The in vitro replication phenotype of hepatitis B virus (HBV) splice variants Sp3 and Sp9 and their impact on wild-type HBV replication.

Prior to nuclear export, the hepatitis B virus (HBV) pregenomic RNA may be spliced by the host cell spliceosome to form shorter RNA sequences known as splice variants. Due to deletions in the open reading frames, splice variants may encode novel fusion proteins. Although not essential for HBV replication, the role of splice variants and their novel fusion proteins largely remains unknown. Some splice variants and their encoded novel fusion proteins have been shown to impair or promote wild-type HBV replication in vitro, and although splice variants Sp3 and Sp9 are two of the most common splice variants identified to date, their in vitro replication phenotype and their impact on wild-type HBV replication are unclear. Here, we utilize greater than genome-length Sp3 and Sp9 constructs to investigate their replication phenotype in vitro, and their impact on wild-type HBV replication. We show that Sp3 and Sp9 were incapable of autonomous replication, which was rescued by providing the polymerase and core proteins in trans. Furthermore, we showed that Sp3 had no impact on wild-type HBV replication, whereas Sp9 strongly reduced wild-type HBV replication in co-transfection experiments. Knocking out Sp9 novel precore-surface and core-surface fusion protein partially restored replication, suggesting that these proteins contributed to suppression of wild-type HBV replication, providing further insights into factors regulating HBV replication in vitro. IMPORTANCE: The role of hepatitis B virus (HBV) splice variants in HBV replication and pathogenesis currently remains largely unknown. However, HBV splice variants have been associated with the development of hepatocellular carcinoma, suggesting a role in HBV pathogenesis. Several in vitro co-transfection studies have shown that different splice variants have varying impacts on wild-type HBV replication, perhaps contributing to viral persistence. Furthermore, all splice variants are predicted to produce novel fusion proteins. Sp1 hepatitis B splice protein contributes to liver disease progression and apoptosis; however, the function of other HBV splice variant novel fusion proteins remains largely unknown. We show that Sp9 markedly impairs HBV replication in a cell culture co-transfection model, mediated by expression of Sp9 novel fusion proteins. In contrast, Sp3 had no effect on wild-type HBV replication. Together, these studies provide further insights into viral factors contributing to regulation of HBV replication.

Hepatobiliary organoids derived from leporids support the replication of hepatotropic lagoviruses.

The genus Lagovirus of the family Caliciviridae contains some of the most virulent vertebrate viruses known. Lagoviruses infect leporids, such as rabbits, hares and cottontails. Highly pathogenic viruses such as Rabbit haemorrhagic disease virus 1 (RHDV1) cause a fulminant hepatitis that typically leads to disseminated intravascular coagulation within 24-72 h of infection, killing over 95 % of susceptible animals. Research into the pathophysiological mechanisms that are responsible for this extreme phenotype has been hampered by the lack of a reliable culture system. Here, we report on a new ex vivo model for the cultivation of lagoviruses in cells derived from the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus). We show that three different lagoviruses, RHDV1, RHDV2 and RHDVa-K5, replicate in monolayer cultures derived from rabbit hepatobiliary organoids, but not in monolayer cultures derived from cat (Felis catus) or mouse (Mus musculus) organoids. Virus multiplication was demonstrated by (i) an increase in viral RNA levels, (ii) the accumulation of dsRNA viral replication intermediates and (iii) the expression of viral structural and non-structural proteins. The establishment of an organoid culture system for lagoviruses will facilitate studies with considerable implications for the conservation of endangered leporid species in Europe and North America, and the biocontrol of overabundant rabbit populations in Australia and New Zealand.

Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection.

