In-house ELISA protocols for capsid p24 detection of diverse HIV isolates.

BACKGROUND: The capsid p24 (CA-p24) antigen is a component of the viral capsid of human immunodeficiency virus (HIV) that has been commonly used for clinical diagnosis and monitoring of HIV infections in Enzyme-linked Immunosorbent Assays (ELISAs). Commercial CA-p24 ELISAs are widely used in research settings, but these kits are costly and have limited breadth for detecting diverse HIV isolates. METHODS: Commercial CA-p24 antibodies were used as capture and detection antibodies. Specific CA-p24 ELISAs were established with these antibodies and tested for the detection of HIV-1 isolates with the aim of developing in-house protocols to recognize HIV-1 infections in vitro for research purposes. RESULTS: Here we present four protocols for in-house ELISAs to detect HIV CA-p24 using commercial antibodies. The assays were able to detect the CA-p24 antigen of different HIV-1 isolates tested. Comparison between the protocols showed that these in-house ELISAs exhibit high specificity, sensitivity, and reproducibility for CA-p24 quantitation but their reactivity varied per HIV-1 isolate and subtype. CONCLUSIONS: These optimized ELISA protocols represent valuable tools to investigate HIV-1 infections in research facilities at a lower price than commercial CA-p24 kits.

Engineered miniature H1 promoters with dedicated RNA polymerase II or III activity.

RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both).

Evaluation of the effect of RNA secondary structure on Cas13d-mediated target RNA cleavage.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13d system was adapted as a powerful tool for targeting viral RNA sequences, making it a promising approach for antiviral strategies. Understanding the influence of template RNA structure on Cas13d binding and cleavage efficiency is crucial for optimizing its therapeutic potential. In this study, we investigated the effect of local RNA secondary structure on Cas13d activity. To do so, we varied the stability of a hairpin structure containing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target sequence, allowing us to determine the threshold RNA stability at which Cas13d activity is affected. Our results demonstrate that Cas13d possesses the ability to effectively bind and cleave highly stable RNA structures. Notably, we only observed a decrease in Cas13d activity in the case of exceptionally stable RNA hairpins with completely base-paired stems, which are rarely encountered in natural RNA molecules. A comparison of Cas13d and RNA interference (RNAi)-mediated cleavage of the same RNA targets demonstrated that RNAi is more sensitive for local target RNA structures than Cas13d. These results underscore the suitability of the CRISPR-Cas13d system for targeting viruses with highly structured RNA genomes.

Efficient CRISPR-Cas13d-Based Antiviral Strategy to Combat SARS-CoV-2.

The current SARS-CoV-2 pandemic forms a major global health burden. Although protective vaccines are available, concerns remain as new virus variants continue to appear. CRISPR-based gene-editing approaches offer an attractive therapeutic strategy as the CRISPR-RNA (crRNA) can be adjusted rapidly to accommodate a new viral genome sequence. This study aimed at using the RNA-targeting CRISPR-Cas13d system to attack highly conserved sequences in the viral RNA genome, thereby preparing for future zoonotic outbreaks of other coronaviruses. We designed 29 crRNAs targeting highly conserved sequences along the complete SARS-CoV-2 genome. Several crRNAs demonstrated efficient silencing of a reporter with the matching viral target sequence and efficient inhibition of a SARS-CoV-2 replicon. The crRNAs that suppress SARS-CoV-2 were also able to suppress SARS-CoV, thus demonstrating the breadth of this antiviral strategy. Strikingly, we observed that only crRNAs directed against the plus-genomic RNA demonstrated antiviral activity in the replicon assay, in contrast to those that bind the minus-genomic RNA, the replication intermediate. These results point to a major difference in the vulnerability and biology of the +RNA versus -RNA strands of the SARS-CoV-2 genome and provide important insights for the design of RNA-targeting antivirals.

Tat has a dual role in simian immunodeficiency virus transcription.

