pairwise-growth-competition-assay-for-determining-replication-fitness

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral populations to those present above ~1%. To improve detection of rare variants, we varied PCR enzymes and conditions to identify those that combined high sensitivity with a low error rate. Substitution errors were found to vary up to 3-fold between the different enzymes tested. The sensitivity of each enzyme, which impacts the number of templates amplified for pyrosequencing, was shown to vary, although not consistently across genes and different samples. We also describe an amplicon-based method to improve the consistency of read coverage over stretches of the HIV-1 genome. Twenty-two primers were designed to amplify 11 overlapping amplicons in the HIV-1 clade B gag-pol and env gp120 coding regions to encompass 4.7 kb of the viral genome per sample at sensitivities as low as 0.01-0.2%.

Improved detection of rare HIV-1 variants using 454 pyrosequencing.

ViroBLAST is a stand-alone BLAST web interface for nucleotide and amino acid sequence similarity searches. It extends the utility of BLAST to query against multiple sequence databases and user sequence datasets, and provides a friendly output to easily parse and navigate BLAST results. ViroBLAST is readily useful for all research areas that require BLAST functions and is available online and as a downloadable archive for independent installation.

ViroBLAST: a stand-alone BLAST web server for flexible queries of multiple databases and user's datasets.

HIV-1 over time: fitness loss or robustness gain?

Human immunodeficiency virus type 1 (HIV-1) mutations that confer escape from cytotoxic T-lymphocyte (CTL) recognition can sometimes result in lower viral fitness. These mutations can then revert upon transmission to a new host in the absence of CTL-mediated immune selection pressure restricted by the HLA alleles of the prior host. To identify these potentially critical recognition points on the virus, we assessed HLA-driven viral evolution using three phylogenetic correction methods across full HIV-1 subtype C proteomes from a cohort of 261 South Africans and identified amino acids conferring either susceptibility or resistance to CTLs. A total of 558 CTL-susceptible and -resistant HLA-amino acid associations were identified and organized into 310 immunological sets (groups of individual associations related to a single HLA/epitope combination). Mutations away from seven susceptible residues, including four in Gag, were associated with lower plasma viral-RNA loads (q < 0.2 [where q is the expected false-discovery rate]) in individuals with the corresponding HLA alleles. The ratio of susceptible to resistant residues among those without the corresponding HLA alleles varied in the order Vpr > Gag > Rev > Pol > Nef > Vif > Tat > Env > Vpu (Fisher's exact test; P < or = 0.0009 for each comparison), suggesting the same ranking of fitness costs by genes associated with CTL escape. Significantly more HLA-B (chi(2); P = 3.59 x 10(-5)) and HLA-C (chi(2); P = 4.71 x 10(-6)) alleles were associated with amino acid changes than HLA-A, highlighting their importance in driving viral evolution. In conclusion, specific HIV-1 residues (enriched in Vpr, Gag, and Rev) and HLA alleles (particularly B and C) confer susceptibility to the CTL response and are likely to be important in the development of vaccines targeted to decrease the viral load.

HLA class I-driven evolution of human immunodeficiency virus type 1 subtype c proteome: immune escape and viral load.

A mean of 9-10 years of human immunodeficiency virus type 1 (HIV-1) infection elapse before clinical AIDS develops in untreated persons, but this rate of disease progression varies substantially among individuals. To investigate host genetic determinants of the rate of progression to clinical AIDS, we performed a multistage genomewide association study.

Multistage genomewide association study identifies a locus at 1q41 associated with rate of HIV-1 disease progression to clinical AIDS.

DIVEIN is a web interface that performs automated phylogenetic and other analyses of nucleotide and amino acid sequences. Starting with a set of aligned sequences, DIVEIN estimates evolutionary parameters and phylogenetic trees while allowing the user to choose from a variety of evolutionary models; it then reconstructs the consensus (CON), most recent common ancestor (MRCA), and center of tree (COT) sequences. DIVEIN also provides tools for further analyses, including condensing sequence alignments to show only informative sites or private mutations; computing phylogenetic or pairwise divergence from any user-specified sequence (CON, MRCA, COT, or existing sequence from the alignment); computing and outputting all genetic distances in column format; calculating summary statistics of diversity and divergence from pairwise distances; and graphically representing the inferred tree and plots of divergence, diversity, and distance distribution histograms. DIVEIN is available at http://indra.mullins.microbiol.washington.edu/DIVEIN.