In HIV-hepatitis B virus (HBV) co-infection, adverse liver outcomes including liver fibrosis occur at higher frequency than in HBV-mono-infection, even following antiretroviral therapy (ART) that suppresses both HIV and HBV replication. To determine whether liver disease was associated with intrahepatic or circulating markers of inflammation or burden of HIV or HBV, liver biopsies and blood were collected from HIV-HBV co-infected individuals (n = 39) living in Bangkok, Thailand and naïve to ART. Transient elastography (TE) was performed. Intrahepatic and circulating markers of inflammation and microbial translocation were quantified by ELISA and bead arrays and HIV and HBV infection quantified by PCR. Liver fibrosis (measured by both transient elastography and liver biopsy) was statistically significantly associated with intrahepatic mRNA for CXCL10 and CXCR3 using linear and logistic regression analyses adjusted for CD4 T-cell count. There was no evidence of a relationship between liver fibrosis and circulating HBV DNA, qHBsAg, plasma HIV RNA or circulating cell-associated HIV RNA or DNA. Using immunohistochemistry of liver biopsies from this cohort, intrahepatic CXCL10 was detected in hepatocytes associated with inflammatory liver infiltrates in the portal tracts. In an in vitro model, we infected an HBV-infected hepatocyte cell line with HIV, followed by interferon-γ stimulation. HBV-infected cells lines produced significantly more CXCL10 than uninfected cells lines and this significantly increased in the presence of an increasing multiplicity of HIV infection. Conclusion: Enhanced production of CXCL10 following co-infection of hepatocytes with both HIV and HBV may contribute to accelerated liver disease in the setting of HIV-HBV co-infection.

Secreted hepatitis B virus splice variants differ by HBV genotype and across phases of chronic hepatitis B infection.

Chronic hepatitis B (CHB) is characterized by progression through different phases of hepatitis B virus (HBV) infection and disease. Although not necessary for HBV replication, there is increasing evidence that HBV splice variants are associated with liver disease progression and pathogenesis. However, there have been no studies till date on the frequency or diversity of splice variants for different HBV genotypes across the phases of CHB. Next generation sequencing data from 404 patient samples of HBV genotype A, B, C or D in Phase I, Phase II or Phase IV of CHB was analysed for HBV splice variants using an in house bioinformatics pipeline. HBV splice variants differed in frequency and type by genotype and phase of natural history. Splice variant Sp1 was the most frequently detected (206/404, 51% of patients), followed by Sp13 (151/404 37% of patients). The frequency of variants was generally highest in Phase II (123/165, 75% of patients), a phase typically associated with enhanced immune activation, followed by Phase I (69/99, 70% of patients). Splice variants were associated with reduced hepatitis B e antigen (HBeAg) levels and statistically reduced likelihood of achieving HBsAg loss (functional cure) in Phase II patients for Sp1 and Sp13 (p = .0014 and .0156, respectively). The frequency of HBV splice variants in patient serum differed markedly by HBV genotype and phase of CHB natural history. The increased levels of HBV splice variants detected in CHB phase II patients compared with the higher replicative Phase I in particular warrants further investigation.

Fine-tuning expression of begomoviral movement and nuclear shuttle proteins confers cell-to-cell movement to mastreviral replicons in Nicotiana benthamiana leaves.

Geminiviruses are a group of small plant viruses responsible for devastating crop damage worldwide. The emergence of agricultural diseases caused by geminiviruses is attributed in part to their high rates of recombination, leading to complementary function between viral components across species and genera. We have developed a mastreviral reporter system based on bean yellow dwarf virus (BeYDV) that replicates to high levels in the plant nucleus, expressing very high levels of GFP. To investigate the potential for complementation of movement function by other geminivirus genera, the movement protein (MP) and nuclear shuttle protein (NSP) from the bipartite begomovirus Bean dwarf mosaic virus (BDMV) were produced and characterized in Nicotiana benthamiana leaves. While overexpression of MP and NSP strongly inhibited GFP expression from the mastreviral reporter and caused adverse plant symptoms, optimizing the expression levels of MP and NSP allowed functional cell-to-cell movement. Hybrid virus vectors were created that express BDMV MP and NSP from mastreviral replicons, allowing efficient cell-to-cell movement comparable to native BDMV replicons. We find that the expression levels of MP and NSP must be fine-tuned to provide sufficient MP/NSP for movement without eliciting the plant hypersensitive response or adversely impacting gene expression from viral replicons. The ability to confer cell-to-cell movement to mastrevirus replicons depended strongly on replicon size: 2.1-2.7 kb replicons were efficiently moved, while 3 kb replicons were inhibited, and 3.9 kb replicons were very strongly inhibited. Optimized expression of MP/NSP from the normally phloem-limited Abutilon mosaic virus (AbMV) allows efficient movement in non-phloem cells.

