Publications by authors named "Eric J Arts"

93 Publications

Dolutegravir response in antiretroviral therapy naïve and experienced patients with M184V/I: Impact in low-and middle-income settings.

Int J Infect Dis 2021 Mar 16;105:298-303. Epub 2021 Mar 16.

Department of Microbiology and Immunology, Western University, London, Canada. Electronic address:

Background: Dolutegravir (DTG) is now recommended to all HIV infected adults, adolescents, and children of right age by WHO. The low cost of $75 per year for generic DTG-based combination, has allowed 3.9 million people living with HIV (PLWH) in low and middle-income countries (LMICs) access to DTG. Lamivudine and emtricitabine associated M184V/I mutation is highly prevalent in PLWH and the majority of HIV infected individuals receiving DTG regimens may already be carrying M184V/I mutation.

Discussion: Despite high prevalence of M184V/I in antiretroviral therapy (ART) experienced patients, DTG treatment outcomes will likely not be adversely affected by this mutation. The use of DTG in ART naïve has been largely characterised by rare emergence of resistance and virological failure. DTG-based regimens have to great extent been effective at maintaining viral suppression in treatment experienced PLWH carrying M184V/I.

Conclusions: Initiating patients on DTG may help preserve more treatment options for HIV infected individuals living in LMICs. High genetic barrier to the development of resistance associated with DTG and progressive viral suppression in patients switched to DTG-based therapy with M184V/I, may encourage better DTG outcomes and help in curbing increasing levels of HIV drug resistance in LMICs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijid.2021.03.018DOI Listing
March 2021

Deep Gene Sequence Cluster Analyses of Multi-Virus-Infected Mucosal Tissue Reveal Enhanced Transmission of Acute HIV-1.

J Virol 2021 01 13;95(3). Epub 2021 Jan 13.

Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada

Exposure of the genital mucosa to a genetically diverse viral swarm from the donor HIV-1 can result in breakthrough and systemic infection by a single transmitted/founder (TF) virus in the recipient. The highly diverse HIV-1 envelope (Env) in this inoculating viral swarm may have a critical role in transmission and subsequent immune response. Thus, chronic (Env) and acute (Env) Env chimeric HIV-1 were tested using multivirus competition assays in human mucosal penile and cervical tissues. Viral competition analysis revealed that Env viruses resided and replicated mainly in the tissue, while Env viruses penetrated the human tissue and established infection of CD4 T cells more efficiently. Analysis of the replication fitness, as tested in peripheral blood mononuclear cells (PBMCs), showed similar replication fitness of Env and Env viruses, which did not correlate with transmission fitness in penile tissue. Further, we observed that chimeric Env viruses with higher replication in genital mucosal tissue (chronic Env viruses) had higher binding affinity to C-type lectins. Data presented herein suggest that the inoculating HIV-1 may be sequestered in the genital mucosal tissue (represented by chronic Env HIV-1) but that a single HIV-1 clone (e.g., acute Env HIV-1) can escape this trapped replication for systemic infection. During heterosexual HIV-1 transmission, a genetic bottleneck occurs in the newly infected individual as the virus passes from the mucosa, leading to systemic infection with a single transmitted HIV-1 clone in the recipient. This bottleneck in the recipient has just been described (K. Klein et al., PLoS Pathog 14:e1006754, https://doi.org/10.1371/journal.ppat.1006754), and the mechanisms involved in this selection process have not been elucidated. However, understanding mucosal restriction is of the utmost importance for understanding dynamics of infections and for designing focused vaccines. Using our human penile and cervical mucosal tissue models for mixed HIV infections, we provide evidence that HIV-1 from acute/early infection, compared to that from chronic infection, can more efficiently traverse the mucosal epithelium and be transmitted to T cells, suggesting higher transmission fitness. This study focused on the role of the HIV-1 envelope in transmission and provides strong evidence that HIV transmission may involve breaking the mucosal lectin trap.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01737-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925087PMC
January 2021

Accumulation of integrase strand transfer inhibitor resistance mutations confers high-level resistance to dolutegravir in non-B subtype HIV-1 strains from patients failing raltegravir in Uganda.

J Antimicrob Chemother 2020 12;75(12):3525-3533

Department of Microbiology and Immunology, Western University, London, Canada.

Background: Increasing first-line treatment failures in low- and middle-income countries (LMICs) have led to increased use of integrase strand transfer inhibitors (INSTIs) such as dolutegravir. However, HIV-1 susceptibility to INSTIs in LMICs, especially with previous raltegravir exposure, is poorly understood due to infrequent reporting of INSTI failures and testing for INSTI drug resistance mutations (DRMs).

Methods: A total of 51 non-subtype B HIV-1 infected patients failing third-line (raltegravir-based) therapy in Uganda were initially selected for the study. DRMs were detected using Sanger and deep sequencing. HIV integrase genes of 13 patients were cloned and replication capacities (RCs) and phenotypic susceptibilities to dolutegravir, raltegravir and elvitegravir were determined with TZM-bl cells. Spearman's correlation coefficient was used to determine cross-resistance between INSTIs.

Results: INSTI DRMs were detected in 47% of patients. HIV integrase-recombinant virus carrying one primary INSTI DRM (N155H or Y143R/S) was susceptible to dolutegravir but highly resistant to raltegravir and elvitegravir (>50-fold change). Two patients, one with E138A/G140A/Q148R/G163R and one with E138K/G140A/S147G/Q148K, displayed the highest reported resistance to raltegravir, elvitegravir and even dolutegravir. The former multi-DRM virus had WT RC whereas the latter had lower RCs than WT.

Conclusions: In HIV-1 subtype A- and D-infected patients failing raltegravir and harbouring INSTI DRMs, there is high-level resistance to elvitegravir and raltegravir. More routine monitoring of INSTI treatment may be advised in LMICs, considering that multiple INSTI DRMs may have accumulated during prolonged exposure to raltegravir during virological failure, leading to high-level INSTI resistance, including dolutegravir resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dkaa355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7662175PMC
December 2020

A targeted reactivation of latent HIV-1 using an activator vector in patient samples from acute infection.

EBioMedicine 2020 Sep 9;59:102853. Epub 2020 Jul 9.

Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada; Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, United States. Electronic address:

Background: During combined anti-retroviral treatment, a latent HIV reservoir persists within resting memory CD4 T cells that initiates viral recrudescence upon treatment interruption. Strategies for HIV-1 cure have largely focused on latency reversing agents (LRAs) capable of reactivating and eliminating this viral reservoir. Previously investigated LRAs have largely failed to achieve a robust latency reversal sufficient for reduction of latent HIV pool or the potential of virus-free remission in the absence of treatment.