DIVEIN: a web server to analyze phylogenies, sequence divergence, diversity, and informative sites.

Despite high potential for HIV-1 genetic variation, the emergence of some mutations is constrained by fitness costs, and may be associated with compensatory amino acid (AA) co-variation. To characterize the interplay between Cytotoxic T Lymphocyte (CTL)-mediated pressure and HIV-1 evolutionary pathways, we investigated AA co-variation in Gag sequences obtained from 449 South African individuals chronically infected with HIV-1 subtype C.

Amino-acid co-variation in HIV-1 Gag subtype C: HLA-mediated selection pressure and compensatory dynamics.

We analyzed HIV-1 genome sequences from 68 newly infected volunteers in the STEP HIV-1 vaccine trial. To determine whether the vaccine exerted selective T cell pressure on breakthrough viruses, we identified potential T cell epitopes in the founder sequences and compared them to epitopes in the vaccine. We found greater distances to the vaccine sequence for sequences from vaccine recipients than from placebo recipients. The most significant signature site distinguishing vaccine from placebo recipients was Gag amino acid 84, a site encompassed by several epitopes contained in the vaccine and restricted by human leukocyte antigen (HLA) alleles common in the study cohort. Moreover, the extended divergence was confined to the vaccine components of the virus (HIV-1 Gag, Pol and Nef) and not found in other HIV-1 proteins. These results represent what is to our knowledge the first evidence of selective pressure from vaccine-induced T cell responses on HIV-1 infection in humans.

Genetic impact of vaccination on breakthrough HIV-1 sequences from the STEP trial.

Wir analysierten HIV-1-Genomsequenzen von 68 neu infizierten Freiwillige im Schritt Impfstudie HIV-1. Um festzustellen, ob der Impfstoff selektiv T-Zellen auf Durchbruch Viren Druck, wir identifiziert m&ouml;gliche T-Zelle Epitopes in den Gr&uuml;nder-Sequenzen und verglichen sie mit Epitopes in den Impfstoff. Wir fanden gr&ouml;&szlig;ere Entfernungen auf die Impfstoff-Sequenz f&uuml;r Sequenzen von Impfstoff-Empf&auml;nger als der Placebo-Empf&auml;nger. Die bedeutendste Signatur-Website Impfstoff von Placebo-Empf&auml;nger zu unterscheiden war Gag Aminos&auml;ure 84, eine Website umfasste durch mehrere Epitope in dem Impfstoff enthalten und eingeschr&auml;nkt durch human Leukocyte Antigen (HLA) Allele h&auml;ufig in der Studie-Kohorte. Dar&uuml;ber hinaus wurde die erweiterte Divergenz beschr&auml;nkt sich auf die Impfstoff-Komponenten des Virus (HIV-1 Gag, Pol und Nef) und in anderen HIV-1 Proteine nicht gefunden. Diese Ergebnisse stellen dar, was ist unseres Wissens der erste Beweis der selektiven Druck von Impfstoff-induzierte T-Zell-Antworten auf HIV-1-Infektion beim Menschen.

Genetische Auswirkungen der Impfung auf Durchbruch-HIV-1 Sequenzen der STEP-Studie.

