitor Therapy and Vaccination.

Emerging evidence in clinical and preclinical studies indicates that success of immunotherapies can be impacted by the state of the microbiome. Understanding the role of the microbiome during immune-targeted interventions could help us understand heterogeneity of treatment success, predict outcomes, and develop additional strategies to improve efficacy. In this review, we discuss key studies that reveal reciprocal interactions between the microbiome, the immune system, and the outcome of immune interventions. We focus on cancer immune checkpoint inhibitor treatment and vaccination as two crucial therapeutic areas with strong potential for immunomodulation by the microbiota. By juxtaposing studies across both therapeutic areas, we highlight three factors prominently involved in microbial immunomodulation: short-chain fatty acids, microbe-associate molecular patterns (MAMPs), and inflammatory cytokines. Continued interrogation of these models and pathways may reveal critical mechanistic synergies between the microbiome and the immune system, resulting in novel approaches designed to influence the efficacy of immune-targeted interventions.

Primary Human Dendritic Cells and Whole-Blood Based Assays to Evaluate Immuno-Modulatory Properties of Heat-Killed Commensal Bacteria.

There is mounting evidence that the microbiome plays a critical role in training and maturation of the host immune system. Pre-clinical and clinical studies have shown that microbiome perturbation is correlated with sub-optimal host responses to vaccines and cancer immunotherapy. As such, identifying species of commensal bacteria capable of modulating immunological outcomes is of considerable interest. Currently, the lack of reliable primary immune cell-based assays capable of differentiating immuno-modulatory properties of various commensal bacteria is a major limitation. Here, we demonstrate that primary human monocyte-derived dendritic cells (MoDC) are capable of stratifying different strains of live and heat-killed commensal bacteria in an in vitro culture system. Specifically, heat-killed bacterial strains were able to differentially modulate co-stimulation/maturation markers CD80, CD83, and HLA-DR, as well as cytokine/chemokine signatures, such as IL-1b, MIP-1a, and TNFa in primary human MoDC. We further validated our observations using the TruCulture® (Myriad RBM, Inc., Austin, TX, USA) whole-blood ex vivo culture system. Using this ex vivo system allowed us to measure immune-altering effects of commensal bacteria in primary human whole-blood. As such, we report that both these primary in vitro and ex vivo systems are robust and enable identification, stratification, and differentiation of various commensal bacteria as potential modulators of host immunity.

Antibody responses to crucial functional epitopes as a novel approach to assess immunogenicity of vaccine adjuvants.

We are interested in developing a vaccine that prevents genital herpes. Adjuvants have a major impact on vaccine immunogenicity. We compared two adjuvants, an experimental Merck Sharp & Dohme lipid nanoparticle (LNP) adjuvant, LNP-2, with CpG oligonucleotide combined with alum for immunogenicity in mice when administered with herpes simplex virus type 2 (HSV-2) glycoproteins C, D and E (gC2, gD2, gE2). The immunogens are intended to produce neutralizing antibodies to gC2 and gD2, antibodies to gD2 and gE2 that block cell-to-cell spread, and antibodies to gE2 and gC2 that block immune evasion from antibody and complement, respectively. Overall, CpG/alum was better at producing serum and vaginal IgG binding antibodies, neutralizing antibodies, antibodies that block virus spread from cell-to-cell, and antibodies that block immune evasion domains on gC2. We used a novel high throughput biosensor assay to further assess differences in immunogenicity by mapping antibody responses to seven crucial epitopes on gD2 involved in virus entry or cell-to-cell spread. We found striking differences between CpG/alum and LNP-2. Mice immunized with gD2 CpG/alum produced higher titers of antibodies than LNP-2 to six of seven crucial epitopes and produced antibodies to more crucial epitopes than LNP-2. Measuring epitope-specific antibodies helped to define mechanisms by which CpG/alum outperformed LNP-2 and is a valuable technique to compare adjuvants.

A Tetravalent Sub-unit Dengue Vaccine Formulated with Ionizable Cationic Lipid Nanoparticle induces Significant Immune Responses in Rodents and Non-Human Primates.

Dengue virus has emerged as an important arboviral infection worldwide. As a complex pathogen, with four distinct serotypes, the development of a successful Dengue virus vaccine has proven to be challenging. Here, we describe a novel Dengue vaccine candidate that contains truncated, recombinant, Dengue virus envelope protein from all four Dengue virus serotypes (DEN-80E) formulated with ionizable cationic lipid nanoparticles (LNPs). Immunization studies in mice, Guinea pigs, and in Rhesus macaques, revealed that LNPs induced high titers of Dengue virus neutralizing antibodies, with or without co-administration or encapsulation of a Toll-Like Receptor 9 agonist. Importantly, LNPs were also able to boost DEN-80E specific CD4+ and CD8+ T cell responses. Cytokine and chemokine profiling revealed that LNPs induced strong chemokine responses without significant induction of inflammatory cytokines. In addition to being highly efficacious, the vaccine formulation proved to be well-tolerated, demonstrating no elevation in any of the safety parameters evaluated. Notably, reduction in cationic lipid content of the nanoparticle dramatically reduced the LNP's ability to boost DEN-80E specific immune responses, highlighting the crucial role for the charge of the LNP. Overall, our novel studies, across multiple species, reveal a promising tetravalent Dengue virus sub-unit vaccine candidate.

