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Array comparative genomic hybridization is available for the evaluation of autism spectrum disorders. The diagnostic yield of testing is 5–18% in children with developmental disabilities, including autism spectrum disorders and multiple congenital anomalies. The yield of array comparative genomic hybridization in the adult autism spectrum disorder population is unknown. We performed a retrospective chart review for 40 consecutive patients referred for genetic evaluation of autism from July 2009 through April 2012. Four pediatric patients were excluded. Medical history and prior testing were reviewed. Clinical genetic evaluation and testing were offered to all patients. The study population comprised 36 patients (age range 18–45, mean 25.3 years). An autism spectrum disorder diagnosis was confirmed in 34 of 36 patients by medical record review. One patient had had an abnormal karyotype; none had prior array comparative genomic hybridization testing. Of the 23 patients with autism who underwent array comparative genomic hybridization, 2 of 23 (8.7%) had pathogenic or presumed pathogenic abnormalities and 2 of 23 (8.7%) had likely pathogenic copy-number variants. An additional 5 of 23 (22%) of autism patients had variants of uncertain significance without subclassification. Including one patient newly diagnosed with fragile X syndrome, our data showed abnormal or likely pathogenic findings in 5 of 24 (21%) adult autism patients. Genetic reevaluation in adult autism patients is warranted. Genet Med 16 1 70–77.

Whether human immunodeficiency virus (HIV) in the female genital tract constitutes a genetically distinct compartment from the blood remains an important question, with both clinical and biologic implications because of the role of the genital tract in mother-to-child and woman-to-man sexual HIV transmission. Therefore, determining if unique viruses develop and persist in the genital tract is important for HIV prophylaxis. Similarly, understanding HIV evolution in tissues that transmit the infection, including the female genital tract, is relevant to vaccine development because vaccine-induced immune responses should target these viral strains. During acute infection, a single HIV genotype predominates in the blood of approximately 70% of individuals [1–5], with several viral variants detected in the remainder [6]. Because of the poor fidelity of the viral reverse transcriptase, HIV diversifies within the new host [7–9]. The variants within a subject form multiple genetically distinct clades with ongoing replication have time-ordered structure [8, 10, 11]. Factors hypothesized to promote compartmentalization of HIV between the blood and genital tract and potentially lead to tissue-specific clades and lineages include differences in CD4 or coreceptor expression [12–14], HIV-specific immune pressure [11, 14–19], and selective antiretroviral pressure due to poor penetration into the genital tissues [20, 21] and inflammation from trauma [22] or sexually transmitted infections [13–16, 23–25]. Examination of HIV sequences from the genital tract and blood of >100 women across 9 cross-sectional and 2 longitudinal studies [11, 13, 14, 16–19, 23, 26–29] observed compartmentalization of virus in more than half of these women. Longitudinal studies of HIV sequences [11, 16] have examined a relatively small number of women. One study of cloned sequences from 3 women reported increasing compartmentalization between genital and blood virus over time [11]. A second study used a heteroduplex tracking assay and identified distinct genital variants in one-third of 12 women's paired plasma and genital tract specimens [16]. Our previous cross-sectional analyses of genital tract and blood viral sequences derived by single genome amplification (SGA) observed subpopulations of identical sequences in the blood and/or genital tract of most women [28, 29]. Among individuals with active viral replication, the observation of closely related sequences suggested that compartmentalization was largely due to sampling of a dominant replicating virus, whereas in women with viral replication suppressed by antiretroviral therapy, we hypothesized that monotypic (identical) HIV DNA sequences were likely due to proliferation of cells containing proviruses, as we [28–30] and others [19, 31, 32] have previously reported. Compartmentalization of HIV in these individuals was due to the clades of monotypic or low-diversity sequences (

Professionals without a social work qualification have been involved in the practice teaching of social work students since the days of CCETSW (the former education and training body for social work in the UK). Historically this has always happened more in the voluntary sector. With the advent of the Social Work Degree in England, the 50% increase in demand for placements in a variety of settings has seen reliance on practice teachers who are not social workers.This raises some interesting questions about how professionals who are not social workers should be trained and supported in the role of practice teacher.We will attempt to explore these questions, drawing on responses to a questionnaire sent to a range of practice teachers in a county in northern England as well as feedback obtained from a focus group drawn from respondents to the questionnaire. The participants in the research came from a range of work backgrounds in the voluntary and statutory sectors. The experiences discussed in the research, in the main, relate to the Diploma in Social Work (the former UK qualification) as the degree only started in 2003- 2004. We will refer to research participants as Practice Teachers. The key criterion for involvement was that all participants in the study had had sole responsibility for at least one social work student.