Methods: We utilize a polyvalent virus-like particle (VLP) formulation called Activator Vector (ACT-VEC) to 'shock' provirus into transcriptional activity. Ex vivo co-culture experiments were used to evaluate the efficacy of ACT-VEC in relation to other LRAs in individuals diagnosed and treated during the acute stage of infection. IFN-γ ELISpot, qRT-PCR and Illumina MiSeq were used to evaluate antigenicity, latency reversal, and diversity of induced virus respectively.

Findings: Using samples from HIV patients diagnosed and treated at acute/early infection, we demonstrate that ACT-VEC can reverse latency in HIV infected CD4 T cells to a greater extent than other major recall antigens as stimuli or even mitogens such as PMA/Iono. Furthermore, ACT-VEC activates more latent HIV-1 than clinically tested HDAC inhibitors or protein kinase C agonists.

Interpretation: Taken together, these results show that ACT-VEC can induce HIV reactivation from latently infected CD4 T cells collected from participants on first line combined antiretroviral therapy for at least two years after being diagnosed and treated at acute/early stage of infection. These findings could provide guidance to possible targeted cure strategies and treatments.

Funding: NIH and CIHR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2020.102853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502668PMC
September 2020

The urgent need for more potent antiretroviral therapy in low-income countries to achieve UNAIDS 90-90-90 and complete eradication of AIDS by 2030.

Infect Dis Poverty 2019 Aug 2;8(1):63. Epub 2019 Aug 2.

Department of Microbiology and Immunology, Western University, 1151 Richmond St., DSB Rm.3007, London, ON, N6A5C1, Canada.

Background: Over 90% of Human Immunodeficiency Virus (HIV) infected individuals will be on treatment by 2020 under UNAIDS 90-90-90 global targets. Under World Health Organisation (WHO) "Treat All" approach, this number will be approximately 36.4 million people with over 98% in low-income countries (LICs).

Main Body: Pretreatment drug resistance (PDR) largely driven by frequently use of non-nucleoside reverse transcriptase inhibitors (NNRTIs), efavirenz and nevirapine, has been increasing with roll-out of combined antiretroviral therapy (cART) with 29% annual increase in some LICs countries. PDR has exceeded 10% in most LICs which warrants change of first line regimen to more robust classes under WHO recommendations. If no change in regimens is enforced in LICs, it's estimated that over 16% of total deaths, 9% of new infections, and 8% of total cART costs will be contributed by HIV drug resistance by 2030. Less than optimal adherence, and adverse side effects associated with currently available drug regimens, all pose a great threat to achievement of 90% viral suppression and elimination of AIDS as a public health threat by 2030. This calls for urgent introduction of policies that advocate for voluntary and compulsory drug licensing of new more potent drugs which should also emphasize universal access of these drugs to all individuals worldwide.

Conclusions: The achievement of United Nations Programme on HIV and AIDS 2020 and 2030 targets in LICs depends on access to active cART with higher genetic barrier to drug resistance, better safety, and tolerability profiles. It's also imperative to strengthen quality service delivery in terms of retention of patients to treatment, support for adherence to cART, patient follow up and adequate drug stocks to help achieve a free AIDS generation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40249-019-0573-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6676518PMC
August 2019

Role of co-expressed APOBEC3F and APOBEC3G in inducing HIV-1 drug resistance.

Heliyon 2019 Apr 16;5(4):e01498. Epub 2019 Apr 16.

University of Saskatchewan, Biochemistry, Microbiology, and Immunology, College of Medicine, Saskatoon, Saskatchewan, S7H 5E5, Canada.

The APOBEC3 enzymes can induce mutagenesis of HIV-1 proviral DNA through the deamination of cytosine. HIV-1 overcomes this restriction through the viral protein Vif that induces APOBEC3 proteasomal degradation. Within this dynamic host-pathogen relationship, the APOBEC3 enzymes have been found to be beneficial, neutral, or detrimental to HIV-1 biology. Here, we assessed the ability of co-expressed APOBEC3F and APOBEC3G to induce HIV-1 resistance to antiviral drugs. We found that co-expression of APOBEC3F and APOBEC3G enabled partial resistance of APOBEC3F to Vif-mediated degradation with a corresponding increase in APOBEC3F-induced deaminations in the presence of Vif, in addition to APOBEC3G-induced deaminations. We recovered HIV-1 drug resistant variants resulting from APOBEC3-induced mutagenesis, but these variants were less able to replicate than drug resistant viruses derived from RT-induced mutations alone. The data support a model in which APOBEC3 enzymes cooperate to restrict HIV-1, promoting viral inactivation over evolution to drug resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.heliyon.2019.e01498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475876PMC
April 2019

First-line HIV treatment failures in non-B subtypes and recombinants: a cross-sectional analysis of multiple populations in Uganda.

AIDS Res Ther 2019 01 22;16(1). Epub 2019 Jan 22.

Department of Microbiology and Immunology, Western University, London, Canada.

Background: Our understanding of HIV-1 and antiretroviral treatment (ART) is strongly biased towards subtype B, the predominant subtype in North America and western Europe. Efforts to characterize the response to first-line treatments in other HIV-1 subtypes have been hindered by the availability of large study cohorts in resource-limited settings. To maximize our statistical power, we combined HIV-1 sequence and clinical data from every available study population associated with the Joint Clinical Research Centre (JCRC) in Uganda. These records were combined with contemporaneous ART-naive records from Uganda in the Stanford HIVdb database.

Methods: Treatment failures were defined by the presence of HIV genotype records with sample collection dates after the ART start dates in the JCRC database. Drug resistances were predicted by the Stanford HIVdb algorithm, and HIV subtype classification and recombination detection was performed with SCUEAL. We used Bayesian network analysis to evaluate associations between drug exposures and subtypes, and binomial regression for associations with recombination.

Results: This is the largest database of first-line treatment failures ([Formula: see text]) in Uganda to date, with a predicted statistical power of 80% to detect subtype associations at an odds ratio of [Formula: see text]. In the subset where drug regimen data were available, we observed that use of 3TC was associated with a higher rate of first line treatment failure, whereas regimens containing AZT and TDF were associated with reduced rates of failure. In the complete database, we found limited evidence of associations between HIV-1 subtypes and treatment failure, with the exception of a significantly lower frequency of failures among A/D recombinants that comprised about 7% of the population. First-line treatment failure was significantly associated with reduced numbers of recombination breakpoints across subtypes.

