Hypervariable Loops Research Articles

Analysis of HIV-1 envelope evolution suggests antibody-mediated selection of common epitopes among Chinese former plasma donors from a narrow-source outbreak

The HIV-1 envelope mutates rapidly to evade recognition and killing, and is a major target of humoral immune responses and vaccine development. Identification of common epitopes for vaccine development have been complicated by genetic variation on both virus and host levels. We studied HIV-1 envelope gp120 evolution in 12 Chinese former plasma donors infected with a purportedly single founder virus, with the aim of identifying common antibody epitopes under immune selection. We found five amino acid sites under significant positive selection in ≥50% of the study participants, and 22 sites consistent with antibody-mediated selection. Despite strong selection pressure, some sites housed a limited repertoire of amino acids. Structural modelling revealed that most of the variable amino acid sites were located on the exposed distal edge of the Gp120 trimer, whilst invariant sites clustered within the centre of the protein complex. Two sites, flanking the V3 hypervariable loop, represent novel antibody sites. Analysis of HIV-1 evolution in hosts infected with a narrow-source virus may provide insight and novel understanding of common epitopes under antibody-mediated selection. If verified in functional studies, such epitopes could be suitable as targets in vaccine development.

OmpA protein sequence-based typing and virulence-associated gene profiles of Pasteurella multocida isolates associated with bovine haemorrhagic septicaemia and porcine pneumonic pasteurellosis in Thailand

BackgroundPasteurella multocida is a Gram-negative bacterium that causes economically significant infections of a broad range of animal species. Pneumonic and septicaemic pasteurellosis caused by this bacterium remain important problems in pigs, cattle, and water buffaloes in Thailand. The aim of this study was to characterise the virulence-associated gene profiles and to develop an OmpA molecular typing scheme for classifying 191 bovine and porcine isolates of P. multocida collected between 1989 and 2012 in Thailand using polymerase chain reactions (PCRs), nucleotide sequencing, and sequence and structural bioinformatics analyses.ResultsPCR screening successfully characterised the profiles of 25 virulence-associated genes in all isolates. The gene profiles separated these isolates into bovine and porcine clusters based on eight genes (hgbB, hsf1, tadD, nanH, pfhA, plpE, pmHAS, and tbpA). Phylogenetic analyses of the nucleotide and protein sequences corresponding to the ompA gene, which encodes a major outer membrane surface protein, showed two major bovine and porcine clusters. Structural prediction and analysis of the dN/dS ratio revealed four hypervariable extracellular loops of the OmpA transmembrane domains. These four loops were used to develop an OmpA typing scheme. This scheme classified 186 isolates into five major loop sequence types (LST8, LST12, LST15, LST18, and LST19), consistent with the phylogenetic results. The loop regions of the bovine isolates were predicted to be more antigenic than those of the porcine isolates. Thus, molecular evolution of the OmpA proteins could be used to classify P. multocida isolates into different capsular types, host types, and, possibly, pathogenicity levels.ConclusionsTogether with the virulence-associated gene profiles, the typing reported in this work provides a better understanding of P. multocida virulence. Effective monitoring and potential strain-specific subunit vaccines could be developed based on these loop oligopeptides.

163 Lox-1-specific affimers block oxldl accumulation in vitro

IntroductionLectin-like oxidised low density lipoprotein receptor 1 (LOX-1, SREC-1, OLR1) is a class E scavenger which binds oxidised low density lipoproteins (oxLDL). It is expressed on endothelial cells, macrophages and...

The structure of a LAIR1-containing human antibody reveals a novel mechanism of antigen recognition.