Hepatitis C virus core protein enhances HIV-1 replication in human macrophages through TLR2, JNK, and MEK1/2-dependent upregulation of TNF-α and IL-6.

Despite their differential cell tropisms, HIV-1 and HCV dramatically influence disease progression in coinfected patients. Macrophages are important target cells of HIV-1. We hypothesized that secreted HCV core protein might modulate HIV-1 replication. We demonstrate that HCV core significantly enhances HIV-1 replication in human macrophages by upregulating TNF-α and IL-6 via TLR2-, JNK-, and MEK1/2-dependent pathways. Furthermore, we show that TNF-α and IL-6 secreted from HCV core-treated macrophages reactivates monocytic U1 cells latently infected with HIV-1. Our studies reveal a previously unrecognized role of HCV core by enhancing HIV-1 infection in macrophages.

MicroRNAs and HIV-1 infection: antiviral activities and beyond.

Cellular microRNAs (miRNAs) are an important class of small, non-coding RNAs that bind to host mRNAs based on sequence complementarity and regulate protein expression. They play important roles in controlling key cellular processes including cellular inception, differentiation and death. While several viruses have been shown to encode for viral miRNAs, controversy persists over the expression of a functional miRNA encoded in the human immunodeficiency virus type 1 (HIV-1) genome. However, it has been reported that HIV-1 infectivity is influenced by cellular miRNAs. Either through directly targeting the viral genome or by targeting host cellular proteins required for successful virus replication, multiple cellular miRNAs seem to modulate HIV-1 infection and replication. Perhaps as a survival strategy, HIV-1 may modulate proteins in the miRNA biogenesis pathway to subvert miRNA-induced antiviral effects. Global expression profiles of cellular miRNAs have also identified alterations of specific miRNAs post-HIV-1 infection both in vitro and in vivo (in various infected patient cohorts), suggesting potential roles for miRNAs in pathogenesis and disease progression. However, little attention has been devoted in understanding the roles played by these miRNAs at a cellular level. In this manuscript, we review past and current findings pertaining to the field of miRNA and HIV-1 interplay. In addition, we suggest strategies to exploit miRNAs therapeutically for curbing HIV-1 infectivity, replication and latency since they hold an untapped potential that deserves further investigation.

A role for microRNA-155 modulation in the anti-HIV-1 effects of Toll-like receptor 3 stimulation in macrophages.

HIV-1 infection of macrophages plays a key role in viral pathogenesis and progression to AIDS. Polyinosine-polycytidylic acid (poly(I:C); a synthetic analog of dsRNA) and bacterial lipopolysaccharide (LPS), the ligands for Toll-like receptors (TLR) TLR3 and TLR4, respectively, are known to decrease HIV-1 infection in monocyte-derived macrophages (MDMs), but the mechanism(s) are incompletely understood. We found that poly(I:C)- and LPS-stimulation of MDMs abrogated infection by CCR5-using, macrophage-tropic HIV-1, and by vesicular stomatitis virus glycoprotein-pseudotyped HIV-1 virions, while TLR2, TLR7 or TLR9 agonists only partially reduced infection to varying extent. Suppression of infection, or lack thereof, did not correlate with differential effects on CD4 or CCR5 expression, type I interferon induction, or production of pro-inflammatory cytokines or ß-chemokines. Integrated pro-viruses were readily detected in unstimulated, TLR7- and TLR9-stimulated cells, but not in TLR3- or TLR4-stimulated MDMs, suggesting the alteration of post-entry, pre-integration event(s). Using microarray analysis and quantitative reverse transcription (RT)-PCR, we found increased microRNA (miR)-155 levels in MDMs upon TLR3/4- but not TLR7-stimulation, and a miR-155 specific inhibitor (but not a scrambled control) partially restored infectivity in poly(I:C)-stimulated MDMs. Ectopic miR-155 expression remarkably diminished HIV-1 infection in primary MDMs and cell lines. Furthermore, poly(I:C)-stimulation and ectopic miR-155 expression did not alter detection of early viral RT products, but both resulted in an accumulation of late RT products and in undetectable or extremely low levels of integrated pro-viruses and 2-LTR circles. Reduced mRNA and protein levels of several HIV-1 dependency factors involved in trafficking and/or nuclear import of pre-integration complexes (ADAM10, TNPO3, Nup153, LEDGF/p75) were found in poly(I:C)-stimulated and miR-155-transfected MDMs, and a reporter assay suggested they are authentic miR-155 targets. Our findings provide evidence that miR-155 exerts an anti-HIV-1 effect by targeting several HIV-1 dependency factors involved in post-entry, pre-integration events, leading to severely diminished HIV-1 infection.

Adenovirus delivered short hairpin RNA targeting a conserved site in the 5' non-translated region inhibits all four serotypes of dengue viruses.

BACKGROUND: Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs). This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi) to attenuate DENV replication may offer one approach to dengue therapy. METHODOLOGY/PRINCIPAL FINDINGS: We screened the non-translated regions (NTRs) of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5' NTR that maps to the 5' upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5) vector to deliver a short-hairpin RNA (shRNA) targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes. CONCLUSION/SIGNIFICANCE: The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection.