In a cohort of HIV-infected individuals and age and sex-matched HIV-uninfected comparators, we assessed soluble (s)CD14, sCD163, interleukin (IL)-6, intestinal fatty acid binding protein (IFAPB) and high-sensitivity C-reactive protein (hs-CRP). The median age was 51 years; and among HIV+, median ART duration was 7 years, median CD4 T-cell count was 433, and 86% had an undetectable viral load. Although HIV+ had higher sCD14, IFABP and hs-CRP, we found evidence of interaction by sex, such that HIV+ women had greater differences versus HIV- compared to men. In models restricted to HIV+, women had higher levels of all five biomarkers than men.

Increased Systemic Inflammation and Gut Permeability Among Women with Treated HIV Infection in Rural Uganda.

Siedner MJ, Zanni M, Tracy RP, Kwon DS, Tsai AC, Kakuhire B, Hunt PW, Okello S. The Journal of infectious diseases. 2018

PURPOSE OF REVIEW:
Despite HIV therapy advances, average life expectancy in HIV-infected individuals on effective treatment is significantly decreased relative to uninfected persons, largely because of increased incidence of inflammation-related diseases, such as cardiovascular disease and renal dysfunction. The enteric microbial community could potentially cause this inflammation, as HIV-driven destruction of gastrointestinal CD4 T cells may disturb the microbiota-mucosal immune system balance, disrupting the stable gut microbiome and leading to further deleterious host outcomes.

RECENT FINDINGS:
Varied enteric microbiome changes have been reported during HIV infection, but unifying patterns have emerged. Community diversity is decreased, similar to pathologies such as inflammatory bowel disease, obesity, and Clostridium difficile infection. Many taxa frequently enriched in HIV-infected individuals, such as Enterobacteriaceae and Erysipelotrichaceae, have pathogenic potential, whereas depleted taxa, such as Bacteroidaceae and Ruminococcaceae, are more linked with anti-inflammatory properties and maintenance of gut homeostasis. The gut viral community in HIV has been found to contain a greater abundance of pathogenesis-associated Adenoviridae and Anelloviridae. These bacterial and viral changes correlate with increased systemic inflammatory markers, such as serum sCD14, sCD163, and IL-6.

SUMMARY:
Enteric microbial community changes may contribute to chronic HIV pathogenesis, but more investigation is necessary, especially in the developing world population with the greatest HIV burden (Video, Supplemental Digital Content 1, http://links.lww.com/COID/A15, which includes the authors’ summary of the importance of the work).

Elevated inflammation in the female genital tract is associated with increased HIV risk. Cervicovaginal bacteria modulate genital inflammation; however, their role in HIV susceptibility has not been elucidated. In a prospective cohort of young, healthy South African women, we found that individuals with diverse genital bacterial communities dominated by anaerobes other than Gardnerella were at over 4-fold higher risk of acquiring HIV and had increased numbers of activated mucosal CD4⁺ T cells compared to those with Lactobacillus crispatus-dominant communities. We identified specific bacterial taxa linked with reduced (L. crispatus) or elevated (Prevotella, Sneathia, and other anaerobes) inflammation and HIV infection and found that high-risk bacteria increased numbers of activated genital CD4⁺ T cells in a murine model. Our results suggest that highly prevalent genital bacteria increase HIV risk by inducing mucosal HIV target cells. These findings might be leveraged to reduce HIV acquisition in women living in sub-Saharan Africa.

Although the development of a fully protective HIV vaccine is the ultimate goal of HIV research, to date only one HIV vaccine trial, the RV144, has successfully induced a weakly protective response. The 31% protection from infection achieved in the RV144 trial was linked to the induction of nonneutralizing antibodies, able to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), suggestive of an important role of Fc-mediated functions in protection. Similarly, Fc-mediated antiviral activity was recently shown to play a critical role in actively suppressing the viral reservoir, but the Fc effector mechanisms within tissues that provide protection from or after infection are largely unknown. Here we aimed to define the landscape of effector cells and Fc receptors present within vulnerable tissues. We found negligible Fc receptor-expressing natural killer cells in the female reproductive and gastrointestinal mucosa. Conversely, Fc receptor-expressing macrophages were highly enriched in most tissues, but neutrophils mediated superior antibody-mediated phagocytosis. Modifications in Fc domain of VRC01 antibody increased phagocytic responses in both phagocytes. These data suggest that non-ADCC-mediated mechanisms, such as phagocytosis and neutrophil activation, are more likely to play a role in preventative vaccine or reservoir-eliminating therapeutic approaches.

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

BACKGROUND:
The use of injectable progestin-only contraceptives has been associated with increased risk of HIV acquisition in observational studies, but the biological mechanisms of this risk remain poorly understood. We aimed to assess the effects of progestins on HIV acquisition risk and the immune environment in the female genital tract.

METHODS:
In this prospective cohort, we enrolled HIV-negative South African women aged 18-23 years who were not pregnant and were living in Umlazi, South Africa from the Females Rising through Education, Support, and Health (FRESH) study. We tested for HIV-1 twice per week to monitor incident infection. Every 3 months, we collected demographic and behavioural data in addition to blood and cervical samples. The study objective was to characterise host immune determinants of HIV acquisition risk, including those associated with injectable progestin-only contraceptive use. Hazard ratios (HRs) were estimated using Cox proportional hazards methods.

FINDINGS:
Between Nov 19, 2012, and May 31, 2015, we characterised 432 HIV-uninfected South African women from the FRESH study. In this cohort, 152 women used injectable progestin-only contraceptives, 43 used other forms of contraception, and 222 women used no method of long-term contraception. Women using injectable progestin-only contraceptives were at substantially higher risk of acquiring HIV (12·06 per 100 person-years, 95% CI 6·41-20·63) than women using no long-term contraception (3·71 per 100 person-years, 1·36-8·07; adjusted hazard ratio [aHR] 2·93, 95% CI 1·09-7·868, p=0·0326). HIV-negative injectable progestin-only contraceptive users had 3·92 times the frequency of cervical HIV target cells (CCR5+ CD4 T cells) compared with women using no long-term contraceptive (p=0·0241). Women using no long-term contraceptive in the luteal phase of the menstrual cycle also had a 3·25 times higher frequency of cervical target cells compared with those in the follicular phase (p=0·0488), suggesting that a naturally high progestin state had similar immunological effects to injectable progestin-only contraceptives.

INTERPRETATION:
Injectable progestin-only contraceptive use and high endogenous progesterone are both associated with increased frequency of activated HIV targets cells at the cervix, the site of initial HIV entry in most women, providing a possible biological mechanism underlying increased HIV acquisition in women with high progestin exposure.

FUNDING:
The Bill and Melinda Gates Foundation and the National Institute of Allergy and Infectious Diseases.

HIV-infected persons are at greater risk of developing tuberculosis (TB) even before profound CD4 loss occurs, suggesting that HIV alters CD4(+) T cell functions capable of containing bacterial replication. An effective immune response to Mycobacterium tuberculosis most likely relies on the development of a balanced CD4 response, in which distinct CD4(+) Th subsets act in synergy to control the infection. To define the diversity of M. tuberculosis-specific CD4(+) Th subsets and determine whether HIV infection impacts such responses, the expression of lineage-defining transcription factors T-bet, Gata3, RORγt, and Foxp3 was measured in M. tuberculosis-specific CD4(+) T cells in HIV-uninfected (n = 20) and HIV-infected individuals (n = 20) with latent TB infection. Our results show that, upon 5-d restimulation in vitro, M. tuberculosis-specific CD4(+) T cells from healthy individuals have the ability to exhibit a broad spectrum of Th subsets, defined by specific patterns of transcription factor coexpression. These transcription factor profiles were skewed in HIV-infected individuals where the proportion of T-bet(high)Foxp3(+) M. tuberculosis-specific CD4(+) T cells was significantly decreased (p = 0.002) compared with HIV-uninfected individuals, a change that correlated inversely with HIV viral load (p = 0.0007) and plasma TNF-α (p = 0.027). Our data demonstrate an important balance in Th subset diversity defined by lineage-defining transcription factor coexpression profiles that is disrupted by HIV infection and suggest a role for HIV in impairing TB immunity by altering the equilibrium of M. tuberculosis-specific CD4(+) Th subsets.

Human immunodeficiency virus (HIV) infection is associated with increased intestinal translocation of microbial products and enteropathy as well as alterations in gut bacterial communities. However, whether the enteric virome contributes to this infection and resulting immunodeficiency remains unknown. We characterized the enteric virome and bacterial microbiome in a cohort of Ugandan patients, including HIV-uninfected or HIV-infected subjects and those either treated with anti-retroviral therapy (ART) or untreated. Low peripheral CD4 T cell counts were associated with an expansion of enteric adenovirus sequences and this increase was independent of ART treatment. Additionally, the enteric bacterial microbiome of patients with lower CD4 T counts exhibited reduced phylogenetic diversity and richness with specific bacteria showing differential abundance, including increases in Enterobacteriaceae, which have been associated with inflammation. Thus, immunodeficiency in progressive HIV infection is associated with alterations in the enteric virome and bacterial microbiome, which may contribute to AIDS-associated enteropathy and disease progression.

AIDS caused by simian immunodeficiency virus (SIV) infection is associated with gastrointestinal disease, systemic immune activation, and, in cross-sectional studies, changes in the enteric virome. Here we performed a longitudinal study of a vaccine cohort to define the natural history of changes in the fecal metagenome in SIV-infected monkeys. Matched rhesus macaques were either uninfected or intrarectally challenged with SIV, with a subset receiving the Ad26 vaccine, an adenovirus vector expressing the viral Env/Gag/Pol antigens. Progression of SIV infection to AIDS was associated with increased detection of potentially pathogenic viruses and bacterial enteropathogens. Specifically, adenoviruses were associated with an increased incidence of gastrointestinal disease and AIDS-related mortality. Viral and bacterial enteropathogens were largely absent from animals protected by the vaccine. These data suggest that the SIV-associated gastrointestinal disease is associated with the presence of both viral and bacterial enteropathogens and that protection against SIV infection by vaccination prevents enteropathogen emergence.

BACKGROUND:
HIV-1 eradication may require reactivation of latent virus along with stimulation of HIV-1-specific immune responses to clear infected cells. Immunization with autologous dendritic cells (DCs) transfected with viral mRNA is a promising strategy for eliciting HIV-1-specific immune responses. We performed a randomized controlled clinical trial to evaluate the immunogenicity of this approach in HIV-1-infected persons on antiretroviral therapy.

METHODS:
Fifteen participants were randomized 2:1 to receive intradermal immunization with HIV-1 Gag- and Nef-transfected DCs (vaccine) or mock-transfected DCs (placebo) at weeks 0, 2, 6, and 10. All participants also received DCs pulsed with keyhole limpet hemocyanin (KLH) to assess whether responses to a neo-antigen could be induced.

RESULTS:
After immunization, there were no differences in interferon-gamma enzyme-linked immunospot responses to HIV-1 Gag or Nef in the vaccine or placebo group. CD4 proliferative responses to KLH increased 2.4-fold (P = 0.026) and CD8 proliferative responses to KLH increased 2.5-fold (P = 0.053) after vaccination. There were increases in CD4 proliferative responses to HIV-1 Gag (2.5-fold vs. baseline, 3.4-fold vs. placebo, P = 0.054) and HIV-1 Nef (2.3-fold vs. baseline, 6.3-fold vs. placebo, P = 0.009) among vaccine recipients, but these responses were short-lived.

CONCLUSION:
Immunization with DCs transfected with mRNA encoding HIV-1 Gag and Nef did not induce significant interferon-gamma enzyme-linked immunospot responses. There were increases in proliferative responses to HIV-1 antigens and to a neo-antigen, KLH, but the effects were transient. Dendritic cell vaccination should be optimized to elicit stronger and long-lasting immune responses for this strategy to be effective as an HIV-1 therapeutic vaccine.