Antibodies are critical components of the human adaptive immune system, providing versatile scaffolds to display diverse antigen-binding surfaces. Nevertheless, most antibodies have similar architectures, with the variable immunoglobulin domains of the heavy and light chain each providing three hypervariable loops, which are varied to generate diversity. The recent identification of a novel class of antibody in humans from malaria endemic regions of Africa was therefore surprising as one hypervariable loop contains the entire collagen-binding domain of human LAIR1. Here, we present the structure of the Fab fragment of such an antibody. We show that its antigen-binding site has adopted an architecture that positions LAIR1, while itself being occluded. This therefore represents a novel means of antigen recognition, in which the Fab fragment of an antibody acts as an adaptor, linking a human protein insert with antigen-binding potential to the constant antibody regions which mediate immune cell recruitment.

PIGSPro: prediction of immunoGlobulin structures v2

PIGSpro is a significant upgrade of the popular PIGS server for the prediction of the structure of immunoglobulins. The software has been completely rewritten in python following a similar pipeline as in the original method, but including, at various steps, relevant modifications found to improve its prediction accuracy, as demonstrated here. The steps of the pipeline include the selection of the appropriate framework for predicting the conserved regions of the molecule by homology; the target template alignment for this portion of the molecule; the selection of the main chain conformation of the hypervariable loops according to the canonical structure model, the prediction of the third loop of the heavy chain (H3) for which complete canonical structures are not available and the packing of the light and heavy chain if derived from different templates. Each of these steps has been improved including updated methods developed along the years. Last but not least, the user interface has been completely redesigned and an automatic monthly update of the underlying database has been implemented. The method is available as a web server at http://biocomputing.it/pigspro.

Structure-Directed and Tailored Diversity Synthetic Antibody Libraries Yield Novel Anti-EGFR Antagonists.

We tested whether grafting an interaction domain into the hypervariable loop of a combinatorial antibody library could promote targeting to a specific epitope. Formation of the epidermal growth factor receptor (EGFR) signaling heterodimer involves extensive contacts mediated by a "dimerization loop." We grafted the dimerization loop into the third hypervariable loop of a synthetic antigen-binding fragment (Fab) library and diversified other loops using a tailored diversity strategy. This structure-directed Fab library and a naı̈ve synthetic Fab library were used to select Fabs against EGFR. Both libraries yielded high affinity Fabs that bound to overlapping epitopes on cell-surface EGFR, inhibited receptor activation, and targeted epitopes distinct from those of cetuximab and panitumumab. Epitope mapping experiments revealed complex sites of interaction, comprised of domains I and II but not exclusively localized to the receptor dimerization loop. These results validate the grafting approach for designing Fab libraries and also underscore the versatility of naı̈ve synthetic libraries.

Investigating Structural Drivers of Antigen Specificity

Cellular immunity and new immunotherapeutic approaches rely on interactions between T‐cell receptors (TCRs) and the peptide‐major histocompatibility complex (pMHC) to initiate successful immune responses. Achieving peptide specificity by the TCR while simultaneously engaging the pMHC is critical for proper recognition and T‐cell activation. Although early studies emphasized the role of CDR3α and CDR3β loops in determining antigen specificity, more recent work has shown that antigen specificity arises from a cooperative interplay between germline and hypervariable loops and the pMHC surface. To better understand the determinants of TCR specificity, we have been studying engineered variants of the human TCR A6, which recognizes the viral peptide Tax presented by the class I MHC HLA‐A2, but has also been re‐engineered to recognize the tumor associated antigen MART‐1 with high specificity. An additional variant shows a surprising loss of specificity, recognizing a large range of structurally and chemically distinct peptide/HLA‐A2 complexes with high affinity. Here, we describe the structural and biochemical features that correlate with this loss in specificity and discuss the implications for how TCRs achieve specificity and how it can be manipulated.

Short Communication: Manα1-2Man-Binding Anti-HIV Lectins Enhance the Exposure of V2i and V3 Crown Neutralization Epitopes on the V1/V2 and V3 Hypervariable Loops of HIV-1 Envelope

This study aimed to explore the contribution of high-mannose glycans in the masking of conserved V3 crown (GPG) and V2i epitopes on the hypervariable loops of most exposed distal surface of HIV-1 Env. Using lectins specific to Manα1-2Man residue containing Man6-9GlcNAc2 glycans extensively decorating HIV-1 Env, we found that Manα1-2Man-binding lectins enhance the exposure of these partially and transiently exposed epitopes and consequentially increase the neutralization strength of antibodies against these epitopes.

