Characterization of HIV-1 virus-like particles and determination of Gag stoichiometry for different production platforms.

The importance of developing new vaccine technologies towards versatile platforms that can cope with global virus outbreaks has been evidenced with the most recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Virus-like particles (VLPs) are a highly immunogenic, safe, and robust approach that can be used to base several vaccine candidates on. Particularly, HIV-1 Gag VLPs is a flexible system comprising a Gag core surrounded by a lipid bilayer that can be modified to present diverse types of membrane proteins or antigens against several diseases, like influenza, dengue, West Nile virus, or human papillomavirus, where it has been proven successful. The size distribution and structural characteristics of produced VLPs vary depending on the cell line used to produce them. In this study, we established an analytical method of characterization for the Gag protein core and clarified the current variability of Gag stoichiometry in HIV-1 VLPs depending on the cell-based production platform, directly determining the number of Gag molecules per VLP in each case. Three Gag peptides have been validated to quantify the number of monomers using parallel reaction monitoring, an accurate and fast, mass-spectrometry-based method that can be used to assess the quality of the produced Gag VLPs regardless of the cell line used. An average of 3617 ± 17 monomers per VLP was obtained for HEK293, substantially varying between platforms, including mammalian and insect cells. This offers a key advantage in quantification and quality control methods to characterize VLP production at a large scale to accelerate new recombinant vaccine production technologies.

Brain is a potential sanctuary for subtype C HIV-1 irrespective of ART treatment outcome.

Subtype C HIV-1 is responsible for the largest proportion of people living with HIV-1 infection. However, there is limited information about the roles of the brain and its cell types as a potential sanctuary for this subtype and how the sanctuary may be affected by the administration of anti-retroviral therapy (ART). To address this issue, we collected postmortem brain tissues from ART treated HIV-1 infected Zambian individuals who experienced complete viral suppression and those who did not. Tissues from various brain compartments were collected from each individual as frozen and formalin-fixed paraffin embedded brain specimens, for detection and quantification of HIV-1 genomes and identification of the infected cell type. Genomic DNA and RNA were extracted from frozen brain tissues. The extracted DNA and RNA were then subjected to droplet digital PCR for HIV-1 quantification. RNA/DNAscope in situ hybridization (ISH) for HIV-1 was performed on formalin-fixed paraffin embedded brain tissues in conjugation with immunohistochemistry to identify the infected cell types. Droplet digital PCR revealed that HIV-1 gag DNA and RNA were detectable in half of the cases studied regardless of ART success or failure. The presence of HIV-1 lacked specific tissue compartmentalization since detection was random among various brain tissues. When combined with immunohistochemistry, RNA/DNAscope ISH demonstrated co-localization of HIV-1 DNA with CD68 expressing cells indicative of microglia or peripheral macrophage. Our study showed that brain is a potential sanctuary for subtype C HIV-1, as HIV-1 can be detected in the brain of infected individuals irrespective of ART treatment outcome and no compartmentalization of HIV-1 to specific brain compartments was evident.

Detection of HTLV-1 proviral DNA in BM mononuclear cells and cultured mesenchymal stromal cells isolated from patients with HTLV-1 infection.

The bone marrow (BM) biology during HTLV-1 infection is obscure. In this study, we investigated BM mononuclear cells and mesenchymal stromal cells (MSC) from HTLV-1 asymptomatic and symptomatic individuals. An infiltration of CD4+ T-cell lymphocytes in the BM of HTLV-1-infected individuals was observed when compared to healthy controls. The provirus detection in the BM CD4+ T cells confirmed the presence of integrated HTLV DNA. In regard to MSC, we observed that the number of fibroblast progenitor cells was lower in HTLV-1 infected individuals than in healthy controls. Isolated HTLV-1 infected BM-MSC demonstrated surface expression markers and in vitro differentiation potential similar to uninfected individuals. The presence of HTLV-1 proviral DNA in the BM-MSC of HTLV-1-infected patients was demonstrated but no p19 antigen was detected in supernatant from cultured MSC. We suppose that HTLV-1 infects human MSC probably by cell-to-cell contact from the infected CD4+ T-lymphocytes infiltrated into the bone marrow.

Colorimetric molecular diagnosis of the HIV gag gene using DNAzyme and a complementary DNA-extended primer.