Conclusions: Expanding access to first-line ART should confer the anticipated public health benefits in Uganda, despite known differences in the pathogenesis of HIV-1 subtypes. Furthermore, the impact of ART may actually be enhanced by frequent inter-subtype recombination in this region.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12981-019-0218-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343277PMC
January 2019

Evolution-Guided Structural and Functional Analyses of the HERC Family Reveal an Ancient Marine Origin and Determinants of Antiviral Activity.

J Virol 2018 07 13;92(13). Epub 2018 Jun 13.

Western University, Schulich School of Medicine and Dentistry, Department of Microbiology and Immunology, London, Ontario, Canada

In humans, homologous to the E6-AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1)-like domain-containing protein 5 (HERC5) is an interferon-induced protein that inhibits replication of evolutionarily diverse viruses, including human immunodeficiency virus type 1 (HIV-1). To better understand the origin, evolution, and function of HERC5, we performed phylogenetic, structural, and functional analyses of the entire human small-HERC family, which includes HERC3, HERC4, HERC5, and HERC6. We demonstrated that the family emerged >595 million years ago and has undergone gene duplication and gene loss events throughout its evolution. The structural topology of the RCC1-like domain and HECT domains from all HERC paralogs is highly conserved among evolutionarily diverse vertebrates despite low sequence homology. Functional analyses showed that the human small HERCs exhibit different degrees of antiviral activity toward HIV-1 and that HERC5 provides the strongest inhibition. Notably, coelacanth HERC5 inhibited simian immunodeficiency virus (SIV), but not HIV-1, particle production, suggesting that the antiviral activity of HERC5 emerged over 413 million years ago and exhibits species- and virus-specific restriction. In addition, we showed that both HERC5 and HERC6 are evolving under strong positive selection, particularly blade 1 of the RCC1-like domain, which we showed is a key determinant of antiviral activity. These studies provide insight into the origin, evolution, and biological importance of the human restriction factor HERC5 and the other HERC family members. Intrinsic immunity plays an important role as the first line of defense against viruses. Studying the origins, evolution, and functions of proteins responsible for effecting this defense will provide key information about virus-host relationships that can be exploited for future drug development. We showed that HERC5 is one such antiviral protein that belongs to an evolutionarily conserved family of HERCs with an ancient marine origin. Not all vertebrates possess all HERC members, suggesting that different HERCs emerged at different times during evolution to provide the host with a survival advantage. Consistent with this, two of the more recently emerged HERC members, HERC5 and HERC6, displayed strong signatures of having been involved in an ancient evolutionary battle with viruses. Our findings provide new insights into the evolutionary origin and function of the HERC family in vertebrate evolution, identifying HERC5 and possibly HERC6 as important effectors of intrinsic immunity in vertebrates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00528-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002735PMC
July 2018

A heterogeneous human immunodeficiency virus-like particle (VLP) formulation produced by a novel vector system.

NPJ Vaccines 2018 19;3. Epub 2018 Jan 19.

1Department of Microbiology and Immunology, University of Western Ontario, London, ON N6A 5C1 Canada.

First identified as the etiological agent behind Acquired Immunodeficiency Syndrome (AIDS) in the early 1980s, HIV-1 has continued to spread into a global pandemic and major public health concern. Despite the success of antiretroviral therapy at reducing HIV-1 viremia and preventing the dramatic CD4 T-cell collapse, infected individuals remain HIV positive for life. Unfortunately, it is increasingly clear that natural immunity is not, and may never be, protective against this pathogen. Therefore, efficacious vaccine interventions, which can either prevent infection or eradicate the latent viral reservoir and effect cure, are a major medical priority. Here we describe the development of a safe vaccine platform, currently being utilized in on-going prophylactic and therapeutic preclinical studies and consisting of highly heterogeneous virus-like particle formulations that represent the virus diversity within infected individuals. These VLPs contain no 5'LTR, no functional integrase, and have a severely mutated stem loop 1-thereby preventing any potential reverse transcription, integration, and RNA packaging. Furthermore, we demonstrate that these VLPs are morphologically identical to wild-type virus with polyvalent Env in a functional form. Finally, we show that the VLPs are antigenic and capable of generating strong immune recall responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41541-017-0040-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775397PMC
January 2018

Absence of HIV-1 Drug Resistance Mutations Supports the Use of Dolutegravir in Uganda.

AIDS Res Hum Retroviruses 2018 05 26;34(5):404-414. Epub 2018 Feb 26.

1 Department of Microbiology and Immunology, Western University , London, Canada .

To screen for drug resistance and possible treatment with Dolutegravir (DTG) in treatment-naive patients and those experiencing virologic failure during first-, second-, and third-line combined antiretroviral therapy (cART) in Uganda. Samples from 417 patients in Uganda were analyzed for predicted drug resistance upon failing a first- (N = 158), second- (N = 121), or third-line [all 51 involving Raltegravir (RAL)] treatment regimen. HIV-1 pol gene was amplified and sequenced from plasma samples. Drug susceptibility was interpreted using the Stanford HIV database algorithm and SCUEAL was used for HIV-1 subtyping. Frequency of resistance to nucleoside reverse transcriptase inhibitors (NRTIs) (95%) and non-NRTI (NNRTI, 96%) was high in first-line treatment failures. Despite lack of NNRTI-based treatment for years, NNRTI resistance remained stable in 55% of patients failing second-line or third-line treatment, and was also at 10% in treatment-naive Ugandans. DTG resistance (n = 366) was not observed in treatment-naive individuals or individuals failing first- and second-line cART, and only found in two patients failing third-line cART, while 47% of the latter had RAL- and Elvitegravir-resistant HIV-1. Secondary mutations associated with DTG resistance were found in 2%-10% of patients failing third-line cART. Of 14 drugs currently available for cART in Uganda, resistance was readily observed to all antiretroviral drugs (except for DTG) in Ugandan patients failing first-, second-, or even third-line treatment regimens. The high NNRTI resistance in first-line treatment in Uganda even among treatment-naive patients calls for the use of DTG to reach the UNAIDS 90:90:90 goals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/AID.2017.0205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934975PMC
May 2018

Higher sequence diversity in the vaginal tract than in blood at early HIV-1 infection.

PLoS Pathog 2018 01 18;14(1):e1006754. Epub 2018 Jan 18.

Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada.