HIV infection is associated with arterial stiffness, but no studies have assessed this relationship in sub-Saharan Africa. We enrolled 205 participants over 40 years old in Uganda: 105 on antiretroviral therapy for a median of 7 years, and a random sample of 100 age and sex-matched HIV-uninfected controls from the clinic catchment area. The prevalence of arterial stiffness (ankle brachial index > 1.2) was 33%, 18%, 19% and 2% in HIV+ men, HIV- men, HIV+ women, and HIV- women. In multivariable models adjusted for cardiovascular risk factors, HIV+ individuals had over double the prevalence of arterial stiffness (adjusted prevalence ratio 2.86, 95% confidence interval 1.41-5.79, P = 0.003).

BACKGROUND:
Human immunodeficiency virus (HIV) infection and associated immune activation predict the risk of cardiovascular disease in resource-rich areas. Less is known about these relationships in sub-Saharan Africa.

METHODS:
Beginning in 2005, we enrolled subjects in southwestern Uganda into a cohort at the time of antiretroviral therapy (ART) initiation. Multiple immune activation measures were assessed before and 6 months after ART initiation. Beginning in 2013, participants aged >40 years underwent metabolic profiling, including measurement of hemoglobin A1c and lipid levels and carotid ultrasonography. We fit regression models to identify traditional and HIV-specific correlates of common carotid intima media thickness (CCIMT).

RESULTS:
A total of 105 participants completed carotid ultrasonography, with a median completion time of 7 years following ART initiation. Age, low-density lipoprotein cholesterol level, and pre-ART HIV load were correlated with CCIMT. No association was found between CCIMT and any pre-ART biomarkers of immune activation. However, in multivariable models adjusted for cardiovascular disease risk factors, lower absolute levels of soluble CD14 and interleukin 6 and greater declines in the CD14 level and kynurenine-tryptophan ratio after 6 months of ART predicted a lower CCIMT years later (P < .01).

CONCLUSIONS:
Persistent immune activation despite ART-mediated viral suppression predicts the future atherosclerotic burden among HIV-infected Ugandans. Future work should focus on clinical correlates of these relationships, to elucidate the long-term health priorities for HIV-infected people in the region.

Viruses that infect the intestine include major human pathogens (retroviruses, noroviruses, rotaviruses, astroviruses, picornaviruses, adenoviruses, herpesviruses) that constitute a serious public health problem worldwide. These viral pathogens are members of a large, complex viral community inhabiting the intestine termed “the enteric virome.” Enteric viruses have intimate functional and genetic relationships with both the host and other microbial constituents that inhabit the intestine, such as the bacterial microbiota, their associated phages, helminthes, and fungi, which together constitute the microbiome. Emerging data indicate that enteric viruses regulate, and are in turn regulated by, these other microbes through a series of processes termed “transkingdom interactions.” This represents a changing paradigm in intestinal immunity to viral infection. Here we review recent advances in the field and propose new ways in which to conceptualize this important area.

Initial events after exposure determine HIV-1 disease progression, underscoring a critical need to understand host mechanisms that interfere with initial viral replication. Although associated with chronic HIV-1 control, it is not known whether interleukin-21 (IL-21) contributes to early HIV-1 immunity. Here we take advantage of tractable primary human lymphoid organ aggregate cultures to show that IL-21 directly suppresses HIV-1 replication, and identify microRNA-29 (miR-29) as an antiviral factor induced by IL-21 in CD4 T cells. IL-21 promotes transcription of all miR-29 species through STAT3, whose binding to putative regulatory regions within the MIR29 gene is enriched by IL-21 signalling. Notably, exogenous IL-21 limits early HIV-1 infection in humanized mice, and lower viremia in vivo is associated with higher miR-29 expression. Together, these findings reveal a novel antiviral IL-21-miR-29 axis that promotes CD4 T-cell-intrinsic resistance to HIV-1 infection, and suggest a role for IL-21 in initial HIV-1 control in vivo.

IL-21 induces antiviral microRNA-29 in CD4 T cells to limit HIV-1 infection.
Adoro S, Cubillos-Ruiz JR, Chen X, Deruaz M, Vrbanac VD, Song M, Park S, Murooka TT, Dudek TE, Luster AD, Tager AM, Streeck H, Bowman B, Walker BD, Kwon DS, Lazarevic V, Glimcher LH.
Nature communications. 2015; 6:7562. NIHMSID: NIHMS693385

Colonization by Lactobacillus in the female genital tract is thought to be critical for maintaining genital health. However, little is known about how genital microbiota influence host immune function and modulate disease susceptibility. We studied a cohort of asymptomatic young South African women and found that the majority of participants had genital communities with low Lactobacillus abundance and high ecological diversity. High-diversity communities strongly correlated with genital pro-inflammatory cytokine concentrations in both cross-sectional and longitudinal analyses. Transcriptional profiling suggested that genital antigen-presenting cells sense gram-negative bacterial products in situ via Toll-like receptor 4 signaling, contributing to genital inflammation through activation of the NF-κB signaling pathway and recruitment of lymphocytes by chemokine production. Our study proposes a mechanism by which cervicovaginal microbiota impact genital inflammation and thereby might affect a woman’s reproductive health, including her risk of acquiring HIV.

Cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital tract.
Anahtar MN, Byrne EH, Doherty KE, Bowman BA, Yamamoto HS, Soumillon M, Padavattan N, Ismail N, Moodley A, Sabatini ME, Ghebremichael MS, Nusbaum C, Huttenhower C, Virgin HW, Ndung’u T, Dong KL, Walker BD, Fichorova RN, Kwon DS.
Immunity. 2015; 42(5):965-76. NIHMSID: NIHMS689549

The proliferation of genetically modified mouse models has exposed phenotypic variation between investigators and institutions that has been challenging to control. In many cases, the microbiota is the presumed cause of the variation. Current solutions to account for phenotypic variability include littermate and maternal controls or defined microbial consortia in gnotobiotic mice. In conventionally raised mice, the microbiome is transmitted from the dam. Here we show that microbially driven dichotomous faecal immunoglobulin-A (IgA) levels in wild-type mice within the same facility mimic the effects of chromosomal mutations. We observe in multiple facilities that vertically transmissible bacteria in IgA-low mice dominantly lower faecal IgA levels in IgA-high mice after co-housing or faecal transplantation. In response to injury, IgA-low mice show increased damage that is transferable by faecal transplantation and driven by faecal IgA differences. We find that bacteria from IgA-low mice degrade the secretory component of secretory IgA as well as IgA itself. These data indicate that phenotypic comparisons between mice must take into account the non-chromosomal hereditary variation between different breeders. We propose faecal IgA as one marker of microbial variability and conclude that co-housing and/or faecal transplantation enables analysis of progeny from different dams.