HIV-1 transmission and viral evolution in the first year of infection were studied in 11 individuals representing four transmitter-recipient pairs and three independent seroconverters. Nine of these individuals were enrolled during acute infection; all were men who have sex with men (MSM) infected with HIV-1 subtype B. A total of 475 nearly full-length HIV-1 genome sequences were generated, representing on average 10 genomes per specimen at 2 to 12 visits over the first year of infection. Single founding variants with nearly homogeneous viral populations were detected in eight of the nine individuals who were enrolled during acute HIV-1 infection. Restriction to a single founder variant was not due to a lack of diversity in the transmitter as homogeneous populations were found in recipients from transmitters with chronic infection. Mutational patterns indicative of rapid viral population growth dominated during the first 5 weeks of infection and included a slight contraction of viral genetic diversity over the first 20 to 40 days. Subsequently, selection dominated, most markedly in env and nef. Mutants were detected in the first week and became consensus as early as day 21 after the onset of symptoms of primary HIV infection. We found multiple indications of cytotoxic T lymphocyte (CTL) escape mutations while reversions appeared limited. Putative escape mutations were often rapidly replaced with mutually exclusive mutations nearby, indicating the existence of a maturational escape process, possibly in adaptation to viral fitness constraints or to immune responses against new variants. We showed that establishment of HIV-1 infection is likely due to a biological mechanism that restricts transmission rather than to early adaptive evolution during acute infection. Furthermore, the diversity of HIV strains coupled with complex and individual-specific patterns of CTL escape did not reveal shared sequence characteristics of acute infection that could be harnessed for vaccine design.

Demographic processes affect HIV-1 evolution in primary infection before the onset of selective processes.

HIV-1-&Uuml;bertragung und virale Evolution im ersten Jahr der Infektion wurden in 11 Personen, vier Sender-Empf&auml;nger-Paare und drei unabh&auml;ngige Seroconverters untersucht. Neun dieser Personen wurden w&auml;hrend der akuten Infektion aufgenommen; Alle waren M&auml;nner, die Sex mit M&auml;nnern (MSM) angesteckt mit HIV-1 Subtyp B. Insgesamt 475 fast in HIV-1 Genom, die Sequenzen generiert wurden, repr&auml;sentieren im Durchschnitt 10 Genome pro Probe 2 bis 12 Besuche im ersten Jahr der Infektion. Einzelne Gr&uuml;ndung Varianten mit fast homogene viralen Populationen wurden in acht der neun Personen festgestellt, die w&auml;hrend der akuten HIV-1 Infektion eingeschrieben waren. Beschr&auml;nkung auf eine einzige Gr&uuml;nder-Variante war nicht aufgrund mangelnder Vielfalt im Sender wie homogene Bev&ouml;lkerungen im Empf&auml;nger von Sendern mit chronischen Infektion gefunden werden konnten. Bezeichnend f&uuml;r schnelle virale Bev&ouml;lkerungswachstum teilhabende Muster dominiert in den ersten 5 Wochen der Infektion und enthalten einen leichten R&uuml;ckgang um virale genetische Vielfalt in den ersten 20 bis 40 Tagen. Anschlie&szlig;end dominierte Auswahl, die meisten deutlich in Env und Nef. Mutanten in der ersten Woche wurden erkannt und wurde bereits Konsens als 21. Tag nach Beginn der Symptome der prim&auml;ren HIV-Infektion. Wir fanden mehrere Indikationen von zytotoxischen T-Lymphozyten (CTL)-Escape-Mutationen, w&auml;hrend Kellergalerie beschr&auml;nkt erschien. Vermeintliche Flucht Mutationen wurden oft rasch mit sich gegenseitig ausschlie&szlig;enden Mutationen in der N&auml;he, angibt, die Existenz eines Reifungs-Escape-Prozesses m&ouml;glicherweise in Anpassung virale Fitness-Einschr&auml;nkungen oder Immunantworten gegen neue Varianten ersetzt. Wir haben gezeigt, dass Einrichtung von HIV-1 Infektion ist wahrscheinlich durch einen biologischen Mechanismus, der &Uuml;bertragung beschr&auml;nkt und nicht zu fr&uuml;h adaptive Evolution w&auml;hrend der akuten Infektion. Dar&uuml;ber hinaus die Vielfalt von HIV-St&auml;mmen gepaart mit komplexen und spezielle Muster der CTL Flucht ergab keine Sequenz Merkmale der akuten Infektion freigegeben, die f&uuml;r Impfstoff-Design genutzt werden k&ouml;nnte.

Demographische Prozesse beeinflussen HIV-1-Entwicklung der prim&auml;ren Infektion vor dem Einbruch des selektiven Prozesse.

The potential for changing HIV-1 virulence has significant implications for the AIDS epidemic, including changing HIV transmission rates, rapidity of disease progression, and timing of ART. Published data to date have provided conflicting results.