Tat has a pivotal role in human and simian immunodeficiency virus (HIV and SIV) replication because it stimulates transcription by binding to the trans-activator response (TAR) element. In addition, several other Tat functions have been proposed. Most studies have focused on HIV-1 Tat and much less is known about SIV Tat. An SIVmac239 variant was constructed previously in which the Tat-TAR transcription mechanism is functionally replaced by the doxycycline-inducible Tet-On gene expression mechanism (SIV-rtTA). In this study, SIV-rtTA variants were used to analyse the functions of SIV Tat. It was shown that Tat-minus SIV-rtTA variants replicated efficiently in PM1 T-cells, ruling out an additional essential Tat function. Nevertheless, replication was suboptimal in other cells, and evolutionary pressure to repair Tat expression was documented. It was demonstrated that SIV-rtTA required Tat for optimal gene expression, despite the absence of the Tat-TAR axis. This Tat effect was lost upon replacement of the long terminal repeat promoter region by a non-related promoter. These results indicate that Tat can activate SIV transcription via TAR RNA and U3 DNA elements but has no other essential function in replication in cultured cells. The experiments were limited to cell lines and PBMCs, and did not exclude an accessory Tat function under specific conditions or in vivo.

Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies.

RNAi-based gene therapy is a powerful approach to treat viral infections because of its high efficiency and sequence specificity. The HIV-1-based lentiviral vector system is suitable for the delivery of RNAi inducers to HIV-1 susceptible cells due to its ability to transduce nondividing cells, including hematopoietic stem cells, and its ability for stable transgene delivery into the host cell genome. However, the presence of anti-HIV short hairpin RNA (shRNA) and microRNA (miRNA) cassettes can negatively affect the lentiviral vector titers. We show that shRNAs, which target the vector genomic RNA, strongly reduced lentiviral vector titers but inhibition of the RNAi pathway via saturation could rescue vector production. The presence of miRNAs in the vector RNA genome (sense orientation) results in a minor titer reduction due to Drosha processing. A major cause for titer reduction of miRNA vectors is due to incompatibility of the cytomegalovirus promoter with the lentiviral vector system. Replacement of this promoter with an inducible promoter resulted in an almost complete restoration of the vector titer. We also showed that antisense poly(A) signal sequences can have a dramatic effect on the vector titer. These results show that not all sequences are compatible with the lentiviral vector system and that care should be taken in the design of lentiviral vectors encoding RNAi inducers.

Diverse HIV-1 escape pathways from broadly neutralizing antibody PGDM1400 in humanized mice.

Recent studies have shown the potential of broadly neutralizing antibodies (bnAbs) for HIV-1 treatment. One of the candidate antibodies moving into clinical trials is the bnAb PGDM1400. Here, we studied the therapeutic potency and escape pathways of bnAb PGDM1400 during monovalent therapy in human immune system (HIS) mice using the BG505, REJO, MJ4 and AMC008 virus isolates. PGDM1400 administered during chronic infection caused a modest decrease in viral load in the first week of administration in 7 out of 10 animals, which correlated with the in vitro neutralization sensitivity of the viruses to PGDM1400. As expected for monotherapy, viral loads rebounded after about a week and different viral escape pathways were observed, involving the deletion of glycans in the envelope glycoprotein at positions 130 or 160. (Pre)clinical trials should reveal whether PGDM1400 is a useful component of an antibody combination treatment or as part of a tri-specific antibody.

Dendritic cells potently purge latent HIV-1 beyond TCR-stimulation, activating the PI3K-Akt-mTOR pathway.

BACKGROUND: The latent HIV-1 reservoir in treated patients primarily consists of resting memory CD4+ T cells. Stimulating the T-cell receptor (TCR), which facilitates transition of resting into effector T cells, is the most effective strategy to purge these latently infected cells. Here we supply evidence that TCR-stimulated effector T cells still frequently harbor latent HIV-1. METHODS: Primary HIV-1 infected cells were used in a latency assay with or without dendritic cells (DCs) and reversion of HIV-1 latency was determined, in the presence or absence of specific pathway inhibitors. FINDINGS: Renewed TCR-stimulation or subsequent activation with latency reversing agents (LRAs) did not overcome latency. However, interaction of infected effector cells with DCs triggered further activation of latent HIV-1. When compared to TCR-stimulation only, CD4+ T cells from aviremic patients receiving TCR + DC-stimulation reversed latency more frequently. Such a "one-two punch" strategy seems ideal for purging the reservoir. We determined that DC contact activates the PI3K-Akt-mTOR pathway in CD4+ T cells. INTERPRETATION: This insight could facilitate the development of a novel class of potent LRAs that purge latent HIV beyond levels reached by T-cell activation.

The evolution of subtype B HIV-1 tat in the Netherlands during 1985-2012.