An Engineered Switch in T Cell Receptor Specificity Leads to an Unusual but Functional Binding Geometry

Utilizing a diverse binding site, Tcell receptors (TCRs) specifically recognize a composite ligand comprised of a foreign peptide and a major histocompatibility complex protein (MHC). To help understand the determinants of TCR specificity, we studied a parental and engineered receptor whose peptide specificity had been switched via molecular evolution. Altered specificity was associated with a significant change in TCR-binding geometry, but this did not impact the ability of the TCR to signal inan antigen-specific manner. The determinants of binding and specificity were distributed among contact and non-contact residues in germline and hypervariable loops, and included disruption of key TCR-MHC interactions that bias αβ TCRs toward particular binding modes. Sequence-fitness landscapes identified additional mutations that further enhanced specificity. Our results demonstrate that TCR specificity arises from the distributed action of numerous sites throughout the interface, with significant implications for engineering therapeutic TCRs with novel and functional recognition properties.

13. Functional Role of Adenovirus Penton in Modulating In Vivo Properties of Liver-Targeted and Liver-Detargeted Adenovirus Variants

Pharmacokinetic studies of adenovirus (Ad) vectors after intravascular delivery demonstrate that the majority of an administered virus dose is rapidly sequestered from the circulation by the liver. The molecular determinants that target Ad to the liver have been found on each of the virus capsid proteins, including hexon, penton, and fiber. The penton protein in the majority of Ad serotypes comprises a hypervariable loop containing an RGD motif that has been shown to interact with a number of cellular integrins upon virus entry into the cell. The binding of penton to integrins triggers virus internalization and relaxes the virus structure for subsequent endosomal disassembly. However, RGD motif interaction with integrins on tissue macrophages leads to production of inflammatory cytokines and significantly contributes to systemic Ad toxicity. Despite its importance for the virus entry into the cell, the penton is not well employed in the development of therapeutic Ad vectors. Therefore, in this study we evaluated strategies for modulating Ad penton interactions with cellular integrins. Ad vectors were created bearing substitutions of the RGD motif with mimetic non-RGD-containing peptides derived from extracellular proteins laminin 1 and laminin 3. These peptides are interacting with a different subset of integrins compared to those binging penton RGD motif. Unlike wild type (WT) Ad5, these penton-modified vectors were able to utilize β4 integrin for cell entry. The in vivo studies demonstrated that penton-modified Ads cannot activate a full inflammatory cascade and their in vivo toxicity, therefore, is dramatically reduced. Although the penton-mutated vectors were infecting a set of cancer cell lines in vitro and hepatocytes in vivo with a similar efficacy as the WT Ad5, interactions of virus with Kupffer cells and virus accumulation in the liver after intravascular vector delivery remain similar for penton-mutated and WT Ad vector. We also developed Ad vectors containing mutations abrogating virus interactions with all types of liver cells. Additionally to penton modifications, we introduced a T425A mutation in the hexon hyper variable loop HVR7 to completely abrogate virus interaction with blood coagulation FX and prevent transduction of hepatocytes. Furthermore, a set of mutations in the hexon HVR1 region were introduced to prevent Ad uptake by Kupffer cells. Due to penton modifications these vectors had very low toxicity and combinations of three mutations diverted the vector away from the liver. These data demonstrate that RGD loop modification represents a useful approach for introducing beneficial properties to the Ad vectors for greatly reducing their systemic toxicity and enable liver de-targeting. CryoEM analyses are currently underway to identify the structural differences in the capsid between the penton mutants and the wild type Ad.