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.

The concentration of human immunodeficiency virus type 1 (HIV-1) is generally lower in breast milk than in blood. Mastitis, or inflammation of the breast, is associated with increased levels of milk HIV-1 and risk of mother-to-child transmission through breastfeeding. We hypothesized that mastitis facilitates the passage of HIV-1 from blood into milk or stimulates virus production within the breast. HIV-1 env sequences were generated from single amplicons obtained from breast milk and blood samples in a cross-sectional study. Viral compartmentalization was evaluated using several statistical methods, including the Slatkin and Maddison (SM) test. Mastitis was defined as an elevated milk sodium (Na + ) concentration. The association between milk Na + and the pairwise genetic distance between milk and blood viral sequences was modeled using linear regression. HIV-1 was compartmentalized within milk by SM testing in 6/17 (35%) specimens obtained from 9 women, but all phylogenetic clades included viral sequences from milk and blood samples. Monotypic sequences were more prevalent in milk samples than in blood samples (22% versus 13%; P = 0.012), which accounted for half of the compartmentalization observed. Mastitis was not associated with compartmentalization by SM testing ( P = 0.621), but Na + was correlated with greater genetic distance between milk and blood HIV-1 populations ( P = 0.041). In conclusion, local production of HIV-1 within the breast is suggested by compartmentalization of virus and a higher prevalence of monotypic viruses in milk specimens. However, phylogenetic trees demonstrate extensive mixing of viruses between milk and blood specimens. HIV-1 replication in breast milk appears to increase with inflammation, contributing to higher milk viral loads during mastitis.

The envelope gene ( env ) of human immunodeficiency virus type 1 (HIV-1) undergoes rapid divergence from the transmitted sequence and increasing diversification during the prolonged course of chronic infection in humans. In about half of infected individuals or more, env evolution leads to expansion of the use of entry coreceptor from CCR5 alone to CCR5 and CXCR4. The stochastic nature of this coreceptor switch is not well explained by host selective forces that should be relatively constant between infected individuals. Moreover, differences in the incidence of coreceptor switching among different HIV-1 subtypes suggest that properties of the evolving virus population drive the switch. We evaluated the functional properties of sequential env clones from a patient with evidence of coreceptor switching at 5.67 years of infection. We found an abrupt decline in the ability of viruses to use CCR5 for entry at this time, manifested by a 1- to 2-log increase in susceptibility to CCR5 inhibitors and a reduced ability to infect cell lines with low CCR5 expression. There was an abnormally rapid 5.4% divergence in env sequences from 4.10 to 5.76 years of infection, with the V3 and V4/V5 regions showing the greatest divergence and evidence of positive selection. These observations suggest that a decline in the fitness of R5 virus populations may be one driving force that permits the emergence of R5X4 variants.

Understanding the characteristics of human immunodeficiency virus (HIV) necessary for infection in a new host is a critical goal for acquired immunodeficiency syndrome (AIDS) research. We studied the characteristics of HIV-1 envelope genes in 38 men in the Multicenter AIDS Cohort Study cohort before seroconversion. We found a range of diversity (0.2%-5.6% [median, 0.86%]), V1-V2 loop length (58-93 aa), and potential N-linked glycosylation sites (n = 2-9). However, at least 46% of the men had replicating virus that appeared to have been derived from a single viral variant. Nearly all variants were predicted to be CCR5 tropic. We found no correlation between these viral characteristics and the HIV outcomes of time to clinical AIDS or death and/or a CD4 cell count

Selecting human immunodeficiency virus (HIV) sequences for inclusion within vaccines has been a difficult problem, as circulating HIV strains evolve relentlessly and become increasingly divergent over time. We report an assessment of this divergence from three perspectives: (i) across different hosts as a function of time of infection, (ii) between donors and recipients in known transmission pairs, and (iii) within individual hosts over time in relation to the initially replicating virus and to the deduced ancestral sequence of the intrahost viral population. Surprisingly, we consistently found less divergence between viruses from different individuals sampled in primary infection than in individuals sampled at more advanced stages of illness. Furthermore, longitudinal analysis of intrahost divergence revealed a 2- to 3-year period of evolution toward a common ancestral sequence at the start of infection, indicating that HIV recovers certain ancestral features when infecting a new host. These results have important implications for the study of HIV population genetics and rational vaccine design, including favoring the inclusion of viral gene sequences taken early in infection.

