Shift In Tropism Research Articles

Tropism-shifted AAV-PHP.eB-mediated bFGF gene therapy promotes varied neurorestoration after ischemic stroke in mice.

AAV-PHP.eB is an artificial adeno-associated virus (AAV) that crosses the blood-brain barrier and targets neurons more efficiently than other AAVs when administered systematically. While AAV-PHP.eB has been used in various disease models, its cellular tropism in cerebrovascular diseases remains unclear. In the present study, we aimed to elucidate the tropism of AAV-PHP.eB for different cell types in the brain in a mouse model of ischemic stroke and evaluate its effectiveness in mediating basic fibroblast growth factor (bFGF) gene therapy. Mice were injected intravenously with AAV-PHP.eB either 14 days prior to (pre-stroke) or 1 day following (post-stroke) transient middle cerebral artery occlusion. Notably, we observed a shift in tropism from neurons to endothelial cells with post-stroke administration of AAV-PHP.eB-mNeonGreen (mNG). This endothelial cell tropism correlated strongly with expression of the endothelial membrane receptor lymphocyte antigen 6 family member A (Ly6A). Furthermore, AAV-PHP.eB-mediated overexpression of bFGF markedly improved neurobehavioral outcomes and promoted long-term neurogenesis and angiogenesis post-ischemic stroke. Our findings underscore the significance of considering potential tropism shifts when utilizing AAV-PHP.eB-mediated gene therapy in neurological diseases and suggest a promising new strategy for bFGF gene therapy in stroke treatment.

Distinct genetic clusters in HIV-1 CRF01_AE-infected patients induced variable degrees of CD4+ T-cell loss.

Retroviruses swiftly adapt, employing error-prone enzymes for genetic and phenotypic evolution, optimizing survival strategies, and enhancing virulence levels. HIV-1 CRF01_AE has persistently undergone adaptive selection, and cluster 1 and 2 infections display lower counts and fast loss of CD4+ T cells than other HIV-1 sub-types and CRF01_AE clusters. Its mechanisms are associated with increased CXCR4 tropism due to an envelope structure change favoring a tropism shift from CCR5 to CXCR4, thereby shaping viral phenotype features and impacting pathogenicity. This underscores the significance of consistently monitoring HIV-1 genetic evolution and phenotypic transfer to see whether selection bias across risk groups alters the delicate balance of transmissible versus toxic trade-offs, since virulent strains such as CRF01_AE clusters 1 and 2 could seriously compromise the efficacy of antiviral treatment. Only through such early warning and diagnostic services can precise antiviral treatments be administered to those infected with more virulent HIV-1 strains.

Every little bit helps: A single-residue switch in a vascular AAV enables blood-brain barrier penetration

Every little bit helps: A single-residue switch in a vascular AAV enables blood-brain barrier penetration

Differential Cell Line Susceptibility to the SARS-CoV-2 Omicron BA.1.1 Variant of Concern.

The unique mutations of the SARS-CoV-2 Omicron variant are associated with increased transmissibility, immune escape, increased binding affinity to ACE-2, and increased viral load. Omicron exhibited a shift in tropism infecting the upper respiratory tract compared to other variants of concern which have tropism for the lower respiratory tract. The tropism of omicron variants in cell lines of different hosts and tissue origins still remains unclear. Considering this, we assessed the susceptibility of different cell lines to the SARS-CoV-2 omicron BA.1.1 variant and permissiveness among different cell lines for omicron replication. Susceptibility and permissiveness of a total of eleven cell lines, including six animal cell lines and five human cell lines for omicron BA.1.1 infection, were evaluated by infecting individual cell lines with omicron BA.1.1 isolate at a 0.1 multiplicity of infection. Virus replication was assessed by observation of cytopathic effects followed by viral load determination by real-time PCR assay and virus infectivity determination by TCID50 assay. The characteristic cytopathic effect, increased viral load, and productive omicron replication was detected in Vero CCL-81, Vero E6, Vero/hSLAM, MA-104, and Calu-3 cells. Although LLC MK-2 cells showed an increased TCID50 titer at the second infection, the viral load did not show much difference in both infections. Caco-2 cells did not show evident CPE, but they supported omicron replication at a low level. A549, RD, MRC-5, and BHK-21 cells supported omicron BA.1.1 replication without the CPE. This is the first study on the comparison of susceptibility of different cell lines to Omicron variant BA.1.1, which might be useful for future studies on emerging SARS-CoV-2 variants.