Distal M domain of cobra ADAM-like metalloproteinase mediates the binding of positively charged cysteine-rich domain to αvβ3 integrin in the suppression of cell migration

We have previously identified two new P-III type ADAM-like snake venom metalloproteinases (SVMPs), i.e., atragin and kaouthiagin-like, from Taiwan cobra venom and determined their 3D structures with a distinct C- and I-shaped metalloproteinase/disintegrin-like/cysteine-rich (MDC) modular architecture. Herein, we investigated their functional targets to elucidate the role of cobra SVMPs in perturbing wound healing in snakebite victims. We showed that the non-RGD (Arg-Gly-Asp) C-shaped SVMP atragin binds about ten-fold stronger than the RGD-containing I-shaped SVMP kaouthiagin-like to αvβ3 integrin in the surface-immobilized form. Atragin binds to αvβ3 integrin through a novel interaction mode involving distal M and C domains via the RRN sequence motif in the hyper variable loop. In a cell adhesion assay, the adhesion of fibroblasts to atragin was mediated by αvβ3 integrin. Furthermore, atragin inhibited wound healing and suppressed cell migration in a αvβ3 integrin-dependent manner. These results, together with our previous demonstration of non-cytotoxic cobra CTX A5 in targeting αvβ3 integrin, suggest that cobra venom consists of several non-RGD toxins with integrin-binding specificity that could perturb wound healing in snakebite victims.

A new EV71 VP3 epitope in norovirus P particle vector displays neutralizing activity and protection in vivo in mice

Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16), as the main agents causing hand, foot and mouth disease (HFMD), have become a serious public health concern in the Asia-Pacific region. Recently, various neutralizing B cell epitopes of EV71 were identified as targets for promising vaccine candidates. Structural studies of Picornaviridae indicated that potent immunodominant epitopes typically lie in the hypervariable loop of capsid surfaces. However, cross-neutralizing antibodies and cross-protection between EV71 and CVA16 have not been observed. Therefore, we speculated that divergent sequences of the two viruses are key epitopes for inducing protective neutralizing responses. In this study, we selected 10 divergent epitope candidates based on alignment of the EV71 and CVA16 P1 amino acid sequences using the Multalin interface page, and these epitopes are conserved among all subgenotypes of EV71. Simultaneously, by utilizing the norovirus P particle as a novel vaccine delivery carrier, we identified the 71-6 epitope (amino acid 176–190 of VP3) as a conformational neutralizing epitope against EV71 in an in vitro micro-neutralization assay as well as an in vivo protection assay in mice. Altogether, these results indicated that the incorporation of the 71-6 epitope into the norovirus P domain can provide a promising candidate for an effective synthetic peptide-based vaccine against EV71.

Recombination of the epsilon determinant and corneal tropism: Human adenovirus species D types 15, 29, 56, and 69

Viruses within human adenovirus species D (HAdV-D) infect epithelia at essentially every mucosal site. Hypervariable loops 1 and 2 of the hexon capsid protein contain epitopes that together form the epsilon determinant for serum neutralization. We report our analyses comparing HAdV-D15, 29, 56, and the recently identified type 69, each with highly similar hexons and the same serum neutralization profile, but otherwise disparate genomes. Of these, only HAdV-D type 56 is associated with epidemic keratoconjunctivitis (EKC), a severe infection of ocular surface epithelium and underlying corneal stroma. In the mouse adenovirus keratitis model, all four viruses induced inflammation. However, HAdV-D56 entry into human corneal epithelial cells and fibroblasts in vitro dramatically exceeded that of the other three viruses. We conclude that the hexon epsilon determinant is not a prime contributor to corneal tropism.