Distinct sequences of human immunodeficiency virus type 1 (HIV-1) have been found between different tissue compartments or subcompartments within a given tissue. Whether such compartmentalization of HIV-1 occurs between different cell populations is still unknown. Here we address this issue by comparing HIV-1 sequences in the second constant region through the fifth hypervariable region (C2 to V5) of the surface envelope glycoprotein (Env) between viruses in purified blood CD14+monocytes and CD4+T cells obtained longitudinally from five infected patients over a time period ranging from 117 to 3,409 days postseroconversion. Viral populations in both cell types at early infection time points appeared relatively homogeneous. However, later in infections, all five patients showed heterogeneous populations in both CD14+monocytes and CD4+T cells. Three of the five patients had CD14+monocyte populations with significantly more genetic diversity than the CD4+T-cell population, while the other two patients had more genetic diversity in CD4+T cells. The cellular compartmentalization of HIV-1 between CD14+monocytes and CD4+T cells was not seen early during infections but was evident at the later time points for all five patients, indicating an association of viral compartmentalization with the time course of HIV-1 infection. The majority of HIV-1 V3 sequences indicated a macrophage-tropic phenotype, while a V3 sequence-predicted T-cell tropic virus was found in the CD4+T cells and CD14+monocytes of two patients. These findings suggest that HIV-1 in CD14+monocytes could disseminate and evolve independently from that in CD4+T cells over the course of HIV-1 infection, which may have implications on the development of new therapeutic strategies.

While worldwide efforts to develop an effective HIV-1 vaccine are underway, the virus continues to spread, particularly in developing countries where the delivery of antiviral therapies presents formidable challenges. Vaccine research has largely focused on three general aspects: vectors, adjuvants, and immunization schedules. Our group favor the use of computational methods to design potential immunogens that capture the genetic and biological features of circulating viruses. These methods allow researchers to predict, in silico, the presence of potential glycosylation sites, humoral immune responses, and epitope coverage. This review shall compare three computational approaches for immunogen design: the consensus sequence, which has at each site the modal nucleotide or amino acid residue across a sequence alignment; the most recent common ancestor, the sequence estimated at the basal node of the clades seen in the HIV-1 phylogeny; and the center of tree method, which minimizes the evolutionary distance to all sequences in the data set.

China is a region of the world with a rapidly spreading HIV-1 epidemic. Studies providing insights into HIV-1 pathogenesis in infected Chinese are urgently needed to support the design and testing of an effective HIV-1 vaccine for this population. HIV-1-specific T cell responses were characterized in 32 HIV-1-infected individuals of Chinese origin and compared to 34 infected caucasians using 410 overlapping peptides spanning the entire HIV-1 clade B consensus sequence in an IFN-gamma ELISpot assay. All HIV-1 proteins were targeted with similar frequency in both populations and all study subjects recognized at least one overlapping peptide. HIV-1-specific T cell responses clustered in seven different regions of the HIV-1 genome in the Chinese cohort and in nine different regions in the caucasian cohort. The dominant HLA class I alleles expressed in the two populations differed significantly, and differences in epitope clustering pattern were shown to be influenced by differences in class I alleles that restrict immunodominant epitopes. These studies demonstrate that the clustering of HIV-1-specific T cell responses is influenced by the genetic HLA class I background in the study populations. The design and testing of candidate vaccines to fight the rapidly growing HIV-1 epidemic must therefore take the HLA genetics of the population into account as specific regions of the virus can be expected to be differentially targeted in ethnically diverse populations.

To evaluate human immunodeficiency virus type 1 (HIV-1) replication and selection of drug-resistant viruses during seemingly effective highly active antiretroviral therapy (HAART), multiple HIV-1 env and pol sequences were analyzed and viral DNA levels were quantified from nucleoside analog-experienced children prior to and during a median of 5.1 (range, 1.8 to 6.4) years of HAART. Viral replication was detected at different rates, with apparently increasing sensitivity: 1 of 10 by phylogenetic analysis; 2 of 10 by viral evolution with increasing genetic distances from the most recent common ancestor (MRCA) of infection; 3 of 10 by selection of drug-resistant mutants; and 6 of 10 by maintenance of genetic distances from the MRCA. When four- or five-drug antiretroviral regimens were given to these children, persistent plasma viral rebound did not occur despite the accumulation of highly drug-resistant genotypes. Among the four children without genetic evidence of viral replication, a statistically significant decrease in the genetic distance to the MRCA was detected in three, indicating the persistence of a greater number of early compared to recent viruses, and their HIV-1 DNA decreased by ≥0.9 log 10 , resulting in lower absolute DNA levels ( P = 0.007). This study demonstrates the variable rates of viral replication when HAART has suppressed plasma HIV-1 RNA for years to a median of