For the production of viral genomic RNA, HIV-1 is dependent on an early viral protein, Tat, which is required for high-level transcription. The quantity of viral RNA detectable in blood of HIV-1 infected individuals varies dramatically, and a factor involved could be the efficiency of Tat protein variants to stimulate RNA transcription. HIV-1 virulence, measured by set-point viral load, has been observed to increase over time in the Netherlands and elsewhere. Investigation of tat gene evolution in clinical isolates could discover a role of Tat in this changing virulence. A dataset of 291 Dutch HIV-1 subtype B tat genes, derived from full-length HIV-1 genome sequences from samples obtained between 1985-2012, was used to analyse the evolution of Tat. Twenty-two patient-derived tat genes, and the control TatHXB2 were analysed for their capacity to stimulate expression of an LTR-luciferase reporter gene construct in diverse cell lines, as well as for their ability to complement a tat-defective HIV-1LAI clone. Analysis of 291 historical tat sequences from the Netherlands showed ample amino acid (aa) variation between isolates, although no specific mutations were selected for over time. Of note, however, the encoded protein varied its length over the years through the loss or gain of stop codons in the second exon. In transmission clusters, a selection against the shorter Tat86 ORF was apparent in favour of the more common Tat101 version, likely due to negative selection against Tat86 itself, although random drift, transmission bottlenecks, or linkage to other variants could also explain the observation. There was no correlation between Tat length and set-point viral load; however, the number of non-intermediate variants in our study was small. In addition, variation in the length of Tat did not significantly change its capacity to stimulate transcription. From 1985 till 2012, variation in the length of the HIV-1 subtype B tat gene is increasingly found in the Dutch epidemic. However, as Tat proteins did not differ significantly in their capacity to stimulate transcription elongation in vitro, the increased HIV-1 virulence seen in recent years could not be linked to an evolving viral Tat protein.

Short Communication: Protective Efficacy of Broadly Neutralizing Antibody PGDM1400 Against HIV-1 Challenge in Humanized Mice.

Broadly neutralizing antibodies (bNAbs) such as PGDM1400 show promise as prophylactic and therapeutic agents against HIV-1. Human immune system mice were passively immunized with different doses of PGDM1400 and challenged 24 h later with a high dose of HIV-1JRCSF. We found that PGDM1400 provided protection against HIV-1 challenge in a concentration dependent manner and that the protective concentration in blood was ∼75-fold higher than the in vitro 50% inhibitory concentration. The results demonstrate that PGDM1400 might be a promising component of strategies to prevent HIV-1 infection and provide support for the pursuit of vaccines that induce PGDM1400-like bNAbs.

HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome.

HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. However, HIV-1 escape variants emerged after prolonged culturing. These RNAi-resistant viruses contain nucleotide substitutions or deletions in or near the targeted sequence. We observed an inverse correlation between the level of resistance and the stability of the siRNA/target-RNA duplex. However, two escape variants showed a higher level of resistance than expected based on the duplex stability. We demonstrate that these mutations induce alternative folding of the RNA such that the target sequence is occluded from binding to the siRNA, resulting in reduced RNAi efficiency. HIV-1 can thus escape from RNAi-mediated inhibition not only through nucleotide substitutions or deletions in the siRNA target sequence, but also through mutations that alter the local RNA secondary structure. The results highlight the enormous genetic flexibility of HIV-1 and provide detailed molecular insight into the sequence specificity of RNAi and the impact of target RNA secondary structure.

From clinical sample to complete genome: Comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing.

The BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project aims to analyse nearly-complete viral genomes from >3000 HIV-1 infected Europeans using high-throughput deep sequencing techniques to investigate the virus genetic contribution to virulence. Following the development of a computational pipeline, including a new de novo assembler for RNA virus genomes, to generate larger contiguous sequences (contigs) from the abundance of short sequence reads that characterise the data, another area that determines genome sequencing success is the quality and quantity of the input RNA. A pilot experiment with 125 patient plasma samples was performed to investigate the optimal method for isolation of HIV-1 viral RNA for long amplicon genome sequencing. Manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) was superior over robotically extracted RNA using either the QIAcube robotic system, the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular), or the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics). We scored amplification of a set of four HIV-1 amplicons of ∼1.9, 3.6, 3.0 and 3.5kb, and subsequent recovery of near-complete viral genomes. Subsequently, 616 BEEHIVE patient samples were analysed to determine factors that influence successful amplification of the genome in four overlapping amplicons using the QIAamp Viral RNA Kit for viral RNA isolation. Both low plasma viral load and high sample age (stored before 1999) negatively influenced the amplification of viral amplicons >3kb. A plasma viral load of >100,000 copies/ml resulted in successful amplification of all four amplicons for 86% of the samples, this value dropped to only 46% for samples with viral loads of <20,000 copies/ml.