An intronless form of the tobacco extensin gene terminator strongly enhances transient gene expression in plant leaves.

KEY MESSAGE: We have found interesting features of a plant gene (extensin) 3' flanking region, including extremely efficient polyadenylation which greatly improves transient expression of transgenes when an intron is removed. Its use will greatly benefit studies of gene expression in plants, research in molecular biology, and applications for recombinant proteins. Plants are a promising platform for the production of recombinant proteins. To express high-value proteins in plants efficiently, the optimization of expression cassettes using appropriate regulatory sequences is critical. Here, we characterize the activity of the tobacco extensin (Ext) gene terminator by transient expression in Nicotiana benthamiana, tobacco, and lettuce. Ext is a member of the hydroxyproline-rich glycoprotein (HRGP) superfamily and constitutes the major protein component of cell walls. The present study demonstrates that the Ext terminator with its native intron removed increased transient gene expression up to 13.5-fold compared to previously established terminators. The enhanced transgene expression was correlated with increased mRNA accumulation and reduced levels of read-through transcripts, which could impair gene expression. Analysis of transcript 3'-ends found that the majority of polyadenylated transcripts were cleaved at a YA dinucleotide downstream from a canonical AAUAAA motif and a UG-rich region, both of which were found to be highly conserved among related extensin terminators. Deletion of either of these regions eliminated most of the activity of the terminator. Additionally, a 45 nt polypurine sequence ~ 175 nt upstream from the polyadenylation sites was found to also be necessary for the enhanced expression. We conclude that the use of Ext terminator has great potential to benefit the production of recombinant proteins in plants.

Chimeric 3' flanking regions strongly enhance gene expression in plants.

Plants represent a promising platform for the highly scalable production of recombinant proteins. Previously, we identified the tobacco extensin terminator lacking its intron as an element that reduced transcript read-through and improved recombinant protein production in a plant-based system. In this study, we systematically compared nonreplicating plant expression vectors containing over 20 commonly used or newly identified terminators from diverse sources. We found that eight gene terminators enhance reporter gene expression significantly more than the commonly used 35S and NOS terminators. The intronless extensin terminator provided a 13.6-fold increase compared with the NOS terminator. Combining terminators in tandem produced large synergistic effects, with many combinations providing a >25-fold increase in expression. Addition of the tobacco Rb7 or TM6 matrix attachment region (MAR) strongly enhanced protein production when added to most terminators, with the Rb7 MAR providing the greatest enhancement. Using deletion analysis, the full activity of the 1193 bp Rb7 MAR was found to require only a 463-bp region at its 3' end. Combined terminators and MAR together provided a >60-fold increase compared with the NOS terminator alone. These combinations were then placed in a replicating geminiviral vector, providing a total of >150-fold enhancement over the original NOS vector, corresponding to an estimated yield of 3-5 g recombinant protein per kg leaf fresh weight or around 50% of the leaf total soluble protein. These results demonstrate the importance of 3' flanking regions in optimizing gene expression and show great potential for 3' flanking regions to improve DNA-based recombinant protein production systems.

High-level expression and enrichment of norovirus virus-like particles in plants using modified geminiviral vectors.