Although use of non-steroidal anti-inflammatory drugs (NSAIDs) generally decreases colorectal cancer (CRC) risk, inherited genetic variation in inflammatory pathways may alter their potential as preventive agents. We investigated whether variation in prostaglandin synthesis and related pathways influences CRC risk in the Colon Cancer Family Registry by examining associations between 192 single nucleotide polymorphisms (SNPs) and two variable nucleotide tandem repeats (VNTRs) within 17 candidate genes and CRC risk. We further assessed interactions between these polymorphisms and NSAID use on CRC risk. Using a case-unaffected-sibling-control design, this study included 1621 primary invasive CRC cases and 2592 sibling controls among Caucasian men and women aged 18-90. After adjustment for multiple comparisons, two intronic SNPs were associated with rectal cancer risk: rs11571364 in ALOX12 [OR(het/hzv) = 1.87, 95% confidence interval (CI) = 1.19-2.95, P = 0.03] and rs45525634 in PTGER2 (OR(het/hzv) = 0.49, 95% CI = 0.29-0.82, P = 0.03). Additionally, there was an interaction between NSAID use and the intronic SNP rs2920421 in ALOX12 on risk of CRC (P = 0.03); among those with heterozygous genotypes, risk was reduced for current NSAID users compared with never or former users (OR(het) = 0.60, 95% CI = 0.45-0.80), though not among those with homozygous wild-type or variant genotypes. The results of this study suggest that genetic variation in ALOX12 and PTGER2 may affect the risk of rectal cancer. In addition, this study suggests plausible interactions between NSAID use and variants in ALOX12 on CRC risk. These results may aid in the development of genetically targeted cancer prevention strategies with NSAIDs.

A great deal of research has been done in the last several years on information centric networking (ICN), where named data items, rather than end host identities, are the primary routable entities. Several prototypes have been proposed, but all face daunting concerns in the areas of performance, complexity, backwards compatibility, and user security and privacy. We propose a protocol which creates globally-unique data item names and embeds these names, plus associated metadata, into an IPv6 header. We then show how this allows the use of the IPsec suite of protocols to mitigate user privacy and security concerns. Next, we show that using RFC-compliant IPv6 datagrams as the named content allows content routing to be done using standard gateway protocols and also ensures backwards compatibility with unmodified networks. Lastly, we give an example of how the CLIP header's structure will simplify the design of automatic caches in the network.

Arachidonate lipoxygenase (ALOX) enzymes metabolize arachidonic acid to generate potent inflammatory mediators and play an important role in inflammation-associated diseases. We investigated associations between colorectal cancer risk and polymorphisms in ALOX5, FLAP, ALOX12, and ALOX15, and their interactions with non-steroidal anti-inflammatory drug (NSAID) use. We genotyped fifty tagSNPs, one candidate SNP, and two functional promoter variable nucleotide tandem repeat (VNTR) polymorphisms in three US population-based case-control studies of colon cancer (1424 cases/1780 controls), rectal cancer (583 cases/775 controls), and colorectal adenomas (485 cases/578 controls). Individuals with variant genotypes of the ALOX5 VNTR had decreased risk of rectal cancer, with the strongest association seen for individuals with one or more alleles of >5 repeats (wildtype=5, OR>5/≥5=0.42, 95% CI 0.20-0.92; p=0.01). Four SNPs in FLAP (rs17239025), ALOX 12 (rs2073438), and ALOX15 (rs4796535 and rs2619112) were associated with rectal cancer risk at p≤0.05. One SNP in FLAP (rs12429692) was associated with adenoma risk. A false discovery rate (FDR) was applied to account for false positives due to multiple testing; the ALOX15 associations were noteworthy at 25% FDR. Colorectal neoplasia risk appeared to be modified by NSAID use in individuals with variant alleles in FLAP and ALOX15. One noteworthy interaction (25% FDR) was observed for rectal cancer. Genetic variability in arachidonate lipoxygenases may affect risk of colorectal neoplasia, particularly for rectal cancer. Additionally, genetic variability in FLAP and ALOX15 may modify the protective effect of NSAID use against colorectal neoplasia.