Comprehensive characterization of the antigen-specific B cells induced during infections or following vaccination would facilitate the discovery of novel antibodies and inform how interventions shape protective humoral responses. The analysis of human B cells and their antibodies has been performed using flow cytometry to evaluate memory B cells and expanded plasmablasts, while microtechnologies have also provided a useful tool to examine plasmablasts/plasma cells after vaccination. Here we present an integrated analytical platform, using arrays of subnanoliter wells (nanowells), for constructing detailed profiles for human B cells comprising the immunophenotypes of these cells, the distribution of isotypes of the secreted antibodies, the specificity and relative affinity for defined antigens, and for a subset of cells, the genes encoding the heavy and light chains. The approach combines on-chip image cytometry, microengraving, and single-cell RT-PCR. Using clinical samples from HIV-infected subjects, we demonstrate that the method can identify antigen-specific neutralizing antibodies, is compatible with both plasmablasts/plasma cells and activated memory B cells, and is well-suited for characterizing the limited numbers of B cells isolated from tissue biopsies (e.g., colon biopsies). The technology should facilitate detailed analyses of human humoral responses for evaluating vaccines and their ability to raise protective antibody responses across multiple anatomical compartments.

Antigen persistence in chronic infections and cancer upregulates inhibitory networks, such as the PD-1 and interleukin-10 (IL-10) pathways, that impair immunity and lead to disease progression. These pathways are attractive targets for immunotherapy, as demonstrated by recent clinical trials of PD-1/PD-L1 blockade in cancer patients. However, in HIV-1 infection not all subjects respond to inhibition of either pathway and the mechanistic interactions between these two networks remain to be better defined. Here we demonstrate that in vitro blockade of PD-L1 and/or IL-10Rα results in markedly different profiles of HIV-1-specific CD4 T cell restoration. Whereas PD-L1 blockade leads to balanced increase in gamma interferon (IFN-γ), IL-2, and IL-13 secretion, IL-10Rα blockade preferentially restores IFN-γ production. In viremic subjects, combined PD-L1/IL-10Rα blockade results in a striking 10-fold increase in IFN-γ secretion by HIV-1-specific CD4 T cells that is not observed in subjects with spontaneous (elite controllers) or therapy-induced control of viral replication. In contrast to the dramatic increase in IFN-γ production, concurrent blockade has a marginal additive effect on IL-2 production, IL-13 secretion, and HIV-1-specific CD4 T cell proliferation. IFN-γ produced by Thelper cells upregulates PD-L1, HLA I/II, and IL-12 expression by monocytes. The effect of combined blockade on IFN-γ was dependent on reciprocal reinforcement through IL-12. These studies provide crucial information on the different immunoregulatory qualities of PD-1 and IL-10 in progressive disease and link exhausted virus-specific CD4 T cells and monocytes in the regulation of IFN-γ and IL-12 secretion.

IMPORTANCE:Infection with HIV results in most people in uncontrolled viral replication and progressive weakening of the body defenses. In the absence of antiviral therapy, this process results in clinical disease, or AIDS. An important reason why HIV continues to multiply is that a population of white blood cells called CD4 T cells that targets the virus fails to work properly. At least part of this impairment is under the control of inhibitory mechanisms that can be blocked to improve the function of these CD4 T cells. In this report, we show that blocking one or two of the molecules involved, called PD-1 and IL-10, has different effects on the individual functions of these cells and that one is strongly improved. We investigate how these effects are caused by interactions between CD4 T cells and antigen-presenting cells. These observations can have implications for new therapeutic approaches in HIV infection.

Critical aspects of HIV-1 infection occur in mucosal tissues, particularly in the gut, which contains large numbers of HIV-1 target cells that are depleted early in infection. We used electron tomography (ET) to image HIV-1 in gut-associated lymphoid tissue (GALT) of HIV-1-infected humanized mice, the first three-dimensional ultrastructural examination of HIV-1 infection in vivo. Human immune cells were successfully engrafted in the mice, and following infection with HIV-1, human T cells were reduced in GALT. Virions were found by ET at all stages of egress, including budding immature virions and free mature and immature viruses. Immuno-electron microscopy verified the virions were HIV-1 and showed CD4 sequestration in the endoplasmic reticulum of infected cells. Observation of HIV-1 in infected GALT tissue revealed that most HIV-1-infected cells, identified by immunolabeling and/or the presence of budding virions, were localized to intestinal crypts with pools of free virions concentrated in spaces between cells. Fewer infected cells were found in mucosal regions and the lamina propria. The preservation quality of reconstructed tissue volumes allowed details of budding virions, including structures interpreted as host-encoded scission machinery, to be resolved. Although HIV-1 virions released from infected cultured cells have been described as exclusively mature, we found pools of both immature and mature free virions within infected tissue. The pools could be classified as containing either mostly mature or mostly immature particles, and analyses of their proximities to the cell of origin supported a model of semi-synchronous waves of virion release. In addition to HIV-1 transmission by pools of free virus, we found evidence of transmission via virological synapses. Three-dimensional EM imaging of an active infection within tissue revealed important differences between cultured cell and tissue infection models and furthered the ultrastructural understanding of HIV-1 transmission within lymphoid tissue.