Recent Zoonotic Spillover and Tropism Shift of a Canine Coronavirus Is Associated with Relaxed Selection and Putative Loss of Function in NTD Subdomain of Spike Protein.

A canine coronavirus (CCoV) has now been reported from two independent human samples from Malaysia (respiratory, collected in 2017–2018; CCoV-HuPn-2018) and Haiti (urine, collected in 2017); these two viruses were nearly genetically identical. In an effort to identify any novel adaptations associated with this apparent shift in tropism we carried out detailed evolutionary analyses of the spike gene of this virus in the context of related Alphacoronavirus 1 species. The spike 0-domain retains homology to CCoV2b (enteric infections) and Transmissible Gastroenteritis Virus (TGEV; enteric and respiratory). This domain is subject to relaxed selection pressure and an increased rate of molecular evolution. It contains unique amino acid substitutions, including within a region important for sialic acid binding and pathogenesis in TGEV. Overall, the spike gene is extensively recombinant, with a feline coronavirus type II strain serving a prominent role in the recombinant history of the virus. Molecular divergence time for a segment of the gene where temporal signal could be determined, was estimated at around 60 years ago. We hypothesize that the virus had an enteric origin, but that it may be losing that particular tropism, possibly because of mutations in the sialic acid binding region of the spike 0-domain.

A rapid phenotype change in the pathogen Perkinsus marinus was associated with a historically significant marine disease emergence in the eastern oyster

The protozoan parasite Perkinsus marinus, which causes dermo disease in Crassostrea virginica, is one of the most ecologically important and economically destructive marine pathogens. The rapid and persistent intensification of dermo in the USA in the 1980s has long been enigmatic. Attributed originally to the effects of multi-year drought, climatic factors fail to fully explain the geographic extent of dermo’s intensification or the persistence of its intensified activity. Here we show that emergence of a unique, hypervirulent P. marinus phenotype was associated with the increase in prevalence and intensity of this disease and associated mortality. Retrospective histopathology of 8355 archival oysters from 1960 to 2018 spanning Chesapeake Bay, South Carolina, and New Jersey revealed that a new parasite phenotype emerged between 1983 and 1990, concurrent with major historical dermo disease outbreaks. Phenotypic changes included a shortening of the parasite’s life cycle and a tropism shift from deeper connective tissues to digestive epithelia. The changes are likely adaptive with regard to the reduced oyster abundance and longevity faced by P. marinus after rapid establishment of exotic pathogen Haplosporidium nelsoni in 1959. Our findings, we hypothesize, illustrate a novel ecosystem response to a marine parasite invasion: an increase in virulence in a native parasite.

Sequential CCR5-Tropic HIV-1 Reactivation from Distinct Cellular Reservoirs following Perturbation of Elite Control.

BackgroundHIV Elite Controllers may reveal insights into virus persistence given they harbour small reservoir sizes, akin to HIV non-controllers treated early with combination antiretroviral therapy. Both groups of patients represent the most promising candidates for interventions aimed at sustained remission or ‘cure’. Analytic treatment interruption (ATI) in the latter group leads to stochastic rebound of virus, though it is unclear whether loss of elite control is also associated with similar rebound characteristics.MethodsWe studied three discrete periods of virus rebound during myeloma related immune disruption over 2.5 years in an elite controller who previously underwent autologous stem cell transplantation (ASCT) in the absence of any antiretroviral therapy. Single genome sequencing of the V1-V4 region of env in PBMC and plasma was performed and phylogenies reconstructed. Average pairwise distance (APD) was calculated and non-parametric methods used to assess compartmentalisation. Coreceptor usage was predicted based on genotypic algorithms.Results122 single genome sequences were obtained (median 26 sequences per rebound). The initial rebounding plasma env sequences following ASCT represented two distinct lineages, and clustered with proviral DNA sequences isolated prior to ASCT. One of the lineages was monophyletic, possibly indicating reactivation from clonally expanded cells. The second rebound occurred 470 days after spontaneous control of the first rebound and was phylogenetically distinct from the first, confirmed by compartmentalisation analysis, with a different cellular origin rather than ongoing replication. By contrast, third rebound viruses clustered with second rebound viruses, with evidence for ongoing evolution that was associated with lymphopenia and myeloma progression. Following ASCT a shift in tropism from CXCR4-tropic viruses to a CCR5-tropic population was observed to persist through to the third rebound.ConclusionsOur data highlight similarities in the viral reservoir between elite and non-controllers undergoing ATI following allogeneic transplantation. The lack of propagation of CXCR4 using viruses following transplantation warrants further study.