In the majority of cases, human immunodeficiency virus type 1 (HIV-1) infection is transmitted through sexual intercourse. A single founder virus in the blood of the newly infected donor emerges from a genetic bottleneck, while in rarer instances multiple viruses are responsible for systemic infection. We sought to characterize the sequence diversity at early infection, between two distinct anatomical sites; the female reproductive tract vs. systemic compartment. We recruited 72 women from Uganda and Zimbabwe within seven months of HIV-1 infection. Using next generation deep sequencing, we analyzed the total genetic diversity within the C2-V3-C3 envelope region of HIV-1 isolated from the female genital tract at early infection and compared this to the diversity of HIV-1 in plasma. We then compared intra-patient viral diversity in matched cervical and blood samples with three or seven months post infection. Genetic analysis of the C2-V3-C3 region of HIV-1 env revealed that early HIV-1 isolates within blood displayed a more homogeneous genotype (mean 1.67 clones, range 1-5 clones) than clones in the female genital tract (mean 5.7 clones, range 3-10 clones) (p<0.0001). The higher env diversity observed within the genital tract compared to plasma was independent of HIV-1 subtype (A, C and D). Our analysis of early mucosal infections in women revealed high HIV-1 diversity in the vaginal tract but few transmitted clones in the blood. These novel in vivo finding suggest a possible mucosal sieve effect, leading to the establishment of a homogenous systemic infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1006754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773221PMC
January 2018

Sensitive detection of HIV-1 resistance to Zidovudine and impact on treatment outcomes in low- to middle-income countries.

Infect Dis Poverty 2017 Dec 4;6(1):163. Epub 2017 Dec 4.

Department of Microbiology and Immunology, University of Western Ontario, 1151 Richmond St., Dental Sciences Bldg., Rm 3014, London, Ontario, N6A 5C1, Canada.

Background: Thymidine analogs, namely AZT (Zidovudine or Retrovir™) and d4T (Stavudine or Zerit™) are antiretroviral drugs still employed in over 75% of first line combination antiretroviral therapy (cART) in Kampala, Uganda despite aversion to prescribing these drugs for cART in high income countries due in part to adverse events. For this study, we explored how the continued use of these thymidine analogs in cART could impact emergence of drug resistance and impact on future treatment success in Uganda, a low-income country.

Methods: We examined the drug resistance genotypes by Sanger sequencing of 262 HIV-infected patients failing a first line combined antiretroviral treatment containing either AZT or d4T, which represents approximately 5% of the patients at the Joint Clinical Research Center receiving a AZT or d4T containing treatment. Next generation sequencing (DEEPGEN™HIV) and multiplex oligonucleotide ligation assays (AfriPOLA) were then performed on a subset of patient samples to detect low frequency drug resistant mutations. CD4 cell counts, viral RNA loads, and treatment changes were analyzed in a cohort of treatment success and failures.

Results: Over 80% of patients failing first line AZT/d4T-containing cART had predicted drug resistance to 3TC (Lamivudine) and non-nucleoside RT inhibitors (NNRTIs) in the treatment regimen but only 45% had resistance AZT/d4T associated resistance mutations (TAMs). TAMs were however detected at low frequency within the patients HIV quasispecies (1-20%) in 21 of 34 individuals who were failing first-line AZT-containing cART and lacked TAMs by Sanger. Due to lack of TAMs by Sanger, AZT was typically maintained in second-line therapies and these patients had a low frequency of subsequent virologic success.

Conclusions: Our findings suggest that continued use of AZT and d4T in first-line treatment in low-to-middle income countries may lead to misdiagnosis of HIV-1 drug resistance and possibly enhance a succession of second- and third-line treatment failures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40249-017-0377-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716384PMC
December 2017

Development of an HIV vaccine using a vesicular stomatitis virus vector expressing designer HIV-1 envelope glycoproteins to enhance humoral responses.

AIDS Res Ther 2017 Sep 12;14(1):55. Epub 2017 Sep 12.

Department of Microbiology and Immunology, Western University, London, Canada.

Vesicular stomatitis virus (VSV), like many other Rhabdoviruses, have become the focus of intense research over the past couple of decades based on their suitability as vaccine vectors, transient gene delivery systems, and as oncolytic viruses for cancer therapy. VSV as a vaccine vector platform has multiple advantages over more traditional viral vectors including low level, non-pathogenic replication in diverse cell types, ability to induce both humoral and cell-mediate immune responses, and the remarkable expression of foreign proteins cloned into multiple intergenic sites in the VSV genome. The utility and safety of VSV as a vaccine vector was recently demonstrated near the end of the recent Ebola outbreak in West Africa where VSV pseudotyped with the Ebola virus (EBOV) glycoprotein was proven safe in humans and provided protective efficacy against EBOV in a human phase III clinical trial. A team of Canadian scientists, led by Dr. Gary Kobinger, is now working with International AIDS Vaccine Initiative (IAVI) in developing a VSV-based HIV vaccine that will combine unique Canadian research on the HIV-1 Env glycoprotein and on the VSV vaccine vector. The goal of this collaboration is to develop a vaccine with a robust and potent anti-HIV immune response with an emphasis on generating quality antibodies to protect against HIV challenges.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12981-017-0179-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5594459PMC
September 2017

Tribute to Mark Wainberg.

Retrovirology 2017 Jun 28;14(1):38. Epub 2017 Jun 28.

Département de microbiologie, infectiologie et immunologie, Université de Montréal, Montreal, Canada.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12977-017-0361-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488361PMC
June 2017

An in vitro Model to Mimic Selection of Replication-Competent HIV-1 Intersubtype Recombination in Dual or Superinfected Patients.

J Mol Biol 2017 07 1;429(14):2246-2264. Epub 2017 May 1.

Department of Molecular Biology and Microbiology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA; Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA; Department of Microbiology and Immunology, Western University, London, Ontario N6A 5C1, Canada. Electronic address:

The low frequency of HIV-1 recombinants within entire viral populations in both individual patients and culture-based infection models impedes investigation of the underlying factors contributing to either the occurrence of recombinants or the survival of recombinants once they are formed. So far, most of the related studies have no consideration of recombinants' functionality. Here, we established a functional recombinant production (FRP) system to produce pure and functional HIV-1 intersubtype Env recombinants and utilized 454 pyrosequencing to investigate the distribution of over 4000 functional and non-functional recombination breakpoints from either the FRP system or dual infection cultures. The results revealed that most of the breakpoints converged in gp41 (62%) and C1 (25.3%) domains of gp120, which has strong correlation with the similarity between the two recombining sequences. Yet, the breakpoints also appeared in C2 (5.2%) and C5 (4.6%) domains not correlated with the recombining sequence similarity. Interestingly, none of the intersubtype gp120 recombinants recombined between C1 and gp41 regions either from the FRP system or from the dual infection culture, and very few from the HIV epidemic were functional. The present study suggests that the selection of functional Env recombinants is one of the reasons for the predominance of C1 and gp41 Env recombinants in the HIV epidemic, and it provides an in vitro model to mimic the selection of replication-competent HIV-1 intersubtype recombination in dual or superinfected patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2017.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202033PMC
July 2017

Infection of rhesus macaques with a pool of simian immunodeficiency virus with the envelope genes from acute HIV-1 infections.