While modulation of regulatory T cell (Treg) function and adoptive Treg transfer are being explored as therapeutic modalities in the context of autoimmune diseases, transplantation and cancer, their role in HIV-1 pathogenesis remains less well defined. Controversy persists regarding their beneficial or detrimental effects in HIV-1 disease, which warrants further detailed exploration. Our objectives were to investigate if functional CD4(+) Tregs can be isolated and expanded from HIV-1-infected individuals for experimental or potential future therapeutic use and to determine phenotype and suppressive capacity of expanded Tregs from HIV-1 positive blood and tissue. Tregs and conventional T cell controls were isolated from blood and gut-associated lymphoid tissue of individuals with HIV-1 infection and healthy donors using flow-based cell-sorting. The phenotype of expanded Tregs was assessed by flow-cytometry and quantitative PCR. T-cell receptor ß-chain (TCR-β) repertoire diversity was investigated by deep sequencing. Flow-based T-cell proliferation and chromium release cytotoxicity assays were used to determine Treg suppressive function. Tregs from HIV-1 positive individuals, including infants, were successfully expanded from PBMC and GALT. Expanded Tregs expressed high levels of FOXP3, CTLA4, CD39 and HELIOS and exhibited a highly demethylated TSDR (Treg-specific demethylated region), characteristic of Treg lineage. The TCRß repertoire was maintained following Treg expansion and expanded Tregs remained highly suppressive in vitro. Our data demonstrate that Tregs can be expanded from blood and tissue compartments of HIV-1+ donors with preservation of Treg phenotype, function and TCR repertoire. These results are highly relevant for the investigation of potential future therapeutic use, as currently investigated for other disease states and hold great promise for detailed studies on the role of Tregs in HIV-1 infection.

Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via the CXCR1 and CXCR2 receptors, influencing tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors.

A small proportion of human immunodeficiency virus-1 (HIV-1) infected individuals, termed HIV-1 controllers, suppress viral replication to very low levels in the absence of therapy. Genetic investigations of this phenotype have strongly implicated variation in the class I major histocompatibility complex (MHC) region as key to HIV-1 control. We collected sequence-based classical class I HLA genotypes at 4-digit resolution in HIV-1-infected African American controllers and progressors (n = 1107), and tested them for association with host control using genome-wide single nucleotide polymorphism data to account for population structure. Several classical alleles at HLA-B were associated with host control, including B*57:03 [odds ratio (OR) = 5.1; P= 3.4 × 10(-18)] and B*81:01 (OR = 4.8; P= 1.3 × 10(-9)). Analysis of variable amino acid positions demonstrates that HLA-B position 97 is the most significant association with host control in African Americans (omnibus P = 1.2 × 10(-21)) and explains the signal of several HLA-B alleles, including B*57:03. Within HLA-B, we also identified independent effects at position 116 (omnibus P= 2.8 × 10(-15)) in the canonical F pocket, position 63 in the B pocket (P= 1.5 × 10(-3)) and the non-pocket position 245 (P= 8.8 × 10(-10)), which is thought to influence CD8-binding kinetics. Adjusting for these HLA-B effects, there is evidence for residual association in the MHC region. These results underscore the key role of HLA-B in affecting HIV-1 replication, likely through the molecular interaction between HLA-B and viral peptides presented by infected cells, and suggest that sites outside the peptide-binding pocket also influence HIV-1 control.

T cell dysfunction in the presence of ongoing antigen exposure is a cardinal feature of chronic viral infections with persistent high viremia, including HIV-1. Although interleukin-10 (IL-10) has been implicated as an important mediator of this T cell dysfunction, the regulation of IL-10 production in chronic HIV-1 infection remains poorly understood. We demonstrated that IL-10 is elevated in the plasma of individuals with chronic HIV-1 infection and that blockade of IL-10 signaling results in a restoration of HIV-1-specific CD4 T cell proliferation, gamma interferon (IFN-γ) secretion, and, to a lesser extent, IL-2 production. Whereas IL-10 blockade leads to restoration of IFN-γ secretion by HIV-1-specific CD4 T cells in all categories of subjects investigated, significant enhancement of IL-2 production and improved proliferation of CD4 T helper cells are restricted to viremic individuals. In peripheral blood mononuclear cells (PBMCs), this IL-10 is produced primarily by CD14(+) monocytes, but its production is tightly controlled by regulatory T cells (Tregs), which produce little IL-10 directly. When Tregs are depleted from PBMCs of viremic individuals, the effect of the IL-10 signaling blockade is abolished and IL-10 production by monocytes decreases, while the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), increases. The regulation of IL-10 by Tregs appears to be mediated primarily by contact or paracrine-dependent mechanisms which involve IL-27. This work describes a novel mechanism by which regulatory T cells control IL-10 production and contribute to dysfunctional HIV-1-specific CD4 T cell help in chronic HIV-1 infection and provides a unique mechanistic insight into the role of regulatory T cells in immune exhaustion.

Regulatory T cells (Tregs) are potent immune modulators, but their role in human immunodeficiency virus type 1 (HIV-1) pathogenesis remains poorly understood. We performed a detailed analysis of the frequency and function of Tregs in a large cohort of HIV-1-infected individuals and HIV-1 negative controls. While HIV “elite controllers” and uninfected individuals had similar Treg numbers and frequencies, the absolute numbers of Tregs declined in blood and gut-associated lymphoid tissue in patients with chronic progressive HIV-1 infection. Despite quantitative changes in Tregs, HIV-1 infection was not associated with an impairment of ex vivo suppressive function of flow-sorted Tregs in both HIV controllers and untreated chronic progressors.

The nature of certain clinical samples (tissue biopsies, fluids) or the subjects themselves (pediatric subjects, neonates) often constrain the number of cells available to evaluate the breadth of functional T-cell responses to infections or therapeutic interventions. The methods most commonly used to assess this functional diversity ex vivo and to recover specific cells to expand in vitro usually require more than 10(6) cells. Here we present a process to identify antigen-specific responses efficiently ex vivo from 10(4)-10(5) single cells from blood or mucosal tissues using dense arrays of subnanoliter wells. The approach combines on-chip imaging cytometry with a technique for capturing secreted proteins–called “microengraving”–to enumerate antigen-specific responses by single T cells in a manner comparable to conventional assays such as ELISpot and intracellular cytokine staining. Unlike those assays, however, the individual cells identified can be recovered readily by micromanipulation for further characterization in vitro. Applying this method to assess HIV-specific T-cell responses demonstrates that it is possible to establish clonal CD8(+) T-cell lines that represent the most abundant specificities present in circulation using 100- to 1,000-fold fewer cells than traditional approaches require and without extensive genotypic analysis a priori. This rapid (<24 h), efficient, and inexpensive process should improve the comparative study of human T-cell immunology across ages and anatomic compartments.