We have developed a novel strategy for the colorimetric detection of PCR products by utilizing a target-specific primer modified at the 5'-end with an anti-DNAzyme sequence. A single-stranded DNAzyme sequence folds into a G-quadruplex structure with hemin and shows strong peroxidase activity. When the complementary strand binds to the DNAzyme sequence, it blocks the formation of the G-quadraduplex structure and loses its peroxidase activity. In the presence of the target gene, PCR amplification proceeds, and anti-DNAzyme sequence modified primers present in the reaction mixture form a double strand through primer extension. Therefore, it does not block the DNAzyme sequence. Further, a colorimetric signal is generated by the addition of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and H2O2 at the end of the reaction. We have successfully detected a single copy of the HIV type 1 gag gene in buffer and 10 copies in human serum. The strategy developed could be used to detect DNA and RNA in complex biological samples by simple primer designing that includes DNAzyme and a DNA extended primer.

A Targeted Mass Spectrometry Assay for Detection of HIV Gag Protein Following Induction of Latent Viral Reservoirs.

During early infection, HIV-1 establishes a reservoir of latently infected cells that persist during antiretroviral therapy. These reservoirs are considered the primary obstacle to eradicating HIV-1 from patients, and multiple strategies are being investigated to eliminate latently infected cells. Measuring the reservoir size using an affordable and scalable assay is critical as these approaches move into clinical trials: the current "gold-standard" viral outgrowth assay is costly, labor-intensive, and requires large numbers of cells. Here, we assessed whether selective reaction monitoring-mass spectrometry (SRM-MS) is sufficiently sensitive to detect latent HIV reservoirs following reactivation of virus. The Gag structural proteins were the most abundant viral proteins in purified virus and infected cells, and tractable peptides for monitoring Gag levels were identified. We then optimized a Gag immunoprecipitation procedure that permitted sampling of more than 107 CD4+ T cells, a requirement for detecting exceedingly rare latently infected cells. Gag peptides were detectable in both cell lysates and supernatants in CD4+ T cells infected in vitro at frequencies as low as ∼1 in 106 cells and in cells from HIV-infected patients on suppressive antiretroviral therapy with undetectable viral loads. To our knowledge, this represents the first detection of reactivated latent HIV reservoirs from patients without signal amplification. Together, these results indicate that SRM-MS is a viable method for measuring latent HIV-1 reservoirs in patient samples with distinct advantages over current assays.

Single-Cell Characterization of Viral Translation-Competent Reservoirs in HIV-Infected Individuals.

HIV cure efforts are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in patients receiving antiretroviral therapy. We combine fluorescent in situ RNA hybridization with detection of HIV protein and flow cytometry, enabling detection of 0.5-1 gag-pol mRNA(+)/Gag protein(+)-infected cells per million. In the peripheral blood of untreated persons, active HIV replication correlated with viremia and occurred in CD4 T cells expressing T follicular helper cell markers and inhibitory co-receptors. In virally suppressed subjects, the approach identified latently infected cells capable of producing HIV mRNA and protein after stimulation with PMA/ionomycin and latency-reversing agents (LRAs). While ingenol-induced reactivation mirrored the effector and central/transitional memory CD4 T cell contribution to the pool of integrated HIV DNA, bryostatin-induced reactivation occurred predominantly in cells expressing effector memory markers. This indicates that CD4 T cell differentiation status differentially affects LRA effectiveness.

Quantification of HIV-1 Gag Localization Within Virus Producer Cells.

Trafficking of newly synthesized Gag protein to the plasma membrane is one of the important steps during HIV-1 assembly. It requires participation of both viral and cellular determinants. Several techniques have been used to measure the amount of Gag that is associated with plasma membrane. Here we describe a microscopy-based method to estimate the distribution of Gag protein within the producer cell. This method can be used in conjunction with other biochemical techniques to quantify the distribution of Gag within a virus-producing cell and its accumulation at the plasma membrane. Since this method is microscopy based, it allows one to quantitate Gag across the cytoplasm, from the nuclear periphery to plasma membrane, at the single-cell level.

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells.

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay allows the rapid identification of HIV-1 nucleocapsid inhibitors.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Sampling a biomarker of the human immunodeficiency virus across a synthetic nanopore.

One primary goal in nanobiotechnology is designing new methodologies for molecular biomedical diagnosis at stages much earlier than currently possible and without use of expensive reagents and sophisticated equipment. In this work, we show the proof of principle for single-molecule detection of the nucleocapsid protein 7 (NCp7), a protein biomarker of the HIV-1 virus, using synthetic nanopores and the resistive-pulse technique. The biosensing mechanism relied upon specific interactions between NCp7 and aptamers of stem-loop 3 (SL3) in the packaging domain of the retroviral RNA genome. One critical step of this study was the choice of the optimal size of the nanopores for accurate, label-free determinations of the dissociation constant of the NCp7 protein-SL3 RNA aptamer complex. Therefore, we systematically investigated the NCp7 protein-SL3 RNA aptamer complex employing two categories of nanopores in a silicon nitride membrane: (i) small, whose internal diameter was smaller than 6 nm, and (ii) large, whose internal diameter was in the range of 7 to 15 nm. Here, we demonstrate that only the use of nanopores with an internal diameter that is smaller than or comparable with the largest cross-sectional size of the NCp7-SL3 aptamer complex enables accurate measurement of the dissociation constant between the two interacting partners. Notably, this determination can be accomplished without the need for prior nanopore functionalization. Moreover, using small solid-state nanopores, we demonstrate the ability to detect drug candidates that inhibit the binding interactions between NCp7 and SL3 RNA by using a test case of N-ethylmaleimide.