AIDS Res Ther 2016 11 25;13(1):41. Epub 2016 Nov 25.

Division of Infectious Diseases, School of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106 USA.

Background: New simian-human immunodeficiency chimeric viruses with an HIV-1 env (SHIVenv) are critical for studies on HIV pathogenesis, vaccine development, and microbicide testing. Macaques are typically exposed to single CCR5-using SHIVenv which in most instances does not reflect the conditions during acute/early HIV infection (AHI) in humans. Instead of individual and serial testing new SHIV constructs, a pool of SHIVenv_B derived from 16 acute HIV-1 infections were constructed using a novel yeast-based SHIV cloning approach and then used to infect macaques.

Results: Even though none of the 16 SHIVenvs contained the recently reported mutations in env genes that could significantly enhance their binding affinity to RhCD4, one SHIVenv (i.e. SHIVenv_B3-PRB926) established infection in macaques exposed to this pool. AHI SHIVenv_B viruses as well as their HIVenv_B counterparts were analyzed for viral protein content, function, and fitness to identify possible difference between SHIVenv_B3-PRB926 and the other 15 SHIVenvs in the pool. All of the constructs produced SHIV or HIV chimeric with wild type levels of capsid (p27 and p24) content, reverse transcriptase (RT) activity, and expressed envelope glycoproteins that could bind to cell receptors CD4/CCR5 and mediate virus entry. HIV-1env_B chimeric viruses were propagated in susceptible cell lines but the 16 SHIVenv_B variants showed only limited replication in macaque peripheral blood mononuclear cells (PBMCs) and 174×CEM.CCR5 cell line. AHI chimeric viruses including HIVenv_B3 showed only minor variations in cell entry efficiency and kinetics as well as replicative fitness in human PBMCs. Reduced number of N-link glycosylation sites and slightly greater CCR5 affinity/avidity was the only distinguishing feature of env_B3 versus other AHI env's in the pool, a feature also observed in the HIV establishing new infections in humans.

Conclusion: Despite the inability to propagate in primary cells and cell lines, a pool of 16 SHIVenv viruses could establish infection but only one virus, SHIVenv_B3 was isolated in the macaque and then shown to repeatedly infected macaques. This SHIVenv_B3 virus did not show any distinct phenotypic property from the other 15 SHIVenv viruses but did have the fewest N-linked glycosylation sites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12981-016-0125-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5124249PMC
November 2016

First Phase I human clinical trial of a killed whole-HIV-1 vaccine: demonstration of its safety and enhancement of anti-HIV antibody responses.

Retrovirology 2016 Nov 28;13(1):82. Epub 2016 Nov 28.

Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, The University of Western Ontario, 1400 Western Road, London, ON, N6G 2V4, Canada.

Background: Vaccination with inactivated (killed) whole-virus particles has been used to prevent a wide range of viral diseases. However, for an HIV vaccine this approach has been largely negated due to inherent safety concerns, despite the ability of killed whole-virus vaccines to generate a strong, predominantly antibody-mediated immune response in vivo. HIV-1 Clade B NL4-3 was genetically modified by deleting the nef and vpu genes and substituting the coding sequence for the Env signal peptide with that of honeybee melittin signal peptide to produce a less virulent and more replication efficient virus. This genetically modified virus (gmHIV-1) was inactivated and formulated as a killed whole-HIV vaccine, and then used for a Phase I human clinical trial (Trial Registration: Clinical Trials NCT01546818). The gmHIV-1 was propagated in the A3.01 human T cell line followed by virus purification and inactivation with aldrithiol-2 and γ-irradiation. Thirty-three HIV-1 positive volunteers receiving cART were recruited for this observer-blinded, placebo-controlled Phase I human clinical trial to assess the safety and immunogenicity.

Results: Genetically modified and killed whole-HIV-1 vaccine, SAV001, was well tolerated with no serious adverse events. HIV-1-specific PCR showed neither evidence of vaccine virus replication in the vaccine virus-infected human T lymphocytes in vitro nor in the participating volunteers receiving SAV001 vaccine. Furthermore, SAV001 with adjuvant significantly increased the pre-existing antibody response to HIV-1 proteins. Antibodies in the plasma of vaccinees were also found to recognize HIV-1 envelope protein on the surface of infected cells as well as showing an enhancement of broadly neutralizing antibodies inhibiting tier I and II of HIV-1 B, D, and A subtypes.

Conclusion: The killed whole-HIV vaccine, SAV001, is safe and triggers anti-HIV immune responses. It remains to be determined through an appropriate trial whether this immune response prevents HIV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12977-016-0317-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126836PMC
November 2016

A Highly Conserved Residue in HIV-1 Nef Alpha Helix 2 Modulates Protein Expression.

mSphere 2016 Nov-Dec;1(6). Epub 2016 Nov 9.

Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

Extensive genetic diversity is a defining characteristic of human immunodeficiency virus type 1 (HIV-1) and poses a significant barrier to the development of an effective vaccine. To better understand the impact of this genetic diversity on the HIV-1 pathogenic factor Nef, we compiled a panel of reference strains from the NIH Los Alamos HIV Database. Initial sequence analysis identified point mutations at Nef residues 13, 84, and 92 in subtype C reference strain C.BR92025 from Brazil. Functional analysis revealed impaired major histocompatibility complex class I and CD4 downregulation of strain C.BR92025 Nef, which corresponded to decreased protein expression. Metabolic labeling demonstrated that strain C.BR92025 Nef has a greater rate of protein turnover than subtype B reference strain B.JRFL that, on the basis of mutational analysis, is related to Nef residue A84. An alanine-to-valine substitution at position 84, located in alpha helix 2 of Nef, was sufficient to alter the rate of turnover of an otherwise highly expressed Nef protein. In conclusion, these findings highlight HIV-1 Nef residue A84 as a major determinant of protein expression that may offer an additional avenue to disrupt or mediate the effects of this key HIV-1 pathogenic factor. The HIV-1 Nef protein has been established as a key pathogenic determinant of HIV/AIDS, but there is little knowledge of how the extensive genetic diversity of HIV-1 affects Nef function. Upon compiling a set of subtype-specific reference strains, we identified a subtype C reference strain, C.BR92025, that contained natural polymorphisms at otherwise highly conserved residues 13, 84, and 92. Interestingly, strain C.BR92025 Nef displayed impaired Nef function and had decreased protein expression. We have demonstrated that strain C.BR92025 Nef has a higher rate of protein turnover than highly expressed Nef proteins and that this higher rate of protein turnover is due to an alanine-to-valine substitution at Nef residue 84. These findings highlight residue A84 as a major determinant of HIV-1 Nef expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mSphere.00288-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5103047PMC
November 2016

Infecting HIV-1 Subtype Predicts Disease Progression in Women of Sub-Saharan Africa.