The human gut mucosa is a major site of human immunodeficiency virus (HIV) infection and infection-associated pathogenesis. Increasing evidence shows that natural killer (NK) cells have an important role in control of HIV infection, but the mechanism(s) by which they mediate antiviral activity in the gut is unclear. Here, we show that two distinct subsets of NK cells exist in the gut, one localized to intraepithelial spaces (intraepithelial lymphocytes, IELs) and the other to the lamina propria (LP). The frequency of both subsets of NK cells was reduced in chronic infection, whereas IEL NK cells remained stable in spontaneous controllers with protective killer immunoglobulin-like receptor/human leukocyte antigen genotypes. Both IEL and LP NK cells were significantly expanded in immunological non-responsive patients, who incompletely recovered CD4+ T cells on highly active antiretroviral therapy (HAART). These data suggest that both IEL and LP NK cells may expand in the gut in an effort to compensate for compromised CD4+ T-cell recovery, but that only IEL NK cells may be involved in providing durable control of HIV in the gut.

Defining the T helper functions impaired by programmed death-1 (PD-1) is crucial for understanding its role in defective HIV control and determining the therapeutic potential of targeting this inhibitory pathway. We describe here the relationships among disease stage, levels of PD-1 expression, and reversibility of CD4 T-cell impairment. PD-L1 blockade in vitro enhanced HIV-specific production of Th0 (IL-2), Th1 (IFN-γ), Th2 (IL-13), and TFH (IL-21) cytokines by CD4 T cells. PD-L1 blockade caused an early increase in cytokine transcription and translation that preceded cell proliferation. Although the impact of PD-L1 blockade on cytokine expression and, to a lesser extent, cell proliferation was associated with markers of disease progression, restoration of cytokine secretion was also observed in most subjects with undetectable viremia. PD-L1 blockade restored cytokine secretion in both PD-1intermediate and PD-1high sorted CD4 T-cell subsets. Compared with PD-1high HIV-specific CD8 T cells, PD-1high HIV-specific CD4 T cells showed lower expression of the inhibitory molecules CD160 and 2B4, demonstrating marked differences in expression of inhibitory receptors between T-cell subsets. These data show that PD-1 impairs HIV-specific T helper responses both by limiting expansion of these cells and by inhibiting effector functions of multiple differentiated CD4 T-cell subsets.

Under persistent antigenic stimulation, virus-specific CD8⁺ T cells become increasingly dysfunctional and up-regulate several inhibitory molecules such as killer lectin-like receptor G1 (KLRG1). Here, we demonstrate that HIV-1 antigen-specific T cells from subjects with chronic-progressive HIV-1 infection have significantly elevated KLRG1 expression (P < .001); show abnormal distribution of E-cadherin, the natural ligand of KLRG1, in the intestinal mucosa; and have elevated levels of systemic soluble E-cadherin (sE-cadherin) that significantly correlate with HIV-1 viral load (R = 0.7, P = .004). We furthermore demonstrate that in the presence of sE-cadherin, KLRG1(hi) HIV-1-specific CD8⁺ T cells are impaired in their ability to respond by cytokine secretion on antigenic stimulation (P = .002) and to inhibit viral replication (P = .03) in vitro. Thus, these data suggest a critical mechanism by which the disruption of the intestinal epithelium associated with HIV-1 leads to increased systemic levels of sE-cadherin, which inhibits the effector functions of KLRG1(hi)-expressing HIV-1-specific CD8⁺ T cells systemically.

Functional defects in cytotoxic CD8(+) T cell responses arise in chronic human viral infections, but the mechanisms involved are not well understood. In mice, CD4 cell-mediated interleukin-21 (IL-21) production is necessary for the maintenance of CD8(+) T cell function and control of persistent viral infections. To investigate the potential role of IL-21 in a chronic human viral infection, we studied the rare subset of HIV-1 controllers, who are able to spontaneously control HIV-1 replication without treatment. HIV-specific triggering of IL-21 by CD4(+) T cells was significantly enriched in these persons (P = 0.0007), while isolated loss of IL-21-secreting CD4(+) T cells was characteristic for subjects with persistent viremia and progressive disease. IL-21 responses were mediated by recognition of discrete epitopes largely in the Gag protein, and expansion of IL-21(+) CD4(+) T cells in acute infection resulted in lower viral set points (P = 0.002). Moreover, IL-21 production by CD4(+) T cells of HIV controllers enhanced perforin production by HIV-1-specific CD8(+) T cells from chronic progressors even in late stages of disease, and HIV-1-specific effector CD8(+) T cells showed an enhanced ability to efficiently inhibit viral replication in vitro after IL-21 binding. These data suggest that HIV-1-specific IL-21(+) CD4(+) T cell responses might contribute to the control of viral replication in humans and are likely to be of great importance for vaccine design.

Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA-viral peptide interaction as the major factor modulating durable control of HIV infection.