O17.2 Characterising clade-specific virus-host interactions in hiv infected clinically asymptomatic and aids presenting subjects

<h3>Introduction</h3> The utilised co-receptor is indicative of the clinical progression in HIV infected subjects. Differences in clades are known to impact the outcome of HIV infection. In this study, we investigated the utilised co-receptor and N-glycosylation sites in clinically asymptomatic and AIDS presenting subjects. <h3>Materials and methods</h3> A total of 1,538 nucleotide sequences encompassing the hyper-variable V3 loop of HIV-1, from clinically asymptomatic and AIDS presenting subjects were downloaded from the Los Alamaos Database, which belonged to clades A, B, C and D of HIV-1. Co-receptor prediction was performed using web-based tools <i>PSSM</i> and <i>(ds) Kernel</i>. Numbers of N-glycosylation sites were also calculated using the ‘<i>N-glycosite</i>’ tool. <h3>Results</h3> CCR5 was the utilised co-receptor in 97% (n = 200) of asymptomatic individuals of clade A and 96.5% (n = 199) of AIDS presenting subjects. In B-clade, 98.9% (n = 194) subjects in asymptomatic group were CCR5 utilising, and 83.5% of AIDS presenting subjects were CCR5 utilising (n = 163, CXCR4 were 22.3%, n = 47). In C-clade the CCR5 was utilised in 193 subjects (asymptomatic, n = 200), and 142 (AIDS presenting, n = 148) utilised both co-receptors (dual co-tropic), and in D clade the co-receptor utilised in 55% subjects was CCR5, n = 154 (CXCR4 in 45% subjects, n = 126), and 81% (n = 198) AIDS presenting subjects utilised CCR5, and 19% utilised CXCR4. Percentage of subjects exhibiting N-glycosylation sites also varied among clades with decrease in number of sites in some and increase in others, when compared between the two clinical categories. <h3>Conclusions</h3> Co-receptor switching and addition of N-glycosylation sites does not seem to occur universally in all clades studied. The number of N-Glycosylation sites is also not increased from clinically asymptomatic to AIDS presenting subjects. In conclusion, co-receptor switching (from CCR5 to CXCR4) and increase in number of N-glycosylation sites, which are predictive of disease progression, does to occur in all clades universally, thus indicating clade specific responses.

The Shark Strikes Twice: Hypervariable Loop 2 of Shark IgNAR Antibody Variable Domains and Its Potential to Function as an Autonomous Paratope.

In this present study, we engineered hypervariable loop 2 (HV2) of the IgNAR variable domain in a way that it solely facilitates antigen binding, potentially functioning as an autonomous paratope. For this, the surface-exposed loop corresponding to HV2 was diversified and antigen-specific variable domain of IgNAR antibody (vNAR) molecules were isolated by library screening using yeast surface display (YSD) as platform technology. An epithelial cell adhesion molecule (EpCAM)-specific vNAR was used as starting material, and nine residues in HV2 were randomized. Target-specific clones comprising a new HV2-mediated paratope were isolated against cluster of differentiation 3ε (CD3ε) and human Fcγ while retaining high affinity for EpCAM. Essentially, we demonstrate that a new paratope comprising moderate affinities against a given target molecule can be engineered into the vNAR scaffold that acts independent of the original antigen-binding site, composed of complementarity-determining region 3 (CDR3) and CDR1.

LYRA, a webserver for lymphocyte receptor structural modeling.

The accurate structural modeling of B- and T-cell receptors is fundamental to gain a detailed insight in the mechanisms underlying immunity and in developing new drugs and therapies. The LYRA (LYmphocyte Receptor Automated modeling) web server (http://www.cbs.dtu.dk/services/LYRA/) implements a complete and automated method for building of B- and T-cell receptor structural models starting from their amino acid sequence alone. The webserver is freely available and easy to use for non-specialists. Upon submission, LYRA automatically generates alignments using ad hoc profiles, predicts the structural class of each hypervariable loop, selects the best templates in an automatic fashion, and provides within minutes a complete 3D model that can be downloaded or inspected online. Experienced users can manually select or exclude template structures according to case specific information. LYRA is based on the canonical structure method, that in the last 30 years has been successfully used to generate antibody models of high accuracy, and in our benchmarks this approach proves to achieve similarly good results on TCR modeling, with a benchmarked average RMSD accuracy of 1.29 and 1.48 Å for B- and T-cell receptors, respectively. To the best of our knowledge, LYRA is the first automated server for the prediction of TCR structure.