Recombinant virus-like particles (VLPs) are proven to be safe and effective vaccine candidates. We have previously described a plant-based recombinant protein expression system based on agroinfiltration of a replicating vector derived from the geminivirus bean yellow dwarf virus (BeYDV). The system has been systematically optimized to improve expression and reduce cell death in Nicotiana benthamiana leaves. Using these modifications, we show that VLPs derived from genotype GII.4 norovirus, the leading cause of acute gastroenteritis worldwide, can be produced at >1 mg/g leaf fresh weight (LFW), over three times the highest level ever reported in plant-based systems. We also produced norovirus GI VLPs at 2.3 mg/g LFW. Treatment of VLP-containing crude leaf extracts with acid, detergent, or heat enhanced recovery and allowed selective enrichment of norovirus VLPs. Optimal treatment conditions allowed removal of >90% of endogenous plant proteins without any loss of norovirus VLPs. Selective enrichment of hepatitis B core antigen (HBcAg) VLPs by acid treatment was also demonstrated, with some losses in yield that were partially mitigated in the presence of detergent. Sedimentation analysis confirmed that acid and detergent did not inhibit proper assembly of norovirus VLPs, although heat treatment had a small negative effect. These results demonstrate that milligram quantities of norovirus VLPs can be obtained and highly enriched in a matter of days from a single plant leaf using the BeYDV plant expression system.

Cross-Reactive Synbody Affinity Ligands for Capturing Diverse Noroviruses.

Noroviruses are the most common cause of acute gastroenteritis in the developed world. Noroviruses are a diverse group of nonenveloped RNA viruses that are continuously evolving. This leads to the rise of immunologically distinct strains of the same genotype on a frequent basis. This diversity presents a unique challenge for detection and tracking of new strains, with the continuous need for new norovirus affinity ligands. Our group developed a family of bivalent synbody affinity ligands using a virus-like particle (VLP) from the 2006 GII.4 Minerva strain of norovirus. We produced more than 20 synbodies with low nanomolar dissociation constants (KD < 10 nM) for GII.4 VLP. We measured binding affinity for four synbodies against VLPs from multiple GI and GII genotypes and found that the synbodies were broadly cross-reactive with affinities that ranged from 0.5 to 8 nM. We tested the ability of these synbodies to capture norovirus from dilute solutions and found that one synbody could capture GII.4 from a 200 000-fold dilution from a norovirus positive stool sample. When these synbodies were tested for the ability to capture of multiple genotypes, we found that four different genotypes were recognized. These data demonstrate that the synbody approach can generate multiple affinity ligands for future use in norovirus detection and possible therapeutic development.

Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro. These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection.

Recombinant Human Acidic Fibroblast Growth Factor (aFGF) Expressed in Nicotiana benthamiana Potentially Inhibits Skin Photoaging.

Responding to the need for recombinant acidic fibroblast growth factor in the pharmaceutical and cosmetic industries, we established a scalable expression system for recombinant human aFGF using transient and a DNA replicon vector expression in Nicotiana benthamiana. Recombinant human-acidic fibroblast growth factor was recovered following Agrobacterium infiltration of N. benthamiana. The optimal time point at which to harvest recombinant human acidic fibroblast growth factor expressing leaves was found to be 4 days post-infiltration, before necrosis was evident. Commassie-stained SDS-PAGE gels of His-tag column eluates, concentrated using a 10 000 molecular weight cut-off column, showed an intense band at the expected molecular weight for recombinant human acidic fibroblast growth factor. An immunoblot confirmed that this band was recombinant human acidic fibroblast growth factor. Up to 10 µg recombinant human-acidic fibroblast growth factor/g of fresh leaves were achieved by a simple affinity purification protocol using protein extract from the leaves of agroinfiltrated N. benthamiana. The purified recombinant human acidic fibroblast growth factor improved the survival rate of UVB-irradiated HaCaT and CCD-986sk cells approximately 89 and 81 %, respectively. N. benthamiana-derived recombinant human acidic fibroblast growth factor showed similar effects on skin cell proliferation and UVB protection compared to those of Escherichia coli-derived recombinant human acidic fibroblast growth factor. Additionally, N. benthamiana-derived recombinant human acidic fibroblast growth factor increased type 1 procollagen synthesis up to 30 % as well as reduced UVB-induced intracellular reactive oxygen species generation in fibroblast (CCD-986sk) cells.UVB is a well-known factor that causes various types of skin damage and premature aging. Therefore, the present study demonstrated that N. benthamiana-derived recombinant human acidic fibroblast growth factor effectively protects skin cell from UVB, suggesting its potential use as a cosmetic or therapeutic agent against skin photoaging.