CD8(+) T cells in chronic viral infections such as HIV develop functional defects including loss of interleukin-2 (IL-2) secretion and decreased proliferative potential that are collectively termed ‘exhaustion’. Exhausted T cells express increased amounts of multiple inhibitory receptors, such as programmed death-1 (PD-1), that contribute to impaired virus-specific T cell function. Although reversing PD-1 inhibition is therefore an attractive therapeutic strategy, the cellular mechanisms by which PD-1 ligation results in T cell inhibition are not fully understood. PD-1 is thought to limit T cell activation by attenuating T cell receptor (TCR) signaling. It is not known whether PD-1 also acts by upregulating genes in exhausted T cells that impair their function. Here we analyzed gene expression profiles from HIV-specific CD8(+) T cells in individuals with HIV and show that PD-1 coordinately upregulates a program of genes in exhausted CD8(+) T cells from humans and mice. This program includes upregulation of basic leucine transcription factor, ATF-like (BATF), a transcription factor in the AP-1 family. Enforced expression of BATF was sufficient to impair T cell proliferation and cytokine secretion, whereas BATF knockdown reduced PD-1 inhibition. Silencing BATF in T cells from individuals with chronic viremia rescued HIV-specific T cell function. Thus, inhibitory receptors can cause T cell exhaustion by upregulating genes–such as BATF–that inhibit T cell function. Such genes may provide new therapeutic opportunities to improve T cell immunity to HIV.

Successful pathogen clearance depends on a finely orchestrated equilibrium between inflammatory immune responses and immunoregulatory mechanisms that limit collateral tissue damage. The cytokine interleukin 10 (IL-10) has been shown to play a critical role in this balance in numerous infectious diseases. Studies in animal models have revealed that IL-10 gene-knockout or signaling blockade can enhance resistance to pathogens, and substantially facilitate viral clearance. These same interventions in other infections however, result in more severe disease due to the inability of the immune system to adequately contain the pathogen load, and to control immune-mediate damage. This IL-10-regulated balance is also apparent in human infectious diseases. This review summarizes evidence that IL-10 impacts many aspects of HIV pathogenesis, including the regulation of HIV-specific CD4 and CD8 T cell functions, as well as modulation of HIV-replication in PBMC subsets. Genetic polymorphisms in the IL-10 gene promoter that lead to decreased IL-10 expression have been associated with more rapid disease progression in late stages of HIV infection, suggesting that the anti-inflammatory effects of IL-10 may be protective in the setting of chronic immune activation. We conclude with a discussion of important questions remaining, and the potential for therapeutic intervention based on manipulation of the IL-10 pathway.

Primary HIV-1 infection (PHI) is marked by a flu-like syndrome and high levels of viremia that decrease to a viral set point with the first emergence of virus-specific CD8+ T-cell responses. Here, we investigated in a large cohort of 527 subjects the immunodominance pattern of the first virus-specific cytotoxic T-lymphocyte (CTL) responses developed during PHI in comparison to CTL responses in chronic infection and demonstrated a distinct relationship between the early virus-specific CTL responses and the viral set point, as well as the slope of CD4+ T-cell decline. CTL responses during PHI followed clear hierarchical immunodominance patterns that were lost during the transition to chronic infection. Importantly, the immunodominance patterns of human immunodeficiency virus type 1 (HIV-1)-specific CTL responses detected in primary, but not in chronic, HIV-1 infection were significantly associated with the subsequent set point of viral replication. Moreover, the preservation of the initial CD8+ T-cell immunodominance patterns from the acute into the chronic phase of infection was significantly associated with slower CD4+ T-cell decline. Taken together, these data show that the specificity of the initial CTL response to HIV is critical for the subsequent control of viremia and have important implications for the rational selection of antigens for future HIV-1 vaccines.

Murine models indicate that interleukin-10 (IL-10) can suppress viral clearance, and interventional blockade of IL-10 activity has been proposed to enhance immunity in chronic viral infections. Increased IL-10 levels have been observed during HIV infection and IL-10 blockade has been shown to enhance T-cell function in some HIV-infected subjects. However, the categories of individuals in whom the IL-10 pathway is up-regulated are poorly defined, and the cellular sources of IL-10 in these subjects remain to be determined. Here we report that blockade of the IL-10 pathway augmented in vitro proliferative capacity of HIV-specific CD4 and CD8 T cells in individuals with ongoing viral replication. IL-10 blockade also increased cytokine secretion by HIV-specific CD4 T cells. Spontaneous IL-10 expression, measured as either plasma IL-10 protein or IL-10 mRNA in peripheral blood mononuclear cells (PBMCs), correlated positively with viral load and diminished after successful antiretroviral therapy. IL-10 mRNA levels were up-regulated in multiple PBMC subsets in HIV-infected subjects compared with HIV-negative controls, particularly in T, B, and natural killer (NK) cells, whereas monocytes were a major source of IL-10 mRNA in HIV-infected and -uninfected individuals. These data indicate that multiple cell types contribute to IL-10-mediated immune suppression in the presence of uncontrolled HIV viremia.

The rising incidence of invasive fungal infections and the emergence of broader fungal resistance have led to the need for novel antifungal agents. Posaconazole is a new member of the triazole class of antifungals. It is available as an oral suspension and has a favorable toxicity profile, has demonstrated clinical efficacy in the treatment of oropharyngeal candidiasis and has shown promise as salvage therapy for invasive aspergillosis, zygomycosis, cryptococcal meningitis and a variety of other fungal infections. In addition, data from randomized controlled studies support its efficacy for use in prophylaxis of invasive fungal infections in patients who are severely immunocompromised. The wide spectrum activity of posaconazole in in vitro studies, animal models and preliminary clinical studies suggest that posaconazole represents an important addition to the antifungal armamentarium.

Fusion of the human immunodeficiency virus (HIV) to the plasma membrane of target cells is mediated by interaction of its envelope glycoprotein, gp120, with CD4 and appropriate chemokine receptors. gp120 additionally binds to DC-SIGN, a C-type lectin expressed on immature dendritic cells. This interaction does not result in viral fusion, but instead contributes to enhanced infection in trans of target cells that express CD4 and chemokine receptors. Here we show that DC-SIGN mediates rapid internalization of intact HIV into a low pH nonlysosomal compartment. Internalized virus retains competence to infect target cells. Removal of the DC-SIGN cytoplasmic tail reduced viral uptake and abrogated the trans-enhancement of T cell infection. We propose that HIV binds to DC-SIGN to gain access to an intracellular compartment that contributes to augmentation or retention of viral infectivity.