Multistage Delivery Technologies: Multifunctional, Interdisciplinary Approaches to Nanomedicine

Nanomedicine frequently employs single particles ranging from very simple1 to highly multifunctional.2 However, the diversity of barriers to effective transport can limit the global optimization of delivery for such systems. Multistage particles present an effective means to overcome such issues, allowing scientists to better segment the delivery mechanism into discrete steps by combining multiple formulations for sequential utility in vivo. A recent article in Nature Biotechnology highlights such a system,3 delivering nanoformulated doxorubicin (Dox) within larger disc-shaped, mesoporous silica particles (MSPs).

Evolution of coreceptor utilization to escape CCR5 antagonist therapy

The HIV-1 envelope interacts with coreceptors CCR5 and CXCR4 in a dynamic, multi-step process, its molecular details not clearly delineated. Use of CCR5 antagonists results in tropism shift and therapeutic failure. Here we describe a novel approach using full-length patient-derived gp160 quasispecies libraries cloned into HIV-1 molecular clones, their separation based on phenotypic tropism in vitro, and deep sequencing of the resultant variants for structure-function analyses. Analysis of functionally validated envelope sequences from patients who failed CCR5 antagonist therapy revealed determinants strongly associated with coreceptor specificity, especially at the gp120-gp41 and gp41-gp41 interaction surfaces that invite future research on the roles of subunit interaction and envelope trimer stability in coreceptor usage. This study identifies important structure-function relationships in HIV-1 envelope, and demonstrates proof of concept for a new integrated analysis method that facilitates laboratory discovery of resistant mutants to aid in development of other therapeutic agents.

Mouse polyomavirus tropism alters magnitude and functionality of virus-specific CD8 T cells (VIR6P.1173)

Abstract Mouse polyomavirus (MPyV) is a ubiquitous persistent natural mouse pathogen. Human JC polyomavirus variants associated with progressive multifocal leukoencephalopathy have discrete amino acid mutations in VP1, the major capsid protein, that affects tropism for glial cells. A glutamic acid (E) to glycine (G) difference at position 92 of VP1, shifts the profile to tumors induced by MPyV from epithelial to mesenchymal cell origin. Here, we asked if this shift in tropism affects the MPyV-specific CD8 T cell response, which controls MPyV infection/tumorigenesis. We found that the A2 (92E-VP1) and RA (92G-VP1) MPyV strains differ sharply in MPyV-specific CD8 T cell responses. A2 virus generates a robust, high magnitude virus-specific CD8 T cell response, whereas RA virus elicits a markedly smaller anti-MPyV CD8 T cell response. Infection by an A2 92G-VP1 mutant virus recapitulates the antiviral CD8 T cell response seen with RA virus. Notably, the antiviral CD8 T cell recall response during persistent infection by this A2 mutant significantly exceeded that of A2 virus. Using immunization with peptide-loaded dendritic cells in the setting of bystander inflammation by A2 or the mutant virus, we found that A2 virus stimulates a higher magnitude MPyV-specific CD8 T cell response; this difference is negated in DC-deficient Flt3-/- mice. Together, these data indicate that viral tropism for DCs is a major determinant of the quantity and quality of the anti-MPyV CD8 T cell response.

Arginine insertion and loss of N-linked glycosylation site in HIV-1 envelope V3 region confer CXCR4-tropism

The third variable region (V3) of HIV-1 envelope glycoprotein gp120 plays a key role in determination of viral coreceptor usage (tropism). However, which combinations of mutations in V3 confer a tropism shift is still unclear. A unique pattern of mutations in antiretroviral therapy-naive HIV-1 patient was observed associated with the HIV-1 tropism shift CCR5 to CXCR4. The insertion of arginine at position 11 and the loss of the N-linked glycosylation site were indispensable for acquiring pure CXCR4-tropism, which were confirmed by cell-cell fusion assay and phenotype analysis of recombinant HIV-1 variants. The same pattern of mutations in V3 and the associated tropism shift were identified in two of 53 other patients (3.8%) with CD4+ cell count <200/mm3. The combination of arginine insertion and loss of N-linked glycosylation site usually confers CXCR4-tropism. Awareness of this rule will help to confirm the tropism prediction from V3 sequences by conventional rules.