The hypervariable region 4 (HV4) and position 93 of the α chain modulate CD1d-glycolipid binding of iNKT TCRs.

TCRs of invariant NKT (iNKT) cells bind α-galactosylceramide (αGC) loaded CD1d in a highly conserved fashion and show a characteristic TCR gene usage: An "invariant" α chain with a canonical AV14/AJ18 rearrangement in mice (AV24/AJ18 in humans) is paired with β chains containing characteristic Vβ segments. In the rat, a multimember AV14 gene family increases the variability within this system. This study characterizes CD1d binding of rat AV14 gene segments in TCR transductants as well as CD1d binding and iNKT TCR expression of expanded polyclonal F344 rat iNKT populations. It defines an important role of position 93 at the V-J transition for TCR avidity and species cross-reactivity of the rat iNKT TCR. Furthermore, for the first time we identified variability within the fourth hypervariable loop (HV4) of the α chain as a modulator of CD1d:αGC binding in rat and mouse. Additionally, we confirmed the importance of the CDR2β for CD1d:αGC binding, but also show that the CDR3β may even have opposite effects on binding depending on the pairing α chain. Altogether, we characterized naturally occurring sources of variability for the iNKT TCR and speculate that they rather level than increase the largely germline encoded differences of iNKT TCR ligand avidity.

45. Adenovirus Variant With a Defined Set of Genetic Mutations in the Penton and Hexon Escapes Liver Sequestration After Intravascular Administration

Adenoviruses (Ad) are promising vectors for therapeutic interventions in humans. Despite significant knowledge regarding the biology of Ad interactions with cells in vitro, the molecular mechanisms governing in vivo Ad infectivity and bio-distribution remain poorly understood. Pharmacokinetic studies of Ad vectors after intravascular delivery demonstrate that the majority of an administered virus dose is rapidly sequestered from the circulation by the liver. Earlier we showed that Ad particles are distributed in liver tissue amongst three distinct cellular compartments, namely i) parenchymal liver cells – hepatocytes, ii) hepatic residential macrophages, Kupffer cells, and iii) hepatic sinusoid endothelial cells. Importantly, we found that the ablation of Ad interaction with only one of these cellular compartments cannot prevent virus trapping the liver shortly after intravascular virus injection and, likely, results in compensatory re-distribution of the virus among two remaining cellular compartments, thus functionally ensuring the quantitative removal of the virus from the blood. We speculated that to prevent Ad sequestration by the liver after intravascular administration, simultaneous ablation of virus interaction with all three cellular compartments of the liver is ultimately required. To evaluate this assumption experimentally, we introduced in a single human Ad5-based vector specific mutations that abrogate virus interactions with one, two, or all three hepatocellular compartments. To ablate Ad interaction with hepatocytes in vivo, we introduced T425A mutation in hexon hyper-variable loop HVR7 that completely abrogates virus interaction with blood coagulation FX. To prevent Ad interaction with liver sinusoid endothelial cells, we introduced a substitution of the RGD-motif-containing penton loop for an iso-functional integrin-interacting non-RGD-containing peptide. We also serendipitously found that the deletion of hexon HVR1 region, in addition to penton RGD-loop substitution and T425A HVR7 mutation, results in the reduction of virus interaction with Kupffer cells. Intravenous injection into mice of Ad variants containing individual mutations either in the penton or hexon or a vector with only two mutations in a single vector does not prevent sequestration of resultant Ad in the liver. However, Ad variant, containing all three mutations simultaneously, completely escaped being sequestered in the liver after intravascular injection and, instead, 20-fold higher amounts of the virus were recovered from the spleen, indicating the major change in virus bio-distribution, compared to unmodified vectors with a wild-type capsid, or vectors with individual mutations in either penton or hexon. To our knowledge this is the first demonstration of a feasibility of constructing Ad variants that would escape liver sequestration after intravascular administration. These variants may prove to be a useful platform for gene delivery to extra-hepatic cells and/or targeting disseminated metastatic cancers.