Is the virulence of HIV changing? A meta-analysis of trends in prognostic markers of HIV disease progression and transmission.

The RV144 trial demonstrated 31% vaccine efficacy at preventing human immunodeficiency virus (HIV)-1 infection. Antibodies against the HIV-1 envelope variable loops 1 and 2 (Env V1 and V2) correlated inversely with infection risk. We proposed that vaccine-induced immune responses against V1/V2 would have a selective effect against, or sieve, HIV-1 breakthrough viruses. A total of 936 HIV-1 genome sequences from 44 vaccine and 66 placebo recipients were examined. We show that vaccine-induced immune responses were associated with two signatures in V2 at amino acid positions 169 and 181. Vaccine efficacy against viruses matching the vaccine at position 169 was 48% (confidence interval 18% to 66%; P = 0.0036), whereas vaccine efficacy against viruses mismatching the vaccine at position 181 was 78% (confidence interval 35% to 93%; P = 0.0028). Residue 169 is in a cationic glycosylated region recognized by broadly neutralizing and RV144-derived antibodies. The predicted distance between the two signature sites (21 ± 7 Å) and their match/mismatch dichotomy indicate that multiple factors may be involved in the protection observed in RV144. Genetic signatures of RV144 vaccination in V2 complement the finding of an association between high V1/V2-binding antibodies and reduced risk of HIV-1 acquisition, and provide evidence that vaccine-induced V2 responses plausibly had a role in the partial protection conferred by the RV144 regimen.

Increased HIV-1 vaccine efficacy against viruses with genetic signatures in Env V2.

Pyrosequencing technology provides an important new approach to more extensively characterize diverse sequence populations and detect low frequency variants. However, the promise of this technology has been difficult to realize, as careful correction of sequencing errors is crucial to distinguish rare variants (∼1%) in an infected host with high sensitivity and specificity.

Indel and Carryforward Correction (ICC): a new analysis approach for processing 454 pyrosequencing data.

Cell culture growth competition assays of human immunodeficiency virus type 1 (HIV-1) are used to estimate viral fitness and quantify the impact of mutations conferring drug resistance and immunological escape. A comprehensive study of growth competition assays was conducted and identified experimental parameters that can impact measurements of relative fitness including multiplicity of infection, viral input ratio, number, timing and interval of time points used to evaluate selective outgrowth, and the algorithm for calculating fitness values. An optimized protocol is developed here that is a multi-point growth competition assay that resolves reproducibly small differences in viral fitness. The optimized protocol uses an MOI of 0.005, a consistent ratio of mutant: parental viruses (70:30), and a multipoint [1+s 4,7] algorithm that uses data points within the logarithmic phase of viral growth for assessing fitness differences.

Factors affecting relative fitness measurements in pairwise competition assays of human immunodeficiency viruses.

The RV144 HIV-1 vaccine trial demonstrated partial efficacy of 31% against HIV-1 infection. Studies into possible correlates of protection found that antibodies specific to the V1 and V2 (V1/V2) region of envelope correlated inversely with infection risk and that viruses isolated from trial participants contained genetic signatures of vaccine-induced pressure in the V1/V2 region. We explored the hypothesis that the genetic signatures in V1 and V2 could be partly attributed to selection by vaccine-primed T cells. We performed a T-cell-based sieve analysis of breakthrough viruses in the RV144 trial and found evidence of predicted HLA binding escape that was greater in vaccine versus placebo recipients. The predicted escape depended on class I HLA A*02- and A*11-restricted epitopes in the MN strain rgp120 vaccine immunogen. Though we hypothesized that this was indicative of postacquisition selection pressure, we also found that vaccine efficacy (VE) was greater in A*02-positive (A*02(+)) participants than in A*02(-) participants (VE = 54% versus 3%, P = 0.05). Vaccine efficacy against viruses with a lysine residue at site 169, important to antibody binding and implicated in vaccine-induced immune pressure, was also greater in A*02(+) participants (VE = 74% versus 15%, P = 0.02). Additionally, a reanalysis of vaccine-induced immune responses that focused on those that were shown to correlate with infection risk suggested that the humoral responses may have differed in A*02(+) participants. These exploratory and hypothesis-generating analyses indicate there may be an association between a class I HLA allele and vaccine efficacy, highlighting the importance of considering HLA alleles and host immune genetics in HIV vaccine trials.