The magnitude of HIV-1 resistance to the CCR5 antagonist maraviroc may impart a differential alteration in HIV-1 tropism for macrophages and T-cell subsets

Human immunodeficiency virus type 1 (HIV-1) resistance to CCR5 antagonists, including maraviroc (MVC), results from alterations in the HIV-1 envelope glycoproteins (Env) enabling recognition of antagonist-bound CCR5. Here, we characterized tropism alterations for CD4+ T-cell subsets and macrophages by Envs from two subjects who developed MVC resistance in vivo, which displayed either relatively efficient or inefficient recognition of MVC-bound CCR5. We show that MVC-resistant Env with efficient recognition of drug-bound CCR5 displays a tropism shift for CD4+ T-cell subsets associated with increased infection of central memory T-cells and reduced infection of effector memory and transitional memory T-cells, and no change in macrophage infectivity. In contrast, MVC-resistant Env with inefficient recognition of drug-bound CCR5 displays no change in tropism for CD4+ T-cell subsets, but exhibits a significant reduction in macrophage infectivity. The pattern of HIV-1 tropism alterations for susceptible cells may therefore be variable in subjects with MVC resistance.

Evolution of proviral DNA HIV-1 tropism under selective pressure of maraviroc-based therapy

To evaluate the evolution of HIV-1 coreceptor tropism in proviral DNA of patients during maraviroc-based therapy. Fourteen heavily high active antiretroviral therapy (HAART)-treated patients with a CCR5 Trofile profile were monitored over a 24 month period from the start of maraviroc therapy. Whole-blood samples were obtained at different timepoints, and coreceptor tropism was determined for proviral DNA from the V3-loop region sequence using the Geno2Pheno algorithm [false positive rate (FPR): 20%]. At the start of maraviroc treatment, 13/14 patients were viraemic (median: 4.33 log copies/mL). Concordance in R5 tropism (R5/R5) was observed between circulating HIV-RNA (Trofile) and HIV-DNA provirus in 10/14 patients (median FPR = 54.0%), while 4 patients showed a CXCR4-tropic R5/X4 variant in their provirus (FPR: 5.8%, 5.7%, 16.6% and 1.1%, respectively). All R5/R5 patients showed a stable HIV-1 DNA coreceptor usage. Two out of four R5/X4 patients showed a tropism shift in their archived provirus and, after 6 months a prevalence of R5-tropic virus was detected in DNA. The other two R5/X4 patients harboured the 11/25 genotype, and maintained X4 tropism in provirus during the study. Virological response did not reveal differences in RNA decay and CD4+ cell recovery in patients with discordant tropism. A relatively good correlation between RNA and DNA tropism was observed at baseline. Proviral DNA tropism remained stable over 24 months of maraviroc-based therapy, indicating that determination of proviral DNA V3 sequence could be used in tropism prediction in clinical practice. The data also confirm the importance of the 11/25 rule in predicting viral tropism.

HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4 + T Cells

We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5, were cross resistant to other small-molecule CCR5 antagonists, and were isolated from the patient's pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4(+) T cells. The V3 loop contained residues essential for viral resistance to APL, while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However, these mutations were context dependent, being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N' terminus of CCR5 in the presence of APL. In addition, the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However, recognition of drug-bound CCR5 was less efficient, resulting in a tropism shift toward effector memory cells upon infection of primary CD4(+) T cells in the presence of APL, with relative sparing of the central memory CD4(+) T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses, then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the T(CM) subset of CD4(+) T cells and result in improved T cell homeostasis and immune function.