EBioMedicine 2016 Nov 12;13:305-314. Epub 2016 Oct 12.

Department of Microbiology and Immunology, Western University, London, ON, Canada; Joint Clinical Research Centre, Kampala, Uganda; Division of Infectious Diseases and HIV Medicine, Department of Medicine, Case Western Reserve University, Cleveland, OH, USA. Electronic address:

Introduction: Long-term natural history cohorts of HIV-1 in the absence of treatment provide the best measure of virulence by different viral subtypes.

Methods: Newly HIV infected Ugandan and Zimbabwean women (N=303) were recruited and monitored for clinical, social, behavioral, immunological and viral parameters for 3 to 9.5years.

Results: Ugandan and Zimbabwean women infected with HIV-1 subtype C had 2.5-fold slower rates of CD4 T-cell declines and higher frequencies of long-term non-progression than those infected with subtype A or D (GEE model, P<0.001), a difference not associated with any other clinical parameters. Relative replicative fitness and entry efficiency of HIV-1 variants directly correlated with virulence in the patients, subtype D>A>C (P<0.001, ANOVA).

Discussion: HIV-1 subtype C was less virulent than either A or D in humans; the latter being the most virulent. Longer periods of asymptomatic HIV-1 subtype C could explain the continued expansion and dominance of subtype C in the global epidemic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2016.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264310PMC
November 2016

Pathogenic infection of Rhesus macaques by an evolving SIV-HIV derived from CCR5-using envelope genes of acute HIV-1 infections.

Virology 2016 12 7;499:298-312. Epub 2016 Oct 7.

Division of Infectious Diseases and HIV Medicine, Case Western Reserve University, Cleveland, OH 44106, USA; Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Western University, London, ON N6A 5C1, Canada. Electronic address:

For studies on vaccines and therapies for HIV disease, SIV-HIV chimeric viruses harboring the HIV-1 env gene (SHIVenv) remain the best virus in non-human primate models. However, there are still very few SHIVenv viruses that can cause AIDS in non-CD8-depleted animals. In the present study, a recently created CCR5-using SHIVenv_B3 virus with env gene derived from acute/early HIV-1 infections (AHI) successfully established pathogenic infection in macaques. Through a series of investigations on the evolution, mutational profile, and phenotype of the virus and the resultant humoral immune response in infected rhesus macaques, we found that the E32K mutation in the Env C1 domain was associated with macaque pathogenesis, and that the electrostatic interactions in Env may favor E32K at the gp120 N terminus and "lock" the binding to heptad repeat 1 of gp41 in the trimer and produce a SHIVenv with increased fitness and pathogenesis during macaque infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2016.09.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899904PMC
December 2016

Low-Frequency Drug Resistance in HIV-Infected Ugandans on Antiretroviral Treatment Is Associated with Regimen Failure.

Antimicrob Agents Chemother 2016 06 23;60(6):3380-97. Epub 2016 May 23.

Center for AIDS Research Uganda Laboratories, Joint Clinical Research Centre, Kampala, Uganda Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA University Hospital Translational Laboratory, University Hospitals Case Medical Center, Cleveland, Ohio, USA

Most patients failing antiretroviral treatment in Uganda continue to fail their treatment regimen even if a dominant drug-resistant HIV-1 genotype is not detected. In a recent retrospective study, we observed that approximately 30% of HIV-infected individuals in the Joint Clinical Research Centre (Kampala, Uganda) experienced virologic failure with a susceptible HIV-1 genotype based on standard Sanger sequencing. Selection of minority drug-resistant HIV-1 variants (not detectable by Sanger sequencing) under antiretroviral therapy pressure can lead to a shift in the viral quasispecies distribution, becoming dominant members of the virus population and eventually causing treatment failure. Here, we used a novel HIV-1 genotyping assay based on deep sequencing (DeepGen) to quantify low-level drug-resistant HIV-1 variants in 33 patients failing a first-line antiretroviral treatment regimen in the absence of drug-resistant mutations, as screened by standard population-based Sanger sequencing. Using this sensitive assay, we observed that 64% (21/33) of these individuals had low-frequency (or minority) drug-resistant variants in the intrapatient HIV-1 population, which correlated with treatment failure. Moreover, the presence of these minority HIV-1 variants was associated with higher intrapatient HIV-1 diversity, suggesting a dynamic selection or fading of drug-resistant HIV-1 variants from the viral quasispecies in the presence or absence of drug pressure, respectively. This study identified low-frequency HIV drug resistance mutations by deep sequencing in Ugandan patients failing antiretroviral treatment but lacking dominant drug resistance mutations as determined by Sanger sequencing methods. We showed that these low-abundance drug-resistant viruses could have significant consequences for clinical outcomes, especially if treatment is not modified based on a susceptible HIV-1 genotype by Sanger sequencing. Therefore, we propose to make clinical decisions using more sensitive methods to detect minority HIV-1 variants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.00038-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879357PMC
June 2016

HIV-1 Group O Genotypes and Phenotypes: Relationship to Fitness and Susceptibility to Antiretroviral Drugs.

AIDS Res Hum Retroviruses 2016 07 16;32(7):676-88. Epub 2016 Mar 16.

1 Division of Infectious Diseases, Case Western Reserve University , Cleveland, Ohio.

Despite only 30,000 group O HIV-1 infections, a similar genetic diversity is observed among the O subgroups H (head) and T (tail) (previously described as subtypes A, B) as in the 9 group M subtypes (A-K). Group O isolates bearing a cysteine at reverse transcriptase (RT) position 181, predominantly the H strains are intrinsically resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs). However, their susceptibility to newer antiretroviral drugs such as etravirine, maraviroc, raltegravir (RAL), and elvitegravir (EVG) remains relatively unknown. We tested a large collection of HIV-1 group O strains for their susceptibility to four classes of antiretroviral drugs namely nucleoside RT, non-nucleoside RT, integrase, and entry inhibitors knowing in advance the intrinsic resistance to NNRTIs. Drug target regions were sequenced to determine various polymorphisms and were phylogenetically analyzed. Replication kinetics and fitness assays were performed in U87-CD4(+)CCR5 and CXCR4 cells and peripheral blood mononuclear cells. With all antiretroviral drugs, group O HIV-1 showed higher variability in IC50 values than group M HIV-1. The mean IC50 values for entry and nucleoside reverse transcriptase inhibitor (NRTI) were similar for group O and M HIV-1 isolates. Despite similar susceptibility to maraviroc, the various phenotypic algorithms failed to predict CXCR4 usage based on the V3 Env sequences of group O HIV-1 isolates. Decreased sensitivity of group O HIV-1 to integrase or NNRTIs had no relation to replicative fitness. Group O HIV-1 isolates were 10-fold less sensitive to EVG inhibition than group M HIV-1. These findings suggest that in regions where HIV-1 group O is endemic, first line treatment regimens combining two NRTIs with RAL may provide more sustained virologic responses than the standard regimens involving an NNRTI or protease inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/AID.2015.0318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931737PMC
July 2016

Defining the fitness of HIV-1 isolates with dual/mixed co-receptor usage.