Objectives and design: A vaccine capable of providing cross-clade, sterilizing protection has been the holy grail of HIV-1 prevention and control since the beginning of the pandemic. A major component of this effort has been the identification and characterization of broadly neutralizing antibodies (bNAbs). Recent advances in bNAb isolation, structure-based engineering, and vector-mediated gene transfer have led to increased interest in bypassing the immune system by expressing neutralizing antibodies directly in muscle. To assess the neutralization potency and coverage of a panel of second-generation bNAbs, we cloned and phenotypically characterized 227 primary HIV-1 envelopes from 23 mother-to-child transmission (MTCT) pairs. Methods: Viral envelopes were tested for in-vitro neutralization sensitivity using a standard pseudotype assay system. A 50% inhibitory concentration (IC50) at least 10 mg/ ml was used to define neutralization resistance. Results: The combination of antibodies PG16 and NIH45‐46 G54W had the broadest activity with the highest neutralization potency, achieving full coverage of 87% of transmission pairs (at a median sampling depth of 10 envelopes per pair) and 96% of recently infected infants in a very conservative analysis. Conclusions: Our data strongly support the inclusion of NIH45‐46 G54W , or a more extensively modified variant, in future proof-of-principle immunoprophylaxis or gene therapy-based trials. Furthermore, until robust sequence-based resistance detection becomes available, it will be necessary to conduct deeper phenotypic screening of primary isolates in order to determine the prevalence of minor resistant variants to help in selecting the best reagents for clinical trials. 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins AIDS 2013, 27:337‐346

Reagents for evaluating non-clade B HIV-specific T cell responses are uncommon. Peptides based on highly conserved HIV-1 consensus group M sequences that are phylogenetically closer to most circulating strains may provide potential alternative reagents in populations with diverse infections, and may be relevant for vaccine design. Recognition of such reagents in clade A1-and D-infected populations has not been previously evaluated. Interferon (IFN)-γ ELISpot assay was used to evaluate T cell recognition of Gag and Nef peptides based on consensus group M sequences in 50 treatment-naive adults predominantly infected with HIV-1 clades A1 and D. Gag-induced T cell responses were correlated with gag sequence diversity. Infecting clades were determined from gag sequences for 45 of the 50 subjects as 40% clade A1 (18/45), 45% clade D (20/45), 2% clade C (1/45), 2% A1/C recombinant (1/45), 2% A1/D (1/45), 7% CRF10_CD (3/45), and 2% U (unclassifiable) (1/45). The mean genetic divergence and diversity of clade A and D gag region compared to group M consensus sequences at synonymous and nonsynonymous nucleotide and amino acid levels were not always significant. Gag peptides were targeted at significantly higher frequency [88% (44/50)] than Nef [64% (32/50)]; p=0.014, although their mean IFN-γ magnitudes were comparable ([3703 (95% CI 2567–4839)] vs. [2120 (95% CI 478–3762)]), respectively. Measurable virus-induced IFN-γ responses were detected in 96% (48/50) individuals, primarily targeting the more conserved Gag p24 and Nef central core regions. Use of these reagents to screen for HIV-specific IFN-γ responses may mitigate the challenge of viral diversity; although this targeting is apparently biased toward a few highly conserved epitopes.

The deoxycytidine analog KP1212, and its prodrug KP1461, are prototypes of a new class of antiretroviral drugs designed to increase viral mutation rates, with the goal of eventually causing the collapse of the viral population. Here we present an extensive analysis of viral sequences from HIV-1 infected volunteers from the first “mechanism validation” phase II clinical trial of a mutagenic base analog in which individuals previously treated with antiviral drugs received 1600 mg of KP1461 twice per day for 124 days. Plasma viral loads were not reduced, and overall levels of viral mutation were not increased during this short-term study, however, the mutation spectrum of HIV was altered. A large number (N = 105 per sample) of sequences were analyzed, each derived from individual HIV-1 RNA templates, after 0, 56 and 124 days of therapy from 10 treated and 10 untreated control individuals (>7.1 million base pairs of unique viral templates were sequenced). We found that private mutations, those not found in more than one viral sequence and likely to have occurred in the most recent rounds of replication, increased in treated individuals relative to controls after 56 (p = 0.038) and 124 (p = 0.002) days of drug treatment. The spectrum of mutations observed in the treated group showed an excess of A to G and G to A mutations (p = 0.01), and to a lesser extent T to C and C to T mutations (p = 0.09), as predicted by the mechanism of action of the drug. These results validate the proposed mechanism of action in humans and should spur development of this novel antiretroviral approach., Koronis Pharmaceuticals

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 .