Modification of the Tet-On regulatory system prevents the conditional-live HIV-1 variant from losing doxycycline-control.

BACKGROUND: We have previously constructed a doxycycline (dox)-dependent HIV-1 variant by incorporating the Tet-On gene regulatory system into the viral genome. Replication of this HIV-rtTA virus is driven by the dox-inducible transactivator protein rtTA, and can be switched on and off at will. We proposed this conditional-live virus as a novel vaccine approach against HIV-1. Upon vaccination, replication of HIV-rtTA can be temporarily activated by transient dox administration and controlled to the extent needed for optimal induction of the immune system. However, subsequent dox-withdrawal may impose a selection for virus variants with reduced dox-dependence. RESULTS: We simulated this on/off switching of virus replication in multiple, independent cultures and could indeed select for HIV-rtTA variants that replicated without dox. Nearly all evolved variants had acquired a typical amino acid substitution at position 56 in the rtTA protein. We developed a novel rtTA variant that blocks this undesired evolutionary route and thus prevents HIV-rtTA from losing dox-control. CONCLUSION: The loss of dox-control observed upon evolution of the dox-dependent HIV-1 variant was effectively blocked by modification of the Tet-On regulatory system.

Selecting the optimal Tet-On system for doxycycline-inducible gene expression in transiently transfected and stably transduced mammalian cells.

The doxycycline (dox)-inducible Tet-On system is widely used to control gene expression in mammalian cells. This system is based on the bacterial Tet operon, which has been modified and improved for its function in eukaryotic cells. To identify the optimal system for different applications, we compared Tet-On variants in frequently used cell types that were either transiently transfected with the relevant plasmids or stably transduced with an "all-in-one" lentiviral vector. The V10 variant performed optimally in the transiently transfected cells and demonstrated no background activity without dox, high dox-induced activity and the highest fold-induction. Because of its very high dox-sensitivity, the V16 system may be preferred if only low intracellular dox concentrations can be reached. V16 performed optimally in the transduced cells and demonstrated the highest activity and dox-sensitivity without background activity. Moreover, V16 demonstrated more robust induction of gene expression after a latency period without dox. This study provides important findings for choosing the optimal Tet-On system for diverse cell culture settings. V10 is the best system for most applications in which the DNA is episomally present in cells, whereas V16 may be optimal when the Tet-On components are stably integrated in the cellular genome.

Alterations of intercellular communication in neonatal cardiac myocytes from connexin43 null mice.

OBJECTIVE: To compare gap junction expression and intercellular coupling in wildtype neonatal cardiac myocytes to those from mice lacking the most abundant cardiac gap junction protein (connexin43, Cx43). METHODS: Northern and Western blots compared connexin mRNA and protein levels, immunocytochemistry evaluated connexin distribution in neonatal Cx43 null(-/-), heterozygous(+/-) and wildtype(+/+) mouse hearts. Ca(2+) imaging, dye coupling and electrophysiological methods evaluated intercellular communication. RESULTS: Similar levels of Cx40 and Cx45 were detected in all genotypes, although in adult cardiac tissue from wildtype mice, Cx43 expression was higher than in heterozygotes. After culturing dissociated cells for 3-4 days, cardiocytes beat spontaneously; in Cx43(+/+) and (+/-) cultures, the beating was generally quite synchronous. In Cx43(-/-) mice, interbeat intervals were on average twice as long and more variable than in Cx43(+/+) or Cx43(+/-) cultures. Junctional conductance was lower by about 60% in Cx43(-/-) as compared to Cx43(+/-) and (+/+) littermates; Lucifer Yellow dye coupling was virtually absent in Cx43(-/-) cardiomyocytes but was comparably strong in wildtype and heterozygous siblings. Macroscopic junctional conductance measurements on Cx43(-/-) cardiocytes showed slightly stronger voltage sensitivity in these cells than in Cx43(+/+) cardiocytes. Unitary junctional conductance measurements revealed distinct populations of channels contributing to macroscopic conductance for Cx43(+/+) and Cx43(-/-) genotypes. CONCLUSIONS: In Cx43-deficient cardiac myocytes, the expression of other connexins only partially compensates for the functional loss, with dye coupling and spontaneous beating being strongly impaired.

Conditional live virus as a novel approach towards a safe live attenuated HIV vaccine.

