S-pecial delivery: Implications of HBV surface antigen subviral particles carrying microRNA payloads

Chronic HBV infection remains a significant public health problem, impacting the lives of nearly 300 million people worldwide. Its unique replication cycle, the primary barrier to cure, is driven from the viral template, covalently closed circular DNA, in the hepatocyte nucleus. Covalently closed circular DNA allows the virus to uncouple viral protein production from the pre-genomic RNA that undergoes reverse transcription to form mature virions. Uncoupling protein production, in particular the HBV surface antigen (HBsAg), from replication allows HBV to produce noninfectious subviral particles that outnumber mature virions 1000–100,000-fold. The majority of subviral particles are comprised of the small HBsAg produced from covalently closed circular DNA or portions of the HBV genome that integrate into the hepatocyte chromosome.1 As a result, HBsAg can be found at concentrations of milligrams per milliliter in the serum of patients with chronic hepatitis B. Because of the sheer abundance of HBsAg in patient serum, it has long been investigated to determine how HBsAg modulates immunity to improve immunotherapies. While we cannot cite every article supporting this observation, it is clear that long-term exposure to HBsAg leads to the exhaustion and deletion of HBs-specific T cells.2 Similarly, persistent exposure to HBsAg leads to the exhaustion of HBs-specific B cells, limiting their ability to produce antibodies and proliferate.3 In contrast to adaptive immunity, the effect of HBsAg on innate immunity has led to a confusing body of literature that associates HBsAg with both immuno-suppressive and immuno-stimulatory properties.4 In some cases, exposure to HBsAg leads to the production of inflammatory/antiviral cytokines and activation of professional antigen-presenting cells, like dendritic cells and monocytes. In other settings, HBsAg causes the production of immune-suppressive cytokines, like IL-10, and inhibits antigen-presenting cell activation by interfering with intracellular signaling cascades. This broad spectrum of outcomes after exposure to a structural viral protein has made it difficult to determine the true impact of HBsAg subviral particles on innate immunity. Unlike adaptive immunity, where a pathway for antigen-specific exhaustion has been extensively studied, modulation of innate immunity by a viral antigen that does not possess a molecularly described motif for innate sensors has been difficult to reconcile. To some extent, the source of HBsAg likely accounts for differences observed after in vitro exposure. HBsAg derived from Escherichia coli or yeast have different glycosylation patterns and lipid composition and may possess contaminating Lipopolysaccharide (LPS) or lipopeptides recognized by Toll-like receptors.5,6 However, some reports have shown differences in dendritic cell or monocyte function directly ex vivo, with little or no in vitro culture, excluding the possibility of LPS contamination.7 Myeloid cells are relatively short-lived in the circulation and sensitive to environmental factors. Therefore, patient heterogeneity, such as stage of disease, degree of cirrhosis, or liver inflammation associated with elevated alanine aminotransferase levels, could account for differences observed ex vivo. The article by Li et al8 provides potential insight into the contradictory, immunomodulatory observations associated with HBsAg and innate immunity in the context of the natural history of chronic HBV infection. Their study demonstrated that subviral particles composed of HBsAg could package hepatocyte-derived microRNAs (miRs). The profile of miRs packaged within subviral particles differed by stage of infection. Hepatocyte-specific miRs, miR-122, miR-194, and miR-192, correlated with HBsAg and viral load, while miR-939, miR-210, and miR-145 significantly associated with alanine aminotransferase during hepatitis. The investigators used density gradient separation and fluorescently labeled miRs to confirm the packaging of miRs in subviral particles versus virions. Using miR-939–loaded HBsAg subviral particles, they showed an increase in monocyte-derived IL-8 production above what could be induced by exposure to either miR-939 or HBsAg alone. Therefore, this study provides a foundation on which to interpret ex vivo studies looking at myeloid function in different patient populations. It suggests immunomodulation is not a consequence of HBsAg itself but the payload within subviral particles that can alter myeloid function. These data also suggest that subviral particles produced in bacteria or yeast might package immunomodulatory components with the potential to skew myeloid activation beyond contamination with LPS/lipopeptides and should be used with caution/awareness of this limitation. These observations expand on previous work from the investigators in HCV infection, showing that exosomes could transmit an Interferon-induced antiviral state to distal cells.9 The question remains: how much subviral particle-mediated delivery of miRs regulates myeloid cells in the periphery? The immunomodulatory miRs were primarily elevated during hepatitis. We, and others, have demonstrated that numerous inflammatory cytokines are detectable in the plasma of patients with chronic hepatitis B with elevated alanine aminotransferase.10 These circulating cytokines will also regulate myeloid cell function. Nonetheless, this report helps interpret the potential caveats of prior studies where contradictory results were obtained from seemingly similar experimental designs. The observation that HBsAg subviral particles can carry miRs that regulate myeloid function may have implications for novel therapies. Small-interfering RNAs and anti-sense oligonucleotides block the production of all HBV proteins, reducing circulating HBsAg 2–4 log10. It will be interesting to see how blocking HBsAg production through RNA interference alters the miR profile and whether this influences response to immunotherapy combinations. Blocking subviral particle release through nucleic acid polymers may have a similar effect to RNA interference. In contrast, anti-HBs monoclonal antibodies could enhance the immunomodulatory effects of subviral particles. The primary mechanism of action for the current generation of monoclonal antibodies promotes immune complex formation to increase antibody-dependent phagocytosis into myeloid cells. One could hypothesize that antibody-driven enhancement of miR delivery in antigen-presenting cells could support or antagonize the immune-stimulatory goal of monoclonal antibody therapies. This could make miR profiles potential biomarkers for new therapies, but it is far too early to tell, and more analyses are needed. Fortunately, these analyses can be performed on plasma, facilitating the use of clinical material collected during trials.

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