AIDS Res Ther 2015 3;12:34. Epub 2015 Oct 3.

Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106 USA ; Virology Program, Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH USA.

Background: CCR5-using (r5) HIV-1 predominates during asymptomatic disease followed by occasional emergence of CXCR4-using (x4) or dual tropic (r5x4) virus. We examined the contribution of the x4 and r5 components to replicative fitness of HIV-1 isolates.

Methods: Dual tropic r5x4 viruses were predicted from average HIV-1 env sequences of two primary subtype C HIV-1 isolates (C19 and C27) and from two patient plasma samples (B12 and B19). Chimeric Env viruses with an NL4-3 backbone were constructed from the B12 and B19 env sequences. To determine replicative fitness, these primary and chimeric dual tropic HIV-1 were then competed against HIV-1 reference isolates in U87.CD4 cells expressing CXCR4 or CCR5 or in PBMCs ± entry inhibitors. Contribution of the x4 and r5 clones within the quasispecies of these chimeric or primary HIV-1 isolates were then compared to the frequency of x4, r5, and dual tropic clones within the quasispecies as predicted by phenotypic assays, clonal sequencing, and 454 deep sequencing.

Results: In the primary HIV-1 isolates (C19 and C27), subtype C dual tropic clones dominated over x4 clones while pure r5 clones were absent. In two subtype B chimeric viruses (B12 and B19), r5 clones were >100-fold more abundant than x4 or r5/x4 clones. The dual tropic C19 and C27 HIV-1 isolates outcompeted r5 primary HIV-1 isolates, B2 and C3 in PBMCs. When AMD3100 was added or when only U87.CD4.CCR5 cells were used, the B2 and C3 reference viruses now out-competed the r5 component of the dual tropic C19 and C27. In contrast, the same replicative fitness was observed with dualtropic B12 and B19 HIV-1 isolates relative to x4 HIV-1 A8 and E6 or the r5 B2 and C3 viruses, even when the r5 or x4 component was inhibited by maraviroc (or AMD3100) or in U87.CD4.CXCR4 (or CCR5) cells.

Conclusions: In the dual tropic HIV-1 isolates, the x4 replicative fitness is higher than r5 clones but the x4 or x4/r5 clones are typically at low frequency in the intrapatient virus population. Ex vivo HIV propagation promotes outgrowth of the x4 clones and provides an over-estimate of x4 dominance in replicative fitness within dual tropic viruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12981-015-0066-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4592561PMC
October 2015

Functional bottlenecks for generation of HIV-1 intersubtype Env recombinants.

Retrovirology 2015 May 23;12:44. Epub 2015 May 23.

Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH, 44106, USA.

Background: Intersubtype recombination is a powerful driving force for HIV evolution, impacting both HIV-1 diversity within an infected individual and within the global epidemic. This study examines if viral protein function/fitness is the major constraint shaping selection of recombination hotspots in replication-competent HIV-1 progeny. A better understanding of the interplay between viral protein structure-function and recombination may provide insights into both vaccine design and drug development.

Results: In vitro HIV-1 dual infections were used to recombine subtypes A and D isolates and examine breakpoints in the Env glycoproteins. The entire env genes of 21 A/D recombinants with breakpoints in gp120 were non-functional when cloned into the laboratory strain, NL4-3. Likewise, cloning of A/D gp120 coding regions also produced dead viruses with non-functional Envs. 4/9 replication-competent viruses with functional Env's were obtained when just the V1-V5 regions of these same A/D recombinants (i.e. same A/D breakpoints as above) were cloned into NL4-3.

Conclusion: These findings on functional A/D Env recombinants combined with structural models of Env suggest a conserved interplay between the C1 domain with C5 domain of gp120 and extracellular domain of gp41. Models also reveal a co-evolution within C1, C5, and ecto-gp41 domains which might explain the paucity of intersubtype recombination in the gp120 V1-V5 regions, despite their hypervariability. At least HIV-1 A/D intersubtype recombination in gp120 may result in a C1 from one subtype incompatible with a C5/gp41 from another subtype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12977-015-0170-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445978PMC
May 2015

SiRNA-induced mutation in HIV-1 polypurine tract region and its influence on viral fitness.

PLoS One 2015 10;10(4):e0122953. Epub 2015 Apr 10.

Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America; Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, United States of America.

Converting single-stranded viral RNA into double stranded DNA for integration is an essential step in HIV-1 replication. Initial polymerization of minus-strand DNA is primed from a host derived tRNA, whereas subsequent plus-strand synthesis requires viral primers derived from the 3' and central polypurine tracts (3' and cPPTs). The 5' and 3' termini of these conserved RNA sequence elements are precisely cleaved by RT-associated RNase H to generate specific primers that are used to initiate plus-strand DNA synthesis. In this study, siRNA wad used to produce a replicative HIV-1 variant contained G(-1)A and T(-16)A substitutions within/adjacent to the 3'PPT sequence. Introducing either or both mutations into the 3'PPT region or only the G(-1)A substitution in the cPPT region of NL4-3 produced infectious virus with decreased fitness relative to the wild-type virus. In contrast, introducing the T(-16)A or both mutations into the cPPT rendered the virus(es) incapable of replication, most likely due to the F185L integrase mutation produced by this nucleotide substitution. Finally, the effects of G(-1)A and T(-16)A mutations on cleavage of the 3'PPT were examined using an in vitro RNase H cleavage assay. Substrate containing both mutations was mis-cleaved to a greater extent than either wild-type substrate or substrate containing the T(-16)A mutation alone, which is consistent with the observed effects of the equivalent nucleotide substitutions on the replication fitness of NL4-3 virus. In conclusion, siRNA targeting of the HIV-1 3'PPT region can substantially suppress virus replication, and this selective pressure can be used to generate infectious virus containing mutations within or near the HIV-1 PPT. Moreover, in-depth analysis of the resistance mutations demonstrates that although virus containing a G(-1)A mutation within the 3'PPT is capable of replication, this nucleotide substitution shifts the 3'-terminal cleavage site in the 3'PPT by one nucleotide (nt) and significantly reduces viral fitness.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122953PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4393142PMC
March 2016

Similar replicative fitness is shared by the subtype B and unique BF recombinant HIV-1 isolates that dominate the epidemic in Argentina.