To control the worldwide spread of HIV, a safe and effective prophylactic vaccine is urgently needed. Studies with the simian immunodeficiency virus demonstrated that a live attenuated virus can be effective as a vaccine, but serious concerns about the safety of such a vaccine virus have arisen. We propose a conditional live virus, of which the replication can be switched on and off at will, as a novel approach for an HIV vaccine.

An AUG codon upstream of rev and env open reading frames ensures optimal translation of the simian immunodeficiency virus Env protein.

The mRNAs encoding the Rev and Env proteins of simian immunodeficiency virus (SIV) are unique because upstream translation start codons are present that may modulate the expression of these viral proteins. We previously reported the regulatory effect of a small upstream open reading frame (ORF) on Rev and Env translation. Here we study this mechanism in further detail by modulating the strength of the translation signals upstream of the open reading frames in subgenomic reporters. Furthermore, the effects of these mutations on SIV gene expression and viral replication are analyzed. An intricate regulatory mechanism is disclosed that allows the virus to express a balanced amount of these two proteins.

Construction of a doxycycline-dependent simian immunodeficiency virus reveals a nontranscriptional function of tat in viral replication.

In the quest for an effective vaccine against human immunodeficiency virus (HIV), live attenuated virus vaccines have proven to be very effective in the experimental model system of simian immunodeficiency virus (SIV) in macaques. However, live attenuated HIV vaccines are considered unsafe for use in humans because the attenuated virus may accumulate genetic changes during persistence and evolve to a pathogenic variant. As an alternative approach, we earlier presented a conditionally live HIV-1 variant that replicates exclusively in the presence of doxycycline (DOX). Replication of this vaccine strain can be limited to the time that is needed to provide full protection through transient DOX administration. Since the effectiveness and safety of such a conditionally live AIDS vaccine should be tested in macaques, we constructed a similar DOX-dependent SIVmac239 variant in which the Tat-TAR (trans-acting responsive) transcription control mechanism was functionally replaced by the DOX-inducible Tet-On regulatory mechanism. Moreover, this virus can be used as a tool in SIV biology studies and vaccine research because both the level and duration of replication can be controlled by DOX administration. Unexpectedly, the new SIV variant required a wild-type Tat protein for replication, although gene expression was fully controlled by the incorporated Tet-On system. This result suggests that Tat has a second function in SIV replication in addition to its role in the activation of transcription.

The genetic stability of a conditional live HIV-1 variant can be improved by mutations in the Tet-On regulatory system that restrain evolution.

Live attenuated human immunodeficiency virus type 1 (HIV-1) vaccines are considered unsafe because more quickly replicating pathogenic virus variants may evolve after vaccination. As an alternative vaccine approach, we have previously presented a doxycycline (dox)-dependent HIV-1 variant that was constructed by incorporating the tetracycline-inducible gene expression system (Tet-On system) into the viral genome. Replication of this HIV-rtTA variant is driven by the dox-inducible transcriptional activator rtTA and can be switched on and off at will. A large scale evolution study was performed to test the genetic stability of this conditional live vaccine candidate. In several long term cultures, we selected for HIV-rtTA variants that no longer required dox for replication. These evolved variants acquired a typical amino acid substitution either at position 19 or 37 in the rtTA protein. Both mutations caused rtTA activity and viral replication in the absence of dox. We designed a novel rtTA variant with a higher genetic barrier toward these undesired evolutionary routes. The corresponding HIV-rtTA variant did not lose dox control in long term cultures, demonstrating its improved genetic stability.

A conditionally replicating HIV-based vector that stably expresses an antiviral shRNA against HIV-1 replication.

Human pathogenic viruses can be targeted by therapeutic strategies based on RNA interference. Whereas the administration of synthetic short interfering RNAs (siRNAs) may transiently inhibit viral replication, long-term inhibition may be achieved through stable intracellular expression of siRNAs or short hairpin RNAs (shRNAs). Both approaches face serious problems with delivery to the right cells in an infected individual. We explored the potential of a replicating HIV-based vector to deliver an antiviral shRNA cassette into HIV-1-susceptible target cells to block chronic HIV-1 infection. The vector is based on a doxycycline (dox)-dependent HIV-1 variant that we previously proposed as a conditional-live HIV-1 vaccine. With dox, this virus spreads efficiently to all HIV-susceptible cells. Subsequent dox withdrawal generates cells with a transcriptionally silent integrated provirus, but with an active shRNA expression cassette. Because the shRNA targets viral sequences that are removed from the vector construct, there is no self-targeting, yet there is specific shutdown of HIV-1 replication.