Analysis of HLA A*02 association with vaccine efficacy in the RV144 HIV-1 vaccine trial.

The modest protection afforded by the RV144 vaccine offers an opportunity to evaluate its mechanisms of protection. Differences between HIV-1 breakthrough viruses from vaccine and placebo recipients can be attributed to the RV144 vaccine as this was a randomized and double-blinded trial. CD8 and CD4 T cell epitope repertoires were predicted in HIV-1 proteomes from 110 RV144 participants. Predicted Gag epitope repertoires were smaller in vaccine than in placebo recipients (p = 0.019). After comparing participant-derived epitopes to corresponding epitopes in the RV144 vaccine, the proportion of epitopes that could be matched differed depending on the protein conservation (only 36% of epitopes in Env vs 84-91% in Gag/Pol/Nef for CD8 predicted epitopes) or on vaccine insert subtype (55% against CRF01_AE vs 7% against subtype B). To compare predicted epitopes to the vaccine, we analyzed predicted binding affinity and evolutionary distance measurements. Comparisons between the vaccine and placebo arm did not reveal robust evidence for a T cell driven sieve effect, although some differences were noted in Env-V2 (0.022≤p-value≤0.231). The paucity of CD8 T cell responses identified following RV144 vaccination, with no evidence for V2 specificity, considered together both with the association of decreased infection risk in RV 144 participants with V-specific antibody responses and a V2 sieve effect, lead us to hypothesize that this sieve effect was not T cell specific. Overall, our results did not reveal a strong differential impact of vaccine-induced T cell responses among breakthrough infections in RV144 participants.

Article	Year
Improved detection of rare HIV-1 variants using 454 pyrosequencing. PloS one\| PubMed ID: 24098517	2013
ViroBLAST: a stand-alone BLAST web server for flexible queries of multiple databases and user's datasets. Bioinformatics (Oxford, England)\| PubMed ID: 17586542	2007
HIV-1 over time: fitness loss or robustness gain? Nature reviews. Microbiology\| PubMed ID: 17703224	2007
HLA class I-driven evolution of human immunodeficiency virus type 1 subtype c proteome: immune escape and viral load. Journal of virology\| PubMed ID: 18434400	2008
Multistage genomewide association study identifies a locus at 1q41 associated with rate of HIV-1 disease progression to clinical AIDS. The Journal of infectious diseases\| PubMed ID: 20064070	2010
DIVEIN: a web server to analyze phylogenies, sequence divergence, diversity, and informative sites. BioTechniques\| PubMed ID: 20569214	2010
Amino-acid co-variation in HIV-1 Gag subtype C: HLA-mediated selection pressure and compensatory dynamics. PloS one\| PubMed ID: 20824187	2010
Genetic impact of vaccination on breakthrough HIV-1 sequences from the STEP trial. Nature medicine\| PubMed ID: 21358627	2011
Demographic processes affect HIV-1 evolution in primary infection before the onset of selective processes. Journal of virology\| PubMed ID: 21593162	2011
Is the virulence of HIV changing? A meta-analysis of trends in prognostic markers of HIV disease progression and transmission. AIDS (London, England)\| PubMed ID: 22089381	2012
Increased HIV-1 vaccine efficacy against viruses with genetic signatures in Env V2. Nature\| PubMed ID: 22960785	2012
Indel and Carryforward Correction (ICC): a new analysis approach for processing 454 pyrosequencing data. Bioinformatics (Oxford, England)\| PubMed ID: 23900188	2013
Factors affecting relative fitness measurements in pairwise competition assays of human immunodeficiency viruses. Journal of virological methods\| PubMed ID: 23933395	2013
Analysis of HLA A*02 association with vaccine efficacy in the RV144 HIV-1 vaccine trial. Journal of virology\| PubMed ID: 24829343	2014
CD8 and CD4 epitope predictions in RV144: no strong evidence of a T-cell driven sieve effect in HIV-1 breakthrough sequences from trial participants. PloS one\| PubMed ID: 25350851	2014

Brandon Maust

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