Background Breast milk transmission of HIV-1 remains a major route of pediatric infection. Defining the characteristics of viral variants to which breastfeeding infants are exposed is important for understanding the genetic bottleneck that occurs in the majority of mother-to-child transmissions. The blood-milk epithelial barrier markedly restricts the quantity of HIV-1 in breast milk, even in the absence of antiretroviral drugs. The basis of this restriction and the genetic relationship between breast milk and blood variants are not well established. Methodology/Principal Findings We compared 356 HIV-1 subtype C gp160 envelope (env) gene sequences from the plasma and breast milk of 13 breastfeeding women. A trend towards lower viral population diversity and divergence in breast milk was observed, potentially indicative of clonal expansion within the breast. No differences in potential N-linked glycosylation site numbers or in gp160 variable loop amino acid lengths were identified. Genetic compartmentalization was evident in only one out of six subjects in whom contemporaneously obtained samples were studied. However, in samples that were collected 10 or more days apart, six of seven subjects were classified as having compartmentalized viral populations, highlighting the necessity of contemporaneous sampling for genetic compartmentalization studies. We found evidence of CXCR4 co-receptor using viruses in breast milk and blood in nine out of the thirteen subjects, but no evidence of preferential localization of these variants in either tissue. Conclusions/Significance Despite marked restriction of HIV-1 quantities in milk, our data indicate intermixing of virus between blood and breast milk. Thus, we found no evidence that a restriction in viral genotype diversity in breast milk accounts for the genetic bottleneck observed following transmission. In addition, our results highlight the rapidity of HIV-1 env evolution and the importance of sample timing in analyses of gene flow.

Human antibody 4E10 targets the highly conserved membrane-proximal external region (MPER) of the HIV-1 transmembrane glycoprotein, gp41, and has extraordinarily broad neutralizing activity. It is considered by many to be a prototype for vaccine development. In this study, we describe four subjects infected with viruses carrying rare MPER polymorphisms associated with resistance to 4E10 neutralization. In one case resistant virus carrying a W680G substitution was transmitted from mother to infant. We used site-directed mutagenesis to demonstrate that the W680G substitution is necessary for conferring the 4E10-resistant phenotype, but that it is not sufficient to transfer the phenotype to a 4E10-sensitive Env. Our third subject carried Envs with a W680R substitution causing variable resistance to 4E10, indicating that residues outside the MPER are required to confer the phenotype. A fourth subject possessed a F673L substitution previously associated with 4E10 resistance. For all three subjects with W680 polymorphisms, we observed additional residues in the MPER that co-varied with position 680 and preserved charged distributions across this region. Our data provide important caveats for vaccine development targeting the MPER. Naturally occurring Env variants described in our study also represent unique tools for probing the structure-function of HIV-1 envelope.

Open Access Oral presentation OA06-06 LB. Evidence of vaccine-induced changes in breakthrough HIV-1 strains from the Step trial M Rolland1, S Tovanabutra2, PB Gilbert3, E Sanders-Buell2, L Heath1, AC deCamp3, CC Magaret3, M Bose2, A Bradfield2, A O'Sullivan2, J Crossler2, W Deng1, H Zhao1, K Wong1, DN Raugi1, J Hural3, S Dubey3, N Frahm3, NL Michael4, J Shiver5, L Corey3, F Li3, SG Self3, J Kim4, S Buchbinder6, DR Casimiro5, MN Robertson5, MJ McElrath3, FE McCutchan4 and JI Mullins*1