Dendritic cells (DC) capture microorganisms that enter peripheral mucosal tissues and then migrate to secondary lymphoid organs, where they present these in antigenic form to resting T cells and thus initiate adaptive immune responses. Here, we describe the properties of a DC-specific C-type lectin, DC-SIGN, that is highly expressed on DC present in mucosal tissues and binds to the HIV-1 envelope glycoprotein gp120. DC-SIGN does not function as a receptor for viral entry into DC but instead promotes efficient infection in trans of cells that express CD4 and chemokine receptors. We propose that DC-SIGN efficiently captures HIV-1 in the periphery and facilitates its transport to secondary lymphoid organs rich in T cells, to enhance infection in trans of these target cells.

A homozygous 24-bp deletion (Delta24) was found in the CC chemokine receptor 5 (CCR5) of 11 out of 15 red-capped mangabeys (RCMs), Cercocebus torquatus torquatus, both in Africa and in an American zoo. The CCR5 Delta24 defect encompassed eight amino acids in frame in the fourth transmembrane region. Unexpectedly, RCM-009, one of 11 homozygotes (Delta24CCR5/ Delta24CCR5), was found to be naturally infected with a divergent simian immunodeficiency virus (SIV) strain, which was not R5-tropic, but used CCR2b (R2b) as its major coreceptor. SIVrcmGab1 was the only R2b-tropic SIV among other divergent SIVs tested. Cells transfected with the Delta24 CCR5 did not support entry of R5-tropic SIVmac, SIVcpz, SIVmne, HIV-2, or HIV-1, and were also inactive in signal transduction mediated by beta-chemokines. At 86.6%, the Delta24 allelic frequency was significantly higher than that of the 32-bp deletion found in humans. The Delta24 frequency was 4.1% in 34 sooty mangabeys (SMs), a geographically isolated subspecies that was naturally infected with R5-tropic SIV. Finding identical deletions in two mangabey subspecies separated for 10,000 years or more dates the Delta24 CCR5 deletion as ancient. However, the source of the selective pressure for the high rate of CCR5 deletion in RCMs remains to be determined. The high allelic frequency of the Delta24 CCR5 in RCMs, in comparison to that of SMs, suggests that R2b-tropism may have been acquired by SIVrcm, as an adaptation to CCR5 genetic defects appeared in its host.

We have tested a panel of pediatric and adult human immunodeficiency virus type 1 (HIV-1) primary isolates for the ability to employ the following proteins as coreceptors during viral entry: CCR1, CCR2b, CCR3, CCR4, CCR5, CCR8, CXCR4, Bonzo, BOB, GPR1, V28, US28, and APJ. Most non-syncytium-inducing isolates could utilize only CCR5. All syncytium-inducing viruses used CXCR4, some also employed V28, and one (DH123) used CCR8 and APJ as well. A longitudinal series of HIV-1 subtype B isolates from an infected infant and its mother utilized Bonzo efficiently, as well as CCR5. The maternal isolates, which were syncytium inducing, also used CXCR4, CCR8, V28, and APJ.

The chemokine receptor CCR5 plays a key role in the CD4-dependent entry of human and simian immunodeficiency viruses into target cells. We have mapped the interaction sites on CCR5 for a number of novel anti-CCR5 monoclonal antibodies and have used these to study the role of the CCR5 N-terminal ectodomain in viral entry and to demonstrate differential CCR5 epitope expression on different cell types. Deletions of the CCR5 amino terminal domain or substitution with equivalent regions from other chemokine receptors did not affect cell surface expression or reactivity with loop-specific antibodies, suggesting that the loop regions remained conformationally intact. Exchanges of the amino terminal segment of CCR5 with the equivalent domains of CCR1, CCR2, and CXCR4 did not significantly affect infection with virus pseudotyped with envelope glycoproteins (Envs) from HIV-2 and SIV, but substitution with the CXCR4 sequence abrogated entry mediated by Env from HIV-1. In contrast, deletion of the amino terminus abrogated CCR5 receptor activity for all viral Envs examined. These data indicate that the amino terminus of CCR5 has an essential role in entry mediated by diverse viral Envs but that the sequence requirements are more relaxed for the HIV-2 and SIV Envs compared to the HIV-1 Env examined. This suggests that different viral Envs make distinct and specific interactions with the amino terminus of CCR5. Viral Env utilization of CCR5 expressed on 293-T cells does not always correlate with the cellular tropism of the virus, and one possible explanation is that Env-accessible interaction sites on CCR5 differ on different cell types. We therefore analyzed binding of several anti-CCR5 monoclonal antibodies to cell lines and primary cells that express this chemokine receptor and found that whereas all antibodies bound to CCR5-transfected 293T cells, several did not bind to PBMC. The results suggest that CCR5 undergoes cell type specific structural modifications which may affect interaction with different HIV and SIV envelope glycoproteins.

All of the DNA cleavage and strand transfer events required for transposition of insertion sequence IS10 are carried out by a 46-kDa IS10-encoded transposase protein. Limited proteolysis demonstrates that transposase has two principal structural domains, a 28-kDa N-terminal domain (N alpha beta; aa 1-246) and a 17-kDa C-terminal domain (C; aa 256-402). The two domains are connected by a 1-kDa proteolytic-sensitive linker region (aa 247-255). The N-terminal domain N alpha beta can be further subdivided into domains N alpha and N beta by a weaker protease-sensitive site located 6 kDa (53 aa) from the N terminus. The N beta and N alpha beta fragments are capable of nonspecific DNA binding as determined by Southwestern blot analysis. None of the fragments alone is capable of carrying out the first step of transposition, assembly of a synaptic complex containing a pair of transposon ends. Remarkably, complete transposition activity can be reconstituted by mixing fragment N alpha beta and fragment C, with or without the intervening linker region. We infer that the structural integrity of transposase during the transitions involved in the chemical steps of the transposition reaction is maintained independent of the linker, presumably by direct contacts between and among the principal domains. Reconstitution of activity in the absence of the linker region is puzzling, however, because mutations that block strand transfer or affect insertion specificity alter linker region residues. Additional reconstitution experiments demonstrate that the N alpha region is dispensable for formation of a synaptic complex but is required for complexes to undergo cleavage.