FLIM FRET technology for drug discovery: automated multiwell-plate high-content analysis, multiplexed readouts and application in situ.

A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated.

Immunization with HIV-1 gp41 subunit virosomes induces mucosal antibodies protecting nonhuman primates against vaginal SHIV challenges.

Human immunodeficiency virus (HIV)-1 is mainly transmitted mucosally during sexual intercourse. We therefore evaluated the protective efficacy of a vaccine active at mucosal sites. Macaca mulatta monkeys were immunized via both the intramuscular and intranasal routes with an HIV-1 vaccine made of gp41-subunit antigens grafted on virosomes, a safe delivery carrier approved in humans with self-adjuvant properties. Six months after 13 vaginal challenges with simian-HIV (SHIV)-SF162P3, four out of five vaccinated animals remained virus-negative, and the fifth was only transiently infected. None of the five animals seroconverted to p27gag-SIV. In contrast, all 6 placebo-vaccinated animals became infected and seroconverted. All protected animals showed gp41-specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or antibody-dependent cellular-cytotoxicity activities. In contrast, plasma IgGs totally lacked virus-neutralizing activity. The protection observed challenges the paradigm whereby circulating antiviral antibodies are required for protection against HIV-1 infection and may serve in designing a human vaccine against HIV-1-AIDS.

Myristate exposure in the human immunodeficiency virus type 1 matrix protein is modulated by pH.

Human immunodeficiency virus type 1 (HIV-1) encodes a polypeptide called Gag that is capable of forming virus-like particles (VLPs) in vitro in the absence of other cellular or viral constituents. During the late phase of HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM) for assembly. A combination of in vivo, in vitro, and structural studies have shown that Gag targeting and assembly on the PM are mediated by specific interactions between the myristoylated matrix [myr(+)MA] domain of Gag and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Exposure of the MA myristyl (myr) group is triggered by PI(4,5)P2 binding and is enhanced by factors that promote protein self-association. In the studies reported here, we demonstrate that myr exposure in MA is modulated by pH. Our data show that deprotonation of the His89 imidazole ring in myr(+)MA destabilizes the salt bridge formed between His89(Hδ2) and Glu12(COO-), leading to tight sequestration of the myr group and a shift in the equilibrium from trimer to monomer. Furthermore, we show that oligomerization of a Gag-like construct containing matrix-capsid is also pH-dependent. Disruption of the His−Glu salt bridge by single-amino acid substitutions greatly altered the myr-sequestered−myr-exposed equilibrium. In vivo intracellular localization data revealed that the H89G mutation retargets Gag to intracellular compartments and severely inhibits virus production. Our findings reveal that the MA domain acts as a "pH sensor" in vitro, suggesting that the effect of pH on HIV-1 Gag targeting and binding to the PM warrants investigation.

Visualizing cell-to-cell transfer of HIV using fluorescent clones of HIV and live confocal microscopy.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

QUASI analysis of host immune responses to Gag polyproteins of human immunodeficiency virus type 1 by a systematic bioinformatics approach.

There is a consensus that Gag-specific cytotoxic T lymphocyte (CTL) response plays a key role in the immune control of human immunodeficiency virus type 1 (HIV-1) infection. In this study, we analyzed all currently available gag sequences in the Los Alamos HIV sequence database and identified positive selection (PS) sites likely restricted by the host immune responses. We found that between 23.4% and 47.4% of PS sites were shared by clades A, B, and C of Gag, indicating similar positive selection pressure on Gag in different subtypes of HIV-1. Furthermore, a significant correlation was observed between the combined CTL and antibody responses and PS sites. The Gag regions of free from PS contained 9 CTL epitopes restricted by 11 HLA class I alleles associated with disease progression to acquired immune deficiency syndrome (AIDS). These analyses provide information important for the identification of cross-clade epitopes and development of a global HIV-1 vaccine.

Young pregnant women living with HIV/AIDS in Criciuma, Southern Brazil, are infected almost exclusively with HIV type 1 clade C.