The extensive diversity of human immunodeficiency virus type 1 (HIV-1) and its capacity to mutate and escape host immune responses are major challenges for AIDS vaccine development. Ancestral sequences, which minimize the genetic distance to circulating strains, provide an opportunity to design immunogens with the potential to elicit broad recognition of HIV epitopes. We developed a phylogenetics-informed algorithm to reconstruct ancestral HIV sequences, called Center of Tree (COT). COT sequences have potentially significant benefits over isolate-based strategies, as they minimize the evolutionary distances to circulating strains. COT sequences are designed to surmount the potential pitfalls stemming from sampling bias with the consensus method and outlier bias with the most-recent-common-ancestor approach. We computationally derived COT sequences from circulating HIV-1 subtype B sequences for the genes encoding the major viral structural protein (Gag) and two regulatory proteins, Tat and Nef. COT genes were synthesized de novo and expressed in mammalian cells, and the proteins were characterized. COT Gag was shown to generate virus-like particles, while COT Tat transactivated gene expression from the HIV-1 long terminal repeat and COT Nef mediated downregulation of cell surface major histocompatibility complex class I. Thus, retrodicted ancestral COT proteins can retain the biological functions of extant HIV-1 proteins. Additionally, COT proteins were immunogenic, as they elicited antigen-specific cytotoxic T-lymphocyte responses in mice. These data support the utility of the COT approach to create novel and biologically active ancestral proteins as a starting point for studies of the structure, function, and biological fitness of highly variable genes, as well as for the rational design of globally relevant vaccine candidates.

Comparative sequence analyses, including such fundamental bioinformatics techniques as similarity searching, sequence alignment and phylogenetic inference, have become a mainstay for researchers studying type 1 Human Immunodeficiency Virus (HIV-1) genome structure and evolution. Implicit in comparative analyses is an underlying model of evolution, and the chosen model can significantly affect the results. In general, evolutionary models describe the probabilities of replacing one amino acid character with another over a period of time. Most widely used evolutionary models for protein sequences have been derived from curated alignments of hundreds of proteins, usually based on mammalian genomes. It is unclear to what extent these empirical models are generalizable to a very different organism, such as HIV-1-the most extensively sequenced organism in existence. We developed a maximum likelihood model fitting procedure to a collection of HIV-1 alignments sampled from different viral genes, and inferred two empirical substitution models, suitable for describing between-and within-host evolution. Our procedure pools the information from multiple sequence alignments, and provided software implementation can be run efficiently in parallel on a computer cluster. We describe how the inferred substitution models can be used to generate scoring matrices suitable for alignment and similarity searches. Our models had a consistently superior fit relative to the best existing models and to parameter-rich data-driven models when benchmarked on independent HIV-1 alignments, demonstrating evolutionary biases in amino-acid substitution that are unique to HIV, and that are not captured by the existing models. The scoring matrices derived from the models showed a marked difference from common amino-acid scoring matrices. The use of an appropriate evolutionary model recovered a known viral transmission history, whereas a poorly chosen model introduced phylogenetic error. We argue that our model derivation procedure is immediately applicable to other organisms with extensive sequence data available, such as Hepatitis C and Influenza A viruses.

Introduction: Inflammation is known to affect the risk of colorectal cancer (CRC). Although use of non-steroidal anti-inflammatory drugs (NSAIDs) is effective in decreasing CRC risk, inherited variation in genes that affect NSAID metabolism may alter their potential as a preventive agent. As NSAIDs function by inhibiting cyclooxygenase (COX) enzymes, which control the conversion of arachidonic acid to prostaglandins and thromboxane, we investigated whether variation in prostaglandin synthesis and related pathways affects the risk of CRC in the Colon Cancer Family Registry. Methods: We examined associations between 221 tagging and candidate single nucleotide polymorphisms (SNPs) within 18 candidate genes (ALOX12, ALOX5, ALOX5AP, CRP, EGFR, GIPC1, HPGD, PARP12, PKN1, PLA2G1B, PTGER1, PTGER2, PTGER3, PTGER4, PTGES, PTGIS, PTGS2, and TBXAS1) and the risk of CRC. We further assessed whether the association between SNPs and CRC risk varied by use of NSAIDs. Using a case-unaffected-sibling-control design, this study included 4,213 Caucasian men and women age 20-74 with no known familial adenomatous polyposis and no personal history of ulcerative colitis or Crohn's disease. Cases (N=1,621) were recruited from six registry centers and were diagnosed with primary invasive CRC from 1998-2002. Controls (N=2,592) were siblings not diagnosed with CRC at the time of ascertainment. We computed odds ratios and 95% confidence intervals using conditional logistic regression and corrected for multiple comparisons using the Conneely and Boehnke method. All models were adjusted for age and sex; NSAID interactions were further adjusted for BMI, smoking, and physical activity. Results: After adjustment for multiple comparisons, two intronic SNPs were associated with a decreased risk of rectal cancer: rs45525634 in PTGER2 (ORhet/hzv 0.49, 95% CI 0.29-0.82) and rs6091005 in PTGIS (ORhet 0.73, 95% CI 0.52-1.03; ORhzv 0.39, 95% CI 0.22-0.68). Additionally, an intronic SNP, rs11571364, in ALOX12 was associated with an increased risk of rectal cancer (ORhet/hzv 1.87, 95% CI 1.19-2.95). The risk of CRC associated with NSAID use differed by genotype at three intronic SNPs: rs10142849 (p = 0.02) in PTGER2 as well as rs11239516 (p = 0.04) and rs2115819 (p = 0.05) in ALOX5. These SNPs in ALOX5 shared low LD among Caucasian controls (r2 = 0.28). There was also evidence for interactions between SNP genotypes and NSAID use for colon cancer for two intronic SNPs, rs11239516 (p = 0.04) in ALOX5 and rs7806848 (p = 0.02) in TBXAS1, and for rectal cancer for rs16870224 (p = 0.05) located in the 3' UTR of PTGER4, the coding SNP rs35416389 (R641Q, p = 0.02) in PKN1, and two intronic SNPs rs17222919 (p = 0.03) in ALOX5 and rs6664305 (p = 0.01) in PTGER3. Conclusion: The results of this study suggest that genetic variation in ALOX12, PTGER2, and PTGIS may affect the risk of rectal cancer. In addition, this study suggests that current use of NSAIDs may modify associations between CRC risk and SNPs in ALOX5, PKN1, PTGER2, PTGER3, PTGER4, and TBXAS1. These findings may aid in the development of genetically targeted cancer prevention strategies with NSAIDs. Citation Format: Alexa J. Resler, Laura Heath, Karen W. Makar, John Whitton, Nina Habermann, Hansong Wang, Polly A. Newcomb, Loic LeMarchand, John D. Potter, Cornelia M. Ulrich. Genetic variation in prostaglandin synthesis and related pathways, NSAID use, and risk of colorectal cancer in the Colon Cancer Family Registry. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 37.