Trimeric HIV Env provides epitope occlusion mediated by hypervariable loops.

Hypervariable loops of HIV-1 Env protein gp120 are speculated to play roles in the conformational transition of Env to the receptor binding-induced metastable state. Structural analysis of full-length Env-based immunogens, containing the entire V2 loop, displayed tighter association between gp120 subunits, resulting in a smaller trimeric diameter than constructs lacking V2. A prominent basal quaternary location of V2 and V3′ that challenges previous reports would facilitate gp41-independent gp120-gp120 interactions and suggests a quaternary mechanism of epitope occlusion facilitated by hypervariable loops. Deletion of V2 resulted in dramatic exposure of basal, membrane-proximal gp41 epitopes, consistent with its predicted basal location. The structural features of HIV-1 Env characterized here provide grounds for a paradigm shift in loop exposure and epitope occlusion, while providing substantive rationale for epitope display required for elicitation of broadly neutralizing antibodies, as well as substantiating previous pertinent literature disregarded in recent reports.

Temporal increase in HIV-1 non-R5 tropism frequency among antiretroviral-naive patients from northern Poland.

IntroductionSequencing of the third hypervariable loop allows to identify genotype-based HIV tropism. R5-tropic viruses associated with early stages of infection are preferentially transmitted, while non-R5 HIV-1 tropism has been associated with severe immunodeficiency and lower lymphocyte CD4 nadir and may reflect delayed HIV diagnosis. In this study, we investigate the changes in tropism frequency from 2007 to 2013.Materials and MethodsStudy included 194 patients with confirmed HIV infection linked to care in 2007–2013. Baseline plasma samples from treatment naive patients were used for HIV-1 genotypic tropism assessment based on triplicate V3 loop sequencing. Non-R5 tropism prediction thresholds were assigned using a false positive rate (FPR) of 10% and 5.75% FPR and associated with clinical and laboratory data (age, gender, date of HIV diagnosis, route of transmission, CDC clinical category at diagnosis, pretreatment HIV viral load, baseline and nadir lymphocyte CD4 counts). For statistics, chi-square and Mann–Whitney U tests were used, time trends were examined using logistic regression (R statistical platform, v. 3.1.0) for binary variables and linear regression for continuous ones.ResultsOverall non-R5 tropism frequency for the 5.75% FPR was 15.5% and 27.8% for 10% FPR. Frequency of the non-R5 tropism predicted using 5.75% FPR increased significantly from 2007 (0%) to 2013 (25%) [OR: 1.44 (95% CI 1.14–1.86), p=0.003, rough slope +3.89%/year] (Figure 1a). With 10% FPR, the frequency changed from 7% (2007) to 33% (2013) [OR: 1.17 (95% CI 0.99–1.39), p=0.054, rough slope +3.0%/year] (Figure 1b). Baseline lymphocyte CD4 count and nadir, as well as pretreatment HIV-1 viral loads were stable over time of observation (r=0.014, p=0.84; r=0.13, p=0.085; r=0.016, p=0.83 for CD4 baseline, nadir and HIV load, respectively). Frequency of AIDS at HIV diagnosis increased from 21.4% in 2007 to 38.0% in 2013, however trend over time was insignificant [OR: 1.1 (95% CI 0.95–1.31), p=0.19]. Temporal trends for HIV transmission route, gender, non-B variant frequencies also were not significant.ConclusionsR5 tropism predominates among the treatment naive individuals but increase in the frequency of non-R5 tropic variants may limit clinical efficacy of the coreceptor inhibitors. Increased prevalence of non-R5 HIV-1 may be related to late care entry and higher number of AIDS diagnoses in the recent years.