Southern Brazil has the highest prevalence rate of AIDS in the country and is the only region in the Americas where HIV-1 C prevails. Metropolitan areas and harbor cities have been evaluated, but limited information is available for small towns and specific populations. We studied women attending the obstetric outpatient clinic of Criciuma, State of Santa Catarina in 2007 to evaluate the molecular epidemiology of HIV-1 among pregnant women living with HIV/AIDS. Forty-two cases had partial pol gene sequenced and additional partial gag and/or env genes from nine women. HIV subtyping was evaluated by phylogenetic methods and antiretroviral (ARV) drug resistance mutations (DRMs) at the Stanford Database. DRMs to one or more ARV class was observed in 20/42, 48% of cases, with 15/41, 37% with viral load <500 copies/ml. Subtype C at pol was identified in 33/42, 78.6% (95% CI: 64-89%), C mosaics (CB, CF) in 2, 4.8% (95% CI: 0.8-19%), F in 4, 9.5% (95% CI: 3-21%), and B in 3, 7.1% (95% CI: 1.8-18%). Discordance in concatenated gag/pol/env or intraregion mosaic was observed in 1/9, 11% of HIV-1 C genomes. The proportion of HIV-1 C in this study is the highest rate described in the Americas. Molecular surveillance in specific populations is instrumental for a better understanding of the Brazilian HIV epidemic.

Reverse micelle encapsulation of membrane-anchored proteins for solution NMR studies.

Perhaps 5%-10% of proteins bind to membranes via a covalently attached lipid. Posttranslational attachment of fatty acids such as myristate occurs on a variety of viral and cellular proteins. High-resolution information about the nature of lipidated proteins is remarkably sparse, often because of solubility problems caused by the exposed fatty acids. Reverse micelle encapsulation is used here to study two myristoylated proteins in their lipid-extruded states: myristoylated recoverin, which is a switch in the Ca(2+) signaling pathway in vision, and the myristoylated HIV-1 matrix protein, which is postulated to be targeted to the plasma membrane through its binding to phosphatidylinositol-4,5-bisphosphate. Both proteins have been successfully encapsulated in the lipid-extruded state and high-resolution NMR spectra obtained. Both proteins bind their activating ligands in the reverse micelle. This approach seems broadly applicable to membrane proteins with exposed fatty acid chains that have eluded structural characterization by conventional approaches.

Molecular characterization of the human immunodeficiency virus type 1 among children in Lima, Peru.

In Peru, there is a lack of information on molecular analysis in pediatric human immunodeficiency virus (HIV) infection. At present, the mother-to-child transmission rate is estimated at approximately 2-4%. The objective of this study was to assess the molecular epidemiology of HIV-1 in infected children. Children with suspected or confirmed pulmonary tuberculosis were evaluated at two public hospitals between 2002 and 2007. Whole blood samples were obtained from 90 HIV-positive children, who were confirmed to be positive by enzyme-linked immunosorbent assay and Western blot. The specimens were subjected to envelope heteroduplex mobility assay (env HMA) followed by gag and pol gene region sequence analysis. Subtype B was found in 88 (98%) of 90 children and 2 (2%) children were subtype BF recombinants. This is the first report of recombinant HIV strains in HIV-infected children in Peru. Understanding the origin, diversity, and spread of HIV strains worldwide will be necessary for the development of an effective vaccine that targets pediatric populations throughout the world.

Comparison of Bayesian and maximum-likelihood phylogenetic approaches in two legal cases involving accusations of transmission of HIV.

We investigated two cases of alleged criminal transmission of HIV-1 using Bayesian and maximum-likelihood phylogenetic approaches to determine whether the inference method used influenced the outcome in these cases. In the first case, Bayesian methods were used to reexamine gag and env sequences from an earlier investigation in which the HIV-1 strains infecting one of several contacts could not be linked phylogenetically to that of the accused despite strongly suggestive epidemiological evidence. In the second case, maximum-likelihood and Bayesian inference methods were used to investigate the relatedness of gag and env sequences from HIV-1 strains infecting a man accused of intentionally transmitting the virus to several contacts. Bayesian analysis of HIV-1 strains from the first case confirmed earlier results obtained by maximum-likelihood analysis. A monophyletic cluster linking viruses from the accused and three of his direct and indirect contacts was supported, but a linkage between these viruses and a fourth epidemiologically linked contact could not be demonstrated. In the second case, a strong virological link between the accused and two of his contacts, and the absence of links with four other contacts, was confirmed by both maximum-likelihood and Bayesian inference methods. It is important that phylogenetic programs applied in a legal setting are conservative in their outcome. Although Bayesian methods offer computational tractability for large data sets and complex evolutionary models, this study demonstrates they do not assist when clear linkages between viruses are demonstrated using maximum-likelihood methods.