Overexpression and self-assembly of virus-like particles in Nicotiana benthamiana by a single-vector DNA replicon system.

Based on recent developments, virus-like particles (VLPs) are considered to be perfect candidates as nanoplatforms for applications in materials science and medicine. To succeed, mass production of VLPs and self-assembly into a correct form in plant systems are key factors. Here, we report expression of synthesized coat proteins of the three viruses, Brome mosaic virus, Cucumber mosaic virus, and Maize rayado fino virus, in Nicotiana benthamiana and production of self-assembled VLPs by transient expression system using agroinfiltration. Each coat protein was synthesized and cloned into a pBYR2fp single replicon vector. Target protein expression in cells containing p19 was fourfold higher than that of cells lacking p19. After agroinfiltration, protein expression was analyzed by SDS-PAGE and quantitative image analyzer. Quantitative analysis showed that BMVCP, CMVCP, and MRFVCP concentrations were 0.5, 1.0, and 0.8 mg · g(-1) leaf fresh weight, respectively. VLPs were purified by sucrose cushion ultracentrifugation and then analyzed by transmission electron microscopy. Our results suggested that BMVCP and CMVCP proteins expressed in N. benthamiana leaves were able to correctly self-assemble into particles. Moreover, we evaluated internal cavity accessibility of VLPs to load foreign molecules. Finally, plant growth conditions after agroinfiltration are critical for increasing heterologous protein expression levels in a transient expression system.

A nonreplicating subunit vaccine protects mice against lethal Ebola virus challenge.

Ebola hemorrhagic fever is an acute and often deadly disease caused by Ebola virus (EBOV). The possible intentional use of this virus against human populations has led to design of vaccines that could be incorporated into a national stockpile for biological threat reduction. We have evaluated the immunogenicity and efficacy of an EBOV vaccine candidate in which the viral surface glycoprotein is biomanufactured as a fusion to a monoclonal antibody that recognizes an epitope in glycoprotein, resulting in the production of Ebola immune complexes (EICs). Although antigen-antibody immune complexes are known to be efficiently processed and presented to immune effector cells, we found that codelivery of the EIC with Toll-like receptor agonists elicited a more robust antibody response in mice than did EIC alone. Among the compounds tested, polyinosinic:polycytidylic acid (PIC, a Toll-like receptor 3 agonist) was highly effective as an adjuvant agent. After vaccinating mice with EIC plus PIC, 80% of the animals were protected against a lethal challenge with live EBOV (30,000 LD(50) of mouse adapted virus). Surviving animals showed a mixed Th1/Th2 response to the antigen, suggesting this may be important for protection. Survival after vaccination with EIC plus PIC was statistically equivalent to that achieved with an alternative viral vector vaccine candidate reported in the literature. Because nonreplicating subunit vaccines offer the possibility of formulation for cost-effective, long-term storage in biothreat reduction repositories, EIC is an attractive option for public health defense measures.

Recombinant immune complexes as vaccines against infectious diseases.

New vaccine technologies are needed to combat many existing infections and prepare better for those that may emerge in the future. The conventional technologies that rely on protein-based vaccines are still severely restricted by the sparsity and poor accessibility of available adjuvants. One possible solution to this problem is to enhance antigen immunogenicity by a more natural means by complexing it with antibodies in the form of immune complexes (ICs). However, natural ICs are impractical as vaccines, and significant research efforts have been made to generate them in recombinant form, with plant bioengineering being at the forefront of these efforts. Here, we describe the challenges and progress made to date to make recombinant IC vaccines applicable to humans.

Enhanced binding and inhibition of SARS-CoV-2 by a plant-derived ACE2 protein containing a fused mu tailpiece.