Background The deoxycytidine analog KP1212, and its prodrug KP1461, are prototypes of a new class of antiretroviral drugs designed to increase viral mutation rates, with the goal of eventually causing the collapse of the viral population. KP1212 will diminish or extinguish HIV-1 replication in cell culture, and has had very good safety profiles in clinical trials. Here we present an extensive analysis of viral sequences from patients from the first “mechanism validation” phase II clinical trial in which “salvage” patients received 1600 mg of drug twice per day for 124 days. The Phase IIa study did not demonstrate significant diminution of viral load. This was not unexpected as prior cell culture studies showed little change in viral titer prior to population collapse [1]. In the present work we sought to identify a subclinical impact of KP1461 therapy [2]. Materials and methods We performed direct gene sequencing of a very large number (>100) of sequences derived from individual HIV-1 RNA templates, after 0, 56 and 124 days of therapy from 10 treated and 10 untreated individuals. NMR studies were performed to identify tautomeric forms of the drug. Results Private mutations, those not found in multiple viruses, were similar in treated and control individuals at day 0 (p=0.28), but were increased in treated individuals after 56 (p=0.02) and 124 (p=0.001) days of drug treatment. Furthermore, the spectrum of mutations observed in the treated group was distinct from that of the controls, with an excess of A to G and G to A mutations (p=0.01), and to a lesser extent T to C and C to T mutations (p=0.09), in the treated group. Tautomeric forms of the drug were detected that are predicted to result in a substantial number of misincorporation events during DNA synthesis. Conclusions The predicted mechanism of action of the drug (transition mutations with a bias toward A G) was demonstrated in vivo. The observed increase in mutations in treated patients and the chemical properties of the new drug support a new mechanism of action by a novel antiretroviral therapy in humans.

Eliciting maximal immune responses to highly divergent viruses is a challenge and a focus in AIDS vaccine development. Another challenge is to identify the immune correlates of protective immunity. Recent AIDS vaccine design approaches attempt to use reconstructed centralized viral sequences that minimize genetic differences to circulating viruses. Using these approaches, we derive and analyze consensus (CON), ancestral (ANC), and center-of-tree (COT) sequences to represent intra-individual HIV-1 env variants encoding a range of diversities and phylogenetic structures. Each reconstructed sequence significantly minimized genetic distances to extant sequences throughout the first 5 years of infection of an individual. Interestingly, ANC sequences diverged and were not significantly better than extant sequences in minimizing genetic distances at later stages of infection and disease, likely due to the development of a substantially asymmetric phylogeny. COT or CON sequences derived from autologous virus samplings may be useful for increasing the sensitivity of assessments of immune reactivity against HIV.