PLoS One 2014 11;9(4):e92084. Epub 2014 Apr 11.

Division of Infectious Diseases and HIV Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America.

The HIV-1 epidemic in South America is dominated by pure subtypes (mostly B and C) and more than 7 BF and BC recombinant forms. In Argentina, circulating recombinant forms (CRFs) comprised of subtypes B and F make up more than 50% of HIV infections. For this study, 28 HIV-1 primary isolates were obtained from patients in Buenos Aires, Argentina and initially classified into subtype B (n = 9, 32.1%), C (n = 1, 3.6%), and CRFs (n = 18, 64.3%) using partial pol and vpu-env sequences, which proved to be inconsistent and inaccurate for these phylogenetic analyses. Near full length genome sequences of these primary HIV-1 isolates revealed that nearly all intersubtype BF recombination sites were unique and countered previous "CRF" B/F classifications. The majority of these Argentinean HIV-1 isolates were CCR5-using but 4 had a dual/mixed tropism as predicted by both phenotypic and genotypic assays. Comparison of the replicative fitness of these BF primary HIV-1 isolates to circulating B, F, and C HIV-1 using pairwise competitions in peripheral blood mononuclear cells (PBMCs) indicated a similarity in fitness of these BF recombinants to subtypes B and F HIV-1 (of the same co-receptor usage) whereas subtype C HIV-1 was significantly less fit than all as previously reported. These results suggest that the multitude of BF HIV-1 strains present within the Argentinean population do not appear to have gained replicative fitness following recent B and F recombination events.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0092084PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3984079PMC
December 2014

Impact of mutations in highly conserved amino acids of the HIV-1 Gag-p24 and Env-gp120 proteins on viral replication in different genetic backgrounds.

PLoS One 2014 8;9(4):e94240. Epub 2014 Apr 8.

Department of Microbiology, University of Washington School of Medicine, Seattle, Washington, United States of America; Department of Medicine, University of Washington School of Medicine, Seattle, Washington, United States of America; Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, Washington, United States of America.

It has been hypothesized that a single mutation at a highly conserved amino acid site (HCS) can be severely deleterious to HIV in most if not all isolate-specific genetic backgrounds. Consequently, potentially universal HIV-1 vaccines exclusively targeting highly conserved regions of the viral proteome have been proposed. To test this hypothesis, we examined the impact of 10 Gag-p24 and 9 Env-gp120 HCS single mutations on viral fitness. In the original founder sequence of the subject in whom these mutations were identified, all Gag-p24 HCS mutations significantly reduced viral replication fitness, including 7 that were lethal. Similar results were obtained at 9/10 sites when the same mutations were introduced into the founder sequences of two epidemiologically unlinked subjects. In contrast, none of the 9 Env-gp120 HCS mutations were lethal in the original founder sequence, and four had no fitness cost. Hence, HCS mutations in Gag-p24 are likely to be severely deleterious in different HIV-1 subtype B backgrounds; however, some HCS mutations in both Gag-p24 and Env-gp120 fragments can be well tolerated. Therefore, when designing HIV-1 immunogens that are intended to force the virus to nonviable escape pathways, the fitness constraints on the HIV segments included should be considered beyond their conservation level.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0094240PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979772PMC
December 2014

Differences in Clinical Manifestations of Acute and Early HIV-1 Infection between HIV-1 Subtypes in African Women.

J Int Assoc Provid AIDS Care 2015 Sep-Oct;14(5):415-22. Epub 2013 Oct 8.

Division of Infectious Diseases and HIV Medicine, Case Western Reserve University, University Hospitals Case Medical Center, Cleveland, OH, USA.

Little is known about the differences in clinical manifestations between women with various HIV-1 subtypes during acute (AI) and early (EI) HIV infection. In a longitudinal cohort study, clinical signs and symptoms among Uganda and Zimbabwe women with AI and EI were compared with HIV-negative controls; symptoms were assessed quarterly for 15 to 24 months. Early HIV infection was defined as the first visit during which a woman tested HIV antibody positive. Women who were HIV negative serologically but DNA polymerase chain reaction positive were considered AI. In all, 26 women were classified AI and 192 EI, with 654 HIV-negative controls. Primary HIV infection (AI and EI) was associated with unexplained fever (P <.01), weight loss (P <.01), fatigue (P <.01), inguinal adenopathy (P <.01), and cervical friability (P =.01). More women with subtype C infection had unexplained fever, fatigue, and abnormal vaginal discharge compared to subtype A or D infection. Inguinal adenopathy occurred less often in women with subtype A infection than those with subtype C or D infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/2325957413504827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4511722PMC
November 2015

Multifaceted mechanisms of HIV inhibition and resistance to CCR5 inhibitors PSC-RANTES and Maraviroc.

Antimicrob Agents Chemother 2013 Jun 25;57(6):2640-50. Epub 2013 Mar 25.

Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA.

Small-molecule CCR5 antagonists, such as maraviroc (MVC), likely block HIV-1 through an allosteric, noncompetitive inhibition mechanism, whereas inhibition by agonists such as PSC-RANTES is less defined and may involve receptor removal by cell surface downregulation, competitive inhibition by occluding the HIV-1 envelope binding, and/or allosteric effects by altering CCR5 conformation. We explored the inhibitory mechanisms of maraviroc and PSC-RANTES by employing pairs of virus clones with differential sensitivities to these inhibitors. Intrinsic PSC-RANTES-resistant virus (YA versus RT) or those selected in PSC-RANTES treated macaques (M584 versus P3-4) only displayed resistance in multiple-cycle assays or with a CCR5 mutant that cannot be downregulated. In single-cycle assays, these HIV-1 clones displayed equal sensitivity to PSC-RANTES inhibition, suggesting effective receptor downregulation. Prolonged PSC-RANTES exposure resulted in desensitization of the receptor to internalization such that increasing virus concentration (substrate) could saturate the receptors and overcome PSC-RANTES inhibition. In contrast, resistance to MVC was observed with the MVC-resistant HIV-1 (R3 versus S2) in both multiple- and single-cycle assays and with altered virus concentrations, which is indicative of allosteric inhibition. MVC could also mediate inhibition and possibly resistance through competitive mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.02511-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3716150PMC
June 2013