Infectious diseases such as Coronavirus disease 2019 (COVID-19) and Middle East respiratory syndrome (MERS) present an increasingly persistent crisis in many parts of the world. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The angiotensin-converting enzyme 2 (ACE2) is a crucial cellular receptor for SARS-CoV-2 infection. Inhibition of the interaction between SARS-CoV-2 and ACE2 has been proposed as a target for the prevention and treatment of COVID-19. We produced four recombinant plant-derived ACE2 isoforms with or without the mu tailpiece (µ-tp) of immunoglobulin M (IgM) and the KDEL endoplasmic reticulum retention motif in a plant expression system. The plant-derived ACE2 isoforms bound whole SARS-CoV-2 virus and the isolated receptor binding domains of SARS-CoV-2 Alpha, Beta, Gamma, Delta, and Omicron variants. Fusion of µ-tp and KDEL to the ACE2 protein (ACE2 µK) had enhanced binding activity with SARS-CoV-2 in comparison with unmodified ACE2 protein derived from CHO cells. Furthermore, the plant-derived ACE2 µK protein exhibited no cytotoxic effects on Vero E6 cells and effectively inhibited SARS-CoV-2 infection. The efficient and rapid scalability of plant-derived ACE2 µK protein offers potential for the development of preventive and therapeutic agents in the early response to future viral outbreaks.

Engineering of sialylated mucin-type O-glycosylation in plants.

Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 ß1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatography-electrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.

A self-binding immune complex vaccine elicits strong neutralizing responses against herpes simplex virus in mice.

Introduction: It has been known for over half a century that mixing an antigen with its cognate antibody in an immune complex (IC) can enhance antigen immunogenicity. However, many ICs produce inconsistent immune responses, and the use of ICs in the development new vaccines has been limited despite the otherwise widespread success of antibody-based therapeutics. To address this problem, we designed a self-binding recombinant immune complex (RIC) vaccine which mimics the larger ICs generated during natural infection. Materials and methods: In this study, we created two novel vaccine candidates: 1) a traditional IC targeting herpes simplex virus 2 (HSV-2) by mixing glycoprotein D (gD) with a neutralizing antibody (gD-IC); and 2) an RIC consisting of gD fused to an immunoglobulin heavy chain and then tagged with its own binding site, allowing self-binding (gD-RIC). We characterized the complex size and immune receptor binding characteristics in vitro for each preparation. Then, the in vivo immunogenicity and virus neutralization of each vaccine were compared in mice. Results: gD-RIC formed larger complexes which enhanced C1q receptor binding 25-fold compared to gD-IC. After immunization of mice, gD-RIC elicited up to 1,000-fold higher gD-specific antibody titers compared to traditional IC, reaching endpoint titers of 1:500,000 after two doses without adjuvant. The RIC construct also elicited stronger virus-specific neutralization against HSV-2, as well as stronger cross-neutralization against HSV-1, although the proportion of neutralizing antibodies to total antibodies was somewhat reduced in the RIC group. Discussion: This work demonstrates that the RIC system overcomes many of the pitfalls of traditional IC, providing potent immune responses against HSV-2 gD. Based on these findings, further improvements to the RIC system are discussed. RIC have now been shown to be capable of inducing potent immune responses to a variety of viral antigens, underscoring their broad potential as a vaccine platform.

HIV DNA persists in hepatocytes in people with HIV-hepatitis B co-infection on antiretroviral therapy.