Molecular Evolution of Adeno-associated Virus for Enhanced Glial Gene Delivery

The natural tropism of most viral vectors, including adeno-associated viral (AAV) vectors, leads to predominant transduction of neurons and epithelia within the central nervous system (CNS) and retina. Despite the clinical relevance of glia for homeostasis in neural tissue, and as causal contributors in genetic disorders such as Alzheimer's and amyotrophic lateral sclerosis, efforts to develop more efficient gene delivery vectors for glia have met with limited success. Recently, viral vector engineering involving high-throughput random diversification and selection has enabled the rapid creation of AAV vectors with valuable new gene delivery properties. We have engineered novel AAV variants capable of efficient glia transduction by employing directed evolution with a panel of four distinct AAV libraries, including a new semi-random peptide replacement strategy. These variants transduced both human and rat astrocytes in vitro up to 15-fold higher than their parent serotypes, and injection into the rat striatum yielded astrocyte transduction levels up to 16% of the total transduced cell population, despite the human astrocyte selection platform. Furthermore, one variant exhibited a substantial shift in tropism toward Müller glia within the retina, further highlighting the general utility of these variants for efficient glia transduction in multiple species within the CNS and retina.

Co‐receptor switch during HAART is independent of virological success

The influence of antiretroviral therapy on co-receptor tropism remains controversial. To verify if co-receptor tropism shift was affected by HAART, the evolution of proviral DNA V3 genotype after 12 months of a new antiretroviral regimen was compared between responder and non-responder patients. Baseline blood samples were collected from 36 patients infected with HIV-1 subtype-B (18 naïve and 18 experienced) for virus isolation and env V3 genotyping from plasma HIV-1 RNA and PBMC DNA. DNA V3 genotyping was repeated after 12 months from initiating HAART. WebPSSM was used for categorizing V3 sequences into X4 or R5; for analysis purposes, dual/mixed viruses were considered as X4. From the 10 (28%) patients changing their proviral DNA V3 genotype during therapy, six shifted from R5-to-X4 and four from X4-to-R5. The lack of reaching virological suppression was not associated with an X4-to-R5 (P = 0.25) or R5-to-X4 (P = 0.14) shift; time-to-viral suppression and CD4 increase were similar in both groups. No association was found between tropism shift and patient baseline characteristics including age, sex, CDC stage, CD4 count, viral load, exposure and length of previous HAART, enfuvirtide use in the new regimen, number of reverse transcriptase and protease resistance-associated mutations. Conversely, CD4 nadir was correlated to emergence of X4 virus in proviral DNA (mean 27.2 +/- 30.6 in R5-to-X4 shifting patients vs. 161.6 +/- 150.6 in non-shifting patients, P = 0.02). The occurrence of a tropism shift in both directions was independent of HAART use, irrespective of its efficacy. The CD4 count nadir was the only baseline characteristic able to predict an R5-to-X4 viral shift.

Proof of Activity with AMD11070, an Orally Bioavailable Inhibitor of CXCR4‐Tropic HIV Type 1

The X4 Antagonist Concept Trial investigates the safety and antiviral activity of AMD11070, a potent inhibitor of X4-tropic human immunodeficiency virus (HIV) in vitro in HIV-infected patients harboring X4-tropic virus. Patients enrolled in the study had an X4 virus population 2000 relative luminescence units (rlu; by the Monogram Trofile Assay) and an HIV-1 RNA level 5000 copies/mL. Patients received AMD11070 monotherapy for 10 days. Coreceptor tropism, plasma HIV-1 RNA level, and CD4 cell count were measured at study entry, on day 5, and on day 10. Daily predose and serial samples on the last day of treatment were obtained for determination of plasma AMD11070 concentration. Ten patients were given AMD11070 monotherapy (200 mg to 8 patients and 100 mg to 2 patients) twice daily for 10 days. The median baseline CD4 cell count was 160 cells/mm(3), and the median HIV-1 RNA level was 91,447 copies/mL. Four of 9 evaluable patients achieved a reduction in X4 virus population of >or= rlu. The median change in X4 virus population at the end of treatment was -0.22 log(10) rlu (range, -1.90 to 0.23 log(10) rlu). Three of 4 patients who responded to therapy showed a tropism shift from dual- or mixed-tropic viruses to exclusively R5 virus by day 10. There were no drug-related serious adverse events, adverse events of greater than grade 2, or laboratory abnormalities. These results demonstrate the activity of AMD11070, the first oral CXCR4 antagonist, against X4-tropic HIV-1. The drug was well tolerated, with no serious safety concerns. AMD11070 is on clinical hold because of histologic changes to the liver observed in long-term animal studies; additional preclinical safety assessments are pending.