BACKGROUND: HIV can infect multiple cells in the liver including hepatocytes, Kupffer cells and infiltrating T cells, but whether HIV can persist in the liver in people with HIV (PWH) on suppressive antiretroviral therapy (ART) remains unknown. METHODS: In a prospective longitudinal cohort of PWH and hepatitis B virus (HBV) co-infection living in Bangkok, Thailand, we collected blood and liver biopsies from 18 participants prior to and following ART and quantified HIV and HBV persistence using quantitative (q)PCR and RNA/DNAscope. Antiretroviral (ARV) drug levels were quantified using mass spectroscopy. FINDINGS: In liver biopsies taken prior to ART, HIV DNA and HIV RNA were detected by qPCR in 53% (9/17) and 47% (8/17) of participants respectively. Following a median ART duration of 3.4 years, HIV DNA was detected in liver in 61% (11/18) of participants by either qPCR, DNAscope or both, but only at very low and non-quantifiable levels. Using immunohistochemistry, HIV DNA was observed in both hepatocytes and liver infiltrating CD4+ T cells on ART. HIV RNA was not detected in liver biopsies collected on ART, by either qPCR or RNAscope. All ARVs were clearly detected in liver tissue. INTERPRETATION: Persistence of HIV DNA in liver in PWH on ART represents an additional reservoir that warrants further investigation. FUNDING: National Health and Medical Research Council of Australia (Project Grant APP1101836, 1149990, and 1135851); This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024.

Utilizing Molecular Epidemiology and Citizen Science for the Surveillance of Lagoviruses in Australia.

Australia has multiple lagoviruses with differing pathogenicity. The circulation of these viruses was traditionally determined through opportunistic sampling events. In the lead up to the nationwide release of RHDVa-K5 (GI.1aP-GI.1a) in 2017, an existing citizen science program, RabbitScan, was augmented to allow members of the public to submit samples collected from dead leporids for lagovirus testing. This study describes the information obtained from the increased number of leporid samples received between 2015 and 2022 and focuses on the recent epidemiological interactions and evolutionary trajectory of circulating lagoviruses in Australia between October 2020 and December 2022. A total of 2771 samples were tested from January 2015 to December 2022, of which 1643 were lagovirus-positive. Notable changes in the distribution of lagovirus variants were observed, predominantly in Western Australia, where RHDV2-4c (GI.4cP-GI.2) was detected again in 2021 after initially being reported to be present in 2018. Interestingly, we found evidence that the deliberately released RHDVa-K5 was able to establish and circulate in wild rabbit populations in WA. Overall, the incorporation of citizen science approaches proved to be a cost-efficient method to increase the sampling area and enable an in-depth analysis of lagovirus distribution, genetic diversity, and interactions. The maintenance of such programs is essential to enable continued investigations of the critical parameters affecting the biocontrol of feral rabbit populations in Australia, as well as to enable the detection of any potential future incursions.

Safety and immunogenicity of bacterial and tobacco plant cell line derived recombinant native and mutant Escherichia coli heat-labile toxin in chickens.

The safety and immunogenicity of the mammalian mucosal adjuvants, Escherichia coli wild-type heat-labile holotoxin (LT) and E. coli mutant LT (LTA-K63/LTB), were examined in 1-day-old chicks and 10-day-old to 21-day-old broilers. Biologically active, E. coli recombinant wild-type LT and recombinant LTA-K63/LTB produced in a transgenic Nicotiana tabacum (NT-1) tobacco cell line (SLT102) were tested for safety and antigenicity following various routes of administration. Safety was assessed by clinical signs, body weight gain, gross organ pathology and wet organ weight, and histopathology. Antigenicity was assessed using LT-B-specific serum IgG enzyme-linked immunosorbent assay. Parenteral administration of E. coli recombinant wild-type LT did not have any discernible effect on bird health and was well tolerated at levels up to 400 µg per dose. Recombinant, SLT102-derived mutant LT derived from SLT102 cells retained in vitro ganglioside binding and was safe and antigenic following repeated mucosal administration to birds. The highest systemic LT-B-specific IgG titres were detected in birds that received three on-feed doses of SLT102-derived mutant LT. Among the various SLT102-derived mutant LT preparations tested, whole, wet cells or whole cell lysates were the most antigenic. These results demonstrate for the first time that E. coli-derived recombinant, wild-type LT holotoxin is well tolerated following multiple administrations to young birds at body weight doses previously reported to be enteropathogenic and toxic in mammalian species. Moreover, these data also demonstrate the feasibility of using recombinant wild-type and mutant LT produced in transgenic NT-1 tobacco cells as safe and potent vaccine adjuvants in poultry.